ischemia–reperfusion impairs ascending vasodilation in feed arteries of hamster skeletal muscle

Ischemia-reperfusion injury

Maintaining skeletal muscle function

and vasomotor control

deWith.indd 1deWith.indd 1 27-7-2009 21:17:0727-7-2009 21:17:07

Ischemia-reperfusion injury

Maintaining skeletal muscle function

and vasomotor control

Ischemie-reperfusie schade

Behoud van skeletspierfunctie en regulatie van spierdoorbloeding

(met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor

aan de Universiteit Utrecht

op gezag van de rector magnifi cus,

prof.dr. J.C. Stoof,

ingevolge het besluit van het college voor promoties

in het openbaar te verdedigen op

donderdag 10 september 2009

des middags te 2.30 uur

door

Miriam Cornélie Joan de With

geboren op 19 februari 1977 te Leerdam


Promotoren: Prof.dr. M. Kon

Prof.dr. P.R. Bär

Co-promotoren: Dr. A.B.A. Kroese

Dr. E.P.A. van der Heijden


voor mijn ouders


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Contents

List of abbreviations 8

Chapter 1General Introduction 9

Chapter 2Ischemia-reperfusion impairs ascending vasodilation in feed arteries of 21

hamster skeletal muscle

Miriam CJ de With, Sara J Haug, EPA Brigitte van der Heijden, Steven S Segal

Microcirculation 2005; 12: 551-561

Chapter 3Ischemia-reperfusion (1h-1h) injury of hamster skeletal muscle in vivo 37

is not due to iron mediated formation of reactive oxygen species but

to Ca2+ overload

Miriam CJ de With, PR Dop Bär, EPA Brigitte van der Heijden, Moshe Kon, Alfons BA Kroese

Submitted

Chapter 4Cooling to 21°C prevents ischemia-reperfusion-induced impairment of 57

ascending vasodilation in feed arteries of hamster skeletal muscle in vivo

Miriam CJ de With, Steven S Segal, PR Dop Bär, Moshe Kon, Alfons BA Kroese

Submitted


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Chapter 5Contractile and morphological properties of hamster retractor muscle 73

and rat cutaneus trunci muscle are dissimilarly affected

by 16 hours of cold preservation

Miriam CJ de With, EPA Brigitte van der Heijden, Matthijs F van Oosterhout, M Kon, Alfons BA Kroese

Cryobiology 2009; conditionally accepted for publication

Chapter 6The vascular anatomy of the hamster retractor muscle with regard 91

to its microvascular transfer

Miriam CJ de With, Anne M de Vries, Alfons BA Kroese, EPA Brigitte van der Heijden, Ronald LAW Bleys,

Steven S Segal, Moshe Kon

European Surgical Research 2009; 42 (2): 97-105

Chapter 7General Discussion 107

Chapter 8Nederlandse samenvatting 117

Dankwoord 123

Curriculum vitae 127


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List of abbreviations:

ACh : acetylcholine ATP : adenosine triphosphateAVD : ascending vasodilationBDM : 2,3-butanedione monoximeCT : cutaneus trunci muscleCTA : composite tissue allograftDef : deferiproneFA : feed artery/iesHTK : HTK-Bretschneider solutionH202 : hydrogen peroxideI-R : ischemia-reperfusionKH : Krebs-Henseleit solutionNO : nitric oxideOH : hydroxyl radicalONOO- : peroxynitriteO2

- : superoxide radicalPE : phenylephrinePSS : physiological salt solutionPt : maximum twitch tensionP0 : maximum tetanus tensionRET : retractor muscleROS : reactive oxygen speciesSR : sarcoplasmic reticulumSNP : sodium nitroprusside

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1

General Introduction

9


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Background

Ischemia- reperfusion injury of skeletal muscle tissue is routinely encountered in a variety of surgical settings such as reconstructive free fl ap surgery, abdominal aortic aneurysm repair, traumatic arterial injuries and thromboembolic events involving the extremities (19; 20; 35; 58). Ischemia (litteraly ‘without blood’ in Greek) is the condition of inadequate blood supply to an organ or tissue, leading to decreased levels of oxygen and glucose, and a subsequent shift towards anaerobic metabolism (12). When the ischemic condition is sustained, tissue damage occurs. Re-establishment of arterial blood fl ow, reperfusion, is necessary for survival of ischemic tissue, but can lead to further damage of the tissue and its vascular supply (28; 35; 47). Such damage occurs because the biochemical and molecular changes that occur during ischemia predispose the affected tissue to the formation of reactive oxygen species (ROS) mediated injury (58).

Skeletal muscle

In relation to free tissue transfers in reconstructive surgery, deliberate interruption of the circulation produces ischemia in composite fl aps containing tissues, such as skin, fat, skeletal muscle and bone. Skeletal muscle components are in comparison to bone, fat and skin highly vulnerable to ischemia and reperfusion (I-R) injury, because of a more active metabolism (58). Notwithstanding this fact, the clinical use of free tissue transfers shows relatively low failure rates of 5-6 % (53; 78), although the failure rate in replantations of traumatically amputated digits or limbs is higher (8-18%, (53)). Apart from a complete loss of the graft, the number of partial failures is often not reported and probably underestimated (75). Therefore, I-R injury in skeletal muscle tissue remains a signifi cant clinical problem and needs further exploration.

An emerging entity dealing with I-R injury in skeletal muscle is the clinical application of composite tissue allografts (CTAs) for reconstructive surgery (50;

63). This involves the allotransplantation of complex composite tissues containing skeletal muscle components, such as hands (18) or facial structures (17; 41) from deceased donors, and provides reconstructive surgeons with analogous tissues without donor-site morbidity in the patient. Another major advantage is the possibility of improved anatomical reconstruction of highly specialized tissues such as the larynx or tongue (9;

10). Reliable failure rates of CTA transplantation are not known yet, because of the small number of procedures performed up till now (50). As in free vascularized muscle fl aps, I-R injury in CTAs is best prevented by minimizing the duration of the ischemic period. Successful transplantation procedures are described following ischemic intervals of 4-12.5 h using irrigation with diverse perfusion solutions at 4°C (17; 18). The more frequent application of CTAs requires the design of a preservation strategy that prevents I-R injury and allows for tissue banking with maintenance of cellular function.


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The defi cit in contractile muscle function following I-R can be attributed to a combination of closely interrelated factors (1), including an overload in intracellular Ca2+ (22; 25), free radical formation (74) and shortage of high energy phosphates (51; 52).

CALCIUM OVERLOAD

The cytosolic concentration free ionized Ca2+ is kept very low under physiological conditions, but in response to I-R elevated intracellular Ca2+ levels occur (32; 76) and lead to loss of membrane integrity and reduction of contractile tension (22; 37). These elevated Ca2+ levels are either due to an augmented Ca2+ infl ux by increased membrane permeability or inadequate ATP levels to extrude the Ca2+ out of the cell or into the sarcoplasmic reticulum (SR; (25; 65; 76)). Such a Ca2+ overload can cause hypercontraction and can lead to a cascade of damaging reactions of the muscle cell by activating proteases and phopholipase A.

REACTIVE OXYGEN SPECIES FORMATION An other important factor involved in the pathogenesis of I-R injury in skeletal

muscle tissue is the formation of reactive oxygen species (ROS, (31; 52)). Mitochondria of skeletal muscle cells, and in fact in all cell types, continuously generate ROS. Under normal conditions cells can manage ROS because of an endogenous antioxidant defense network (29). During I-R however, the amount of ROS is increased while natural scavenging systems fall short. In particular the highly reactive hydroxyl radical (OH) causes lipid peroxidation, leading to failure of membrane structures.

DEPLETION OF HIGH ENERGY PHOSPHATES

During ischemia, adenosine triphospate (ATP) stores are progressivelydepleted (46; 73). As a result ATP-dependent ion pumps cannot function properly, leading to intracellular Na+ and Ca2+ accumulation. Ultimately, these accumulations cause membrane depolarization, cell swelling and necrosis (38; 43)).

Microvasculature

Microvascular integrity is essential for the successful application of both free muscle fl aps and CTAs. The main reasons for total fl ap failure remain vasospasm (2; 34) and thrombosis of the anastomosis (78). However, partial fl ap failure is mainly initiated by local ischemia in hypoperfused areas. The latter is often referred to as the no-refl ow phenomenon (66), which is following I-R caused by disturbance in active vasomotility of arteries and arterioles (45).

(Micro)vascular injury is mainly assigned to the endothelium, which has been found more susceptible to the effects of I-R than smooth muscle function (3; 11; 13; 33). Upon reperfusion, injury to the endothelium is attributable to the production of ROS by leukocytes as well as by the endothelium itself (26; 30; 60). It is known that endothelial


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cells intrinsically generate OH, (64; 80) via the Haber-Weiss and Fenton reactions. Upon generation this radical reacts very rapidly with various molecules such as lipids, proteins and DNA, thereby destroying their structure and function (14; 29). Besides, ROS- induced lipid peroxidation of membrane components correlates with ultrastructural changes, including cell swelling and increased permeability (42). An early manifestation of I-R injury to endothelial cell function is the loss of endothelium dependent vasodilation. Until now this was only investigated in experiments where responses to application of acetylcholine were measured in in vitro preparations, such as rings of coronary arteries and isolated mesenteric arteries (3; 59). The effect of I-R on the vasomotor function of ‘in vivo’ arteries controlling blood fl ow to skeletal muscle (‘feed arteries’) is not known.

Feed arteries originate from conduit arteries and provide substantial resistance to blood fl ow before giving rise to arterioles within the muscle (15; 54). In response to contractile activity, vasodilator signals ‘ascend’ from the microcirculation/arterioles in the muscle to encompass the feed arteries upstream (ascending vasodilation; AVD; (54;

77). This AVD enables these proximal vessels to play a key role in regulating blood fl ow delivery into intermediate and terminal arteriolar networks, which control the local distribution and magnitude of blood fl ow in skeletal muscle (15; 54). The endothelium of feed arteries serves as the cellular pathway for conducting the vasodilator response (i.e. hyperpolarization) along the vessel wall and into the surrounding smooth muscle cells to produce relaxation (21). Preservation of the endothelial cell layer is essential to attain peak levels of muscle blood fl ow during contractile activity following an I-R insult.

The general aim of the Utrecht Plastic Surgery Research Laboratory is to develop preservation strategies for CTAs with maintenance of skeletal muscle function and microvascular integrity following ischemia and reperfusion. To this aim, in the past decade a number of investigations have been performed. These studies focused on the effect of cold storage (up to 16 h) on contractile muscle function of isolated rat soleus and cutaneus trunci muscles in various preservation solutions (67-70). Other studies focused on the microvascular bed of the rat cremaster muscle during cold I-R insults (4-

7). The experimental models mentioned above are not suitable for combined functional measurements of skeletal muscle and vasculature. Consequently, a new experimental model was introduced: the hamster retractor muscle.

The hamster retractor muscle model

The hamster cheek pouch retractor (RET) muscle is a long, thin, strap-shaped muscle, running from the lumbodorsal fascia and last three thoracic vertebrae to the cheek pouch (49). The RET serves as an antagonist to the longitudinal musculature of the cheek pouch wall and retains the cheek pouch above the scapula when it is fi lled with material, as well as prevents eversion of the pouch when its content is ejected (49). Its superfi cial location and unique anatomical organization have enabled the muscle


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to be used for intravital studies of oxygen transport (62) and blood fl ow regulation (56;

61; 72) in the microcirculation of mammalian skeletal muscle. The central region of the RET muscle is supplied by posterior retractor feed arteries, which arise from the thoracodorsal artery, a branch of the subscapular artery (27; 48; 49). These feed arteries vary in number, are accompanied by collecting veins, and anastomose with each other at the arteriolar level via arcading segments (27; 48; 61). The RET is unique in that the muscle can be exteriorized while preserving its primary vascular supply (44), enabling muscle force production to be monitored concomitant with the study of microvascular responses at defi ned locations within the peripheral circulation {AVD (see above; (72;77)). This preparation has thereby provided novel insights into the regulation of skeletal muscle blood fl ow (for review see (54; 55). The unique potential of the RET to measure both skeletal muscle function and endothelium dependent ascending vasodilation simultaneously was the primary reason to develop this model for investigations of the effects of I-R and CTA preservation techniques on skeletal muscle tissue and its vasculature.

Experimental parameters

Isometric contractile properties were assessed by fi eld stimulation of in vivo RET muscles (Chapters 2, 3 and 4) or direct stimulation of isolated RET muscles (Chapter 5). The key functional parameter evaluated in this way was maximum tetanus tension (P0; Chapter 5). Ascending vasodilation (AVD) was measured by assessment of RET feed artery diameter increase in response to standardized periods of rhythmic muscle contractions (duty cycles; Chapters 2, 3 and 4). Vascular responses in feed arteries were observed by intravital microscopy.

Interventions

The following interventions to reduce I-R injury were employed in this thesis:

REDUCTION OF ROS- The iron chelator deferiprone was used to investigate the role of iron mediated

OH formation in derangement of RET contractile muscle and endothelial cell function following I-R. Deferiprone has previously been shown to prevent the loss in contractile function of rat heart muscle function during warm I-R (71) and rat skeletal muscles following cold ischemia and reoxygenation (68).

- The vitamin E analogue trolox was examined to determine the contribution of peroxyl mediated loss of RET contractile muscle function during cold storage at 4°C. Trolox has shown to reduce peroxidation in phopholipid bilayers by scavenging peroxyl radicals (16) and contractile defi cit in rat skeletal muscles following cold storage (68).


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REDUCTION OF CALCIUM OVERLOAD

The Ca2+ release inhibitor 2,3-butanedione monoxime (BDM) was tested to diminish the effects of Ca2+ overload on muscle function during I-R and cold storage of the RET. BDM was selected because in skeletal muscle BDM reduces Ca2+ release from the sarcoplasmic reticulum and reversibly inhibits the contractile apparatus (23;

57). Besides, BDM improved contractile function and cytorachitecture of rat skeletal muscles following cold storage (68).

HYPOTHERMIA

The mechanism by which hypothermia protects tissues from ischemic injury is only partly understood. With decrease of temperature, oxygen consumption is reduced because all energy demanding processes are slowed by a factor of 1.5 - 3(Q 10; (8; 24; 79)). The delayed depletion of ATP and phosphocreatine stores associated with hypothermia, and the resulting preservation of cellular ion homeostasis contribute to prolonged maintenance of cellular viability and function during anoxic insults. Besides, it was recognized recently that hypothermia is associated with decreased production of ROS such as superoxide (O2

-) and hydroxyl radicals, as measured in neural and liver tissues (36; 39; 40).

Aim of this thesis

The aim of this thesis was to establish a new experimental model (RET) that allows for combined evaluation of functional parameters of skeletal muscle tissue and (micro)vasculature during I-R insults.

Key questions in this thesis

Does a relatively short period of I-R (1h-1h) affect RET skeletal muscle function and endothelium dependent vasomotor control? (Chapter 2)

What is the cause of the observed attenuation of AVD following I-R? (Chapter 2)

Can substances that limit Ca2+ overload and ROS formation prevent I-R injury to skeletal muscle function and AVD? (Chapter 3)

Does mild hypothermia (21°C) prevent IR- induced impairment of skeletal muscle function and AVD? (Chapter 4)

Do prolonged anoxic cold storage (16h at 4°C) and reoxygenation affect contractile function of isolated RET muscles? (Chapter 5)


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Are skeletal muscles of hibernators more tolerant to hypoxia and hypothermia than muscles of non-hibernators? (Chapter 5)

Can the RET be isolated on its vascular pedicle and be used as a transplantation model? (Chapter 6)

Outline of this thesis

To address the above questions the following investigations are described in this thesis:

In Chapter 2 the effect of I-R (1h-1h) on the ability of hamster RET in vivo feed arteries to dilate in response to contractile skeletal muscle activity (ascending vasodilation) is investigated. Furthermore, the postischemic functional integrity of the endothelium with respect to initiation and conduction of vasodilation independent of muscle fi ber activity is measured.

Using the hamster RET in vivo preparation, the relative contributions of Ca2+ overload and of the formation of OH radicals to the injury of contractile muscle function are studied following I-R (1h-1h) in Chapter 3. Additionally, it is determined whether the formation of OH radicals is a causative factor in the impaired initiation of AVD.

In Chapter 4 the protective effect of hypothermia (21°C) on contractile muscle and AVD in the RET in vivo preparation following I-R (1h-1h) is documented.

Chapter 5 describes the effects of 16 h storage at 4°C in HTK-Bretschneider solution (HTK) on the contractile muscle function and cytoarchitecture of the in vitro hamster retractor muscle (RET) in the non-hibernating state. Furthermore, it is investigated whether the cold preservation of the RET could be improved by the addition of the Ca2+ release inhibitor BDM and the antioxidants trolox and deferiprone. The results are compared to those found previously in the rat cutaneus trunci (CT) muscle, to evaluate if skeletal muscles of hibernators display increased tolerance to hypoxic hypothermia in comparison to muscles of non-hibernators.

Chapter 6 provides anatomic information concerning the RET and its vascular anatomy which was essential for the development of microsurgical methods of vascular isolation required for transplantation of the RET.

Chapter 7 features the general discussion of this thesis, which includes a summary, address to the aims and future perspectives.


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21

2

Ischemia-reperfusion impairs ascending

vasodilation in feed arteries of hamster

skeletal muscle

Miriam CJ de With 1,2 , Sara J Haug 1, EPA Brigitte van der Heijden 2

and Steven S Segal 1

1 The John B Pierce Laboratory and Department of Cellular and Molecular Physiology, Yale University,

New Haven, Connecticut 06519 USA

2 Department of Plastic Reconstructive and Hand Surgery, University Medical Center Utrecht,

Utrecht, The Netherlands

Microcirculation 2005; 12: 551-561


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Abstract

Objective Vasodilation originating within the microcirculation ascends into proximal feed

arteries during muscle contraction to attain peak levels of muscle blood fl ow. Ascending vasodilation (AVD) requires an intact endothelium, as does conducted vasodilation in response to acetylcholine (ACh). Whereas ischemia-reperfusion (I-R) can affect endothelial cell function, the effect of I-R on AVD is unknown. The authors tested the hypothesis that I-R (1h-1h) would impair AVD.

Materials and Methods Using the retractor muscle of anaesthetized hamsters, contractions were evoked

using fi eld stimulation (200 ms at 40 Hz every 2 s for 1 min) and ACh was delivered using microiontophoresis (1 μm tip, 500-4000 ms pulse at 800 nA). Feed artery responses were monitored 500 to 1500 μm upstream.

Results Neither resting (51±4 μm) nor maximal diameter (815 μm; 10 μM sodium

nitroprusside) following I-R (n=8) were different from time-matched controls (n=10). With peak active tension of 234 mNmm-2, control AVD was 262 μm. Following I-R, active tension fell by 48% (p<0.05) and AVD by 57% (p<0.05). Stimulation at 70 Hz restored active tension but AVD remained depressed by nearly half (p<0.05), as did local and conducted responses to ACh. Nevertheless, control responses to 500 ms ACh were restored by increasing stimulus duration to 4000 ms.

ConclusionsIschemia-reperfusion impairs the initiation of feed artery dilation with muscle

contraction and with ACh while conduction along the vessel wall is preserved. Respective components of endothelial cell signaling events may differ in their susceptibility to I-R.


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Introduction

Functional vasodilation in response to skeletal muscle contraction begins in the arterioles supplying the active muscle (18). Once initiated, vasodilation spreads along arteriolar branches to coordinate the magnitude and distribution of muscle blood fl ow in accord with local requirements for capillary perfusion (3; 34). As metabolic demand increases, vasodilation ‘ascends’ into the proximal feed arteries (17; 41) which are located external to the muscle and can provide nearly half of the total resistance to muscle blood fl ow (11). Thus, dilation of feed arteries in concert with arterioles is essential to attain peak levels of muscle blood fl ow during contractile activity (26; 35).

Structural and functional studies have demonstrated a key role for the endothelium

as the cellular pathway for initiating and conducting vasodilator responses in arterioles and feed arteries of skeletal muscle (12; 15; 23). Experiments in isolated pressurized feed arteries of the hamster retractor muscle have shown that acetylcholine (ACh, an endothelium-dependent vasodilator) delivered from a micropipette (15) or direct intracellular injection of electrical current (14) initiates hyperpolarization at the site of stimulation. This electrical response is then conducted rapidly from cell to cell along the endothelium and into the surrounding smooth muscle through gap junction channels, producing relaxation and dilation of the entire vessel (14). Complementary experiments performed in anesthetized hamsters have shown that the integrity of the endothelium is essential for ascending vasodilation (AVD) of feed arteries in response to muscle contraction as well as the conduction of vasodilation in response to ACh (35).

The integrity of endothelial cells can be disrupted by ischemia and reperfusion (I-R) (5; 19) and I-R is experienced routinely during surgical procedures. For example, during reconstructive surgery, interruption of the peripheral circulation produces ischemia of skeletal muscle, resulting in a sequence of events that disrupts cellular function throughout the tissue (6; 43). Reperfusion with arterial blood can lead to further damage within the muscle and its vascular supply (21). Nevertheless, the restoration of tissue blood fl ow and its control by the resistance vasculature following an I-R insult are essential to the survival of reconstructed tissue (6). Remarkably, little is known of how I-R affects blood fl ow control to skeletal muscle in the context of AVD or cell-to-cell conduction into and along feed arteries.

The purpose of this study was to determine the effect of I-R on the ability of feed arteries to dilate in response to muscle contraction. We tested the hypothesis that I-R would impair AVD through disrupting the initiation of vasodilation and its conduction by the endothelium. Experiments were performed using the hamster retractor preparation in order to evaluate AVD in relation to contractile function (41) and ACh was used to evaluate the functional integrity of the endothelium with respect to the initiation and the conduction of vasodilation independent of muscle fi ber activity (35).


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Materials and Methods

Animal care and preliminary surgeryAll procedures were approved by the Institutional Animal Care and Use Committee

of The John B Pierce Laboratory and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Male Syrian golden hamsters (n=18, 96-121 g; Charles River Breeding Laboratories, Kingston, NY, USA) were maintained at 24°C on a 14h/10h light/dark cycle and provided rodent chow and water ad libitum. Hamsters were anaesthetized with pentobarbital sodium (60 mg kg-1, intraperitoneal injection) and tracheotomized with polyethylene tubing (PE 190) to maintain airway patency. The left femoral vein was cannulated (PE 50) to replace fl uids and maintain anaesthesia during the experiment (10 mg pentobarbital ml–1 sterile saline, infused at 410 μl h-1). The depth of anaesthesia was maintained according to the rate of spontaneous ventilation and lack of withdrawal to toe pinch with supplemental anaesthetic infused as needed. Esophageal temperature was maintained at 35-38°C with heat conducted from a warm copper plate positioned beneath the hamster. At the end of each day’s experimental procedures (typically 6-7 h), a hamster was euthanized with an overdose of pentobarbital delivered through the intravenous cannula.

Retractor muscle preparationFollowing preliminary surgery, the hamster was positioned on a transparent acrylic

platform and the right retractor muscle was prepared for study (42). Briefl y, a 2-3 cm incision was made through the overlying skin, which was retracted to expose the underlying tissue. The surgical fi eld was irrigated continuously with bicarbonate-buffered physiological salt solution (PSS, pH 7.4, 34-36°C) of the following composition (mM): NaCl 131.9; KCl 4.7; CaCl2 2.0; MgSO4 1.2; NaHCO3 18; dissolved in deionized purifi ed water (dH2O) and equilibrated with 95% N2/ 5% CO2. Reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) or J.T. Baker (Philipsburg, NJ, USA). Using microdissection while viewing through a stereomicroscope, the muscle and its feed arteries (with paired collecting veins) were cleared of connective tissue and visible nerve branches; great care was taken to minimize trauma. The origin and insertion of the muscle were severed and each was secured in a clamp. While carefully maintaining the integrity of the vascular supply, the muscle was refl ected away from the body, positioned in a chamber (volume ~10 ml) that was integral to the platform, and respective muscle clamps were connected to micrometer drives for control of muscle length (42). Surgical procedures typically required 3-4 h.

The completed preparation was transferred to the stage of an intravital microscope (modifi ed ACM; Zeiss, Thornwood, NY, USA) and superfused continuously (10 ml min-1) with fresh PSS (34-35°C) while the effl uent was removed with a vacuum line to maintain a constant fl uid level and a stable optical image.

Video microscopy and vessel diameter measurementsFeed arteries were observed using brightfi eld illumination (Zeiss ACH/APL


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condenser, numerical aperture (NA)=0.32; Leitz UM32 objective, NA=0.20). The image was coupled to a video camera (C2400; Hamamatsu, Japan) and monitor (PVM 1343 MD, Sony, Japan) with total magnifi cation = 860X. The internal diameter of vessels was measured with a video caliper at the widest point of the vessel lumen or as the width of the column of red blood cells with spatial resolution <2 m (35; 41). To normalize vasodilations across feed arteries with different diameters (range at rest, 31-84 μm; Figure 1), peak responses were expressed as:

% increase = [(peak diameter – resting diameter) / resting diameter] x 100.

The level (%) of spontaneous vasomotor tone was calculated as:

[(resting diameter / maximum diameter) X 100].

Maximum diameter was recorded during the addition of 10-5 M sodium nitroprusside (SNP) to the PSS.

MicroiontophoresisMicropipettes with a tip internal diameter of ~1 m were prepared (Model P-97

pipette puller; Sutter Instruments, Novato, CA, USA) from borosilicate glass capillaries (1.2 mm OD, 0.69 mm ID) and fi lled with ACh (1 M in dH2O). A micropipette was secured in a micromanipulator (MN-151; Narishige, Japan) and connected via an Ag-AgCl wire to a constant current source for microiontophoresis (Model 260; World Precision Instruments, Sarasota, FL, USA) with a platinum electrode in the muscle chamber used for reference. The micromanipulator was mounted on the acrylic platform that contained the hamster, enabling all components of the preparation to be moved as a unit. Thus, with the tip of the micropipette positioned adjacent to the distal end of the feed artery (i.e., just above the site of vessel entry into the muscle), vasodilation to ACh was recorded at the local site of delivery (i.e., at distance = 0) and at 500, 1000 and 1500 m along the vessel upstream from the stimulus site (Figure 2, inset). Retain current (~0.2 A) was adjusted to prevent leakage of ACh from the stimulus micropipette as confi rmed by the lack of vasodilation at rest. To obtain a range of vasodilator responses, ejection current was held at 800 nA with stimulus durations of 500, 1000, 2000 and 4000 ms. Each stimulus was repeated at the local site for recording responses at respective sites along the vessel, which were held constant throughout each experiment. The order in which respective stimulus durations were delivered was varied across experiments. At least 2 min elapsed between each stimulus to allow the vessel to recover resting diameter and minimize any tachyphylaxis to ACh.

Electrical stimulation and muscle contractionPlatinum electrodes (50 mm x 3 mm x 0.2 mm) were positioned along either side

of the retractor muscle and isometric contractions were evoked using fi eld stimulation


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to depolarize motor end plates of skeletal muscle fi bers (26; 42). Stimulus pulses were 0.1 ms at 140 V (~120 mA) and delivered via a SIU-5 stimulus isolation unit driven by an S48 monopolar square wave stimulator (Grass Instruments, Quincy, MA, USA). Muscle tension at rest and during contractions was recorded using a load beam (LCL-1136; Omega, Stamford, CT, USA; 0-100 g; resolution, 0.1 g) mounted in series with a muscle clamp. The active tension developed in response to each stimulus was calculated as the difference between resting tension and the corresponding peak response. These values were normalized to muscle cross-sectional area (calculated from muscle length and wet mass, assuming tissue density = 1.056 mgmm-3) and expressed in mNmm-2 (41).

Ischemia and reperfusionIschemia was induced by gently compressing the feed arteries and collecting veins

using a piece of silicon tubing secured along the gate separating the hamster from the retractor muscle and its feed arteries (42). Cessation of blood fl ow was confi rmed by visual inspection and verifi ed at least every 10 min during the 60 min period of ischemia. As a further precaution during ischemia, the muscle bath was covered with Saran® to prevent oxygen entering the fl uid from room air. The PO2 in the muscle bath under these conditions was <10 torr (MI 730 oxygen electrode and OM-4 meter; Microelectrodes, Inc., Bedford, NH, USA). After 1 h of ischemia, the silastic occluder was removed. As verifi ed by inspection, blood fl ow was restored immediately. This period of ischemia was chosen to limit the degree of endothelial injury and prevent a total loss of vascular tone. Criterion measurements were taken following 1 h of reperfusion, as our preliminary experiments indicated that this period allowed feed arteries to recover resting tone. These durations for I-R are supported by previous investigations (28).

Experimental protocolOnce the preparation was secured on the intravital microscope it was verifi ed that

blood fl ow was intact along feed arteries and throughout the muscle microcirculation. Optimal muscle length for developing twitch tension (L0) was determined and all subsequent procedures were performed at L0. The hamster was then assigned to one of two groups: time controls (n=10), in which resting blood fl ow and superfusion were maintained for 2 h; or as I-R (n=8), where 1 h of ischemia was followed by 1 h of reperfusion. For feed arteries in both groups, the ‘initial’ resting diameter was measured at this 2 h time point. Local and conducted vasodilator responses to ACh were then studied, which required ~1 h. The relationship between stimulus frequency and peak active tension was then evaluated using fi eld stimulation, whereby a 400 ms train was delivered at 40-150 Hz (see Figure 4) to verify a plateau in maximum tetanic tension (P0). A 3-min period of recovery was allowed between consecutive stimulus trains.

Ascending vasodilation was then evaluated in response to rhythmic muscle contractions performed for 200 ms every 2 s for 1 min, corresponding to duty cycle


27

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(DC) of 10% (26). With tissue displacement during each contraction, feed artery diameter was measured between contractions. Previous fi ndings have shown that, in response to muscle contraction, the entire feed artery segment dilates to the same extent (35). For the present experiments, responses were evaluated at the midpoint of the exposed vessel, which was located at least 500 μm upstream from where it entered the muscle. Ascending vasodilation was fi rst evaluated using a stimulation frequency of 40 Hz. Resting tone was allowed to recover for 10 min and AVD was reevaluated at 70 Hz (to restore peak tension – see Results). Outputs from the video caliper (feed artery diameter) and load beam (muscle tension) were sampled at 100 Hz using a MacLab 8s (AD Instruments, Australia) coupled to a personal computer running Chart software (version 4.0.1).

One artery was studied per hamster. In one preparation from each experimental group, spontaneous tone was weak and phenylephrine (PE, 10-8 M) was added to the superfusion solution to obtain a typical level of resting tone. Vasomotor responses for both of these preparations were otherwise similar to those of the vessels studied without PE; therefore the data for each of these vessels were pooled with respective groups. At the end of each experiment, maximum vessel diameter was determined by exposing the preparation to 10 μM SNP. Muscle length (L0) was measured (±0.1 mm) and the muscle was excised, blotted of excess moisture, and weighed (±0.1 mg).

Data analysisTo evaluate the effect of I-R relative to control, responses to ACh microiontophoresis

were measured at four sites along the vessel (0, 500, 1000 and 1500 μm from the site of delivery, respectively). An analysis of variance (ANOVA) was performed to compare the experiments in which responses to all four stimulus durations of ACh were recorded at each site (n=4 vessels per group). Exponential curves were fi tted (least squares) to the change in vessel diameter with respect to distance. A decay constant ‘ d’ (expressed in mm) was calculated to describe the rate at which the amplitude of vasodilation decreased with distance along the feed artery according to the relationship: Ix = I0 e-x/d, where ‘I0’ represents the initial response (expressed as % increase of resting diameter) and ‘x’ is the distance (mm) from the site of application. We tested the null hypothesis that d would not differ between control and I-R treatments or with ACh pulse durations. Values of I0 and d for respective groups were compared using a Greenhouse-Geisser test. With the shortest ACh stimulus (500 ms), the loss of responses from I-R vessels (Results) precluded the determination of a corresponding value for d. In response to electrical fi eld stimulation of the retractor muscle, active tension and AVD were compared between groups using Student’s unpaired t-tests and ANOVA followed by Tukey post hoc comparisons (Sigma Stat; version 3.0, SPSS, Chicago, IL, USA). Figures were prepared using Sigma Plot (version 8.0, SPSS). Summary data are reported as means ± S.E.M., with ‘n’ indicating the number of feed arteries or muscles studied from as many hamsters. Differences were accepted as statistically signifi cant with p<0.05.


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Results

There were no differences between control and I-R groups (n=18) in the initial peak twitch tension when establishing L0 (131 mN mm-2), the muscle length at L0 (28.20.6 mm) or wet muscle mass (1146 mg).

Resting tone recovers following Ischemia-Reperfusion Upon restoration of perfusion following ischemia, feed arteries constricted

transiently and resting diameter recovered over ~ 40 min. Following 1 h of reperfusion (i.e., at the 2 h time point for initial measurements), the resting diameters of feed arteries in control preparations (515 m; n=10) were not different from those following I-R (525 m; n=8), nor was the level of spontaneous resting tone (control, 623 %; I-R, 652 % of maximum diameters obtained with SNP: control, 826 μm; I-R, 797 μm; P=0.4). Figure 1 illustrates the similarity in resting and maximal diameters of individual vessels that comprised respective groups. For both groups, resting diameters recovered consistently within 2 min following ACh stimulation, within 3 min following muscle contraction, and otherwise remained stable during experiments.

Resting diameter (μm)

20 40 60 80 100

Max

imum

dia

met

er (μ

m)

40

60

80

100

120

140

controlI-R

Fig. 1.:Feed artery tone recovers

following ischemia and reperfusion.

For each vessel studied, maximum diameter

(with 10 μM SNP) is plotted against resting

diameter. Note similarity in range of

respective values for control (n=10) and I-R

(n=8) groups. Spontaneous tone averaged

623 and 652 %, respectively.

Ischemia-Reperfusion impairs vasodilation to ACh while conduction persists

The conduction of vasodilation in response to ACh is integral to the pathway of AVD and ACh has consistently proven to be a reliable stimulus for evoking conducted responses (35). Microiontophoresis of ACh produced transient dilations at local and remote sites along every feed artery in this study (Figure 2). After a lag of ~2 s, the entire vessel dilated in unison, which is consistent with rapid, electrical conduction of hyperpolarization along the vessel wall (14). Withdrawing the ACh micropipette >50 μm from the vessel eliminated all responses to microiontophoresis, confi rming the localized nature of this stimulus and verifying that vasodilations observed at remote sites were not caused by diffusion or convection of ACh from the site of delivery. The change in diameter increased with stimulus duration at local (p<0.001) and remote


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sites (p<0.05; Figure 3). Relative to maximal diameters obtained with SNP, the peak local dilation to ACh (4000 ms stimulus) was 918% for control and 484% following I-R (p=0.001).

Following I-R, vasodilator responses to ACh were depressed at all sites compared to time controls (Figures 2 and 3). However, as confi rmed by values for d (Table 1), the decay of vasodilation with distance along vessels was not signifi cantly different (p=0.244) between control and I-R groups. Further, a stimulus of 4000 ms restored local and conducted responses in I-R preparations to those seen in control preparations with a 500 ms stimulus (Figure 3).

30

40

50

60 local 500 μm 1500 μm

30 s

control

I-R

control

control

I-R I-Rretractor muscle

ACh

hamster

1500

1000

500

local

Fig. 2: Local and conducted vasodilation in feed arteries of retractor muscle.

Inset (right panel) illustrates the experimental design; a single feed artery is shown for clarity. Ischemia was

produced with a silastic occluder (black bar) positioned upstream near the hamster. In time controls and

following (I-R), acetylcholine (ACh) was delivered from a micropipette at the downstream end of the feed artery

near the muscle. Diameter was recorded locally at the site of stimulation and 500, 1000 and 1500 m upstream.

Representative traces from control and I-R groups illustrate local responses to ACh (800 nA, 4000 ms; delivered

at arrows) and conducted responses at 500 and 1500 m upstream. Note attenuation of vasodilation at each site

following I-R.

Ischemia-Reperfusion impairs active tension and ascending vasodilation

Active tension produced by the retractor muscle increased with stimulus frequency and was depressed at each frequency following I-R (p<0.05; Figure 4). Nevertheless, P0 was attained consistently at ~100 Hz in both groups. Respective values for P0 were 73±6 and 45±6 mNmm-2 for control and I-R, respectively (p<0.05). Stimulation at 40 Hz evoked contractions that were ~50% of P0 for both groups of muscles and this intermediate level of activation was used to evaluate AVD (26). Active tension was stable during each period of muscle contractions.


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Fig. 3: Ischemia-Reperfusion depresses local and conducted vasodilation to ACh.

Acetylcholine was delivered onto the distal end of a feed artery using microiontophoresis (see Figure 2, inset).

Responses were recorded locally (A) and at remote sites along the vessel located (B) 500 μm, (C) 1000 m, and (D)

1500 m upstream. At each site, the change in diameter (expressed as % increase from rest) is shown in relation

to stimulus duration (ejection current was held at 800 μA). Symbols indicate means S.E.M. with corresponding

number of observations (n). * p<0.05, control vs. I-R.

I0 I0 d d

ACh stimulus (ms) Control I-R Control I-R

500 2 4 ± 7 NR 2.1 ± 0.2 NR

1000 34 ± 7 16 ± 0* 2.2 ± 0.2 2.9 ± 0.2

2000 46 ± 9 21 ± 1* 2.5 ± 0.2 3.2 ± 0.3

4000 53 ± 9 28 ± 2* 2.7 ± 0.1 2. 4 ± 0.1

Table 1: Effect of Ischemia-Reperfusion on local and conducted vasodilation.

Abbreviations: I0, % increase of resting diameter at local site of stimulation; d, decay constant (expressed in mm,

see Methods); I-R, ischemia-reperfusion; NR, no response. Analysis of Variance confi rmed that I0 was impaired at

all stimulus intensities following I-R (p<0.001) while d was not signifi cantly different across stimulus durations

(p=0.522) or between groups (p=0.244).

ACh pulse duration (ms)

0 1000 2000 3000 4000

Cha

nge

in d

iam

eter

(%)

0

20

40

60controlI-R


0 1 000 2000 3000 4000

Cha

nge

in d

iam

eter

(%)

0

20

40

60controlI-R

A B

(9)

(9)

(6)(4)

(5)(5)

(8)(8)

(9)

(9)

(6)

(4)

(5)

(5)

(8)(8)

*

*

*

*

Local 500 μm

* * *

*


0 1 000 2000 3000 4000

Cha

nge

in d

iam

eter

(%)

0

20

40

60controlI-R

(8)(8)

(6) (4)

(5) (5)(7)

(7)

**

*

*


0 1000 2000 3000 4000

Cha

nge

in d

iam

eter

(%)

0

20

40

60controlI-R

C 1000 μm

(9)

(9)

(6)(4)

(5)(5)

(7)(7)

**

**

D 1500 μm


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Stimulus frequency (Hz)

40 60 80 100 120 140 160

0

20

40

60

80

controlI-R

*

**

Act

ive

ten

sio

n(m

N.m

m)

* *

Fig. 4: Ischemia-Reperfusion depresses active

tension of retractor muscle.

Tetanic tension increased with stimulus frequency

and plateaued at 100 Hz for control (n=10) and

I-R (n=8) muscles. Following I-R, active tension

was depressed by a similar extent at each stimulus

frequency. Symbols indicate means S.E.M. *

p<0.05, control vs. I-R.

Ascending vasodilation was recorded approximately midway along the vessel region studied for conduction to ACh; these distances were 71052 and 78826 μm proximal to the edge of the muscle for control and I-R groups, respectively. Diameter typically began to increase within 10 s after the onset of contractile activity and reached a steady state within ~40 s (Figure 5A). With 40 Hz stimulation, feed artery diameter increased by 262 m in control muscles (n=8) and by 131 m following I-R (n=6; p<0.05). To determine whether the loss of active tension following I-R was responsible for the impairment of AVD, preparations that were subjected to I-R rested for an additional 10 min and then stimulated at 70 Hz (200 ms every 2 s for 1 min; n=5). Although active tension was restored to values obtained at 40 Hz in control muscles (Figure 5B), AVD remained depressed by 40% (p<0.05; Figure 5C).

Fig. 5: Ischemia-Reperfusion depresses ascending

vasodilation despite restoration of active tension. (A)

Representative recordings of AVD in feed arteries of hamster

retractor muscle. The 60 s period of muscle contractions (40 Hz

for control; 70 Hz for I-R; see panels B and C for summary data) is

indicated by the bar. Despite similar levels of active tension, AVD

was depressed by ~40%. (B) Active tension during 10% duty cycles

(200 ms every 2 s for 1 min) at 40 Hz for control (n=8) and I-R (n=6)

and at 70 Hz for I-R (n=5). Increasing stimulus frequency to 70 Hz

following I-R restored active tension to a level not different from that

obtained at 40 Hz in control muscles. (C) Ascending vasodilation

during 1 min of muscle contractions with a 10% duty cycle. At the

same stimulation frequency (40 Hz), AVD was depressed by 57%

following I-R. Increasing stimulus frequency to 70 Hz following

I-R restored active tension (panel B) but not AVD, which remained

depressed by 40%. Bars indicate means S.E.M. * P< 0.05 for

comparison indicated.

Cha

nge

in d

iam

eter

(%)

0

20

40

60

30

40

50

60

IR 40 Hz IR 70 Hzcontrol 40 Hz

control 40 Hz IR 40 Hz IR 70 Hz

0

10

20

30

A

C

B

**

*

60 s

control

I-R

*

Act

ive

tens

ion

(mN

.mm

-2)

Dia

met

er (μ

m)


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Discussion

The present study is the fi rst to investigate the effects of ischemia and reperfusion on ascending vasodilation in response to the contractile activity of skeletal muscle. In the hamster retractor muscle preparation, 1 h of ischemia followed by 1 h of reperfusion impaired the ability of contractile activity to produce AVD even when the fall in active tension following I-R was reversed by raising stimulation frequency. In response to ACh, local and conducted responses were also reduced following I-R. Nevertheless, increasing the duration of the ACh stimulus (to 4000 ms) restored local and conducted vasodilation following I-R to levels obtained in control muscles using a brief (500 ms) stimulus. Moreover, the constant (d) for describing the decay of vasodilation with distance from the site of initiation was not signifi cantly affected by I-R. Resting tone and vasodilation to sodium nitroprusside also remained intact. Thus, while the initiation of vasodilation by the endothelium was impaired by I-R, the conduction of vasodilation along the vessel was preserved. These fi ndings suggest that disruption of endothelial cell function following I-R occurs in a graded manner, with impairment in the ability to initiate a signal (e.g., hyperpolarization (15)) preceding impairment in the ability to conduct the signal from cell to cell along the vessel wall.

The functional defi cit in AVD following I-R was clearly manifest with muscle

contractions at 40 Hz, where electrical fi eld stimulation was associated with a pronounced drop in active tension (Figure 5). However, even when stimulation frequency was increased to 70 Hz and active tension was restored to control levels obtained with 40 Hz, AVD remained depressed by nearly half. Stimulating muscles at even higher frequencies in the attempt to further restore AVD was without effect due to muscle fatigue, which can suppress AVD (26). The reduction in active tension produced by the retractor muscle following I-R is consistent with previous reports in skeletal muscle, although the decrement seen here (45% of control) is somewhat greater than reported (10-40%) in rat and rabbit models (7; 9). This functional defi cit can be attributed to a combination of effects, including the shortage of high energy phosphates (32), free radical formation (44), and an overload in intracellular Ca2+ concentration ((10), for review see (33)). However, no difference in resting tone of feed arteries was observed between groups, which was maintained at a level consistent with that reported previously (35; 41). Further, maximum vasodilator responses to SNP (which acts independent of endothelium) were preserved. These fi ndings argue against a loss in smooth muscle function as an explanation for the diminished response of feed arteries to ACh or to muscle contraction following the I-R protocol used here.

Across species and preparations, endothelial cell function has been found to

be more susceptible to the effects of I-R as compared to that of smooth muscle (2; 5; 8;

27). Upon reperfusion, injury to the endothelium is attributable to the production of reactive oxygen species by leukocytes as well as the endothelium (20; 25; 39). These highly reactive molecules induce lipid peroxidation of membrane components which correlate


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with ultrastructural changes including cell swelling and increased permeability (29). Consistent with the present fi ndings, ring preparations of the superior mesentery artery from mice displayed disruption of endothelium-dependent vasodilation to ACh following I-R (45 min-45 min), while relaxation to SNP remained intact (2). Moreover, the attenuation of ACh-induced vasodilation we observed at the site of stimulation is in agreement with that observed for dog coronary arteries (37) and resistance vessels (31). While the present study did not determine whether the loss of endothelium-dependent vasodilation to ACh was reversible, such impairment induced by I-R (1h-1h) in dog coronary arteries had recovered after 4 weeks (28).

The effects of I-R observed here suggest that disruption of endothelial cell function in feed arteries was graded. When taken by itself, the fi nding that AVD was compromised following I-R is of limited value because it does not resolve whether the initiation and/or the conduction of vasodilation was most affected. However, the impairment of local vasodilation to ACh following I-R indicates that the ability of the endothelium to initiate a vasodilator response was affected adversely (Figure 3). Nevertheless, because d was not signifi cantly different between respective groups (Table 1), the ability to conduct the signal along the vessel appeared to be relatively unaffected by I-R. This interpretation is supported by the fi nding that both local and conducted responses to a short (500 ms) ACh stimulus in control preparations could be restored following I-R by increasing the stimulus duration to 4000 ms (Figure 3). Indeed, this outcome suggests that once local vasodilation has been reestablished, conduction of the response along the endothelium and into the smooth muscle (15) is preserved.

At the endothelial cell plasma membrane, the binding of ACh to muscarinic receptors is coupled to G-proteins, which leads to a rise in intracellular Ca2+ and the opening of calcium-activated K+ (KCa) channels to initiate hyperpolarization (4; 30). In feed arteries of the hamster retractor muscle, this hyperpolarization spreads along the endothelium and into surrounding smooth muscle cells via gap junction channels (14; 15; 35). We have previously excluded a crucial role for NO in both the initiation and the conduction of ACh induced vasodilation in these vessels (36). In accord with the present fi ndings, we hypothesize that impairment in the ability to initiate conduction following I-R refl ects damage to the endothelial cell membrane by reactive oxygen species (1; 22; 24; 29). Such damage interferes with G-protein coupled receptor signaling (16) and the ability to initiate hyperpolarization through activation of KCa channels (38; 40). In contrast, the maintenance of conduction suggests that cell-to-cell coupling through gap junction channels (14) remains intact. Indeed this ‘selective’ loss of signal transduction is consistent with an early phase of I-R injury that occurs prior to morphological changes of the vascular wall (27). In turn, we hypothesize that the defi cit in AVD in response to muscle contraction following I-R may be explained by a reduction in the ability to initiate responses in the endothelium of intramuscular arterioles (12; 13). In light of the present and previous fi ndings (26; 35; 41), key questions for future studies include how the coupling between muscle fi ber contraction and the initiation of AVD is affected by I-R, oxygen stress, and reactive oxygen species.


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Conclusion

The present study demonstrates that the initiation of conducted vasodilation in response to ACh and of ascending vasodilation in response to muscle contractions are impaired signifi cantly after 1 h of ischemia followed by 1 h of reperfusion. These functional decrements in the dilation of feed arteries will limit muscle blood fl ow and can be attributed to a derangement in the initiation of the dilator signal (e.g., hyperpolarization) by the endothelium at the site of stimulation. In contrast, once vasodilation is initiated, conduction along the vessel is preserved. Thus, I-R can selectively disrupt particular signaling pathways in the vessel wall while others remain intact. This distinction raises the possibility of developing selective interventions to maximize the recovery of vascular function following an ischemic insult to tissue.

Acknowledgements

This study was supported by grant RO1- HL56786 from the National Institutes of Health, United States Public Health Service and the Jan Dekker and Ludgardine Bouwman Foundation of The Netherlands. The authors thank Dr. Alfons BA Kroese for his essential help with data analysis, valuable discussions and critical review of the manuscript.

References

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2. Banda MA, Lefer DJ and Granger DN. Postischemic endothelium-dependent vascular reactivity is preserved

in adhesion molecule-defi cient mice. Am J Physiol 273: H2721-H2725, 1997.

3. Berg BR, Cohen KD and Sarelius IH. Direct coupling between blood fl ow and metabolism at the capillary

level in striated muscle. Am J Physiol 272: H2693-H2700, 1997.

4. Busse R, Fichtner H, Luckhoff A and Kohlhardt M. Hyperpolarization and increased free calcium in

acetylcholine-stimulated endothelial cells. Am J Physiol 255: H965-H969, 1988.

5. Carden DL and Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol 190: 255-266,

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6. Carroll WR and Esclamado RM. Ischemia/reperfusion injury in microvascular surgery. Head Neck 22: 700-

713, 2000.

7. Carvalho AJ, McKee NH and Green HJ. Metabolic and contractile responses of fast and slow twitch rat

skeletal muscles to ischemia and reperfusion. Plast Reconstr Surg 99: 163-171, 1997.

8. Chen G, Kelly C, Chen H, Leahy A and Bouchier-Hayes D. Thermotolerance preserves endothelial vasomotor

function during ischemia/reperfusion. J Surg Res 94: 13-17, 2000.


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37

3

Ischemia-reperfusion (1h-1h) injury of hamster

skeletal muscle in vivo is not due to iron mediated

formation of reactive oxygen species but to Ca2+

overload

Miriam CJ de With1, PR Dop Bär2, EPA Brigitte van der Heijden1, Moshe Kon1,

Alfons BA Kroese3,4

1,2,3 Departments of Plastic Reconstructive and Hand Surgery1, Biomedical Sciences2 and Surgery3,

University Medical Center Utrecht, Utrecht, The Netherlands

4 Institute for Risk Assessment Sciences, Utrecht University, The Netherlands

Submitted


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Abstract

BackgroundIn the hamster retractor muscle (RET) ischemia-reperfusion (I-R; 1h-1h) leads to

impairment of both the contractile function and the initiation of ascending vasodilatation (AVD). These impairments supposedly result from the action of reactive oxygen species on the myocytes and the endothelial cells in the vessel wall, respectively. Therefore, we investigated the contribution of the highly reactive hydroxyl radical (OH) to the contractile defi cit and the AVD impairment as well as the contribution of Ca2+ overload to the contractile defi cit.

Materials and MethodsUsing electrical fi eld stimulation of the RET in anaesthetized hamsters, contractile

tension (maximum tetanus P0) was assessed. AVD along feed arteries was induced by contractions of intermediate level (40 Hz; duty cycle 10%) and quantifi ed by measuring arterial diameter by intravital microscopy. The effects of I-R (1h-1h) on P0 and AVD were determined in muscles supplemented with the iron chelating antioxidant deferiprone (10-5 M) or the Ca2+ release inhibitor BDM (10-2 M).

ResultsI-R reduced P0 and AVD to 574 and 4913% of control values, respectively. Addition

of BDM prevented the I-R induced reduction of P0 (to 895% of control). The contractile muscle function and AVD were not protected by the addition of deferiprone (447% and 499% of control, respectively).

ConclusionThe protection by BDM and lack of protective effect of deferiprone show that the defi cit

in contractile muscle function resulting from I-R (1h-1h) injury can be attributed to a Ca2+ overload and not to injury resulting from iron mediated OH formation. Moreover, not OH, but most likely other reactive oxygen species are a causative factor for impairment of endothelium dependent AVD.


39

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Introduction

Ischemia-reperfusion (I-R) of skeletal muscle tissue occurs routinely during surgical procedures, such as vascular surgery, replantations and free tissue transfers. In these procedures, the obligatory interruption of the peripheral circulation results in a sequence of events that disrupt cellular function throughout the muscle (5; 56). Restoration of tissue blood fl ow is essential for the survival and recovery of skeletal muscle (5), but can lead to additional damage of the muscle and of its vascular supply (22; 24).

The defi cit in contractile muscle function following I-R can be attributed to a combination of closely interrelated factors (2), including an overload of intracellular Ca2+ (17; 20), free radical formation (57) and shortage of high-energy phosphates (41); for review see (42). The cytosolic concentration free Ca2+ ions is kept very low under physiological conditions, but in response to I-R, elevated intracellular Ca2+ levels occur (25; 58) and lead to loss of membrane integrity and reduction of contractile tension (17; 27). These elevated Ca2+

levels are either due to an augmented Ca2+ infl ux by increased membrane permeability, or inadequate ATP levels to extrude the Ca2+ out of the cell or into the sarcoplasmatic reticulum (SR; (20; 51; 58)). Such a Ca2+ overload can cause hypercontraction and also lead to a cascade of damaging reactions of the muscle cell by activating proteases and phopholipase A. The three main causative factors for I-R injury mentioned above have been considered as separate players, but it has been recognized recently that these processes are interrelated extensively (2; 9). Reactive oxygen species (ROS), and especially hydroxyl (OH), interfere with Ca2+ channels such as the Ca2+ release channel (RyR) in the sarcoplasmatic reticulum (SR) and with L-type Ca2+ channels in the skeletal muscle membrane (34; 61). Thus the elevated OH levels in skeletal muscle following I-R (39) may contribute to the functional defi cit by interfering with the Ca2+ homeostasis.

Disturbance in active vasomotility of feed arteries (21) is a major factor in development of the no-refl ow phenomenon in skeletal muscle following I-R (35). Feed arteries originate from conduit arteries and provide substantial resistance to blood fl ow, before giving rise to arterioles within the muscle (8; 43). In response to contractile muscle activity, vasodilator signals ‘ascend’ from the arterioles to encompass the feed arteries upstream (ascending vasodilation; AVD (43; 60)). These signals enable the feed arteries to serve a key role in regulating blood fl ow delivery into arteriolar networks. In feed arteries of the hamster RET, the endothelium serves as the cellular pathway for conducting the vasodilator response (ie hyperpolarization) along the vessel wall (15). We previously demonstrated that following I-R (1h-1h; 37°C) the AVD in RET feed arteries was diminished to ~50% of control values (10). This reduction of AVD was due to impairment of the local endothelial response to the vasodilatory trigger distally in the vascular tree of the muscle (10). I-R did not affect the endothelial cell-to-cell coupling through gap junction channels (14). We assumed (10) that the impairment in the ability to initiate AVD refl ected damage to the endothelial cell membrane by ROS (1; 19; 32).


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When subjected to I-R, endothelial cells generate ROS, such as the highly reactive hydroxyl radical (OH; (50; 63)). This radical is formed via the Haber-Weiss and Fenton reactions and upon generation, it reacts very rapidly with various molecules such as lipids, proteins and DNA, thereby destroying their structure and function (7; 23). Such damage interferes with the action of KCa channels (47; 49), of which the opening serves in AVD to initiate endothelial hyperpolarization (12). For both the Haber-Weiss and Fenton reactions free iron is essential as a transition metal. In isolated arteries, the presence of iron chelators during I-R has been shown to prevent the OH-induced decrease in operative KCa channels, as well as the reduced vasodilation in response to ACh (47; 50). With respect to endothelium dependent AVD in vivo, however, the involvement of ROS or OH in the effects of I-R has not been demonstrated so far (10).

The aim of this study was to assess the relative contributions of Ca2+ overload and of the formation of OH radicals to the injury of contractile muscle function. Additionally, we aimed to determine whether the formation of OH radicals is a causative factor in the impaired initiation of AVD. Therefore, contractile parameters and feed artery AVD were measured in the in vivo hamster retractor preparation following I-R (1h-1h). Pharmacological interventions were employed by addition during I-R of BDM, which reduces Ca 2+ release by the SR and thereby prevents hypercontraction (18; 46) or deferiprone, which inhibits iron-catalyzed OH mediated lipidperoxidation (33).


Animal care and preliminary surgeryMale Syrian golden hamsters (n=32, 1242 g; Harlan-Winkelmann, Borchen,

Germany) were housed in groups of 3-4 animals on a 12h/12h (light-dark) cycle and provided with standard diet food and water ad libitum. The animals received care in compliance with the European Convention guidelines (86/ 609/EC). All procedures were approved by the Utrecht University Committee for experiments on animals. Hamsters were injected with buprenorfi nehydrocloride (Temgesic®; Schering-Plough, Utrecht, The Netherlands) 5 mg/kg subcutaneously 30 minutes prior to surgery. Isofl urane® (ABBOTT Animal Health, Oudewater, The Netherlands), supplied by a vaporizer (Isotec 5, Datex Ohmeda, Madison, WI, USA) through a ventilation mask, at 5% with 100% oxygen (1 l/ minute) was used to induce anaesthesia induction (~5 minutes) and at 1,5 - 2 % with oxygen (0.25 l/min) and air (0.5 l/min) to maintain anaesthesia. Excess vapor was scavenged by a vacuum system. Sterile saline was delivered through a cannula (PE 50) secured in the left femoral vein to replace fl uids during the experiment (300 μl h-1;Fresenius Orchestra, Fresenius Kabi, Utrecht, The Netherlands). Rectal temperature was maintained at 36-38°C with heat conducted from a warm copper plate positioned beneath the hamster. At the end of each day’s experimental procedures (typically 7-8 h), the hamster was euthanized with an overdose of pentobarbital (Nembutal®; CEVA Sante Animale BV, Maassluis, The Netherlands) delivered through the intravenous cannula.


41

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platform and the right RET muscle was prepared for study (10; 55). Briefl y, a 2-3 cm incision was made through the overlying skin, which was retracted to expose the underlying tissue. The surgical fi eld was irrigated continuously with bicarbonate-buffered physiological salt solution (PSS, pH 7.4, 34-36°C) of the following composition (mM): NaCl 131.9; KCl 4.7; CaCl2 2.0; MgSO4 1.2; NaHCO3 18; dissolved in deionized purifi ed water (dH2O) and equilibrated with 95% N2/ 5% CO2. All reagents were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Using microdissection while viewing through a stereomicroscope (Zeiss OPMI 6 DF), the muscle and its feed arteries (with paired collecting veins) were cleared of connective tissue and visible nerve branches; great care was taken to minimize trauma during microdissection. The origin and insertion of the muscle were severed and each was secured in a clamp. While carefully maintaining the integrity of the vascular supply, the muscle was refl ected away from the body, positioned in a chamber (volume, ~10 ml) that was integral to the platform. Respective muscle clamps were connected to micrometer drives for control of muscle length (55). Surgical procedures typically required 3-4 h.

The completed preparation was transferred to the stage of an intravital microscope (Axiotech 100 HD; Zeiss, Sliedrecht, The Netherlands) and superfused continuously (20 ml min-1) with fresh PSS (34-36°C) while the effl uent was removed with a vacuum line to maintain a constant fl uid level and a stable optical image.

Video microscopy and vessel diameter measurementsFeed arteries were observed using a Zeiss LD Achroplane (20X, numerical aperture,

0.40) objective lens. The image was coupled to a color video camera (Sony Hyper HAD; CCD-IRIS/RGB; Sony, Japan) and monitor (Hitachi, Japan) with total magnifi cation = 980X. The internal diameter of vessels was measured with a video caliper at the widest point of the vessel lumen or as the width of the column of red blood cells. To normalize vasodilations across feed arteries with different diameters (range at rest: 16-69 μm), peak responses were expressed as:


The level (%) of spontaneous vasomotor tone was calculated as:


Maximum diameter was recorded during superfusion with 10-5 M sodium nitroprusside (SNP) to the PSS.


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Load beam

Muscle

PSS

MicrometerAspirationMicroscope

SuperfusionB

MuscleChamber

Occluder

Hamster

A

Fig. 1: Experimental setup.

(A) (top view), the RET muscle is refl ected away from the anaesthetised hamster, immersed in a chamber (10 ml

volume) integral to the acrylic platform, and secured in clamps connected to micrometer spindles at each end to

control muscle length. Fresh PSS is introduced at the proximal end (10 ml min-1) and aspirated at the distal end to

maintain constant fl uid level with superfusion along the muscle. (B) (side view), the PSS is introduced through ports

positioned above and below the muscle to minimize unstirred layers. A load beam (LCL-1136, Omega; Stamford,

CT, USA; resolution: 0.1 g) is mounted in series with the muscle records tension. Platinum electrodes are positioned

(within the chamber shown in (A)) along both sides of the muscle for activating skeletal muscle fi bres through fi eld

stimulation. A silastic occluder (as shown in (A)) was used to induce ischemia. Figure 1 is adapted from (55).


of the retractor muscle and isometric contractions were evoked using fi eld stimulation to depolarize motor end plates of skeletal muscle fi bers (26; 55). Stimulus pulses (0.1 ms) were delivered via a S4 stimulator driven by a S88 stimulator (both Grass Instruments, Quincy, MA, USA). Muscle tension at rest and during contractions was recorded using a load beam (LCL-113G; Omega, Stamford, CT, USA; 0-100 g; resolution, 0.1 g) mounted in series with a muscle clamp. The active tension developed in response to each stimulus was calculated as the difference between resting tension and the corresponding peak response. These values were normalized to muscle cross-sectional area (calculated from muscle length and wet mass, assuming tissue density = 1.056 mgmm-3) and expressed in mNmm-2 (10; 54).

Ischemia and reperfusionIschemia was induced by gently compressing the feed arteries and collecting veins using

a piece of silicon tubing secured along the gate separating the hamster from the retractor muscle and its feed arteries (see Figure 1,(10)). Cessation of blood fl ow was confi rmed by visual inspection and verifi ed at least every 10 min during the 60 min period of ischemia.


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As a further precaution during ischemia, the muscle bath was covered with Saran® to prevent oxygen entering the fl uid from room air. The PO2 in the muscle bath under these conditions has been shown <10 torr (10). After 1h ischemia, the silastic occluder was removed. As verifi ed by inspection, blood fl ow was restored immediately. This period of ischemia was chosen to limit the degree of endothelial injury and prevent a total loss of vascular tone. Criterion measurements were taken following 1 h of reperfusion, as we have shown that this period allowed feed arteries to recover resting tone (10).

Addition of drugsThe Ca2+ release inhibitor 2,3-butanedione monoxime (BDM; Sigma-Aldrich

Chemie, Zwijndrecht, NL) was added to the PSS to diminish the effects of Ca2+ overload on muscle function during IR. BDM (10-2 M) was dissolved directly in warm PSS. This concentration prevents the loss in rat contractile muscle function following cold ischemia and reoxygenation (52). Since BDM has been demonstrated to inhibit smooth muscle contraction (18; 38) and to cause vasodilation (48), BDM was washed out from the PSS during the second experimental hour (ie ‘normal blood fl ow’ in controls and ‘reperfusion’ in I-R) (52).As expected, feed arteries dilated in immediate response to the addition of BDM to the PSS. However, resting tone of feed arteries did not recover upon removal of BDM, and even application of PE 10-8 M could not resume normal resting tone. Tone was lost completely, as exposing the preparations to SNP did not result in diameter increase. Therefore, only contractile muscle function and not AVD was assessed in these preparations.

The iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (deferiprone; CP20; L1; Sigma-Aldrich Chemie, Zwijndrecht, NL) was evaluated for indicating the role of iron mediated OH formation in derangement of RET contractile muscle and endothelial cell function following I-R. Deferiprone was prepared as a concentrate in warm d2O daily and diluted in fresh PSS to the fi nal working concentration (‘def PSS’; 10-5 M). This concentration prevents the loss in contractile muscle function of the RET following cold ischemia and reoxygenation (11) and rat heart muscle function during warm I-R (53). Deferiprone was present in the superfusion media throughout the experimental course. Additionally, in a separate series of experiments, deferiprone was dissolved in warm sterile saline and administered systemically through the intravenous femoral canula (‘def IV’; infusion at 300 μl h-1). This was done to optimize the availability of deferiprone to the tissue. We aimed to reach an intravascular steady state concentration of 10-5 M during I-R. Therefore, hamsters were given a loading dose of 510-6 mol/kg of deferiprone at least 4 h prior to induction of ischemia, followed by a continuous infusion of 510-6 mol/kg/h for the duration of the experiment. The protocol used was based upon the pharmacokinetics of deferiprone in the rat (16).

Experimental protocolOnce the preparation was secured on the intravital microscope it was verifi ed that

blood fl ow was intact along feed arteries and throughout the muscle microcirculation within the muscle. Muscle length was adjusted to optimal length for developing twitch


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tension (L0), which is 115% of in vivo resting length, corresponding to a preload of ~5-10 mN (59). All subsequent procedures were performed at L0. Each muscle preparation was equilibrated in PSS for 60 minutes at 370.5°C to establish stable baseline conditions. Subsequently, the RET was subjected to one of two procedures: ‘time control’, in which resting blood fl ow and superfusion were maintained for 2 h; or ‘I-R’, in which 1 h of ischemia was followed by 1 h of reperfusion. In all experimental groups, the ‘initial’ resting diameter of feed arteries was measured at this 2-h time point and then contractile function of the RET was evaluated.

In control experiments it was verifi ed that deferiprone in PSS (n=3) or IV (n=2) did not infl uence resting tone, contractile properties and ascending vasodilation in time controls (data not shown; p-values range from 0.7 to 1.0). To obtain a twitch curve, stimulation voltage of a single pulse was increased incrementally, until twitch tension reached its maximum (Pt; see Figure 2A). A one-minute rest period was allowed between successive stimuli. After measurement of Pt, a muscle rested for 5 minutes. All further stimulation was performed at 1.5X the Pt stimulation intensity. The relationship between stimulus frequency and peak active tension was then evaluated, to verify a plateau in maximum tetanic tension (P0; see Figure 2B). For this procedure, a 400 ms stimulus train was delivered at 40-150 Hz. A 2-min period of recovery was allowed between consecutive stimulus trains.

Ascending vasodilation was then evaluated in response to rhythmic muscle contractions performed for 200 ms every 2 s, for 1 minute, corresponding to duty cycle (DC) of 10% (26). With tissue displacement during each contraction, feed artery diameter was measured between contractions. Previous fi ndings have shown that, in response to muscle contraction, the entire feed artery segment dilates to the same extent (44). For the present experiments, responses were evaluated at the midpoint of the feed artery, which was located approximately 1000 μm upstream from where it entered the muscle. Ascending vasodilation was fi rst evaluated using a stimulation frequency of 40 Hz. Resting tone was allowed to recover for 10 min and AVD was reevaluated at 55 Hz (to restore peak tension – see Results). Outputs from the video caliper (feed artery diameter) and load beam (muscle tension; low-pass fi ltered at 5 Hz) were sampled at 250 Hz using a 1401 Micro CED system (Cambridge Electronic Design, Cambridge, UK) coupled to a personal computer running CED software (Spike 2).

One feed artery was studied per hamster. In seven preparations (n=2 control; n=2 I-R; n=2 I-R def PSS; n=1 def IV), spontaneous tone was weak and phenylephrine (PE, 10-

8 M) was added to the superfusion solution to obtain a level of resting tone typical for these vessels (10; 44; 54). Vasomotor responses for these preparations were otherwise similar to those of the other 15 vessels studied without PE; therefore the data of these vessels were pooled with respective groups. At the end of each experiment, maximum vessel diameter was determined by exposing the preparation to 10-5 M SNP. Muscle length (L0) was measured (± 0.1 mm) and the muscle was excised, blotted of excess moisture, and weighed (± 0.1 mg).


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Data analysisStatistical analysis of the experimental data was performed using ANOVA followed

by Holm-Sidak post hoc comparisons (Sigma Stat; version 3.0, SPSS, Chicago, IL, USA). Figures were prepared using Sigma Plot (version 8.0, SPSS). Summary data for all parameters are reported as means ± S.E., with ‘n’ indicating the number of feed arteries or RET preparations studied. Differences were accepted as statistically signifi cant with p<0.05. The lines plotted in Figure 3 were fi t to the data using linear regression (Origin 7.5; Microcal Software Inc, Northampton, MA, USA).

Results

Hamsters remained stable throughout each day’s experiments as demonstrated by a stable heart rate (~350 beats per minute) and spontaneous ventilation (~32 breathings per minutes). There were no differences in the muscle length at L0 (29.7. 0.3 mm; n=32) or wet muscle mass (1153 mg; n=32) between groups. A total of 32 hamsters were used in the present experiments.

Effect of I-R and additives on RET contractile parametersAs shown in Figure 2A, a maximum for twitch tension (Pt) of 6.40.7 mNmm-2 was

reached at 20-30 V in control muscles (n=6). The amplitude of the tetanus increased with stimulus frequency until a maximum (P0) of 19612 mNmm-2 was reached at ~ 120 Hz (Figure 2B). This value is comparable to that reported previously ((26; 54); ~140mNmm-2 ). Following I-R, Pt and P0 were consistently attained at 20-30 V and 120 Hz, but depressed to 3.90.3 and 1117 mNmm-2 (Table 1 and Figure 2). This I-R induced decrease of active muscle tension is similar to values reported in a previous study (10).

n Pt

(% of control)

p-value P0

(% of control)

p-value

control 6 100 11 - 100 6 -

I-R 5 61 4 0.01 57 4 ≤0.001

I-R deferiprone in PSS 5 44 11 ≤0.001 44 7 ≤0.001

I-R deferiprone intravenously 4 61 16 0.02 54 5 ≤0.001

I-R BDM 4 96 6 0.8 89 5 0.2

Table 1: Effect of BDM and deferiprone on the contractile properties of the RET.

Values for twitch (Pt) and tetanus tension (P0) are expressed as % of control values SE; n is indicated. Muscles were

subjected to control or I-R procedures. P-values represent differences compared to control. Deferiprone and BDM did

not have effect on contractile properties of control muscles (0.6<p<0.8). BDM but not deferiprone effectively prevented

I-R induced impairment of Pt and P0.

The Ca2+ release inhibitor BDM was added to the PSS (n=4) to determine the involvement of Ca2+ overload to the defi cit in contractile parameters resulting from I-R.


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Figure 2 and Table 1 show that the presence of BDM in PSS completely preserves Pt and P0 of the RET to control level, indicating that Ca2+ overload is the key factor in contractile defi cit following 1 h of reperfusion. This preservation of contractile function by BDM can not be attributed to the enhanced intramuscular blood fl ow induced by BDM, because control experiments showed that addition of BDM to control RET did not affect P0 (n=2; p=0.6).

The iron chelating antioxidant deferiprone was added to the PSS (n=5) in order to assess the role of iron-mediated formation of OH to muscular and microvascular derangement due to I-R. To optimize the availability of deferiprone, in a separate group (n=4) the compound was administered systemically by continuous intravenous infusion. Pt and P0 values, which were reduced by I-R to 614 and 574% of control values respectively, were not recovered by either the addition to the PSS (4411% and 447%) or intravenous administration (6116 and 545%) of deferiprone (Table 1 and Figure 2). Control experiments showed that addition of deferiprone to control RET (bath and IV) did not affect P0 (n=5; p=0.75).

Fig. 2: Effect of BDM and deferiprone on

maximum twitch (Pt; A) and tetanus

(P0; B) responses of RET muscles

subjected to I-R.

(A) Maximum twitch (Pt) and (B) tetanus (P0)

tensions are decreased following I-R (; n= 5)

to 61 4 and 57 4% respectively of controls

(; n = 6). The addition of Ca2+ release

inhibitor BDM

(; n = 4) prevents I-R induced contractile

impairment.

The addition to the PSS

(; n = 4) or intravenous administration

(; n = 5) of deferiprone does not lead to

improvement of contractile function. Symbols

represent mean values S.E. * values differ

signifi cantly from control muscles.

A

B

****

***

**

*

********

Stimulus voltage (V)

Act

ive

tens

ion

(mN

.mm

)

0200 30 40

2

4

6

Twitch tension (Pt)

controlI-R BDM

I-R

I-R def PSS

I-R def IV

*

*

*

**

*

*

**

*

*

**

*

*


40 60 80 100 120 140 1600

50

100

150

200 control

I-R BDM

I-R

I-R def PSSI-R def IV

0 1

Act

ive

tens

ion

(mN

.mm

)

Tetanus tension(P )


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Effect of I-R and additives on artery diameter and AVD Upon restoration of perfusion following I-R, feed arteries constricted transiently

and resting diameter recovered over ~40 min. Following 1 h of reperfusion, the resting diameters of feed arteries following I-R (479 m; n=5; p=0.92), were not different from those in control preparations (439 m; n=5), nor was the level of spontaneous resting tone (I-R, 603 %; control, 573 % of maximum diameters obtained with SNP: I-R, 7712 μm; control, 7513 μm; p=1.0 and 0.9 respectively). These values are comparable to those previously reported (10; 26). The addition to the PSS or intravenous administration of deferiprone did not infl uence resting tone in control or I-R feed arteries (0.9<p<1.0). Figure 3 illustrates the similarity in resting and maximal diameters of individual vessels that comprised control and I-R groups. Resting diameters recovered consistently within 3 min following muscle contractions, and otherwise remained stable during experiments.

Fig. 3: Relation between resting and maximum

vessel diameter.

For each vessel, maximum diameter (10 M SNP) is

plotted against resting diameter. Control (n=9) and

I-R (n=13) groups comprise RET supplemented with

deferiprone (control n=4; I-R n=8), as tone did not

differ between RET without or supplemented with

deferiprone (0.7<p<0.9). The uninterrupted line

represents a linear fi t (Origin 7.5; Microcal Software

Inc, Northampton, MA, USA) for all control vessels

(intercept 14.8; slope 1.32; p<0.0001; R=0.910).

The dotted line represents a linear fi t for all vessels

subjected to I-R (intercept 11.6; slope 1.49;

p<0.0001; R=0.962). Comparison of the two lines

shows similarity of resting and maximum diameters

of control and I-R feed arteries (p=0.966).

Stimulation at 40 Hz evoked contractions with stable active tensions that were ~45% of P0 and this intermediate level of activation was used to evaluate ascending vasodilation (AVD, (26)). Ascending vasodilation was recorded 954 17 m (n=23) proximal to the edge of the muscle. The diameter typically began to increase ~10 seconds after onset of contractile activity and reached a steady state within ~40 seconds (Figure 4A). With 40 Hz stimulation, feed artery diameter increased by 59 3% in control muscles (n=5) and by 29 8% following I-R (n=5; p<0.01). To determine whether the loss of active tension was responsible for the impairment of AVD, preparations that were subjected to I-R were also stimulated at 55 Hz. Although active tension was restored to values obtained at 40 Hz in control muscles (Figure 5 and Table 2), AVD remained depressed (to 618% of control). These I-R induced effects of AVD are similar to those previously reported (10).

Resting diameter (μm)

10 20 30 40 50 60 70 80

Max

imum

dia

met

er (μ

m)

20

40

60

80

100

120

140

controlI-RcontrolI-R


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In RET exposed to I-R and deferiprone, stimulation at 40 Hz produced an active tension far below control level (494% for def PSS and 384% for def IV). Although in response to stimulation with 55 Hz active tension was restored to values obtained at 40 Hz in control muscles (p=0.92; Figure 5), AVD remained depressed to 6720% with deferiprone added to the PSS (n=4; p=0.10) and to 529% when deferiprone was administered intravenously (n=3; p<0.01; for representative sample see Figure 4C).

Dia

met

er (m

m)

30

35

40

45

50

Dia

met

er (m

m)

25

30

35

40

45

50

Dia

met

er (m

m)

40

45

50

55

60

65

70

75

80A

B

C

control

I-R

I-R def IV

60 s

Fig. 4: Lack of effect of deferiprone on AVD in feed arteries of the RET.

Representative feed artery diameter changes evoked by 60 s (bar) muscle contractions of the RET. The stimulus

protocols were such (10% duty cycle; control at 40 Hz (A); I-R (B) and I-R def IV (C) at 55 Hz) that similar levels of

active muscle tension (about 55 mNmm-2) were evoked (see Figure 5). In comparison to control responses (A), the

AVD was typicallly depressed by ~40% in I-R (B) and I-R def IV (C) muscles.


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on to ol ed ly

f

Cha

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(%)

0

10

20

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70 controlI-RI-R def PSSI-R def IV

Act

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(mN

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0

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60

70 controlI-RI-R def PSSI-R def IV

40 Hz 55 H z

40 Hz 55 H z

5 5 35 43 3(n = )

5 5 35 43 3(n = )

Contractile tension

Ascending vasodilation

p<0.01

* *

*

p<0.01

* **

p<0.01

**

p<0.01

p=0.10

p=0.92

A

B

Fig. 5: I-R induced depression of AVD is not prevented by deferiprone.

(A) Active tension during 10% muscle contractions of 60 s at 40 Hz for control, I-R, I-R def PSS and I-R def IV and at

55 Hz for I-R, I-R def PSS and I-R def IV. Increasing stimulus frequency from 40 to 55 Hz following I-R restored active

tension to a level not different from that obtained at 40 Hz in control muscles (about 55 mNmm-2). The addition

to PSS or intravenous administration of deferiprone did not cause an increase of active tension in I-R muscles. Bars

indicate means S.E. Relevant p-values for comparisons with control values are indicated.

(B) AVD during 60 s of muscle contractions. At stimulation frequency 40 Hz, AVD was depressed by 49% following

I-R. Increasing stimulus frequency to 55 Hz restored active tension of I-R muscles (see A) but not AVD, which remained

depressed by 39%. The addition to PSS or intravenous administration of deferiprone did not cause an increase of AVD

when compared to I-R muscles. Bars indicate means S.E. Relevant p-values for comparisons with control values are

indicated.


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Stimulus

Frequency

n Tension

(% of control)

p -value Diameter increase

(% of control)

p -value

40 Hz control 5 100 18 - 100 6 -

I-R 5 51 7 ≤0.01 49 13 ≤ 0.01

I-R def PSS 5 49 9 ≤0.01 49 9 ≤ 0.01

I-R def IV 3 38 4 ≤0.01 40 12 ≤ 0.01

Active tension similar to control at 40 Hz

55 Hz I-R 3 99 15 0.92 61 8 ≤ 0.01

I-R def PSS 4 87 16 0.92 67 20 0.10

I-R def IV 3 89 18 0.92 52 9 ≤ 0.01

Table 2: Effect of the addition of deferiprone on ascending vasodilation in RET feed arteries during electrical

stimulation.

Values for active tension and ascending vasodilation (increase in resting diameter) during electrical stimulation (duty

cycle 10%) are expressed as % of control values SE; n is indicated. Muscles were subjected to control or I-R procedures.

P-values represent differences compared to control. Abbreviations for experimental groups are: def PSS: deferiprone added

to the physiologic salt solution (10-5 M); def IV: deferiprone administered intravenously (10-5 M). As I-R impairs active

tension (upper panel), all I-R muscles were additionally stimulated at a higher frequency that restored active tension to

control level (55 vs 40 Hz, lower panel). Despite restoration of active tension, ascending vasodilation remained depressed.

The addition of deferiprone did not prevent I-R induced impairment of either active tension or ascending vasodilation.

Discussion

Both the contractile function and the ascending vasodilation (AVD) of the RET were severely impaired following I-R (1h-1h). The present results show that presence of the Ca2+ release inhibitor BDM causes a complete maintenance of muscle contractile function following I-R (1h-1h) and thereby indicate that an overload in intracellular Ca2+ is an important factor contributing to the muscle tension defi cit. Such an accumulation of intracellular Ca2+ in muscle fi bres has been demonstrated in several isolated rat muscle preparations (17; 25; 58) and results from increased Ca2+ release from the sarcoplasmatic reticulum (SR), decreased reuptake of Ca2+ by SR Ca 2+ATPase and an increased Ca2+ infl ux (51). The putative action of BDM in skeletal muscle is reduction of Ca2+ release from the SR and a reversible inhibition of the contractile apparatus, which together prevent hypercontraction-induced damage to the sarcolemma (18; 46). Moreover, BDM also reversibly blocks L-type Ca2+ channels in skeletal muscle (13). The preservation of contractile function by BDM can not be attributed to the enhanced intramuscular blood fl ow induced by BDM, because P0 was not increased in RET supplemented with BDM.

In skeletal muscle intracellular Ca2+ overload and oxygen free radical formation are supposed to be interrelated extensively (reviewed in (2; 9)). Especially the highly reactive hydroxyl radical (OH), of which the formation is catalyzed by iron, is thought to contribute


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to the destruction of the SR and impairment of contractile myocyte function following I-R (39). The complete lack of protective action of the potent iron chelating antioxidant deferiprone on the contractile properties of the RET indicates, however, that the I-R (1h-1h) induced contractile defi cit and Ca2+ overload in myocytes is not due to OH formation. The contractile defi cit may rather be due to depletion of high energy phosphates (ie phospocreatine and ATP; (6)), which is associated with changes in intracellular Ca2+ (58) and decreased contractile function in early phases of I-R.The protection by BDM and lack of protective effect of deferiprone lead to the conclusion that the defi cit in contractile muscle function resulting from I-R (1h-1h) injury can be solely attributed to a Ca2+ overload and is not caused by injury resulting from iron mediated OH formation.

In a previous study (10) we showed, by measuring vessel diameter responses to local application of acetylcholine, that the cell-to-cell conduction of vasodilation along the endothelial cell layer of the feed arteries was not affected by the IR (1h-1h) procedure. Thus the impairment of AVD (to 49% of control) results from a derangement of initiation of the vasodilatory signal by the endothelium of the intramuscular arterioles. The general notion is that the endothelium is highly sensitive to oxygen radicals, especially OH. Most likely, this is because OH has a destructive action on KCa ion channels (47; 49), which serve to initiate endothelial hyperpolarization, resulting in vasodilation (12). However, the functional defi cit in AVD following I-R remained in the RET unaltered by the addition of deferiprone, a powerful inhibitor (23) of the formation of OH. Even when the stimulus frequency was raised to 55 Hz and active muscle tension was restored to control levels obtained at 40 Hz, the AVD remained depressed by ~40% in control experiments but also in presence of deferiprone. Although it cannot be excluded that deferiprone in the superfusion fl uid might not have been effective because it failed to reach the endothelium by diffusion, this lack off effect was also observed in experiments where intravenous administration of deferiprone (10-5 M) did not restore AVD.

The role of Ca2+ overload in endothelial cell dysfunction (29) was not assessed, as AVD measurements were impeded by BDM. The application of other agents interfering with intracellular Ca2+ was prevented by the fact that in vivo arterial resting tone is dependent on the level of Ca2+-mediated smooth muscle contraction. The results suggest that the IR-induced endothelial cell dysfunction leading to impaired AVD cannot be attributed to iron-mediated formation of OH. It should be noted, however, that the current approach to inhibit formation of OH by reducing the iron availability does not entirely exclude the possibility that OH is a causative factor in the endothelial cell dysfunction. This is because in vascular endothelium OH can be produced also independently of iron, from protonated ONOO- (peroxynitrite) that is formed from NO (nitric oxide) and O2

-.(superoxide; (3; 30)). It seems unlikely, however, that iron independent OH formation plays an important role in endothelial cell dysfunction following I-R, because the O2

- -induced reduction of vasodilation in coronary and cerebral arteries can be prevented by addition of the iron scavenger deferoxamine (36; 50). For other ROS such as O2

- (28; 40), hydrogen peroxide (H2O2; (4; 62)) and ONOO-; (31; 37), stimulatory as well as inhibitory effects on endothelial


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KCa channels and on the endothelium dependent vasodilation have been described, so potentially these might in the RET contribute to the AVD defi cit. Finally, it should be mentioned that a crucial and major role for NO in both the initiation and conduction of Ach-induced vasodilation in RET feed arteries has been excluded (12; 45). Thus, most likely other ROS than OH are a causative factor for impairment of endothelium dependent AVD.

In conclusion, we show that during early phases of I-R such as currently used (1h-1h), the full extent of contractile muscle defi cit is attributable to the detrimental action of Ca2+

and that iron-mediated formation of OH is not involved in impairment of contractile muscle function. Additionally, we show that iron mediated formation of OH does not play a role in the disruption of endothelium dependent mechanisms of blood fl ow control. We hypothesize that other reactive oxygen species, such as superoxide, may be involved in the IR-induced impairment of the initiation of AVD.

Acknowledgements

The authors thank André Verheem, Jan Dirk Tiggelman and Hans Vosmeer for expert technical assistance. Professor Dick de Wildt is gratefully acknowledged for assistence in calculation of the intravenous dose of deferiprone.

References 1. Ali MH, Schlidt SA, Chandel NS, Hynes KL, Schumacker PT and Gewertz BL. Endothelial permeability and IL-6

production during hypoxia: role of ROS in signal transduction. Am J Physiol 277: L1057-L1065, 1999.

2. Allen DM, Chen LE, Seaber AV and Urbaniak JR. Pathophysiology and related studies of the no refl ow phenomenon

in skeletal muscle. Clin Orthop Relat Res 122-133, 1995.

3. Beckman JS, Beckman TW, Chen J, Marshall PA and Freeman BA. Apparent hydroxyl radical production by

peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A 87:

1620-1624, 1990.

4. Brakemeier S, Eichler I, Knorr A, Fassheber T, Kohler R and Hoyer J. Modulation of Ca2+-activated K+ channel in

renal artery endothelium in situ by nitric oxide and reactive oxygen species. Kidney Int 64: 199-207, 2003.

5. Carroll WR and Esclamado RM. Ischemia/reperfusion injury in microvascular surgery. Head Neck 22: 700-713,

2000.

6. Carvalho AJ, McKee NH and Green HJ. Metabolic and contractile responses of fast and slow twitch rat skeletal

muscles to ischemia and reperfusion. Plast Reconstr Surg 99: 163-171, 1997.

7. Chiou WF, Lin LC and Chen CF. Acteoside protects endothelial cells against free radical-induced oxidative stress. J

Pharm Pharmacol 56: 743-748, 2004.


9. Clanton TL. Hypoxia-induced reactive oxygen species formation in skeletal muscle. J Appl Physiol 102: 2379-

2388, 2007.


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57

4

Cooling to 21°C prevents ischemia-reperfusion-

induced impairment of ascending vasodilation in

feed arteries of hamster skeletal muscle in vivo

Miriam CJ de With1, Steven S Segal2, PR Dop Bär3, Moshe Kon1, Alfons BA Kroese4,5

1,3,5 Departments of Plastic Reconstructive and Hand Surgery1, Biomedical Sciences3 and Surgery5, University

Medical Center Utrecht, Utrecht, The Netherlands

2 Department of Medical Pharmacology and Physiology & Dalton Cardiovascular Research Center,

University of Missouri – Columbia, Columbia, MO, USA


Submitted


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Abstract

BackgroundAt 37°C ischemia-reperfusion (I-R) leads to impairment of not only contractile

skeletal muscle function but also of the ascending vasodilation (AVD) which is essential to increasing blood fl ow delivery to contracting skeletal muscle. Following I-R at 37°C, impairment of AVD is attributed to failure of initiating vasodilation via the endothelium. We investigated whether cooling of skeletal muscle to room temperature (21°C) during I-R in vivo could preserve AVD.

Materials and Methods Using electrical fi eld stimulation, in the retractor muscle (RET) of anaesthetized

male golden hamsters (n=18), rhythmic contractions of intermediate tension levels (~60 mNmm-2; duty cycle 10%; 40 Hz at 37°C; 15 Hz at 21°C) were used to produce AVD in feed arteries. Diameter changes were measured ~1 mm upstream from the RET in preparations maintained at either 37°C or 21°C (n=3-6 per group) and subjected to 1 hour of ischemia followed by 1 h of reperfusion (I-R; 1h-1h). Respective control groups maintained normal fl ow during the same 2 h period.

ResultsPeak tetanic tension (P0) was 196 mNmm-2 at 37°C and 139 mNmm-2 at 21°C.

Contractions at intermediate tension induced an AVD of 255 m at 37°C and 272 m at 21°C, respectively. Following I-R at 37°C, active tension and AVD at 40 Hz were reduced by half relative to control. Stimulation at 55 Hz restored active tension but AVD remained depressed (61% of control). In contrast, both active tension (89%) and AVD (102%) were maintained following I-R at 21°C.

Conclusion Cooling to 21°C prevents impairment of AVD following I-R in hamster skeletal

muscle. These fi ndings demonstrate a valuable role for cooling in maintaining endothelium-dependent signalling underlying AVD in vivo.


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Introduction

Ischemia reperfusion (I-R) of skeletal muscle occurs routinely during surgical procedures, such as free tissue transfers. Interruption of the peripheral circulation produces ischemia, resulting in disruption of cellular function throughout the tissue (5; 39). Reperfusion with arterial blood leads to additional damage to the muscle and its vascular supply (19). A major factor in development of the ‘no-refl ow’ phenomenon in skeletal muscle following I-R is the disruption of vasomotor control of microvessels (29). In response to contractile activity, vasodilator signals originating within the muscle ascend from the microcirculation to encompass the feed arteries upstream (ascending vasodilation; AVD (31; 41). Dilation of these proximal arteries serves to increase the total blood fl ow delivered into arteriolar networks (9; 31). In feed arteries, the endothelium serves as the cellular pathway by which the vasodilator signal (i.e. hyperpolarization) is conducted along the vessel wall and into the surrounding smooth muscle cells (15). Therefore, preserving the function of this endothelial signalling pathway following an I-R insult is vital to maintaining appropriate blood fl ow to active skeletal muscle.

In the retractor muscle preparation (RET) of anesthetized hamsters studied at normal body temperature, we have demonstrated that I-R (1h-1h) diminished both contractile function and the ability to initiate AVD by ~50% (11). The reduction of AVD coincided with an impaired ability to initiate vasodilation via the endothelium within the affected muscle (11). Based upon fi ndings that the postischemic skeletal muscle contractile defi cit can be prevented by hypothermia (18), we questioned whether cooling would prevent the endothelial dysfunction associated with impaired AVD (20). There is a paucity of in vivo or in vitro studies evaluating the effect of temperature on preserving AVD following an I-R insult (16). Therefore, experiments were performed in vivo using the RET to test the hypothesis that AVD evoked by muscle contraction would be preserved by cooling during I-R.


Animal care and preliminary surgeryMale Syrian golden hamsters (n=18, 1202 g; Harlan-Winkelmann, Borchen,

Germany) were housed in groups of 3-4 animals on a 12h-12h (light/dark) cycle and provided with standard diet food and water ad libitum. The animals received care in compliance with the European Convention guidelines (86/ 609/EC). All procedures were approved by the Utrecht University Committee for experiments on animals. Hamsters were injected with buprenorfi ne hydrocloride (Temgesic®; Schering-Plough, Utrecht, The Netherlands) 5 mg/kg subcutaneously 30 minutes prior to surgery. Isofl urane® (ABBOTT Animal Health, Oudewater, The Netherlands), supplied by a vaporizer (Isotec 5, Datex Ohmeda, Madison, WI, USA) through a ventilation mask, at 5% with 100% oxygen (1 l/ minute) was used to induce anaesthesia (~5 minutes) and at


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1.5 - 2 % with oxygen (0.25 l/min) and air (0.5 l/min) to maintain anaesthesia. Excess vapor was scavenged by a vacuum system. Sterile saline was delivered through a cannula (PE 50) secured in the left femoral vein to replace fl uids during the experiment (300 μl h-1; Fresenius Orchestra, Fresenius Kabi, Utrecht, The Netherlands). Rectal temperature was maintained at 36-38°C with heat conducted from a warm copper plate positioned beneath the hamster. At the end of each day’s experimental procedures (typically 7-8 h), the hamster was euthanized with an overdose of pentobarbital ((Nembutal®; CEVA Sante Animale BV, Maassluis, The Netherlands) delivered through the intravenous cannula.


platform and the right RET was prepared for study (11; 37). Briefl y, a 2-3 cm incision was made through the overlying skin, which was retracted to expose the underlying tissue. The surgical fi eld was irrigated continuously with bicarbonate-buffered physiological salt solution (PSS, pH 7.4, 34-36°C) of the following composition (mM): NaCl 131.9; KCl 4.7; CaCl2 2.0; MgSO4 1.2; NaHCO3 18; dissolved in deionized purifi ed water (dH2O) and equilibrated with 95% N2/ 5% CO2. All reagents were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Using microdissection while viewing through a stereomicroscope (Zeiss OPMI 6 DF), the muscle and its feed arteries (with paired collecting veins) were cleared of connective tissue and visible nerve branches; great care was taken to minimize trauma during microdissection. The origin and insertion of the muscle were severed and each was secured in a clamp. While carefully maintaining the integrity of the vascular supply, the muscle was refl ected away from the body, positioned in a chamber (volume, ~10 ml) that was integral to the platform (37). Respective muscle clamps were connected to micrometer drives for control of muscle length and recording isometric tension (37). Surgical procedures typically required 3-4 h.

The completed preparation was transferred to the stage of an intravital microscope (Axiotech 100 HD; Zeiss, Sliedrecht, The Netherlands) and superfused continuously (20 ml min-1) with fresh PSS (37°C) while the effl uent was removed with a vacuum line to maintain a constant fl uid level and a stable optical image.

Video microscopy and vessel diameter measurementsFeed arteries were observed using a Zeiss LD Achroplane (20X, numerical aperture,

0.40) objective lens. The image was coupled to a color video camera (Sony Hyper HAD; CCD-IRIS/RGB; Sony, Japan) and monitor (Hitachi, Japan) with total magnifi cation = 980X. The internal diameter of vessels was measured with a video caliper at the widest point of the vessel lumen or as the width of the column of red blood cells. To normalize vasodilations across feed arteries with different diameters (range at rest: 20-69 μm), peak responses were expressed as:



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The level of spontaneous vasomotor tone, expressed as % of maximum diameter, was calculated as:


Maximum diameter was recorded during superfusion with 10-5 M sodium nitroprusside (SNP) in the PSS.


of the retractor muscle and isometric contractions were evoked using fi eld stimulation to depolarize motor end plates of skeletal muscle fi bers (21; 37). Stimulus pulses (0.1 ms) were delivered via a S4 stimulator driven by a S88 stimulator (both Grass Instruments, Quincy, MA, USA). Muscle tension at rest and during contractions was recorded using a load beam (LCL-113G; Omega, Stamford, CT, USA; 0-100 g; resolution, 0.1 g) mounted in series with a muscle clamp. The active tension developed in response to each stimulus was calculated as the difference between resting tension and the corresponding peak response. These values were normalized to muscle cross-sectional area (calculated from muscle length and wet mass, assuming tissue density = 1.056 mgmm-3) and expressed in mNmm-2 (11; 36).

Ischemia and reperfusionIschemia was induced by gently compressing the feed arteries and collecting veins

using a piece of silicon tubing secured along the gate separating the hamster from the retractor muscle and its feed arteries (11; 37). Cessation of blood fl ow was confi rmed by visual inspection and verifi ed at least every 10 min during the 60 min period of ischemia. As a further precaution during ischemia, the muscle bath was covered with Saran® to prevent oxygen entering the fl uid from room air. The PO2 in the muscle bath under these conditions has been shown <10 torr (11). After 1h ischemia, the silastic occluder was removed. As verifi ed by inspection, blood fl ow was restored immediately. This period of ischemia was chosen in accord with previous experiments (11) to limit the degree of endothelial injury and prevent a total loss of vascular tone. Criterion measurements were taken following 1 h of reperfusion, as we have shown that this period allowed feed arteries to recover resting tone (11). To evaluate the effect of cooling during I-R, preparations (control n=3; I-R n=4) were kept at 21°C.

Experimental protocolOnce the preparation was secured on the intravital microscope it was verifi ed visually

that blood fl ow was intact along feed arteries and throughout the microcirculation within the muscle. Muscle length was adjusted to optimal length for developing twitch tension (L0), which is 115% of in vivo resting length, corresponding to a preload of ~5-10 mN (40). All subsequent procedures were performed at L0. Each muscle preparation was equilibrated in PSS for 60 minutes at 370.5°C to establish stable baseline conditions.


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Subsequently, the RET was subjected to one of two procedures, either maintaining the preparation at 37°C or after cooling it to 21°C: ‘time control’, in which resting blood fl ow and superfusion was maintained for 2 h; or ‘I-R’, in which 1 h of ischemia was followed by 1 h of reperfusion. In all experimental groups, the ‘initial’ resting diameter of feed arteries was measured at this 2-h time point and then contractile function of the RET was evaluated.

To obtain a peak twitch tension (Pt), stimulation voltage of a single pulse was increased until Pt reached its maximum. A one-minute rest period was allowed between successive stimuli. After measurement of Pt, a muscle rested for 5 minutes. All further stimulation was performed at 1.5X stimulus intensity used for Pt. The relationship between stimulus frequency and peak active tension was then evaluated to verify a plateau in maximum tetanic tension (P0). For this procedure, a 400 ms stimulus train was delivered at 40-150 Hz for preparations maintained at 37°C (see Figure 1) or at 10-70 Hz for preparations at 21°C because of cooling- induced changes in the relationship between active tension and stimulation frequency (32). A 2-min period of recovery was allowed between consecutive stimulus trains.

Ascending vasodilation (AVD) was then evaluated in response to rhythmic muscle contractions performed for 200 ms every 2 s, corresponding to duty cycle (DC) of 10% (21). With tissue displacement during each contraction, feed artery diameter was measured between contractions. Previous fi ndings have shown that, in response to muscle contraction, the entire feed artery segment dilates to the same extent (33). For the present experiments, responses were evaluated at the midpoint of the feed artery, which was located nearly 1 mm upstream from the proximal edge of the muscle. For preparations maintained at 37°C, the duration of rhythmic contractions was 1 minute. AVD was fi rst evaluated using a stimulation frequency of 40 Hz. Resting tone was allowed to recover for 10 min and AVD was reevaluated at 55 Hz (to restore peak tension – see Results). In contrast, preparations maintained at 21°C were stimulated at a lower frequency (15 Hz) for a longer period (2.5 minutes) because of temperature- induced changes in contractile properties as well as the reactivity of feed arteries (see Results). Outputs from the video caliper (feed artery diameter) and load beam (muscle tension; low-pass fi ltered at 5 Hz) were sampled at 250 Hz using a 1401 Micro CED system (Cambridge Electronic Design, Cambridge, UK) coupled to a personal computer running CED software (Spike 2).

One feed artery was studied per hamster. In 4 preparations studied at (all 37°C; n=2 control; n=2 I-R), spontaneous tone was weak and phenylephrine (PE, 10-8 M) was added to the superfusion solution to obtain a level of resting tone typical for these vessels (11; 33;

36). Vasomotor responses for these preparations were otherwise similar to those of the other 12 vessels studied without PE; therefore the data from these vessels were pooled with respective groups. At the end of each experiment, maximum vessel diameter was determined by exposing the preparation to 10 μM SNP. The vasodilatory response to SNP has previously been shown not to be temperature sensitive in cerebral arteries (38). Muscle


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length (L0) was measured with a caliper (± 0.1 mm) and the muscle was excised, blotted of excess moisture, and weighed (± 0.1 mg).

Data analysisStatistical analysis of the experimental data was performed using t-tests (to compare

P0), ANOVA (active force and AVD during rhythmic contractions) and two-way ANOVA (vessel diameter and tone) followed by Holm-Sidak post hoc comparisons (Sigma Stat; version 3.0, SPSS, Chicago, IL, USA). Figures were prepared using Sigma Plot (version 8.0, SPSS). Summary data for all parameters are reported as means ± S.E., with ‘n’ indicating the number of feed arteries or RET preparations studied. Differences were accepted as statistically signifi cant with p< 0.05. Regression lines (Figure 2) were fi t using linear regression (Origin 7.5; Microcal Software Inc, Northampton, MA, USA).

Results

Hamsters remained stable throughout each day’s experiments as demonstrated by the maintenance of heart rate (~350 beats/ minute) and spontaneous ventilation (~32 breaths/minute). There were no differences in the muscle length at L0 (29.00.3 mm; n=18) or wet muscle mass (1144 mg; n=18) between groups. A total of 18 hamsters were used in the present experiments.

Effect of I-R and temperature on contractile parameters of the RETThe effects of I-R (1h-1h) at 37°C and of cooling to 21°C on the tension: frequency

responses of the RET are depicted in Figure 1. In control preparations at 37°C, tetanic tension increased with stimulus frequency until a maximum (P0) of 19612 mNmm-2 (n=6) was reached at ~120 Hz. Following I-R, P0 was attained at 120 Hz, but was reduced to 11117 mNmm-2 (n=5; p<0.001 vs. control), consistent with previous observations (10; 11). In control preparations (n=3) maintained at 21°C, P0 (13912 mNmm-2) was

Fig. 1: Effect of I-R on frequency-

force response curves of the RET

at 37°C and 21°C.

In time control preparations, active

tension increased with stimulus frequency

until a maximum tetanic tension (P0)

was reached at ~120 Hz at 37°C (n=6)

and at ~55 Hz (n=3) at 21°C. Following

I-R at 37°C, P0 was signifi cantly reduced

(n=5; t-test; p<0.001) while I-R at 21°C

did not affect P0 (n=4).


1000 20 40 60 80 120 140 160

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less than values obtained at 37°C (p=0.02) and was achieved at lower stimulus frequencies (~50 Hz). These effects of cooling on P0 are in agreement with temperature effects on skeletal muscle contractility reported previously (1; 32). Following I-R at 21°C, the frequency-force response curve of the RET (n=4) was not different from that of control preparations maintained at that temperature (P0 =1329 mNmm-2).

Effect of I-R and temperature on RET feed artery resting diametersFor preparations maintained at 37°C, restoration of perfusion following ischemia

resulted in transient constriction of feed arteries and the original resting diameter recovered over ~40 minutes. Thus, resting diameters of feed arteries (n=5) following I-R (479 m) were not different from those in control preparations (439 m; p=0.93). Further, maximum diameters with SNP (I-R, 7713 μm; control 7513 μm; p=0.85) and respective levels of spontaneous vasomotor tone (I-R, 603 %; control, 573 %) were not different (p=0.82). These values, depicted in Figure 2, are similar to reported values previously for the RET (11; 21; 32).

Resting diameter (mm)

10 20 30 40 50 60 70

Max

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dia

met

er (m

m)

20

40

60

80

100

control 21°CI-R 21°C

Fig. 2: I-R and hypothermia do not affect feed artery resting and maximum diameters.

For each vessel, maximum diameter (during exposure to 10 M SNP) is plotted against resting diameter. At both

temperatures, resting (p=0.93) and maximum (p=0.85) diameters of feed arteries following I-R were not different

from those of controls. The dotted line represents a linear fi t (Origin 7.5; Microcal Software Inc, Northampton, MA,

USA) for all vessels at 21°C (n=6; intercept 34; slope 1.32; p=0.095; R=0.736). The uninterrupted line represents a

linear fi t for all vessels at 37°C (n=10; intercept 13.2; slope 1.39; p=0.0001; R=0.736). Comparison of the two lines

(Origin7.5) showed that the linear fi ts at 21°C and 37°C are not signifi cantly different (p=0.637).

For preparations maintained at 21°C, feed arteries also constricted transiently upon restoration of perfusion following ischemia. Consistent with observations at 37°C, there were no differences between resting (p=0.93) or maximum (p=0.85) diameters (see Figure 2). However, resting tone (452%) at 21°C was stronger than at 37°C (552%; p=0.001).


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Fig. 3: Representative recordings

of AVD in feed arteries of the RET

at 37°C and 21°C.

Feed artery diameter increases in

response to muscle contractions at

37°C (A) and 21°C (B). Bars indicate

the period of muscle contractions: at

37°C (A) 60 seconds at 40 Hz for

control; 60 seconds at 55 Hz for I-R;

At 21°C (B) 150 s at 15 Hz for control

and for I-R. Note that the onset of

diameter increase at 21°C is slower

than at 37°C. Following I-R at 37°C

AVD was depressed to ~60%, despite

similar levels of active tension when

stimulation frequency was increased

from 40 to 55 Hz (see Figure 4).

In contrast, AVD was not affected

following I-R at 21°C.

Following muscle contractions at either temperature, resting diameters recovered consistently within 3 minutes and otherwise remained stable during experiments.

Effect of temperature on I-R-induced impairment of RET feed artery AVDIn preparations maintained at 37°C, stimulation at 40 Hz (10% duty cycle, see

Methods) evoked contractions that were ~45% of P0 (5510 mNmm-2; see Figure 1). This intermediate level of activation, where active tension remained stable during each contraction period, was used to evaluate AVD (cf. (21)). The AVD in feed arteries was recorded 92925 m (n=16) upstream from the proximal edge of the muscle.

For RET preparations maintained at 37°C, the diameter of feed arteries typically began to increase ~10 seconds after onset of contractile activity and typically reached a stable peak value within ~40 seconds (Figure 3A). Feed artery diameter increased by 593% above rest in control muscles (n=5) and by 298% in muscles exposed to I-R (n=5; p<0.001). Since the loss of active tension could be partially responsible for this impairment of AVD (11), preparations that were subjected to I-R were subsequently stimulated at 55 Hz. Although

I-R

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21°C


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at 55 Hz active tension was restored to values that were not different from those obtained at 40 Hz in control muscles (558 mN; Figure 4A), AVD remained signifi cantly depressed (618% of control, Figure 4B). This reduction of AVD following I-R is consistent with previously reported results (11).

Fig. 4: I-R induced impairment of AVD is

prevented by cooling to 21°C.

(A) Mean active tension during 10% duty cycles

(200 ms every 2 s) of 60 seconds at 40 Hz for

control, at 40 Hz and 55 Hz for I-R at 37°C and

of 150 seconds at 15 Hz for control and for I-R at

21°C. Following I-R at 37°C the active tension

in response to 40 Hz stimulation was impaired.

Increasing stimulus frequency from 40 to 55 Hz

following I-R at 37°C restored the active tension

to a level not different from that obtained at 40 Hz

in control muscles (55 mNmm-2). Active tension

at 21°C was less than that at 37°C but was not

depressed further by I-R.

(B). Mean AVD during 10% duty cycles. At 37°C

and 40 Hz, I-R reduced AVD to 4913% of control.

Increasing stimulus frequency to 55 Hz restored

active tension (see 1A and 1D) but not AVD, which

remained depressed to 61 8% of control. At 21°C,

15 Hz stimulation resulted in AVD responses that

were similar to those induced by 40 Hz stimulation

at 37°C in control preparations. Following I-R at

21°C, AVD was not affected (1024% of control

AVD at 21°C). Summary values are means S.E.

Signifi cant P-values (ANOVA) are indicated.

For RET preparations maintained at 21°C, stimulation at 15 Hz (~45 % of P0; see Figure 1) evoked contractions with levels of active tension (658 mN) that were similar to those obtained during intermediate activation at 37°C (40 Hz) and remained stable during each contraction period. Therefore, 15 Hz stimulation (10% duty cycle, as above) was used to evaluate AVD at 21°C. Representative tracings are depicted in Figure 3B. As compared to responses at 37°C, the onset of AVD was delayed (to ~40 seconds) and stable peak values for diameter were reached after ~ 2 minutes. Remarkably, and in contrast to the behavior observed at 37°C, the amplitude of AVD was well preserved for preparations maintained at 21°C (1024% of control; p=0.90; Figure 4B).

control40 Hz

I-R40 Hz

I-R55 Hz

control15 Hz

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control40 Hz

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I-R55 Hz

control15 Hz

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.mm

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p = 0.024

Cha

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(%)

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p = 0.011

Ascending vasodilation

37 °C 21°C

37 °C 21°C

A

B


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Discussion

The present study is the fi rst to investigate the effects of cooling on preserving ascending vasodilation (AVD) following ischemia-reperfusion of skeletal muscle. Our main fi nding is that cooling skeletal muscle to 21°C preserved AVD following I-R (1h-1h) in feed arteries of the hamster RET studied in vivo. In contrast, maintaining the muscle at normal body temperature (37°C) reduced AVD by nearly half.

The impairments in contractile muscle function and AVD at 37°C observed here are consistent with those reported previously (10; 11). The reduction in active tension following I-R (to 43% of corresponding time controls maintained with intact blood fl ow at the same temperature) is also consistent with previous reports following I-R insults of equal duration in rat and rabbit models (10-40%; (6; 7)). A recent study (10) in which additives (Ca2+ release inhibitor 2,3-butanedione monoxime (BDM); inhibitor of iron-catalyzed OH mediated lipid peroxidation deferiprone) were applied to the RET preparation indicated that the reduction in tension produced by contracting muscle fi bers following I-R (1h-1h) is due to Ca2+ overload rather than to injury of the myocytes from iron-mediated OH formation.

The decrement AVD observed in feed arteries is attributed to a derangement in the initiation of the dilator signal (e.g. hyperpolarization) by the endothelium within the muscle. In contrast, once the signal was initiated, conduction along the vessel was preserved (11). The function of smooth muscle cells along the vessel wall appeared to be relatively unaffected by I-R, since resting tone and maximum vasodilator responses to SNP remained intact following I-R at either 37°C or 21°C (this study; (10; 11). This difference in resilience between respective cell types is in agreement with the notion that endothelial cells are more susceptible to the effects of I-R than smooth muscle cells (2; 4; 22). Our recent fi ndings suggest that impairment of AVD following I-R likely results from the actions of reactive oxygen species other than OH formation (10).

The reduction in Po as a result of cooling the RET to 21°C is consistent with other reports (1; 32) and likely refl ects partial impairment of excitation-contraction coupling in skeletal muscle fi bers. That cooling prevents the I-R-induced impairment of contractile properties is also well known (cf. (18)). Further, our fi nding that resting tone in RET feed arteries is enhanced when preparations are maintained at 21°C vs. 37°C is also in agreement with in vivo observations of arterioles in hamster striated (dorsal skinfold chamber, (35)) and rat extensor digitorum longus muscle (30). The increase in resting tone may be attributed to enhanced2 adrenoreceptor activation (8) and is in accordance with the notion that superfi cial vessels (such as feed arteries of the RET) constrict whereas deep vessels dilate in response to cooling (28). At 21°C, the AVD response exhibited a longer lag time before onset and developed more slowly than compared to responses at 37°C. This difference in response kinetics may be due to the effects of lower temperature on the


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speed of smooth muscle relaxation (Q10 = 2-3; (14; 34)) as well as on tension development (and the corresponding production of vasodilatory signals (32)).

To date, investigations on the effects of cooling on endothelium-dependent vasodilatation have typically used ring preparations of arteries that are taken from deeper regions of the body (16; 23; 27; 28). Unfortunately, vasomotor responses to endothelium-dependent vasodilators such as acetylcholine (23) and carbachol (42) of arteries taken from deep within the body and studied in vitro cannot be compared to in vivo recordings of AVD of the RET. In addition to maintaining more physiological conditions in vivo, the mechanisms of endothelium-dependent vasodilation differ between deep (16; 23; 27; 28) and superfi cial (13) vessels as well as between large conduit arteries and resistance microvessels. Although cooling is generally used in clinical surgery for protection from hypoxic damage (3), there is a paucity of information concerned with the combined effects of cooling and hypoxia/ischemia on the integrity of vasomotor control. Furthermore, studies on the effects of I-R or ischemia on AVD in skeletal muscle are scarce (11; 35). Nevertheless, prevention of I-R-induced impairment of AVD by cooling the RET muscle to 21°C observed in the present experiments is in accord with the fi nding that enhanced leukocyte adhesion and microvascular permeability (which both refl ect endothelial cell trauma) following I-R in hamster striated muscle can be prevented by cooling to 8°C (35).

The mechanism by which cooling from 37°C to 21°C protects the endothelium from I-R-induced injury is most likely related to the overall reduction of oxygen consumption and slowing down of energy demanding processes by a factor of 1.5 - 3 (17). The delayed depletion of ATP and phosphocreatine stores associated with hypothermia will contribute to prolonged maintenance of cellular viability and function. As we have excluded a major role for iron-catalyzed formation of hydroxyl radicals (OH) as a causative factor in the impairment of AVD in RET feed arteries following I-R at 37°C (10), we hypothesize that impairment of AVD at 37°C refl ects damage to endothelial cell membranes by other reactive oxygen species such as O2

- (superoxide). The decreased levels of reactive oxygen species (ROS) measured in neural and liver tissues maintained at 15-33°C in comparison to those at 37°C support this hypothesis (24-26). Further, the decreased expression of chemokines and upregulation of the antiapoptotic Bcl-2 protein such as found in human umbilical vein endothelial cells (HUVECs (12)) following exposure to hypothermia could further contribute to preservation of endothelial cell function.

In conclusion, the present fi ndings illustrate that maintaining skeletal muscle at 21°C (vs. 37°C) during a 2-hour period of I-R maintains vasomotor tone, contractile function, and the ability of vasodilation to ascend from the microcirculation within skeletal muscle into the proximal feeding arteries. In turn, the preservation of ascending vasodilation refl ects the maintenance of endothelial signaling integrity and enables muscle blood fl ow to increase to greater levels than are possible with endothelial dysfunction (21). We suggest that the lack of effect of I-R on AVD at 21°C can be explained by the lower production of ROS due to reduced metabolism and oxygen requirements as temperature is reduced.


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Acknowledgements

The authors thank André Verheem, Jan Dirk Tiggelman and Hans Vosmeer for expert technical assistance.

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35. Thorlacius H, Vollmar B, Westermann S, Torkvist L and Menger MD. Effects of local cooling on microvascular

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38. Wagerle LC, Russo P, Dahdah NS, Kapadia N and Davis DA. Endothelial dysfunction in cerebral microcirculation

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42. Zygmunt PM, Sorgard M, Petersson J, Johansson R and Hogestatt ED. Differential actions of anandamide, potassium

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73

5

Contractile and morphological properties of hamster

retractor muscle and rat cutaneus trunci muscle are

dissimilarly affected by 16 hours of cold preservation

Miriam CJ de With1, EPA Brigitte van der Heijden1, Matthijs F van Oosterhout2, M Kon1, Alfons BA Kroese3,4

1,2,3 Departments of Plastic Reconstructive and Hand Surgery1, Pathology2 and Surgery3, University Medical

Center Utrecht, Utrecht, The Netherlands


Cryobiology 2009; conditionally accepted for publication


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Abstract

IntroductionCold hypoxia is a common factor in cold tissue preservation and mammalian

hibernation. The purpose of this study was to determine the effects of cold preservation on the function of the retractor (RET) muscle of the non-hibernating hamster and compare these with previously published data on the rat cutaneus trunci (CT) muscle.

Materials and MethodsAfter cold storage (16 h at 4°C), muscles were stimulated electrically to measure

maximum tetanus tension (P0) and histologically analyzed. The protective effects of addition of the antioxidants trolox and deferiprone and the calcium release inhibitor BDM to the storage fl uid were determined.

ResultsAfter storage, the twitch threshold current was increased (from 60 to 500 μA) and

P0 was decreased to 27% of control. RET morphology remained unaffected. RET muscle function was protected by trolox and deferiprone (P0 resp 43% and 59% of control). Addition of BDM had no effect on the RET.

Conclusions The effects of cold preservation and of trolox and deferiprone on the RET and

CT muscle function were comparable. Both hamster RET and rat CT muscles show considerable functional damage due to actions of reactive oxygen species. In contrast to the CT, in the RET cold preservation-induced functional injury could not be prevented by BDM and was not accompanied by morphological damage such as necrosis and edema. This suggests that the RET myocytes possess a specifi c adaptation to withstand the Ca2+ overload induced by cold ischemia.


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Introduction

The clinical application of composite tissue allografts (CTAs) is an emerging fi eld in reconstructive surgery (47). This involves the allotransplantation of complex composite tissues, such as hands (13) or facial structures (11; 31) from deceased donors. CTAs contain skeletal muscle components which are in comparison to bone, fat and skin highly vulnerable to ischemia and reperfusion (I-R) injury (43). The most important factors involved in the pathogenesis of I-R injury in skeletal muscle fi bres are increased intracellular calcium levels ([Ca2+] i) (15; 18) and the formation of reactive oxygen species (ROS, (22; 36)). In CTA transplantations and also in replantations of traumatically amputated limbs, cold storage (~4°C) of the involved tissue is the current practice. Hypothermia reduces the metabolic rate by decreasing ATP turnover, but this can ultimately result in edema and disruption of cellular function (37). The more frequent application of CTAs requires the design of a preservation strategy that allows for tissue banking with maintenance of cellular function.

Hibernators such as hamsters and thirteen lined - or arctic ground squirrels show superior survival when compared to rats and mice during hypothermia (23; 32) and hypoxia (3). In isolated hearts of hibernators, contractility is maintained better during decreased temperatures than in those of non-hibernators (30). Similarly, after cold perfusion and rewarming and after I-R the recovery of contractile function in hibernator hearts is greater than in rat hearts (1; 17). There are a few reports on isolated solid organs (liver and kidney) and these show that functional and metabolic parameters (7; 19; 28) of hibernators in the non-hibernating state (state of arousal) do not display superior resistance to cold ischemia/ warm reperfusion compared to these organs of rats and rabbits. The tolerance to storage of these organs is, however, clearly enhanced in ground squirrels that are in the hibernating state (19; 28). Several cellular adaptations of hibernators aimed at protection and stabilization of cell functions have been described, such as metabolic rate depression (45), reorganisation of the priorities for ATP consumption, changes in expression of selected genes, and enhancement of antioxidant defence (33). At present, the notion is that such specifi c metabolic adaptations to withstand cold hypoxia are not continuously apparent in tissues of hibernators, but are triggered by transition into the hibernating state (5; 45). About the tolerance of isolated skeletal muscle of hibernators to cold preservation, however, both in the hibernating state and in the non-hibernating state, no information is available.

The purpose of this study was to determine the effect of 16 h storage at 4°C in HTK (49) on contractile muscle function and cytoarchitecture of the hamster retractor muscle (RET) in the non-hibernating state. The RET has been used extensively in studies of muscle contraction, control mechanisms of muscular blood fl ow (for reviews see (38; 39)) and the effect of warm I-R (10). We investigated whether the cold preservation of the RET could be improved by the addition of the Ca2+ release inhibitor BDM and the antioxidants trolox and deferiprone. The results are compared to those found previously (50) in the rat cutaneus trunci (CT) muscle.


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Animal care Male golden hamsters (Harlan) weighing 85-110 gram were housed in groups on a

12h /12h (light-dark) cycle and provided with standard diet and water ad libitum. The animals received care in compliance with the European Convention guidelines (86/ 609/ EC). All procedures used were approved by the Utrecht University Committee for experiments on animals and were in accordance with the Dutch federal law on experimental animals. For isolation of the retractor muscles (RET), the hamsters were anaesthetized with intraperitoneal pentobarbital sodium, 60 mg/kg and additional injections were given when necessary. The body temperature was maintained at 37ºC. At the end of the operation the animals were euthanized by an intracardial overdose of Nembutal.

Retractor isolationThe animal was positioned on one side to isolate the contralateral retractor. To expose

the RET, a 2- 3 cm incision was made through the overlying skin from cheek pouch to the lower thoracic vertebrae. One ligature (Vicryl 5.0) was attached to the origin; the other ligature was secured to the muscle ~ 5 mm from the cheek pouch. The distance between the two ligatures was recorded as resting length (~ 20 mm). Subsequently, the RET, comprising intact muscle fi bers (40), was removed. Both retractor muscles of each hamster were used.

StorageEach muscle was secured on a sturdy plastic card at resting length and preserved at

4°C for 16 h in 25 ml of HTK- Bretschneider solution (HTK) containing in mmol/l: 15 NaCl, 9 KCl, 4 MgCl2, 0.015 CaCl2, 1 K-2-oxoglutarate, 180 histidine, 18 histidine HCl, 30 mannitol and 2 tryptophan, pH 7.1 (2), in which skeletal muscles are best preserved (49; 51). After storage at 4°C, the muscles were allowed to acclimatize at room temperature (21°C) for 90 minutes in Krebs- Henseleit solution (KH) containing in mmol/l 118.1 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, 1.2MgSO4 and 5.6 glucose, pH 7.4, carbogenated with 95% O2 and 5% C02.

The substances added to protect the RET muscle function during cold storage were: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), 1,2-dimethyl-3-hydroxypyrid-4-one (deferiprone) and 2,3-butanedione monoxime (BDM). All substances were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands) and added to HTK over the relevant concentration ranges. All compounds were present in the media throughout the experimental course, except BDM. Since BDM has been demonstrated to inhibit muscle contraction (16), after 30 min of acclimatization (see above) BDM was deleted from the KH (see (50)).


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Functional analysisExperiments were performed at room temperature (21°C) because at this temperature

the contractile properties of muscles tested in vitro are more stable than at 37°C (14; 41) The muscle was mounted vertically in air, with one end attached to a fi xed post and the other end tied to the lever arm of a force transducer (Harvard Universal Force Transducer®; Harvard Apparatus, Edenbridge, Kent, U.K.). The isometric properties of the muscles were measured at optimal length, which is 115% of resting length (57), corresponding with a preload of ~5-10 mN. Muscles were stimulated directly by two stainless steel needle electrodes of 200 μm diameter, which were inserted horizontally into the muscle, at a distance of ~1.5 cm (51). The muscle was stimulated with pulses of 0.5 ms duration produced by a stimulus isolation unit connected to a stimulator (Grass Medical Instruments, Quincy, MA, U.S.A.). The transducer signal was amplifi ed, low-pass fi ltered (5 Hz) and digitized at a sampling rate of 250 Hz (1401 micro CED; Cambridge Electronic Design, Cambridge, U.K.). The data were collected with standard software (CED- Spike 2) and analyzed with computer programs written in the CED SCRIPT language.

To obtain a twitch curve, the stimulation current of single pulses was increased incrementally until the twitch tension reached its maximum (Pt). After measurement of Pt each muscle was rested for 10 minutes. All further stimulation was performed at 1.5X the Pt stimulation intensity. A tetanus curve was obtained by applying stimulus trains of 1.4 s duration with increasing pulse frequencies (increments of 4 Hz) until the tetanic tension reached a maximum (P0). The Pt and P0 values were expressed in mN/mg. During the entire stimulation protocol, which took about 40-50 minutes, the muscle was superfused with carbogenated KH. Muscles were weighed and prepared for histological analysis. Values for P0 were normalized to muscle cross sectional area, calculated from muscle length and wet mass, assuming tissue density = 1.056 mgmm-3.

Histological analysisTransverse 5 mm slices (n6) were taken from the central portion of each muscle (51).

The slices were mounted on cork discs between two thin slices of Agar (Agar-Agar 4% in aqua dest) and frozen with isopentane in liquid nitrogen (-160°C) and stored at -75°C. For evaluation, at least two sections of 8 μm thickness were prepared from each slice in a cryostat at -25°C and stained with haematoxylin/eosin and ATP-ase after alkaline incubation (pH 9.4).

For muscle fi ber analysis, all sections were examined by light microscopy (Nikon Eclipse E800; 40 x objective) The most central fi eld and two standardized peripheral fi elds of each muscle (0.14 mm2 per fi eld, each containing ~90 fi bers) were photographed at a fi xed magnifi cation (500X) using a Nikon DXM-1200 camera, connected to a personal computer.

The images were evaluated and scored for four criteria: 1) the percentage of hypercontracted fi bers, 2) the percentage of necrotic fi bers, 3) the occurrence of interstitial oedema and 4) the shape of the muscle fi ber. The histomorphometric counts were


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performed using an image analyzing system (Image J 1.33u, National Institutes of Health, USA; free download at http://rsb.info.nih.gov/ij/).

For evaluation of the hypercontracted and necrotic fi bers the images of HE- stained

slices were used. In each fi eld the numbers of hypercontracted and necrotic fi bers were counted as well as the total amount of fi bers. Myofi brillar ATPase staining (pH 9.4) was used for evaluation of the occurrence of interstitial edema, because of the enhanced contrast between the muscle fi bers and the background tissue. Each image could be scored with 1, 2 or 3: a score of 3 refl ected no edema and a score of 1 refl ected much edema. The images of ATPase stained slices were also used to evaluate the integrity of the muscle fi ber shape (26). The numbers of rounded fi bers were counted in addition to the total number and percentages of rounded fi bers were calculated per fi eld.

Two investigators independently scored each sample that was blinded for treatment protocol. After tests of the between-observer variability (Wilcoxon matched-pairs signed-rank test; signifi cance limit of p<0.05), the scores were averaged.

Statistical analysisStatistical analysis of the experimental data was performed using ANOVA followed

by Tukey post hoc comparisons or Kruskal-Wallis ANOVA on ranks followed by Dunn’s post hoc test (Sigma Stat; version 3.0, SPSS, Chicago, IL, USA). Figures were prepared using Sigma Plot (version 8.0, SPSS). Summary data for all parameters are reported as means ± S.E., with ‘n’ indicating the number of muscles studied. Differences were accepted as statistically signifi cant with p<0.05.

The dose-response relations in Figures 3 and 5 were fi t using a Boltzmann function (Origin 7.5; Microcal Software Inc, Northampton, MA, USA).

Results

There was no difference between control (non-stored) and stored RET muscles in in vivo length (200 mm; p=0.4) or mean wet muscle mass (841 mg; p=0.6).

Twitch and tetanus responses of control musclesMean control twitch and tetanus responses of the RET (n=12) are shown in Figures

1A and B. Typically, the fi rst twitch was observed at a current of 0.060.00 mA (threshold current) and the amplitude of the response increased with increasing current. A maximum for twitch tension (Pt) of 0.800.05 mN/mg was reached at 2-3 mA. The amplitude of the tetanus increased with stimulus frequency until a maximum (P0) of 2.90.1 mN/mg was reached at ~ 16 Hz. After normalization to muscle cross-sectional area (see Methods), the value for P0 is 58.2 3.4 mNmm-2. This value is similar to that reported by us previously for RET muscles (74 mNmm-2 at 37°C ; (10)), when the difference in temperature is taken into consideration.


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Effect of storage on twitch and tetanus responsesMean twitch and tetanus responses of HTK stored RET muscles (n=8) are shown

in Figures 1A and B. After 16 h of storage in HTK at 4°C, RET muscles showed a major reduction in Pt to 122% of control values. The threshold current in HTK stored muscles was increased to 0.50.1 mA. Pt was attained consistently at a current of ~3 mA. Tetanus responses in stored muscles were depressed at all stimulus frequencies. P0 was decreased to 275 % of control and reached at an increased stimulus frequency of ~24 Hz. Following 16 h of storage the Pt/P0 ratio (twitch-to-tetanus ratio) was decreased from 0.280.01 (control) to 0.130.02, indicating that the effect of storage was more profound on Pt than on P0.

Fig. 1: Effect of 16 h storage

in HTK at 4°C on twitch (A)

and tetanus (B) responses of

the RET muscle.

Maximum twitch (Pt ) and

tetanus (P0) tensions are

decreased following storage to

122 and 275% of control

respectively. Note that the

stimulus frequency to attain P0

has increased following storage.

For the added compounds

deferiprone, trolox and

BDM, results are shown of

the concentrations that had

maximum effects on Pt and P0.

Symbols represent mean SEM

(control : n=12; 16h HTK

: n=8; 16h HTK + def 10-5 M:

n=6; 16h HTK + trox 10-3 M:

n=7; 16h HTK + BDM 10-2 M

: n=4; 16h HTK + def 10-5 M +

trox 10-3 M : n=12).

Effect of storage on muscle cytoarchitectureIn the cross sections of control RET muscles (Figures 2A and C) the muscle fi bers had

a polygonal outline and the nuclei were located at the periphery of the fi ber. Little to no edema was observed. ATPase staining showed that the RET is composed of 11.30.4% of type I fi bers and 88.70.4% of type II fi bers, which is comparable to values reported for the RET by others (46). Irrespective of location (central or peripheral), no signs of hypercontraction and necrosis were seen following 16 h of storage at 4°C in HTK (Figures

A

B

HTK (16h)

HTK + trox/def (16h)

Twitch tension

Stimulus current (mA)0 1 2 3 5

tens

ion

(mN

/mg)

0,0

0,2

0,4

0,6

0,8

HTK + def (16h)HTK + trox (16h)HTK + BDM (16h)HTK + trox/def (16h)

Tetanus tension


0 4 8 12 16 20 24 28 32 36

tens

ion

(mN

/mg)

0,0

0,5

1,0

1,5

2,0

2,5

3,0

control

controlHTK (16h)

HTK (16h)


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2B and D). Storage did not result in any increase of interstitial edema within the muscle (p=0.4). The percentage of rounded fi bers did not differ between control (50 %) and stored muscles (51 %; p=0.7).

A B

C D

Fig. 2: Well preserved RET morphology following storage in HTK at 4 °C for 16 h.

Photomicrographs of representative histological cross sections of ATP stained control (A) and stored (B) RET muscles

and HE stained control (C) and stored (D) RET muscles.

RET morphology remained intact following storage, as shown by the unaffected fi ber shape and the absence of edema,

hypercontraction and necrosis. Bar represents 100 μm. The asterisks indicate rounded fi bers.

Pt

(% of control)

P0

(% of control)

Pt/P0 threshold

current (mA)

freq max

(Hz)

n

control 100 ± 6 100 ± 4 0.28 ± 0.01 0.06 ± 0.00 17 ± 1 12

HTK 12 ± 2* 27 ± 5* 0.13 ± 0.02* 0.53 ± 0.11* 25 ± 2* 8

trolox 28 ± 2* 43 ± 3* 0.18 ± 0.01* 0.17 ± 0.04* 28 ± 2* 6

deferiprone 50 ± 6* 58 ± 5* 0.24 ± 0.02 0.17 ± 0.02* 23 ± 1* 6

trox/def 41 ± 3* 63 ± 2* 0.18 ± 0.01* 0.15 ± 0.02* 22 ± 2* 12

BDM 13 ± 3* 30 ± 7* 0.14 + 0.00 * 0.58 ± 0.25 * 29 ± 3* 4

Table 1: Effect of 16 h storage at 4ºC in HTK on contractile properties of the RET.

Values are expressed as mean SEM; n≥6, except BDM: n=4. All muscles except controls were stored for 16 h at 4°C in

HTK. Values for additives to HTK, trolox, deferiprone, trox/def and BDM, represent the maximum effect. Deferiprone

provides the most effective preservation of the contractile properties of the RET.

* Values differ signifi cantly from control.


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Fig. 3: Effect of additives

on the maximum tetanus tension (P0) of

hamster RET muscles stored for 16h at 4°C.

The addition of deferiprone (; n≥6) and trolox

(; n≥6 for each concentration)

induced a concentration dependent improvement

of P0 compared to HTK (; n=8).

BDM (; n=2-4) did not result in improvement

of preservation of P0. Symbols represent mean

values SEM, expressed as percentages of control. *

Values differ signifi cantly of muscles stored in HTK

(p<0.05).

Effect of additives on muscle function and cytoarchitectureThe effi cacy of the three substances that were added to HTK to reduce muscle damage

during cold storage is shown in Figure 3 as the dose –response relationships for P0.

P0 (% of control)

0 20 40 60 80 1 00

Pt (%

of c

ontr

ol)

0

20

40

60

80

100control

def

trox/def

trox

16 htk

Fig. 4: Correlation between RET

maximum tetanus tension (P0) and

maximum twitch tension (Pt) of

muscles stored for 16 h at 4°C.

A positive correlation exists between Pt

and P0 (r=0.98; p<0.01) of the different

experimental groups. Note that the Pt of

the muscles is more susceptible to cold

storage than the P0. Symbols represent

mean SEM (n=6-12). r = correlation

coeffi cient; HTK = HTK only; def = HTK +

deferiprone 10-5 M, trox = HTK + trolox

10-3 M, trox/def = HTK + deferiprone

10-5 M + trolox 10-3 M.

Addition of trolox (10-8-10-3 M) caused a concentration dependent increase of P0 in the RET muscle. The presence of 10-3 M of trolox in the HTK solution resulted in an increase of P0 to 433% of control. Values for Pt and Pt/P0 increased accordingly (Figure 1 and Table 1). Addition of deferiprone (10-12-10-4 M) also caused a concentration-dependent increase in P0 (Figure 3). The maximum effect was reached at 10–5 M and resulted in an improvement of P0 to 593% of control. Accordingly, deferiprone enhanced Pt and Pt/P0 (Figure 1 and Table 1). The improved preservation of contractile force by trolox and

Log concentration (M)

P 0 (% o

f con

trol

)

0

10

20

30

40

50

60

**

*

HTK

def

trox

BDM

* *

*

-12 -10 -8 -6 -4 -2


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deferiprone was accompanied by a reduction of the twitch threshold current (~0.2 mA) compared to muscles stored in HTK only (0.5 mA; P<0.05). Addition of the combination (trox/def) of the optimal concentrations of trolox (10-3 M) and deferiprone (10-5 M) caused no additional improvement (p=0.3) of muscle preservation (Po 632% of control) compared to addition of deferiprone alone (Figure 1 and Table 1). Addition of the Ca2+ release inhibitor BDM (3·10-3 – 3·10-2 M) did not have any effect on P0, Pt or Pt/Po (Figure 1 and Table 1). The values for Pt and P0 of the different experimental groups were linearly correlated (Figure 4). The additives showed no effect on RET cytoarchitecture.

P0 control

(mN/mg)

P0 HTK

(% of control)

Threshold control

(mA)

Threshold HTK

(mA)

RET 2.9 ± 0.1 27 ± 5 0.1 0.5

CT 1,3 ± 0.1 32 ± 4 0.2 0.8

Table 2: Comparison of the effect of 16 h storage at 4ºC in HTK on contractile parameters of RET and

CT muscles.

Values are expressed as mean ± SEM; n≥8. Storage in HTK shows similar effects on P0 and threshold values for both

muscles.

Discussion

Muscle function and cytoarchitecture of the RET following cold storageAll contractile parameters were profoundly affected after 16h of storage in HTK at

4°C. This considerable reduction in muscle function was not accompanied by any sign of necrosis or hypercontraction of the muscle fi bers which implicates that not a decreased number of participating fi bers, but a deterioration of the condition of the individual fi bers led to the decreased muscle function. Although the present data do not reveal the exact mechanism of damage, the increased threshold current and decreased Pt/Po ratio indicate damage to the electrical component of the excitation-contraction coupling process (E-C coupling). The higher stimulus frequency needed to reach a smooth tetanic contraction in stored muscles suggests that the duration of the active state is decreased, leading to a reduction in the amount of Ca2+ that is released from the sarcoplasmatic reticulum and thus a reduction of tension developed during contraction. When taken together, this implies that damage of the cell membrane was an important factor that limited the recovery of muscle function after cold storage. A partly, and presumably reversible, depolarization of the muscle cells seems a likely cause of the observed impairment. Increased [Ca2+]i and resulting hypercontraction (36; 50) and oxidative stress (50; 53) are thought to be responsible for membrane damage. The possible contributions of these two mechanisms to the membrane damage during storage are discussed below, based on the effects that additives intervening with these mechanism had on preservation of muscle function.


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Effect of antioxidants on the stored RET During hypoxia/reoxygenation or ischemia/reperfusion (20) the production of oxygen

free radicals is enhanced while endogenous antioxidant defense mechanisms are exhausted (21), which leads to membrane damage and subsequent functional disturbances. Much of the oxidative injury in postischemic tissue is a result of the highly reactive hydroxyl radical (OH). This radical is formed via the Haber-Weiss reaction or via the Fenton reaction and upon generation it destroys very rapidly the structure and function of molecules such as lipids, proteins and DNA (20). Deferiprone chelates both extra- and intracellular iron, which is essential as a transition metal in both reactions. The concentration of deferiprone that we found to preserve muscle function most effectively (10-5 M) is the same as that previously found to prevent postischemic cardiac injury in the rat (52). The prevention by deferiprone of the storage-induced increase in threshold current presumably results from the ability of deferiprone to reduce lipid peroxidation (34) and damage to the membrane (48).The current fi ndings indicate that deferiprone protects RET muscles effectively during cold storage and reoxygenation.

Trolox, which is a vitamin E analog, is an antioxidant that inhibits peroxidation in phopholipid bilayers by extra- and intracellularly scavenging of peroxyl radicals (8). The dose-dependent improvement of preservation of muscle function by trolox, accompanied by restoration of the twitch threshold current , is in accordance with the hypothesis that oxidant-induced lipid peroxidation of the sarcolemma is involved in the pathogenesis of muscle injury during cold storage and reoxygenation (25). The concentration of 10–3 M trolox that improved RET function is similar to that found to accelerate functional recovery in reperfused porcine hearts (27). Trolox appeared less effective in protection of RET muscle function than deferiprone, which indicates that in the hamster RET damage caused by the highly reactive hydroxyl radical plays a more important role than lipid peroxidation per se.

Effect of BDM on the stored RET In response to ischemia/reperfusion or hypoxia/reoxygenation a rise in intracellular

free Ca2+ occurs (15; 18), which can cause hypercontraction and can lead to a cascade of damaging reactions of the muscle cell by activating proteases and phopholipase A. BDM reduces Ca2+ release by the sarcoplasmic reticulum (16; 42). Our observation that BDM did not have any effect on the function of RET muscles stored for 16 h at 4°C is thought to implicate that Ca2+ overload does not play a large role in cold storage induced injury of the RET muscle. This is in accordance with the absence of hypercontracted fi bers after cold storage. Since the injury can be partly prevented by addition of antioxidants it seems that formation of reactive oxygen species is the main cause of the loss of function.

Dissimilarities in the effects of cold storage on hamster RET and rat CT muscleIn heart cells of hibernators during the non-hibernating state, differences in

intracellular Ca2+ handling are thought to be involved in the better tolerance to ischemia


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and hypothermia in comparison to those of non-hibernators (55; 56), but for skeletal muscles nothing is known about possible differences in tolerance of ischemia and cold storage. Therefore, the data for the hamster RET will be compared with those for the rat cutaneus trunci (CT) muscle, which has been previously tested under the same circumstances (50). The rat CT has the same fi ber type composition as the RET (70% type IIB, 16% type IIA and 14% type I for RET; 72% type IIB, 20% IIA and 8% type I for CT; (46; 51)).

The observation that the contractile function of the RET is not better preserved than that of the CT following 16 h storage in HTK at 4°C is in agreement with our earlier fi nding that the P0 values in the RET were similarly affected by an I-R insult in vivo (1h-1h;37°C; (10)) as those of skeletal muscles of rabbit (6). This leads to the conclusion that the tolerance to ischemia and storage of muscle contractility is not different between non-hibernators (i.e. rat CT) and hibernators (i.e. hamster RET) during the non-hibernating state. This is in agreement with the results obtained in comparative studies on solid organs of hibernators in the non-hibernating state (7; 19). The thickness of both muscles (CT 1.2 mm; RET 0.5 mm) is smaller than the so called ‘critical diffusion distance’ (about 4 mm at 4°C) defi ned by Hill (24) as ‘critical depth to which oxygen penetrates resting muscle’. (see also (49)). Thus, the difference in muscle thickness most likely does not play a role in the differential morphological effects of cold storage on CT (rounded and necrotic fi bers (50)) and RET (unaffected) morphology.

Figure 5 depicts the dose- response relationship of the effects of BDM on the P0 of stored RET and CT muscles. The fi nding that the CT but not the RET benefi ts from the addition of BDM, suggests that Ca2+ overload does not play an important role in the storage induced injury of the RET. There is a paucity of information on Ca2+ handling of skeletal muscles of hibernators, but it is known that heart cells of hibernators have a remarkable capability to maintain homeostatic intracellular Ca2+ levels (35; 56). The superior Ca2+ handling capacity displayed by hibernator hearts in the non-hibernating state is achieved by a number of adaptations (reviewed in (12; 55)). The greater amounts of SR (44) and an enhanced SR Ca2+ uptake (29; 54) by increased activity of SR Ca2+-ATPase are examples of such adaptations. The lack of effect of BDM on the RET and also the lack of damage to the morphology suggest that similar adaptative Ca2+ strategies as in hibernator heart cells are present in their skeletal muscles. Thus, skeletal muscles of hibernators in the non-hibernating state appear to have an alternative Ca2+ handling compared to skeletal muscles of non-hibernators.

Not only differences in Ca2+ handling between hibernators and non-hibernators, but also differences in endogenous defense mechanisms against reactive oxygen species have been reported (5). There is evidence for increased ROS production and management (33) particularly during arousal from the hibernating state. This suggested that hibernators intrinsically have more powerful antioxidant systems than non-hibernators (4). Our experiments, however, indicate that, under non-hibernating conditions, there are between hamster RET and rat CT no marked differences in endogenous defence mechanisms


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against reactive oxygen species, since cold storage induced a similar reduction in function in the rat CT and hamster RET. Also, the antioxidants trolox and deferiprone caused a comparable increase of P0 values of RET and CT. This leads to the conclusion that the main difference between the responses of the CT and RET to cold storage is in the Ca2+ handling, which is superior in the RET.

Fig. 5: Comparison of the effect of

additives on the maximum tetanus

tension (P0) of the hamster RET and

rat CT muscles stored for 16h at 4°C.

(A) BDM has no effect on the P0 of the

RET

(n=2-4); however for the CT (n≥4), the P0

was improved signifi cantly.

(B) Deferiprone causes a concentration

dependent increase of the P0 for the RET

(n≥6) and the CT (n≥4).

(C) Trolox induces a concentration

dependent increase of the P0 for RET (n ≥

6 for each concentration). The maximum

effect is similar to that found in rat CT

(n≥4).

The CT data have been published

previously by Van der Heijden et al (50).

Symbols represent mean values SEM,

expressed as percentages of control

( RET and CT). * Values differ

signifi cantly of muscles stored in original

HTK ( for RET and for CT; p<0.05).


-4,0 -3,5 -3,0 -2,5 -2,0 -1,5

P0

(% o

f con

trol

)

0

20

40

60

*

*

*BDM

CT

RET

A


-12 -1 0 -8 - 6 -4 -2

P0

(% o

f con

trol

)

0

20

40

60

*

**

*

trolox

CT

RET

CLog concentration (M)

-16 -14 -12 -10 -8 -6 -4

P0

(% o

f con

trol

)

0

20

40

60*

*

**deferiprone

RET

CT

B


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Conclusions

The present study demonstrates that after 16 h of storage at 4°C the function but not the morphology of the RET in the non-hibernating state was affected. This decline in function can be attributed to damage to the sarcolemma of the fi bers by reactive oxygen species. Although the cold storage-induced functional damage of hamster RET is of comparable magnitude as that of the rat CT, the underlying mechanisms are dissimilar. The responses of RET and CT to addition of additives point to a hibernation related species difference in myocyte Ca2+ handling. In this aspect, the Ca2+ handling of the RET seems comparable to that in the hibernator heart (29; 54). Further exploration of such adaptive mechanisms of hibernator skeletal muscle may offer new insights in the management of cold preservation prior to transplantation of CTAs. In this light, the value of the hamster RET might be even more evident through its recent introduction as a free fl ap model (9).

Acknowledgements

The authors thank Henk Veldman (Laboratory for Experimental Neurology, University Medical Center Utrecht) for advice and assistance during histological preparation. Sara van Velsen is acknowledged for her contribution in scoring the histological preparations. The authors do not have any fi nancial interests in products, devices and drugs used in this study.

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91

6

The vascular anatomy of the hamster retractor

muscle with regard to its microvascular transfer

Miriam CJ de With1, Anne M de Vries1, Alfons BA Kroese2,4, EPA Brigitte van der Heijden1, Ronald LAW Bleys3, Steven S Segal5, Moshe Kon1

1,2,3 Departments of 1Plastic Reconstructive and Hand Surgery, 2Surgery & 3Pharmacology and Anatomy,

University Medical Center Utrecht, Utrecht, The Netherlands

4 Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands

5 Department of Medical Pharmacology and Physiology & Dalton Cardiovascular Research Center, University

of Missouri – Columbia, Columbia, MO, USA

European Surgical Research 2009; 42 (2): 97-105


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Abstract

BackgroundThe hamster retractor muscle (RET) is used as an in vivo model in studies of skeletal

muscle ischemia-reperfusion injury. The RET is unique in that the muscle can be isolated while preserving the primary vascular supply so that its contractile function can be measured simultaneously with local microvascular responses to experimental interventions. The goal of this study was to understand the anatomical origin of the vascular supply to the RET and determine whether the RET can be used as a free fl ap after surgical isolation of the thoracodorsal vessels.

MethodsMicrodissection was performed to determine the anatomy of the vasculature that

supplies and drains the RET.

ResultsDistinct numbers and patterns of feed arteries (2-4) and collecting veins (1-3) were

identifi ed (n=26 animals). Dye injection (n=8) of the thoracodorsal artery demonstrated that the RET remains perfused following its isolation on the thoracodorsal pedicle. Heterotopic allograft transplantation of the RET (n=2) was performed by anastomosing the thoracodorsal vessels to the femoral vessels using the end-to-side technique.

ConclusionsThe anatomical relationships indicate that the RET can be used as a free fl ap model

for evaluating the effect of preservation strategies and transplantation on skeletal muscle microcirculation and contractile function.


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Introduction

The hamster cheek pouch retractor (RET) muscle is a long, thin strap-shaped muscle, running from the lumbodorsal fascia and last three thoracic vertebrae to the cheek pouch (25). The RET serves as an antagonist to the longitudinal musculature of the cheek pouch wall and retains the cheek pouch above the scapula when it is fi lled with material as well as prevents eversion of the pouch when its contents are ejected (25). Its superfi cial location and unique anatomical organization have enabled the muscle to be used for intravital studies of oxygen transport (32) and blood fl ow regulation (30; 31; 37) in the microcirculation of mammalian skeletal muscle. The central region of the RET muscle is supplied by posterior retractor ‘feed’ arteries, which arise from the thoracodorsal artery, a branch of the subscapular artery (11; 24; 25). These feed arteries vary in number, are accompanied by ‘collecting veins’, and anastomose with each other at the arteriolar level via arcading segments (11; 24; 31). The RET is unique in that the muscle can be exteriorized while preserving its primary vascular supply (18), enabling muscle force production to be monitored concomitant with the study of microvascular responses at defi ned locations within the peripheral circulation (37; 40). This preparation has thereby provided novel insight into the regulation of skeletal muscle blood fl ow (for review see (28; 29)).

Feed arteries originate proximally from a larger conduit artery (e.g. thoracodorsal artery in the RET) and provide substantial resistance to blood fl ow before giving rise to the arterioles within the muscle (6; 28). In response to contractile activity, vasodilator signals ‘ascend’ from the microcirculation to encompass the feed arteries upstream (28; 40) and thereby enable these proximal vessels to serve a key role in regulating blood fl ow delivery into intermediate and terminal arteriolar networks, which control the distribution and magnitude of blood fl ow to skeletal muscle fi bers (6; 28). In feed arteries of the RET, the endothelium serves as the cellular pathway for conducting the vasodilator response along the vessel wall and into the surrounding smooth muscle cells to produce relaxation (9). Accordingly, direct observation of conducted vasodilation along RET feed arteries following an ischemia-reperfusion (I-R) insult is integral to assessing endothelial dysfunction (8).

The effect of transplantation on the microcirculation of skeletal muscle is poorly understood, in large part due to the lack of a suitable experimental model. Recently, we reported that both contractile function and endothelium-dependent blood fl ow regulation of the RET were impaired by I-R (8). These fi ndings suggested that the RET may prove useful as a free fl ap model to evaluate the effi cacy of preservation strategies aimed to protect skeletal muscle and its vascular supply during cold preservation and reperfusion (3; 7; 34). With the goal of providing further insight into how transplantation affects microvascular integrity, the aim of the present study was to understand the anatomical origin of the vascular supply to the RET and determine whether it can be isolated on a thoracodorsal vascular pedicle, preserving the feed arteries and collecting veins. Because restoration of fl ow is essential to the survival of a free fl ap, we assessed the vascular territory within


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the RET that was actually perfused by the feed arteries. Further, the most suitable site for performing microsurgical anastomoses was determined, which requires vessel diameter of at least ~300 m (14).


Animal careMale golden hamsters (Harlan-Winkelmann, Borchen, Germany), weighing 1203 g,

were housed in groups of three to four animals on a 12h-12h (light-dark) cycle at 23-24°C and provided with standard diet food and water ad libitum. The animals received care in compliance with the European Convention guidelines (86/ 609/EC). All procedures were approved by the Utrecht University Committee for Experiments on Animals. Hamsters were injected with buprenorfi nehydrochloride (Temgesic®; Schering-Plough, Utrecht, The Netherlands) 5 mg/kg subcutaneously 30 minutes prior to surgery. Isofl urane® (Abbott Animal Health, Oudewater, The Netherlands), supplied through a ventilation mask, at 5% with 100% oxygen (1 l/ minute) was used to induce anaesthesia (~5 minutes). Isofl urane® at 2% with oxygen (0.3 l/min) and air (0.6 l/min) was subsequently used to maintain anaesthesia. Excess vapor was scavenged by vacuum. Sterile saline was delivered through an intraperitoneal cannula (Abbocath–T 18 G, Abbott, Sligo, Ireland) to replace body fl uids during the experiment (1 ml/h; Fresenius Orchestra, Fresenius Kabi, Utrecht, The Netherlands).

During the surgery for isolation of the RET on its vascular pedicle, body temperature was maintained at 37.0-38.0 °C with heat conducted from a warm copper plate positioned beneath the hamster. The surgical fi eld was irrigated continuously with preheated Ringers lactate solution containing in mmol/L: Na+ 131; Ca2+ 1.8; K+ 5.4; Cl- 111; lactate 111; 273 mOsm/L (Baxter, Utrecht, The Netherlands). At the end of the experiment, animals were euthanized by an intracardial overdose of sodium pentobarbital (Nembutal®; CEVA Sante Animale BV, Maassluis, The Netherlands).

Retractor muscle and vascular dissectionA hamster was positioned on its left side to isolate the RET on the right side. To

expose the RET, a ~3-4 cm incision was made through the overlying skin, which was retracted to expose the underlying tissue. Using microdissection while viewing through a stereomicroscope (OPMI6-DF Zeiss, Germany), the muscle was cleared of connective tissue and visible nerve branches. Stainless steel wires (diameter 0.5 mm) were sutured to the origin and insertion of the RET, exactly at the caudal margin of the cheek pouch, representing the rostral muscle end, and at the musculo-tendinious transition point at the lumbodorsal (caudal) end (see dotted lines in Figure 1A). This was done to enhance muscle manipulation during surgery and prevent folding of muscles during the staining process (see Vascular Territory). The resting length of the muscle was measured between the stainless steel wires in situ.


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The RET was then severed from its origin and insertion and refl ected dorsally to visualize the feed arteries and collecting veins on its ventral surface. During this isolation, small vessels supplying the cranial and lumbodorsal ends of the muscle were cauterized and cut (shown in Figure 1A and see Results). While carefully maintaining the integrity of the feed arteries and collecting veins at the ventral surface, the nerve branches, fat pads and connective tissue that covered the transition point of the feed arteries and collecting veins into the thoracodorsal vessels were removed to optimize visibility.

The number and routing of feed arteries and collecting veins of the RET were documented in 26 animals. All side branches of vessels that supplied other muscles were ligated and cut until the RET was connected to the body exclusively by the thoracodorsal pedicle. As the diameters of the thoracodorsal vessels were too small for performing microsurgical anatomoses, the vascular pedicle running between the latissimus dorsi (LM) and teres major (TM) muscles (see Figure 1A), was dissected further upstream until a suitable vessel diameter (~0,3 mm) was reached. Both the anatomically diffi cult accessible location within the armpit and the density of side branches make the axillary vessels unfi t as an alternative to the smaller thoracodorsal vessels. Surgical procedures typically required ~5 h. At the end of the study, preparation time had decreased towards 4 h.

Vascular TerritoryIn contrast to previous I-R experiments(8) the entire RET was used in this study.

Therefore, in 8 of 26 preparations the vascular territory supplied by the feed arteries of the RET was investigated by intravascular injection with plastic as described (35; 36). Briefl y, 12 parts of Araldite F® with 7.5% Microlith-T pigment was mixed with 4 parts of diluent DY 026 SP and 9 parts of hardener HY 2967 (all from Ciba-Geigy BV, Arnhem, The Netherlands). At the end of the dissection, the thoracodorsal artery was ligated as far proximally possible. Following arteriotomy, a cannula (outer diameter ~0.25 mm) was inserted and secured to the vessel with two ligatures. Vessels were fl ushed with 1 ml of Fraxiparine® (~100 units, Glaxo Smith Kline, Zeist, The Netherlands) to prevent clotting. After that, the hamster was euthanized as described above and the Araldite F® mixture was injected through the cannula at a perfusion pressure of 210 mmHg. The relatively high injection pressure was required because of the small diameter of the cannula. By a closed valve, the vasculature was maintained under constant pressure overnight in order to allow the plastic to polymerize. Subsequently, the muscle fl ap was removed from the hamster. The fl ap was then suspended in graded concentrations of ethanol (50%-70%-80%-96%-absolute; incrementing every 24 hours) and then in methyl benzoate (Sigma-Aldrich Chemie bv, Zwijndrecht, The Netherlands) to clear the muscle tissue. The entire preparation was examined under a microscope (Makroskop M420, Wild, Heerbrugg, Switzerland) to distinguish the vascular territory containing Araldite F®. Photomicrographs were taken (E450 camera, Nikon, Tokyo, Japan; total magnifi cation X10) and the total muscle length, as well as that of border areas at each end of the muscle that were not fi lled with Araldite F® were measured to the closest mm. To normalize the size of unfi lled border areas across preparations with different absolute lengths, the length of unfi lled areas was expressed as percentage of the respective total muscle length.


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Allograft Muscle TransplantationIn a pilot study for allograft transplantation (n=2), the right RET fl ap was isolated in

donor hamsters as described above (see Figure 2A). The thoracodorsal artery and vein were cut obliquely in order to increase vascular diameter and thus facilitate the subsequent procedure for performing anastomoses. Volume controlled fl ush of the vascular bed was performed with 1 ml warmed Ringer’s lactate solution containing 100 units of Fraxiparine®, using a ~0.15 mm cannula inserted into the thoracodorsal artery (~0.1 ml/min). The washout fl uid was heparinized, because this has been shown to increase ischemic tolerance in free fl aps (4; 22; 26). The tissue was kept moist by continuous irrigation with preheated Ringers lactate solution. During the same period of surgery, an oblique incision was made through the skin overlying the left inguinal region of a recipient hamster anesthetized as described above. The femoral artery and vein were dissected free from connective tissue to create a suitable acceptor site for the muscle fl ap, as shown in Figure 2B. The femoral vessels were clamped using microvascular clamps (S&T AG, Neuhausen, Switzerland). Arteriotomy and venotomy were performed with microscissors. The RET free fl ap from the donor hamster was transferred to the recipient hamster and blood supply was re-established by end-to-side microanastomosis of the donor thoracodorsal artery and vein to the recipient femoral artery and vein (see Figures 2C and D). The end-to-side method was preferred because of the signifi cant size discrepancy between the donor and recipient vessels (1). Four to fi ve 11-0 sutures (Ethicon, Johnson and Johnson, Amersfoort, The Netherlands) were used for each microanastomosis. Immediately and during 1 hour of reperfusion, anastomotic patency was tested using visual inspection of muscle color and vessel shape. Besides, the ‘milking test’ was performed (23; 38). In short, a Dumont no. 5 microforceps was used to occlude the lumen of the thoracodorsal artery approximately 2 mm downstream from the anastomosis. A second forceps was moved downward from the occluding forceps to clear a ~ 2 mm vascular segment from blood. Anastomotic patency was confi rmed by immediate anterograde refi lling after removal of the most proximal (fi rst) forceps.

Data PresentationRepresentative illustrations were prepared to describe the vascular supply to the RET.

Summary data are reported as the percentage of hamsters (out of n=26) which exhibited the designated number of feed arteries or collecting veins. Photomicrographs were taken to illustrate the vascular anastomoses prepared surgically and to demonstrate the perfused vascular territory.

Results

Isolation of the Retractor MuscleThe average resting length of the RET muscle measured from cheek pouch to

lumbodorsal fascia was 36 ± 1 mm (n=26). Muscles typically shortened by 20-30% when cut, as reported previously (18).


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Vascular isolationThe blood supply to the RET enters via several routes (Figure 1A). The feed arteries,

which branch off from the thoracodorsal artery to enter the ventral surface of the muscle, provide the main blood supply of the RET. In addition, the posterior auricular artery, a branch of the external carotid artery, gives off the anterior RET artery, which enters the muscle at the level of the scapula and vascularizes the rostral region of the RET (24; 25; 31). An additional small perforating artery was found to supply the lumbodorsal (i.e. caudal) region of the muscle (Figure 1A). These vessels supplying the rostral and caudal regions were cut in order to clearly visualize the ventral surface of the RET.

Anatomical Variation in Arterial SupplyThe branches of the thoracodorsal artery which form the RET feed arteries were

found to have both consistent and variable anatomical patterns (Figures 1B and C). In all preparations (n = 26) the main thoracodorsal artery bifurcated into two major branches. The fi rst, ‘deep branch’ ran underneath the spinotrapezius muscle; the other, ‘superfi cial branch’, ran over the latissimus dorsi muscle, and consistently bifurcated into one branch supplying the latissimus dorsi muscle while the other branch supplied the overlying skin. The length of the main thoracodorsal pedicle before the bifurcation was approximately 5 mm. The total length of the vascular pedicle to the muscle border varied from ~13 mm at the side of the most rostral feed artery to ~25 mm at the location of the most caudal feed artery.

Across preparations (n=26), the number of feed arteries varied from one to four, with their distribution displayed in Figure 3. Generally, three (50%; Figure 1B) or two (38%; Figure 1C) feed arteries supplied the RET. With one exception, all RET had at least one feed artery that branched off before the fi rst bifurcation or from the ‘deep branch’ (96%) itself. The remaining feed arteries originate from the ‘superfi cial branch’. A solitary feed artery, i.e. without paired collecting vein, was found in many preparations (42%).

In order to isolate the RET on the thoracodorsal vessels, a variable pattern of smaller vessel branches to surrounding muscles and fat pads needed to be ligated and cut. For the ‘deep branch’ this was done distal from the point where the feed arteries originate from the thoracodorsal artery. While this was diffi cult due to lack of space, it was simplifi ed by making an incision in the spinotrapezius muscle (STP, Figure 1A). The diameter of the main thoracodorsal artery was ~0.3 mm in the region used for creating anastomoses. Although the more proximal arterial segments were larger, they did not offer a better alternative for microsurgical anastomosis because of their many side branches.

Anatomical Variation in Venous DrainageDistinct types of venous patterns were identifi ed (Figures 1B and C). Similar to the

anatomy of the arterial supply, the main thoracodorsal vein consistently (n=26) bifurcated into ‘deep’ and ‘superfi cial’ thoracodorsal branches which received blood from the collecting veins draining the RET. In all but two cases, at least one collecting vein drained


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2

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RET

A

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*

**

Fig. 1: Vascular anatomy of the hamster RET muscle.

In the exposed RET in situ (A) the blood supply is indicated to be via three routes (1, 2, and 3), of which the thoracodorsal

(1) is the primary source. The feed arteries and collecting veins are indicated by 4 and can not clearly be seen from

this perspective of the dorsal surface. The two dotted lines represent the locations at which the rostral (*) and caudal

(**) margins of the RET were sutured to stainless steel wires and cut in order to fl ip the muscle over and reveal the

vasculature in more detail from the ventral surface as illustrated in B, C and D.

The feed arteries and collecting veins on the ventral surface of the RET are depicted in B and C, where the most

frequently encountered arterial and venous pedicle patterns are shown. The main thoracodorsal artery emerges from

the axilla and runs between the teres major and latissimus dorsi muscles. It bifurcates at the level of the spinotrapezius

muscle, giving off a branch running underneath the spinotrapezius muscle, which is defi ned as the ‘deep’ thoracodorsal

artery. The other branch runs over the latissimus dorsi muscle and is defi ned as the ‘superfi cial’ thoracodorsal artery.

The same nomenclature is used for the accompanying venous branches. In (B) three feed arteries branching off the

thoracodorsal artery are accompanied by two collecting veins (35% of total). Note that the most caudal artery does

not have a paired collecting vein. In (C) two feed arteries are each accompanied by a paired collecting vein (31%). The

darker coloured arterial and venous segments in (B) and (C) indicate – in case of performing a free fl ap - the full extent

of the vascular pedicles. The arrows point out the exact sites used for anastomosis. In (C) a dual thoracodorsal vein is

shown. The enlargement in (D) shows a detail of (B). The side branches of the most proximal feed artery and collecting

vein supplying a part of the spinotrapezius muscle (present in 65% of preparations) are particularly diffi cult to dissect

and ligate.

Abbreviations and defi nitions: CP, cheek pouch; RET, retractor muscle; STP, spinotrapezius muscle; LD, latissimus dorsi

muscle; TM, teres major muscle. (1) thoracodorsal artery and vein; (2) lumbodorsal branch; (3) posterior auricular

artery; (4) feed arteries and collecting veins; (5) ‘deep’ thoracodorsal branch; (6) ‘superfi cial’ thoracodorsal branch;

(7) dual ‘main’ thoracodorsal vein; (8) spinotrapezius branch of the thoracodorsal artery.


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into the main or into the ‘deep’ thoracodorsal vein while in the other two preparations all collecting veins drained into the ‘superfi cial’ thoracodorsal vein.

In eight preparations (31%) the main thoracodorsal vein was dual over its entire course between the latissimus dorsi and teres major muscles (see Figure 1C). In all eight cases, these twin veins converged just before reaching the most rostral collecting vein of the RET and then bifurcated into the ‘deep’ and ‘superfi cial’ vein. When dual main thoracodorsal veins were present, they appeared smaller as compared to preparations having a single thoracodorsal vein of ~0.3 mm diameter. However, in such cases the ‘superfi cial’ vein, which has a similar diameter, provides a suitable alternative for performing a vascular anastomosis (see Figure 1C).

The distribution of the number of collecting veins in individual RET muscles is depicted in Figure 3. Across hamsters (n=26), the number of collecting veins varied from one to four, with two present in most (73%) cases. In fi fteen preparations (57%), all feed arteries had paired collecting veins and their routing was identical. As seen with small arterial branches, a variable pattern of small venous branches to surrounding muscles and fat pads were ligated and cut to prepare a free fl ap that was isolated to the thoracodorsal artery and vein. Branches running from the spinotrapezius muscle often drained in the most proximal collecting vein (69%).


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Vascular TerritoryFollowing injection with Araldite F via the thoracodorsal artery (n=8), the vascular bed

of the RET was nearly completely stained except for the lumbodorsal and anterior borders (17±3 and 6±3 % of total muscle length, respectively). Figure 4 shows a representative example.

Allograft TransplantationIn a pilot study, we examined whether the RET, with preserved feed arteries and

collecting veins, could be isolated on the thoracodorsal pedicle for use as a free fl ap model in future studies. Thus, two transplantations of the RET from a donor hamster to the femoral artery and vein of a recipient hamster were performed as described in Materials and Methods. The duration of ischemia during these transplantations was ~50 minutes. Both of the two transplantations that were performed were considered successful, as confi rmed visually through the microscope. Observations immediately after restoration of blood fl ow included true fi lling of the vascular system by change of muscle colour from grey-blue to pink-red and natural rounded appearances of the thoracodorsal and recipient femoral veins. Besides, microvascular patency was verifi ed by a positive milking test throughout the 1 hour testing period.

Discussion

The goal of this study was to determine whether the RET muscle of the hamster cheek pouch could be utilized as a model for studying the effects of transplantation on the microcirculation of mammalian skeletal muscle. Complete surgical isolation of the RET and its vessels was performed, such that only the primary arterial supply and venous drainage remained attached to the muscle. Measurement of the perfused vascular territory of free muscle fl aps following Araldite F® injection showed that, after transplantation, the muscle is adequately perfused via feed arteries and veins arising from the thoracodorsal vessels, which function as the donor vessels. Further, the results of this study verify that the caliber of the proximal section of the thoracodorsal artery and vein are suitable for microvascular anastomosis at a suitable location, e.g. the femoral artery and vein.

As shown by injection with Araldite F®, relatively small regions of the caudal and the rostral borders of the RET may be excluded from the free fl ap preparation, as blood fl ow is unlikely to be restored in these areas following transplantation. The fi nding that respective end-regions are no longer perfused once the RET is cut from its origin and insertion is in agreement with our observation that, in addition to the feed arteries that branch off the thoracodorsal artery, there were two other sites of vascularization at respective muscle borders. Whereas the perforating lumbodorsal arterial branch at the caudal end has not been described in detail previously, vascularization of the rostral region of the RET has been well defi ned (24). Within the muscle, the anterior RET artery and (to a lesser extent) the saccular arteries of the cheek pouch are interconnected to the


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posterior RET ‘feed’ arteries by several anastomoses at arteriolar level. The presence of these anastomoses suggests that, with time, the vascular supply to each end of the muscle may be re-established through remodeling of the microcirculation to restore blood fl ow into ischemic regions. For experiments designed to measure the force developed by RET muscle fi bers during contractions, these non-perfused border areas at each end are used to secure the muscle to an experimental platform while observing the microcirculation with intravital microscopy (8; 37; 40).

Apart from a somewhat variable number of feed arteries and collecting veins, we found the gross vascular anatomy to be relatively consistent across preparations. These fi ndings are consistent with an earlier study of blood vessels to the rat cremaster muscle (17). For RET with unequal numbers of arteries and veins, it should be noted that solitary feed arteries can provide an advantage for experimental observations in that these vessels are more easily visualized for intravital microscopy as compared to arteries that run parallel to (and can thereby be obscured by) a paired collecting vein. The venous anatomy of the RET can be complicated by the presence of dual main thoracodorsal veins that are too small for effective surgical manipulations. Nevertheless, in such cases we found that the more caudal ‘superfi cial’ thoracodorsal vein provides a viable alternative of suffi cient diameter. Moreover, the vascular pedicle of the RET is consistently of suffi cient length to perform microsurgical anastomoses.

The use of the RET as a model for free fl ap transplantation has several restrictions. Firstly, the diameter of the donor artery and vein measure ~0.3 mm. This dimension is considered to be near the smallest vessel size that can be surgically attached to another (14). As anastomotic patency decreases with diameter, such a challenging technical procedure is recommended to be performed by a highly experienced microsurgeon. Secondly, due to the small size of the vascular branches, and the complexity of their relationship with each other, dissection of the pedicle to the RET requires extreme care and patience. Ligating all of the side branches can also cause damage to the feeding and draining vessels. With practice, however, dissection of the RET as a vascularised free fl ap can be readily performed in a reproducible manner.

Contrary to the sole parameter (survival) that can be measured in the majority of

free fl ap animal models (for examples see (42)), only few adequate animal skeletal muscle models exist that allow detailed intravital microcirculatory analysis. The primary reason for developing the hamster RET free fl ap model was that both contractile muscle (34) and microcirculatory measurements (3) can be used to evaluate the effi cacy of preservation techniques in future studies. For example, the potential for recovery of muscle function after cold ischemia depends on recovery of both the parenchymal (muscle) cells and their vascular supply (7; 10). The use of intravital microscopy allows for direct visualization of the microvascular network within the RET along with its feed arteries (37; 40), including manifestations of impaired endothelial cell function and vasomotor control (8; 30) or leukocyte/endothelial interactions (8; 12; 33). An important advantage of the RET over other


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muscle preparations used to study the microcirculation, such as the free vascularized rat or mouse cremaster muscle models (3; 21), is that the parallel arrangement of the RET muscle fi bers enables a quantitative and reliable serial evaluation of the contractile function of the muscle fi bres (8; 37; 40).

Fig. 2: Transplantation procedure of the RET muscle.

(A) The RET is isolated on its thoracodorsal pedicle (TDA + TDV). Note that the ‘superfi cial’ venous branch (TDV)

is also dissected free and available for anastomosis. (B) The femoral artery and vein (FMA + FMV) are dissected free

to create the acceptor site. (C) End-to-side anastomoses are performed between thoracodorsal and femoral vessels,

microvascular clamps are in situ (D). Successful restoration of blood fl ow following release of the microvascular

clamps. The asterisk indicates the microvascular anastomoses. Abbreviations and defi nitions: RET: retractor muscle;

FA: feed arteries; CV: collecting veins; TDA: thoracodorsal artery; TDV: thoracodorsal vein; FMA: femoral artery; FMV:

femoral vein. Pieces of blue (A, C and D) and white (B) surgical gloves were used to enhance tissue contrast during

surgery. The scale bars represent 2 mm.

Fig. 3: Distribution of number of feed arteries and collecting veins in individual hamster RET.

For all 26 muscles studied the distribution of the number of feed arteries and collecting veins is depicted. Overall, there

are two or three (88%) feed arteries vs. two (73%) collecting veins.

Number of feed arteries (n)

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Number of collecting veins (n)

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0

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Fig. 4: Photomicrograph of the RET muscle fl ap after injection of Araldite F® into the thoracodorsal

artery.

The rostral muscle end is oriented to the right. This representative photomicrograph of vascular fi lling with Araldite

F® illustrates that the RET is primarily vascularized by three branches (which form the feed arteries) arising from the

thoracodorsal artery. The muscle border at the cranial end was almost completely perfused, whereas 21% of muscle

length bordering the caudal end was not. Fragments of blue sutures, that connect the muscle ends to stainless steel wires,

are visible on both lateral sides of the photograph. Dotted green lines represent the borderlines of the non-perfused

regions. Note the presence of multiple arcading anastomoses between distinct feed arteries at arteriolar level, indicated

by an asterisk. The scale bar represents 3 mm. FA: feed arteries.

In terms of clinical relevance, the RET model has the potential to augment our understanding of microvascular and skeletal muscle dysfunction following ischemic insults of various varieties. Currently, the clinical use of free tissue transfers shows relatively low failure rates of 5-6% (27; 41). The failure rate in replantations is much higher (8-18%, (27)). Apart from complete graft loss, the number of partial failures is not often reported and probably underestimated (39). The main reasons for total fl ap failure remain thrombosis of the anastomosis (41) and vasospasm (2; 16). Partial fl ap failure, however, is mainly initiated by local ischemia in hypoperfused areas. The latter is often referred to as the no-refl ow phenomenon (for review see (33)). Recently, it was reported that disturbance in active vasomotility is a major factor in development of the no-refl ow phenomenon in free fl aps following I-R (19). Most evidence for the role of endothelial vasomotor dysfunction however results from animal studies in particular in cardiac muscle tissue (13). We previously concluded that following I-R ascending vasodilation in the RET feed arteries was diminished due to impairment of local response to the vasodilatory trigger distally in the vascular tree of the muscle (8). We hypothesize that interventions


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that lead to reestablishment of local vasodilator signals will contribute to prevention of hypoperfusion and the no-refl ow phenomenon. Interventions that could be employed in future research include cold preservation and several pharmacological approaches (34).

Anticipating on future experiments with the RET model, including cold ischemic events and subsequent reperfusion, it must be emphasized that success percentages of ~90% have been reported in microanastomoses with similar diameters (0.3-0.4 mm) following 24 h of storage at 4° C (5). Therefore, we expect that irrespective of temperature, the RET can be used reliably with gentle tissue handling, good visualisation and the meticulous use of fi ne instruments. Given the anatomical complexity and technical constraints of the surgical preparation, the verifi cation of reperfusion was in the present study limited to visual inspection and the milking test. In future RET transplantation experiments vascular integrity will be assessed by direct observation of the microvascular network through intravital microscopy, enabling quantifi cation of reperfusion by blood fl ow measurement (15). Functional parameters could then include evaluation of diameter changes during ascending vasodilation and skeletal muscle twitch and tetanus tensions (8). For such an investigation inbred hamster strains need to be used, since it is not possible to reliably study the sole effect of prolonged reperfusion of the RET fl ap in genetically non-identical animals, as earlier reports from limb allotransplantations show microcirculatory changes within 24 hours after transplantation (20). An additional advantage of the RET transplantation model is that it has the potential to display allograft related immunological responses in survival studies on outbred strains.

In conclusion, the present fi ndings provide essential new anatomic information concerning the hamster RET muscle and its vascular pedicle in light of using this preparation as a model to study the microcirculation and contractile muscle function in free muscle fl ap transplantation. We demonstrate the relative consistency of it vascular anatomy while accounting for subtle variations in arterial and venous branches. Moreover, in a pilot study, we showed that the RET can be prepared as a viable allograft.

Acknowledgements

The authors thank Ingrid Janssen for skilfully producing the illustrations in Adobe Illustrator CS and Quinten Ruhé, MD PhD for optimizing the graphical quality of the photomicrographs. Willem van Wolferen and Simon Plomp are gratefully acknowledged for their technical assistance during the Araldite F study. This study was supported by the Jan Dekker and Ludgardine Bouwman Foundation, The Netherlands. Steven Segal PhD is supported by NIH grants RO1-HL41026 and RO1-HL56786. The authors do not have any fi nancial interests in products, devices and drugs used in this study.


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107

7

General Discussion


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Summary

In reconstructive surgery, ischemia-reperfusion (I-R) injury of skeletal muscle tissue occurs during replantations, free vascularized transfers of muscle fl aps and following composite tissue allograft (CTA) transplantations. The latter is a newly emerging fi eld and involves the allotransplantation of complex composite tissues from deceased donors (25). Complete and partial failures of free vascularized muscle fl aps and replantations remain a signifi cant clinical problem (27). The more frequent application of CTAs requires the design of a preservation strategy that prevents I-R injury and allows tissue banking with maintenance of cellular function. Full recovery following I-R insults requires both skeletal muscle- and microvascular integrity.

The hamster retractor muscle model (RET; (21; 37)) was introduced by the author in the Utrecht Plastic Surgery Research Laboratory and developed for investigations of I-R and CTA preservation techniques. The RET is unique in that the muscle can be exteriorized while preserving its vascular supply (21), enabling in vivo muscle force production to be monitored concomitant with microvascular responses of the peripheral circulation. This model has provided novel insights into the regulation of skeletal muscle blood fl ow by ‘ascending vasodilation’ (AVD) during contractile activity (28; 29). AVD entails the ascend of the initial vasodilation of the intramuscular arteriolar networks to the proximally located ‘feed arteries’. These proximal vessels were shown to play a key role in the vasomotor control of skeletal muscles (28; 38). The studies described in this thesis are the fi rst reports on the effects of I-R on AVD. In this chapter the results and conclusions of our studies on the RET will be summarized and discussed, the questions raised in Chapter 1 will be answered and directions for future research will be presented.

In Chapter 2 we showed that I-R (1h-1h) reduced skeletal muscle function and AVD of the RET to ~50% of control values. The loss in contractile muscle function was only partially responsible for the impairment of AVD, as stimulating muscles with a higher stimulus frequency restored contractile tension, but AVD remained markedly depressed. To investigate in more detail the mechanism behind the I-R-induced compromise of the AVD, vasodilatory responses to acetylcholine (ACh) were measured at the site of application and at three remote sites along the feed artery. The responses at all four sites were equally affected by I-R, but all responses recovered by an eightfold increase of the ACh stimulus. These fi ndings demonstrate that the initiation of AVD in response to muscle contractions is impaired after I-R, while conduction of the dilatory signal along the vessel wall remains intact. The functional decrement in the dilation of feed arteries following I-R can thus be attributed to a derangement in the initiation of the dilator signal (e.g. hyperpolarization) by the endothelium at the site of stimulation. Once vasodilation is initiated, conduction along the vessel by cell-to-cell-coupling through gap junction channels is preserved. Such a functional decrement following I-R will limit muscle blood fl ow and vasomotor control. We hypothesize that the impairment in the ability to initiate conduction following I-R refl ects damage to the endothelial cell membrane by reactive


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oxygen species (ROS). This is in agreement with the reported interference of ROS with receptor signalling and the ability to initiate hyperpolarization through activation of Ca2+-sensitive potassium channels (KCa), which are essential for initiation of AVD in RET feed arteries (7; 32; 33).

In Chapter 3 we assessed the relative contributions of Ca2+ overload and the formation of hydroxyl radicals (OH), a ROS species, to the I-R injury of contractile muscle function. Additionally, we aimed to determine whether the formation of OH is a causative factor in the impaired initiation of AVD. Therefore, two pharmacological interventions were employed during I-R. The addition of BDM, which reduces Ca 2+ release by the sarcoplasmic reticulum (SR; (8; 30)) entirely prevented the I-R-induced reduction of contractile muscle function. In skeletal muscle intracellular Ca2+ overload and ROS formation are supposed to be interrelated extensively (1; 4). Especially OH is thought to contribute to the destruction of the SR and impairment of contractile myocyte function following I-R (24). The complete lack of protective action of deferiprone, which inhibits iron-catalyzed OH-mediated lipid peroxidation (20), on the contractile properties of the RET indicates that the I-R-induced contractile defi cit and Ca2+ overload in myocytes is not due to OH formation. We concluded that during early phases of I-R such as currently used (1h-1h), the full extent of contractile muscle defi cit is attributable to the detrimental action of Ca2+ and iron-mediated formation of OH is not involved in impairment of contractile muscle function.

The functional defi cit in AVD following I-R remained unaltered by the addition of deferiprone, although the general notion is that, of all ROS, OH in particular has a destructive action on the endothelium (32; 33). Yet, iron-mediated formation of OH does not seem to play a role in the disruption of endothelium-dependent mechanisms of blood fl ow control. Since for other ROS, such as superoxide (O2

-), hydrogen peroxide (H2O2)and peroxynitrite (ONOO-), inhibitory effects on endothelium dependent vasodilation have been described (14; 16; 26), we hypothesize that other reactive oxygen species than OH may be involved in the I-R-induced impairment of the initiation of AVD.

In Chapter 4 we investigated whether cooling to 21°C would prevent the endothelial dysfunction associated with impaired AVD. The results showed that following I-R at 37°C, contractile function and AVD were reduced to ~50% of control values, whereas both were fully preserved at 21°C.

The mechanism by which cooling from 37°C to 21°C protects the endothelium from I-R-induced injury is most likely related to the overall reduction of oxygen consumption and slowing down of energy demanding processes by a factor of 1.5 - 3 (2; 9; 39). The delayed depletion of ATP and phosphocreatine stores associated with hypothermia will contribute to prolonged maintenance of cellular viability and function. The decreased levels of ROS measured in neural and liver tissues during mild hypothermia support the hypothesis (13; 17;

18) that the impairment of AVD at 37°C refl ects damage to the endothelial cell membranes by ROS such as superoxide (O2

-).


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The preserving effect on AVD of cooling presents the fi rst intervention that completely maintains AVD following I-R (1h-1h). This may have immediate implications for clinical surgery. We propose that under clinical circumstances, where prolonged ischemia of skeletal muscle tissue is anticipated, tissue cooling should be applied to optimally preserve skeletal muscle function and vasomotor control.

In Chapter 5 the effect of cold storage and subsequent reoxygenation on the contractile function and morphology of the in vitro RET was evaluated. This investigation was done in the context of CTA preservation, but also because hibernators such as hamsters are known to show superior survival when compared to rats and mice during hypothermia (10; 19) and hypoxia (3). About the tolerance of isolated skeletal muscle of hibernators to cold preservation, however, no information was available. The contractile function of isolated RET muscles stored in HTK-Bretschneider solution (HTK) for 16 h at 4°C was reduced to 27% of that of unstored control muscles. RET morphology remained unaffected. The fi nding that the considerable reduction in muscle function was not accompanied by any sign of necrosis or hypercontraction of the muscle fi bres, implicates that not a decreased number of participating fi bres, but a deterioration of the condition of individual fi bres led to the decrease in muscle function. The possible contributions of Ca2+ overload and oxidative stress triggered by ROS to damage of the cell membrane were investigated by the addition of the Ca2+ release inhibitor BDM and the antioxidants trolox and deferiprone. The fi nding that RET muscle function was partly preserved by trolox and deferiprone and not by BDM indicates that ROS formation, but not Ca2+ overload is a causative factor in cold preservation- induced myocyte injury.

The current results of cold storage of the hamster RET were compared to results published previously (34) for rat cutaneus trunci (CT) muscle stored under identical circumstances. The cold storage-induced functional damage of hamster RET is of comparable magnitude as that of the rat CT, however CT morphology was severely affected by storage. Similar to the RET, functional damage of the CT was partly attributed to ROS, because the addition of trolox and deferiprone during storage showed comparable functional improvements. In contrast to the RET, the CT showed improved contractile function following storage in the presence of BDM, which suggests that Ca2+ overload does not play an important role in the storage induced injury of the RET and points towards a hibernation- related species difference in myocyte Ca2+ handling. In this aspect, the Ca2+ handling of the RET seems comparable to that in the hibernator heart (15; 35), which is achieved by adaptations such as greater amounts of SR (31) and an enhanced SR Ca2+ uptake (15; 35) by increased activity of SR Ca2+-ATPase (6; 36). It is expected that further exploration of such adaptive mechanisms of hibernator skeletal muscle may offer new insights in the management of cold preservation prior to transplantation of CTAs.

Although the ischemic insults during cold storage experiments described in Chapter 5 do not only differ from those in I-R experiments (Chapters 2, 3 and 4) with regard to temperature, but also differ much in duration of ischemia (16 h vs. 1 h), testing conditions


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(reoxygenation vs. reperfusion) and intactness of the preparation (in vitro vs. in vivo), it is remarkable that opposite contributions of Ca2+ overload and ROS are observed in respective experiments. With regard to this observation it could be speculated that the mechanism of cellular injury depends on the temperature to which the tissue or cells are exposed during ischemia.

In Chapter 6, microdissections of the RET and the vasculature that supplies and drains the muscle were performed in order to develop the RET as a new transplantation model. It was found that blood supply enters the RET via several routes. The main blood supply is provided by the feed arteries and accompanying collecting veins, which branch off of the thoracodorsal artery and vein. By the nearly complete staining of the RET following dye injection of the thoracodorsal artery, it was demonstrated that the main part of the RET remains adequately perfused following its isolation on the thoracodorsal pedicle. The diameters of the thoracodorsal artery and vein are ~0.3 mm in the regions used for anastomoses. These dimensions are considered to be near the smallest vessel size that can be surgically attached to one another (11) although new techniques that facilitate such small ‘supermicrosurgical” anastomoses are currently introduced (22). The vascular pedicle was of suffi cient length (13-25 mm) to use the RET as a free fl ap. We isolated two RET muscles on their thoracodorsal pedicles and performed allotransplantation by microvascular transfer to the femoral vessels of two recipient hamsters. Both transplantations were considered successful. The fi ndings of this chapter provide essential anatomical information concerning the RET and its vascular pedicle and provide a foundation for using this preparation as a model to study the microcirculation and contractile muscle function in free muscle fl ap and allograft transplantation.

Address to the aims

The aim of this thesis was to establish the hamster retractor muscle model (RET) as a new experimental model that allows combined evaluation of functional parameters of skeletal muscle tissue and (micro)vasculature during I-R insults. Furthermore, we aimed to investigate if the RET could be used as a free fl ap model to design preservation strategies for CTAs. In this prospect a number of questions were raised in Chapter 1. Answers to these questions are provided below.

Does a relatively short period of I-R (1h-1h) affect RET skeletal muscle function and endothelium dependent vasomotor control?

In the RET, I-R (1h-1h) results in a reduction by half of skeletal muscle function and AVD. This implies that vasomotor control is considerably decreased following I-R(Chapter 2).

What is the cause of the observed attenuation of AVD following I-R The attenuation of AVD is due to impaired initiation of the vasodilatory signal in the


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endothelium of the intramuscular arterioles. In contrast, conduction of the vasodilatory signal along the vessel wall remains intact (Chapter 2).

Can substances that limit Ca2+ overload and ROS formation prevent I-R injury to skeletal muscle function and AVD?

Limitation of Ca2+ overload by the Ca2+ release inhibitor BDM entirely prevents the I-R-induced defi cit in skeletal muscle following I-R. Reduction of hydroxyl radical (OH) formation by the iron chelating antioxidant deferiprone does not preserve contractile muscle function nor AVD (Chapter 3).

Does mild hypothermia (21°C) prevent I-R induced impairment of skeletal muscle function and AVD?

AVD and contractile muscle function, which show a ~50% reduction following I-R (1h-1h) at 37°C, are maintained completely at 21°C (Chapter 4).

Does prolonged anoxic cold storage (16h at 4°C) and reoxygenation affect contractile function of isolated RET muscles?

RET muscle function is severely affected by 16h of cold storage at 4°C. The contractile defi cit is partly attributed to the formation of ROS (Chapter 5).

Are skeletal muscles of hibernators more tolerant to hypoxia and hypothermia than muscles of non-hibernators?

The hamster RET, representing a hibernator muscle, does not display superior tolerance to 16 h of cold storage at 4°C, when compared to a similar muscle of a non-hibernator, such as the rat cutaneous trunci (CT) muscle. However, the mechanism that causes functional derangement is different in RET and CT muscles (Chapter 5).

Can the RET be isolated on its vascular pedicle and be used as a transplantation model?The RET can be isolated on its thoracodorsal pedicle and can be used as a free fl ap model for future evaluation of the effect of preservation strategies and transplantation on skeletal muscle microcirculation and contractile function (Chapter 6).

Future perspectives

During the current I-R experiments, the RET remained attached to the body during ischemia and therefore, ischemic periods were limited by the hamsters anaesthetic tolerance. As a result, the relative contributions of Ca2+ overload and ROS following longer ischemic periods at 21°C (for example 4h) could not be assessed in the current setup. As future experiments will comprise the RET as the free fl ap model (Chapter 6), the supposed temperature dependence of damaging mechanisms contributing to I-R injury will be a point of interest. In transplantation experiments vascular integrity can be assessed by direct observation of the microvascular network through intravital microscopy, enabling


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quantifi cation of reperfusion by blood fl ow measurement (12). Functional parameters could then include evaluation of diameter changes during ascending vasodilation and muscle tetanus tensions (5). For such investigations inbred hamster strains need to be used, since it is not possible to reliably study the sole effect of prolonged reperfusion of the RET fl ap in genetically non-identical animals, as earlier reports from limb allotransplantations show microcirculatory changes within 24 hours after transplantation (23). Alternatively, when using outbred strains, the RET transplantation model has the potential to display allograft related immunological responses in survival studies.

Since the current thesis proposes that ROS such as superoxide and peroxynitrite might be involved in the pathogenesis of reduced AVD and skeletal muscle function, future pharmacological interventions should include scavengers or inhibitors of these ROS.

The main conclusions of this thesis are that in the in vivo hamster RET during I-R (1h-1h) inhibition of Ca2+ overload and hypothermia (21°C) contribute to maintenance of skeletal muscle function, and hypothermia contributes to maintenance of vasomotor control. Further investigations using the RET free fl ap model could provide a foundation for better understanding of I-R injury in skeletal muscle fi bres and blood vessels and optimize preservation strategies for CTAs.

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Samenvatting

Binnen de reconstructieve chirurgie is ischemie-reperfusie (I-R) schade van skeletspierweefsel een veelvuldig optredend fenomeen. I-R-schade wordt deels veroorzaakt door het zuurstoftekort (ischemie) in weefsel of een orgaan als de doorbloeding daarvan is gestopt en deels door schadelijke stoffen die vrijkomen als de doorbloeding op een later tijdstip (reperfusie) weer op gang komt. I-R-schade treedt bijvoorbeeld op tijdens replantaties van (traumatisch) geamputeerde lichaamsdelen, vrije lap chirurgie en bij het gebruik van zogenaamde samengestelde weefseltransplantaten (CTA’s: composite tissue allografts). Het laatstgenoemde is een nieuw werkgebied binnen de reconstructieve chirurgie en betreft transplantatie van samengestelde weefsels van overleden donoren, zoals handen of aangezichtsstructuren. Het geheel of gedeeltelijk falen van vrije gevasculariseerde spierlappen en gereplanteerde lichaamsdelen is een belangrijk klinisch probleem. Daarnaast is het, vanwege de steeds frequentere toepassing van CTA’s, gewenst om (bewaar) methodes te ontwikkelen waardoor weefsels kunnen worden getransplanteerd met behoud van functie. Voor het behoud van functie van skeletspieren is het van groot belang dat niet alleen de kontraktieigenschappen behouden blijven, maar dat het (micro-)vaatbed ook intact en functioneel blijft.

De auteur van dit proefschrift introduceerde het hamster retractor spier (RET) model in het Laboratorium Plastische Chirurgie van het UMC Utrecht met het doel dit te gebruiken voor onderzoek naar I-R schade en preservatie technieken voor CTA’s. De RET is een uniek model, omdat de spier buiten het lichaam van de hamster kan worden gebracht, terwijl de vaatvoorziening intact blijft. Onder in vivo condities kunnen dan tegelijkertijd de contractiekracht van de spier en de reactie van het vaatbed op de spierkontraktie worden gemeten. In het verleden heeft onderzoek aan dit model geleid tot nieuwe inzichten in de regulatie van de doorbloeding van skeletspieren. Met name de zogenaamde “ascending vasodilation” (AVD: opstijgende vasodilatatie) speelt in deze regulatie een belangrijke rol. Tijdens spiercontracties treedt niet alleen bloedvatverwijding op in de intramusculaire arteriolen, maar ook in de grotere stroomopwaarts gelegen voedende arteriën (‘feed arteries’) van de spier. Het electrisch signaal dat de grote voedende arteriën activeert tot verwijding ontstaat in de spier en wordt langs de wand van de arterie stroomopwaarts voortgeleid. Dit proefschrift bevat de resultaten van de eerste studie naar de effecten van I-R op de AVD.

De experimenten beschreven in Hoofdstuk 2 tonen aan dat na 1 uur ischemie en 1 uur reperfusie (I-R; 1h-1h) niet alleen de contractiekracht van de RET, maar ook de AVD met 50% is afgenomen. Deze afname in AVD na I-R werd ook gemeten in RET spieren waarin, door sterkere stimulatie, contracties werden opgewekt vergelijkbaar met die van spieren die niet zijn blootgesteld aan I-R. Hieruit volgt de conclusie dat de afname van de AVD niet een direct gevolg is van de verminderde spierfunktie. Het mechanisme dat verantwoordelijk is voor de reductie van AVD werd nader onderzocht door de bloedvatverwijding (vasodilatatie) te meten, veroorzaakt door lokale toediening van de


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vasodilatator acetylcholine. De acetylcholine werd toegediend op de intreedplaats van het vat in de spier en de vaatverwijding werd gemeten op vier plaatsen stroomopwaarts langs het vat. Aangezien de vasodilatatie op alle vier de plaatsen in gelijke mate was aangedaan door I-R, werd geconcludeerd dat niet een defect in de voortgeleiding van de AVD langs de vaatwand, maar een defect in in de totstandkoming van de AVD in de arteriolen verantwoordelijk is voor de afname na I-R. Uit de literatuur was bekend dat zuurstofradicalen een destructieve werking hebben op de functie van de ionkanalen in endotheelcellen die essentieel zijn voor de initiatie van AVD. Op grond van de bevindingen in dit hoofdstuk is de hypothese opgesteld dat de schade aan het initiatie-mechanisme van de AVD veroorzaakt wordt door effecten van zuurstofradicalen.

In aansluiting op het voorgaande, is in Hoofdstuk 3 onderzocht of een verhoogde intracellulaire Ca2+ concentratie en de vorming van een zeer reactief zuurstofradicaal, hydroxyl (OH), verantwoordelijk zijn voor de vermindering van contractiekracht na I-R. Verder is de invloed van OH op het verval van AVD tijdens I-R bestudeerd. Twee farmacologische interventies werden hiervoor gebruikt: 1.) 2,3- butanedione monoxime (BDM) om de stijging van intracellulaire Ca2+ concentratie te voorkomen en 2.) deferiprone om de ijzer-afhankelijke vorming van OH te voorkomen.

De toevoeging van BDM gaf een volledige bescherming tegen het verlies van contractiekracht tijdens I-R. Uit de literatuur is bekend dat in skeletspierweefsel een verhoogde intracellulaire Ca2+ concentratie ten gevolge van I-R gerelateerd kan zijn aan de vorming van zuurstofradicalen, en met name OH. De toevoeging van deferiprone had echter geen enkel effect op de contractiekracht van de RET tijdens I-R. Op grond van deze uitkomsten wordt geconcludeerd dat een verhoogde intracellulaire Ca2+ concentratie de belangrijkste oorzaak is voor het verlies van contractiekracht tijdens I-R (1h-1h) en dat OH radicalen hierbij geen rol spelen.

Ook de vermindering van AVD na I-R kon niet worden voorkomen door de aanwezigheid van deferiprone. Op basis van deze resultaten wordt geconcludeerd dat OH dus ook geen belangrijke rol speelt bij de beschadiging van het initiatiemechanisme van AVD in het endotheel tijdens I-R. Aangezien bekend is dat ook andere zuurstofradicalen, zoals superoxide, waterstof peroxide en peroxynitriet nadelige effecten kunnen hebben op de werking van ionkanalen in endotheelcellen die belangrijk zijn voor AVD, veronderstellen we dat deze zuurstofradicalen betrokken zijn bij het verval van AVD tijdens I-R.

De experimenten die beschreven zijn in Hoofdstuk 4 laten zien dat endotheelschade, die verantwoordelijk is voor de vermindering van AVD tijdens I-R op 37°C, geheel voorkomen wordt door een verlaging van de temperatuur tot 21°C. Ook de contractiekracht van de spier, die na I-R (1h-1h) bij 37°C was gehalveerd, is volledig intact na I-R op 21°C.


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Het mechanisme waardoor koeling het endotheel beschermt voor I-R schade is hoogst waarschijnlijk gerelateerd aan een reductie van zuurstofconsumptie en energieverbruik van de cellen. Alle celprocessen verlopen op 21°C ongeveer anderhalf tot drie maal trager dan het geval is op 37°C. Hierdoor raken de energiebronnen veel later uitgeput. Dit draagt bij aan een langer behoud van celfunctie. Aangezien er lagere concentraties zuurstofradicalen worden gevonden in zenuw- en leverweefsel tijdens milde hypothermie, veronderstellen we dat de vermindering van AVD tijdens I-R bij 37°C wordt veroorzaakt door zuurstofradicalen zoals superoxide (O2

-). In Hoofdstuk 5 worden de effecten van ischemische koude preservatie (4°C) en

reoxygenatie (21°C) op de functie en morfologie van de RET gepresenteerd. Deze studie is uitgevoerd in het kader van de ontwikkeling van preservatiestrategieën voor CTA’s, maar ook omdat bekend is dat winterslapers, zoals de hamster, beter bestand zijn tegen moeilijke omstandigheden zoals hypothermie en hypoxie. Tot op heden is geen literatuur beschikbaar waaruit blijkt dat geïsoleerde skeletspieren van winterslapers beter bestand zijn tegen koude preservatie dan spieren van niet-winterslapers. De spierfunctie van de RET was na 16 uur bewaren bij 4°C in een preservatievloeistof (HTK-Bretschneider: HTK) gereduceerd tot ongeveer een kwart van de functie van controle spieren. Het morfologisch beeld van de RET bleef daarentegen volledig intact. Het feit dat de aanzienlijke afname in contractiekracht niet gepaard ging met necrose of hypercontractie van de spieren, betekent dat niet een verminderd aantal spiervezels, maar een verslechterde conditie van de afzonderlijke vezels de belangrijkste oorzaak is voor de vermindering van spierfunctie. De mogelijke bijdrage van een verhoogde intracellulaire Ca2+ concentratie en zuurstofradicalen aan deze schade werd onderzocht door de toevoeging van BDM, deferiprone (zie Hoofdstuk 3) en de antioxidant trolox. Aangezien de spierfunctie van de RET tijdens koude preservatie gedeeltelijk werd beschermd door trolox en deferiprone, maar niet door BDM, is de conclusie dat niet een verhoogde intracellulaire Ca2+ concentratie, maar zuurstofradicalen bijdragen aan de schade aan skeletspiercellen tijdens koude preservatie.

De resultaten voor de RET spier van de hamster werden verder vergeleken met eerder gepubliceerde resultaten voor de cutaneus trunci (CT) spier van de rat, die onder identieke omstandigheden bewaard was. Het verlies van contractiekracht na koude preservatie was gelijk in de RET en de CT. De morfologie van de CT was echter ernstig beschadigd na bewaren. Zoals ook bij de RET werd gezien, was de destructieve werking van zuurstofradicalen debet aan de functionele schade in de CT. Echter, in tegenstelling tot de RET, werd de spierfunctie van de CT wel beschermd door de aanwezigheid van BDM in HTK. Dit suggereert dat een verhoogde intracellulaire Ca2+ concentratie niet van belang is in de hamster RET spier maar wel in de rat CT spier. Dat zou kunnen wijzen op een verschil in de Ca2+ huishouding in de skeletspiercellen van winterslapers en niet-winterslapers. In dit opzicht is het interessant, dat van hartspierweefsel van winterslapers verschillende adaptatiemechanismen bekend zijn, die bijdragen aan een stabielere Ca2+ - huishouding. Wellicht spelen dergelijke processen ook een rol in de RET. De verwachting


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is dat verdere bestudering van de adaptatiemechanismen van winterslapers een rol kan spelen bij de optimalisering van preservatiestrategieën voor CTA’s.

In Hoofdstuk 6 worden de microchirurgische dissecties van de vaatvoorziening van de RET beschreven, die zijn uitgevoerd om de RET te ontwikkelen tot transplantatiemodel. De RET wordt gevasculariseerd door bloedvaten die op meerdere plaatsen intreden. Verreweg de belangrijkste bloedvaten zijn de voedende arteriën en venen die afstammen van de arteria en vena thoracodorsalis. Middels injectie van een kleurstof werd gezien dat inderdaad het volledige vaatbed van de RET gevasculariseerd wordt via deze thoracodorsale vaatsteel. De diameter van de arteria en vena thoracodorsalis zijn ongeveer 0.3 mm op de plaatsen die gebruikt kunnen worden voor het maken van de anastomosen in geval van transplantatie. Dit is ongeveer de kleinste diameter waarbij bloedvaten chirurgisch kunnen worden verbonden, hoewel er momenteel nieuwe methoden in ontwikkeling zijn die dergelijke ‘supramicrochirurgie’ vergemakkelijken. De vaatsteel is van voldoende lengte om de RET als een vrij gevasculariseerde spierlap te transplanteren (13-25 mm).

Naast de anatomische studie werden twee transplantaties uitgevoerd, waarbij RET spieren van twee donor hamsters geïsoleerd werden op de thoracodorsale vaten en naar de arteria en vena femoralis van twee ontvangers werden getransplanteerd. Beide transplantaties waren succesvol. Op basis van de anatomische kennis beschreven in dit hoofdstuk kan worden geconcludeerd dat het RET model geschikt is als transplantatiemodel, waarin tegelijkertijd de functie van skeletspierweefsel en (micro-) vaatbed kan worden bestudeerd.

De belangrijkste conclusies van dit proefschrift zijn dat in de hamster RET spier hypothermie (21°C) en beperking van een overschot aan Ca2+ bijdragen aan het behoud van skeletspier functie en dat hypothermie bijdraagt aan het behoud van regulatie van de doorbloeding van skeletspieren. Verder kan de RET worden gebruikt als transplantatiemodel. Toekomstig onderzoek aan de RET kan bijdragen tot een beter begrip over het ontstaan van I-R schade in skeletspierweefsel en het (micro-)vaatbed en uiteindelijk leiden tot de optimalisering van preservatiestrategieën voor CTA’s.


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DankwoordHet wordt vaak gezegd en is een cliché, maar de totstandkoming van een proefschrift

is echt alleen mogelijk als je hulp en advies krijgt van anderen. Ik ben iedereen die heeft bijgedragen aan het slagen van dit project dan ook zeer dankbaar! Een aantal mensen wil ik in het bijzonder bedanken.

Lieve Quinten. Hier is deel twee. Jouw proefschrift is al lang voltooid. Het afronden van mijn werk heeft op zich laten wachten. Je hebt het hele proces meegemaakt; vanaf het begin van mijn onderzoekstijd tot nu. Jouw onvoorwaardelijke liefde, steun en relativeringsvermogen hebben mij enorm geholpen om door te zetten, vooral ook wanneer het moeilijk was. Hiervoor ben ik je ontzettend dankbaar. Maar bovendien ben ik je dankbaar voor wie je bent: elke dag geniet ik van jouw aanwezigheid in mijn leven. Je maakt me gelukkig. Ik had me geen beter iemand kunnen wensen! We hebben samen een heerlijk leven en ik verheug me enorm op november en de tijd daarna, als we met z’n drieën zullen zijn.

Prof. Dr. M. Kon, mijn promotor en opleider. Professor, ik wil u bedanken voor het vertrouwen en de vrijheid die ik heb gekregen om mijn eigen onderzoeksplan te maken. Ik waardeer het enorm dat u mij zonder twijfels hebt laten doen wat ik voor ogen had, ook toen het tegenzat, of toen het betekende dat ik naar New Haven wilde gaan om het retractormodel bij Steven Segal te gaan leren. In de periode van anderhalf jaar die ik na mijn vooropleiding weer terug ben in het UMCU heb ik niet alleen gelegenheid gekregen om mijn proefschrift af te schrijven, maar heb ik ook veel van u geleerd op klinisch gebied. Ik waardeer onze gesprekken over het vak en andere zaken in het leven enorm.

Dr. A.B.A. Kroese, beste Alfons. Ere wie ere toekomt! Co-promotor, maar de motor achter dit proefschrift. Vanaf het begin tot het eind ben jij een zeer waardevolle begeleider geweest. Je bent een top-wetenschapper en - mens. Je hebt me leren onderzoeken en schrijven. Jij was het, die altijd begreep dat het allemaal niet zo snel gaat met fundamenteel onderzoek en dat kwaliteit tijd kost. Regelmatig ging het mes in mijn lange stukken tekst en maakte jij er een adequaat en compact geheel van. Onze neuzen stonden altijd dezelfde kant op. Je hebt buitengewoon veel tijd in dit project gestoken en zonder jou was het me absoluut niet gelukt om het tot een goed einde te brengen! Ik heb genoten van de plezierige sfeer tijdens de experimentele jaren en ook weer tijdens de afgelopen anderhalf jaar, toen we in een sneltreinvaart de hoofdstukken hebben afgerond. Ik ben je erg dankbaar voor alles wat je voor mij gedaan hebt.

Dr. S.S. Segal, dear Steve. You welcomed me into your lab, to learn the retractor preparation. It was a great honour for me to be part of your Microcirculation crew at the JB Pierce Lab and Yale University. You respected my ideas and allowed me to do it my way. You taught me lots of research tricks. I have enjoyed our collaboration very much and hope we will somehow continue our research on the retractor.


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Dr. E.P.A. van der Heijden, co-promotor, beste Brigitte. Het begon in 1997, toen ik als student een jaar lang meewerkte aan jouw promotie. Je hebt me zo enthousiast gemaakt voor het onderzoek, dat ik er mee verder ben gegaan. Hoewel ons contact minder intensief is geworden, ben ik je dankbaar voor alles wat je me geleerd hebt op onderzoeksgebied en dat je me hebt binnengeleid in de wereld van de plastische chirurgie.

Prof. Dr. P.R. Bär, promotor, beste Dop. Als supervisor aan de zijlijn was je altijd zeer geïnteresseerd en heb je de grote lijnen van het project in de gaten gehouden. Veel dank dat je er was op momenten dat er belangrijke beslissingen genomen moesten worden en voor je punctuele correcties van de manuscripten.

Prof. Dr. P.M.N. Werker, beste Paul. Jij was het die mij in 1997 als student binnenhaalde in het laboratorium van de plastische chirurgie en in contact bracht met Brigitte. Al in een zeer vroege fase heb je je vertrouwen in mij uitgesproken, waar ik je voor dank. Ik waardeer onze huidige gesprekken over het vak en de wetenschap. Ik ben blij dat je plaats wilde nemen in de manuscriptcommissie.

Overige leden van de commissie: Prof. Dr. I.H.M. Borel Rinkes, Prof. Dr. J.R. Lahpor, en Prof. Dr. F. L. Moll, dank voor het kritisch lezen en beoordelen van het manuscript.

Dr. J. W. Van Teeffelen, beste Jurgen. Dankzij jou kwam ik in contact met Steve en heb ik de mogelijkheid gekregen het retractor model te leren en verder te ontwikkelen. Ik ben zeer vereerd dat je komt opponeren bij mijn verdediging.

Sara van Velsen, dank voor je hulp bij de histologische beoordelingen en Anne Martien de Vries, bedankt voor je onmisbare assistentie bij de anatomische studie. Ik wens jullie veel succes met jullie verdere onderzoek en opleidingen.

Dr. R.L.A.W. Bleijs, beste Ronald. Bedankt dat de deuren van jouw afdeling Anatomie altijd openstonden voor raad en daad. Anatomie blijft de basis! Met name ook veel dank aan Willem van Wolferen en Simon Plomp; Met veel geduld hebben jullie mij soms tot laat in de middag nog geholpen bij het opspuiten van het vaatbed van de spieren.

Dr. M.F. van Oosterhout, beste Matthijs. Dank voor je adviezen op het gebied van de histologische kleuringen en je co-auteurschap bij het preservatie artikel.

Dear collegue reseachers of the Segal Lab in 2003: Sara Haug, Geoff Payne, Alia Chisty, Robin Looft-Wilson, Tim Domeier, David Chu and Shawn Bearden. My stay at the Pierce Lab and Yale University has provided an essential basis for this thesis. Although during my stay in New Haven I spent most of my time amongst sleeping hamsters and in the Yale Gym, we shared effi cient lab meetings and time outside the lab. Thanks for helping me to fi nd my way in the lab. Verena Cap, André Rego, Heun Soh, Azusa Shito, Ikuo Katayama, and You Ree, thanks for your pleasant company during my stay in New Haven.

Dan

kwoo

rd


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Beste Margreet Blokland, Bea Sopacua en Angelique van Dijk, (voormalig) secretaresses van de afdeling plastische chirurgie in het UMC. Veel dank voor jullie gezelligheid en dat jullie altijd bereid zijn te helpen op allerlei gebied.

Alle (voormalig) stafl eden, assistenten en onderzoekers van de afdeling plastische chirurgie in het UMC. Beste Yvette, Pascal, Arnold, Anne Floor, Aebele, Corstiaan, Menso, Wilco, Pien, Aveline, Reinier, Laura, Duco, Max, Ralph, Erik, Erik, Edwin, Bart, Pascal, Marieke, Dalibor, Sven, Jacqueline, Bas, Eline en Wies. Bedankt voor de goede sfeer, jullie bijdrage aan mijn opleiding en interesse in mijn onderzoek. In het bijzonder wil ik Marieke Vossen bedanken voor de gezelschap tijdens ons gezamenlijke jaar in de ‘cellen’. Bart, veel dank voor je uitzonderlijke interesse. Paul van Minnen, bijzonder bedankt voor de plezierige tijd die we samen in het GDL doorbrachten en Eveline Corten, bedankt voor je vriendschap; nu eindelijk quatre- mains!

(Bio) technici en medewerkers van het GDL: met name Jan-Dirk Tiggelman, Hans Vosmeer, Anja van der Sar, Jannie Visser, Helma Avezaat, Kees Brandt, Nico Attevelt, Elly van Zwol, Fred Visch, Toon Hesp en Herman Koning. Jullie hulp was onmisbaar. Ik ben jullie zeer dankbaar voor alle raad en daad op het gebied van experimenteren. Ik vind het heel prettig dat ik ook na mijn onderzoekstijd nog altijd kan binnenlopen voor vragen op het gebied van technieken, microchirurgie of onderwijs.

André Verheem. Enorm bedankt voor het oplossen van alle praktische problemen, maar ook je prettige gezelschap in alle dagen, weken en maanden dat ik in mijn eentje het lab was. Jij fi xt alles!

Phil Buckley. Dank voor het oplossen van talloze computerprobelemen.

Henk Veldman van het voormalig laboratorium Neuromusculaire Ziekten in het UMC. Bedankt voor al je hulp en goede adviezen op het gebied van de histologie.

Willem Renooij en Martin de Smet. Bedankt dat jullie altijd bereid waren te helpen en mee te denken over oplossingen van kleine en grote problemen.

Ingrid Janssen van de afdeling Multimedia, bedankt voor je prachtige anatomische illustraties.

Lieve Janneke, Charlotte en Eleonoor. Lang leve de JCLM! Jullie zijn fantastische vriendinnen. De tijd in het studentenhuis op de Mgr. ligt achter ons, maar onze band is nog net zo hecht als in die tijd. Ik ben jullie dankbaar voor jullie hulp, begrip, interesse, maar vooral voor jullie humor, gezelligheid, relativering, eerlijkheid, enthousiasme, gezamenlijke dates en weekendjes. Jullie zijn mij heel dierbaar! En eigenlijk had ik vier paranimfen moeten hebben!


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Lieve vrienden en vriendinnen, Willemijn, René, Hanneke, Flip, Lot, Matthijs, Marieke, Ian, Carolien, Matthieu, Ieneke en Ben. Veel dank voor jullie fi jne vriendschappen, interesse en goede gesprekken. De laatste jaren heb ik jullie aangename gezelschap regelmatig moeten missen.

Lopers van de lange-afstands groep van atletiekvereniging Phoenix, met name Henk en Elvira, dank voor de gezelligheid en alle gezamenlijke kilometers door de jaren heen. Het duurt nog wat maandjes voordat ik eindelijk weer met jullie meega...

Mijn paranimfen. Lieve Eleonoor, als geen ander weet jij als violiste hoe het is om op het podium te staan. Ik ben blij dat je me bijstaat tijdens mijn ‘optreden’. Lieve Hanneke, samen aan de studie begonnen en we hebben sinds het eerste jaar een hechte vriendschap. Onze wegen in de geneeskunde en daarbuiten zijn altijd parallel blijven lopen. Bedankt dat ook jij me als ‘gepromoveerde ervaringsdeskundige’ bij wilt staan tijdens de verdediging.

Lieve schoonouders, bedankt voor jullie voortdurende steun en uitzonderlijke interesse in de voortgang van dit project en al het andere. Bij jullie vind ik altijd een luisterend oor en een warm onthaal. Ondanks de drukte vergeten jullie nooit te informeren als er belangrijke dingen zijn. Lieve Eric, Katelijne, Toine, Andrea, Laura en Merle, Finn en Luka, wat ben ik blij met zo’n lieve schoonfamilie; jullie gezelligheid en support had ik niet willen missen!

Tenslotte mijn lieve ouders. Zolang ik me kan herinneren hebben jullie ons gesteund in alles wat we deden. Het beste uit jezelf halen, maar het moet wel leuk blijven, is altijd jullie advies geweest. Discussies in onze jeugd aan de keukentafel op allerlei terreinen zijn de basis voor mijn kritische blik op de wereld en de wetenschap. Wat hebben we een fi jne band met elkaar en wat ben ik jullie dankbaar voor de onvoorwaardelijke liefde, steun en hulp! Jullie beschouwen dat als vanzelfsprekend, maar het is heel bijzonder. Lieve Thijmen, Quirine, Arie Dirk en Mirthe. Bedankt voor alles en vooral voor het ‘samen groot worden’. Jullie humor, gezelligheid, interesse, èn bijdragen aan discussies zijn onmisbaar. We zijn een fi jne familie. Ik hoop nog lang met elkaar te kunnen genieten!

Dan

kwoo

rd


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Curriculum vitaeMiriam de With werd op 19 februari 1977 geboren te

Leerdam. In 1994 behaalde zij het diploma op het Gymnasium Camphusianum te Gorinchem. Zij startte datzelfde jaar met de studie geneeskunde aan de Rijks Universiteit Utrecht. In 1999 behaalde zij het doctoraalexamen, waarbij zij van 1997 tot 1998 wetenschappelijke stage liep op de afdeling Plastische Chirurgie in het AZU. Tijdens de studie werkte zij onder andere als student-assistent bij de afdelingen Medische Genetica, Fysiologie en Sportgeneeskunde en Functionele Anatomie. In 2001 werden de co-schappen afgesloten. Tijdens deze periode verbleef zij enkele maanden in Zürich, Zwitserland, waar ze een co-schap Dermatologie/ Dermatochirurgie in de Dermatologische Klinik van het UniversitätsSpital volgde.

Van 2002 tot en met 2005 werd promotieonderzoek verricht in het Research Laboratorium Plastische Chirurgie van het Universitair Medisch Centrum te Utrecht. In het kader van dit promotieonderzoek werkte zij van maart tot en met september 2003 als research fellow in het Microcirculatory Laboratory van het John B. Pierce Laboratory, Yale University New Haven, CT, VS (hoofd: S.S. Segal, PhD).Daar leerde zij te werken met het retractormodel en ontwikkelde het tot een ischemie-reperfusie model. Dit proefschrift kwam tot stand op basis van experimenten met dit model.

Van januari 2006 tot en met 2008 vervulde zij de vooropleiding Heelkunde in het Jeroen Bosch Ziekenhuis te ’s Hertogenbosch (opleider: Dr. K. Bosscha). Sinds januari 2008 is zij terug in het UMCU, waar ze wordt opgeleid tot plastisch chirurg (opleider: Prof. Dr. M. Kon).

Miriam woont samen met Quinten Ruhé. Zij verwachten in november dit jaar hun eerste kind.


Colofon

Basisontwerp en omslag: Floris Peeters

Opmaak en druk: Gildeprint Drukkerijen - Enschede, The Netherlands

© M.C.J. de With

ISBN: 978-90-393-51338


ischemia–reperfusion impairs ascending vasodilation in feed arteries of hamster skeletal muscle

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