Levels of brain-derived neurotrophic factor and neurotrophin-4 in lumbar motoneurons after low-thoracic spinal cord hemisection

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    1. Introduction Several studies suggested that neurotrophinsmay also play

    (2004Neurotrophins are a family of chemically related pro-

    teins, which play an important role in development, survival

    and maintenance of neuronal function in the peripheral and

    central nervous system [12,13,19,25]. Brain-derived neuro-

    trophic factor (BDNF) and neurotrophin-4 (NT-4) have a

    wide variety of effects on spinal motoneurons. During

    development and in the adult rat [12,25], they may represent

    target-derived and activity-dependent trophic signals for

    motoneurons by regulating several neural functions such

    as fiber sprouting, electrical and metabolic properties and

    cell size [5,8]. Recently, it has been demonstrated that

    recent investigations showed neurotrophin expression in

    motoneurons [2] indicating that endogenous BDNF and

    NT-4 may be involved in functional changes induced by

    exercise onmotor unit properties [6,7,21,26]. It is also known

    that spinal injury or deafferentation induce plastic changes in

    the spinal circuitry, which could compensate for the alteration

    of modulatory inputs and promote functional recovery [4,23].

    In addition to changes that occur at the lesion site, spinal and

    supraspinal circuits undergo substantial reorganization by

    two different mechanisms: (1) anatomical reorganization of

    circuits and formation of new pathways by sprouting; (2)a crucial role in spinal cord (SC) plasticity. For example,involving synaptic plasticity, which are responsible for the rapid recovery of hindlimb motility after hemisection, in the rat. In order to gain

    further insight, we evaluated the changes in BDNF and NT-4 expression by lumbar motoneurons after low-thoracic spinal cord hemisection.

    Early after lesion (30 min), the immunostaining density within lumbar motoneurons decreased markedly on both ipsilateral and contralateral

    sides of the spinal cord. This reduction was statistically significant and was then followed by a significant recovery along the experimental

    period (14 days), during which a substantial recovery of hindlimb motility was observed. Our data indicate that BDNF and NT-4 expression

    could be modulated by activity of spinal circuitry and further support putative involvement of the endogenous neurotrophins in mechanisms

    of spinal neuroplasticity.

    D 2004 Elsevier B.V. All rights reserved.

    Theme: Development and regeneration

    Topic: Neurotrophic factors: expression and regulation

    Keywords: Spinal cord hemisection; Motoneuron; Plasticity; Neuromuscular activity; Neurotrophin; RatAbstract

    Neuroplasticity represents a common phenomenon after spinal cord (SC) injury or deafferentation that compensates for the loss of

    modulatory inputs to the cord. Neurotrophins play a crucial role in cell survival and anatomical reorganization of damaged spinal cord, and

    are known to exert an activity-dependent modulation of neuroplasticity. Little is known about their role in the earliest plastic events, probablyResear

    Levels of brain-derived neurotrophi

    motoneurons after low-thor

    Rosario Gulinoa, Salvatore AndreGiampiero Leanzab,

    aDepartment of Physiological Sciences, UniversitybDepartment of Physiology and Pathology, Univ

    Accepted

    Brain Research 1013neurotrophins may act via autocrineparacrine as well as

    anterograde mechanisms, in an activity-dependent manner

    [1,17,22].

    0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

    doi:10.1016/j.brainres.2004.03.055

    * Corresponding author. Tel.: +39-95-339095; fax: +39-95-330645.

    E-mail address: perciava@unict.it (V. Perciavalle).report

    actor and neurotrophin-4 in lumbar

    ic spinal cord hemisection

    ombardoa, Antonino Casabonaa,cenzo Perciavallea,*

    tania, Viale Andrea Doria, 6-95125, Catania, Italy

    of Trieste, Via Fleming, 22-34127, Trieste, Italy

    rch 2004

    www.elsevier.com/locate/brainres

    ) 174181modification of pre-existing circuits by modulation of syn-

    aptic strength [4,23]. Anatomical reorganization requires

    long time, so the earliest signs of functional recovery are

    likely attributable tomodifications of excitability of the spinal

    circuitry. Althoughmany studies have investigated the role of

    neurotrophins in preventing neuronal death or promoting

  • Hemisected rats were divided in four experimental groups

    (four animals each), which were sacrificed 30 min, 4 h, 12

    R. Gulino et al. / Brain Research 1013 (2004) 174181 175h (short-term) and 14 days after lesion (long-term), respec-

    tively. Long-term animals were allowed to recover sponta-

    neously after injury, without administration of any

    treatments. Finally, three intact animals served as normal

    controls. All rats were sacrificed by intracardiac perfusion,

    using saline followed by cold 4% paraformaldehyde in 0.1 M

    phosphate buffer (PB).

    2.2. Immunohistochemistry

    After perfusion, the thoracic-lumbar parts of SCs wereanatomical reorganization after SC injury [14,16,18], the

    involvement of endogenous neurotrophins in the dynamic

    modulation of local circuitry remains to be elucidated.

    In the present study, the expression of BDNF and NT-4 in

    lumbar motoneurons was investigated at various time inter-

    vals after low-thoracic SC hemisection. This way we could

    evaluate the basal production of these neurotrophins and

    observe the time course of their expression immediately

    after the disruption of spinal and supraspinal pathways and

    during the spontaneous post-lesion recovery of hindlimb

    motility, that normally starts within few days after surgery.

    2. Materials and methods

    2.1. Animals and treatment

    Young adult male rats (Wistar, 200250 g, n = 22) were

    used in this study. They were given free access to water and

    food and were housed under standard conditions of humidity

    and temperature with 12-h light/dark cycle. Animal care and

    handling were carried out in accordance with guidelines

    issued by the Committee of Experimental Animals of Uni-

    versity of Catania; all efforts were made to minimize animal

    suffering and to reduce the number of animal used. Sixteen

    animals were deeply anaesthetised with chloral hydrate (400

    mg/kg, Fluka, Germany) andmounted on a stereotaxic device

    (David Kopf Instruments, USA).We stabilized the column by

    suspending it with vertebral clamps and drilled a hole on the

    dorsal surface of the ninth thoracic vertebra (corresponding to

    spinal segments T10T11). Then, we removed the dura

    mater from the dorsal surface of SC and transected the right

    side of the cord by using a microsurgical knife attached to the

    arm of the stereotaxic frame, under visual guidance via an

    operating microscope. Care was taken in order to avoid

    damage to the median blood vessels. This operation has the

    advantage of producing consistent lesion, with virtually no

    bleeding and causes minimal if any local operative discom-

    fort. The hemisection site was filled with sterile gelfoam and

    muscles and skin were sutured. Three other animals were

    operated but not hemisected and they were used as sham

    operated controls, which were euthanized 4 h after surgery.removed, postfixed for 2 h in the same fixative andcryoprotected by overnight immersion in 0.1 M PB con-

    taining 20% sucrose. Two series of SC horizontal sections

    (40 Am) were cut on a freezing microtome and used for free-floating BDNF and NT-4 immunostaining. Sections were

    rinsed in 0.05 M Tris buffer saline (TBS) and soaked in TBS

    containing 3% H2O2 and 10% methanol, for 10 min, in

    order to quench the endogenous peroxidase activity. Sec-

    tions were then preincubated at room temperature for 1 h in

    TBS containing 5% normal goat serum and 0.3% Triton.

    After pre-incubation, the two series of sections were incu-

    bated at 4 jC for 1 week with either anti-BDNF (1:1000;Chemicon, USA, Cat. No. AB1534) or anti-NT-4 (1:1000;

    Chemicon, Cat. No. AB1781) rabbit polyclonal antibodies.

    This was followed by incubation with a biotinylated goat

    anti-rabbit antibody (1:200; Vector Laboratories, USA) for 2

    h, and by reaction with Vectastain ABC complex (Vector

    Laboratories). BDNF or NT-4 immunoreactivity (IR) was

    visualised as a brown colour by using diaminobenzidine and

    H2O2 as substrate. All sections belonging to control and

    injured animals were incubated in the same antibody and

    DAB solutions, under identical incubation time and temper-

    ature conditions.

    Control of immunostaining specificity was performed by

    omitting the primary antibody or by substitution of the anti-

    rabbit secondary antibody. These controls did not exhibit

    any specific immunostaining.

    A cresyl violet staining was performed on alternate

    sections from the normal group and the 14 days group.

    This staining allowed us to count the number of motoneuron

    profiles and verify whether the hemisection caused moto-

    neuronal depletion due to neuronal death or only a down-

    regulation of the marker.

    2.3. Morphometrical analyses

    The sections were examined using a Zeiss light micro-

    scope coupled with a computer assisted video camera

    (Sony). The Scion Image software (ScionCorp., NIH,

    USA) was used to count immunopositive motoneuron

    profiles and for densitometric and morphometric analyses.

    In order to ensure consistency in the analyses, all sections

    were stained at the same time. Also, we used the same

    illumination level, microscope and digital camera settings

    throughout the image capturing session. Three SC sections

    from each animal were used for these analyses and all

    sections were separately examined by two investigators

    uninformed about the experimental procedures performed

    on the animals. These sections were cut horizontally through

    the ventral horn in a region corresponding to the dorsolateral

    part of lamina IX and to the segments L4L5 (Fig. 1). This

    motoneuron pool is also known as retrodorsolateral nucleus

    (RDLN) and contains motoneurons that innervate the hin-

    dlimb via the sciatic nerve [20,27]. Only unambiguous

    immunopositive profiles characterized by evident nucleus

    and well-defined motoneuronal features were considered.Average soma diameter and optical density (OD) were

  • R. Gulino et al. / Brain Research 1013 (2004) 174181176measured from motoneuron bodies outlined manually by the

    experimenter from greyscale images (400 magnification),using a protocol modified from Skup et al. [26]. The OD

    measured in the grey matter of the control sections obtained

    by omitting the primary antibody was used as background

    staining and subtracted from each measurement. The immu-

    nopositivity of inter-perikaryal grey matter was evaluated by

    measuring the average OD of three circular areas (3 cm

    diameter at 400 magnification) randomly selected withineach SC side, at L4L5 level.

    2.4. Statistical analysis

    Quantitative variations of the number of motoneuron

    profiles and of the intra-motoneuron OD with respect to

    the time-point after lesion and to the SC side were evaluated

    by means of a two-way ANOVA and Bonferronis post-hoc

    3.1. Changes of the number of positive profiles

    Fig. 1. Schematic representation of the SC area where the motoneurons

    included in the study were located. (A) The rectangle represents the area

    from which the horizontal sections were collected. Note that, when the SC

    is placed horizontally on its ventral surface, the RDLN is present at the

    same level along L4 and L5 segments, thus almost all horizontal sections

    show this motoneuronal pool for all the length of measured area. (B)

    Example of an horizontal section showing the longitudinal extent of these

    motoneuronal pools.In all groups, the average soma diameters for BDNF-like

    or NT-4-like IR profiles were distributed bimodally, with antest. Students t-test were also used within each animal

    group to compare the left and right SC sides. All analyses

    were performed by using SYSTAT software package.

    3. Results

    All rats survived surgery throughout the experimental

    period. Spinal cords were inspected after removal in order to

    verify the position and extent of the hemisection. We found

    that all cords were hemisected correctly at low thoracic level

    except for two animals, belonging to the 14 days and 4

    h groups, respectively. These two animals were excluded

    from the study due to incomplete lesion. The extent of the

    lesion in each animal was assessed by reconstruction from

    40-Am-thick serial longitudinal sections taken through theentire dorso-ventral aspect of the spinal cord at the level of

    the lesion. Hemisections were considered complete when

    they included the ipsilateral dorsal column and corticospinal

    tract, and did not encroach on the contralateral side for more

    than 10% of its width, as assessed on digitized images by

    the Scion Image software. Only individuals with complete

    hemisections were considered for the study.

    Hemisected animals exhibited a pronounced paralysis of

    the right hindlimb. The group of rats that were allowed to

    survive for 14 days showed visible improvement of hin-

    dlimb support and stepping ability within 34 days after

    injury. This recovery was also more evident at the end of the

    survival period.

    In normal animals, a number of large multipolar profiles

    showed intense BDNF-like and NT-4-like IR (Fig. 2A and B,

    normal). Dark BDNF-like IR was also observed inside and

    around the cell nuclei (Fig. 2A, normal, arrows) and within

    dendritic arborizations and axons (Fig. 2A, arrow heads).

    These profiles were located in the ventral portion of SC

    corresponding to RDLN and were classifiable as motoneur-

    ons. Smaller cells, such as interneurons or glial cells, also

    showed some staining.

    The number of BDNF-like and NT-4-like positive pro-

    files as well as their staining density did not differ between

    intact and sham lesioned rats (data not shown); thus, these

    animals were pooled in a single normal group.

    Both BDNF-like and NT-4-like IR of inter-perikaryal

    grey matter was found to be constant among groups and

    between sides of the SC (data not shown).

    To evaluate the modification of neurotrophin expression

    during the post surgery period, we analysed the variations of

    the number of immunopositive profiles and the changes of

    intra-motoneuronal OD 30 min, 4 h, 12 h and 14 days after

    lesion.apparent boundary at about 23 Am (data not shown). The

  • R. Gulino et al. / Brain Research 1013 (2004) 174181 177profiles with average soma diameter ranging between 11

    and 23 Am were presumed to be g-motoneurons, whereaslarger ones were considered a-motoneurons [10]. The meansoma diameter of both populations of motoneurons was not

    apparently affected by the hemisection, since no significant

    Fig. 2. High magnification (150 ) photomicrographs showing examples of BDappears very intense in the motoneurons belonging to normal animals, and a larg

    dendritic arborizations and axons are also greatly stained (arrow heads). Both the

    but they gradually recovered until reaching near-normal levels of optical density, 1

    within cytoplasm and there are not any accumulations of NT-4 IR around the neu

    optical density that returned to normal levels 12 h after lesion. Axons and dendridifference was observed between left and right sides or

    between groups (data not shown).

    No changes in the total number of motone...

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