Intestinal inflammation caused by magnesium deficiency altersbasal and oxidative stress-induced intestinal function

Bradford J. Scanlan Æ Blaine Tuft Æ Justin E. Elfrey ÆAllen Smith Æ Aiping Zhao Æ Motoko Morimoto ÆJoanna J. Chmielinska Æ Maria Isabel Tejero-Taldo ÆIu Tong Mak Æ William B. Weglicki Æ Terez Shea-Donohue

Received: 13 April 2007 / Accepted: 12 July 2007 / Published online: 27 July 2007

� Springer Science+Business Media, LLC 2007

Abstract The aim of this study was to determine the

effect of magnesium deficiency on small intestinal mor-

phology and function. Rats were assigned to 4 groups and

placed on magnesium sufficient or deficient diet for 1 or

3 weeks. Infiltration of neutrophils and mucosal injury

were assessed in stained sections of small intestine. Mag-

nesium deficiency alone induced a significant increase in

neutrophil infiltration and increased vascular ICAM-1

expression, in the absence of changes in mucosal injury or

expression of proinflammatory mediators. Magnesium

deficiency was associated with hyposecretory epithelial

cell responses and vascular macromolecular leak in the

small intestine and lung, which was attributed partly to

reduced expression of NOS-3. To determine the effect of

hypomagnesmia on the intestinal responses to a known

oxidative stress, groups of rats were randomized to either

sham operation or superior mesenteric artery occlusion for

10 (non-injurious) or 30 (injurious) minutes followed by a

1- or 4-hour reperfusion period. In response to mesenteric

ischemia/reperfusion, deficient rats showed exaggerated

PMN influx, but similar mucosal injury. Intestinal ischemia

in sufficient animals induced vascular macromolecular leak

in the small intestine and lung at 4 hours of reperfusion,

with levels similar to those observed in untreated deficient

rats. Acute magnesium repletion of deficient rats 24 h

before surgery attenuated the exaggerated inflammation in

deficient rats. These data show that magnesium deficiency

induced a subclinical inflammation in the small intestine in

the absence of mucosal injury, but with significant func-

tional changes in local and remote organs and increased

sensitivity to oxidative stress.

Keywords Hypomagnesemia � Oxidative stress �Neutrophil � Inflammation � Nitric oxide

Introduction

Hypomagnesemia is encountered frequently in hospitalized

patients and is seen most often in patients admitted to

intensive care units and is a primary cause of significant

morbidity and mortality [1]. Clinically, magnesium defi-

ciency (MgD) occurs often as a consequence of restricted

dietary intake (e.g. alcoholism), Mg2+-wasting drug thera-

pies, or diseases that impair intestinal absorption including

inflammatory bowel disease and celiac disease [2]. There is

The opinions contained herein are those of the authors and are not to

be construed as official policy or reflecting the views of the

Department of Defense

B. J. Scanlan

Department of Surgery, Walter Reed Army Medical Center,

Washington, DC 20307, USA

B. Tuft

Department of Pediatrics, Walter Reed Army Medical Center,

Washington, DC 20307, USA

J. E. Elfrey � A. Zhao � M. Morimoto � T. Shea-Donohue (&)

Department of Medicine & The Mucosal Biology Research

Center, University of Maryland, School of Medicine, Baltimore,

MD 21201, USA

e-mail: tdonohue@medicine.umaryland.edu

A. Smith

Beltsville Human Nutrition Research Center, Nutrient

Requirements and Functions Laboratory, ARS, USDA,

Beltsville, MD 20705, USA

J. J. Chmielinska � M. I. Tejero-Taldo � I. T. Mak �W. B. Weglicki

Division of Experimental Medicine, Department of

Biochemistry and Molecular Biology, George Washington

University Medical Center, Washington, DC 20037, USA

123

Mol Cell Biochem (2007) 306:59–69

DOI 10.1007/s11010-007-9554-y

a renewed interest in Mg2+ since deficiency is proposed to

be a novel factor in the pathogenesis of the complications

of diabetes. In diabetes, Mg2+ is not only an outcome, but is

now considered also to be a potential risk factor in the

actual development of the disease [3]. Cardiac and other

surgeries are associated with an increased incidence of

MgD. Indeed, the cardiovascular complications associated

with MgD are often the driving force behind Mg2+ reple-

tion in hospitalized patients [4]. In rodents, dietary

restriction of Mg2+ for 3 weeks was associated with the

production of reactive oxygen species and the development

of cardiomyopathy that may result from a synergism or

cascade of inflammatory events expressed by these tissues

[5–8]. The effects of MgD on cardiac pathology in these

animal models are well-documented, yet there is little

information on the impact of MgD on the intestine.

Tissue injury due to ischemia and subsequent reperfu-

sion events (IR) is a common event associated with mul-

tiple clinical pathologies. The mesenteric circulation is

especially sensitive to systemic hypoperfusion and while

hypoxia alone is detrimental, reperfusion of the affected

area dramatically exacerbates the damage [9]. Significant

neutrophil (PMN) infiltration and mucosal injury are clas-

sic features of the oxidative stress induced by mesenteric

IR [10–13]. The recruitment of leukocytes to the affected

tissue is a critical step in the inflammatory response and

involves a complex series of adhesive interactions and

signaling events [13–15]. Leukocytes also play a prominent

role in the events that link the post-ischemic gut to acute

lung injury, the earliest manifestation of ARDS. The

recruitment of leukocytes into an area of inflammation

begins with the activation of PMNs and/or endothelial

cells, followed by the bonding of white blood cells to the

endothelium and their subsequent transmigration into tis-

sues [16]. These processes are governed by the interaction

between adhesion molecules on the surface of both

leukocytes (CD11/CD18, L-selectin) and endothelial cells

(ICAM-1, E-selectin, P-selectin) [17].

The aims of the present study, therefore, were to deter-

mine (1) the effect of MgD on intestinal morphology and

mucosal function; (2) the effect of MgD on the intestinal

responses to an oxidative stress; and (3) the impact of acute

magnesium repletion (MgR) on MgD-induced changes in

the basal state and in response to an oxidative stress.

Materials and methods

Animal preparation

All experiments were conducted in accordance to the

principles for the care and use of laboratory animals as

recommended by the US Department of Health and Human

Services and approved by the Institutional Animal Care

and Use Committee. Male Sprague–Dawley rats (250–

350 gm) were kept under 12:12 h light:dark cycle, food

and distilled water were provided ad libitum. Animals were

fed either a modified diet (Teklad Laboratory, Madison,

WI) containing 2 mmole Mg2+/kg diet (Mg2+ -deficient

group [MgD], 9% Recommended Daily Allowance

[RDA]); or the same diet supplemented with 25 mmoles of

magnesium/diet (Mg2+-sufficient group [MgS], 100%

RDA). Rats were fed the MgD or MgS diet for 1 or

3 weeks.

On the day of study, rats from both diet groups were

anesthetized with ketamine (80 mg/kg im) and xylazine

(16 mg/kg im), weighed, and randomly assigned to one of

the following treatment groups: sham operation (sham),

10 min of ischemia followed by 1 h of reperfusion (IR-10/

1), or 30 min of ischemia followed by 1 h of reperfusion

(IR-30/1). Groups of rats fed the MgD or MgS diet for

3 weeks were subjected also to 30 min of ischemia

followed by 4 h of reperfusion (IR-30/4).

In all animals, a midline laparotomy was performed and

the superior mesenteric artery (SMA) was carefully

isolated as it originated from the abdominal aorta. After a

30-min stabilization period, the animals underwent their

assigned treatment. Prior to euthanasia samples of jejunum,

10 cms from the ligament of Treitz, were harvested for

microscopic evaluation. Serum levels of Mg2+, calcium

(Ca2+), and potassium (K+) were measured by atomic

absorption spectroscopy. Sham operations, consisting of a

midline laparotomy without occlusion of the SMA, were

performed in all dietary groups.

Measurement of epithelial cell function

Four 1 cm segments of mucosa were stripped of muscle

and mounted in Ussing chambers that exposed 0.126 cm2

to 10 ml Krebs’ buffer. Agar-salt bridges and electrodes

were used to measure potential difference. Concentration-

dependent changes in short-circuit current (Isc) were

determined for the cumulative addition of the neurotrans-

mitter, acetylcholine (ACH) added to the serosal side, as an

index of MgD effect on secretory function and to glucose,

added to the mucosal side, as an index of MgD effects on

mucosal absorption.

Acute Mg2+ replacement

About 24 h prior to study, some MgD animals were

assigned randomly to receive acute Mg2+ replacement.

Animals assigned to the MgR protocol were placed on the

Mg2+-supplemented diet 24 h prior to study and received 3

60 Mol Cell Biochem (2007) 306:59–69

123

doses of 10% magnesium sulfate (50 mg/kg im) 18, 6, and

2 h prior to surgery. This paradigm is based on the current

practice of repletion of magnesium deficiency in critically

ill patients prior to surgery. Serum Mg2+ levels from the

rats undergoing replacement therapy were determined to

confirm normal serum levels.

RBC glutathione determination

Blood samples were drawn from the inferior vena cava into

Na-citrate (1%) solution. Red blood cells (RBC) were

centrifuged at 200 · g for 10 min, washed twice with

Dulbecco’s PBS, and pelleted at 200 · g for 10 min. The

packed RBCs were diluted 50-fold, hemolyzed in 10%

PBS, and acidified by 5% 5-sulfosalicyclic acid immedi-

ately to preserve the reduced glutathione (GSH). Total

glutathione [unoxidized glutathione (GSH) + ½ oxidized

glutathione (GSSG)] was determined by the ‘enzymatic

recycling method’ involving GSSG reductase-catalyzed

cycling of GSH with 5,5¢-dithiobis(2-nitrobenzoic acid) as

described [18, 19]. GSSG was determined by the same

procedure except for the prior masking of GSH by 1%

vinyl pyridine. RBC hemoglobin was determined by the

cyanmethemoglobin technique using Sigma Diagnostics

hemoglobin reagents (Sigma, St. Louis, MO).

Histology

Intestinal sections were rinsed in cold saline and fixed in

4% paraformaldehyde. Tissues were embedded in paraffin

and 5 lm transverse sections were stained with H&E and

Giemsa for light microscopic evaluation of tissue archi-

tecture and differentiation of leukocytes to clearly identify

neutrophilic infiltration, respectively. Under 10· magnifi-

cation, two blinded examiners graded the mucosal injury of

each slide based on a previously established six-tiered scale

[0 (normal)-5 (severe)]. Villous height was also obtained

using a digital micrometer and reported in micrometers.

Under 40· magnification, the number of PMNs was

counted in 20 separate fields per slide immediately superior

to the muscularis mucosae. The mean number of PMNs per

high-power field (HPF) was determined for each animal.

Immunohistochemistry

Frozen tissue sections (5–10 lm) were air-dried, washed

3 times with phosphate buffered saline (PBS), and trans-

ferred to the blocking reagent 10% goat serum, 1% BSA,

and 0.03% Triton ·100 in PBS for 60 min to reduce non-

specific binding. Sections were incubated overnight at 4�C

with primary antibody, diluted in blocking buffer, washed

3 times, and incubated with secondary antibody conjugated

with FITC or Texas Red (CD11b) for 1 h at room

temperature. Tissues were protected by mounting medium

and glass cover slips. Multiple digital images were ob-

tained at different magnifications for further analysis and

fluorescence intensity and area were measured (Nikon).

CD11b, ICAM-1, and NOS-3 staining were evaluated

using this technique and appropriate primary and second-

ary antibodies (Santa Cruz Biotechnology, Inc., Santa

Cruz, CA).

Vascular macromolecular leak

In some animals, Evan’s blue dye (0.2 ml, 1.5%) was in-

jected intravenously through the tail vein 1 h prior to

harvest to allow for determination of systemic vascular

leak. At euthanasia, small bowel lavage (SBL) was per-

formed by irrigating the terminal 20 cm of small intestine

with 5 ml of normal saline and collecting the effluent. The

heart and lungs were removed en bloc via median stern-

otomy and a brocheoalveolar lavage (BAL) was performed

by infusing 5 ml of normal saline via tracheotomy, waiting

30 s, and then aspirating the effluent. Prior to the extirpa-

tion of the heart and lungs, a blood sample was obtained

from the right ventricle by cardiac puncture. All collected

samples were centrifuged and serum samples were diluted

20 fold. The concentration of dye was assessed spectro-

photometrically (Eflab, Titerrek, Multiscan Plus, Helsinki,

Finland) at a wavelength of 630 nm. SBL to serum and

BAL to serum AB630 ratios were calculated as measures of

in vivo intestinal macromolecular capillary and pulmonary

leak. Baseline leak was defined as the mean leak in sham-

operated animals.

RNA extraction and cDNA synthesis

Full thickness sections of mid jejunum were placed

immediately in TRIzol reagent (Invitrogen, Grand Island,

NY). Total RNA was extracted as per the manufacture’s

instructions and RNA quantity was assessed using the

Beckman Coulter DU530 UV/Vis Sprectrophotometer

(Beckman Coulter, Fullerton, CA). RNA samples (2 lg)

were reverse-transcribed to cDNA using the First Strand

cDNA Synthase Kit (MBI Fermentas, Hanover, MD) with

random hexamer primer.

Real-time quantitative PCR

Real-time PCR was performed with a BioRad iCycler se-

quence detection system (Hercules, CA). Primer sequences

were designed using computer program Beacon Designer

4.00 (Premier Biosoft International, Palo Alto, CA), and

synthesized by The Biopolymer Lab (University of Mary-

land, Dept. of Microbiology/Immunology, Baltimore, MD).

PCR was performed in a 25-ll volume using BioRad iQ

Mol Cell Biochem (2007) 306:59–69 61

123

SYBR Green Supermix (BioRad Hercules, CA). Assays

were optimized as follows: primer concentrations were

varied until highest signal intensity and lowest threshold of

detection were achieved; cDNA templates were diluted to

determine linearity of optimum primer and probe concen-

tration. Amplification conditions were: 50�C for 2 min,

95�C for 10 min, 50 cycles of 95�C for 15s and 60�C for

1 min. The fold-changes in mRNA expression for NOS-2,

NOS-3, NFjB, and COX-2 were relative to the respective

vehicle groups of mice after normalization to 18s rRNA

(Applied Biosystems, Foster City, CA).

Statistical analysis

Results are expressed as means ± SEM. Statistical analysis

was performed using a paired t-test or one-way ANOVA

followed by Neuman–Keuls tests for determining differ-

ences between individual means using GraphPad Prism

(v4, Graphpad Software Inc., San Diego, CA). A value of

p < 0.05 was considered significant.

Results

MgD effects on serum electrolyte levels

Rats fed the deficient diet for 3 weeks had significantly

lower levels of serum Mg2+ than sham controls (Table 1).

There were no differences in survival among the treatment

groups. Acute Mg2+ repletion significantly elevated serum

Mg2+ levels above those in the MgD, however, the level

remained lower than those in the sham control groups

(Table 1). Of interest is that 4 hours after IR, there was a

significant increase in serum Mg2+ that was also observed

even in the MgD group suggesting that mesenteric IR

mobilizes Mg2+ (Table 1). Serum levels of potassium (K+)

or calcium (Ca2+) were not altered significantly by either

diet or treatment (Table 1).

Effects of MgD on red blood cell (RBC) glutathione

To confirm previous results showing that endogenous

antioxidant defenses were impaired by MgD, we observed

that total glutathione levels of the RBC samples from the

MgD rats reduced gradually over time. Blood glutathione

levels were unchanged after 1 week (6.30 ± 0.38 vs.

5.28 ± 0.29 lM)), but were decreased significantly to 57%

of levels in MgS rats at the end of the second week

(6.22 ± 0.33 vs. 3.61 ± 0.21 lM, p < 0.01), and to 49% by

the end of third week of deficiency (6.11 ± 0.30 vs.

3.09 ± 0.22 lM, p < 0.01). In data not shown, the levels of

GSSG in either the Mg deficient or Mg control RBC

samples were less than 1% of the total glutathione.

MgD effects on intestinal morphology and function

Histology

When compared to the control diet (MgS), the MgD alone

did not alter mucosal injury (Fig. 1A, B). In contrast, MgD

alone induced significant PMN infiltration after 1 week

that was still evident at 3 weeks (Fig. 2A). PMN infiltra-

tion in the intestine involves the leukocyte b2 integrins,

especially CD11b/CD18 and is associated with increased

expression of ICAM. Immunohistochemistry of jejunal

tissue sections obtained from MgS rats stained with

anti-CD11b antibody showed the presence of PMNs and

confirmed the MgD-increased PMN infiltration (Fig. 2C

vs. 2D) that was observed in Giemsa-stained sections under

light microscopy (Fig. 2B). Staining with ICAM-1 anti-

body showed a specific, but low level of, staining in the

endothelium along the vascular wall in MgS rats (Fig. 3A),

demonstrating that ICAM-1 is constitutively and moder-

ately expressed. There was evidence of marked vasodila-

tion in the MgD group associated with increased ICAM-1

staining within the wall of submucosal vessels (Fig. 3B).

ICAM-1 is an adhesion molecule involved in PMN infil-

tration and therefore, the increased staining for ICAM-1 in

blood vessels in MgD rats (Fig. 3A vs. 3B) is consistent

with the increased numbers of PMN (Fig. 2).

mRNA expression of inflammatory mediators

To determine if this PMN infiltration was associated with

changes in the proinflammatory mediators, we determined

mRNA expression of NOS-2, COX-2, and NFjB in full

Table 1 Changes in Serum magnesium (Mg2+), potassium (K+) and calcium (Ca2+) levels in response to mesenteric IR in rats fed Mg2+

sufficient (MgS) or Mg2+ deficient (MgD) diets or in MgD rats after acute Mg2+ repletion (MgR)

MgS MgD MgR

SHAM IR-30 IR-30/4 SHAM IR-30 IR-30/4 SHAM IR-30 IR-30/4

Mg2+ 1.8 ± 0.1 2.3 ± 0.2* 2.9 ± 0.2* 0.7 ± 0.1* 0.8 ± 0.1 1.3 ± 0.1 ** 1.5 ± 0.1 1.5 ± 0.6 2.5 ± 0.3**

K+ 8.9 ± 0.4 10.3 ± 0.7 9.5 ± 0.7 7.4 ± 0.5 10.9 ± 0.9 12.2 ± 2.5 7.7 ± 0.6 8.6 ± 0.1 12.2 ± 1.6

Ca2+ 11.2 ± 0.5 10.7 ± 0.4 11.9± .6 11.2 ± 0.4 11.4 ± 0.2 11.5 ± 0.6 11.5 ± 0.2 11.9 ± 0.2 11.0 ± 0.4

n ‡ 5 for each group; *p < 0.05 vs MgS; **p < 0.05 vs. respective SHAM; K+ in mEq/l; Ca2+ in mg/dl

62 Mol Cell Biochem (2007) 306:59–69

123

thickness sections of mid-jejunum. MgD alone did not

significantly alter expression of NOS-2 (1.0 ± 1.5 vs.

1.0 ± 1.1 fold), COX-2 (1.0 ± 0.2 vs. 0.6 ± 0.2 fold), or

NFjB (1.0 ± 0.2 vs. 0.7 ± 0.1 suggesting that MgD in-

duces a subclinical inflammation.

Vascular macromolecular leak

PMN infiltration in the small intestine is associated with

reduced levels of NOS-3 activity and evidence of increased

vascular permeability to macromolecules [20]. When

compared to MgS, full thickness sections of small intestine

taken from MgD (3 week) showed decreased NOS-3

mRNA expression in (Fig. 4A) coincident with decreased

staining for NOS-3 in the submucosal nerves as well as in

the walls of blood vessels in the submucosa (Fig. 4C vs.

4D). The decrease in NOS-3 expression (Fig. 4A) was

associated with an increase in vascular leak in both the

small intestine and lung (Fig. 4B).

Mucosal function

Secretory responses to acetylcholine were unchanged after

1 week, but were significantly inhibited after 3 weeks

(Fig. 5A). Glucose absorption was unaltered by MgD

(Fig. 5B) and is consistent with the lack of effect of

3 weeks of MgD on mucosal injury and on body weight

(301.1 ± 19.7 vs. 295.6 ± 8.6 gms).

Effects of MgD and acute MgR on responses to

mesenteric ischemia/reperfusion

Mucosal injury

Since MgD alone induced a proinflammatory state in the

gut, we next determined if MgD altered the response of the

small intestine to mesenteric IR, a known inducer of

oxidative stress. Neither MgS nor 1 or 3 weeks of the MgD

diet resulted in development of mucosal injury after a

Fig. 1 Sections of fixed tissue

taken from mid-jejunum in MgS

(A) and MgD (B) rats were cut

and mounted onto slides and

stained with Giemsa. MgD did

not alter intestinal morphology.

One representative picture from

each group of at least 6–8/group

rats is shown (Magnification,

·20)

A

MgS 1WMgD 3WMgD0

10

20

30

** **)fph/#(

NM

PB

C D

Fig. 2 Rats were fed a Mg2+

sufficient diet (MgS) or a Mg2+

deficient diet for either 1 week

(1W MgD) or 3 weeks (3W

MgD). (A) Both 1W MgD and

3W MgD increased the number

of PMNs; (B) Giemsa-stained

section of 3WMgD rat showing

increased PMN infiltration

(arrowhead); (C–D) Frozen

tissue blocks of mid-jejunum

were prepared and the sections

were cut for immunofluoresence

staining for CD11b. The

intensity of the staining was

determined by establishing

settings for the samples from the

individual vehicle groups and

using the same conditions to

evaluate the samples from the

treated groups. MgD increased

neutrophil infiltration (D) when

compared to MgS (C)

Mol Cell Biochem (2007) 306:59–69 63

123

10 minute period of ischemia and 1 hour of perfusion (IR-

10/1), but all groups exhibited a comparable and significant

mucosal injury after IR-30/1 (Fig. 6A) or IR-30/4 (Fig. 6C)

of reperfusion. Acute Mg2+ repletion (MgR) did not

diminish mucosal injury induced by IR-30/4 (Fig. 6D).

Inflammation

In the MgS rats, IR-10/1 induced PMN influx in the

absence of mucosal damage (Fig. 6A, C). In contrast, there

was a significant increase in PMN infiltration following

IR-30/1 (Fig. 6B) and IR-30/4 (Fig. 6D) in the MgS group

that was associated with the mucosal damage (6A, C).

MgD exaggerated the inflammatory response following

IR30/1 (Fig. 6B) and IR30/4 (Fig. 6D). MgR blunted the

MgD-induced enhancement of PMN infiltration to levels

observed in the MgS group (Fig. 6D).

mRNA expression of inflammatory mediators

Since MgD enhanced PMN infiltration in response to

mesenteric IR, we determined if this was associated with

altered expression of inflammatory mediators. In MgS rats,

IR-30/1 significantly increased mRNA expression of COX-

2 and NOS-2 consistent with IR-induced injury and PMN

infiltration (Fig. 7A, B). Expression of COX-2 remained

elevated in IR-30/4 (Fig. 7A) while expression of NOS-2

increased further after IR-30/4 (Fig. 7B) in MgS rats. In the

MgD group, the expression of COX-2 (Fig. 7A) was sim-

ilar to that in the MgS rats after IR-30/1. In contrast, after

IR-30/4 COX-2 expression was significantly less in the

MgD, than in MgS, rats suggesting that MgD shortens the

duration of the upregulation of COX-2 in response to IR.

MgR did not alter the effects of MgD on COX-2 expression

(Fig. 7A). The expression of NOS-2 in response to IR was

both delayed and attenuated in the MgD group (Fig. 7B).

MgR did not alter the MgD-induced delay in NOS-2

expression (Fig. 7B), but the level of expression at IR30/4

was similar to that in the MgS group.

Vascular macromolecular leak

When compared to sham-operated MgS rats, IR increased

intestinal vascular leak in both the small intestine (SBL)

and lung (BAL), but only after 4 h (Fig. 8A, B). In con-

trast, MgD alone induced significant vascular leak in the

small intestine and lung that remained elevated, but was

not further enhanced, after 1 or 4 h of reperfusion. Acute

Mg2+ repletion of MgD rats did not improve basal or

IR-induced changes in vascular permeability in either

region (Fig. 8B).

Discussion

The intestine is the site of Mg2+ absorption, yet little is

known about the effects of hypomagnesmia on the small

intestine. In the present study, rats were fed a Mg2+ defi-

cient diet to induce a gradual hypomagnesmia over a

3 week period. Significant inflammation in the absence of

mucosal injury in the small intestine was evident after

1 week and remained significant after 3 weeks on the diet.

The intestinal inflammation after 3 weeks of MgD was

Fig. 3 Rats were fed a Mg2+ sufficient diet (MgS) or a Mg2+ deficient

diet for 3 weeks (3W MgD). Sections (5 l) of frozen tissue blocks of

mid-jejunum taken from rats after 3 weeks on the MgS and MgD diet

were prepared for immunofluoresent staining of ICAM-1 (arrows).

The intensity of the staining was determined by establishing settings

for the samples from the individual vehicle groups and using the same

conditions to evaluate the samples from the infected or treated groups.

ICAM-1 positive staining was very specific for the endothelium of the

vascular walls (arrows). There is a low-level, constitutive expression

of ICAM-1 in MgS, however, 3W MgD greatly enhanced the intensity

of ICAM-1 staining of rat endothelium

64 Mol Cell Biochem (2007) 306:59–69

123

associated with significant changes in intestinal and pul-

monary vascular permeability as well as intestinal function.

MgD also induced an increased sensitivity to mesenteric

ischemia reperfusion characterized by inflammation in re-

sponse to a normally non-injurious period of intestinal

ischemia (10 min) and amplification of PNM infiltration

after an injurious period (30 min) of ischemia. Acute Mg2+

repletion was only partially effective in reversing the MgD-

induced changes in intestinal morphology and function.

In the present study, we showed that rats fed Mg2+-

deficient diets for 3 weeks exhibited significant reductions

in serum Mg2+ without alterations in Ca2+ or K+. These

data indicate, however, that homeostasis of Ca2+ or K+ can

be maintained during a gradual onset of Mg2+ deficiency.

A NOS-3

MgS 3W MgD MgR0.00

0.25

0.50

0.75

1.00

1.25

* *

oitaR

noisserpxE

Vascular Leak

0.0

0.1

0.2

0.3

0.4

0.5 MgSMgD**

**

Intestine Lung

)mures/

LB

Sr

oL

AB (

k aeL

B

C D

Fig. 4 Rats were fed a Mg2+ sufficient diet (MgS) or a Mg2+ deficient

diet for 3 weeks (3W MgD). Separate groups of 3w MgD rats were

repleted with magnesium before study (MgR). Segments of intestine

were taken from each animal in each group to measure NOS-3 mRNA

expression using real-time PCR. Expression of NOS-3 was dimin-

ished by 3W MgD and MgR in rat small intestine (A). Acute Mg2+

repletion did not improve expression of NOS-3. *p < 0.05 vs. MgS.

Sections of frozen tissue blocks of mid-jejunum were prepared for

immunofluorescent staining for NOS-3. NOS-3 positive staining is

visible throughout the section and is similar in both groups in

myenteric plexus neurons (arrowheads). In contrast, staining for

NOS-3 in MgS (C) was greater in the submucosal plexus neurons and

blood vessels (arrows) compared with 3W MgD (D). The intensity of

the staining was determined by establishing settings for the samples

from the individual vehicle groups and using the same conditions to

evaluate the samples from the treated groups. About 1 h before tissue

harvest, a group of MgS, MgD, and MgR rats were injected with

Evan’s Blue dye. Small bowel and bronchio-alveolar lavage were

performed on each rat to measure vascular macromolecular leak (B).

MgD alone induced significant leak in both the small intestine and

lung compared to MgS. **p < 0.01 vs.MgS

Acetylcholine

-8 -7 -6 -5 -4 -3

0

20

40

60

80

100A B

MgS

3WMgD

*

1wMgD

ACETYLCHOLINE LOG [M]

(csI

NIE

GN

AH

Cµ

mc/A

2 )

Glucose

1 10 1000

100

200

300

MgS

3WMgD1WMgD

GLUCOSE [mM]

(csI

NIE

GN

AH

Cµ

mc /A

2 )

Fig. 5 Rats were fed a Mg2+

sufficient diet (MgS) or a Mg2+

deficient diet for either 1 week

(1W MgD) or 3 weeks (3W

MgD). Segments of muscle-free

jejuna were mounted in Ussing

chambers to measure secretion

in response to acetylcholine (A)

and absorption in response to

glucose (B)

Mol Cell Biochem (2007) 306:59–69 65

123

0

1

2

3

4

5

φ

φ φφ φ

ShamIR30/4

MgS MgRMgD

jury

nIlasocuM

A B

C D

0

25

50

75

φ

* *

φγ

γ

φ

φφ

ShamIR10/1IR30/1

MgS 1WMgD 3WMgD

)fph/#(N

MP

0

25

50

75

*

φγ

φ

*φ

ShamIR30/4

MgS MgD MgR

)fph/#(N

MP

0

1

2

3

4

5

φφ

φ

ShamIR10/1IR30/1

MgS 1WMgD 3WMgD

yrujnIlasocuM

Fig. 6 Rats were fed a Mg2+ sufficient diet (MgS) or a Mg2+ deficient

diet for either 1 week (1W MgD) or 3 weeks (3W MgD) and were

subsequently challenged with either a sham surgery or ischemia of the

superior mesenteric artery (SMA) for 10 min (IR10), or 30 min

(IR30), followed by one hour of reperfusion. Maximum injury

occurred in the IR30 treatment for all diet groups and 1WMgD, and

3W MgD diets did not exacerbate mucosal injury in response to IR10

or IR30 compared to MgS (A).A group of 3W MgD rats were given

intramuscular injections of Mg2SO4 (50 mg/kg) 18, 6, and 2 h before

surgery (MgR). Rats from MgS, 3W MgD, and MgR groups received

a sham operation or IR30 followed by 4 hours of reperfusion

[IR30(4)]. MgR did not attenuate injury caused by IR (C).

Polymorphonuclear neutrophils (PMNs) were counted at ·40

magnification (n = 5 for each group). The number of PMNs

significantly increased after IR10(1) in MgS and 3W MgD rats, and

after IR30(1) in all groups. 3W MgD caused the greatest influx of

PMNs following IR, and was significantly higher than the corre-

sponding treatments in MgS (B). A group of 3W MgD rats was given

intramuscular injections of Mg2SO4 (50 mg/kg) 18, 6, and 2 h before

surgery (MgR). Rats from MgS, 3W MgD and MgR groups received a

sham operation or IR30 followed by 4 h of reperfusion [IR30(4)]*p < 0.05, compared to respective sham for each group (D)./p < 0.05, compared to respective sham for each group, *p < 0.05

compared to MgS Sham, cp < 0.05 compared to MgS IR10(1), MgS

IR30(1), and MgS IR30(4) (n ‡ 5 for each group)

NOS-2

0

5

10

15

20

25

**

*

**

*

ShamIR30/1IR30/4

MgS MgD MgR

oitaR

noisserpxE

COX-2

0

1

2

3

4A B

* *

*

*

Sham

IR30/1

IR30/4

MgS MgD MgR

oitaR

noisserpxE

Fig. 7 Intestinal segments were taken from the rats for total RNA

extraction. After reverse-transcription, real-time PCR was performed

to measure COX-2 and NOS-2 mRNA expression. Expression of

COX-2 and NOS-2 was increased by IR30(1) and IR30(4) compared

to MgS Sham (A). For A, and B, the fold increases were relative to

MgS Sham groups after normalization to 18s rRNA. For C the fold

increases were relative to the respective Sham for each group.

*p < 0.05 vs. respective Sham, **p < 0.01 vs. respective Sham

66 Mol Cell Biochem (2007) 306:59–69

123

There is a slight increase in K+ in response to IR in all

groups that was slightly higher in the MgD and MGR

groups. This may be related to Mg2+ ability to regulate K+

transport in cardiac cells and nerves such that low Mg2+

increases the movement of K+ out of cells. The balance

between Ca2+ and Mg2+ is important as others showed that

diets deficient in both Ca2+ and Mg2+ protected against the

proinflammatory effects of Mg2+ deficiency alone [21, 22].

We also noted a significant increase in serum Mg2+ in re-

sponse to mesenteric IR in all groups regardless of the

initial Mg2+ status. There is a considerable amount of Mg2+

complexed to ATP and the reduction in ATP production or

increase in ATP hydrolysis allows intracellular Mg2+ to

increase. This increase in Mg2+ in all groups may be due to

the rise in intracellular Mg2+ with its subsequent efflux into

serum in response to mesenteric IR-induced hydrolysis of

ATP [23].

The MgD diet alone induced a proinflammatory state

characterized by prominent intestinal PMN infiltration that

was evident at 1 week and remained significant at 3 weeks.

Previous studies have shown that after 8 days of Mg2+

deficiency, there is a significant reduction of RBC gluta-

thione [24] and elevation of complement (C3) in the liver

[22, 25] and with longer durations of the diet, there was a

increased production of NOS-2 activity by circulating

neutrophils. The reduction in RBC glutathione was con-

firmed in the present study showing that MgD induced a

systemic oxidative stress.

PMN transepithelial migration involves a complex ser-

ies of adhesive interactions and signaling events and is

mediated in part, by ICAM expression [26]. This proin-

flammatory state was associated with decreased secretion

in response to acetylcholine consistent with results from

other studies using different means to induce inflammation.

MgD alone did not alter glucose absorption, a finding that

contrasts with previous studies in which inflammation is

accompanied by significant mucosal injury [27]. The

absence of tissue damage, changes in intestinal COX-2 or

NOS-2 mRNA expression, glucose absorption, or body

weight supports the conclusion that Mg2+ deficiency

induces a subclinical inflammation of the small intestine.

The small intestine is disproportionately sensitive to

oxidative stress. PMN recruitment begins within minutes of

intestinal reperfusion and firm adhesion of PMN in pos-

tischemic vessels. Even short periods of ischemia (10 min),

which did not result in mucosal injury, induced significant

influx of PMN associated with an increased expression of

ICAM on endothelial cells of the submucosal blood ves-

sels. As we showed previously [20, 28], longer periods of

ischemia (30 min) resulted in significant mucosal injury

and inflammation after one hour of reperfusion that persists

even after 4 h of reperfusion. In the present study, this was

associated with an increase in the intestinal expression of

COX-2 and NOS-2 mRNA after 1 hr, with a further

increase in NOS-2 at 4 h post reperfusion. Prostaglandin E2

(PGE2) generation is markedly enhanced by mesenteric IR

[11] and COX-2 has been identified as an important

mediator of ischemia/reperfusion-induced inflammation

[29]. Although upregulation of NOS-2 and COX-2 is

generally considered to be part of the proinflammatory

response, their metabolic products also exert beneficial

effects on the intestinal mucosa in the reperfusion period.

In addition, emerging evidence supports a role for COX-2

in mucosal homeostasis [30]. PGE2 has well-documented

cytoprotective properties [31] and inhibition of NOS-2 was

shown previously to impair mucosal healing [32]. The

A

B

0 1 2 3 4

0.00

0.25

0.50

0.75

1.00MgS

MgD

** φ

**

Hours of Reperfusion

mureS/L

BS

0 1 2 3 4

0.00

0.25

0.50

0.75

1.00

MgS

MgD

MgR

**φ

**

**

Hours of Reperfusion

mureS/L

AB

Fig. 8 About 1 h before tissue harvest, a group of MgS, MgD, and

MgR rats were injected with Evan’s Blue dye. MgS, MgD, and MgR

rats were allowed to reperfuse for 0, 1, or 4 h post ischemia. Small

bowel (SBL) and bronchio-alveolar (BAL) lavage were performed on

each rat to measure vascular macromolecular leak (A and B). MgD

alone induced significant leak in both the small intestine and lung

compared to MgS. MgR did not attenuate vascular leak at 1 or 4 hours

post-reperfusion

Mol Cell Biochem (2007) 306:59–69 67

123

maximal induction of NOS-2 occurs at 4 h into the reper-

fusion period, correlating with the observed initiation of

mucosal restitution [20, 33]. Taken together these data

suggest that the MgD attenuates IR-induced upregulation

of COX-2 and NOS-2, whose products may act to balance

the inflammation-induced injury and to promote sub-

sequent healing and restitution of the gut.

Mg2+ deficiency significantly enhanced the sensitivity of

the intestine to IR-induced inflammation. After 1 week of

the deficient diet there was an evident subclinical inflam-

mation with no change in mucosal morphology, when

compared to the MgS group. Rats on the MgD diet for

3 weeks exhibited a significant PMN infiltration after only

10 min of ischemia and an augmented response to 30 min

of ischemia at 1 and 4 h of reperfusion. An acute Mg2+

repletion paradigm, similar to that utilized for repletion in

the trauma patients, did not alter PMN infiltration, but

significantly blunted the exaggerated IR-induced PMN

influx observed in MgD rats; PMN infiltration is a hallmark

feature of mesenteric IR, but its role in the associated

mucosal injury may be limited. Recent studies have

implicated other factors including macrophages [34],

complement activation and deposition [28, 35] and/or

natural antibodies [36] as having a more direct effect.

These data indicate that Mg2+ has a specific anti-inflam-

matory effect in the intestinal response to oxidative stress

and that clinical repletion of Mg2+ may be beneficial in

preventing further inflammation in response to intestinal

trauma.

We showed previously that the post ischemic intestine

serves as a priming bed for circulating PMNs that

contribute to the development of a systemic inflammatory

response (SIRS) and pulmonary injury [12, 20, 33, 37]. In

the MgS group, there was an emergence of significant

endothelial permeability within the small intestine and lung

that developed after 4 h. The inflammation induced by

MgD alone was sufficient to induce increased endothelial

permeability that was, surprisingly, not worsened by

intestinal IR. Constitutive production of NO plays a key

role in the maintenance of endothelial as well as epithelial

permeability. MgD inhibited constitutive NOS-3 expres-

sion particular in the submucosal area, where the large

mucosal blood vessels are located. Neither NOS-3

expression nor the enhanced vascular permeability were

prevented by MgR.

In summary, this data shows that gradual development

of severe MgD over 3 weeks induces a subclinical

inflammation characterized by PMN infiltration and

enhanced vascular permeability without mucosal injury or

upregulation of expression of proinflammatory pathways.

MgD, however, enhanced the inflammatory responses to

mesenteric IR and altered the amplitude and the duration

of the expression of key mediators that contribute to

inflammation. Acute Mg2+ repletion of MgD rats was

ineffective in alleviating the proinflammatory effects

induced by MgD alone, but was able to blunt the MgD-

induced alteration in molecular and functional responses to

mesenteric IR. These data demonstrate the importance of

Mg2+ status and the limited benefits of therapeutic repletion

of Mg2+ serum levels in trauma patients.

Acknowledgements This work was supported by NIH grants

National Institutes of Health grants R01-HL-62282 and HL-65718

awarded to W.B.W. and AI 49316 to TSD, and USDA CRIS project

#1235-52000-055. The opinions and assertions in this article are those

of the authors and do not necessarily represent those of the U. S.

Department of Defense or the U. S. Department of Agriculture.

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