Magnesium Sulfate Mitigates Lung Injury Induced by Bilateral Lower Limb Ischemia-Reperfusion in Rats

Journal of Surgical Research 171, e97–e106 (2011) doi:10.1016/j.jss.2011.03.028

Magnesium Sulfate Mitigates Lung Injury Induced by Bilateral Lower Limb Ischemia-Reperfusion in Rats Ming-Chang Kao, M.D.,*,† Woan-Ching Jan, Ph.D.,‡ Pei-Shan Tsai, Ph.D.,§ Tao-Yeuan Wang, M.D.,k and Chun-Jen Huang, MD, Ph.D.*,†,1 *Department of Anesthesiology, Buddhist Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan; †School of Medicine, Tzu Chi University, Hualien, Taiwan; ‡Department of Nursing, Mackay Medicine, Nursing and Management College, Taipei, Taiwan; §College of Nursing, Taipei Medical University, Taipei, Taiwan; and kDepartment of Pathology, Mackay Memorial Hospital, Taipei, Taiwan Submitted for publication January 13, 2011

Background. Lower limb ischemia-reperfusion (I/R) elicits oxidative stress and causes inflammation in lung tissues that may lead to lung injury. Magnesium sulfate (MgSO4) possesses potent anti-oxidation and anti-inflammation capacity. We sought to elucidate whether MgSO4 could mitigate I/R-induced lung injury. As MgSO4 is an L-type calcium channel inhibitor, the role of the L-type calcium channels was elucidated. Materials and Methods. Adult male rats were allocated to receive I/R, I/R plus MgSO4 (10, 50, or 100 mg/ kg), or I/R plus MgSO4 (100 mg/kg) plus the L-type calcium channels activator BAY-K8644 (20 mg/kg) (n [ 12 in each group). Control groups were run simultaneously. I/R was induced by applying rubber band tourniquets high around each thigh for 3 h followed by reperfusion for 3 h. After euthanization, degrees of lung injury, oxidative stress, and inflammation were determined. Results. Arterial blood gas and histologic assays, including histopathology, leukocyte infiltration (polymorphonuclear leukocytes/alveoli ratio and myeloperoxidase activity), and lung water content, confirmed that I/R caused significant lung injury. Significant increases in inflammatory molecules (chemokine, cytokine, and prostaglandin E2 concentrations) and lipid peroxidation (malondialdehyde concentration) confirmed that I/R caused significant inflammation and oxidative stress in rat lungs. MgSO4, at the dosages of 50 and 100 mg/kg but not 10 mg/kg, attenuated the oxidative stress, inflammation, and lung injury

1 To whom correspondence and reprint requests should be addressed at Department of Anesthesiology, Buddhist Tzu Chi General Hospital, Taipei Branch, No. 289, Jianguo Road, Sindian City, Taipei County 231, Taiwan. E-mail: [email protected].

induced by I/R. Moreover, BAY-K8644 reversed the protective effects of MgSO4. Conclusions. MgSO4 mitigates lung injury induced by bilateral lower limb I/R in rats. The mechanisms may involve inhibiting the L-type calcium channels. Ó 2011 Elsevier Inc. All rights reserved. Key Words: MIP-2; IL-6; NO; MDA; L-type calcium channels.

INTRODUCTION

Several clinical situations, such as traumatic arterial injury, atherosclerotic thrombosis/embolism, and aortic clamping during abdominal aortic aneurysm repair, can result in acute lower limb(s) ischemia [1–3]. To resume perfusion to the ischemic lower limb(s) in a timely manner, surgical and/or medical interventions are usually performed [1–3]. Nevertheless, resume perfusion (i.e., reperfusion) to the acutely ischemic lower limb(s) can in turn elicit oxidative stress and induce inflammatory response that may lead to the development of remote vital organ injuries, especially the lungs [4]. Magnesium sulfate (MgSO4) is used clinically for the treatment of severe pre-eclampsia and eclampsia [5]. In addition, cellular data from our group [6] and those from the other group [7] revealed that MgSO4 possesses potent anti-inflammation capacity, as endotoxin-induced upregulation of inflammatory molecules could be inhibited by MgSO4. Moreover, MgSO4 has been shown to possess potent anti-oxidation capacity, as preeclamptic women treated with MgSO4 were found to have less lipid peroxidation [8]. This concept is further supported by previous data that hypoxia-induced brain damage in fetal guinea pig [9] and radiation-induced

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oxidative stress in rat spinal cord [10] could both be attenuated by MgSO4. As oxidative stress and inflammatory response are crucial in mediating the development of acute lung injury induced by lower limb ischemia-reperfusion (I/R) [4], we speculated that MgSO4 supplement could exert certain therapeutic effects against acute lung injury induced by lower limb I/R. To elucidate further, we thus conducted this animal study with the hypothesis that MgSO4 could mitigate acute lung injury in bilateral lower limb I/R rats. Moreover, MgSO4 is a potent inhibitor of the L-type calcium channels [11]. The possible role of the L-type calcium channels in this regard was thus also investigated. MATERIALS AND METHODS A total of 96 adult male Sprague-Dawley rats (200 to 250 g; BioLASCO Taiwan Co., Ltd., Taipei, Taiwan) were used for the experiments. All animal studies were approved by the Institutional Animal Use and Care Committee, Buddhist Tzu Chi General Hospital, Taipei Branch. The care and handling of the animals were in accordance with National Institutes of Health guidelines. All rats were fed a standard laboratory chow and were provided water ad libitum until the day of experiment.

Animal Preparation Under halothane anesthesia, tracheostomy was performed and a 14-gauge angiocatheter was inserted as a tracheostomy tube. Rats were then mechanically ventilated (tidal volume: 10 mL of room air; rate: 50 breaths/min; peak airway pressure: approximately 20 cm H2O) with a small animal ventilator (SAR-830/P ventilator; CWE Inc., Ardmore, PA). All rats were anesthetized with halothane (0.75%) throughout the experiment. The right external jugular vein (for intravenous injection) and the right carotid artery (for continuous blood pressure monitoring) were cannulated with polyethylene (PE50) catheters and mean arterial pressure (MAP) and heart rate (HR) were continuously monitored (BIOPAC System, Santa Barbara, CA) until the end of the experiments.

Lower Limb I/R Protocol To achieve bilateral lower limb I/R, rubber band tourniquet was applied high around each thigh for 3 h followed by reperfusion for 3 h, according to a previous report [4].

Experimental Protocols Rats were randomly allocated to receive sham instrumentation (Sham), Sham plus MgSO4 (100 mg/kg, i.v.; Sigma-Aldrich, St. Louis, MO), I/R, or I/R plus MgSO4 (10, 50, or 100 mg/kg, i.v.) and designated as the Sham, Sham-M(100), I/R, I/R-M(10), I/R-M(50), or I/R-M(100), respectively (n ¼ 12 in each group). To elucidate further the possible role of the L-type calcium channels in this regard, another two groups of rats (n ¼ 12 in each group) were allocated to receive I/R plus the L-type calcium channel activator BAY-K8644 (20 mg/kg, i.v.; SigmaAldrich) or I/R plus MgSO4 (100 mg/kg, i.v.) plus BAY-K8644 and designated as the I/RþK or I/R-M(100)þK group, respectively. MgSO4 (dissolved in 0.5 mL normal saline) was intravenously injected immediately after reperfusion or at comparable time point in those received sham instrumentation. BAY-K8644 was administered at 5 min prior to reperfusion followed by MgSO4 in the I/R-M(100)þK group or at the comparable time point in the I/RþK group. To control for the volume

effect of vehicle, rats of the Sham and I/R groups also received 0.5 mL normal saline intravenous injection at comparable time point. After reperfusion for 3 h, all rats were euthanized with intravenous injection of high dose pentobarbital (300 mg/kg). The dosages of MgSO4 employed in this study were chosen to match the clinical dosages of MgSO4 used in the treatment of pre-eclampsia [12]. Our previous cellular data demonstrated that 1 mM BAY-K8644 could reverse the effects of 20 mM MgSO4 [6]. According to these data, we chose to employ 20 mg/kg of BAY-K8644 to elucidate the possible roles of the L-type calcium channels in this regard, as we believe the dosage of 20 mg/kg should be potent enough to allow BAY-K8644 to exhibit its effects, if they exist, on counteracting the effects of 100 mg/kg MgSO4.

Blood Sample Collection and Arterial Blood Gas (ABG) Analysis Immediately before euthanization, arterial blood (0.5 mL) was drawn and ABG levels were measured immediately with a blood gas analyzer (Rapidlab 348; Bayer Healthcare LLC, East Walpole, MA). Immediately after euthanization, another 5 mL of blood was drawn. After centrifugation, plasma were separated and stored at –80 C for subsequent analysis, including nitric oxide (NO, the indicator of oxidative stress) and interleukin-6 (IL-6, the indicator of inflammatory response).

Lung Tissues Collection and Bronchoalveolar Lavage (BAL) After euthanization, the left main bronchus was tied and left lung was removed. After dividing the upper and lower lobes of left lung, the left upper lobe lung tissues were used for subsequent wet/dry weight ratio assay and the left lower lobe lung tissues were snap frozen in liquid nitrogen and stored at 80 C for subsequent analysis. For six rats of each group, the right lung tissues were infused with 4% formaldehyde through the tracheostomy tube and then removed. For the other six rats of each group, the right lung tissues were lavaged with sterile saline (3 mL) for five times as we have previously reported [13]. An aliquot of the BAL fluid (BALF) was diluted 1:1 with trypan blue dye (Life Technologies, Grand Island, NY) and the total cell number was calculated. Then, cytospin-prepared slides were stained with Wright-Giemsa stain and a total of 500 cells were counted per sample to determine the percentage of polymorphonuclear leukocytes (PMN) in the BALF samples. The number of PMN in BALF was then calculated as the total cell count times the percentage of PMN in the BALF samples. In addition, the remaining BALF was centrifuged and the protein concentration of the supernatant was determined using a BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL).

Histologic Analysis The formaldehyde-infused right lungs were stained with hematoxylin and eosin, after the process of paraffin wax embedding and serial section. Histologic characteristics of lung injury, including edematous changes of the alveolar wall, hemorrhage, vascular congestion, and polymorphonuclear leukocytes (PMN) infiltration, were evaluated under a light microscope, as we have previously reported [13]. Each histologic characteristic was scored (0-normal to 5-severe), and overall lung injury was then categorized according to the sum of the score (0–5: normal to minimal injury; 6–10: mild injury; 11–15: moderate injury; 16–20: severe injury). Evaluation of histologic characteristics and scoring of lung injury was performed by a pathologist (T-YW), who was blinded to the grouping. Degree of leukocyte infiltration was assayed by determining PMN/ alveoli ratio, as we have previously reported [13]. In brief, PMNs and alveoli per high-power field (HPF, 4003) in 10 randomly selected areas of each sample were counted. The PMNs/alveoli ratio was determined by dividing the sum of the PMNs in 10 HPFs by that of the alveoli.

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Wet/dry Weight Ratio and Myeloperoxidase (MPO) Activity Assay For wet/dry weight ratio assay to determine lung water content, the freshly harvested left upper lobe was weighed and then placed in the oven for 24 h at 60 C and weighed again when it was dry, as we have previously reported [13]. The wet/dry weight ratio was then determined. Lung MPO activity was also quantified to measure lung injury. For MPO activity assay, snap frozen tissue samples (80 C) were homogenized, re-suspended, sonicated, and centrifuged. After separation, the supernatant was incubated in a water bath for 2 h at 60 C and MPO activity was then measured, as we have previously reported [13].

Inflammatory Molecules Snap-frozen lung tissues were processed, as we have previously reported [13]. Inflammatory molecules concentrations of the harvested rat lung tissues, including chemokine (e.g., macrophage inflammatory protein-2, MIP-2), cytokine (e.g., IL-6), and prostaglandin E2 (PGE2), were then measured using enzyme-linked immunosorbent assay (ELISA) (MIP-2 ELISA kit; R&D Systems, Inc., Minneapolis, MN; ELISA kits for IL-6 and PGE2; Pierce).

Reverse Transcription and Polymerase Chain Reaction (RT-PCR) COX-2 (i.e., the enzyme mediates PGE2 production) [14] transcriptional expression of the harvested lung tissues was measured using RT-PCR. Primer sequences and amplification protocols for COX-2 and b-Actin (as an internal standard) were adapted from previous reports [15, 16]. After amplification and separation, cDNA band densities were quantified using densitometric techniques (Scion Image for Windows; Scion Corp., Frederic, MD).

the Sham and Sham-M(100) groups remained stable throughout the experiment. HR and MAP measured at the end of the experiment (i.e., end HR and MAP) of the Sham and Sham-M(100) groups were also comparable (Table 1). End HR of the I/R group was significantly higher than that of the Sham group (P ¼ 0.026) whereas end MAP of the I/R group was significantly lower than that of the Sham group (P ¼ 0.022; Table 1). End HR and MAP of the I/R-M(10) and I/RþK groups were not significantly different from those of the I/R group (Table 1). In contrast, end HR of the I/R-M(50) and I/R-M(100) groups were significantly lower than that of the I/R group (P ¼ 0.033 and 0.030) whereas end MAP of the I/R-M(50) and I/R-M(100) groups were significantly higher than that of the I/R group (P ¼ 0.035 and 0.028; Table 1). End HR and MAP of the I/R-M(50) and I/R-M(100) groups were comparable. End HR of the I/R-M(100)þK group, in contrast, was significantly higher than that of the I/R-M(100) group (P ¼ 0.037) and end MAP of the I/R-M(100)þK group was significantly lower than that of the I/R-M(100) group (P ¼ 0.032; Table 1). ABG Data

Data were evaluated by one-way analysis of variance with the posthoc Tukey test. Data were presented as mean 6 standard errors. The significance level was set at 0.05. A commercial software package (SigmaStat for Windows; SPSS Science, Chicago, IL) was used for data analysis.

Baseline ABG, including pH, PaO2, PaCO2, and base excess (BE), among these eight groups were comparable (data not shown). End ABG of the Sham, and ShamM(100) groups were also not significantly different (Table 1). End pH, PaO2, and BE of the I/R group were significantly lower than those of the Sham group (P ¼ 0.022, 0.015, and 0.020, respectively) whereas end PaCO2 of the I/R group was significantly higher than that of the Sham group (P ¼ 0.028; Table 1). End ABG among the I/R, I/R-M(10), and I/RþK groups were not significantly different. End ABG of the I/RM(50) and I/R-M(100) groups were also comparable. In contrast, end pH, PaO2, and BE of the I/R-M(50) and I/R-M(100) groups were significantly lower than those of the I/R group (all P < 0.035, respectively) whereas end PaCO2 of the I/R-M(50) and I/R-M(100) groups were significantly higher than that of the I/R group (P ¼ 0.026 and 0.029; Table 1). Moreover, end pH, PaO2, and BE of the I/R-M(100)þK group were significantly lower than those of the I/R-M(100) group (P ¼ 0.034, 0.035, and 0.022, respectively) and end PaCO2 of the I/R-M(100)þK group was significantly higher than that of the I/R-M(100) group (P ¼ 0.037; Table 1).

RESULTS

Plasma and BALF Data

NO Assay for Oxidative Status Evaluation Lung tissues samples were processed as we have previously reported [13]. Then the concentrations of stable NO metabolites, nitrite, and nitrate, of the processed lung tissue samples together with the plasma samples were measured using a colorimetric assay kit (Cayman Chemical, Ann Arbor, MI) to determine the lung and plasma NO concentrations.

Malondialdehyde (MDA) Assay for Lipid Peroxidation Status Evaluation The protocol of MDA assay was modified from our previous report [17]. In brief, snap-frozen lung tissues were thawed and tissues homogenates were collected. Then, phosphoric acid and thiobarbituric acid solution were added to homogenates (0.5 mL) and the mixture was heated in boiling water (45 min). After cooling, the absorbance was measured to determine the amounts of lipid peroxides.

Statistical Analysis

Hemodynamics

Baseline HR and MAP among these eight groups were comparable (data not shown). HR and MAP of

Plasma NO of the Sham and Sham-M(100) groups were low (Fig. 1A). Plasma NO of the I/R group was significantly higher than that of the Sham group (P < 0.001; Fig. 1A). Plasma NO of the I/R, I/R-M(10),

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TABLE 1 Hemodynamics and Arterial Blood Gas Data at the End of Experiment Hemodynamics

Arterial blood gas

Group (n ¼ 12)

HR (beats/min)

MAP (mm Hg)

pH

PaO2 (mm Hg)

PaCO2 (mm Hg)

Base Excess (mM)

Sham Sham-M(100) I/R I/R-M(10) I/R-M(50) I/R-M(100) I/RþK I/R-M(100)þK

305 6 18 312 6 16 351 6 17* 348 6 15* 331 6 20y,z 322 6 16y,z 340 6 14* 343 6 19*,x

101 6 9 104 6 12 80 6 7* 82 6 9* 90 6 11*,y,z 92 6 9*,y,z 75 6 11* 82 6 6*,x

7.40 6 0.07 7.42 6 0.08 7.22 6 0.07* 7.24 6 0.06* 7.30 6 0.07*,y,z 7.32 6 0.05*,y,z 7.20 6 0.08* 7.26 6 0.04*,x

105 6 7 101 6 5 67 6 7* 70 6 8* 89 6 7*,y,z 92 6 5*,y,z 69 6 6* 75 6 8*,x

39 6 6 42 6 5 58 6 6* 56 6 7* 48 6 5*,y,z 46 6 7*,y,z 60 6 9* 55 6 6*,x

2.261.4 1.562.0 8.962.3* 7.562.0* 4.161.9*,y,z 3.561.6*,y,z 9.362.5* 7.861.7*,x

Sham ¼ the sham instrumentation group; Sham-M(100) - the Sham plus magnesium sulfate (MgSO4, 100 mg/kg) group; I/R ¼ the lower limb ischemia-reperfusion group; I/R-M(10) ¼ the I/R plus MgSO4 (10 mg/kg) group; I/R-M(50) ¼ the I/R plus MgSO4 (50 mg/kg) group; I/R-M(100) ¼ the I/R plus MgSO4 (100 mg/kg) group; I/RþK ¼ the I/R plus the L-type calcium channel activator BAY-K8644 (20 mg/kg) group; I/R-M(100)þ K ¼ the I/R plus MgSO4 (100 mg/kg) plus BAY-K8644 (20 mg/kg) group; HR ¼ heart rate; MAP ¼ mean arterial pressure. Data are means 6 standard errors. * P < 0.05 versus the Sham group. y P < 0.05 versus the I/R group. z P < 0.05 the I/R-M(100) or I/R-M(50) group versus the I/R-M(10) group. x P < 0.05 the I/R-M(100)þK group versus the I/R-M(100) group.

and I/RþK groups were comparable. Plasma NO of the I/R-M(50) and I/R-M(100) groups were also comparable. In contrast, plasma NO of the I/R-M(50) and I/ R-M(100) groups were significantly lower than that of the I/R group (P ¼ 0.024 and 0.018; Fig. 1A). However, plasma NO of the I/R-M(100)þK group was significantly higher than that of the I/R-M(100) group (P ¼ 0.021; Fig. 1A). Between-group differences regarding the data of plasma IL-6 (Fig. 1B) as well as total protein concentration in BALF (Fig. 2A), total cell numbers in BALF (Fig. 2B), and PMN in BALF (Fig. 2C) paralleled the plasma NO data. However, plasma IL-6 and total protein in BALF of the I/R-M(100) group were significantly

lower than those of the I/R-M(50) group (P ¼ 0.029 and 0.034; Fig. 1B and 2A). Histology and Lung Injury Score

Histologic analysis revealed normal to minimal injury in lung tissues of the Sham and Sham-M(100) groups (Fig. 3A and B). Histologic analysis also revealed moderate injury in lung tissues of the I/R, I/RM(10), I/RþK, and I/R-M(100)þK groups (Fig. 3C, D, G, and H) and mild injury in those of the I/R-M(50) and I/R-M(100) groups (Fig. 3E and F). Moreover, findings of the lung injury score (Fig. 4A) paralleled findings of the histologic analysis.

FIG. 1. Plasma concentrations of (A) nitric oxide (NO) and (B) interleukin-6 (IL-6). Sham: the sham instrumentation group. Sham-M(100): the Sham plus magnesium sulfate (MgSO4, 100 mg/kg) group. I/R: the lower limb ischemia-reperfusion group. I/R-M(10): the I/R plus MgSO4 (10 mg/kg) group. I/R-M(50): the I/R plus MgSO4 (50 mg/kg) group. I/R-M(100): the I/R plus MgSO4 (100 mg/kg) group. I/RþK: the I/R plus the Ltype calcium channel activator BAY-K8644 (20 mg/kg) group. I/R-M(100)þK: the I/R plus MgSO4 (100 mg/kg) plus BAY-K8644 (20 mg/kg) group. NS: normal saline (served as vehicle control). Data are mean 6 standard errors. *P < 0.05 versus the Sham group. yP < 0.05 versus the I/R group. zP < 0.05 the I/R-M(100) or I/R-M(50) group versus the I/R-M(10) group. xP < 0.05 the I/R-M(100) group versus the I/R-M(50) group. { P < 0.05 the I/R-M(100)þK group versus the I/R-M(100) group.

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FIG. 2. (A) The total protein concentrations, (B) the total cell numbers, and (C) the polymorphonuclear leukocyte (PMN) numbers in bronchoalveolar lavage fluid (BALF). Sham: the sham instrumentation group. Sham-M(100): the Sham plus magnesium sulfate (MgSO4, 100 mg/kg) group. I/R: the lower limb ischemia-reperfusion group. I/R-M(10): the I/R plus MgSO4 (10 mg/kg) group. I/R-M(50): the I/R plus MgSO4 (50 mg/ kg) group. I/R-M(100): the I/R plus MgSO4 (100 mg/kg) group. I/RþK: the I/R plus the L-type calcium channel activator BAY-K8644 (20 mg/kg) group. I/R-M(100)þK: the I/R plus MgSO4 (100 mg/kg) plus BAY-K8644 (20 mg/kg) group. NS: normal saline (served as vehicle control). Data are mean 6 standard errors. *P < 0.05 versus the Sham group. yP < 0.05 versus the I/R group. zP < 0.05 the I/R-M(100) or I/R-M(50) group versus the I/R-M(10) group. xP < 0.05 the I/R-M(100) group versus the I/R-M(50) group. {P < 0.05 the I/R-M(100)þK group versus the I/R-M(100) group.

Lung Wet/Dry Weight Ratio, PMNs/Alveoli Ratio, and MPO Activity

Wet/dry weight ratio, PMNs/alveoli ratio, and MPO activity in lung tissues of the Sham and Sham-M(100) groups were low (Fig. 4B, C, and D). Wet/dry weight ratio, PMNs/alveoli ratio, and MPO activity of the I/R group were significantly higher than those of the Sham group (P ¼ 0.028, 0.016, and 0.009, respectively; Fig. 4B, C, and D). Wet/dry weight ratio of the I/RM(10), I/R-M(50), I/R-M(100), I/RþK, and I/R-M(100)þ K groups were comparable to that of the I/R group (Fig. 4B). PMNs/alveoli ratio and MPO activity of the I/R and I/R-M(10) groups were comparable, whereas the PMNs/alveoli ratio and MPO activity of the I/RþK group were significantly higher than those of the I/R group (P ¼ 0.036 and 0.033, Fig. 4C and D). However, PMNs/alveoli ratio and MPO activity of the I/R-M(50) and I/R-M(100) groups were significantly lower than those of the I/R group (PMNs/alveoli ratio: P ¼ 0.020 and 0.015; Fig. 4C; MPO: P ¼ 0.031 and 0.024; Fig. 4D). PMNs/alveoli ratio and MPO activity of the I/R-M(100) group were significantly lower than those

of the I/R-M(50) group (P ¼ 0.034 and 0.037; Fig. 4C and D). Moreover, PMNs/alveoli ratio and MPO activity of the I/R-M(100)þK group were significantly higher than those of the I/R-M(100) group (P ¼ 0.026 and 0.023, respectively; Fig. 4C and D).

Lung Inflammatory Molecules

Lung COX-2 mRNA and PGE2 concentrations of the Sham and Sham-M(100) groups were low (Fig. 5A). Lung COX-2 mRNA and PGE2 of the I/R group were significantly higher than those of the Sham group (both P < 0.001; Fig. 5A). Lung COX-2 mRNA and PGE2 among the I/R, I/R-M(10), and I/RþK groups were comparable (Fig. 5A). Lung COX-2 mRNA and PGE2 between the I/R-M(50) and I/R-M(100) groups were not significantly different (Fig. 5A). In contrast, lung COX-2 mRNA and PGE2 of the I/R-M(50) and I/ R-M(100) groups were significantly lower than those of the I/R group (COX-2: P ¼ 0.023 and 0.016; PGE2: P ¼ 0.017 and 0.020; Fig. 5A). However, lung COX-2 mRNA and PGE2 of the I/R-M(100)þK group were

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FIG. 3. Representative microscopic findings of lung tissues stained with hematoxylin-eosin (2003). (A) The sham instrumentation (Sham) group. Microscopic findings reveal normal to minimal lung injury. (B) The Sham plus magnesium sulfate (MgSO4, 100 mg/kg) [Sham-M(100)] group. Microscopic findings reveal normal to minimal lung injury. (C) The lower limb ischemia-reperfusion (I/R) group. Microscopic findings reveal moderate lung injury. (D) The I/R plus MgSO4 (10 mg/kg) [IR-M(10)] group. Microscopic findings reveal moderate lung injury. (E) The I/R plus MgSO4 (50 mg/kg) [IR-M(50)] group. Microscopic findings reveal mild lung injury. (F) The I/R plus MgSO4 (100 mg/kg) [IRM(100)] group. Microscopic findings reveal mild lung injury. (G) The I/R plus the L-type calcium channel activator BAY-K8644 (20 mg/kg) [IRþK] group. Microscopic findings reveal moderate lung injury. (H) The I/R plus MgSO4 (100 mg/kg) plus BAY-K8644 (20 mg/kg) [IRM(100)þK] group. Microscopic findings reveal moderate lung injury.

significantly higher than those of the I/R-M(100) group (P ¼ 0.023 and 0.028, respectively; Fig. 5A). Moreover, between-group differences regarding the data of lung MIP-2 (Fig. 5B) and lung IL-6 (Fig. 5C) paralleled the data of lung COX-2 mRNA and PGE2. In addition, lung MIP-2 concentration of the I/R-M(100) group was significantly lower than that of the I/R group (P ¼ 0.038; Fig. 5B). Lung NO and MDA

Lung NO concentrations of the Sham and ShamM(100) groups were low (Fig. 6A). Lung NO of the I/R

group was significantly higher than that of the Sham group (P ¼ 0.007; Fig. 6A). Lung NO of the I/R, I/RM(10), and I/RþK groups were comparable (Fig. 6A). In contrast, lung NO of the I/R-M(50) and I/R-M(100) groups were significantly lower than those of the I/R group (P ¼ 0.024 and 0.016; Fig. 6A). Lung NO of the I/R-M(100) group was significantly lower than that of the I/R-M(50) group (P ¼ 0.028; Fig. 6A). However, lung NO of the I/R-M(100)þK group was significantly lower than that of the I/R-M(100) group (P ¼ 0.026; Fig. 6A). Moreover, between-group differences regarding the data of lung MDA (Fig. 6B) paralleled the data of lung NO.

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FIG. 4. (A) Injury scores, (B) wet/dry weight ratio, (C) polymorphonuclear leukocyte (PMN)/alveoli ratio, and (D) myeloperoxidase (MPO) activity in lung tissues. Sham: the sham instrumentation group. Sham-M(100): the Sham plus magnesium sulfate (MgSO4, 100 mg/kg) group. I/ R: the lower limb ischemia-reperfusion group. I/R-M(10): the I/R plus MgSO4 (10 mg/kg) group. I/R-M(50): the I/R plus MgSO4 (50 mg/kg) group. I/R-M(100): the I/R plus MgSO4 (100 mg/kg) group. I/RþK: the I/R plus the L-type calcium channel activator BAY-K8644 (20 mg/kg) group. I/RM(100)þK: the I/R plus MgSO4 (100 mg/kg) plus BAY-K8644 (20 mg/kg) group. NS: normal saline (served as vehicle control). Data are mean 6 standard errors. *P < 0.05 versus the Sham group. yP < 0.05 versus the I/R group. zP < 0.05 the I/R-M(100) or I/R-M(50) group versus the I/RM(10) group. xP < 0.05 the I/R-M(100) group versus the I/R-M(50) group. {P < 0.05 the I/R-M(100)þK group versus the I/R-M(100) group.

DISCUSSION

Data from the present study, in concert with those previous ones [4], confirmed that lower limb I/R could cause acute lung injury. Data from this study also provided the first evidence to demonstrate the therapeutic potentials of MgSO4 against injuries caused by I/R, as our data revealed that MgSO4 administered immediately after reperfusion could mitigate acute lung injury in rats experiencing bilateral lower limb I/R. MgSO4 is commonly used in clinical situations. Data from this study, thus, should have profound clinical implications and warrant further investigation. It has been shown that oxidative stress and inflammatory response are crucial in mediating the development of lung injury induced by lower limb I/R [4], as oxidants, infiltrated leukocytes, and inflammatory molecules can work synergistically to damage pulmonary microvascular endothelial cell and result in microvascular integrity loss and eventually gas exchange impairment and lung dysfunction [18]. Data from this study confirmed previous findings that lower limb I/R elicited oxidative stress and induced

inflammatory response in rats [4]. It is well established that bursts of reactive oxygen and nitrogen species induced by oxidative stress can result in disarrangement of vital pathways, including energy metabolism, survival/stress responses, and apoptosis [19, 20]. However, the mechanisms by which oxidative stress triggers inflammatory response in animals experiencing lower limb I/R have not been fully elucidated. It is well established that expression of inflammatory molecules is tightly regulated by the transcription factor nuclear factor kB (NF-kB) [21]. Moreover, activation of NF-kB is mediated by toll-like receptor (TLR) signaling pathways [22]. Judging from these data, we thus speculate that oxidative stress may act through activating TLRs and subsequent NF-kB to induce inflammatory response during lower limb I/R. This concept is supported by previous data that oxidative stress, caused by hemorrhagic shock/resuscitation or oxidants, could increase TLRs expression [23]. Moreover, inhibition of TLRs and NF-kB pathways was reported to significantly attenuate hepatic injury and inflammatory response in liver in mice experiencing hepatic I/R [24].

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FIG. 5. Lung concentrations of (A) cyclooxygenase 2 (COX-2) mRNA and prostaglandin E2 (PGE2), (B) macrophage inflammatory protein-2 (MIP-2), and (C) interleukin-6 (IL-6). Sham: the sham instrumentation group. Sham-M(100): the Sham plus magnesium sulfate (MgSO4, 100 mg/kg) group. I/R: the lower limb ischemia-reperfusion group. I/R-M(10): the I/R plus MgSO4 (10 mg/kg) group. I/R-M(50): the I/R plus MgSO4 (50 mg/kg) group. I/R-M(100): the I/R plus MgSO4 (100 mg/kg) group. I/RþK: the I/R plus the L-type calcium channel activator BAY-K8644 (20 mg/kg) group. I/R-M(100)þK: the I/R plus MgSO4 (100 mg/kg) plus BAY-K8644 (20 mg/kg) group. NS: normal saline (served as vehicle control). Data are mean 6 standard errors. *P < 0.05 versus the Sham group. yP < 0.05 versus the I/R group. zP < 0.05 the I/R-M(100) or I/R-M(50) group versus the I/R-M(10) group. xP < 0.05 the I/R-M(100) group versus the I/R-M(50) group. {P < 0.05 the I/R-M(100)þK group versus the I/R-M(100) group.

MgSO4 possesses significant anti-oxidation and antiinflammation capacities [6–10]. Data from this study revealed that rats of the I/R-M(50) and I/R-M(100)

groups had lower concentrations of plasma NO as well as pulmonary NO and MDA than those of the I/R group, indicating that the oxidative stress induced by

FIG. 6. Lung concentrations of (A) nitric oxide (NO) and (B) malondialdehyde (MDA). Sham: the sham instrumentation group. ShamM(100): the Sham plus magnesium sulfate (MgSO4, 100 mg/kg) group. I/R: the lower limb ischemia-reperfusion group. I/R-M(10): the I/R plus MgSO4 (10 mg/kg) group. I/R-M(50): the I/R plus MgSO4 (50 mg/kg) group. I/R-M(100): the I/R plus MgSO4 (100 mg/kg) group. I/RþK: the I/R plus the L-type calcium channel activator BAY-K8644 (20 mg/kg) group. I/R-M(100)þK: the I/R plus MgSO4 (100 mg/kg) plus BAYK8644 (20 mg/kg) group. NS: normal saline (served as vehicle control). Data are mean 6 standard errors. *P < 0.05 versus the Sham group. y P < 0.05 versus the I/R group. zP < 0.05 the I/R-M(100) or I/R-M(50) group versus the I/R-M(10) group. xP < 0.05 the I/R-M(100) group versus the I/R-M(50) group. {P < 0.05 the I/R-M(100)þK group versus the I/R-M(100) group.

KAO ET AL.: MgSO4 AND LOWER LIMB I/R INJURY

lower limb I/R was significantly attenuated by MgSO4. Our data also revealed that the levels of leukocyte infiltration and the concentrations of plasma IL-6 and pulmonary inflammatory molecules of the I/R-M(50) and I/R-M(100) groups were significantly lower than those of the I/R group. These data suggested that the inflammatory response induced by lower limb I/R was mitigated by MgSO4. Considering the crucial roles of the oxidative stress and inflammatory response in mediating the development of acute lung injury [4], these above-mentioned data seem to support the concept that MgSO4 might act through anti-oxidation and/or antiinflammation capacities to exert its protective effects against acute lung injury caused by lower limb I/R. MgSO4 is a potent inhibitor of the L-type calcium channels [11]. Data from this study demonstrated that the protective effects of MgSO4 on mitigating lung injury as well as oxidative stress and inflammatory response induced by lower limb I/R were counteracted by the L-type calcium channel activator BAY-K8644. These data seem to support the concept that MgSO4 might act through antagonizing the L-type calcium channels to exhibit its protective effects against lower limb I/R. The L-type calcium channels are the crucial members of trans-membrane ion channel proteins that mediate the calcium influx signal in immune cells [25]. Previous data also highlighted the involvement of the L-type calcium channels on mediating the I/R-induced increases in intra-cellular calcium levels [26]. Transient increases in intra-cellular calcium levels can activate the crucial signaling pathways, e.g., NF-kB and TLR, and subsequently induce inflammation response [23, 27]. Moreover, transient increases in intra-cellular calcium levels can lead to cell death, mainly through mechanisms involving mitochondrial function impairment, necrosis, and/or apoptosis [28, 29]. Judging from these data, we thus speculate that therapies aiming at antagonizing the L-type calcium channel activity could be beneficial in situations associated with I/R injury. This concept is supported by previous data that the L-type calcium channel antagonist verapamil could mitigate lung injury caused by unilateral lung I/R [30]. This concept is also supported by previous data that the other L-type calcium channel antagonist nicardipine could ameliorate the deleterious effects caused by cerebral I/R [31]. MgSO4 is a potent vasodilator [5]. One would expect that MgSO4 administration might cause significant decreases in blood pressure. Data from this study confirmed this notion, as our data revealed that the MAP measured immediately after MgSO4 administration in rats of the Sham-M(100), I/R-M(50), and I/R-M(100) groups were significantly lower than those of the Sham and I/R groups (data not shown). However, our data also revealed that the MAP measured at 1 h after

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MgSO4 administration in rats of the Sham-M(100), I/RM(50), and I/R-M(100) groups were comparable to those of the Sham and I/R groups (data not shown). These data reveal that the hypotensive effect of MgSO4 is transient and thus its administration in situations associated with lower limb I/R should be safe. Certain study limitations do exist. First, MgSO4 was administered immediately after reperfusion in this study. As ischemia per se does not significantly increase pulmonary inflammatory molecules [4], timing for MgSO4 administration in this study, thus, should be considered as co-treatment. However, effects of different timings of MgSO4 administration, i.e., pre- or post-treatment, remain to be elucidated. Second, MgSO4 is a bronchodilator [32]. Though we did not observe significant between-group differences regarding the peak airway pressure immediately after MgSO4 administration, it is likely that the bronchodilation effects of MgSO4 might contribute to the protective effects of MgSO4 observed in this study. More studies are needed before further conclusions can be drawn. Third, this study focused on the acute phase of reperfusion. The long-term effects of MgSO4 in this regard remain unstudied. Fourth, this study focused on the lungs. Lower limb I/R may also result in deleterious effects on the other vital organs, including liver, kidney, intestine, and brain [4, 33]. The question of whether MgSO4 could protect the other vital organs in situations associated with lower limb I/R remains unstudied. However, previous data that MgSO4 could attenuate the effect of sepsis on damaging blood-brain barrier integrity [34] seem to suggest that MgSO4 might protect vital organs from the deleterious effects caused by lower limb I/R. In summary, MgSO4 significantly mitigated lung injury as well as oxidative stress and inflammatory response imposed by bilateral lower limb I/R in rats. The mechanisms may involve inhibiting the L-type calcium channels. ACKNOWLEDGMENTS This work was mainly performed at the Buddhist Tzu Chi General Hospital, Taipei Branch and supported by grants from the Buddhist Tzu Chi General Hospital, Taipei Branch (TCRD-TPE-100-09) and the National Science Council, Taiwan (NSC 98-2314-B-303-012MY3) awarded to C-JH.

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