Age-related differences of neutrophil activation in a skeletal muscle ischemia-reperfusion model

Free tissue transfers and replantation of amputated limbs are better tolerated by young adolescents than mature adults. The authors hypothesized that this observation may be, in part, because of an attenuated ischemia-reperfusion (IR) injury in younger patients. Because neutrophils have been identified as a critical cell line responsible for IR injury, the authors investigated the effects of animal age on the degree of neutrophil activation in a rat model. Activation was evaluated by monitoring expression of integrin surface markers (mean fluorescence intensity [MFI] of CD11b) and oxidative burst potential (MFI of dihydrorhodamine [DHR] oxidation) by flow cytometry in neutrophils analyzed after 4 hours of ischemia and 1, 4, and 16 hours of reperfusion in a gracilis muscle flap model in mature adult and young adolescent rats. Neutrophil activation was also evaluated in control sham-operated animals, which underwent elevation of gracilis muscle flaps without exposure to an ischemic insult. Muscle edema, determined by wet-to-dry muscle weight ratio, and muscle viability, determined by nitro blue tetrazolium (NBT) staining, were completed for gracilis muscles exposed to ischemia after 24 hours of reperfusion for each of the groups. Integrin expression, assessed by MFI of CD11b, was increased significantly in ischemic muscles of mature adult rats at 4 hours of reperfusion (71.10+/-3.53 MFI vs. 54.88+/-12.73 MFI, p=0.025). Neutrophil oxidative potential, assessed by MFI of DHR oxidation, was increased significantly in ischemic muscles of mature adult rats compared with young adolescent rats at 1 hour of reperfusion (78.10+/-9.53 MFI vs. 51.78+/-16.91 MFI, p=0.035) and 4 hours of reperfusion (83.69+/-15.29 MFI vs. 46.55+/-8.09 MFI, p=0.005). Increased edema formation was observed in the ischemic muscles of mature adult rats when compared with young adolescent rats (1.25+/-0.04 vs. 1.12+/-0.05, p=0.031) after 24 hours of reperfusion. A trend toward decreased muscle viability was observed in the mature adult rats when compared with young adolescent rats (23.7+/-3.1% NBT staining vs. 32.3+/-13.7% NBT staining, p=0.189) after 24 hours of reperfusion. The authors present evidence of an attenuated IR injury in young adolescent animals when compared with mature adult rats. These findings emphasize the importance that studies involving IR injury should be performed with consideration of animal age.     

Analysis of nitric oxide activity in prevention of reperfusion injury

This project was designed to determine the role of nitric oxide (NO) in the prevention of ischemia-reperfusion injury. Inferiorly based rectus abdominis muscle flaps were elevated in pigs and subjected to 6 hours of ischemia followed by 4 hours of reperfusion. Group I animals received a bolus of L-arginine before reperfusion, and a continuous infusion once flow was restored. Group II animals served as controls and received an equal volume of saline as a bolus and subsequent continuous infusion. Microdialysis was used to measure tissue NO levels, and these were correlated with muscle survival determined by vital staining with nitroblue tetrazolium. The results demonstrated a significant increase in tissue NO levels in L-arginine-supplemented animals (p < 0.05), which in turn correlated with a significant increase in muscle survival (p = 0.0051). These results suggest that administration of supplemental L-arginine to ischemic skeletal muscle during reperfusion results in increased NO production and decreased tissue damage.     

Study of L—arginine—nitric oxide pathway in ischemiareperfusion injured limbs in rats

OBJECTIVE: To observe the change of nitric oxide (NO) levels in the blood and the morphological change of the muscles in the limbs of rats during the (IR) injury and after being intervened by L-arginine (L-Arg) and L-nitroarginine (L-NNA). METHODS: Sixty-six male Sprague-Dawley (SD) rats were used an d grouped into the normal controls, the sham injury controls, the IR injury group and the intervention groups (L-Arg group and L-NNA group). After 6 hours of ischemia, followed by reperfusion for 3, 12 or 24 hours, the samples in the IR injury group were obtained. The rats in the intervention groups were given L-Ar g (100 mmol/L) and L-NNA (10 mmol/L), respectively, through the abdominal cavity. Then the anterior tibial muscle in the right limb was obtained for histological examination, the anterior tibial muscle in the left limb for ultrastructure observation and the blood for assay of NO in all the rats. NO was assayed by indirect measurement of NO(2)(-)/NO(3)(-) with Griess method. RESULTS: There was no significant difference of NO between the normal controls and the sham injury controls (P>0.05). But NO significantly decreased in the IR injury group (P<0.01), and further decreased with reperfusion (P<0.01) and reached the lowest point at 12 hours after reperfusion. The level of NO in the L-Arg group was significantly higher than that in the IR injury group ( P<0.01), but was not significantly different from that in the controls (P>0.05). In the L-NNA group, NO decreased to the undetectable level (P<0.01). Histological examination and ultrastructure observation showed the muscles were normal in the control groups. After 6 hours of ischemia, the skeletal muscles displayed injuries, and they were most severely injure d after 12 hours of reperfusion. In the L-Arg group, the skeletal muscles were less injured, while in the L-NNA group, the injury was similar to that in the I R injury group. CONCLUSIONS: When the limbs of the rats sustain IR, NO in the blood decreases. Meanwhile, the muscles in the limbs are injured. When L-Arg is given, NO in the blood is restored and the muscles are protected. When L-NNA completely inhibits NO, no protection of the muscles is shown.     

Effects of supplementation of BH4 after prolonged ischemia in skeletal muscle

PURPOSE: To determine whether the supplementation of tetrahydrobiopterin (BH(4), an essential cofactor of nitric oxide synthase; NOS) could attenuate endothelial dysfunction and improve NOS activity and cell viability in skeletal muscle after ischemia/reperfusion (I/R). METHODS:A vascular pedicle isolated rat cremaster muscle model was used. Cremaster muscles were subjected to 4 h of ischemia followed by 2 h of reperfusion. Rats were given either normal saline or BH(4) by intravenous injection at 1 min prior to reperfusion. After reperfusion, average arteriole diameter, capillary perfusion, endothelial-dependent/-independent vasodilatation, NOS activity, and muscle cell viability were evaluated. RESULTS: Supplementation of BH(4) prior to reperfusion significantly attenuated reperfusion-induced vasoconstriction, poor capillary perfusion, and endothelial dysfunction and enhanced cNOS activity and slightly improved cell viability in the skeletal muscle after I/R. CONCLUSION: Supplementation of BH(4) during reperfusion provided a significant protection against I/R injury in rat skeletal muscle.     

雌激素在大鼠肝移植缺血再灌注损伤中的保护作用

目的 了解雌激素对大鼠肝移植缺血再灌注损伤是否具有保护作用及其可能的作用机制.方法 将大鼠分为三组:雄性组、雌性组以及给予外源性雌激素的雄性组,分别应用两袖套法实施肝移植手术.术后6 h麻醉后处死,取血及肝组织标本.观察外周血丙氨酸氨基转移酶(ALT)和谷氨酸氨基转移酶(AST)以及一氧化氮(NO)水平,免疫组化染色分析内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)在各组肝组织中的表达.结果 与雄性组和雌性组比较给予外源雌激素的雄性大鼠血清ALT水平和AST水平显著降低(分别为561.69 U/L比730.78 U/L和678.82 U/L,726.44 U/L比914.21 U/L和861.86 U/L);血清NO水平明显升高,平均为63.54 μmol/L;肝组织eNOS表达显著增加,iNOS表达显著降低.结论 雌激素对于大鼠肝移植缺血再灌注损伤具有保护作用,这种保护作用一部分是通过调节eNOS和iNOS表达、提高NO水平实现的.

Abstract：

Objective To investigate the protective effects of 17β-estradiol on ischemia reperfusion injury in rat liver transplantation. Methods The rats were divided into three groups: male to male group(MG), female to female group (FG), and male to male group which were given 17β-estradiol 4000 μg/kg 24 hours before liver transplantation intraperitoneally (M + EG). Then transplantation was performed. At 6 hours after portal vein reperfusion, blood samples were obtained to determine the levels of alanine aminotransferase(ALT), aspartate aminotransferase (AST), and nitric oxide(NO). The expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in liver were observed by immunohistochemistry. Results ALT and AST levels in the M+EG group were lower than those in MG and FG (561. 69 U/L vs 730. 78 U/L and 678. 82 U/L; 726. 44 U/L vs 914. 21 U/L and 861. 86 U/L). The NO level (63. 54 μmol/L) was much higher than those in the MG and FG groups. The expression of eNOS in the M+EG group was higher than those in MG and FG, while the expression of iNOS in M+EG were lower than those in MG. and FG. Conclusion 17β-estradiol can attenuate ischemia reperfusion injury in rat liver transplantation by improving the balance of eNOS and iNOS.     

Initiation of microvascular protection by nitric oxide in late preconditioning

The authors hypothesized that nitric oxide is induced by a brief period of ischemia/reperfusion (ischemic preconditioning, IPC) on postoperative day (POD) 1, and that this released nitric oxide is responsible for initiating a delayed microvascular protection against a prolonged period of ischemia in skeletal muscle on POD day 2. The cremaster muscle of male Sprague-Dawley rats underwent 4 hr of ischemia, and then 60 min of reperfusion. IPC consisted of 45 min of ischemia but was done 24 hr before the prolonged ischemia. Local intraarterial infusion of sodium nitroprusside (SNP, a donor of nitric oxide) or Nw-nitro-L-arginine (L-NA, a nonselective nitric oxide synthase antagonist) were also given 24 hr before prolonged ischemia. Arteriole diameters and capillary perfusion were measured using intravital microscopy. Four groups were compared: 1) control; 2) IPC; 3) SNP + sham IPC; and 4) L-NA + IPC. Four hours of ischemia followed by reperfusion created a significant vasoconstriction and capillary no-reflow in the microcirculation of cremaster muscles. These alterations were largely prevented by IPC. Local intraarterial infusion of SNP without IPC created a similar microvascular protection to that induced by IPC alone. In contrast, intraarterial infusion of L-NA prior to IPC eliminated the IPC-induced microvascular protection. In conclusion, in late preconditioning, nitric oxide contributes to the initiation of a delayed microvascular protection against prolonged ischemia in skeletal muscle.     

Effect of nitric oxide on the contractile function of rat reperfused skeletal muscle

BACKGROUND: The involvement of nitric oxide (NO) in ischemia-reperfusion injury remains controversial and has been reported to be both beneficial and deleterious. The purpose of this study was to examine the contribution of NO and superoxide to skeletal muscle function using an ischemic revascularized hind limb model in rats. PATIENTS AND MATERIALS: Warm ischemia produced by vascular pedicle clamping was sustained for 3 h. The animals were divided into four groups according to the solution administrated: (1) saline, (2) N-methyl-L-arginine acetate (L-NMMA), (3) L-NMMA + N-(N-L-g-glutamyl-S-nitroso-l-cysteinyl)glycine (S-nitrosoglutathione), or (4) superoxide dismutase (SOD). Saline, L-NMMA, or L-NMMA + S-nitrosoglutathione was infused for the first 2 h of reperfusion. The SOD was administered as an intravenous bolus 5 min before the onset of reperfusion. Postischemic blood flow was measured by a Doppler flow meter. Muscle contractile function was determined after 24 h of reperfusion. RESULTS: Postischemic blood flow was significantly decreased by the L-NMMA infusion compared with that in the saline-treated group. No significant difference in postischemic blood flow was noted in the saline-, L-NMMA + S-nitrosoglutathione-, and SOD-treated groups. Contractile function of the gastrocnemius muscle in the L-NMMA-and SOD-treated groups, but not in the L-NMMA + S-nitrosoglutathione group, was significantly better than that in the saline-treated group. CONCLUSION: Limiting postischemic blood flow and SOD infusion are both beneficial in decreasing the ischemia-reperfusion injury of skeletal muscle. S-Nitrosoglutathione infusion following suppression of endogenous NO production does not reduce ischemia-reperfusion injury.     

L-Arginine transport is augmented through up-regulation of tubular CAT-2 mRNA in ischemic acute renal failure in rats

BACKGROUND: Ischemic acute renal failure (iARF) is associated with increased nitric oxide (NO) production during the reperfusion period, as endothelial nitric oxide synthase (eNOS) is maximally activated, and renal tubular inducible NOS (iNOS) is stimulated. Increased NO production leads to augmented tubular injury, probably through the formation of peroxynitrite. l-Arginine (l-Arg), the only precursor for NO, is transported into cells by cationic amino acid transporters, CAT-1 and CAT-2. We hypothesized that the increased NO production observed in iARF may result from increased l-Arg uptake, which would be reflected in the augmented expression of l-Arg transporter(s). METHODS: Ischemic acute renal failure was induced in rats by right nephrectomy + left renal artery clamping for 60 minutes. l-Arg uptake was examined in freshly harvested glomeruli and tubuli from control, sham operated, and animals subjected to 15, 30, and 60 minutes, and 24 hours of reperfusion, following 60 minutes of ischemia. Using RT-PCR, renal tissues were examined further for the expression of iNOS, CAT-1, CAT-2, arginase I and arginase II. RESULTS: Tubular expression of iNOS mRNA was initiated by ischemia, continued to increase after 60 minutes of reperfusion, and decreased after 24 hours. l-Arg transport into glomeruli was similar in all experimental groups. l-Arg uptake into tubuli was markedly augmented following the 60-minute reperfusion, while it moderately increased after 24 hours of reperfusion. This was accompanied by a parallel, preferential increase in tubular CAT-2 mRNA expression at 60 minutes of reperfusion. CAT-1 mRNA expression was unchanged, as detected by RT-PCR. In addition, the expression of arginase II and arginase I mRNA was attenuated by 30 minutes and one hour of reperfusion, and returned to baseline values after 24 hours of reperfusion. CONCLUSIONS: Ischemic ARF is associated with augmented tubular CAT-2 mRNA expression, which leads to enhanced l-Arg transport and increased NO production. This may contribute to the renal injury exhibited in iARF.     

Statins inhibit neutrophil infiltration in skeletal muscle reperfusion injury

BACKGROUND: Neutrophil infiltration is a major determinant of ischemia-reperfusion injury (IRI). Statins improve endothelial function by elevating nitric oxide synthase activity and inhibiting adhesion molecule expression and may, therefore, inhibit IRI-induced neutrophil extravasation. Although statins are protective against myocardial IRI and stroke, a role for statins in ameliorating skeletal muscle IRI has not yet been confirmed. This study, therefore, addressed the hypothesis that simvastatin would attenuate the severity of tissue damage during skeletal muscle IRI. METHODS: Rats were administered simvastatin for 6 d before 4 h hind limb ischemia and 24 h reperfusion. Neutrophil infiltration was assessed using myeloperoxidase (MPO) assays and tissue damage by quantitative immunohistochemical analysis of collagen IV. The effect of reducing nitric oxide levels on the severity of IRI was assessed by administering the NOS inhibitor, N-Imino-L-ornithine (L-NIO), before ischemia. RESULTS: Simvastatin significantly inhibited IRI-induced MPO activity but not collagen degradation in postischemic skeletal muscle. Inhibition of nitric oxide synthase by L-NIO markedly inhibited neutrophil infiltration and protected against IRI-induced collagen degradation. When both simvastatin and L-NIO were administered before IRI, the IRI-induced elevation in MPO activity was completely inhibited. However, paradoxically, simvastatin counteracted the protective effect of L-NIO against IRI-induced collagen IV degradation. CONCLUSIONS: The inhibition by simvastatin of IRI-induced neutrophil infiltration in skeletal muscle suggests that statins may be a useful therapy to attenuate the severity of IRI but their precise mechanisms of action remains to be determined. Nitric oxide also plays a cytotoxic, rather than protective, role in mediating IRI in this model.     

Platelet-activating factor contributes to postischemic vasospasm

BACKGROUND: The purpose of the present study was to determine if platelet-activating factor is an important mediator that produces vasospasm during reperfusion after ischemia in skeletal muscle. MATERIALS AND METHODS: A vascular isolated cremaster muscle in male Sprague-Dawley rats was coupled with local intraarterial drug infusion as a model to study microcirculation responses to ischemia/reperfusion injury. Arteriole diameters and capillary perfusion were measured using intravital microscopy. Group 1: platelet-activating factor dose response. Group 2: Effects of a cyclooxygenase inhibitor; indomethacin, and a thromboxane synthetase inhibitor, imidazole, on the response to platelet-activating factor. Group 3: Effects of nitric oxide synthesis inhibitor; N(omega)-nitro-L-arginine methyl ester, on the response to platelet-activating factor. Group 4: Effects of a platelet-activating factor receptor antagonist, CV-3988, indomethacin, and imidazole after 4 h of warm ischemia and reperfusion. RESULTS: Intraarterial infusion of platelet-activating factor produced a dose-related but mild vasoconstriction. Pretreatment with indomethacin or imidazole resulted in significant vasodilation actually emanating from platelet-activating factor infusion. Nitric oxide inhibition (with N(omega)-nitro-L-arginine methyl ester) enhanced the vasoconstriction produced by platelet-activating factor. Pretreatment with CV-3988, indomethacin, or imidazole significantly attenuated ischemia/reperfusion-induced vasospasm and capillary no-reflow in the cremaster muscles. CONCLUSIONS: Ischemia/reperfusion-induced vasoconstriction is at least in part mediated by platelet-activating factor and thromboxane A(2).     

Technetium 99m pyrophosphate quantitation of skeletal muscle ischemia and reperfusion injury

The study of ischemia and reperfusion injury in the extremity has been hampered by lack of an accurate method of measuring skeletal muscle injury. We used a bilateral isolated in vivo canine gracilis muscle model in 15 anesthetized dogs. The experimental muscles had 4, 6, or 8 hours of ischemia and 1 hour of reperfusion. The contralateral gracilis muscle served as a control. Technetium 99m pyrophosphate (99mTc-PYP), an agent which localizes in injured muscle cells, was used to quantitate canine skeletal muscle damage. After 6 hours of ischemia and 1 hour of reperfusion, there was a significant increase of 215% of 99mTc-PYP uptake in the experimental vs the control muscle. Experimental muscle uptake was 8% greater than control after 4 hours and 405% more after 8 hours of ischemia and reperfusion. Segmental distribution of 99mTc-PYP uptake showed localization to be greatest in the middle of the muscle at the entry site of the gracilis artery. Electron microscopic evaluation also documented this area to have undergone the most severe injury. Distal portions of the muscle did not show increased damage. Our results show that 99mTc-PYP effectively quantitates skeletal muscle ischemia and reperfusion injury. The pattern of 99mTc-PYP uptake suggests that considerable injury is caused during reperfusion.     

Effect of a nitric oxide donor on microcirculation of acutely denervated skeletal muscle during reperfusion

The authors have shown that exogenous nitric oxide (NO) protects innervated skeletal muscle against reperfusion injury. This study further evaluated the effects of exogenous NO donor on denervated skeletal muscle. Forty-eight denervated rat cremaster muscles underwent 3 hr of ischemia, followed by 90 min of reperfusion, and received systemic infusion of 100 nmol/min s-nitroso-n-acetylcysteine (SNAC) or an equal amount of phosphate-buffered saline (PBS). Results showed that the average diameter in 10 to 20 microm arterioles was between 107 percent and 123 percent of baseline in the SNAC group, and between 55 percent and 84 percent in the PBS group during 90 min of reperfusion. These values in 21 to 40 microm and 41 to 70 microm arteries were between 100 percent and 110 percent in the SNAC group, and between 70 percent and 90 percent in the PBS group from 20 to 90 min of reperfusion. Compared to the PBS group, the SNAC group had a statistically significantly greater vessel diameter in both 10 to 20 microm (p<0.001) and 21 to 40 microm arterioles (p<0.01) during 90 min of reperfusion, and in 41 to 70 microm arteries (p<0.02) from 20 to 90 min of reperfusion. The overall blood flow of the muscle in the SNAC group increased from 37 percent of baseline at 10 min to 108 percent at 40 min of reperfusion, and remained above baseline thereafter. In contrast, this value in the PBS group was only between 27 percent and 68 percent of baseline during 90 min of reperfusion. The blood flow was statistically significantly (p<0.03) greater in the SNAC group than in the PBS group from 40 to 90 min of reperfusion. Among the conclusions were: (1) NO donor SNAC improves the microcirculation of denervated skeletal muscle during early reperfusion; and (2) this protection against reperfusion injury is independent of innervation in skeletal muscle.     

缺血后处理对大鼠骨骼肌缺血再灌注损伤的影响

目的 探讨缺血后处理对大鼠骨骼肌缺血再灌注损伤的影响以及应用缺血后处理的时机.方法 将32只大鼠随机分成四组,采用切断患肢全部皮肤、肌肉和神经,保留患肢股动静脉的动物模型,通过夹闭和开放股动静脉造成骨骼肌缺血和再灌注损伤.采用测定骨骼肌缺血4 h.再灌注1 h后血清丙二醛(MDA)、骨骼肌髓过氧化物酶(MPO),再灌注6 h后骨骼肌的死亡程度来观察缺血后处理对大鼠骨骼肌缺血再灌注损伤的影响,以及再灌注5 min后应用缺血后处理是否对骨骼肌缺血再灌注损伤有保护作用.结果 对骨骼肌缺血4 h再灌注6 h的损伤,再灌注开始后即刻应用30 s缺血、30 s再通,三次循环的缺血后处理对骨骼肌的缺血再灌注损伤即有保护作用,不仅减少了骨骼肌再灌注区域中性粒细胞浸润(MPO)和血清氧自由基水平(MDA)水平,而且减少了骨骼肌的死亡程度;再灌注5 min后应用缺血后处理并没有降低骨骼肌缺血再灌注区域的MPO和血清MDA水平,也没有降低骨骼肌缺血再灌注后的死亡程度,与直接缺血再灌注组相同,对骨骼肌缺血再灌注损伤并没有保护作用.结论 骨骼肌缺血后再灌注开始前立刻应用缺血后处理对大鼠骨骼肌缺血再灌注损伤有一定的保护效果,可以减少骨骼肌缺血再灌注损伤后的死亡程度;缺血后处理应用时机非常重要,再灌注5 min后应用缺血后处理则失去对骨骼肌缺血再灌注损伤的保护作用.     

Postconditioning protects skeletal muscle from ischemia‐reperfusion injury

Ischemia‐reperfusion (I/R) injury caused by abrupt restoration of the circulation after prolonged ischemic insult induces significant morbidity after reconstructive microsurgery. The authors investigated whether a postconditioning (post‐con) procedure attenuated skeletal muscle I/R injury and protected muscular function. Three hours of complete ischemia was induced by occluding the muscular branches of rat extensor digitorum longus (EDL) muscle. The post‐con procedure was started at the end of ischemia and involved six cycles of 15 seconds of reperfusion followed by 15 seconds of re‐occlusion (3 minutes of total intervention) prior to initiating unlimited reperfusion. EDL muscle contractilities were compared with those of normal sides (no ischemic exposure), and experimental group results were also compared with control group results (3 hours of ischemia followed by full reperfusion without post‐con) at 3 hours and 5 days postreperfusion. Muscle wet weights, myeloperoxidase (MPO) activities, and histological results were also evaluated. The muscle contractilities in the post‐con group were significantly preserved at both 3 hours and 5 days postreperfusion as compared with ischemic controls. Decreased inflammatory cell infiltration, MPO activity, and wet weight of postconditioned EDL muscle suggested that post‐con attenuated acute inflammatory reactions induced by I/R. This study demonstrates that post‐con provides effective functional protection to skeletal muscles from I/R injury. © 2010 Wiley‐Liss, Inc. Microsurgery 2010.     

Inhibition of white blood cell adhesion at reperfusion decreases tissue damage in postischemic striated muscle

Purpose: To determine the impact of white blood cell (WBC)-endothelium adhesion on tissue damage in the setting of ischemia-reperfusion injury in striated muscle.Methods: The cremaster muscle of four groups of anesthetized Sprague-Dawley rats was subjected to 4 hours of global, warm (37° C) ischemia and 2 hours of reperfusion. At reperfusion two groups of animals received intravenous injections of monoclonal antibodies directed against either CD11b/CD18 (1B6) or ICAM-1 (1A29). The remaining two groups of animals received saline injections (NoR_x) or nonreactive IgG₁. In vivo light microscopic techniques were used to determine WBC adherence (number of WBCs per 100 μm postcapillary venules) at different intervals of reperfusion. Muscle viability was assessed with computer-assisted image analysis by measuring the optical intensity of transilluminated muscles after incubation with nitroblue tetrazolium.Results: Our results (mean + SEM) demonstrate a significant increase in the number of adherent WBCs relative to baseline (8.0 + 0.5) after 4 hours of global ischemia in animals receiving NoR_x or IgG₁. The significant increase occurred at 30 minutes of reperfusion (17.6 + 0.6 and 17.4 + 0.4 for NoR_x or IgG₁, respectively) and was sustained for the duration of the experiment. This increase in adherence was attenuated by 1B6 and 1A29 (12.2 + 2.2 and 12.4 + 0.8, respectively; p < 0.05 compared with NoR_x and IgG₁). The decrease in WBC adhesion was associated with a decrease in reperfusion injury to the muscle, as indicated by lower optical intensity values for the 1B6 and 1A29 groups (123 + 3 and 129 + 2) compared with the NoR_x and IgG₁ groups (151 + 2 and 158 + 4).Conclusions: Our data support an important role for WBCs in the pathogenesis of ischemia-reperfusion injury. Interfering with the WBC-endothelium interactions by using monoclonal antibodies directed against WBCs and endothelial cell adhesion molecules may help to limit ischemia-reperfusion injury. (J Vasc Surg 1996;24:187-93.)     

Resveratrol neuroprotective effects during focal cerebral ischemia injury via nitric oxide mechanism in rats

BACKGROUND: Our prior study showed that resveratrol could suppress infarct volume and exert neuroprotective effect on rats subjected to focal cerebral ischemia (FCI) injury. Recently, it has been reported in some literature that resveratrol protects the spinal cord, kidney, and heart from ischemia-reperfusion injury through upregulation of nitric oxide (NO). Therefore, this study was designed to investigate the role of nitric oxide on the neuroprotective mechanisms of resveratrol on rats after FCI injury. METHODS: The FCI injury was induced by the middle cerebral artery (MCA) occlusion for 1 hour and then a 24-hour reperfusion followed in the anesthetized Long-Evans rats. Resveratrol was intravenously injected after 1 hour MCA occlusion. RESULTS: Treatment of resveratrol (0.1 and 1 microg/kg) decreased the lactate dehydrogenase (LDH) in plasma and malondialdehyde (MDA) in FCI injury brain tissue, whereas the level of NO in plasma was increased. In addition, resveratrol downregulated protein and mRNA expression of inducible nitric oxide synthase (iNOS), and upregulated protein and mRNA expression of endothelial nitric oxide synthase (eNOS), while the expression of protein and mRNA of neuronal nitric oxide synthase (nNOS) was unchanged. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, the nonselective NOS inhibitor) or L-N(5)-(1-iminoethyl)-ornithine (L-NIO, the eNOS selective inhibitor) completely blocked the effect of resveratrol in decreasing infarction volumes. CONCLUSIONS: This study demonstrated the important role of NO in the neuroprotective effect of resveratrol in FCI injury.     

Modulation of the NO pathway during short or prolonged blood reperfusion following ischaemia in a heterotopic rat heart transplantation model

Ischemia-reperfusion injury plays a major role in graft dysfunction following transplantation. Extensive research has demonstrated that nitric oxide (NO) plays a fundamental role to protect the heart against this injury. Consequently, we quantified NO synthase (NOS) isoform protein levels in a rat heart transplant model during short and prolonged reperfusion following ischemia. Experiments were performed using a modified Lewis to Lewis heterotopic abdominal heart transplantation with a total ischemic time of 3 hours followed by 1 or 24 hours of blood reperfusion (n = 12). Heart function, as represented by the rate pressure product, increased from 7912 +/- 489 to 27067 +/- 9982 mm Hg/min (mean +/- SEM, short vs prolonged reperfusion, P = .0027). NOS isoform protein levels determined using Western blotting of freeze-clamped hearts were compared to baseline values. eNOS protein levels were significantly lower during short reperfusion compared to the basal value (P = .0077) or to prolonged reperfusion (P = .004), returning to the basal value after 24 hours of reflow. iNOS protein was not detected in the basal condition or after 1 hour of reflow, but was present after 24 hours of reflow (P = .0001 vs basal value and 1-hour reflow). nNOS protein was 69% lower after 1 hour of reflow compared with the baseline value (P = .0001), it was not restored after 24 hours of reflow (P = .002). These results suggest involvement of the NO pathway in ischemia-reperfusion injury with distinctive roles of NOS isoforms during short and prolonged reperfusion following ischemia.     

Viability of ischemia/reperfused muscles in rat: a new evaluation method by RNA degradation.

The rat's skeletal muscle viability was evaluated using the muscle viability index (MVI) which reflects the mRNA degradation. To evaluate ischemic injury of the muscle, 24 hind limbs of Fischer rats (three subgroups of eight rats each) were preserved at normothermia for 1, 3 and 6 h and then tibialis anterior muscle was harvested. To investigate ischemia/reperfusion injury, another 48 limbs were transplanted to recipient Fischer rats after the ischemia at normothermia for 1, 3 and 6 h, respectively. The transplanted muscles were harvested on day 3 and day 7 after transplantation. Eight fresh muscles were also harvested and used as control. Total RNA isolated from each muscle was fractionated by electrophoresis and hybridized with 32P-labelled cDNA of GAPDH, and the radioactivity of intact and degraded GAPDH mRNA was measured. MVI was calculated as follows, MVI = [X/(X + Y)] x 100, where X and Y represent the radioactivities corresponding to intact GAPDH and degraded GAPDH mRNA band, respectively. In 1-h ischemia group, the MVI indices of both ischemic insult and ischemia/reperfusion group were comparable to control. In the 3-h ischemia group, the index of ischemia/reperfused group was comparable to control although the index of ischemic insult group was significantly lower than control. However, in the 6-h ischemia group, both indices of ischemic insult and ischemia/reperfusion group were significantly lower than control. These results show that the muscle damage was detected in ischemia at normothermia even after 3 h. However, this damage was overcome by reperfusion. There was no recovery from damage in muscles that had been preserved for more than 6 h which had resulted in irreversible degeneration. Therefore, in clinical muscle transplantation, one has to transplant the muscle at least within 3-h ischemia.