Effects of Dantrolene on Extracellular Glutamate Concentration and Neuronal Death in the Rat Hippocampal CA1 Region Subjected to Transient Ischemia

Background: Excessive extracellular glutamate produced by cerebral ischemia has been proposed to initiate the cascade toward neuronal cell death. Changes in extracellular glutamate concentration are closely linked to changes in intracellular calcium ion concentration. Dantrolene inhibits calcium release from intracellular calcium stores. In this study, the authors investigated the effects of dantrolene on extracellular glutamate accumulation and neuronal degeneration in a rat model of transient global forebrain ischemia.

Methods: Male Wistar rats weighing 230-290 g were anesthetized with halothane in nitrous oxide-oxygen and were subjected to 10 min of transient forebrain ischemia using a four-vessel occlusion technique. Fifteen minutes before ischemic injury, dantrolene sodium (5 mm), dimethyl sulfoxide as a vehicle for dantrolene, or artificial cerebrospinal fluid as a control was intracerebroventricularly administered (n = 8 in each group). In the hippocampal CA1 subfield, the extracellular glutamate concentration in vivo was measured during the periischemic period with a microdialysis biosensor, and the number of intact neurons was evaluated on day 7 after reperfusion.

Results: Both dantrolene and dimethyl sulfoxide significantly reduced the ischemia-induced increase in glutamate concentration to a similar extent, i.e., by 53 and 51%, respectively, compared with artificial cerebrospinal fluid (P < 0.01). The number of intact hippocampal CA1 neurons (mean +/- SD; cells/mm) in dantrolene-treated rats (78 +/- 21) was significantly higher than that in artificial cerebrospinal fluid- (35 +/- 14;P < 0.001) and dimethyl sulfoxide-treated (56 +/- 11;P < 0.05) animals. Dimethyl sulfoxide also significantly increased the number of preserved neurons in comparison with artificial cerebrospinal fluid (P < 0.05).     

Protective Effect of Infliximab on Ischemia/Reperfusion-Induced Damage in Rat Kidney

Objective: To investigate the protective effect of infliximab on ischemia–reperfusion (I/R) injury of the rat kidney. Methods: Twenty-eight male Wistar albino rats were divided into four groups: sham-operated, I/R, I/R with infliximab administered before ischemia [I/R + infliximab (bi)], and I/R with infliximab administered before reperfusion [I/R + infliximab (br)]. After a right nephrectomy to produce damage, the left renal vessels were occluded for 60 min, followed by 24-h reperfusion in rats. Changes in the rat kidney were observed by measuring the tissue levels of malondialdehyde (MDA), myeloperoxidase (MPO), glutathione (GSH), and superoxide dismutase (SOD) and by evaluating hematoxylin–eosin (H&E)-stained and periodic acid–Schiff (PAS) sections. Results: The MDA and MPO levels in the I/R group were significantly higher than in the other groups (p < 0.05), and the SOD and GSH levels in the I/R + infliximab (bi) and I/R + infliximab (br) groups were significantly higher than in the I/R group (p < 0.05). However, histological examination revealed that the I/R + infliximab (bi) group and the I/R + infliximab (br) group had significantly fewer tubular changes and interstitial inflammatory cell infiltration than the I/R group. Conclusion: These results show that infliximab may protect against I/R injury in the rat I/R model.     

参麦注射液抗肝缺血再灌注损伤的研究

目的：研究参麦注射液对肝缺血再灌注损伤的保护作用。方法：将３７只成年雄性ＳＤ大鼠随机分成假手术对照组（ＳＯＣ）、缺血再灌注组（Ｉ／Ｒ）、缺血再灌注加参麦组（Ｉ／Ｒ＋ｓｈｅｎｍａｉ）。通过阻断大鼠肝门３０ｍｉｎ后再开放建立肝缺血再灌注损伤模型,在肝脏再灌注９０ｍｉｎ时测肝组织丙二醛（ＭＤＡ）、超氧化物歧化酶（ＳＯＤ）和测血ＡＬＴ、ＡＳＴ、ＬＤＨ,并取肝组织作光镜及电镜观察。结果：再灌注９０ｍｉｎ时Ｉ／Ｒ＋Ｓｈｅｎｍａｉ组的肝组织ＭＤＡ生成,ＳＯＤ消耗,血清ＡＬＴ、ＡＳＴ、ＬＤＨ升高值均少于Ｉ／Ｒ组（Ｐ＜０．０１）,且Ｉ／Ｒ＋Ｓｈｅｎｍａｉ组的肝细胞显微、超微结构损害的改变较Ｉ／Ｒ组轻。结论：参麦注射液能清除肝缺血再灌注过程中产生的氧自由基,对鼠肝缺血再灌注所致肝细胞的结构和功能损伤有保护作用     

Propofol with N-Acetylcysteine Reduces Global Myocardial Ischemic Reperfusion Injury More than Ketamine in a Rat Model

Objective: We examined the cardioprotective effects of propofol and ketamine with and without N-acetylcysteine (NAC). Methods: 60 rats were divided into six groups of 10 rats each. Anesthesia induction was produced with an intraperitonal injection of ketamine in Groups 1–3 and propofol in Groups 4–6. NAC (200 mg kg^{? 1}) was given intraperitonally during anesthesia induction in Groups 3 and 6. Groups 2, 3, 5, and 6 were subjected to 90 s of myocardial ischemia by clamping the ascending aorta, and then reperfusion was begun by unclamping the ascending aorta. After 60 min of reperfusion, blood samples were taken from the ascending aorta for biochemical analyses, and heart tissue samples were taken for biochemical and histopathological analyses. Results: Creatine kinase (CK), myocardial band of creatine kinase (CK-MB), and troponin-I (Tn-I) levels were significantly higher in the ischemia–reperfusion groups (2, 3, 5, 6) compared to the nonischemic groups (1, 4). CK, CK-MB, and Tn-I levels did not differ significantly between the ketamine groups (1–3) and the propofol groups (4–6) p >. 05). Malondialdehyde levels were significantly higher in Groups 2 and 3 than in Group 1 and were significantly lower in Groups 4 and 6 than in Group 5 (p <. 05). Malondialdehyde levels in the propofol groups (4–6) were significantly lower than in the ketamine groups (1–3; p <. 05). Catalase levels in propofol groups were higher than ketamine groups. Superoxide dismutase levels were significantly higher in Group 6 than in Group 3 (p <. 05). Conclusions: In this rat model of global cardiac ischemia, propofol with NAC attenuates myocardial injury more than ketamine (with or without NAC).     

Nebivolol Reduces Experimentally Induced Warm Renal Ischemia Reperfusion Injury in Rats

Ischemia/reperfusion injury, which is commonly seen in the field of renal surgery or transplantation, is a major cause of acute renal failure. The objective of the present study was to examine the role of nebivolol in modulating peroxynitrite species-induced inflammation and apoptosis after renal warm ischemia/reperfusion injury in rats. The present study was designed to investigate the effects of nebivolol on the renal warm ischemia/reperfusion injury in rats treated with the nitric oxide synthase inhibitor, N^ω-nitro-L-arginine methyl ester. After right nephrectomy, nebivolol was administered for 15 days. On the 16^th day, ischemia was induced in contra lateral kidney for 45 min, followed by reperfusion for 24 hr. Renal function, inflammation, and apoptosis were estimated at the end of 24 hr reperfusion. Nebivolol improved the renal dysfunction and reduced inflammation and apoptosis after renal ischemia/reperfusion injury. In conclusion, nebivolol shows potent anti-apoptotic and anti-inflammatory properties due to its NO-releasing property. These findings may have major implications in the treatment of human ischemic acute renal failure.     

超前缺血对培养乳鼠心肌细胞缺血再灌注损伤保护作用的实验研究

观察超前缺血（ｐｒｅｃｏｎｄｉｔｉｏｎｉｎｇｗｉｔｈｉｓｃｈｅｍｉａ，ＰＣＩ）对培养乳鼠心肌细胞缺血再灌注（ｉｓｃｈｅｍｉａ－ｒｅｐｅｒｆｕｓｉｏｎ，Ｉ－Ｒ）损伤的影响。发现（１）心肌细胞经ＰＣＩ后，其ＡＴＰ含量及培养液ＬＤＨ无明显变化；（２）经ＰＣＩ诱导的心肌细胞，持续３ｈ缺糖、缺氧，其ＡＴＰ消耗较对照组显著减少；（３）复灌期ＰＣＩ组ＡＴＰ含量增加，ＬＤＨ下降；（４）在各时相［Ｃａ＋＋］ｉ与对照组比较无明显变化，但ＬＰＯ于复糖、复氧后显著降低。结果提示：有限次数短暂ＰＣＩ对心肌细胞代谢及膜通透性无损伤作用；ＰＣＩ能提高细胞对长时间缺氧的耐受能力，减轻缺氧心肌Ｉ－Ｒ损伤。其机理可能是：心肌细胞作为一独立的基本功能单位，经ＰＣＩ诱导，细胞内抗氧自由基系统酶的活性增加，减轻了氧自由基的损伤效应。     

Ultrastructural Changes in the Contralateral Lung Tissue Following Unilateral Lung Ischemia: An Experimental Study in Rabbits

Purpose. To investigate the acute ultrastructural changes that may occur in the contralateral nonischemic lung tissue after unilateral ischemia of a lung in a rabbit model. Methods. The animals were divided into three main groups of eight; namely, a 2-h procedure group, a 4-h procedure group, and an 8-h procedure group. Each of these groups was further divided into two subgroups of four rabbits each; namely, a control group, given a sham operation without any ischemic insult, and an ischemia group, in which the main pulmonary arteries, the pulmonary veins, and the main bronchi of the left lungs were ligated after thoracotomy. Tissue samples were taken from the left and right lungs to examine the ultrastructural changes after 2, 4, and 8h of ischemia. Each sample was given a semiquantitative histological injury score. Statistical analysis was done by the Mann-Whitney U-test. Results. Contralateral ultrastructural damage, evident by heterochromatin in the nuclei, mitochondrial degeneration, cisternal widening of the endoplasmic reticulum, increased lipid droplets, and lysosomes, was determined by electron microscopy after unilateral lung ischemia. The contralateral lung injury was significantly correlated with the duration of ischemia. Conclusions. Unilateral lung ischemia affected the bilateral lungs in a rabbit model. Therefore, in operations such as single-lung transplantation, pulmonectomy, or lobectomy, if the procedure is unnecessarily prolonged, the contralateral lung may be damaged, which could seriously affect the prognosis of the patient.     

Remote Ischaemic Pre-Conditioning Reduces Intestinal Ischaemia Reperfusion Injury in a Newborn Rat

ObjectiveRemote ischaemic conditioning (RIC) has been shown to reduce ischaemia-reperfusion injury(IRI) in multiple organ systems. IRI is seen in multiple bowel pathologies in the newborn, including NEC. We investigated the potential of RIC as a novel therapy for various intestinal pathologies in the newborn.MethodsWe used an established intestinal IRI model in rat pups which results in similar intestinal injury to necrotising enterocolitis (NEC). Animals were randomly allocated to IRI only(n = 14), IRI + RIC(n = 13) or sham laparotomy(n = 10). The macroscopic extent of intestinal injury is reported as a percentage of total small bowel. Injury severity was measured using Chiu-Park scoring. Neutrophil infiltration/activation was assayed by myeloperoxidase activity. Immunohistochemistry was used to assess the expression of hypoxia-inducible factor alpha (HIF-1α). Data are median (interquartile range).ResultsAnimals that underwent RIC showed a decreased extent of macroscopic injury from 100%(85–100%) in the IRI only group to 58%(15–84%, p = 0.003) in the IRI + RIC group. Microscopic injury score was significantly lower in animals that underwent RIC compared to IRI alone (3.5[1.25–5] vs 5.5[4–6], p = 0.014). Intestinal myeloperoxidase activity in animals exposed to IRI was 3.4 mU/mg of tissue (2.5–3.7) and 2.1 mU/mg(1.5–2.8) in the IRI + RIC group(p = 0.047). HIF-1α expression showed a non-significant trend towards reduced expression in the IRI + RIC group.ConclusionsRIC reduces the extent and severity of bowel injury in this animal model, supporting the hypothesis that RIC has therapeutic potential for intestinal diseases in the newborn.     

Highly Reproducible Rat Model of Reversible Forebrain Ischemia – Modified Four-Vessel Occlusion Model and its Metabolic Feature

Summary.  Background: The four-vessel occlusion method introduced by Pulsinelli et al. is widely used as an experimental model for reversible forebrain ischemia in rats.  Method: In this study, we further developed highly reproducible model of reversible forebrain ischemia. Under the microscope the visible vertebral arteries at the second vertebra could be easily electrocauterized and completely cut to yield complete cessation of circulation of both vertebral arteries. After 24 hours, male Wistar rats were subjected to 15, 30 and 45 minutes of forebrain ischemia by occluding both common carotid arteries with Sugita's temporary clips. ³¹P-magnetic resonance spectra (³¹P-MRS) and ¹H-magnetic resonance images (¹H-MRI) were obtained with a 6.3-T spectrometer to investigate sequential change of the in vivo brain metabolism. Electroencephalogram and the cortical blood flow by laser Doppler flowmetry were measured during ischemia and recirculation. Determination of endogenous superoxide scavenging activity in the brain cortex was performed by electron spin resonance spectrometry.  Findings: Brain water contents evaluated by the dry-wet weight method were increased at 1 hour and 48 hours after recirculation, which were demonstrated by ¹H-MRI. The superoxide scavenging activity showed a significant decrease at 45 minutes of recirculation and a significant increase at 12 hours of recirculation. The present modified model demonstrated that the mortality rates by 72 hours were 8.3% (15 minutes ischemia), 15.0% (30 minutes ischemia), and 42.9% (45 minutes ischemia), all of which were higher than that of the original method described by Pulsinelli et al.  Interpretation: In conclusion, this modified four-vessel occlusion method gives a high level of success in producing reversible forebrain ischemia. Published online December 5, 2002  Correspondence: Shigeki Toda, M.D., D.M.Sc, Department of Neurosurgery, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, TOKYO. 113-8603. Japan.     

Nitrite Reduces Acute Lung Injury and Improves Survival in a Rat Lung Transplantation Model

Ischemia/reperfusion injury (IRI) is the most common cause of early mortality following lung transplantation (LTx). We hypothesized that nitrite, an endogenous source of nitric oxide (NO), may protect lung grafts from IRI. Rat lung grafts were stored in preservation solution at 4°C for 6 hours. Both grafts and recipients were treated with nitrite. Nitrite treatment was associated with significantly higher levels of tissue oxygenation, lower levels of cytokines and neutrophil/macrophage infiltration, lower myeloperoxidase activity, reduced oxidative injury and increased cGMP levels in grafts than in the controls. Treatment with either a nitric oxide scavenger or a soluble guanylyl cyclase (sGC) inhibitor diminished the beneficial effects of nitrite and decreased cGMP concentrations. These results suggest that nitric oxide, generated from nitrite, is the molecule responsible for the effects of nitrite via the nitric oxide/sGC/cGMP pathway. Allopurinol, a xanthine oxidoreductase (XOR) inhibitor, abrogated the protective effects of nitrite, suggesting that XOR is a key enzyme in the conversion of nitrite to nitric oxide. In vitro experiments demonstrated that nitrite prevented apoptosis in pulmonary endothelial cells. Nitrite also exhibits longer survival rate in recipients than control. In conclusion, nitrite inhibits lung IRI following cold preservation and had higher survival rate in LTx model.     

Early Anastomotic Repair in the Rat Intestine is Affected by Transient Preoperative Mesenteric Ischemia

Introduction  During bowel surgery, perioperative blood loss and hypotension can lead to transient intestinal ischemia. Recent preclinical studies reveal that the strength of intestinal anastomoses can be compromised after reperfusion. So far, this phenomenon has not been investigated in the very first days of healing when wound strength is lowest. Material and Method  Ischemia was induced in rats by clamping both the superior mesenteric artery and ileal branches for 30 min. Immediately after declamping, anastomoses were constructed in both terminal ileum and descending colon. The same was done in control groups after sham-ischemia. Anastomotic bursting pressure and breaking strength were measured immediately after operation (day 0) and after 1, 2, or 3 days. Anastomotic hydroxyproline content, gelatinase activity, and histology were analyzed. Results and Discussion  In ileal anastomoses, at day 1, both the breaking strength and bursting pressure were significantly (p < 0.05) lower in the ischemic group, while at day 2, this was the case for the bursting pressure only. In the colon, the bursting pressure in the ischemic group was lower at day 1. Anastomotic hydroxyproline content remained unchanged. Increased presence of the various gelatinase activities was found in ileum only at day 0 and in colon at days 1 and 2. Histological mucosal damage was found in ischemia–reperfusion groups. Conclusion  Transient mesenteric ischemia can negatively affect anastomotic strength during the very first days of healing, even if the tissue used for anastomotic construction looks vital.     

茶多酚对大鼠肾缺血再灌注损伤的保护作用

目的：探讨茶多酚（teapolyphenols,TP）预处理对大鼠肾缺血再灌注损伤（ischemia reperfusion injury,IRI）的保护作用及其机制。方法：雄性SD大鼠42只,随机分为假手术组、IRI组和TP预处理组。IRI组及TP组手术建立肾IRI模型,TP预处理组在此基础上加用TP治疗。IRI组及TP预处理组在缺血1h恢复灌注开始时刻、2h后、24h后分别检测血清肌酐（Scr）、血清超氧化物岐化酶（SOD）、血清丙二醛（MDA）、肾组织SOD和肾组织MDA水平,并观察肾组织的病理变化。结果：TP预处理组肾组织病理变化轻于IRI组;与假手术组相比较,IRI组的Scr水平升高（P〈0.05）,肾组织及血清中的SOD降低及MDA水平增加（P〈0.05）。而TP预处理组的Scr、MDA和肾组织的MDA均比IRI组低（P〈0.05）,而血清SOD水平及肾组织中的SOD水平升高（P〈0.05）。结论：TP对肾脏缺血再灌注损伤具有保护作用,其机制可能是通过抗氧化作用实现的。     

Leukocyte-Endothelium-Interaction in Pial Vessels Following Global, Cerebral Ischaemia

Summary ? Background. Investigations have shown an increase of leukocyte-endothelium-interaction in a variety of organs following an ischaemic insult. To elucidate the role of leukocyte-endothelium-interaction following global, cerebral ischaemia the present study was performed. Methods. Global, cerebral ischaemia was induced for twenty minutes by four-vessel-occlusion (PULSINELLI). Leukocyte-endothelium-interaction was studied in the cerebral microcirculation using a rat closed cranial window and intravital microscopy. Leukocytes were stained intravenously using rhodamine 6G. Diameters of pial vessels, leukocyte centreline velocity and number of rolling or adhering leukocytes were determined off-line up to 2 h following global cerebral ischaemia. To confirm these results immunohistochemistry of the brain was performed. Findings. Four-vessel-occlusion induced an iso-electric EEG, venular stasis and minimal rest flow in arterioles. Reperfusion yielded a significant increase of the arteriolar (p<0.001) and a smaller increase of the venular diameters (p<0.01). Up to 2 h after ischaemia no significant increase of the number of rolling or adhering leukocytes was measured which was confirmed by immunohistochemistry. Interpretation. In contrast to other studies, in particular regarding focal cerebral ischaemia, an increase of leukocyte-endothelium-interaction in rat brain following 20 min of global cerebral ischaemia was not observed despite histological evidence of ischaemic damage. Thus in our model leukocytes seem not to contribute to the brain damage following global ischaemia.     

骨髓间充质干细胞治疗慢性后肢缺血机理的初步研究

目的利用细胞示踪技术探讨骨髓间充质干细胞（MSCs）治疗慢性后肢缺血的相关机理。方法采用密度梯度离心法结合直接贴壁法分离和培养大鼠MSCs,并以5-溴脱氧尿嘧啶核苷（BrdU）标记。采用线栓法制备8只Lewis大鼠慢性后肢缺血模型,将其随机均分为MSCs移植组和对照组,分别于患侧后肢肌肉注射MSCs和生理盐水。分别于移植术后第7天和第14天,对其进行临床观察、后肢血流量测定及后肢血管造影,再于相应时间点处死大鼠,取患侧后肢股四头肌和腓肠肌,行HE染色及BrdU免疫组化染色。结果移植术后14 d,8只大鼠全部成活,移植部位均无坏死和肿瘤形成;MSCs移植组大鼠患侧/健侧后肢的血流灌注比值明显增高（1.773比1.279）,而血管造影结果提示2组大鼠的侧支血管数量比值未见显著增加（0.908比0.835）。HE染色结果示2组大鼠的股四头肌及腓肠肌并未发生特殊的病理学变化。BrdU免疫组化结果显示,阳性颗粒定位为股四头肌及腓肠肌的间质细胞和血管内皮细胞;且MSCs的分布存在差异,移植术后7 d腓肠肌内阳性细胞所占比例明显高于股四头肌,而14 d时则相反。结论 MSCs移植在术后早期可以提高血流灌注量,但这并非为增加了侧支血管数量使然,MSCs移植后所引起的旁分泌效应可能在术后早期起着重要的作用。