Late Reperfusion Period Research Articles

Histological changes of the arterial bed of the hind limbs of the rats under condition of the acute ischemia-reperfusion and correction with the carbacetam

The ischemic-reperfusion lesion is a complex multifactorial damage of the primary ischemic tissues as a result of restoration of the arterial blood circulation in them, which is accompanied by local morpho-functional reorganization of the vascular bed of the hind limbs of the rats. One of the promising means in the treatment and prevention of the reperfusion disorders is a carbacetam, which smooths the phenomena of hypo- and hyperperfusion in the post-ischemic period. The aim of the study was to established the manifestations of the morpho-functional remodeling of the vascular bed of the hind limbs of the rats in ischemia-reperfusion and under conditions of correction with carbacetam. Histological examination of the vascular bed of the hind limbs of 30 rats under conditions of ischemia-reperfusion (group I) and 30 rats in the simulation of ischemia-reperfusion, which in the post-ischemic period administered carbacetam once a day (5 mg/kg) for 14 days (group II) were done. There were 6 intact animals in the control group. Simulation of ischemia was performed by applying SWAT rubber tourniquets on the hind limbs for 2 hours, and reperfusion – by removing of the tourniquet. The animals of the experimental groups were divided into 5 subgroups with reperfusion terms after 1, 2 hours and 1 day, as well as after 7 and 14 days. Histological examination was performed according to generally accepted methods. The vascular bed in the middle third of the thigh and the shin below the tourniquet was examined using a Bresser Trino Researcher 40x–1000x microscope. Analyzing of the obtained results, was established that after 1 hour of the reperfusion the histological changes became a systemic, and after 1 day it were more significant. It should be noted that the thickness of the vessel walls increased, and the elastic membranes were partially eligned, thinned and torned. The stepwise clarity of the arterials walls structure was lost. The edema acquired a total nature. The histological examination of the vessels after 7 days revealed that the swelling of the walls decreased and the condition of the elastic frame was improved. There was a proliferation of collagen fibers in the adventitia, which was a response to ischemic effects. It is noted that after 14 days in all wall membranes the proliferative activity of fiboblasts was remained. Under the conditions of the correction with the carbacetam after 2 hours, the structural positive dynamics became more pronounced and increased to a maximum level after 7 days of the experiment. The number of the modified and exfoliated endothelial cells decreased, and the condition of smooth myocytes increased. Histologically, the gradual restoration of endothelial coverage of the intima was established. As follows, ischemia and reperfusion cause vascular remodeling after 1 hour with a peak of the manifestations after 1 day of the reperfusion, which includes edematous syndrome, dystrophic-degenerative changes with an inflammatory response to the damage, and in the late reperfusion period increased a fibroblasts activity. Gradual return of morphological changes occurs after 14 days of the experiment. Under the conditions of correction, the acceleration of the remodeling with stabilization of the process and the most possible structural restoration after 7 days of the study was noted.

Comparative analysis of histological and morphometric changes of the arterial bed of the hind limbs of the rats in acute ischemia-reperfusion and correction with carbacetam

Resume. Ischemic-repefusion injury is a complex multifactorial tissue damage as the result of restoration of blood circulation, after the period of ischemia or lack of the oxygen, accompanied by the local and systemic disorders. Recently, more and more attention in treatment and prevention of postischemic disorders is paid to nootropic medicine, which smooths the phenomena of hypo- and hyperperfusion, and also improves tissue microcirculation.The aim of the study was to establish the manifestations of morpho-functional remodeling of the vascular bed of the hind limbs of the rats during ischemia-reperfusion and under conditions of carbacetam correction using histological and morphometric іnvestigations methods.Materials and methods. There was histological and morphometric examination of the arterial bed of the hind limbs of 30 rats under conditions of ischemia-reperfusion injury (group I) and 30 rats in the simulation of ischemia-reperfusion injury, which in the post-ischemic period was administered carbacetam once a day (5 mg / kg) for 14 days (group II) was performed. There were 6 intact animals in the control group.Simulation of ischemia was performed by applying SWAT rubber tourniquets on the hind limbs for 2 hours, and reperfusion - by removing the tourniquet. The animals were divided into 5 subgroups with reperfusion terms after 1, 2 hours and 1 day, as well as after 7 and 14 days. Histological examination was performed according to generally accepted methods. The vascular bed in the middle third of the thigh and shin, below the level of the application of the tourniquet was examined by Bresser Trino Researcher 40x – 1000x microscope. The morphometric parameters were calculated using SEO Image Lab software from Sumy Electron Optics.Research results and their discussion. Analyzing of the obtained results, it was founded that after 1 hour of the repefusion the structural changes became systemic, and after 1 day they were the most significant. Histologically, the elastic membranes were thinned and torned up, the swelling became a total character. In the late reperfusion period, a gradual return of morphological changes to the initial state and the increased in the proliferative activity of the fibroblasts were revealed. Under the conditions of correction after 1 day, the positive dynamics became more pronounced and reached its maximum after 7 days of the study. Morphometrically, the differences between the groups are especially pronounced 7 days after the removal of the tourniquet. At the level of the femoral artery revealed a lower value of external (D1) by 1.9% (p<0,005) and internal (D2) diameters by 3,0% (p<0,005) and an increased in Vi by 3,54% (p<0,005)) in the specified experimental period in the animals with the correction, compared with the group without it.In small-caliber arteries, on the contrary, there was a less pronounced vasoconstriction level, when applying the correction, because the value of T decreased by 5,4% (p<0,005) and Vi by 20,28% (p<0,005), but there were higher indicator of D1 by 1,89 % (p<0,005) and D2 by 11.55% (p<0,005) compared to the group of animals without the correction.Conclusions. Ischemia and repfusion cause vascular remodeling after 1 hour with a peak of manifestations after 1 day of reperfusion. Under the conditions of the correction, the acceleration of the remodeling with stabilization of the process and the most possible structural restoration after 7 days of the study was noted. Morphometric data indicate less pronounced vasodilation at the level of large caliber arteries and spasm of arteries of muscular branches during that experimental period in the group with correction.

Effect of Ischemic Postconditioining on Change of Immunoreactivity Level to PECAM-1/CD31 in Rat Neocortex Structures after Global Brain Ischemia

Introduction. Ischemic postconditioning (IPostC) of the brain can be considered as a promising approach to limit reperfusion injury in the ischemic area of the brain. Objective – to study the effect of IPostC after global cerebral ischemia on the level of immunoreactivity to PECAM-1/CD31 in the structures of layers II, III and V of the neocortex of rats at different periods of the reperfusion period.Material and methods. In male Wistar rats, a 10-minute global cerebral ischemia was modeled followed by IPostC in the form of reperfusion-ischemia at 15sec/15sec. In the early (2 days) and late (7 days) reperfusion periods after damaging ischemia, the number of morphologically unchanged neurons and the level of immunoreactivity to PECAM-1/CD31 in the structures of layers II, III and V of the neocortex were estimated.Results. It is shown that the use of IPostC by 2 days of reperfusion contributed to the increase in the number of unchanged neurons in layers II and III of 25.8 and 28.2 % (P<0.05), which was not accompanied by changes in the level of immunoreactivity to PECAM-1/CD31, to 7 days of reperfusion there was an increase in the number of unchanged neurons in layers II, III and V of 19.2, 22,1, 21,4 % (P<0.05) was observed a decrease in the level of immunoreactivity to PECAM-1/CD31 in the structures of these layers of 27.4, 39.4, and 16.7 % (P<0.05), respectively, when compared with similar indicators in groups without the use of IPostC.Conlusions. In the mechanisms of physiological reaction formed in the application of ischemic postconditioning after cerebral ischemia and leading to the preservation of the number of unchanged neurons in the late reperfusion period involved PECAM-1/CD31, which suggests that the protective potential of the phenomenon is realized by possible inhibiting the migration of neutrophils, monocytes and lymphocytes and extravasation of leukocytes from the systemic blood flow into the damaged area of the brain, i.e. through suppression of inflammatory response.

The effect of Betanin parenteral pretreatment on Jejunal and pulmonary tissue histological architecture and inflammatory response after Jejunal ischemia-reperfusion injury

Intestinal ischemic-reperfusion (IR) injury has detrimental effects on both local and distant organs in the body. Betanin is known for its antioxidant properties, and it is found mostly in vegetables. Therefore, the aim of the present study was to test the hypothesis that betanin administration prior intestinal IR, may be beneficial in protecting jejunal mucosa and lung parenchyma against IR damage. Male specific pathogen-free Charles River Wistar rats were used (n = 42). Betanin (50 mg/kg) was administered intraperitoneally 30 min before ischemia of the superior mesenteric artery lasting 1 h, followed by 1, 4 and 24 h of reperfusion. Immunohistochemical as well as histomorphometrical analysis indicated a protective effect of betanin pretreatment on jejunal tissue. Regarding morphometrical analysis betanin significantly (p < 0.01) augments intestinal villus height after 24 of reperfusion comparing to early stages. Betanin application reduced number of mast cells population in early reperfusion periods (p < 0.05). The protective effect of betanin on lung parenchyma, was detected in late reperfusion period (24 h) with improvement of histopathological injury index and morphometric analysis (p < 0.001 for both). The improvement of histopathological injury index (p < 0.001) and morphometric analysis (p < 0.001) during the late reperfusion period, suggests a protective effect of betanin on lung parenchyma. Moreover, suppression of the inflammatory response was mirrored by the reduction of myeloperoxidase (MPO) positive cells within lung parenchyma after 1 and 4 h of reperfusion (p < 0.001). Especially, during the first 4 h of reperfusion after betanin administration, a reduction of 74% of the polymorphonuclear neutrophils infiltration (MPO positive cell population) and of a nearly 46% of active MCs was observed. Upon morphometric examination, the lung histological architecture after 24 h of reperfusion appeared to be almost 100% better following betanin treatment, with 25% thinner interalveolar septa and 20% larger alveolar surface for respiratory gas exchange. The results suggest that betanin pretreatment protects the jejunal mucosa and the lung parenchyma, as well as reduces the inflammatory cell density after intestinal IR injury.

Morphofunctional Characteristics of the Microcirculatory Bed and Neurons in the Neocortex after Ischemic Postconditioning

Expression of PECAM-1/CD31 protein (a biomarker for endothelial function and neovascularization processes) was studied in microvessels in layers II, III, and V of the cerebral cortex in Mongolian gerbils (Meriones unguiculatus) in the early (day 2) and late (day 7) parts of the reperfusion period after 7-min forebrain ischemia, ischemic postconditioning (iPostC), and sham-operated animals (n = 60). In the latter, the lowest level of PECAM-1/CD31 immunoreactivity was seen in structures in layer III of the cerebral cortex. Reversible ischemic brain injury decreased the number of morphologically unaltered neurons in the neocortex with increases in the duration of the reperfusion period; cortical layers II, III, and V also showed increased levels of PECAM-1/CD31 immunoreactivity with significant increases in the late reperfusion period. iPostC consisting of three stimulatory episodes of reperfusion-ischemia (15/15 sec) led to significant increases in the number of morphologically unaltered neurons and PECAM-1/CD31 immunoreactivity in layers II and III in the early reperfusion period. In the later reperfusion period following iPostC, the number of unaltered neurons in layers II, III, and V of the cortex increased, while the level of PECAM-1/CD31 immunoreactivity decreased significantly. The results lead to the conclusion that the cytoprotective effect of iPostC in forebrain ischemia is mediated by a physiological adaptive mechanism leading to increases in PECAM-1/CD31 immunoreactivity in cortical microvessels in the early reperfusion period and a decrease in the later reperfusion period.

Changes in the Expression of Bcl-2 Protein in Neurons in the Hippocampal Fields after Use of Ischemic Postconditioning of the Brain

The expression of Bcl-2 protein in pyramidal neurons in hippocampal fields CA1, CA2, CA3, and CA4 was studied in Mongolian gerbils (Meriones unguiculatus) in the early (two days) and late (seven days) reperfusion periods after 7-min ischemia of the forebrain, after use of ischemic postconditioning (IPostC) and in sham-operated animals (n = 60). The highest level of Bcl-2 expression in the latter group was seen in neurons in field CA4 and the lowest in neurons in field CA1 (p < 0.01). Reversible ischemic injury led to an increase in the deficit of morphologically unaltered neurons in the hippocampus at the later period of reperfusion and a significant decrease in neuronal Bcl-2 expression at the early reperfusion period, this decrease being significantly smaller in the late reperfusion period. IPostC consisting of three episodes of reperfusion-ischemia (15/15 sec) promoted a significant increase in the number of morphologically unaltered neurons in fields CA1 and CA3 in the early reperfusion period. An increase in the level of Bcl-2 expression was seen in the cytoplasm of morphologically unaltered neurons in all hippocampal fields. In the late reperfusion period after IPostC, the number of unaltered neurons was increased in fields CA1, CA3, and CA4 (p < 0.05); only hippocampal field CA1 neurons showed a significant increase in Bcl-2 expression (by 12.7%, p < 0.01). These results lead to the conclusion that the cytoporotective effect of IPostC for hippocampal field CA1 is mediated by a mechanism leading to an increase in Bcl-2 expression, i.e., via blockade of apoptosis.

The role of AMPA-receptors in mechanisms of neuroprotective effect of cerebral ischemic postconditioning

Objective. The purpose of the study is to explore the role of AMPA receptors in the neuroprotective effect of ischemic postconditioning (IРost) in the model of reversible global forebrain ischemia in Mongolian gerbils. Design and methods. Reversible global cerebral ischemia was induced by bilateral occlusion of common carotid arteries in Mongolian gerbils for 7 minutes. IРost was induced by three 15-s episodes of reperfusion/reocclusion. Antagonist of AMPA receptors NBQX was administered intraperitoneally at 2 nd minute of ischemia at a dose of 30 mg/kg. In the early and delayed reperfusion period, the number of viable neurons in the CA1, CA2, CA, and CA4 fields of the hippocampus was estimated. Results. Reversible 7 minutes ischemia followed by 48 hours of reperfusion resulted in a significant reduction in the number of viable neurons in the fields CA1 and СА3 of the hippocampus, while in the late reperfusion period a significant reduction in the number of viable neurons was observed in fields CA1, СА3 and CA4. Application of IРost has led to an increase in the number of viable neurons in fields CA1 and CA3 in the early reperfusion period and in fields CA1, CA3 and CA4 — in delayed period. AMPA receptor antagonist NBQX attenuated neuroprotective effect of IPost to a level comparable with the effect of a separate application of NBQX for neurons in the CA1 and CA3 fields of the hippocampus. Conclusions . Along with other mechanisms, the activation of AMPA receptors might be implicated in the mechanisms of cerebral IPost.

Influence of cerebral global ischemia-reperfusion on succinate dehydrogenase activity in neurons of different neocortical layers

The aim of the study was to investigate changes in activity of succinate dehydrogenase (SDH) in cytoplasm of neurons of different cortical layers in early and late reperfusion period after global cerebral ischemia in rats. Reversible global cerebral ischemia was modeled by occlusion of the brachiocephalic trunk, left subclavian artery and left common carotid artery for 10 minutes and following reperfusion during 2 or 7 days. The SDH activity in cytoplasm of neurons of II, III and V cortical layers was determined histoenzymatically. It is shown that the SDH activity in neurons of the studied cortical layers was characterized by the increased reperfusion period to the 2 days with a subsequent increased activity of the reperfusion period to the 7 days. The change in the SDH activity in cytoplasm of cortical neurons depends on the particular cerebral layer and duration of postischemic reperfusion.

Knockdown of glucose-6-phosphate dehydrogenase (G6PD) following cerebral ischemic reperfusion: The pros and cons

NADPH derived from glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, has been implicated not only to promote reduced glutathione (GSH) but also enhance oxidative stress in specific cellular conditions. In this study, the effects of G6PD antisense oligodeoxynucleotides (AS-ODNs) was examined on the CA1 pyramidal neurons following transient cerebral ischemia. Specifically knockdown of G6PD protein expression in hippocampus CA1 subregion at early reperfusion period (1–24h) with a strategy to pre-treated G6PD AS-ODNs significantly reduced G6PD activity and NADPH level, an effect correlated with attenuation of NADPH oxidase activation and superoxide anion production. Concomitantly, pre-treatment of G6PD AS-ODNs markedly reduced oxidative DNA damage and the delayed neuronal cell death in rat hippocampal CA1 region induced by global cerebral ischemia. By contrast, knockdown of G6PD protein at late reperfusion period (48–96h) increased oxidative DNA damage and exacerbated the ischemia-induced neuronal cell death in hippocampal CA1 region, an effect associated with reduced NADPH level and GSH/GSSG ratio. These findings indicate that G6PD not only plays a role in oxidative neuronal damage but also a neuroprotective role during different ischemic reperfusion period. Therefore, G6PD mediated oxidative response and redox regulation in the hippocampal CA1 act as the two sides of the same coin and may represent two potential applications of G6PD during different stage of cerebral ischemic reperfusion.

Effect of vitamin E on reperfusion injuries during reconstructive vascular operations on lower limbs

The challenge against reperfusion injury and tissue oxidative stress, especially in vascular surgical interventions has an essential importance to reach the optimal clinical result. Numerous experimental attempts have proved the positive antioxidant effect of vitamin E in both chronic and acute phase models. In our study we monitored the effect of continuous preoperative treatment with vitamin E, on oxidative stress and tissue inflammation reactions developed after reconstructive operations. 32 patients have been involved in a randomized, prospective study, all suffering from AFS occlusion proved by angiography, and all undergone supragenual reconstruction. Duration of ischemia and amount of tissues under vascular clamping were almost the same in all patients. In the group treated with E-vitamin, we administered 1 x 200 mg of vitamin E p/o from the preoperative day till the 7th post operative day. Patients of the second group did not receive vitamin E. Peripheral blood samples were collected immediately before operation and at the end of the second reperfusion hour (early reperfusion period). Late reperfusion period has been monitored by analyzing blood samples taken at 24th hour and 7th day next to the operative ischemia. Among oxidative stress parameters, direct measurement of reactive oxygen intermediator (ROI) and determination of antioxidant state (GSH, Total-SH group, SOD) have been performed. Malondialdehyde was chosen as marker for lipidperoxidation. Inflammation reactions were monitored up on expression of adhesion molecules (CD11a and CD18). We also controlled the oscillation of myeloperoxidase (MPO) activity. Our study has proved that preoperative (from the preoperative day till the 7th post operative day) administration of 200 mg vitamin E could reduce the level of oxidative stress developed after ischemic-reperfusion insult (lipidproxidation, antioxidant enzymes). According to our results, the prooxidant-antioxidant imbalance also diminished in the group with E-vitamin treatment. We proved that elective administration of vitamin E could decrease the WBC activity (MPO activity, free radicals production, expression of adhesion molecules) and its consequential local inflammation process, during early reperfusion.

In Vivo Fluorescence Microscopic Imaging for Dynamic Quantitative Assessment of Intestinal Mucosa Permeability in Mice

Herein, we introduce a novel intravital microscopic method to quantitatively assess mucosal permeability in mouse intestine. To this end, the time course of changes in the accumulation of fluorescent markers (sodium fluorescein [MW 376] and fluorescent dextran [FD4000, MW 4000; Merck, Darmstadt, Germany]) was examined in the perivascular interstitium of the mesentery of sham-operated animals and animals undergoing 30 min intestinal ischemia and 90 min postischemic reperfusion. An increased permeation of intraluminally installed sodium fluorescein was found only in the late reperfusion period, while permeability was enhanced during the entire postischemic examination period when renal excretion of the fluorescent marker was excluded by kidney ligation. Similarly, the late reperfusion phase was associated with enhanced tissue fluorescence to FD 4000 when the kidneys were ligated. Fluorescence values in the plasma, as measured by standard fluorimetry, showed a significant correlation with tissue fluorescence determined by intravital microscopy. Further, villus tip denudation at 90 min reperfusion correlated with increased permeability of sodium fluorescein in the early (10-20 min) and FD 4000 in the late reperfusion phase (90 min). Thus, intravital microscopic measurement of mesenteric perivascular fluorescence provides a simple and easily applicable on-line method to quantitatively assess epithelial permeability, allowing to detect altered mucosal barrier function and to evaluate salvage therapies which target mucosal integrity.

Effects of emulsified isoflurane induced preconditioning on cardiac hemodynamics duing myocardiac ischemin-reperfusion injury in rabbits

近年来研究表明吸入麻醉药异氟醚可模拟缺血预处理发挥心肌保护作用,减轻再灌注期心肌功能抑制[1,2].以脂肪乳为载体静脉注射的乳化异氟醚可产生麻醉作用[3],但是否也具有心肌保护作用,尚无报道.为了进一步研究乳化异氟醚预处理对心肌缺血的保护效应,本实验在兔心肌缺血再灌注模型上测定不同时点血流动力学的有关指标,旨在探讨乳化异氟醚预处理对兔缺血再灌注心肌的血流动力学影响。

Effect of Ketamine on Apoptosis by Energy Deprivation in Astroglioma Cells using Flow Cytometry System

Apoptosis is a programmed, physiologic mode of cell death that plays an important role in tissue homeostasis. As for the central nervous system, ischemic insults can induce pathophysiologic cascade of apoptosis in neurophils. Impairment of astroctye functions during brain ischemia can critically influence neuron survival by neuronglia interactions. We aimed to elucidate the protective effect of ketamine on apoptosis by energy deprivation in astrocytes. Ischemic insults was induced with iodoacetate/carbonylcyanide m-chlorophenylhydrazone (IAA/CCCP) 1.5 mM/20 µM or 150 µM/2 µM for 1 hr in the HTB-15 and CRL-1690 astrocytoma cells. Then these cells were reperfused with normal media or ketamine (0.1 mM) containing media for 1 hr or 24 hr. FITC-annexin-V staining and propidium iodide binding were determined by using flow cytometry. Cell size and granularity were measured by forward and side light scattering properties of flow cytometry system, respectively. An addition of ketamine during reperfusion increased the proportion of viable cells. Ketamine alleviated cell shrinkage and increased granularity during the early period, and ameliorated cell swelling during the late reperfusion period. Ketamine may have a valuable effect on amelioration of early and late apoptosis in the astrocytoma cells, even though the exact mechanism remains to be verified.

Late reversal of cerebral perfusion and water diffusion after transient focal ischemia in rats.

Region-specific cerebral blood flow (CBF) and the apparent diffusion coefficient (ADC) of tissue water in the rat brain were quantified by high-field magnetic resonance imaging at 9.4 T in the rat suture occlusion model. Cerebral blood flow and ADC were compared during the short- (4.5 hours) and long-term (up to 6 days) reperfusion after 80 minutes of transient middle cerebral artery occlusion, and correlated with the histology analysis. On occlusion, average CBF fell from approximately 100 to less than 50 mL x 100 g(-1) x min(-1) in the cortex, and to less than 20 mL x 100 g(-1) x min(-1) in the caudate putamen (CP). Corresponding ADC values decreased from (6.98 +/- 0.82) x 10(-4) to (5.49 +/- 0.54) x 10(-4) mm2/s in the cortex, and from (7.16 +/- 0.58) x 10(-4) to (4.86 +/- 0.62) x 10(-4) mm2/s in the CP. On average, CBF recovered to approximately 50% of baseline in the first 24 hours of reperfusion. After 2 to 4 days, a strong hyperperfusion in the ipsilateral cortex and CP, up to approximately 300 mL x 100 g(-1) x min(-1), was observed. The ADC ratio in the ipsilateral and contralateral CP was also inverted in the late reperfusion period. Histology revealed more severe tissue damage at the late stage of reperfusion than at 4.5 hours. Significant reversal of CBF and ADC during the late reperfusion period may reflect the impairment of autoregulation in the ischemic regions. Vascular factors may play an important role in the infarct development after 80-minute focal ischemia.

Leukocyte-endothelium interactions in pial venules during the early and late reperfusion period after global cerebral ischemia in gerbils.

The contribution of leukocytes to secondary brain damage after cerebral ischemia is still under discussion. The purpose of the present study was to examine the pial microcirculation after global cerebral ischemia while focusing on leukocyte-endothelium interactions during the early and late reperfusion period of up to 4 days. A closed cranial window technique that leaves the dura mater intact was used. Global cerebral ischemia of 15 minutes' duration was induced in male Mongolian gerbils (n = 91). Pial microcirculation was observed by intravital fluorescence microscopy. Leukocyte-endothelium interactions (LEIs) in pial venules, vessel diameters, capillary density, and regional microvascular blood flow measured by laser Doppler flowmetry were quantified during 3 hours of reperfusion and in intervals up to 4 days after ischemia. Within 3 hours of reperfusion, the number of leukocytes (cells/100 microm x minute) rolling along or adhering to the venular endothelium increased from 0.1 +/- 0.2 to 28.4 +/- 17.4 (P < 0.01 vs. control) and from 0.2 +/- 0.2 to 4.0 +/- 3.8 (P < 0.05), respectively. There was no capillary plugging by leukocytes; capillary density remained unchanged. In the late reperfusion period, at 7 hours after ischemia, LEIs had returned to baseline values. Furthermore, from 12 hours to 4 days after ischemia, no LEIs were observed. Changes in regional microvascular blood flow did not correlate with LEIs. Global cerebral ischemia of 15 minutes' duration induces transient LEIs that reach a maximum within 3 hours of reperfusion and return to baseline at 7 hours after ischemia. LEIs are not related to changes in microvascular perfusion, which suggests mainly that the expression of adhesion receptors is necessary to induce LEIs rather than rheologic factors. It seems unlikely that this short-lasting activation of leukocytes can play a role in the development of secondary brain damage.

Combination of intraischemic and postischemic hypothermia provides potent and persistent neuroprotection against temporary focal ischemia in rats.

It is not known whether a combination of intraischemic and postischemic mild hypothermia provides extra neuroprotection and if so, whether the neuroprotection is persistent. Sixty-eight Sprague-Dawley rats were used. In group 1, ischemia and reperfusion were performed under normothermic (N) conditions (control, N-N). In group 2, ischemia was induced and maintained under hypothermic conditions (33 degrees C for 2 hours) and reperfusion was performed under normothermic conditions, H-N. In group 3, both ischemia and reperfusion were performed under hypothermic conditions for an additional 21 hours after the surgery, H-22H. In group 4, ischemia was induced and maintained under hypothermic conditions and reperfusion was performed under hypothermic conditions only for the initial 3 hours (H-3H). In group 5, ischemia was induced and maintained under normothermic conditions and reperfusion was performed under hypothermic conditions (33 degrees C) (N-22H). All rats were perfused 48 hours after the induction of ischemia. In addition, the normothermic or hypothermic therapy used for groups 1, 3, and 4 was performed again, and these rats were killed 30 days after the induction of ischemia. Furthermore, neurological deficits were monitored in groups N-N and H-22H for 4 weeks. In the H-3H and H-22H groups, the total infarct volume was significantly reduced by 41% or 66%, respectively, assessed 48 hours after ischemia. The significant reduction in group H-22H was again confirmed 30 days after ischemia, ie, 50% reduction was observed. In contrast, the reduction in group H-3H (31%) was not significant. The neurological deficits were significantly more severe in the N-N group than in the H-22H group during week 4. The neuroprotective effects against temporary focal ischemia evaluated by infarct volume and neurological functions by the combination therapy with intraischemic and prolonged postischemic mild hypothermia were persistent in rats. Appropriate design of mild hypothermia therapy extending into the late reperfusion period is important to maximize the neuroprotective effects of hypothermia.

Effects of L-carnitine and its acetyl and propionyl esters on ATP and PCr levels of isolated rat hearts perfused without fatty acids and investigated by means of 31P-NMR spectroscopy.

31P-NMR in vivo spectroscopy is a non-invasive and non-hazardous technique which investigates chemical composition and metabolism of living objects, for example by determining phosphocreatine (PCr) and ATP concentrations. In the present study we investigated the influence of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine on the energetic state of the Langendorff rat heart subjected to an ischemic period of 20 min followed by a reperfusion period of 60 min. To avoid an overlapping of the effects of fatty acids and glucose, the hearts were perfused with a Tyrode solution containing no fatty acids. Ischemia causes a rapid decrease in the PCr signal, followed by a decrease in the ATP signal after a prolonged period of ischemia. At the same time, a drastic increase in the Pi signal was observed. A partial recovery of the ATP and PCr signals was observed in the reperfusion period. With L-carnitine a markedly improved recovery of the high energy phosphates (e.g. increased PCr/P ratios) was found. With acetyl-L-carnitine this effect was enhanced in the first postischemic phase. It was followed, however, by a more rapid decrease in the PCr/Pi ratio in the late reperfusion period. The effect of propionyl-L-carnitine was not significantly improved in the first minutes of the reperfusion period, but during the whole reperfusion phase a stabilization of the PCr/Pi ratio was observed. Intracellular pH can be calculated from determination of the Pi-chemical shift. This shows that L-carnitine and its derivatives have a protective effect against intracellular pH decrease during ischemia. L-carnitine improves the energetic state of the heart, which leads to increased ischemia tolerance. Hearts under L-carnitine were able to tolerate up to four ischemia-reperfusion periods in succession, whereas the controls were not able to do so. These NMR results confirm the hypothesis that L-carnitine and its esters have a protective effect in the reperfusion period of the ischemic rat heart. This could be of importance for the treatment of ischemic cardiac diseases.

The nitric oxide donor sodium nitroprusside is protective in ischemia/reperfusion injury of the pancreas.

The role of nitric oxide in the ischemia/reperfusion injury of the pancreas is still unclear. In other organs, protective as well as aggravating effects have been described. We have, therefore, investigated the effect of the nitric oxide donor sodium nitroprusside on pancreatic ischemia/reperfusion injury. In Landrace pigs, after transsection of the pancreas, complete vascular isolation of the pancreatic tail was performed. The tail was subjected to 3 hr of warm ischemia and thereafter reperfusion (6 hr). The animals were divided into a control group (n=7) and a treatment group (n=7) that received 15 mg of sodium nitroprusside after reperfusion intra-arterially into the splenic artery. The morphological tissue damage and lipase activity in the venous effluent of the pancreas were significantly lower in the treatment group. Partial oxygen tension in the tissue after reperfusion was markedly reduced in the control group, indicating an impairment of microcirculation. In the treatment group, however, partial oxygen tension in the tissue was significantly higher (43 vs. 20 mmHg; P<0.014). Furthermore, total blood flow through the pancreatic tail in the treatment group was found to be significantly higher in the late reperfusion period (14 vs. 9.5 ml/min at 5 hr after reperfusion; P<0.05). There is a marked impairment of pancreatic microcirculation after reperfusion. Sodium nitroprusside counteracts this impairment and has a protective effect on ischemia/reperfusion injury of the pancreas.

Nucleotide hydrolysis in rats submitted to global cerebral ischemia: A possible link between preconditioning and adenosine production

Adenosine, an endogenous neuroprotective agent, can be produced in the synaptic cleft from adenosine triphosphate (ATP) hydrolysis via the concerted action of two enzymes: ATP diphosphohydrolase and 5'-nucleotidase. The aim of the present study was to investigate such enzymatic activities in the hippocampus of rats subjected to single (2- or 10-minute) or double (2+10 minute, with a 24-hour interval in between, named preconditioned group) ischemic episodes. Ischemia was produced by four-vessel occlusion method. Histological analysis showed no cell death in 2-minute ischemia, and up to 90% of pyramidal CA(1) cell loss in the 10-minute ischemic group. As predicted, double ischemic rats displayed a significant cytoprotective effect (around 60%). Preconditioned rats presented a delayed enhancement in ATP diphosphohydrolase activity (for ATP and adenosine diphosphate hydrolysis) after 48 hours of reperfusion. 5'-nucleotidase activity was increased immediately after ischemic insult (for all groups) and after a late reperfusion period (48 hours). We suggest that preconditioning causes delayed changes in enzymatic activities that would conceivably lead to increased adenosine production. This effect could be related to cytoprotection seen in preconditioned rats.

The Protective Effects of α-Melanocyte Stimulating Hormone on Canine Brain Stem Ischemia

OBJECTIVE: To evaluate the influence of α-melanocyte stimulating hormone(α-MSH), an anti-inflammatory antagonist of the production and action of proinflammatory cytokines, 26 dogs were divided into four groups and exposed to isolated, reversible brain stem ischemia in the presence or absence of α-MSH treatment. METHODS: Brain stem auditory evoked potentials (BAEPs) and regional cerebral blood flow were measured during ischemia and for 5 hours after reperfusion. Group I was composed of five dogs that underwent surgical preparation only. Group II was composed of seven dogs that were exposed to 20 minutes of ischemia without treatment. Group III was comprised of seven dogs exposed to 20 minutes of ischemia with α-MSH treatment before and during ischemia. Group IV was composed of seven dogs exposed to 20 minutes of brain stem ischemia with α-MSH treatment only during reperfusion. RESULTS: During the ischemic period, BAEPs were abolished in all animals within 10 minutes. With reperfusion, the BAEPs increased to approximately 36% of baseline in Group II dogs that received no treatment. However, this increase was approximately 63% in animals that received α-MSH both before and during ischemia (Group III). In Group IV dogs that received α-MSH only during reperfusion, BAEPs were increased approximately 10 to 14% more than in Group II during the late reperfusion period. CONCLUSION: The improved recovery of BAEPs in dogs treated with α-MSH suggests that this peptide may have neuroprotective effects in brain stem ischemia and reperfusion injury. This effect may be caused by an antagonistic action ofα-MSH on cytokine-induced ischemic brain damage.