Arginine-Vasopressin V1 but not V2 Receptor Antagonism Modulates Infarct Volume,Brain Water Content,and Aquaporin-4 Expression Following Experimental Stroke

Background  

Aquaporin-4 (AQP4) plays an important role in the evolution of ischemia-evoked cerebral edema. Experimental studies have also demonstrated anti-edema effects of arginine-vasopressin (AVP) antagonists. In a well-characterized murine model of ischemic stroke, we tested the hypotheses that treatment with selective AVP V₁ but not V₂ receptor antagonist (1) attenuates injury volume and ischemia-evoked cerebral edema; and (2) modulates ischemia-evoked AQP4 expression.     

An experimental study of arginine vasopressin on acute ischemic brain edema in gerbils (1)]

The purpose of this experiments is to study the role of arginine vasopressin (AVP) in acute ischemic brain edema of mongolian gerbils. The results showed that the contents of AVP in ischemic cortex, hypothalamus and striatum increased remarkably in 15-120 minutes after ischemia, while the contents of AVP had no change in pons-medulla which was not affected from ischemia, and there was relationship between the contents of AVP in cortex and the ischemic cortical edema. Intracerebroventricular injection (ICV) of AVP exacerbated the ischemic cortical edema and it showed dose-response correlation. While ICV of AVP antiserum significantly decreased ischemic cortical edema. These suggested that AVP was involved in the pathophysiologic process of acute ischemic brain edema. The increasing of AVP contents in ischemic brain regions could exacerbate the formation of ischemic brain edema.     

The effects of selective A1 and A2a adenosine receptor antagonists on cerebral ischemic injury in the gerbil

Cerebral ischemia of 5 min duration was induced in unanesthetized Mongolian gerbils by bilateral occlusion of the carotid arteries. The extent of ischemic injury was assessed behaviorally by measuring the increases in locomotor activity following ischemia and by a histopathological assessment of the extent of CA1 hippocampal pyramidal cell injury and loss 5 days after ischemia. The A_2a adenosine receptor selective antagonists 8-(3-chlorostyryl) caffeine (CSC; 0.1 mg/kg i.p.) and 4-amino-l-phenyl[1,2,4]-triazolo[4,3-a] quinoxaline (CP 66,713; 0.1 mg/kg i.p.) reduced the extent of ischemia-induced injury. An A1 selective receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1.0 mg/kg i.p.), enhanced ischemia-evoked injury. These results suggest that adenosine A_2a receptor antagonists may be useful for the prevention of cerebral injuries resulting from stroke or cardiac arrest.     

Blood-brain barrier sodium transport limits development of brain edema during partial ischemia in gerbils

Sodium derived from the blood is known to accumulate in brain tissue during the early stages of incomplete ischemia. Our present studies were undertaken to determine the relation between blood-brain barrier sodium transport and the development of ischemic brain edema. Incomplete cerebral ischemia was produced in gerbils by ligation of the left common carotid artery under ether anesthesia. Following recovery from the anesthetic, the gerbis were evaluated for the presence of neurologic symptoms and were divided into symptomatic (n = 77) and asymptomatic (n = 94) groups. Tissue water, sodium, and potassium contents, tissue plasma volume, and brain uptake of 22Na were measured in both groups 1.5, 3, 6, 12, and 24 hours after carotid ligation. There was a progressive accumulation of sodium and water in the ipsilateral cerebral cortex of the symptomatic group compared with either the corresponding contralateral cortex of the same gerbils or with the asymptomatic group. Net changes in brain sodium and potassium concentrations appeared to be the main determinants of fluid accumulation. Brain edema was not due to opening of the blood-brain barrier because the unidirectional transport of 22Na remained low and was even reduced by 35-55% in the ischemic cortex. Nevertheless, this sodium transport activity appeared to be rate-limiting in the development of brain edema during the first 3 hours of ischemia because the rate of sodium accumulation in the tissue was the same as the rate of 22Na transport from the blood to the brain. We conclude that blood-brain barrier sodium transport is an important factor in the formation of ischemic brain edema.     

5-HT_2受体拮抗剂赛庚啶对沙土鼠单侧脑缺血的影响

目的：观察５－ＨＴ_2受体拮抗剂赛庚啶对沙土鼠单侧颈总动脉结扎所致单侧脑缺血的影响。方法：将动物随机分为５组。动物模型采用单侧颈总动脉夹闭并再灌注的方法。结果：赛庚啶对沙土鼠单侧脑缺血能减少脑组织水及钠含量改善脑水肿，并减少脑组织Ｃａ~(2 )含量。结论：５－ＨＴ_2可通过５－ＨＴ_2介导加重脑缺血时脑水肿和脑组织Ｃａ~(2 )聚积，５－ＨＴ_2受体拮抗剂可减轻缺血性脑损伤。     

腺苷A1受体激动剂CHA对缺血性脑水肿的影响

在沙土鼠脑半球缺血模型上，观察了腺苷Ａ１受体激动剂ＣＨＡ（Ｃｙｃｌｏｈｅｘｙｌａｄｅｎｏｓｉｎｅ）对缺血８ｈ内的卒中发生率、卒中症状、以及脑水肿的影响。分析了脑水肿程度与卒中症状的关系。结果显示：ＣＨＡ可以延迟沙土鼠缺血后卒中发生时间，降低卒中发生率，改善卒中症状，减轻缺血性脑水肿。卒中指数与脑组织比重呈明显的负相关。提示腺苷减轻脑水肿，可能是其抗缺血性脑损伤的保护作用之一。     

Proton NMR relaxation times in ischemic brain edema

The state of water in cerebral ischemia was studied by using the proton nuclear magnetic resonance (1H-NMR) method. Cerebral ischemia was induced experimentally in Mongolian gerbils by unilateral ligation of the common carotid artery. Longitudinal (T1) and transverse (T2) relaxation times of the ischemic brain were measured with a pulse FT-NMR spectrometer and the water content was determined by the wet/dry method. Quantitative analysis of the relaxation times was performed sequentially during the initial 7 hours following ligation and the data were compared with those of brain edema previously reported by S. Naruse in the rat. Characteristic findings in brain ischemia include prolongation of the slow component of T2 and increase in the water content. A quantitative comparison of relaxation rate and water content demonstrates that ischemic brain edema in Mongolian gerbils is different from cytotoxic and vasogenic types of brain edema. When R2 (1/T2) was plotted against the water content, the slope value of ischemia in the gerbil was between the slope values of the TET intoxication and cold injury induced edemas reported previously. From these results, it might be said that ischemic brain edema includes both the cytotoxic and vasogenic types of brain edema. Glycerol was demonstrated to affect brain ischemia by decreasing the water content and by shortening the slow component of T2. By analysis of the relaxation times and water content, we examined the pathophysiological characteristics of water molecules in ischemic brain tissue.     

Three-Dimensional metabolic mapping of the freeze-trapped brain: Effects of ischemia in the mongolian gerbil

The effects of unilateral carotid artery (left or right) occlusion on the three-dimensional metabolic mapping were studied in the Mongolian gerbil (Meriones unguiculatus). The brain was freeze-trapped by the liquid nitrogen funnel technique and was analyzed for the two-dimensional distribution of the reduced pyridine nucleotides and oxidized flavoprotein using a time-sharing micro-light guide surface fluorometer/reflectometer. The results could be summarized as follows. Bilateral carotid artery occlusion induced uniform effects in terms of redox state in the cerebral cortex and the olfactory bulbs. After unilateral carotid occlusion, the redox state of the ischemic ipsilateral hemisphere was higher and was not affected by blood volume changes as evaluated from the reflectance signal scanned in several brains. The narrow band of tissue near the midline of the ischemic hemisphere connected to the contralateral hemisphere blood supply appeared in a large number of gerbils. The anomaly in the redox state of the intracellular space also appeared in subcortical structures, as seen in all the two-dimensional mappings measured. It seems that blood circulation to the two olfactory bulbs is connected to the same blood vessel. It is suggested that unilateral carotid artery-occluded gerbils may show large variability inside the same hemisphere and therefore results must be evaluated very carefully.     

Role of arginine vasopressin V1 and V2 receptors for brain damage after transient focal cerebral ischemia.

Brain edema formation is one of the most important mechanisms responsible for brain damage after ischemic stroke. Despite considerable efforts, no specific therapy is available yet. Arginine vasopressin (AVP) regulates cerebral water homeostasis and has been involved in brain edema formation. In the current study, we investigated the role of AVP V1 and V2 receptors on brain damage, brain edema formation, and functional outcome after transient focal cerebral ischemia, a condition comparable with that of stroke patients undergoing thrombolysis. C57/BL6 mice were subjected to 60-min middle cerebral artery occlusion (MCAO) followed by 23 h of reperfusion. Five minutes after MCAO, 100 or 500 ng of [deamino-Pen(1), O-Me-Tyr(2), Arg(8)]-vasopressin (AVP V1 receptor antagonist) or [adamantaneacetyl(1), O-Et-D-Tyr(2), Val(4), Abu(6), Arg(8,9)]-vasopressin (AVP V2 receptor antagonist) were injected into the left ventricle. Inhibition of AVP V1 receptors reduced infarct volume in a dose-dependent manner by 54% and 70% (to 29+/-13 and 19+/-10 mm3 versus 63+/-17 mm3 in controls; P<0.001), brain edema formation by 67% (to 80.4%+/-1.0% versus 82.7%+/-1.2% in controls; P<0.001), blood-brain barrier disruption by 75% (P<0.001), and functional deficits 24 h after ischemia, while V2 receptor inhibition had no effect. The current findings indicate that AVP V1 but not V2 receptors are involved in the pathophysiology of secondary brain damage after focal cerebral ischemia. Although further studies are needed to clarify the mechanisms of neuroprotection, AVP V1 receptors seem to be promising targets for the treatment of ischemic stroke.     

Thrombin inhibition attenuates neurodegeneration and cerebral edema formation following transient forebrain ischemia

The disturbance of microcirculation following cerebral ischemia leads to an enlargement of cerebral infarct volume. Endogenous thrombin may play a role in this disturbance of microcirculation following cerebral ischemia. Therefore, the inhibition of thrombin may improve neurodegeneration and the accumulation of cerebral edema following cerebral ischemia in gerbils. The effects of thrombin inhibitor (argatroban) on cerebral ischemia were investigated in comparison with thromboxane A2 synthase inhibitor (ozagrel) and cyclooxygenase inhibitor (aspirin) following bilateral common carotid artery occlusion and reperfusion (CCA:O/R) in male Mongolian gerbils. This study consisted of three experiments: (1) morbidity and survival ratio (n=40 for each), (2) histopathology (n=12 for each), and (3) mean arterial blood pressure, local cerebral blood flow (CBF), and cerebral specific gravity (n=8 for each). Argatroban treatment improved survival ratio and stroke index, and decreased ischemically injured cell numbers in cortex and hippocampus and cerebral edema in cortex compared with aspirin and saline, in concert with the fast recovery of local CBF without reactive hyperemia following bilateral CCA:O/R. Ozagrel treatment also improved those factors compared with saline, in concert with the fast recovery of local CBF with reactive hyperemia. Aspirin treatment improved survival ratio and stroke index, and decreased ischemically injured cell numbers in cortex. Thrombin inhibition with argatroban decreases neurodegeneration and cerebral edema following bilateral CCA:O/R in gerbils.     

Ornithine decarboxylase activity and immunohistochemical location in postischemic brain

Ornithine decarboxylase, rate-limiting in polyamine formation, has been found to be necessary for the development of vasogenic edema after cryogenic cerebral injury and is postulated to be of importance in late ischemic brain edema formation. Ornithine decarboxylase activity and accompanying edema was studied after transient cerebral ischemia in Mongolian gerbils. Bilateral carotid artery occlusion was utilized to produce dense forebrain ischemia. After 4 h of reperfusion a significant elevation in ornithine decarboxylase activity was present (72.5 +/- 24.7 vs 8.5 +/- 2 pmoles/mg protein/h, p less than 0.05). Immunohistochemical localization of ornithine decarboxylase indicated its presence in cortical neurons of ischemic gerbils. This was typically located in the perinuclear cytoplasm and extended into proximal dendrites. Nonischemic animals did not contain ornithine decarboxylase immunoreactivity. These studies show the presence and location of ornithine decarboxylase in cerebral tissue subjected to transient ischemia. The increase in this marker of polyamine activity paralleled previous studies in this model of cerebral edema formation and reperfusion deficit in blood flow and evoked potential, suggesting that ornithine decarboxylase is a marker for and may be associated with those late metabolic events leading to progressive functional deterioration after incomplete cerebral ischemia.     

Increased neuroglobin levels in the cerebral cortex and serum after ischemia-reperfusion insults

Neuroglobin (NGB) is a newly discovered protein localized in neurons of the central and peripheral nervous systems in vertebrates. It functions to bind, store, and facilitate the utilization of oxygen in neuronal cells. Recent studies suggest that it may modulate hypoxic and ischemic injury. The major goal of the present study is to characterize the dynamic changes of NGB protein in the brain and serum in a global forebrain ischemia-reperfusion model using gerbils. The sensitivity and validity of serum NGB as a potential biomarker for brain injury were further evaluated. Global cerebral ischemia-reperfusion models were induced by bilateral carotid occlusion for 20 min followed with 2-, 8-, 16-, 24-, 48-, or 72-h reperfusion in forty-six Mongolian gerbils. Sham-operated and operated animals were sacrificed at the designated time after reperfusion. Brains were fixed for immunocytochemical study to evaluate the time-dependent expression of NGB, and the concentration of NGB in serum was measured by enzyme-linked immunosorbent assay. Our results showed that the expression of NGB was upregulated in the cerebral cortex but significantly downregulated in the hippocampus from 2 to 72 h of reperfusion after 20 min of bilateral common carotid arteries occlusion. The concentration of NGB in serum was significantly increased at 8 h and reached a peak at 48 h of reperfusion. There is a significant correlation between NGB levels in the serum and severity of neuronal damage in the gerbil brain. In summary, the upregulation of NGB in cerebral cortex and downregulation in hippocampus after reperfusion insults in the gerbil brain are consistent with the fact that cerebral cortex is more tolerant to hypoxic or ischemic injury than the hippocampus. Moreover, the changes of NGB levels in serum may be used to monitor the extent of brain damage in ischemic brain diseases.     

Experimental cerebral ischemia in Mongolian gerbils

The behavior of the BBB in cerebral ischemia was studied in symptom-positive Mongolian gerbils subjected to left common carotid artery occlusion using Evans Blue dye as indicator of BBB injury. The BBB damage was demonstrable grossly by the presence of areas of blue discoloration, and microscopically by the presence of a bright red fluorescent tracer, localized mostly in the neurons. The survey of various groups of animals revealed a direct relationship between the incidence and time of appearance of the BBB lesions and the duration of the ischemic occlusion. This relationship can be interpreted as another example of the previously described "maturation" phenomenon. A relatively late occurrence of the BBB injury in cerebral ischemia, at the time when the affected brain tissue shows severe, edematous histopathologic changes indicates that the brain edema, as the main complication of ischemia, could be regarded as being primarily of the cytotoxic type.     

Learning impairment and microtubule-associated protein 2 decrease in gerbils under chronic cerebral hypoperfusion

A coiled stainless steel wire clip was made that allowed us to chronically reduce cerebral blood flow in Mongolian gerbils. After 6 weeks of reduced cerebral blood flow in 15 experimental gerbils, we evaluated their learning ability and found it to be impaired relative to that in 15 control gerbils. Eight weeks after surgery, regional cerebral blood flow in the parietal cortex measured by the hydrogen clearance method in the experimental gerbils was 73-76% of that in the control gerbils. Light microscopy showed minimal histologic changes in the brains of the experimental gerbils. Concentrations of brain proteins analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that among water-soluble brain proteins, the concentrations of cytoskeletal proteins (microtubule-associated protein 2, calspectin, and clathrin) declined in the experimental gerbils. In particular, the concentration of microtubule-associated protein 2 declined significantly. Our findings show that the reduction of cerebral blood flow via carotid stenosis impairs the learning behavior in gerbils, with an associated decrease in the concentration of microtubule-associated protein 2. We believe that Mongolian gerbils with chronically reduced cerebral blood flow are a useful animal model of chronic brain hypoperfusion.     

Changes of neuropeptides and their receptors in experimental stroke gerbil brains.

Eight kinds of neuropeptides and four kinds of neuropeptide receptors were examined in the right and left hemispheres of mongolian gerbils after unilateral carotid ligation-induced stroke and in normal controls. Five hours after ligation of the right common carotid artery, beta-endorphin concentration in the right hemisphere (ischemic side) of the stroke group was significantly increased compared with that in the contralateral hemisphere (non-ischemic side), but there were no differences between sides in other neuropeptides either with or without stroke. Furthermore, although there were no differences in [3H]naloxone binding, [3H]thyrotropin-releasing hormone binding or 125I-vasoactive intestinal polypeptide binding in the brain in this model of stroke, [3H]enkephalin binding was significantly lower on the ischemic side than on the non-ischemic side in the stroke group. These results suggest that increased activity in the beta-endorphinergic system in the brain might be partly caused by ischemic brain failure.     

Alanine:Glutamate ratios as an index of reversibility of cerebral ischemia in gerbils

We examined whether or not increases in alanine:glutamine ratios correlate with the severity of brain damage during ischemia and reperfusion. Concentrations of selected metabolites for energy metabolism and several amino acids were measured in quick-frozen brains of Mongolian gerbils after either unilateral or bilateral common carotid artery occlusion. Unilateral arterial occlusion (10 min) resulted in neurological symptoms that were minor in 30% of the animals and marked in 20%. During unilateral ischemia, alanine:glutamate (A:G) ratios in cerebral cortex correlated directly with increases in lactate and cAMP concentrations in the ipsilateral cerebral cortex. Alterations in phosphocreatine, lactate, cAMP, and NAD:NADH ratios induced by bilateral occlusion of the carotid arteries from 1 to 20 min returned to normal values in all animals after 30 min of recirculation. Normal cerebral A:G ratios were recorded after 1 h of recirculation in animals with 1-min bilateral occlusion whereas gerbils that were subjected to 20-min bilateral occlusion did not show a decrease in the cerebral A:G ratio. This longer ischemic injury was accompanied by a mortality rate of about 90%. Correlations between A:G ratios and morbidity-mortality of this injury suggest that this ratio may be a useful index to predict tissue survival after cerebral ischemia.     

Monoamines and related enzymes in cerebral cortex and basal ganglia following transient ischemia in gerbils

Summary The post-ischemic effects on cerebral cortex and basal ganglia monoamine levels and monoamine oxidase (MAO A and B) and catechol-O-methyl transferase (COMT) activities were evaluated in Mongolian gerbils (Meriones unguiculatus) subjected to bilateral common carotid arteries occlusion for 15 min and reflow for 7 days. Disorders of monoamine metabolism was found in ischemic brain which persisted during the long-term post-ischemia. A rebound increase of norepinephrine and serotonin appeared in early stages (up to 1 h) of post-ischemia both in cerebral cortex and basal ganglia; a rebound increase of dopamine was found only in cerebral cortex. Thereafter, the serotonin level was enhanced over the control level during the whole post-ischemic period whereas the levels of catecholamines were reduced particularly in basal ganglia. With respect to monoamine content and activities of monoamine degraded enzymes an oscillatory behavior was observed in post-ischemia. Disorder of the monoamine metabolism found during post-ischemic period possibly contributes to neurological dysfunction after an ischemic insult.These studies were supported by a grant from the Union of Sciences of Republic Serbia (No. 40404-14) to B.B.M.     

Microvascular disturbances and edema formation after repetitive ischemia of gerbil brain

Summary Three transient episodes of 5 min ischemia spaced at 1-h intervals were produced in Mongolian gerbils by bilateral carotid artery occlusion with an implanted vascular occlusion device. The interval of 1 h was chosen to allow for the development of postischemic hypoperfusion between the ischemic episodes. Three minutes and 1 h after each ischemic episode, and 6 and 24 h after the third occlusion, Evan's blue (EB) was injected intravenously to trace circulating blood, and the number of perfused capillaries was determined in various brain regions by fluorescence microscopy. Brain edema was evaluated by measuring specific gravity in tissue samples taken from adjacent areas. Repetitive ischemia caused progressively increasing brain edema and a progressive reduction of the number of perfused capillaries. Immediately after each ischemic episode, transient recruitment of capillaries occurred, thus excluding noreflow as a main pathogenetic factor of microcirculatory disturbances. The pattern of microcirculation 6 and 24 h after the last occlusion revealed a redistribution of circulating blood, characterized by a reduction in the number of EB-filled capillaries associated with a noticeable dilatation of the larger vascular channels. Our studies suggest a close interrelationship between post-ischemic microcirculatory hypoperfusion and the development of brain edema, the degree and extent of which progresses with the repetition of ischemic episodes when they are carried out during the periods of hypoperfusion.     

Neurochemical correlates of selective neuronal loss following cerebral ischemia: role of decreased Na, K-ATPase activity

In order to investigate the role of Na+,K(+)-ATPase in the development of neuronal necrosis following cerebral ischemia, ischemia was induced in gerbils by occluding the common carotid artery unilaterally for 10 min. A time-course analysis revealed that significant reductions of the Na+,K(+)-ATPase activity in the cerebral cortex and hippocampus were manifested at 15 min, 30 min, and 1 h, and returned to the control level one day following recirculation. No apparent alterations of the Mg(2+)-ATPase activity, on the other hand, were obtained throughout the experimental period. Furthermore, Scatchard analyses of [3H]ouabain binding to the cerebral cortex membranes disclosed that the Bmax values invariably decreased without any change of Kd values following ischemia. It has also been shown that treatment of the animals with an agent known to mitigate ischemic neuronal necrosis, i.e. BY-1949, significantly reversed such derangements. These results suggest that the recovery of decreased Na+,K(+)-ATPase activity shortly after ischemia exerts a protective effect against ischemic brain damage.     

Are blood platelets involved in the pathogenesis of ischemic brain edema in gerbils?

Edema formation following severe permanent or temporary cerebral ischemia in gerbils with an artificially reduced platelet count was investigated. Acute focal cerebral ischemia was produced by extracranial carotid ligation, and the local cerebral blood flow was estimated using the hydrogen clearance method. Brain tissue water and sodium and potassium contents were taken as indexes of brain edema. The platelet count was reduced in some gerbils by intravenous injection of neuraminidase. After 60 minutes of ischemia, a marked increase in tissue water and sodium contents accompanied by a decrease in potassium content was observed in untreated gerbils. However, gerbils with a reduced platelet count revealed similar but significantly smaller changes in all the measured parameters. Restoration of blood flow after 60 minutes of ischemia resulted in further accumulation of water and sodium and in depletion of potassium in both groups. These changes were significantly smaller in the gerbils with a reduced platelet count. It is concluded that platelets, activated by cerebral ischemia, may be involved in the development of ischemic brain edema in gerbils.