Permanent Focal Ischemia Research Articles

Up-regulation of syntaxin1 in ischemic cortex after permanent focal ischemia in rats

Syntaxin1 and synaptotagmin are located in the pre-synaptic terminals and play central roles in Ca 2+-triggered neurotransmitter release. Because excessive synaptic transmission has been implicated in neuronal cell death after ischemia, we investigated the effects of cerebral ischemia on the levels of these proteins using a rat permanent focal ischemia model. Western blot analysis revealed that the protein level of syntaxin1 was significantly up-regulated in the ischemic core cortex and peri-ischemic cortex at 1 day after ischemia, while the protein level of synaptotagmin was not. Immunohistochemical analysis revealed that the protein level of syntaxin1 was markedly up-regulated in the ischemic areas where immunoreaction for MAP2 was lost. Furthermore, we showed that resident microglial cells were quite vulnerable to ischemia. Our data provide novel insights into the molecular mechanism of cerebral ischemia at the pre-synaptic terminals.

Defining the Macroscopic and Microscopic Findings of Experimental Focal Brain Ischemia in Rats From a Forensic Scientist's Point of View

Approximately 10% of all deaths in the world occur as a result of stroke. Determination of the time schedule of the pathologic events in a stroke patient is invaluable for a forensic specialist. The aim of this study was to define the schedule of the macroscopic and microscopic changes that occurred in a rat model of permanent focal ischemia for providing useful clues for the evaluation of stroke patients. Male Wistar rats weighing 250 to 350 g were used in this study. Permanent focal brain ischemia was applied by the suture occlusion method. The animals were divided into 7 experimental groups (n = 6) with time schedules including 1.5, 3, 6, 12, 24, 72 hours, and the sham. Brains were harvested at the end of the determined time schedule. Lesions in the frontoparietal cortex were evaluated macroscopically first and later hematoxylin eosin stained sections from the infarct core were investigated microscopically. Macroscopically, enlargement of the ipsilateral hemisphere was mild at 6 hour, apparent at 12 and 24 hours, and mild again at 72 hours. Microscopically, ischemic changes were apparent even at 1.5 hour. Red neurons and infiltration of the parenchyma with neutrophil leukocytes were observed at 12 hours. Pannecrosis and massive leukocyte infiltration were observed at 72 hours. Macroscopic and microscopic findings obtained from a rat model may provide clues for determination of the time-dependent changes due to brain ischemia in human subjects. Finally, the benefits of determination of time course of pathologic changes in the brain for forensic scientists were discussed.

Fluoxetine and Sertraline Attenuate Postischemic Brain Injury in Mice

This study aimed to investigate whether selective serotonin reuptake inhibitors (SSRIs) attenuate brain injury and facilitate recovery following photothrombotic cortical ischemia in mice. Male ICR mice were anesthetized and systemically administered Rose Bengal. Permanent focal ischemia was induced in the medial frontal and somatosensory cortices by irradiating the skull with cold light laser. The animals were treated with fluoxetine or sertraline once a day for 14 d starting 1 h after ischemic insult. Treatment with fluoxetine and sertraline significantly reduced the infarct size. The Evans blue extravasation indices of the fluoxetine- and sertraline-treated groups were significantly lower than that of the vehicle group. Treatment with fluoxetine and sertraline shifted the lower limit of the mean arterial blood pressure for cerebral blood flow autoregulation toward normal, and significantly increased the expression of heme oxygenase-1 (HO-1) and hypoxia-inducible factor-1alpha (HIF-1alpha) proteins in the ischemic region. These results suggest that SSRIs, such as fluoxetine and sertraline, facilitate recovery following photothrombotic cortical ischemia via enhancement of HO-1 and HIF-1alpha proteins expression, thereby providing a benefit in therapy of cerebral ischemia.

Augmentation of nitrite therapy in cerebral ischemia by NMDA receptor inhibition

Our recent work suggested that early infusion of nitrite might represent a novel therapeutic approach for acute ischemic stroke. In this study, we sought to examine the therapeutic time window of nitrite in an experimental stroke model, and to develop combined strategies for augmenting its protective effects. Nitrite was infused at various times after ischemia to rats subjected to transient or permanent focal ischemia. Nitrite was infused with memantine to prevent the potential toxicity. Infarct volumes, functional outcomes, microhypoxic areas, and oxidative stress were measured. Nitrite reduced the infarction volume and enhanced functional recovery when administered within 3 and 1.5 h in the transient and permanent model, respectively. Combined therapy with nitrite and memantine prolonged the time window up to 4.5 h. The potential oxidative toxicities of nitrite were significantly inhibited by memantine. The combination therapy of nitrite and memantine may be a feasible therapeutic approach for acute ischemic stroke.

A Functional Role for Sodium-Dependent Glucose Transport across the Blood-Brain Barrier during Oxygen Glucose Deprivation

In the current study, we determined the functional significance of sodium-dependent/-independent glucose transporters at the neurovasculature during oxygen glucose deprivation (OGD). Confluent brain endothelial cells cocultured with astrocytes were exposed to varying degrees of in vitro stroke conditions. Glucose transporter (GLUT) 1 and sodium glucose cotransporter (SGLT) activity were investigated by luminal membrane uptake and transport studies using [(3)H]D-glucose and also by [(14)C]alpha-methyl D-glucopyranoside (AMG), a specific, nonmetabolized substrate of SGLT. In vivo middle cerebral artery occlusion experiments were tested to determine whether blood-brain barrier (BBB) SGLT activity was induced during ischemia. Increases in luminal D-glucose and AMG uptake and transport were observed with in vitro stroke conditions. Specific inhibitor experiments suggest a combined role for both SGLT and GLUT1 at the BBB during OGD. A time-dependent increase in the uptake of AMG was also seen in mice exposed to permanent focal ischemia, and this increase was sensitive to the SGLT inhibitor, phlorizin. Infarct and edema ratio during ischemia were significantly decreased by the inhibition of this transporter. These results show that both GLUT1 and SGLT play a role at the BBB in the blood-to-brain transport of glucose during ischemic conditions, and inhibition of SGLT during stroke has the potential to improve stroke outcome. Pharmacological modulation of this novel BBB transporter could prove to be a brain vascular target in stroke.

Dynamic changes in cortical NADH fluorescence in rat focal ischemia: Evaluation of the effects of hypothermia on propagation of peri-infarct depolarization by temporal and spatial analysis

Suppression of peri-infarct depolarizations (PIDs) is one of the major mechanisms of hypothermic protection against transient focal cerebral ischemia. Previous studies have shown the lack of hypothermic protection against permanent focal ischemia. We hypothesized the lack of hypothermic protection was due to the poor efficacy in suppression of PIDs. To examine the hypothesis, we elucidated the effects of hypothermia on the manner of propagation of PIDs with temporal and spatial resolutions using NADH (reduced nicotinamide adenine dinucleotide) fluorescence images by illuminating the parietal–temporal cortex with ultraviolet light. Spontaneously hypertensive rats ( n = 14) were subjected to permanent focal ischemia by occlusion of the middle cerebral and left common carotid arteries. 2-h hypothermia (30 °C) was initiated before ischemia. Although hypothermia delayed the appearance of PIDs, it did not suppress their appearance. Furthermore, 54% of the PIDs enlarged the high-intensity area of NADH fluorescence in the hypothermia group, similar to the normothermia group (53%). The high-intensity area of NADH fluorescence widened by each PID was larger in the hypothermia group than in the normothermia group. These findings suggest that PIDs even in hypothermia are one of the major factors causing growth of infarction, emphasizing the importance of therapy that targets suppression of PIDs even during hypothermia.

Effectiveness of PSD95 Inhibitors in Permanent and Transient Focal Ischemia in the Rat

Postsynaptic density-95 inhibitors reduce ischemic brain damage without inhibiting excitatory neurotransmission, circumventing the negative consequences of glutamatergic inhibition. However, their efficacy in permanent ischemia and in providing permanent neuroprotection and neurobehavioral improvement in a practical therapeutic window is unproven. These were tested here under conditions that included fever, which is a common occurrence in clinical stroke. Six studies were performed in unfasted Sprague-Dawley rats. Two involved permanent pial vessel occlusion in male and female rats. Two involved permanent middle cerebral artery occlusion, which induced severe hyperthermia, and 2 involved transient middle cerebral artery occlusion. Animals were treated with a single intravenous injection of postsynaptic density-95 inhibitors (Tat-NR2B9c([SDV]) or Tat-NR2B9c([TDV])) 1 hour or 3 hours after stroke. Infarct volumes and neurobehavior were assessed in a blinded manner at 24 hours (pial vessel occlusion and permanent middle cerebral artery occlusion) or at 62 days (transient middle cerebral artery occlusion). Postsynaptic density-95 inhibitors dramatically reduced infarct size in male and female animals exposed to pial vessel occlusion (>50%), in hyperthermic animals with fever exceeding 39 degrees C exposed to permanent middle cerebral artery occlusion (approximately 50%), and at 62 days poststroke in animals exposed to transient middle cerebral artery occlusion (approximately 80%). Effectiveness of postsynaptic density-95 inhibitors was achieved without the drugs affecting body temperature. In transient middle cerebral artery occlusion, a single dose of postsynaptic density-95 inhibitor given 3 hours after stroke onset permanently maintained reduced infarct size and improved neurobehavior. Postsynaptic density-95 inhibitors administrated 3 hours after stroke onset reduced infarct volumes and improved long-term neurobehavioral functions in a wide therapeutic window. This raises the possibility that they may have future clinical usefulness.

Differential neuroprotective effects of carnosine, anserine, and N-acetyl carnosine against permanent focal ischemia.

Carnosine (beta-alanyl-L-histidine) has been shown to exhibit neuroprotection in rodent models of cerebral ischemia. In the present study, we further characterized the effects of carnosine treatment in a mouse model of permanent focal cerebral ischemia and compared them with its related peptides anserine and N-acetylated carnosine. We also evaluated the efficacy of bestatin, a carnosinase inhibitor, in ameliorating ischemic brain damage. Permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery (pMCAO). Mice were subsequently randomly assigned to receive an intraperitoneal injection of vehicle (0.9% saline), carnosine, N-acetyl carnosine, anserine, bestatin alone, or bestatin with carnosine. Infarct size was examined using 2,3,5-triphenyltetrazolium chloride staining 1, 3, and 7 days following pMCAO, and neurological function was evaluated using an 18-point-based scale. Brain levels of carnosine were measured in treated mice using high-performance liquid chromatography 1 day following pMCAO. We demonstrated that treatment with carnosine, but not its analogues, was able to significantly reduce infarct volume and improve neurological function compared with those in vehicle-treated mice. These beneficial effects were maintained for 7 days post-pMCAO. In contrast, compared with the vehicle-treated group, bestatin-treated mice displayed an increase in the severity of ischemic lesion, which was prevented by the addition of carnosine. These new data further characterize the neuroprotective effects of carnosine and suggest that carnosine may be an attractive candidate for testing as a stroke therapy.

Reduction of ischemic brain damage and increase of glutathione by a liposomal preparation of quercetin in permanent focal ischemia in rats

Oxidative stress is implicated in the pathogenesis of cerebral ischemia injury, and the flavonoids have shown to be neuroprotective in experimental models of cerebral ischemia. Previously, we have shown that an aqueous preparation of quercetin did not reach the brain while a liposomal preparation produced measurable cerebral amounts of quercetin that reduced significantly the cerebral damage provoked by permanent middle cerebral artery occlusion (pMCAo) of rats. In this context, the protective effects of liposomal quercetin (LQ) were investigated in the same model after 1 and 4 hours of arterial occlusion. LQ was administered in a single dose (30 mg/kg), at 30 min, 1 and 4 h after pMCAo, and the brain was studied 24 h later. Cerebral damage and the oedema volume were assessed with a tetrazolium salt (TTC). The status of brain tissue, the neuronal population, the global motor behaviour as well as the antioxidant, endogenous reduced glutathione (GSH), were also assessed in the brain. Thirty min after LQ there was a significantly protective effect against ischemic lesion demonstrated by a significant increase in numbers of cells in striatum and cortex, together with a partial reversal of motor deficits. GSH levels decreased after ischemia in ipsilateral striatum and cortex, and the LQ preparation reversed these effects 24 h after the occlusion. Our results suggest that endogenous brain GSH is critical in the defense mechanisms after ischemia, as a significant mediator of the protective effects of the LQ preparation.

Comparison of 12, 24 and 48 h of systemic hypothermia on outcome after permanent focal ischemia in rat

Mild hypothermia reduces injury in models of global and focal cerebral ischemia even when initiated after the insult. Neuroprotection depends critically upon the duration of hypothermia with longer treatments often being more efficacious. However, the ideal treatment duration is not known for most insults and this knowledge would facilitate clinical studies. Thus, we compared 12, 24 and 48 h of systemic hypothermia (33 °C vs. normothermia) initiated 1 h after permanent middle cerebral artery occlusion (pMCAO), which was produced by permanent occlusion of the carotid arteries and cauterization of the distal MCA in rat. Behavioral recovery and lesion volume were determined 7 days after pMCAO. All three treatments significantly and equally attenuated neurological deficits (e.g., forelimb placing response). Conversely, stepping error rate in the horizontal ladder test was significantly reduced only by the 24-h (18.7%) and 48-h treatments (11.7%) compared to normothermic rats (34.4%), and the 48-h treatment was significantly better than the 12-h treatment (28.8%). Similarly, brain injury was significantly reduced by 24-h (78.8 mm 3 lesion volume) and 48-h (66.8 mm 3) treatments compared to normothermia (142.6 mm 3), and the 48-h treatment was significantly better than the 12-h duration (114.6 mm 3). In separate experiments cerebral edema was measured via wet–dry weight measurements and significantly reduced by hypothermia (e.g., from 83.7% water in the injured cortex of normothermic rats to 81.4% in rats cooled for one day), but for this there were no significant duration effects. In summary, prolonged hypothermia treatment provides superior protection overall, but this is not explained by reductions in edema.

EVALUATION OF THE ANTI-ISCHEMIC EFFECT OF THE PPARy AGONIST, (ROSIGLITAZONE) IN PERMANENT FOCAL CEREBRAL ISCHEMIA IN DIABETIC RATS.

Diabetes mellitus (DM) is a clear risk factor for stroke. Furthermore, di­abetes has been shown to be a strong determinant for the presence of multiple lacunar infarcts in stroke patients. There has been a recent ap­preciation that peroxisome prolifera-tor-activated receptors (PPARs) and their ligands may play an important role in the brain. An increasing num­ber of studies have reported on the effects of PPAR agonists in animal models of neurological damage and disease, including the excitatory dam­age that occurs in stroke. The PPARy agonist, rosiglitazone, a synthetic li-gand of the thiazolidinedione class was currently used as an antidiabetic agent because of its insulin- sensitizing effect. So, the purpose of this study was to determine whether rosigiitazone may be neuroprotective in a model of permanent focal cerer-bral ischemia in diabetic rats. Meth­ods: This study was carried on 96 al­bino rats. Rats were divided into 8 equal groups: Group (1): Control group, Group (2): Diabetic control group, Group (3): Non-treated is-chemic group, Group (4): Diabetic and non-treated sham operated group, Group (5): Diabetic and non-treated permanent left middle cere­bral artery occlusion group, Group (6): Treated ischemic group, Group (7): Diabetic and treated sham oper­ated group, Group (8): Diabetic and treated permanent middle cerebral ar­tery occlusion group.

Caffeic acid phenethyl ester protects rabbit brains against permanent focal ischemia by antioxidant action: A biochemical and planimetric study

The present study was conducted to investigate whether caffeic acid phenethyl ester (CAPE), an active component of propolis extract, has a protective effect on brain injury after focal permanent cerebral ischemia, and to determine the possible antioxidant mechanisms. Cerebral infarction in adult male New Zealand rabbits was induced by microsurgical procedures producing right focal permanent middle cerebral artery occlusion (pMCAO). CAPE was administered to the treatment group after pMCAO at a dose of 10 μmol kg − 1 once a day intraperitoneally for 7 days. Neurological deficits were evaluated, using a modified six-point scale. Spectrophotometric assay was used to determine the contents of malondialdehyde (MDA), glutathione (GSH), catalase (CAT), nitric oxide (NO) and xanthine oxidase (XO). In the ipsilateral hemisphere, the infarct volume of the brain was assessed in brain slices stained with heamatoxylen and eosin. The results showed that treatment with CAPE significantly reduced the percentage of infarction in the ipsilateral hemisphere compared with the ischemia group. CAPE treatment significantly attenuated the elevation of plasma MDA, CAT and XO content ( p < 0.05), whereas it significantly increased the levels of plasma GSH and NO ( p < 0.05). Therefore, subacute CAPE administration plays a protective role in focal pMCAO due to attenuation of lipid peroxidation and its antioxidant activity. All of these findings suggest that CAPE provides neuroprotection against cerebral ischemia injury through its antioxidant action.

Refined Qing Kai Ling, Traditional Chinese Medicinal Preparation, Reduces Ischemic Stroke-Induced Infarct Size and Neurological Deficits and Increases Expression of Endothelial Nitric Oxide Synthase

Refined Qing Kai Ling (RQKL) is an improved injectable multi-component preparation derived from Qing Kai Ling, which could offer the neuroprotection effect in middle cerebral artery occlusion (MCAO) model of rats by relieving the damage of vascular endothelial cell as well as inhibiting the process of inflammation. Herein, we observed whether RQKL could exert influence on the expression of endothelial nitric oxide synthase (eNOS), as a mechanism of its protective effects against ischemia. Sprague-Dawley rat model of focal cerebral ischemia was established by permanent filament occlusion of the left middle cerebral artery. We found that the administration of RQKL could reduce the ischemic infarct size as well as neurological deficit of model rats. Furthermore, it was showed that the eNOS level was consistently increased in endothelium of blood vessels of the ischemic penumbra after 2 to 72 h of permanent MCAO, and the expression of eNOS increases more in animals treated with RQKL. Our results suggested that eNOS levels in penumbral zone were enhanced after permanent focal ischemia, and RQKL could stimulate postischemic eNOS expression, which may be an important mechanism in RQKL's protection against cerebral ischemia.

Spatial and temporal distribution of laminins in permanent focal ischemic brain damage of the adult rat

Laminins are extracellular matrix glycoproteins with multiple functions in the central nervous system, including maintenance of the blood-brain barrier. Because ischemic brain damage results in rapid degradation of extracellular matrix, we used immunocytochemistry on rat central nervous system after permanent focal ischemia to identify laminins involved in pathophysiology of stroke. At 24 hr after stroke, laminin-1 is transiently expressed by neurons inside the ischemic core, but from 2-3 days to 28 days it is expressed only in basement membrane structures. During the first 24 hr, alpha1, alpha5, beta1, and gamma1 laminins are transiently expressed in neurons within the ischemic core as an acute reaction of the brain to ischemia. Rapid induction of gamma1 laminin but no other laminin in reactive astrocytes surrounding the ischemic core is clear at 24 hr, and importantly, expression of gamma1 laminin in astrocytes surrounding the ischemic core intensifies during the first days and persists up to 28 days after stroke. At 2-3 days, gamma1 laminin immunoreactive barrier of reactive astrocytes is already fully formed, isolating the ischemic area from the healthy brain. Similar to gamma1 laminin, its KDI domain localizes in reactive astrocytes isolating the ischemic core. Results indicate that gamma1 laminin and its KDI domain are rapidly induced in glial cells after stroke and their expression persists, forming a molecular barrier between the healthy and the damaged brain. Thus, gamma1 laminin is involved in pathology of stroke and is likely to serve a protective function, considering its potent neuroprotective role after spinal cord injury and in neurodegenerative disorders.

Evaluation of the neuroprotective effect of ketoprofen on rats submitted to permanent focal brain ischemia

To study the neurobehavioral, biochemical and histopathological consequences of permanent focal brain ischemia, and the putative neuroprotective action of ketoprofen. One-hundred-and-three Wistar rats divided into groups A and B were respectively submitted to 48 hours and 15 days of ischemia. Each group was divided into 4 subgroups: ischemic not treated, ischemic treated, sham not treated, and sham treated. Ischemic animals had the left middle cerebral artery coagulated. Ketoprofen was administered to treated subgroups 15 minutes before arterial coagulation (manipulation in the sham group). Exploratory activity and defecation were reduced in all ischemic animals in the first postoperative days and constant histopathological changes were observed in each group. The total brain glutamate levels were higher in treated animals 48 hours after surgery. No clear parallelism among behavioral, biochemical and histopathological findings was observed. Ketoprofen demonstrated no neuroprotective effect on the behavioral or histopathological aspects of focal permanent brain ischemia.

General versus Specific Actions of Mild-Moderate Hypothermia in Attenuating Cerebral Ischemic Damage

Mild or moderate hypothermia is generally thought to block all changes in signaling events that are detrimental to ischemic brain, including ATP depletion, glutamate release, Ca(2+) mobilization, anoxic depolarization, free radical generation, inflammation, blood-brain barrier permeability, necrotic, and apoptotic pathways. However, the effects and mechanisms of hypothermia are, in fact, variable. We emphasize that, even in the laboratory, hypothermic protection is limited. In certain models of permanent focal ischemia, hypothermia may not protect at all. In cases where hypothermia reduces infarct, some studies have overemphasized its ability to maintain cerebral blood flow and ATP levels, and to prevent anoxic depolarization, glutamate release during ischemia. Instead, hypothermia may protect against ischemia by regulating cascades that occur after reperfusion, including blood-brain barrier permeability and the changes in gene and protein expressions associated with necrotic and apoptotic pathways. Hypothermia not only blocks multiple damaging cascades after stroke, but also selectively upregulates some protective genes. However, most of these mechanisms are addressed in models with intraischemic hypothermia; much less information is available in models with postischemic hypothermia. Moreover, although it has been confirmed that mild hypothermia is clinically feasible for acute focal stroke treatment, no definite beneficial effect has been reported yet. This lack of clinical protection may result from suboptimal criteria for patient entrance into clinical trials. To facilitate clinical translation, future efforts in the laboratory should focus more on the protective mechanisms of postischemic hypothermia, as well as on the effects of sex, age and rewarming during reperfusion on hypothermic protection.

Upregulation of EMMPRIN after permanent focal cerebral ischemia

Elevated activities of matrix metalloproteinases (MMPs) following ischemic stroke have been shown to mediate ischemic injury as well as neurovascular remodeling. The extracellular MMP inducer (EMMPRIN) is a 58-kDa cell surface glycoprotein, which has been known to play a key regulatory role for MMP activities. The roles of EMMPRIN in stroke injury are not clearly understood. In this study, we investigated changes of EMMPRIN in a mouse model of permanent focal cerebral ischemia, and examined potential association between EMMPRIN and MMP-9 expression. Adult male CD-1 mice were subjected to permanent focal ischemia by intraluminal occlusion of the left middle cerebral artery (MCAO) under anesthesia. EMMPRIN expression was markedly upregulated in the peri-infarct area at 2–7 days after ischemia compared to the contralateral non-ischemic hemisphere by Western blot analysis. Immunofluorescent double staining demonstrated that EMMPRIN signals co-localized with vwF-positive endothelial cells and GFAP-positive peri-vascular astrocytes. In contrast, EMMPRIN signal did not co-localize with NeuN-positive neurons, or MPO-positive neutrophils. Dual fluorescent staining revealed that EMMPRIN co-localized with MMP-9. Our data also demonstrated that increased EMMPRIN expression correlated with increased MMP-9 levels in a temporal manner. In summary, we report for the first time that EMMPRIN expression was significantly increased in a mouse model of permanent focal cerebral ischemia. The spatial and temporal association between increased EMMPRIN expression and elevated MMP-9 levels suggest that EMMPRIN may modulate MMP-9 activity, and participate in neurovascular remodeling after ischemic stroke.

Antithrombin Reduces Ischemic Volume, Ameliorates Neurologic Deficits, and Prolongs Animal Survival in Both Transient and Permanent Focal Ischemia

Antithrombin (AT), a glycoprotein belonging to the serpin family, blocks thrombin formation and activity at several steps. Thrombin, beside its relevant role in the coagulation cascade, exerts neurodetrimental effects through the activation of a family of protease-activated receptors, which can be implicated in stroke pathophysiology. The aims of the present study were to evaluate whether AT could reduce brain damage, ameliorate neurologic deficits, and prolong animal survival. Two different doses of AT (10 and 30 IU/kg IP) were administered 3 hours, 6 hours, or 3 and 6 hours after an ischemic insult to mice and rats subjected to either transient or permanent focal ischemia. Ischemic volume was evaluated 24 hours or 7 days after the ischemic insult. Neurologic deficits were also scored. In mice, 10 or 30 IU/kg AT administered twice, at 3 and 6 hours after transient ischemia, and 30 IU/kg AT administered 3 hours only after transient ischemia substantially reduced total ischemic volume, significantly improved neurologic deficits evaluated 24 hours after the insult, and prolonged animal survival. In rats, the same doses given at the same time intervals significantly reduced ischemic volume, evaluated 24 hours after permanent ischemia. These results indicate that AT remarkably reduces infarct volume, ameliorates neurologic deficit scores, and prolongs animal survival in 2 rodent models of brain ischemia. Taken together, our data suggest that AT, delivered via systemic administration, an easily achievable route of administration and in a clinically useful time window, could represent a new therapeutic strategy to be validated for the clinical treatment of human stroke.

Regulation of A2A adenosine receptor expression and functioning following permanent focal ischemia in rat brain

Ischemia, through modulation of adenosine receptors (ARs), may influence adenosine-mediated-cellular responses. In the present study, we investigated the modulation of rat A(2A) receptor expression and functioning, in rat cerebral cortex and striatum, following in vivo focal ischemia (24 h). In cortex, middle cerebral artery occlusion did not induce any alterations in A(2A) receptor binding and functioning. On the contrary, in striatum, a significant decrease in A(2A) ligand affinity, associated with an increase in receptor density, were detected. In striatum, ischemia also induced a significant reduction both in G protein pool and in A(2A) receptor-G protein coupling. On the contrary, A(2A) receptor functional responsiveness, measured as stimulation of adenylyl cyclise, was not affected by ischemia, suggesting receptor up-regulation may represent a compensatory mechanism to maintain receptor functioning during cerebral damage. Immunohistochemical study showed that following 24 h middle cerebral artery occlusion, A(2A) ARs were definitely expressed both on neurons and activated microglia in ischemic striatum and cortex, but were not detected on astrocytes. In the non-ischemic hemisphere and in sham-operated rats A(2A) ARs were barely detected. Modifications of ARs may play a significant role in determining adenosine effects during ischemia and therefore should be taken into account when evaluating time-dependent protective effects of specific A(2A) active compounds.

Estrogen receptor beta agonist diarylpropiolnitrile (DPN) does not mediate neuroprotection in a rat model of permanent focal ischemia

Selective estrogen receptor (ER) agonists can indicate which receptor subtypes are implicated in neuroprotection. This study investigated the contribution of ERβ, using the selective agonist diarylpropiolnitrile (DPN), in a rat model of stroke. Lister Hooded rats were ovariectomized and implanted with mini-pumps containing either DPN (8 mg kg − 1 day − 1 ) ( n = 7) or vehicle ( n = 5). Sensorimotor function was assessed using a neurological score and the spontaneous forelimb use asymmetry (cylinder) test. One week later the animals received a middle cerebral artery occlusion (MCAO), and T 2-weighted MRI at 48 h post-MCAO quantified ischemic damage. Functional recovery was tested for 7 days post-MCAO and brains processed for histological verification of infarct size. The MRI images revealed no significant differences in hemispheric lesion volumes between vehicle- and DPN-treated groups (35.6 ± 3.5% and 30.8 ± 1.7%, respectively [mean ± SEM]; Student's unpaired t-test df = 10, t = − 1.357, p = 0.453); this was confirmed histologically at 7 days. MCAO induced significant decline in neurological score performance (from 22 to 11 at 2 h post-MCAO) in the vehicle-treated animals, which was not significantly influenced by DPN. MCAO also induced significant changes in forelimb use in the cylinder test (10% reduction in contralateral, 20% reduction in both, and 30% increase in ipsilateral forelimb use) but this response was not significantly different between groups [ F(1,1) =2.929, p = 0.118, repeated-measures ANOVA]. In conclusion, pretreatment with the ERβ agonist DPN did not influence infarct size or sensorimotor function in rats exposed to MCAO.