Comparison of pre- and post-ischemic treatment of telmisartan and nimodipine combination in experimentally induced cerebral ischemia.

Time dependent intervention plays a crucial role in preventing neurodegeneration after ischemic insult. The intensity of excitotoxicity is greater in the secondary reperfusion phase (2-4 h) compared to the primary occlusion phase (2 h), which could be attributed to secondary elevation of excitatory amino acids (EAA) in cerebral ischemia. In the present study, we tried to assess the neuroprotective effects of telmisartan and nimodipine (TM-NM) combination on the secondary reperfusion phase. The drug treatments were made immediately after reperfusion and their effects were compared with pre-treatment. The neuroprotective effect was studied using middle cerebral artery occlusion (MCAo) transient ischemic model in rats. On the 7th day after reperfusion, the rats were subjected to behavioral studies. The brain was dissected out on the 9th day to measure neurobiochemical alterations and for histopathological observations. The results have shown that TM-NM (5 mg/kg) attenuated the EAA release in different brain regions with partial restoration of energy levels in secondary reperfusion phase. Similarly, it normalized the behavioral alteration and the effect was comparable to pre-ischemic treatment (2.5 mg/kg). Pre-ischemic treatment of TM-NM (2.5 mg/kg) protected the neurons from ischemic reperfusion injury by energy dependent EAA regulation. It can be concluded from the study that, even though the pre- and post-treatment of TM-NM show similar results, the post-ischemic treatment of TM-NM combination is beneficial due to better EAA control. Since hypertension is the primary risk factor for stroke, clinical incidents of stroke in hypertensive patients receiving angiotensin receptor blockers (ARBs) can be further investigated to understand the present study in the clinical situation.

The Neuroprotective Effects of Carnosine in Early Stage of Focal Ischemia Rodent Model

OBJECTIVE: This study was conducted to elucidate neuroprotective effect of carnosine in early stage of stroke. METHODS: Early stage of rodent stroke model and neuroblastoma chemical hypoxia model was established by middle cerebral artery occlusion and antimycin A. Neuroprotective effect of carnosine was investigated with 100, 250, and 500 mg of carnosine treatment. And antioxidant expression was analyzed by enzyme linked immunosorbent assay (ELISA) and western blot in brain and blood. RESULTS: Intraperitoneal injection of 500 mg carnosine induced significant decrease of infarct volume and expansion of penumbra (p<0.05). The expression of superoxide dismutase (SOD) showed significant increase than in saline group in blood and brain (p<0.05). In the analysis of chemical hypoxia, carnosine induced increase of neuronal cell viability and decrease of reactive oxygen species (ROS) production. CONCLUSION: Carnosine has neuroprotective property which was related to antioxidant capacity in early stage of stroke. And, the oxidative stress should be considered one of major factor in early ischemic stroke.

Achyrocline satureioides (Lam.) DC. (marcela) reduces brain damage in permanent focal ischemia in rats / Achyrocline satureioides (Lam.) DC. (marcela) reduce el daño cerebral en la isquemia focal permanente en ratas

Introduction: Achyrocline satureioides is a plant which has been widely used in popular medicine and experimental studies confirm its antioxidant and anti-inflammatory effects, attributable to the presence of flavonoids, mainly quercetin. Objectives: to evaluate the neuroprotective effects of a chronic oral pre-administration to rats with an Achyrocline satureioides decoction (2 %). Methods: for decoction, dried flowers of Achyrocline satureioides were used. The consumption of food and AS decoction/water of the rats was evaluated daily and weight gain weekly; quercetin content in the decoction and in the plasma of the rats was evaluated by high performance liquid chromatography. The cerebral damage was assessed with a tetrazolium salt (TTC) and a behavioral test was performed previously. Nissl staining and Fluoro-Jade histochemistry were used. Results: the pre-treatment with Achyrocline satureioides in all groups reverted the functional deficit and, during 21 days, the infarction volume also decreased significantly. Nissl staining showed a higher percentage of preserved neuronal population and the Fluoro-Jade showed a decreased of the neurons in degeneration. Conclusions: the quercetin levels in the decoction and plasma of rats could explain the preventive benefits of Achyrocline satureioides due to the antioxidant and anti-inflammatory properties described for this flavonoid.

Introducción: Achyrocline satureioides es una planta que ha sido ampliamente utilizada en la medicina popular y los estudios experimentales confirman sus efectos antioxidantes y antiinflamatorios, atribuibles a la presencia de flavonoides, principalmente quercetina. Objetivos: evaluar los efectos neuroprotectores de la pre-administración oral crónica a ratas con una decocción de Achyrocline satureioides 2 %. Métodos: para la decocción se utilizaron flores secas de Achyrocline satureioides. Se cuantificaron, diariamente, el consumo de alimentos, la decocción y el agua; y cada semana, la ganancia de peso. El contenido de quercetina en la decocción y en el plasma de las ratas se evaluó utilizando la técnica de cromatografía líquida de alta resolución. El daño cerebral se cuantificó con una sal de tetrazolio y antes se realizó una prueba de comportamiento. Se utilizaron la tinción de Nissl y el fluoro Jade. Resultados: el pretratamiento con Achyrocline satureioides en todos los grupos revirtió el déficit funcional, y la decocción durante 21 días también decreció de modo significativo el volumen del infarto. La tinción de Nissl mostró alto porcentaje de población neuronal conservada y el fluoro Jade presentó un decrecimiento en las neuronas en degeneración. Conclusiones: los niveles de quercetina en la decocción y el plasma de las ratas podrían explicar los beneficios preventivos de Achyrocline satureioides, debido a las propiedades antioxidantes y antiinflamatorias descritas para este flavonoide.

Hyperthermia induced after recirculation triggers chronic neurodegeneration in the penumbra zone of focal ischemia in the rat brain

Chronic neurodegenerative processes have been identified in the rat forebrain after prolonged survival following hyperthermia (HT) initiated a few hours after transient global ischemia. Since transient global ischemia and ischemic penumbra share pathophysiological similarities, this study addressed the effects of HT induced after recirculation of focal brain ischemia on infarct size during long survival times. Adult male Wistar rats underwent intra-luminal occlusion of the left middle cerebral artery for 60 min followed by HT (39.0-39.5°C) or normothermia. Control procedures included none and sham surgery with and without HT, and middle cerebral artery occlusion alone. Part I: 6-h HT induced at recirculation. Part II: 2-h HT induced at 2-, 6-, or 24-h recirculation. Part III: 2-h HT initiated at recirculation or 6-h HT initiated at 2-, 6- or 24-h recirculation. Survival periods were 7 days, 2 or 6 months. The effects of post-ischemic HT on cortex and striatum were evaluated histopathologically by measuring the area of remaining tissue in the infarcted hemisphere at -0.30 mm from bregma. Six-hour HT initiated from 6-h recirculation caused a significant decrease in the remaining cortical tissue between 7-day (N = 8) and 2-month (N = 8) survivals (98.46 ± 1.14 to 73.62 ± 8.99 percent, respectively). When induced from 24-h recirculation, 6-h HT caused a significant reduction of the remaining cortical tissue between 2- (N = 8) and 6-month (N = 9) survivals (94.97 ± 5.02 vs 63.26 ± 11.97 percent, respectively). These data indicate that post-ischemic HT triggers chronic neurodegenerative processes in ischemic penumbra, suggesting that similar fever-triggered effects may annul the benefit of early recirculation in stroke patients over the long-term.

Correlation Between Neuronal Apoptosis and Expression of Inducible Nitric Oxide Synthase after Transient Focal Cerebral Ischemia

BACKGROUND: Neuronal death in acute-phase cerebral ischemic injury is caused by necrosis. However, neuronal injury after reperfusion can be associated with apoptosis. METHODS: We used Sprague-Dawley rats whose brains were reperfused after middle cerebral artery occlusion for either 30 min or 2 h. We examined a relationship between apoptosis and the expression of inducible nitric oxide synthase (iNOS) in the brain tissue from 3 h to 14 days after reperfusion in both groups. RESULTS: TUNEL and iNOS positivity were closely related in both groups. The 2-h ischemia group exhibited increases in the amount of TUNEL and iNOS-positive cells for up to 3 days after reperfusion, at which the TUNEL and iNOS-positive cells decreased. The 30-min ischemia group exhibited peak positivity 24 h after reperfusion, followed by a similar decrease. iNOS mRNA expression peaked 3 h after reperfusion in the 30-min ischemia group, at which time it decreased. In the 2-h ischemia group, iNOS mRNA increased 3 h after reperfusion, peaked 24 h after reperfusion, and then decreased. CONCLUSION: These results indicated the occurrence of delayed apoptosis in transient cerebral ischemia. Increased expression of iNOS is closely associated with this apoptosis, and oxygen free radical-producing materials, such as nitric oxide, may play an important role in the induction of this apoptosis.

The Effect of Albumin Therapy for Reperfusion Injury Following Transient Focal Cerebral Ischemia in Rats

OBJECTIVE: Albumin is a very useful drug for the improving of cerebral blood volume and the oncotic effect in cerebral ischemia or cerebral vasospasm. The purpose of this study was to examine the morphological and neurological effect of albumin therapy on reperfusion injury following transient focal cerebral ischemia. MATERIALS AND METHODS: 18 Male Sprague-Dawley rats weighing 270-320g were used. The ischemia model was produced by 2-hour period of transient middle cerebral artery occlusion with a poly-L-lysin coated intraluminal suture. The agent(20% human serum albumin[HSA]) or control solution(NaCl 0.9%) was administered intravenously at a dosage of 1% of body weight immediate after reperfusion following a 2-hour period occlusion. Neurological function was evaluated by the postural reflex and the forlimb placing test during occlusion(at 60 min) and daily for 3 days thereafter. The brain was perfusion-fixed, and infarct volumes and brain edema were measured. RESULTS: The HSA significantly improved the neurological score in treated group. The rats of albumin treatment group showed significantly reduced total infarct volume(by 34%) and brain edema(by 81%) compared with saline-treated rats. CONCLUSION: HSA showed a substantial effect on the transient focal cerebral ischemia and reperfusion injury model. These results may indicate its usefulness in treating reperfusion injury patients after thrombolysis treatment for the thrombo-embolic major cerebral artery occlusions.

Neuroprotective Effect of Focal Ischemic Preconditioning in Transient Focal Ischemia Animal Model

BACKGROUND: The term 'ischemic preconditioning', which implies the first, brief, sublethal ischemia before the next ischemia, is widely accepted to have protective effect in the myocardium, and recently with a specified circumstances, in the brain also. However, the existence of this 'ischemic tolerance' phenomenon is not yet clarified in the repeated transient focal cerebral ischemia model. This study was performed to test whether the ischemic preconditiong has protective effect also in this TIA-mimicking condition. METHODS: Using intraluminar suture technique, initial transient focal ischemia was maintained for 30 minutes in the rat brain. After this ischemic preconditioning, second ischemia of 120 minutes duration was performed using the same method at 1, 3, 5, and 7 days after the 1st ischemia (n=20). The resulting brain infarct volume was assessed and compared to that of previously sham-operated paired controls(n=20). RESULTS: Using the infarct volume as parameters, there was no significant difference between the ischemia and control group in all pairs. But when the percent of infarct volume compared to the hemispheric volume was used instead, neocortical infarct percent was significantly smaller at day 3 after preconditiong (p<0.05). But such difference was not found at 1, 5, and 7th day in the neocotex. Neither the percent of total infarct nor the subcortical infarct showed any statistical difference. CONCLUSION: It could be concluded that transient focal cerebral ischemic preconditioning have neuroprotective effect. The optimal interval between ischemia for this 'ischemic tolerance' to happen is 3 days, and this phenomenon seems to be the function of cerebral cortex, but not the subcortex.

The Significance of Induction of Heat Shock Protein-70 and Glial Fibrillary Acidic Protein Messenger RNA by Delayed Postischemic Hyperthermia Following Transient Focal Ischemia

The heat shock protein, especially 70 class(HSP-70) and glial fibrillary acidic protein have been postulated to participate either in the injury process or in adaptive or protective tissue responses after induction by injurious events. We studied to find out the relationships between the regional expression of message for heat shock protein and glial fibrillary acidic protein and cellular survival in the repetitive stress/injury model. Sixty minutes of transient middle cerebral artery occlusion was produced in rats by insertion of an intraluminal filament. Twenty-four hours after reperfusion, awaken rats were subjected to normothermic(37-38degreesC) or hyperthermic(40degreesC) temperature modulation for 3 hours in a heating chamber. At times of either 2 or 24 hours after temperature modulation, brains were examined for mRNA expression of heat shock protein and glial fibrillary acidic protein by dot blot method. Four regions of interest were chosen: fronto-cingulate, sensorimotor, and piriform-insular cortex; and caudoputamen. The analysis demonstrated that HSP-70 at 2 hours after temperature modulation was expressed at low levels in all groups. However, at 24 hours, HSP-70 mRNA expression was upregulated by hyperthermia alone in frontocingulate(penumbral) cortex, and to an equal degree by sham hyperthermia, ischemic normothermia and ischemic hyperthermia in regions of core-ischemia. Moreover, HSP-70 mRNA was expressed unevenly in the same territory of infarction. Ischemic stress induced marked glial fibrillary acidic protein expression in both penumbra and ischemic core regions; in contrast, hyperthermic stress failed to make any significant difference between animal groups. Consequently, these data suggest that heat shock protein and glial fibrillary acidic protein upregulation after stress/injury are unlikely to be involved in cellular survival and to reflect injury severity also.

Expression of Immediate Early Genes Messenger RNAs in Focally Ischemic Rat Brain Following Delayed Postischemic Hyperthermia

Previous studies have indicated that hyperthermic modulation in the delayed postischemic period, even when occurring days after an ischemic insult, may augment ischemic injury. In order to evaluate the molecular mechanisms of the detrimental effects of hyperthermia on ischemic outcome, we performed a study to assess the effects of delayed postischemic hyperthermia on the regional expression of message for the immediate early genes c-fos and c-jun. 60 minutes of transient middle cerebral artery occlusion was produced in rats by insertion of an intraluminal filament. 24 hours after reperfusion, rats which were awake were subjected to normothermic(37-38degreesC) or hyperthermic(40degreesC) temperature modulation for 3 hours in a heating chamber. Either 2 or 24 hours after temperature modulation, brains were examined for mRNA expression of c-fos and c-jun by the dot blot method. Four regions of interest were chosen: fronto-cingulate, sensorimotor, piriform-insular cortex, and caudoputamen. Dot blot analysis revealed that c-fos mRNA at 2 hours after temperature modulation was significantly upregulated in the ipsilateral fronto-cingulate cortex-the site of the ischemic penumbra- in rats exposed to ischemia+delayed hyperthermia, in comparison to rats exposed to sham+normothermia(one-way ANOVA with post-hoc Bonferroni's test, p<0.05). In contrast, c-jun mRNA was significantly upregulated by ischemia+delayed hyperthermia within regions of core ischemia-i.e., sensorimotor and piriform-insular cortex, and caudoputamen. These findings have extended those of our earlier histopathological study10) to show that a significant increase in c-fos or c-jun expression may be closely linked to cellular survival or death after delayed moderate hyperthermia following ischemia.

Effect of stimulation of cerebellar fastigial Nucleus on focal Cerebral Infaretion in Rat

Electrical stimulation of the cerebellar fastigial nucleus(FN) increases cerebral blood flow(CBF) and reduces brain damage after focal cerebral ischemia. The authors studied whether the neuroprotection elicited from electrical stimulation of the cerebellar FN is attibutable to the elevation in regional CBF(rCBF) or reduction in release of excitatory amino acid sprague-Dawley rats were anesthtized with a mixture of halothane(3% for the indurction and 1% for maintenance) and oxygen and artificially ventilated through a tracheal cannula. Arterial pressure, blood gases and body temperature were monitored. The middle cerebral artery(MCA) was occluded distal to the lenticulostriate branches. The FN was then for 2 hours, over the regions corresponding to the ischemic core and penumbra. Postiischemic release of glutamate and aspartate were measured by microdialysis for 2 hours at the same site of measurement of rCBF. Infarct volume was determined 8 hours later in 2,3,5-triphenyl tetrazolium chloride(TTC)-stained sections FN stimulation(n=12) increased mean arterial pressure by 28+/-16mmHg. In nonstimulated control rats(n=12), mean AP was not changed significantly during the experimental procedures. Compared with nonstimulated animal, stimulation of FN for 1 hour following MCA occlusion siginficantly increased rCBF in ischemic core and penumbra by 53.6% and 67.6% respectively. And the volume of infarction decreased by 42% at 8 hours after MCA occlusion. The concentration of glutamate and aspartate in ischemic core after MCA occlusion increased both in the control group(to 12.2+/-3.3 folds and 10.4+/-4.1 folds respectively) and in the stimulation group(10.5+/-2.8 and 11.2+/-4.1 folds, respectively). The concentration of glutamate and aspartate in penumbra did change significantly neither in the control group(to 2.5+/-1.3 folds and 1.8+/-0.6 folds respectively) nor in the stimulation group(1.9+/-0.5 folds and 2.1+/-0.4 folds, respectively). There was no significant difference between the two groups.