Study To The Effect Of Pretreatment With Irbesartan On The Neuroprotection In Rat With Cerebral/reprefusion Injury

Study to the effect of pretreatment with Irbesartan on the neuroprotection in rat with cerebral /reprefusion injurySUMMARYIschaemic stroke is associated with significant morbidity and mortality. Few effective treatments are available once stroke has occurred; Only the therapeutic approach, showing improved neurological function, has been that of thrombolysis with recombinant tissue plasminogen activator (rtPA). The restrictions on time and potential adverse effects have limited the use of rtPA to approximately 2%-5% of stroke patients in the United States. Moreover, although thrombolysis treatment can recover blood supply, which cannot provent ischemia-reperfusion injury. To prevent or reduce ischemic brain damage, the prophylaxis treatment regimen should be put the first step. Secondly, development of new interventions geared toward a wider therapeutic window is necessary to meet the large need for this important and undertreated disorder.Biochemical, physiological and functional studies suggest that the brain renin-angiotensin system (RAS) is regulated independently of the peripheral RAS. The effector peptide of the RAS, angiotensinâ…¡(Angâ…¡) binds at least to two receptor subtypes, referred to as the angiotensinâ…¡type 1 receptor (AT₁R) and the angiotensinâ…¡type 2 receptor (AT₂R) . During the past decade, a number of studies have indicated that the brain RAS may be involved in the initiation and regulation of processes occurring during and after brain ischaemia. Selective non-peptide AT₁R blockers have been shown to inhibit both peripheral and brain AT₁R. In experimental studies, angiotensinâ…¡type 1 receptor blockers (ARB) , applied systemically before or after stroke, decrease mortality, ischemic area and neurologic deficit after acute ischaemic stroke. These experimental results have been corroborated by recent major clinical study, such as the Life trial, Access study and Jikei Heart Study. This benefit may be BP-independent and non AT₁R-mediated, suggesting an important role for AT₂R stimulation in cerebroprotection, involving both neuronal and vascular protection. However, the physiologic and pathophysiologictive properties of Angâ…¡via its receptor in the brain are only poorly understood. There is still an enigma concerning the mechanisms of ARB modulating such pathologic conditions. To get more information about the molecular regulating role of pretreatment with ARB in neuroprotective phenomenon against ischemic-induced injury, we investigated the influences of pretreatment with ARB, Irbesartan on apoptosis, angiogenesis and neurogenesis in the rats with cerebral ischemic/ reperfusion injury. Our studies include the following three parts. Partâ… Changes of the rennin-angiotensin system in rat brain withfocal ischemia- reperfusion injury and the effects ofchronic pretreatment with Irbsartan on itObjective To explore the changes of the rennin-angiotensin system in rat brain with focal ischemia-reperfusion injury and the neuroprotective effect and mechanisms of chronic pretreatment with irbsartan. Methods The male SD rats were randomly assigned to sham operated group, ischemia group and Irbsartan pretreatment group( 30mg/kg.d, for 3 weeks). The focal ischemia-reperfusion (IR) model was made by suture occlusion of right middle cerebral artery (MCAO). At 24h and 72h following IR, the nerve function scores and the infarction volume were evaluated, the mRNA expressions of angiotensin II type 1 receptor (AT₁R) and angiotensinâ…¡type 2 receptor (AT₂R) were detected by RT-PCR, Angâ…¡levels and Renin activity were examined by radioimmuno-assay. Results (1) Pretreatment with Irbsartan can significantly improved the neurological outcome and reduced the infarction size. (2) In bilateral cerebral cortex, hypothalamus, brain stem and peripharial blood cell, the mRNA expressions of the AT₁R and AT₂R were significantly increased after either 24h or 72h of IR (all P < 0. 01); AT₁R mRNA expression was downregulated, while AT₂R mRNA expression was upregulated by Irbsartan pretreatment in the above-mentioned content. (3) Compared with those in the sham group, the levels of angiotensinâ…¡in brain and peripherial blood as well as renin in peripherial blood in ischemia group were significantily elevated 24h and 72h after reperfusion, but there was no significant diffierence between Irbsartan group and ischemia group. Conclusions The AngII and expression of AT₁R and AT₂R were elevated in rat brain with focal IR. The pretreatment with Irbsartan has a protective effect on ischemic brain injury, which may be attributed to blocking AT₁R, downregulating AT₁R mRNA expressions and upregulateing AT₂R mRNA expression.Partâ…¡Effects of pretreatment with Irbensartan on neurocyte apoptosis and its molecular regulation followingcerebral ischemic in ratObjective To get more information on the molecular regulating role of pretreatment with ARB in ischemic-induced apoptosis, we examined the influence of ARB, Irbesartan on different regulators of apoptosis, bcl-2/bax, BDNF, VEGF , CREB/p-CREB and Cleaved Caspase-3 in the rats with cerebral ischemic/reperfusion (IR) injury. Methods The male SD rats were randomly assigned to sham group, Irbsartan sham group , MCAO group and Irbsartan pretreatment + MCAO group (Irb- MCAO group). The focal IR model was made by suture MCAO. At 24h , 72h and 7d following IR, the rat was killed, the apoptosis neurocyte was identified by the TUNEL method. The mRNA expressions of bcl-2 and box were detected by RT-PCR. The levels of the protein of Bal-2, Bax, Cleaved Caspase-3, BDNF, VEGF, CREB andp-CREB in peri-infarct were detected by Western blotting. Results TUNEL⁺ cells were predominnatly seen in the cortex at the border of infarct core. Compare to sham group, cerebral ischemia significantly induced neurocyte apoptosis and increasd the levels of cleaved caspase-3 at 24h, 72h and 7d after IR ( all P<0.01), and decreased the mRNA ratio of bcl-2/bax at 24h, 72h after IR (P<0.01, P<0.05). Meanwhile, cerebral ischemia also significantly induced anti- apoptotic protein BDNF, VEGF and p-CREB at early phase after IR, but no change was observed in neurocyte apoptosis and its molecular regulators in Irb-sham group. Compare to MCAO group, pretreatmen with Irbsartan could significantly inhibit neurocyte apoptosis from 24h to 7d, downregulated the level of cleaved caspase-3 and upregulated the ratio of bcl-2 mRNA /bax mRNA and p-CREB/ CREB as well as anti-apoptotic protein BDNF, VEG at 24h after IR. The ratio of Bcl-2 /Bax proteins were similar to those of bcl-2/bax mRNA. Conclusions Cerebarl isehemia signifienarly induced neurocyte apoptosis. Pretreatment with Irbsartan could supperss ischemia-induced neurocyte apoptosis in rat with cerebral ischemia/ repurfision injury, whose mechniasms were related to downregulating proapoptosis molecular caspase-3, upregulating the ratio of Bcl-2/Bax and p-CREB/ CREB, and anti-apoptosis moleculars of BDNF,VEGF via AT₂R signalling pathway. Part III Effects of pretreatment with Irbesartan on the angiogenesis and neurogenesis in rat with cerebral ischemic/reperfusion injuryObjective To investigate the effect of pretreatment with irbsartan on the angiogenesis and neurogenesis in rat with cerebral ischemic/reperfusion (I/R) injury. Methods Seventy-five adult male SD rats were randomly divided into sham operated group, MCAO group and irbsartan group. The focal I/R model was made by suture occlusion of right middle cerebral artery. Neurogenesis was detected by immunohistochemistry staining with Brdu at 3, 7, 14 and 21 days after reperfusion, and the proliferation of cerebral microvessels were identified by immunostaining with antibodies to CD31. Results (1) There were a small quantities of Brdu⁺ cells in DG of rats in sham operated group. The number of Brdu⁺ cells was increased bilaterally from 3 to 21d after I/R (peak at day 7). The number of Brdu⁺ cells in Irbesartan group respectively increased 50%(P < 0. 01),54% (P< 0. 01),44%(P < 0. 05) compared with those in MCAO group at 3, 7, 14 days after I/R, and a paralleled change was seen in the contralateral DG at 3 and 7d. (2) There were a small amount of CD31⁺ cells in peri-infarct of sham operated group. The numbers of CD31⁺cells in MCAO group boundary increased in peri-infarct at 3 days after I/R. In irbsartan pretreated rats the numbers of CD31⁺ cells in peri-infarct were much more than those in rats of MCAO group ( P < 0. 01). Conclusions Pretreatment with irbsartan can promote proliferation of endogenous NSC in DG of hippocampus and microvessels in peri-infarct in the rat with cerebral I/R injury.