Stroke-prone Renovascular Hypertensive Rats Research Articles

Neurogenesis and Angiogenesis within the Ipsilateral Thalamus with Secondary Damage after Focal Cortical Infarction in Hypertensive Rats

Neurogenesis and angiogenesis in the subventricular zone and peri-infarct region have been confirmed. However, newly formed neuronal cells and blood vessels that appear in the nonischemic ipsilateral ventroposterior nucleus (VPN) of the thalamus with secondary damage after stroke has not been previously studied. Twenty-four stroke-prone renovascular hypertensive rats were subjected to distal right middle cerebral artery occlusion (MCAO) or sham operation. 5'-Bromo-2'-deoxyuridine (BrdU) was used to label cell proliferation. Rats were killed at 7 or 14 days after the operation. Neuronal nuclei (NeuN), OX-42, BrdU, nestin, laminin(+), BrdU(+)/nestin(+), BrdU(+)/NeuN(+), nestin(+)/GFAP(+)(glial fibrillary acidic protein), and BrdU(+)/laminin(+) immunoreactive cells were detected within the ipsilateral VPN. The primary infarction was confined to the right somatosensory cortex. Within the ipsilateral VPN of the ischemic rats, the number of NeuN(+) neurons decreased, the OX-42(+) microglia cells were activated, and BrdU(+) and nestin(+) cells were detected at day 7 after MCAO and increased in number at day 14. Moreover, BrdU(+)/nestin(+) cells and BrdU(+)/NeuN(+) cells were detected at day 14 after MCAO. In addition, the ischemic rats showed a significant increase in vascular density in the ipsilateral VPN compared with the sham-operated rats. These results suggest that secondary damage with neurogenesis and angiogenesis of the ipsilateral VPN of the thalamus occurs after focal cortical infarction.

Enhanced angiogenesis with dl-3n-butylphthalide treatment after focal cerebral ischemia in RHRSP

Appropriate restoration of blood flow via angiogenesis is critical for the recovery from ischemic stroke. Previously, we reported that treatment with dl-3n-butylphthalide (NBP) increases the number of local potent cerebral microvessels. However, the underlying mechanism remained unclear. The present study was conducted to test whether NBP enhances post-ischemic cerebral angiogenesis via vascular endothelial growth factor (VEGF) and hypoxia induced factor-1α (HIF-1α). Stroke-prone renovascular hypertensive rats (RHRSP) were used to create middle cerebral artery occlusion (MCAO) model. NBP was given 80 mg/kg per d for 10 consecutive days, starting 12, 24, 48 and 72 h respectively after MCAO. Neurological function was assessed daily and infarct volume as well as the expressions of CD31, VEGF, HIF-1α and bFGF was detected 13 days after MCAO. The administration of NBP starting within 24 h after MCAO enhanced recovery of neurobehavioral function, reduced infarct volume, increased the quantity of CD31 positive vessels, and up-regulated expressions of VEGF and HIF-1α. These findings suggest that treatment with NBP within 24 h post-ischemic stroke rescues brain tissue by enhancing angiogenesis associated with up-regulation of VEGF and HIF-1α expressions.

Exogenous kallikrein enhances neurogenesis and angiogenesis in the subventricular zone and the peri-infarction region and improves neurological function after focal cortical infarction in hypertensive rats

Kallikrein, a serine proteinase, has been identified as an angiogenic growth factor recently. We investigated whether delayed treatment with exogenous kallikrein enhances neurogenesis and angiogenesis after focal cortical infarction in stroke-prone renovascular hypertensive rats. Human tissue kallikrein (1.6 × 10 − 2 PNAU/kg) or vehicle was given through a tail vein daily for 6 consecutive days starting 24 h after distal middle cerebral artery occlusion (MCAO). Cell proliferation was examined by using 5′-bromo-2′-deoxyuridine (BrdU, 50 mg/kg). Rats were sacrificed at 3, 7, 14 or 28 d after MCAO, respectively. Treatment with kallikrein significantly increased the number of BrdU + cells in the subventricular zone (SVZ) and the peri-infarction region initiating 3 d after MCAO compared with the vehicle group (all p < 0.05). Kallikrein significantly increased the number of BrdU +/DCX + cells and BrdU +/nestin + cells in the SVZ as well as vascular density in the peri-infarction region compared with the vehicle group (all p < 0.05), which increased at 3 d, peaked at 7–14 d after MCAO, and then gradually decreased. Kallikrein markedly increased the number of BrdU +/NeuN + cells in the peri-infarction region compared with the vehicle group at 14 d and 28 d after MCAO (all p < 0.05). The kallikrein group showed better functional improvement after stroke (all p < 0.05). Our study demonstrates that delayed administration of kallikrein at 24 h after cortical infarction promotes the SVZ neuroblasts proliferation, migration, and selective differentiation. Moreover, kallikrein enhanced endogenous neurogenesis is associated with angiogenesis, both attributing to functional improvement after stroke. Therefore, kallikrein may have a potential therapeutic perspective on ischemic stroke.

Delayed Decompressive Craniectomy Improves the Long-term Outcomes in Hypertensive Rats with Space-occupying Cerebral Infarction

No experimental data has been published on the long-term effects of decompressive craniotomy in hypertensive rats with space-occupying cerebral infarction. The aim of the present study was to investigate the efficacy of decompressive craniectomy in a middle cerebral artery occlusion (MCAO) model of hypertensive rats in a prolonged period. Totally 92 stroke-prone renovascular hypertensive rats (RHRSP) were subjected to left MCAO by an endovascular occlusion technique. The decompressive craniectomy was performed on 26 RHRSP at 1 and 24 h after MCAO, respectively. Infarct volume, neurological performance, and mortality were evaluated at 1, 2, 4, and 8 weeks after MCAO. The mortality was reduced from 52.5% in controls to 7.7% and 23.1% in the rats underwent craniectomy at 1 and 24 h after MCAO, respectively (P < 0.05, respectively). All of the treated rats presented smaller infarct volume from 1 week to 8 weeks and better neurological performance at 4-8 weeks after MCAO compared to the controls (P < 0.05, respectively). The craniectomy at early stage was more effective than that at late stage in reducing infarct volume and improving neurological performances at 1 and 2 weeks (P < 0.05, respectively). However, there was no significant difference in infarct volume and neurological scores between the treated groups of rats at 4 and 8 weeks after MCAO (P > 0.05). Although the early craniectomy is more effective than delayed craniectomy in improving short-term outcome, the latter has the similar beneficial effects as early craniectomy on long-term outcome in hypertensive rats with space-occupying cerebral infarction.

Nogo-A is involved in secondary axonal degeneration of thalamus in hypertensive rats with focal cortical infarction

We investigate whether Nogo-A is involved in the secondary axonal degeneration in the thalamus after distal middle cerebral artery occlusion (MCAO) in stroke-prone renovascular hypertensive rats (RHRSP). The expression of Nogo-A in ipsilateral ventroposterior nucleus (VPN) of the thalamus in RHRSP was observed at 1, 2 and 4 weeks after distal MCAO. In addition, intracerebroventricular infusion of NEP1-40, a Nogo-66 receptor (NgR) antagonist peptide, was administered starting 24 h after MCAO and continued for 1, 2 and 4 weeks, respectively. Axonal damage and regeneration were evaluated by analysis of the immunoreactivity (IR) of amyloid βA4 precursor protein (APP), growth associated protein 43 (GAP-43) and microtubule associated protein 2 (MAP-2) in ipsilateral VPN of the thalamus at 1, 2 and 4 weeks after distal MCAO. Following ischemia, the expression of Nogo-A in oligodendrocytes increased persistently and its localization became redistributed around damaged axons and dendrites. Administration of NEP1-40 downregulated the expression of Nogo-A, reduced axonal injury and enhanced axonal regeneration. Our data suggest that Nogo-A is involved in secondary axonal degeneration and that inhibition of Nogo-A can reduce neuronal damage in the thalamus after distal MCAO.

INHIBITION OF NAD(P)H OXIDASE REDUCES FIBRONECTIN EXPRESSION IN STROKE‐PRONE RENOVASCULAR HYPERTENSIVE RAT BRAIN

1. The aim of the present study was to test the hypothesis that in vivo chronic inhibition of NAD(P)H oxidase reduces cerebrovascular fibronectin expression in stroke-prone renovascular hypertensive rats (RHRSP). 2. The RHRSP model was induced by two clips and NAD(P)H oxidase was inhibited with apocynin. The mRNA and protein expression of NAD(P)H oxidase subunit p22(phox) in brains of RHRSP and Sprague-Dawley (control) rats was determined using real-time reverse transcription-polymerase chain reaction, western blot and immunohistochemistry. The expression of fibronectin protein was localized immunohistochemically in cerebral vessels and then quantified by western blot. 3. Cerebrovascular fibronectin levels in RHRSP (n = 6) were significantly higher than control (n = 5) levels 8 weeks after operation (1.29 +/- 0.04 vs 1.15 +/- 0.02, respectively; P = 0.007). The p22(phox) immunopositive reactivity was localized in the cerebral vasculature of control rats and RHRSP. Furthermore, chronic treatment of RHRSP with apocynin, a selective NAD(P)H oxidase inhibitor, in the drinking water for 4 weeks (1.5 mmol/L, 5 weeks after operation) resulted in a significant decrease in the expression of p22(phox) protein (0.85 +/- 0.01 vs 0.93 +/- 0.01 in non-treated RHRSP; n = 5; P = 0.002), with a concomitant reduction of fibronectin levels in the cerebral vasculature (1.31 +/- 0.03 vs 1.56 +/- 0.05 in non-treated RHRSP; n = 5; P = 0.002). No significant differences were detected in the expression of p22(phox) mRNA and protein between RHRSP (4 and 8 weeks after renal artery constriction) and the control group. 4. These findings suggest that the chronic inhibition of NAD(P)H oxidase in vivo by apocynin reduces cerebrovascular fibronectin levels, which may lessen hypertensive cerebrovascular fibrosis.

EphB2-Fc promotes activation of endogenous neural stem cells after cerebral cortex infarction: experimental with hypertensive rats

To explore the effects of intraventricular injection of EphB2-Fc on activation of inherent neural stem cells after cerebral cortex infarction. Stroke-prone renovascular hypertension model was established in 96 SD rats by two-kidney, two-clip method. Middle cerebral artery occlusion (MCAO) model was established in 72 of these 96 stroke-prone renovascular hypertensive rats and the other 24 rats were used as sham operation group. Then the 72 rats were randomly divided into 3 equal groups: cerebral infarction group without any treatment after the MCAO, MCAO + EphB2-Fc group undergoing stereotaxical infusion of EphB2-Fc at the dose of 20 microl x 200 microg/ml into the lateral ventricle 4 days after the distal ligation of right middle cerebral artery, and MCAO + IgG-Fc group undergoing stereotaxical infusion of IgG-Fc at the dose of 20 microl x 200 microg/ml into the lateral ventricle 4 days after the distal ligation of right middle cerebral artery. By the ends of the first and fourth weeks after the MCAO procedure 12 rats from each group were killed and their brains were taken out to undergo in situ hybridization, immunohistochemistry and Western blotting analysis in order to determine the expression of EphB2 protein and mRNA, nestin and polysialic acid-neural cell adhesion molecule (PSA-NCAM). One week after the distal ligation of right middle cerebral artery, the EphB2 protein and mRNA expression levels in the ipsilateral cortex and subventricular zone (SVZ) of the cerebral infraction group were both lower than those of the sham operation group (P < 0.05), such levels of the MCAO + EphB2-Fc group were higher than those of the MCAO + IgG-Fc group (both P < 0.05), but there was no significant difference between the cerebral infraction group and IgG-Fc group (both P > 0.05), and there were no differences in such levels between the cerebral infarction group and MCAO + IgG-Fc group (both P > 0.05); the nestin and PSA-NCAM expression levels in the ipsilateral SVZ of the cerebral infraction group were both higher than those of the sham operation group (both P < 0.05), such levels of the MCAO + EphB2-Fc group were both higher than those of the MCAO + IgG-Fc group (both P < 0.05), and migration of PSA-NCAM positive cells to corpus callosum could be seen. Four weeks after, there were no significant differences in the expression levels of EphB2 protein and mRNA among different groups (all P > 0.05), the nestin and PSA-NCAM expression levels in the ipsilateral SVZ decreased in all groups, there were no significant differences in the expression of nestin among all groups, but the PSA-NCAM expression in the ipsilateral SVZ of the cerebral infraction group was still higher than that of the sham operation group. Disruption of EphB2 signal promotes the proliferation and migration of endogenous neural stem cells in the SVZ after cerebral cortex infarction.

In vivo chronic inhibition of NADPH oxidase reduces cerebrovacular fibronectin expression in stroke-prone renovascular hypertensive rats

In vivo chronic inhibition of NADPH oxidase reduces cerebrovacular fibronectin expression in stroke-prone renovascular hypertensive rats

Two-kidney, two clip renovascular hypertensive rats can be used as stroke-prone rats.

The cerebrovascular lesions in stroke-prone spontaneously hypertensive rats are not only dependent on high blood pressure but partly related to pressure-independent genetic factors. The aim of the present study was to observe whether spontaneous stroke occurred in renovascular hypertensive rats without a genetic deficiency. The 1-kidney, 1 clip (1k1c); 2-kidney, 1 clip (2k1c); and 2-kidney, 2 clip (2k2c) methods were used to induce hypertension in male Sprague-Dawley rats with a ring-shaped silver clip. Sham-operated rats were used as controls. Blood pressure and neurological symptoms were observed in the rats without any artificial inducement. Brain sections stained with hematoxylin-eosin and phosphotungstic acid-hematoxylin were examined under a microscope to determine stroke foci. The attack rate of stable hypertension was 100% (55/55) in the 2k2c group, which was significantly higher than that in the 1k1c (23/30, 76.7%) and 2k1c (21/30, 70%) groups (P<0.01). None of the rats in the 2k2c group died of acute renal failure or suffered from diffuse cerebral lesions postoperatively. Forty weeks after renal artery constriction, the incidence of spontaneous stroke in the 2k2c group was 61.8% (34/55), which was significant higher than that in the 1k1c (7/30, 23.3%) and 2k1c (5/30,16.7%) groups (P<0.01). Stroke foci were not observed in normotensive controls. We conclude that 2k2c renovascular hypertensive rats with proper renal artery constriction can be used as stroke-prone renovascular hypertensive rats independent of a genetic deficiency.