Stroke-prone Renovascular Hypertensive Rats Research Articles

Cerebral Perfusion Characteristics and Dynamic Brain Structural Changes in Stroke-Prone Renovascular Hypertensive Rats: A Preclinical Model for Cerebral Small Vessel Disease.

Hypertension is a leading cause of cerebral small vessel disease (CSVD) and vascular dementia in elderly individuals. We aimed to assess cerebral perfusion and dynamic changes in brain structure in stroke-prone renovascular hypertensive rats (RHRSPs) with different durations of hypertension and to investigate whether they have pathophysiological features similar to those of humans with CSVD.The RHRSP model was established using the two-kidney, two-clip (2k2c) method, and the Morris water maze (MWM) test, MRI, immunohistochemistry, and biochemical analysis were performed at multiple time points for up to six months following the 2k2c operation.Systolic blood pressure was significantly greater in the RHRSP group than in the sham-operated group at week 4 post-surgery and continued to increase over time, leading to cognitive decline by week 20. Arterial spin labeling revealed cerebral hypoperfusion in the RHRSP group at 8 weeks, accompanied by vascular remodeling and decreased vessel density. Diffusion tensor imaging and Luxol fast blue staining indicated that white matter disintegration and demyelination gradually progressed in the corpus callosum and that myelin basic protein levels decreased. Eight weeks after surgery, blood-brain barrier (BBB) leakage into the corpus callosum was observed. The albumin leakage area was negatively correlated with the myelin sheath area (r=-0.88, p<0.001). RNA-seq analysis revealed downregulation of most angiogenic genes and upregulation of antiangiogenic genes in the corpus callosum of RHRSPs 24 weeks after surgery.RHRSPs developed cerebral hypoperfusion, BBB disruption, spontaneous white matter damage, and cognitive impairment as the duration of hypertension increased. RHRSPs share behavioral and neuropathological characteristics with CSVD patients, making them suitable animal models for preclinical trials related to CSVD.

Enhanced angiogenesis in the thalamus induced by a novel TSPO ligand ameliorates cognitive deficits after focal cortical infarction

Neuronal loss in the ipsilateral thalamus after focal cortical infarction participates in post-stroke cognitive deficits, and enhanced angiogenesis in the thalamus is expected to reduce neuronal damage. We hypothesize that novel translocator protein (TSPO) ligand, 2-Cl-MGV-1, can promote angiogenesis, attenuate neuronal loss in the thalamus, and ameliorate post-stroke cognitive deficits. Cortical infarction was induced by distal middle cerebral artery occlusion (dMCAO) in stroke-prone renovascular hypertensive rats. 2-Cl-MGV-1 or dimethyl sulfoxide was administered 24 h after dMCAO and then for 6 or 13 days. Spatial learning and memory were assessed using the Morris water maze. Neuronal loss, TSPO expression, angiogenesis, and intrinsic pathway were determined by immunofluorescence and immunoblotting 7 and 14 days after dMCAO. Cortical infarction caused post-stroke cognitive deficits and secondary neuronal loss with gliosis in the ipsilateral thalamus within 14 days of dMCAO. Increased angiogenesis and elevated expression of vascular TSPO were detected in the ipsilateral thalamus, and treatment with 2-Cl-MGV-1 enhanced angiogenesis by stimulating the PI3K-AKT-mTOR pathway. The effects of 2-Cl-MGV-1 on angiogenesis coincided with reduced neuronal loss in the thalamus and contributed to improvements in post-stroke cognitive deficits. Our findings suggest that 2-Cl-MGV-1 stimulates angiogenesis, ameliorates neuronal loss in the thalamus, and improves post-stroke cognitive deficits.

Toll-like receptors (TLR2 and TLR4) antagonist mitigates the onset of cerebral small vessel disease through PI3K/Akt/GSK3β pathway in stroke-prone renovascular hypertensive rats

ABSTRACT To examine the effect and mechanism of Toll-Like Receptors (TLR2, TLR4) antagonist in CSVD. The rat model of stroke-induced renovascular hypertension (RHRSP) was constructed. TLR2 and TLR4 antagonist was administrated by Intracranial injection. Morris water maze was used to observe the behavioral changes of rat models. HE staining, TUNEL staining and Evens Blue staining were performed to evaluate the permeability of the blood-brain barrier (BBB) and examine the CSVD occurrence and neuronal apoptosis. The inflammation and oxidative stress factors were detected by ELISA. Hypoxia-glucose-deficiency (OGD) ischemia model was constructed in cultured neurons. Western blot and ELISA were used to examine the related protein expression in TLR2/TLR4 signaling pathway and PI3K/Akt/GSK3β signaling pathway. The RHRSP rat model was successfully constructed, and the blood well and BBB permeability were altered. The RHRSP rats showed cogitative impairment and excessive immune response. After TLR2/TLR4 antagonist administration, the behavior of model rats were improved, cerebral white matter injury was reduced, and the expression of several key inflammatory factors including TLR4, TLR2, Myd88 and NF-kB were decreased, as well as the ICAM-1, VCAM-1, inflammation and oxidative stress factors. In vitro experiments showed that TLR4 and TLR2 antagonist increased the cell viability, inhibited the apoptosis, and decreased p-Akt and p-GSK3β expression. Moreover, the PI3K inhibitors resulted in decreased anti-apoptotic and anti-inflammatory effects of TLR4 and TLR2 antagonist. These results suggested that TLR4 and TLR2 antagonist achieved protective effect on the RHRSP through the PI3K/Akt/GSK3β pathway.

Mesenchymal Stem Cells Improve Cognitive Impairment and Reduce Aβ Deposition via Promoting AQP4 Polarity and Relieving Neuroinflammation in Rats With Chronic Hypertension-Induced Cerebral Small-Vessel Disease.

Cerebral small-vessel disease (CSVD) is the main cause of vascular cognitive impairment (VCI), and the accumulation of amyloid β-protein (Aβ) may be significantly involved in CSVD-induced VCI. The imbalance between Aβ production and clearance is believed to be an important pathological mechanism of Aβ deposition in Alzheimer disease. In this study, we aimed to disclose the roles of aquaporin 4 (AQP4) and neuroinflammation in CSVD, which were the key factors for Aβ clearance and production, respectively, and the effect of mesenchymal stem cells (MSCs) on Aβ deposition and these two factors. The stroke-prone renovascular hypertensive (RHRSP) rats were grouped and received MSC and MSC + AS1517499 (an inhibitor of pSTAT6). The latter was used to explore the underlying mechanism. The cognitive function, white matter lesions, Aβ expression, expression, and polarity of AQP4, neuroinflammation and the STAT6 pathway were investigated. Compared with sham-operated rats, RHRSP rats showed spatial cognitive impairment, white matter lesions and Aβ deposition. Moreover, AQP4 polarity disorder and neuroinflammatory activation were found, which were linked to Aβ deposition. Treatment with MSCs markedly improved cognitive tasks and reduced Aβ deposition but failed to reduce white-matter lesions. Furthermore, MSCs not only promoted AQP4 polarity but also alleviated neuroinflammation probably through the STAT6 pathway. The present study demonstrated that Aβ deposition, AQP4 polarity disorder and neuroinflammation might be involved in CSVD and the regulatory effects of MSCs on them suggested potential therapeutic value for CSVD.

Allisartan isoproxil reduces mortality of stroke-prone rats and protects against cerebrovascular, cardiac, and aortic damage.

Stroke is a common cause of death and disability. Allisartan isoproxil (ALL) is a new angiotensin II receptor blocker and a new antihypertensive drug discovered and developed in China. In the present study we investigated the therapeutic effects of ALL in stroke-prone renovascular hypertensive rats (RHR-SP) and the underlying mechanisms. The model rats were generated via two-kidney two-clip (2K2C) surgery, which led to 100% of hypertension, 100% of cerebrovascular damage as well as 100% of mortality 1 year after the surgery. Administration of ALL (30 mg · kg-1 · d-1 in diet, for 55 weeks) significantly decreased stroke-related death and prolonged lifespan in RHR-SP, but the survival ALL-treated RHR-SP remained of hypertension and cardiovascular hypertrophy compared with sham-operated normal controls. In addition to cardiac, and aortic protection, ALL treatment for 10 or 12 weeks significantly reduced cerebrovascular damage incidence and scoring, along with a steady reduction of blood pressure (BP) in RHR-SP. Meanwhile, it significantly decreased serum aldosterone and malondialdehyde levels and cerebral NAD(P)H oxidase expressions in RHR-SP. We conducted 24 h continuous BP recording in conscious freely moving RHR-SP, and found that a single intragastric administration of ALL produced a long hypotensive effect lasting for at least 12 h on systolic BP. Taken together, our results in RHR-SP demonstrate that ALL can be used for stroke prevention via BP reduction and organ protection, with the molecular mechanisms related to inhibition of angiotensin-aldosterone system and oxidative stress. This study also provides a valuable scoring for evaluation of cerebrovascular damage and drug efficacy.

Rosuvastatin Improves Cognitive Function of Chronic Hypertensive Rats by Attenuating White Matter Lesions and Beta-Amyloid Deposits.

Hypertensive white matter lesion (WML) is one of common causes of vascular cognitive impairment. In this study, we aimed to investigate the effect of rosuvastatin on cognitive impairment and its underlying mechanisms in chronic hypertensive rats. From the 8th week after establishment of stroke-prone renovascular hypertensive rats (RHRSPs), rosuvastatin (10 mg/kg) or saline as a control was administrated once daily for consecutive 12 weeks by gastric gavage. Cognitive function was assessed with the Morris water maze test and novel object recognition test. WML was observed by Luxol fast blue staining. Aβ deposits, Claudin-5, Occludin, and ZO-1 were determined by immunofluorescence. After rosuvastatin treatment, the escape latencies were decreased and the time of crossing the hidden platform was increased in the Morris water maze, compared with the vehicle-treated RHRSP group. In a novel object recognition test, the recognition index in the rosuvastatin-treated RHRSP group was significantly larger than that in the vehicle-treated RHRSP group. Rosuvastatin treatment presented with the effects of lower WML grades, higher expression of tight junction proteins Claudin-5, Occludin, and ZO-1 in the corpus callosum, and less Aβ deposits in the cortex and hippocampus. The data suggested that rosuvastatin improved the cognitive function of chronic hypertensive rats partly by attenuating WML and reducing Aβ burden.

Intermittent Theta Burst Stimulation Improves the Spatial Cognitive Function of Rats with Chronic Hypertension-induced Cerebral Small Vessel Disease

We investigated whether intermittent theta burst stimulation (iTBS) can improve the spatial cognitive function of rats with hypertension-induced cerebral small vessel disease. To prove our hypothesis, stroke-prone renovascular hypertensive rats (RHRSPs) were treated with iTBS beginning at postoperative week 22. The Morris water maze was performed to assess spatial cognitive function. The expression of the N-methyl-d-aspartate receptor (NMDAR) subunits NR1, NR2A and NR2B, calcium/calmodulin-dependent protein kinase IIα (CaMKIIα), p-CaMKIIα and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit 1 (GluR1) in the hippocampus were evaluated by western blot analysis. The distribution of GluR1, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (IBa-1) in the CA1 and CA3 regions and dentate gyrus (DG) of the hippocampus were evaluated by immunofluorescence analysis. Treatment with iTBS significantly improved the spatial cognitive function of RHRSPs, increased the expression of NR2B, p-CaMKIIα and GluR1 in the hippocampus, and decreased the proliferation of astrocytes and microglia. Our results showed that iTBS treatment had a beneficial effect on the cognitive impairments induced by cerebral small vessel disease, potentially through the activation of the NR2B-CaMKII pathway, an increase in GluR1 expression and the suppression of astrocyte and microglial activation.

Cerebral Venous Collagen Remodeling in a Modified White Matter Lesions Animal Model

To mimic the expected pathological changes of white matter lesions (WMLs) and increase the stability, we applied modified two-vessel occlusion (modified 2VO) (1-week interval bilateral carotid artery occlusion) in stroke-prone renovascular hypertensive rats (RHRSP) and established a modified WMLs model (RHRSP/modified 2VO) that compared their phenotypes with RHRSP and sham-operated rats. In addition, we tried to differentiate small veins from small arteries through the presence of smooth muscle to study the pathological changes of small veins detailed in the model. RHRSP/modified 2VO rats showed higher stability and more extensive white matter damage without an obvious increase in mortality rate at 12 weeks after the modified 2VO operation compared to RHRSP rats. RHRSP/modified 2VO rats showed more severe small venous collagen deposition than RHRSP rats, and the majority of the deposition was collagen I and IV rather than collagen III. In addition, RHRSP/modified 2VO rats possessed cognitive impairment, mild wall thickness and blood–brain barrier disruption. Our findings suggest that the modified WMLs model (RHRSP/modified 2VO) mimics cognitive impairment and small vessel pathological changes similar to WMLs in humans. Differentiating small veins from small arteries through smooth muscle is feasible, and marked small venous deposition may play an important role in the hypertensive white matter lesions.

Different effects of running wheel exercise and skilled reaching training on corticofugal tract plasticity in hypertensive rats with cortical infarctions

The approaches that facilitate white matter plasticity may prompt functional recovery after a stroke. The effects of different exercise methods on motor recovery in stroke rats have been investigated. However, it is not clear whether their effects on axonal plasticity different. The aim of this study was to compare the effects of the forced running wheel exercise (RWE) and skilled reaching training (SRT) on axonal plasticity and motor recovery. Cortical infarctions were generated in stroke-prone renovascular hypertensive rats. The rats were randomly divided into the following three groups: the control (Con) group, the RWE group, and the SRT group. A sham group was also included. The mNSS and forelimb grip strength tests were performed on days 3, 7, 14, 21, 28, 35, and 42 after ischemia. The anterograde tract tracer biotinylated dextran amine (BDA) was injected into the rats to trace the axonal plasticity of the contralesional corticofugal tracts. Compared with the Con group, the mNSS scores in the SRT and RWE groups decreased on day 28 (P<0.05) and on days 35 and 42 (P<0.01). The grip strength in the SRT group increased relative to that in the RWE group at 42day post-ischemia (P<0.01). Both the RWE and SRT groups exhibited enhanced plasticity of the contralesional corticofugal tract axons at the level of the red nucleus (P<0.01) and the cervical enlargement (P<0.01). More contralateral corticorubral tract remodeling was observed at the red nucleus level in the SRT group than in the RWE group (P<0.001). Taken together, these results suggest that SRT may enhance axon plasticity in the corticorubral tract more effectively than the forced RWE and is associated with better motor recovery after cerebral ischemia.

Unfolded protein response is activated in the ipsilateral thalamus following focal cerebral infarction in hypertensive rats.

Focal cerebral cortical infarction causes secondary neurodegeneration in the remote regions, such as the ventroposterior nucleus of the thalamus. Retrograde degeneration of thalamocortical fibers is considered as the principle mechanism, but the exact molecular events remain to be elucidated. This study aimed to investigate whether unfolded protein response (UPR) is activated in thalamic neurons following distal middle cerebral artery occlusion (MCAO) in stroke-prone renovascular hypertensive rats. Immunostaining and immunoblotting were performed to evaluate the expression of Grp78 and its downstream effectors in the thalamus at 3, 7 and 14days after MCAO. Secondary thalamic degeneration was assessed with Nissl staining and NeuN immunostaining. Neuronal death was not apparent at 3days post-ischaemia but was evident in the thalamus at 7 and 14days after MCAO. Grp78 level was reduced in the ipsilateral thalamus at 3 and 7days after MCAO. In parallel, phosphorylated eIF2α and ATF4 levels were elevated, indicating the activation of UPR. In contrast, ATF6α and CHOP levels were not changed. These results suggest that UPR is activated before neuronal death in the ipsilateral thalamus after MCAO and may represent a key early event in the secondary thalamic degeneration.

Exercise Training Inhibits the Nogo-A/NgR1/Rho-A Signals in the Cortical Peri-infarct Area in Hypertensive Stroke Rats.

The aim of this study was to test the hypothesis that exercise training promotes motor recovery after stroke by facilitating axonal remodeling via inhibition of the Nogo-A/NgR1 and Rho-A pathway. A distal middle cerebral artery occlusion model was generated in stroke-prone renovascular hypertensive rats. Stroke-prone renovascular hypertensive rats were randomly divided into a control group, an exercise training group, and a sham group. Motor function was measured using the grip strength test. Axon and myelin remodeling markers, growth-associated protein 43, myelin basic protein, Tau, and amyloid precursor protein were detected by immunofluorescence. The expression of Nogo-A, NgR1, and Rho-A was demonstrated by immunofluorescence and Western blotting in the peri-infarction area at 7, 14, 28, and 52 days after distal middle cerebral artery occlusion. Grip strength was higher in the exercise training group (P < 0.05). Exercise training increased the expression of growth-associated protein 43, myelin basic protein (at 7, 14, and 28 days), and Tau (at 7 and 14 days), and decreased the expression of axonal damage amyloid precursor protein (at 7 and 14 days), compared with the control group. The protein levels of Nogo-A (at 7 and 14 days), NgR1 (at 7, 14, and 28 days), and Rho-A (at 14 and 28 days) were reduced after exercise training. Exercise training promotes axonal recovery, which is associated with functional improvement after cerebral infarction. Down-regulation of the Nogo-A/NgR1/Rho-A may mediate the axonal remodeling induced by exercise training.

Spontaneous white matter lesion in brain of stroke-prone renovascular hypertensive rats: a study from MRI, pathology and behavior.

Hypertension is considered one of the most important controllable risk factors for white matter lesion (WML). Our previous work found that stroke-prone renovascular hypertensive rats (RHRSP) displayed a high rate of WML. This study aimed to investigate the WML in RHRSP from MRI, pathology and behavior. RHRSP model was established by two-kidney, two-clipmethod and kept for 20 weeks. WML was decteted by magnetic resonance imaging (MRI) and loyez staining. Cognition was tested by morris water maze (MWM). Vascular changes were observed by HE staining on brain and carotid sections. Ultrastucture of blood brain barrier (BBB) were observed by transmission electron microscope. Immunofluorescence was used to detect albumin leakage and cell proliferation. T(2)-weighted MRI scans of RHRSP displayed diffuse, confluent white-matter hyperintensities. Pathological examination of the same rat showed marked vacuoles, disappearence of myelin and nerve fibers in white matter, supporting the neuroimaging findings. Spatial learning and memory impairment were observed in RHRSP. The small arteries in brain exhibited fibrinoid necrosis, hyalinosis and vascular remodeling. BBB disruption and plasma albumin leakage into vascular wall was observed in RHRSP. Increased cell proliferation in subventricular zone was seen in RHRSP. RHRSP demonstrated spontaneous WML and cognitive impairment. Hypertensive small vessel lesions and BBB disruption might paly causative factors for the onset and development of WML. The characteristic features of WML in RHRSP suggested it a valid animal model for WML.

Ebselen reduces autophagic activation and cell death in the ipsilateral thalamus following focal cerebral infarction

Previous studies have demonstrated that both oxidative stress and autophagy play important roles in secondary neuronal degeneration in the ipsilateral thalamus after distal middle cerebral artery occlusion (MCAO). This study aimed to investigate whether oxidative stress is associated with autophagy activation within the ipsilateral thalamus after distal MCAO. Sixty stroke-prone renovascular hypertensive rats were subjected to distal MCAO or sham operation, and were killed at 14 days after MCAO. Mn-SOD, LC3-II, Beclin-1 and p62 expression were evaluated by immunostaining and immunoblotting. Secondary damage in the thalamus was assessed with Nissl staining and immunostaining. The association of oxidative stress with autophagy activation was investigated by the antioxidant, ebselen. We found that treatment with ebselen at 24h after MCAO significantly reduced the expression of Mn-SOD in the ipsilateral thalamus at 14 days following focal cerebral infarction. In parallel, it prevented the elevation of LC3-II and Beclin-1, and the reduction of p62. Furthermore, ebselen attenuated the neuronal loss and gliosis in the ipsilateral thalamus. These results suggested that ebselen reduced oxidative stress, autophagy activation and secondary damage in the ipsilateral thalamus following MCAO. There are associations between oxidative stress, autophagy activation and secondary damage in the thalamus after MCAO.

Peroxisome proliferator-activated receptor-γ agonist pioglitazone ameliorates white matter lesion and cognitive impairment in hypertensive rats.

Cerebrovascular white matter lesion (WML) is a major subtype of cerebral small vessel disease. Clinical drugs are not available for WML. We investigated whether peroxisome proliferator-activated receptor-γ agonist pioglitazone, with properties of vascular protection and antiinflammation, exerts beneficial effect in hypertensive WML rats. Stroke-prone renovascular hypertensive rats (RHRSP) were treated with pioglitazone for 12 weeks. Morris water maze experiment was conducted to assess cognition. WML was observed by Luxol fast blue staining. Smooth muscle actin-alpha, collagen I, collagen IV, glial fibrillary acidic protein, and ionized calcium-binding adaptor molecule-1 were evaluated by immunohistochemistry. Interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in brain and soluble intercellular adhesion molecule-1 (sICAM-1) in serum were detected. Pioglitazone significantly attenuated WML in corpus callosum, caudate putamen, external capsule, and internal capsule. Cognitive impairment in RHRSP was ameliorated by pioglitazone. Pioglitazone attenuated arteriolar remodeling and reduced sICAM-1 level in serum. Pioglitazone decreased the proliferation of microglia and astrocyte and lowered the expression of proinflammatory cytokines IL-1β and TNF-α in the white matter. Long-term treatment of pioglitazone has beneficial effect on hypertension-induced WML and cognition decline, which may partly through its effect on attenuation of arteriolar remodeling, endothelial activation, and brain inflammation.

Effects of physical training on pyramidal tract regeneration in hypertensive rats with focal cerebral infarction

To explore the effects of exercise training on motor functional recovery and pyramidal tract regeneration in hypertensive rats with focal cerebral infarction. Middle cerebral artery occlusion (MCAO) model was generated by electric coagulation on stroke-prone renovascular hypertensive Sprague-Dawley rats. The rats were randomly allocated to three groups of sham (n = 15), exercise training (n = 10) and control (n = 12). The exercise training group had running wheel exercise. The neurological function was evaluated with the modified neurological severity scores (mNSS) and forelimb grip strength test at Days 3, 7, 14, 21, 28, 35 and 42 post-ischemia. The pathological changes of neurons around infarct cortex were measured by Nissl staining. At Day 42 post-ischemia, anterograde tract tracers of biotinylated dextran amine (BDA) and cascade blue-labeled dextran amine (CB) were injected into cortex. And axonal extension of ipsi- and contra-lesional pyramidal tract was observed at Day 14 post-injection. The mNSS score declined in the exercise training group (4.0 ± 1.1, 2.7 ± 0.7, 2.6 ± 0.5) versus those in the control group (6.0 ± 1.3, 5.6 ± 1.0, 5.6 ± 1.1) at Days 28, 21 and 35 post-ischemia (all P < 0.01). The grip strength of paralytic forelimb increased in the exercise training group ((379 ± 41, 344 ± 15, 430 ± 48, 471 ± 47, 454 ± 17)g) versus those in the control group ((276 ± 8, 170 ± 5, 236 ± 12, 283 ± 14, 317 ± 15)g) at Days 14, 21, 28, 35 and 42 post-infarction (all P < 0.05). Compared with the control group (0.571 ± 0.060) , exercise training (0.734 ± 0.035) reduced neuron loss (P < 0.05). Moreover, the percentage of the number of BDA-positive, midline-crossing fibers over the number of labeled fibers in cerebral peduncle ipsilateral to injection site increased in the exercise training group (0.096 8 ± 0.022 6) versus those in the control group (0.014 2 ± 0.003 9) at the level of cervical enlargement (P < 0.016 7). The percentage of CB immunofluorescence in striatum ipsilateral to lesion-sided motor cortex over those in ischemic cortex was lower in the control group (0.521 ± 0.020) and the exercise training group (0.499 ± 0.034) than that in the sham group (0.824 ± 0.017) (all P < 0.01). However, no significant difference existed between control and exercise training groups (P > 0.05). Exercise training promotes the recovery of motor function and contralesional pyramidal tract regeneration after cerebral infarction.

Electroacupuncture attenuates cervical spinal cord injury following cerebral ischemia/reperfusion in stroke-prone renovascular hypertensive rats.

Cerebral ischemia induces injury, not only in the ischemic core and surrounding penumbra tissues, but also in remote areas such as the cervical spinal cord. The aim of the present study was to determine the effects of electroacupuncture (EA) on cervical spinal cord injury following cerebral ischemia/reperfusion in stroke-prone renovascular hypertensive (RHRSP) rats. The results demonstrated that neuronal loss, which was assayed by Nissl staining in the cervical spinal cords of RHRSP rats subjected to transient middle cerebral artery occlusion (MCAO), was markedly decreased by EA stimulation at the GV20 (Baihui) and GV14 (Dazhui) acupoints compared with that in rats undergoing sham stimulation. Quantitative polymerase chain reaction and western blot analysis demonstrated that EA stimulation blocked the MCAO-induced elevated protein expression levels of glial fibrillary acidic protein and amyloid precursor protein in the cervical spinal cord at days 24 and 48. To further investigate the mechanism underlying the neuroprotective role of EA stimulation, the protein expression levels of Nogo-A and Nogo-66 receptor-1 (NgR1), two key regulatory molecules for neurite growth, were recorded in each group. The results revealed that EA stimulation reduced the MCAO-induced elevation of Nogo-A and NgR1 protein levels at day 14 and 28 in RHRSP rats. Therefore, the results demonstrated that EA reduced cervical spinal cord injury following cerebral ischemia in RHRSP rats, indicating that EA has the potential to be developed as a therapeutic treatment agent for cervical spinal cord injury following stroke.

A Method of Evaluating the Lower Limit of Cerebral Autoregulation and Its Correlation with Blood Pressure by Transcranial Doppler in Rats

The aim of present study was to validate the assessment of lower limit of cerebral autoregulation (LLCA) as derived from mean artery blood pressure (MABP) and cerebral zero flow pressure (ZFP) by means of transcranial Doppler (TCD) and to determine the accurate relationship between LLCA and MABP in stroke-prone renovascular hypertensive rats (RHRSP). We studied two groups of rats: RHRSP and normal controls. Blood flow velocity of middle cerebral artery was monitored by TCD and arterial blood pressure was recorded in right femoral artery to compute the ZFP. The value of LLCA was determined as the difference between MABP and ZFP and validated by the value determined by blood withdrawal-induced cerebral autoregulation. In normal rats, the LLCA derived from the new method was 69.8 ± 8.7 mm Hg, from the change of blood velocity was 69.4 ± 9.8 mmHg and from blood volume flow after blood withdrawal was 68.8 ± 9.7 mmHg. In the RHRSP group, the corresponding values of LLCA were 109.1 ± 17.2 mm Hg, 110.0 ± 18.0 mm Hg and 109.0 ± 19.3 mm Hg, respectively. In each group, there was no statistically significant difference among the three values. LLCA in RHRSP began to increase 6 weeks after hypertension-induced operation, significantly higher than controls (p < 0.05), and stabilized at 110 mm Hg, 10 weeks after operation. The increase of LLCA was positively correlated with MABP, following an “S” curve, demonstrating that the change of LLCA was more obvious in the middle range of MABP in RHRSP (R2 = 0.8848, p < 0.05). In conclusion, TCD is a valid and noninvasive method for determination of LLCA compared with the classic method in rats. Our data demonstrated that the change of LLCA may be correlated with MABP, following an “S” curve relationship.

Changes in DNA repair enzymes in rat ventroposterior nucleus of the thalamus after cerebral cortex infarction

To investigate the damage within the ventroposterior nucleus (VPN) of the thalamus after focal cortical infarction and its mechanism, and explore the effect of ebselen on the oxidative damage after cerebral cortex infarction in hypertensive rats. Middle cerebral artery occlusion (MCAO) was induced in stroke-prone renovascular hypertensive rats (RHRSP), and the rats were divided into four groups by table of random number: sham operation group, model group, vehicle group and ebselen group, each group consisted of 8 rats. In animals subjected to sham surgery the middle cerebral artery was exposed only. Ebselen (5 ml/kg) or vehicle (a mixed solvent consisting of 0.5% carboxymethyl cellulose and 0.02% Tween 20, 5 ml/kg) was given by gastric gavage starting 24 hours after cerebral cortical infarction. Two weeks after the MCAO, the rats were sacrificed, and VPN from each group was sectioned and stained with hematoxylin-eosin (HE), and apurinic/apyrimidinic endonuclease (APE) and Escherichia coli MutY DNA glycosylase (MYH) were determined by immunohistochemistry. HE staining showed that ebselen ameliorated the VPN damage induced by ischemia. Immunohistochemical imaging analysis revealed a distinct nuclear staining of APE and nuclear and cytoplasm distribution of MYH in the entire region of the VPN. Compared with sham operation group, the number of APE and MYH positive cells decreased in model group and vehicle group (APE: 57.0±14.7, 49.4±12.5 vs. 101.0±13.6, MYH: 15.0±4.7, 10.4±2.5 vs. 56.0±13.2, all P<0.05). Compared with model group and vehicle group, the number of APE and MYH positive cells increased significantly in ebselen group (APE: 72.2±7.6 vs. 57.0±14.7, 49.4±12.5, MYH: 32.2±7.6 vs. 15.0±4.7, 10.4±2.5, all P<0.05); the difference of the number of APE and MYH positive cells between model group and vehicle group showed no statistical significance. After 2 weeks of MCAO, there is a marked decrease of APE and MYH in VPN; ebselen can obviously increase the level of APE and MYH, and ebselen may protect the VPN of the thalamus from damage after focal cortical infarction in rats.

Reduction of β-Amyloid Deposits by γ-Secretase Inhibitor is Associated with the Attenuation of Secondary Damage in the Ipsilateral Thalamus and Sensory Functional Improvement after Focal Cortical Infarction in Hypertensive Rats

Abnormal β-amyloid (Aβ) deposits in the thalamus have been reported after cerebral cortical infarction. In this study, we investigated the association of Aβ deposits, with the secondary thalamic damage after focal cortical infarction in rats. Thirty-six stroke-prone renovascular hypertensive rats were subjected to distal middle cerebral artery occlusion (MCAO) and then randomly divided into MCAO, vehicle, and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) groups and 12 sham-operated rats as control. The DAPT was administered orally at 72 hours after MCAO. Seven days after MCAO, sensory function, neuron loss, and glial activation and proliferation were evaluated using adhesive removal test, Nissl staining, and immunostaining, respectively. Thalamic Aβ accumulation was evaluated using immunostaining and enzyme-linked immunosorbent assay (ELISA). Compared with vehicle group, the ipsilateral thalamic Aβ, neuronal loss, glial activation and proliferation, and the mean time to remove the stimulus from right forepaw significantly decreased in DAPT group. The mean time to remove the stimulus from the right forepaw and thalamic Aβ burden were both negatively correlated with the number of thalamic neurons. These findings suggest that Aβ deposits are associated with the secondary thalamic damage. Reduction of thalamic Aβ by γ-secretase inhibitor may attenuate the secondary damage and improve sensory function after cerebral cortical infarction.

Endostatin expression in neurons during the early stage of cerebral ischemia is associated with neuronal apoptotic cell death in adult hypertensive rat model of stroke

Endostatin (ES) has been recognized as a potent anti-angiogenic factor. We here investigated the expression of ES in ischemic brain and the consequence of cells expressing ES after stroke in adult stroke-prone renovascular hypertensive rats. A single dose of Ca-074ME, a membrane-permeable cathepsin B (CB) specific inhibitor, or vehicle was given by intraperitoneal injection immediately after distal middle cerebral artery occlusion (dMCAO), ES expression was evaluated using fluorescent immunohistochemistry staining, and CB enzyme activity was tested by measuring the free 7-amino-4-methylcoumarin (AMC) released by CB from its' specific substrate, the Z-Arg-Arg-7-amido-4-methylcoumarin. ES immunoreactivity (IR) was significantly up-regulated as early as 6 h and returned to baseline level at 3 days in peri-infarct area following dMCAO. Double-staining experiment revealed that the majority of ischemia-induced ES positive cells were neurons. Furthermore, ES was co-labeled with CB and Cleaved Caspase-3(Asp175) whereas treatment with Ca-074ME reduced up-regulation of ES expression and attenuated apoptosis in peri-infarct neurons. Collectively, our data suggest that peri-infarct neurons express ES during the early stage of cerebral ischemia and treatment with Ca-074ME attenuates ES expression and apoptosis in peri-infarct neurons.