Bilateral Carotid Artery Ligation Research Articles

NADPH Oxidase 1, a Novel Molecular Source of ROS in Hippocampal Neuronal Death in Vascular Dementia

Chronic cerebral hypoperfusion (CCH) is a common pathological factor that contributes to neurodegenerative diseases such as vascular dementia (VaD). Although oxidative stress has been strongly implicated in the pathogenesis of VaD, the molecular mechanism underlying the selective vulnerability of hippocampal neurons to oxidative damage remains unknown. We assessed whether the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, a specialized superoxide generation system, plays a role in VaD by permanent ligation of bilateral common carotid arteries in rats. Male Wistar rats (10 weeks of age) were subjected to bilateral occlusion of the common carotid arteries (two-vessel occlusion [2VO]). Nox1 expression gradually increased in hippocampal neurons, starting at 1 week after 2VO and for approximately 15 weeks after 2VO. The levels of superoxide, DNA oxidation, and neuronal death in the CA1 subfield of the hippocampus, as well as consequential cognitive impairment, were increased in 2VO rats. Both inhibition of Nox by apocynin, a putative Nox inhibitor, and adeno-associated virus-mediated Nox1 knockdown significantly reduced 2VO-induced reactive oxygen species generation, oxidative DNA damage, hippocampal neuronal degeneration, and cognitive impairment. We provided evidence that neuronal Nox1 is activated in the hippocampus under CCH, causing oxidative stress and consequential hippocampal neuronal death and cognitive impairment. This evidence implies that Nox1-mediated oxidative stress plays an important role in neuronal cell death and cognitive dysfunction in VaD. Nox1 may serve as a potential therapeutic target for VaD.

Translational realistic expectations of chronic cerebral hypoxemia in rat model after bilateral commom carotid artery ligation. Neurocognitive aspects

To evaluate the effects of chronic cerebral hypoxia on memory of rats submitted to bilateral common carotid artery ligation (BCCL). Every each week, for 16 weeks, 31 rats were tested for memory using a water and land mazes and compared with 30 normal rats (control group A). The variables were expressed by their mean and standard error of the mean (SEM). p<0.05 was used for rejecting the null hypothesis. The study was approved by the Ethics Committee for animal investigation. There was a significant increase in the latency time, in the survival water and land mazes, after four weeks (study group B) follow-up. However, without any medication or therapeutically induced measures, after 16 weeks (study group C) follow-up the latency mean times tend to be similar to control group (A) in the neurocognitive tests. Neurocognitive deficits after 16 weeks post-operative follow-up of rats that underwent bilateral common carotid artery ligation is a natural adaptive phenomenon. Thus, is not realistic to allow translational information from this animal model for therapeutically approaches aiming at to prevent, or to improve brain damage in human beings suffering from chronic deprivation of adequate blood supply.

Chronic brain ischemia induces the expression of glial glutamate transporter EAAT2 in subcortical white matter

Glutamate plays a central role in brain physiology and pathology. The involvement of excitatory amino acid transporters (EAATs) in neurodegenerative disorders including acute stroke has been widely studied, but little is known about the role of glial glutamate transporters in white matter injury after chronic cerebral hypoperfusion. The present study evaluated the expression of glial (EAAT1 and EAAT2) and neuronal (EAAT3) glutamate transporters in subcortical white matter and cortex, before and 3–28days after the ligation of bilateral common carotid arteries (LBCCA) in rat brain. K-B staining showed a gradual increase of demyelination in white matter after ischemia, while there was no cortical involvement. Between 3 and 7days after LBCCA, a significant increase in EAAT2 protein levels was observed in the ischemic brain and the number of EAAT2-positive cells also significantly increased both in the cortical and white matter lesions. EAAT2 was detected in glial-fibrillary acidic protein (GFAP)-positive astrocytes in both the cortex and white matter, but not in neuronal and oligodendroglial cells. EAAT1 was slightly elevated after ischemia only in the white matter, but EAAT3 was at almost similar levels both in the cortex and white matter after ischemia. A significant increase in EAAT2 expression level was also noted in the deep white matter of chronic human ischemic brain tissue compared to the control group. Our findings suggest important roles for up-regulated EAAT2 in chronic brain ischemia especially in the regulation of high-affinity of extracellular glutamate and minimization of white matter damage.

Abstract TMP72: L-carnitine Enhances Axonal Plasticity Via Akt/mammalian Target Of Rapamycin Pathway In A Rat Chronic Cerebral Hypoperfusion

Background and Purpose: Chronic cerebral hypoperfusion (CCH) induces white matter lesions with oligodendrocytic damage and causes cognitive impairment. Oxidative stress has been elucidated as a pivotal role of its pathophysiology. Damage of axon can be also involved in CCH, however, mechanisms that associated with axonal damage under CCH have not been fully studied. In the present study, we analyzed axonal plasticity and damage of oligodendrocytes in a rat CCH model using L-carnitine, antioxidant agent. Methods: Adult rats subjected to ligation of bilateral common carotid artery (LBCCA), were randomly divided into two groups: vehicle-treated rats; and L-carnitine-treated rats (600mg/kg/day), and were sacrificed at 7, 14, 21 and 28 days after LBCCA (n=7/group). Sham operated rats (n=7) were used as a control group. The Morris water maze was performed to evaluate spatial learning and memory tasks, and immunohistochemical and Western blot analysis were studied. Results: Rats subjected to LBCCA substantially increased escape latency on the Morris water maze in a time-dependent fashion. L-carnitine-treated rats significantly reduced escape latency compared to vehicle-treated rats at 28 days after LBCCA (P&lt;0.05). Protein levels of phosphorylated neurofilament heavy protein (pNFH), a marker of axons, were reduced after LBCCA, however, L-carnitine significantly increased pNFH levels compared to levels in vehicle-treated rats at 28 days after LBCCA (P&lt;0.01). Concurrently, increases of phosphorylated Akt (P&lt;0.05) and mammalian target of rapamycin (mTOR) (P&lt;0.05) were detected in L-carnitine-treated rats compared to vehicle-treated rats at 28 days after LBCCA. 4-hydroxy-2-nonenal (HNE)+ and 8-hydroxy-deoxyguanosine (8-OHdG)+, markers of oxidative stress, oligodendrocytes substantially increased until 28 days after LBCCA (P&lt;0.001). L-carnitine-treated rats significantly reduced HNE+ and 8-OHdG+ oligodendrocytes at each time point after LBCCA (P&lt;0.001). Conclusion: Our results indicated that L-carnitine increased axonal plasticity via Akt/mTOR pathway and ameliorated oxidative stress in oligodendorocytes damaged by LBCCA, and thereby resulting an improvement of cognitive impairment in a rat CCH model.

An enriched environment elevates corticosteroid receptor levels in the hippocampus and restores cognitive function in a rat model of chronic cerebral hypoperfusion

An enriched environment (EE) is beneficial for modifying certain behaviors, particularly those involving complex cognitive functions. In models of chronic cerebral hypoperfusion (CCH), the ability of an EE to restore cognition depends on hippocampal synaptic plasticity and brain-derived neurotrophic factor. The mechanisms for this effect have not, however, been adequately studied. Here we investigated the effects of CCH and an EE on serum corticosteroid concentrations and the levels of the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the hippocampus. Rats were randomly divided into four treatment groups that received either permanent bilateral ligation of the common carotid arteries or sham surgery. Following this procedure, rats were exposed to 4weeks of either an EE or standard housing. After the environmental intervention, their spatial learning and memory abilities were examined using the Morris water maze. In addition, the levels of MR and GR proteins in the hippocampus were determined. CCH impaired spatial cognitive function in rats, and exposure to an EE diminished these spatial learning and memory deficits. CCH also reduced the levels of MR and GR proteins in the hippocampus, but an EE restored the levels. Our results demonstrate that EE exposure restores cognitive impairments induced by CCH and up-regulates MR and GR expression. As such, MR and GR may contribute to the diminished effects of an EE in rats with CCH.

Bilateral internal carotid arteries ligation temporary impairs brain vasculaturev in young rats

Bilateral internal carotid artery ligation (BICL) rat model is one of the chronic cerebral hypoperfusion animal models used for investigating brain dysfunction related diseases. Cerebral blood flow decreases in different cerebral regions in a time-dependent manner after the BICL. However little is known about the cerebral vasculature change in the brain after the BICL. In the current study, the bilateral internal carotid arteries of the juvenile rats were permanently ligated and the change of the cerebral vasculature was studied 7, 14 and 21 days after the BICL. In the juvenile rats, 7 days after the BICL, the functional vascular area was decreased significantly in the anterior half of the cerebral cortex, but it had only little decrease in the posterior half of the cerebral cortex and hippocampus. However, at the time points of 14 and 21 days after the surgery, the functional vascular area throughout the whole cerebral cortex and hippocampus was almost similar to those in the sham control rats. In conclusion, the results from our current study showed that in the BICL hypoperfusion model in young rats, the brain functional vascular area was impaired initially in certain brain regions after the artery ligation, but likely to be quickly self-recovered late after. The results suggest that the brain vasculature in young rats has plasticity to external insult caused by cerebral hypoperfusion.

Synthesis and pharmacological properties of a new 3-hydroxypyridine derivative

The new 3-hydroxypyridine derivative 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-L-glutamate (EMOP-AG) was synthesized. It was established that EMOP-AG (10 and 30 mg/kg, single administration) exhibited antihypoxic activity on various models of acute hypoxia (baric hypoxia with hypercapnia and hemic and histotoxic hypoxia) in mice and produced an equal or stronger effect than reference drugs (amtizol, mexidol, and emoxipin). EMOP-AG (10 and 30 mg/kg) had a marked neuroprotective effect on rats with bilateral ligation of common carotid arteries and was more effective in the treatment of neurologic deficiency than amtizol (10 and 30 mg/kg/day), mexidol (90 and 120 mg/kg/day), and emoxipin (120 mg/kg/day). It was also established that EMOP-AG (30 mg/kg) could prevent amnesia in a step-down passive avoidance situation caused by different factors (maximal electroshock, scopolamine, acute baric hypoxia with hypercapnia, and the combined action of extreme factors).

Abstract 2513: Intraventricular Administration of Cystatin C Attenuates White Matter Injury In Rat Chronic Ischemia

Background and Purpose Cerebral white matter lesions (WMLs) contribute to elderly cognitive deterioration and are usually caused by chronic ischemic insult. Experimental studies have shown that proteases are highly expressed in activated astrocyte and microglia, which are thought to be crucial in the development of WMLs. Cystatin C (CSTC) functions as a major extracellular cysteine protease inhibitor in mammals. Since the concentration of CSTC is 5 times higher in CSF than in plasma, CSTC is thought to be a major inhibitor of cysteine proteases in the central nervous system. We have shown that polymorphisms in the CSTC gene, which results in a decrease of plasma CSTC level, are significantly associated with prevalence of WMLs. However, direct impact of CSTC on the development of WMLs has not been elucidated yet. Here we studied whether direct administration of CSTC into brain has a protective effect on the pathophysiology of WMLs produced by chronic cerebral hypoperfusion in a rat model of permanent bilateral common carotid artery occlusion. Methods Adult Wister rats underwent ligation of bilateral common carotid arteries (LBCCA) and were divided into three groups; the CSTC group in which CSTC was injected continuously into the ventricle 14 consecutive days after LBCCA (n=10), the PBS group in which PBS was injected continuously into the ventricle 14 consecutive days after LBCCA (n=10), and the sham group (n=10). All rats were perfused on 14 days after LBCCA. The severity of WMLs and accumulation of microglia and astrocytes in the corpus callosum (CC) and optic nerve (ON) were evaluated. Furthermore, the activity of cathepsin B, one of the major proteases, was measured in the tissue lysates of CC and ON using the assay kit. Results Continuous intraventricular infusion of CSTC significantly reduced the severity of WMLs (CC; p&lt;0.001 vs. PBS, ON; p&lt;0.001 vs. PBS) and inhibited the accumulation and activation of microglia (CC; p=0.03 vs. PBS, ON; p&lt;0.001 vs. PBS) and astrocytes (CC; p=0.003 vs. PBS, ON; p=0.04 vs. PBS) both in CC and ON. There were no alterations in cathepsin B activity in these regions. Conclusions The present study indicates that CSTC could suppress chronic ischemia-induced WMLs and the inhibition of proteases other than cathepsin B may be implicated in this beneficial effect.

Protective effects of acetyl-L-carnitine on neurodegenarative changes in chronic cerebral ischemia models and learning-memory impairment in aged rats

This study investigated the effects of acetyl-L-carnitine (ALC) in secondarily-induced cerebral chronic ischemia models using rats with permanent ligation of bilateral common carotid arteries (BCCL) and spontaneously hypertensive rats (SHR). Additionally, we used normal aged rats as a primary dementia model. Chronic ALC administration at 100 mg/kg (p.o.) for 4 weeks significantly attenuated neurodegenerative changes. In groups receiving 50 mg/kg or 100 mg/kg, ALC inhibited the active astrocyte increase in cerebral tissues of both BCCL and SHR models. In BCCL rats, ALC administration (50 mg/kg or 100 mg/kg, p.o.) resulted in significant promotion of glutathione levels in brain tissues. We also confirmed behavioral improvement after ALC treatment (100 mg/kg for 8 weeks, p.o.) on learning-memory function using aged rats (18 months old) in a passive avoidance task and preservation of CA1 pyramidal neurons was coincided on histopathological observation. In conclusion, chronic ALC administration may ameliorate cerebral ischemia progress after a cerebrovascular disorder as well as spontaneous ageing-related cerebral dysfunction via hippocampal protection.

Endothelial cells express a unique transcriptional profile under very high wall shear stress known to induce expansive arterial remodeling

Chronic high flow can induce arterial remodeling, and this effect is mediated by endothelial cells (ECs) responding to wall shear stress (WSS). To assess how WSS above physiological normal levels affects ECs, we used DNA microarrays to profile EC gene expression under various flow conditions. Cultured bovine aortic ECs were exposed to no-flow (0 Pa), normal WSS (2 Pa), and very high WSS (10 Pa) for 24 h. Very high WSS induced a distinct expression profile compared with both no-flow and normal WSS. Gene ontology and biological pathway analysis revealed that high WSS modulated gene expression in ways that promote an anti-coagulant, anti-inflammatory, proliferative, and promatrix remodeling phenotype. A subset of characteristic genes was validated using quantitative polymerase chain reaction: very high WSS upregulated ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motif-1), PLAU (urokinase plasminogen activator), PLAT (tissue plasminogen activator), and TIMP3, all of which are involved in extracellular matrix processing, with PLAT and PLAU also contributing to fibrinolysis. Downregulated genes included CXCL5 and IL-8 and the adhesive glycoprotein THBS1 (thrombospondin-1). Expressions of ADAMTS1 and uPA proteins were assessed by immunhistochemistry in rabbit basilar arteries experiencing increased flow after bilateral carotid artery ligation. Both proteins were significantly increased when WSS was elevated compared with sham control animals. Our results indicate that very high WSS elicits a unique transcriptional profile in ECs that favors particular cell functions and pathways that are important in vessel homeostasis under increased flow. In addition, we identify specific molecular targets that are likely to contribute to adaptive remodeling under elevated flow conditions.

Recombinant human erythropoietin for repair of white matter damage

Erythropoietin has been shown to exhibit neuroprotective effects in animal models. A neonatal rat model of hypoxic-ischemic white matter damage was established via bilateral carotid artery ligation in 4-day-old Sprague-Dawley rats. The rats were subsequently treated with recombinant human erythropoietin to observe pathological changes in the brain and long-term neurobehavioral functions before and after intervention. Results showed that the number of myelin basic protein-positive cells, which reflected myelin/oligodendrocyte damage, significantly increased, although the number of amyloid precursor protein-positive cells, which reflected axonal injury, significantly decreased in periventricular white matter at 72 hours and 7 days following erythropoietin intervention. The number of glial fibrillary acidic protein-positive cells, indicating astrocytic damage, significantly decreased in periventricular white matter of erythropoietin-treated rats at 48 hours, 72 hours, 7 days, and 26 days. Following erythropoietin intervention in the 30-day-old rats, head-turning time in the slope test was shortened and open-field test scores increased. These results suggested that erythropoietin promoted repair of white matter damage, as well as improved neurobehavioral functions in a rat model of hypoxic-ischemic injury.

Effects of nimodipine on rabbits with symptomatic cerebral vasospasm

To investigate the effects of nimodipine on symptomatic cerebral vasospasm in rabbits. Twenty four japanese white rabbits which ligation of bilateral common carotid arteries and no neurological deficits were randomized to sham-operation, subarachnoid hemorrhage (SAH) and nimodipine which injected of nimodipine 0.1 mg/kg, continuous vein administration 5 day. The behavior scores, neurological scores were observed everyday and cerebral angiography changes were measured twice by 3D-CTA, and basilar artery was removed for pathological examination after last CTA examination. In SAH group, The basilar artery were significantly vasoconstrictive on 5 days, neurological scores were increased, and the basilar artery was found apoptosis-like changes under light microscopic and electron microscope. Nimodipine group could not dilated the basilar artery arteriospasm after SAH, but it could attenuate neurological deficit, and obviously alleviate the pathological changes of basilar artery. Nimodipine could not vasodilation of basilar artery in SCVS, but obviously could alleviate neurological changes and pathological changes of basilar artery in rabbits with symptomatic cerebral vasospasm.

The modified 2VO ischemia protocol causes cognitive impairment similar to that induced by the standard method, but with a better survival rate

Permanent bilateral occlusion of the common carotid arteries (2VO) in the rat has been established as a valid experimental model to investigate the effects of chronic cerebral hypoperfusion on cognitive function and neurodegenerative processes. Our aim was to compare the cognitive and morphological outcomes following the standard 2VO procedure, in which there is concomitant artery ligation, with those of a modified protocol, with a 1-week interval between artery occlusions to avoid an abrupt reduction of cerebral blood flow, as assessed by animal performance in the water maze and damage extension to the hippocampus and striatum. Male Wistar rats (N = 47) aged 3 months were subjected to chronic hypoperfusion by permanent bilateral ligation of the common carotid arteries using either the standard or the modified protocol, with the right carotid being the first to be occluded. Three months after the surgical procedure, rat performance in the water maze was assessed to investigate long-term effects on spatial learning and memory and their brains were processed in order to estimate hippocampal volume and striatal area. Both groups of hypoperfused rats showed deficits in reference (F(₈,₁₇₂) = 7.0951, P < 0.00001) and working spatial memory [2nd (F(₂,₄₄) = 7.6884, P < 0.001), 3rd (F(₂,₄₄) = 21.481, P < 0.00001) and 4th trials (F(₂,₄₄) = 28.620, P < 0.0001)]; however, no evidence of tissue atrophy was found in the brain structures studied. Despite similar behavioral and morphological outcomes, the rats submitted to the modified protocol showed a significant increase in survival rate, during the 3 months of the experiment (P < 0.02).

Environmental Enrichment Influences BDNF and NR1 Levels in the Hippocampus and Restores Cognitive Impairment in Chronic Cerebral Hypoperfused Rats

An enriched environment (EE) is beneficial in modifying behaviors, particularly in tasks involving complex cognitive functions. However, the impact of EE on cognitive impairment induced by chronic cerebral hypoperfusion (CCH) has not been studied. We investigated the effects of EE on cognitive impairment caused by CCH and examined whether CCH altered the protein levels of brain-derived neurotrophic factor (BDNF) and N-methyl-D-aspartate (NMDA) receptor subunit 1 (NR1) and subunit 2B (NR2B) in the hippocampus of rats and whether EE exposure attenuated the effects. Rats were divided into four groups that received either permanent bilateral ligation of the common carotid arteries (2-vessel occlusion) surgery or sham surgery followed by either EE housing or standard environment housing for 4 weeks. We examined non-spatial recognition memory in the novel object recognition task, spatial learning, and memory ability in the Morris water maze as well as the protein levels of BDNF, NR1, and NR2B in the hippocampus. CCH impaired both spatial and non-spatial cognitive functions, and EE exposure reversed the spatial cognitive performance and improved non-spatial memory performance. CCH resulted in decreased levels of BDNF and NR1 protein in the hippocampus, and EE exposure restored the decreased expression. Our results demonstrate for the first time that EE exposure restores cognitive impairment induced by CCH and up-regulates the decreased protein levels of BDNF and NR1. Inversely, BDNF and NR1 may contribute to the beneficial effects of EE on CCH in rats.

Neuroprotective effects of tanshinone ⅡA on vascular dementia in rats

To investigate the underlying neuroprotective mechanisms of Tanshinone II A (TSA) on rat cerebral ischemia in vivo. Study of TSA on rat cerebral ischemia in vivo: Male SD rats were divided into four groups (sham-operated, ischemic and treated group (lower dose and higher dose). Chronic cerebral ischemmia after permanent bilateral carotid artery ligation was introduced as an in vivo ischemic model. After ischemia impairment, TSA (2, 4 mg x kg(-1) x d(-1)) was administrated by ip for 30 days in treated group. We used Morris water maze to investigate the learning and memory. Levels of malondialdehyde (MDA), activity of superoxide dismetase (SOD) and glutathione peroxidase (GPX) in brain tissue were detected by spectrophotometer. High-performance liquid chromatography (HPLC) with fluorescence detection was applied to measure the contents of glutamate and gamma-aminobutyric acid (GABA) in cortex and hippocampus. TSA can improve learning and memory deficits in vascular dementia. An elevation of SOD and GPX activity and decrease of MDA level were shown in TSA treated group after brain ischemia. Decreased glutamate and gamma-aminobutyric acid induced by chronic brain ischemia were markedly inhibited by TSA pretreatment. The neuroprotective effect of TSA are partly due to its functions as follow: anti-free radical injury; regulating the content of glutamate and gamma-aminobutyric acid.

Edaravone attenuates white matter lesions through endothelial protection in a rat chronic hypoperfusion model

A multicenter randomized clinical trial demonstrated that acute ischemic stroke patients treated with edaravone, a scavenger of hydroxyl radicals, had significant functional improvement. We tested the hypothesis that edaravone has protective effects against white matter lesions (WML) and endothelial injury, using a rat chronic hypoperfusion model. Adult Wistar rats underwent ligation of bilateral common carotid artery (LBCCA) and were divided into the edaravone group (injected once only immediately after LBCCA [ n=39, ED 1]; and injected on three consecutive days [ n=39, ED 3]), the vehicle group ( n=39), and the sham group ( n=15). Cerebral blood flow, Morris water maze performance, footprint test for locomotor function, immunohistochemical analyses and Western blot analysis were performed before and after LBCCA. The ED 3 group upregulated endothelial nitric oxide synthase and attenuated Evans Blue extravasation at day 3 after LBCCA ( P<0.05). Edaravone markedly suppressed accumulation of 4-hydroxy-2-nonenal-modified protein and 8-hydroxy-deoxyguanosine ( P<0.01), and loss of oligodendrocytes ( P<0.05) in the cerebral white matter at days 3, 7, 14, 21 and 28 after LBCCA. These results were more evident in the ED 3 group. Moreover, at day 21 after LBCCA, spatial memory but not motor function, and axonal damage were significantly improved by three-time treatment of edaravone ( P<0.05). Our results indicated that 3-day treatment with edaravone provides protection against WML through endothelial protection and free radical scavenging and suggested that edaravone is potentially useful for the treatment of cognitive impairment.

Multiparameter Analysis of EEG in Old Wistar Rats after Bilateral Carotid Artery Ligation

We showed that 302 of 840 EEG parameters in old Wistar rats significantly change 3 h after bilateral carotid artery ligation. All animals (n=7) died 250+/-47 min after ligation. EEG power spectra had signs of suppression (increased d-activity, decreased t-activity, and increased low-frequency alpha-activity) or activation of CNS (decreased high-frequency alpha-activity and paroxysmal increase in beta-activity). The coherence, synchrony, and entropy of potentials were reduced in the majority of brain regions. This method and scheme of experiments are suitable for the search and study of anti-stroke drugs.

Influence of chronic cerebral hypoperfusion on gap junction ultrastructure and expression of connexin (Cx) 36, Cx32, and Cx26 in hippocampus: experiment with rats

To investigate the Influence of chronic cerebral hypoperfusion on gap junction ultrastructure and expression of connexin (Cx)36, Cx32, and Cx26 in hippocampus the microstructure of gap junction and the expression of gap junction subunits in the hippocampus of cognition impaired animals. Cognition impairment was induced by the permanent occlusion of the carotid arteries (2-VO). Twenty-seven Wistar rats underwent two-vessel occlusion (2-VO, ligation of bilateral common carotid arteries) and then were randomly divided into 2 equal groups: 2-VO 3 week, 2-VO 8 week and 2-VO 12 week groups to undergo Morris water maze test 3, 8, and 12 weeks later to observe the spatial learning and memory ability. Another 9 rats underwent sham operation to be used as control group. After Morris water maze test the rats were killed. The brains were taken out from 3 rats from each group to undergo thin-section transmission electron microscopy to observe the ultrastructure of the gap junctions in the CA1 area of hippocampus. RT-PCR and Western blotting were used to detect the mRNA and protein expression of Cx 36, Cx32, and Cx26 in the hippocampus of the other 27 rats, 6 from each group. Compared to the control group, the density of cytomembrane of gap junction was lower, the interspace of gap junction were larger in the 2-VO 3 week group, and these abnormalities were gradually improved in the 2-VO 8 and 12 week groups. The mRNA and protein levels of Cx36 and Cx32 of the three 2-VO groups were the lowest in the 2-VO 3 week group, and then gradually increased, however, all significantly lower than those of the control group (all P < 0.05). The mRNA level of Cx26 was not altered significantly, while the protein expression levels of Cx26 in the three 2-VO groups were all higher compared with the control group (all P < 0.05). The altered neuronal gap junction may participate in the cognition impairment caused by chronic cerebral hypoperfusion.

DDPH: Improving cognitive deficits beyond its α 1-adrenoceptor antagonism in chronic cerebral hypoperfused rats

DDPH (1-(2, 6-dimethylphenoxy)-2-(3, 4-dimethoxyphenylethylamino) propane hydrochloride), a candidate drug known to be an α 1-adrenoceptor antagonist, can efficiently penetrate through blood brain barrier and inhibit the contraction of vascular smooth muscle in the brain. In rats with chronic cerebral hypoperfusion after permanent bilateral carotid artery ligation, we found that DDPH treatment at 6 or 12 mg/kg per day for 30 days significantly reversed pathological changes such as glial cell proliferation and nuclei shrinkage and reduced neuronal cell loss. In vivo electrophysiological studies revealed that DDPH increased long-term potentiation that was inhibited in these animals. In water maze tests, the percentage of time spent in the target quadrant (Q3) for ischemic rats (20.17 ± 2.87%) was much shorter than that for the sham rats (45.39 ± 3.68%), but DDPH at 12 mg/kg increased the time (39.58 ± 3.77%) spent in Q3 in ischemic rats by 96.23%. These data suggested that DDPH improved the learning and memory performance significantly in rats with ischemia induced by bilateral carotid artery ligation. DDPH also lowered the levels of malondialdehyde (MDA), which was increased in the hypoperfused rats, and enhanced the activities of superoxide dismutase (SOD) and glutathione peroxidase, which were decreased in these rats. Further more, immunohistochemistry, RT-PCR assays and Western blot study demonstrated that DDPH attenuated the decreased expression of NMDAR2B (NR2B) in cortex and hippocampal CA1 region of the rats after bilateral carotid artery ligation. Our results suggest that DDPH may have favorable effects for the subjects in cerebrovascular insufficiency state following ischemic stroke.

Concurrent administration of cilostazol with donepezil effectively improves cognitive dysfunction with increased neuroprotection after chronic cerebral hypoperfusion in rats

In the present study, we assessed whether concurrent treatment with low doses of cilostazol and donepezil effectively improve memory deficits in association with amelioration of the pathological changes in the white matter of rats subjected to permanent ligation of bilateral common carotid arteries (BCCAL). The escape latency in Morris water maze test was significantly increased at 7, 14 and 21 days in rats subjected to BCCAL. At 21 days after ligation, the white matter lesions including vacuole formation with rarefaction were increased in the optic tract and corpus callosum accompanied by a large increase in glial fibrillary acidic protein (GFAP) immunoreactivity with significantly decreased CNPase-positive oligodendrocytes, all of which were significantly alleviated by the combination therapy with suboptimal doses of cilostazol (30 mg/kg orally) and donepezil (0.3 mg/kg intraperitoneally). The phosphorylated cyclic AMP response element-binding protein (p-CREB)- and Bcl-2-positive cells were significantly decreased following BCCAL, which were completely restored by the combination therapy, whereas the monotherapy with cilostazol or donepezil showed marginal effect. In conclusion, concurrent treatment with cilostazol and donepezil effectively prevented the occurrence of neuropathological alterations in the white matter by activation of p-CREB and Bcl-2, thereby resulting in improvement of spatial learning memory in rats subjected to chronic cerebral hypoperfusion.