Different effects of mechanical embolus removal and intra-arterial thrombolysis on neural functional recovery in patients with acute middle cerebral artery occlusion

Objective:To study the different effects of mechanical embolus removal and intra-arterial thrombolysis on neural functional recovery in patients with acute middle cerebral artery occlusion.Methods: Patients with acute middle cerebral artery occlusion who were treated in the First Hospital of Yulin between September 2013 and October 2017 were selected and retrospectively studied, and the differences in reperfusion therapies in history data were referred to divide them into study group A and study group B who underwent mechanical embolus removal and intra-arterial thrombolysis respectively. The levels of neurocyte damage markers, apoptosis markers and stress markers in serum as well as the expression of Wnt pathway molecules in peripheral blood were determined before treatment and 24 h after treatment.Results: Compared with those of same group before treatment, serum NSE, S100B, VILIP1, sFas, sFasL, ET-1 and MDA levels as well as peripheral blood GSK3β, LC3-II and Beclin1 expression intensity of both groups were decreasing whereas serum BDNF, NTF, sLivin and SOD levels as well as peripheral bloodβ-catenin and mTOR expression intensity were increasing, and serum NSE, S100B, VILIP1, sFas, sFasL, ET-1 and MDA levels as well as peripheral blood GSK3β, LC3-II and Beclin1 expression intensity of study group A after treatment were lower than those of study group B whereas serum BDNF, NTF, sLivin and SOD levels as well as peripheral bloodβ-catenin and mTOR expression intensity were higher than those of study group B.Conclusion: Mechanical embolus removal for acute middle cerebral artery occlusion can be more effective than intra-arterial thrombolysis to reduce the nerve function damage as well as the corresponding oxidative stress and apoptosis.

Therapeutic effects of different durations of acupuncture on rats with middle cerebral artery occlusion

Acupuncture is regarded as an effective therapy for cerebral ischemia. Different acupuncture ma- nipulations and durations may result in different therapeutic effects. In the present study, the Neig uan （PC6） acupoint of rats with occluded middle cerebral arteries was needled at a fixed frequency （3 Hz） with different durations, i.e., 5, 60 and 180 seconds under a twisting-rotating acupuncture method. Results showed that different durations of acupuncture had different therapeutic effects, with 60 seconds yielding a better therapeutic effect than the other two groups. This duration of treatment demonstrated rapid cerebral blood flow, encouraging recovery of neurological function, and small cerebral infarct volume. Experimental findings indicated that under 3 Hz frequency, the treatment of needling Neiguan for 60 seconds is effective for ischemic stroke.

Modified constraint-induced movement therapy alters synaptic plasticity of rat contralateral hippocampus following middle cerebral artery occlusion

Modified constraint-induced movement therapy is an effective treatment for neurological and motor impairments in patients with stroke by increasing the use of their affected limb and limiting the contralateral limb.However,the molecular mechanism underlying its efficacy remains unclear.In this study,a middle cerebral artery occlusion(MCAO)rat model was produced by the suture method.Rats received modified constraint-induced movement therapy 1 hour a day for 14 consecutive days,starting from the 7^th day after middle cerebral artery occlusion.Day 1 of treatment lasted for 10 minutes at 2r/min,day 2 for 20 minutes at 2 r/min,and from day 3 onward for 20 minutes at 4 r/min.CatWalk gait analysis,adhesive removal test,and Y-maze test were used to investigate motor function,sensory function as well as cognitive function in rodent animals from the 1st day before MCAO to the 21^st day after MCAO.On the 21^st day after MCAO,the neurotransmitter receptor-related genes from both contralateral and ipsilateral hippocampi were tested by micro-array and then verified by western blot assay.The glutamate related receptor was shown by transmission electron microscopy and the glutamate content was determined by high-performance liquid chromatography.The results of behavior tests showed that modified constraint-induced movement therapy promoted motor and sensory functional recovery in the middle cerebral artery-occluded rats,but had no effect on cognitive function.The modified constraint-induced movement therapy upregulated the expression of glutamate ionotropic receptor AMPA type subunit 3(Gria3)in the hippocampus and downregulated the expression of the beta3-adrenergic receptor gene Adrb3 and arginine vasopressin receptor 1 A,Avprla in the middle cerebral artery-occluded rats.In the ipsilateral hippocampus,only Adra2 a was downregulated,and there was no significant change in Gria3.Transmission electron microscopy revealed a denser distribution the more distribution of postsynaptic glutamate receptor 2/3,which is an a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor,within 240 nm of the postsynaptic density in the contralateral cornu ammonis 3 region.The size and distribution of the synaptic vesicles within 100 nm of the presynaptic active zone were unchanged.Western blot analysis showed that modified constraint-induced movement therapy also increased the expression of glutamate receptor 2/3 and brain-derived neurotrophic factor in the hippocampus of rats with middle cerebral artery occlusion,but had no effect on Synapsin I levels.Besides,we also found modified constraint-induced movement therapy effectively reduced glutamate content in the contralateral hippocampus.This study demonstrated that modified constraint-induced movement therapy is an effective rehabilitation therapy in middle cerebral artery-occluded rats,and suggests that these positive effects occur via the upregulation of the postsynaptic membrane a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor expression.This study was approved by the Institutional Animal Care and Use Committee of Fudan University,China(approval No.201802173 S)on March 3,2018.

An enriched environment promotes synaptic plasticity and cognitive recovery after permanent middle cerebral artery occlusion in mice

Cerebral ischemia activates an endogenous repair program that induces plastic changes in neurons. In this study, we investigated the effects of environmental enrichment on spatial learning and memory as well as on synaptic remodeling in a mouse model of chronic cerebral ischemia, produced by subjecting adult male C57 BL/6 mice to permanent left middle cerebral artery occlusion. Three days postoperatively, mice were randomly assigned to the environmental enrichment and standard housing groups. Mice in the standard housing group were housed and fed a standard diet. Mice in the environmental enrichment group were housed in a cage with various toys and fed a standard diet. Then, 28 days postoperatively, spatial learning and memory were tested using the Morris water maze. The expression levels of growth-associated protein 43, synaptophysin and postsynaptic density protein 95 in the hippocampus were analyzed by western blot assay. The number of synapses was evaluated by electron microscopy. In the water maze test, mice in the environmental enrichment group had a shorter escape latency, traveled markedly longer distances, spent more time in the correct quadrant(northeast zone), and had a higher frequency of crossings compared with the standard housing group. The expression levels of growth-associated protein 43, synaptophysin and postsynaptic density protein 95 were substantially upregulated in the hippocampus in the environmental enrichment group compared with the standard housing group. Furthermore, electron microscopy revealed that environmental enrichment increased the number of synapses in the hippocampal CA1 region. Collectively, these findings suggest that environmental enrichment ameliorates the spatial learning and memory impairment induced by permanent middle cerebral artery occlusion. Environmental enrichment in mice with cerebral ischemia likely promotes cognitive recovery by inducing plastic changes in synapses.

Predictors of short-term outcome in patients with acute middle cerebral artery occlusion: unsuitability of fluid-attenuated inversion recovery vascular hyperintensity scores

Fluid-attenuated inversion recovery （FLAIR） vascular hyperintensity （FVH） is used to assess leptomeningeal collateral circulation, but clinical outcomes of patients with FVH can be very different. The aim of the present study was to assess a FVH score and explore its relationship with clinical outcomes. Patients with acute ischemic stroke due to middle cerebral artery M1 occlusion underwent magnetic resonance imaging and were followed up at 10 days （National Institutes of Health Stroke Scale） and 90 days （modified Rankin Scale） to determine short-term clinical outcomes. Effective collateral circulation indirectly improved recovery of neurological function and short-term clinical outcome by extending the size of the pial penumbra and reducing infarct lesions. FVH score showed no correlation with 90-day functional clinical outcome and was not sufficient as an independent predictor of short-term clinical outcome.

Screen-imaging guidance using a modified portable video macroscope for middle cerebral artery occlusion

The use of operating microscopes is limited by the focal length.Surgeons using these instruments cannot simultaneously view and access the surgical field and must choose one or the other.The longer focal length （more than 1 000 mm） of an operating telescope permits a position away from the operating field,above the surgeon and out of the field of view.This gives the telescope an advantage over an operating microscope.We developed a telescopic system using screen-imaging guidance and a modified portable video macroscope constructed from a Computar MLH-10 × macro lens,a DFK-21AU04 USB CCD Camera and a Dell laptop computer as monitor screen.This system was used to establish a middle cerebral artery occlusion model in rats.Results showed that magnification of the modified portable video macroscope was appropriate （5-20 ×） even though the Computar MLH-10 × macro lens was placed 800 mm away from the operating field rather than at the specified working distance of 152.4 mm with a zoom of 1-40 ×.The screen-imaging telescopic technique was clear,life-like,stereoscopic and matched the actual operation.Screen-imaging guidance led to an accurate,smooth,minimally invasive and comparatively easy surgical procedure.Success rate of the model establishment evaluated by neurological function using the modified neurological score system was 74.07%.There was no significant difference in model establishment time,sensorimotor deficit and infarct volume percentage.Our findings indicate that the telescopic lens is effective in the screen surgical operation mode referred to as ＂long distance observation and short distance operation＂ and that screen-imaging guidance using an modified portable video macroscope can be utilized for the establishment of a middle cerebral artery occlusion model and micro-neurosurgery.

Effect of Fujian tablet on the expression of Nogo-A mRNA in the cervical spinal cord of middle cerebral artery occlusion model rats

Inhibiting the expression of Nogo-A in cervical spinal cord by use of interaction of antigen and antibody can help the remodeling of corticospinal projection of focal cerebral ischemia model rats to facilitate neurological recovery, which provides a new possible mechanism for drugs to promote neurological recovery. However, the effects of drugs on the expression of Nogo-A in cervical spinal cord are still unclear. OBJECTIVE： To observe the effect of Fujian tablet on the expression of Nogo-A mRNA in cervical spinal cords of middle cerebral artery occlusion （MCAO） rats, and to investigate the possible regulatory effect of Fujian tablet on the regenerated microenvironment of spinal conduction bundle. DESIGN： A randomized and controlled trial taking Wistar rats as experimental animals. SETTING： Department of Neurology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine. MATERIALS： This experiment was carried out in the laboratory of Shandong Academy of Medical Science between June 2005 and July 2006. A total of 40 healthy male Wistar rats, aged 12 weeks, weighing 250 - 300 g, were provided by the Experimental Animal Center of Shandong University. Fujian tablets （main components： Heshouwu, Yinyanghuo, etc） were provided by office of Pharmaceutics of Shandong University of traditional Chinese medicine. Nogo-A detection kit was provided by Wuhan Boster Biotechnology Co.,Ltd., and batch number was 040309009. This experiment was approved by Local Animal Ethics Committee. METHODS： Forty male rats were randomly divided into 4 groups, with 10 in each： normal group, sham-operation group, model group and administration group. Rats in the administration group and model group were subjected to MCAO. Rats in the sham-operation group underwent the same craniotomy, and their middle cerebral arteries （MCA） were not occluded. Rats in the normal group were untouched. Rats in administration group were intragastrically administrated with the solution of Fujian tablet at a dose of 9 g/kg and others were given equal dosage of normal saline two days later. The treatments were done once a day and the course totaled 2 weeks. MAIN OUTCOME MEASURES： The expression of Nogo-A mRNA in slices of cervical spinal cords. RESULTS： Forty rats were involved in the final analysis. The expression of Nogo-A mRNA in the cervical spinal cord of rats in the administration group and model group was significantly decreased as compared with that in the normal group （P 〈 0.01 and P 〈 0.05, respectively）. The expression of Nogo-A mRNA in the administration group was also significantly weaker than that in the model group （P 〈 0.05 ） . CONCLUSION： Fujian tablet can inhibit the expression of Nogo-A mRNA in cervical spinal cords of MCAO rats, which facilitates regeneration and remodeling of cervical spinal cords.

In vivo tracking of human adipose-derived stem cells labeled with ferumoxytol in rats with middle cerebral artery occlusion by magnetic resonance imaging

Ferumoxytol, an iron replacement product, is a new type of superparamagnetic iron oxide ap- proved by the US Food and Drug Administration. Herein, we assessed the feasibility of tracking transplanted human adipose-derived stem cells labeled with ferumoxytol in middle cerebral artery occlusion-injured rats by 3.0 T MRI in vivo. 1 × 104 human adipose-derived stem cells labeled with ferumoxytol-heparin-protamine were transplanted into the brains of rats with middle cerebral artery occlusion. Neurologic impairment was scored at 1, 7, 14, and 28 days after transplantation. T2-weighted imaging and enhanced susceptibility-weighted angiography were used to observe transplanted cells. Results of imaging tests were compared with results of Prussian blue staining. The modified neurologic impairment scores were significantly lower in rats transplanted with cells at all time points except I day post-transplantation compared with rats without transplantation. Regions with hypointense signals on T2-weighted and enhanced susceptibility-weighted angiography images corresponded with areas stained by Prussian blue, suggesting the presence of superparamagnetic iron oxide particles within the engrafted cells. Enhanced susceptibility-weighted angiography image exhibited better sensitivity and contrast in tracing ferumoxytol-heparin-protamine-labeled human adipose-derived stem ceils compared with T2-weighted imaging in routine MRI.

Apparent diffusion coefficient evaluation for secondary changes in the cerebellum of rats after middle cerebral artery occlusion

Supratentorial cerebral infarction can cause functional inhibition of remote regions such as the cerebellum, which may be relevant to diaschisis. This phenomenon is often analyzed using positron emission tomography and single photon emission CT. However, these methods are expensive and radioactive. Thus, the present study quantified the changes of infarction core and remote regions after unilateral middle cerebral artery occlusion using apparent diffusion coefficient values. Diffu- sion-weighted imaging showed that the area of infarction core gradually increased to involve the cerebral cortex with increasing infarction time. Diffusion weighted imaging signals were initially in- creased and then stabilized by 24 hours. With increasing infarction time, the apparent diffusion co- efficient value in the infarction core and remote bilateral cerebellum both gradually decreased, and then slightly increased 3-24 hours after infarction. Apparent diffusion coefficient values at remote regions （cerebellum） varied along with the change of supratentorial infarction core, suggesting that the phenomenon of diaschisis existed at the remote regions. Thus, apparent diffusion coefficient values and diffusion weighted imaging can be used to detect early diaschisis.

Effects of Echinacoside on Histio-central Levels of Active Mass in Middle Cerebral Artery Occlusion Rats

Objective To investigate the effects of echinacoside on the extracellular striatal levels of norepinephrine（NE）,dopamine（DA）,homovanillic acid（HVA）,3,4-dihydroxyphenylethanoid acid（DOPAC）,5-hydroxyindoleacetic acid（HIAA）,and 5-hydroxytryptamine（5-HT） in middle cerebral artery occlusion（MCAO rats.Methods The middle cerebral artery was occluded in male Sprague-Dawley rats.Three days later microdialysis probes were placed into the right striatum of MCAO rat brains and the brains were perfused with Ringer＇s solution at a rate of 1.5 μL/min.Cerebral microdialysates were collected every 30 minutes from awake and freely moving rats before assaying for NE,DA,HVA,DOPAC,HIAA,and 5-HT levels by reverse phase HPLC with electrochemistry.Results Three days after MCAO,the extracellular striatal levels of NE,DA,DOPAC,HIAA,HVA,and 5-HT of the MCAO rats increased significantly（at least P0.05 vs.control）.However,simultaneous treatment with echinacoside（30.0 or 15.0 mg/kg） attenuated these increases（at least P0.05 vs.non-treated model rats）.Conclusion These results imply that echinacoside may protect striatal dopa minergic neurons from the injury induced by MCAO and may help prevent and treat cerebral ischemic diseases.

Establishment of a rhesus monkey model of middle cerebral artery ischemia and reperfusion using a microcatheter embolization method

Nonhuman primates are closest to humans in terms of lineage, and middle cerebral artery ischemia/reperfusion responses of nonhuman primates are most similar to ischemic stroke in humans. Therefore, nonhuman primates could be utilized to simulate the process of ischemic stroke in the human. Few studies, however, have reported the use of endovascular technology to establish a rhesus monkey stroke model. In the present study, seven adult, male, rhesus monkeys were selected and, following anesthesia, a microcatheter was inserted into one side of the middle cerebral artery via the femoral artery to block blood flow, thereby resulting in middle cerebral artery occlusion. After 2 hours, the microcatheter was withdrawn to restore the middle cerebral artery blood flow and to establish ischemia/reperfusion. Results from angiography and magnetic resonance angiography confirmed occlusion and reopening of the middle cerebral artery. Magnetic resonance imaging revealed the existence of ischemic brain lesions, and neurological examination showed sustained functional deficits following surgery. The rhesus monkey middle cerebral artery ischemia/reperfusion models established by microcatheter embolization had the advantage of non-craniotomy invasion and reproducibility. The scope and degree of ischemic damage using this model was controllable. Therefore, this nonhuman primate model is an ideal model for cerebral ischemia and reperfusion.

Neuroprotective effect of Longshengzhi capsule following permanent middle cerebral artery occlusion in rats

Objective:Longshengzhi capsule(LSZC)is an optimized preparation based on the traditional Chinese Medicine formula Buyanghuanwu Decoction(BYHWD),and is approved by the China Food and Drug Administration for treating stroke-induced disability and vascular diseases.Herein,we examined the pharmacodynamics,anti-apoptotic and anti-oxidant actions,and potential mechanisms of action of LSZC following stroke in rats.Methods:Permanent middle cerebral artery occlusion(MCAO)was used as an ischemic stroke model.LSZC was administered intragastrically.We examined the survival rate,bodyweight,and neurological deficits in stroke rats.Brain infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining.Brain pathology was examined using hematoxylin and eosin staining,Nissl staining,and TdT-mediated dUTP Nick-End Labeling staining.Malondialdehyde,catalase,superoxide dismutase,and glutathione levels were examined by commercial kits.Expression of Nrf2,heme oxygenase-1,Bax,Bcl-2,cleaved caspase-3,and caspase-3 proteins in brain tissue was measured by Western blot.Results:LSZC markedly improved the survival rate and bodyweight,and reduced infarct volume and neurological deficit scores,in MCAO stroke rats.LSZC also significantly attenuated oxidative stress,as indicated by decreased expression of malondialdehyde,and upregulation of Nrf2,heme oxygenase-1,catalase,superoxide dismutase,and glutathione.Moreover,LSZC significantly decreased apoptosis,including a decrease in Bax and cleaved caspase-3 expression,and an increase in Bcl-2,as well as a reduction in numbers of apoptotic neurons.Conclusion:LSZC treatment is neuroprotective against ischemic stroke,potentially via reducing oxidative stress and apoptosis.The Nrf2 and apoptotic signaling pathways may play important roles in the antioxidant and anti-apoptotic actions of LSZC.

Occlusion of the middle cerebral artery Guidance by screen imaging using an EDA-H portable medium-soft electronic endoscope

The present study aimed to verify the practicability of performing screen-based surgical operations under the guidance of a real-time viewing and recording system （RTVRS） using a portable medium-soft electronic endoscope （pmsEE）. The middle cerebral artery in rats was occluded under screen-imaging guidance using a pmsEE RTVRS to reproduce an animal model of human cerebral infarction. The screen imaging of the pmsEE RTVRS was clear, life-like, stereoscopic and synchronous with the actual operation. Screen-imaging guidance led to an accurate, smooth, minimally invasive and comparatively easy surgical procedure. The surgical success rate, time of model establishment, neurological function scores, and infarct volume were similar to those using an operating microscope. These results indicate that the self-designed pmsEE RTVRS could be utilized for portable endoscopic screen-based surgical operations.

Middle cerebral artery occlusion methods in rat versus mouse model of transient focal cerebral ischemic stroke

Experimental stroke research commonly employs focal cerebral ischemic rat models （Bederson et al., 1986a; Longa et al., 1989）. In human patients, ischemic stroke typically results from thrombotic or embolic occlusion of a major cerebral artery, usually the mid- dle cerebral artery （MCA）. Experimental focal cerebral ischemia models have been employed to mimic human stroke （Durukan and Tatlisumak, 2007）. Rodent models of focal cerebral ischemia that do not require craniotomy have been developed using intraluminal suture occlusion of the MCA （MCA occlusion, MCAO） （Rosamond et al., 2008）. Furthermore, mouse MCAO models have been wide- ly used and extended to genetic studies of cell death or recovery mechanisms （Liu and McCullough, 2011）. Genetically engineered mouse stroke models are particularly useful for evaluation of isch- emic pathophysiology and the design of new prophylactic, neuro- protective, and therapeutic agents and interventions （Armstead et al., 2010）. During the past two decades, MCAO surgical techniques have been developed that do not reveal surgical techniques for mouse MCAO model engineering. Therefore, we compared MCAO surgical methods in rats and mice.

Superficial Temporal Artery to Middle Cerebral Artery Bypass and Endovascular Parent Artery Occlusion in the Treatment of Giant Intracranial Aneurysms

The authors are reporting on a study drawn from unpublished dissertation done by the corresponding author when he completed his neurosurgical training in Paris, France in 2004, few years before the advent of flow diverters. The study was a retrospective review of giant intracranial aneurysms treated by superficial temporal artery to middle cerebral artery bypass combined with endovascular occlusion of the parent artery. From 1990 to 2003, 29 consecutive cases of giant cerebral aneurysms, not suitable to selective treatment were managed in that way. Twenty-one medical records had enough data to allow objective evaluation. Sixteen female and five male patients bearing 21 giant aneurysms were involved. Their mean age was 46 years. The aneurysm was revealed by mass effect in 13 cases and subarachnoid hemorrhage in one case. On admission 19 patients presented with unruptured aneurysms and two have sustained a subarachnoid hemorrhage. The balloon occlusion test before the bypass operation was not tolerated in 18 patients. The treatment was completed in 19 patients and 17 of them had parent artery occlusion with latex detachable balloons. The only death of the series occurred before the endovascular treatment. The mean follow-up period was 30 months. After completion of the treatment, 16 (84%) patients had no symptom. Aneurysm recanalization or rupture was not observed after the parent artery occlusion. With the combination of superficial temporal artery to middle cerebral artery bypass + endovascular parent artery occlusion, 90% of giant intracranial aneurysms untreatable selectively were permanently excluded with a good outcome in 95%.

Tissue plasminogen activator via cross-collateralization for tandem internal carotid and middle cerebral artery occlusion

Tandem internal carotid and middle cerebral artery occlusion after carotid dissection predicts poor outcome after systemic thrombolysis. Current treatments include the use of endovascular carotid stenting, which carries with it a high risk of propagating further embolic events and worsening the dissection. New strategies for avoiding the aforementioned side-effects include recanalization using cross-collaterals for delivery of intra-lesional tissue plasminogen activator(t PA). We present two cases that provide further support for this novel approach. Both patients presented with a National Institute of Health Stroke Scale of 20, received intra-arterial t PA via cross-collateralization, and made full recoveries without the need for stenting.

Neurite Orientation Dispersion and Density Imaging of Rat Brain Microstructural Changes due to Middle Cerebral Artery Occlusion at a 3T MRI

The purpose of this work was to demonstrate the feasibility of neurite orientation dispersion and density imaging(NODDI)in characterizing the brain tissue microstructural changes of middle cerebral artery occlusion(MCAO)in rats at 3T MRI,and to validate NODDI metrics with histology.A multi-shell diffusion MRI protocol was performed on 11 MCAO rats and 10 control rats at different post-operation time points of 0.5,2,6,12,24 and 72 h.NODDI orientation dispersion index(ODI)and intracellular volume fraction(V_(ic))metrics were compared between MCAO group and control group.The evolution of NODDI metrics was characterized and validated by histology.Infarction was consistent with significantly increased ODI and V_(ic)in comparison to control tissues at all time points(P<0.001).Lesion ODI increased gradually from 0.5 to 72 h,while its V_(ic)showed a more complicated and fluctuated evolution.ODI and V_(ic)were significantly different between hyperacute and acute stroke periods(P<0.001).The NODDI metrics were found to be consistent with the histological findings.In conclusion,NODDI can reflect microstructural changes of brain tissues in MCAO rats at 3T MRI and the metrics are consistent with histology.This study helps to prepare NODDI for the diagnosis and management of ischemic stroke in translational research and clinical practice.

Ginkgo biloba extract-761 improves the motor functions in permanent middle cerebral artery occlusion rats

OBJECTIVE To study the role of Ginkgo biloba extract-761(EGb-761)in the recovery of gait abnormality and its neuroprotective effect against the brain injury induced by permanent middle cerebral artery occlu-sion in rats.METHODS Male Sprague Dawley rats(n=200,240-305 g)were anesthetized with 0.2%pentobarbital sodium diluted in physiological saline(2.0 m L·kg-1,ip).Then a monofilament coated with poly-L-lysine,was used to occlude the origin of the middle cerebral artery.It was inserted into the internal carotid artery lumen until it met mild resistance,approximately 20mm beyond the common carotid artery bifurcation.The suture was secured with a ligature and maintained in place until sacrifice.The same surgical procedure was conducted in sham-operated rats in which the middle cerebral artery was not occluded.Motor and behavioral changes were assessed after surgery using a five point scale.The rats securing the point scale above 2 were included in the study.The rats were randomly divided into control,and treated groups:EGb-761(20,50,and 100 mg·kg-1).The treated groups were oral y administered(10 mL·kg-1)for 28 d.On 7th,14th,21st,and 28th day the neurological scores,rotar rod test and gait assessment(the automated computer-assisted method)were performed.The brains were collected for TTC staining and histopathological analysis.RESULTS 1)Weight:On 28th day,EGb-761(20 mg·kg-1,)significantly increased the weight of the rat by^8%as compared to control(~300 g).However,at 50 mg·kg-1,and 100 mg·kg-1,a significant increase of^7-7.6%(control:~232 g),and^7.3-7%,respectively from 14 to 28 days was noted.2)Neurological scores:On 28thday,EGb-761(20,50,and 100 mg·kg-1)significantly decreased the neurological scores by^18%,~22%,~21%,respectively as compared to control(~2.07).3)Rotar rod test:On 28thday,EGb-761(50,and100 mg·kg-1)significantly increased by^69.1%,~74.1%,respectively as compared to control(~28.2).4)Gait assessment:On 7th,14th,21st,and 28thday,EGb-761(20,50,and 100 m·kg-1)significantly reduced the average body angle,on 7th,14th,21st,and 28thday,EGb-761(100 mg·kg-1)significantly increased the walk speed and reduced the average walking cycle,EGb-761(50,and 100 mg·kg-1)significantly the area of the left brain/right brain area percentage and reduced tissue pathologic neuron injury.CONCLUSION Ginkgo biloba extract EGb-761 has obvious improve behavior disorders,and has a protective neuroprotective effect against the brain injury induced by permanent middle cerebral artery occlusion.

Expression of insulin-like growth factor-1 mRNA and protein level of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkeys

BACKGROUND： Insulin-like growth factor-I（IGF-1）, as one of the important members of growth factor family, participants in the regulation of many physiological functions and behaviors, having very strong neuroprotective effect. However, the expression of IGF-1 following cerebral ischemia/reperfusion is still disputed. OBJECTIVE： To observe the expression of IGF-1 and protein of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkey. DESIGN ： A completely randomized grouping design, controlled animal experiment SETTING ： Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University MATERIALS： ① Totally 17 rhesus monkeys , of either gender, aged 4 to 5 years, were enrolled . Seven rhesus monkeys observed with gene chip were randomly divided into 2 groups： sham operation group （n=3） and ischemia/reperfusion group 〈n=4〉. Ten rhesus monkeys observed with in situ hybridization and immunohistochemistry method were randomly divided into 2 groups： sham operation group 〈n=3 〉and ischemia/reperfusion group （n=7）. Rhesus monkeys observed under microscope were divided into 2 groups： sham operation group （n=6） and ischamia/reperfusion group （n=-11）.②Materials used in the experiment： cresyl violet （Sigma Company, America）; immunohistochemical reagent kit （ Huamei Bio-engineering Company）; In situ hybridization reagent kit （Boshide Bio-engineering Co.Ltd, Wuhan）; 12 800 dots chip （Boxing Company, Shanghai）. METHODS ： This experiment was carried out at the Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University from January 2001 to December 2003.① The onset area of middle cerebral artery was blocked for 2 hours, middle cerebral artery ischemia/reperfusion models were created.② After ischemia/reperfusion for 24 hours, cerebral tissue sections of rhesus monkeys were prepared and stained with cresyl violet. Image analysis was performed with 5001W image analysis software. Morphological change of corpora striata of operative side was observed in the rhesus monkeys between two groups. Total RNA was extracted from cerebral tissue. ③ Detection of gene chip： Cy3-duTP and Cy5-duTP were used to respectively perform reverse transcription labeling. The sample was reversely transcribed into cDNA, then hybridized with cDNA of cerebral tissue. Genes with the separate absolute value of cy3 and cy5〉800, cY3/cy5 〉 2（high expression） or 〈 0.5 （low expression） were found out. Those were genes with differential expression. ④ The expressions of IGF-1 mRNA and protein level of corpora striata in ischemic side of rhe- sus monkeys were detected between sham operation group and ischemia/reperfusion group at 9 and 24 hours after ischemia/reperfusion with in situ hybridization method and immunohistochemical method. Brown granules were IGF-1 protein positive cells. ⑤ Analysis of variance was used in the difference comparison of measurement data among groups. MAIN OUTCOME MEASURES ： ① Change of morphological structure of corpora striata at ischemic side in rhesus monkeys. ② Change of cerebral gene expression profiles at ischemia/reperfusion in rhesus monkeys between two groups.③ Expression of IGF-1 mRNA and protein level of corpora striata at ischemia/reperfu- sion in rhesus monkeys between two groups. RESULTS ： ① Pathological change ： Obvious pathological change of cerebral infarction appeared in the ischemia and reperfusion group, while there was no such pathological change in the sham operation group.② Change of gene expression profile ： There were 4480 genes with difference expression in the ischemia/reperfusion group and sham-operation group, in which, 260 genes had high expression and their absolute value was over 800, and 63 genes had low expression, cy3/cy5 of IGF-1 was 0.379, being relative low ex- pression. ③ IGF-1 mRNA and protein positive cell counts in corpora striata at cerebral ischemic side[IGF-1 mRNA： 〈9.72±1.18）,（9.11 ±0.76）,（14.77±0.60） counts/field：lGF-1 protein： （15.11 ±1.83）,（15.39±0.78）, （34.62±0.97）counts/field, P 〈 0.05-0.01]. CONCLUSION： IGF-1 mRNA and protein are lowly expressed in middle cerebral artery of rhesus monkeys at ischemia/reperfusion.