The Effects Of Escin And Donepezil On The Cerebral Ischemia Injury And Vascular Dementia In The Rodent

Objective::Cerebrovascular diseases have the characteristics of high morbidity, high rate of mutilation and high mortality.They are the main causes resulting in cripplehood and death in middle and elder man,therefore has given rise to an enormous socioeconomic burden.Vascular dementia is a degenerative cerebrovascular disease that leads to a progressive decline in memory and cognitive functioning.It occurs when the blood supply carrying oxygen and nutrients to the brain is interrupted by a blocked or diseased vascular system.Signs and symptoms are varied and usually reflect increasing difficulty to perform everyday activities,such as eating,dressing,shopping, etc.Alzheimer's disease(AD) and vascular dementia(VD) are the two main causes of dementia.The incidence of VD is the second most common form of dementia in the elderly after AD.Pharmacological experiments and controlled clinical trials with cholinesterase inhibitors,such as donepezil,galantamine and rivastigmine in VD and AD,have demonstrated improvements in cognition,behavior and activities of daily living.However,the cholinesterase inhibitor agents may cause a broad spectrum of adverse events-nausea,vomiting,and diarrhoea,and so on,which make many patients withdrawn themselves from taking CHI agents.Vascular dementia is associated with the inflammation,edema and neural lesion resulted from cerebral ischemia.Escin(Sodium aescinate) is prescribed to treat ischemic stroke and traumatic brain injury.Donepezil,a cholinesterase inhibitor,is administered to the patients with dementia in the world.In this study,we investigated the effects of both escin and donepezil on cerebral ischemic injury and dementia in rodent animals,and the prevention of adverse action of these drugs.Methods:There are two parts of our study.One is about the effects and it's mechanism of escin on cerebral ischemic injury;the other is about the effects of donepezil on vascular dementia,and prevention of adverse action of donepezil.1.The effect of escin on cerebral ischemic injury and dementiaIn rats,two animal models were used to study the effect of escin on the cerebral ischemic injury and dementia.The first rat model,typical rat thread model,performed by middle cerebral artery occlusion(MCAO),was used to induce the reversible focal cerebral ischemia/reperfusion injury.Focal cerebral ischemia/reperfusion injury group which were subject to 2 hours of ischemia followed by 22 hours of reperfusion.The behavioral tests were used to evaluate the damage to central nervous system.2,3, 5-triphenyl tetrazolium chloride(TTC) staining method was used to assess the percentage of brain infarct area.H&E staining was used to observe the pathologic histological changes of hippocampus.The second rat model,rats were subjected to 2VO surgery.Briefly,under anesthesia,the bilateral common carotid arteries of rats were exposed and carefully separated from the carotid sheath and cervical sympathetic and vagus nerves through a ventral cervical incision,and ligated with silk thread.The animals in sham group received the same surgical operation without ligation of the carotid arteries served as sham-operated controls.The drugs were administered and Morris performance was carried out.In another experiment,six hours after occlusion,escin was given by vein every day for 3 days.To isolate brain mitochondria,rats were anesthetized with 10% chloral hydrate and killed by cardiac perfusion.The forebrain tissue was removed immediately and homogenized.The homogenate was immediately centrifuged to separate mitochondria.Mitochondrial swelling was assayed by measuring the decrease in absorbance at 540 nm.Mitochondrial respiratory control ratio(RCR) was measured. In mice,transient cerebral ischemia was induced by bilateral common carotid occlusion.The mice were anesthetized.Then the common carotid arteries were exposed and occluded with artery clips for 20 min.While the arteries were clamped, 0.3 ml of blood was withdrawn from the tail vein.Sham-operated mice were subjected to the same procedure without carotid clamping and withdrawal of blood.Mice alive 24 hr after operation were used in the Morris water maze task.Dally learning consisted of four trials in which the mouse was placed in the water from four different starting points and the latency of escape onto the platform was recorded. This was conducted for 5 consecutive days.A maximum of 60 sec was allowed during which the mouse had to find the platform and climb onto it.When the mouse reached the platform,it was allowed to remain on it for 20 sec.If the mouse failed to find the platform within 60 sec,it was removed from the water and placed on the platform for 20 sec.All latency for a given day was calculated by averaging the four trials.For cholinesterase activity assay,mice were killed by decapitation after the Morris water maze task had been carried out.The cerebral cortex from the right hemisphere was dissected on ice and homogenized.Cholinesterase activity in the homogenates was measured using the spectrophotometric methods.The brain edema was evaluated by the water content in the brain.The anti-inflammation characteristics of escin administered by intravenous injection,was observed by carrageenan-induced paw edema model in rats and vascular permeability induced by acetic acid in mice.The relationship between the effect of escin and adrenal gland was investigated by observing the anti-inflammatory effect of it in rats with adrenalectomy.The neurotransmitter receptors and regulators microarray were used to observe the change of gene regulation in the course of inflammation and anti-inflammation. The signal pathway of NFκB was also observed by ELISA.2.Neuroprotection of Acetylcholinesterase inhibitor donepezil and prevention of it's adverse actionIn mice,transient cerebral ischemia was induced by bilateral common carotid occlusion as above. Mice alive 24 hr after operation were used in the Morris water maze task.Daily learning consisted of four trials in which the mouse was placed in the water from four different starting points and the latency of escape onto the platform was recorded. This was conducted for 5 consecutive days.A maximum of 60 sec was allowed during which the mouse had to find the platform and climb onto it.When the mouse reached the platform,it was allowed to remain on it for 20 sec.If the mouse failed to find the platform within 60 sec,it was removed from the water and placed on the platform for 20 sec.All latency for a given day was calculated by averaging the four trials.For cholinesterase activity assay,mice were killed by decapitation after the Morris water maze task had been carded out.The cerebral cortex from the right hemisphere was dissected on ice and homogenized.Cholinesterase activity in the homogenates was measured using the spectrophotometric methods.In order to investigate how attenuate the side reaction of CHI agent while without affecting its efficacy,the effects of propantheline bromide(PB) co-administered with donepezil,such as the gastric emptying(GE) and gastrointestinal transit(GIT),the markers of gastrointestinal motility,and brain cholinesterase(ChE) activities,maze tasks of mice with vascular dementia induced by transient ischemia,were observed.The effects of ischemia and drugs(donepezil,propantheline alone,or co-administration of them) on the gene associated with neurotransmitter and receptor were investigated by real time PCR.Results:1.Compared with sham-operate group,rats with MCAO showed significant increase of neurological scores,significant increase of infarct area ratio.Escin could attenuate these changes.It suggested that escin could have a protection against ischemic injury.Twenty eight days after occlusion,in rats subjected to 2VO surgery,the memory of rat was decreased by ischemia and ameliorated by escin.Six hour after occlusion, escin could protect the respiratory energy system and increase the RCR.Mice subjected to transient cerebral ischemia took a longer time to locate the hidden platform than the sham-operated control mice during the learning trials, although the ischemia did not affect the swimming ability of the mice in the pretraining trial in the water maze.Treatment of mice with escin shortened the latency of escape onto the platform during the learning,as compared with the ischemia model control.Escin could attenuate the brain edema significantly for 12 h.In acute inflammatory animal models,escin inhibited carrageenan-induced paw edema in rats and vascular permeability induced by acetic acid in mice.The results demonstrated that the anti-inflammatory effect of escin was similar to dexamethasone,beginning from 4 h to 24 h after it was given.The anti-inflammatory effect of escin would be abolished by adrenalectomy in rats.The Corticosterone in sera was not increased by escin administered intravenously. These findings suggest the effect of escin depend on the adrenal gland and may amplify,the anti-inflammatory effect of glucocorticoids in vivo.2.Neuroprotection of acetylcholinesterase inhibitor donepezil and prevention of adverse action of donepezilGIT was accelerated significantly by donepezil 0.625 mg/kg during 30-60 min after it was given.The maximal acceleration of 26%was achieved 60 min after administration.Thus in experiments the effects of donepezil were observed 60 min after it was administered.Donepezil increased GIT,but GIT was inhibited by PB. When donepezil and PB were administered simultaneously,PB attenuated the GIT promoted by donepezil.At a dose of 2 mg/kg,PB restored GIT to the same level as in the saline group.This dose was equivalent to the therapeutic one in patients based on body surface area.Thus in the measurement of GE,the dose of PB was 2 mg/kg.Donepezil 0.625 mg/kg accelerated GE.PB co-administered with donepezil attenuated the increase of in GE.At dose of 2 mg/kg,PB restored GE to nearly normal level.About 30%of mice with transient cerebral ischemia were died within 24 hr after operation.Mice subjected to transient cerebral ischemia took a longer time to locate the hidden platform than the sham-operated control mice during the learning.(?) although the ischemia did not affect the swimming ability of the mice(?) pretraining trial in the water maze.Treatment of mice with donepezil signifi(?) shortened the latency of escape onto the platform during the learning,especia(?) the 5th learning day,as compared with the ischemia model control.PB alone di(?) have any effect on the latency.Co-administration of PB and donepezil did not(?) the effects of donepezil on the latency of escaping onto the platform in the(?) maze performance,showing that PB had no effect on the therapeutic eff(?) donepezil.After administration of donepezil or both donepezil and PB for 8 days,(?) activity in the brain was decreased compared with that in the ischemia model(?) However,there was no difference between the donepezil alone and donepezil pl(?) groups.This suggested that PB could not change the ability of donepezil to pen(?) through the blood-brain barrier and the inhibitory effects of donepezil on brain(?) activity.The muscarinic receptor blocker PB is unable to penetrate the blood-brain(?) and when co-administered with a cholinesterase inhibitor attenuates its side(?) without affecting its therapeutic effects,and thus may be beneficial for patients(?) VD.The gene of acetylcholinesterase was not changed by drugs mentioned above.(?) effects of co-administered with PB and donepezil was different from that of don(?) given alone,which suggested that clinical trial shoud be carried out to know the(?) effect of co-administration.Conclusion:1.Escin could protect rat brains against ischemia injury and attenuate the vas(?) dementia in the rodemt.2.The anti-inflammatory effect of eacin is similar to dexamethasone.(?) anti-inflammatory mechanism of escin may be associated with amplifying(?) anti-inflammatory effect of glucocorticoids in vivo. 3.The muscarinic receptor blocker PB is unable to penetrate the blood-brain barrier and when co-administered with a cholinesterase inhibitor attenuates its side effects without affecting its therapeutic effects,and thus may be beneficial for patients with VD.