| Background Ischemic stroke is one of the leading causes of death in the world,and continues to be the leading causes of long-term disability.The research for a good repeating.reliable animal model which is similiar to the pathophysiology of human ischemic stroke has been prompted by many scholars. With the development of the neuron injuring and repairment,a better ischemic animal model is wanted.How to develop a standarized ischemic animal model under the condition and choose the proper one for the research of the ischemic stroke is a good question to us now.To development a cheaper.simple and reliable animal model is very important for the research of the ischemic stroke. Many ischemic stroke happen owing to the occlusion of middle cerebral artery(MCA).So middle cerebral artery occlusion/reperfusion(MCAO/R)model has been widely used today.A direct microsurgical technique for permanently occlusion the MCA through a craniectomy had also been done.Since Traura invented the MCAO model through a craniectomy,it has been studied and modified extensively.Now the model of reversible MCAO without craniectomy is accepted widely.Objective To establish a classical model with MCAO/R in ratMethods We choosed 200 healthy male Wister rats,weighing between240-260g.All rats were anesthetized with hydrochloride ketemine.The nylon sutures were coated with silica gel at one end.Then we introduced the nylon sutures into the cervical internal carotid artery and advanced them intracranically to block blood flow into the MCA at 0h.0.5h,1.0h,1.5h and 2.0h.then drawed out the suture.So the occlused MCA were reperfused.Neurologic deficit,morality rate,brain wet content and infarct volume were evaluated in all surving rats. Results 165 rats survived and 35 rats were dead.Of all surving rats,the grades of neurologic defict were 0.00±0.00,2.07±0.51,2.91±0.44,3.24±0.49and 3.61±0.39 at the different groups which were divided according the ischemic time from 0h to 2h one by one.The brain wet contents were 78.93±1.22,81.44±1.41,83.63±1.39,84.15±1.44,84.81±1.37 and the infarct volumes were 0%,65.63±5.1%,72.76±4.2%,75.39±4.8%and 80.21±4.2%at different groups.Conclusion The MCAO/R model is stable with accurate timing control of occlusion and reperfusion.It is the classical one for researching of pathophysiology in ischemic stroke. Background The matrix metalloproteinases(MMPs),may degrade extracellular matrix and trigger anoikis-like cell death in neurons.MMPs comprise an important family of proteases associated with basement membrane and extracellular matrix remodeling and are involved in both physiological and pathological CNS processes.In particular,the gelatinases(MMP-2 and MMP-9) have been implicated specifically in cerebral ischemia.Knock-out mice deficient in MMP-9 are protected against brain trauma and focal cerebral ischemia.The mechanisms of injury may involve MMP-9-mediated disruption of blood-brain barrier integrity,edema,and hemorrhagic conversion.Neuregulins are signalling proteins and the NRG1 family encodes at least 15 different splice variants,classified into four isoforms.They play an important role in cell differentiation,migration, myelination and proliferation of oligodendrocytes and neurons.In animal models of ischemia/hypoxia,NRG1 has been shown to act as a therapeutic,neuroprotective agent and should be investigated in more detail in transgenic animal models.A recent study demonstrated that NRG-1 blocked delayed neuronal death and pro-inflammatory responses following focal ischemic stroke in the rat.However,the mechanisms that underlie the neuroprotective effects of NRG-1 are unclear.Objective To observe the expression of matrix metalloproteinase-9 (MMP-9) and the influence of neuregulin-1β(NRG-1β) on brain tissue of rats following cerebral ischemia/reperfusion.Methods The animal models of middle cerebral artery occlusion/reperfusion (MCAO/R) were established by a monofilament method from left external-internal carotid artery in 200 adult healthy male Wister rats.The rat models in the treatment group were injected 1.5%NRG-1β5μL from internal carotid artery (ICA).The cerebral infarct volume was measured by TTC stain,the apoptosis was determined wit in situ TUNEL method,the expressions of MMP-9 were determined by immunohistochemical and immumofluorescent double staining and western-blotting assay.Results Cerebral ischemia reperfusion can induce apoptosis and expression of MMP-9 in cerebral cortex and striatum.With the ischemic time prolonging,the number of apoptotic cells in cortex from ischemic 0h,0.5h,1.0h,1.5h to 2.0h increased from 1.78±0.15,5.78±0.51,10.35±0.77,21.50±1.19 to 32.00±1.78,while the number of apoptotic cells in striatum from ischemic 0h,0.5h,1.0h,1.5h to 2.0h also increased successfully from 1.46±0.21,4.12±0.54,7.33±0.71,16.54±1.63 to 19.03±1.44(t=5.44～21.80,P＜0.05) and the expression of MMP-9 increased successfully(t=3.363～25.523,P＜0.01) in the control group. With NRG-1βtreatment,the number of apoptotic cells incortex from ischemic 0h,0.5h,1.0h,1.5h to 2.0h reduced from 1.66±0.11,4.80±0.61,5.63±0.56,9.75±1.22 to 13.54±1.26;while the number of apoptotic cells in striatum from ischemic 0h,0.5h,1.0h,1.5h to 2.0h also decreased successfully from 1.34±0.14,3.35±0.32,4.55±0.50,7.63±1.41 to 10.46±0.98(t=3.68～18.93,P＜0.05),the expression of MMP-9 decreased(t=2.678～8.535,P＜0.05),and the infarct volume decreased significantly(t=4.645～17.837,P＜0.01 ) compared with that in the control group at the same timepoint and the corresponding region.Conclusion The expression of MMP-9 is high after cerebral ischemia/reperfusion,that contribute to the inflammatory reaction.NRG-1βmight down-regulate the expression of MMP-9 to inhibit apoptosis inducing by inflammatory reaction in cerebral ischemic reperfusion. Background Nuclear factor-kappaB(NF-κB) is a transcription factor that is activated after cerebral ischemia.NF-κB activation leads to the expression of many inflammatory genes involved in the pathogenesis of stroke,causing enhanced release of proinflammatory cytokines,which are known to be associated with delayed neuronal death following ischemia.Gelatinous fiber acid protein (GFAP) is the main fiber of astrocytes in brain.It can be expressed only by astrocytes.Astrocytes can be actived after ischemia which is regard as character of the inflammatory response after ischemia.In MACO rats,GFAP was found around the infarct tissue while its expression can decrease after treatment of NRG-1β. Neuregulins are signalling proteins and the NRG1 family encodes at least 15 different splice variants,classified into four isoforms.They play an important role in cell differentiation,migration,myelination and proliferation of oligodendrocytes and neurons.In animal models of ischemia/hypoxia,NRG1 has been shown to act as a therapeutic,neuroprotective agent and should be investigated in more detail in transgenic animal models.A recent study demonstrated that NRG-1 blocked delayed neuronal death and pro-inflammatory responses following focal ischemic stroke in the rat.However,the mechanisms that underlie the neuroprotective effects of NRG-1 are unclear.Objective To observe the expression of nuclear factorκB,NF-κB),gelatinous fiber acid protein(GFAP) and the influence of neuregulin-1β(NRG-1β) on brain tissue of rats following cerebral ischemia/reperfusion.Methods The animal models of middle cerebral artery occlusion/reperfusion (MCAO/R) were established by a monofilament method from left external-internal carotid artery in 200 adult healthy male Wister rats.The rat models in the treatment group were injected 1.5%NRG-1β5μL from internal carotid artery (ICA).The cerebral infarct volume was measured by TTC stain,the apoptosis was determined wit in situ TUNEL method,the expressions of NF-κB and GFAPwere determined by immtmohistochemical and immumofluorescent double staining and western-blotting assay.Results Cerebral ischemia reperfusion can induce apoptosis and expression of NF-κB and GFAP in cerebral cortex and striatum.With the ischemic time prolonging,the number of apoptotic cells(t=5.44～21.80,P＜0.05) and the expression of NF-κB(t=3.363～25.523,P＜0.01) and GFAP(t=3.16～21.38, P＜0.01) increased successfully in the control group.With NRG-1βtreatment, the expression of NF-κB(t=2.46～5.46,P＜0.05) and GFAP(t=3.03～5.89, P＜0.01) decreased,the number of apoptotic cells reduced(t=3.68～18.93,P＜0.05), and the infarct volume decreased significantly(t=4.645～17.837,P＜0.01) compared with that in the control group at the same timepoint and the corresponding region.Conclusion The expression of NF-κB and GFAP is high after cerebral ischemia/reperfusion,that contribute to the inflammatory reaction.NRG-1βmight down-regulate the expression of NF-κB and GFAP to inhibit apoptosis inducing by inflammatory reaction in cerebral ischemic reperfusion.
|
PDF Full Text Request