| BackgroundStroke is considered to be one of the major public health problems around the world,the health expenditure of which is the third in the developed countries.It is common cause of death and disability for adults.It was one case died of stroke among sixteen death cases of patients.Most of the stroke events are ischemic stroke,hemorrhagic stroke is only about 15%.Epidemiological statistics showed that the number of people died of acute ischemic stroke was more than cancer and cardiovascular disease annually in our country at present.Acute ischemic stroke becomes the most common disease,still growing at a rate of 9%per year.Survivors still needed long-term and expensive rehabilitation.About seventy-five percent stroke survivors lost the ability to work,of which about 40%were severely disabled.At present,evidence-based medicine had been confirmed that early thrombolysis and interventional therapy were effective,but due to narrow treatment time window,high cost and technic,only a small number of patients benefited from the revascularization after intravenous thrombolysis.Up to now,there is no effective treatment strategy for cerebral infarction.The pathological process of ischemic cerebral is very complex.Various factors lead to the activation of cell death signaling pathway after cerebral infarction.Therefore,a variety of studies had been carried out on the cell death pathway after cerebral ischemia.Although the research and development of neuroprotective drugs had achieved gratifying results in the animal experiment,there was no success in the clinical trials.Considering the failure of many candidate drugs target for single signal transduction pathway,the preferred protective strategy of ischemic injury may be blocking multiple critical pathways at the same time,including multiple cells,complicated activities and events related to the pathological process of cerebral ischemia.Cell death signal and inflammatory response play an important role in cerebral ischemia injury.The heat shock protein family contains a group of heat-sensitive cell stress proteins which are characterized by molecular chaperone,especially with heat stress.Heat shock protein was the focus of research for decades.Heat shock proteins are divided into several sub-families based on molecular weight,domain protection and function.The small heat shock proteins(sHSP),including HSP27,are widely expressed in a variety of species which molecular weight is between 15 and 42 kDa.Recent experimental studies have begun to emphasize the protective effect of Hsp27 on cellular stress.More and more evidence indicated that Hsp27 inhibits cell death signals.In addition,Hsp27 is modified after transcription,resulting in different control functions.The difference expression of HSP27 is expressed in the subtype of neurons,the high constitutive expression is mainly limited to the spinal ganglion and brain stem,the basal expression is expressed in the subtype of cerebellar purkinje cells.Whether constitutive expression or induced by cellular stress,Hsp27 is expressed in neuronal injury.There was sufficient experimental evidence to show that Hsp27 has the ability to reduce neuronal damage in many neurological diseases and plays an important neuroprotective role.Although the constitutive expression level of Hsp27 in the brain is low,it can be induce by many central nervous system stress,including heat stress,ischemia and seizure.Immunohistochemistry showed that Hsp27 expressed more in glial cells and a limited number of neurons.It was reported that Hsp27 was delivered to the brain via herpes simplex virus vector which prevented hippocampus neurons apoptosis induced by red alga effectively.Over-expression of human Hsp27 could effectively reduce hippocampal neurons apoptosis induced by red alginic acid in transgenic mice.After cerebral ischemia,the expression of endogenous Hsp27 increased mainly in glial cells.Viral transduction or gene overexpression of Hsp27 significantly reduces the focal ischemic lesion.The full-length protein transduction HSP27 could effectively reduce hippocampal CA1 area injury in the whole cerebral ischemia model.Apoptosis signal regulating kinase 1(ASK 1)is a key molecule in the signal conduction of cell death and plays a key role in the activation of microglial cell.Many studies had found that Hsp27 could directly inhibit ASK1 and protect brain ischemia by blocking cell death signals.Large molecular protein drugs are not easy to pass through the blood brain barrier and have immunogenicity which greatly restricts its application.The polypeptide drugs have become the focus of medical research in recent years.Many polypeptide drugs had been widely used in tumor,inflammation,cardiovascular disease,central nervous system diseases and immune diseases.Polypeptide drugs have biological function of the whole protein,the molecular weight is small,easy to synthesize and the side effect is low,so it is more valuable in clinical research and development.The rapid development of polypeptide drugs has attracted the attention of neuroscientists.The progress had been made on polypeptide drugs targeting the ischemic nerve injury.According to the standard of international pharmacology,protein drugs are composed of more than one hundred amino acids,such as insulin,interferon etc.Polypeptide drugs are composed of less than one hundred amino acids.The polypeptide drugs of clinical application are more than eighty kinds,involving endocrine,blood vessel,tumor,urinary and immune system disease.Compared with full-length protein and chemical drugs,polypeptide drugs have advantages following:(1)The immunogenicity of polypeptide drugs is weaker and less side effects.(2)Polypeptide drugs have higher biological activity and specificity.(3)It is easy to synthesize and purify.(4)It has good affinity in vivo and the few drug interactions relatively.The protective effect of Hsp27 mimic peptide(Hsp27 MP)on cerebral infarction is not reported.Objective1.The distribution of HSP27MP in the neurons,microglia and ASK1 molecules was observed after the intravenous administration at the state of normal physiological and experimental cerebral infarction.2.To investigate the protective effect of HSP27MP on experimental cerebral infarction.3.To study the protective mechanism of HSP27MP on experimental cerebral infarction.Methods1.Experimental animalsSPF male Kunming mice weighting 25-35g were provided by Shandong animal laboratory animal center[animal license number:SCXK(Beijing)2012-0001].The laboratory animal operation was conducted in accordance with the principles of laboratory animal use and management of the brain institute of Taishan medical university.2.Experimental cerebral infarction model in mice.The focal cortical ischemic model was induced by photochemical method in mice.The mice were anaesthetized with 5%isoflurane and maintained anesthesia with 2%isoflurane.During the operation,keeping the rectal temperature(37±0.5℃)with constant temperature blanket,2%rose bengal(100mg/kg weight)was injected into the femoral vein,while HSP27MP(or HSP27MP-FITC)(10mg/kg body weight)was injected in no less than two minutes.After injection,the mouse were fixed to a stereoscopic instrument,the anterior and posterior fontanel were in a horizontal position by adjusting the paramet.After disinfection and preparation,the parietal midline skin was cut open about one centimeter long,blunt dissection the subcutaneous tissue,removed the periosteum,fully exposed anterior and posterior fontanelle,sagittal suture and lambdoid suture.According to the mouse stereospecific coordinate map,the sensory motor cortex was selected as the light region,and the specific parameters were:two millimeter on the left side of the sagittal seam,two point four millimeter in the anterior fontanelle and two millimeter in the posterior crown seam.The light source was placed as close to the skull as possible so that the optical fiber probe of the cold light source was perpendicular to the skull about one point five millimeter,the full bone was exposed to the cold light source for 4 minutes.3.HSP27MP distribution in brain.The distribution of HSP27MP in the brain was evaluated by NeuN,Iba-1 and ASK1 immunofluorescence staining at 24h after HSP27MP-FITC administration.4.Determination of regional cerebral blood flow(rCBF).At 0 and 24h after the postoperative,the rCBF of mice was measured by 2D doppler laser blood flow instrument.5.Neurological scoresAfter 24h,the mice in each group were selected to evaluate the neurological function by using the beam balance test,the cylinder test and the foot fault test.6.To determine the volume of cerebral infarction by TTC staining.At 24h after operation,the mice were killed by excessive anesthesia,fresh brain tissue sections were taken and stained in 1%TTC solution.After scanning,ImageJ software was used to measure the volume of cerebral infarction.7.To detect the permeability of blood brain barrier by Evans Blue(EB)extravasation.At 23h after operation,2%EB(5ml/kg weight)was injected into the femoral vein.After circulating for 1h,the brain was removed after cardiac perfusion of 50ml PBS,and the permeability of BBB was evaluated qualitatively and quantitatively.The dry and wet weight method was used to evaluate the brain edema.8.To evaluate the activation of microglia cell,activated microglia cell by imunofluorescence staining.After frozen sections,Iba-1 and CD68 staining were used to evaluate microglia and activated microglia.9.Neuronal cell apoptosis.The neuron apoptosis was evaluated by NeuN+TUNEL immunofluorescence staining in ischemic penumbra.10.Western blot.ASK1 and p-ASK1 expression levels were detected after cerebral infarction.Results1.HSP27 MP distribution in brainAt 24h after the FITC labeled HSP27MP intravenous administration,the distribution of HSP27MP in the mouse brain was observed by laser confocal microscopy and fluorescence microscopy.We found that in the state of physical and cerebral infarction,HSP27MP could go through the blood brain barrier,diffuse in the brain,enter neurons and microglia cells,colocate with target molecule ASK1 and aggregate on the side of cerebral infarction.2.The protective effect of HSP27MP on experimental cerebral infarction.Our study results showed that the HSP27MP could effectively reduce cerebral infarction volume and improve the early neurological deficits after cerebral infarction,but failed to improve rCBF.The protective effect of HSP27MP on experimental cerebral infarction was confirmed.3.The protective mechanism of HSP27MP on experimental cerebral infarction.We used EB extravasation to detect BBB permeability and dry-wet weighting method to evaluate cerebral edema.The results showed that the HSP27 MP can effectively protect the BBB damage caused by cerebral infarction and reduce brain edema.Immunofluorescence showed that HSP27 MP decreased the activation of astrocytes and microglia cells and reduced the apoptosis of neurons in the ischemic penumbra.Conclusion1.The HSP27 MP could go through BBB and enter the central nervous system by intravenous administration.2.HSP27 MP had protective effect on experimental cerebral infarction.3.HSP27MP protected the experimental cerebral infarction through reducing cerebral edema,inhibiting the activation of microglial cells,protecting blood brain barrier and reducing neuron apoptosis.BackgroundSubarachnoid hemorrhage is an acute cerebrovascular event caused by intracranial aneurysm ruptured which accounts for about 5%-10%of all stroke.In all patients with SAH,sudden death rate is as high as 20%,approximate 50%of the survivors suffered from cognitive impairment and chronic neurologic decline.SAH and its complications are one of the major causes of adult death and disability among worldwide.Although the diagnosis and surgical treatment of SAH continues to progress,effective intervention measures are still limited.Nowdays it is still difficult to obtain satisfactory clinical effect.In the past decades,cerebral vasospasm and aneurysm rebleeding were considered major determinants of poor prognosis.Although many animal studies found that many drugs could inactivate the spasm or blocked artery smooth muscle contraction,there is no one drug that could improve clinical outcome greatly in SAH patients.Kusaka et al reported early brain injury after SAH which defined as the stage between bleeding instantly and the beginning of the CVS.It is within 72 h after SAH acute brain injury,including oxidative stress,nerve inflammation,ion disruption and nerve inflammation etc.The detailed mechanism has remained unknown,the potential pathophysiologic mechanism mainly includes the increasing of intracranial pressure,cerebral blood flow reducing,cerebral oxygen content descending,cerebral perfusion pressure reducing,neuronal apoptosis,the destruction of the blood brain barrier and brain edema,etc.Neurologist found that EBI was a major cause of death and disability after SAH.Nowdays EBI is considered for the potential treatment of SAH patients.It can abate some long-term secondary fatal brain injury after SAH.Therefore,to seek new strategy of the treatment for EBI after SAH is a problem to be solved urgently in the world of neuroscience.Recently many studies had shown that many inflammatory components were be involved in the progress of brain injury after aneurysm rupture.Microglia is phagocyte pos-sesseing immunologic activity of the central nervous system in situ which plays a crucial role in nerve inflammation.Like microglia,astrocyte can synthesize and secrete inflammatory factors,such as cytokines and chemokine.Previous studies showed that numerous factors were associated with the development of inflammatory lesions.After SAH,subarachnoid hemoglobin stimulated white blood cell and glial cell proliferation,and then those cells secrete cytokines leading to brain damage.Growing evidence from research suggested that nerve inflammation plays an important role in the pathogenesis of EBI after SAH.Elevated inflammatory factors participated in the occurrence and development of EBI after SAH.Many research results highlighted that the inflammatory reaction was attributed to EBI.Therefore,to seek safe and effective anti-inflammatory drug is a potential treatment strategy of improving the prognosis of SAH patients.Since the 1870s,phosphodiesterase(PDE)inhibitors had been a research goal as anti-inflammatory drug.In a large amount of immune cells,the PDE-4 is the main degradation enzyme for cAMP which is the second messenger.The PDE-4 is widely expressed in the cerebral cortex and hippocampus.Inhibitting PDE-4 plays an important role in animal models of neurological diseases and injuries where inflammation plays an essential role,such as spinal cord injury,traumatic brain injury,alzheimers disease,severe depression,multiple sclerosis and ischemic stroke,producing beneficial effect.Neuroscientists found that cilostazol as a selective PDE3 inhibitor could attenuate the CVS after SAH through the animal experiment studies.Encouragingly,a clinical systematic review and meta-analysis suggested that cilostazol could significantly reduce the incidence of cerebral vasospasm,serious cerebral vasospasm,cerebral vasospasm associated with cerebral infarction in the aneurysmal SAH patients,with no significant adverse reactions.As a consequence,the evidence supported the application of cilostazol to the patients with aneurysmalSAH.In vitro,roflumilast N-oxide inhibited PDE-4 in a variety of cells,including neutrophils,endothelial cells,monocytes/macrophages,Cd4+ and CD8+ T cells,and smooth muscle cells.In vivo,roflumilast mitigated the tobacco smoke induced lung inflammation,pulmonary vascular remodeling and hypertension.Roflumilast is the brain penetrant,its concentration in the brain increases in a dose-dependent manner.It is effective on hypertension-induced impairment of learning and memory and the cognitive deficit in rodents at non-emetic doses.The present study tests the hypothesis that roflumilast decreases proinflammatory cytokines,blood-brain barrier permeability,and neuronal apoptosis following SAH in rats.Objective1.To investigate protective effect of roflumilast on EBI after SAH.2.To study the effect of roflumilast on neuroinflammation after SAH.3.To investigate the effect of roflumilast on the early CVS of SAH.Method1.Experimental animal12 weeks old male SPF SD rats(weight 250-350g)were purchased from the animal experimental center of Shandong Province.Rats were fed in the animal room brain institute of taishan medical university,circadian rhythm,feeding freely.All operations conformed to the management of laboratory animal life science research center of taishan Medical University in Shandong province.After one week of feeding,the experiment was carried out.Rats were randomly divided into sham,SAH and SAH+ roflumilast groups.2.SAH model and drug administrationAccording to previous study methods,a modified intracisternal injection of autologous arterial blood was used in rats.Rats were anesthetized by intraperitoneal injection of Chloral hydrate(400 mg/kg BW).And then,the head was fixed to the stereotaxic apparatus 30°head-down.The head and skin were exposed to the occipital membrane,after that non-heparinized autologous arterial blood(0.1 ml/100g BW)was injected uniformly into the occipital cistern by microinjection pump within 3min.After the blood injection was completed,retained 10min before suture.During the operation,the constant temperature blanket was used to maintain the temperature of the rat at 37+1℃.3.Neurological behavior scoresThe neurological behavior scores(appetite,activity and defects)in three groups of rats were performed at 48 and 72 h after SAH.4.Measurement of brain water content and blood-brain barrier permeabilityThe brain water content of three groups was measured at 48 and 72 h after SAH according to the wet/dry method.The BBB permeability was measured by evans blue dye extravasation at 48 and 72 h following SAH.5.Immunofluorescence staining of IL-1β,IL-6 and TNF-aImmunofluorescence staining of IL-1β,IL-6 and TNF-a were performed at 48 and 72 h following SAH.All images were captured on a fluorescence microscope.6.Enzyme-Linked Immunosorbent AssayAt 48h and 72h after SAH,the levels of IL-1β,IL-6 and TNF-a were measured using an enzyme-linked immunosorbent assay(ELISA)kit following the manufacturer’s instructions.7.Immunofluorescence staining of microgliaAt 72h after SAH,the expression of microglia in the cerebral cortex was observed by immunofluorescent staining.8.Cortical neurons apoptosisImmunofluorescence and TUNELstaining were performed at 48 and 72h following SAH.TUNEL staining of sections were performed using the in situ Cell Death Detection Kit with Fluorescein.9.HE staining of basilar arteryAfter SAH 72h,the brain paraffin section was made and the morphology of the basilar artery was observed by HE staining.The image analyzer was used to determine the cross-sectional area of the lumen.10.Statistical analysisAll experimental data obtained was expressed as mean ±SD(standard deviation).Comparisons among the sham,SAH,and SAH+roflumilast group were made by two-way analysis of variance(ANOVA)using the GraphPad Software Prism 6.0.Statistical significance was considered at the 95%confidence interval.Results1.Roflumilast improved neurological deficits following SAHTo evaluate possible neuroprotective role of roflumilast on experimental SAH,neurological assessments were conducted at 48 and 72 h after SAH using a battery of three behavioral tests.The statistical analysis results showed that compared with the sham group,the neurological score of the SAH group was significantly increased.Compared with SAH group,the neurological score of roflumilast treatment group was significantly reduced.2.Roflumilast inhibited blood-brain barrier permeability and brain edema following SAHAt 48h and 72h after SAH,Evans blue extravasation was used to detect the effect of roflumilast on the permeability of blood brain barrier and brain edema.SAH group of Evans blue extravasation and water content were higher than sham group.After treatment with roflumilast,the permeability of blood brain barrier and cerebral edema decreased significantly.3.Roflumilast decreased the expression of IL-1β,IL-6 and TNF-a after SAHAt 48h and 72h after SAH,immunofluorescence staining was used to evaluate the effect of roflumilast on the expression level of IL-1β,IL-6 and TNF-a.Fluorescence microscopy was used to observe the results of immunofluorescence staining.The statistical results showed that compared with sham group,the number of IL-1β,IL-6 and TNF-a positive cells in SAH group increased significantly.Compared with the SAH group,IL-1β,IL-6 and TNF-a positive cells were significantly reduced after roflumilast administration.ELISA results showed that compared with sham group,the level of IL-1β,IL-6 and TNF-a in serum and cortex increased significantly in SAH group,and the level of IL-1β,IL-6 and TNF-a was significantly decreased after treatment with roflumilast.4.Roflumilast reduced the expression of microglia after SAHAt 72h after,SAH,the results of immunofluorescence staining showed that the number of microglia positive cells in the SAH group was significantly increased compared with the sham group.However,the number of microglia positive cells in the roflumilast treatment group decreased significantly.5.Roflumilast inhibited neuronal apoptosis after SAHAt 48h and 72h after SAH,active caspase-3/NeuN immunofluorescence staining and TUNEL/DAPI staining were used to quantify neuronal apoptosis.The statistical results showed that the active caspase-3 positive cells in SAH group were significantly increased compared with the sham group.However,compared with the SAH group,the active caspase-3 positive cells in the roflumilast treatment group were significantly reduced.Compared with the SAH group,the TUNEL positive cells in the roflumilast treatment group were significantly reduced.6.Roflumilast attenuated early CVS after SAHAt 72h after SAH,HE staining results showed that the area of the basilar artery lumen decreased obviously in the SAH group compared with the sham group.However,the basilar artery spasm in the roflumilast treatment group was improved significantly.ConclusionRoflumilast improved the neurological deficits after experimental SAH significantly in rats,reduced the permeability of blood brain barrier,reduced the expression of IL-1β,IL-6,TNF-α and microglia,inhibited the apoptosis of neurons,alleviated the early CVS after SAH. |
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