The Mechanism Of Interleukin-10 Protects Blood-brain Barrier Permeability After Intracerebral Hemorrhage

BackgroundIntracerebral hemorrhage(ICH)has a high mortality and disability rate,accounting for about 10-15% of all strokes.More and more evidences show that ICH can induce excessive neuroinflammatory response due to the activation of immune cells and the release of inflammatory cytokines,leading to the degradation of blood-brain barrier(BBB)and the death of neurons.ICH-induced BBB destruction further aggravates inflammatory response and destroys BBB function.Therefore,maintaining BBB integrity and inhibiting brain inflammation are key strategies for ICH treatment.Interleukin-10(IL-10),an anti-inflammatory cytokine,is secreted by alternative activated MM?.The expression of IL-10 in brain increases with the pathological process of central nervous system(CNS),promotes the survival of neurons and glial cells,and inhibits inflammation through many signal transduction pathways.Studies also have shown that IL-10 has obvious vascular endothelial protection characteristics.However,whether IL-10 has protective effect on BBB after ICH has not been reported.The blood brain barrier(BBB)is a dynamic structure between the peripheral circulation and the central nervous system(CNS),which has dual functions of maintaining the microenvironment of the nervous system.BBB is composed of vascular endothelial cells and their tight junction proteins,basement membrane,pericytes and astrocyte terminals.It forms a powerful physical barrier at the dynamic interface between blood,cerebrospinal fluid and brain.BBB plays a key role in the clinical practice of hemorrhagic stroke.On the one hand,the permeability of blood-brain barrier increases during ICH,and generally opens at the initial and late stages.On the other hand,the relative impermeability of blood-brain barrier limits the access of many beneficial drugs to the central system at treatment-related concentrations,making blood-brain barrier one of the main obstacles to the treatment of hemorrhagic stroke.In order to study the potential therapeutic targets of ICH,it is particularly important to establish an appropriate animal model of ICH.At present,the animal models of ICH include the whole-blood ICH model and the collagenase-induced ICH model,microsphere inflation model and the disease-induced spontaneous ICH model.The first two ICH models are most commonly used.Collagenase digested the extracellular matrix of small vessels,which resulted in blood exosmosis in a dose-dependent manner.In the autologous blood model,the blood from the tail artery is injected into the striatum of the mouse brain.The stability of cerebral hemorrhage modeling is closely related to the dynamic changes of blood-brain barrier in clinical patients,which is directly related to the scientificity and practicability of experimental research.However,up to now,there are no comparative studies on the molecular mechanism of blood-brain barrier in two animal models of cerebral hemorrhage.Therefore,we first compared the dynamic changes of BBB permeability including TJ,astrocyte,matrix metalloproteinases 9(MMP)and aquaporin 4(AQP4)after ICH both in c-ICH and b-ICH animal models;The second part of this experiment mainly verifies whether IL-10 has protective effect on BBB after ICH and further explores its molecular mechanism.This study will improve the understanding of BBB after ICH and provide reference value for the study of time window and target drugs for ICH patients.Purposes(1)Two animal models of c-ICH and b-ICH were established to evaluate the dynamic changes of BBB injury and to provide a reliable animal model for simulating ICH patients.(2)To study the regulatory function of IL-10 on blood-brain barrier after ICH,and further study its molecular mechanism,so as to provide potential therapeutic targets for blood-brain barrier after cerebral hemorrhage.Methods(1)Establishment of collagenase and autologous blood caudate nucleus ICH model,evaluating motor function after ICH.Mice were divided into sham group,c-ICH group and b-ICH group.By comparing the hematoma size of the two groups with optimized dosage,the concentration of collagenase and the volume of autologous blood were screened out when the hematoma volume of the two models was the same.In c-ICH group,collagenase VII was injected into the left caudate nucleus of mice by stereotaxic frame.The location coordinates were 0.6 mm in front of the fontanel,2.1 mm in the left side,and 3.1 mm in the depth of the skull surface.In b-ICH group,mice were injected into the left caudate nucleus in situ by autologous tail vein blood.After the model was established and stabilized,a series of neurobehavioral changes including body weight,neurological function score,forelimb and hind limb placement and corner test were compared among the three groups at 6h,12 h,D1,D3 and D5 after ICH.(2)Comparing the morphological changes of BBB after operation in c-ICH and b-ICH models,we choose a hemorrhage model which is close to the serious damage of BBB in patients with ICH.At 6h,12 h,D1,D3 and D5 after ICH,medium molecular dye Evans blue and IgG(160 Da)were used to observe the dynamic changes of the severity of BBB in sham,c-ICH and b-ICH mice after ICH,and to compare the severity of BBB damage at the most severe time points in c-ICH and b-ICH models.Western blot and transmission electron microscopy were used to compare the BBB damage.The integrity of tight junction of blood model,the activity of MMP9 in c-ICH and b-ICH models were compared by gelatin zymogram test,and the dynamic expression of aquaporin 4(AQP4)in sham,c-ICH and b-ICH groups was detected by qPCR.(3)To observe the protective effect of IL-10 on neurological function and BBB after ICH.IL-10(concentration: 0.02 ug/?L,0.1 g/mouse in 5 L PBS)was nasogastric fed 2h after ICH,twice a day until sampling.The experimental animals were divided into four groups: sham group,ICH group,ICH + PBS group and ICH + IL-10 group.On D3 after ICH,the changes of motor function in mice were observed,including neurological function score and corner turn test.Fluorescence tracer technique,Evans blue and WB were used to detect the changes of a series of molecular structures of BBB after nasal feeding IL-10.(4)To analyze whether IL-10 protects the blood-brain barrier by reducing glial cell activation.D3 after ICH,the activation of microglia and astrocytes around the hematoma was analyzed by immunofluorescence test,and the activation of microglia and astrocytes was analyzed by fluorescence counting.Results(1)At 6h after ICH,the neuromotor deficit score(NDS)of b-ICH group was higher than that of c-ICH group(P < 0.05).There was no difference in motor function between the c-ICH and b-ICH group at other time points.(2)At 6h after ICH,Evans blue began to leak at 6 hours which indicate that BBB of the two groups began to break down,but the most EB and IgG leakage was found in c-ICH group around D3 hematoma,and the brain water content was significantly higher than that in b-ICH group(P < 0.05),while the peak value was found in b-ICH group(P < 0.05).(3)The expression of tight junction protein occludin and cadherin-10 in c-ICH group decreased significantly at D3 and b-ICH group at D5(P < 0.05).Transmission electron microscopy showed that the tight junction became shorter and severely damaged after operation in c-ICH group at D3,and that of D5 in b-ICH group was more severely damaged(P < 0.05).There was a statistical difference in the degree of tight junction damage between c-ICH and b-ICH groups at D3 and D5(P < 0.05);The activity of MMP9 in brain tissue were increased both in c-ICH group and b-ICH group at 6h and were the highest at D3 and D5 respectively(P < 0.05).The results of q-PCR showed that AQP4 expression peaked on D3 in c-ICH group and on D5 in b-ICH group(P < 0.05).(4)IL-10 decreased the hematoma size and mitigated the neurological deficits on D3 after ICH in the collagenase model(P < 0.05);IL-10 aslo decreased BBB permeability,decreased the tight junction protein expression in the perihematomalbrain tissue(P < 0.05).(5)Intranasal administration of IL-10 inhibited microglia and astrocyte activation around hematoma and decreased the proinflammatory cytokine TNF-a in the perihematomal brain tissue on day 3 after ICH in the collagenase model(P < 0.05).Conclusions:(1)In our study,it was revealed that the most severe BBB injury occurred on D3 in the c-ICH mdoel and on day 5 in the b-ICH model respectively;According to clinical reports,brain edema in patients with cerebral hemorrhage increases gradually within 24 hours after hemorrhage and reaches a peak rapidly on 3 days after onset,and slowly decreases the incidence of brain edema after on 3 days,suggesting that c-ICH may be more suitable for studying BBB damage mechanism than b-ICH.(2)IL-10 could reduce BBB permeability in the c-ICH model through up-regulating the expression of cadherin-10 and occludin,inhibiting the activation of glial cells including microglia and astrocytes in the perihematomal brain tissue around hematoma and the expression of TNF-alpha of peripheral blood.