Ischemic Preconditioning Alleviates Cerebral Ischemia-reperfusion Injury By Interfering With Endothelial Glycocalyx

Objective: Thrombolysis and endovascular treatment(EVT)are the most effective treatments for acute ischemic stroke(AIS),but only parts of the AIS patients receiving them get a good prognosis.As one of the main reasons for poor prognosis,ischemia-reperfusion injury(IRI)after recanalization involved multiple mechanisms,in which the destruction of the blood-brain barrier(BBB)plays a key role,which is followed by cerebral edema,hemorrhage transformation,and neuroinflammation.In addition to the known cell structure,including vascular endothelial cells(VECs),pericytes,and astrocyte end-feet,the extracellular matrix(ECM)also plays an important role in maintaining the integrity and permeability of BBB.As an important component of ECM,glycocalyx located on the luminal side of VECs is regarded as the first defense of BBB.The major components of glycocalyx consist of a negatively charged mesh of proteoglycans,glycosaminoglycans,and glycoproteins.Proteoglycans are composed of core proteins and covalently linked glycosaminoglycan chains,and the former predominantly includes syndecans(SYND)and glypican,while glycosaminoglycans predominantly include chondroitin sulfate(CS)and heparan sulfate(HS).Another important glycosaminoglycan is hyaluronic acid(HA),which does not attach to core protein but the HA receptor.The glycocalyx has important physiological functions,including the maintenance of vascular permeability and intravascular homeostasis,the regulation of blood flow,mechanical sensing,signal transduction,prevention of leukocyte adhesion to endothelium,and storage of growth factors and enzymes.The glycocalyx was degraded into segments and shed into the blood under the pathological state of IRI,therefore,the changes of glycocalyx components in the blood are commonly used to indirectly reflect the integrity of the vessel wall.Collectively,the targeted intervention on glycocalyx may provide a possible treatment strategy for IRI.The neuroprotection of ischemic preconditioning(IPC)on IRI has been confirmed in animal and cellular studies,but the underlying mechanisms are not fully understood.One of the mechanisms may be attributed to its protection against BBB damage.In this context,we hypothesized that IPC may alleviate IRI by protecting glycocalyx,which has not been fully investigated.The current study was designed to test the effect of cerebral IRI on glycocalyx,and whether IPC played a neuroprotective role by interfering with glycocalyx in preclinical study.Furthermore,we also investigated the effect of IRI on glycocalyx in patients with anterior circulatory AIS after EVT.Methods:The experiment was divided into part 1 and part 2.In part 1,Sprague-Dawley(SD)rats were randomly divided into sham operation group(18 rats),ischemia-reperfusion injury(IRI)group(30 rats),and ischemia preconditioning + ischemia-reperfusion injury(IPC+IRI)group(30 rats).In part 2,SD rats were randomly divided into sham group(30rats),IRI group(63 rats),and IPC+IRI group(57 rats),and each group was randomly divided into 6 independent time points(2 h,6 h,1 d,2 d,3 d,and 7 d)subgroups.1.The neuroprotective effect of IPC on IRIIn part 1,the neurological deficit scale was used to evaluate the neurological impairment.Infarct volume was evaluated by 2,3,5-triphenyl tetrazolium chloride(TTC)staining.The water content of brain was assessed by the wet-dry method.Hematoxylin and eosin(HE)staining was performed to evaluate brain tissue edema.Neuronal cells apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL).BBB tight junction protein expressions were assessed using western blot(WB).The proliferation and activation of astrocytes and microglia were assessed by immunofluorescence staining.2.The dynamic changes of serum biomarkers and association with brain edema and neurological outcomesThe blood samples were collected from two parts of the experiment.In part 1,the blood samples were collected from rats 15 min before surgery,6 h and 3 d after surgery.The concentrations of HS,HA,SYND1,matrix metalloproteinase(MMP-2),and inducible nitric oxide synthase(i NOS)were detected by enzyme-linked immunosorbent assay(ELISA).The correlations between i NOS,MMP-2 and HS,HA,SYND1 were analyzed.In part 2,blood samples were collected 15 min before surgery and corresponding time points after surgery,and the concentrations of HS,HA,SYND1,MMP-2,and i NOS were detected by ELISA.The correlations between blood biomarkers and behavioral deficit score,and the water content of brain tissue were analyzed.3.Cellular and molecular mechanisms of IPC affecting GlxBrain tissue samples collected from part 1 were used in this part.The m RNA levels of MMP-2 and i NOS were detected by reverse transcription-polymerase chain reaction(RT-PCR).The protein levels of MMP-2,i NOS,heparanase(HPSE),hyaluronidase(Hyal-1),and hyaluronate synthase-1(HAS-1)were detected by WB.Immunofluorescence staining was used to detect cellular expression of MMP-2,HAS-1,and Hyal-1 through double-labeling with neuron marker Neu N,astrocyte marker GFAP,microglia marker Iba-1,or vascular endothelial cell marker CD31,respectively.4.The dynamic changes of serum factors of AIS patients and association with functional prognosisThis study was a single-center,prospective clinical trial.Forty anterior circulation AIS patients with EVT and 10 control patients whose baseline matched with AIS patients were recruited.All the enrolled subjects were required to sign informed consents and complete demographic characteristics and clinical data.In the AIS group,blood samples were collected 15 min before surgery,2 h,24 h,and 3 d after surgery,while blood samples were collected at admission in the control group.The concentrations of HA,HS,SYND1,MMP-2,and i NOS were measured by ELISA.Endpoints included early neurological improvement(ENI)and modified Rankin Scale(m RS)at 90 days.Results:1.Compared with IRI group,IPC+IRI group reduced behavioral deficit score,infarct volume,neuronal cells apoptosis,water content of brain,tissue edema,BBB damage,and increased the number of reactive astrocytes and reduced the number of activated microglia.2.The results in part 1 showed that the ratios of HA,HS,SYND1,i NOS,and MMP-2(postoperative concentration/preoperative concentration)in the sham group did not change over time.In the IRI group,the ratios of HA,SYND1,and MMP-2 increased 6 h and 3 d after surgery,while the ratios of HS and i NOS increased 3 d after surgery.IPC increased ratios of HA and MMP-2 but reduced ratios of HS and i NOS on day 3,compared with the IRI group.MMP-2 ratio was positively correlated to HA ratio and SYND1 ratio in IRI and IPC+IRI groups,and i NOS ratio was positively correlated to HS ratio in the IRI group.The results of part 2 showed that compared with the sham group,the ratios of HA,SYND1 and MMP-2(2 h-7 d),and HS and i NOS(2 d and 3 d)increased in the IRI group.Compared with the IRI group,the IPC+IRI group increased the ratio of HA from 6 h to 3 d and ratio of MMP-2 on day 3,but decreased the ratio of HS and i NOS on day 2 and day 3.The ratio of HA was negatively correlated with cerebral edema and behavioral deficit score.3.IRI group downregulated the protein levels of HAS-1 and upregulated that of Hyal-1.IPC upregulated the protein levels of HAS-1 and downregulated that of Hyal-1 vs the IRI group.The IRI group upregulated the protein and m RNA levels of MMP-2 and i NOS.IPC upregulated the protein and m RNA levels of MMP-2,while downregulated that of i NOS vs the IRI group.Compared with the IRI group,IPC promoted the expression of HAS-1 in astrocytes and neurons,inhibited the expression of Hyal-1 in microglia and VECs,and had a bidirectional regulation of MMP-2.4.The baseline values of HA,HS,SYND1,MMP-2,and i NOS in the AIS group were not different from those of the control group.The concentrations of these factors showed a continuous upward tendency for 3 days after surgery.The patients in the AIS group were divided into the high ratio group and the low ratio group according to the median of 2h-ratio.The results showed that the incidence of ENI in the high HA ratio group was higher than that in the low HA ratio group,and the other factors were not found to be correlated with functional prognosis.Conclusion:1.IPC played a neuroprotective role by reducing infarct volume,alleviating cerebral edema,protecting the BBB,and regulating inflammatory response.2.IRI damaged Glx in brain tissue and released its shedding into blood,and Glx destruction is related to the increase of MMP-2 and i NOS.IPC had selective intervention on Glx components.IPC may reduce cerebral edema and behavioral deficits by increasing HA ratio.3.IPC promoted the expression of HAS-1 in astrocytes and neurons,inhibited the expression of Hyal-1 in microglia and VECs,and had a bidirectional regulation of MMP-2,which mediated HA metabolism.4.The concentrations of HA,HS,SYND1,MMP-2,and i NOS in AIS patients undergoing EVT showed a continuous increase from 2 h to 3 d after surgery.The 2h-HA ratio was related to ENI.