| Over the past decade,stroke has been one of the leading causes of adult morbidity and mortality globally and in China,both on ischemic and hemorrhagic basis.Ischemic strokes,however,account for the largest proportion,about 75-80% of all strokes.Previous studies have shown that 30% to 60% of the occurrence of ischemic cerebrovascular disease can be attributed to stenosis of the carotid artery.Strokes due to carotid stenosis account for 15%of ischemic strokes,and the annual stroke rate can be as high as 13% in patients with carotid stenosis >70%.In clinical practice,revascularization via intravenous thrombolysis and mechanical thrombectomy is the main therapeutic target in acute ischemic stroke.Traditional drug therapy has limited efficacy in severe carotid stenosis,so surgical treatment and interventional therapy become the main treatment modalities.Carotid endarterectomy is limited in its application because of the high surgical injury and the high operator skill required.Percutaneous balloon dilation has a high rate of postoperative restenosis and is now less commonly used.In contrast,stenting has a higher success rate and fewer complications,so it has a greater advantage in the treatment of carotid stenosis and has been promoted in clinical practice.In the acute early phase of ischemia,a dramatic reduction in cerebral blood flow causes a cascade of events,including depletion of energy supply,cessation of metabolic processes,subsequent cellular damage,and disruption of the blood-brain barrier.Damaged and dead cells at the site of injury release pro-inflammatory mediators and cellular debris that induce neuroinflammation and recruit peripheral immune cells.Rapid progressive degeneration and dysfunction of neurons and other cells caused by vascular obstruction are key to the treatment of ischemic stroke in the acute phase.After an ischemic stroke event,two more distinct regions can gradually develop: the infarct core and the ischemic penumbra zone.The ischemic penumbra zone is thought to be an area with many dormant or reversibly damaged brain cells that may remain viable for several hours after the stroke event due to the availability of collateral circulation as well as collateral arteries supplying this area after the ischemic event.The Neurovascular Unit(NVU),which is composed mainly of neurons,astrocytes,microglia,endothelial cells,pericytes,basement membrane and extracellular matrix,plays an important role in maintaining normal physiological functions and repairing damaged cells during the development of acute phase ischemic stroke.In past studies,it has been proposed to consider the neurovascular unit as a whole for the treatment of stroke as well as neurodegenerative diseases.However,several cell types within the neurovascular unit have distinctly different responses in the early phase of acute ischemic stroke,so it is particularly unclear which cell type dominates in response to ischemic stroke.Furthermore,the ability to target early interventions with targeted drugs based on the cell types that change most predominantly within the neurovascular unit is what we are exploring in this study.However,the reasons for the heterogeneity of different cell types within the neurovascular unit in the ischemic stroke penumbra zone have remained elusive for a long time,partly because of technical limitations to study a particular cell type in vivo,and partly because of previous studies focusing on single cell types in vivo or in vitro under stroke conditions or at the level of common RNA-seq for ischemic stroke.On the other hand,previous studies have focused on single cell types or general RNA-seq levels in the ischemic stroke region under stroke conditions.However,the advent of single-cell RNA sequencing(sc RNA-seq)has made it possible to analyze cell population heterogeneity at the single-cell level.To our knowledge,our study is the first single-cell sequencing study to investigate the pathological processes in the ischemic stroke hemispheric zone among thousands of physiological and pathological brain cells,providing a cellular map of the ischemic hemispheric zone of the cerebral cortex under ischemic stroke conditions.This study was first conducted in a mouse model of transient focal cerebral ischemia.A higher resolution analysis of the cells in the stroke and sham groups revealed the heterogeneity,death and survival of cells in the ischemic penumbra zone of the cerebral cortex under ischemic stroke conditions.To investigate the heterogeneity of each cell type in the early phase of ischemic stroke and to identify early therapeutic targets that contribute to cell survival,we applied sc RNA-seq combined with bulk RNA-seq analysis together to study the phenotypic changes in the ischemic penumbra zone of the mouse brain in the early phase of stroke.To better understand the early stages of ischemic stroke,we regrouped specific cell types to uncover the heterogeneity between different cell types and between different cell subpopulations of that cell type in the face of ischemic stroke.In addition,we applied RNAscope multiplex in situ hybridization and immunofluorescence to confirm the presence of these cell subpopulations and the molecular changes in the acute phase of ischemic stroke.Our study shows that microglia make up the largest proportion of these cell types in terms of cell numbers after ischemic stroke induction.Whether microglia polarization promotes inflammation or injury repair currently varies depending on the pathological conditions.There is still a debate as to when microglia polarization starts after stroke.Previous studies claim that there is no significant microglial polarization within 24 hours of ischemia induction.In contrast to these studies,our single-cell sequencing results suggest that microglia already start polarizing 24 hours after ischemic stroke and differentiate in the direction of two different progression trajectories,implying that this time point may be a better time point for intervention to help cell survival.In clinical practice,it is generally accepted that there is an optimal time window for the treatment of intravenous thrombolytic revascularization.Intervention of revascularization within the time window can save brain tissue in the ischemic-ischemic penumbra zone [30].Undoubtedly,targeted interventions in the early stages of stroke are a better preventive measure.Therefore,we focused our sequencing efforts on the early stages of stroke in order to identify therapeutic targets and characterize early cellular changes.As microglial activation is traditionally thought to play a deleterious role in ischemic stroke,inhibition of microglial activation can alleviate ischemic brain injury[17].Currently,there is a growing view that microglia activation leads to differentiation to both M1 and M2 types.Our experimental results suggest that the M1-M2 dichotomy may be an oversimplified classification that represents only two extreme activation states.In the acute phase of ischemic stroke,subpopulations 4,9,10 are likely to be M1 microglia,mainly enriched in hypoxic pathways,Tnfα,IL6,and IL2 enriched in inflammation-related genes and pathways.In contrast,subpopulations 6,8 exhibit a different response variation,with no significant overexpression of M2-type marker genes(e.g.,Arg1,Ym1,and IL10)in these subpopulations.The above data suggest that M2-type microglia may differentiate more slowly than M1-type microglia.In addition to the classical marker genes of M1 and M2 types,we identified many novel subcluster-specific genes(e.g.,Gadd45 b in subcluster 4,9),which was reported to relieve infarct volume via the TGFβ-smad3 pathway after ischemic stroke induction.Furthermore,GSVA results for subpopulations 6,8(most likely M2 type)suggest that these subpopulations are mainly enriched in the Kras signaling pathway,which is closely associated with cancer cell survival.Through this pathway,we inferred that these microglia may be more likely to survive under ischemic stroke.Therefore,in the acute phase of ischemic stroke,there is an urgent need to inhibit M1-type activation while inducing microglia polarization to the protective type.In particular,M2-type microglia may be associated with repair processes,neurogenesis,axonal remodeling and angiogenesis.Microglia were also abundant in the ischemic stroke group compared to the Control group in terms of phagocytosis[17].However,it remains controversial whether microglial phagocytes play a protective or a damaging role in neurodegenerative diseases.Microglial phagocytosis is a "double-edged sword";on the one hand,it facilitates the repair of neurological damage;on the other hand,it can induce or aggravate neurodegenerative lesions under certain circumstances.In addition,peripheral macrophages are recruited to reach the lesion through the damaged blood-brain barrier.This process occurs within 24 hours.The polarization of peripheral macrophages appears to be more complete compared to microglia,suggesting a different activation period of resident microglia in the brain and immune cells in the blood after ischemic stroke.Macrophage infiltration and polarization can be detected early in stroke,at a time point that favors the progression of inflammation.Therefore,interfering with the recruitment of peripheral immune cells early in stroke might be a good measure to alleviate inflammation in stroke.Unfortunately,although this measure did alleviate inflammation to some extent,previous studies did not produce significant positive effects.In addition,previous studies have shown that Mmp9 is highly expressed in microglia,thereby hydrolyzing and disrupting blood vessels,leading to blood-brain barrier damage.Matrix metalloproteinases can achieve physical destruction of the BBB by digesting BBB matrix proteins.Our analysis showed that Mmp9 and Mmp8 were overexpressed in macrophages in the ischemic stroke group,suggesting that blood-brain barrier disruption may also be accomplished by peripherally recruited macrophages and not only by microglia in the ischemic penumbra zone.disruption of the BBB appears to be a delayed secondary phase of stroke lesions.However,it is still controversial whether peripheral macrophages destroy or protect the blood-brain barrier.A pathophysiological hallmark of ischemic stroke is the activation and gliosis of astrocytes,which may help to limit the ischemic core region in the early stages of ischemic stroke and favor post-stroke outcomes.Astrocytes and oligodendrocytes,as part of the glial system,provide many important biological functions,such as blood-brain barrier(BBB)formation,energy metabolism of neurons,maintenance of structures and intercellular communication.In contrast to microglia and macrophages,subpopulations of these cells did not appear so clearly polarized in early stroke in sc RNA-seq analysis.In contrast,when astrocyte gap junctions are impaired during stroke,inflammatory mediators such as cytokines and chemokines are released extracellularly via the BBB[18].In our findings,Cyr61 in astrocytes and Sgk3 in oligodendrocytes were both overexpressed in ischemic stroke and may be potential therapeutic targets at this stage.Although ischemic stroke has been understudied,previous studies have shown that the Cyr61 gene is closely associated with the survival of stress and tumor cells.overexpression of the Cyr61 gene may contribute to the survival of these astrocytes under acute ischemic stroke conditions.Furthermore,GSVA showed that subpopulation 9 is enriched for IL-6,complement,TNFαpathway and Kras signaling,suggesting that Sgk3 in oligodendrocytes may play an important role in regulating oligodendrocyte viability and inflammatory response during the acute phase of ischemic stroke.Traditional studies have suggested that reactive astrocytes may have a greater neurological impact after stroke.However,our sc RNA-seq results suggest that some reactive astrocytes and oligodendrocytes play a beneficial role simultaneously under stroke conditions.In addition to astrocytes and oligodendrocytes,endothelial cells are also involved in the composition of the blood-brain barrier.sc RNA-seq analysis showed that endothelial cells during ischemic stroke have a function in revascularization and extracellular communication after injury.In addition,endothelial cells upregulated oxidative phosphorylation,ROS detoxification,glutathione metabolism and cyclic nucleotide metabolism.The above results suggest that these endothelial cells may contribute to the positive response to stroke,which also suggests that endothelial cells may be one of the early therapeutic targets.Taken together,we have demonstrated that the ischemic half-dark band mapping in ischemic stroke presented here contains much information at the molecular and cellular level.Whether therapeutic strategies targeting these points can be developed as effective treatments for cerebral ischemic diseases remains to be further investigated,as other cells expressing the same targets are also affected.However,further understanding of the activity of the targets expressed on each cell type will facilitate the development of novel and effective therapeutic strategies for the treatment of ischemic stroke.Also,single-cell level studies will help us to find more precise therapeutic targets and applications.Our single-cell studies open a new perspective on the pathological process of ischemic stroke.In addition,we try to use one of the target molecules with subpopulation specificity on the drug coating of drug-eluting carotid artery stents to further the clinical treatment of severe carotid artery stenosis in both mechanical dilation and rethrombolytic recanalization,allowing the drug to act in a slow-release effect on the ischemic penumbra zone in the brain.At present,after the adjustment of testing methods,drug stent processing and exploration of drug stability,some target drug coatings of drug-eluting stents now have a slow-release effect,and the maximum expansion diameter of the stent on the balloon is about 6.54 mm,and the diameter after retraction is about 6.05 mm,and the stent length is adjusted to 40 mm,which can already meet the requirements of expansion to,but further animal experiments are still needed for validation to better apply to clinical ischemic stroke patients.Part 1 Single-cell study of the ischemic penumbra zone neurovascular unit in acute ischemic strokeObjectiveTo investigate the heterogeneity among cell types in the early phase of ischemic stroke,to explore which cell types occupy the major role in early stroke,and to identify early therapeutic targets that contribute to cell survival,we applied sc RNA-seq together with general RNA-seq analysis to explore in depth the phenotypic changes in the ischemic penumbra zone of the mouse brain in the early phase of stroke.To better understand the early stages of ischemic stroke,we regrouped specific cell types to uncover the heterogeneity between different cell types and between different cell subpopulations of that cell type in the face of ischemic stroke.MethodsIn this study,a mouse model of transient focal cerebral ischemia was used as the study object.Firstly,MCAO model mice were made by wire bolus method,and blood flow was restored after temporary blockage of the middle cerebral artery for 60 min[12],and after 24 h of blood reperfusion,the ischemic penumbra zone area of mouse cerebral cortex was taken by severing,and single cells were prepared into single cell suspension using mouse brain tissue dissociation reagent,and after judging the cell activity,single cells in the penumbra zone area of cerebral cortex were captured by high-throughput microfluidic technology,and subsequently the single cells were The c DNA library was created by reverse transcription of the m RNA molecules in the single cells.After high-throughput sequencing,the original data are subjected to quality control,normalization,data correction,feature selection,and dimensionality reduction,and the cell cluster is obtained for cell type identification.After cell type identification,the cell types with significant differences were further analyzed downstream at the cell/gene level to complete the cell mapping of the ischemic half dark zone area in the mouse cerebral cortex during the acute phase of ischemic stroke.Changes in gene expression patterns and cell subpopulation-specific molecular genes were compared between the MCAO and control groups to identify molecular targets with subpopulation specificity and reveal the heterogeneity,death and survival of cells in the ischemic hemispheric zone of the cerebral cortex under ischemic stroke conditions.For differentially expressed genes,the DESeq and DESeq2 packages were used to determine the underlying distribution of reads.We used Benjamini-Hochberg correction for false discovery rate(FDR)and Bonferroni correction for p-value based tests.Statistical differences between the two groups in batch RNA-seq were examined using Student’s t-test(unpaired,two-tailed).p < 0.05 was considered statistically significant.Statistical analysis was performed using Graph Pad Prism 8.0 software(Graph Pad,Inc.,San Diego,CA,USA).Results1.We specified 24 cell clusters,each containing similar gene expression patterns.And then the obtained subcluster-specific gene molecules were compared with known Markers of mouse cortical cell types reported in previous studies as well as the Cell Marker database(http://biocc.hrbmu.edu.cn/Cell Marker).2.The major cell types were identified,including neurons,astrocytes,microglia,oligodendrocytes,oligodendrocyte progenitor cells(OPC),endothelial cells,pericytes,macrophages,B cells,T cells,monocytes,epithelial cells,and fibroblasts.3.New genetic markers for some major cell types in the early course of acute ischemic stroke were identified,and these may be valuable MCAO cell-specific target molecules,providing data to support future analyses.4.Notably,the cell types that account for the majority of the 13 cell types identified are microglia,astrocytes and oligodendrocytes.5.When comparing samples from the acute ischemia(MCAO)and control groups,we noted that many microglia,astrocytes,macrophages,oligodendrocytes,and endothelial cell subpopulations were more abundant in the MCAO samples,suggesting some significant alterations in these cells in the MCAO group.We further clustered these cell types(i.e.,microglia,astrocytes,macrophages,neurons,endothelial cells,and oligodendrocytes)to assess the heterogeneity of the subpopulations.6.At the end of this part of the experiment,we determined the subpopulation-specific expression patterns in the penumbra.We also further verified the presence of expression specificity of the new markers by RNA scope immunogenic hybridization and immunofluorescence staining.7.Since only a few cells had cell proliferation markers,we did not cell cycle for correction and subjected them to the next step of data analysis.Indeed,the enzymatic method used in lysing brain tissue to obtain single cell suspensions may result in the loss of neuronal cells,possibly leading to underrepresentation of neurons in the total cell population obtained as well as changes in enzymatic digestion-related genes.A previous study showed that the cell isolation process may present cell dissociation artifacts.Therefore,to exclude the effect of enzymatic dissociation and to reduce method-specific errors in single-cell sequencing,we added bulk RNA-seq as a complementary method to corroborate the findings.Transcripts per million(TPM)values were used as a criterion to determine gene expression by bulk RNA-seq.These combined data demonstrate the high intercellular heterogeneity of cortical penumbra cells during MCAO and suggest that these 13 cell subtypes encompass most of the cellular heterogeneity in the mouse cortical brain.ConclusionStroke has been the leading cause of adult morbidity and mortality for the past several years.After an ischemic stroke episode,many dormant or reversibly damaged brain cells are present in the penumbra region.However,the pathological processes and unique cellular information of the penumbra region in acute ischemic stroke remain elusive.We applied unbiased single-cell sequencing combined with general RNA-seq analysis to investigate the heterogeneity of each cell type in the early stages of ischemic stroke and to detect possible early therapeutic targets to aid cell survival,and we used these analyses to study the mouse brain penumbra at this stage.Our results reveal the effects of ischemic stroke on specific genes and pathways in different cell types and altered cellular differentiation trajectories,suggesting potential pathological mechanisms and therapeutic targets.In addition to classical genetic markers,single-cell genomics has demonstrated unique information on subpopulations and metabolic changes in several cell types in ischemic stroke.These findings suggest that Gadd45 b in microglia,Cyr61 in astrocytes,and Sgk3 in oligodendrocytes may play subpopulation-specific roles in cell death or survival in the early stages of ischemic stroke.At the end of this part of the experiment,we applied RNA-scope immunogenic hybridization and immunofluorescence staining to validate selected target gene markers,confirming the presence of these cellular subtypes and molecular changes during the acute phase of ischemic stroke.Among the 13 cell types identified early in the onset of acute ischemic stroke,microglia,astrocytes,peripheral macrophages,endothelial cells,and oligodendrocytes dominate the early cellular response.In further subpopulation subdivision of these major cell types,we found by comparing the MCAO group as well as the sham group.However,whether microglia polarization promotes inflammation or injury repair varies depending on the pathological condition.In addition,there is still controversy regarding the moment of initial microglia polarization after stroke.Previous studies have found no significant microglial polarization within 24 hours of ischemia induction.In contrast,our single-cell sequencing results suggest that microglia already show two different progression trajectories of polarization and differentiation at 24 hours after MCAO.These findings imply that the first 24 hours may be a good window of intervention to help the cells survive.In clinical practice,it is generally accepted that there is a limited optimal time window for the treatment of intravenous thrombolytic revascularization.Interventions for revascularization within this time window may save ischemic penumbra brain tissue and targeting the early stages of stroke is the best preventive measure.Therefore,we focused our sequencing efforts on the early stages of stroke to identify therapeutic targets and characterize early cellular changes.Part 2 Study on the olfactory dysfunction after endoscopic endonasal transsphenoidal surgeryObjectiveTo explore early targets in acute ischemic stroke in drug-coated carotid artery stents.In the first part of the experiment,we sequenced and experimentally validated the major cellular and molecular changes in the ischemic half dark zone of the cerebral cortex in the early phase of ischemic stroke.Subsequently,we subjected anti-stenosis drug targets as well as these specific molecular targets to cellular experimental validation and exploratory studies in drug-coated carotid artery stents.In addition,the formation and progression of carotid atherosclerotic plaques,followed by rupture,thrombosis and vascular obstruction,leading to ischemic tissue damage,are the clinical underlying conditions of a large proportion of vascular diseases.Therapeutic agents for the prevention of atherosclerosis are directed at epidemiologically identified and relatively easy-to-measure risk factors(e.g.,lipids and blood pressure).This strategy has proven to be somewhat effective,but its efficacy is less than optimal because the prevalence of blood-brain vascular disease remains high.Treatment targeting the mechanisms of atherosclerosis in the vessel wall is a conceptually attractive proposition that could complement risk factor-oriented strategies.Vascular smooth muscle cells(VSMC)are the main cellular component of the vascular media,and their migration and proliferation lead to the formation of neointima,the development of which makes the vessel particularly sensitive to atherosclerosis[19].Numerous hormones and growth factors act on VSMC to cause migration,proliferation,and extracellular matrix secretion[20],and regulation or dysfunction of these processes may be important in the development of atherosclerosis.Endothelin-1(ET-1)is a 21-amino acid peptide that acts on seven transmembrane G protein-coupled receptors and elicits a large number of responses that can modulate the behavior of VSMCs and thus influence the development of atherosclerosis.We attempted to use ET-1 and target molecules with subgroup specificity therein on the drug coating of drug-coated carotid stents to further the clinical treatment of severe carotid stenosis in both mechanical dilatation and rethrombolytic recanalization,allowing the drug to act with a slow-release effect on the ischemic penumbra zone in the brain.MethodsIn the second part of the study,we examined the effects of Endothelin-1 and its antagonists on the proliferation and migration of vascular smooth muscle cells by culturing and treating vascular smooth muscle primary cells and cell lines.The proliferation of Endothelin-1 and its antagonist ACT-132577 was first investigated by CCK8 reagent at different concentrations and time points,and the migration of vascular smooth muscle after stent implantation was simulated by scratch assay.To further validate the role of Endothelin-1,we interfered with the expression level of Endothelin-1 by means of small interfering RNA and quantified the protein by blotting electrophoresis experiments.The results showed that after silencing expression of small interfering RNA[16],there was a significant reduction of downstream molecules(e.g.α-sm-actin and Osteopontin)in cells where Endothelin-1 promotes cell proliferation.On the other hand,basic data on carotid artery stent implantation and release were determined by measuring parameters such as carotid artery vessel diameter,intima,and blood flow velocity in Parmesan pigs.Results(1)The ELISA reagents of 20 clinical cases revealed that the ET-1 concentration in the serum of patients after stent implantation was significantly higher than that of the control group.(2)The proliferative effect of Endothelin-1 on vascular smooth muscle at different concentrations and different time points.(3)Inhibition of proliferation of vascular smooth muscle by the Endothelin-1 antagonist ACT-132577 at different concentrations and time points;(4)Inhibition of proliferation of vascular smooth muscle by the Endothelin-1 antagonist ACT-132577 at different concentrations and time points.(4)The migration-promoting and inhibitory effects of Endothelin-1 and its antagonists on vascular smooth muscle.(5)The expansion diameter of ET-1 target drug-coated stent and drug retardation target.After the adjustment of testing method,drug stent processing and drug stability exploration,the drug stent now has a retardation effect.The stent expansion diameter was adjusted to 6.5 mm and the stent length was adjusted to 40 mm,which has been able to meet the requirements of expansion.ConclusionCurrently,there is no commercial application of drug-coated stents for carotid arteries,and the treatment of atherosclerotic mechanisms in the vessel wall by drug-coated carotid artery stents is a direction worth exploring and researching.In addition,vascular smooth muscle cells are the main cellular component of the carotid vascular media,and their migration and proliferation lead to the formation of scars,the development of which makes the vessels particularly sensitive to atherosclerosis.Numerous hormones and growth factors act on VSMC to cause migration,proliferation,and secretion of extracellular matrix,and regulation or dysfunction of these processes is an important cause of restenosis after stent implantation.By the action of ET-1 antagonist ACT-132577,the value addition and migration of VSMCs after stent implantation can be inhibited,thus reducing the possibility of in-stent restenosis development.Elevated plasma levels of ET-1 have also been shown in cases after stent implantation.Therefore,the target of ET-1 may have more positive implications for restenosis after carotid artery atherosclerosis stent implantation. |
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