The Role And Mechanism Of Depletion Of Hippocampal Neural Stem Cell Pool In Cognitive Impairment After Intracerebral Hemorrhage

Background and purposesIntracerebral hemorrhage(ICH),a subtype of stroke,comprises 10-15% of all stroke incidents.ICH presents with acute onset,rapid disease progression,conspicuous intracranial space-occupying effects,and higher risks compared to ischemic stroke.ICH has a case fatality rate of 30-40% within a month,and over 70-90% of patients suffer from various neurological deficits,including sensory,motor,cognitive,emotional,and psychological functions.Since the 1990 s,extensive clinical research has aimed to improve ICH prognosis via treatment modalities,such as STICH and MISTIE.Clinical scholars strive to enhance surgical techniques and neurological intensive care to enhance patient outcomes.Despite declining mortality and disability rates over the past few decades due to improved medical technology,clinicians have overlooked the recovery of cognitive function in ICH patients.Currently,ICH treatment has reached a bottleneck,and researchers are increasingly focusing on how to enhance ICH patient cognition and quality of life.The probability of cognitive impairment within 3 years after intracerebral hemorrhage is as high as 23%.ICH is associated with a high risk of cognitive impairment,which can significantly decrease patients’ quality of life and lifespan.However,clinicians often overlook cognitive dysfunction in ICH patients,and the pathogenesis and treatment measures for cognitive dysfunction are still in their early stages of exploration.While past studies have focused on the effects of blood-brain barrier damage,inflammatory reactions,oxidative stress,excitotoxicity,and white matter fiber bundle injury on motor function,there is limited understanding of cognitive function in ICH.To address this gap,we propose exploring ICH from a different perspective and identifying new therapeutic targets.Cell replacement therapy,particularly stem cell transplantation,holds promise as a potential treatment for central nervous system injuries,including ICH.While stem cell therapy has been applied to clinical treatments,such as hematopoietic stem cell therapy for leukemia,its application in the central nervous system is still unresolved.Ethical issues,immune rejection,and the tumorigenicity of stem cells are some of the primary challenges.Before considering stem cell therapy,it is important to understand the evolution of endogenous neural stem cells.Endogenous neural stem cells are concentrated in the dentate gyrus and subependymal region of the hippocampus,where they play a critical role in maintaining neurogenesis,learning,and memory.Exercise,learning,and antidepressant therapy are believed to enhance neurogenesis,while aging,oxidative stress,and inflammatory responses negatively regulate neurogenesis.Damage to hippocampal neurons is linked to cognitive impairment,dementia,depression,epilepsy,and schizophrenia.Our previous studies indicate that hemorrhagic stroke combined with oxidative stress and inflammatory reactions can lead to neuronal apoptosis and inhibit the proliferation of neural stem cells.Therefore,exploring the impact of hemorrhagic stroke on hippocampal neurogenesis and the underlying mechanisms could improve our understanding of cognitive function in ICH patients and identify new therapeutic targets.Inflammation and oxidative stress have been implicated in the pathogenesis of ICH,and hematomas release hemoglobin that undergoes oxidative decomposition,leading to the release of iron ions and significant changes in the intracranial microenvironment.Whether these changes in the microenvironment affect endogenous neural stem cells and cognition after ICH remains an area of investigation.In this study,we aim to elucidate the potential repair mechanism of iron ion accumulation on endogenous neurogenesis after ICH,following closely to clinical practice.Our investigation involves three aspects:(1)analyzing the pattern of iron accumulation in the hippocampus following ICH and its impact on neurogenesis;(2)exploring the target mechanism of iron accumulation that affects neurogenic impairment and cognition;and(3)examining the effect of tetrahydrofolate on the proliferation of neural stem cells after oxidative stress and its role in cognitive function after ICH.Part Ⅰ Iron deposition in the hippocampus after intracerebral hemorrhage depletes the neural stem cell pool and leads to cognitive impairmentObjectiveCognitive dysfunction is a common occurrence in patients with intracerebral hemorrhage(ICH),but clinical attention on this issue is still insufficient.Previous studies suggest that hippocampal neurogenesis is involved in regulating cognitive behavior such as learning and memory in animals,but it is unclear whether ICH affects hippocampal neurogenesis and the potential role of iron ions released from ICH hematoma.In this study,we investigate the changes of hippocampal neurons following ICH and examine the patterns of iron ion deposition in the hippocampus after ICH.The objective is to unravel the impact of iron deposition in the hippocampus on neurogenesis after ICH.MethodsIn this study,we employed both clinical and experimental approaches to investigate the impact of iron deposition on hippocampal neurogenesis and cognitive impairment following ICH.Clinical data were collected from patients with cerebral hemorrhage and cognitive impairment,and susceptibility-weighted imaging(SWI)was utilized to assess iron deposition in the hippocampus.In animal models,we established an autologous blood-induced ICH model by injecting 20 μ L of autologous blood into the right striatum of Nestin GFP transgenic mice.We then employed immunofluorescence to observe the proliferation of hippocampal neural stem cells after ICH.Furthermore,we employed immunohistochemistry,Western blotting,and enzyme-linked immunosorbent assay to study the temporal pattern of iron deposition in the hippocampus of mice after ICH.Additionally,we investigated the effect of iron salt injection into the dentate gyrus of the hippocampus on neural stem cell proliferation using immunofluorescence and behavioral and cognitive function tests.We also established a Nestin Cre ERT2:: ROSA26CAG(td Tomato WPRE)mouse strain using the Cre-Loxp system to track neurogenesis after ICH.Furthermore,we investigated the effects of iron ions on neural stem cell proliferation and differentiation in vitro using experimental methods such as immunofluorescence,cell spheroidization,and enzyme-linked immunosorbent assay.Results1.Iron deposition occurs in the hippocampus of patients with ICH and cognitive impairment.2.After ICH in mice,hippocampal neural stem cells are activated early and neural stem cell pools are depleted late.3.After ICH,iron deposition occurred in the hippocampus,and the expression of ferritin reached its peak on the seventh day,followed by a decrease in ferritin content,which continued until 28 days.The ferritin content was still higher than that of the control group.4.After injection of bivalent iron salt into the hippocampus,the expression is the same as that of ICH,with early activation of neural stem cells,late depletion of neural stem cell pools,and the same cognitive impairment caused by ICH.5.Using Nestin Cre ERT2:: ROSA26CAG(td Tomato WPRE)mice to track the development of neural stem cells,we found that ICH and iron salts can overactivate hippocampal neural stem cells in the early stage,resulting in an increase in the number of newly born neurons.However,when the neural stem cell pool is depleted in the late stage,the number of neural stem cells is depleted,and there are fewer self-activated stem cells,resulting in a decrease in the number of newly born and mature neurons,which impairs neurogenesis.In vitro experiments,Fe2+inhibited the proliferation of neural stem cells and promoted their differentiation towards astrocytes and fewer neurons.ConclusionIn this section,our focus was to investigate the spatiotemporal dynamics of iron deposition in the hippocampus of patients who experienced intracerebral hemorrhage,and further explore its impact on hippocampal neurogenesis and associated cognitive outcomes.Our observations reveal that persistent iron deposition occurs in the hippocampus following intracerebral hemorrhage.In the early stages,this deposition activates neural stem cells which undergo abnormal differentiation towards newborn neurons.However,in the late stages,this process leads to a depletion of neural stem cell pools and subsequently disrupts normal neural development,thereby affecting cognition.Part Ⅱ Oxidative stress induced by iron deposition after intracerebral hemorrhage and down-regulation of integrinα3 affects neurogenesisObjectiveIntracerebral hemorrhage(ICH)can lead to secondary brain injury,which is a crucial determinant of neurological function prognosis in ICH patients.Among various factors contributing to secondary damage,the accumulation of iron released from red blood cell breakdown and metabolism is particularly detrimental.Iron overload around the hematoma generates reactive oxygen species,causes lipid peroxidation,and induces ROS accumulation,leading to significant changes in the local microenvironment.Our previous study has revealed that iron deposition also occurs in the hippocampus after ICH,which is a site of neurogenesis in the body.The impact of iron deposition on hippocampal neurogenesis after ICH remains to be elucidated.Thus,this section aims to investigate the molecular mechanisms underlying neurogenesis impairment after ICH,providing potential therapeutic targets for maintaining homeostasis and improving cognition.MethodsIn this study,we used a series of experimental methods to explore the impact of iron deposition on neural stem cells in the hippocampus after ICH.Initially,neural stem cells were isolated from the hippocampus of mice 28 days post-ICH and sham group using flow cytometry,followed by RNA-seq sequencing.The gene Itga3 was identified through GO function enrichment and PCR experiments.The accumulation of ROS in stem cells after ICH was observed using immunofluorescence,in order to establish the relationship between iron and ROS.Furthermore,the impact of Itga3 on neural stem cell growth was examined using overexpression and knockdown techniques through lentivirus transfection.Finally,we investigated the neurogenesis and cognitive recovery of ICH using conditional overexpression mice,and assessed the effects of Itga3 overexpressing neural stem cells through immunofluorescence and behavioral studies.Results1.R-seq sequencing,PPI gene network,and GO function enrichment screen out fibronectin binding entries.Subsequent m RNA validation revealed that Itga3 is a potential target gene.2.Through immunofluorescence,we found that iron deposition in hippocampus induced ROS production after ICH,and neural stem cells accumulated ROS.Through cell experiments,we have demonstrated that Fe2+inhibits the proliferation of neural stem cells through ROS.3.Using lentivirus transfection to knock down Itga3 on stem cells can observe a decrease in the number and diameter of cell spheres,a decrease in cell viability,and a decrease in the number and proportion of neural stem cells in the proliferative phase.Overexpression of Itga3 on neural stem cells can reverse the inhibitory effect of Fe2+on neural stem cells and promote the growth of neural stem cells.4.Conditional overexpression of Itga3 can restore effective hippocampal neurogenesis after ICH,maintain the stability of stem cell pools,promote the growth of neuronal dendrites,and improve cognition after ICH.ConclusionFollowing the identification of Itga3 as a potential gene involved in hippocampal neurogenesis through R-seq sequencing,we conducted lentivirus transfection and conditional overexpression experiments in vivo.Our findings revealed that Itga3 promotes the proliferation of neural stem cells in vitro and maintains the stability of the neural stem cell pool in vivo,thus ensuring the balance of neurogenesis.Moreover,AAV-Retro injection tracing experiments demonstrated that overexpression of Itga3 promotes the growth of neuronal dendrites.These findings suggest that iron deposition in the hippocampus following intracerebral hemorrhage may downregulate the depletion of stem cell pools by Itga3,thereby affecting neurogenesis.Our study provides new insights and a potential therapeutic target for understanding endogenous neurogenesis.Part Ⅲ Tetrahydrofolate alleviates oxidative stress and promotes proliferation of neural stem cells through Akt/m TOR signaling pathwayObjectiveIntracerebral hemorrhage(ICH)often results in oxidative stress reactions.Previous chapters have established that iron accumulation triggers ROS production,suppresses neural stem cell proliferation,and causes aberrant neurogenesis after ICH.In this section,our focus is on exploring the impact of ROS-induced oxidative stress on neural stem cell proliferation in vitro.Specifically,we investigate whether tetrahydrofolate(THF)can promote neural stem cell proliferation by alleviating oxidative stress.We also examine the regulation of the PTEN/Akt/m TOR signaling pathway on stem cells after oxidative stress,and explore whether THF can enhance neural stem cell proliferation by alleviating oxidative stress in vivo,ultimately leading to cognitive improvement after intracerebral hemorrhage.By offering new strategies and perspectives for ICH treatment,our study aims to improve patient outcomes.MethodsEmbryonic neural stem cells were cultured,and cell spheroidization and immunofluorescence were used to identify the stem cells.Hydrogen peroxide was utilized to induce ROS production in the stem cells,thereby simulating oxidative stress in vitro.The presence of oxidative stress was confirmed through protein blotting and mitochondrial hydroxyl radical detection.Tetrahydrofolate was then administered as an intervention,and the growth status and proliferative phase of the cells were assessed through various experiments,including cell balling,cell counting,cell viability tests,and immunofluorescence.Following this,we assessed the activation of the PTEN/Akt/m TOR signaling pathway through Western blotting and observed the effects of lentivirus transfection and PTEN knockdown on neural stem cell proliferation,oxidative stress,and changes in the Akt/m TOR signal.Finally,we investigated the impact of tetrahydrofolate on neural stem cells and its influence on cognitive improvement after ICH in vivo using immunofluorescence and behavioral assessments.Results1.THF can reduce mitochondrial hydroxyl free radicals and alleviate oxidative stress in neural stem cells.2.THF promotes the proliferation of neural stem cells after alleviating oxidative stress through PTEN/Akt/m TOR signaling pathway.3.Knocking down PTEN can counteract the inhibitory proliferation effect of oxidative stress on NSCs and promote the proliferation of NSCs.4.THF can alleviate hippocampal ROS after ICH,promote neural stem cell proliferation,and improve cognitive impairment after ICH.ConclusionOur study investigated the effect of THF on the proliferation of neural stem cells following oxidative stress in an in vitro model.Our mechanistic investigations identified the involvement of the PTEN/Akt/m TOR pathway,as knockdown of PTEN using lentivirus transfection reversed the observed effects of THF.Furthermore,we demonstrated that THF can alleviate oxidative stress associated with intracerebral hemorrhage,enhance neural stem cell proliferation,and improve cognitive impairment following intracerebral hemorrhage.These findings suggest that THF may hold therapeutic potential for improving oxidative stress and promoting cognitive recovery after intracerebral hemorrhage.