Modeling subcortical ischemic white matter injury in rodents:unmet need for a breakthrough in translational research

Subcortical ischemic white matter injury(SIWMI),pathological correlate of white matter hyperintensities or leukoaraiosis on magnetic resonance imaging,is a common cause of cognitive decline in elderly.Despite its high prevalence,it remains unknown how various components of the white matter degenerate in response to chronic ischemia.This incomplete knowledge is in part due to a lack of adequate animal model.The current review introduces various SIWMI animal models and aims to scrutinize their advantages and disadvantages primarily in regard to the pathological manifestations of white matter components.The SIWMI animal models are categorized into 1)chemically induced SIWMI models,2)vascular occlusive SIWMI models,and 3)SIWMI models with comorbid vascular risk factors.Chemically induced models display consistent lesions in predetermined areas of the white matter,but the abrupt evolution of lesions does not appropriately reflect the progressive pathological processes in human white matter hyperintensities.Vascular occlusive SIWMI models often do not exhibit white matter lesions that are sufficiently unequivocal to be quantified.When combined with comorbid vascular risk factors(specifically hypertension),however,they can produce progressive and definitive white matter lesions including diffuse rarefaction,demyelination,loss of oligodendrocytes,and glial activation,which are by far the closest to those found in human white matter hyperintensities lesions.However,considerable surgical mortality and unpredictable natural deaths during a follow-up period would necessitate further refinements in these models.In the meantime,in vitro SIWMI models that recapitulate myelinated white matter track may be utilized to study molecular mechanisms of the ischemic white matter injury.Appropriate in vivo and in vitro SIWMI models will contribute in a complementary manner to making a breakthrough in developing effective treatment to prevent progression of white matter hyperintensities.

Protective Effects of Activated Protein C on Neurovascular Unit in a Rat Model of Intrauterine Infection-Induced Neonatal White Matter Injury

Summary： Activated protein C （APC）, a natural anticoagulant, has been reported to exert direct vascu- loprotective, neural protective, anti-inflammatory, and proneurogenic activities in the central nervous system. This study was aimed to explore the neuroprotective effects and potential mechanisms of APC on the neurovascular unit of neonatal rats with intrauterine infection-induced white matter injury. In- traperitoneal injection of 300 ~tg/kg lipopolysaccharide （LPS） was administered consecutively to preg- nant Sprague-Dawley rats at embryonic days 19 and 20 to establish the rat model of intrauterine infec- tion-induced white matter injury. Control rats were injected with an equivalent amount of sterile saline on the same time. APC at the dosage of 0.2 mg/kg was intraperitoneally injected to neonatal rats imme- diately after birth. Brain tissues were collected at postnatal day 7 and stained with hematoxylin and eo- sin （H＆E）. Immunohistochemistry was used to evaluate myelin basic protein （MBP） expression in the periventricular white matter region. Blood-brain barrier （BBB） permeability and brain water content ~were measured using Evens Blue dye and wet/dry weight method. Double immunofluorescence staining and real-time quantitative PCR were performed to detect microglial activation and the expression of protease activated receptor 1 （PAR1）. Typical pathological changes of white matter injury were ob- served in rat brains exposed to LPS, and MBP expression in the periventricular region was significantly decreased. BBB was disrupted and the brain water content was increased. Microglia were largely acti- vated and the mRNA and protein levels of PAR1 were elevated. APC administration ameliorated the pathological lesions of the white matter and increased MBP expression. BBB permeability and brain water content were reduced. Microglia activation was inhibited and the PAR1 mRNA and protein ex- pression levels were both down-regulated. Our results suggested that APC exerted neuroprotective ef- fects on multiple components of the neurovascular unit in neonatal rats with intrauterine infec- tion-induced white matter injury, and the underlying mechanisms might involve decreased expression of PAR1.

Compound from Magnolia officinalis Ameliorates White Matter Injury by Promoting Oligodendrocyte Maturation in Chronic Cerebral Ischemia Models

Chronic cerebral hypoperfusion leads to white matter injury(WMI),which subsequently causes neurodegeneration and even cognitive impairment.However,due to the lack of treatment specifically for WMI,novel recognized and effective therapeutic strategies are urgently needed.In this study,we found that honokiol and magnolol,two compounds derived from Magnolia officinalis,significantly facilitated the differentiation of primary oligodendrocyte precursor cells(OPCs)into mature oligodendrocytes,with a more prominent effect of the former compound.Moreover,our results demonstrated that honokiol treatment improved myelin injury,induced mature oligodendrocyte protein expression,attenuated cognitive decline,promoted oligodendrocyte regeneration,and inhibited astrocytic activation in the bilateral carotid artery stenosis model.Mechanistically,honokiol increased the phosphorylation of serine/threonine kinase(Akt)and mammalian target of rapamycin(mTOR)by activating cannabinoid receptor 1 during OPC differentiation.Collectively,our study indicates that honokiol might serve as a potential treatment for WMI in chronic cerebral ischemia.

Loss of monocarboxylate transporter 1 aggravates white matter injury after experimental subarachnoid hemorrhage in rats

Monocarboxylic acid transporter 1(MCT1)maintains axonal function by transferring lactic acid from oligodendrocytes to axons.Subarachnoid hemorrhage(SAH)induces white matter injury,but the involvement of MCT1 is unclear.In this study,the SAH model of adult male Sprague-Dawley rats was used to explore the role of MCT1 in white matter injury after SAH.At 48 h after SAH,oligodendrocyte MCT1 was significantly reduced,and the exogenous overexpression of MCT1 significantly improved white matter integrity and long-term cognitive function.Motor training after SAH significantly increased the number of ITPR2+SOX10+oligodendrocytes and upregulated the level of MCT1,which was positively correlated with the behavioral ability of rats.In addition,miR-29b and miR-124 levels were significantly increased in SAH rats compared with non-SAH rats.Further intervention experiments showed that miR-29b and miR-124 could negatively regulate the level of MCT1.This study confirmed that the loss of MCT1 may be one of the mechanisms of white matter damage after SAH and may be caused by the negative regulation of miR-29b and miR-124.MCT1 may be involved in the neurological improvement of rehabilitation training after SAH.

Lipocalin-2-Mediated Insufficient Oligodendrocyte Progenitor Cell Remyelination for White Matter Injury After Subarachnoid Hemorrhage via SCL22A17 Receptor/Early Growth Response Protein 1 Signaling

Insufficient remyelination due to impaired oligodendrocyte precursor cell(OPC)differentiation and maturation is strongly associated with irreversible white matter injury(WMI)and neurological deficits.We analyzed whole transcriptome expression to elucidate the potential role and underlying mechanism of action of lipocalin-2(LCN2)in OPC differentiation and WMI and identified the receptor SCL22A17 and downstream transcription factor early growth response protein 1(EGR1)as the key signals contributing to LCN2-mediated insufficient OPC remyelination.In LCN-knockdown and OPC EGR1 conditional-knockout mice,we discovered enhanced OPC differentiation in developing and injured white matter(WM);consistent with this,the specific inactivation of LCN2/SCl22A17/EGR1 signaling promoted remyelination and neurological recovery in both atypical,acute WMI due to subarachnoid hemorrhage and typical,chronic WMI due to multiple sclerosis.This potentially represents a novel strategy to enhance differentiation and remyelination in patients with white matter injury.

Recovery of corticospinal tract injured by traumatic axonal injury at the subcortical white matter:a case report

The corticospinal tract （CST） is a major neural tract for mo- tor function in the human brain. In addition, CST is mainly concerned with execution of movement of the hand （Jang, 2014）. However, few studies are reported on the mecha- nism underlying CST recovery after traumatic brain injury （Seo and Jang, 2015）. In this study, we report on a case that showed recovery of an injured CST by traumatic axonal injury （TAI） at subcortical white matter, as detected on fol- low-up diffusion tensor tractography （DTT）.

Bone morphogenetic protein signaling：a promising target for white matter protection in perinatal brain injury

Prematurely born newborns,as well as those born at term,may suffer from several types of brain injury including hypoxic-ischemic injury,intracranial hemorrhage,both intraventricular and parenchymal,and injury that is the consequence of intrauterine growth restriction（IUGR）.Injury of all types can impact the motor and cognitive abilities of survivors.The mechanisms leading to disability are not completely understood.

Decellularized optic nerve functional scaffold transplant facilitates directional axon regeneration and remyelination in the injured white matter of the rat spinal cord

Axon regeneration and remyelination of the damaged region is the most common repair strategy for spinal cord injury.However,achieving good outcome remains difficult.Our previous study showed that porcine decellularized optic nerve better mimics the extracellular matrix of the embryonic porcine optic nerve and promotes the directional growth of dorsal root ganglion neurites.However,it has not been reported whether this material promotes axonal regeneration in vivo.In the present study,a porcine decellularized optic nerve was seeded with neurotrophin-3-overexpressing Schwann cells.This functional scaffold promoted the directional growth and remyelination of regenerating axons.In vitro,the porcine decellularized optic nerve contained many straight,longitudinal channels with a uniform distribution,and microscopic pores were present in the channel wall.The spatial micro topological structure and extracellular matrix were conducive to the adhesion,survival and migration of neural stem cells.The scaffold promoted the directional growth of dorsal root ganglion neurites,and showed strong potential for myelin regeneration.Furthermore,we transplanted the porcine decellularized optic nerve containing neurotrophin-3-overexpressing Schwann cells in a rat model of T10 spinal cord defect in vivo.Four weeks later,the regenerating axons grew straight,the myelin sheath in the injured/transplanted area recovered its structure,and simultaneously,the number of inflammatory cells and the expression of chondroitin sulfate proteoglycans were reduced.Together,these findings suggest that porcine decellularized optic nerve loaded with Schwann cells overexpressing neurotrophin-3 promotes the directional growth of regenerating spinal cord axons as well as myelin regeneration.All procedures involving animals were conducted in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Sun Yat-sen University(approval No.SYSU-IACUC-2019-B034)on February 28,2019.

Recombinant human erythropoietin for repair of white matter damage

Erythropoietin has been shown to exhibit neuroprotective effects in animal models. A neonatal rat model of hypoxic-ischemic white matter damage was established via bilateral carotid artery ligation in 4-day-old Sprague-Dawley rats. The rats were subsequently treated with recombinant human erythropoietin to observe pathological changes in the brain and long-term neurobehavioral functions before and after intervention. Results showed that the number of myelin basic protein-positive cells, which reflected myelin/oligodendrocyte damage, significantly increased, although the number of amyloid precursor protein-positive cells, which reflected axonaf injury, significantly decreased in periventricular white matter at 72 hours and 7 days following erythropoietin intervention. The number of glial fibrillary acidic protein-positive cells, indicating astrocytic damage, significantly decreased in periventricular white matter of erythropoietin-treated rats at 48 hours, 72 hours, 7 days and 26 days. Following erythropoietin intervention in the 30-day-old rats, head-turning time in the slope test was shortened and open-field test scores increased. These results suggested that erythropoietJn promoted repair of white matter damage, as well as improved neurobehavioral functions in a rat model of hypoxic-ischemic injury.

Embracing oligodendrocyte diversity in the context of perinatal injury

Emerging evidence is fueling a new appreciation of oligodendrocyte diversity that is overturning the traditional view that oligodendrocytes are a homogenous cell population.Oligodendrocytes of distinct origins,maturational stages,and regional locations may differ in their functional capacity or susceptibility to injury.One of the most unique qualities of the oligodendrocyte is its ability to produce myelin.Myelin abnormalities have been ascribed to a remarkable array of perinatal brain injuries,with concomitant oligodendrocyte dysregulation.Within this review,we discuss new insights into the diversity of the oligodendrocyte lineage and highlight their relevance in paradigms of perinatal brain injury.Future therapeutic development will be informed by comprehensive knowledge of oligodendrocyte pathophysiology that considers the particular facets of heterogeneity that this lineage exhibits.

Bystanders or not?Microglia and lymphocytes in aging and stroke

As the average age of the world population increases,more people will face debilitating aging-associated conditions,including dementia and stroke.Not only does the incidence of these conditions increase with age,but the recovery afterward is often worse in older patients.Researchers and health professionals must unveil and understand the factors behind age-associated diseases to develop a therapy for older patients.Aging causes profound changes in the immune system including the activation of microglia in the brain.Activated microglia promote T lymphocyte transmigration leading to an increase in neuroinflammation,white matter damage,and cognitive impairment in both older humans and rodents.The presence of T and B lymphocytes is observed in the aged brain and correlates with worse stroke outcomes.Preclinical strategies in stroke target either microglia or the lymphocytes or the communications between them to promote functional recovery in aged subjects.In this review,we examine the role of the microglia and T and B lymphocytes in aging and how they contribute to cognitive impairment.Additionally,we provide an important update on the contribution of these cells and their interactions in preclinical aged stroke.

Piezo1 suppression reduces demyelination after intracerebral hemorrhage

Piezo1 is a mechanically-gated calcium channel.Recent studies have shown that Piezo1,a mechanically-gated calcium channel,can attenuate both psychosineand lipopolysaccharide-induced demyelination.Because oligodendrocyte damage and demyelination occur in intracerebral hemorrhage,in this study,we investigated the role of Piezo1 in intracerebral hemorrhage.We established a mouse model of cerebral hemorrhage by injecting autologous blood into the right basal ganglia and found that Piezo1 was largely expressed soon(within 48 hours)after intracerebral hemorrhage,primarily in oligodendrocytes.Intraperitoneal injection of Dooku1 to inhibit Piezo1 resulted in marked alleviation of brain edema,myelin sheath loss,and degeneration in injured tissue,a substantial reduction in oligodendrocyte apoptosis,and a significant improvement in neurological function.In addition,we found that Dooku1-mediated Piezo1 suppression reduced intracellular endoplasmic reticulum stress and cell apoptosis through the PERK-ATF4-CHOP and inositol-requiring enzyme 1 signaling pathway.These findings suggest that Piezo1 is a potential therapeutic target for intracerebral hemorrhage,as its suppression reduces intracellular endoplasmic reticulum stress and cell apoptosis and protects the myelin sheath,thereby improving neuronal function after intracerebral hemorrhage.

Potential physiological and pathological roles for axonal ryanodine receptors

Clinical disability following trauma or disease to the spinal cord often involves the loss of vital white matter elements including axons and glia.Although excessive Cais an established driver of axonal degeneration,therapeutically targeting externally sourced Cato date has had limited success in both basic and clinical studies.Contributing factors that may underlie this limited success include the complexity of the many potential sources of Caentry and the discovery that axons also contain substantial amounts of stored Cathat if inappropriately released could contribute to axonal demise.Axonal Castorage is largely accomplished by the axoplasmic reticulum that is part of a continuous network of the endoplasmic reticulum that provides a major sink and source of intracellular Cafrom the tips of dendrites to axonal terminals.This“neuron-within-a-neuron”is positioned to rapidly respond to diverse external and internal stimuli by amplifying cytosolic Calevels and generating short and long distance regenerative Cawaves through Cainduced Carelease.This review provides a glimpse into the molecular machinery that has been implicated in regulating ryanodine receptor mediated Carelease in axons and how dysregulation and/or overstimulation of these internodal axonal signaling nanocomplexes may directly contribute to Ca-dependent axonal demise.Neuronal ryanodine receptors expressed in dendrites,soma,and axonal terminals have been implicated in synaptic transmission and synaptic plasticity,but a physiological role for internodal localized ryanodine receptors remains largely obscure.Plausible physiological roles for internodal ryanodine receptors and such an elaborate internodal binary membrane signaling network in axons will also be discussed.

Roles of NG2 Glia in Cerebral Small Vessel Disease

Cerebral small vessel disease(CSVD)is one of the most prevalent pathologic processes affecting 5%of people over 50 years of age and contributing to 45%of dementia cases.Increasing evidence has demonstrated the pathological roles of chronic hypoperfusion,impaired cerebral vascular reactivity,and leakage of the blood–brain barrier in CSVD.However,the pathogenesis of CSVD remains elusive thus far,and no radical treatment has been developed.NG2 glia,also known as oligodendrocyte precursor cells,are the fourth type of glial cell in addition to astrocytes,microglia,and oligodendrocytes in the mammalian central nervous system.Many novel functions for NG2 glia in physiological and pathological states have recently been revealed.In this review,we discuss the role of NG2 glia in CSVD and the underlying mechanisms.

Digoxin Ameliorates Glymphatic Transport and Cognitive Impairment in a Mouse Model of Chronic Cerebral Hypoperfusion

The glymphatic system plays a pivotal role in maintaining cerebral homeostasis.Chronic cerebral hypoperfusion,arising from small vessel disease or carotid stenosis,results in cerebrometabolic disturbances ultimately manifesting in white matter injury and cognitive dysfunction.However,whether the glymphatic system serves as a potential therapeutic target for white matter injury and cognitive decline during hypoperfusion remains unknown.Here,we established a mouse model of chronic cerebral hypoperfusion via bilateral common carotid artery stenosis.We found that the hypoperfusion model was associated with significant white matter injury and initial cognitive impairment in conjunction with impaired glym・phatic system function.The glymphatic dysfunction was associated with altered cerebral perfusion and loss of aquaporin 4 polarization.Treatment of digoxin rescued changes in glymphatic transport,white matter structure,and cognitive function.Suppression of glymphatic functions by treatment with the AQP4 inhibitor TGN-020 abolished this protective effect of digoxin from hypoperfusion injury.Our research yields new insight into the relationship between hemodynamics,glymphatic transport,white matter injury,and cognitive changes after chronic cerebral hypoperfusion.

MFG-E8 Alleviates Cognitive Impairments Induced by Chronic Cerebral Hypoperfusion by Phagocytosing Myelin Debris and Promoting Remyelination

Chronic cerebral hypoperfusion is one of the pathophysiological mechanisms contributing to cognitive decline by causing white matter injury.Microglia phagocytosing myelin debris in a timely manner can promote remyelination and contribute to the repair of white matter.However,milk fat globule-epidermal growth factor-factor 8(MFG-E8),a microglial phagocytosis-related protein,has not been well studied in hypoperfusion-related cognitive dysfunction.We found that the expression of MFG-E8 was significantly decreased in the brain of mice after bilateral carotid artery stenosis(BCAS).MFG-E8 knockout mice demonstrated more severe BCAS-induced cognitive impairments in the behavioral tests.In addition,we discovered that the deletion of MFG-E8 aggravated white matter damage and the destruction of myelin microstructure through fluorescent staining and electron microscopy.Meanwhile,MFG-E8 overexpression by AAV improved white matter injury and increased the number of mature oligodendrocytes after BCAS.Moreover,in vitro and in vivo experiments showed that MFG-E8 could enhance the phagocytic function of microglia via theαVβ3/αVβ5/Rac1 pathway and IGF-1 production to promote the differentiation of oligodendrocyte progenitor cells into mature oligodendrocytes.Interestingly,we found that MFG-E8 was mainly derived from astrocytes,not microglia.Our findings suggest that MFG-E8 is a potential therapeutic target for cognitive impairments following cerebral hypoperfusion.