Mesenchymal Stem Cell (MSC)–Derived Extracellular Vesicles Protect from Neonatal Stroke by Interacting with Microglial Cells

Mesenchymal stem cell (MSC)-based therapies are beneficial in models of perinatal stroke and hypoxia–ischemia. Mounting evidence suggests that in adult injury models, including stroke, MSC-derived small extracellular vesicles (MSC-sEV) contribute to the neuroprotective and regenerative effects of MSCs. Herein, we examined if MSC-sEV protect neonatal brain from stroke and if this effect is mediated via communication with microglia. MSC-sEV derived from bone marrow MSCs were characterized by size distribution (NanoSight™) and identity (protein markers). Studies in microglial cells isolated from the injured or contralateral cortex of postnatal day 9 (P9) mice subjected to a 3-h middle cerebral artery occlusion (tMCAO) and cultured (in vitro) revealed that uptake of fluorescently labeled MSC-sEV was significantly greater by microglia from the injured cortex vs. contralateral cortex. The cell-type–specific spatiotemporal distribution of MSC-sEV was also determined in vivo after tMCAO at P9. MSC-sEV administered at reperfusion, either by intracerebroventricular (ICV) or by intranasal (IN) routes, accumulated in the hemisphere ipsilateral to the occlusion, with differing spatial distribution 2 h, 18 h, and 72 h regardless of the administration route. By 72 h, MSC-sEV in the IN group was predominantly observed in Iba1⁺ cells with retracted processes and in GLUT1⁺ blood vessels in ischemic-reperfused regions. MSC-sEV presence in Iba1⁺ cells was sustained. MSC-sEV administration also significantly reduced injury volume 72 h after tMCAO in part via modulatory effects on microglial cells. Together, these data establish feasibility for MSC-sEV delivery to injured neonatal brain via a clinically relevant IN route, which affords protection during sub-acute injury phase.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01076-9.     

Gliosis and brain remodeling after treatment of stroke in rats with marrow stromal cells

The long-term (4-month) responses to treatment of stroke in the older adult rat, using rat bone marrow stromal cells (MSCs), have not been investigated. Retired breeder rats were subjected to middle cerebral artery occlusion (MCAo) alone, or injected intravenously with 3 x 10(6) MSCs, at 7 days after MCAo. Functional recovery was measured using an adhesive-removal patch test and a modified neurological severity score. Bromodeoxyuridine, a cell proliferation marker, was injected daily for 14 before sacrifice. Animals were sacrificed 4 months after stroke. Double immunostaining was used to identify cell proliferation and cell types for axons, astrocytes, microglia, and oligodendrocytes. MSC treatment induced significant improvement in neurological outcome after MCAo compared with control rats. MSC treatment reduced the thickness of the scar wall (P < 0.05) and reduced the numbers of microglia/macrophages within the scar wall (P < 0.01). Double staining showed increased expression of an axonal marker (GAP-43), among reactive astrocytes in the scar boundary zone and in the subventricular zone in the treated rats. Bromodeoxyuridine in cells preferentially colocalized with markers of astrocytes (GFAP) and oligodendrocytes (RIP) in the ipsilateral hemisphere, and gliogenesis was enhanced in the subventricular zone of the rats treated with MSCs. This is the first report to show that MSCs injected at 7 days after stroke improve long-term neurological outcome in older animals. Brain tissue repair is an ongoing process with reactive gliosis, which persists for at least 4 months after stroke. Reactive astrocytes responding to MSC treatment of ischemia may also promote axonal regeneration during long-term recovery.     

Combined fetal inflammation and postnatal hypoxia causes myelin deficits and autism‐like behavior in a rat model of diffuse white matter injury

Diffuse white matter injury (WMI) is a serious problem in extremely preterm infants, and is associated with adverse neurodevelopmental outcome, including cognitive impairments and an increased risk of autism‐spectrum disorders. Important risk factors include fetal or perinatal inflammatory insults and fluctuating cerebral oxygenation. However, the exact mechanisms underlying diffuse WMI are not fully understood and no treatment options are currently available. The use of clinically relevant animal models is crucial to advance knowledge on the pathophysiology of diffuse WMI, allowing the definition of novel therapeutic targets. In the present study, we developed a multiple‐hit animal model of diffuse WMI by combining fetal inflammation and postnatal hypoxia in rats. We characterized the effects on white matter development and functional outcome by immunohistochemistry, MRI and behavioral paradigms. Combined fetal inflammation and postnatal hypoxia resulted in delayed cortical myelination, microglia activation and astrogliosis at P18, together with long‐term changes in oligodendrocyte maturation as observed in 10 week old animals. Furthermore, rats with WMI showed impaired motor performance, increased anxiety and signs of autism‐like behavior, i.e. reduced social play behavior and increased repetitive grooming. In conclusion, the combination of fetal inflammation and postnatal hypoxia in rats induces a pattern of brain injury and functional impairments that closely resembles the clinical situation of diffuse WMI. This animal model provides the opportunity to elucidate pathophysiological mechanisms underlying WMI, and can be used to develop novel treatment options for diffuse WMI in preterm infants.     

Transplantation with hypoxia‐preconditioned mesenchymal stem cells suppresses brain injury caused by cardiac arrest–induced global cerebral ischemia in rats

Cardiac arrest–induced global cerebral ischemia is a main cause of neurological dysfunction in emergency medicine. Transplantation with bone marrow mesenchymal stem cells (MSCs) has been used in stroke models to repair the ischemic brain injury, but it is little studied in models with global cerebral ischemia. In the present study, a hypoxia precondition was used to improve the efficacy of MSC transplantation, given the low survival and migration rates and limited differentiation capacities of MSCs. We found that hypoxia can increase the expansion and migration of MSCs by activating the PI3K/AKT and hypoxia‐inducible factor‐1α/CXC chemokine receptor‐4 pathways. By using a cardiac arrest–induced global cerebral ischemic model in rats, we found that transplantation of hypoxia‐preconditioned MSCs promoted the migration and integration of MSCs and decreased neuronal death and inflammation in the ischemic cortex. © 2017 Wiley Periodicals, Inc.     

N‐methyl‐D‐aspartate receptor‐mediated axonal injury in adult rat corpus callosum

Damage to white matter such as corpus callosum (CC) is a pathological characteristic in many brain disorders. Glutamate (Glut) excitotoxicity through AMPA receptors on oligodendrocyte (OL) was previously considered as a mechanism for white matter damage. Recent studies have shown that N‐methyl‐D‐aspartate receptors (NMDARs) are expressed on myelin sheath of neonatal rat OL processes and that activation of these receptors mediated demyelization. Whether NMDARs are expressed in the adult CC and are involved in excitotoxic axonal injury remains to be determined. In this study, we demonstrate the presence of NMDARs in the adult rat CC and their distributions in myelinated nerve fibers and OL somata by means of immunocytochemical staining and Western blot. Incubation of the CC slices with Glut or NMDA induced axonal injury as revealed by analyzing amplitude of CC fiber compound action potentials (CAPs) and input–output response. Both Glut and NMDA decreased the CAP amplitude and input–output responses, suggesting an involvement of NMDARs in Glut‐ and NMDA‐induced axonal injury. The involvement of NMDAR in Glut‐induced axonal injury was further assayed by detection of β‐amyloid precursor protein (β‐APP) in the CC axonal fibers. Treatment of the CC slices with Glut resulted in β‐APP accumulation in the CC fibers as detected by Western blot, reflecting an impairment of axonal transport function. This injurious effect of Glut on CC axonal transport was significantly blocked by MK801. Taken together, these results show that NMDARs are expressed in the adult CC and are involved in excitotoxic activity in adult CC slices in vitro. © 2012 Wiley Periodicals, Inc.     

Time‐dependent change of in vivo optical imaging of oxidative stress in a mouse stroke model

Nuclear factor erythroid 2‐related factor 2 (Nrf2) plays a pivotal role in cellular defense against oxidative stress damage after ischemic stroke. In the present study, we examined the time‐dependent change of in vivo optical imaging of oxidative stress after stroke with Keap1‐dependent oxidative stress detector (OKD) mice. OKD mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 45 min, and in vivo optical signals were detected during the pre‐operative period, 12 h, 1 d, 3 d, and 7 d after tMCAO. Ex vivo imaging was performed immediately after obtaining in vivo optical signals at 1 d after tMCAO. Immunohistochemical analyses and infarct volume were also examined after in vivo imaging at each period. The in vivo signals showed a peak at 1 d after tMCAO that was slightly correlated to infarct volume. The strong ex vivo signals, which were detected in the peri‐ischemic area, corresponded to endogenous Nrf2 expression. Moreover, endogenous Nrf2 expression was detected mainly in neurons followed by oligodendrocytes and pericytes, but only slightly in astrocytes, microglia, endothelial cells. The present study successfully demonstrated the temporal change of in vivo imaging of oxidative stress after tMCAO, which is consistent with strong expression of endogenous Nrf2 in the peri‐ischemic area with a similar time course. © 2017 Wiley Periodicals, Inc.     

Reactive astrocyte COX2‐PGE2 production inhibits oligodendrocyte maturation in neonatal white matter injury

Inflammation is a major risk factor for neonatal white matter injury (NWMI), which is associated with later development of cerebral palsy. Although recent studies have demonstrated maturation arrest of oligodendrocyte progenitor cells (OPCs) in NWMI, the identity of inflammatory mediators with direct effects on OPCs has been unclear. Here, we investigated downstream effects of pro‐inflammatory IL‐1β to induce cyclooxygenase‐2 (COX2) and prostaglandin E2 (PGE2) production in white matter. First, we assessed COX2 expression in human fetal brain and term neonatal brain affected by hypoxic‐ischemic encephalopathy (HIE). In the developing human brain, COX2 was expressed in radial glia, microglia, and endothelial cells. In human term neonatal HIE cases with subcortical WMI, COX2 was strongly induced in reactive astrocytes with “A2” reactivity. Next, we show that OPCs express the EP1 receptor for PGE2, and PGE2 acts directly on OPCs to block maturation in vitro. Pharmacologic blockade with EP1‐specific inhibitors (ONO‐8711, SC‐51089), or genetic deficiency of EP1 attenuated effects of PGE2. In an IL‐1β‐induced model of NWMI, astrocytes also exhibit “A2” reactivity and induce COX2. Furthermore, in vivo inhibition of COX2 with Nimesulide rescues hypomyelination and behavioral impairment. These findings suggest that neonatal white matter astrocytes can develop “A2” reactivity that contributes to OPC maturation arrest in NWMI through induction of COX2‐PGE2 signaling, a pathway that can be targeted for neonatal neuroprotection.     

Cerebral tissue repair and atrophy after embolic stroke in rat: A magnetic resonance imaging study of erythropoietin therapy

Using magnetic resonance imaging (MRI) protocols of T₂‐, T₂*‐, diffusion‐ and susceptibility‐weighted imaging (T2WI, T2*WI, DWI, and SWI, respectively) with a 7T system, we tested the hypothesis that treatment of embolic stroke with erythropoietin (EPO) initiated at 24 hr and administered daily for 7 days after stroke onset has benefit in repairing ischemic cerebral tissue. Adult Wistar rats were subjected to embolic stroke by means of middle cerebral artery occlusion (MCAO) and were randomly assigned to a treatment (n = 11) or a control (n = 11) group. The treated group was given EPO intraperitoneally at a dose of 5,000 IU/kg daily for 7 days starting 24 hr after MCAO. Controls were given an equal volume of saline. MRI was performed at 24 hr and then weekly for 6 weeks. MRI and histological measurements were compared between groups. Serial T2WI demonstrated that expansion of the ipsilateral ventricle was significantly reduced in the EPO‐treated rats. The volume ratio of ipsilateral parenchymal tissue relative to the contralateral hemisphere was significantly increased after EPO treatment compared with control animals, indicating that EPO significantly reduces atrophy of the ipsilateral hemisphere, although no significant differences in ischemic lesion volume were observed between the two groups. Angiogenesis and white matter remodeling were significantly increased and occurred earlier in EPO‐treated animals than in the controls, as evident from T2*WI and diffusion anisotropy maps, respectively. These data indicate that EPO treatment initiated 24 hr poststroke promotes angiogenesis and axonal remodeling in the ischemic boundary, which may potentially reduce atrophy of the ipsilateral hemisphere. © 2010 Wiley‐Liss, Inc.     

Ethyl pyruvate improves white matter remodeling in rats after traumatic brain injury

BackgroundSevere traumatic brain injury (TBI) results in long‐term neurological deficits associated with white matter injury (WMI). Ethyl pyruvate (EP) is a simple derivative of the endogenous energy substrate pyruvate with neuroprotective properties, but its role in recovery from WMI has not been explored.AimsThis study examines the effect of EP treatment on rats following TBI using behavioral tests and white matter histological analysis up to 28 days post‐injury.Materials and MethodsAnaesthetised adult rats were subjected to TBI by controlled cortical impact. After surgery, EP or Ringers solution (RS) was administrated intraperitoneally at 15 min after TBI and again at 12, 24, 36, 48, and 60 h after TBI. Sensorimotor deficits were evaluated up to day 21 after TBI by four independent tests. Immunofluorescence and transmission electron microscopy (TEM) were performed to assess white matter injury. Microglia activation and related inflammatory molecules were examined up to day 14 after TBI by immunohistochemistry or real‐time PCR.ResultsHere, we demonstrate that EP improves sensorimotor function following TBI as well as improves white matter outcomes up to 28 d after TBI, as shown by reduced myelin loss. Furthermore, EP administration during the acute phase of TBI recovery shifted microglia polarization toward the anti‐inflammatoryM2 phenotype, modulating the release of inflammatory‐related factors.ConclusionEP treatment may protect TBI‐induced WMI via modulating microglia polarization toward M2.     

Dynamic Changes in Brain Iron Metabolism in Neonatal Rats after Hypoxia-Ischemia

ObjectivesThe pathogenesis of hypoxic-ischemic white matter injury (WMI) in premature infants is still unclear, and the imbalance of cerebral iron metabolism may play an important role. Our study set out to investigate the changes in iron distribution, iron content and malondialdehyde (MDA) in disparate brain regions (parietal cortex, corpus callosum, hippocampus) within 84 days after hypoxia-ischemia (HI) in neonatal rats and to clarify the role of iron metabolism in WMI.Materials and MethodsWe adopted a rat model of hypoxic-ischemic WMI. Alterations in iron metabolism were detected by iron staining and iron assay kits, and the degree of brain injury was determined by MDA assays.ResultsOur results showed that different degrees of brain iron deposition occurred within 28 days after HI, and iron staining was the most obvious 3 days after HI. The iron content increased remarkably at 1–7 d after HI in the mixed tissues, especially at 3 d after HI. While the iron content in the parietal cortex and corpus callosum elevated obviously 14 days after HI. And the change trend of MDA was almost consistent with that of the iron content.ConclusionsOur findings revealed that brain iron metabolism changed dynamically in 3-day-old neonatal rats suffering from HI, which may cause lipid peroxidation damage to brain tissues. This process may be one of the pathogeneses of hypoxic-ischemic WMI.     

磁共振在大鼠脑缺血再灌注模型中的应用研究

【摘要】
目的 利用磁共振成像（magnetic resonance imaging,MRI）筛选合格的大鼠大脑中动脉闭塞（middle cerebral artery occlusion,MCAO）模型,分析大鼠脑缺血再灌损伤后表观弥散系数（apparent diffusion coefficient,ADC）及部分各向异性（fractional anisotropy,FA）指标的变化特征。
方法 制作SD大鼠MCAO模型,根据是否再灌注分为永久闭塞组和缺血再灌组,每组各9只,另设假手术组2只作为对照。对大鼠行磁共振T2、弥散加权成像（diffusion weighted imaging,DWI）、弥散张量成像（diffusion tensor imaging,DTI）和磁共振血管成像（magnetic resonance angiography,MRA）扫描,重建脑血管及计算相对ADC值和FA值,比较两组的相对ADC值和FA值。
结果 MRA可以显示大鼠脑血管的堵塞与再通,T2加权像可以观测到蛛网膜下腔出血。永久闭塞组比缺血再灌组的相对ADC值高,差异有显著性（0.657±0.140 vs 0.588±0.132,t＝2.27,P=0.048）;相对FA值高,差异有显著性（1.010±0.143 vs 0.915±0.111,t＝2.89,P=0.02）。
结论 利用MRA和T2加权像扫描可以筛选出合格的MCAO模型,MCAO永久闭塞组相对ADC值和FA值均高于缺血再灌注组。     

Human mesenchymal factors induce rat hippocampal‐ and human neural stem cell dependent oligodendrogenesis

The generation of new oligodendrocytes is essential for adult brain repair in diseases such as multiple sclerosis. We previously identified the multifunctional p57kip2 protein as a negative regulator of myelinating glial cell differentiation and as an intrinsic switch of glial fate decision in adult neural stem cells (aNSCs). In oligodendroglial precursor cells (OPCs), p57kip2 protein nuclear exclusion was recently found to be rate limiting for differentiation to proceed. Furthermore, stimulation with mesenchymal stem cell (MSC)‐derived factors enhanced oligodendrogenesis by yet unknown mechanisms. To elucidate this instructive interaction, we investigated to what degree MSC secreted factors are species dependent, whether hippocampal aNSCs respond equally well to such stimuli, whether apart from oligodendroglial differentiation also tissue integration and axonal wrapping can be promoted and whether the oligodendrogenic effect involved subcellular translocation of p57kip2. We found that CC1 positive oligodendrocytes within the hilus express nuclear p57kip2 protein and that MSC dependent stimulation of cultured hippocampal aNSCs was not accompanied by nuclear p57kip2 exclusion as observed for parenchymal OPCs after spontaneous differentiation. Stimulation with human MSC factors was observed to equally promote rat stem cell oligodendrogenesis, axonal wrapping and tissue integration. As forced nuclear shuttling of p57kip2 led to decreased CNPase‐ but elevated GFAP expression levels, this indicates heterogenic oligodendroglial mechanisms occurring between OPCs and aNSCs. We also show for the first time that dominant pro‐oligodendroglial factors derived from human fetal MSCs can instruct human induced pluripotent stem cell‐derived NSCs to differentiate into O4 positive oligodendrocytes.     

Reductions in kinesin expression are associated with nitric oxide‐induced axonal damage

Axonal injury is often characterized by axonal transport defects and abnormal accumulation of intra‐axonal components. Nitric oxide (NO) has a key role in mediating inflammatory axonopathy in many neurodegenerative diseases, but little is known about how nitrosative/oxidative stress affects axonal transport or whether reductions in kinesin superfamily protein (KIF) expression correlate with axon pathology. KIFs are molecular motors that have a key role in axonal and dendritic transport, and impairment of these mechanisms has been associated with a number of neurological disorders. This study shows that rat cortical neurons exposed to NO display both a time‐dependent decrease in KIF gene/protein expression and neurofilament phosphorylation in addition to a reduction in axonal length and neuronal survival. Because mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for neuronal/axonal repair, this study analyzes the capacity of MSCs to protect neurons and axonal transport mechanisms from NO damage. Results show that coculture of MSCs with NO‐exposed neurons results in the preservation of KIF expression, axonal length, and neuronal survival. Altogether, these results suggest a potential mechanism involved in the disruption of axonal transport and abnormal accumulation of proteins in axons during nitrosative insult. We hypothesize that impaired axonal transport contributes, per se, to progression of injury and provide further evidence of the therapeutic potential of MSCs for neurodegenerative disorders. © 2015 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.     

Reduction of rat brain CD8+ T‐cells by levodopa/benserazide treatment after experimental stroke

The activation of inflammatory cascades in the ischemic hemisphere impairs mechanisms of tissue reorganization with consequences for recovery of lost neurological function. Recruitment of T‐cell populations to the post‐ischemic brain occurs and represents a significant part of the inflammatory response. This study was conducted to investigate if treatment with levodopa, potentially acting as an immunomodulator, affects the T‐cell accumulation in the post‐ischemic brain. Male Sprague–Dawley rats were subjected to transient occlusion of the middle cerebral artery (tMCAO) for 105 min followed by levodopa/benserazide treatment (20 mg/kg/15 mg/kg) for 5 days initiated on day 2 post‐stroke. One week after tMCAO, T‐cell populations were analysed from brains, and levels of interleukin (IL)‐1β, chemokine (C‐X‐C motif) ligand 1, IL‐4, IL‐5, interferon gamma and IL‐13 were analysed. After levodopa/benserazide treatment, we found a significant reduction of cytotoxic T‐cells (CD3⁺CD8⁺) in the ischemic hemisphere together with reduced levels of T‐cell‐associated cytokine IL‐5, while other T‐cell populations (CD3⁺, CD3⁺CD4⁺, CD3⁺CD4⁺CD25⁺) were unchanged compared with vehicle‐treated rats. Moreover, a reduced number of cells was associated with reduced levels of intercellular adhesion molecule 1, expressed in endothelial cells, in the infarct core of levodopa/benserazide‐treated animals. Together, we provide the first evidence that dopamine can act as a potential immunomodulator by attenuating inflammation in the post‐ischemic brain.     

Increased oligodendrogenesis by humanin promotes axonal remyelination and neurological recovery in hypoxic/ischemic brains

Oligodendrocytes are the predominant cell type in white matter and are highly vulnerable to ischemic injury. The role of oligodendrocyte dysfunction in ischemic brain injury is unknown. In this study, we used a 24‐amino acid peptide S14G‐Humanin (HNG) to examine oligodendrogenesis and neurological functional recovery in a hypoxic/ischemic (H/I) neonatal model. Intraperitoneal HNG pre‐treatment decreased infarct volume following H/I injury. Delayed HNG treatment 24 h after H/I injury did not reduce infarct volume but did decrease neurological deficits and brain atrophy. Delayed HNG treatment did not attenuate axonal demyelination at 48 h after H/I injury. However, at 14 d after H/I injury, delayed HNG treatment increased axonal remyelination, the thickness of corpus callosum at the midline, the number of Olig2⁺/BrdU⁺ cells, and levels of brain‐derived neurotrophic factor (BDNF). Our results suggest that targeting oligodendrogenesis via delayed HNG treatment may represent a promising approach for the treatment of stroke. © 2014 Wiley Periodicals, Inc.     

Neuroimmune processes associated with Wallerian degeneration support neurotrophin‐3‐induced axonal sprouting in the injured spinal cord

Lesions of the spinal cord cause two distinctive types of neuroimmune responses, a response at the lesion site that leads to additional tissue destruction and a more subtle response, termed Wallerian degeneration (WD), that occurs distal to the lesion site. We have evidence that the neuroimmune response associated with WD may support tissue repair. Previously, we found that overexpression of neurotrophin‐3 (NT‐3) induced axonal growth in the spinal cord after a unilateral corticospinal tract (CST) lesion, but only if the immune system was intact and activated. We reasoned that a neuroimmune response associated with WD was involved in this neuroplasticity. To test this, we compared NT‐3‐induced axonal sprouting in athymic nude rats that lack functional T cells with rats with functional T cells and in nude rats grafted with CD4⁺ T cells or CD8⁺ T cells. There was no sprouting in nude rats and in nude rats grafted with CD8⁺ T cells. However, nude rats grafted with CD4⁺ T cells mounted a sprouting response. To determine which CD4⁺ subtype, type 1 T helper (Th1) or type 2 T helper (Th2) cells, was responsible, we grafted Th1 and Th2 cells into nude rats and tested whether they would support sprouting. Axonal sprouting was greater in rats grafted with Th2 cells, demonstrating that the Th2 subtype was responsible for supporting axonal sprouting. These data suggest that WD activates Th2 cells that, along with the direct effects of NT‐3 on CST axons, act to support axonal sprouting in the lesioned spinal cord. © 2013 Wiley Periodicals, Inc.     

Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats

Contusive spinal cord injury (SCI) produces large fluid-, debris- and inflammatory cell-filled cystic cavities that lack structure to support significant axonal regeneration. The recent discovery of stem cells capable of generating central nervous system (CNS) tissues, coupled with success in neurotransplantation strategies, has renewed hope that repair and recovery from CNS trauma is possible. Based on results from several studies using bone marrow stromal cells (MSCs) to promote CNS repair, we transplanted MSCs into the rat SCI lesion cavity to further investigate their effects on functional recovery, lesion morphology, and axonal growth. We found that transplanted MSCs induced hindlimb airstepping--a spontaneous locomotor movement associated with activation of the stepping control circuitry--but did not alter the time course or extent of overground locomotor recovery. Using stereological techniques to describe spinal cord anatomy, we show that MSC transplants occupied the lesion cavity and were associated with preservation of host tissue and white matter (myelin), demonstrating that these cells exert neuroprotective effects. The tissue matrix formed by MSC grafts supported greater axonal growth than that found in specimens without grafts. Moreover, uniform random sampling of axon profiles revealed that the majority of neurites in MSC grafts were oriented with their long axis parallel to that of the spinal cord, suggesting longitudinally directed growth. Together, these studies support further investigation of marrow stromal cells as a potential SCI repair strategy.     

White matter extension of the Melbourne Children's Regional Infant Brain atlas: M‐CRIB‐WM

Brain atlases providing standardised identification of neonatal brain regions are key in investigating neurological disorders of early childhood. Our previously developed Melbourne Children's Regional Infant Brain (M‐CRIB) and M‐CRIB 2.0 neonatal brain atlases provide standardised parcellation of 100 brain regions including cortical, subcortical, and cerebellar regions. The aim of this study was to extend M‐CRIB atlas coverage to include 54 white matter (WM) regions. Participants were 10 healthy term‐born neonates that were used to create the initial M‐CRIB atlas. WM regions were manually segmented based on T₂ images and co‐registered diffusion tensor imaging‐based, direction‐encoded colour maps. Our labelled regions imitate the Johns Hopkins University neonatal atlas, with minor anatomical modifications. All segmentations were reviewed and approved by a paediatric radiologist and a neurosurgery research fellow for anatomical accuracy. The resulting neonatal WM atlas comprises 54 WM regions: 24 paired regions, and six unpaired regions comprising five corpus callosum subdivisions, and one pontine crossing tract. Detailed protocols for manual WM parcellations are provided, and the M‐CRIB‐WM atlas is presented together with the existing M‐CRIB cortical, subcortical, and cerebellar parcellations in 10 individual neonatal MRI data sets. The novel M‐CRIB‐WM atlas, along with the M‐CRIB cortical and subcortical atlases, provide neonatal whole brain MRI coverage in the first multi‐subject manually parcellated neonatal atlas compatible with atlases commonly used at older time points. The M‐CRIB‐WM atlas is publicly available, providing a valuable tool that will help facilitate neuroimaging research into neonatal brain development in both healthy and diseased states.     

Doxycycline treatment in a neonatal rat model of hypoxia-ischemia reduces cerebral tissue and white matter injury: a longitudinal magnetic resonance imaging study

Doxycycline may potentially be a neuroprotective treatment for neonatal hypoxic-ischemic brain injury through its anti-inflammatory effects. The aim of this study was to examine any long-term neuroprotection by doxycycline treatment on cerebral gray and white matter. Hypoxic-ischemic brain injury was induced in 7-day-old rats. Pups were treated with either doxycycline (HI+doxy) or saline (HI+vehicle) by intraperitoneal injection at 1 h after hypoxia-ischemia (HI). At 6 h after HI, MnCl(2) was injected intraperitoneally for later manganese-enhanced magnetic resonance imaging (MRI). MRI was performed with diffusion-weighted imaging on day 1 and T(1) -weighted imaging and diffusion tensor imaging at 7, 21 and 42 days after HI. Animals were killed after MRI on day 42 and histological examinations of the brains were performed. There was a tendency towards lower lesion volumes on diffusion maps among HI+doxy than HI+vehicle rats at 1 day after HI. Volumetric MRI showed increasing differences between groups with time after HI, with less cyst formation and less cerebral tissue loss among HI+doxy than HI+vehicle pups. HI+doxy pups had less manganese enhancement on day 7 after HI, indicating reduced inflammation. HI+doxy pups had higher fractional anisotropy on diffusion tensor imaging in major white matter tracts in the injured hemisphere than HI+vehicle pups, indicating less injury to white matter and better myelination. Histological examinations supported the MRI results. Lesion size on early MRI was highly correlated with final injury measures. In conclusion, a single dose of doxycycline reduced long-term cerebral tissue loss and white matter injury after neonatal HI, with an increasing effect of treatment with time after injury.