alpha-Phenyl-n-tert-butyl-nitrone attenuates hypoxic-ischemic white matter injury in the neonatal rat brain

White matter of the neonatal brain is highly sensitive to hypoxic-ischemic insult. The susceptibility of premature oligodendrocytes (OLs) to free radicals (FRs) produced during hypoxia-ischemia (HI) has been proposed as one of the mechanisms involved. To test this hypothesis, and to further investigate if the FR scavenger alpha-phenyl-N-tert-butyl-nitrone (PBN) attenuates hypoxic-ischemic white matter damage (WMD), postnatal day 4 (P4) SD rats were subjected to bilateral common carotid artery ligation (BCAL), followed by 8% oxygen exposure for 20 min. Pathological changes were evaluated on P6 and P9, 2 and 5 days after the HI insult. HI caused severe WMD including rarefaction, necrosis and cavity formation in the corpus callosum, external and internal capsule areas. OL injury was evidenced by degeneration of O4 positive OLs on P6. Disrupted myelination was verified by decreased immunostaining of myelin basic protein (MBP) on P9. Axonal injury was demonstrated by increased amyloid precursor protein (APP) immunostaining on both P6 and P9. Two lipid peroxidation end products, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), showed a one-fold elevation within 1-24 h following HI. 4-HNE immunostaining was found to specifically localize in the white matter area. Furthermore, pyknotic O4+ OLs were double-labeled with 4-HNE. These findings suggest that FRs are involved in the pathogenesis of neonatal WMD. PBN (100 mg/kg, i.p.) treatment alleviated the pathological changes of WMD following HI. It improved the survival of O4 positive OLs, attenuated hypomyelination and reduced axonal damage. PBN treatment also decreased the brain concentration of MDA/4-HNE and positive 4-HNE staining in the white matter area. These findings indicate that in the current WMD model, PBN protects both OLs and axons, the two main components in the white matter, from neonatal HI insult. FR scavenging appears to be the primary mechanism underlying its neuroprotective effect.     

Chronic ischemia preferentially causes white matter injury in the neonatal rat brain

Chronic ischemic brain injuries were studied in 7- and 14-day-old rat pups, which were subjected to bilateral carotid artery occlusion (BCAO) on postnatal day 1. BCAO preferentially injured white matter in the corpus callosum, subcortex and internal capsule areas while largely spared cortical neurons. White matter rarefaction in the corpus callosum was observed in 12 out of the 17 BCAO rat brains and significantly enlarged lateral ventricles were found in five out of seven P14 BCAO rat brains. These white matter changes were similar to injuries found in newborn infants with periventricular leukomalacia (PVL). White matter injuries in the 7-day-old BCAO rat brain were accompanied with increased activation of microglia/macrophages, as indicated by ED1 and OX42 positive immunostaining. Immature oligodendrocytes in the 7-day-old BCAO rat brain, as indicated by O4+/O1+ staining, were much fewer than in the sham-operated rat brain. Immunostaining for myelin basic protein (MBP) at the fimbria hippocampus and the internal capsule areas in the 7-day-old BACO rat brain was also much less than in the control rat brain. Consistent with the immunostaining data, MBP mRNA expression in the 7-day-old, but not in the 14-day-old, BCAO rat brain was significantly less than in the control rat brain. The overall results suggest that pre-oligodendrocytes and immature oligodendrocytes might be major targets for chronic ischemic insults and activated microglia/macrophages are possibly involved in the process of white matter injury.     

Myelin loss and oligodendrocyte pathology in white matter tracts following traumatic brain injury in the rat

Axonal injury is an important contributor to the behavioral deficits observed following traumatic brain injury (TBI). Additionally, loss of myelin and/or oligodendrocytes can negatively influence signal transduction and axon integrity. Apoptotic oligodendrocytes, changes in the oligodendrocyte progenitor cell (OPC) population and loss of myelin were evaluated at 2, 7 and 21 days following TBI. We used the central fluid percussion injury model (n = 18 and three controls) and the lateral fluid percussion injury model (n = 15 and three controls). The external capsule, fimbriae and corpus callosum were analysed. With Luxol Fast Blue and RIP staining, myelin loss was observed in both models, in all evaluated regions and at all post‐injury time points, as compared with sham‐injured controls (P ≤ 0.05). Accumulation of β‐amyloid precursor protein was observed in white matter tracts in both models in areas with preserved and reduced myelin staining. White matter microglial/macrophage activation, evaluated by isolectin B4 immunostaining, was marked at the early time points. In contrast, the glial scar, evaluated by glial fibrillary acidic protein staining, showed its highest intensity 21 days post‐injury in both models. The number of apoptotic oligodendrocytes, detected by CC1/caspase‐3 co‐labeling, was increased in both models in all evaluated regions. Finally, the numbers of OPCs, evaluated with the markers Tcf4 and Olig2, were increased from day 2 (Olig2) or day 7 (Tcf4) post‐injury (P ≤ 0.05). Our results indicate that TBI induces oligodendrocyte apoptosis and widespread myelin loss, followed by a concomitant increase in the number of OPCs. Prevention of myelin loss and oligodendrocyte death may represent novel therapeutic targets for TBI.     

Apcdd1 stimulates oligodendrocyte differentiation after white matter injury

Wnt signaling plays an essential role in developmental and regenerative myelination of the CNS, therefore it is critical to understand how the factors associated with the various regulatory layers of this complex pathway contribute to these processes. Recently, Apcdd1 was identified as a negative regulator of proximal Wnt signaling, however its role in oligodendrocyte (OL) differentiation and reymelination in the CNS remain undefined. Analysis of Apcdd1 expression revealed dynamic expression during OL development, where its expression is upregulated during differentiation. Functional studies using ex vivo and in vitro OL systems revealed that Apcdd1 promotes OL differentiation, suppresses Wnt signaling, and associates with β‐catenin. Application of these findings to white matter injury (WMI) models revealed that Apcdd1 similarly promotes OL differentiation after gliotoxic injury in vivo and acute hypoxia ex vivo. Examination of Apcdd1 expression in white matter lesions from neonatal WMI and adult multiple sclerosis revealed its expression in subsets of oligodendrocyte (OL) precursors. These studies describe, for the first time, the role of Apcdd1 in OLs after WMI and reveal that negative regulators of the proximal Wnt pathway can influence regenerative myelination, suggesting a new therapeutic strategy for modulating Wnt signaling and stimulating repair after WMI. GLIA 2015;63:1840–1849     

The effects of hypoxic preconditioning on white matter damage following hypoxic-ischaemic injury in the neonatal rat brain

Myelination is an essential process in human development that is carried out by oligodendrocytes in the central nervous system. Hypoxic-ischaemic (HI) brain injury can disrupt myelination by causing oxidative stress, inflammation and excitotoxicity, resulting in the loss of myelin as well as cells of the oligodendrocyte lineage. We have previously shown that hypoxic preconditioning (HP) can protect against HI injury, however, to date there have been no reports of its effects on white matter injury. Sprague-Dawley rat pups (postnatal day (P) 6) were placed into control and HP groups. On P7, pups were further separated into HI and sham surgery groups. HI pups underwent a unilateral common carotid artery occlusion and then exposed to 8% oxygen for 3 h. Sham pups underwent the same procedure without occlusion and were maintained in room air. Brains were removed 5 days post-surgery for analysis. In HI-only pups there was a significant reduction in brain volume observed. Consequently, when HP was performed prior to HI, the loss of brain tissue was prevented. The number of early and late oligodendrocyte progenitors (preOLs) in the corpus callosum was unaffected by HI, however, HI reduced the amount of myelin basic protein, indicating that HI may inhibit the maturation of preOLs. Whilst HP did not affect preOL density, it was found to prevent the loss of myelin caused by HI. This indicates that HP may either protect myelin directly or possibly promote the maturation of preOLs to regenerate the lost or damaged myelin.     

Tissue tears in the white matter after lateral fluid percussion brain injury in the rat: relevance to human brain injury

A characteristic feature of severe diffuse axonal injury in man is radiological evidence of the “shearing injury triad” represented by lesions, sometimes haemorrhagic, in the corpus callosum, deep white matter and the rostral brain stem. With the exception of studies carried out on the non-human primate, such lesions have not been replicated to date in the multiple and diverse rodent laboratory models of traumatic brain injury. The present report describes tissue tears in the white matter, particularly in the fimbria of Sprague-Dawley rats killed 12, 24, and 48 h and 7 days after lateral fluid percussion brain injury of moderate severity (2.1–2.4 atm). The lesions were most easily seen at 24 h when they appeared as foci of tissue rarefaction in which there were a few polymorphonuclear leucocytes. At the margins of these lesions, large amounts of accumulated amyloid precursor protein (APP) were found in axonal swellings and bulbs. By 1 week post-injury, there was macrophage infiltration with marked astrocytosis and early scar formation. This lesion is considered to be due to severe deformation of white matter and this is the first time that it has been identified reproducibly in a rodent model of head injury under controlled conditions. Received: 25 February 1999 / Revised: 7 June 1999 / Accepted: 22 June 1999     

Glial activation in white matter following ischemia in the neonatal P7 rat brain

This study examines cell death and proliferation in the white matter after neonatal stroke. In postnatal day 7 injured rat, there was a marked reduction in myelin basic protein (MBP) immunostaining mainly corresponding to numerous pyknotic immature oligodendrocytes and TUNEL-positive astrocytes in the ipsilateral external capsule. In contrast, a substantial restoration of MBP, as indicated by the MBP ratio of left-to-right, occurred in the cingulum at 48 (1.27 +/- 0.12) and 72 (1.30 +/- 0.18, P < 0.05) h of recovery as compared to age-matched controls (1.03 +/- 0.14). Ki-67 immunostaining revealed a first peak of newly generated cells in the dorsolateral hippocampal subventricular zone and cingulum at 72 h after reperfusion. Double immunofluorescence revealed that most of the Ki-67-positive cells were astrocytes at 48 h and NG2 pre-oligodendrocytes at 72 h of recovery. Microglia infiltration occurs over several days in the cingulum, and a huge quantity of macrophages reached the subcortical white matter where they engulfed immature oligodendrocytes. The overall results suggest that the persistent activation of microglia involves a chronic component of immunoinflammation, which overwhelms repair processes and contributes to cystic growth in the developing brain.     

Semaphorin 6D regulate corralling,hematoma compaction and white matter injury in mice after intracerebral hemorrhage

ObjectivesThe Semaphorin 6D (SEMA6D) shows important roles in cell guidance and lipid metabolism, but the effects and mechanisms of SEMA6D on tissue repair, white matter injury and the recovery of neurological function after intracerebral hemorrhage have not been well studied.Materials and MethodsIn this study, the autologous whole blood injection model of intracerebral hemorrhage was established in C57 male mice. SEMA6D knockout CRISPR utilized in the study. Assessments included neurological score evaluation and immunofluorescence.ResultsSEMA6D increased and peaked at 7d after intracerebral hemorrhage, and mainly located in neurons, microglia and astrocytes. SEMA6D knockout CRISPR aggravated neurological function and showed signs of poorer corralling and hematoma resolution, with more compartments of well-established physical barrier and more extensive GFAP positive astrocytic border. Furthermore, SEMA6D can prevent the decrease of NF-H in the peri-hematoma region, while SEMA6D knockout aggravated WMI.ConclusionsOur study suggested that SEMA6D could influence the recovery of neurological function by regulating the corralling, hematoma compaction and WMI in mice after intracerebral hemorrhage.     

Oxymatrine ameliorates white matter injury by modulating gut microbiota after intracerebral hemorrhage in mice

Introduction

White matter injury (WMI) significantly affects neurobehavioral recovery in intracerebral hemorrhage (ICH) patients. Gut dysbiosis plays an important role in the pathogenesis of neurological disorders. Oxymatrine (OMT) has therapeutic effects on inflammation-mediated diseases. Whether OMT exerts therapeutic effects on WMI after ICH and the role of gut microbiota involved in this process is largely unknown.

Methods

Neurological deficits, WMI, gut microbial composition, intestinal barrier function, and systemic inflammation were investigated after ICH. Fecal microbiota transplantation (FMT) was performed to elucidate the role of gut microbiota in the pathogenesis of ICH.

Results

OMT promoted long-term neurological function recovery and ameliorated WMI in the peri-hematoma region and distal corticospinal tract (CST) region after ICH. ICH induced significant and persistent gut dysbiosis, which was obviously regulated by OMT. In addition, OMT alleviated intestinal barrier dysfunction and systemic inflammation. Correlation analysis revealed that gut microbiota alteration was significantly correlated with inflammation, intestinal barrier permeability, and neurological deficits after ICH. Moreover, OMT-induced gut microbiota alteration could confer protection against neurological deficits and intestinal barrier disruption.

Conclusions

Our study demonstrates that OMT ameliorates ICH-induced WMI and neurological deficits by modulating gut microbiota.     

Ethyl pyruvate protects against lipopolysaccharide-induced white matter injury in the developing rat brain

The neuroprotective effects of ethyl pyruvate (EP) have been proved in several brain injury models, yet very little is known about its action on neonatal white matter injury. To investigate the effect of EP on white matte damage, a stereotactic intracerebral injection of lipopolysaccharide (LPS, 1 mg/kg) was performed on postnatal day 5 Sprague–Dawley rat pups, and EP was administrated intraperitoneally at a dose of 40 mg/kg immediately, 1 h and 12 h after LPS exposure. Significantly, treatment with EP reduced LPS-induced ventricle dilation, loss of O4+ and O1+ oligodendrocytes, apoptosis of oligodendrocytes, and hypomyelination. The protective effect of EP was associated with suppressed inflammatory responses, indicated by the inhibition of activation of microglia and astrocytes, as well as the decreased expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in rat brains. Also, EP prevented the elevation of cleaved caspase-3 in periventricular white matter tissue after LPS insult. Taken together, these results suggest that EP confers potent protection against LPS-induced white matter injury via its anti-inflammatory and anti-apoptotic properties.     

Experimental cerebral hypoperfusion induces white matter injury and microglial activation in the rat brain

Though cerebral white matter injury is a frequently described phenomenon in aging and dementia, the cause of white matter lesions has not been conclusively determined. Since the lesions are often associated with cerebrovascular risk factors, ischemia emerges as a potential condition for the development of white matter injury. In the present study, we induced experimental cerebral hypoperfusion by permanent, bilateral occlusion of the common carotid arteries of rats (n=6). A sham-operated group served as control (n=6). Thirteen weeks after the onset of occlusion, markers for astrocytes, microglia, and myelin were found to be labeled by means of immunocytochemistry in the corpus callosum, the internal capsule, and the optic tract. The ultrastructural integrity and oligodendrocyte density in the optic tract were investigated by electron microscopy. Quantitative analysis revealed that chronic cerebral hypoperfusion caused mild astrogliosis in the corpus callosum and the internal capsule, while astrocytic disintegration in the optic tract increased by 50%. Further, a ten-fold increase in microglial activation and a nearly doubled oligodendrocyte density were measured in the optic tract of the hypoperfused rats as compared with the controls. Finally, vacuolization and irregular myelin sheaths were observed at the ultrastructural level in the optic tract. In summary, the rat optic tract appears to be particularly vulnerable to ischemia, probably because of the rat brains angioarchitecture. Since the detected glial changes correspond with those reported in vascular and Alzheimer dementia, this model of cerebral hypoperfusion may serve to characterize the causal relationship between ischemia and white matter damage.     

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.     

Imaging white matter diffusion changes with development and recovery from brain injury

PURPOSE: This study reviews the application of diffusion tensor imaging (DTI) to the study of developmental and pathological changes in brain white matter. The ability to measure and monitor such changes in vivo would provide important opportunities for charting disease progression and monitoring response to therapeutic intervention. This study first reviews the use of DTI in studying normal human brain development. It goes on to illustrate how DTI has been used to provide insights into recovery from damage in selected brain disorders. CONCLUSIONS: It is concluded that potential clinical applications of DTI include: (i) monitoring pathological change, (ii) providing markers that predict recovery and allow for individual targeting of therapy, (iii) providing outcome measures, (iv) providing measures of potentially compensatory structural changes and (v) improving understanding of normal brain anatomy to aid in interpretation of the consequences of localized damage.     

Changes in white matter late after severe traumatic brain injury in childhood

Severe traumatic brain injury in childhood, particularly that complicated by raised intracranial pressure, has significant long-term effects on the brain. Since magnetic resonance imaging provides a means of visualizing neuroanatomic structure in exquisite detail, the scope of this review is to revisit the pathology of traumatic brain injury described in recent clinical imaging studies. Acute imaging provides insight into the acute mechanism of focal and diffuse injury. There is some reduction in threshold for white matter pathology in the hemisphere ipsilateral to injury. After injury, there may be long-term effects on white matter architecture and the potential for brain growth. In this context, the pattern of hippocampal rather than parahippocampal gyrus tissue loss provides insight into the likely cause of white matter injury being cerebral hypoperfusion.     

Cyclooxygenase 2 mRNA expression in rat brain after peripheral injection of lipopolysaccharide

Inducible cyclooxygenase 2 (COX 2) converts arachidonic acid to prostaglandins, which are thought to mediate various peripheral lipopolysaccharide (LPS)-induced central effects, including generation of fever and activation of the hypothalamic–pituitary–adrenal axis. To localize prostaglandin production in the brain following peripheral LPS administration, COX 2 mRNA expression was examined by in situ hybridization histochemistry in rats injected intraperitoneally (i.p.) or intravenously (i.v.) with various doses of LPS or saline. Constitutive expression of COX 2 mRNA was found in neurons of cortex, hippocampus, and amygdala, but not in cells of the blood vessels. COX 2 mRNA levels were not altered in saline-injected animals as compared to non-injected controls. In LPS-injected animals, no consistent changes of neuronal COX 2 mRNA expression were observed. COX 2 mRNA expression appeared ex novo at 0.5-h post-injection in cells closely associated with blood vessels, however, ex novo labeling of the number of labeled cells increased to a peak at 2 h and subsided gradually to basal levels by 24 h. Initially, labeling was observed in cells comprising major surface-lying blood vessels and meninges. Later, vascular and perivascular cells associated with smaller penetrating blood vessels were labeled. This pattern of COX 2 mRNA induction is independent of the route and dose of the LPS injection. The induced COX 2 mRNA producing cells are identified as endothelial and leptomeningeal cells. Changes in COX 2 mRNA expression were not observed in circumventricular organs. These results suggest that peripheral LPS induces a rapid increase in COX 2 production throughout the vasculatures of the brain, which could affect the neuronal activity of widespread brain regions by elevating the levels of prostaglandins.     

Microglia/macrophage polarization dynamics in white matter after traumatic brain injury

Mononuclear phagocytes are a population of multi-phenotypic cells and have dual roles in brain destruction/reconstruction. The phenotype-specific roles of microglia/macrophages in traumatic brain injury (TBI) are, however, poorly characterized. In the present study, TBI was induced in mice by a controlled cortical impact (CCI) and animals were killed at 1 to 14 days post injury. Real-time polymerase chain reaction (RT–PCR) and immunofluorescence staining for M1 and M2 markers were performed to characterize phenotypic changes of microglia/macrophages in both gray and white matter. We found that the number of M1-like phagocytes increased in cortex, striatum and corpus callosum (CC) during the first week and remained elevated until at least 14 days after TBI. In contrast, M2-like microglia/macrophages peaked at 5 days, but decreased rapidly thereafter. Notably, the severity of white matter injury (WMI), manifested by immunohistochemical staining for neurofilament SMI-32, was strongly correlated with the number of M1-like phagocytes. In vitro experiments using a conditioned medium transfer system confirmed that M1 microglia-conditioned media exacerbated oxygen glucose deprivation–induced oligodendrocyte death. Our results indicate that microglia/macrophages respond dynamically to TBI, experiencing a transient M2 phenotype followed by a shift to the M1 phenotype. The M1 phenotypic shift may propel WMI progression and represents a rational target for TBI treatment.