Timing of appearance of late oligodendrocyte progenitors coincides with enhanced susceptibility of preterm rabbit cerebral white matter to hypoxia-ischemia

Emerging evidence supports that premature infants are susceptible to both cerebral white and gray matter injury. In a fetal rabbit model of placental insufficiency, preterm rabbits at embryonic day 22 (E22) exhibited histologic evidence of gray matter injury but minimal white matter injury after global hypoxia-ischemia (H-I). We hypothesized that the dissociation between susceptibility to gray and white matter injury at E22 was related to the timing of appearance of late oligodendrocyte progenitors (preOLs) that are particularly vulnerable in preterm human white matter lesions. During normal rabbit oligodendrocyte (OL) lineage progression, early OL progenitors predominated at E22. PreOL density increased between E24 and E25 in major forebrain white matter tracts. After H-I at E22 and E25, we observed a similar magnitude of cerebral H-I, assessed by cortical microvascular blood flow, and gray matter injury, assessed by caspase activation. However, the increased preOL density at E25 was accompanied by a significant increase in acute white matter injury after H-I that coincided with enhanced preOL degeneration. At E29, significant white matter atrophy developed after H-I at E25 but not E22. Thus, the timing of appearance of preOLs coincided with onset of a developmental window of enhanced white but not gray matter susceptibility to H-I.     

Crosstalk between oligodendrocytes and cerebral endothelium contributes to vascular remodeling after white matter injury

After stroke and brain injury, cortical gray matter recovery involves mechanisms of neurovascular matrix remodeling. In white matter, however, the mechanisms of recovery remain unclear. In this study, we demonstrate that oligodendrocytes secrete matrix metalloproteinase-9 (MMP-9), which accelerates the angiogenic response after white matter injury. In primary oligodendrocyte cultures, treatment with the proinflammatory cytokine interleukin-1β (IL-1β) induced an upregulation and secretion of MMP-9. Conditioned media from IL-1β-stimulated oligodendrocytes significantly amplified matrigel tube formation in brain endothelial cells, indicating that MMP-9 from oligodendrocytes can promote angiogenesis in vitro. Next, we asked whether similar signals and substrates operate after white matter injury in vivo. Focal white matter injury and demyelination was induced in mice via stereotactic injection of lysophosphatidylcholine into corpus callosum. Western blot analysis showed that IL-1β expression was increased in damaged white matter. Immunostaining demonstrated MMP-9 signals in myelin-associated oligodendrocytic basic protein-positive oligodendrocytes. Treatment with an IL-1β-neutralizing antibody suppressed the MMP-9 response in oligodendrocytes. Finally, we confirmed that the broad spectrum MMP inhibitor GM6001 inhibited angiogenesis around the injury area in this white matter injury model. In gray matter, a neurovascular niche promotes cortical recovery after brain injury. Our study suggests that an analogous oligovascular niche may mediate recovery in white matter.     

Netrin-1 overexpression promotes white matter repairing and remodeling after focal cerebral ischemia in mice

Damage of oligodendrocytes after ischemia has negative impact on white matter integrity and neuronal function. In this work, we explore whether Netrin-1 (NT-1) overexpression facilitates white matter repairing and remodeling. Adult CD-1 mice received stereotactic injection of adeno-associated virus carrying NT-1 gene (AAV-NT-1). One week after gene transfer, mice underwent 60 minutes of middle cerebral artery occlusion. The effect of NT-1 on neural function was evaluated by neurobehavioral tests. Proliferated oligodendrocyte progenitor cells (OPCs), newly matured oligodendrocytes, and remyelination were semi-quantified by immunohistochemistry. The role of NT-1 in oligodendrogenesis was further explored by examining specific NT-1 receptors and their function. Netrin-1 overexpression was detected in neurons and astrocytes 2 weeks after AAV-NT-1 gene transfer and significantly improved the neurobehavioral outcomes compared with the control (P<0.05). In comparison with the control, proliferated OPCs, newly matured oligodendrocytes, and remyelination were greatly increased in the ipsilateral hemisphere of AAV-NT-1-transduced mice. Furthermore, both NT-1 receptors deleted in colorectal carcinoma and UNC5H2 were expressed on OPCs whereas only UNC5H2 was expressed in myelinated axons. Our study indicated that NT-1 promoted OPC proliferation, differentiation, and increased remyelination, suggesting that NT-1 is a promising factor for white matter repairing and remodeling after ischemia.     

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.     

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.     

Origin and dynamics of oligodendrocytes in the developing brain: Implications for perinatal white matter injury

Infants born prematurely are at high risk to develop white matter injury (WMI), due to exposure to hypoxic and/or inflammatory insults. Such perinatal insults negatively impact the maturation of oligodendrocytes (OLs), thereby causing deficits in myelination. To elucidate the precise pathophysiology underlying perinatal WMI, it is essential to fully understand the cellular mechanisms contributing to healthy/normal white matter development. OLs are responsible for myelination of axons. During brain development, OLs are generally derived from neuroepithelial zones, where neural stem cells committed to the OL lineage differentiate into OL precursor cells (OPCs). OPCs, in turn, develop into premyelinating OLs and finally mature into myelinating OLs. Recent studies revealed that OPCs develop in multiple waves and form potentially heterogeneous populations. Furthermore, it has been shown that myelination is a dynamic and plastic process with an excess of OPCs being generated and then abolished if not integrated into neural circuits. Myelination patterns between rodents and humans show high spatial and temporal similarity. Therefore, experimental studies on OL biology may provide novel insights into the pathophysiology of WMI in the preterm infant and offers new perspectives on potential treatments for these patients.     

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.     

Axon-glia synapses are highly vulnerable to white matter injury in the developing brain

The biology of cerebral white matter injury has been woefully understudied, in part because of the difficulty of reliably modeling this type of injury in rodents. Periventricular leukomalacia (PVL) is the predominant form of brain injury and the most common cause of cerebral palsy in premature infants. PVL is characterized by predominant white matter injury. No specific therapy for PVL is presently available, because the pathogenesis is not well understood. Here we report that two types of mouse PVL models have been created by hypoxia-ischemia with or without systemic coadministration of lipopolysaccharide (LPS). LPS coadministration exacerbated hypoxic-ischemic white matter injury and led to enhanced microglial activation and astrogliosis. Drug trials with the antiinflammatory agent minocycline, the antiexcitotoxic agent NBQX, and the antioxidant agent edaravone showed various degrees of protection in the two models, indicating that excitotoxic, oxidative, and inflammatory forms of injury are involved in the pathogenesis of injury to immature white matter. We then applied immunoelectron microscopy to reveal fine structural changes in the injured white matter and found that synapses between axons and oligodendroglial precursor cells (OPCs) are quickly and profoundly damaged. Hypoxia-ischemia caused a drastic decrease in the number of postsynaptic densities associated with the glutamatergic axon-OPC synapses defined by the expression of vesicular glutamate transporters, vGluT1 and vGluT2, on axon terminals that formed contacts with OPCs in the periventricular white matter, resulted in selective shrinkage of the postsynaptic OPCs contacted by vGluT2 labeled synapses, and led to excitotoxicity mediated by GluR2-lacking, Ca(2+) -permeable AMPA receptors. Overall, the present study provides novel mechanistic insights into the pathogenesis of PVL and reveals that axon-glia synapses are highly vulnerable to white matter injury in the developing brain. More broadly, the study of white matter development and injury has general implications for a variety of neurological diseases, including PVL, stroke, spinal cord injury, and multiple sclerosis.     

Heterogeneity of microglia and their differential roles in white matter pathology

Microglia are resident immune cells that play multiple roles in central nervous system (CNS) development and disease. Although the classical concept of microglia/macrophage activation is based on a biphasic beneficial‐versus‐deleterious polarization, growing evidence now suggests a much more heterogenous profile of microglial activation that underlie their complex roles in the CNS. To date, the majority of data are focused on microglia in gray matter. However, demyelination is a prominent pathologic finding in a wide range of diseases including multiple sclerosis, Alzheimer's disease, and vascular cognitive impairment and dementia. In this mini‐review, we discuss newly discovered functional subsets of microglia that contribute to white matter response in CNS disease onset and progression. Microglia show different molecular patterns and morphologies depending on disease type and brain region, especially in white matter. Moreover, in later stages of disease, microglia demonstrate unconventional immuno‐regulatory activities such as increased phagocytosis of myelin debris and secretion of trophic factors that stimulate oligodendrocyte lineage cells to facilitate remyelination and disease resolution. Further investigations of these multiple microglia subsets may lead to novel therapeutic approaches to treat white matter pathology in CNS injury and disease.     

Phosphodiesterase III inhibition promotes differentiation and survival of oligodendrocyte progenitors and enhances regeneration of ischemic white matter lesions in the adult mammalian brain

Vascular dementia is caused by blockage of blood supply to the brain, which causes ischemia and subsequent lesions primarily in the white matter, a key characteristic of the disease. In this study, we used a chronic cerebral hypoperfusion rat model to show that the regeneration of white matter damaged by hypoperfusion is enhanced by inhibiting phosphodiesterase III. A rat model of chronic cerebral hypoperfusion was prepared by bilateral common carotid artery ligation. Performance at the Morris water-maze task, immunohistochemistry for bromodeoxyuridine, as well as serial neuronal and glial markers were analyzed until 28 days after hypoperfusion. There was a significant increase in the number of oligodendrocyte progenitor cells in the brains of patients with vascular dementia as well as in rats with cerebral hypoperfusion. The oligodendrocyte progenitor cells subsequently underwent cell death and the number of oligodendrocytes decreased. In the rat model, treatment with a phosphodiesterase III inhibitor prevented cell death, markedly increased the mature oligodendrocytes, and promoted restoration of white matter and recovery of cognitive decline. These effects were cancelled by using protein kinase A/C inhibitor in the phosphodiesterase III inhibitor group. The results of our study indicate that the mammalian brain white matter tissue has the capacity to regenerate after 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 tradi-tional 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 abnormali-ties 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.     

Neuronal NAMPT is released after cerebral ischemia and protects against white matter injury

Nicotinamide phosphoribosyltransferase (NAMPT) has been implicated in neuroprotection against ischemic brain injury, but the mechanism underlying its protective effect remains largely unknown. To further examine the protective effect of NAMPT against ischemic stroke and its potential mechanism of action, we generated a novel neuron-specific NAMPT transgenic mouse line. Transgenic mice and wild-type littermates were subjected to transient occlusion of the middle cerebral artery (MCAO) for 60 minutes. Neuron-specific NAMPT overexpression significantly reduced infarct volume by 65% (P=0.018) and improved long-term neurologic outcomes (P≤0.05) compared with littermates. Interestingly, neuronal overexpression of NAMPT increased the area of myelinated fibers in the striatum and corpus callosum, indicating that NAMPT protects against white matter injury. The mechanism of protection appeared to be through extracellular release of NAMPT. First, NAMPT was secreted into the extracellular medium by primary cortical neurons exposed to ischemia-like oxygen–glucose deprivation (OGD) in vitro. Second, conditioned medium from NAMPT-overexpressing neurons exposed to OGD protected cultured oligodendrocytes from OGD. Third, the protective effects of conditioned medium were abolished by antibody-mediated NAMPT depletion, strongly suggesting that the protective effect is mediated by the extracellular NAMPT released into in the medium. These data suggest a novel neuroprotective role for secreted NAMPT in the protection of white matter after ischemic injury.     

Associations between T1 white matter lesion volume and regional white matter microstructure in aging

White matter lesions, typically manifesting as regions of signal intensity abnormality (WMSA) on MRI, increase in frequency with age. However, the role of this damage in cognitive decline and disease is still not clear, as lesion volume has only loosely been associated with clinical status. Diffusion tensor imaging (DTI) has been used to examine the quantitative microstructural integrity of white matter, and has applications in the examination of subtle changes to tissue that appear visually normal on conventional imaging. The primary goal of this study was to determine whether major macrostructural white matter damage, (total WMSA volume), is associated with microstructural integrity of normal appearing white matter, and if these macrostructural changes fully account for microstructural changes. Imaging was performed in 126 nondemented individuals, ages 43–85 years, with no history of cerebrovascular disease. Controlling for age, greater WMSA volume was associated with decreased fractional anisotropy (FA) in widespread brain regions. Patterns were similar for FA and radial diffusivity but in contrast, WMSA was associated with axial diffusivity in fewer areas. Age was associated with FA in several regions, and many of these effects remained even when controlling for WMSA volume, suggesting the etiology of WMSAs does not fully account for all age‐associated white matter deterioration. These results provide evidence that WMSA volume is associated with the integrity of normal‐appearing white matter. In addition, our results suggest that overt lesions may not account for the association of increasing age with decreased white matter tissue integrity. Hum Brain Mapp 35:1085–1100, 2014. © 2013 Wiley Periodicals, Inc.     

Interleukin-4 improves white matter integrity and functional recovery after murine traumatic brain injury via oligodendroglial PPARγ

Long-term neurological recovery after severe traumatic brain injury (TBI) is strongly linked to the repair and functional restoration of injured white matter. Emerging evidence suggests that the anti-inflammatory cytokine interleukin-4 (IL-4) plays an important role in promoting white matter integrity after cerebral ischemic injury. Here, we report that delayed intranasal delivery of nanoparticle-packed IL-4 boosted sensorimotor neurological recovery in a murine model of controlled cortical impact, as assessed by a battery of neurobehavioral tests for up to five weeks. Post-injury IL-4 treatment failed to reduce macroscopic brain lesions after TBI, but preserved the structural and functional integrity of white matter, at least in part through oligodendrogenesis. IL-4 directly facilitated the differentiation of oligodendrocyte progenitor cells (OPCs) into mature myelin-producing oligodendrocytes in primary cultures, an effect that was attenuated by selective PPARγ inhibition. IL-4 treatment after TBI in vivo also failed to stimulate oligodendrogenesis or improve white matter integrity in OPC-specific PPARγ conditional knockout (cKO) mice. Accordingly, IL-4-afforded improvements in sensorimotor neurological recovery after TBI were markedly impaired in the PPARγ cKO mice compared to wildtype controls. These results support IL-4 as a potential novel neurorestorative therapy to improve white matter functionality and mitigate the long-term neurological consequences of TBI.