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.     

Early postnatal development of rat brain: in vivo diffusion tensor imaging

Perinatal hypoxia is a major cause of neurodevelopmental deficits. Neuronal migration patterns are particularly sensitive to perinatal hypoxia/ischemia and are associated with the clinical deficits. The rat model of hypoxia/ischemia at P7 mimics that of perinatal injury in humans. Before assessing the effects of postnatal injury on brain development, it is essential to determine the normal developmental trajectories of various brain structures in individual animals. In vivo longitudinal diffusion tensor imaging (DTI) was performed from postnatal day 0 (P0) to P56 on Wistar rats. The DTI metrics, mean diffusivity (MD), fractional anisotropy (FA), axial (lambdal) and radial (lambdat) diffusivities, were determined for four gray matter and eight white matter structures. The FA of the cortical plate and the body of corpus callosum decreased significantly during the first 3 weeks after birth. The decrease in the cortical plate's FA value was associated mainly with an increase in lambdat. The initial decrease in FA of corpus callosum was associated with a significant decrease in lambdal. The FA of corpus callosum increased during the rest of the observational period, which was mainly associated with a decrease in lambdat. The FA of gray matter structures, hippocampus, caudate putamen, and cortical mantle did not show significant changes between P0 and P56. In contrast, the majority of white matter structures showed significant changes between P0 and P56. These temporal changes in the DTI metrics were related to the neuronal and axonal pruning and myelination that are known to occur in the developing brain.     

Hypoxia/ischemia depletes the rat perinatal subventricular zone of oligodendrocyte progenitors and neural stem cells

Cerebral hypoxia/ischemia of the newborn has a frequency of 4/1,000 births and remains a major cause of cerebral palsy, epilepsy, and mental retardation. Despite progress in understanding the pathogenesis of hypoxic-ischemic injury, the data are incomplete regarding the mechanisms leading to permanent brain injury. Here we tested the hypothesis that cerebral hypoxia/ischemia damages stem/progenitor cells in the subventricular zone (SVZ), resulting in a permanent depletion of oligodendrocytes. We used a widely accepted rat model and examined animals at recovery intervals ranging from 4 h to 3 weeks. Within hours after the hypoxic-ischemic insult 20% of the total cells were deleted from the SVZ. The residual damaged cells appeared necrotic. During 48 h of recovery deaths accumulated; however, these later deaths were predominantly apoptotic. Many apoptotic SVZ cells stained with a marker for immature oligodendrocytes. At 3 weeks survival, the SVZ was smaller and markedly less cellular, and it contained less than 1/4 the normal complement of neural stem cells. The corresponding subcortical white matter was dysmyelinated, relatively devoid of oligodendrocytes and enriched in astrocytes. We conclude that neural stem cells and oligodendrocyte progenitors in the SVZ are vulnerable to hypoxia/ischemia. Consequently, the developmental production of oligodendrocytes is compromised and regeneration of damaged white matter oligodendrocytes does not occur resulting in failed regeneration of CNS myelin in periventricular loci. The resulting dysgenesis of the brain that occurs subsequent to perinatal hypoxic/ischemic injury may contribute to the cognitive and motor dysfunction that results from asphyxia of the newborn.     

Amyloid beta peptide-induced corpus callosum damage and glial activation in vivo

The effects of stereotaxic injection of amyloid beta-peptide (Abeta1-42) into rat brain to induce white matter damage have been studied. Administration of 1 nmol Abeta1-42 into corpus callosum resulted in considerable damage to axons as evidenced by the loss of neurofilament-immunoreactive (NF-ir) fibers 6 h and 3 and 7 days post-injection. Significant damage was also evident to myelin (using Luxol fast blue myelin staining) and oligodendrocytes (using CC1 immunocytochemistry); in the latter case marked caspase-3 immunoreactivity was evident in oligodendrocytes. Additionally, the numbers of GFAP-ir astrocytes and OX-42/OX-6-ir microglia were markedly increased following Abeta1-42 injection. These results suggest that Abeta plays an important pathophysiological role in white matter damage and that inflammatory responses may contribute to Abeta-induced demyelination and oligodendrocyte injury in corpus callosum. Loss of function of cells in corpus callosum could provide a potential new model for the study of white matter damage in Alzheimer's disease.     

Acute oligodendrocyte loss with persistent white matter injury in a third trimester equivalent mouse model of fetal alcohol spectrum disorder

Alcohol exposure during central nervous system (CNS) development can lead to fetal alcohol spectrum disorder (FASD). Human imaging studies have revealed significant white matter (WM) abnormalities linked to cognitive impairment in children with FASD; however, the underlying mechanisms remain unknown. Here, we evaluated both the acute and long‐term impacts of alcohol exposure on oligodendrocyte number and WM integrity in a third trimester‐equivalent mouse model of FASD, in which mouse pups were exposed to alcohol during the first 2 weeks of postnatal development. Our results demonstrate a 58% decrease in the number of mature oligodendrocytes (OLs) and a 75% decrease in the number of proliferating oligodendrocyte progenitor cells (OPCs) within the corpus callosum of alcohol‐exposed mice at postnatal day 16 (P16). Interestingly, neither mature OLs nor OPCs derived from the postnatal subventricular zone (SVZ) were numerically affected by alcohol exposure, indicating heterogeneity in susceptibility based on OL ontogenetic origin. Although mature OL and proliferating OPC numbers recovered by postnatal day 50 (P50), abnormalities in myelin protein expression and microstructure within the corpus callosum of alcohol‐exposed subjects persisted, as assessed by western immunoblotting of myelin basic protein (MBP; decreased expression) and MRI diffusion tensor imaging (DTI; decreased fractional anisotropy). These results indicate that third trimester‐equivalent alcohol exposure leads to an acute, albeit recoverable, decrease in OL lineage cell numbers, accompanied by enduring WM injury. Additionally, our finding of heterogeneity in alcohol susceptibility based on the developmental origin of OLs may have therapeutic implications in FASD and other disorders of WM development.     

ST8SIA2 promotes oligodendrocyte differentiation and the integrity of myelin and axons

ST8SIA2 is a polysialyltransferase that attaches polysialic acid to the glycoproteins NCAM1 and CADM1. Polysialylation is involved in brain development and plasticity. ST8SIA2 is a schizophrenia candidate gene, and St8sia2 ^?/? mice exhibit schizophrenia‐like behavior. We sought to identify new pathological consequences of ST8SIA2 deficiency. Our proteomic analysis suggested myelin impairment in St8sia2 ^?/? mice. Histological and immune staining together with Western blot revealed that the onset of myelination was not delayed in St8sia2 ^?/? mice, but the content of myelin was lower. Ultrastructure analysis of the corpus callosum showed thinner myelin sheaths, smaller and irregularly shaped axons, and white matter lesions in adult St8sia2 ^?/? mice. Then we evaluated oligodendrocyte differentiation in vivo and in vitro . Fewer OLIG2+ cells in the cortex and corpus callosum, together with the higher percentage of undifferentiated oligodenroglia in St8sia2 ^?/? mice suggested an impairment in oligodendrocyte generation. Experiment on primary cultures of oligodendrocyte precursor cells (OPCs) confirmed a cell‐autonomous effect of ST8SIA2 in oligodendroglia, and demonstrated that OPC to oligodendrocyte transition is inhibited in St8sia2 ^?/? mice. Concluding, ST8SIA2‐mediated polysialylation influences on oligodendrocyte differentiation, and oligodendrocyte deficits in St8sia2 mice are a possible cause of the demyelination and degeneration of axons, resembling nerve fiber alterations in schizophrenia. GLIA 2016;65:34–49     

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.     

Toll-like receptor 4 participates in the myelin disruptions associated with chronic alcohol abuse

Alcohol abuse and alcoholism can cause brain damage, loss of white matter, myelin fiber disruption, and even neuronal injury. The underlying mechanisms of these alterations remain elusive. We have shown that chronic ethanol intake, by activating glial toll-like receptor 4 (TLR4) receptors, triggers the production of inflammatory mediators and can cause brain damage. Because neuroinflammation may be associated with demyelination and neuronal damage, we evaluate whether the ethanol-induced TLR4-dependent proinflammatory environment in the brain could be involved in the myelin disruptions observed in alcoholics. Using brains from wild-type (WT) and TLR4 knockout (KO, TLR4(-/-) ) mice, we demonstrate that chronic ethanol treatment downregulated proteins involved in myelination [proteolipid protein (PLP), myelin basic protein (MBP), myelin-oligodendrocyte glycoprotein, 2,3-cyclic-nucleotide-3-phosphodiesterase, and myelin-associated glycoprotein], while increased chondroitin sulfate proteoglycan NG2 (NG2)-proteoglycan in several brain regions of ethanol-treated WT mice. The immunohistochemistry analysis also revealed that ethanol-treatment-altered myelin morphology reduced the number of MBP-positive fibers and caused oligodendrocyte death, as demonstrated by an increase in caspase-3-positive oligodendrocytes. The in vivo imaging system further confirmed that chronic ethanol intake markedly reduced the PLP in WT mice. Most myelin alterations were not observed in brains from ethanol-treated TLR4(-/-) mice. Electron microscopy studies revealed that although 41-47% of axons showed myelin sheath disarrangements in the cerebral cortex and corpus callosum of WT ethanol-treated mice, respectively, small focal fiber disruptions were noticed in these brain areas of ethanol-treated TLR4(-/-) mice. In summary, the present results suggest that ethanol-induced neuroinflammation might be involved in myelin disruptions and white matter loss observed in human alcoholics.     

White matter changes in HIV-1 infected brains: A combined gross anatomical and ultrastructural morphometric investigation of the corpus callosum

Objective

The HIV-1 associated cognitive/motor complex is characterized by cognitive, motor and behavioral disturbances. Besides a significant loss of neurons in the cerebral cortex and subcortical nuclei, a possible morphological substrate of this complex is also given by changes of the white matter as seen in HIV-1 leucoencephalopathy (HIVL), which is characterized by widespread diffuse pallor of myelin and the presence of gliomesenchymal nodules with multinucleated giant cells.

Methods

The corpus callosum as a sensitive marker for damage of the cerebral white matter was investigated by morphometry both at the macroscopic and electronmicroscopic level.

Results

In HIV-1 infected brains, a significant decrease of the profile area of the whole corpus callosum as well as of its different parts was noted. The absolute number of nerve fibers was significantly decreased, in particular in the frontal and occipital parts of the corpus callosum. Moreover, several morphometric parameters for nerve fibers, axons and myelin sheaths indicate in some areas a reduction of nerve fibers and axons, as well as a diminished myelin sheath thickness, whereas, in other regions, swelling of axons and myelin sheaths was observed.

Conlusions

The observed changes are considered to represent subtle changes affecting nerve fibers before histological evidence of HIVL, and might represent one aspect of the morphological substrates preceeding the development of the HIV-1 related cognitive/motor complex.     

Juvenile striatal white matter is resistant to ischemia‐induced damage

White matter injury following ischemic stroke is a major cause of functional disability. Injury to both myelinated axons and oligodendrocytes, the myelin producing cells in the central nervous system, occurs in experimental models of ischemic stroke. Age‐related changes in white matter vulnerability to ischemia have been extensively studied and suggest that both the perinatal and the aged periods are times of increased white matter vulnerability. However, sensitivity of white matter following stroke in the juvenile brain has not been evaluated. Interestingly, the late pediatric period is an important developmental stage, as it is the time of maximal myelination. The current study demonstrates that neurons in late pediatric/juvenile striatum are vulnerable to ischemic damage, with neuronal injury being comparable in juvenile and adult mice following ischemia. By contrast, actively myelinating striatal oligodendrocytes in the juvenile brain are resistant to ischemia, whereas adult oligodendrocytes are quite sensitive. As a result, myelin sheaths are remarkably intact and axons survive well in the injured striatum of juvenile mice. In addition to relative resistance of juvenile white matter, other glial responses were very different in juvenile and adult mice following cerebral ischemia, including differences in astrogliosis, fibrosis, NG2‐cell reactivity, and vascular integrity. Together, these responses lead to long‐term preservation of brain parenchyma in juvenile mice, compared to severe tissue loss and scarring in adult mice. Overall, the current study suggests that equivalent ischemic insults may result in less functional deficit in children compared to adults and an environment more conducive to long‐term recovery. GLIA 2016;64:1972–1986     

甲基维生素B12对拟血管性痴呆大鼠记忆功能及脑白质病理变化的影响

目的 观察甲基维生素B12对拟血管性痴呆大鼠记忆功能及脑白质病理变化的影响。方法 反复前脑缺血制备大鼠拟血管性痴呆模型，造模后甲基维生素B12治疗1周、2周、4周时Morris水迷宫检测大鼠学习记忆功能、LFB染色观察白质髓鞘变化、电镜下观察少突胶质细胞、髓鞘超微结构，并与假手术组及对照组对比。结果 治疗4周大鼠游迷宫时间明显短于对照组，造模2周、4周时LFB染色均可见白质髓鞘脱失，但治疗组明显较对照组轻。造模4周电镜下少突胶质细胞、髓鞘改变治疗组轻于对照组。结论 甲基维生素B12能改善拟血管性痴呆大鼠记忆功能，并能减轻脑白质病理变化。     

Selective reduction in microglia density and function in the white matter of colony‐stimulating factor‐1–deficient mice

It is still debated whether microglia play a beneficial or harmful role in myelin disorders such as multiple sclerosis and leukodystrophies as well as in other pathological conditions of the central nervous system. The osteopetrotic (op/op) mouse has reduced numbers of cells of monocyte lineage as a result of an inactivating mutation in the colony stimulating factor‐1 gene. To determine whether this mutant mouse might be used to study the role of microglia in myelin disorders, we quantified the number of microglia in the central nervous system of op/op mice and explored their ability to respond to brain injury created by a stab wound. Microglial density in the 2‐month‐old op/op mice was significantly decreased in the white matter tracts compared with the ‐ge matched wild‐type controls (by 63.6% in the corpus callosum and 86.4% in the spinal dorsal column), whereas the decrease was less in the gray matter, cerebral cortex (24.0%). A similar decrease was seen at 7 months of age. Morphometric studies of spinal cord myelination showed that development of myelin was not affected in op/op mice. In response to a stab wound, the increase in the number of microglia/macrophages in op/op mice was significantly less pronounced than that in wild‐type control. These findings demonstrate that this mutant is a valuable model in which to study roles of microglia/macrophages in the pathophysiology of myelin disorders. © 2009 Wiley‐Liss, Inc.     

Protective effects of carnosine on white matter damage induced by chronic cerebral hypoperfusion

Carnosine is a dipeptide that scavenges free radicals,inhibits inflammation in the central nervous system,and protects against ischemic and hypoxic brain damage through its anti-oxidative and anti-apoptotic actions.Therefore,we hypothesized that carnosine would also protect against white matter damage caused by subcortical ischemic injury.White matter damage was induced by right unilateral common carotid artery occlusion in mice.The animals were treated with 200,500 or 750 mg/kg carnosine by intraperitoneal injection 30 minutes before injury and every other day after injury.Then,37 days later,Klüver-Barrera staining,toluidine blue staining and immunofluorescence staining were performed.Carnosine(200,500 mg/kg) substantially reduced damage to the white matter in the corpus callosum,internal capsule and optic tract,and it rescued expression of myelin basic protein,and alleviated the loss of oligodendrocytes.However,carnosine at the higher dose of 750 mg/kg did not have the same effects as the 200 and 500 mg/kg doses.These findings show that carnosine,at a particular dose range,protects against white matter damage caused by chronic cerebral ischemia in mice,likely by reducing oligodendroglial cell loss.     

A modified flavonoid accelerates oligodendrocyte maturation and functional remyelination

Myelination delay and remyelination failure following insults to the central nervous system (CNS) impede axonal conduction and lead to motor, sensory and cognitive impairments. Both myelination and remyelination are often inhibited or delayed due to the failure of oligodendrocyte progenitor cells (OPCs) to mature into myelinating oligodendrocytes (OLs). Digestion products of the glycosaminoglycan hyaluronan (HA) have been implicated in blocking OPC maturation, but how these digestion products are generated is unclear. We tested the possibility that hyaluronidase activity is directly linked to the inhibition of OPC maturation by developing a novel modified flavonoid that functions as a hyaluronidase inhibitor. This compound, called S3, blocks some but not all hyaluronidases and only inhibits matrix metalloproteinase activity at high concentrations. We find that S3 reverses HA-mediated inhibition of OPC maturation in vitro, an effect that can be overcome by excess recombinant hyaluronidase. Furthermore, we find that hyaluronidase inhibition by S3 accelerates OPC maturation in an in vitro model of perinatal white matter injury. Finally, blocking hyaluronidase activity with S3 promotes functional remyelination in mice with lysolecithin-induced demyelinating corpus callosum lesions. All together, these findings support the notion that hyaluronidase activity originating from OPCs in CNS lesions is sufficient to prevent OPC maturation, which delays myelination or blocks remyelination. These data also indicate that modified flavonoids can act as selective inhibitors of hyaluronidase activity and can promote OPC maturation, making them excellent candidates to accelerate myelination or promote remyelination following perinatal and adult CNS insults.     

Sequential myelin protein expression during remyelination reveals fast and efficient repair after central nervous system demyelination

To understand the mechanisms of remyelination and the reasons for regeneration failure is one of the major challenges in multiple sclerosis research. This requires a good knowledge and reliable analysis of experimental models. This work was undertaken to characterize the pattern of myelin protein expression during experimental remyelination. Acute demyelination of the corpus callosum was induced by feeding of 0.3% cuprizone for 6 weeks, followed by a 10-week remyelination period. We used a combination of Luxol fast blue (LFB) myelin staining, electron microscopy (EM) and immunohistochemistry for the myelin proteins 2',3'-cyclic nucleotide 3' phosphodiesterase (CNPase), myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). Early remyelination was detected by the re-expression of CNPase, MBP and PLP as early as 4 days. MOG, as a marker for late differentiation of oligodendrocytes, was not detectable until 2 weeks of remyelination. EM data correlated well with the LFB myelin staining and myelin protein expression, with 50% of the axons being rapidly remyelinated within 2 weeks. While particularly MBP but also PLP and CNPase are re-expressed very early before significant remyelination is observed by EM, the late marker MOG shows a lag behind the remyelination detected by EM. The presented data indicate that immunohistochemistry for various myelin proteins expressed early and late during myelin formation is a suitable and reliable method to follow remyelination in the cuprizone model. Furthermore, investigation of early remyelination confirms that the intrinsic repair programme is very fast and switched on within days.     

The role of glutamate receptor maturation in perinatal seizures and brain injury

The perinatal age window is characterized by vulnerability to age-specific patterns of injury. Hypoxia/ischemia occurs in a number of settings both in term and preterm neonates, yet the patterns of response appear dependent upon the age of the infant. In the preterm neonate, hypoxic/ischemic insults result in selective white matter injury, termed periventricular leukomalacia (PVL), with little or no cortical pathology. However, in term babies, hypoxic encephalopathy is the most common cause of seizures, and also can result in cortical infarction. Extracellular glutamate accumulates in the setting of hypoxia/ischemia, and excess activation of glutamate receptors has been implicated in hypoxic/ischemic cellular death. Glutamate receptors are developmentally regulated in both neuronal and glial cells within the brain. Using rodent models, we have shown that hypoxia/ischemia results in selective white matter injury in postnatal day (P) seven rat pups, while hypoxia causes seizures in P10-12 rats, but not at younger or older ages. We have further demonstrated that antagonists of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor subtype block white matter injury at P7 and seizures at P10. We have shown that AMPA receptors are relatively overexpressed in oligodendrocytes (OLs) within white matter at P7 and in neurons in cortex and hippocampus at P10. Hence maturational patterns of glutamate receptor expression correlate with age-specific regional susceptibility to injury to hypoxia/ischemia. While glutamate receptor blockade represents a rational strategy in the treatment of perinatal hypoxic/ischemic brain injury, it is unclear what role variations in their expression play in normal development and plasticity. Further investigation of patterns of glutamate receptor subunit expression in human brain and in experimental animal models is necessary to determine potential age specific strategies as well as adverse effects.     

In vivo MRI analysis of an inflammatory injury in the developing brain

Cerebral periventricular white matter injury stands as a leading cause of cognitive, behavioral and motor impairment in preterm infants. There is epidemiological and histopathological evidence demonstrating the role of prenatal or neonatal inflammation in brain injury in preterm infants. In order to define the effect of an inflammatory insult in the developing brain on magnetic resonance (MR) imaging, we obtained high resolution conventional and diffusion MR images of the brain of rat pups after an inflammatory injury. Rat pups were subjected on postnatal day 5 (P5) to a stereotaxic injection of lipopolysaccharide in the corpus callosum and then imaged at 11.7 T on days 0, 2 and 4 following the injury. They were subsequently sacrificed for immunohistochemistry. Diffusion tensor imaging (DTI) acquired at high spatial resolution showed an initial reduction of the apparent diffusion coefficient (ADC) in the white matter. This was followed by an increase in ADC value and in T2 relaxation time constant in the white matter, with an associated increase of radial diffusivity of the corpus callosum, and a 10-fold increase in ventricular size. On histology, these MR changes corresponded to widespread astrogliosis, and decreased proportion of the section areas containing cresyl violet positive stain. The increase in radial diffusivity, typically attributed to myelin loss, occurred in this case despite the absence of myelin at this developmental stage.