Temporal progression of kainic acid induced changes in vascular laminin expression in rat brain with neuronal and glial correlates

We recently demonstrated a dramatic up regulation of laminin expression within the blood vessels of brain regions vulnerable to excitotoxin mediated neuronal degeneration. Although this effect was clearly demonstrable at 2 days post kainic acid exposure, its expression at shorter and longer post-dosing intervals has not been reported. Therefore, a primary goal of the present study was to characterize the laminin labeling at intervals ranging from 4 hours to 2 months following i.p. injection of kainic acid. To better characterize the nature and possible underlying mechanism of action of the changes in laminin expression, both Fluoro-Jade C and GFAP immunohistochemistry were employed respectively at all survival intervals. At the shortest intervals examined (4hr, 8hr), Fluoro-Jade C positive cells could be detected in the hippocampus, thalamus, and piriform cortex. In these same regions, both vascular laminin and astrocytic GFAP expression were up regulated. At intermediate survival intervals (2, 5, 14, and 21 days), the respective labeling of degenerating neurons, astrocytes and capillaries were all maximal. Morphologically, Fluoro-Jade C labeled degenerating neurons were labeled in their entirety, GFAP positive astrocytes appeared hypertrophic and blood vessels took on a fragmented appearance. Hypertrophied GFAP positive astrocytes were conspicuous around periphery of the lesion but absent within the core of the lesion at these times. At longer survival intervals (1-2 months), the number of FJ-C labeled degenerating neurons was greatly reduced, while GFAP staining essentially returned to base line and laminin expression remained noticeably elevated, although the vessels appeared to be intact morphologically. These data allow for speculation on possible mechanisms underlying these events.     

Estradiol--induced redistribution of glial fibrillary acidic protein immunoreactivity in the rat brain

The immunohistochemical distribution of the glial fibrillary acidic protein (GFAP), a marker of glial filaments, was studied on coronal sections of the globus pallidus, the area CA4 of the hippocampus and the arcuate nucleus of the hypothalamus, 3 estrogen-sensitive areas of the rat brain. The number and the surface density of the GFAP-immunoreactive cells were evaluated in 6 adult ovariectomized rats injected with a single dose (20 mg/kg) of estradiol valerate (OVX + E2 rats) and in 6 ovariectomized littermates injected with vehicle (OVX rats). Two days after the injection, a similar distribution of the GFAP was observed in the arcuate nucleus of OVX + E2 rats when compared to OVX rats, whereas a significantly (P less than 0.001) increased surface density of GFAP immunoreactive material was observed in the globus pallidus and hippocampus of estradiol-treated rats. Since the number of GFAP-positive cells was unchanged by the estradiol injection, the enhanced surface density of GFAP immunoreactive material in the hippocampus and globus pallidus suggest a possible influence of estradiol on GFAP-immunoreactive glial processes.     

Brain lesion correlates of fatigue in individuals with traumatic brain injury

ABSTRACT

The purpose of this study was to investigate the neurological correlates of both subjective fatigue as well as objective fatigability in individuals with traumatic brain injury (TBI). The study has a cross-sectional design. Participants (N?=?53) with TBI (77% male, mean age at injury 38 years, mean time since injury 1.8 years) underwent a structural magnetic resonance imaging (MRI) scan and completed the Fatigue Severity Scale (FSS), while a subsample (N?=?36) was also tested with a vigilance task. While subjective fatigue (FSS) was not related to measures of brain lesions, multilevel analyses showed that a change in the participants’ decision time was significantly predicted by grey matter (GM) lesions in the right frontal lobe. The time-dependent development of the participants’ error rate was predicted by total brain white matter (WM) lesion volumes, as well as right temporal GM and WM lesion volumes. These findings could be explained by decreased functional connectivity of attentional networks, which results in accelerated exhaustion during cognitive task performance. The disparate nature of objectively measurable fatigability on the one hand and the subjective experience of fatigue on the other needs further investigation.     

Glutamine synthetase immunoreactivity in two types of mouse brain glial cells

The localization and distribution of glutamine synthetase (GS) in the adult mouse brain were studied by immunohistochemistry. GS immunoreactivity was found in two morphologically distinct types of glial cells apart from Bergmann glia, one asteroid and the other ovoid. The light and electron microscopic features of the GS-positive asteroid and ovoid cells were well consistent with those of astrocytes and oligodendrocytes, respectively. The GS-positive asteroid cells were present in the hippocampus, cerebral cortex, neostriatum, and cerebellar granular layer, where many synapse receptors for excitatory amino acids such as glutamate are densely distributed. Weakly GS-positive asteroid cells were also scattered in the white matter. The GS-positive ovoid cells were present throughout the gray matter regions of the brain except for the hippocampus, and they were the predominant type of GS-positive cells in the thalamus and brainstem gray matter where excitatory amino acid receptors are relatively sparse. No GS-positive ovoid cells were found in the white matter. These results suggest that, in the mouse brain, GS is localized in oligodendrocytes of the gray matter and in astrocytes. These two types of GS-positive glial cells may play different roles in the metabolism of glutamate.     

S-100 immunoreactivity in rat brain glial cultures is associated with both astrocytes and oligodendrocytes

S-100 protein, a Ca²⁺-binding protein of the EFhand type, is most abundant in the brain, and is involved in cell differentiation and the molecular mechanisms underlying cytoskeletal organization. We have investigated the immunocytochemical localization of S-100 protein in rat brain glial cultures prepared from the cerebral hemispheres of newborn rats. In mixed glial cultures, containing astrocytes type I and II and oligodendrocytes at various stages of differentiation, S-100 immunoreactivity was detected in all three cell types. Double immunofluorescence analysis revealed that in astrocytes, S-100 immunoreactivity was mainly colocalized with glial fibrillary acidic protein (GFAP), while in oligodendrocytes a close association with microtubular structures was observed. For immunoblot analysis, highly enriched oligodendrocytes and astrocytes were separately cultured for another week, and their extracts were analyzed by immunoblotting. The immunoblots of the cell extracts of both cell types showed a single S-100-immunoreactive polypeptide with an apparent molecular weight of approximately 12,000 daltons. Thus, the data presented here demonstrate that S-100 protein is not confined to astrocytes but occurs also in oligodendrocytes of rat brain. The close association with the oligodendroglial cytoskeleton suggests that this protein could also play a regulatory role in the organization of microtubules in oligodendrocytes and hence may be involved in the formation and maintenance of the myelin-containing membrane sheets. © 1995 Wiley-Liss, Inc.     

The repair of brain lesion by implantation of hyaluronic acid hydrogels modified with laminin

The hyaluronic acid (HA) hydrogels modified with laminin were used for implantation in rat brain in present study, in order to investigate its effects in reparation of injury in the CNS. Cross-linked HA hydrogels were synthesized and their characteristics were analyzed. Laminin, an extracellular matrix protein, which participates in neuronal development and survival, was immobilized on the backbone of the hydrogels. Hydrogels unmodified and modified with laminin were implanted into cortical defects mechanically created in rats and their ability to improve tissue reconstruction was then evaluated. After 6 and 12 weeks of implantation, sections of brains were processed with Nissl and Glees staining for revealing neural cell bodies and fibers, with DAB histochemistry for detecting the blood vessels, as well as with immunocytochemistry for recognizing GFAP. The sections were also taken to SEM and TEM for ultrastructral examination. The results showed that the HA hydrogels synthesized had mechanical properties and rheological behavior similar to the brain tissue. After being implanted into the lesion of the cortex, the porous hydrogels created a scaffold, which could support cell infiltration and angiogenesis, and simultaneously inhibit the formation of glial scar. In addition, HA hydrogels modified with laminin could promote neurite extension. It seems possible that the tissue engineering technique may pave the way to repair injury in the CNS as suggested by the results in present study.     

Hydrocortisone administration alters glial reaction to entorhinal lesion in the rat dentate gyrus

Young adult male rats received subcutaneous implants of Alzet osmotic minipumps which delivered 400 micrograms hydrocortisone per day. Untreated rats received no pumps or pumps containing the vehicle. Five days after receiving the implantation, both groups of rats were subjected to unilateral entorhinal lesion. Seven days after surgery, brains were analyzed quantitatively for glial changes in the denervated dentate outer molecular layer. Numerical densities of astrocytes and nonastrocytic glia were calculated by cell counting using 1.0-micron toluidine blue-stained sections. Glial acid phosphatase staining was quantitated using computer-assisted cytophotometric measurement of individual glial cells. Hydrocortisone-treated animals demonstrated 31% more astrocytes and 22.4% less nonastrocytes in the dentate outer molecular layer compared with untreated animals. Glia in the treated animals also showed a 33% decrease in average optical density of cytoplasmic acid phosphatase staining. These findings suggest that hydrocortisone treatment prior to and following an entorhinal lesion accelerates lesion-induced migration of astrocytes to the outer molecular layer, and reduces the increase in microglial number resulting from the lesion. The observed effect on microglia may result from a direct hormonal inhibition of local proliferation of microglia or from the well known systemic anti-inflammatory action of glucocorticoids on monocytes, the putative precursors of brain microglia. Our findings suggest that glucocorticoid hormones significantly alter the response of non-neuronal cells to neural tissue damage.     

轻型颅脑损伤后患者血清生物标志物GFAP含量的临床研究

目的通过分别检测并比较轻型颅脑损伤(m TBI)患者、格拉斯哥昏迷量表(GCS)评分在13~15分的中型颅脑损伤患者和健康对照组血清胶质细胞纤维酸性蛋白(GFAP)含量,为临床诊断m TBI患者提供依据。方法用ELISA法检测43例轻、35例中型颅脑损伤患者和20例健康对照组血清GFAP含量并采用ROC曲线分析,对比m TBI患者和中型颅脑损伤患者血清GFAP含量与头颅CT的关系。结果血清GFAP含量对鉴别m TBI患者和健康人群有一定准确性(AUC=0.726);对鉴别GCS评分在13~15分的中型颅脑损伤患者和健康人群有较高准确性(AUC=0.914);血清GFAP含量对鉴别头颅CT阳性和阴性患者有一定准确性(AUC=0.762)。结论血清GFAP可作为m TBI患者诊断的生物标志物,对鉴别颅脑损伤后头颅CT阳性和阴性患者有一定准确性,可以为减少m TBI患者头颅CT检查提供依据。     

MK-801 affects the potassium-induced increase of glial fibrillary acidic protein immunoreactivity in rat brain

Exposure of a limited brain surface to a high potassium (K+) concentration produces an injury limited to the underlying cortex, without apparently affecting other brain areas. Such a treatment produces an increased expression of glial fibrillary acidic protein (GFAP) in astrocytes, as assessed by immunohistochemical techniques, throughout the cortex ipsilateral to K+ exposure. This effect is evident 2 days after treatment and persists up to, at least, day 7. Thirty days after K+ exposure GFAP immunostaining is similar in both hemispheres. Administration of the non-competitive NMDA antagonist MK-801 (4 mg/kg i.p.) prior to the injury prevented the rise in GFAP immunoreactivity (IR) at 2 but not 7 days after the treatment. Administration of MK-801 after the injury appeared to have no effect on GFAP expression. This work confirms that brain injury, associated with spreading depression, can induce a glial response far from the lesion site. Furthermore, the fact that this phenomenon can be modified by an NMDA receptor antagonist suggests that glutamate may play a role, in vivo, in the regulation of astrocytic response to injury and introduces the possibility that brain injury-induced gliosis may be pharmacologically manipulated.     

Isolated cystic lesion of the callosal genu after traumatic brain injury

We report the case of a 17-month-old infant who developed an isolated cystic lesion of the callosal genu as a unique lesion of traumatic axonal injury (TAI). Although one of the most common sites of TAI is the corpus callosum, there have been no reports describing the lesion seen in our patient. Brain computed tomography findings were normal on the day of the traffic accident. After 3 months, brain magnetic resonance imaging showed an isolated cystic lesion of the callosal genu that had the appearance of a cystic cavity. This lesion decreased in size 16 months later. The neuroimaging findings of this patient suggest that an isolated cystic lesion of the callosal genu could appear as a unique form of TAI in infants after traumatic brain injury (TBI), but it is nevertheless important to attend to such lesions in children with TBI.     

Cellular reaction to an acute demyelinating/remyelinating lesion of the rat brain stem: Localisation of GD3 ganglioside immunoreactivity

We describe a simple and reproducible acute demyelinating lesion of the rat brain stem induced by injection of ethidium bromide into the cisterna magna of young adult rats. Using immunofluorescence with a panel of antibodies to cell-specific antigens we have studied the changes in cell populations that occur at various stages during lesion progression and repair. In particular we localized the expression of ganglioside G_D3 immunoreactivity, a marker for oligodendroglial progenitors in developing brain. Both astroglia (GFAP⁺) and oligodendroglia (CNP⁺) were destroyed during the early response to the ethidium bromide although axons were spared. Splitting of myelin lamellae occurred as early as 4 days post-injection (DPI), with extensive demyelination of the inferior cerebellar peduncle following by 6 DPI. Large numbers of ED1⁺ and OX-42⁺ macrophages were present in the lesion site at this stage. Astrogliosis occurred around the perimeter of the lesions. Two populations of G_D3⁺ cells appeared within and around the lesion sites during the demyelination. One population was identified by the phenotype G_D3⁺ ED1⁺ and thus probably belonged to the macrophage/microglial lineage. In these cells both antigens appeared cytoplasmic. The second population of G_D3⁺ cells exhibited cell membrane G_D3 immunoreactivity but did not express the ED1 antigen. These cells are suggested to be oligodendroglial progenitors generated in response to the demyelination. No such cells were seen in control tissue. G_D3⁺ cells were present within the lesion sites from 6 DPI until 10–12. Following the clearance of myelin debris from the lesions, remyelination was a relatively rapid event with thin MBP⁺ myelin sheaths first seen at 11–12 DPI. Remyelination, which was extensive by 25 DPI, was predominantly oligodendroglial in origin (MBP⁺Po^? myelin) with only small pockets of peripheral myelin (MBP⁺Po⁺ myelin) observed. The present study, in addition to identifying putative glial progenitors within a demyelinated lesion, also demonstrates the difficulties in unambiguously identifying such cells in the normal and damaged adult CNS. © 1993 Wiley-Liss, Inc.     

Neural correlates of apathy revealed by lesion mapping in participants with traumatic brain injuries

Apathy, common in neurological disorders, is defined as disinterest and loss of motivation, with a reduction in self‐initiated activity. Research in diseased populations has shown that apathy is associated with variations in the volume of brain regions such as the anterior cingulate and the frontal lobes. The goal of this study was to determine the neural signatures of apathy in people with penetrating traumatic brain injuries (pTBIs), as to our knowledge, these have not been studied in this sample. We studied 176 male Vietnam War veterans with pTBIs using voxel‐based lesion‐symptom mapping (VLSM) and apathy scores from the UCLA Neuropsychiatric Inventory (NPI), a structured inventory of symptoms completed by a caregiver. Our results revealed that increased apathy symptoms were associated with brain damage in limbic and cortical areas of the left hemisphere including the anterior cingulate, inferior, middle, and superior frontal regions, insula, and supplementary motor area. Our results are consistent with the literature, and extend them to people with focal pTBI. Apathy is a significant symptom since it can reduce participation of the patient in family and other social interactions, and diminish affective decision‐making. Hum Brain Mapp 35:943–953, 2014. © 2013 Wiley Periodicals, Inc.     

Implanted neural progenitor cells regulate glial reaction to brain injury and establish gap junctions with host glial cells

Transplantation of neural stem/progenitor cells (NPCs) in the lesioned brain is able to restore morphological and physiological alterations induced by different injuries. The local microenvironment created at the site of grafting and the communication between grafted and host cells are crucial in the beneficial effects attributed to the NPC implants. We have previously described that NPC transplantation in an animal model of central axotomy restores firing properties and synaptic coverage of lesioned neurons and modulates their trophic factor content. In this study, we aim to explore anatomical relationships between implanted NPCs and host glia that might account for the implant‐induced neuroprotective effects. Postnatal rat subventricular zone NPCs were isolated and grafted in adult rats after transection of the medial longitudinal fascicle. Brains were removed and analyzed eight weeks later. Immunohistochemistry for different glial markers revealed that NPC‐grafted animals displayed significantly greater microglial activation than animals that received only vehicle injections. Implanted NPCs were located in close apposition to activated microglia and reactive astrocytes. The gap junction protein connexin43 was present in NPCs and glial cells at the lesion site and was often found interposed within adjacent implanted and glial cells. Gap junctions were identified between implanted NPCs and host astrocytes and less frequently between NPCs and microglia. Our results show that implanted NPCs modulate the glial reaction to lesion and establish the possibility of communication through gap junctions between grafted and host glial cells which might be involved in the restorative effects of NPC implants. GLIA 2014;62:623–638     

Connexin immunoreactivity in glial cells of the rat retina

The rat retina contains two types of macroglial cells, Müller cells, radial glial cells that are the principal macroglial cells of vertebrate retinas, and astrocytes associated with the surface vasculature. In addition to the often-described gap-junctional coupling between astrocytes, coupling also occurs between astrocytes and Müller cells. Immunohistochemistry and confocal microscopy were used to identify connexins in the retinas of pigmented rats. Several antibodies directed against connexin43 stained astrocytes, identified using antibodies directed against glial fibrillary acidic protein (GFAP). In addition, two connexin43 antibodies stained Müller cells, identified with antibodies directed against S100 or glutamine synthetase. Connexin30-immunoreactive puncta were confined to the vitreal surface of the retina and colocalized with GFAP-immunoreactive astrocyte processes. Connexin45 immunoreactivity was associated with both astrocytes and Müller cells. We conclude that retinal glial cells express multiple connexins, and the patterns of immunostaining that we observe in this study are consistent with the expression of connexins30, -43, and possibly -45 by astrocytes and the expression of connexins43 and -45 by Müller cells. As gap-junction channels may be formed by both homotypic and heterotypic hemichannels, and the hemichannels may themselves be homomeric or heteromeric, there exists a multitude of possible gap-junction channels that could underlie the homotypic coupling between retinal astrocytes and the heterotypic coupling between astrocytes and Müller cells.     

Neurons and glial cells of the embryonic human brain and spinal cord express multiple and distinct isoforms of laminin

We have identified by immunocytochemistry, Western blotting, and RT-PCR the isoforms of laminin expressed by glial cells and neurons cultured from human embryonic brain and spinal cord. We show that most of the known laminins are present in human neurons and glial cells. Importantly, Western analysis demonstrates that the isoforms of laminin present in embryonic human brain differ from those expressed in human spinal cord. Neurons of the brain and spinal cord also express their distinct and characteristic isoforms of laminin compared to the glial cells of the same CNS regions. These results suggest that, in addition to the known laminins, several novel isoforms may exist in the human embryonic CNS. The observed differences between the isoforms of laminin in brain and spinal cord neurons and glial cells may result from primary structural changes or from posttranslational modifications, e.g., variations in glycosylation. Thus, identification of these novel laminins and determination of their function(s) should further our understanding of the mechanisms of aging, disease, and trauma in the human CNS.     

Glial fibrillary acidic protein and vimentin in the experimental glial reaction of the rat brain

Glial reaction has been studied in the rat by the immunohistochemical demonstration of glial fibrillary acidic protein (GFAP) and vimentin (VIM) in two experimental conditions. The first was represented by a necrotic cerebral lesion obtained by laser irradiation and the second by the development of experimental tumors induced by transplacental ethylnitrosourea. Reactive astrocytes develop not only in the proximity of the lesion but also distant from it. The intensity of the glial response seems to depend upon the normal distribution of astrocytes and the perilesional edema. GFAP decorates all the reactive astrocytes, whereas VIM is positive only in those at the edges of the lesion. The significance of the different responses in the two models and between the two intermediate filaments is discussed.     

Trauma and tumor: malignant glioma in the area of a traumatic lesion of the brain with metal scales

Report of a 75 year-old male who sustained severe head injury by shell splinters during the second world war. 38 years later he died due to a malignant glioma which was localized exactly in the left and right frontal areas damaged by the shell. Multiple metal scales were still localized within these areas and the glioma. The reasons for the presumption of a causal relation between the trauma and the tumor will be discussed.     

小胶质细胞在颅脑外伤后胶质瘢痕形成中的实验研究

目的研究小胶质细胞在颅脑外伤后胶质瘢痕形成中的作用。方法选取8~10周龄SD大鼠48只,雌雄各半。按体质量相近原则,随机分为观察组和对照组,观察组建立大鼠颅脑外伤模型,对照组不作任何处理,正常饲养。分别在造模后1d和7d收集各组大鼠的血液,检测血清中TNF?α、IL-1β、IL-6、IL-4以及IL-10表达量,并进行统计分析,并于2组造模后1d、7d分别取每组12只大鼠大脑组织进行病理检查,观察颅脑外伤后胶质瘢痕的形态学。结果观察组造模1d血清中TNF?α、IL-1β、IL-6、IL-4以及IL-10的表达量分别为79.8±8.7、83.6±8.3、83.3±10.2、83.3±7.8以及82.7±6.8。对照组饲养1d血清中TNF?α、IL-1β、IL-6、IL-4以及IL-10的表达量分别为69.3±7.2、68.6±7.4、71.4±7.3、69.5±7.6以及65.4±8.6。观察组造模7d后血清中TNF?α、IL-1β、IL-6、IL-4以及IL-10的表达量分别为96.8±12.4、92.6±11.2、96.8±12.2、95.8±12.5以及90.4±12.2。对照组饲养7d后,血清中TNF?α、IL-1β、IL-6、IL-4以及IL-10的表达量分别为82.1±9.5、70.3±6.8、82.2±12.6、82.2±8.6以及67.1±6.4。观察组TNF?α、IL-1β、IL-6、IL-4以及IL-10的表达量明显高于对照组,差异有统计学意义(P0.05)。结论小胶质细胞在颅脑外伤后胶质瘢痕形成中具有双重作用,小胶质细胞激发后可产生TNF?α、IL-1β、IL-6、等炎症因子,又同时可以释放L-4以及IL-10等抑制炎症反应的因子。干预小胶质细胞的活化,抑制炎症反应因子的释放,可有效预防颅脑外伤后胶质瘢痕形成,同时,也能指导临床治疗颅脑外伤后胶质瘢痕形成。