NF-kappaB and IkappaBalpha expression following traumatic brain injury to the immature rat brain

NF-kappaB is one of the most important modulators of stress and inflammatory gene expression in the nervous system. In the adult brain, NF-kappaB upregulation has been demonstrated in neurons and glial cells in response to experimental injury and neuropathological disorders, where it has been related to both neurodegenerative and neuroprotective activities. Accordingly, the aim of this study was to evaluate the cellular and temporal patterns of NF-kappaB activation and the expression of its endogenous inhibitor IkappaBalpha following traumatic brain injury (TBI) during the early postnatal weeks, when the brain presents elevated levels of plasticity and neuroprotection. Our results showed that cortical trauma to the 9-day-old rat brain induced a very fast upregulation of NF-kappaB, which was maximal within the first 24 hours after injury. NF-kappaB was mainly observed in neuronal cells of the degenerating cortex as well as in astrocytes located in the corpus callosum adjacent to the injury, where a pulse-like pattern of microglial NF-kappaB activation was also found. In addition, astrocytes of the corpus callosum, and microglial cells to a lower extent, also showed de novo expression of IkappaBalpha within the time of NF-kappaB activation. This study suggests an important role of NF-kappaB activation in the early mechanisms of neuronal death or survival, as well as in the development of the glial and inflammatory responses following traumatic injury to the immature rat brain.     

Neuronal, astroglial and microglial cytokine expression after an excitotoxic lesion in the immature rat brain

Cytokines are important intercellular messengers involved in neuron-glia interactions and in the microglial-astroglial crosstalk, modulating the glial response to brain injury and the lesion outcome. In this study, excitotoxic lesions were induced by the injection of N-methyl-D-aspartate in postnatal day 9 rats, and the cytokines interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), tumour necrosis factor alpha (TNFalpha) and transforming growth factor beta 1 (TGF-beta1) analysed by ELISA and/or immunohistochemistry. Moreover, cytokine-expressing glial cells were identified by means of double labelling with glial fibrillary acidic protein or tomato lectin binding. Our results show that both neurons and glia were capable of cytokine expression following different patterns in the excitotoxically damaged area vs. the nondegenerating surrounding grey matter (SGM). Excitotoxically damaged neurons showed upregulation of IL-6 and downregulation of TNFalpha and TGF-beta1 before they degenerated. Moreover, in the SGM, an increased expression of neuronal IL-6, TNFalpha and TGF-beta1 was observed. A subpopulation of microglial cells, located in the SGM and showing IL-1beta and TNFalpha expression, were the earliest glial cells producing cytokines, at 2-10 h postinjection. Later on, cytokine-positive glial cells were found within the excitotoxically damaged area and the adjacent white matter: some reactive astrocytes expressed TNFalpha and IL-6, and microglia/macrophages showed mild IL-1beta and TGF-beta1. Finally, the expression of all cytokines was observed in the glial scar. As discussed, this pattern of cytokine production suggests their implication in the evolution of excitotoxic neuronal damage and the associated glial response.     

Glial expression of the 90-kDa heat shock protein (HSP90) and the 94-kDa glucose-regulated protein (GRP94) following an excitotoxic lesion in the mouse hippocampus

Heat shock proteins (HSPs) are immediately expressed in neuronal and glial cells under various stressful conditions and play a protective role through molecular chaperones. Although several studies have been focused on the expression of HSPs, little is known about HSP90s expression in glial cells under neuropathological conditions. In this study, we evaluated the expression pattern of the glial cell-related HSP90 and GRP94 proteins, following the induction of an excitotoxic lesion in the mouse brain. Adult mice received an intracerebroventricular injection of kainic acid; the brain tissue was then analyzed immunohistochemically for HSPs and double labeling using glial markers. HSPs expression was quantified by Western blot analysis. Excitotoxic damage was found to cause pyramidal cell degeneration in the CA3 region of the hippocampus. In the injured hippocampus, reactive microglia/macrophages expressed HSP90 from 12 h until 7 days postlesion (PL), showing maximal levels at day 1. In parallel, hippocampal reactive astrocytes showed the expression of GRP94 from 12 h until 7 days PL. In general, HSPs expression was transient, peaked at 1-3 days PL and reached basal levels by day 7. For the first time, our data demonstrate the injury-induced expression of HSP90 and GRP94 in glial cells, which may contribute to the mechanism of glial cell protection and adaptation in response to damage, thereby playing an important role in the evolution of the glial response and the excitotoxic lesion outcome. HSP90 may provide antioxidant protective mechanisms against microglia/macrophages, whereas GRP94 may stabilize the astroglial cytoskeleton and participate in astroglial antioxidant mechanisms.     

Expression of 27 kDa heat shock protein (Hsp27) in immature rat brain after a cortical aspiration lesion.

The 27 kDa heat shock protein (Hsp27) is a well-known member of the astroglial response to injury, playing a protective role against oxidative stress, apoptosis, and cytoskeletal destruction. Although several studies have been focused on the damaged adult brain, little is known about Hsp27 expression in the immature brain. In this work, we have examined the spatiotemporal pattern of Hsp27 expression in the normal postnatal rat brain following a cortical aspiration lesion at postnatal day 9. In the immature brain, Hsp27 is mainly observed in the internal capsule, although some scattered cells are also found in the ependyma, the corpus callosum, the septum, and hypothalamic glia limitans. In the internal capsule, Hsp27 expression is developmentally regulated, being significantly decreased from postnatal day 14. After a cortical aspiration lesion, de novo expression of Hsp27 is observed in cortical injured areas as well as in the secondary affected thalamus. In the cortex, expression of Hsp27 is first seen at day 1 postlesion (PL) surrounding the neurodegenerative area, becoming restricted to the glial scar at longer survival times. Although a pulse-like expression of Hsp27 is observed in some microglial cells at day 1 PL, most Hsp27-labeled cells are reactive astrocytes, which show GFAP overexpression and coexpress vimentin from day 3 PL. In the thalamus, astroglial Hsp27 expression is delayed, being first observed at day 5 PL. Thalamic Hsp27-labeled astrocytes do not show vimentin expression. Our observations demonstrate astroglial expression of Hsp27 in areas of tissue damage following postnatal traumatic injury, suggesting an involvement of this cytoskeleton-stabilizing protein in the remodeling processes following postnatal brain damage.     

Differential expression of heat shock protein HSP27 in human neurons and glial cells in culture

HSP27 expression was investigated in cultured neurons and glial cells isolated from fetal human brains using immunoblotting and immunocytochemistry. Under unstressed conditions, HSP27 was identified at a high level in astrocytes (>99%), at a low level in neurons (7%), and at a minimally detectable level in microglia (<1%), whereas it was undetectable in oligodendrocytes. Under these conditions, HSP27 was located in the cytoplasm, fractionated into the Triton X-100-soluble phase, and composed chiefly of the basic isoform (HSP27a). After exposure to heat stress (43°C90 min), the level of HSP27 exproion ryas not altered in astrocytes but was elevated significantly in neurons (11–21%) and microglia (4–7%) during 8–48 hr postrecovery periods, while it remained undetectable in oligodendrocytes. In addition, various human neural cell lines exhibited differential patterns of HSP27 expression under unstressed and heat-stressed conditions. Following heat shock treatment (45°C/30 min), granular aggregates of HSP27 were identified in the cytoplasm of astrocytes. Under heat-stressed conditions, HSP27 was distributed within the Triton X-100-insoluble fraction associated with an increase in two more acidic isoforms (HSP27b and HSP27c). HSP27 and αβ-crystallin were coexpressed in astrocytes under unstressed and heat-stressed conditions. When astrocytes were exposed to known HSP27 inducers, hydrogen peroxide and cysteamine reduced the synthesis of HSP27, while estradiol showed no effects. The differential patterns of constitutive and heat-induced expression of HSP27 in cultured human neurons and glial cells suggest that the cellular mechanisms by which HSP27 expression is regulated are different among various cell types in the human central nervous system. © 1995 Wiley-Liss, Inc.     

Expression of inducible nitric oxide synthase and cyclooxygenase-2 after excitotoxic damage to the immature rat brain

It is well established that after adult brain damage the enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) play an important role in the inflammatory processes and oxidative stress, which are considered to be the leading factors contributing to delayed cell death. The contribution of these enzymes to postnatal brain damage, however, is poorly understood. In our study, excitotoxic lesions were induced by the injection of N-methyl-D-aspartate in the cortex of postnatal day 9 rats. After different survival times ranging from 4 hr to 7 days post-lesion, brain sections were processed for the immunocytochemical demonstration of COX-2 and iNOS and double labeling with neuronal, glial and neutrophil markers. First and maximal de novo induction of iNOS and COX-2 expression was found at 10 hr post-lesion. Expression of both enzymes started to diminish at 24 hr, reaching basal levels at day 3. iNOS-expressing cells were mainly identified as infiltrated neutrophils as well as highly ramified protoplasmic astrocytes closely associated with blood vessels. Moreover, scattered iNOS-positive neurons were found at the lesion borders. In contrast, COX-2 was mainly observed in reactive microglial cells and neuronal cells. COX-2-positive neurons were found within the degenerating area at 10 hr and at the borders of the lesion later on. This study shows that maximal iNOS and COX-2 expression precedes the period of massive neuronal death observed at 24 hr post-lesion, and may therefore contribute to the evolution of the inflammatory response and the neurodegenerative process after an excitotoxic lesion to the postnatal brain.     

Distinct spatial and temporal activation of caspase pathways in neurons and glial cells after excitotoxic damage to the immature rat brain

Although cleaved caspase-3 is known to be involved in apoptotic cell death mechanisms in neurons, it can also be involved in a nonapoptotic role in astrocytes after postnatal excitotoxic injury. Here we evaluate participation of upstream pathways activating caspase-3 in neurons and glial cells, by studying the intrinsic pathway via caspase-9, the extrinsic pathway via caspase-8, and activation of the p53-dependent pathway. N-methyl-D-aspartate (NMDA) was injected intracortically in 9-day-old postnatal rats, which were sacrificed at several survival times between 4 hr postlesion (pl) and 7 days pl. We analyzed temporal and spatial expression of caspase-8, caspase-9, and p53 and correlation with neuronal and glial markers and caspase-3 activation. Caspase-9 was significantly activated at 10 hpl, strongly correlating with caspase-3. It was present mainly in damaged cortical and hippocampal neurons but was also seen in astrocytes and oligodendrocytes in layer VI and corpus callosum (cc). Caspase-8 showed a diminished correlation with caspase-3. It was present in cortical neurons at 10-72 hpl, showing layer specificity, and also in astroglial and microglial nuclei, mainly in layer VI and cc. p53 Expression increased at 10-72 hpl but did not correlate with caspase-3. p53 Was seen in neurons of the degenerating cortex and in some astrocytes and microglial cells of layer VI and cc. In conclusion, after neonatal excitotoxicity, mainly the mitochondrial intrinsic pathway mediates neuronal caspase-3 and cell death. In astrocytes, caspase-3 is not widely correlated with caspase-8, caspase-9, or p53, except in layer VI-cc astrocytes, where activation of upstream cascades occurs.     

Neuroprotective preconditioning of rat brain cultures with ethanol: potential transduction by PKC isoforms and focal adhesion kinase upstream of increases in effector heat shock proteins

Preconditioning rat hippocampal-entorhinocortical (HEC) slice or cerebellar cell cultures with moderate concentrations of ethanol (20-30 mm) neuroprotects against pro-inflammatory proteins such as HIV-1 glycoprotein 120 (gp120) or amyloid-β. The neuroprotective mechanism of ethanol is unclear, but it conceivably involves sensors→transducers→effectors, analogous to other preconditioning modalities. We initially found that the preconditioning augmented two likely heat shock protein (HSP) 'effectors', HSP70 and HSP27, and that precluding HSP upregulation abolished neuroprotection. Here we examined whether pro-survival kinases are transducers potentially leading to HSP effectors. In cerebellar cultures, protein kinase C (PKC) activity increased modestly after 2 days of 30 mm ethanol and was significantly induced after 6 days, when neuroprotection against gp120 becomes manifest. After 4 and particularly after 6 days of preconditioning, immunoblots showed highly elevated PKCε levels and moderately increased PKCα and PKCδ, accompanied by increased membrane translocation (activation) of these isoforms. Also, at the latter preconditioning duration, focal adhesion kinase (FAK), an important actin-associated kinase, and its Y397-phosphorylated form (p-FAK) were elevated, along with parallel increases in HSP27, S85p-HSP27 and HSP70. Furthermore, while confirming increased HSP27 and HSP70 in HEC slices ethanol-preconditioned for 6 days, we detected elevations in PKC isoforms, FAK, p-FAK and p-HSP27 in these organotypic cultures. Importantly, PKC inhibition with GF109203X suppressed FAK, HSP70 and HSP27 amplification/activation in ethanol-preconditioned cerebellar cultures, indicating that PKC is an upstream transducer of FAK and the HSP effectors. Neuroprotection associated with increases in HSP27/HSP70 from ethanol preconditioning entails upregulation/activation of PKC isoforms and FAK, the latter kinase implicating actin cytoskeletal prosurvival pathways in brain preconditioning.     

实验性大鼠脑出血血肿周围组织胶质细胞纤维酸性蛋白的动态表达

目的:动态观察胶质细胞纤维酸性蛋白(GFAP)在脑出血动物模型脑组织中的表达及其意义。方法应用脑立体定向技术,建立大鼠脑出血模型,假手术作为对照组。分别测定脑组织含水量,观察血肿周围GFAP的表达规律。结果:大鼠脑出血后脑水肿于48h达到高峰,与对照组比较有统计学意义(P<0.05)。脑出血后脑内GFAP阳性细胞数6h表达最强,在72h时表达最少,7d的阳性细胞数量比72h增加(P<0.05),但数量少于6h(P<0.05)。相应时间点出血组阳性细胞数与对照组比较,有明显统计学意义,(P<0.05)。结论:脑出血后脑内GFAP的表达与脑水肿相关,提示星形胶质细胞在脑出血的病理变化过程中有重要作用。星形胶质细胞在损伤的修复作用具有二重性,即可以抑制也可以促进损伤修复。     

Regional expression of c-fos and heat shock protein-70 mRNA following hypoxia-ischemia in immature rat brain.

Cerebral ischemia induces the expression of a number of proteins that may have an important influence on cellular injury. The purpose of this study was to compare the regional effects of hypoxia-ischemia on the expression of the proto-oncogene, c-fos, and the heat shock protein-70 (HSP-70) gene in developing brain. Unilateral hypoxia-ischemia was produced in the brain of immature rats (7, 15, and 23 days after birth) using a combination of carotid artery ligation and systemic hypoxia (8% O2). After recovery for 2 and 24 h, the regional expression of c-fos and HSP-70 mRNA was determined using in situ hybridization. Littermates were permitted to recover for 1 week for assessment of histologic injury. Hypoxia-ischemia increased the expression of both c-fos and HSP-70 mRNA, but the topography of expression varied with the age of the animal as well as the mRNA species. In the 7-day-old group, expression of c-fos at 2 h increased in multiple regions of the ipsilateral hemisphere in nearly one-half of the animals, while HSP-70 mRNA was not expressed until 24 h and, then, predominantly in the hippocampus. In 15- and 23-day-old rats, expression of c-fos was increased at 2 h in the entorhinal cortex and in the dendritic field of the upper blade of the hippocampal dentate gyrus, while HSP-70 mRNA was prominently expressed in neocortex and the cell layers of the hippocampus. Interestingly, the strong expression of HSP-70 mRNA in dentate granule cells did not occur in the innermost layer of cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression and induction of small heat shock proteins in rat brain and cultured hippocampal neurons

The so-called stress response involving up-regulation of heat shock proteins (Hsps) is a powerful mechanism of cells to deal with harmful conditions to which they are exposed throughout life, such as hyperthermia, hypoxia, or oxidative stress. Some members of the group of small Hsps (sHsps) seem to play a neuroprotective role in the brain. Here we analyzed the expression of all 11 sHsps in the rat brain by using RNA in situ hybridization and quantitative real-time RT-PCR. Additionally, we investigated sHsps in cultured neurons exposed to heat shock. We found seven sHsps to be expressed in the rat brain, with HspB5 (αB-crystallin), HspB6 (Hsp20), and HspB11 (Hsp16.2) showing the highest expression levels (4-24% of reference genes) followed by HspB1 (Hsp25) and HspB8 (Hsp22; 0.1-2% of reference genes), all being widely expressed in the brain areas investigated. HspB2 (MKBP) and HspB3, however, showed selective expression in only some regions (B2: cortex and hippocampus, B3: cortex and cerebellum). Whereas HspB5 was expressed mainly in the white matter, HspB6 showed the greatest expression in the cerebellar cortex, and HspB11 was widely distributed over the whole brain. In cultured hippocampal neurons, heat shock led to an increase of HspB1 and HspB8 mRNA and additionally HspB5 protein. Our data indicate that the sHsps induced by heat shock, HspB1, B5, and B8, might be especially involved in neuroprotection under stress conditions. The other sHsps showing constant neuronal expression may play a constitutive role or may be up-regulated and important in types of stresses other than heat shock.     

Glial coverage of the small cell somata in the rat nucleus of tractus solitarius during postnatal development

Astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. Previous studies suggested that axosomatic synapses decreased in number on the small cells of the rat caudal nucleus of tractus solitarius (cNTS) toward the end of the first postnatal week. Astrocytes might be involved in this phenomenon. We examined the morphological development of astrocytic processes around the small cell soma in the rat cNTS using light and electron microscopy. Glial fibrillary acidic protein (GFAP), glutamate-aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1)-positive structures within the cNTS became more intensely stained as development proceeded. GLAST-positive structures encompassed calbindin-positive small cell somata after postnatal day 10. Electron microscopic observations indicated that astrocytic processes encompass the small cell soma, while the number of axosomatic synapses decreases as development proceeds. The timing for glial coverage of the small cell soma appears to be consistent with the decrease in axosomatic synapses on the small cells. These observations imply that astrocytes may participate actively in regulating the decrease of axosomatic synapses on small cells in the cNTS during postnatal development.     

戊四氮致痫大鼠脑内热休克蛋白70的表达

目的：探讨热休克蛋白70（HSP70）在癫痫大鼠脑内的表达情况及其意义。方法：采用戊四氮致痫模型。应用免疫组织化学及常规清理检查的方法进行研究。结果：在正常大鼠脑内未见HSP70免疫反应（IR）阳性细胞,成四氮致痫12小时后脑内开始出现HSP70IR阳性细胞,24小对IR达高峰．3天后开始下降．7天后消失。HSP70主要在边缘系统（尤其是海马的CA1、CA3、CA4区）、大脑皮质（尤其是颞叶皮质,梨状皮质）等区域表达。同时常规病理检查发现,上述区域散在出现受损的异常神经元。结论：癫痫发作可诱导HSP70在大鼠脑内广泛表达。HSP70表达可作为神经元受损的一个早期指标。     

Glial expression of estrogen and androgen receptors after rat brain injury

Estrogens and androgens can protect neurons from death caused by injury to the central nervous system. Astrocytes and microglia are major players in events triggered by neural lesions. To determine whether glia are direct targets of estrogens or androgens after neural insults, steroid receptor expression in glial cells was assessed in two different lesion models. An excitotoxic injury to the hippocampus or a stab wound to the parietal cortex and hippocampus was performed in male rats, and the resultant expression of steroid receptors in glial cells was assessed using double-label immunohistochemistry. Both lesions induced the expression of estrogen receptors (ERs) and androgen receptors (ARs) in glial cells. ERalpha was expressed in astrocytes immunoreactive (ERalpha-ir) for glial fibrillary acidic protein or vimentin. AR immunoreactivity colocalized with microglial markers, such as Griffonia simplicifolia lectin-1 or OX-6. The time course of ER and AR expression in glia was studied in the stab wound model. ERalpha-ir astrocytes and AR-ir microglia were observed 3 days after lesion. The number of ERalpha-ir and AR-ir glial cells reached a maximum 7 days after lesion and returned to low levels by 28 days postinjury. The studies of ERbeta expression in glia were inconclusive; different results were obtained with different antibodies. In sum, these results suggest that reactive astrocytes and reactive microglia are a direct target for estrogens and androgens, respectively.     

Induction of 27-kDa heat shock protein following cerebral ischemia in a rat model of ischemic tolerance

Preconditioning the brain with sublethal cerebral ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). The purpose of this study is to investigate the role of low-molecular weight stress proteins, 27-kDa heat shock protein (HSP27) and αB crystallin, in ischemic tolerance. We measured the content of these proteins with enzyme immunoassay in the rat hippocampus and cerebral cortex following 6 min of ischemia with and without preconditioning with 3 min of ischemia and 3 days of reperfusion. We also visualized the localization of HSP27 immunohistochemically in comparison with that of HSP70. A 3-min period of ischemia caused a 2.4-fold increase in HSP27 content in the hippocampus after 3 days. Immunohistochemical localization of HSP27 was found in glial cells in all subregions of the hippocampus, whereas HSP70 immunostaining was seen only in CA1 pyramidal neurons. HSP27 content in the hippocampus decreased 2 h after 6 min of ischemia. HSP27 content progressively increased in the unpreconditioned hippocampus after 1 and 3 days, but returned to preischemic levels in the preconditioned hippocampus. HSP27 and HSP70 immunostaining was seen in CA1 pyramidal neurons after 1 day both with and without preconditioning. After 3 and 7 days, an intense HSP27 staining was observed in reactive glial cells in the CA1 without preconditioning, whereas the staining decreased in the preconditioned hippocampus. HSP70 staining was seen only in neurons at these time points. We observed no significant changes in HSP27 content in the cerebral cortex although neurons in the third and fifth layers were immunostained after 1 and 3 days. We observed no alterations in αB crystallin content after ischemia both in the hippocampus and the cortex. The present study demonstrated that cerebral ischemia induces HSP27 expression but not αB crystallin. Both HSP27 and HSP70 induction had a good temporal correlation with the induction of ischemic tolerance. However, different sites of action were suggested because the localization and cell types of HSP27 induction were quite different from those of HSP70 induction. The result suggests that it is unlikely that HSP27 is directly involved in the protection afforded by ischemic preconditioning.     

Developmental and cell type-specific expression of thyroid hormone transporters in the mouse brain and in primary brain cells

Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins. One of these, monocarboxylate transporter 8 (MCT8) is mutated in Allan-Herndon-Dudley syndrome, a severe mental retardation associated with abnormal thyroid hormone constellations. Since mice deficient in Mct8 exhibit a milder neurological phenotype than patients, we hypothesized that alternative thyroid hormone transporters may compensate in murine brain cells for the lack of Mct8. Using qPCR, Western Blot, and immunocytochemistry, we investigated the expression of three different thyroid hormone transporters, i.e., Mct8 and L-type amino acid transporters Lat1 and Lat2, in mouse brain. All three thyroid hormone transporters are expressed from corticogenesis and peak around birth. Primary cultures of neurons and astrocytes express Mct8, Lat1, and Lat2. Microglia specifically expresses Mct10 and Slco4a1 in addition to high levels of Lat2 mRNA and protein. As in vivo, a brain microvascular endothelial cell line expressed Mct8 and Lat1. 158N, an oligodendroglial cell line expressed Mct8 protein, consistent with delayed myelination in MCT8-deficient patients. Functional T(3)- and T(4)-transport assays into primary astrocytes showed K(M) values of 4.2 and 3.7 μM for T(3) and T(4). Pharmacological inhibition of L-type amino acid transporters by BCH and genetic inactivation of Lat2 reduced astrocytic T(3) uptake to the same extent. BSP, a broad spectrum inhibitor, including Mct8, reduced T(3) uptake further suggesting the cooperative activity of several T(3) transporters in astrocytes.     

Differential inductions of small heat shock protein 27 and 1-Cys peroxiredoxin in reactive astrocytes in sulfatide-deficient mouse spinal cord

In myelinated fibers, various interactions among axons, oligodendrocytes, and astrocytes are present, particularly around the node of Ranvier. In the present study, we examined the protein composition of cerebroside sulfotransferase knockout (CST KO) mouse spinal cord by two-dimensional gel electrophoresis to examine the molecular changes resulting from the disruption of paranodal junctions in addition to the sulfatide-deficient condition. Interestingly, heat shock protein 27 (Hsp27) and 1-cys peroxiredoxin (1-Cys Prx) were both elevated in CST KO mice. Hsp27 was increased specifically in reactive astrocytes in the white matter, and the elevation was well correlated to the progression of neurologic symptoms. In contrast, 1-Cys Prx was elevated both in white and gray matter astrocytes in CST KO mice. These results suggest that astrocytes do not always respond stereotypically, as they display differences in their activation in these two regions. To determine whether these changes are specific to the sulfatide-deficient condition, spinal cords from CST KO mice and the hypomyelinating mutant shiverer mice were compared. The same distribution patterns of Hsp27 and 1-Cys Prx were found in reactive astrocytes in both CST KO and shiverer mice, suggesting that paranodal disruption with progressive nodal changes may underlie the similar reaction of white matter astrocytes. In contrast, CST KO and shiverer mice showed distinctly different localization patterns of connexin 43 and connexin 47, suggesting that intercellular communication between astrocytes and oligodendrocytes was different in these mutants. These results suggest that astrocytes may respond differentially to individual white matter abnormalities and may modulate specific axonal functions.     

Glial cell reactions in the spinal cord after sensory nerve stimulation are associated with axonal injury

Astroglial and microglial reactions in the dorsal and ventral horns of the adult rat spinal cord were studied after graded electrical stimulation of the rat sciatic nerve and after topical application of mustard oil to the hindlimb foot. Antibodies to glial fibrillary acidic protein and complement receptor 3 (OX-42) were used as markers for astroglia and microglia, respectively. The results showed that electrical nerve stimulation resulted in increased immunoreactivity for GFAP and OX-42 in the spinal cord dorsal and ventral horns only after the use of stimulation strengths which were associated with nerve fiber degeneration in the stimulated nerve. Application of mustard oil to the foot caused no changes in GFAP or OX-42 immunoreactivity. These findings indicate that peripheral nerve stimulation in itself is insufficient to induce astroglial and microglial responses in the spinal cord. The signal(s) mediating these responses, regularly seen after nerve injury, are therefore most probably not related to the afferent barrage of action potentials evoked by the injury.     

Transient expression of the NG2 proteoglycan by a subpopulation of activated macrophages in an excitotoxic hippocampal lesion

Cells that express the NG2 proteoglycan (NG2+ cells) constitute a large glial population in the normal mature rodent brain. They can differentiate into oligodendrocytes but are distinct from mature oligodendrocytes, astrocytes, microglia, and neurons. Changes in NG2+ cells were examined in kainic acid-induced excitotoxic lesions of the hippocampus, and the relationship between NG2+ cells and reactive astrocytes and microglia was investigated between 1 and 90 days after lesioning. Two types of reactive NG2+ cells with altered morphology and increased NG2 immunoreactivity were observed in the lesion. Early changes, consisting of an increase in NG2 immunoreactivity and the number of processes, were apparent 24 h after lesioning and persisted through 3 months. These cells were distinct from reactive astrocytes or activated microglia/macrophages. A second type of reactive NG2+ cells appeared 2 weeks after injection, following an influx of macrophages. They had large, round cell bodies with short processes and expressed the microglia/macrophage antigens OX42 and ED1. Single cells coexpressing NG2 and macrophage/microglial antigens could be isolated from the lesion. The number of NG2+/OX42+ cells gradually declined and disappeared by 3 months after injection. They did not express glial fibrillary acidic protein or the alpha receptor for platelet-derived growth factor, indicating that they are distinct from astrocytes or oligodendrocyte progenitor cells. Cells that coexpressed NG2 and OX42 were never observed in hippocampal slice cultures treated with kainic acid, suggesting that NG2+/OX42+ cells are not derived from endogenous resident brain cells. These findings demonstrate that NG2 expression is transiently upregulated on activated macrophages/microglia that appear during the chronic stage in an excitotoxic lesion in the adult CNS.