GFAP-immunoreactive Processes Research Articles

Immunohistochemical evidence of tissue hypoxia and astrogliosis in the rostral ventrolateral medulla of spontaneously hypertensive rats

Increased activity of the sympathetic nervous system has been highlighted as a key factor that contributes to the development and maintenance of arterial hypertension. However, the factors that precipitate sustained increases in sympathetic activity remain poorly understood. Resting tissue oxygen partial pressure (PtO2) in the brainstem of anesthetized spontaneously hypertensive rats (SHRs) has been shown to be lower than in normotensive rats despite normal levels of arterial PO2. A hypoxic environment in the brainstem has been postulated to activate astroglial signalling mechanisms in the rostral ventrolateral medulla (RVLM) which in turn increase the excitability of presympathetic neuronal networks. In this study, we assessed the expression of indirect markers of tissue hypoxia and astroglial cell activation in the RVLM of SHRs and age-matched normotensive Wistar rats. Immunohistochemical labelling for hypoxia-induced factor-1α (HIF-1α) and bound pimonidazole adducts revealed the presence of tissue hypoxia in the RVLM of SHRs. Double immunostaining showed co-localization of bound pimonidazole labelling in putative presympathetic C1 neurons and in astroglial cells. Quantification of glial fibrillary acidic protein (GFAP) immunofluorescence showed relatively higher number of astrocytes and increased GFAP mean grey value density, whilst semi-quantitative analysis of skeletonized GFAP-immunoreactive processes revealed greater % area covered by astrocytic processes in the RVLM of adult SHRs. In conclusion, the morphological findings of tissue hypoxia and astrogliosis within brainstem presympathetic neuronal networks in the SHR support previous observations, showing that low brainstem PtO2 and increased astroglial signalling in the RVLM play an important role in pathological sympathoexcitation associated with the development of arterial hypertension.

Components of the basal lamina and dystrophin-dystroglycan complex in the neurointermediate lobe of rat pituitary gland: different localizations of beta-dystroglycan, dystrobrevins, alpha1-syntrophin, and aquaporin-4.

The so-called neurointermediate lobe is composed of the intermediate and neural lobes of the pituitary. The present immunohistochemical study investigated components of the basal lamina (laminin, agrin, and perlecan), the dystrophin-dystroglycan complex (dystrophin, beta-dystroglycan, alpha1-dystrobrevin, beta-dystrobrevin, utrophin, and alpha1-syntrophin), and the aquaporins (aquaporin-4 and -9). Glia markers (GFAP, S100, and glutamine synthetase) and components of connective tissue (collagen type I and fibronectin) were also labeled. In the neurohypophysis, immunostaining of basal lamina delineated meningeal invaginations. In these invaginations, vessels were seen to penetrate the organ without submerging into its parenchyma. On the parenchymal side of the invaginations, beta-dystroglycan was detected, whereas utrophin was detected in the walls of vessels. Immunostaining of alpha1-dystrobrevin and alpha1-syntrophin did not delineate the vessels. The cells of the intermediate lobe were fully immunoreactive to alpha1-dystrobrevin and alpha1-syntrophin, whereas components of the basal lamina delineated the contours of the cells. GFAP-immunoreactive processes surrounded them. Aquaporin-4 localized at the periphery of the neurohypophysis, mainly adjacent to the intermediate lobe but not along the vessels. It colocalized only partially with GFAP and not at all with alpha1-syntrophin. Aquaporin-9 was not detected. These results emphasize the possibility that the components of the dystrophin-dystroglycan complex localize differently and raise the question about the roles of dystrobrevins, alpha1-syntrophin, and aquaporin-4 in the functions of the intermediate and neural lobes, respectively.

Morphological changes in white matter astrocytes in response to hypoxia/ischemia in the neonatal pig

White matter damage is a significant problem in the human pre-term baby. Damage to white matter is usually associated with injury or insults to babies born prematurely, typically before 32 weeks' gestation, however there is increasing evidence of both grey and white matter damage occurring after 32 weeks' gestation. Astrocytes play a vital role in white matter, regulating molecules such as glutamate in the extracellular space and preventing excitotoxic damage to neighbouring oligodendrocytes and axons. We have previously described dramatic changes in grey matter astrocytes in response to a hypoxic/ischemic (H/I) insult around the time of birth. In this study, we have used GFAP immunohistochemistry and Golgi–Kopsch staining to examine the morphology of white matter astrocytes in control neonatal pig brains, and in the brains of animals exposed to the same (perinatal) H/I insult. We demonstrate that the areal percentage of the section occupied by GFAP-immunoreactive processes and cell bodies is significantly decreased (by 46%, P < 0.0001) in subcortical white matter from H/I brains. This loss of GFAP was accompanied by alterations in astrocyte morphology and an overall decrease in the size (field of section occupied by an individual astrocyte) of white matter astrocytes from 649 μm 2 to 426 μm 2, as revealed by Golgi–Kopsch staining and image analysis. These data suggest that astrocytes may contribute to the pathology of white matter damage following an H/I insult around the time of birth, and suggest that astrocytes may offer a novel target for therapies to improve outcomes after H/I.

Blockade of Endothelinergic Receptors Prevents Development of Proliferative Vitreoretinopathy in Mice

Proliferative vitreoretinopathy (PVR) is characterized by severe glial remodeling. Glial activation and proliferation that occur in brain diseases are modulated by endothelin-1 (ET-1) and its receptor B (ETR-B). Because retinal astrocytes contain ET-1 and express ETR-B, we studied the changes of these molecules in an experimental mouse model of PVR and in human PVR. Both ET-1 and ETR-B immunoreactivities increased in mouse retina after induction of PVR with dispase. Epi- and subretinal outgrowths also displayed these immunoreactivities in both human and experimental PVR. Additionally, myofibroblasts and other membranous cell types showed both ET-1 and ETR-B immunoreactivities. In early stages of experimentally induced PVR, prepro-ET-1 and ETR-B mRNA levels increased in the retina. These mRNA levels also increased after retinal detachment (RD) produced by subretinal injection. Treatment of mice with tezosentan, an antagonist of endothelinergic receptors, reduced the histopathological hallmarks of dispase-induced PVR: retinal folding, epiretinal outgrowth, and gliosis. Our findings in human and in dispase-induced PVR support the involvement of endothelinergic pathways in retinal glial activation and the phenotypic transformations that underlie the growth of membranes in this pathology. Elucidating these pathways further will help to develop pharmacological treatments to prevent PVR. In addition, the presence of ET-1 and ETR-B in human fibrous membranes suggests that similar treatments could be helpful after PVR has been established.

Basic fibroblast growth factor, neurofilament, and glial fibrillary acidic protein immunoreactivities in the myenteric plexus of the rat esophagus and colon.

The enteric nervous system consists of a number of interconnected networks of neuronal cell bodies and fibers as well as satellite cells, the enteric glia. Basic fibroblast growth factor (bFGF) is a mitogen for a variety of mesodermal and neuroectodermal-derived cells and its presence has been described in many tissues. The present work employs immunohistochemistry to analyze neurons and glial cells in the esophageal and colic enteric plexus of the Wistar rat for neurofilament (NF) and glial fibrillary acidic proteins (GFAP) immunoreactivity as well as bFGF immunoreactivity in these cells. Rats were processed for immunohistochemistry; the distal esophagus and colon were opened and their myenteric plexuses were processed as whole-mount preparations. The membranes were immunostained for visualization of NF, GFAP, and bFGF. NF immunoreactivity was seen in neuronal cell bodies of esophageal and colic enteric ganglia. GFAP-immunoreactive enteric glial cells and processes were present in the esophageal and colic enteric plexuses surrounding neuronal cell bodies and axons. A dense net of GFAP-immunoreactive processes was seen in the ganglia and connecting strands of the myenteric plexus. bFGF immunoreactivity was observed in the cytoplasm of the majority of the neurons in the enteric ganglia of esophagus and colon. The two-color immunoperoxidase and immunofluorescence methods revealed bFGF immunoreactivity also in the nucleus of GFAP-positive enteric glial cells. The results suggest that immunohistochemical localization of NF and GFAP may be an important tool in the study of the plasticity in the enteric nervous system. The presence of bFGF in neurons and glia of the myenteric plexus of the esophagus and the colon indicates that this neurotrophic factor may exert autocrine and paracrine actions in the enteric nervous system.

Age-related Changes in Astrocytes and Microvasculature in the Median Eminence of the Rat

Age-related changes in astrocytes, tanycytes and the microvasculature were investigated in the median eminence (ME) of the anterior hypothalamo-hypophyseal system of 2- and 24-month old rats by means of immunocytochemistry and scanning electron microscopy. A computer-assisted image processing system showed a significant increase of the glial fibrillary acidic protein (GFAP) immunoreactivity in the internal layer (IL) and the perivascular region of the external layer (EL) of the ME with age. However, no remarkable changes in either layer could be detected employing vimentin immunoreactivity. An immunoelectron microscopic study revealed that the number of the GFAP-immunoreactive processes markedly increased with age in regions surrounding the capillaries of the EL. Therefore, it may be considered that, in both layers, the astrocytic cell bodies and associated glial processes exhibit a significant increase with age. Investigation of the microvasculature under a scanning electron microscope demonstrated the development and the sinusoidal progress of the external capillary plexus with age, but these changes were not observed in the internal capillary network of the ME. In conclusion, the results of the present study showed a close correlation between the increase of the GFAP-immunoreactive astrocytes and the development of the external capillary plexus. In addition, there was a close correlation between the increase in the GFAP-immunoreactive astrocytes and the sinusoidal progress.

Development of Astroglial Elements in the Suprachiasmatic Nucleus of the Rat: With Special Reference to the Involvement of the Optic Nerve

The development of astroglial cells and the effect of the retinohypothalamic tract on it were studied by vimentin and glial fibrillary acidic protein (GFAP) immunocytochemistry in the suprachiasmatic nucleus (SCN) of the rat. At the embryonic stage, vimentin-immunoreactive (VIM-IR) radial glia, precursors of astrocytes, were dominant. However, their filaments vanished in the first few postnatal days. Instead of VIM-IR glial filaments, GFAP-immunoreactive (GFAP-IR) astrocytes appeared at E20 and grew rapidly from the P3 stage. GFAP immunoreactivity in the ventrolateral portion of the SCN (VLSCN) was measured using a computer-assisted image analyzing system. In normal rats, GFAP immunoreactivity showed a stepwise pattern with two slopes at P3–P4 and P20–P25. Bilaterally eye-enucleated rats operated on the day of birth showed lower GFAP immunoreactivity than normal rats and the GFAP immunoreactivity did not increase between P20 and P25 when GFAP-IR glial processes rapidly expand. Electron microscopic investigation at P50 (adult stage) revealed that neurons in the VLSCN had often direct apposition without astroglial processes and the frequency of this finding was significantly higher in eye-enucleated rats than in the control rats. These findings strongly suggest that the postnatal development of astroglial elements, particularly the expansion of GFAP-IR processes in the SCN, is regulated by retinohypothalamic projection.

Neurosteroids modulate the reaction of astroglia to high extracellular potassium levels.

The extracellular concentration of potassium ([K+]o) in brain tissue is modified by neuronal activity and is increased under several pathological conditions. The influence of neurosteroids on the astroglia response to high [K+]o was assessed on cultured slices from rat hippocampus. Exposure to [K+]o above physiological (3 mM) levels resulted in the progressive appearance of cell processes immunoreactive for glial fibrillary acidic protein (GFAP). The maximal effect was observed at 50 mM [K+]o, and further increases of [K+]o did not increase the extension of GFAP-immunoreactive processes. The effect was observed as early as 10 min after increasing [K+]o, was independent of new protein synthesis, and was reversible, reaching control conditions by 15 h after resetting [K+]o to physiological levels. Gonadal hormones and neurosteroids had prominent and variable effects on the stimulatory influence of high [K+]o on astroglia morphology. At physiological [K+]o, 17beta-estradiol and pregnenolone, as well as its sulfate derivative, increased the extension of GFAP-immunoreactive processes. However, at high [K+]o, testosterone, pregnenolone, and dehydroepiandrosterone and its sulfate derivative decreased the extension of GFAP-immunoreactive processes. Effects of gonadal hormones and neurosteroids were blocked by the protein synthesis inhibitor cycloheximide. These results suggest that non-genomic effects of high [K+]o on glial cells interact with genomic effects of steroids to modulate astroglia morphology.

Neurosteroids modulate the reaction of astroglia to high extracellular potassium levels

The extracellular concentration of potassium ([K+]0) in brain tissue is modified by neuronal activity and is increased under several pathological conditions. The influence of neurosteroids on the astroglia response to high [K+]0 was assessed on cultured slices from rat hippocampus. Exposure to [K+]0 above physiological (3 mM) levels resulted in the progressive appearance of cell processes immunoreactive for glial fibrillary acidic protein (GFAP). The maximal effect was observed at 50 mM [K+]0, and further increases of [K+]0 did not increase the extension of GFAP-immunoreactive processes. The effect was observed as early as 10 min after increasing [K+]0, was independent of new protein synthesis, and was reversible, reaching control conditions by 15 h after resetting [K+]0 to physiological levels. Gonadal hormones and neurosteroids had prominent and variable effects on the stimulatory influence of high [K+]0 on astroglia morphology. At physiological [K+]0, 17β-estradiol and pregnenolone, as well as its sulfate derivative, increased the extension of GFAP-immunoreactive processes. However, at high [K+]0, testosterone, pregnenolone, and dehydroepiandrosterone and its sulfate derivative decreased the extension of GFAP-immunoreactive processes. Effects of gonadal hormones and neurosteroids were blocked by the protein synthesis inhibitor cycloheximide. These results suggest that non-genomic effects of high [K+]0 on glial cells interact with genomic effects of steroids to modulate astroglia morphology. © 1996 Wiley-Liss, Inc.

Localisation of NADPH diaphorase activity and NOS immunoreactivity in astroglia in normal adult rat brain

This study demonstrates the co-localisation of NADPH diaphorase activity and GFAP immunoreactivity in non-neuronal cells in weakly fixed brain sections from normal adult rats. The presence of GFAP immunoreactivity in these cells indicates that they are astroglia. In addition, cells possessing the morphological characteristics of astroglia were weakly inununoreactive for the endothelial isoform of nitric oxide synthase (eNOS) — these cells also co-localised NADPH diaphorase activity. Furthermore, cells immunoreactive for eNOS displayed GFAP immunoreactive processes. This cytochemical evidence strongly suggests that resting astroglia are potential sources of nitric oxide — a powerful modulator of cell activity.

Patterns of GFAP-immunoreactivity parallel the tonotopic axis in the developing dorsal cochlear nucleus

The role of glia in the development of tonotopic and laminar organization in the auditory central nervous system has not been well characterized. In other systems, glia immunoreactive for glial fibrillary acidic protein (GFAP) appear to function in development of radial, laminar and topographic organization. Using a polyclonal antibody to GFAP, we have characterized the development of GFAP-immunoreactivity in the dorsal cochlear nucleus (DCN), a laminated and tonotopically organized central auditory system structure. Results suggest that in this nucleus, the GFAP-immunoreactive processes are not found within or between developing laminae, rather glial processes are observed parallel to presumptive isofrequency sheets before primary afferents have invaded the nucleus. Thus, GFAP-immunoreactive processes are positioned to play an early role in establishing the tonotopic axis of the DCN.

Neuroactive steroids regulate astroglia morphology in hippocampal cultures from adult rats.

Recent evidence indicates that astroglia may be involved in the synthesis of endogenous neurosteroids. The extension of glial fibrillary acidic protein (GFAP)-immunoreactive astroglial cell processes was assessed in hippocampal slice cultures from adult gonadectomized male rats under the influence of the neurosteroids dehydroepiandrosterone, dehydroepiandrosterone sulfate, dehydroepiandrosterone estereate, pregnenolone, pregnenolone sulfate, and pregnenolone oleate. The effects of neurosteroids were compared to those induced by the gonadal steroids testosterone, 17 beta-estradiol and progesterone. Astrocytes in hippocampal slice cultures had a morphology that was indistinguishable from that observed in the hippocampus fixed in situ. Castration of adult male rats resulted in a significant decrease in the extension of GFAP-immunoreactive processes, both in tissues fixed in situ and in slice cultures. In contrast, incubation of slice cultures from gonadectomized animals with pregnenolone, pregnenolone sulfate, 17 beta-estradiol, and testosterone enhanced the extension of GFAP-immunoreactive processes. While other steroids tested did not affect this parameter, dehydroepiandrosterone and its sulfate and estereate derivatives induced the transformation of astroglial cells into hypertrophic and highly GFAP immunoreactive cells with the morphological characteristics of reactive astroglia. We conclude that neurosteroids regulate the morphology and/or GFAP distribution of astrocytes in hippocampal slice cultures from adult rats.

Rapid and reversible astrocytic reaction to afferent activity blockade in chick cochlear nucleus

We describe here rapid proliferation of astrocytic processes immunoreactive for glial fibrillary acidic protein (GFAP) in the chick cochlear nucleus following blockade of action potentials in the afferent nerve. Unilateral eighth nerve activity blockade was achieved through intralabyrinthine injection of TTX. Within 1 hr of activity blockade, a 56% increase in area density of GFAP-immunoreactive processes was found in the ipsilateral cochlear nucleus as compared to the contralateral side of the brain. This increase reached 152% by 3 hr. When eighth nerve activity was allowed to recover and animals were studied 1 week after TTX injection, no difference was found in GFAP immunoreactivity between the ipsilateral and contralateral cochlear nuclei. This is the first report of a glial reaction to documented neuronal inactivity in the absence of neuropathology. These results indicate that neuronal activity may regulate the structure of astrocytic processes.