Glial Fibrillary Acidic Protein Cells Research Articles

Adipose Tissue-Derived Mesenchymal Stem Cell Concentrated Conditioned Medium Alters the Expression Pattern of Glutamate Regulatory Proteins and Aquaporin-4 in the Retina after Mild Traumatic Brain Injury.

Concentrated conditioned media from adipose tissue-derived mesenchymal stem cells (ASC-CCM) show promise for retinal degenerative diseases. In this study, we hypothesized that ASC-CCM could rescue retinal damage and thereby improve visual function by acting through Müller glia in mild traumatic brain injury (mTBI). Adult C57Bl/6 mice were subjected to a 50-psi air pulse on the left side of the head, resulting in an mTBI. After blast injury, 1 μL (∼100 ng total protein) of human ASC-CCM was delivered intravitreally and followed up after 4 weeks for visual function assessed by electroretinogram and histopathological markers for Müller cell-related markers. Blast mice that received ASC-CCM, compared with blast mice that received saline, demonstrated a significant improvement in a- and b-wave response correlated with a 1.3-fold decrease in extracellular glutamate levels and a concomitant increase in glutamine synthetase (GS), as well as the glutamate transporter (GLAST) in Müller cells. Additionally, an increase in aquaporin-4 (AQP4) in Müller cells in blast mice received saline restored to normal levels in blast mice that received ASC-CCM. In vitro studies on rMC-1 Müller glia exposed to 100 ng/mL glutamate or RNA interference knockdown of GLAST expression mimicked the increased Müller cell glial fibrillary acidic protein (a marker of gliosis) seen with mTBI, and suggested that an increase in glutamate and/or a decrease in GLAST might contribute to the Müller cell activation in vivo. Taken together, our data suggest a novel neuroprotective role for ASC-CCM in the rescue of the visual deficits and pathologies of mTBI via restoration of Müller cell health.

LncRNA-UCA1 inhibits the astrocyte activation in the temporal lobe epilepsy via regulating the JAK/STAT signaling pathway.

This article aimed to reveal the mechanism of long noncoding RNA (lncRNA) urothelial cancer-associated 1 (UCA1) regulated astrocyte activation in temporal lobe epilepsy (TLE) rats via mediating the activation of the JAK/STAT signaling pathway. A model of TLE was established based on rats via kainic acid (KA) injection. All rats were divided into the Sham group (without any treatments), KA group, normal control (NC; injection with empty vector) + KA group, and UCA1 + KA group. The Morris water maze was used to test the learning and memory ability of rats, and the expression of UCA1 in the hippocampus was determined by quantitative real time polymerase chain reaction (qRT-PCR). Surviving neurons were counted by Nissl staining, and expression levels of glial cells glial fibrillary acidic protein (GFAP), p-JAK1, and p-STAT3 and glutamate/aspartate transporter (GLAST) were analyzed by immunofluorescence and Western blot analysis. A rat model of TLE was established by intraperitoneal injection of KA. qRT-PCR and fluorescence analyses showed that UCA1 inhibited astrocyte activation in the hippocampus of epileptic rats. Meanwhile, the Morris water maze analysis indicated that UCA1 improved the learning and memory in epilepsy rats. Moreover, the Nissl staining showed that UCA1 might have a protective effect on neuronal injury induced by KA injection. Furthermore, the immunofluorescence and Western blot analysis revealed that the overexpression of UCA1 inhibited KA-induced abnormal elevation of GLAST, astrocyte activation of the JAK/STAT signaling pathway, as well as hippocampus of epilepsy rats. UCA1 inhibited hippocampal astrocyte activation and JAK/STAT/GLAST expression in TLE rats and improved the adverse reactions caused by epilepsy.

Culture and identification of the tumor stem-like cells isolated from rat prolactinoma MMQ cell lines

Objectives To explore the in vitro culture condition of the tumor stem-like cells isolated from rat prolactinoma MMQ cell lines and to observe their characteristics of proliferation, differentiation, and formation of planted tumor. Methods MMQ cells were cultured with serum-free medium suspension of containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), and the tumor stem cell spheres (briefly cell spheres) were obtained. Clone culture was conducted and its proliferative capacity was observed. Then flow cytometry was used to detect the CD133-positive cell ratio. Western blot was used to detect cell sphere CD133 and nestin expressions. Immunofluorescence technique was used to detect the expressions of cell sphere CD133, nestin, homing cell adhesion molecules (HCAM/CD44), octamer binding transcription factor 4 (OCT4), stem cell antigen 1 (SCA1), as well as their expressions of differentiated cell glial fibrillary acidic protein (GFAP), β-tublinIII, and S100 protein. Twenty-seven female nude mice were randomly divided into 3 groups: a cell sphere planting group, a MMQ cell sphere planting group, and a normal control group (n=9 in each group). Six weeks after subcutaneous plantation, the tumor volume and serum prolactin (PRL) levels were measured. The sections of tumor tissue were detected by the HE staining and immunohistochemistry. Results Under the condition of suspension culture of serum-free medium, only a few cells survived and formed cell spheres. After being blown away, cells could clone and proliferate into cell spheres. The proportion of CD133 positive cells was 1.10±0.17%. CD133, nestin, HCAM/CD44, OCT4, and SCA1 on the cell spheres showed positive expression. After the differentiation of cell spheres, the cells expressed GFAP, β-tublinIII, and S10 protein. The volume of tumor formation in the cell sphere planting group was 1262.2±279.1 mm3, and the MMQ cell sphere planting group was 495.9±158.9 mm3 (P<0.05). The serum PRL of the tumor-bearing nude rats in the cell sphere planting group (median, P25, P75) was 36.08 (34.23, 41.42) ng/ml, and the MMQ cell sphere planting group was 21.51 (20.39, 22.98) ng/ml respectively, and they both higher than 0.26 (1.03-0.15) ng/ml in the normal control group (P<0.05). The HE staining of tumor tissue showed the similar characteristics of the rat pituitary adenoma. Immunohistochemical staining showed that the PRL and nerve synaptophysin (Syn) expressions were positive. Conclusions In MMQ cell lines of rat pituitary prolactin adenoma, a small amount cells with tumor stem-like cell characteristics exist, and they can be cultured successfully In vitro. Key words: Prolactinoma; In vitro; In vivo; MMQ cell line; Tumor stem-like cells; Rats

Environmental Enrichment Protects the Retina from Early Diabetic Damage in Adult Rats

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Available treatments are not completely effective. We analyzed the effect of environmental enrichment on retinal damage induced by experimental diabetes in adult Wistar rats. Diabetes was induced by an intraperitoneal injection of streptozotocin. Three days after vehicle or streptozotocin injection, animals were housed in enriched environment or remained in a standard environment. Retinal function (electroretinogram, and oscillatory potentials), retinal morphology, blood-retinal barrier integrity, synaptophysin, astrocyte and Müller cell glial fibrillary acidic protein, vascular endothelial growth factor, tumor necrosis factor-α, and brain-derived neurotrophic factor levels, as well as lipid peroxidation were assessed in retina from diabetic animals housed in standard or enriched environment. Environmental enrichment preserved scotopic electroretinogram a-wave, b-wave and oscillatory potential amplitude, avoided albumin-Evan's blue leakage, prevented the decrease in retinal synaptophysin and astrocyte glial fibrillary acidic protein levels, the increase in Müller cell glial fibrillary acidic protein, vascular endothelial growth factor and tumor necrosis factor-α levels, as well as oxidative stress induced by diabetes. In addition, enriched environment prevented the decrease in retinal brain-derived neurotrophic factor levels induced by experimental diabetes. When environmental enrichment started 7 weeks after diabetes onset, retinal function was significantly preserved. These results indicate that enriched environment could attenuate the early diabetic damage in the retina from adult rats.

Post-ischemic environmental enrichment protects the retina from ischemic damage in adult rats

The aim of this study was to elucidate whether post-ischemic enriched environment (EE) housing protects the retina from ischemic damage in adult rats, and the involvement of glutamate in retinal protection induced by EE housing. For this purpose, ischemia was induced by increasing intraocular pressure to 120mmHg for 40min. After ischemia, animals were housed in a standard environment (SE) or EE and subjected to electroretinography and histological analysis. EE housing afforded significant functional protection in eyes exposed to ischemia/reperfusion injury. A marked reduction in retinal thickness and ganglion cell number, and an increase in Müller cell glial fibrillary acidic protein (GFAP) levels were observed in ischemic retinas from SE-housed animals, which were reversed by EE housing. A deficit in anterograde transport from the retina to the superior colliculus was observed in SE- but not in EE-housed animals. In SE-housed animals, ischemia induced a significant decrease in retinal glutamate uptake and glutamine synthetase activity, whereas EE housing reversed the effect of ischemia on these parameters. The intravitreal injection of supraphysiological levels of glutamate partially reproduced retinal alterations induced by ischemia/reperfusion, which were abrogated by EE housing. These results indicate that EE housing significantly protected retinal function and histology from ischemia/reperfusion injury in adult rats, likely through a glutamate-dependent mechanism.

Ischemic conditioning protects the rat retina in an experimental model of early type 2 diabetes

Diabetic retinopathy is a leading cause of acquired blindness in adults, mostly affected by type 2 diabetes mellitus (T2DM). We have developed an experimental model of early T2DM in adult rats which mimics some features of human T2DM at its initial stages, and provokes significant retinal alterations. We investigated the effect of ischemic conditioning on retinal changes induced by the moderate metabolic derangement. For this purpose, adult male Wistar rats received a control diet or 30% sucrose in the drinking water, and 3weeks after this treatment, animals were injected with vehicle or streptozotocin (STZ, 25mg/kg). Retinal ischemia was induced by increasing intraocular pressure to 120mmHg for 5min; this maneuver started 3weeks after vehicle or STZ injection and was weekly repeated in one eye, while control eyes were submitted to a sham procedure. Fasting and postprandial glycemia, and glucose, and insulin tolerance tests were analyzed. At 12weeks of treatment, animals which received a sucrose-enriched diet and STZ showed significant differences in metabolic tests, as compared with control groups. Brief ischemia pulses in one eye and a sham procedure in the contralateral eye did not affect glucose metabolism in control or diabetic rats. Ischemic pulses reduced the decrease in the electroretinogram a-wave, b-wave, and oscillatory potential amplitude, and the increase in retinal lipid peroxidation, NOS activity, TNFα, Müller cells glial fibrillary acidic protein, and vascular endothelial growth factor levels observed in diabetic animals. In addition, ischemic conditioning prevented the decrease in retinal catalase activity induced by T2DM. These results indicate that induction of ischemic tolerance could constitute a fertile avenue for the development of new therapeutic strategies to treat diabetic retinopathy associated with T2DM.

Therapeutic efficacy of melatonin in reducing retinal damage in an experimental model of early type 2 diabetes in rats

Diabetic retinopathy (DR) is a leading cause of acquired blindness in adults, mostly affected by type 2 diabetes mellitus (T2DM). We have developed an experimental model of early T2DM in adult rats which mimics some features of human T2DM at its initial stages and provokes significant retinal alterations. The aim of this work was to analyze the effect of melatonin on retinal changes induced by the moderate metabolic derangement. For this purpose, adult male Wistar rats received a control diet or 30% sucrose in the drinking water. Three weeks after this treatment, animals were injected with vehicle or streptozotocin (STZ, 25mg/kg). One day or 3wk after vehicle or STZ injection, animals were subcutaneously implanted with a pellet of melatonin. Fasting and postprandial glycemia, and glucose, and insulin tolerance tests were analyzed. At 12wk of treatment, animals which received a sucrose-enriched diet and STZ showed significant differences in metabolic tests, as compared with control groups. Melatonin, which did not affect glucose metabolism in control or diabetic rats, prevented the decrease in the electroretinogram a-wave, b-wave, and oscillatory potential amplitude, and the increase in retinal lipid peroxidation, NOS activity, TNFα, Müller cells glial fibrillary acidic protein, and vascular endothelial growth factor levels. In addition, melatonin prevented the decrease in retinal catalase activity. These results indicate that melatonin protected the retina from the alterations observed in an experimental model of DR associated with type 2 diabetes.

Expression of connexin57 in mouse development and in harmaline-tremor model

Connexin57 (Cx57) was previously reported in retinal cells but not in brain nerve cells. This occurrence was tested in this study, by searching for the expression of Cx57 RNA and protein transcripts during the postnatal development of the mouse CNS. Both the Cx57 RNA (investigated by reverse transcriptase-polymerase chain reaction (RT-PCR)) and the protein (Western-Blot and immunohistochemistry using a polyclonal antibody generated in chicken) transcripts were firstly expressed in the late postnatal development (P12). The expression of Cx57 in adult life (studied at P28, by in situ hybridization and immunohistochemical analysis) concerned few regions of the brain stem (inferior olive, lateral reticular nucleus and motor trigeminal nucleus), the cerebellum (Purkinje cells and cerebellar nuclei) and the spinal cord (alpha-motoneurons). Double immunohistochemical studies using the Cx57 antibody and antibodies, which specifically labelled neuronal nuclei (NeuN) and astrocyte cells glial fibrillary acidic protein (GFAP), showed the expression of Cx57 segregated in neuronal cells. The study also confirmed the expression of Cx57 in the horizontal cells of the retinal outer plexiform layer, reported in previous investigations. Given the expression of Cx57 in the cerebellum and pre-cerebellar nuclei, such as olivary and lateral reticular nuclei, a possible role of Cx57 was hypothesized in the electrical coupling of the cerebellum. This hypothesis was tested by searching for the expression of the Cx57 transcripts in the mouse cerebellum of the harmaline-tremor model. The up-regulation of the Cx57 transcripts reported in this model suggested a possible involvement of Cx57 in the electrotonic coupling of the cerebellar system.

Involvement of glutamate in retinal protection against ischemia/reperfusion damage induced by post‐conditioning

Retinal ischemia could provoke blindness and there is no effective treatment against retinal ischemic damage. Brief intermittent ischemia applied during the onset of reperfusion (i.e., post-conditioning) protects the retina from ischemia/reperfusion injury. Multiple evidences support that glutamate is implicated in retinal ischemic damage. We investigated the involvement of glutamate clearance in post-conditioning-induced protection. For this purpose, ischemia was induced by increasing intra-ocular pressure for 40 min, and 5 min after reperfusion, animals underwent seven cycles of 1 min/1 min ischemia/reperfusion. One, three, or seven days after ischemia, animals were subjected to electroretinography and histological analysis. The functional and histological protection induced by post-conditioning was evident at 7 (but not 1 or 3) days post-ischemia. An increase in Müller cell glial fibrillary acidic protein (GFAP) levels was observed at 1, 3, and 7 days after ischemia, whereas post-conditioning reduced GFAP levels of Müller cells at 3 and 7 days post-ischemia. Three days after ischemia, a significant decrease in glutamate uptake and glutamine synthetase activity was observed, whereas post-conditioning reversed the effect of ischemia. The intravitreal injection of supraphysiological levels of glutamate mimicked electroretinographic and histological alterations provoked by ischemia, which were abrogated by post-conditioning. These results support the involvement of glutamate in retinal protection against ischemia/reperfusion damage induced by post-conditioning.

Retinal Neuroprotection against Ischemia-Reperfusion Damage Induced by Postconditioning

Retinal ischemia may provoke blindness. There is no effective treatment against retinal ischemic damage. The authors investigated whether brief intermittent ischemia applied during the onset of reperfusion (i.e., postconditioning) protects the retina from ischemia-reperfusion damage. Ischemia was induced by increasing intraocular pressure (120 mm Hg for 40 or 60 minutes). Five minutes after reperfusion, animals underwent 3, 7, or 10 cycles of 1-minute ischemia/1-minute reperfusion or 7 minutes of ischemia. In other experiments, seven ischemia-reperfusion cycles were applied 10, 30, and 60 minutes or 24 hours after ischemia. A group of animals received intraperitoneal injections of cycloheximide (CHX) 1 minute before or 6 hours after postconditioning. Seven or 14 days after ischemia, animals were subjected to electroretinography and histologic analysis. Seven ischemia-reperfusion cycles applied 5 minutes after reperfusion afforded significant functional protection in eyes exposed to ischemia-reperfusion injury. A marked reduction in retinal thickness and an increase in Müller cell glial fibrillary acidic protein (GFAP) levels were observed in ischemic retinas, whereas postconditioning preserved retinal structure and reduced GFAP levels in Müller cells. Postconditioning initiated between 5 and 60 minutes after reperfusion protected against ischemic injury. Retinal protection depended on the number of ischemia-reperfusion cycles. One 7-minute pulse applied 5 minutes after ischemia induced significant protection against ischemic damage. Retinal protection induced by postconditioning was reversed by CHX (injected 1 minute before but not 6 hours after postconditioning). These results indicate that postconditioning significantly protected retinal function and histology from ischemia-reperfusion injury through a mechanism that involved de novo synthesis of protein.

Sustained upregulation of glial fibrillary acidic protein in Müller cells in pigeon retina following disruption of the parasympathetic control of choroidal blood flow

Choroidal blood flow in pigeon eyes is light driven and controlled by a parasympathetic input from ciliary ganglion (CG) neurons that receive input from the medial subdivision of the ipsilateral nucleus of Edinger–Westphal (EWM). EWM lesions diminish basal ChBF and irreversibly prevent ipsilateral light-evoked increases in ChBF, presumably rendering the retina mildly ischemic. To characterize the location, severity, and time course of the retinal abnormality caused by an EWM lesion, we quantitatively analyzed the cellular and regional extent of Müller cell glial fibrillary acidic protein (GFAP) immunolabeling up to nearly a year after an EWM lesion. We found that unilateral EWM lesions greatly increased Müller cell GFAP throughout the entire retinal depth and topographic extent of the affected eye, up to nearly a year post lesion. By contrast, destruction of the pupilloconstrictive pretectum or of the pupilloconstrictive part of lateral EW (EWL) did not appreciably increase Müller cell GFAP. Thus, the large increase in Müller cell GFAP following an EW lesion is attributable to an ongoing defect in choroidal vasodilatory function rather than to chronic pupil dilation. The Müller cell GFAP increase was greater ipsilateral than contralateral to the EWM destruction for the retinal territory deep to the heavily CG-innervated superior and temporal choroid, but not for the retinal territory deep to the poorly CG-innervated inferior and nasal choroid. The GFAP increase was light-dependent, since it did not occur in EW-lesioned birds housed in dim illumination. Our results show that the chronic vascular insufficiency caused by the loss of the EWM-mediated parasympathetic control of choroidal blood flow leads to a significant and sustained increase in retinal Müller cell GFAP. This increase could be a sign of a disturbance in retinal homeostasis that eventually leads to retinal injury and impaired visual function.

Focus on Molecules: Glial fibrillary acidic protein (GFAP)

The aim of this study was to elucidate whether post-ischemic enriched environment (EE) housing protects the retina from ischemic damage in adult rats, and the involvement of glutamate in retinal protection induced by EE housing. For this purpose, ischemia was induced by increasing intraocular pressure to 120 mm Hg for 40 min. After ischemia, animals were housed in a standard environment (SE) or EE and subjected to electroretinography and histological analysis. EE housing afforded significant functional protection in eyes exposed to ischemia/reperfusion injury. A marked reduction in retinal thickness and ganglion cell number, and an increase in Müller cell glial fibrillary acidic protein (GFAP) levels were observed in ischemic retinas from SE-housed animals, which were reversed by EE housing. A deficit in anterograde transport from the retina to the superior colliculus was observed in SE- but not in EE-housed animals. In SE-housed animals, ischemia induced a significant decrease in retinal glutamate uptake and glutamine synthetase activity, whereas EE housing reversed the effect of ischemia on these parameters. The intravitreal injection of supraphysiological levels of glutamate partially reproduced retinal alterations induced by ischemia/reperfusion, which were abrogated by EE housing. These results indicate that EE housing significantly protected retinal function and histology from ischemia/reperfusion injury in adult rats, likely through a glutamate-dependent mechanism.

The Ins2AkitaMouse as a Model of Early Retinal Complications in Diabetes

This study tested the Ins2(Akita) mouse as an animal model of retinal complications in diabetes. The Ins2(Akita) mutation results in a single amino acid substitution in the insulin 2 gene that causes misfolding of the insulin protein. The mutation arose and is maintained on the C57BL/6J background. Male mice heterozygous for this mutation have progressive loss of beta-cell function, decreased pancreatic beta-cell density, and significant hyperglycemia, as early as 4 weeks of age. Heterozygous Ins2(Akita) mice were bred to C57BL/6J mice, and male offspring were monitored for hyperglycemia, beginning at 4.5 weeks of age. After 4 to 36 weeks of hyperglycemia, the retinas were analyzed for vascular permeability, vascular lesions, leukostasis, morphologic changes of micro- and macroglia, apoptosis, retinal degeneration, and insulin receptor kinase activity. The mean blood glucose of Ins2(Akita) mice was significantly elevated, whereas the body weight at death was reduced compared with that of control animals. Compared with sibling control mice, the Ins2(Akita) mice had increased retinal vascular permeability after 12 weeks of hyperglycemia (P < 0.005), a modest increase in acellular capillaries after 36 weeks of hyperglycemia (P < 0.0008), and alterations in the morphology of astrocytes and microglia, but no changes in expression of Muller cell glial fibrillary acidic protein. Increased apoptosis was identified by immunoreactivity for active caspase-3 after 4 weeks of hyperglycemia (P < 0.01). After 22 weeks of hyperglycemia, there was a 16.7% central and 27% peripheral reduction in the thickness of the inner plexiform layer, a 15.6% peripheral reduction in the thickness of the inner nuclear layer (P < 0.001), and a 23.4% reduction in the number of cell bodies in the retinal ganglion cell layer (P < 0.005). In vitro insulin receptor kinase activity was reduced (P < 0.05) after 12 weeks of hyperglycemia. The retinas of heterozygous male Ins2(Akita) mice exhibit vascular, neural, and glial abnormalities generally consistent with clinical observations and other animal models of diabetes. In light of the relatively early, spontaneous onset of the disease and the popularity of the C57BL/6J inbred strain as a background for the generation and study of other genetic alterations, combining the Ins2(Akita) mutation with other engineered mutations will be of great use for studying the molecular basis of retinal complications of diabetes.

Immunocytochemical localization of basic fibroblast growth factor and glial fibrillary acidic protein after laser photocoagulation in the Royal College of Surgeons rat

Purpose: Argon laser photocoagulation slows photoreceptor degeneration in the Royal College of Surgeons (RCS) rat, as does intravitreal injection of basic fibroblast growth factor (bFGF). We hypothesize that up-regulation of retinal bFGF is a consequence of laser lesioning in RCS rats. Therefore, we examined the localization of bFGF after laser and correlated this with Müller cell glial fibrillary acidic protein (GFAP) expression, which is known to increase after injury. Methods: A total of 34 RCS rats at postnatal day 23 were anaesthetized (ketamine 40 mg/kg) and their retinas were irradiated with a grid pattern of 40 non-overlapping argon green lesions with a power of 120 mW for 0.2 s using a 50 μm spot size. At 0,6, 12,24 and 48 h and 7, 14 and 21 days post-lesion, rats were anaesthetized and their eyes were enucleated and cryostat sectioned and the sections were processed using either an antibody to bFGF or GFAP using the standard avidin-biotinylated peroxidase complex method. Five age-matched RCS rats without laser lesions served as controls. Results: Basic fibroblast growth factor immunoreactivity (IR) was normally located within cells in the ganglion cell layer, inner nuclear layer and in retinal pigment epithelium cells and in the extracellular matrix/cell membranes of the outer nuclear layer (ONL). In lasered retinas, there was elevated bFGF-IR in the coagulated outer segments for the first 24 h. Retinal blood vessels/Müller cells/astrocytes were moderately labelled in and near each lesion immediately after lesion and became more intense after 48 h and persisted for at least 21 days. There was an elevation of bFGF-IR in the ONL on the lesion flanks at 14 days. Müller cell GFAP-IR was first detected at 6 h post-lesion and spread for a considerable distance beyond the lesion site. At 7 and 14 days, Müller cells at the lesion site had sprouted, while those on the flanks were still GFAP-IR. Conclusions: Following laser lesion there was an increase in bFGF at the lesion core only for the first 24 h. However elevated levels of bFGF were observed in the ONL at 14 days, which extended into the lesion flanks for a similar distance to that over which increased photoreceptor survival is found. These results provide support for the hypothesis that laser lesions induce bFGF and this may be the mechanism whereby photoreceptors are spared. Müller cell activation is consistent with growth factor stimulation, but was more widespread than the bFGF changes in ONL. However blood vessel labelling was similarly widespread and so the responses may be linked between Müller cell GFAP reaction and blood vessel bFGF localization after laser lesions.

Immunocytochemical localization of basic fibroblast growth factor and glial fibrillary acidic protein after laser photocoagulation in the Royal College of Surgeons rat

Argon laser photocoagulation slows photoreceptor degeneration in the Royal College of Surgeons (RCS) rat, as does intravitreal injection of basic fibroblast growth factor (bFGF). We hypothesize that up-regulation of retinal bFGF is a consequence of laser lesioning in RCS rats. Therefore, we examined the localization of bFGF after laser and correlated this with Mailer cell glial fibrillary acidic protein (GFAP) expression, which is known to increase after injury. A total of 34 RCS rats at postnatal day 23 were anaesthetized (ketamine 40 mg/kg) and their retinas were irradiated with a grid pattern of 40 non-overlapping argon green lesions with a power of 120 mW for 0.2 s using a 50 microm spot size. At 0, 6, 12, 24 and 48 h and 7, 14 and 21 days post-lesion, rats were anaesthetized and their eyes were enucleated and cryostat sectioned and the sections were processed using either an antibody to bFGF or GFAP using the standard avidin-biotinylated peroxidase complex method. Five age-matched RCS rats without laser lesions served as controls. Basic fibroblast growth factor immunoreactivity (IR) was normally located within cells in the ganglion cell layer inner nuclear layer and in retinal pigment epithelium cells and in the extracellular matrix/cell membranes of the outer nuclear layer (ONL). In lasered retinas, there was elevated bFGF-IR in the coagulated outer segments for the first 24 h. Retinal blood vessels/Müller cells/astrocytes were moderately labelled in and near each lesion immediately after lesion and became more intense after 48 h and persisted for at least 21 days. There was an elevation of bFGF-IR in the ONL on the lesion flanks at 14 days. Muller cell GFAP-IR was first detected at 6 h post-lesion and spread for a considerable distance beyond the lesion site. At 7 and 14 days, Müller cells at the lesion site had sprouted, while those on the flanks were still GFAP-IR. Following laser lesion there was an increase in bFGF at the lesion core only for the first 24 h. However, elevated levels of bFGF were observed in the ONL at 14 days, which extended into the lesion flanks for a similar distance to that over which increased photoreceptor survival is found. These results provide support for the hypothesis that laser lesions induce bFGF and this may be the mechanism whereby photoreceptors are spared. Müller cell activation is consistent with growth factor stimulation, but was more widespread than the bFGF changes in ONL. However, blood vessel labelling was similarly widespread and so the responses may be linked between Müller cell GFAP reaction and blood vessel bFGF localization after laser lesions.

Trigeminal ganglion neuronal activity and glial fibrillary acidic protein immunoreactivity after inferior alveolar nerve crush in the adult rat

Nerve injury to the mandibular division of the trigeminal nerve has been shown to cause satellite cell reactions that extend beyond the mandibular division of the trigeminal ganglion into the maxillary and ophthalmic divisions. The goal of this study was to determine whether any physiological abnormalities correlated with this dispersal of satellite cell reaction. We investigated the electrophysiological and satellite cell glial fibrillary acidic protein immunoreactivity (GFAP-IR) changes that occur within the trigeminal ganglion 3, 10 and 59 days after a crush injury of the inferior alveolar nerve (IAN). At 3 days after IAN crush, there were no mechanically-evoked responses to ipsilateral stimulation of the skin and intraoral structures (e.g., mandibular incisor, lower lip and rostral mandibular gingiva) innervated by the IAN. However, the peripheral representations of the auriculotemporal, mylohyoid, lingual and maxillary nerve were intact and no abnormal responses to mechanical stimulation were detected to stimulation of tissue innervated by these nerves. By 10 days after the IAN crush, mandibular neurons responded to mechanical and electrical stimuli of the peripheral receptive field of the IAN, but with slower conduction velocities and higher electrical thresholds compared to control values. These abnormal electrophysiological response characteristics persisted 59 days following nerve injury. At 3, 10 and 59 days after IAN crush, 3–4% of the recorded mandibular neurons displayed spontaneous activity that was never observed in rats without nerve injury. Spontaneous activity was also never observed in neurons recorded in the maxillary or ophthalmic divisions of the trigeminal ganglion. Intense GFAP-IR in satellite cells was observed surrounding a mean of 131.7 neurons/section within the mandibular division of the trigeminal ganglion 3 days after nerve injury and around 50.3 neurons/section at 10 days. GFAP-IR was also present surrounding 16.5 and 10.3 neurons/section in the maxillary division of the trigeminal nerve at 3 and 10 days, respectively. At 59 days after IAN crush, GFAP-IR satellite cells were found around 22.9 neurons/section in the mandibular division of the trigeminal nerve, but were not found elsewhere in the trigeminal ganglion. The more extensive distribution of neurons encircled by satellite cell GFAP-IR compared to the trigeminal ganglion region containing abnormal electrophysiological responses demonstrates that abnormal neuronal signaling may not be characteristic of trigeminal ganglion neurons that are surrounded by GFAP injury reactions. However, the persistence of GFAP-IR 59 days after nerve injury suggests that satellite cell GFAP is involved in the long-term recovery of injured neurons.

Induction of undifferentiated tumors by JC virus in the cerebrum of rats.

Newborn Sprague-Dawley rats were inoculated intracranially with JC virus (Tokyo-1), a human polyomavirus, which had been isolated by Nagashima et al. from the autopsied brain of a patient with progressive multifocal leukoencephalopathy in Japan. Twenty-one to 70 weeks later, 21 of 27 rats developed brain tumors in the cerebrum, but not in the cerebellum. Most of the tumor cells were of an undifferentiated neuroectodermal nature and showed nuclear palisades and pseudorosettes. In some tumor cells glial fibrillary acidic protein was positive immunohistochemically, and many glial filaments were demonstrated ultrastructurally. Neuronal differentiation was not proved. Two continuous lines of cultured tumor cells were established, and T antigen of JCV (Tokyo-1) was present in both cell lines. Glial differentiation was confirmed also in the tumors produced by subcutaneous transplantation of cultured tumor cells.