Distribution of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes in the rat brain

Summary The topographical mapping of glial fibrillary acidic protein (GFAP)-immunoreactivity was performed in coronal serial sections of the rat mesencephalon, rhombencephalon and spinal cord. Relative to a background of poor or moderate overall staining of the mesencephalon, the interpeduncular nucleus, substantia nigra and the periaqueductal grey matter were prominent by their intense GFAP-immunoreactivity. The pons and particularly the medulla contained more GFAP-labelled elements compared with the mesencephalon. The spinal trigeminal nucleus and Rolando substance were distinguished by their intense staining. Large fibre tracts were usually poor in immunoreactive GFAP. In a concluding discussion, findings relevant to the GFAP-mapping of the whole rat CNS are evaluated with regard to possible reasons underlying the observed differential distribution of GFAP-immunoreactivity.     

Evidence for glial-mediated inflammation in aged APP(SW) transgenic mice

Chronic expression of inflammatory cytokines, including interleukin-1β, tumor necrosis factor , and interleukin-6, by glia may underlie the neurodegenerative events that occur within the brains of patients with Alzheimer’s disease (AD). The present study determined whether these markers of inflammation could be observed within the brains of Tg(HuAPP695.K670N/M671L)2576 transgenic mice (Tg2576) that have recently been shown to mimic many features of AD. Interleukin-1β- and tumor necrosis factor -immunopositive microglia were localized with thioflavine-positive (fibrillar) Aβ deposits. Moreover, interleukin-6 immunoreactive astrocytes surrounded fibrillar Aβ deposits. These findings provide evidence that Tg2576 mice exhibit features of the inflammatory pathology seen in AD and suggest that these mice are a useful animal model for studying the role inflammation may play in this disease.     

Kappa opioid receptors are expressed by interneurons in the CA1 area of the rat hippocampus: a correlated light and electron microscopic immunocytochemical study

A local GABA-system is known to have a mediatory function between several afferents and the principal cells of the hippocampus. This study examines the distribution and fine structure of kappa opioid receptor-immunoreactive elements in the CA1 subfield and reveals some new aspects concerning the structural basis of opioid-GABA interaction in the rat hippocampal formation. Kappa receptors were visualized immunocytochemically with a previously produced and characterized monoclonal antibody, the mAb KA8 (Maderspach, K., Németh, K., Simon, J., Benyhe, S., Szûcs, M., Wollemann, M., 1991. A monoclonal antibody recognizing kappa-, but not mu- and delta-opioid receptors. J. Neurochem. 56, 1897–1904). The antibody selectively recognizes the kappa opioid receptor with preference to the kappa₂ subtype. Neuronal cell bodies, proximal dendrites and occasionally glial processes surrounding neuronal perikarya were labelled in the CA1 area. The immunopositive cells were present mainly in the stratum oriens, followed by the stratum pyramidale in a rostrocaudally increasing number. Their shape was fusiform, or multipolar. Occasionally kappa receptor-immunoreactive boutons surrounding weakly immunopositive somata were also observed. Electron microscopy of immunopositive neurons showed that the DAB labelling was intensive in the perinuclear cytoplasm. The widths and electron densities of the postsynaptic densities of some axosomatic synapses were remarkably increased. Similar increase of postsynaptic densities were observable at some axodendritic and axospinous synapses. On the basis of their location and fine structural properties the labelled cells are suggested to be GABAergic inhibitory interneurons, probably belonging to the somatostatinergic sub-population. The axons of these inhibitory interneurons are known to arborize in the stratum lacunosum-moleculare where the entorhinal afferents terminate. A modulatory effect of opioids on the entorhinal input, mediated by somatostatinergic interneurons is suggested     

Molecular cloning and cellular expression of the cholesterol synthesizing enzymes during the prenatal development of the optic nerve in the dromedary camel (Camelus Dromedarius)

The Cholesterol-synthesizing proteins (HMGCS1 and HMGCS2) are mitochondrial enzymes that believed to catalyze the first reaction of ketogenesis, the process by which energy is provided from fats in the absence of carbohydrates. Typically, astrocytes developed from its progenitor cells in the embryonic optic nerve and enriched with HMGCS1 and 2. However, the detailed histomorphology of camel HMGCS1 and 2 remains to be clearly defined. Here, we investigated the changes that associate with astrocytes differentiation within the developing camel optic nerve. Firstly, we isolated cDNAs encoding HMGCS1 and 2 from the optic nerve. Then, we found that HMGCS1 shared high similarity to human, while HMGCS2 showed a lower similarity and was more diverse. Immunohistochemical studies revealed that distinct correlation of astrocytes differentiation with HMGCS1 and 2 expressions in the developing camel optic nerve. Both encoded proteins were localized throughout the cytoplasm, as well as the nuclei of the astrocytes. In addition, semi-quantitative PCR analysis and western analysis confirmed that both HMGCS1 and 2 were highly expressed in camel optic nerve as well as other tissue, but they were lower in both skeletal and heart muscles. Moreover, various stains such as Sudan black and florescence filipin stains were used to visualize the free cholesterol in the astrocytes, indicating the enzymatic activity of HMGCS1 and 2. Together, our study reported the first comprehensive investigation of the molecular cloning and cellular expression of HMGCS1 and 2 in the optic nerve of dromedary camel.     

Evidence for glial-mediated inflammation in aged APPSW transgenic mice

Chronic expression of inflammatory cytokines, including interleukin-1β, tumor necrosis factor α, and interleukin-6, by glia may underlie the neurodegenerative events that occur within the brains of patients with Alzheimer’s disease (AD). The present study determined whether these markers of inflammation could be observed within the brains of Tg(HuAPP695.K670N/M671L)2576 transgenic mice (Tg2576) that have recently been shown to mimic many features of AD. Interleukin-1β- and tumor necrosis factor α-immunopositive microglia were localized with thioflavine-positive (fibrillar) Aβ deposits. Moreover, interleukin-6 immunoreactive astrocytes surrounded fibrillar Aβ deposits. These findings provide evidence that Tg2576 mice exhibit features of the inflammatory pathology seen in AD and suggest that these mice are a useful animal model for studying the role inflammation may play in this disease.     

Modulation of immune-associated surface markers and cytokine production by murine retinal glial cells

Murine retinal glia are normally negative for major histocompatibility complex (MHC) Class II antigens and express low levels of MHC Class I and intercellular adhesion molecule-1 (ICAM-1) as detected by avidin-biotin-peroxidase immunohistochemistry. These surface molecules associated with immune function were either induced (Class II) or upregulated (Class I and ICAM-1) on cultured retinal glial cells in a dose- and time-dependent manner following exposure to recombinant Interferonγ (rIFN-γ). MHC Class I and II expression by passaged and primary cells was maximal ( > 90% positive) after incubation with 100 U/ml of rlFN-y for 48 h. ICAM-1 expression by primary and passaged cells tripled between 48 and 72 h after exposure to 25 or 50 U/ml of rIFN-γ. By 72 h after exposure to 100 U/ml of rIFN-y, 62% of the retinal glia were positive for ICAM-1, whereas under normal culture conditions these molecules were detected on < 3% of the retinal glia. Bacterial lipopolysaccharide (LPS), a known stimulator of central nervous system (CMS) astrocytes, increased ICAM-1 expression only 3-fold to 9% of cells staining positively, but neither MHC Class I nor Class II expression was altered from baseline levels. Surface expression of ICAM-1, MHC Class I, and MHC Class II was unaffected by exposure to either rTNF-α (1000 U/ml) or rIL-6 (100 U/ml) for 24 h. Under normal culture conditions, intracellular interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected immunohistochemically. Exposure to either rIFN-γ or LPS induced more intense staining which correlated with increased secreted levels of both cytokines in culture supernatants. Levels of secreted TNF-α increased 6-fold after stimulation with LPS for 24 h, while secreted IL-6 increased over 9-fold. These results support the hypothesis that retinal glia may participate in intraretinal immune processes following stimulation during inflammatory and infectious processes via either cell surface- or soluble mediator-dependent mechanisms or a combination of both.     

Reduced degeneration of dopaminergic terminals and accentuated astrocyte activation by high dose methamphetamine administration in nociceptin receptor knock out mice

A major pathology of methamphetamine abuse is loss of dopaminergic function due to destruction of dopaminergic terminals, especially in the striatum. This process is accompanied by gliosis by astrocytes and microglia. Here, we evaluated the function of endogenous nociceptin/orphanin FQ in these events using nociceptin receptor (NOP) knockout mice. Wild-type and knockout mice were injected systemically either saline vehicle or 5 mg/kg methamphetamine four times interspersed by 2 h intervals. Three days later, brains were immunohistochemically processed to visualize methamphetamine-induced loss of tyrosine hydroxylase (as a marker of damage to dopamine terminals), glial fibrillary acidic protein (GFAP, as a marker of astrocytes), and ionized calcium-binding adapter molecule 1 (lba-1, as a marker of microglia) in the striatum. Methamphetamine treatment induced an approximately 80% loss of tyrosine hydroxylase-immunoreactivity, and this effect was mildly attenuated in NOP receptor knockout mice. There was a large increase (approximately 15-fold) in GFAP-immunoreactivity in methamphetamine-treated wild-type mice, which was almost two times larger still in NOP receptor knockout mice. In contrast, Iba-1 immunostaining was only modestly increased (approximately 30%) by methamphetamine treatment, and there were no difference between genotypes. Finally, there were no genotype-dependent differences in hyperthermic responses to methamphetamine. These results indicate that endogenous nociceptin/orphanin FQ exacerbates the neurotoxic effects of methamphetamine on striatal dopamine neurons, and suggests this is due in part to an astrocyte-mediated event.     

Therapeutic concentration of morphine reduces oxidative stress in glioma cell line

Morphine is a potent analgesic opioid used extensively for pain treatment. During thelast decade, global consumption grew more than 4-fold. However, molecular mechanismselicited by morphine are not totally understood. Thus, a growing literature indicatesthat there are additional actions to the analgesic effect. Previous studies aboutmorphine and oxidative stress are controversial and used concentrations outside therange of clinical practice. Therefore, in this study, we hypothesized that atherapeutic concentration of morphine (1 μM) would show a protective effect in atraditional model of oxidative stress. We exposed the C6 glioma cell line to hydrogenperoxide (H₂O₂) and/or morphine for 24 h and evaluated cellviability, lipid peroxidation, and levels of sulfhydryl groups (an indicator of theredox state of the cell). Morphine did not prevent the decrease in cell viabilityprovoked by H₂O₂ but partially prevented lipid peroxidationcaused by 0.0025% H₂O₂ (a concentration allowing more than 90%cell viability). Interestingly, this opioid did not alter the increased levels ofsulfhydryl groups produced by exposure to 0.0025% H₂O₂, openingthe possibility that alternative molecular mechanisms (a direct scavenging activityor the inhibition of NAPDH oxidase) may explain the protective effect registered inthe lipid peroxidation assay. Our results demonstrate, for the first time, thatmorphine in usual analgesic doses may contribute to minimizing oxidative stress incells of glial origin. This study supports the importance of employing concentrationssimilar to those used in clinical practice for a better approximation betweenexperimental models and the clinical setting.     

Cerebral complement C1q activation in chronic Toxoplasma infection

Exposure to the neurotropic parasite, Toxoplasma gondii, causes significant brain and behavioral anomalies in humans and other mammals. Understanding the cellular mechanisms of T. gondii-generated brain pathologies would aid the advancement of novel strategies to reduce disease. Complement factor C1q is part of a classic immune pathway that functions peripherally to tag and remove infectious agents and cellular debris from circulation. In the developing and adult brain, C1q modifies neuronal architecture through synapse marking and pruning. T. gondii exposure and complement activation have both been implicated in the development of complex brain disorders such as schizophrenia. Thus, it seems logical that mechanistically, the physiological pathways associated with these two factors are connected. We employed a rodent model of chronic infection to investigate the extent to which cyst presence in the brain triggers activation of cerebral C1q. Compared to uninfected mice, cortical C1q was highly expressed at both the RNA and protein levels in infected animals bearing a high cyst burden. In these mice, C1q protein localized to cytoplasm, adjacent to GFAP-labeled astrocytes, near degenerating cysts, and in punctate patterns along processes. In summary, our results demonstrated an upregulation of cerebral C1q in response to latent T. gondii infection. Our data preliminarily suggest that this complement activity may aid in the clearance of this parasite from the CNS and in so doing, have consequences for the connectivity of neighboring cells and synapses.     

Molecular mechanisms and cellular events involved in the neuroprotective actions of estradiol. Analysis of sex differences

Estradiol, either from peripheral or central origin, activates multiple molecular neuroprotective and neuroreparative responses that, being mediated by estrogen receptors or by estrogen receptor independent mechanisms, are initiated at the membrane, the cytoplasm or the cell nucleus of neural cells. Estrogen-dependent signaling regulates a variety of cellular events, such as intracellular Ca²⁺ levels, mitochondrial respiratory capacity, ATP production, mitochondrial membrane potential, autophagy and apoptosis. In turn, these molecular and cellular actions of estradiol are integrated by neurons and non-neuronal cells to generate different tissue protective responses, decreasing blood-brain barrier permeability, oxidative stress, neuroinflammation and excitotoxicity and promoting synaptic plasticity, axonal growth, neurogenesis, remyelination and neuroregeneration. Recent findings indicate that the neuroprotective and neuroreparative actions of estradiol are different in males and females and further research is necessary to fully elucidate the causes for this sex difference.