TGF-beta(2)- and H2O2-Induced Biological Changes in Optic Nerve Head Astrocytes Are Reduced by the Antioxidant Alpha-Lipoic Acid

Background/Aims: The goal of the present study was to determine whether transforming growth factor-beta(2) (TGF-beta(2))- and oxidative stress-induced cellular changes in cultured human optic nerve head (ONH) astrocytes could be reduced by pretreatment with the antioxidant alpha-lipoic acid (LA). Methods: Cultured ONH astrocytes were treated with 1.0 ng/ml TGF-beta(2) for 24 h or 200 mu M hydrogen peroxide (H2O2) for 1 h. Lipid peroxidation was measured by a decrease in cis-pari-naric acid fluorescence. Additionally, cells were pretreated with different concentrations of LA before TGF-beta 2 or H2O2 exposure. Expressions of the heat shock protein (Hsp) alpha B-crystallin and Hsp27, the extracellular matrix (ECM) component fibronectin and the ECM-modulating protein connective tissue growth factor (CTGF) were examined with immunohistochemistry and real-time PCR analysis. Results: Both TGF-beta(2) and H2O2 increased lipid peroxidation. Treatment of astrocytes with TGF-beta(2) and H2O2 upregulated the expression of alpha B-crystallin, Hsp27, fibronectin and CTGF. Pretreatment with different concentrations of LA reduced the TGF-beta(2)- and H2O2-stimulated gene expressions. Conclusion: We showed that TGF-beta(2)- and H2O2-stimulated gene expressions could be prevented by pretreatment with the antioxidant LA in cultured human ONH astrocytes. Therefore, it is tempting to speculate that the use of antioxidants could have protective effects in glaucomatous optic neuropathy. Copyright (C) 2012 S. Karger AG, Base

Deletion of Munc18-1 in 5-HT Neurons Results in Rapid Degeneration of the 5-HT System and Early Postnatal Lethality

The serotonin (5-HT) system densely innervates many brain areas and is important for proper brain development. To specifically ablate the 5-HT system we generated mutant mice carrying a floxed Munc18-1 gene and Cre recombinase driven by the 5-HT-specific serotonin reuptake transporter (SERT) promoter. The majority of mutant mice died within a few days after birth. Immunohistochemical analysis of brains of these mice showed that initially 5-HT neurons are formed and the cortex is innervated with 5-HT projections. From embryonic day 16 onwards, however, 5-HT neurons started to degenerate and at postnatal day 2 hardly any 5-HT projections were present in the cortex. The 5-HT system of mice heterozygous for the floxed Munc18-1 allele was indistinguishable from control mice. These data show that deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and suggests that the 5-HT system is important for postnatal survival

Cellular injury and neuroinflammation in children with chronic intractable epilepsy

<p>Abstract</p> <p>Objective</p> <p>To elucidate the presence and potential involvement of brain inflammation and cell death in neurological morbidity and intractable seizures in childhood epilepsy, we quantified cell death, astrocyte proliferation, microglial activation and cytokine release in brain tissue from patients who underwent epilepsy surgery.</p> <p>Methods</p> <p>Cortical tissue was collected from thirteen patients with intractable epilepsy due to focal cortical dysplasia (6), encephalomalacia (5), Rasmussen's encephalitis (1) or mesial temporal lobe epilepsy (1). Sections were processed for immunohistochemistry using markers for neuron, astrocyte, microglia or cellular injury. Cytokine assay was performed on frozen cortices. Controls were autopsy brains from eight patients without history of neurological diseases.</p> <p>Results</p> <p>Marked activation of microglia and astrocytes and diffuse cell death were observed in epileptogenic tissue. Numerous fibrillary astrocytes and their processes covered the entire cortex and converged on to blood vessels, neurons and microglia. An overwhelming number of neurons and astrocytes showed DNA fragmentation and its magnitude significantly correlated with seizure frequency. Majority of our patients with abundant cell death in the cortex have mental retardation. IL-1beta, IL-8, IL-12p70 and MIP-1beta were significantly increased in the epileptogenic cortex; IL-6 and MCP-1 were significantly higher in patients with family history of epilepsy.</p> <p>Conclusions</p> <p>Our results suggest that active neuroinflammation and marked cellular injury occur in pediatric epilepsy and may play a common pathogenic role or consequences in childhood epilepsy of diverse etiologies. Our findings support the concept that immunomodulation targeting activated microglia and astrocytes may be a novel therapeutic strategy to reduce neurological morbidity and prevent intractable epilepsy.</p

Astrocyte-Derived Tissue Transglutaminase Interacts with Fibronectin: A Role in Astrocyte Adhesion and Migration?

An important neuropathological feature of neuroinflammatory processes that occur during e.g. Multiple Sclerosis (MS) is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that contribute to the interaction between astrocytes and ECM proteins. Tissue Transglutaminase (TG2) is a multifunctional enzyme with an ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can enhance TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-fibronectin interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. Astrocyte-derived TG2 interacts with fibronectin and is involved in astrocyte adhesion onto and migration across fibronectin. TG2 is involved in stimulating focal adhesion formation which is necessary for the interaction of astrocytes with ECM proteins. We conclude that astrocyte-derived TG2 contributes to the interaction between astrocytes and fibronectin. It might thereby regulate ECM remodeling and possibly glial scarring

Neuroanatomical Study of the A11 Diencephalospinal Pathway in the Non-Human Primate

BACKGROUND: The A11 diencephalospinal pathway is crucial for sensorimotor integration and pain control at the spinal cord level. When disrupted, it is thought to be involved in numerous painful conditions such as restless legs syndrome and migraine. Its anatomical organization, however, remains largely unknown in the non-human primate (NHP). We therefore characterized the anatomy of this pathway in the NHP. METHODS AND FINDINGS: In situ hybridization of spinal dopamine receptors showed that D1 receptor mRNA is absent while D2 and D5 receptor mRNAs are mainly expressed in the dorsal horn and D3 receptor mRNA in both the dorsal and ventral horns. Unilateral injections of the retrograde tracer Fluoro-Gold (FG) into the cervical spinal enlargement labeled A11 hypothalamic neurons quasi-exclusively among dopamine areas. Detailed immunohistochemical analysis suggested that these FG-labeled A11 neurons are tyrosine hydroxylase-positive but dopa-decarboxylase and dopamine transporter-negative, suggestive of a L-DOPAergic nucleus. Stereological cell count of A11 neurons revealed that this group is composed by 4002±501 neurons per side. A 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) intoxication with subsequent development of a parkinsonian syndrome produced a 50% neuronal cell loss in the A11 group. CONCLUSION: The diencephalic A11 area could be the major source of L-DOPA in the NHP spinal cord, where it may play a role in the modulation of sensorimotor integration through D2 and D3 receptors either directly or indirectly via dopamine formation in spinal dopa-decarboxylase-positives cells

Gene Expression and Functional Studies of the Optic Nerve Head Astrocyte Transcriptome from Normal African Americans and Caucasian Americans Donors

To determine whether optic nerve head (ONH) astrocytes, a key cellular component of glaucomatous neuropathy, exhibit differential gene expression in primary cultures of astrocytes from normal African American (AA) donors compared to astrocytes from normal Caucasian American (CA) donors.We used oligonucleotide Affymetrix microarray (HG U133A & HG U133A 2.0 chips) to compare gene expression levels in cultured ONH astrocytes from twelve CA and twelve AA normal age matched donor eyes. Chips were normalized with Robust Microarray Analysis (RMA) in R using Bioconductor. Significant differential gene expression levels were detected using mixed effects modeling and Statistical Analysis of Microarray (SAM). Functional analysis and Gene Ontology were used to classify differentially expressed genes. Differential gene expression was validated by quantitative real time RT-PCR. Protein levels were detected by Western blots and ELISA. Cell adhesion and migration assays tested physiological responses. Glutathione (GSH) assay detected levels of intracellular GSH.Multiple analyses selected 87 genes differentially expressed between normal AA and CA (P<0.01). The most relevant genes expressed in AA were categorized by function, including: signal transduction, response to stress, ECM genes, migration and cell adhesion.These data show that normal astrocytes from AA and CA normal donors display distinct expression profiles that impact astrocyte functions in the ONH. Our data suggests that differences in gene expression in ONH astrocytes may be specific to the development and/or progression of glaucoma in AA

Enhanced Functional Recovery in MRL/MpJ Mice after Spinal Cord Dorsal Hemisection

Adult MRL/MpJ mice have been shown to possess unique regeneration capabilities. They are able to heal an ear-punched hole or an injured heart with normal tissue architecture and without scar formation. Here we present functional and histological evidence for enhanced recovery following spinal cord injury (SCI) in MRL/MpJ mice. A control group (C57BL/6 mice) and MRL/MpJ mice underwent a dorsal hemisection at T9 (thoracic vertebra 9). Our data show that MRL/MpJ mice recovered motor function significantly faster and more completely. We observed enhanced regeneration of the corticospinal tract (CST). Furthermore, we observed a reduced astrocytic response and fewer micro-cavities at the injury site, which appear to create a more growth-permissive environment for the injured axons. Our data suggest that the reduced astrocytic response is in part due to a lower lesion-induced increase of cell proliferation post-SCI, and a reduced astrocytic differentiation of the proliferating cells. Interestingly, we also found an increased number of proliferating microglia, which could be involved in the MRL/MpJ spinal cord repair mechanisms. Finally, to evaluate the molecular basis of faster spinal cord repair, we examined the difference in gene expression changes in MRL/MpJ and C57BL/6 mice after SCI. Our microarray data support our histological findings and reveal a transcriptional profile associated with a more efficient spinal cord repair in MRL/MpJ mice

Network analysis of human glaucomatous optic nerve head astrocytes

<p>Abstract</p> <p>Background</p> <p>Astrocyte activation is a characteristic response to injury in the central nervous system, and can be either neurotoxic or neuroprotective, while the regulation of both roles remains elusive.</p> <p>Methods</p> <p>To decipher the regulatory elements controlling astrocyte-mediated neurotoxicity in glaucoma, we conducted a systems-level functional analysis of gene expression, proteomic and genetic data associated with reactive optic nerve head astrocytes (ONHAs).</p> <p>Results</p> <p>Our reconstruction of the molecular interactions affected by glaucoma revealed multi-domain biological networks controlling activation of ONHAs at the level of intercellular stimuli, intracellular signaling and core effectors. The analysis revealed that synergistic action of the transcription factors AP-1, vitamin D receptor and Nuclear Factor-kappaB in cross-activation of multiple pathways, including inflammatory cytokines, complement, clusterin, ephrins, and multiple metabolic pathways. We found that the products of over two thirds of genes linked to glaucoma by genetic analysis can be functionally interconnected into one epistatic network via experimentally-validated interactions. Finally, we built and analyzed an integrative disease pathology network from a combined set of genes revealed in genetic studies, genes differentially expressed in glaucoma and closely connected genes/proteins in the interactome.</p> <p>Conclusion</p> <p>Our results suggest several key biological network modules that are involved in regulating neurotoxicity of reactive astrocytes in glaucoma, and comprise potential targets for cell-based therapy.</p

Bioinformatic and statistical analysis of the optic nerve head in a primate model of ocular hypertension

<p>Abstract</p> <p>Background</p> <p>The nonhuman primate model of glaucomatous optic neuropathy most faithfully reproduces the human disease. We used high-density oligonucleotide arrays to investigate whole genome transcriptional changes occurring at the optic nerve head during primate experimental glaucoma.</p> <p>Results</p> <p>Laser scarification of the trabecular meshwork of cynomolgus macaques produced elevated intraocular pressure that was monitored over time and led to varying degrees of damage in different samples. The macaques were examined clinically before enucleation and the myelinated optic nerves were processed post-mortem to determine the degree of neuronal loss. Global gene expression was examined in dissected optic nerve heads with Affymetrix GeneChip microarrays. We validated a subset of differentially expressed genes using qRT-PCR, immunohistochemistry, and immuno-enriched astrocytes from healthy and glaucomatous human donors. These genes have previously defined roles in axonal outgrowth, immune response, cell motility, neuroprotection, and extracellular matrix remodeling.</p> <p>Conclusion</p> <p>Our findings show that glaucoma is associated with increased expression of genes that mediate axonal outgrowth, immune response, cell motility, neuroprotection, and ECM remodeling. These studies also reveal that, as glaucoma progresses, retinal ganglion cell axons may make a regenerative attempt to restore lost nerve cell contact.</p