Oral microbiome link to neurodegeneration in glaucoma.

BACKGROUND: Glaucoma is a progressive optic nerve degenerative disease that often leads to blindness. Local inflammatory responses are implicated in the pathology of glaucoma. Although inflammatory episodes outside the CNS, such as those due to acute systemic infections, have been linked to central neurodegeneration, they do not appear to be relevant to glaucoma. Based on clinical observations, we hypothesized that chronic subclinical peripheral inflammation contributes to neurodegeneration in glaucoma. METHODS: Mouthwash specimens from patients with glaucoma and control subjects were analyzed for the amount of bacteria. To determine a possible pathogenic mechanism, low-dose subcutaneous lipopolysaccharide (LPS) was administered in two separate animal models of glaucoma. Glaucomatous neurodegeneration was assessed in the retina and optic nerve two months later. Changes in gene expression of toll-like receptor 4 (TLR4) signaling pathway and complement as well as changes in microglial numbers and morphology were analyzed in the retina and optic nerve. The effect of pharmacologic blockade of TLR4 with naloxone was determined. FINDINGS: Patients with glaucoma had higher bacterial oral counts compared to control subjects (p<0.017). Low-dose LPS administration in glaucoma animal models resulted in enhancement of axonal degeneration and neuronal loss. Microglial activation in the optic nerve and retina as well as upregulation of TLR4 signaling and complement system were observed. Pharmacologic blockade of TLR4 partially ameliorated the enhanced damage. CONCLUSIONS: The above findings suggest that the oral microbiome contributes to glaucoma pathophysiology. A plausible mechanism by which increased bacterial loads can lead to neurodegeneration is provided by experiments in animal models of the disease and involves activation of microglia in the retina and optic nerve, mediated through TLR4 signaling and complement upregulation. The finding that commensal bacteria may play a role in the development and/or progression of glaucomatous pathology may also be relevant to other chronic neurodegenerative disorders.

Complement expression in the retina is not influenced by short-term pressure elevation.

PURPOSE: To determine whether short-term pressure elevation affects complement gene expression in the retina in vitro and in vivo. METHODS: Muller cell (TR-MUL5) cultures and organotypic retinal cultures from adult mice and monkeys were subjected to either 24-h or 72-h of pressure at 0, 15, 30, and 45 mmHg above ambient. C57BL/6 mice were subjected to microbead-induced intraocular pressure (IOP) elevation for 7 days. RNA and protein were extracted and used for analysis of expression levels of complement component genes and complement component 1, q subcomponent (C1q) and complement factor H (CFH) immunoblotting. RESULTS: mRNA levels of complement genes and C1q protein levels in Muller cell cultures remained the same for all pressure levels after exposure for either 24 or 72 h. In primate and murine organotypic cultures, pressure elevation did not produce changes in complement gene expression or C1q and CFH protein levels at either the 24-h or 72-h time points. Pressure-related glial fibrillary acidic protein (GFAP) mRNA expression changes were detected in primate retinal organotypic cultures (analysis of variance [ANOVA]; p<0.05). mRNA expression of several other genes changed as a result of time in culture. Eyes subjected to microbead-induced IOP elevation had no differences in mRNA expression of complement genes and C1q protein levels (ANOVA; p>0.05 for both) with contralateral control and naïve control eyes. CONCLUSIONS: Short-term elevation of pressure in vitro as well as short-term (1 week) IOP elevation in vivo does not seem to dramatically alter complement system gene expression in the retina. Prolonged expression to elevated pressure may be necessary to affect the complement system expression.

Gene expression changes in steroid-induced IOP elevation in bovine trabecular meshwork.

PURPOSE: To determine whether gene expression changes occur in the trabecular meshwork (TM) of cow eyes with steroid-induced intraocular pressure (IOP) elevation. METHODS: Adult female Braford cows (n = 4) were subjected to uniocular prednisolone acetate treatment for 6 weeks. IOP was monitored with an applanation tonometer. At the conclusion of the experiment, animals were euthanized, eyes were enucleated, and the TM was dissected and stored in an aqueous nontoxic tissue storage reagent. RNA was extracted and subjected to microarray analysis using commercial oligonucleotide bovine arrays. Some of the genes differentially expressed between control and experimental eyes were confirmed by quantitative RT-PCR and some of the respective proteins were studied by immunoblotting. RESULTS: IOP began to increase after 3 weeks of treatment, reaching a peak 2 weeks later. IOP differences between corticosteroid-treated and fellow control eyes were 6 ± 1 mm Hg (mean ± SD) at the conclusion of the study. Microarray analysis revealed that expression of 258 genes was upregulated, whereas expression of 187 genes was downregulated in the TM of eyes with steroid-induced IOP elevation. Genes identified to be differentially expressed include genes coding for cytoskeletal proteins, enzymes, growth and transcription factors, as well as extracellular matrix proteins and immune response proteins. A number of relevant gene networks were detected by bioinformatic analysis. CONCLUSIONS: Steroid-induced IOP elevation alters gene expression in the bovine TM. Identification of genes with changing expression in this model of open-angle glaucoma may help elucidate the primary changes occurring at the molecular level in this condition.

Retinal gene expression changes related to IOP exposure and axonal loss in DBA/2J mice.

PURPOSE: To determine the effects of cumulative IOP exposure and axonal damage on retinal gene expression in DBA/2 mice. METHODS: DBA/2J, DBA/2J(pe) (pearl), and C57BL/6 mice from 3 to 12 months of age were used. IOP was measured with a rebound tonometer, and optic nerve (ON) damage was determined by grading of ON sections. Retinal RNA was subjected to microarray analysis. Comparisons explored the effects of cumulative IOP exposure (cIOPx) as well as ON damage (ONd) in the DBA/2J animals compared with that in the C57BL/6 and pearl mice. RT-PCR was performed to confirm some of the genes and bioinformatic analysis to identify affected gene networks. RESULTS: Microarrays revealed that an increasing number of genes were up- or downregulated in 9- and 12-month DBA/2J mice with various degrees of ONd. A smaller number of genes were expressed differentially between eyes with different cIOPx at the same age, from 6 months on. Expression of 1385 and 1133 genes differed between DBA/2J animals and C57BL/6 or pearl mice, respectively, and some them were confirmed by RT-PCR. Bioinformatics analysis identified functional gene networks, including members of the complement system, that appeared to be related to cIOPx, ONd, or both. CONCLUSIONS: Gene expression changes occur in retinas of DBA/2 mice with various amounts of cIOPx as well as ONd. Genes involved, code for proteins with diverse cellular functions and include among others the complement system. cIOPx and ONd affect common as well as unique gene sets.

A role for complement in glaucoma?

Chronic open angle glaucoma is a degenerative optic neuropathy that can lead to blindness. We have shown that one of the major genes with altered expression in the glaucomatous retina is complement component C1q in both animal models of the disease as well as in humans. These observations together with evidence of upregulation of other complement components within the retina suggest a role for complement in the pathogenesis of this disease. We review the current evidence that supports such a role and discuss possible mechanisms through which complement may act. A thorough understanding of these mechanisms is important in allowing us to rationally design new therapeutic approaches.

Gene expression changes in areas of focal loss of retinal ganglion cells in the retina of DBA/2J mice.

Purpose. To determine whether differences in gene expression occur between areas of focal retinal ganglion cell (RGC) loss and of relative RGC preservation in the DBA/2 mouse retina and whether they can provide insight into the pathophysiology of glaucoma. Methods. Areas of focal RGC loss (judged by lack of Fluorogold labeling; Fluorochrome, Denver, CO), adjacent areas with relative RGC preservation in DBA/2 retina, and Fluorogold-labeled retina from DBA/2(-pe) (pearl) mice were dissected and used for microarray analysis. RT-PCR and immunoblot analysis were used to confirm differential gene expression. Bioinformatic analysis was used to identify gene networks affected in the glaucomatous retina. Results. Microarray analysis identified 372 and 115 gene chip IDs as up- and downregulated, respectively, by 0.5-fold in areas of RGC loss. Differentially expressed genes included those coding for cytoskeletal proteins, enzymes, transport proteins, extracellular matrix (ECM) proteins, and immune response proteins. Several genes were confirmed by RT-PCR. For at least two genes, differential protein expression was verified. Bioinformatics analysis identified multiple affected functional gene networks. Pearl mice appeared to have significantly different gene expression, even when compared with relatively preserved areas of the DBA/2 retina. Conclusions. Regional gene expression changes occur in areas of focal RGC loss in the DBA/2 retina. The genes involved code for proteins with diverse cellular functions. Further investigation is needed to determine the cellular localization of the expression of these genes during the development of spontaneous glaucoma in the DBA/2 mouse and to determine whether some of these gene expression changes are causative or protective of RGC loss.

Ceruloplasmin upregulation in retina of murine and human glaucomatous eyes.

PURPOSE: Ceruloplasmin (Cp) expression is increased locally as a response to many neurodegenerative conditions. The purposes of this study were to confirm findings of Cp upregulation in glaucoma, detect the time course of this upregulation in a glaucoma model, and better localize its expression in the retina. METHODS: mRNA and protein were extracted from the retina and brain of DBA/2 and C57BL/6 mice and were subjected to analysis by RT-PCR and immunoblotting. In addition, eyes from the same mouse strains were subjected to immunohistochemistry using antibodies specific for Cp. Eyes from human subjects with or without glaucoma were also subjected to immunohistochemical analysis for Cp. RESULTS: Cp mRNA and Cp protein were upregulated in the retinas of glaucomatous DBA/2 mice. Upregulation of Cp occurred at approximately the time of extensive retinal ganglion cell (RGC) death and increased with increasing age to 15 months in the retinas but not in the brains of these animals. No age-related Cp upregulation was detected in the reference normal mouse strain (C57BL/6), which can develop significant nonglaucomatous RGC loss toward the end of the same time frame. Cp upregulation was also detected in most eyes from the patients with glaucoma. Cp upregulation was localized to the Müller cells within the retinas and in the area of the inner limiting membrane. CONCLUSIONS: Cp is upregulated in the retina of a commonly used glaucoma model (the DBA/2 mouse) and in most human glaucomatous eyes. The timing of this upregulation suggests that it may represent a reactive change of the retina in response to a noxious stimulus or to RGC death. Such Cp upregulation may represent a protective mechanism within the retina.

Apoptotic retinal ganglion cell death in the DBA/2 mouse model of glaucoma.

The DBA/2 mouse has been used as a model for spontaneous secondary glaucoma. We attempted to determine the in vivo time course and spatial distribution of retinal ganglion cells (RGCs) undergoing apoptotic death in DBA/2 mice. Female DBA/2 mice, 3, 9-10, 12, 15, and 18 months of age, received intravitreal injections of Annexin-V conjugated to AlexaFluor 1h prior to euthanasia. Retinas were fixed and flat-mounted. Annexin-V-positive RGCs in the hemiretina opposite the site of injection were counted, and their locations were recorded. Positive controls for detection of apoptotic RGCs by Annexin-V labeling included rats subjected to optic nerve ligation, and C57BL/6 mice subjected to either optic nerve ligation or intravitreal injection of NMDA. To verify that Annexin-V-labeled cells were RGCs, intravitreal Annexin-V injections were also performed on retinas pre-labeled retrogradely with FluoroGold or with DiI. Annexin-V-positive RGC locations were analyzed to determine possible clustering and areas of preferential loss. Annexin-V labeled apoptotic RGCs in eyes after optic nerve ligation, intravitreal NMDA injection, as well as in aged DBA/2 animals. In glaucomatous DBA/2 mice 95-100% of cells labeled with Annexin-V were also FluoroGold- and DiI-positive. This confirms that Annexin-V can be used to specifically detect apoptotic RGCs in rodent retinas. In DBA/2 mice, apoptotic RGC death is maximal from the 12th to the 15th month of age (ANOVA, p<0.001, Fisher's post hoc test) and occurs in clusters. These clusters are initially located in the midperipheral retina and progressively occur closer to the optic nerve head with increasing age. Retrograde axonal transport of FluoroGold in the glaucomatous mouse retina is functional until at least 2-3days prior to initiation of apoptotic RGC death.

Complement component 1Q (C1Q) upregulation in retina of murine, primate, and human glaucomatous eyes.

PURPOSE: Complement has been implicated in the pathogenesis of neurodegenerative diseases. The purpose of this study was to investigate whether complement activation is part of the pathogenesis of retinal ganglion cell (RGC) loss in glaucoma. METHODS: mRNA and protein was extracted from the retina and brain of DBA/2 and C57/BL6 mice and subjected to RT-PCR and immunoblot analysis, respectively. In addition, eyes from the same mouse strains were subjected to immunohistochemistry with antibodies specific to complement component 1q (C1q). Eyes from monkeys with unilateral experimental glaucoma were also subjected to immunohistochemical analysis, as were eyes from human subjects with or without glaucoma. RESULTS: C1q mRNA and C1q protein were found to be upregulated in the retina of glaucomatous DBA/2 mice. Upregulation of C1q preceded the time of extensive RGC death and increased with increasing age to 15 months in the retina, but not in the brain. No age-related C1q upregulation was detected in the reference mouse strain (C57BL/6), which develops significant nonglaucomatous RGC loss toward the end of the same time frame. C1q upregulation was also detected in laser-induced glaucomatous monkey eyes and in some (but not all) eyes of patients with glaucoma. C1q upregulation was localized to the Müller cells within the retina and in the area of the inner limiting membrane. CONCLUSIONS: Complement expression is upregulated in the retina of two commonly used glaucoma models (in the DBA/2 mouse and the monkey) and in some human glaucomatous eyes. The timing of this upregulation suggests that complement activation plays a significant role in the pathogenesis of glaucoma.

Predictability and limitations of non-invasive murine tonometry: comparison of two devices.

Our purpose was to evaluate the accuracy, reproducibility and predictive ability of two non-invasive tonometers developed for intraocular pressure (IOP) measurements in the mouse. The prototype impact-rebound tonometer (I-R) and a prototype optical interferometry tonometer (OIT) utilizing a fiberoptic pressure sensor, were compared. Enucleated eyes from C57/BL6 mice were used for the calibration. The anterior chamber was cannulated and the IOP was adjusted in increments of 5 cm of H2O (open stopcock method). A calibration curve was generated for each individual eye along with a master calibration curve for all eyes. Two operators measured the IOP. The instruments were then used in alternating order to measure the IOP in C57/BL6 and in DBA2/J animals. The same eyes were subsequently cannulated and the error of the non-invasive tonometers was determined. Both tonometers yielded almost equivalent ex vivo calibration curves with individual R2 of 0.9878 and 0.9902 respectively. Both instruments were highly reproducible. In vivo the I-R tonometer underestimated while the OIT overestimated the IOP. This error was systematic and therefore predictable. The confidence intervals of this error were determined by comparing the IOP estimates provided by each tonometer with the measurements obtained invasively by cannulation in vivo. The 95% CI of the error were 2.36 mmHg for the I-R and 2.62 mmHg for the OIT respectively. Non-invasive tonometry in the mouse is feasible. Both non-invasive instruments provide accurate and reproducible measurements with the OIT requiring calibration curves for each individual investigator.