- Title
- An investigation of the role of the microbiome in the development of glaucoma
- Creator
- McPherson, Zachary Edward
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2020
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Glaucoma is a neurodegenerative illness of the optic nerve with only one treatment pathway due to the lack of clear modifiable factors. Amongst its pathophysiological mechanisms, neurotrophic factor deprivation [particularly of Brain Derived Neurotrophic Factor (BDNF)] and inflammation are mechanisms that may present therapeutic opportunity. Safely modulating the endogenous neurotrophic mechanisms or immune pathways may be suitable therapeutic pathways in future. The microbiome is now clearly understood to be crucial to the development of the host. In animal research the links between microbiome status and host physiology are becoming increasingly clear. It is now known that the microbiome plays an important role in the central nervous system with the ability to regulate neurotrophins and the neuro-immune system (amongst other mechanisms). As these mechanisms are important in glaucoma pathophysiology, the central hypothesis of this study is that the microbiome contributes to glaucoma. This thesis presents a series of studies that begin the process of linking glaucoma to the microbiome. The research presented in this thesis falls broadly into two categories: human observational epidemiology and experimental animal research. Epidemiological Research: As human microbiome research represents a data analysis problem, illnesses that are clearly related to abnormal microbiome should be useful markers of altered microbiome in epidemiological research. Irritable Bowel Syndrome (IBS) and dental illness are both very strongly correlated to abnormal microbiome in the gastrointestinal tract and the oral cavity, respectively. The first study (Chapter 3) aimed to quantify the prevalence of IBS in an Australian cohort of glaucoma sufferers as compared to the general Australian population. Participants from the Australia and New Zealand Registry of Advanced Glaucoma (n=1021) and a population representative cohort, the Hunter Community Study (n=2251), returned a mailed survey with the ROME-III criteria for the diagnosis of IBS. The participants with glaucoma were also significantly more likely to have ROME-III defined IBS [Odds Ratio (OR) 1.93, 95% Confidence Interval (CI) 1.52-2.44]. The second study (Chapter 4) aimed to identify and quantify an increased incidence of glaucoma in people with IBS in two large population based European cohorts. In the 1958 UK Birth Cohort, participants (n=9091) were surveyed regularly regarding their health. Amongst people who had IBS at 42 who continued to report their illness at age 50, the adjusted odds ratio of developing glaucoma in this period was 5.84 (95% CI 2.26-15.13). In the Danish National Patient Register (n=62,541 with IBS, 625,410 general population controls), people with IBS had a hazard ratio (HR) of 1.35 for developing physician-diagnosed glaucoma (95%CI 1.15-1.59), a HR of 1.34 for undergoing surgery for glaucoma (95%CI 1.04-1.74), and a HR of 1.19 for initiating use of glaucoma medication (95%CI 1.02-1.40). These effects were similar in lagged analyses, and when Cholelithiasis was used as a negative control. A third investigation (Chapter 5) was undertaken to identify and quantify the size of an association between dental illness (periodontitis and incidental tooth loss) and the incidence of glaucoma. In the Health Professionals Follow-up Study participants (40,536 men) followed biennially from 1986 to 2012, the number of natural teeth, teeth lost, periodontal disease and root canal treatments was followed with assessment of glaucoma incidence as its outcome. Incident tooth loss was associated with receiving a glaucoma diagnosis in the following two years (Risk Ratio (RR) 1.45, 95% CI 1.06-1.97), especially if the tooth loss was in the context of periodontal disease (RR: 1.85, 95% CI 1.07-3.18). The total number of teeth, periodontal disease (alone) and root canal treatment were not related to glaucoma incidence. Animal Research: Although there are several microbiome manipulation models, Germ Free (GF) mice [when compared to Specific Pathogen Free (SPF) mice and Conventionalized GF (CON) mice] are the best model for assessing the role of the normal microbiome. Similarly, the Optic Nerve Crush (ONC), is a reproducible optic nerve injury model of glaucoma, that allows researchers to investigate the pressure independent mechanisms at work in retinal ganglion cell (RGC) neurodegeneration in mice. In the study presented in Chapter 6, GF, SPF and CON mice were subjected to ONC, and allowed to survive until their retinae were harvested for analysis (up to 3 days for protein analysis, 1 week for qPCR and 5 weeks for cell survival analysis). Immunohistochemistry was used to examine the cell survival, and qPCR and ELISA protein analysis were used to quantify the BDNF levels in the retina, at various time points after the ONC. A further cohort of GF mice were treated with live or heat-killed Lactobacillus probiotic, and its effects on cell survival after ONC were quantified. Finally, a cohort of GF and SPF mice that also received an injection of BDNF protein at the time of ONC and its effects were compared to mice who received a placebo injection. GF mice had significantly worse RGC survival at 7 days (RGC survival of 40.5% compared to 50.4% and 48.4% for SPF and CON mice, respectively, p<0.05) and at 35 days (RGC survival of 11.8% compared to 18.1% and 18.8% for SPF and CON mice, respectively, p<0.05) after initiation of ONC. Probiotic supplementation for GF mice with Lactobacillus plantarum PS128 was able to increase cell survival after ONC. At day 35 after ONC, cell survival in live probiotic treated mice was 16.2% compared to GF mice with 11.8% survival (p=0.04). When the probiotic was heat-killed the RGC cell survival was insignificantly elevated compared to GF mice (12.5%). At day 3 after ONC, it was shown that SPF mice had 34.6% greater expression of BDNF protein as compared to GF mice (p<0.001), however protein levels at baseline and mRNA levels at all timepoints were no different. To evaluate if the differentially expressed BDNF may be responsible for differential cell survival between SPF and GF mice, a single intraocular injection of recombinant BDNF was administered at the time of ONC. The BDNF injection was protective in both SPF and GF mice, and importantly it normalised the cell survival rates between SPF and GF mice after ONC [at day 35, cell survival was 22.4% and 19.9%, respectively (p=0.61)]. Conclusions and Discussion: These epidemiological studies together show that IBS and perhaps dental illness (both illnesses associated with abnormal microbiome), are risk factors for glaucoma. Although the microbiome is not certainly the mechanism linking these entities, as there is limited plausible overlap in the physiology of these illnesses aside from the microbiome these findings are evidence towards the hypothesis that the microbiome is relevant to glaucoma's pathology. The animal research presented demonstrated conclusively that the absence of microbiome leads to poorer outcomes after ONC, an optic nerve injury model of glaucoma. These findings also suggest that microbiome dependant effects on retinal BDNF levels after ONC may be the reasons for this protective effect. Although these findings require further investigation, they also support the hypothesis that the microbiome is involved in neuroprotective mechanisms in glaucoma. In summary, this thesis provides epidemiological evidence that the microbiome may be clinically relevant to glaucoma incidence; also, animal research suggests that a BDNF mediated mechanism could underly this effect.
- Subject
- glaucoma; microbiome; irritable bowel syndrome; germ free; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1438237
- Identifier
- uon:40563
- Rights
- Copyright 2020 Zachary Edward McPherson
- Language
- eng
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