John P. M. Wood

Genome-wide association study identifies susceptibility loci for open angle glaucoma at TMCO1 and CDKN2B-AS1

We report a genome-wide association study for open-angle glaucoma (OAG) blindness using a discovery cohort of 590 individuals with severe visual field loss (cases) and 3,956 controls. We identified associated loci at TMCO1 (rs4656461[G] odds ratio (OR) = 1.68, P = 6.1 × 10−10) and CDKN2B-AS1 (rs4977756[A] OR = 1.50, P = 4.7 × 10−9). We replicated these associations in an independent cohort of cases with advanced OAG (rs4656461 P = 0.010; rs4977756 P = 0.042) and two additional cohorts of less severe OAG (rs4656461 combined discovery and replication P = 6.00 × 10−14, OR = 1.51, 95% CI 1.35–1.68; rs4977756 combined P = 1.35 × 10−14, OR = 1.39, 95% CI 1.28–1.51). We show retinal expression of genes at both loci in human ocular tissues. We also show that CDKN2A and CDKN2B are upregulated in the retina of a rat model of glaucoma.

A hypothesis to explain ganglion cell death caused by vascular insults at the optic nerve head: possible implication for the treatment of glaucoma

Some apparent characteristics of ganglion cell death in glaucoma Glaucoma is a progressive optic neuropathy with characteristic optic disc changes and associated visual field defects.1 2 The pattern and progression of visual field loss due to ganglion cell death varies between glaucoma patients suggesting that there is some variability in the magnitude of the insult responsible for the cell loss. Ganglion cell death with a spatial and temporal distribution typical of “glaucoma” can be experimentally induced in animals.3-5 One way to try and mimic glaucoma in experimental animals is to raise the intraocular pressure (IOP).3-6 It is clear from such studies that ganglion cells do not all die at the same time.4-8Furthermore, the rate of deterioration of ganglion cells is proportional to the magnitude of the insult.9 The reason for the initiation of ganglion cell death in glaucoma is unknown, but a number of explanatory theories have been proposed with the vasogenic theory perhaps the most widely accepted hypothesis.2 10-12 Raised IOP is not the sole factor responsible for glaucomatous retinal damage but an important one of a number that have been implicated (Fig 1).12-17 Only 10% of patients with increased IOP (⩾22 mm Hg) have glaucoma and between one third and one half of patients with glaucoma initially do not have elevated IOP.1 18 19 Furthermore, as many as one sixth of patients with glaucomatous damage do not appear to have elevated IOP.1 It is therefore clear that raised IOP is not synonymous with having glaucoma.1 20 21 Nevertheless, high IOP is arguably the most important risk factor (see Fig 1), and it is clearly associated with ganglion cell death in glaucoma patients. Figure 1 Possible causes of ganglion cell death in glaucoma. It is suggested that activation of …

Ganglion cell death in glaucoma: what do we really know?

Glaucomatous optic neuropathy is a chronic process which progresses over many years. Data derived from clinical observations and from animal experiments suggest that the axons of the optic nerve and the retinal ganglion cell somata do not die at the same time but that death can vary between months and many years.1 2 Glaucoma patients have characteristic fields of visual loss which enlarge as the disease progresses. Thus, glaucomatous optic neuropathy may not be a chronic degeneration of the whole of the optic nerve and ganglion cell somata but rather a series of acute losses of individual, or groups of, ganglion cells. It seems therefore reasonable to assume that when a patient is diagnosed initially as having glaucoma, only some ganglion cells are dead, whereas others may range from being “unhealthy” to being “slightly sick” while others are “perfectly normal”. It seems also reasonable to argue that if a neuroprotectant (a substance which reaches the retina to elicit an effect: Table 1) can be applied at some stage before blindness occurs it could be of benefit to the glaucoma patient by either “slowing down” the death process of neurons that has already been initiated to die or perhaps by preventing the initiation of death signals to perfectly healthy ganglion cells. This argument can be made despite the lack of success in the use of neuroprotectants for a variety of other diseases (stroke, epilepsy, Parkinson’s disease, AIDS dementia) where neuronal death is a characteristic.1 3 It is the apparent nature of ganglion cell death in glaucoma, very slow and variable, that makes it more likely that the use of neuroprotectants could be successful. View this table: Table 1 The term neuroprotection in glaucoma implies that the substance used to “protect” the ganglion cells reaches the retina (“direct neuroprotection”) to have an effect. In contrast, …

Electrophysiological, Electroanatomical, and Structural Remodeling of the Atria as Consequences of Sustained Obesity

BACKGROUND Obesity and atrial fibrillation (AF) are public health issues with significant consequences. OBJECTIVES This study sought to delineate the development of global electrophysiological and structural substrate for AF in sustained obesity. METHODS Ten sheep fed ad libitum calorie-dense diet to induce obesity over 36 weeks were maintained in this state for another 36 weeks; 10 lean sheep with carefully controlled weight served as controls. All sheep underwent electrophysiological and electroanatomic mapping; hemodynamic and imaging assessment (echocardiography and dual-energy x-ray absorptiometry); and histology and molecular evaluation. Evaluation included atrial voltage, conduction velocity (CV), and refractoriness (7 sites, 2 cycle lengths), vulnerability for AF, fatty infiltration, atrial fibrosis, and atrial transforming growth factor (TGF)-β1 expression. RESULTS Compared with age-matched controls, chronically obese sheep demonstrated greater total body fat (p < 0.001); LA volume (p < 0.001); LA pressure (p < 0.001), and PA pressures (p < 0.001); reduced atrial CV (LA p < 0.001) with increased conduction heterogeneity (p < 0.001); increased fractionated electrograms (p < 0.001); decreased posterior LA voltage (p < 0.001) and increased voltage heterogeneity (p < 0.001); no change in the effective refractory period (ERP) (p > 0.8) or ERP heterogeneity (p > 0.3). Obesity was associated with more episodes (p = 0.02), prolongation (p = 0.01), and greater cumulative duration (p = 0.02) of AF. Epicardial fat infiltrated the posterior LA in the obese group (p < 0.001), consistent with reduced endocardial voltage in this region. Atrial fibrosis (p = 0.03) and TGF-β1 protein (p = 0.002) were increased in the obese group. CONCLUSIONS Sustained obesity results in global biatrial endocardial remodeling characterized by LA enlargement, conduction abnormalities, fractionated electrograms, increased profibrotic TGF-β1 expression, interstitial atrial fibrosis, and increased propensity for AF. Obesity was associated with reduced posterior LA endocardial voltage and infiltration of contiguous posterior LA muscle by epicardial fat, representing a unique substrate for AF.

Microglial Activation in the Visual Pathway in Experimental Glaucoma: Spatiotemporal Characterization and Correlation with Axonal Injury

PURPOSE Glia are the main cellular CNS elements initiating defense mechanisms against destructive influences and promoting regenerative processes. The aim of the current work was to characterize the microglial response within the visual pathway in a rat model of experimental glaucoma and to correlate the microglial response with the severity of axonal degeneration. METHODS Experimental glaucoma was induced in each right eye of adult Sprague-Dawley rats by translimbal laser photocoagulation of the trabecular meshwork. Rats were subsequently killed at various times from 3 days to 6 weeks. Tissue sections were obtained from globes, optic nerves, chiasmata, and optic tracts for immunohistochemistry and toluidine blue staining. RESULTS This model of experimental glaucoma led to a marked activation of microglia in the retina, optic nerve, and tract. Indeed, microglial activity remained elevated, even after intraocular pressure returned to basal levels. It is postulated that this process accompanies ongoing axonal degeneration. The degree of activation in the optic nerve correlated with axonal damage. Activation was characterized by increased density and morphologic changes. Both major histocompatibility complex (MHC) class I and MHC class II surface proteins were persistently upregulated in optic nerves and localized to microglial cells; however, this did not correlate with any significant T-cell infiltration. Interestingly, MHC class II expression was not detected in the retina. CONCLUSIONS The present data may have implications for the study of the pathology associated with the visual pathway in diseases such as glaucoma.

Pharmacological Neuroprotection for Glaucoma

Glaucoma represents a group of neurodegenerative diseases characterised by structural damage to the optic nerve and slow, progressive death of retinal ganglion cells (RGCs). Elevated intraocular pressure is traditionally considered to be the most important risk factor for glaucoma, and treatment options for the disease have hitherto been limited to its reduction. However, visual field loss and RGC death continue to occur in patients with well controlled intraocular pressures and, thus, a consensus has recently emerged that additional treatment strategies are needed.One such strategy is pharmacological neuroprotection, which in the context of glaucoma, refers to the situation in which a drug is deployed to interact with neuronal or glial elements within the retina/optic nerve head and thereby facilitate the survival of RGCs. The advent of animal models of chronic glaucoma has enhanced our understanding of many of the pathological processes occurring in glaucoma and, in doing so, described logical targets for pharmacological intervention. Such targets, which have been manipulated with varying degrees of success in relevant animal paradigms include glutamate receptors, autoimmune elements, neurotrophin deprivation, nitric oxide synthesis, oxidative stress products, sodium and calcium channels, heat shock proteins and apoptotic pathways.With exciting data now emerging from many research laboratories, it is obvious that pharmacological neuroprotection for glaucoma without doubt represents an exciting development in the search for a treatment modality for this debilitating disease.

A comparison of differentiation protocols for RGC-5 cells.

PURPOSE. Although the RGC-5 cell line is widely used in retinal ganglion cell (RGC) research, recent data have raised questions about the nature of these cells. The authors performed a systematic analysis of RGC-5 cells to determine which RGC or neuronal markers are expressed after treatment with known differentiating agents, thus providing further insight into the nature of these cells and assisting in defining their future use. METHODS. RGC-5 cells were treated for 5 days with staurosporine (STSN; 316 nM), trichostatin A (TSA; 500 nM), or succinyl-concanavalin A (sConA; 50 microg/mL), after which they were assayed for specific marker antigen/mRNA expression. Treated cells were also assayed for excitotoxic responsiveness. RESULTS. Neither treated nor untreated RGC-5 cells expressed any specific RGC marker mRNAs or proteins (Brn-3, neurofilaments, Thy-1) or calbindin, calretinin, synaptophysin, PKCalpha, or glial fibrillary acidic protein. However, control RGC-5 cells did express the neuronal markers tau, betaIII-tubulin, microtubule-associated protein (MAP)-1b, MAP2, and PGP9.5. Although treatment with sConA had no effect on the expression of these markers, STSN and (dose dependently) TSA increased their expression and induced excitotoxic responsiveness. All cells, treated or not, expressed high levels of nestin but no other progenitor cell markers. All cells also expressed cone-specific, but not rod-specific, opsin indicative of cone photoreceptor lineage. CONCLUSIONS. RGC-5 cells expressed neuronal, but not RGC-specific, markers that were dose dependently upregulated by TSA. Hence, TSA provided the best tested means to terminally differentiate the cells to a neuronal phenotype from a precursor-like lineage.

Expression of osteopontin in the rat retina: effects of excitotoxic and ischemic injuries.

PURPOSE The cytokine osteopontin (OPN) has been localized to the retinal ganglion cell layer in the normal rodent retina, prompting the suggestion that it could serve as a useful marker for identifying and quantifying such neurons in models of retinal and optic nerve neurodegeneration. In the present study, we characterized the time course and cellular localization of OPN expression in the rat retina after excitotoxic and ischemic injuries. METHODS Excitotoxicity and ischemia-reperfusion experiments were performed by using standard techniques. Rats were killed at various time points, and the retinas were removed either for mRNA analysis or to be processed for immunohistochemistry. RESULTS In the normal retina, double-labeling immunofluorescence indicated that OPN is expressed by the majority of, if not all, RGCs, since OPN was associated with more cells than Brn-3, but was colocalized with Thy1.1. NMDA, kainic acid, and ischemia-reperfusion all caused decreases in the total retinal levels of Thy1 and Brn-3 mRNAs, reflecting injury to RGCs, but a dramatic, short-lived upregulation in OPN mRNA. The source of the increased OPN signal after excitotoxic-ischemic insults is unlikely to be injured RGCs, as no alteration in the intensity of OPN immunostaining in RGCs was apparent. Instead, additional cells, mostly contained within the IPL, were identified as positive for OPN. Double-labeling immunofluorescence showed that ED1 always colocalized with OPN in these cells, indicating their status as activated microglia. CONCLUSIONS OPN is exclusively expressed by RGCs in the physiological retina, but in response to retinal neurodegeneration is synthesized de novo by endogenous, activated microglia.

Expectations in the treatment of retinal diseases: neuroprotection.

Loss of vision leading to blindness is commonly due to photoreceptor or ganglion cell malfunction. No evidence exists to suggest that a lesion associated with other retinal celltypes (Müller cells, horizontal cells, bipolar cells and amacrine cells) can directly lead to blindness. However, in ischaemia for example, impairment of Müller cell and astrocyte functions could lead to the death of retinal neurones including photoreceptors and ganglion cells. A challenge in ophthalmology is therefore to find ways of attenuating photoreceptor or ganglion cell injury as occurs in various diseases. With the increased knowledge of genetics and the finding that gene malfunctions are associated with certain retinal diseases, the use of gene therapy as a method for attenuating loss of photoreceptor function in retinitis pigmentosa and age-related macular degeneration has become a possibility. Great progress has been made in this area in animal studies and it is only a matter of time before gene therapy is tested on humans. Retinal transplantation technology has also advanced over recent years and the results of animal studies have shown some promise, but the visual results from limited human studies have thus far been disappointing. The use of stem cells may provide new opportunities in this area. Many studies have suggested that altered blood flow dynamics in defined ocular blood vessels are a cause for ganglion cell or photoreceptor death in diseases such as glaucoma, ischaemic optic neuropathy, diabetic retinopathy, age-related macular degeneration and retinitis pigmentosa. As a consequence, great efforts are being made to correlate changes in ocular blood flow characteristics with the loss of ganglion cell and/or photoreceptor function in defined diseases so that appropriate treatments can be undertaken. Advancement in this area, however, continues to be slow and controversial. This is because measuring ocular blood dynamics in defined microvessels needs to be performed on human subjects and thus is often indirect, so that interpretation of information is difficult. The use of chemicals or pharmacological agents to attenuate whatever causes the death of photoreceptors or ganglion cells in the various blinding diseases is another procedure worthy of consideration and is the subject of this article. Support for this approach is very strong but is based almost entirely on animal studies. The word “neuroprotection” in ophthalmology has been primarily associated with ganglion cell survival and is hardly used in connection with photoreceptor survival, perhaps because photoreceptors lack certain neurone characteristics (they do not stain positive for neurone-specific proteins like PGP 9.5). The term neuroprotection is applied here to both photoreceptors and ganglion cells.

Translational neuroprotection research in glaucoma: a review of definitions and principles

The maintenance of vision, through prevention and attenuation of neuronal injury in glaucoma, forms the basis of current clinical practice. Currently, the reduction of intraocular pressure is the only proven method to achieve these goals. Although this strategy enjoys considerable success, some patients progress to blindness; hence, additional management options are highly desirable. Several terms describing treatment modalities of neuronal diseases with potential applicability to glaucoma are used in the literature, including neuroprotection, neurorecovery, neurorescue and neuroregeneration. These phenomena have not been defined within a coherent framework. Here, we suggest a set of definitions, postulates and principles to form a foundation for the successful translation of novel glaucoma therapies from the laboratory to the clinic.