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From CellBiology

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Methodology

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Preliminary Results

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Introduction

Glaucoma

Glaucoma is a type of optic neuropathy involving the loss of retinal ganglion cells (RGCs) with concomitant loss of visual field. It is the second most common cause of reversible vision loss, after cataracts, but the leading cause of irreversible vision loss. Prevalence of glaucoma lies around 1.7-3.0% of the general population.

There are generally two types of glaucoma: open angle glaucoma and closed angle. The two differ in their typical presentations. The most common type of glaucoma is open angle, also known as Primary Open Angle Glaucoma (POAG). It accounts for around 90% of glaucoma cases. POAG typically demonstrates insidious onset and is often asymptomatic. It affects peripheral vision first then central vision. Patients can lose up to 40% of retinal cell axons before measurable visual defects become apparent. the other type of glaucoma is closed angle. It generally presents with acute onset of pain and is a medical emergency that requires prompt treatment.

The pathophysiology behind glaucoma is not well known. It is typically associated with raised intraocular pressure (IOP) (>22mmHg) and management often involves reducing IOP. However. raised IOP though is not a requirement for glaucoma. Non-raised IOP glaucoma is referred to as Normal Tension Glaucoma (NTG)

Sleep Apnea

Sleep Apnea is a type of sleep disorder characterised by episodic airflow cessation resulting in multiple microarousals from sleep. Different subtypes exist, though generally refers to Obstructive Sleep Apnea. Each episode is referred to as an Apnea. It is an often underappreciated disorder that can induce systemic changes. Pulmonary hypertension, cardiovascular events and all cause mortality are some of the few sequelae that results depending on severity of Apnea.

Commonly presents with snoring and daytime somnolence.

Association between Glaucoma and Sleep Apnea

Controversy exists as to whether there is an association. Recent studies conducted by Mojon et al suggest increased prevalence of Glaucoma in Sleep Apnea population and vice versa compared to general population.

Despite the links shown in studies, however, the pathophysiological mechanisms between them remains unknown. We propose that changes in PCO2 induced through disturbances in respiratory function in apnea can subsequently affect pH levels through formation of carbonic acid. These changes may induce apoptosis in RGCs.

Literature suggests that under certain oxygen conditions, pH can have a neurotoxic effect on neural cells. We explore the effects different levels of pH and exposure times can have on RGC survival.

Main Study Findings

Due to the time limitations of the ILP, this was mainly run as a pilot study with discussion of preliminary results. The main findings of these results are discussed below.

Morphological Apoptosis Detection

Examination of fixed and stained cells revealed morphology consistent with apoptosis: reduced cell size, nuclear condensation, membrane blebbing and process retraction. These were seen in positive controls and largely absent in negative controls. In addition, varying levels of this was seen across the experiment pH groups suggesting a spectrum of apoptotic activity across them. This was consistent with concomitant levels of fluorescent cleaved-caspase 3 staining.

Variations in Control Apoptotic Indices

AI for pH groups were largely within the AI between the positive and negative controls. This was expected given that controls represented the extremities of apoptotic activity. However, of concern was the AI range between the controls. Some negative controls were observed with AIs in the range of 10-50%, with positive control AIs consistently around 40-50% higher than their negative counterpart. The variations were disconcerting and ultimately were ascribed to methodology, specifically variations in staining detection introduced through custom pixel intensity thresholds used for each separate time-period run. This has implications for how results can be interpreted. Although any patterns or trends observed across pH groups in each experiment time group is still meaningful, apoptosis detection through intensity thresholding limits our ability to compare between experiment time groups.

Increased RGC AI with Low pH Conditions

Certainly this trend appeared in our preliminary results suggesting pH conditions resulted in noticeable, proportional changes in the RGC apoptosis. This is an important finding as it supports changes in pH as a potential mechanism linking sleep apnea and glaucoma.

Reductions in Total Cell Number

Total cells were found to decrease dramatically with lower pH's, with this trend becoming much more apparent with longer exposure times. Cell numbers dropped significantly between the 2 and 3-hour time periods. There could be a number of possible reasons but we consider the following to be specifically pertinent: duration of apoptosis for RGCs have not been comprehensively characterised. It is also known that this duration can vary depending on the type of apoptotic stimulus -- in this case pH changes. The results seem to suggest RGCs are completing apoptosis, disintegrating and becoming increasingly difficult to detect between 2-3 hours. This was supported by the fact that cell fragments were observed in greater frequency and were more evident as the exposure time increased.

Recommendations

Increased sample sizes

Our preliminary data suffered from small sample sizes and hence it was difficult to draw statistical conclusions. Further studies would benefit from larger sample sizes, especially given that the patterns and trends seem in the preliminary results seem encouraging.

Change in Apoptosis Detection Method

Some of the variations in control AIs pointed to possible underlying concerns in the current apoptosis detection method, specifically the use of custom intensity threshold values for each time-period run. This ultimately reduced our ability to compare inter-time AI results (though intra-time period results and trends were still meaningful). We suggest using a stathmo-apoptotic assay for future studies, which slow down and halt progress of apoptosis in cells. Carboxyfluorescein-labeled peptide fluoromethyl ketone (FAM-VAD-FMK) is used. This is a generic type of fluorescent inhibitor of caspase (FLICA) that binds irreversibly to multiple active caspases (casp-1, -3, -4, -5, -6, -7, -8 and -9). FAM-VAD-FMK remains steadily bound even after fixation, which makes it ideal for arresting cells entering apoptosis in live cultures. Cells can be drawn off at regular time intervals and analysed through flow cytometry. The proportion of apoptotic cells can be plotted against time to construct a cumulative apoptotic index (CAI). This gives us information not only on the total proportion of cells undergoing apoptosis, but also on cell entry rates into apoptosis. Importantly, this also eschews many of the shortcomings of using cells where the apoptosis characteristics are not well known -- such as duration with pH stimuli in this instance.

Use of Differentiated RGCs

Variations in cell culture density were observed in preliminary results with some suspicion that this could be caused by possible variations in cell cycle phases among the RGCs. A differentiation protocol had been carried out with success during the study (though we did not have time to include them in the study). This was carried out through temporary serum deprivation with later addition of succinyl concanavalin A (sConA). Using differentiated RGCs would reduce the introduction of a potential extra variable.