Discussion

4.9.1

Main findings

This study has revealed differences in the response of the retinal vasculature to flicker light between mild newly diagnosed AD patients and healthy age matched controls. Newly diagnosed AD patients took significantly longer to reach the point of maximum arterial dilation on two out of the three occasions in which their retinal vessels were challenged by flickering light and this alteration in response was found to correlate with their degree of cognitive impairment. Furthermore AD patients demonstrated a

consistently increased fluctuation in venous baseline diameter prior to the onset of flicker across all three cycles in comparison to healthy controls. No significant

differences however were found in systemic endothelial dysfunction between groups.

4.9.2

Systemic endothelial function

No significant differences in systemic vascular endothelial function, as measured by FMD, were found between groups in this study. This is in contradiction to previous research by Dede et al (2007) who demonstrated impaired brachial artery FMD in a group of diagnosed AD patients compared to healthy controls 631. Such contradiction in findings between previous research and our present study could partly be accounted for by differences in the measurement protocol and/or differences in the severity of the AD patients assessed. Our present study included only mild newly diagnosed AD patients and therefore it could be hypothesised that, as endothelial dysfunction is known to occur much earlier at the microvascular level than at the macrovascular level in a disease process 734, signs of systemic endothelial dysfunction, although not yet detectable in our newly diagnosed AD patients on FMD, could become more apparent as the disease progresses. In line with this Dede et al 631 found that the degree of endothelial

dysfunction as indicated by FMD correlated with the severity of cognitive impairment in their AD patients, with endothelial dysfunction getting worse as the severity of the

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disease increased. Further research however would be required to confirm this

hypothesis before any firm conclusions can be drawn. Alternatively, it is possible that the small sample size of AD patients included in this study did not provide sufficient power to detect a difference in FMD between groups. Indeed on power analysis it was

identified that a sample size of 12 would be required to provide 80% power at an α level of 0.05, however only 10 AD participants were able to be recruited. Post-hoc analysis, conducted using G-power 3, reveals the sample sizes achieved in this study were in fact only able to provide a power of 69%. This could potentially explain these non-significant findings.

4.9.3

Retinal vessel reactivity

A large body of evidence exists linking AD to the presence of cerebral vascular

dysfunction and highlighting the involvement of the ocular circulation in the AD disease process 3, 567, 617, 620, 621, 668, 669. Furthermore, the easy access to the neural and vascular tissue at the retinal level, as well as the many anatomical and physiological similarities shared by the ocular and cerebral microcirculation, makes the retina an ideal screening target in cerebrovascular disease. The use of techniques such as the DVA method used in the present study, which are aimed at assessing retinal microvascular function, could therefore not only offer information regarding general microvascular function in AD patients but also offer an assessment of the potential risk for future decline and

development of systemic vascular disease. In the present study, DVA analysis revealed how our newly diagnosed mild AD patients without manifest systemic vascular disease took significantly longer to reach the point of maximum dilation on two out of the three occasions in which their retinal vessels were challenged by flickering light. These results indicate that some form of microvascular dysfunction, detectable at the retinal level, does indeed appear to exist in AD patients; the cause of this vascular dysfunction, however, can only be hypothesised at this point.

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The retinal vascular response to flicker light occurs due to an increase in retinal metabolic demand and is predominantly a neurovascular coupling driven response 226,

685, 686, 691, 692_{. It could therefore be hypothesised that the altered retinal vessel reactivity}

demonstrated in our AD patients is indicative of a disturbed neurovascular coupling mechanism, possibly related to endothelial dysfunction, a decreased bioavailability of the vasodilator NO or an alteration in the activity of astrocytes in these patients, all of which are known to be key mediators of the neurovascular response 600. Interestingly, both disturbed neurovascular coupling and dysfunction of the vascular endothelium have been previously linked to the aetiology of AD 631, 632, 735, 736, as have alterations in

astrocyte activity 737-739 and in NO production/release 565, 740. Such alterations in the production/release of NO could additionally be attributed to either cholinergic receptor degeneration in AD 741 and the subsequent reduction in acetylcholine mediated NO release, or to Aß deposition and the subsequent impairment of neuronal NO production. Indeed acetylcholine receptor stimulation and subsequent release of NO has been previously identified to play a role in the retinal vasodilation response to flicker light in rabbits 742, 743 and the accumulation of Aß, a key feature of AD, has been previously identified to reduce NO production in retinal neuronal cultures 730, adding plausibility to this theory.

Aside from disturbed neurovascular coupling mechanisms it could alternatively be hypothesised that factors such as relative arterial inertia due to either increased vascular stiffness or vasospasm, similar to that documented in AD and cognitively impaired patients at the cerebral level 636, 744, 745 may also contribute to the observed abnormalities in retinal vascular function. Such an alternative hypothesis could also partly explain the dissimilarities in reaction time observed in our AD patients on consecutive flicker cycles, in that the presence of limited arterial elasticity could theoretically lead to an incomplete baseline recovery after the initial stimulation cycle, reducing the time taken to then reach the point of maximum dilation on the subsequent flicker cycle. Exhaustion of vasoactive

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mediator reserves, such as that of NO, which is a feature commonly associated with vascular endothelial dysfunction 201, could then theoretically override this and prolong the reaction time again on the final cycle. Further research however would be required to validate these assumptions.

With regard to the venous dynamic retinal vessel response profile our AD patients demonstrated an increased fluctuation in baseline vessel diameter across all three cycles, prior to the onset of flicker, which was not replicated by our healthy controls. Consideration of baseline diameter fluctuation (BDF) was first recommended by Nagel et al 686 as a way of taking into account the effect of the spontaneous variations in vessel diameter that occur under normal resting conditions on the observed response of the vasculature to flicker light stimulation, however it is a parameter which is not commonly reported in the literature and which has, to date, mainly been considered in regard to the retinal arteries, where it has been tentatively linked to vascular disturbance in the form of instability or increased variation in vascular tone or rigidity 695, 746-748. As such the cause and relevance of increased BDF in the venous circulation is currently unclear. Retinal veins are generally thought to play a more secondary role in retinal autoregulation, perhaps providing a fine tuning of the regulation response following the active reaction of the retinal arteries and instigating a regulatory contribution passively in response to increased blood flow 685. Interestingly increased retinal venous diameters have been previously linked to impaired cerebral blood flow and have been suggested as a marker of both retinal and cerebral ischemia and hypoxia 652, 749-751. The finding here is

somewhat different as fluctuations in diameter have been assessed dynamically as opposed to vessel diameter measurements being taken statically from photographs. Nevertheless, as both ischemia and hypoxia have been well linked to the development of AD, it could be hypothesised that the increased fluctuation in baseline diameter observed in these patients could be reflective of early hypoxic changes and perhaps an

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increased risk of future damage. Further investigation however would be necessary to confirm these hypotheses.

4.9.4

Vascular function vs. Cognitive impairment

A significant positive correlation was found between the abnormal retinal arterial reaction to flickering light and the level of cognitive impairment in AD patients,

suggesting a possible link may exist between the degree of vascular dysfunction and disease severity even in the early stages of the disease process. This is supported by previous research in which vascular dysregulation at the cerebral level has been shown to become more pronounced with increasing severity of AD 629 and by the finding of a correlation between cognition and the geometry of the retinal vessels in elderly people after correcting for age, visual acuity and apolipoprotein E status 752. The possibility that the degree of vascular dysfunction at the retinal level could be a sensitive predictor of cognitive decline in mild AD patients emphasises the important role that vascular factors might play in the aetiology of the disease. In addition, it is tempting to propose that examining the function of retinal microvasculature could predict future cognitive decline in patients suffering from AD; nevertheless, more research is necessary to validate our presumption.