Corneal Confocal Microscopy Detects Alterations in Corneal Endothelial Cell Morphology in Patients Admitted with Acute Ischemic Stroke
Authors: Khan Adnan 
Published: June 2019

Background The major risk factors for stroke include diabetes, hypertension, smoking, dyslipidemia 1 and metabolic syndrome 2. Endothelial dysfunction is central to promoting vasoconstriction and thrombosis and limited angiogenesis 3 and may also contribute to enhanced plaque vulnerability, triggering plaque rupture, and thrombus formation. There are many methods to assess endothelial dysfunction including brachial flow-mediated dilation, cerebrovascular reactivity to L-arginine and alterations in endothelium dependent dilatation using laser Doppler. We have previously shown significant abnormalities in gluteal resistance vessel endothelium dependent dilatation in patients with obesity 4, diabetes and hypertension 5. Patients admitted with an acute ischemic stroke had reduced forearm flow mediated dilatation and increased circulating levels of P-selectin, a marker of endothelial dysfunction, suggesting widespread vascular abnormalities 6. These measures of endothelial dysfunction are evaluated in vascular territory which is a distance from the brain. Direct imaging of the cerebral blood vessels can identify atherosclerosis 7 and Magnetic resonance imaging can identify silent infarcts, cerebral microbleeds, periventricular white matter hyperintensities and perivascular spaces, which have been shown to predict a higher risk of stroke 8. Subtle alterations in the microstructure of normal-appearing white matter, independent of prevalent vascular lesions also predicts the risk of stroke 9. However, these techniques cannot directly image endothelial cells. We have pioneered corneal confocal microscopy as a rapid non-invasive ophthalmic imaging technique to image the corneal nerves. Whilst we have predominantly demonstrated an abnormality in the corneal nerves in a range of peripheral neuropathies 10, more recently we have shown an abnormality in central neurodegenerative conditions including Parkinson's disease 11 and multiple sclerosis 12. Furthermore, in our recent study we showed that people with acute ischemic stroke also had a reduction in corneal nerve fibers 13. In the present study, we have undertaken corneal confocal microscopy and automated quantification of endothelial cell density, area and perimeter as well as the degree of polymegathism and pleomorphism and related it to corneal nerve morphology and vascular risk factors in a cohort of patients admitted with acute ischemic stroke. Aim Corneal confocal microscopy can identify alterations in corneal endothelial cell morphology and neuronal deficit in patients presenting with an acute ischemic stroke. Methods One hundred and forty six patients admitted with an acute stroke with NGT (n = 62); IGT (n = 34) and T2DM (n = 50) and 18 age-matched healthy control participants underwent corneal confocal microscopy. There was a significant reduction in corneal endothelial cell density and an increase in endothelial cell area and perimeter in stroke patients with NGT (P = 0.002, P = 0.001, P = 0.002), IGT (P = 0.030, P = 0.028, P = 0.06) and T2DM (P&lt;0.001, P&lt;0.001, P = 0.001) compared to controls, respectively, with no significant difference in polymegathism and pleomorphism in stroke patients compared to healthy controls. There was a significant reduction in CNFD, CNBD and CNFL in stroke patients with NGT (P = 0.016, P = 0.001, P = 0.016), IGT (P = 0.007, P = 0.005, P = 0.007) and T2DM (P = 0.002, P = 0.008, P = 0.002) compared to controls, respectively. Diastolic blood pressure correlated with endothelial cell density (P = 0.01), endothelial cell area (P = 0.02) and endothelial cell perimeter (P = 0.01). Endothelial cell density, endothelial cell area and perimeter correlated with corneal nerve fiber density (P = 0.03, P = 0.02, P = 0.02) and corneal nerve fiber length (P = 0.02, P = 0.02, P = 0.023), respectively. Conclusion We show a reduction in corneal endothelial cell density and an increase in size which relates to diastolic blood pressure and corneal nerve loss, independent of glucose tolerance status in patients with an acute stroke. CCM allows rapid non-invasive imaging of endothelial cells to enable risk stratification of patients with stroke. References 1. Shuaib A. Alteration of blood pressure regulation and cerebrovascular disorders in the elderly. Cerebrovasc Brain Metab Rev. 1992;4:329-345 2. Heymann EP, Goldsmith D. Best approaches in the battle against globesity? Learning lessons from our experience tackling hiv-aids and tobacco smoking. JRSM short reports. 2012;3:45 3. Rajendran P, Rengarajan T, Thangavel J, Nishigaki Y, Sakthisekaran D, Sethi G, et al. The vascular endothelium and human diseases. International journal of biological sciences. 2013;9:1057 4. Aghamohammadzadeh R, Greenstein AS, Yadav R, Jeziorska M, Hama S, Soltani F, et al. Effects of bariatric surgery on human small artery function: Evidence for reduction in perivascular adipocyte inflammation, and the restoration of normal anticontractile activity despite persistent obesity. Journal of the American College of Cardiology. 2013;62:128-135 5. Malik RA, Schofield IJ, Izzard A, Austin C, Bermann G, Heagerty AM. Effects of angiotensin type-1 receptor antagonism on small artery function in patients with type 2 diabetes mellitus. Hypertension. 2005;45:264-269 6. Blum A, Vaispapir V, Keinan-Boker L, Soboh S, Yehuda H, Tamir S. Endothelial dysfunction and procoagulant activity in acute ischemic stroke. Journal of vascular and interventional neurology. 2012;5:33 7. Imam YZ, D'Souza A, Malik RA, Shuaib A. Secondary stroke prevention: Improving diagnosis and management with newer technologies. Translational stroke research. 2016;7:458-477 8. Debette S, Markus H. The clinical importance of white matter hyperintensities on brain magnetic resonance imaging: Systematic review and meta-analysis. British Medical Journal. 2010;341:c3666 9. de Groot M, Verhaaren BF, de Boer R, Klein S, Hofman A, van der Lugt A, et al. Changes in normal-appearing white matter precede development of white matter lesions. Stroke. 2013;44:1037-1042 10. Alam U, Jeziorska M, Petropoulos IN, Asghar O, Fadavi H, Ponirakis G, et al. Diagnostic utility of corneal confocal microscopy and intra-epidermal nerve fibre density in diabetic neuropathy. PloS one. 2017;12:e0180175 11. Kass-Iliyya L, Javed S, Gosal D, Kobylecki C, Marshall A, Petropoulos IN, et al. Small fiber neuropathy in parkinson»s disease: A clinical, pathological and corneal confocal microscopy study. Parkinsonism and Related Disorders. 2015;21:1454-1460 12. Petropoulos IN, Kamran S, Li Y, Khan A, Ponirakis G, Akhtar N, et al. Corneal confocal microscopy: An imaging endpoint for axonal degeneration in multiple sclerosis. Investigative Ophthalmology &amp; Visual Science. 2017 13. Khan A, Akhtar N, Kamran S, Ponirakis G, Petropoulos IN, Tunio NA, et al. Corneal confocal microscopy detects corneal nerve damage in patients admitted with acute ischemic stroke. Stroke. 2017:STROKEAHA. 117.018289