Corneal Nerve Loss in Patients with Acute Ischemic Stroke: A Surrogate Marker for Poor Pial Collaterals

Background: In patients with ischemic stroke, pial collaterals play a key role in limiting neurological disability by maintaining blood ow to ischemic penumbra. We hypothesized that patient with poor pial collateral status will have greater corneal nerve and endothelial cell abnormalities. Method: 35 patients with acute ischemic stroke secondary to middle cerebral artery occlusion with poor (n=12) and moderate/good (n=23) pial collaterals and 35 healthy controls underwent corneal confocal microscopy and quantication of corneal nerve and endothelial cell morphology. Results: In patients with MCA stroke, corneal nerve bre length (CNFL) (P=0.000), density (CNFD) (P=0.025) and branch density (CNBD) (P=0.002) were lower compared to controls. Age, BMI, cholesterol, triglycerides, HDL, LDL, systolic blood pressure, NIHSS and endothelial cell parameters did not differ but mRS was higher (p=0.023) and CNFL (p=0.026) and CNBD (p=0.044) were lower in patients with poor compared to moderate-good collaterals. CNFL and CNBD distinguished subjects with poor from good pial collaterals with an AUC of 72% (95% CI: 53-92%) and 71% (95% CI: 53-90%), respectively. Conclusion: Corneal nerve loss is greater in patients with poor compared to good pial collaterals and may act as a surrogate marker for pial collateral status in patients with ischemic stroke. automated image analysis system 47 . Endothelial cell density (ECD, cells/mm 2 ), endothelial cell area (ECA, µm 2 ), endothelial cell perimeter (ECP, µm), endothelial cell polymegathism (%) and endothelial cell pleomorphism (%) were


Background
Stroke is a major cause of disability and the second leading cause of death 1 . Ischemic stroke typically occurs following occlusion of a cerebral artery due to in situ thrombosis or an embolus from the heart or neck vessels 2 . Several studies have shown that the leptomeningeal collateral circulation, or pial collateral status determines the neurological outcomes following acute ischemic stroke 3 . Indeed, patients with poor pial collaterals sustain larger infarcts, respond poorly to reperfusion, have increased risk for and severity of intracerebral hemorrhage and suffer increased morbidity and mortality [3][4][5][6] .
There is considerable variability in the pial collateral status amongst patients with acute ischemic stroke 7,8 . Along with genetics factors, several modi able risk factors such as hypertension 9 , metabolic syndrome, hyperuricemia, age 10 , smoking 11 and hyperglycemia 12 are associated with poor collaterals. Animal studies have shown that "rarefaction of collaterals" is associated with multiple cardiovascular risk factors 13 . Additionally, MR imaging has also shown that the presence of white matter hyperintensities is associated with a poor pial collateral circulation 14 and their severity is associated with poor outcomes after stroke 15 .
Corneal confocal microscopy (CCM) is a rapid non-invasive ophthalmic imaging technique that has been used to demonstrate axonal loss in patients with impaired glucose tolerance 16 , diabetes 17,18 , and other peripheral neuropathies 19 . Recent studies have also demonstrated a signi cant reduction in corneal nerves [20][21][22] and abnormalities in endothelial cells in patients with TIA 23 and acute ischemic stroke 22 . We have also shown that corneal nerve loss is associated with age, HbA 1c , lipids and blood pressure 18 and the presence of white matter hyperintensities 24 in patients with acute ischemic stroke.
This study aimed to assess whether corneal nerve and endothelial cell abnormalities could act as surrogate markers of the pial collateral status in patients with acute ischemic stroke.    (Fig. 2, Table 2).

Results
Diagnostic accuracy for distinguishing Poor from Moderate-Good Collateral Patients Table 3 and Fig. 4 show the diagnostic accuracy of CCM measures for identifying patients with poor compared to moderate-good collaterals. CNFL and CNBD distinguished subjects with poor from good collaterals with 72% AUC (95% CI: 53-92%) and 71% AUC (95% CI: 53-90%), respectively. Using an abnormal cutoff of CNFL ≤ 16 mm/mm 2 the sensitivity and speci city were 96% and 58%, respectively, and using an abnormal cutoff of CNBD ≤ 62 mm/mm 2 the sensitivity and speci city were 65% and 75% according to Youden index, respectively.  22 , the current study showed no differences between patients with MCA stroke and controls or between patients with good compared to poor pial collaterals.
Pial collateral status can only be ascertained after ischemic stroke due to large vessel occlusion. White matter hyperintensities (WMH) predict poor stroke outcomes 90 days [25][26][27] after thrombectomy. In a recent study, the presence of WMH was associated with greater cerebrovascular dysfunction in patients with large vessel occlusion 14 and favorable outcomes in the ASPECT score in those without WMH 27 . The severity of WMH is associated with endothelial dysfunction 28 , and poor pial collateral circulation 14 . Many of the risk factors and comorbidities associated with WMHs have also been associated with poor pial collateral status. WMH increase with age 29 , hypertension 30,31 and diabetes 32,33 and may improve with antiplatelet therapy 34 and improved management of hypertension 30,31 and diabetes 32,33 . These same risk factors have also been related to corneal nerve degeneration 35 and indeed improvement in blood pressure, lipids and glycemic control is associated with corneal nerve regeneration 36, 37 . Recently we showed that CCM may act as a surrogate imaging marker for the presence and severity of WMHs in patients with acute ischemic stroke 24 .
CCM has emerged as a powerful non-invasive ophthalmic imaging endpoint to identify corneal nerve loss in patients with multiple sclerosis 38 , Parkinson's disease 39 , dementia 40 and patients with acute ischemic stroke 20 and recurrent stroke 21 . We now show that CCM identi es greater corneal nerve loss in patients with poor compared to moderate/good pial collaterals. This ophthalmic imaging method may act as a surrogate imaging marker for poor pial collaterals and allow the identi cation of patients who require greater risk factor reduction and more urgent reperfusion for ischemic stroke. Indeed, in the present study patients with poor pial collaterals had a higher mRS and a previous study showed a larger infarct volume and higher mRS and NIHSS in patients with worse pial collateral scores 41 .
Limitations of the current study include the moderate sample size and the assessment of only patients with moderate disability who could undergo CCM. However, our study broadens the clinical utility of CCM in patients with neurodegenerative disease and in the assessment of patients with ischemic stroke. These data warrant larger studies utilising CCM in patients with or at risk of ischemic stroke.

Materials And Methods
Thirty-ve patients with middle cerebral artery occlusion and 35 age-matched healthy control participants were recruited. Exclusion criteria included patients with stroke secondary to non-vascular disorder, intracerebral hemorrhage, a known history of ocular trauma or surgery, high refractive error, and glaucoma.
Acute ischemic stroke was con rmed clinically and radiologically using American Heart Association (AHA) criteria 42 . The pial collateral status was established using multi-modal/dynamic CTA according to the criteria of Tan et al. 43 . The ordinal collateral score ranges from 0 to 3: 0= absent collateral supply to the occluded MCA territory (de ned as "poor"), 1= collateral supply lling ≤50% but >0% of the occluded MCA territory, 2= collateral supply lling >50% but <100% of the occluded MCA territory and 3= 100% collateral supply of the occluded MCA territory (de ned as "good").
Clinical and demographic data along with blood pressure, HbA 1c and lipid pro le were obtained at admission. The National Institutes of Health Stroke Scale (NIHSS) 44 and modi ed Rankin Scale (mRS) 45 was obtained for all patients at admission and at discharge from hospital.

Corneal Confocal Microscopy
All patients underwent CCM (Heidelberg Retinal Tomograph III Rostock Cornea Module; Heidelberg Engineering GmbH, Heidelberg, Germany). CCM uses a 670 nm wavelength helium neon diode laser, which is a class I laser and therefore does not pose any ocular safety hazard. A ×63 objective lens with a numeric aperture of 0.9 and a working distance, relative to the applanating cap (TomoCap; Heidelberg Engineering GmbH) of 0.0 to 3.0 mm, is used. The size of each 2-dimensional image produced is 384×384 pixels with a 15°×15° eld of view and 10 μm/pixel transverse optical resolutions. To perform the CCM examination, local anesthetic (0.4% benoxinate hydrochloride; Chauvin Pharmaceuticals, Chefaro, United Kingdom) was used to anesthetize both eyes, and Viscotears (Carbomer 980, 0.2%, Novartis, United Kingdom) was used as the coupling agent between the cornea and the cap. Patients were asked to xate on an outer xation light throughout the CCM scan and a CCD camera was used to correctly position the cap onto the cornea. 19 The examination took approximately 10 minutes for both eyes. The examiners captured images of the central sub-basal nerve plexus using the section mode. On the basis of depth, contrast, focus, and position, 6 images per patient were selected. 46 All CCM images were manually analysed using validated, purpose-written software. Corneal nerve ber density (CNFD: total number of nerves /mm 2 ), corneal nerve branch density (CNBD: number of branches emanating from major nerve trunks /mm 2 ), corneal nerve ber length (CNFL: total length of all nerve bers and branches mm/mm 2 ) and inferior whorl length (IWL: total length of all nerve bers in inferior whorl area mm/mm 2 ) were analyzed using CCMetrics (M. A. Dabbah, ISBE, University of Manchester, Manchester, United Kingdom) 17 . Corneal endothelial cell images were analyzed using the Corneal Endothelium Analysis System (CEAS), an automated image analysis system 47 . Endothelial cell density (ECD, cells/mm 2 ), endothelial cell area (ECA, µm 2 ), endothelial cell perimeter (ECP, µm), endothelial cell polymegathism (%) and endothelial cell pleomorphism (%) were quanti ed. Polymegathism was de ned as the standard deviation of the cell area divided by the mean cell area, while pleomorphism was de ned as the hexagonality coe cient. Adequate corneal endothelial cells images were available in seventeen patients with poor (n=6) and moderate-good (n=11) collaterals and sixteen healthy controls.

Statistical Analysis
All statistical analyses were performed using IBM SPSS Statistics software Version 25. Normality of the data was assessed using the Shapiro-Wilk test and by visual inspection of the histogram and a normal Q-Q plot. Data are expressed as mean ± standard deviation (SD). Mann Whitney test (for non-normally distributed variables) and t-test (for normally distributed variables) were performed to nd the differences between two groups. Receiver operating characteristic (ROC) curve analysis was performed for corneal nerve parameters to identify patients with poor compared to moderate-good collateral status.

Declarations
Funding Supported by Qatar National Research Fund Grant BMRP20038654. The funders had no role in study design, data collection and analysis, decision to publish, or publish, or preparation of the manuscript.