Retinal vessel multifractals predict pial collateral status in patients with acute ischemic stroke.

OBJECTIVES: Pial collateral blood flow is a major determinant of the outcomes of acute ischemic stroke. This study was undertaken to determine whether retinal vessel metrics can predict the pial collateral status and stroke outcomes in patients.
METHODS: Thirty-five patients with acute stroke secondary to middle cerebral artery (MCA) occlusion underwent grading of their pial collateral status from computed tomography angiography and retinal vessel analysis from retinal fundus images.
RESULTS: The NIHSS (14.7 ± 5.5 vs 10.1 ± 5.8, p = 0.026) and mRS (2.9 ± 1.6 vs 1.9 ± 1.3, p = 0.048) scores were higher at admission in patients with poor compared to good pial collaterals. Retinal vessel multifractals: D0 (1.673±0.028vs1.652±0.025, p = 0.028), D1 (1.609±0.027vs1.590±0.025, p = 0.044) and f(α)max (1.674±0.027vs1.652±0.024, p = 0.019) were higher in patients with poor compared to good pial collaterals. Furthermore, support vector machine learning achieved a fair sensitivity (0.743) and specificity (0.707) for differentiating patients with poor from good pial collaterals. Age (p = 0.702), BMI (p = 0.422), total cholesterol (p = 0.842), triglycerides (p = 0.673), LDL (p = 0.952), HDL (p = 0.366), systolic blood pressure (p = 0.727), HbA1c (p = 0.261) and standard retinal metrics including CRAE (p = 0.084), CRVE (p = 0.946), AVR (p = 0.148), tortuosity index (p = 0.790), monofractal Df (p = 0.576), lacunarity (p = 0.531), curve asymmetry (p = 0.679) and singularity length (p = 0.937) did not differ between patients with poor compared to good pial collaterals.
CONCLUSIONS: This is the first translational study to show increased retinal vessel multifractal dimensions in patients with acute ischemic stroke and poor pial collaterals. A retinal vessel classifier was developed to differentiate between patients with poor and good pial collaterals and may allow rapid non-invasive identification of patients with poor pial collaterals.

Introduction

Acute ischemic stroke is the second most common cause of death, and survivors are left with significant disability [1]. Ischemic stroke typically occurs due to occlusion of a cerebral artery or an embolus from the heart or neck vessels [2]. Irrespective of the cause of ischemia, adequate pial collateral flow can offset the severity of ischemic brain injury [3]. In experimental studies of stroke, the infarct volume correlated more strongly with collateral number, diameter and penetrating arteriole number than with middle cerebral artery territory [4].

The pial collateral circulation is a network of leptomeningeal arteries that cross-connect the outer-most branches of adjacent arterial trees [5], but their extent is determined by genetic and environmental factors that vary widely in the population [6]. Rarefaction of collaterals is associated with ageing and multiple cardiovascular risk factors [7]. Indeed, individuals with ischemic stroke and 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 [8-11]. In this respect, the identification of patients with poor pial collaterals may allow risk stratification and targeted strategies to reduce risk factors associated with rarefaction of collaterals in patients at risk of acute ischemic stroke.

Currently, a direct assessment of the pial collaterals status can only be undertaken after presentation with a stroke, using Computed Tomography Angiography or magnetic resonance imaging of the brain. In this context, the retinal and neocortical vasculature share many anatomic similarities during development and maturation [12] and retinal fundus images have been used to identify alterations in the retinal vasculature to predict stroke [13, 14]. Indeed, retinal vessel width differs between subtypes of stroke [13-15] and in those with recurrent stroke [16, 17]. Interestingly, Prabhakar et al. [18] showed that retinal vessel metrics e.g. retinal vessel diameter, tortuosity and fractal dimensions, which capture the complexity of the vascular tree may predict pial collateral status and neurological outcomes in mice models of acute ischemic stroke.

This proof-of-concept study has assessed for the first time whether retinal fundus imaging can be utilized to quantify retinal vessel metrics and geometric complexity using fractal dimensions to identify patients with acute ischemic stroke and poor pial collaterals.

Materials and methods

Thirty-five patients with MCA occlusion and 21 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 confirmed clinically and radiologically using American Heart Association (AHA) criteria [19]. The pial collateral status was established using multi-modal/dynamic CTA according to the criteria of Tan et al. [20]. The ordinal collateral score ranges from 0 to 3: 0 = absent collateral supply to the occluded MCA territory (defined as “poor”), 1 = collateral supply filling ≤50% but >0% of the occluded MCA territory, 2 = collateral supply filling >50% but <100% of the occluded MCA territory and 3 = 100% collateral supply of the occluded MCA territory (defined as “good”).

Clinical and demographic (blood pressure, HbA1c, lipid profile) data were obtained at admission. Clinical outcome measures National Institutes of Health Stroke Scale (NIHSS) [21] and modified Rankin Scale (mRS) [22] were obtained for all patients at admission and discharge from the hospital. This assessed the quantity of the impairment caused by stroke, and degree of disability or dependence in daily activities, respectively. This study adhered to the tenets of the declaration of Helsinki and was approved by the Institutional Review Board of Weill Cornell Medicine (15–00021) and Hamad General Hospital (15304/15). Informed, written consent was obtained from all patients before participation in the study after assessment of speech, comprehension, and reasoning by a consultant neurologist.

Retinal imaging and vascular measurements

A spectral-domain optical coherence tomography (OCT) system (Spectralis OCT; Heidelberg Engineering GmbH, Heidelberg, Germany) with a 30° field-of-view was used to obtain non-mydriatic optic disc (OD) centered high resolution retinal fundus images (768x768 or 1536x1536) of both eyes. A trained grader masked to the participant’s characteristics, analyzed 1–3 retinal images from each eye using semi-automated MONA REVA vessel analysis software (version 2.1.1) developed by VITO (Mol, Belgium; http:\\mona.health) [23]. The FracLac plugin in FIJI software was used to calculate multifractal metrics. Vessel parameters from both eyes were averaged.

Selection of consistent and similar retinal regions across all fundus images was obtained in MONA REVA by defining an annular region centered on the optic disc, with the inner and outer radii of the annulus set at 1.5 and 3.0 times the radius of the optic disc, respectively. Next, the MONA REVA algorithm automatically segmented the retinal vessels. The segmentation algorithm is based on a multiscale line filtering algorithm inspired by Nguyen and coworkers [24]. Post-processing steps such as double thresholding, blob extraction, removal of small connected regions and filling holes were performed. Retinal vessel diameters, tortuosity, monofractal dimension and lacunarity were calculated in the software using predetermined regions of interest (ROIs) ().

Calculation of retinal vessel metrics with MONA REVA software and FracLac plugin in FIJI software.

The concentric circles on the segmented images indicate the regions of interest (ROIs) in which the metrics were calculated. R is defined as the radius of the optic disc (inner red circle). For ROI 1 and ROI 2, radii of the larger circles are a multiple of R. The entire segmented without the optic disc is ROI 3. Note: the segmented image is a representative example and not the segmented image of the shown greyscale image.

The diameters of the retinal arterioles and venules that passed completely through the circumferential zone 0.5 to 1 disc diameter from the optic disc margin (ROI 1) were calculated automatically. The trained grader verified and corrected vessel diameters and vessel labels (arteriole or venule) with the MONA REVA vessel editing toolbox. The diameters of the 6 largest arterioles and 6 largest venules were used in the revised Parr-Hubbard formula for calculating the Central Retinal Artery Equivalent (CRAE) and Central Retinal Venular Equivalent (CRVE) [25]. The arteriovenous ratio (AVR) is the ratio between CRAE and CRVE. The tortuosity index was computed as the mean tortuosity of the branch segments, where the tortuosity of a branch segment is the ratio of the line traced on each vascular tree along the vessel axis from 1.5 to 3.0 times the OD radius (ROI 2) and the line connecting the end points. Tortuosity of individual vessel segments was calculated as reported by Lisowska and co-workers [26]. The tortuosity index for the vessel network is similar to Prabhakar et al. [18].

The monofractal dimension Df was computed using the sliding box-count method on ROI 2 of the segmented image. Boxes with side length δ slide across the image and for each δ the number of boxes (N) required to cover the segmented image is recorded. The fractal dimension Df is expressed as follows [27].

Lacunarity is computed like Df but uses the standard deviation of the pixel count for boxes with side length δ that slide across the image [18].

Multifractals give additional and more fine-grained insights into the retinal vessel structure as they can provide information on its geometrical features and spatial distribution [27]. The generalized dimension Dq can be expressed as follows [28]. with Z(q, ε) the partition function, q the order of the moment of the measure and ε is the side length of the boxes used to cover the image.

Multifractal analysis was performed on the entire segmented vessel image () [29]. Settings for the FracLAc plugin of ImageJ were as follows: box-counting method, 10 grid positions, scaled series for calculating grid calibers, and Q range of −10, +10, and 0.1. We calculated the generalized dimensions (Dq) for q = 0, 1, and 2, which are the capacity dimension (D0), which captures how much the data fills the physical space and provides global information about the structure; the information dimension (D1), which captures the data density and quantifies the degree of disorder in the distribution; and correlation dimension (D2), which measures how scattered the data is with a higher value indicating more compact data. For a multifractal structure, the following applies: D0≥D1≥D2 [30, 31].

Three additional multifractal metrics were derived: the curve asymmetry, which indicates the degree of fluctuation in different fractal exponents, the singularity length (Δα) and f (α)max.

The curve asymmetry:

A = 1 Symmetric

A > 1 Left skewed → stronger presence of high fractal exponent and significant fluctuations

A < 1 Right skewed → greater presence of low fractal exponent and low fluctuations

The singularity length Δα = α−α represents multifractality and when the singularity length increases, the pixel distribution of the image gets more complex and the multifractality gets stronger. f(α)max represents the maximum of the singularity spectrum and approximates the fractal dimension at Q = 0 (Q is the set of points where the Holder exponent is zero).

Statistical analysis

All statistical analyses were performed in Orange (version 3.27.0; https://orange.biolab.si/). Orange is an open source machine learning, data mining and data visualization package [32]. The components of Orange are called widgets that run Python modules in the background. Analyses range from simple data visualization, subset selection, and preprocessing, to empirical evaluation of learning algorithms and modeling. The visual programming is implemented through an interface in which workflows are created by linking widgets.

Data are expressed as mean ± standard deviation (SD). Group comparisons were performed using independent t tests with p ≤ 0.05 as threshold for statistical significance. Spearman’s ρ was calculated as a nonparametric measure of rank correlation between two variables. Exploratory data mining was done using Principal Component Analysis (PCA), which computes the PCA linear transformation of the input data to identify clusters of similar samples (individuals with poor collaterals and individuals with good collaterals) based on the retinal metrics. Supervised learning was performed to develop a classification model to differentiate individuals with poor collaterals from individuals with good collaterals. Selection of retinal metric features was based on group comparisons. Goodness of fit of logistic regression and support vector machines were evaluated based on classification accuracy, sensitivity and specificity using leave-one-out cross-validation. This splits the dataset according to the number of subjects in the dataset. One subject is randomly selected for testing purpose while the other subjects are used to train the model. This procedure is iterated until all subjects have been used as test dataset. This methodology is efficient for replicability assessment in small data sets and is relevant for clinically relevant use-case scenario of diagnosis [33, 34]. The data used for statistical analysis in this study is available (https://doi.org/10.6084/m9.figshare.16574498.v1).

Results

Thirty-five patients with acute ischemic stroke were age-matched with twenty-one healthy controls (48.1 ± 10.6 vs 44.3 ± 10.6 yrs., p = 0.200). Patients with acute ischemic stroke were classified into those with poor (n = 15) and good (n = 20) pial collaterals.

Clinical, metabolic and neurological disability according to pial collateral status

Clinical and metabolic parameters and neurological disability are given in Table 1. Age (48.9 ± 10.5 vs 47.5 ± 10.4, p = 0.702), BMI (27.5 ± 3.2 vs 28.5 ± 4.3, p = 0.422), total cholesterol (4.70 ± 1.12 vs 4.62 ± 0.96, p = 0.842), triglycerides (1.57 ± 0.68 vs 1.47 ± 0.62, p = 0.673), LDL (3.06 ± 0.91 vs 3.08 ± 0.87, p = 0.952), HDL (0.93 ± 0.26 vs 0.85 ± 0.17, p = 0.366), systolic blood pressure (143.2 ± 18.4 vs 145.9 ± 27.7, p = 0.727), HbA1c (7.0 ± 3.3 vs 5.9 ± 1.1, p = 0.261) and number of smokers (p = 0.205) did not differ significantly between patients with poor compared to good pial collaterals. The National Institute of Health Stroke Scale (NIHSS) (14.7 ± 5.5 vs 10.1 ± 5.8, p = 0.026) and modified Rankin Scale (mRS) (2.9 ± 1.6 vs 1.9 ± 1.3, p = 0.048) scores were higher at admission, but not at discharge (NIHSS: 7.5 ± 5.7 vs 3.9 ± 4.3, p = 0.082, mRS: 2.1 ± 1.7 vs 1.5 ± 2.1, p = 0.384), in patients with poor compared to good pial collaterals.

Table 1

Demographic, metabolic and clinical characteristics of the participants (n = 35) with acute ischemic stroke with good and poor pial collaterals expressed as mean ± SD.

Parameters	All stroke (n = 35)	Good collaterals (n = 20)	Poor collaterals (n = 15)	p-value
Age (years)	48.1 ± 10.6	47.5 ± 10.4	48.9 ± 10.5	0.702
BMI (kg/m2)	28.0 ± 3.9	28.5 ± 4.3	27.5 ± 3.2	0.422
Systolic blood pressure (mmHg)	144.8 ± 24.6	145.9 ± 27.7	143.2 ± 18.4	0.727
HbA1c (%)	6.4 ± 2.4	5.9 ± 1.1	7.0 ± 3.3	0.261
mRS at admission	2.3 ± 1.5	1.9 ± 1.3	2.9 ± 1.6	0.048 *
mRS at discharge	1.7 ± 2.0	1.5 ± 2.1	2.1 ± 1.7	0.384
NIHSS at admission	12.1 ± 6.2	10.1 ± 5.8	14.7 ± 5.5	0.026 *
NIHSS at discharge	5.6 ± 5.4	3.9 ± 4.3	7.5 ± 5.7	0.082
Total cholesterol (mmol/l)	4.66 ± 1.05	4.62 ± 0.96	4.70 ± 1.12	0.842
Triglycerides (mmol/l)	1.52 ± 0.66	1.47 ± 0.62	1.57 ± 0.68	0.673
LDL (mmol/l)	3.08 ± 0.90	3.08 ± 0.87	3.06 ± 0.91	0.952
HDL (mmol/l)	0.89 ± 0.22	0.85 ± 0.17	0.93 ± 0.26	0.366

* Statistically significant differences between groups tested using t-test at p ≤ 0.05 (data in bold). Clinical data has some missing values: mRS at admission (n = 34), mRS at discharge (n = 30), NIHSS at admission (n = 35; no missing values), NIHSS at discharge (n = 27).

Retinal vessel metrics in patients with acute ischemic stroke compared to controls

The arterio-venular ratio (0.675 ± 0.049 vs 0.703 ± 0.042, p = 0.026) and tortuosity index (0.890 ± 0.019 vs 0.903 ± 0.011, p = 0.002) were lower and CRVE (275.9 ± 21.4 vs 259.1 ± 25.6, p = 0.016) was higher in patients with acute ischemic stroke compared to controls. CRAE (p = 0.313), monofractal Df (p = 0.067), capacity dimension (D0) (p = 0.821), information dimension (D1) (p = 0.640), correlation dimension (D2) (p = 0.477), curve asymmetry (p = 0.139), singularity length (p = 0.108) and f(α)max (p = 0.784) did not differ between patients with acute ischemic stroke compared to healthy controls (Table 2).

Table 2

Retinal vessel metrics comparing controls to patients with acute ischemic stroke and between patients with good and poor collaterals expressed as mean ± SD.

Retinal Parameters	Control (n = 21)	All stroke (n = 35)	P-value	Good collaterals (n = 20)	Poor collaterals (n = 15)	P-value
Vessel width
CRAE (μm)	181.4 ± 15.3	185.4 ± 11.6	0.313	182.6 ± 13.0	189.1 ± 8.0	0.084
CRVE (μm)	259.1 ± 25.6	275.9 ± 21.4	0.016 *	276.1 ± 19.9	275.6 ± 23.2	0.946
Arteriovenous ratio	0.703 ± 0.042	0.675 ± 0.049	0.026 *	0.664 ± 0.041	0.690 ± 0.055	0.148
Tortuosity index	0.903 ± 0.011	0.890 ± 0.019	0.002 *	0.891 ± 0.020	0.889 ± 0.017	0.790
Monofractal D f	1.368 ± 0.038	1.389 ± 0.045	0.067	1.392 ± 0.048	1.384 ± 0.041	0.576
Lacunarity	0.986 ± 0.024	0.997 ± 0.025	0.088	0.995 ± 0.023	1.001 ± 0.028	0.531
Multifractal
D 0	1.660 ± 0.019	1.661 ± 0.028	0.821	1.652 ± 0.025	1.673 ± 0.028	0.028 *
D 1	1.601 ± 0.019	1.598 ± 0.027	0.640	1.590 ± 0.025	1.609 ± 0.027	0.044 *
D 2	1.577 ± 0.024	1.572 ± 0.027	0.477	1.564 ± 0.025	1.581± 0.027	0.071
Curve asymmetry	0.361 ± 0.072	0.389 ± 0.061	0.139	0.385 ± 0.049	0.395 ± 0.073	0.679
Singularity length	0.902 ± 0.078	0.934 ± 0.061	0.108	0.934 ± 0.041	0.933 ± 0.080	0.937
f(α) max	1.660 ± 0.019	1.662 ± 0.027	0.784	1.652 ± 0.024	1.674 ± 0.027	0.019 *

* Statistically significant differences between groups tested using t-test at p ≤ 0.05 (data in bold).

Retinal vessel metrics in patients with poor compared to good pial collaterals

CRAE (p = 0.084), CRVE (p = 0.946), AVR (p = 0.148), tortuosity index (p = 0.790), monofractal Df (p = 0.576), curve asymmetry (p = 0.679) and singularity length (p = 0.937) did not differ between patients with poor compared to good pial collaterals. Capacity dimension (D0) (1.673 ± 0.028 vs 1.652 ± 0.025, p = 0.028), information dimension (D1) (1.609 ± 0.027 vs 1.590 ± 0.025, p = 0.044), correlation dimension D2 (1.581 ± 0.027 vs 1.564 ± 0.025, p = 0.071) and f(α)max (1.674 ± 0.027 vs 1.652 ± 0.024, p = 0.019) were higher in patients with poor compared to good pial collaterals. Because mRS and NIHSS differed significantly between patients with poor and good collaterals, possible correlation with retinal metrics was tested. Spearman’s ρ was <0.2 in all cases and values were not significant.

Machine learning to differentiate patients with poor and good pial collaterals using retinal vessel metrics

Unsupervised Principal Component Analysis was used to assess which metrics can differentiate patients with poor from good pial collaterals. Three principal components explained a cumulative variance of 75.1% and achieved a fair separation between patients with good and poor collaterals (). Principal components are linear combinations of individual retinal metrics that capture better variance in the dataset than individual metrics. Possible correlation of each of the principal components with mRS and NIHSS were therefore evaluated. Spearman’s ρ ranged between 0.13 and 0.22 for mRS at admission, between 0.19 and 0.28 for mRS at discharge, between 0.02 and 0.33 for NIHSS at admission; and between 0.01 and 0.11 for NIHSS at discharge. All correlations had p-values >0.05.

Planar projection of the Principal Component Analysis with 3 principal components represented explaining a total variance of 75.1%.

Patients with good collaterals are represented with blue circles and poor collaterals with red circles.

Further, a classification model utilizing the features (f(α)max, D0, and D1) which differed significantly between the two groups was developed to differentiate between individuals with poor and good collaterals. Models were built with f(α)max and D1 because f(α)max and D0 had an almost perfect direct correlation. The logistic regression model did not converge and was not further considered. However, the support vector machine learner with a linear kernel already resulted in a classification accuracy of 0.686, sensitivity of 0.686 and a specificity of 0.631. The use of a sigmoid kernel function in the support vector machine learner further improved the classification accuracy (0.743), sensitivity (0.743) and specificity (0.707). All results are based on leave-one-out cross-validation that tests the collateral status of each individual on the learner developed by using all other individuals. This procedure is iterated until all subjects have been used as test dataset. visualizes the results of the scenario with the sigmoid kernel function as a scatter plot of the individuals and the confusion matrix with the performance of that classification model. There are 9 patients misclassified and data of these patients were inspected to check if specific clinical profiles were overrepresented, but there was no indication to support this.

Scatterplot for the support vector machine learner and the corresponding confusion matrix based on leave-one-out cross validation of the classification.

Patients with good collaterals are represented with blue circles and poor collaterals with red circles.

Discussion

This translational study demonstrates increased retinal vessel multifractal dimensions in patients with acute ischemic stroke and poor pial collaterals and presents a retinal vessel classifier that differentiates patients with poor from good pial collaterals. Patients with acute ischemic stroke and worse pial collateral scores have evidence of a larger infarct volume and higher modified Rankin scale score and NIHSS at discharge [35]. In contrast, a good pial collateral status is associated with lower rates of symptomatic intracranial hemorrhage and mortality in patients with acute ischemic stroke following reperfusion [36]. We also show that patients with poor pial collaterals had a higher modified Rankin score and NIHSS at admission. However, standard risk factors for stroke e.g., age, hypertension, lipids and HbA1c did not differ between patients with good and poor pial collaterals. Previous studies have shown that metabolic syndrome and older age [37] but not diabetes [38] predict poor pial collateral status.

Imaging techniques such as cerebral angiography (gold standard for the evaluation of pial collateral status), CT Angiography, MRI- FLAIR, MRI-SWI and contrast-enhanced magnetic resonance angiography have been used to visualize the pial collaterals [39]. While some of these techniques require the use of contrast (CTA, contrast MRA, cerebral angiography), others require patient cooperation and have limited availability. Furthermore, for the aforementioned techniques to show the pial collateral status, a large vessel stenosis or occlusion, as a consequence of which pial collaterals channels open, is a pre-requisite. Therefore, there exists a need for a reliable and non-invasive imaging biomarker that can assess the collateral status before the vessel is occluded and/or stroke happens. In 2015, a seminal study undertook detailed morphometric quantification of 21 retinal vessel parameters in flat-mounted retina of mice models of ischemic stroke and identified 10 retinal vessel metrics that predicted the pial collateral status, infarct volume and neurological outcomes [18]. Utilizing retinal fundus images acquired after admission of patients with acute ischemic stroke, we show that an increase in retinal vessel multifractal dimensions was associated with poor pial collaterals. Furthermore, discriminative analysis identified patients with poor and good pial collaterals with fair sensitivity and specificity. The higher multifractal values are indicative of more compact and complex retinal vessels in patients with poor compared to good pial collaterals. A structural change in the retinal vessel tree, as quantified by fractal metrics, can indicate abnormalities in vasculogenesis and angiogenesis. In diabetic retinopathy increased fractal values are a marker for angiogenesis secondary to retinal hypoxia. Thus, changes in retinal fractal dimensions in patients with ischemic stroke could reflect a surrogate response to hypoxic stimuli observed in the cerebral vessels [40].

We have also shown that standard retinal vessel metrics such as venular widening and lower arterio-venular ratio and tortuosity differ in patients with acute ischemic stroke compared to healthy controls [41, 42], but do not differentiate patients with poor from good pial collaterals. Previous studies utilizing retinal vessel imaging in large population-based studies have demonstrated somewhat mixed associations between standard and more advanced metrics of retinal vessel complexity and stroke [43]. Stroke and the occurrence of white matter lesions have been associated with retinopathy, retinal arteriolar narrowing, venular widening and suboptimal retinal bifurcation [44-46]. Retinal arteriolar narrowing, venular widening and increased retinal vascular fractal dimension have been associated with lacunar infarcts [47], and recurrent stroke risk [17]. Standard retinal vessel metrics and arteriolar fractal dimension are also associated with cerebral blood flow in healthy older adults [48]. However, whilst a decrease in retinal arteriolar fractal dimension has been associated with cerebral microbleeds [44], increased retinal fractal dimension has been associated with a 4-fold increased risk of lacunar stroke [40]. Furthermore, in a large study of 557 patients with ischemic stroke decreased arteriolar and venular fractal dimension were associated with lacunar, cardioembolic and large vessel occlusion stroke [41]. A recent systematic review has confirmed an overall reduction in retinal vessel fractals in patients with stroke and dementia [49]. However, they showed variability in fractal outcomes in different studies which was attributed to differences in populations studied, the equipment used to capture retinal images and segmentation tools used to define and calculate the fractals [50]. We have recently shown that retinal vessel multifractals are associated with glucose, hypertension and the WHO/ISH cardiovascular risk score [31] and that retinal vessel analysis is uniquely associated with blood pressure, glycemic status and obesity [51]. The current data suggests that the differences in retinal vessel fractals in patients with acute ischemic stroke may be further related to the pial collateral status.

Limitations of the current study include the sample size of this pilot study and the assessment of patients with moderate neurological disability as patients with more severe stroke could not undergo retinal fundus imaging. However, the retinal vessel analysis was performed double blinded on high-definition retinal fundus images using semi-automated methods with minimal manual grading input, making the analysis rapid and with minimal bias.

We show that retinal fundus imaging can be utilized to differentiate ischemic stroke patients with poor from good pial collaterals. Larger studies are needed to confirm the current data and to establish the clinical utility of retinal vessel fractal analysis in patients with intra or extracranial vascular stenosis or occlusion, prior to ischemic stroke.

14 Dec 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Aurel Popa-Wagner

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please describe in your methods section how capacity to provide consent was determined for the participants whom have suffered a stroke in this study. Please also state whether your ethics committee or IRB approved this consent procedure. If you did not assess capacity to consent please briefly outline why this was not necessary in this case.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

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.

Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

The author(s) received no specific funding for this work.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. Please note that in order to use the direct billing option the corresponding author must be affiliated with the chosen institute. Please either amend your manuscript to change the affiliation or corresponding author, or email us at plosone@plos.org with a request to remove this option.

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study “Retinal Vessel Multifractals Predict Pial Collateral Status in Patients with Acute Ischemic Stroke” by Khan et al, represents a very thorough image analysis of the retinal blood vessels aiming to assess their morphology as a fast and accessible predictor tool for pial collateral circulation availability in stroke patients.

Leptomeningeal collaterals of the pia vessels are small arterial connections of the terminal cortical branches of major cerebral arteries along the surface of the brain. These vessels are closed under normal conditions when blood flow from all major cerebral arteries is not affected, but are recruited when one major artery is occluded.

Minor issues

- The authors should clearly present the phisiopathological connection between the retinal vessels pattern and pial collateral circulation, and whether this is or not an independent risk factor. Many prior publications have demonstrated that retinal vessel morphology can be a diagnostic marker for early detection and monitoring of ischemic stroke, based on the fact that ophthalmic artery originates in the internal carotid artery, the same as for MCA, and thus both vessels might reflect a common pressure and motility change.

Major issues

- Binarized images were analysed utilising the Frac Lac plugin for ImageJ. This software applies the sliding box algorithm and looks only at the silhouettes of the defined Regions of Interest (ROI), and does not consider the inner holes of the ROIs. Were multifractal and lacunarity analyses enough to evaluate the complete filling patterns of the ROIs. The authors might also consider utilising a sliding box algorithm (for example Image ProPlus from Media Cybernetics) that considers the full patterns and not only the profile lines, this could improve the monofractal discriminating efficiency.

- How did the main physiopathological denominators of a direct vascular pathology influenced the connection between retinal vessels, pial vessels, and stroke in these patients? Besides the presence/absence of hypertension, the analysis needs to consider diabetes, atheromatosis and vasculitis in these patients. These parameters should in fact be evaluated for both the control and the study group.

- How did the retina blood vessels correlate with the area of the core of the infarct and penumbra, on CT scans?

- Where the patients analysed during the ischemic thrombolysis window? Was thrombolysis performed on these patients? If yes, how was the recovery considering the retina vascular pattern as a predictor?

Altogether, this is a very valuable study in the field, and I will fully endorse its publication after answering the above questions.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

12 Apr 2022

Dear Editor,

We thank you and the reviewers for their constructive and valuable comments, which have helped us to improve the quality of our manuscript. A point-by-point response (blue) to you and the reviewers’ comments is provided below along with the revised manuscript.

Responses to Editor Questions:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Answer: Thank you. We have followed the PLOS ONE’s style requirements and amended the documents.

2. Please describe in your methods section how capacity to provide consent was determined for the participants whom have suffered a stroke in this study. Please also state whether your ethics committee or IRB approved this consent procedure. If you did not assess capacity to consent please briefly outline why this was not necessary in this case.

Answer: Thank you. Each participant was consented for the study. The study was approved from Weill Cornell medicine-Qatar and Hamad General Hospital IRBs. It has been stated on page 4, last paragraph.

“This study adhered to the tenets of the declaration of Helsinki and was approved by the Institutional Review Board of Weill Cornell Medicine-Qatar (15–00021) and Hamad General Hospital (15304/15). Informed, written consent was obtained from all patients/guardians before participation in the study.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Answer: Thank you. We have provided the correct grant number BMRP20038654.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

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.

Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

The author(s) received no specific funding for this work.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Answer: Thank you. We would like to add following funding statement.

“Supported by Qatar National Research Fund Grant BMRP20038654. The funders had no role in study design, data collection, analysis and interpretation, and decision to prepare the manuscript and the publication process.”

4. Please note that in order to use the direct billing option the corresponding author must be affiliated with the chosen institute. Please either amend your manuscript to change the affiliation or corresponding author, or email us at plosone@plos.org with a request to remove this option.

Answer: Thank you. Billing will be paid by Weill Cornell Medicine-Qatar.

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Answer: Thank you. We have provided the data availability statement on page 8, 1st paragraph.

“The data used for statistical analysis in this study is available at repository with URL https://doi.org/10.6084/m9.figshare.16574498.v1 .”

Reviewers' comments:

Reviewer's Responses to Questions

Reviewer:

Reviewer #1: The study “Retinal Vessel Multifractals Predict Pial Collateral Status in Patients with Acute Ischemic Stroke” by Khan et al, represents a very thorough image analysis of the retinal blood vessels aiming to assess their morphology as a fast and accessible predictor tool for pial collateral circulation availability in stroke patients.

Leptomeningeal collaterals of the pia vessels are small arterial connections of the terminal cortical branches of major cerebral arteries along the surface of the brain. These vessels are closed under normal conditions when blood flow from all major cerebral arteries is not affected, but are recruited when one major artery is occluded.

We thank the reviewer for his/her positive evaluation of our study.

Minor issues

The authors should clearly present the phisiopathological connection between the retinal vessels pattern and pial collateral circulation, and whether this is or not an independent risk factor.

Many prior publications have demonstrated that retinal vessel morphology can be a diagnostic marker for early detection and monitoring of ischemic stroke, based on the fact that ophthalmic artery originates in the internal carotid artery, the same as for MCA, and thus both vessels might reflect a common pressure and motility change.

Answer: Thank you for suggesting that a common anatomical source for the origin of the ophthalmic and middle cerebral arteries may explain the comparable changes in the structure of the retinal and pial collaterals vessels. Given that standard retinal vessel metrics did not differ between ischemic stroke patients with good and poor pial collaterals, while retinal vessel complexity did . We have suggested (page 13) that hypoxia may lead to altered vessel complexity due to alterations in vasculogenesis and angiogenesis.

Major issues

Binarized images were analysed utilising the Frac Lac plugin for ImageJ. This software applies the sliding box algorithm and looks only at the silhouettes of the defined Regions of Interest (ROI), and does not consider the inner holes of the ROIs. Were multifractal and lacunarity analyses enough to evaluate the complete filling patterns of the ROIs. The authors might also consider utilising a sliding box algorithm (for example Image ProPlus from Media Cybernetics) that considers the full patterns and not only the profile lines, this could improve the monofractal discriminating efficiency.

Answer: We thank the reviewer for the suggestion to consider using ImageProPlus software. However, this software was not available to us and we believe that the use of another pipeline would complicate the interpretation of essentially a proof of concept study because of different image pre-processing and segmentation pipelines.

Nevertheless, Df, lacunarity and additional multifractal parameters (Fig.1) were quantified on the MONA REVA segmented images using open-source FracLac. The fractal dimension Df is the most widely used metric for quantifying vascular complexity. We have also used lacunarity, a proxy for heterogeneity and additional multifractality metrics to identify patterns of complexity that cannot be identified with the single Df. We have now clarified on page 5 (last paragraph), page 6 (1st, 2nd and 3rd paragraphs) of the revised manuscript the complementary role of the metrics we have assessed. In the revised manuscript on page 13 we have referred to the issue of using multiple different outcomes, especially in a proof of concept translational study: “A recent systematic review has confirmed an overall reduction in retinal vessel fractals in patients with stroke and dementia [52]. However, they showed variability in fractal outcomes in different studies attributed to differences in populations studied, the equipment used to capture retinal images, and segmentation tools used to define and calculate the fractals [53].”

To further clarify this we have added the following sentence in the revised manuscript on page 13: “Other image capture and analysis techniques may help to further differentiate patients with poor and good pial collaterals.”

How did the main physiopathological denominators of a direct vascular pathology influenced the connection between retinal vessels, pial vessels, and stroke in these patients? Besides the presence/absence of hypertension, the analysis needs to consider diabetes, atheromatosis and vasculitis in these patients. These parameters should in fact be evaluated for both the control and the study group.

Answer: Thank you. We excluded patients with vasculitis as stated on page 4, 1st paragraph. Moreover, the systolic blood pressure and HbA1c did not differ between patients with ischemic stroke and good compared to poor pial collaterals (Table 1). We have also updated our material and method section, and stated the numbers with diabetes and hypertension on page 4, 3rd Paragraph. “In the ischemic stroke group, seven had diabetes and twenty had hypertension, whilst none of the healthy control participants had diabetes or hypertension.”

How did the retina blood vessels correlate with the area of the core of the infarct and penumbra, on CT scans?

Unfortunately, we did not have the image analysis software to measure the area of the core infarct volume and penumbra. Going forward we will try and incorporate these metrics.

Where the patients analysed during the ischemic thrombolysis window? Was thrombolysis performed on these patients? If yes, how was the recovery considering the retina vascular pattern as a predictor?

Thank you. Retinal imaging was performed outside the ischemic thrombolysis window. Thrombolysis was performed on 19 patients. We have measured the structural retinal metrics which we believe would not change after thrombolysis.

18 Apr 2022

Retinal Vessel Multifractals Predict Pial Collateral Status in Patients with Acute Ischemic Stroke

PONE-D-21-30340R1

Dear Dr. Malik,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Aurel Popa-Wagner

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

26 Apr 2022

PONE-D-21-30340R1

Retinal Vessel Multifractals Predict Pial Collateral Status in Patients with Acute Ischemic Stroke

Dear Dr. Malik:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Aurel Popa-Wagner

Academic Editor

PLOS ONE