Diabetic Neuropathy: Function-Structure of Corneal Nerves to Assess Injury-Repair
Phase: NA
Conditions: Diabetic Peripheral Neuropathy
Sponsor: VA Office of Research and Development

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Diabetic Neuropathy: Function-Structure of Corneal Nerves to Assess Injury - Repair 
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Running title:  corneal sensitivity and diabetic neuropathy 
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The study is catalogued NCT04222660. 
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April 23, 2024

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Study Protocol 
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The purpose of this study is to test a new approach for the early detection of DPN.  
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Diabetes patients meeting the criteria were recruited from the Diabetes Clinics and non-diabetic 
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subjects from the general veteran population.  A written and informed consent was obtained 
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from all participants. The study adhered to the Declaration of Helsinki and was approved by the 
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Institutional Review Board (#201610799).  Participants were divided into two groups: diabetic 
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with peripheral neuropathy and non-diabetic with no history of peripheral neuropathy. 
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Inclusion Criteria:  1) Type 2 diabetes diagnosed based on the opinion of an Endocrinologist, 
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the absence of a history of ketoacidosis, and a C-peptide > 0.8 ng/ml. 2) At least 10 year known 
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duration of diabetes. 3) Age ≥ 50 yr. 4) HbA1c < 9.0%. 5) Mild to moderate PN based on 
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response to Michigan Neuropathy Screening Instrument (MNSI),18 nerve conduction and 
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amplitude determined using DPNCheck, medical record, presence or absence of ankle reflex, 
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vibration threshold and 10g monofilament sensation. Exclusion Criteria: 1) History of neurologic, 
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muscular, genetic, or other condition known to affect muscle function or exercise tolerance. 2) 
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Electrolyte abnormalities, untreated hypothyroidism, abnormalities in calcium, phosphate, or 
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magnesium concentrations, or any other metabolic disturbance affecting neural function. 3) 
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Cigarette smoking in the past year. 4) Peripheral vascular, cardiac, pulmonary, or any other 
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disorder affecting blood or tissue oxygenation. 5) Use of any medication known to alter 
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peripheral nerve function. 6) History of diabetic foot ulcer. 7) History of corneal disease including 
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dry eye. 8) History of wearing contacts. 9) Any other medical or psychological condition judged 
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to limit compliance with the protocol or interpretation of results.  
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The study included 28 patients with type 2 diabetes and peripheral neuropathy (24 male 
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and 4 female).  The average age of the diabetes patients was 66.8 ± 1.9, with duration of 
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diabetes 20.0 ± 2.6 and hemoglobin A1C 8.2 ± 0.3.  The non-diabetic subjects (11 male and 5 
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female) were younger 49.8 ± 5.0. 
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Clinical neuropathy assessment:  The MNSI questionnaire was self-administered.  
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Each applicant was encouraged to answer yes or no to each of the 15 questions.  A score of ≥ 4 
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was considered abnormal.  During the MNSI examination a health professional examined the 
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feet, for deformities, dry skin, calluses, infections, and ulcers.  Quantitative sensory testing 
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included the presence or absence of ankle reflexes and vibration and monofilament sensation at 
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the dorsum of the great toe.19  Nerve conduction values were determined using a point of care 
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device DPNCheck (DPNCheck, NeuroMetrix, Inc., Waltham, MA).  Manufacture instructions 
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were followed, briefly after skin preparation, the probes were coated with the conductive gel and 
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applied directly to the skin, posterior to the lateral malleolus.20  Up to four recordings were made 
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as individual tolerance allowed.  The recordings were averaged. 
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Ophthalmic assessment:  All patients underwent a visual acuity assessment.  Provided 
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answers to two questionnaires the Ocular Surface Disease Index (OSDI) and Dry Eye 
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Questionnaire (DEQ-5).  To evaluate cornea sensitivity each patient underwent a Cochet-
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Bonnet aesthesiometer examination and blinking response determination following application 
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of a drop of isotonic saline (308 mosmol/liter) or Muro-128 solution 5% sodium chloride (1710 
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mosmol/liter) (Rugby Laboratories, Livonia, MI).  This study was performed by having the 
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subject rest their chin comfortably on a support.  Three CMOS cameras (Imaging Development 
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Systems GmbH, Obersulm, Germany), one positioned in front of the subject and two positioned 
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laterally, were used to observe both eyes simultaneously.  Custom software was used to 
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synchronize video streams and obtain images (MATLAB R2012a, The MathWorks Inc., Natick, 
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MA).  Video was taken continuously at 30fps of each subject as an isotonic solution was 
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delivered to the left eye.  The response was video recorded for 60 sec after a 10 sec delay.  
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After a 15 min wash and rest period the procedure was repeated using a drop of Muro-128 
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solution 5% sodium chloride.  An image collector was used offline to retrieve video frames every 
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10 seconds from each eye drop concentration epoch; therefore 15 images were analyzed 
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subject per solution.  Fiji image analysis software was used by a masked technician to measure 
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the visible surface area of both eyes between the upper and lower eyelids.  In each recording 
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epoch, the ratio of time the eyelids were closed are presented as a percentage of the subjects’ 
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maximum visible eye surface area between the eyelids during that epoch. 
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Data Analysis 
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Results are presented as mean ± S.E.M.  Comparisons between the control and diabetic 
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subjects were conducted using Student t-test (Prism software; GraphPad, San Diego, CA).  A p 
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value of less than 0.05 was considered significant. 
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