Corneal Nerves Function and Structure

Study Overview
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Brief Summary
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Peripheral neuropathy affects about 50% of the diabetic population and there is no treatment other than good blood glucose control, which is ineffective in subjects with type 2 diabetes. Part of the problem for the lack of an effective treatment is the inability to detect peripheral neuropathy in its early stage. The hypotheses to be addressed in the first phase of this study is that changes in cornea sensitivity (blinking and squinting) following addition of a hyperosmotic solution will provide a novel screening tool for early diagnosis of peripheral neuropathy. For the second phase of the study the investigators will examine the effect of fish oil treatment of diabetic subjects with neuropathy on corneal nerve density and sensitivity. Corneal nerves are the most highly innervated part of the human body with great sensitivity. The first phase will use this property and determine whether sensitivity is lost in diabetic patients with neuropathy. Preclinical studies have supported this hypothesis and now this will be tested in human subjects. Preclinical studies have also shown that treating diabetic rodents with fish oil improves nerve regeneration and outcome measures of peripheral in diabetic rodents. In the second phase the investigators will perform preliminary studies in human subjects with diabetic neuropathy and determine whether treating them with fish oil increases corneal nerve density and sensitivity.

Detailed Description
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Recruitment has been suspended because of the COVID-19 crisis and the Iowa City VA is just beginning to have face to face appointments. The hypotheses to be studied in the following specific aims is that changes in cornea sensitivity to a hyperosmotic solution will provide a novel screening tool for early diagnosis of diabetic peripheral neuropathy. The investigators also propose that clinical feasibility studies will demonstrate that treatment with fish oil will improve outcome measures of diabetic peripheral neuropathy. Specific aims: Determine if the reflex squinting and blinking response to the application of a hyperosmotic solution to the cornea is impaired in patients with type 2 diabetes with or without peripheral neuropathy. a. Determine whether changes in corneal sensitivity correlate with loss of cornea nerves in the sub-epithelial layer of the cornea and symptom scores for PN. Determine if treating human subjects with type 2 diabetic peripheral neuropathy with an oral fish oil supplement increases circulating levels of resolvin D1, restores cornea sensitivity to a hyperosmotic eye drop challenge and improves corneal nerve density and symptom scores for peripheral neuropathy. For this first phase the investigators will enroll 75 subjects to participate (Fifty subjects with type 2 diabetes; 25 with clinically detectable peripheral neuropathy and 25 without neuropathy will be enrolled as well as 25 age-matched control subjects). In the second phase of the study the investigators will investigate whether increased intake of fish oil improves diabetic peripheral neuropathy. Subjects will receive 4g (2g twice a day) of fish oil per day in capsule form. The investigators first introduced fish oil as a successful treatment of peripheral neuropathy in pre-clinical studies with diabetic rodents and will now expand those studies , culminating in this feasibility study of treatment using human subjects with type 2 diabetes and neuropathy. In this study non diabetic subjects (control) and subjects with type 2 diabetes with mild to moderate peripheral neuropathy will be recruited from phase 1 of this study to participate in the phase 2 treatment phase of the study. A baseline determination of their neuropathy (Subjective Peripheral Neuropathy Screen Questionnaire) including evaluation of corneal nerve structure and sensitivity and blood sample will be performed. Tests besides the questionnaire to be used to screen the subjects for peripheral neuropathy will include 10 g mono filament, vibration threshold, ankle reflex and sensitivity to warm and cold stimulus. Subjects will then be instructed to take 2 fish oil capsules twice a day. Subjects will return at 4, 8 and 12 months for re-evaluation. If successful, the feasibility study will provide a proof of concept to advance fish oils to a larger clinical trial for diabetic peripheral neuropathy. Visual acuity (subject's vision) will be measured by having the subject read the smallest letters on an eye chart with their glasses or best correction. This takes about 5 minutes or less. Slit lamp examination: A standard slit lamp examination will be performed to insure that no exclusion criteria are present and no corneal abrasion is present. The corneal sensitivity procedure will consist of applying a drop of 5% Muro 128 to the eye and recording the blinking and squinting response over the next 5 minutes. Based on preclinical studies it is anticipated that normal subjects will have an increased blinking and squinting response seconds after the addition of the hyperosmotic solution (5% Muro 128), whereas diabetic subjects with neuropathy will have a attenuated response. Corneal sensation threshold: Corneal sensation will also be measured in each eye using a handheld Luneau Cochet -Bonnet aesthesiometer. This device contains a thin, retractable, nylon monofilament in which the length (stiffness) is varied between 6.0 mm and 1.0 mm until the subject can detect the touch on their central cornea, resulting in a blink reflex. In each eye, testing will begin with the 6.0 mm filament, after which the length will be progressively shortened by 0.5 mm until the patient reports sensing the corneal contact. Each eye will be tested three times and the best performance (in terms of longest filament that is perceived to touch the cornea) will be recorded as the corneal sensation threshold. Ocular Coherence Tomography (OCT): The thickness of the optic nerve and macula will also be measured inside of the eye using a special camera that forms an image of the layers of the retina. The imaging is harmless and measures the thickness or structural health of retinal layers and optic nerve. A new feature of OCT is eye-tracking. Eye tracking does not take any longer since it is done simultaneously with the imaging. Eye tracking during OCT may give us new insight into some eye disorders. This test takes approximately 10 minutes. A hand-held pupillometer/electroretinogram device (RETeval, LKC) will be held in front of the subject s eye, but will not touch the eye. The device will provide a brief, a series of brief light stimuli and then record the pupil response and the elicited electrical response from the retina from a surface skin patch (electrode) placed below each eye, from the light as a measure of whether the inherent sensitivity of the eye in the retina is normal. The investigators will repeat this in the left eye. The visible light stimulus is safe and is given at an intensity experienced in normal daily light exposures. The test takes about 2 minutes per eye.

Official Title
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Diabetic Neuropathy: Function-Structure of Corneal Nerves to Assess Injury-Repair

Conditions
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Diabetic Peripheral Neuropathy

Intervention / Treatment
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* Dietary Supplement: Blackmores Omega Daily (4 1g capsules per day)


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Type 2 diabetes patients diagnosed based on the opinion of an Endocrinologist (Dr. Correia), the absence of a history of ketoacidosis, and a C-peptide > 0.8 ng/ml At least 5 year known duration of diabetes. HbA1c < 9.0% None or mild to moderate peripheral neuropathy based on medical record, Michigan Neuropathy Screening Instrument including response to monofilament test, ankle reflex test, vibration perception threshold examination using a 128 Hz tuning fork as well as hot and cold sensation evaluation Exclusion Criteria: Any neurologic, muscular, genetic, or other condition known to affect nerve or muscle function Electrolyte abnormalities, untreated hypothyroidism, abnormalities in calcium, phosphate, or magnesium concentrations, or any other metabolic disturbance affecting neural function Cigarette smoking in the past year Peripheral vascular, cardiac, pulmonary, or any other disorder affecting blood or tissue oxygenation Risk of bleeding disorder or on medication known to increase risk of bleeding History for use of aspirin Any other medical or psychological condition judged to limit compliance with the protocol or interpretation of results History of diabetic foot ulcer LDL > 100 mg/100ml, HDL < 30 mg/100ml, Triglyceride > 400 mg/100ml.* 10) BP < 140 systolic and 80 diastolic BMI 45 Insulin use to manage diabetes 200 units History of taking fish oil supplements The investigators are limiting subject population to those over 50 thus pregnancy should not be an issue nonetheless females who are pregnant, looking to become pregnant or are breast feeding will be excluded (* After overnight fast of 8-12 h; Average of three determinations in the sitting position after at least 5 minutes rest.)

Ages Eligible for Study
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Minimum Age: 50 Years
Maximum Age: 75 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Non-Randomized
Intervention Model: Single Group Assignment
Interventional Model Description: The study will be done in two phases as described in tab 5. In the second phase we will investigate whether increased intake of fish oil improves diabetic peripheral neuropathy including improvement in corneal nerve morphology and sensitivity. Subjects will receive 4g (2g twice a day) of fish oil per day in capsule form. The second phase of the study will be done in 2 of the 3 groups. The two groups for this phase of the study will be the control subjects and the diabetic subjects with neuropathy.
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Type 2 diabetes without neuropathy<br>Subjects with type 2 diabetes will be enrolled and determination if they have neuropathy will be determined from their clinical record and evaluation. | Dietary Supplement: Blackmores Omega Daily (4 1g capsules per day)<br>* Subjects enrolled in phase two of the study will be instructed to take 4 capsules per day (2 in the morning and 2 in the evening). Each capsule contains 1000 mg of triglyceride; 35% eicosapentaenoic acid and 25% docosahexaenoic acid.<br>* Other names: fish oil;|
| Experimental: Type 2 diabetes with neuropathy<br>Subjects with type 2 diabetes will be enrolled and determination if they have neuropathy will be determined from their clinical record and evaluation. | Dietary Supplement: Blackmores Omega Daily (4 1g capsules per day)<br>* Subjects enrolled in phase two of the study will be instructed to take 4 capsules per day (2 in the morning and 2 in the evening). Each capsule contains 1000 mg of triglyceride; 35% eicosapentaenoic acid and 25% docosahexaenoic acid.<br>* Other names: fish oil;|
| Experimental: Normal subjects, aged match with no symptoms of diabetes<br>Healthy, aged matched control subjects will be enrolled and determination if they have neuropathy will be determined from their clinical record and evaluation. | Dietary Supplement: Blackmores Omega Daily (4 1g capsules per day)<br>* Subjects enrolled in phase two of the study will be instructed to take 4 capsules per day (2 in the morning and 2 in the evening). Each capsule contains 1000 mg of triglyceride; 35% eicosapentaenoic acid and 25% docosahexaenoic acid.<br>* Other names: fish oil;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in corneal sensitivity to hyperosmotic eye drop | A recording of the eyes will be performed using a multi-camera video platform for a period of 150 seconds at four different time points. The first will be a baseline, second after a drop of isotonic saline and 3 and 4 after drops of 2 increasing concentrations of Muro 128 (2% sodium chloride, and 5% sodium chloride). Each of these will be separated by a 5 minute rest period. After, this is completed each subject will also be asked to record their sense of pain to each eye drop solution on a scale of 1 to 10. Squinting (an analysis of the amount of time a extent that the eye is closed) and blinking (number of blinks during the procedure) will be quantified during each time point using an image analysis program which analyzes the recorded digital video frames. | Done once as the primary outcome of phase 1 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in cornea nerve density | This is a primary endpoint for phase 2 of the study. Prior to beginning treatment with fish oil and 1 year later after fish oil intervention is complete the subjects cornea nerve density will be determined using cornea confocal microscopy. This is a non-invasive procedure that takes images on the sub-epithelial layer of the cornea. Six images will be collected at each of the two visits. From these images the total corneal nerve length will be determined and reported as mm/mm2 | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in corneal sensation threshold using Cochet Bonnet filament | This test records the mechanical sensitivity of the cornea to a filament that is touched to the cornea. The rigidity of the filament can be adjusted and the outcome is the length of the filament (6 to 1 cm) when the subjects blinks. The data will be recorded as cm. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in Michigan neuropathy screening instrument | All subjects will answer a 15 question survey that relates to symptoms of peripheral neuropathy. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in ocular coherence tomography. | Ocular coherence tomography is a special camera that measures the neural structure of the retina. This device is often used as standard of care. The subject will rest their chin on a supporting device and the camera will be positioned to image the neural retina. The test will take about 5 minutes and will be performed twice at baseline and 12 months later. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in slit lamp examination of the eye. | This examination will be the last test performed by each subject enrolled in the study. This device will shine light in the eye to examine the front and back portions of the eye. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in 10 g monofilament test | The 10 g monofilament test is a routine evaluation used to screen the diabetic foot for loss of sensory sensation and part of the standard of care for any diabetic patient. The subject will remove their footwear and lie down on a table. The filament will be applied perpendicular to the skin surface on the bottom of the feet will sufficient force to allow the filament to bend. Each subject will be asked to tell the examiner if they feel it. A lack of sensation is a marker for diabetic neuropathy. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in vibratory sensation of the great toe. | Vibratory sensation is a standard of care test used with patients with diabetes to test sensory nerve sensation. A 129 Hz tuning fork is used and placed over the dorsum of the great toe on the boney prominence of the distal interphalangeal joint. The subject is asked to tell the examiner if they feel the object touching their toe. A lack of sensation is a marker for diabetic neuropathy. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in reflex testing | Reflex testing is commonly used to detect sensory neuropathy in diabetic patients. The ankle reflex is examined by aligning the subjects ankle into a neutral position and the examiner strikes the Achilles tendon with a neurological hammer. An abnormal result is recorded if the subject does not display any ankle plantarflexion. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in hot or cold sensation. | Cold and warm thresholds measure response of A-delta and C fibers of sensory nerves. A Velcro strap containing a metal circular pad about the size of a half dollar is attached to the dorsal part of the right foot. The subject is asked to push a button on a hand held device when they feel hot or cold from the circular pad attached to their foot. The device is set to default before any damaging temperature is reached. First warm followed by cold will be tested and the temperature of the pad will be recorded when the patient pushes the button. The commercial TSA-II Neurosensory Analyzer will be used. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in visual acuity (subject's vision) | A routine visual acuity eye examination will be used. Subjects vision will be measured by having the subject read the smallest letters on an eye chart with their glasses or best correction. This takes about 5 minutes or less. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in omega-3 polyunsaturated fatty acids and their metabolites in serum | Blood will be collected at baseline and end of the phase 2 part of the study. Blood will be used to determine the omega-6 to omega-3 ratio in blood a marker of inflammatory stress. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Change in pupil responsiveness to light | A hand-held pupillometer/electroretinogram device (RETeval, LKC) will be held in front of the subject s eye, but will not touch the eye. The device will provide a brief, a series of brief light stimuli and then record the pupil response and the elicited electrical response from the retina from a surface skin patch (electrode) placed below each eye, from the light as a measure of whether the inherent sensitivity of the eye in the retina is normal. The investigators will repeat this in the left eye. The visible light stimulus is safe and is given at an intensity experienced in normal daily light exposures. The test takes about 2 minutes per eye. | Done once as a baseline measurement for phase 2 of the study and again at the end of the study after 12 months of fish oil treatment. | 
| Questionnaires for corneal sensitivity | There will be two questionnaires, Ocular Surface Disease Index (OSDI which contains 12 questions eye sensitivity to light, vision acuity, dryness, and sensitivity when reading, using the computer, and watching TV) and the Dry Eye Questionnaire (DEQ which contains 5 questions relating to discomfort, dryness and watery eyes). | Done once as baseline for phase 1 of the study and also will be repeated at the end of phase 2 of the study. | 
| Determination of tibial nerve conduction velocity | This is a non-invasive method of determining conduction velocity of the tibial nerve. The subject will be instructed to lie down on a examination table with shoes and socks removed. Afterwards, the lower ankle will be swabbed with an alcohol patch and allowed to air dry. A device containing 2 posts (about 3 inches apart) will be placed on the lower ankle in the region of the Achilles and activated. For a period of 5-10 sec the device sends a pulse stimulating the nerve distally (post located at the lower part of the ankle) and the response recorded by the post located distally on the skin of the upper part of the ankle. The nerve conduction is recorded electronically by the device and data down loaded to a secured computer. During the time of the analyses the subject will feel dual pulsating sensation that will last up to 10 sec. | Done once as baseline for phase 1 of the study and also will be repeated at the end of phase 2 of the study. | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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diabetes, neuropathy, fish oil, cornea, omega 3 polyunsaturated fatty acids