COMPARATIVE EFFECT OF A HIGH FAT WITH OR WITHOUT HIGH LEVELS OF SUCROSE DIETS ON PERIPHERAL NEUROPATHY IN C57BL/6J MICE.

Objective: Feeding mice a diet containing high fat and high sucrose has been promoted as a good model for type 2 diabetes. This study sought to determine the effect of feeding mice a high fat and high sucrose diet on neuropathy compared to mice fed only a high fat diet and mice fed a high diet and treated with streptozotocin. Methods: C57Bl/6J mice were divided into five groups and fed the following diets for 20 weeks: Normal (Control); Sucrose enriched (Control + Sucrose), High Fat (Diet-induced obesity (DIO)), High Fat and High Sucrose (DIO + sucrose) and High Fat diet/streptozotocin treated (Diabetic). The endpoints evaluated included motor and sensory nerve conduction velocity, thermal and mechanical sensitivity and innervation of sensory nerves in the cornea and skin. Results: Diabetic mice were hyperglycemic at the end of the study and along with DIO mice with or without Sucrose had impaired glucose utilization. DIO mice had slowed sensory nerve conduction velocity, mechanical allodynia and decreased innervation of the cornea and skin. DIO + Sucrose and to a greater extent diabetic mice were thermal hypoalgesic, had mechanical allodynia, reduced motor and sensory nerve conduction velocities and decrease innervation of the cornea and skin. Conclusions: Development of peripheral neuropathy was more severe in High Fat and High Sucrose fed mice compared to high fat fed mice but fasting hyperglycemia and impaired glucose utilization was similar for these two models. Peripheral neuropathy was most severe in diabetic mice.

Dietary fats modify vascular fat composition, eNOS localization within lipid rafts and vascular function in obesity.

We tested whether dietary fatty acids alter membrane composition shifting localization of signaling pathways within caveolae to determine their role in vascular function. Wild type (WT) and caveolin-1-deficient mice (cav-1 KO), required for vascular caveolae formation, were fed low fat (LF), high saturated fat (HF, 60% kcal from lard), or high-fat diet with 50:50 lard and n-3 polyunsaturated fatty acid-enriched menhaden oil (MO). HF and MO increased body weight and fat in WT but had less effect in cav-1 KO. MO increased unsaturated fatty acids and the unsaturation index of aorta from WT and cav-1 KO. In LF WT aorta, endothelial nitric oxide synthase (eNOS) was localized to cav-1-enriched low-density fractions which shifted to actin-enriched high-density fractions with acetylcholine (ACh). HF and MO shifted eNOS to high-density fractions in WT aorta which was not affected by ACh. In cav-1 KO aorta, eNOS was localized in low-density non-caveolar fractions but not shifted by ACh or diet. Inducible NOS and cyclooxygenase 1/2 were not localized in low-density fractions or affected by diet, ACh or genotype. ACh-induced dilation of gracilis arteries from HF WT was similar to dilation in LF but the NOS component was reduced. In WT and cav-1 KO, dilation to ACh was enhanced by MO through increased role for NOS and cyclooxygenase. We conclude that dietary fats affect vascular fatty acid composition and membrane localization of eNOS but the contribution of eNOS and cyclooxygenase in ACh-mediated vascular responses is independent of lipid rafts.

Effect of Dietary Content of Menhaden Oil with or without Salsalate on Neuropathic Endpoints in High-Fat-Fed/Low-Dose Streptozotocin-Treated Sprague Dawley Rats.

In this study, we wanted to extend our investigation of the efficacy of fish oil with or without salsalate on vascular and neural complications using a type 2 diabetic rat model. Four weeks after the onset of hyperglycemia, diabetic rats were treated via the diet with 3 different amounts of menhaden oil with or without salsalate for 12 weeks. Afterwards, vascular reactivity of epineurial arterioles and neuropathy-related endpoints were examined. The addition of salsalate to high-fat diets enriched with 10% or 25% kcal of menhaden oil protected vascular reactivity to acetylcholine and calcium gene-related peptide, motor and sensory nerve conduction velocity, thermal nociception, intraepidermal nerve fiber density, and cornea sensitivity to a greater extent than 10% or 25% menhaden oil alone. Vascular and neural function was maximally protected with diet containing 45% kcal as menhaden oil, and adding salsalate did not provide any additional benefit. Salsalate alone in the high-fat diet of diabetic rats provided minimal protection/improvement of vascular and neural dysfunction. These studies imply that dietary salsalate in combination with lower amounts of menhaden oil can provide greater benefit toward diabetes-induced vascular and neural impairment than menhaden oil alone.

Vascular and Neural Complications in Type 2 Diabetic Rats: Improvement by Sacubitril/Valsartan Greater Than Valsartan Alone.

Previously, we had shown that a vasopeptidase inhibitor drug containing ACE and neprilysin inhibitors was an effective treatment for diabetic vascular and neural complications. However, side effects prevented further development. This led to the development of sacubitril/valsartan, a drug containing angiotensin II receptor blocker and neprilysin inhibitor that we hypothesized would be an effective treatment for diabetic peripheral neuropathy. Using early and late intervention protocols (4 and 12 weeks posthyperglycemia, respectively), type 2 diabetic rats were treated with valsartan or sacubitril/valsartan for 12 weeks followed by an extensive evaluation of vascular and neural end points. The results demonstrated efficacy of sacubitril/valsartan in improving vascular and neural function was superior to valsartan alone. In the early intervention protocol, sacubitril/valsartan treatment was found to slow progression of these deficits and, with late intervention treatment, was found to stimulate restoration of vascular reactivity, motor and sensory nerve conduction velocities, and sensitivity/regeneration of sensory nerves of the skin and cornea in a rat model of type 2 diabetes. These preclinical studies suggest that sacubitril/valsartan may be an effective treatment for diabetic peripheral neuropathy, but additional studies will be needed to investigate these effects further.

Determination of peripheral neuropathy in high-fat diet fed low-dose streptozotocin-treated female C57Bl/6J mice and Sprague-Dawley rats.

AIMS/INTRODUCTION: Peripheral neuropathy is a common complication of diabetes and also occurs in 30% of human obese individuals with impaired glucose tolerance. Even though peripheral neuropathy affects both sexes, most pre-clinical studies have been carried out using male rodents. The aim of the present study was to create diet-induced obesity and type 2 diabetes in female rats and mice in order to examine the development of peripheral neuropathy. MATERIALS AND METHODS: At 12 weeks-of-age, rats and mice were separated into three groups. Two groups or rats and mice were fed a 60-kcal% high-fat diet for 12 weeks (rats) or 8 weeks (mice). To induce type 2 diabetes, one group of high-fat diet-fed rats and mice were treated with a low dose of streptozotocin. Analyses of multiple neural end-points were carried out 12 weeks later. RESULTS: Glucose utilization was impaired in diet-induced obese female rats and mice, as was a number of neurological end-points including nerve conduction velocity, intraepidermal and subepithelial corneal nerve fiber densities, and thermal and mechanical sensitivity. When female diet-induced obese rats or mice were made hyperglycemic, glucose utilization and sensory nerve density of the skin and cornea, as well as thermal and mechanical sensitivity, were more significantly impaired compared with diet-induced obese female rodents. CONCLUSIONS: These studies show that diet-induced obese and type 2 diabetic female rodents develop peripheral neuropathy that is similar to that occurring in male rodents. However, for female rats, more aggressive treatment is required to induce dietary obesity.

Impaired Corneal Sensation and Nerve Loss in a Type 2 Rat Model of Chronic Diabetes Is Reversible With Combination Therapy of Menhaden Oil, α-Lipoic Acid, and Enalapril.

PURPOSE: This study investigated the efficacy of monotherapy versus combination of menhaden oil, α-lipoic acid, and enalapril on corneal sensation and morphometry and other neuropathy-related endpoints in a rat model of type 2 diabetes. METHODS: Male Sprague-Dawley rats (aged 12 weeks) were fed a high-fat diet for 8 weeks followed by 30 mg/kg streptozotocin. After 16 weeks of hyperglycemia, 12-week treatments consisting of menhaden oil, α-lipoic acid, enalapril, or their combination were initiated. Before and after treatments, we performed analyses of multiple neural and vascular endpoints including corneal sensitivity, corneal nerve density, vascular reactivity of epineurial arterioles, motor and sensory nerve conduction velocity, intraepidermal nerve fiber density, and thermal nociception. RESULTS: Before treatment, all the neural and vascular endpoints in diabetic rats were impaired. Treating diabetic rats with monotherapy was effective in improving neural and vascular deficits with menhaden oil being most efficacious. However, the combination therapy provided the greatest benefit and improved/reversed all nerve and vascular deficits. The effect of combination therapy on corneal relative sensitivity and structure (in mm/mm), primary endpoints for this study, for control, diabetic, and diabetic treated rats was 4.2 ± 1.4 and 7.5 ± 0.5, 12.1 ± 1.3* and 3.8 ± 0.2*, and 6.6 ± 2.3 and 7.3 ± 0.5, respectively (*P < 0.05 compared with control rats; P < 0.05 compared with diabetic rats). CONCLUSIONS: These studies suggest that a combination therapeutic approach may be most effective for treating vascular and neural complications of type 2 diabetes.

Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (l50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1α mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function.

Effect of tempol on peripheral neuropathy in diet-induced obese and high-fat fed/low-dose streptozotocin-treated C57Bl6/J mice.

In this study, we sought to determine the efficacy of tempol on multiple neuropathic endpoints in a diet-induced obese mouse, a model of pre-diabetes, and a high-fat fed low-dose streptozotocin treated mouse, a model of type 2 diabetes. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperdine -1-oxyl) is a low molecular weight, water soluble, membrane permeable, and metal-independent superoxide dismutase mimetic that has been widely used in cellular studies for the removal of intracellular and extracellular superoxide. This in vivo study was designed to be an early intervention. Fourteen weeks post-high-fat diet (6 weeks post-hyperglycemia) control, obese, and diabetic mice were divided into no treatment and treatment groups. The treated mice received tempol by gavage (150 mg/kg in water), while the untreated mice received vehicle. The diet-induced obese and the diabetic mice were maintained on the high-fat diet for the duration of the study, while the control group was maintained on the standard diet. Obesity and diabetes caused slowing of motor and sensory nerve conduction, reduction in intraepidermal nerve fiber density, thermal hypoalgesia, and mechanical allodynia. Treatment with tempol partially or completely protected obese and diabetic mice from these deficits. These studies suggest that tempol or other effective scavengers of reactive oxygen species may be a viable option for treating neural complications associated with obesity or type 2 diabetes.

Effect of Fish Oil vs. Resolvin D1, E1, Methyl Esters of Resolvins D1 or D2 on Diabetic Peripheral Neuropathy.

OBJECTIVE: Fish oil is enriched in omega-3 polyunsaturated fatty acids primarily eicosapentaenoic and docosahexaenoic fatty acids. Metabolites of these two polyunsaturated fatty acids include the E and D series resolvins. Omega-3 polyunsaturated fatty acids and resolvins have been reported to have anti-inflammatory and neuroprotective properties. The objective of this study was to evaluate the efficacy of menhaden oil, a fish oil derived from the menhaden, resolvins D1 and E1 and the methyl esters of resolvins D1 and D2 on diabetic peripheral neuropathy. Hypothesis being examined was that the methyl esters of resolvins D1 and D2 would be move efficacious than resolvins D1 or E1 due to an extended half-life. METHODS: A model of type 2 diabetes in C57BL/6J mice was created through a combination of a high fat diet followed 8 weeks later with treatment of low dosage of streptozotocin. After 8 weeks of untreated hyperglycemia type 2 diabetic mice were treated for 8 weeks with menhaden oil in the diet or daily injections of 1 ng/g body weight resolvins D1, E1 or methyl esters of resolvins D1 or D2. Afterwards, multiple neurological endpoints were examined. RESULTS: Menhaden oil or resolvins did not improve hyperglycemia. Untreated diabetic mice were thermal hypoalgesic, had mechanical allodynia, reduced motor and sensory nerve conduction velocities and decreased innervation of the cornea and skin. These endpoints were significantly improved with menhaden oil or resolvin treatment. However, the methyl esters of resolvins D1 or D2, contrary to our hypothesis, were generally less potent than menhaden oil or resolvins D1 or E1. CONCLUSION: These studies further support omega-3 polyunsaturated fatty acids derived from fish oil via in part due to their metabolites could be an effective treatment for diabetic neuropathy.

Early vs. late intervention of high fat/low dose streptozotocin treated C57Bl/6J mice with enalapril, α-lipoic acid, menhaden oil or their combination: Effect on diabetic neuropathy related endpoints.

We have previously demonstrated that enalapril, α-lipoic acid and menhaden (fish) oil has potential as a treatment for diabetic peripheral neuropathy. In this study we sought to determine the efficacy of these treatments individually or in combination on multiple neuropathic endpoints in a high fat fed low dose streptozotocin treated mouse, a model of type 2 diabetes, following early or late intervention. Four or twelve weeks after the onset of hyperglycemia, diabetic mice were treated with enalapril, α-lipoic acid, menhaden oil or their combination for 12 weeks. Afterwards, endpoints including glucose tolerance, motor and sensory nerve conduction velocity, thermal nociception, and intraepidermal and cornea nerve fiber density was determined. Glucose clearance was impaired in diabetic mice and significantly improved only with combination treatment and early intervention. Diabetes caused steatosis, slowing of motor and sensory nerve conduction velocity, thermal hypoalgesia and reduction in intraepidermal and cornea nerve fiber density. Treating diabetic mice with enalapril, α-lipoic acid or menhaden oil partially protected diabetic mice from these deficits, whereas the combination of these three treatments was more efficacious following early or late intervention. These studies suggest that a combination therapy may be more effective for treating neural complications of type 2 diabetes.

Effect of Treatment with Salsalate, Menhaden Oil, Combination of Salsalate and Menhaden Oil, or Resolvin D1 of C57Bl/6J Type 1 Diabetic Mouse on Neuropathic Endpoints.

Aims. In this study a streptozotocin induced type 1 diabetes mouse model was used to assess the effectiveness of salsalate, menhaden oil, the combination of salsalate and menhaden oil, or resolvin D1 on neuropathic endpoints. Materials and Methods. Changes in body weight, blood glucose, serum markers for triglycerides, free fatty acids, cholesterol, and resolvin D1, motor and sensory nerve conduction velocities and thermal sensitivity were assessed, as well as performing in vivo confocal microscopy of subepithelial corneal nerves and immunohistochemistry of nerves in the cornea and foot pad. Results. Diabetic animals failed to gain weight and had elevated blood glucose levels. Diabetic mice had slowed nerve conduction velocity, reduced innervation of the foot pad and cornea subepithelial and epithelial layers, and reduced thermal sensitivity. Monotherapy treatment with salsalate, menhaden oil, and resolvin D1 reduced the pathological signs of diabetic neuropathy. The combination of salsalate and menhaden oil also reduced signs of pathology and generated elevated plasma levels of resolvin D1 compared to other groups. Conclusions. Additional studies are needed to determine whether the combination of salsalate and menhaden oil may be more efficacious than monotherapy alone for the treatment of diabetic peripheral neuropathy.

Corneal Sensitivity to Hyperosmolar Eye Drops: A Novel Behavioral Assay to Assess Diabetic Peripheral Neuropathy.

PURPOSE: Diagnosis of peripheral neuropathy (PN), which affects approximately 50% of the diabetic population, is subjective, with many patients seeking a diagnosis only after presenting with symptoms. Recently, in vivo confocal microscopy of subepithelial corneal nerve density has been promoted as a surrogate marker for early detection of PN, but imaging of corneal nerves requires sophisticated instrumentation, expertise in confocal imaging, cooperative patients, and automated analysis tools to derive corneal nerve density. As an alternative, we developed a simple screening method that is based on the sensitivity of corneal nerves to cause reflex eyelid squinting in response to hyperosmolar eye drops. METHODS: Eyes of control and type 2 diabetic rats were given an eye drop of a 290- to 900-mOsm solution, and the ocular response was video recorded. Other neuropathic end points including nerve conduction velocity and subepithelial cornea nerve density were determined. RESULTS: Motor and sensory nerve conduction velocity and total nerve fiber length of corneal nerves in the subepithelial layer were significantly decreased in diabetic rats. Applying the hyperosmotic solutions to the ocular surface caused an osmolarity-dependent increase in squinting of the treated eye in control rats. Squinting was almost totally blocked by preapplication of proparacaine or N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide, a transient receptor potential melastatin-8 channel blocker. Squinting in response to the 900-mOsm solution was significantly reduced in diabetic rats. CONCLUSIONS: Preclinical studies show that evaluation of corneal sensitivity may be an alternative method for the early detection of PN and has potential for translation to clinical studies.

Nicotinamide Riboside Opposes Type 2 Diabetes and Neuropathy in Mice.

Male C57BL/6J mice raised on high fat diet (HFD) become prediabetic and develop insulin resistance and sensory neuropathy. The same mice given low doses of streptozotocin are a model of type 2 diabetes (T2D), developing hyperglycemia, severe insulin resistance and diabetic peripheral neuropathy involving sensory and motor neurons. Because of suggestions that increased NAD(+) metabolism might address glycemic control and be neuroprotective, we treated prediabetic and T2D mice with nicotinamide riboside (NR) added to HFD. NR improved glucose tolerance, reduced weight gain, liver damage and the development of hepatic steatosis in prediabetic mice while protecting against sensory neuropathy. In T2D mice, NR greatly reduced non-fasting and fasting blood glucose, weight gain and hepatic steatosis while protecting against diabetic neuropathy. The neuroprotective effect of NR could not be explained by glycemic control alone. Corneal confocal microscopy was the most sensitive measure of neurodegeneration. This assay allowed detection of the protective effect of NR on small nerve structures in living mice. Quantitative metabolomics established that hepatic NADP(+) and NADPH levels were significantly degraded in prediabetes and T2D but were largely protected when mice were supplemented with NR. The data justify testing of NR in human models of obesity, T2D and associated neuropathies.

Effect of combination therapy consisting of enalapril, α-lipoic acid, and menhaden oil on diabetic neuropathy in a high fat/low dose streptozotocin treated rat.

We have previously demonstrated that treating diabetic rats with enalapril, an angiotensin converting enzyme (ACE) inhibitor, α-lipoic acid, an antioxidant, or menhaden oil, a natural source of omega-3 fatty acids can partially improve diabetic peripheral neuropathy. In this study we sought to determine the efficacy of combining these three treatments on vascular and neural complications in a high fat fed low dose streptozotocin treated rat, a model of type 2 diabetes. Rats were fed a high fat diet for 8 weeks followed by a 30 mg/kg dose of streptozotocin. Eight weeks after the onset of hyperglycemia diabetic rats were treated with a combination of enalapril, α-lipoic acid and menhaden oil. Diabetic rats not receiving treatment were continued on the high fat diet. Glucose clearance was impaired in diabetic rats and significantly improved with treatment. Diabetes caused steatosis, elevated serum lipid levels, slowing of motor and sensory nerve conduction, thermal hypoalgesia, reduction in intraepidermal nerve fiber profiles, decrease in cornea sub-basal nerve fiber length and corneal sensitivity and impairment in vascular relaxation to acetylcholine and calcitonin gene-related peptide in epineurial arterioles of the sciatic nerve. Treating diabetic rats with the combination of enalapril, α-lipoic acid and menhaden oil reversed all these deficits to near control levels except for motor nerve conduction velocity which was also significantly improved compared to diabetic rats but remained significantly decreased compared to control rats. These studies suggest that a combination therapeutic approach may be most effective for treating vascular and neural complications of type 2 diabetes.

Rat Models of Diet-Induced Obesity and High Fat/Low Dose Streptozotocin Type 2 Diabetes: Effect of Reversal of High Fat Diet Compared to Treatment with Enalapril or Menhaden Oil on Glucose Utilization and Neuropathic Endpoints.

We examined whether reversal of high fat diet, stimulating weight loss, compared to two treatments previously shown to have beneficial effects, could improve glucose utilization and peripheral neuropathy in animal models of obesity and type 2 diabetes. Rats were fed a high fat diet and treated with a low dose of streptozotocin to create models of diet induced obesity or type 2 diabetes, respectively. Afterwards, rats were transferred to a normal diet or treated with enalapril or dietary enrichment with menhaden oil for 12 weeks. Obesity and to a greater extent type 2 diabetes were associated with impaired glucose utilization and peripheral neuropathy. Placing obese rats on a normal diet improved glucose utilization. Steatosis but not peripheral neuropathy was improved after placing obese or diabetic rats on a normal diet. Treating obese and diabetic rats with enalapril or a menhaden oil enriched diet generally improved peripheral neuropathy endpoints. In summary, dietary improvement with weight loss in obese or type 2 diabetic rats was not sufficient to correct peripheral neuropathy. These results further stress the need for discovery of a comprehensive treatment for peripheral neuropathy.

Effect of enriching the diet with menhaden oil or daily treatment with resolvin D1 on neuropathy in a mouse model of type 2 diabetes.

The purpose of this study was to determine the effect of supplementing the diet of a mouse model of type 2 diabetes with menhaden (fish) oil or daily treatment with resolvin D1 on diabetic neuropathy. The end points evaluated included motor and sensory nerve conduction velocity, thermal sensitivity, innervation of sensory nerves in the cornea and skin, and the retinal ganglion cell complex thickness. Menhaden oil is a natural source for n-3 polyunsaturated fatty acids, which have been shown to have beneficial effects in other diseases. Resolvin D1 is a metabolite of docosahexaenoic acid and is known to have anti-inflammatory and neuroprotective properties. To model type 2 diabetes, mice were fed a high-fat diet for 8 wk followed by a low dosage of streptozotocin. After 8 wk of hyperglycemia, mice in experimental groups were treated for 6 wk with menhaden oil in the diet or daily injections of 1 ng/g body wt resolvin D1. Our findings show that menhaden oil or resolvin D1 did not improve elevated blood glucose, HbA1C, or glucose utilization. Untreated diabetic mice were thermal hypoalgesic, had reduced motor and sensory nerve conduction velocities, had decreased innervation of the cornea and skin, and had thinner retinal ganglion cell complex. These end points were significantly improved with menhaden oil or resolvin D1 treatment. Exogenously, resolvin D1 stimulated neurite outgrowth from primary cultures of dorsal root ganglion neurons from normal mice. These studies suggest that n-3 polyunsaturated fatty acids derived from fish oil could be an effective treatment for diabetic neuropathy.

Enriching the diet with menhaden oil improves peripheral neuropathy in streptozotocin-induced type 1 diabetic rats.

The purpose of this study was to determine the effect of supplementing the diet of type 1 diabetic rats with menhaden oil on diabetic neuropathy. Menhaden oil is a natural source for n-3 fatty acids, which have been shown to have beneficial effects in cardiovascular disease and other morbidities. Streptozotocin-induced diabetic rats were used to examine the influence of supplementing their diet with 25% menhaden oil on diabetic neuropathy. Both prevention and intervention protocols were used. Endpoints included motor and sensory nerve conduction velocity, thermal and mechanical sensitivity, and innervation and sensitivity of the cornea and hindpaw. Diabetic neuropathy as evaluated by the stated endpoints was found to be progressive. Menhaden oil did not improve elevated HbA1C levels or serum lipid levels. Diabetic rats at 16-wk duration were thermal hypoalgesic and had reduced motor and sensory nerve conduction velocities, and innervation and sensitivity of the cornea and skin were impaired. These endpoints were significantly improved with menhaden oil treatment following the prevention or intervention protocol. We found that supplementing the diet of type 1 diabetic rats with menhaden oil improved a variety of endpoints associated with diabetic neuropathy. These results suggest that enriching the diet with n-3 fatty acids may be a good treatment strategy for diabetic neuropathy.

Characterization of diabetic neuropathy in the Zucker diabetic Sprague-Dawley rat: a new animal model for type 2 diabetes.

Recently a new rat model for type 2 diabetes the Zucker diabetic Sprague-Dawley (ZDSD/Pco) was created. In this study we sought to characterize the development of diabetic neuropathy in ZDSD rats using age-matched Sprague-Dawley rats as a control. Rats were examined at 34 weeks of age 12 weeks after the onset of hyperglycemia in ZDSD rats. At this time ZDSD rats were severely insulin resistant with slowing of both motor and sensory nerve conduction velocities. ZDSD rats also had fatty livers, elevated serum free fatty acids, triglycerides, and cholesterol, and elevated sciatic nerve nitrotyrosine levels. The corneas of ZDSD rats exhibited a decrease in subbasal epithelial corneal nerves and sensitivity. ZDSD rats were hypoalgesic but intraepidermal nerve fibers in the skin of the hindpaw were normal compared to Sprague-Dawley rats. However, the number of Langerhans cells was decreased. Vascular reactivity of epineurial arterioles, blood vessels that provide circulation to the sciatic nerve, to acetylcholine and calcitonin gene-related peptide was impaired in ZDSD rats. These data indicate that ZDSD rats develop many of the neural complications associated with type 2 diabetes and are a good animal model for preclinical investigations of drug development for diabetic neuropathy.

Differences and similarities in development of corneal nerve damage and peripheral neuropathy and in diet-induced obesity and type 2 diabetic rats.

PURPOSE: Peripheral neuropathy has been shown to exist in prediabetic and diabetic patients and animal models. However, the development of peripheral neuropathy in prediabetes and posthyperglycemia is likely different. The purpose of this study was to examine the progression of peripheral neuropathy in diet-induced obese rats and high-fat-fed rats treated with a low dose of streptozotocin, a model for type 2 diabetes, using standard endpoints as well as corneal sensitivity and innervation. METHODS: Diet-induced obese rats and high-fat/low-dose streptozotocin diabetic rats were used to examine standard peripheral neuropathy endpoints and innervation of the cornea and corneal epithelium using corneal and standard confocal microscopy, respectively, and corneal sensitivity using a Cochet-Bonnet esthesiometer at three different time points. RESULTS: Obese rats and to a greater extent diabetic rats were insulin resistant. Obese and diabetic rats had developed sensory nerve deficits, but only diabetic rats had motor nerve dysfunction as determined by measuring nerve conduction velocity, thermal nociception, and intraepidermal nerve fiber density. In the cornea there was a decrease in corneal nerve fiber length, innervation of the corneal epithelium, and corneal sensitivity in both diet-induced obese and diabetic rats. CONCLUSIONS: These studies demonstrate that changes in corneal nerve innervation and sensitivity occur in both obese and type 2 diabetic rat models that are consistent with development of peripheral neuropathy. Examination of corneal nerve changes may be valuable endpoints for exploring potential treatments for peripheral neuropathy in both prediabetes with insulin resistance and diabetes.

Early loss of innervation of cornea epithelium in streptozotocin-induced type 1 diabetic rats: improvement with ilepatril treatment.

PURPOSE: Cornea confocal microscopy is emerging as a clinical tool to evaluate the development and progression of diabetic neuropathy. The purpose of these studies was to characterize the early changes in corneal sensitivity and innervation in a rat model of type 1 diabetes in relation to standard peripheral neuropathy endpoints and to assess the effect of Ilepatril, a vasopeptidase inhibitor which blocks angiotensin converting enzyme and neutral endopeptidase, on these endpoints. METHODS: Streptozotocin-diabetic rats 8 weeks duration were treated with or without Ilepatril for the last 6 weeks of the experimental period. Afterwards, standard diabetic neuropathy endpoints, subbasal corneal nerves and innervation of the epithelium, corneal sensitivity using a Cochet-Bonnet esthesiometer, and vascular reactivity of the posterior ciliary artery were examined. RESULTS: Diabetes caused a decrease in nerve conduction velocity, thermal hypoalgesia, and a reduction in intraepidermal nerve fiber profiles. In the cornea there was a decrease in corneal nerve fibers innervating the epithelium and corneal sensitivity, but subbasal corneal nerve fibers was not changed. Vascular relaxation in response to acetylcholine was decreased in the posterior ciliary artery. These defects were partially to completely prevented by Ilepatril treatment. CONCLUSIONS: These studies suggest that in type 1 diabetic rats decreased innervation of the cornea epithelium occurs early in diabetes and prior to a detectable decrease in subbasal corneal nerves and that these and other diabetic neuropathy-related defects can be partially to completely prevented by a vasopeptidase inhibitor.