Effects And Mechanism Of Dyslipidemia On Peripheral Neuropathy In C57BL/6Mice

BackgroundNowadays, the prevalence of diabetes mellitus is growing rapidly. Depending on the diagnostic criteria used, at least50%up to90%of individuals with diabetes will develop diabetic neuropathy. Diabetic peripheral neuropathy is one of the most common chronic complications of diabetes mellitus. The most common complication of diabetes is diabetic neuropathy. The most common form of diabetic neuropathy is diabetic polyneuropathy, a symmetric loss of nerve function beginning in the toes and progressing in a distal to proximal fashion, yielding what is commonly called a stocking/glove pattern of sensory loss. Twenty-five years after the diagnosis of diabetes, the cumulative risk of a lower extremity amputation is22%and, in the general population,60%of all lower extremity amputations are secondary to diabetic neuropathy. This represents a cost of over$22billion per year and a significant loss of quality of life for diabetic patients. Thus diabetes peripheral neuropathy is not only a serious threat to human health but also a heavy economic burden to the whole society. Therefore, the prevention and treatment of diabetes peripheral neuropathy have been the important and urgent public health issues, which are dependent on the intensive study about the pathogenesis of diabetes peripheral neuropathy.Hyperglycemia is the driving force underlying the development of diabetic neuropathy and clearly leads to peripheral nerve injury through the development of systemic and neuronal oxidative stress. Improved blood glucose control reduces the risk of diabetic peripheral neuropathy,but the data suggest glycemic control alone is insufficient to prevent complications in type2diabetes.Emerging data establish dyslipidemia as a significant contributor to the development of diabetic neuropathy. The emerging idea may explain the earlier incidence of diabetic neuropathy in individuals with type2compared to type1diabetes. Lipid profiles are commonly abnormal early in the course of type2diabetes in a temporal pattern that correlates with the presence of diabetic neuropathy In contrast, lipid profiles are nearly always normal in type1patients at the time of diabetes diagnosis. Dyslipidemia develops later in the course of type1diabetes, and these abnormal lipid profiles coincide with the delayed onset and progression of diabetic neuropathy. Accumulating data from several large scale trials of patients with type2diabetes also point to early dyslipidemia as a major independent risk factor for the development of diabetic neuropathy.Due to complex pathogenesis, the etiology of diabetic peripheral neuropathy is hardly unveiled and effective treatments are still lacking.There is a growing consensus, driven by both clinical and basic studies, that oxidative stress underlies the development of the microvascular complications of diabetes, including diabetic neuropathy. In type1diabetic patients, the severity of microvascular complications parallels the degree of systemic oxidative stress. With disease and diabetic neuropathy progression, antioxidant potential decreases while lipid peroxidation products increase. Type2diabetic patients have a similar oxidative stress profile which directly relates the onset and progression of microvascular complications. Enhanced oxidative stress has been documented in peripheral nerve, dorsal root and sympathetic ganglia, and vasculature of the peripheral nervous system and has been implicated in neurovascular dysfunction and motor and sensory nerve conduction velocity (MNCV and SNCV) deficits, impaired neurotrophic support, nerve metabolic and signal transduction changes, and morphologic abnormalities characteristic for diabetes.12/15-lipoxygenase (12/15-LOX) is a cytosolic nonheme iron-containing dioxygenase that oxidizes esterified arachidonic acid in lipoproteins and phospholipids with formation of the bioactive lipids termed eicosanoids. Up-regulation of12/15-LOX, causes impaired cell signaling, oxidative-nitrosative stress, and inflammation. Recent studies implicated the12/15-LOX pathway in diabetic neuropathy. Scholar Stavniichuk found that12/15-LOX overexpression and activation contribute to nitrosative stress in the peripheral nervous system and development of neuropathic changes associated with prediabetes and overt diabetes. The results are consistent with another study demonstrating an important role of12/15-LOX in motor and sensory nerve conduction slowing, small sensory nerve fiber dysfunction, and axonal atrophy of large myelinated fibers characteristic for PDN.There is increasing clinical evidence linking dyslipidemia with the development of diabetic peripheral neuropathy. Further cell culture and animal models are necessary. This study is designed to investigate effects of dyslipidemia on peripheral neuropathy in C57BL/6mice and the underlying mechanism, thus provide new ideas for studies on preventing and treatment of diabetic peripheral neuropathy. We measured the thermal pain threshold and nerve conduction velocity to investigate the relationship between dyslipidemia and peripheral neuropathy in HFD-fed C57BL/6mice. Moreover, we observed the oxidative stress indexes of malondialdehyde and total superoxide dismutase in serum and12/15-lipoxygenase protein expression in sciatic nerve to explore the underlying mechanism of dyslipidemia on peripheral neuropathy.Chapter1Effects of Dyslipidemia on Peripheral Neuropathy in C57BL/6MiceObjectiveTo evaluate the peripheral nerve functional changes in C57BL/6mice with dyslipidemia induced by high-fat diet.Methods1. C57BL/6mice at3weeks age were randomly assigned to receive a control or high-fat diet (HFD) and had ad libitum access to water, with lOmice/group.2. Record body weight every week.3. An intraperitoneal glucose tolerance test (IPGTT) was performed every other week following6weeks. After6h of fasting, a fasting glucose level was obtained from one drop of tail blood and analyzed using a standard glucometer. lmg/g glucose was injected intraperitoneally to conscious mice. Blood glucose values was obtained at30,60and120min. Glucose tolerance was indicated as area under the curve of blood glucose. Mice with area under the curve of blood glucose bigger than the average of the reference group plus three times the standard deviation was diagnosed diabetes.4. Functional Studies of peripheral nerve were taken when the HFD-fed mice displayed glucose intolerance. To determine the sensitivity to noxious heat, mice were placed on a hot plate maintained at (55±0.5℃). Reaction time taken as the thermal pain threshold was recorded when the mice licked hindpaw in response to the noxious heat. Four tests separated by at least10minutes were performed for each animal, and the mean value of these tests was calculated.5. Mice were anesthetized with100/10mg/kg ketamine/acepromazine by peritoneal injection and body temperature were maintained at32-34℃. NCV was measured by powerlab/8s. MNCV was calculated by subtracting the distal latency from the proximal latency, and the result was divided into the distance between the stimulating and recording electrode. SNCV was calculated by measuring the latency to the onset/peak of the initial negative deflection and the distance between stimulating and recording electrodes. Three tests separated by at least15minutes were performed for each animal, and the mean value of these tests was calculated.6. Mice were euthanized with an overdose of sodium pentobarbital. A blood sample was collected into eppendorf tubes and centrifuged at1500r/min for15min at4℃. Serum was collected and stored at-80℃until analysis. Serum triglyceride (TG), total cholesterol(TC), high density lipoprotein cholesterol(HDL-C) and low density lipoprotein cholesterol(LDL-C) were detected with automatic biochemical meter.7. Using SPSS13.0software for statistical treatment, the results were expressed as mean±standard deviation. Comparison between groups was performed using t test. Differences were considered significant at P<0.05.Results1. Following14weeks on a high fat diet, weight was increased by29.09%compared to mice on a control diet (t=-5.804,P=0.000) while random random blood glucose remained normal (t=-2.055,P=0.055).2. Following14weeks, the blood glucose concerntration in HFD-fed mice were much higher than those fed normal chow at30min and60min after glucose was injected (t=-3.844,P<0.001; t=-2.511,P=0.022), but were similar with the control at Omin and120min(t=-1.989,P=0.062; t=-2.023,P=0.058). The area under the curve of blood glucose showed glucose tolerance was impaired in mice fed HFD compared with those fed normal chow(t=-3.625, P=0.002).3. In the hot-plate test, the latency of hindpaw response to a heat stimulus was significantly decreased in the HFD-fed mice (t=2.313,P=0.033vs. mice fed normal chow).4. MNCV and SNCV were slowed in HFD-fed mice in contrast to the control mice(t=2.430,P=0.026;t=2.527,P=0.021).5. Serum total cholesterol(TC), high density lipoprotein cholesterol(HDL-C) and low density lipoprotein cholesterol(LDL-C) were respectively increased by86.99%,38.0%and85.42%(t=-12.676,P=0.000; t=-8.061,P=0.000; t=-12.794,P=0.000). There was no obvious difference in serum triglyceride between the two groups (t=-1.502,P=0.151).Conclusions1. An animal model of hyperlipidemia induced by high-fat diet in C57BL/6mice is established successfully.2. Dyslipidemia in C57BL/6mice produces peripheral neuropathy prior to the development of frank diabetes. The characteristics of peripheral neuropathy in the animal model is similar to human subjects with pre-diabetes and obesity. Dyslipidemia is a risk factor for diabetic peripheral neuropathy. Lipid lowering therapies at the earliest stage of the disease will help to halt or delay the progression of diabetic peripheral neuropathy. Chapter2Mechanism of Peripheral Neuropathy in Dyslipidaemic C57BL/6MiceObjectiveTo observe the changes of oxidative stress indexes in serum and12/15-lipoxygenase in the sciatic nerve and explore the underlying mechanism by which peripheral neuropathy was incuced in dyslipidaemic C57BL/6mice.Methods1. Total superoxide dismutase (T-SOD) activities and malondialchelyche (MDA) contents in serum were examined with TBA and nitrite respectively.2. Sciatic nerves were rapidly dissected, frozen in liquid nitrogen and stored at-80℃until analysis.12/15-lipoxygenase protein expression was determined by western blot analysis.3. Using SPSS13.0software for statistical treatment, the results were expressed as mean±standard deviation. Comparison between groups was performed using t test. Differences were considered significant at P<0.05.Results1. Compared with the normal control, the activities of T-SOD and contents of MDA in the serum were markedly increased in the HFD-fed mice (t=-2.278,P=0.035; t=-2.6967,P=0.020).2. The contents of12/15-lipoxygenase in the sciatic nerve in the HFD-fed mice were significantly higher than that of the normal control (t=-8.135,P=0.001).Conclusions1. Oxidative stress was induced in high-fat diet-fed mice, which suggests oxidative stress may play a role in the development of peripheral neuropathy in dyslipidaemic C57BL/6mice. The strategic use of antioxidants in diabetic peripheral neuropathy may have important significance.2.12/15-lipoxygenase overexpression and activation in dyslipidaemic C57BL/6mice may contribute to the progress of peripheral neuropathy, which provides a new research direction and therapeutic target for diabetic peripheral neuropathy.