Effect of glycemic control on corneal nerves and peripheral neuropathy in streptozotocin‐induced diabetic C57Bl/6J mice

We sought to determine the impact that duration of hyperglycemia and control has on corneal nerve fiber density in relation to standard diabetic neuropathy endpoints. Control and streptozotocin‐diabetic C57Bl/6J mice were analyzed after 4, 8, 12, and 20 weeks. For the 20‐week time point, five groups of mice were compared: control, untreated diabetic, and diabetic treated with insulin designated as having either poor glycemic control, good glycemic control, or poor glycemic control switched to good glycemic control. Hyperglycemia was regulated by use of insulin‐releasing pellets. Loss of corneal nerves in the sub‐epithelial nerve plexus or corneal epithelium progressed slowly in diabetic mice requiring 20 weeks to reach statistical significance. In comparison, slowing of motor and sensory nerve conduction velocity developed rapidly with significant difference compared with control mice observed after 4 and 8 weeks of hyperglycemia, respectively. In diabetic mice with good glycemic control, average blood glucose levels over the 20‐week experimental period were lowered from 589 ± 2 to 251 ± 9 mg/dl. All diabetic neuropathy endpoints examined were improved in diabetic mice with good glycemic control compared with untreated diabetic mice. However, good control of blood glucose was not totally sufficient in preventing diabetic neuropathy.     

Repeated monitoring of corneal nerves by confocal microscopy as an index of peripheral neuropathy in type‐1 diabetic rodents and the effects of topical insulin

We developed a reliable imaging and quantitative analysis method for in vivo corneal confocal microscopy (CCM) in rodents and used it to determine whether models of type 1 diabetes replicate the depletion of corneal nerves reported in diabetic patients. Quantification was reproducible between observers and stable across repeated time points in two rat strains. Longitudinal studies were performed in normal and streptozotocin (STZ)‐diabetic rats, with innervation of plantar paw skin quantified using standard histological methods after 40 weeks of diabetes. Diabetic rats showed an initial increase, then a gradual reduction in occupancy of nerves in the sub‐basal plexus so that values were significantly lower at week 40 (68 ± 6%) than age‐matched controls (80 ± 2%). No significant loss of stromal or intra‐epidermal nerves was detected. In a separate study, insulin was applied daily to the eye of control and STZ‐diabetic mice and this treatment prevented depletion of nerves of the sub‐basal plexus. Longitudinal studies are viable in rodents using CCM and depletion of distal corneal nerves precedes detectable loss of epidermal nerves in the foot, suggesting that diabetic neuropathy is not length dependent. Loss of insulin‐derived neurotrophic support may contribute to the pathogenesis of corneal nerve depletion in type 1 diabetes.     

Protection from neuropathy in extreme duration type 1 diabetes

A proportion of individuals with type 1 diabetes mellitus for more than 50 years (medallists) may be protected from developing nephropathy, retinopathy and neuropathy. Detailed neuropathy phenotyping was undertaken in a cohort of 33 medallists aged 63.7 ± 1.4 years with diabetes for 58.5 ± 0.8 years and HbA1c of 65.9 ± 2.1 mmol/mmol. Medallists had a significantly higher HbA1c (P < .001), lower estimated glomerular filtration rate (eGFR) (P = .005) and higher albumin creatinine excretion ratio (ACR) (P = .01), but a lower total cholesterol (P < .001), triacylglycerols (P = .001), low density lipoprotein‐cholesterol (P < .001) and higher high density lipoprotein‐cholesterol (P = .03), compared to controls. Twenty‐four percent of participants were identified as “escapers” without confirmed diabetic neuropathy. They had a lower neuropathy symptom profile (P = .002), vibration perception threshold (P = .02), warm threshold (P = .05), higher peroneal amplitude (P = .005), nerve conduction velocity (P = .03), heart rate variability (P = .001), corneal nerve fibre density (P = 0.001), branch density (P < .001) and length (P = .001), compared to medallists with diabetic neuropathy. Escapers had a shorter duration of diabetes (P = .006), lower alcohol consumption (P = .04), lower total cholesterol (P = .04) and LDL (P = .02), higher eGFR (P = .001) and lower ACR (P < .001). Patients with extreme duration diabetes without diabetic neuropathy have a comparable HbA1c, blood pressure and body mass index, but a more favourable lipid profile and consume less alcohol compared to those with diabetic neuropathy.     

同型半胱氨酸与糖尿病周围神经病变的关系

目的：了解血浆同型半胱氨酸(Hcy)与2型糖尿病周围神经病变间的关系及叶酸对Hcy水平的影响和对糖尿病周围神经病变的防治作用。方法：24只糖尿病KKAy小鼠分为3组：糖尿病诱发饮食组(KA组)；高蛋氨酸饮食组(KB组)；喂养高蛋氨酸饮食的基础上加用叶酸组(Kc组)。16只C57小鼠为正常对照组，分为2组：糖尿病诱发饮食组(CA组)；高蛋氨酸饮食组(CB组)。测定血糖、体重、血浆HCY、叶酸浓度，并观察光镜下及电镜下坐骨神经结构变化。结果：糖尿病小鼠喂饲蛋氨酸后出现高Hcy血症，对照组无此改变。糖尿病小鼠的坐骨神经出现不同程度的缺血性改变，且与Hcy水平相关。叶酸可减轻坐骨神经损害的程度。结论：高同型半胱氨酸为糖尿病微血管病变的危险因素，叶酸可起到干预作用。     

Risks for sensorimotor peripheral neuropathy and autonomic neuropathy in non-insulin-dependent diabetes mellitus (NIDDM)

Identification of risk factors for development of diabetic sensorimotor peripheral neuropathy (DSPN) and diabetic autonomic neuropathy (DNA) may help to prevent or modify these complications. The ABCD Trial, a prospective study of diabetic complications, has identified risk factors of the presence and staging of peripheral neuropathy based on neurological symptom scores, neurological disability scores, autonomic function testing and quantitative sensory examination. DSPN is independently associated with diabetes duration [odds ratio (OR) = 1.5 per 10 years], body weight (OR = 1.1 per 5 kg), age (OR = 1.8 per 10 years), retinopathy (OR = 2.3), overt albuminuria (OR = 2.5), height (OR = 1.2 per 10 cm), duration of hypertension (OR = 1.1 per 10 years), insulin use (OR = 1.4), and race/ethnicity [African American vs. non-Hispanic white (OR = 0.4) and Hispanic vs. non-Hispanic white (OR = 0.8)]. DAN is independently associated with diabetes duration (OR = 1.2 per 10 years), body weight (OR = 1.1 per 5 kg), glycosylated hemoglobin (OR = 1.1 per 2.5%), overt albuminuria (OR = 1.6), and retinopathy (OR = 1.8). © 1998 John Wiley & Sons, Inc. Muscle Nerve, 21: 72–80, 1998.     

Cardiovascular autonomic neuropathy in patients with type 2 diabetes with and without sensorimotor polyneuropathy

Background and Aims

Cardiovascular autonomic neuropathy (CAN) in patients with diabetes is associated with poor prognosis. We aimed to assess signs of CAN and autonomic symptoms and to investigate the impact of sensorimotor neuropathy on CAN by examining type 2 diabetes patients with (DPN [distal sensorimotor polyneuropathy]) and without distal sensorimotor polyneuropathy (noDPN) and healthy controls (HC). Secondarily, we aimed to describe the characteristics of patients with CAN.

Methods

A population of 374 subjects from a previously described cohort of the Danish Centre for Strategic Research in Type 2 Diabetes (DD2) were included. Subjects were examined with the Vagus™ device for the diagnosis of CAN, where two or more abnormal cardiovascular autonomic reflex tests indicate definite CAN. Autonomic symptoms were assessed with Composite Autonomic Symptom Score 31 (COMPASS 31) questionnaire. DPN was defined according to the Toronto consensus panel definition.

Results

Definite CAN was present in 22% with DPN, 7% without DPN and 3% of HC, and 91% of patients with definite CAN had DPN. Patients with DPN and definite CAN reported higher COMPASS 31 scores compared to patients with noDPN (20.0 vs. 8.3, p < 0.001) and no CAN (22.1 vs. 12.3, p = 0.01). CAN was associated with HbA1c and age in a multivariate logistic regression analysis but was not associated with IEFND or triglycerides.

Interpretation

One in five patients with DPN have CAN and specific CAN characteristics may help identify patients at risk for developing this severe diabetic complication. Autonomic symptoms were strongly associated with having both DPN and CAN, but too unspecific for diagnosing CAN.     

2型糖尿病并发糖尿病周围神经病变与代谢综合征的关系

目的 探讨2型糖尿病(T2DM)并发糖尿病周围神经病变(DPN)与代谢综合征(MS)的关系。方法 选取我院收治的T2DM患者95例,按是否并发MS进行分组,并发MS 45例为研究组,无MS为对照组50例。2组均进行电生理检查、MS相关指标的血液、尿液标本测定,统计2组DPN发生率,比较2组SCV和MCV及MS相关指标水平,并进行影响DPN的多因素Logistic回归分析。结果 研究组DPN发生率为62.22%,对照组为12.00%,差异有统计学意义(P0.01)。2组左右腓神经SCV、MCV比较有显著差异(P0.01)。2组TC、TG、LDL-C比较差异无统计学意义(P0.05),2hINS、UA、HbA1c、CRP、UAER水平比较有明显差异(P0.01)。多因素Logistic回归分析结果显示,HbA1c、UAER、2hINS、CRP等MS相关指标异常均为DPN的危险因素。结论 T2DM并发DPN与MS密切相关,MS相关指标,如HbA1c、UAER、2hINS等水平过高是导致DPN发生的高危因素,故临床在控制血糖的时候应注重患者MS相关指标的检测与调控。     

Metformin exacerbates and simvastatin attenuates myelin damage in high fat diet‐fed C57BL/6 J mice

Diabetic neuropathy is one of the most deleterious complications of diabetes mellitus in humans. High fat diet (HFD)‐fed C57BL/6 J mice are a widely used animal model for type 2 diabetes mellitus and metabolic syndrome. We investigated the effects of metformin and simvastatin on the ultrastructural characteristics of sciatic nerve fibers in these mice. Metformin treatment increased the number of structural defects of the myelin sheet surrounding these fibers in already affected nerves of HFD fed mice, and simvastatin treatment reduced these numbers to the levels seen in control mice. These results warrant further research on the effects of metformin and statins in patients developing diabetic neuropathy and advise caution when deciding about optimal treatment modalities in these patients.     

Peripheral neuropathy in type 2 diabetes mellitus in Isfahan, Iran: prevalence and risk factors

BACKGROUND: To estimate the prevalence and risk factors of peripheral neuropathy (PN) in people with type 2 diabetes mellitus. METHODS: A total of 810 patients with type 2 diabetes (289 men and 521 women) from Isfahan Endocrinology and Metabolism Research Centre outpatient clinics, Iran, were examined. Part of examination included an assessment of neurological function including neuropathic symptoms and physical signs and nerve conduction velocity. RESULTS: The prevalence of PN was 75.1% (95% confidence interval 72.1, 78.0). PN was associated with age, proteinuria, and duration of diabetes, insulin-treatment, and presence of any retinopathy and ischaemic heart disease (IHD). The age-adjusted prevalence rate of PN was 78% higher among patients with IHD, 64% higher among patients with any retinopathy, 66% higher among insulin-treated type 2 diabetes, and it was greater with duration of diabetes. Using a stepwise binary logistic regression model, age, duration of diabetes and proteinuria were significant independent predictors of PN. CONCLUSION: Peripheral neuropathy is a common complication in this population of Iranian type 2 diabetic patients. It increases with age, duration of diabetes and proteinuria.     

Anti‐ganglioside autoantibodies in type 1 diabetes

Type 1 diabetes is an autoimmune disease that is accompanied by an immune response against pancreatic cells. As gangliosides are expressed in both peripheral nerves and pancreatic cells, we examined the possibility of correlation between type 1 diabetes, anti‐ganglioside autoantibodies, and neuropathy. Fifty diabetic patients and 30 controls with other autoimmune diseases underwent neurological examination and search for antibodies to gangliosides, glutamic acid decarboxylase (GAD₆₅), and tyrosine phosphatase (IA2). Sixteen (32%) diabetic patients had neuropathy. Twelve diabetic sera were found to have anti‐ganglioside autoantibodies. Twenty sera were positive for anti‐GAD₆₅ and nine for anti‐IA2 antibody. Sera from three control patients had anti‐ganglioside autoantibodies. No significant correlation was found between autoantibodies, neuropathy, and disease duration. Disease duration was shorter in patients with antibodies to GAD₆₅ and IA2 and longer in neuropathic patients. The higher prevalence of antibodies in diabetic patients compared to controls may reflect the common pattern of antigens shared by peripheral nerve and pancreatic islet cells. Muscle Nerve, 2009     

Association of corneal nerve loss with markers of axonal ion channel dysfunction in type 1 diabetes

ObjectiveCorneal confocal microscopy (CCM) has been identified as a non-invasive technique to assess corneal nerve fiber morphology. It is not known how corneal nerve changes relate to measures of peripheral nerve function in diabetic peripheral neuropathy (DPN). The present study investigates the relationship between nerve structure and function in DPN.MethodsFifty participants with type 1 diabetes (T1DM) and 29 healthy controls underwent CCM to assess corneal nerve fiber density (CNFD), branch density (CNBD), fiber length (CNFL), total branch density (CTBD), nerve fractal dimension (CNFrD) and inferior whorl length (IWL). The severity of DPN was assessed as Total Neuropathy Score (TNS). Motor nerve axonal excitability tests were conducted to assess axonal function.ResultsSignificant correlations were noted between CNFD (rho = −0.783; P < 0.01) or superexcitability (rho = 0.435; P < 0.01) and TNS. CNFrD was significantly correlated with peak response to stimulus (r = 0.414; P < 0.01) and superexcitability (r = −0.467; P < 0.01) measurements.ConclusionCorneal nerve loss demonstrates a significant association with axonal ion channel dysfunction in T1DM.SignificanceDetection of altered corneal nerve morphology may lead to the earlier diagnosis of DPN.     

Temporal and site-specific brain alterations in CB1 receptor binding in high fat diet-induced obesity in C57Bl/6 mice

The cannabinoid CB1 receptor has been implicated in the regulation of appetite and the consumption of palatable foods. This experiment aimed to explore the involvement of the CB1 receptor in the early and late stages of high fat diet-induced obesity in C57BL/6 mice. The C57Bl/6 mice were placed on a high fat (HF) or low fat/high carbohydrate (LF) diet for 3 or 20 weeks. Quantitative autoradiography revealed that binding of [3H] CP-55,940 (CB1 receptor ligand) was elevated following 3 weeks of HF feeding in areas including the medial/ventral anterior olfactory nucleus (22.1%), agranular insular cortex (24.0%) and the hypothalamus (31.5%) compared to LF controls. This increased level of binding was correlated with an increase in plasma leptin in the hypothalamus, raising the possibility that this hormone may exert inhibitory control over endocannabinoid signalling at this stage of obesity. Mice fed a HF diet for 20 weeks were obese, hyperphagic and had decreased CB1 receptor binding levels in the substantia nigra (12.8%) and ventral tegmental area (17.1%) compared to LF controls. The low [3H] CP-55,940 binding density seen in these reward-related areas in the late stage of obesity may be indicative of increased endocannabinoid release due to the chronic HF diet consumption.     

HLA class Ⅱ alleles and risk for peripheral neuropathy in type 2 diabetes patients

The potential impact of human leukocyte antigen (HLA) genotype variations on development of diabetic peripheral neuropathy (DPN) is not well determined. hTis study aimed to identify the association of HLA class II alleles with DPN in type 2 diabetes (T2D) patients. Totally 106 T2D patients, 49 with DPN and 57 without DPN, and 100 ethnic-matched healthy controls were analyzed. Both groups of the patients were matched based on sex, age, body mass index (BMI) and duration of T2D. Polyneuropathy was diagnosed using electrodiagnostic methods. HLA-DRB1 and DQB1 genotyping was performed in all subjects by the polymerase chain reaction with sequence-specific primers (PCR-SSP) method. T2D patients with DPN showed higher frequencies of HLA-DRB1*10 and DRB1*12 alleles compared to control group (P = 0.04). HLA-DQB1*02 allele and HLA-DRB1*07-DQB1*02 haplotype were associated with a decreased risk for developing DPN in T2D patients (P = 0.02 andP = 0.05 respectively). Also, patients with severe neurop-athy showed higher frequencies of DRB1*07 (P = 0.003) and DQB1*02 (P = 0.02) alleles than those with mild-to-moderate form of neuropathy. The distribution of DRB1 and DQB1 alleles and haplotypes were not statistically different between all patients and healthy controls. Our ifndings implicate a possible protective role of HLA-DQB1*02 allele and HLA-DRB1*07-DQB1*02 haplotype against development of peripheral neuropathy in T2D patients. Therefore, variations in HLA genotypes might be used as genetic markers for prediction and potentially management of neuropathy in T2D patients.     

Small and large fiber sensory polyneuropathy in type 2 diabetes: Influence of diagnostic criteria on neuropathy subtypes

Diabetic polyneuropathy (DPN) can be classified based on fiber diameter into three subtypes: small fiber neuropathy (SFN), large fiber neuropathy (LFN), and mixed fiber neuropathy (MFN). We examined the effect of different diagnostic models on the frequency of polyneuropathy subtypes in type 2 diabetes patients with DPN. This study was based on patients from the Danish Center for Strategic Research in Type 2 Diabetes cohort. We defined DPN as probable or definite DPN according to the Toronto Consensus Criteria. DPN was then subtyped according to four distinct diagnostic models. A total of 277 diabetes patients (214 with DPN and 63 with no DPN) were included in the study. We found a considerable variation in polyneuropathy subtypes by applying different diagnostic models independent of the degree of certainty of DPN diagnosis. For probable and definite DPN, the frequency of subtypes across diagnostic models varied from: 1.4% to 13.1% for SFN, 9.3% to 21.5% for LFN, 51.4% to 83.2% for MFN, and 0.5% to 14.5% for non‐classifiable neuropathy (NCN). For the definite DPN group, the frequency of subtypes varied from: 1.6% to 13.5% for SFN, 5.6% to 20.6% for LFN, 61.9% to 89.7% for MFN, and 0.0% to 6.3% for NCN. The frequency of polyneuropathy subtypes depends on the type and number of criteria applied in a diagnostic model. Future consensus criteria should clearly define sensory functions to be tested, methods of testing, and how findings should be interpreted for both clinical practice and research purpose.     

2型糖尿病并发周围神经病的危险因素及其发病机制探讨

目的 :研究 2型糖尿病合并周围神经病 ( DPN)的危险因素 ,探讨其发病机制。方法 :测定 1 0 6例伴或不伴有周围神经病的 2型糖尿病患者的空腹血糖 ( FBG)、糖化血红蛋白 ( Hb AIC)、甘油三酯 ( TG)、总胆固醇 ( TC)、高密度脂蛋白 -胆固醇 ( HDL-C)、低密度脂蛋白 -胆固醇 ( LDL-C)、脂蛋白 ( a) [Lp( a) ]、载脂蛋白 A( Apo-A)、载脂蛋白 B( Apo-B)以及身高、体重、血压 ,行 Logistic多元逐步回归 ,筛选 DPN的危险因素。结果 :糖尿病病程、身高、舒张压、Hb AIC、TC、Lp( a)是 DPN的独立危险因素 ,Apo-A是 DPN的保护因素。结论 :血糖、血压、血脂控制不良导致 DPN的发生和发展 ,代谢因素和血管因素在其发病机制中起重要作用。     

美吡哒对2型糖尿病周围神经病变影响的实验研究

目的：观察降糖药物美吡哒对2型糖尿病周围神经病变的预防和治疗作用。方法：用高糖高脂饮食加小剂量的STZ注射诱导2型糖尿病Wistar大鼠模型,然后给予治疗组美吡哒5mg/kg·d,分别于用药1、3个月后测定坐骨神经运动潜伏速率、传导速度和动作电位的波幅,并观察腓肠神经病理改变。结果：美吡哒治疗组大鼠坐骨神经运动潜伏速率和动作电位的波幅较糖尿病组明显改善(P＜0．01),腓肠神经有髓神经纤维数量和纤维总截面积较糖尿病组亦明显改善(P＜0．01)。结论：美吡哒对实验性2型糖尿病周围神经损害有一定的防治作用。     

Reduced association between dendritic cells and corneal sub‐basal nerve fibers in patients with fibromyalgia syndrome

In our study, we aimed at investigating corneal langerhans cells (LC) in patients with fibromyalgia syndrome (FMS) and small fiber neuropathy (SFN) as potential contributors to corneal small fiber pathology. We enrolled women with FMS (n = 134) and SFN (n = 41) who underwent neurological examination, neurophysiology, prostaglandin analysis in tear fluid, and corneal confocal microscopy (CCM). Data were compared with those of 60 age‐matched female controls. After screening for dry eye disease, corneal LC were counted and sub‐classified as dendritic (dLC) and non‐dendritic (ndLC) cells with or without nerve fiber association. We further analyzed corneal nerve fiber density (CNFD), length (CNFL), and branch density (CNBD). Neurological examination indicated deficits of small fiber function in patients with SFN. Nerve conduction studies were normal in all participants. Dry eye disease was more prevalent in FMS (17%) and SFN (28%) patients than in controls (5%). Tear fluid prostaglandin levels did not differ between FMS patients and controls. While corneal LC density in FMS and SFN patients was not different from controls, there were fewer dLC in association with nerve fibers in FMS and SFN patients than in controls (P < .01 each). Compared to controls, CNFL was lower in FMS and SFN patients (P < .05 each), CNFD was lower only in FMS patients (P < .05), and CNBD was lower only in SFN patients (P < .001). There was no difference in any CCM parameter between patients with and without dry eyes. Our data indicate changes in corneal innervation and LC distribution in FMS and SFN, potentially based on altered LC signaling.     

Nicotine-taking and nicotine-seeking in C57Bl/6J mice without prior operant training or food restriction

The ability to examine genetically engineered mice in a chronic intravenous (IV) nicotine self-administration paradigm will be a powerful tool for investigating the contribution of specific genes to nicotine reinforcement and more importantly, to relapse behavior. Here we describe a reliable model of nicotine-taking and -seeking behavior in male C57BL/6J mice without prior operant training or food restriction. Mice were allowed to self-administer either nicotine (0.03mg/kg/infusion) or saline in 2-h daily sessions under fixed ratio 1 (FR1) followed by FR2 schedules of reinforcement. In the nicotine group, a dose-response curve was measured after the nose-poke behavior stabilized. Subsequently, nose-poke behavior was extinguished and ability of cue presentations, priming injections of nicotine, or intermittent footshock to reinstate responding was assessed in both groups. C57BL/6J mice given access to nicotine exhibited high levels of nose-poke behavior and self-administered a high number of infusions as compared to mice given access to saline. After this acquisition phase, changing the unit-dose of nicotine resulted in a flat dose-response curve for nicotine-taking and subsequently reinstatement of nicotine-seeking behavior was achieved by both nicotine-associated light cue presentation and intermittent footshock. Nicotine priming injections only triggered significant reinstatement on the second consecutive day of priming. In contrast, mice previously trained to self-administer saline did not increase their responding under those conditions. These results demonstrate the ability to produce nicotine-taking and nicotine-seeking behavior in naive C57BL/6J mice without both prior operant training and food restriction. Future work will utilize these models to evaluate nicotine-taking and relapsing behavior in genetically-altered mice.