Animal Models Of Diabetic Neuropathy Research Articles

CEBP\u03b2 regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth

Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer’s disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P < 0.05) reduced in liver and DRG from streptozotocin (STZ)-induced type 1 diabetic rats, Zucker diabetic fatty (ZDF) rats, mice on a high-fat/ high-sugar diet and db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPβ, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPβ overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPβ can be a promising therapeutic approach.

Mitochondrial dysfunction in an animal model of diabetic neuropathy is associated with a reduction of neurosteroid synthesis.

Background: Recent work in a model of diabetic neuropathy revealed that layer 2/3 cortical pyramidal neurones of the pain pathway exhibited reduced endogenous neurosteroid modulation of the GABA AR and exogenously applied neurosteroids had an exaggerated impact. It is postulated that this is related to reduced precursor synthesis, due to mitochondrial dysfunction in diabetic neuropathy. Benzodiazepines are also known to activate neurosteroidogenesis by binding to mitochondrial translocator protein (TSPO). This study explored the differential effect of diazepam on GABA AR modulation via neurosteroidogenesis in diabetic and wild type (WT) mice. Methods: Whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from layer 2/3 cortical pyramidal neurons of the pain pathway from mice with type-II diabetic neuropathy ( ob/ob) and WT controls aged 60-80 days. Results: There was a key difference in the response of the WT and ob/ob cortical neurons to simultaneous incubation with diazepam and flumazenil. In contrast, diazepam and the 5a-reductase inhibitor finasteride, individually or in combination, produced the same response in both strains. Conclusions: The exaggerated effect of diazepam on GABAergic inhibitory tone in the ob/ob, despite the presence of the GABA AR benzodiazepine antagonist flumazenil is likely observed due to physiological upregulation of key neurosteroidogenic enzymes in response to the reduced pregnenolone synthesis by the mitochondria. By increasing pregnenolone via TSPO activation, it is possible to promote enhanced neurosteroidogenesis and increase GABAergic inhibitory tone via an alternate route. In diabetic neuropathy, mitochondrial dysfunction may play an important role. Enhancing the GABAergic neurosteroid tone could be of potential therapeutic benefit.

Mitochondrial dysfunction in an animal model of diabetic neuropathy is associated with a reduction of neurosteroid synthesis.

Background : Recent work in a model of diabetic neuropathy revealed that layer 2/3 cortical pyramidal neurones of the pain pathway exhibited reduced endogenous neurosteroid modulation of the GABA AR and exogenously applied neurosteroids had an exaggerated impact. It is postulated that this is related to reduced precursor synthesis, due to mitochondrial dysfunction in diabetic neuropathy. Benzodiazepines are also known to activate neurosteroidogenesis by binding to mitochondrial translocator protein (TSPO). This study explored the differential effect of diazepam on GABA AR modulation via neurosteroidogenesis in diabetic and wild type (WT) mice. Methods : Whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from layer 2/3 cortical pyramidal neurons of the pain pathway from mice with type-II diabetic neuropathy ( ob/ob) and WT controls aged 60-80 days. Results : There was a key difference in the response of the WT and ob/ob cortical neurons to simultaneous incubation with diazepam and flumazenil. In contrast, diazepam and the 5a-reductase inhibitor finasteride, individually or in combination, produced the same response in both strains. Conclusions : The exaggerated effect of diazepam on GABAergic inhibitory tone in the ob/ob, despite the presence of the GABA AR benzodiazepine antagonist flumazenil is likely observed due to physiological upregulation of key neurosteroidogenic enzymes in response to the reduced pregnenolone synthesis by the mitochondria. By increasing pregnenolone via TSPO activation, it is possible to promote enhanced neurosteroidogenesis and increase GABAergic inhibitory tone via an alternate route. In diabetic neuropathy, mitochondrial dysfunction may play an important role. Enhancing the GABAergic neurosteroid tone could be of potential therapeutic benefit.

Neurosteroids are reduced in diabetic neuropathy and may be associated with the development of neuropathic pain

Introduction: Peripheral and central sensitisation are implicated in the development of neuropathic pain. Hypersensitivity of pain pathway neurons has been described in animal models of diabetic neuropathy, which is postulated to be related to an imbalance between inhibitory and excitatory signals within the spinal cord. GABAergic neurons within the pain pathway are vital for the transmission of painful stimuli to higher centres. A developmental change in the rate of exponential decay of GABAergic synaptic events has been observed in other types of neurons and this may be associated with fluctuations in endogenous neurosteroid tone. Methods: The whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from wild type mice between the ages of 6 and 80 days in the spinal cord, the nucleus reticularis of the thalamus and the cerebral cortex. Recordings were also obtained from mice with diabetic neuropathy (ob/ob and db/db) between the ages of 60 and 80 days. Behavioural experiments were performed to examine mechanical and thermal nociception. Results: Electrophysiological recordings from cortical pain pathway neurons from mature type-2 diabetic mice revealed that the endogenous neurosteroid tone is reduced compared to control. However, selected neurosteroid compounds had a more pronounced effect on the GABA A receptors of these diabetic mice. ob/ob mice exhibit mechanical hyperalgesia and allodynia, which was reduced by neurosteroids applied exogenously. Conclusions: The reduced endogenous neurosteroid tone in ob/ob mice may be linked to their hypersensitivity. Neurosteroids may exert analgesic effects in pathological pain states by attempting to restore the physiological GABAergic inhibitory tone.

Neurosteroids are reduced in diabetic neuropathy and may be associated with the development of neuropathic pain.

Peripheral and central sensitisation are implicated in the development of neuropathic pain. Hypersensitivity of pain pathway neurons has been described in animal models of diabetic neuropathy, which is postulated to be related to an imbalance between inhibitory and excitatory signals within the spinal cord. GABAergic neurons within the pain pathway are vital for the transmission of painful stimuli to higher centres. A developmental change in the rate of exponential decay of GABAergic synaptic events has been observed in other types of neurons and this may be associated with fluctuations in endogenous neurosteroid tone. Methods: The whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from wild type mice between the ages of 6 and 80 days in the spinal cord, the nucleus reticularis of the thalamus and the cerebral cortex. Recordings were also obtained from mice with diabetic neuropathy (ob/ob and db/db) between the ages of 60 and 80 days. Behavioural experiments were performed to examine mechanical and thermal nociception. Electrophysiological recordings from cortical pain pathway neurons from mature type-2 diabetic mice revealed that the endogenous neurosteroid tone is reduced compared to control. However, selected neurosteroid compounds had a more pronounced effect on the GABA A receptors of these diabetic mice. ob/ob mice exhibit mechanical hyperalgesia and allodynia, which was reduced by neurosteroids applied exogenously. The reduced endogenous neurosteroid tone in ob/ob mice may be linked to their hypersensitivity. Neurosteroids may exert analgesic effects in pathological pain states by attempting to restore the physiological GABAergic inhibitory tone.

Endovanilloid control of pain modulation by the rostroventromedial medulla in an animal model of diabetic neuropathy

The involvement of transient receptor vanilloid type-1 (TRPV1) channels in pain modulation by the brain remains understudied. The rostroventromedial medulla (RVM) plays a key role in conveying to the spinal cord pain modulatory influences triggered in higher brain centres, with co-existence of inhibitory (antinociceptive) and facilitatory (pronociceptive) effects. In spite of some reports of TRPV1 expression in the RVM, it remains unknown if endovanilloid signalling plays a direct role in local pain modulation. Here we used a model of diabetic neuropathy, the streptozotocin (STZ)-diabetic rat, to study the role of endovanilloid signalling in RVM-mediated pain modulation during chronic pain. Four weeks after diabetes induction, the levels of TRPV1 mRNA and fatty acid amide hydrolase (FAAH), a crucial enzyme for endovanilloid catabolism, in the RVM of STZ-diabetic rats were higher than control. The RVM of STZ-diabetic rats presented decreased levels of several TRPV1 endogenous ligands, namely anandamide (AEA), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). Administration of capsaicin (a TRPV1 agonist) into the RVM decreased nociceptive behavioural responses in the inflammatory phase of the formalin test (phase 2). These findings suggest that diabetic neuropathy induces plastic changes of RVM endovanilloid signalling, indicating that TRPV1 may be a putative target for pain modulation in this chronic pain condition.

An Introduction to the History and Controversies of Animal Models of Diabetic Neuropathy.

An Introduction to the History and Controversies of Animal Models of Diabetic Neuropathy.

Comment on “The Combined Extract ofZingiber officinaleandZea mays(Purple Color) Improves Neuropathy, Oxidative Stress, and Axon Density in Streptozotocin Induced Diabetic Rats”

In a recent study, Wattanathorn et al. identified that the combined extract of purple waxy corn and ginger was effective in improving functional and structural changes in STZ-diabetic rats submitted to chronic constriction of the sciatic nerve, an effect that is potentially mediated by the antioxidant properties of these combined extracts. Although the results have been quite interesting, we would like to present a complementary conceptual perspective to improve the interpretation of the findings considering the disease model investigated. In the natural course of nontreated diabetes mellitus, diabetic neuropathy represents a chronic metabolic neuropathic disorder secondary to accumulated molecular and microvascular damage that impairs nerve structure and function [1, 2]. Fundamentally, diabetic neuropathy consists of a nontraumatic condition with prolonged time-course in which functional impairment overcomes the nerve structural damage, especially in the early stages of neuropathy [1–3]. Thus, even when applied to diabetic animals, the model of nerve constriction represents a traditional model of traumatic neuropathy and neuropathic pain [4, 5], which manifests a completely different pathophysiologic mechanism compared to diabetic neuropathy. In this trauma model, nerve ligation causes variable degrees of injury, which is difficult to measure directly and can range from neuropraxia (less severe functional nerve lesion) to neurotmesis (extensive and complex morphofunctional nerve lesion) [4, 5]. Furthermore, the injured nerve fibers cause chromatolysis and distal Wallerian degeneration, processes that are associated with intense focal inflammation and fibrosis, which limits axonal reparation [4, 5]. All of these structural changes are typical of traumatic lesions, but less frequent in diabetic neuropathy [1–3]. From this conceptual basis, the current interpretation of the findings reflects a construct bias since the results described cannot be used as an empirical basis to explain the improvements in diabetic neuropathy. Thus, a proper interpretation of the findings should consider the effect of treatments on healing/repair of traumatic nerve injury in diabetic animals, which recognizably presents an impaired healing process [1, 3]. When reviewing animal models of diabetic neuropathy, Klusakova and Dubový [5] indicated that rats with partial sciatic nerve ligation present minor pathogenesis of diabetic neuropathy, which is hardly representative of the human condition. This argument is reinforced by the short time course for neuropathy evolution, which showed a significant impairment of sciatic function index 24 hours after nerve ligation. This finding indicates a typical acute effect of mechanical nerve injuries [4, 5], which is not consistent with the chronic and progressive clinical course of diabetic neuropathy [1–3]. Although the relevance of the results is evident in traumatic nerve injury, it is wise to discuss the limitations of the model used and the interpretation of the findings. Whereas diabetic neuropathy is a condition of difficult clinical management that demands high cost to health care, we are looking forward to further studies by Wattanathorn et al. investigating whether the extracts tested can also bring similar benefits in specific and robust models, such as genetic or genetically modified models [3, 5, 6] of diabetic neuropathy.

Phenotyping animal models of diabetic neuropathy: a consensus statement of the diabetic neuropathy study group of the EASD (Neurodiab)

NIDDK, JDRF, and the Diabetic Neuropathy Study Group of EASD sponsored a meeting to explore the current status of animal models of diabetic peripheral neuropathy. The goal of the workshop was to develop a set of consensus criteria for the phenotyping of rodent models of diabetic neuropathy. The discussion was divided into five areas: (1) status of commonly used rodent models of diabetes, (2) nerve structure, (3) electrophysiological assessments of nerve function, (4) behavioral assessments of nerve function, and (5) the role of biomarkers in disease phenotyping. Participants discussed the current understanding of each area, gold standards (if applicable) for assessments of function, improvements of existing techniques, and utility of known and exploratory biomarkers. The research opportunities in each area were outlined, providing a possible roadmap for future studies. The meeting concluded with a discussion on the merits and limitations of a unified approach to phenotyping rodent models of diabetic neuropathy and a consensus formed on the definition of the minimum criteria required for establishing the presence of the disease. A neuropathy phenotype in rodents was defined as the presence of statistically different values between diabetic and control animals in 2 of 3 assessments (nocifensive behavior, nerve conduction velocities, or nerve structure). The participants propose that this framework would allow different research groups to compare and share data, with an emphasis on data targeted toward the therapeutic efficacy of drug interventions.

PGC-1α regulation of mitochondrial degeneration in experimental diabetic neuropathy

Mitochondrial degeneration is considered to play an important role in the development of diabetic peripheral neuropathy in humans. Mitochondrial degeneration and the corresponding protein regulation associated with the degeneration were studied in an animal model of diabetic neuropathy. PGC-1α and its-regulated transcription factors including TFAM and NRF1, which are master regulators of mitochondrial biogenesis, are significantly downregulated in streptozotocin diabetic dorsal root ganglion (DRG) neurons. Diabetic mice develop peripheral neuropathy, loss of mitochondria, decreased mitochondrial DNA content and increased protein oxidation. Importantly, this phenotype is exacerbated in PGC-1α (−/−) diabetic mice, which develop a more severe neuropathy with reduced mitochondrial DNA and a further increase in protein oxidation. PGC-1α (−/−) diabetic mice develop an increase in total cholesterol and triglycerides, and a decrease in TFAM and NRF1 protein levels. Loss of PGC-1α causes severe mitochondrial degeneration with vacuolization in DRG neurons, coupled with reduced state 3 and 4 respiration, reduced expression of oxidative stress response genes and an increase in protein oxidation. In contrast, overexpression of PGC-1α in cultured adult mouse neurons prevents oxidative stress associated with increased glucose levels. The study provides new insights into the role of PGC-1α in mitochondrial regeneration in peripheral neurons and suggests that therapeutic modulation of PGC-1α function may be an attractive approach for treatment of diabetic neuropathy.

Animal Models of Diabetes and Its Associated Complications

Diabetes is a major threat to global public health. According to the latest data from the International Diabetes Federation (IDF), at least 366 million people are living with diabetes and this number is projected to be 552 million by 2030. At least 50% of people with diabetes suffer from one or two major diabetic complications such as diabetic cardiomyopathy, nephropathy, neuropathy, retinopathy, and diabetic foot diseases. Development of an authentic model of each type of diabetes and their associated complications, which exactly mimic the human pathogenesis, is very crucial not only for a better understanding of the core causes of the diseases but also for the development and routine pharmacological screening of novel anti-diabetic drugs. A number of genetic and nongenetic animal models of the different types of diabetes and their associated complications have been developed in last three decades but none of them are without limitations. This special issue of the Journal of Diabetes Research has been designed to publish original and review articles with novel approaches of the development of animal model of diabetes and its associated complications. Almost all areas of the animal models of diabetes and its associated complications have been covered by this issue. Among the reviews: the animal models of diabetic retinopathy by A. K. W. Lai and A. Lo, animal models of diabetic neuropathy by M. S. Islam, murine models of diabetic neuropathy by L. L. Kong et al., methods and models for metabolic assessment by G. Pacini et al., and porcine models of accelerated coronary atherosclerosis by D. Hamamdzic et al. contributed significantly by summarizing the advantages, disadvantages, and suitability of animal models developed to study the above-mentioned diabetic complications. Many other original papers such as role of adrenomedulin in the renal NADPH oxidase and (pro)renin in diabetic mice by M. Hayashi et al., development of glomerulopathy in the KK.Cg-Ay/J mouse with the pathology of diabetic nephropathy by P. Stephen et al., and the role of ERK/MAPK and TGF-beta/Smad signaling pathways in the kidney fibrosis of diabetic mice by X. Cheng et al. contributed novel information in the pathogenesis of diabetic nephropathy, a major and common complications of diabetes mellitus. Finally, an exceptional paper has been contributed by E. Hillhouse et al. on the effect of nearby construction on the progress to overt autoimmune diabetes in NOD mice. As a whole, papers from almost every aspects of the animals models of diabetes and its associated complications have been published in this special issue so it can be a special interest of diabetes researchers particularly those who are working in the different areas of diabetic complications. Md. Shahidul Islam Daisuke Koya Bernard Portha

Animal Models of Diabetic Neuropathy: Progress Since 1960s

Diabetic or peripheral diabetic neuropathy (PDN) is one of the major complications among some other diabetic complications such as diabetic nephropathy, diabetic retinopathy, and diabetic cardiomyopathy. The use of animal models in the research of diabetes and diabetic complications is very common when rats and mice are most commonly used for many reasons. A numbers of animal models of diabetic and PDN have been developed in the last several decades such as streptozotocin-induced diabetic rat models, conventional or genetically modified or high-fat diet-fed C57BL/Ks (db/db) mice models, streptozotocin-induced C57BL6/J and ddY mice models, Chinese hamster neuropathic model, rhesus monkey PDN model, spontaneously diabetic WBN/Kob rat model, L-fucose-induced neropathic rat model, partial sciatic nerve ligated rat model, nonobese diabetic (NOD) mice model, spontaneously induced Ins2 Akita mice model, leptin-deficient (ob/ob) mice model, Otsuka Long-Evans Tokushima Fatty (OLETF) rat model, surgically-induced neuropathic model, and genetically modified Spontaneously Diabetic Torii (SDT) rat model, none of which are without limitations. An animal model of diabetic or PDN should mimic the all major pathogeneses of human diabetic neuropathy. Hence, this review comparatively evaluates the animal models of diabetic and PDN which are developed since 1960s with their advantages and disadvantages to help diabetic research groups in order to more accurately choose an appropriate model to meet their specific research objectives.

Impaired adenosine monophosphate-activated protein kinase signalling in dorsal root ganglia neurons is linked to mitochondrial dysfunction and peripheral neuropathy in diabetes

Mitochondrial dysfunction occurs in sensory neurons and may contribute to distal axonopathy in animal models of diabetic neuropathy. The adenosine monophosphate-activated protein kinase and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signalling axis senses the metabolic demands of cells and regulates mitochondrial function. Studies in muscle, liver and cardiac tissues have shown that the activity of adenosine monophosphate-activated protein kinase and PGC-1α is decreased under hyperglycaemia. In this study, we tested the hypothesis that deficits in adenosine monophosphate-activated protein kinase/PGC-1α signalling in sensory neurons underlie impaired axonal plasticity, suboptimal mitochondrial function and development of neuropathy in rodent models of type 1 and type 2 diabetes. Phosphorylation and expression of adenosine monophosphate-activated protein kinase/PGC-1α and mitochondrial respiratory chain complex proteins were downregulated in dorsal root ganglia of both streptozotocin-diabetic rats and db/db mice. Adenoviral-mediated manipulation of endogenous adenosine monophosphate-activated protein kinase activity using mutant proteins modulated neurotrophin-directed neurite outgrowth in cultures of sensory neurons derived from adult rats. Addition of resveratrol to cultures of sensory neurons derived from rats after 3-5 months of streptozotocin-induced diabetes, significantly elevated adenosine monophosphate-activated protein kinase levels, enhanced neurite outgrowth and normalized mitochondrial inner membrane polarization in axons. The bioenergetics profile (maximal oxygen consumption rate, coupling efficiency, respiratory control ratio and spare respiratory capacity) was aberrant in cultured sensory neurons from streptozotocin-diabetic rats and was corrected by resveratrol treatment. Finally, resveratrol treatment for the last 2 months of a 5-month period of diabetes reversed thermal hypoalgesia and attenuated foot skin intraepidermal nerve fibre loss and reduced myelinated fibre mean axonal calibre in streptozotocin-diabetic rats. These data suggest that the development of distal axonopathy in diabetic neuropathy is linked to nutrient excess and mitochondrial dysfunction via defective signalling of the adenosine monophosphate-activated protein kinase/PGC-1α pathway.

Antiplatelet Therapy, Diabetic Neuropathy and Peripheral Vascular Disease: A Unitary Approach

Although pathologically not necessarily the same, germane to both diabetic neuropathy and peripheral vascular disease is the morbid vascular complication of diabetes mellitus. As it appears, strict glycemic control alone is unable to neither prevent nor promote recovery of diabetic microvascular complications, thus the need to seek other treatment strategies. In addressing the vascular complications, a unitary approach between diabetic neuropathy, peripheral vascular disease and antiplatelet therapy is in order. Notably, the fine balance between platelet/coagulation and fibrinolysis systems are dominantly shifted to acceleration of the platelet/coagulation system in diabetes mellitus. Not only platelet homophilic aggregation, but also platelet heterophilic aggregation with neutrophils or monocytes in inflammatory conditions is observed. Hyperglycemia also induces overproduction of reactive oxygen species by multiple pathomechanisms that injure endothelial cells and reduce nitric oxide which eventually reduce the blood flow in microvessels. Among other antiplatelet agents, cilostazol is a novel antiplatelet agent that has been found to have beneficial effects in macrovascular events like stroke and peripheral vascular diseases. In peripheral vascular disease clinical trials, there is robust data that cilostazol is efficacious in improving walking speed. Also, cilostazol increases nerve blood flow/nerve conduction, and inhibits reduction in pericyte area of endoneurial microvessels in animal models of diabetic neuropathy. Clinical trials on diabetic polyneuropathy alone or in combination with peripheral vascular disease indicate clinical improvement in blood flow, but not necessarily in neuropathy parameters. Since a neurodegenerative process is equally as important a pathomechanism, the effect of antiplatelet therapy in diabetic neuropathy should be examined in long-term clinical trials that may potentially unfold its benefits over time.

The effects of anesthesia on measures of nerve conduction velocity in male C57Bl6/J mice

Animal models, particularly mice, are used extensively to investigate neurological diseases. Basic research regarding animal models of human neurological disease requires that the animals exhibit hall mark characteristics of the disease. These include disease specific anatomical, metabolic and behavioral changes. Nerve conduction velocity (NCV) is the predominant method used to assess peripheral nerve health. Normative data adjusted for age, gender and height is available for human patients; however, these data are not available for most rodents including mice. NCV may be affected by animal age and size, body temperature, stimulus strength and anesthesia. While the effects of temperature, age and size are documented, the direct and indirect effects of anesthesia on NCV are not well reported. Our laboratory is primarily concerned with animal models of diabetic neuropathy (DN) and uses NCV to confirm the presence of neuropathy. To ensure that subtle changes in NCV are reliably assayed and not directly or indirectly affected by anesthesia, we compared the effects of 4 commonly used anesthetics, isoflurane, ketamine/xylazine, sodium pentobarbital and 2-2-2 tribromoethanol on NCV in a commonly used rodent model, the C57Bl6/J mouse. Our results indicate that of the anesthetics tested, isoflurane has minimal impact on NCV and is the safest, most effective method of anesthesia. Our data strongly suggest that isoflurane should become the anesthetic of choice when performing NCV on murine models of neurological disease.

Mitochondrial biogenesis and fission in axons in cell culture and animal models of diabetic neuropathy

Mitochondrial-mediated oxidative stress in response to high glucose is proposed as a primary cause of dorsal root ganglia (DRG) neuron injury in the pathogenesis of diabetic neuropathy. In the present study, we report a greater number of mitochondria in both myelinated and unmyelinated dorsal root axons in a well-established model of murine diabetic neuropathy. No similar changes were seen in younger diabetic animals without neuropathy or in the ventral motor roots of any diabetic animals. These findings led us to examine mitochondrial biogenesis and fission in response to hyperglycemia in the neurites of cultured DRG neurons. We demonstrate overall mitochondrial biogenesis via increases in mitochondrial transcription factors and increases in mitochondrial DNA in both DRG neurons and axons. However, this process occurs over a longer time period than a rapidly observed increase in the number of mitochondria in DRG neurites that appears to result, at least in part, from mitochondrial fission. We conclude that during acute hyperglycemia, mitochondrial fission is a prominent response, and excessive mitochondrial fission may result in dysregulation of energy production, activation of caspase 3, and subsequent DRG neuron injury. During more prolonged hyperglycemia, there is evidence of compensatory mitochondrial biogenesis in axons. Our data suggest that an imbalance between mitochondrial biogenesis and fission may play a role in the pathogenesis of diabetic neuropathy.

Assessment of Anti-Diabetic Activity of Syzygium jambolanum Using In vitro Model

Problem statement: Poor glycemic control and oxidative stress is implicated as a common pathway in the development of diabetic neuropathy. Approach: In the present study, we investigated the protective effects of Enicostemma littorale Blume (EL) (2.5 g kg-1), a hypoglycemic and antioxidant herbal medicine in alloxan-induced diabetic neuropathy in male Charles foster rats. Results: Tail flick latency in hot immersion test and the formalin test were used to evaluate nociceptive responses in diabetic rats compared to non-diabetic rats. Preventive treatment of EL (for 45 days) and standard drug glibenclamide (Glib) significantly improved nociception in diabetic rats. The changes in lipid peroxidation status and anti-oxidant enzymes (super-oxide dismutase, glutathione peroxidase and catalase) levels observed in diabetic rats, were significantly restored by EL and glib treatment. Decrease in Na-K+ ATPase activity was also significantly restored by EL. Glibenclamide was used as standard drug in present study. Conclusion: This study provided experimental evidence to preventive effect of EL on nerve function and oxidative stress in animal model of diabetic neuropathy. Hence EL may be tried clinically for the treatment of diabetic neuropathy since it was used as folk medicine in diabetic patients.

SOD2 protects neurons from injury in cell culture and animal models of diabetic neuropathy

Hyperglycemia-induced oxidative stress is an inciting event in the development of diabetic complications including diabetic neuropathy. Our observations of significant oxidative stress and morphological abnormalities in mitochondria led us to examine manganese superoxide dismutase (SOD2), the enzyme responsible for mitochondrial detoxification of oxygen radicals. We demonstrate that overexpression of SOD2 decreases superoxide (O 2 − ) in cultured primary dorsal root ganglion (DRG) neurons and subsequently blocks caspase-3 activation and cellular injury. Underexpression of SOD2 in dissociated DRG cultures from adult SOD2 +/− mice results in increased levels of O 2 − , activation of caspase-3 cleavage and decreased neurite outgrowth under basal conditions that are exacerbated by hyperglycemia. These profound changes in sensory neurons led us to explore the effects of decreased SOD2 on the development of diabetic neuropathy (DN) in mice. DN was assessed in SOD2 +/− C57BL/6J mice and their SOD2 +/+ littermates following streptozotocin (STZ) treatment. These animals, while hyperglycemic, do not display any signs of DN. DN was observed in the C57BL/6Jdb/db mouse, and decreased expression of SOD2 in these animals increased DN. Our data suggest that SOD2 activity is an important cellular modifier of neuronal oxidative defense against hyperglycemic injury.

Preventive and curative effect of edaravone on nerve functions and oxidative stress in experimental diabetic neuropathy

Oxidative stress is implicated as a final common pathway in the development of diabetic neuropathy and pharmacological interventions targeted at inhibiting free radical production have shown beneficial effects. In the present study, we have investigated the effects of edaravone (3 mg/kg; 3-Methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger (relatively selective to hydroxyl radicals) in streptozotocin (50 mg/kg i.p.) induced diabetic neuropathy in male Sprague-Dawley rats. Significant reduction (18%) in motor nerve conduction velocity, nerve blood flow (55%) and tail flick latency in cold (53%) and hot (50%) immersion test was observed in diabetic rats compared to age matched non-diabetic rats. Preventive (8 week) and curative (2 week) treatment of edaravone significantly improved the nerve conduction velocity and nociception but not nerve blood flow in diabetic rats. The changes in lipid peroxidation status and anti-oxidant enzymes (Superoxide dismutase and Catalase) levels observed in diabetic rats were significantly restored by edaravone treatment. Increase in blood pressure and vascular resistance was also significantly attenuated by edaravone treatment. This study provides experimental evidence to preventive and curative effect of edaravone on nerve function and oxidative stress in animal model of diabetic neuropathy. Hence edaravone may be tried clinically for the treatment of diabetic neuropathy since it is clinically used in stroke patients.

Inhibitors of Advanced Glycation End Product Formation and Neurovascular Dysfunction in Experimental Diabetes

Advanced glycation and lipoxidation end products (AGEs/ALEs) have been implicated in the pathogenesis of the major microvascular complications of diabetes mellitus: nephropathy, neuropathy, and retinopathy. This article reviews the evidence regarding the peripheral nerve and its vascular supply. Most investigations done to assess the role of AGEs/ALEs in animal models of diabetic neuropathy have used aminoguanidine as a prototypic inhibitor. Preventive or intervention experiments have shown treatment benefits for motor and sensory nerve conduction velocity, autonomic nitrergic neurotransmission, nerve morphometry, and nerve blood flow. The latter depends on improvements in nitric oxide-mediated endothelium-dependent vasodilation and is responsible for conduction velocity improvements. A mechanistic interpretation of aminoguanidine's action in terms of AGE/ALE inhibition is made problematic by the relative lack of specificity. However, other unrelated compounds, such as pyridoxamine and pyridoxamine analogues, have recently been shown to have beneficial effects similar to aminoguanidine, as well as to improve pain-related measures of thermal hyperalgesia and tactile allodynia. These data also stress the importance of redox metal ion-catalyzed AGE/ALE formation. A further approach is to decrease substrate availability by reducing the elevated levels of hexose and triose phosphates found in diabetes. Benfotiamine is a transketolase activator that directs these substrates to the pentose phosphate pathway, thus reducing tissue AGEs. A similar spectrum of improvements in nerve and vascular function were noted when using benfotiamine in diabetic rats. Taken together, the data provide strong support for an important role for AGEs/ALEs in the etiology of diabetic neuropathy.