Treatment for diabetic peripheral neuropathy: What have we learned from animal models?

Abstract

Introduction:

Animal models have been widely used to investigate the etiology and potential treatments for diabetic peripheral neuropathy. What we have learned from these studies and the extent that this information has been adaptable to the human condition will be the subject of this review article.

Methods:

A comprehensive search of the PubMed database was performed and relevant articles on the topic were included in this review.

Results:

Extensive study of diabetic animal models has shown that the etiology of diabetic peripheral neuropathy is complex with multiple mechanisms affecting neurons, Schwann cells and the microvasculature all contributing to the phenotypic nature of this most common complication of diabetes. Moreover, animal studies have demonstrated that the mechanisms related to peripheral neuropathy occurring in type 1 and type 2 diabetes are likely different with hyperglycemia being the primary factor for neuropathology in type 1 diabetes and contributing to a lesser extent in type 2 diabetes where insulin resistance, hyperlipidemia and other factors may have a greater role. Two of the earliest mechanisms described from animal studies as a cause for diabetic peripheral neuropathy were the activation of the aldose reductase pathway and increased non-enzymatic glycation. However, continuing research has identified numerous other potential factors that may contribute to diabetic peripheral neuropathy including; oxidative and inflammatory stress, dysregulation of protein kinase C and hexosamine pathways and decreased neurotrophic support. In addition, recent studies have demonstrated that peripheral neuropathy like symptoms are present in animal models representing pre-diabetes in the absence of hyperglycemia.

Conclusions:

This complexity complicates the identification of a successful treatment of diabetic peripheral neuropathy and has likely been a factor in the poor outcome of translating successful treatments from animal studies to human clinical trials.

1. Introduction:

Diabetic peripheral neuropathy:

Diabetic peripheral neuropathy is a heterogenous condition that can manifest as many different symptoms and are the most common complications of diabetes mellitus with an estimated prevalence ranging up to 50% and possibly higher depending on the diagnostic criteria, whether the subjects have type 1 or type 2 diabetes and duration of diabetes [1–3]. Diabetic peripheral neuropathy affects both sensorimotor and autonomic parts of the peripheral nervous system [1]. The most common clinically recognized form is diabetic sensorimotor polyneuropathy which is characterized by the progressive loss of nerve fibers, both large and small [1,4]. In this progressive disorder the most distal nerve segments of the feet and hands are affected first and involve retraction of terminal sensory axons in the periphery with relative preservation of the perikarya. This phenomenon is often referred to as ‘dying back syndrome’ or the ‘stocking and glove’ pattern reflects damage to the longest sensory axons first and thus is considered a length-dependent neuropathy [4,5]. This decrease in sensory perception is the most common and earliest form of diabetic peripheral neuropathy and is gradual with symptoms of tingling, pain and loss of sensation in the toes [6,7]. Clinical evidence of motor dysfunction is less prevalent with only 1-6% of diabetic patients displaying clinical symptoms and generally occurs in patients with established diabetic peripheral neuropathy [7]. A decrease in motor nerve conduction velocity early in diabetic animal models is a common finding but there is also evidence in animal models and humans of a decrease in compound muscle action potential amplitudes and reduced muscle strength [7,8]. Even though hyperglycemia is a contributing factor to the onset and progression of nerve damage, recent evidence also suggests that small nerve fiber damage occurs in individuals with impaired glucose tolerance, independent of hyperglycemia and the diagnosis of diabetes [9,10]. Furthermore, good glycemic control, known to partially prevent and/or delay the onset and progression of peripheral neuropathy in individuals with type 1 diabetes, provides little benefit in those with type 2 diabetes [1,10]. Thus, other conditions, in addition to hyperglycemia, must contribute to the onset and progression of diabetic peripheral neuropathy in subjects with type 2 diabetes. Because of the multiple clinical manifestations associated with diabetic peripheral neuropathy that can include pain in 15 to 30% of diabetic patients with neuropathy determining the pathophysiology and therapy for diabetic neuropathies is challenging [9,11]. Studies using diabetic animal models, primarily rodents, have provided insight to the pathophysiology of diabetic peripheral neuropathy in mammals [1]. The mechanisms identified from these studies include metabolic dysregulation leading to protein alterations, mitochondrial defects and oxidative and inflammatory stress, endothelial and vascular dysfunction, ion channel irregularities and decreased neurotrophic support [11,12–14]. However, in spite of many years of research discovery of a successful treatment translational to humans other than to mask the painful symptoms has eluded investigators [4,15,16]. There are many potential reasons that can account for this failure including animal models of diabetes and subsequent treatments for peripheral neuropathy poorly represent the human condition, endpoints that rely on clinical changes that occur late in the progression of the disease are not readily reversible, duration of clinical trials being too short to adequately determine the potential success of a treatment, and the reliance on a monotherapy as a treatment for of a disease with multiple etiologies [12]. Another concern for the failure of translational therapies for the treatment of diabetic peripheral neuropathy is the role of environmental risk factors in the development and progression of disease. Commonly in animal studies environmental risk factors such as exercise, diet, climate and genetic variability are well controlled. Current studies suggest that the risk factors for diabetic peripheral neuropathy are the duration of diabetes, age, glycosylated hemoglobin A1c, diabetic retinopathy, smoking, and body mass Index [17]. However, accurately duplicating these factors in animal studies in addition to exercise, diet and genetic variability are difficult and thus could compromise results of animal studies thereby making comparisons to the human diabetes condition questionable.

As studies continue on identifying earlier markers for diabetic peripheral neuropathy that can be applied in clinically trials, work also needs to continue on identifying potential new treatments that are safe and can be taken for long period of time likely the duration of the patient’s life [18]. This review will focus on the mechanisms discovered from studies of animal models of diabetes thought to contribute to diabetic peripheral neuropathy as well as on the treatments that have arisen from the identification of these mechanisms and their outcome when translated to human subjects.

2. Diabetic peripheral neuropathy: Mechanisms and related treatment:

Animal models primarily rodents have been widely used to examine the etiology of diabetic peripheral neuropathy. This has included rodents genetically pre-disposed to develop hyperglycemia as well as animal models chemically treated to induce diabetes. The origin, background and limitations of these different models for the study of diabetic neuropathy have been previously reviewed and will not be discussed in detail in this article [19–22]. The different mechanisms that have been discovered from the studies to be discussed linking the etiology of diabetic peripheral neuropathy to dysfunctional and abnormal pathways have been used to design many of the treatments that pre-clinically have been shown to be successful in preventing and/or reversing diabetic peripheral neuropathy.

2.1. Aldose Reductase Pathway

Hyperglycemia activates the aldose reductase or polyol pathway leading to the increased production of sorbitol and fructose [23]. This pathway, which was first documented almost 50 years ago, utilizes NAD(P)H to convert glucose to sorbitol through the enzymatic action of aldose reductase [24]. In the process consumption of NAD(P)H compromises antioxidant capacity through depletion of reduced glutathione and glutathione peroxidase activity, elevation of sorbitol, a polyol leads to a compensatory depletion of myo-inositol and taurine, as well as a decrease in the activity of Na/K ATPase and activation of this pathway also leads to an excess of NADH, which is a substrate for complex I in the mitochondrial electron transport chain leading to the production of superoxide radicals and increased oxidative stress. Therefore, activation of this pathway causes both oxidative and osmotic stress. Use of different aldose reductase inhibitors in animal models of diabetes improved diabetic peripheral neuropathy related endpoints such as nerve conduction velocity as well as corrected the other metabolic derangements associated with activation of the aldose reductase pathway [25–30]. Treating normal rats with high levels of galactose in the diet also induces a diabetes-like peripheral neuropathy that can be prevented by treating these rats with an aldose reductase inhibitor further suggesting that increased polyol accumulation and subsequent disturbance of oxidative and osmotic pathways lead to nerve damage [24,31–33]. Other studies have demonstrated that restoring myo-inositol or taurine levels of diabetic rats through dietary supplementation also improved diabetic peripheral neuropathy without reducing sorbitol levels [34–38]. These studies implied that normal levels of these two important metabolites/osmolytes have a role in maintaining normal nerve activity and depletion causes neuropathology. In studies by my laboratory we demonstrated that treating normal rats with L-fucose in the diet, a competitive inhibitor of myo-inositol, caused a decrease in myo-inositol levels in vivo and diabetes-like peripheral neuropathy that was prevented by replenishing myo-inositol [39,40]. Our study further demonstrated that myo-inositol perhaps due to its role in the synthesis of phosphatidylinositides, which are a component of an important signaling pathway, contributes to normal neural function.

In clinical trials for diabetic peripheral neuropathy the outcome to date has been less than encouraging. The early clinical trials with sorbinil did not live up to expectations following successful studies in animal models demonstrating a modest increase in nerve conduction velocity [41–43]. In Phase III trials the outcome was less favorable and concerns with toxicity terminated its advancement to general use [17]. Epalrestat has found greater success. It is the only aldose reductase inhibitor to be licensed use (Japan and India) based on randomized controlled trials [17]. Long-term treatment with epalrestat has been shown to be well tolerated and it has been shown to effectively delay the progression of diabetic neuropathy especially in subjects with good glycemic control [17,44–46]. In human subjects it has been proposed that the favorable effects are due to improvement in the polyol pathway and reducing production of advanced glycation endproducts [46]. More recently clinical trials using ranirestat demonstrated that is was well tolerated and improved nerve conduction velocity both sensory and motor [47–49]. Thus, the final story for aldose reductase inhibitors as a treatment for diabetic peripheral neuropathy remains to be written.

2.2. Nonenzymatic Glycation and Advanced Glycation Endproducts:

Another common pathway to be recognized early to contribute to diabetic peripheral neuropathy was nonenzymatic glycation and the formation of advanced glycation endproducts. Advanced glycation endproducts are created from nonenzymatic reactions of glucose and amino groups of proteins through a process termed Maillard reaction [50,51]. Diabetes induced hyperglycemia leads to the increased production of advanced glycation endproducts that are represented by many different products [51]. These compounds insert onto proteins, lipids, membranes and the extracellular matrix altering function, structure, turnover and cell to cell interactions, and in the nerve can also impact myelin synthesis [51,52]. Advanced glycation endproducts can also indirectly affect peripheral nerves by altering vascular structure and function and ultimately affecting blood flow causing localized ischemia. Another way that advanced glycation endproducts exert their pathological effects is through binding to cellular receptors [52]. The most recognized receptor for advanced glycation endproducts is commonly referred to RAGE or receptor for advanced glycation endproducts. Animal studies have been the primary source of data implicating advanced glycation endproducts and RAGE in pathology of diabetes complications including diabetic peripheral neuropathy. Therapies that lower levels of advanced glycation endproducts such as pyridoxamine, aminoguanidine and benfotiamine or increase activity of the glyoxalase system, to detoxify advanced glycation endproducts, have been shown to improve diabetic peripheral neuropathy [53–58]. Studies with mice deficient in RAGE and use of a competitive decoy for advanced glycation endproducts, soluble RAGE, have contributed to our understanding of the role of advanced glycation endproducts in the development of diabetic peripheral neuropathy [55,59,60]. Therefore, much like the study of the polyol pathway in animal models of diabetes there is considerable support from preclinical studies with diabetic rodents for a pathologic role of advanced glycation endproducts in the development and progression of diabetic peripheral neuropathy. There have been few clinical trials testing the effects of advanced glycation endproducts on diabetic peripheral neuropathy. One of the more promising treatments based on animal studies is benfotiamine, a fat-soluble derivative of thiamine [61]. In a phase III trial clinical trial benfotiamine improved patient-reported symptoms but no difference was found in peripheral nerve function compared to placebo [62]. Other studies have been conducted using thiamine and benfotiamine for diabetic nephropathy and the results were also reported to be less than successful [63,64].

2.3. Oxidative and Inflammatory Stress:

This section combines two important pathways downstream of the polyol pathway and nonenzymatic glycation that contribute to diabetic peripheral neuropathy. Discussion of oxidative and inflammatory stress has been combined into one sub-section since in almost all cases you cannot have one without the other being present [65,67].

A common definition of oxidative stress is an imbalance between the production of reactive oxygen species and their removal by antioxidant mechanisms. Thus, enhanced oxidative stress is the result of over production of reactive oxygen species with or without reduced reactive oxygen species scavenging ability, which over time can lead to tissue and organ damage [67]. The most common forms of reactive oxygen species are superoxide (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (OH⁻), and peroxynitrite (ONOO⁻) [70,71]. Cellular pathways and enzymes that includes plasma membrane, cytosol, mitochondria, and peroxisomes have been shown to produce these compounds under both basal and disease-like conditions [68,69]. Superoxide is produced primarily by the electron transport chain of the mitochondria, and by NADH oxidase, NAD(P)H oxidase, xanthine oxidase, cyclooxygenase, lipoxygenase, cytochrome P-450, and, during periods of tetrahydrobiopterin deficiency, by nitric oxide synthase [68]. Superoxide can be converted into H₂O₂ by spontaneously acquiring an electron. The formation of H₂O₂ from O₂⁻ can also occur via an enzyme catalyzed reaction requiring superoxide dismutase (SOD). There are three isoforms of this enzyme: Mn-SOD is found in the mitochondria and the other two isoforms are Cu, Zn-SOD and they are found in the cytosol or extracellularly [68]. Hydrogen peroxide is converted to water by catalase or glutathione peroxidase in the presence of reduced glutathione [68]. However, if trace metals are present such as Fe, H₂O₂ can form OH⁻ by what is commonly known as the Fenton reaction [68]. Finally, the formation of ONOO⁻ is the product of a reaction between O₂⁻ and nitric oxide (NO). Peroxynitrite is known to cause damage to neural and vascular tissues and has been shown to be enhanced by diabetes [68,69].

In diabetes, multiple sources exist for overproduction of reactive oxygen species including increased activity of several enzymes such as nitric oxide synthase and NADH/NAD(P)H oxidase [70]. Nitric oxide synthase occurs in three isoforms; inducible NOS (iNOS), neuronal NOS (nNOS) and endothelial NOS (eNOS). In vascular endothelial cells, NO is produced from arginine through the reaction catalyzed by eNOS. eNOS and nNOS are constitutive to endothelial tissue and neurons, respectively, and produce small amounts of NO in a short time [71]. After formation, NO acts as a signaling molecule leading to the activation of guanylate cyclase in target tissues, which in the smooth muscle, leads to its relaxation and increased blood flow [71]. Much of the NO produced by eNOS is neutralized by hemoglobin and eventually reduced to nitrate [71]. NO even when produced in small amounts can be reduced or oxidized to more reactive nitrogen species that can initiate lipid peroxidation in cell membranes [71]. An overabundance of NO can lead to a more harmful reactive compound by combining with O₂⁻ producing ONOO⁻ [71]. Peroxynitrite is responsible for the majority of the cytotoxicity of NO. Even though ONOO⁻ has a short half-life it can readily diffuse across cell membranes and depending on the cell environment can induce nitrosylation of proteins that can result in the reduction of enzyme activity, oxidation of glutathione, an important antioxidant, and increased peroxidation of lipids [71].

Hyperglycemia, through an overproduction of electron donors via glycolysis and the Krebs cycle, has been shown to increase the proton gradient across the mitochondrial inner membrane above a threshold level inducing a prolonged period of O₂⁻ generation [72–74]. Increased flux of glucose through the polyol pathway, increased formation of advanced glycation endproducts, increased activity of protein kinase C, and increased flux through the hexosamine pathway are all mechanisms/pathways that are activated by hyperglycemia that contribute to the overproduction of O₂⁻ by the mitochondrial electron-transport chain [75,76]. Our studies in diabetes support this paradigm demonstrating that the mitochondria are a major source for O₂⁻ formation in the vasculature [77]. Our studies of epineurial arterioles, blood vessels that provide circulation to the region of the sciatic nerve, derived from diabetic rats has determined that complex 1 of the electron-transport chain is a site for O₂⁻ formation in the mitochondria [77]. We have also shown that a decrease in vascular reactivity of these blood vessels precedes slowing of nerve conduction velocity in diabetic rats linking early vascular dysfunction to diabetic peripheral neuropathy [78].

In the endothelium of vascular tissue a primary result of hyperglycemia-induced increase in formation of reactive oxygen species is activation of the transcription factor nuclear factor-κB (NF-κB) [79–83]. NF-κB, upon activation translocates from the cytosol to the nucleus of the cell where it impacts on the regulation of many genes associated with oxidative stress. In endothelial cells, one of the more documented effects of NF-κB activation is the enhanced expression of adhesion molecules, which causes increased attachment of monocytes to the wall of the endothelium [79,80,84,85]. My laboratory has shown that the effect of increased glucose levels on the activation of NF-κB and subsequent increased adhesion of monocytes to endothelial cells is blocked by the antioxidant α-lipoic acid [79]. We have also shown that α-lipoic acid is an effective treatment for vascular dysfunction and diabetic peripheral neuropathy in rodent models of diabetes [86,87]. These studies demonstrate that increased oxidative stress in vascular tissue and endothelial dysfunction is important since diabetic vascular disease is a contributing factor in the development of diabetic peripheral neuropathy. Additional evidence for vascular dysfunction contributing to diabetic peripheral neuropathy came from studies by Cameron and Cotter who demonstrated that reduced nerve perfusion was evident in diabetic rodents displaying signs of diabetic peripheral neuropathy [88]. The free radicals O₂⁻ and OH⁻, causes damage to the vascular endothelium that reduces NO-mediated vasodilation. Blockage of advanced glycosylation and autoxidation, primary sources of free radicals, by aminoguanidine and transition metal chelators, or antioxidants and free radical scavengers have been shown to correct the diabetes-induced decrease in endoneurial blood flow and improve neural dysfunction [89–97]. Some of the causes of diabetes-induced endothelium dysfunction are impaired signal transduction pathways or substrate availability, disrupted release or increased metabolism of vasodilatory mediators, enhanced release of vascular constricting factors and decreased reactivity of the smooth muscle to vasodilatory mediators [98,99]. Studies from my laboratory have provided evidence that the generation of oxidative stress through the formation of O₂⁻ and ONOO⁻ impairs vascular reactivity and endothelium-dependent vascular relaxation of epineurial arterioles of the sciatic nerve from diabetic rats, which occurs prior to slowing of motor nerve conduction velocity [78,90–92]. My laboratory has also shown that treating diabetic rodents with three different types of antioxidants attenuated the diabetes-induced increase in O₂⁻ and ONOO⁻ formation in aorta and epineurial arterioles of the sciatic nerve and diabetes-induced vascular and neural dysfunction. This provides further evidence that increased oxidative stress contributes to diabetes-induced neural disease [89,91]. Cameron and colleagues have also reported similar findings demonstrating that treating diabetic rats with α-lipoic acid or the metal chelators hydroxyethyl starch deferoxamine or trientine prevented the diabetes-induced impairment in vascular relaxation associated with hyperalgesia and neurovascular deficits [93,94,100–102].

As previously stated persistent hyperglycemia is the driving force for increasing oxidative stress through the activation of the polyol pathway, increased production of advanced glycation endproducts, activation of protein kinase C, and increased flux through the hexosamine pathway. Activation of these pathways is also directly or indirectly responsible for chronic production of inflammatory mediators [65,66]. Acute inflammation is an important component of innate immunity that is activated in response to a threat to tissue homeostasis [52]. This form of inflammation is the body’s process of fighting against pathogens or events that damage it, such as infections, injuries, and toxins. This response includes the release of antibodies, cytokines and proteins, as well as increased blood flow to the damaged area. The whole process usually lasts for a few hours or days in the case of acute inflammation. Chronic inflammation happens when this response lingers and over time may have a negative impact on tissues and organs. Chronic inflammation plays a role in number of disease states including type 2 diabetes and is thought to be a characteristic feature seen at sites of diabetes complications [52]. In regard to diabetic neuropathy anti-inflammatory agents have been widely tested in diabetic animal models. Ma et al. demonstrated in a streptozotocin-induced diabetic rat model that the proinflammatory cytokines tumor necrosis factor-α (TNF-α), and interleukin 1β (IL-1β) and 6 (IL-6) in serum were increased as was the mRNA expression levels in the sciatic nerve of TNF-α, IL-1β, IL-6, and the chemokine monocyte chemoattractant protein-1 (MCP-1), adhesion molecule intercellular adhesion molecule-1 (ICAM-1) [103]. There studies also demonstrated that protein expression of phosphorylated p38 mitogen activated protein kinase (MAPK) and NF-κB were increased. Treating these rats with the glucagon-like peptide 1 receptor agonist, liraglutide, attenuated these changes while improving several markers of peripheral nerve dysfunction including nerve conduction velocity and density of myelin nerve fibers. They attributed the effects of liraglutide in their study to improvement in multiple markers of inflammation. Chen et al. reported that exposing cultured Schwann cells to a hyperglycemic condition and treating rats with streptozotocin to induce diabetes caused an increase in the expression of cyclooxygenase-2 (COX-2), IL-1β, IL-6 and caspase-3 as well as Schwann cell apoptosis and decreased nerve conduction velocity and demyelination of the sciatic nerve in diabetic rats [104]. All these defects were attenuated by treating the culture medium of Schwann cells or diabetic rats with thymoquinone. They attributed the effects of thymoquinone in part to modulation of the inflammatory reaction. The function of Schwann cells is to maintain peripheral nerve structure by ensheathment of unmyelinated axons, myelination of myelinated axons, and secretion of neurotrophic factors [105]. Animal studies have demonstrated that unmyelinated and small myelinated axons decrease earlier than changes in structure of large myelinated fibers in diabetic peripheral neuropathy. In studies of humans with diabetic neuropathy electron microscopy has demonstrated edematous cell cytoplasm, aggregates of glycogen particles, and hyperplasia of the surrounding basal lamina in Schwann cells [105]. Therefore, maintaining a healthy Schwann cell environment is another important target for the treatment of diabetic peripheral neuropathy.

Other studies utilizing diabetic rodents have demonstrated that a variety of treatments having anti-inflammatory properties improve diabetic peripheral neuropathy including berberine, tocotrienol, fish oil and mesenchymal stem cells [106–109]. Our group has focused on the activation of poly(ADP-ribose) polymerase (PARP) as a downstream mediator of oxidative-nitrosative/inflammatory stress in experimental diabetic neuropathy. We have demonstrated that inhibitors of PARP activation improved diabetes vascular and neural complications in streptozotocin-treated animal models but also attenuated the effects of hyperglycemia on cultured dorsal root ganglion neurons and Schwann cells [110–113]. Combined these studies demonstrate the crosstalk that exists between oxidative-nitrosative/inflammatory stress that exists in the pathology of diabetic peripheral neuropathy ranging from increased levels of markers of oxidative and inflammatory stress, and downstream activation of protein kinase C, NF-κB and toll-like receptors and other pathways that contributes to the complex etiology of this diabetes complication [114].

Human studies have also pointed to an association between the occurrence of oxidative stress and increased markers of inflammation and incidence and progression of diabetic polyneuropathy. Oxidative stress is enhanced in diabetes patients before the development of polyneuropathy and to a higher degree in those with polyneuropathy [115]. D. Ziegler and colleagues demonstrated that subclinical inflammation as shown by increased levels of C-reactive peptide, IL-6, and TNF-α, and soluble intercellular adhesion molecule-1 and lower adiponectin levels were associated with both onset and progression of distal sensorimotor polyneuropathy [116–118]. Even though there is evidence for oxidative and inflammatory stress being contributing factors to peripheral neuropathy in human diabetes subject’s treatments with anti-oxidative or anti-inflammatory agents shown to be efficacious in pre-clinical studies have not had great success in clinical trials [13,119]. The most highly studied anti-oxidant for the treatment of diabetic peripheral neuropathy in human subjects has been α-lipoic acid [120,121]. Several trials have demonstrated that treatment with α-lipoic acid is safe and significantly improves neuropathic symptoms and deficits in diabetic subjects with symptomatic polyneuropathy [122–124]. α-Lipoic acid is currently used to treat some diabetic peripheral neuropathy in some countries of the United Kingdom.

As discussed above aldose reductase inhibitors and α-lipoic acid are being used in several countries. We were interested in determining the effect of combining an aldose reductase inhibitor with α-lipoic acid on vascular and neural in a diabetic rat. Lipoic acid in the diet is quickly absorbed, transported to the intracellular compartments, and reduced to dihydrolipoic acid [125]. However, in diabetes the availability of reducing equivalents is impaired due in part to the increased activity of the polyol pathway. The hypothesis driving our study was by treating diabetic rats with an aldose reductase inhibitor the availability of reducing equivalents would be increased and formation of dihydrolipoic acid from α-lipoic acid would be increased. We found that treating diabetic rats with 0.25% α-lipoic acid, a sub-optimal dose, and fidarestat (3 mg/kg body weight) was more effective in preventing vascular and neural dysfunction than monotherapy. In this study serum levels of dihydrolipoic acid were increased with the combination therapy vs. α-lipoic acid alone [86]. This approach for treatment of diabetic peripheral neuropathy has also been examined in human subjects. A recent meta-analysis of 20 randomized controlled trials testing the efficacy of epalrestat and α-lipoic acid vs. monotherapy in patients with diabetic peripheral neuropathy revealed that the combination therapy was superior to α-lipoic acid and epalrestat monotherapies for clinical efficacy and nerve conduction velocities [126].

2.4. Insulin and Neurotrophic Support:

Studies dating back more than 25 years ago with diabetic rodents have implicated impairment of neurotrophic support contributing to diabetic peripheral neuropathy. Insulin has been shown to be a potent neuronal growth factor through binding to receptors on sensory neurons [11]. Insulin along with insulin-like growth factor-1 activates signaling cascades that are shared with other neurotrophin growth factors [11]. In an interesting study an investigative team led by D. Zochodne demonstrated that directly treating peripheral neurons of streptozotocin-induced diabetic rats with low doses of insulin through intrathecal injection, insufficient to reduce hyperglycemia, improved slowing of motor and sensory nerve conduction velocity and reversed atrophy in myelinated sensory axons in the sural nerve [127]. In this study it was shown that similar doses of insulin delivered by subcutaneous injection were ineffective. This same team has also demonstrated that treating diabetic mice with intranasal insulin, in doses insufficient to correct hyperglycemia, suppressed the development of diabetic peripheral neuropathy in streptozotocin-treated male and female mice as demonstrated by reduced nerve conduction slowing and improved mechanical and thermal sensitivity [128]. Another exemplarily research team led by P. Fernyhough has focused on the role of mitochondrial dysfunction in diabetes complications including peripheral neuropathy [129]. In a recent review article they described mitochondrial dysfunction in diabetic neuropathy to be a series of unfortunate metabolic events in part due to disrupted insulin signaling [130]. In that article they conclude that the diabetic state is driving a transition in neuronal metabolism toward anaerobic glycolysis leading to less efficient ATP generation. This ultimately leads to an inability to maintain and/or repair distal nerve endings and the result is neurodegeneration [130].

Dysregulation of the neurotrophin family of growth factors or their receptors have also received significant attention as a likely contributor to diabetic peripheral neuropathy. Studies have shown that many of the neuronal abnormalities observed in diabetic animal models can be duplicated by artificial disruption of neurotrophic factors expression, receptors or signaling pathways [131]. The most studied of the neurotrophin family of growth factors has been nerve growth factor and its high-affinity receptor tropomyosin receptor kinase A (trkA). Early studies in rodent models of diabetes showed that there were expression deficits in nerve growth factor and trkA leading to decreased support of sensory neurons and reduced expression of their neuropeptides, substance P and calcitonin gene-related peptide [132]. We have shown that epineurial arterioles that provide blood flow to the sciatic nerve are innervated by sensory neurons that express calcitonin gene-related peptide and that vascular relaxation to calcitonin gene-related peptide is decreased by diabetes [133]. This demonstrated that decreased neurotrophin support for sensory neurons can also contribute to neurovascular dysfunction through decreased expression of calcitonin gene-related peptide. Treating diabetic rodents with neurotrophins has been shown to restore growth factor levels in dorsal root ganglion sensory neurons and sciatic nerve, rescue mitochondrial dysfunction and increase intraepidermal nerve fiber density [134–136]. These and numerous other studies have generated interest for endogenous replenishment of growth factors as a therapy for diabetic peripheral neuropathy [137]. However, additional studies will be needed to determine which growth factors would provide the most efficacious treatment and it is unlikely that a single compound will be sufficient and that a combination of growth factors will be most beneficial [138].

2.5. Obesity and Hyperlipidemia:

Obesity and hyperlipidemia are common components of the metabolic syndrome and often a comorbidity seen in individuals with type 2 diabetes [52]. Smith and Singleton have demonstrated that obesity and hypertriglyceridemia significantly increase the risk for peripheral neuropathy, independent of glucose control [139]. They have also demonstrated that impaired glucose tolerance is a common feature in patients with peripheral neuropathy with preferential injury to small nerve fibers [10]. Miscio et al. have demonstrated that non-diabetic obese patients showed early signs of sensory neuropathy that was related to hyperinsulinemia and insulin sensitivity [140]. However, unlike type 1 diabetes improving glucose control in individuals with type 2 diabetes has only a marginal effect preventing peripheral neuropathy suggesting that other factors such as hyperlipidemia, impaired glucose tolerance and insulin resistance are contributing to nerve damage [141]. The high level of obesity has created a new disease-like burden on health care professionals. Obesity triggers many of the same pathological pathways as diabetes and there is a distinct need for investigations to identify efficacious interventions. The obvious answer is lifestyle changes through diet and exercise but this approach is not acceptable for many of these new patients. Studies in animal models have aided in the identification of the pathology associated with obesity including peripheral neuropathy. Feeding rodents a high fat diet creates obesity like condition not unlike over eating and sedentary lifestyle that accompany obesity in the human population. Our studies in diet-induced obese rats have demonstrated a development of impaired glucose tolerance and vascular dysfunction with progression of sensory neuropathy demonstrated by slowing of sensory nerve conduction velocity and decrease in intraepidermal and corneal nerve fiber density and sensitivity [142,143]. We have obtained similar results with obese Zucker rats [144]. When diet-induced obese rats were treated with low dose of streptozotocin to make them hyperglycemic as well as analysis of Zucker Diabetic Fatty rats revealed that the onset of hyperglycemia rapidly increases the pathology of peripheral neuropathy with the most notable impact being slowing of motor nerve conduction velocity being most prominent [144,145]. Replacing the high fat diet in diet-induced obese or type 2 diabetic rats following 12 weeks of the high fat diet with or without 4 weeks of hyperglycemia in the type 2 diabetic rats with the normal diet for 12 weeks improved glucose utilization in diet-induced obese but not diabetic rats [146]. Reversal of the high fat diet in these two models did not significantly improve peripheral neuropathy related endpoints [146]. In contrast, Hinder et al., demonstrated that reversal of a high fat diet in wild type C57Bl/6J mice following 16 weeks of a high fat diet completely normalized peripheral neuropathy related pathological endpoints [147]. Obrosova et al. also demonstrated that dietary reversal for 6 weeks after 16 weeks of a high fat diet alleviated tactile allodynia and improved thermal hypoalgesia and sensory nerve conduction deficit without affecting motor nerve conduction slowing in C57Bl/6J mice [148]. In this study the authors also demonstrated that treating high fat fed obese mice with normal chow containing an aldose reductase inhibitor corrected all changes associated with obesity-induced neuropathy. The reason for the complete differences in reversal of peripheral neuropathy in rat and mouse models are unknown but may be related to species or age of animals at the beginning of the studies. In addition to dietary correction exercise has been shown to be a potential therapy for diabetic and pre-diabetic neuropathy including neuropathic pain [149,150]. In human subjects exercise has been shown to improve symptoms of neuropathy and promote re-growth of cutaneous small-diameter fibers [151]. D. Wright and colleagues have shown that mice fed a high fat diet develop hyperalgesia, decreased neurotrophin protein expression and epidermal innervation with all being normalized with exercise [152]. Studies performed in animal models as well as human subjects have demonstrated that obesity causes a number of neuropathic symptoms commonly associated with diabetic peripheral neuropathy. Often referred to as pre-diabetes this condition should be a warning sign of things to come and offers a time when early intervention may provide a significant benefit.

2.6. Angiotensin-converting enzyme and neutral endopeptidase inhibition:

We have extensively studied the effect of inhibition of angiotensin-converting enzyme or blocking of the angiotensin receptor and inhibition of neutral endopeptidase or neprilysin on peripheral neuropathy in our pre-clinical models [153]. These drugs have antioxidant properties, neuroprotective potential and may reduce the accumulation of advanced glycation endproducts [154–158]. Angiotensin II causes endothelium dysfunction by increasing NAD(P)H oxidase-mediated vascular O₂⁻ production [159,160]. Extensive evidence exists demonstrating that blocking the renin-angiotensin system reduces the progression of diabetic nephropathy in human subjects. However, information is lacking if treatment of human subjects with diabetes with angiotensin converting enzyme inhibitors or angiotensin receptor antagonists improves or slows the progression of diabetic vascular and neural disease.

We have demonstrated that treating type 1 diabetic rats and mice with an angiotensin converting enzyme inhibitor and to a lesser extent an angiotensin II receptor blocker improved vascular relaxation, by epineurial arterioles, endoneurial blood flow and multiple endpoints associated with peripheral neuropathy [161]. We have also demonstrated that treating obese Zucker rats or Zucker diabetic fatty rats with an angiotensin converting enzyme inhibitor is efficacious toward neural vascular and neural pathology [89,162]. Cameron and colleagues also demonstrated that treatment of diabetic rats with an angiotensin converting enzyme inhibitor or with an angiotensin II receptor antagonist improved motor and sensory nerve conduction velocities, nerve blood flow and stimulated endoneurial angiogenesis [163,164]. Aggarwal et al. have also demonstrated that treating streptozotocin-induced diabetic rats with lisinopril, an angiotensin converting enzyme inhibitor, improved diabetic peripheral neuropathy [165].

In addition to these pre-clinical studies there have also been a few small clinical trials showing that diabetic peripheral neuropathy can be improved by treatment of diabetes subjects with neuropathy with trandolapril or lisinopril [165,166]. In the former study treatment with trandolapril for 12 months improved peroneal motor nerve conduction velocity and M-wave amplitude were increased, F-wave latency decreased, and sural nerve action potential amplitude increased significantly. However, vibration-perception threshold, autonomic function and the neuropathy symptom and deficit score showed no improvement in either group. From this study the authors concluded that that trandolapril may improve peripheral neuropathy in normotensive patients with diabetes although they state that larger clinical trials are needed before clinical practice can be advocated [165]. In the other study 13 diabetic patients with hypertension were treated with lisinopril for 12 weeks [166]. In this study lisinopril treatment significantly improved median motor, median sensory, peroneal motor and sural sensory nerve conduction velocities. In this study there was also significant improvement in warm temperature discrimination and vibration perception thresholds. These data lead to a review article to highlight the potential of angiotensin converting enzyme inhibitors as a treatment for diabetic peripheral neuropathy [167].

Due to the complex etiology of diabetic neuropathy we have investigated the effect of combination therapies on vascular and neural endpoints in obese and diabetic rodents. The rationale for these studies was that treatments that would correct or improve more than one mechanism contributing to vascular and neural complications may have a better outcome. We have focused on several different therapeutic combinations. First, we have examined the effect of a vasopeptidase inhibitor. Vasopeptidase inhibitors simultaneously inhibit neutral endopeptidase and angiotensin converting enzyme activity [168]. Neutral endopeptidase is a protease found in many tissues including vascular and renal tissue and its activity is increased by fatty acids and glucose in human microvascular cells [169–173]. Interestingly, neutral endopeptidase activity has been shown to be activated by protein kinase C, which is increased in vascular tissues by diabetes including endothelial cells [174,175]. Neutral endopeptidase degrades natriuretic peptides, adrenomedullin, bradykinin, endothelin and calcitonin gene-related peptide [168]. Therefore, use of vasopeptidase inhibitors would likely protect expression of C type natriuretic peptide and calcitonin gene-related peptide by blocking degradation and thus, improving vascular functions. We have demonstrated that C type natriuretic peptide is expressed on the membrane of endothelial cells of epineurial arterioles and that sensory nerves that innervate these epineurial arterioles contain calcitonin gene-related peptide and both peptides potently vasodilate epineurial arterioles that provide circulation to the sciatic nerve [132,176]. Furthermore we have shown that diabetes causes an increased expression of neutral endopeptidase in endothelial cells lining epineurial arterioles thus, inhibition of this protease would protect the vasodilatory properties of C type natriuretic peptide and calcitonin gene-related peptide [176]. Multiple studies using ilepatril, a vasopeptidase inhibitor, and different models of diabetic and obese rats demonstrated that the combination therapeutic was overall more efficacious than inhibitors of angiotensin converting enzyme or neutral endopeptidase alone toward maintained normal vascular function and neural activity [176–183]. These results were further verified using neutral endopeptidase deficient mice. In those studies we demonstrated that mice deficient in neutral endopeptidase were protected from the development of neuropathology and loss of corneal nerves in diet-induced obesity and type 2 diabetes [184,185]. Unfortunately, clinical trials with ilepatril revealed an unacceptable risk for angioedema and further development of this drug was discontinued. However, this approach was rescued by production of sacubitril/valsartan (LCZ696, Entresto®), a neutral endopeptidase inhibitor and angiotensin II receptor blocker. Clinical studies with this combination drug for heart failure demonstrated efficacy and a good safety profile [186]. We were able to obtain this drug from Novartis who sponsored a study we performed using rats modeling type 2 diabetes. In this study diabetic rats were treated for 12 weeks with sacubitril/valsartan compared to valsartan alone beginning at 4 weeks and 12 weeks post-hyperglycemia. Results from this study showed that sacubitril/valsartan improved vascular and neural function to a greater extent than valsartan alone. In the early intervention sacubitril/valsartan slowed progression of neural deficits and in the late intervention this 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 [187]. We concluded that sacubitril/valsartan since it has already been shown to be clinically safe may be a promising new treatment for diabetic peripheral neuropathy.

Another combination therapy my laboratory has focused on for treatment of vascular and neural complications is α-lipoic acid, enalapril (angiotensin converting enzyme inhibitor) and menhaden (fish) oil. α-Lipoic acid and inhibitors of angiotensin converting enzyme have been discussed above. Fish oil is enriched in the omega-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid. Omega-3 polyunsaturated fatty acids are essential nutritional lipids that must be obtained from the diet and are essential for normal development including membrane organization and many studies have proposed wide ranging benefits from their increased consumption [188]. Eicosapentaenoic acid and docosahexaenoic acid are the precursors of E and D series resolvins, respectively, which have anti-inflammatory and neuroprotective properties. In our studies we have conducted early and late intervention protocols using monotherapies and comparing their effect to the combination therapy of α-lipoic acid, enalapril and menhaden oil. Results from these studies demonstrated that the combination therapy is superior to monotherapy in regard to improving vascular and neural function in type 2 diabetic mice and rats [189–191]. Treatment with enalapril alone and in the combination therapy modestly improved glucose utilization but overall hyperglycemia was not corrected by this combination therapy. We have also demonstrated that this combination therapy improves both oxidative and inflammatory stress. Since the α-lipoic acid and fish oil in this combination therapy are available commercially and enalapril has an extensive history of safe clinical use advancing this combination to clinical trials for diabetic peripheral neuropathy should be safe and affordable.

Another combination therapy we have examined in diabetic rodent models that I would like to discuss has been menhaden oil and salsalate. We have shown that menhaden oil used as a monotherapy partially but significantly improves vascular and neural complications in type 1 and type 2 diabetic rats [192,193]. As discussed above eicosapentaenoic and docosahexaenoic acids are precursors for E and D series resolvins. We have shown that resolvins elicit repair of nerve damage caused by diabetes when administered endogenously in vivo and stimulate neurite growth in vitro [192,193]. Our studies have also demonstrated that combining menhaden oil with salsalate, a highly effective agent in blocking proinflammatory chemokines and cytokines, with a large margin of safety and low cost, increases the production of resolvins and has greater efficacy toward improving diabetic peripheral neuropathy outcomes than menhaden oil alone [194,195]. Salicylate derived anti-inflammatory agents, including acetylsalicylic acid (aspirin) and salsalate, have been shown to improve insulin sensitivity, an additional important risk factor for diabetic peripheral neuropathy [196,197]. Unlike aspirin, whose side effects spectrum in the required dose side prohibit its use, salsalate is well tolerated and demonstrated to be safe for extended use in patients with T2D [197]. Thus, the success of our pre-clinical studies with menhaden oil and salsalate showing greater efficacy when combined and the safety record of both agents in humans with type 2 diabetes provide the rationale to extend this work clinical trials with the ultimate goal of finding a safe and effective disease modifying treatment for diabetic peripheral neuropathy.

2.7. Additional treatments:

Methylcobalamin is used to treat vitamin B12 deficiency [198]. Vitamin B12 is important for the brain and nerves, and for the production of red blood cells. Metformin a common treatment for type 2 diabetes has been shown to cause vitamin B12 deficiency in some patients. In animal studies methylcobalamin treatment has been shown to improve multiple endpoints associated with diabetic peripheral neuropathy [198–201]. This was reportedly associated with improvement of oxidative stress, activation of protein kinase C, and up-regulation of neural IFG-1 gene expression [198–201]. Clinical studies have also demonstrated promise for methylcobalamin as a potential treatment for diabetic peripheral neuropathy [202–207]. However, in many of these human studies it was reported that combination therapy with methylcobalamin and different secondary compounds including α-lipoic acid, probucol, or prostaglandin E1 was more effective than monotherapy of methylcobalamin [202,205–207]. The rationale for the selection of a range of compounds used in different clinical studies for diabetic peripheral neuropathy in combination with methylcobalamin is confusing but supports the growing understanding of the complexity of the etiology of diabetic peripheral neuropathy and the future need of multiple compounds to target these different mechanisms.

Presented above was the potential role of essential omega-6 and omega-3 polyunsaturated fatty for the treatment of diabetic peripheral neuropathy. Other lipids such as prostaglandin E1 or analogues have also been shown to be a potential treatment for diabetic peripheral neuropathy. Some years ago Cotter et al. [208] and Ohno et al. [209] reported that treating streptozotocin-diabetic rats with iloprost prevented and reversed motor and sensory peripheral neuropathy. More recently Mo et al. [210] have shown that treatment of streptozotocin-diabetic rats with prostaglandin E1 prevented peripheral nerve damage through preventing microvascular permeability. Two independent studies have shown that treating two different models of diabetic rats with the prostaglandin E1 analogue (TFC-612) improved experimental diabetic peripheral neuropathy [211,212]. Two small clinical trials have also demonstrated that treating type 2 diabetic patients with prostaglandin E1 in lipid microspheres improved diabetic peripheral neuropathy [213,214].

3.0. Summary:

The following Table provides information relating to the complex etiology and underlying causes for the development and progression of diabetic peripheral neuropathy identified using animal models. As shown in the Table (right column) many therapeutic approaches for the prevention of diabetic peripheral neuropathy have been successfully applied in animal models of diabetes. This has led investigators to propose a wide range of mechanisms (Table center column) for the cause of diabetic peripheral neuropathy. Aldose reductase inhibitors and α-lipoic acid are being used clinically in some countries but there is no Federal Drug Administration approved treatment for diabetic peripheral neuropathy other than drugs to relieve pain, which are often not totally successful. This raises the question of why these treatments based on verified mechanisms discovered in pre-clinical studies generally failed in human trials.

Diabetic Peripheral Neuropathy Etiology, Mechanistic Disturbances, and Treatment

ETIOLOGY	MECHANISMS	TREATMENTS
Early Diagnosis	Oxidative Stress	Glycemic Control
Hyperglycemia	Inflammatory Stress	Exercise and Life Style Changes
Obesity/Dyslipidemia	Nitrergic Stress	Pancreas Transplantation
Insulin Deficiency/Resistance	Endoplasmic Reticulum Stress	Antioxidant Agents
Hypertension	Mitochondrial Stress	Antiinflammatory Agents
Aldose Reductase Pathway	DNA Damage	Aldose Reductase Inhibitors
Non-enzymatic Glycation/Glycoxidation	NF-κB Activation	ACE Inhibitors
Protein Kinase C Activation	PARP Activation	Angiotensin Receptor Blockers
Neurotrophic Support	Apoptosis	Protein Kinase C Inhibitors
Mitochondrial Dysfunction	MAP Kinase and PIP3 Kinase Activation	DPP IV Inhibitors
Microvascular Dysfunction	Cyclooxygenase and Lipoxygenase Activation	Neurotrophic Support
Ion Channel Dysregulation	Na+ and Ca++ Homeostasis	C-Peptide
Renin-Angiotensin-Aldosterone System	Ischemia	RAGE Antibody
Dipeptidyl Peptidase IV	Essential Polyunsaturated Fatty acid Deficiency	GLP-1 Agonists
Neutral Endopeptidase Overexpression		Thiamine/Benfotiamine
Electrolyte Imbalance		Omega-3 PUFA
		Omega-6 PUFA
		Taurine/myo-Inositol
		Neutral Endopeptidase Inhibitors
		PARP Inhibitors
		Prostaglandin E1 or analogues
		Methylcobalamin
		Acetyl-L-carnitine

There are a several possibilities that could explain this outcome. First, due to side effects or other concerns the dosage of the drugs used in human studies may have been inadequate and the efficacy too low to be effective against the targeted mechanism compared to animal studies. This is a valid concern and outcomes from studies using aldose reductase inhibitors, nerve growth factor and antioxidants may have been influenced by this problem. Second, the targeted mechanisms identified from animal studies may have no contribution to diabetic peripheral neuropathy in humans. This seems unlikely since each of the therapeutics discussed above and their targeted mechanism are based on valid pre-clinical scientific data and for some of these interventions there are some supportive clinical data. However, whether each of these targeted mechanisms plays a significant role in the development of diabetic peripheral neuropathy as it occurs in humans is a point of conjecture. Another serious concern is that it is unlikely that a single mechanism is solely responsible for the development and progression of diabetic peripheral neuropathy and behavioral changes such as exercise and diet combined with therapies that target two or more have the identified mechanism will likely be needed to successfully treat diabetic peripheral neuropathy in human subjects. We have provided several examples for this in this article. One possible advantage of combination therapies may be that sub-optimal dosing may be sufficient since multiple targets are being treated. Third, the human subjects studied in earlier clinical trials may have been less than ideal because their neuropathy may have progressed to the point that the symptoms or endpoints being examined are not readily reversible within the time frame of the treatment. Drug interventions for a short period of time in human subjects with diabetic peripheral neuropathy with advanced clinical symptoms will likely have minimal benefit since many of these patients may likely have nerve damage that is either irreversible or only slowly reversible that may not occur during the treatment period applied in the clinical trial. For instance, many of the clinical trials conducted with aldose reductase inhibitors were designed with a year or less of treatment. Considering the duration of diabetes and the progression of diabetic neuropathy in the patients used in these studies this treatment period was likely too short. Lastly, outcomes were based on invalid endpoints. For instance, measurement of sorbitol for interventions using aldose reductase inhibitors may not be a good outcome predictor for the patient. Measurement of sorbitol may provide good evidence of the current metabolism but may not reflect the severity or progression of the disease and its reversibility. In the case of human diabetic peripheral neuropathy, treatment with aldose reductase inhibitors may provide control of glucose flux through the aldose reductase pathway and measurement of the level of sorbitol in tissue samples or cells may provide information on the efficacy of aldose reductase inhibitor treatment, but likely this measurement will not provide any indication of the present extent of nerve damage or progression of the disease. In order to better analyze the progression of diabetic neuropathy several relevant markers of nerve function and biological status including oxidative and inflammatory stress will probably be required. Investigators are also developing new and earlier endpoints to clinically evaluate diabetic peripheral neuropathy. The developing analysis of small nerve fibers in the skin or cornea along with sensitivity currently provides hope for more successful clinical trials in the future.

I foresee a successful treatment for diabetic peripheral neuropathy being a combination of exercise and lifestyle changes along with dietary and pharmaceutical interventions that target different mechanism that are deemed to be safe in order to be taken for the life of the patient. This approach has successfully been used to treat hypertension and a similar strategy is needed for the treatment of diabetic peripheral neuropathy.