Introduction:A ketogenic diet (KD) can benefit a range of neurological conditions, including different types of peripheral neuropathy. Bortezomib (BTZ), a first-line chemotherapy for treating multiple myeloma, has neurotoxic effects and often induces chemotherapy-induced peripheral neuropathy. Objectives:Here, we tested whether a KD could prevent the onset of small fiber neuropathy and behavioral hypersensitivity associated with bortezomib-induced peripheral neuropathy (BIPN). Methods:Male C57BL/6 mice were fed either a standard chow diet or a KD for 10 days. During the first 5 days of KD administration, mice received daily intraperitoneal injections of BTZ or vehicle. Mice entered a BTZ washout period on day 6 while remaining on their specific diets. Hindpaw responses to mechanical (von Frey) and cold (acetone) stimuli were measured to determine how KD and BTZ treatment affected behavioral sensitivity. Footpads were harvested for analysis of intraepidermal nerve fiber density (IENFD), and lumbar dorsal root ganglia were collected for in vitro analysis of neurite outgrowth and cellular bioenergetics. Results:Bortezomib-treated, KD-fed mice exhibited decreased mechanical hypersensitivity, normalized cold hypersensitivity, preserved IENFD, and decreased extracellular acidification rate compared with BTZ-treated, chow-fed mice. In vitro analysis of ketones' ability to protect against BTZ-induced reductions in neurite growth revealed that cultured neurons pretreated with ketones were protected from BTZ-induced neurite degeneration. Conclusion:Findings suggest that KD protects against reductions in IENFD, behavioral hypersensitivity, and bioenergetic shifts induced by BIPN, and provides evidence that KD provides protection against BTZ and could be a potential therapeutic for BIPN.

Methylglyoxal is a highly reactive by-product of glycolysis that is elevated in diabetes and contributes to the development of diabetic peripheral neuropathy (DPN). DPN is characterized by nerve degeneration, typically manifesting in patients' extremities. This leads to patients experiencing numbness, burning, and pain. It has been established that elevated methylglyoxal levels lead to nociception, but the broader cellular effects of methylglyoxal on neurons in the dorsal root ganglia (DRG) remain poorly understood. This review provides mechanistic insight regarding methylglyoxal's impact on various cell types and disease contexts. Five main mechanisms were identified: protein glycation, proteostasis change, oxidative stress, metabolic changes, and increased inflammation. These mechanisms are thoroughly interconnected, contributing to cellular dysfunction associated with DPN. We propose that methylglyoxal functions as a central mediator in cellular stress, linking hyperglycemia and elevated glycolysis to neuronal dysfunction in DPN. There is extensive evidence that these mechanisms are methylglyoxal-driven in other cell types and diseases, but a gap in the field remains in determining whether and how they occur in DRG neurons. This is particularly important, as DPN is a frequent comorbidity in diabetes and metabolic diseases and greatly affects patients' quality of life. Understanding the effect of methylglyoxal on DRG in relation to these mechanisms will provide novel insights into the development of DPN and lead to new therapeutic targets.

Diabetic peripheral neuropathy (DPN) is a prevalent complication of diabetes, significantly impairing quality of life and lacking effective disease-modifying treatments. Chronic inflammation involving toll-like receptor 4 (TLR4) has been implicated in diabetes and neuropathy development. TLR4 is an innate immune receptor that triggers an inflammatory response, leading to the production of pro-inflammatory cytokines. While TLR4 has been studied in various neuropathy models, its role in diabetic neuropathy and its effect on nerve fiber integrity remains unclear. To investigate the impact of TLR4 on DPN, we induced diabetes in wild-type and TLR4 knockout (TLR4-/-) mice using streptozotocin. Groups contained 8-18 animals and were approximately half male and half female. Over six weeks, we assessed blood glucose, weight changes, thermal and mechanical sensory function, hind paw intraepidermal nerve fiber density (IENFD), dermal macrophage accumulation, and serum cytokines. In males, TLR4 deletion protected against STZ-induced thermal hyposensitivity, decreases in IENFD, and dermal macrophage accumulation. Female mice developed less severe hyperglycemia and were resistant to neuropathic changes, making the protective effects of TLR4 deletion less pronounced in females than in males. Our findings confirm that TLR4 plays a role in DPN pathogenesis in a diabetic mouse model, demonstrating that its deletion promotes sensory function and preserves IENFD in males. These results highlight TLR4 as a potential therapeutic target for slowing the progression of neuropathy in diabetes. Our data also emphasizes the need for further research into the role of sex-specific disease mechanisms in DPN.

Ketones, or ketone bodies, are organic molecules produced via ketogenesis in the liver in response to changing energy demands. Three ketones are generated that can act as metabolic messengers and a fuel source for the body, typically forming when glucose levels decrease within the bloodstream. A ketogenic diet, a form of low-carbohydrate, high-fat diet, stimulates ketogenesis and forces ketone utilization as an energy source by nonhepatic tissues. Currently, ketones, along with the ketogenic diet, have been of interest to many as a therapeutic mechanism for multiple conditions, including epilepsy, numerous neurodegenerative diseases, and diabetes. Emerging preclinical evidence suggests that ketones may play a powerful role in modulating acute and chronic pain. Here, we summarize the known benefits of ketones on neurological disease and nociceptive systems associated with pain. We discuss possible mechanisms identified from preclinical studies underlying the identified benefits of ketones in reducing pain.

Nociceptors that innervate the epidermis convey afferent transmission of pain information. When damaged, c-fibers become sensitized and can increase spontaneous activity. Typically, pain is not thought of when studying Alzheimer’s Disease (AD); however, 45% of AD patients have chronic pain, and widespread pain is associated with a 47% increase in Alzheimer’s disease dementia risk. To identify mechanisms of pain and c-fiber damage in AD, we are exploring the hypothesis that amyloid-beta accumulation is toxic to c-fibers via toll-like receptor(TLR)4/NF-kB signaling pathways. To test this hypothesis, we examined a 5xFAD transgenic mouse model that overexpress human mutant APP and PS1. The Barnes Maze test assessed cognitive dysfunction and memory in these mice at eight weeks of age. Homozygous 5XFAD mice had significantly reduced spatial memory and learning compared to wildtype (WT) mice (n=4-5). Experiments also examined the density of c-fibers in the hind paw. Homozygous 5XFAD mice significantly reduced intraepidermal nerve fiber (IENF) density compared to WT mice consistent with a small fiber neuropathy. Homozygous 5XFAD mice had significantly more NF-KB-positive DRG neurons compared to WT mice. Ongoing studies examine pain responsiveness, pro-inflammatory cytokine levels, assessing amyloid and tau expression and deposition, and activation of spinal microglia. Collectively, these studies are consistent with a small fiber neuropathy in 5XFAD mice that likely contributes to elevations in pain similar to the human condition. These studies should identify if DRG neurons are impacted by amyloid-beta deposition and if inflammation and TLR4 signaling pathways mediate these effects. Funding: R01NS043314-17, 5P20GM103418.

Nociceptors that innervate the epidermis convey afferent transmission of pain information. When damaged, c-fibers become sensitized and can increase spontaneous activity. Typically, pain is not thought of when studying Alzheimer's Disease (AD); however, 45% of AD patients have chronic pain, and widespread pain is associated with a 47% increase in Alzheimer's disease dementia risk. To identify mechanisms of pain and c-fiber damage in AD, we are exploring the hypothesis that amyloid-beta accumulation is toxic to c-fibers via toll-like receptor(TLR)4/NF-kB signaling pathways. To test this hypothesis, we examined a 5xFAD transgenic mouse model that overexpress human mutant APP and PS1. The Barnes Maze test assessed cognitive dysfunction and memory in these mice at eight weeks of age. Homozygous 5XFAD mice had significantly reduced spatial memory and learning compared to wildtype (WT) mice (n=4-5). Experiments also examined the density of c-fibers in the hind paw. Homozygous 5XFAD mice significantly reduced intraepidermal nerve fiber (IENF) density compared to WT mice consistent with a small fiber neuropathy. Homozygous 5XFAD mice had significantly more NF-KB-positive DRG neurons compared to WT mice. Ongoing studies examine pain responsiveness, pro-inflammatory cytokine levels, assessing amyloid and tau expression and deposition, and activation of spinal microglia. Collectively, these studies are consistent with a small fiber neuropathy in 5XFAD mice that likely contributes to elevations in pain similar to the human condition. These studies should identify if DRG neurons are impacted by amyloid-beta deposition and if inflammation and TLR4 signaling pathways mediate these effects. Funding: R01NS043314-17, 5P20GM103418.

Small fiber neuropathy (SFN) is a widespread complication that leads to pain, burning, or loss of sensation in the distal limbs. A hallmark of SFN is a loss of intraepidermal nerve fibers (IENF). Currently, therapeutic options for humans with SFN are severely lacking. However, preclinical studies have identified numerous drugs that stimulate IENF regeneration in rodents but have not yet translated to human use. Here, we leveraged existing information from published articles to identify all drugs that have reported IENF regenerative effects in rodents. As part of a larger scoping review on IENF, we identified a subset of articles that reported increased IENF density in rodent models. Using PubMed, 323 initial articles were narrowed down using specific inclusion criteria. We analyzed 72 articles and categorized the successful drugs into seven groups based on implied mechanisms of action. The mechanistic categories include metabolic (38%), antioxidant (23%), hormonal (18%), anti-inflammatory (11%), stem cell (6%), electrophysiological (3%), and unknown mechanisms (1%). The models used to test these drugs include diabetic, chemotherapeutic, and toxic models, suggesting a narrow set of preclinical models to address IENF regeneration. There are additional diseases and conditions in which IENF loss has been reported, including neurodegenerative and autoimmune models that should be explored and may provide new perspectives. This information will identify critical roadblocks, allow us to focus on specific mechanisms, and narrow the areas where we should exert effort to identify new treatments or modify existing drugs to use in humans with SFN. Funding: R01NS043314-17, 5P20GM103418.

Ketogenic diets are emerging as protective interventions in preclinical and clinical models of somatosensory nervous system disorders. Additionally, dysregulation of succinyl-CoA 3-oxoacid CoA-transferase 1 (SCOT, encoded by Oxct1), the fate-committing enzyme in mitochondrial ketolysis, has recently been described in Friedreich's ataxia and amyotrophic lateral sclerosis. However, the contribution of ketone metabolism in the normal development and function of the somatosensory nervous system remains poorly characterized. We generated sensory neuron-specific, Advillin-Cre knockout of SCOT (Adv-KO-SCOT) mice and characterized the structure and function of their somatosensory system. We used histological techniques to assess sensory neuronal populations, myelination, and skin and spinal dorsal horn innervation. We also examined cutaneous and proprioceptive sensory behaviors with the von Frey test, radiant heat assay, rotarod, and grid-walk tests. Adv-KO-SCOT mice exhibited myelination deficits, altered morphology of putative Aδ soma from the dorsal root ganglion, reduced cutaneous innervation, and abnormal innervation of the spinal dorsal horn compared to wildtype mice. Synapsin 1-Cre-driven knockout of Oxct1 confirmed deficits in epidermal innervation following a loss of ketone oxidation. Loss of peripheral axonal ketolysis was further associated with proprioceptive deficits, yet Adv-KO-SCOT mice did not exhibit drastically altered cutaneous mechanical and thermal thresholds. Knockout of Oxct1 in peripheral sensory neurons resulted in histological abnormalities and severe proprioceptive deficits in mice. We conclude that ketone metabolism is essential for the development of the somatosensory nervous system. These findings also suggest that decreased ketone oxidation in the somatosensory nervous system may explain the neurological symptoms of Friedreich's ataxia.

Chronic pain is a substantial health burden and options for treating chronic pain remain minimally effective. Ketogenic diets are emerging as well-tolerated, effective therapeutic strategies in preclinical models of chronic pain, especially diabetic neuropathy. We tested whether a ketogenic diet is antinociceptive through ketone oxidation and related activation of ATP-gated potassium (KATP) channels in mice. We demonstrate that consumption of a ketogenic diet for one week reduced evoked nocifensive behaviors (licking, biting, lifting) following intraplantar injection of different noxious stimuli (methylglyoxal, cinnamaldehyde, capsaicin, or Yoda1) in mice. A ketogenic diet also decreased the expression of p-ERK, an indicator of neuronal activation in the spinal cord, following peripheral administration of these stimuli. Using a genetic mouse model with deficient ketone oxidation in peripheral sensory neurons, we demonstrate that protection against methylglyoxal-induced nociception by a ketogenic diet partially depends on ketone oxidation by peripheral neurons. Injection of tolbutamide, a KATP channel antagonist, prevented ketogenic diet-mediated antinociception following intraplantar capsaicin injection. Tolbutamide also restored the expression of spinal activation markers in ketogenic diet-fed, capsaicin-injected mice. Moreover, activation of KATP channels with the KATP channel agonist diazoxide reduced pain-like behaviors in capsaicin-injected, chow-fed mice, similar to the effects observed with a ketogenic diet. Diazoxide also reduced the number of p-ERK+ cells in capsaicin-injected mice. These data support a mechanism that includes neuronal ketone oxidation and activation of KATP channels to provide ketogenic diet-related analgesia. This study also identifies KATP channels as a new target to mimic the antinociceptive effects of a ketogenic diet.

As common complication of prediabetes, type I and type II diabetes, diabetic peripheral neuropathy (DPN) includes a series of sensory and motor changes associated with slow nerve conduction, nerve degeneration, gate disturbances, pain, and loss of sensation. Although proper glycemic control can prevent DPN progression, these complications remain difficult to clinically treat. Current pharmacological medications have limited effectiveness, creating the need for additional clinical options. Lifestyle interventions hold great promise as the broad spectrum of improvements derived from certain lifestyle changes appears promising to improve diabetes management and DPN. In this chapter, we highlight research that illustrates the consequences of poor diet on DPN and discuss the benefits of lifestyle changes associated with dietary change and/or exercise. Reversal of dietary changes appears to have positive impact on DPN, and we highlight new studies in which a low-carbohydrate/high-fat diet has been used to prevent and/or reverse DPN. In addition, a growing number of basic and clinical studies are revealing how exercise can improve symptoms of DPN. These interventions affect a broad range of cellular and metabolic changes that can lead to improvements in DPN symptoms. These interventions likely involve overlapping cellular pathways but could also improve DPN through unique mechanisms. As approaches using personalized medicine increase, clinical treatments for DPN will need to determine the most impactful interventions that are relevant to specific symptoms in patients suffering from DPN. Lifestyle and dietary interventions should play an important role in these treatment plans and the convergence of shared mechanisms should be a focus of preclinical and clinical research.

Methylglyoxal (MGO) is a reactive dicarbonyl byproduct of glycolysis implicated in a growing number of neuropathic pain conditions, including chemotherapy-induced peripheral neuropathy, diabetic peripheral neuropathy, and radiculopathy with lumbar disk herniation. Recent studies show success in preclinical models treating these disorders with an interventional ketogenic diet. Here, we tested the hypothesis that a ketogenic diet modifies pathological MGO signaling as a mechanism underlying neuropathy improvement. We found that mice injected with MGO displayed nocifensive behaviors, whereas mice prefed a ketogenic diet were resistant to mechanical allodynia elicited by MGO. In addition, levels of circulating MGO were reduced in ketogenic diet-fed mice and negatively correlated with levels of the ketone body beta-hydroxybutyrate (beta-HB). Methylglyoxal is normally scavenged by the glyoxalase system, and ketogenic diet-fed mice displayed increased glyoxalase 1 activity compared with chow-fed control mice. Recent studies also suggest that ketone bodies contribute to MGO detoxification, consistent with a negative correlation between beta-HB and MGO. To assess whether ketone bodies modified MGO-evoked nociception through direct MGO detoxification, we coincubated either acetoacetate or beta-HB with MGO before injection. Mice receiving intraplantar MGO injection exhibit increased nociceptive behavior (lifting, licking, biting, and scratching), which was significantly reduced by coincubation with either acetoacetate or beta-HB. Methylglyoxal increased phospho-extracellular signal-regulated kinase-positive cells in the spinal dorsal horn, and this evoked spinal activation was ameliorated by preincubation with acetoacetate or beta-HB. These results suggest that a ketogenic diet and ketone bodies ameliorate MGO-evoked nociception, partially through detoxification of MGO, and provide rationale for therapeutic intervention with a ketogenic diet in MGO-driven pathologies.

Significance: Diabetic peripheral neuropathy (DPN), a complication of metabolic syndrome, type I and type II diabetes, leads to sensory changes that include slow nerve conduction, nerve degeneration, loss of sensation, pain, and gate disturbances. These complications remain largely untreatable, although tight glycemic control can prevent neuropathy progression. Nonpharmacologic approaches remain the most impactful to date, but additional advances in treatment approaches are needed. Recent Advances: This review highlights several emerging interventions, including a focus on dietary interventions and physical activity, that continue to show promise for treating DPN. We provide an overview of our current understanding of how exercise can improve aspects of DPN. We also highlight new studies in which a ketogenic diet has been used as an intervention to prevent and reverse DPN. Critical Issues: Both exercise and consuming a ketogenic diet induce systemic and cellular changes that collectively improve complications associated with DPN. Both interventions may involve similar signaling pathways and benefits but also impact DPN through unique mechanisms. Future Directions: These lifestyle interventions are critically important as personalized medicine approaches will likely be needed to identify specific subsets of neuropathy symptoms and deficits in patients, and determine the most impactful treatment. Overall, these two interventions have the potential to provide meaningful relief for patients with DPN and provide new avenues to identify new therapeutic targets.

ABSTRACT:Dietary interventions are promising approaches to treat pain associated with metabolic changes because they impact both metabolic and neural components contributing to painful neuropathy. Here, we tested whether consumption of a ketogenic diet could affect sensation, pain, and epidermal innervation loss in type 1 diabetic mice. C57Bl/6 mice were rendered diabetic using streptozotocin and administered a ketogenic diet at either 3 weeks (prevention) or 9 weeks (reversal) of uncontrolled diabetes. We quantified changes in metabolic biomarkers, sensory thresholds, and epidermal innervation to assess impact on neuropathy parameters. Diabetic mice consuming a ketogenic diet had normalized weight gain, reduced blood glucose, elevated blood ketones, and reduced hemoglobin-A1C levels. These metabolic biomarkers were also improved after 9 weeks of diabetes followed by 4 weeks of a ketogenic diet. Diabetic mice fed a control chow diet developed rapid mechanical allodynia of the hind paw that was reversed within a week of consumption of a ketogenic diet in both prevention and reversal studies. Loss of thermal sensation was also improved by consumption of a ketogenic diet through normalized thermal thresholds. Finally, diabetic mice consuming a ketogenic diet had normalized epidermal innervation, including after 9 weeks of uncontrolled diabetes and 4 weeks of consumption of the ketogenic diet. These results suggest that, in mice, a ketogenic diet can prevent and reverse changes in key metabolic biomarkers, altered sensation, pain, and axon innervation of the skin. These results identify a ketogenic diet as a potential therapeutic intervention for patients with painful diabetic neuropathy and/or epidermal axon loss.

Acute pain is prevalent following burn injury and can often transition to chronic pain. Prolonged acute pain is an important risk factor for chronic pain and there is little preclinical research to address this problem. Using a mouse model of second-degree burn, we investigated whether pre-existing stress influences pain(sensitivity) after a burn injury. We introduced a contribution of stress in two different ways: (1) the use of foot-shock as a pre-injury stressor or (2) the use of A/J mice to represent higher pre-existing stress compared to C57Bl/6 mice. C57Bl/6 and A/J mice were exposed to repeated mild foot shock to induce stress for 10 continuous days and mice underwent either burn injury or sham burn injury of the plantar surface of the right hind paw. Assessments of mechanical and thermal sensitivities of the injured and uninjured paw were conducted during the shock protocol and at intervals up to 82-day post-burn injury. In both strains of mice that underwent burn injury, thermal hypersensitivity and mechanical allodynia appeared rapidly in the ipsilateral paw. Mice that were stressed took much longer to recover their hind paw mechanical thresholds to baseline compared to non-stressed mice in both burn and non-burn groups. Analysis of the two mouse strains revealed that the recovery of mechanical thresholds in A/J mice which display higher levels of baseline anxiety was shorter than C57Bl/6 mice. No differences were observed regarding thermal sensitivities between strains. Our results support the view that stress exposure prior to burn injury affects mechanical and thermal thresholds and may be relevant to as a risk factor for the transition from acute to chronic pain. Finally, genetic differences may play a key role in modality-specific recovery following burn injury.

Patients with a history of early life stress (ELS) exposure have an increased risk of developing chronic pain and mood disorders later in life. The severity of ELS in patients with urologic chronic pelvic pain syndrome (UCPPS) is directly correlated with symptom severity and increased comorbidity, and is inversely related to likelihood of improvement. Voluntary exercise improves chronic pain symptoms and our group and others have shown that voluntary wheel running can improve outcomes in stress-induced UCPPS models, suggesting that exercise may negate some of the outcomes associated with ELS. Here we provide further evidence that voluntary wheel running can attenuate increased perigenital mechanical sensitivity, bladder output, and mast cell degranulation in the bladder and prostate in male mice that underwent neonatal maternal separation (NMS). Sedentary male NMS mice had reduced serum corticosterone, which was not impacted by voluntary wheel running, although stress-related regulatory gene expression in the hypothalamus and hippocampus was significantly increased following exercise. Neurogenesis in the dentate gyrus of the hippocampus was diminished in sedentary NMS mice and significantly increased in both exercised naïve and NMS mice. Sucrose consumption increased in exercised naïve but not NMS mice, and anxiety behaviors measured on an elevated plus maze were increased following exercise. Together these data suggest that voluntary wheel running is sufficient to normalize many of the UCPPS-related outcomes resulting from NMS. Exercise also increased hippocampal neurogenesis and stress-related gene expression within the hypothalamic-pituitary-adrenal axis, further supporting exercise as a non-pharmacological intervention for attenuating outcomes related to ELS exposure.