Modulation of diet-induced mechanical allodynia by metabolic parameters and inflammation.

Dietary-associated diseases have increased tremendously in our current population, yet key molecular changes associated with high-fat diets that cause clinical pre-diabetes, obesity, hyperglycemia, and peripheral neuropathy remain unclear. This study examines molecular and metabolic aspects altered by voluntary exercise and a high-fat diet in the mouse dorsal root ganglion. Mice were examined for changes in mRNA and proteins encoding anti-inflammatory mediators, metabolic-associated molecules, and pain-associated ion channels. Proteins involved in the synaptosomal complex and pain-associated TRP ion channels decrease in the dorsal root ganglion of high-fat exercise animals relative to their sedentary controls. Exercise reversed high-fat diet induced mechanical allodynia without affecting weight gain, elevated blood glucose, and utilization of fat as a fuel source. Independent of weight or fat mass changes, high-fat exercised mice display reduced inflammation-associated mRNAs. The benefits of exercise on abnormal peripheral nerve function appear to occur independent of systemic metabolic changes, suggesting that the utilization of fats and inflammation in the peripheral nervous system may be key for diet-induced peripheral nerve dysfunction and the response to exercise.

In vivo peripheral nervous system insulin signaling.

Alterations in peripheral nervous system (PNS) insulin support may contribute to diabetic neuropathy (DN); yet, PNS insulin signaling is not fully defined. Here, we investigated in vivo insulin signaling in the PNS and compared the insulin responsiveness to that of muscle, liver, and adipose. Non-diabetic mice were administered increasing doses of insulin to define a dose-response relationship between insulin and Akt activation in the dorsal root ganglion (DRG) and sciatic nerve. Resulting EC50 doses were used to characterize the PNS insulin signaling time course and make comparisons between insulin signaling in the PNS and other peripheral tissues (i.e., muscle, liver, and adipose). The results demonstrate that the PNS is responsive to insulin and that differences in insulin signaling pathway activation exist between PNS compartments. At a therapeutically relevant dose, Akt was activated in the muscle, liver, and adipose at 30 min, correlating with the changes in blood glucose levels. Interestingly, the sciatic nerve showed a similar signaling profile as insulin-sensitive tissues; however, there was not a comparable activation in the DRG or spinal cord. These results present new evidence regarding PNS insulin signaling pathways in vivo and provide a baseline for studies investigating the contribution of disrupted PNS insulin signaling to DN pathogenesis.

ATP-Gated Potassium Channels Contribute to Ketogenic Diet-Mediated Analgesia in Mice.

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.

ATP-gated potassium channels contribute to ketogenic diet-mediated analgesia in mice.

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.

Ketolysis is Required for the Proper Development and Function of the Somatosensory Nervous System.

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.

Ketolysis is required for the proper development and function of the somatosensory nervous system.

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.

Ketogenic diet prevents methylglyoxal-evoked nociception by scavenging methylglyoxal.

ABSTRACT: 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 ß-hydroxybutyrate (ß-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 ß-HB and MGO. To assess whether ketone bodies modified MGO-evoked nociception through direct MGO detoxification, we coincubated either acetoacetate or ß-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 ß-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 ß-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.

The impact of foot shock-induced stress on pain-related behavior associated with burn injury.

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.

Intrinsic Activity of C57BL/6 Substrains Associates with High-Fat Diet-Induced Mechanical Sensitivity in Mice.

Pain is significantly impacted by the increasing epidemic of obesity and the metabolic syndrome. Our understanding of how these features impact pain is only beginning to be developed. Herein, we have investigated how small genetic differences among C57BL/6 mice from 2 different commercial vendors lead to important differences in the development of high-fat diet-induced mechanical sensitivity. Two substrains of C57BL/6 mice from Jackson Laboratories (Bar Harbor, ME; C57BL/6J and C57BL/6NIH), as well as C57BL/6 from Charles Rivers Laboratories (Wilmington, MA; C57BL/6CR) were placed on high-fat diets and analyzed for changes in metabolic features influenced by high-fat diet and obesity, as well as measures of pain-related behaviors. All 3 substrains responded to the high-fat diet; however, C57BL/6CR mice had the highest weights, fat mass, and impaired glucose tolerance of the 3 substrains. In addition, the C57BL/6CR mice were the only strain to develop significant mechanical sensitivity over the course of 8 weeks. Importantly, the C57BL/6J mice were protected from mechanical sensitivity, which may be based on increased physical activity compared with the other 2 substrains. These findings suggest that activity may play a powerful role in protecting metabolic changes associated with a high-fat diet and that these may also be protective in pain-associated changes as a result of a high-fat diet. These findings also emphasize the importance of selection and transparency in choosing C57BL/6 substrains in pain-related research. PERSPECTIVE: Obesity and the metabolic syndrome play an important role in pain. This study identifies key differences in the response to a high-fat diet among substrains of C57BL/6 mice and differences in intrinsic physical activity that may influence pain sensitivity. The results emphasize physical activity as a powerful modulator of obesity-related pain sensitivity.

A ketogenic diet reduces metabolic syndrome-induced allodynia and promotes peripheral nerve growth in mice.

Current experiments investigated whether a ketogenic diet impacts neuropathy associated with obesity and prediabetes. Mice challenged with a ketogenic diet were compared to mice fed a high-fat diet or a high-fat diet plus exercise. Additionally, an intervention switching to a ketogenic diet following 8 weeks of high-fat diet was performed to compare how a control diet, exercise, or a ketogenic diet affects metabolic syndrome-induced neural complications. When challenged with a ketogenic diet, mice had reduced bodyweight and fat mass compared to high-fat-fed mice, and were similar to exercised, high-fat-fed mice. High-fat-fed, exercised and ketogenic-fed mice had mildly elevated blood glucose; conversely, ketogenic diet-fed mice were unique in having reduced serum insulin levels. Ketogenic diet-fed mice never developed mechanical allodynia contrary to mice fed a high-fat diet. Ketogenic diet fed mice also had increased epidermal axon density compared all other groups. When a ketogenic diet was used as an intervention, a ketogenic diet was unable to reverse high-fat fed-induced metabolic changes but was able to significantly reverse a high-fat diet-induced mechanical allodynia. As an intervention, a ketogenic diet also increased epidermal axon density. In vitro studies revealed increased neurite outgrowth in sensory neurons from mice fed a ketogenic diet and in neurons from normal diet-fed mice given ketone bodies in the culture medium. These results suggest a ketogenic diet can prevent certain complications of prediabetes and provides significant benefits to peripheral axons and sensory dysfunction.

Deletion of the insulin receptor in sensory neurons increases pancreatic insulin levels.

Insulin is known to have neurotrophic properties and loss of insulin support to sensory neurons may contribute to peripheral diabetic neuropathy (PDN). Here, genetically-modified mice were generated in which peripheral sensory neurons lacked the insulin receptor (SNIRKO mice) to determine whether disrupted sensory neuron insulin signaling plays a crucial role in the development of PDN and whether SNIRKO mice develop symptoms of PDN due to reduced insulin neurotrophic support. Our results revealed that SNIRKO mice were euglycemic and never displayed significant changes in a wide range of sensorimotor behaviors, nerve conduction velocity or intraepidermal nerve fiber density. However, SNIRKO mice displayed elevated serum insulin levels, glucose intolerance, and increased insulin content in the islets of Langerhans of the pancreas. These results contribute to the growing idea that sensory innervation of pancreatic islets is key to regulating islet function and that a negative feedback loop of sensory neuron insulin signaling keeps this regulation in balance. Our results suggest that a loss of insulin receptors in sensory neurons does not lead to peripheral nerve dysfunction. The SNIRKO mice will be a powerful tool to investigate sensory neuron insulin signaling and may give a unique insight into the role that sensory neurons play in modifying islet physiology.

Pro-NGF, sortilin, and p75NTR: potential mediators of injury-induced apoptosis in the mouse dorsal root ganglion.

The nerve growth factor precursor (pro-NGF) may function as a death-inducing ligand that mediates its apoptotic effects via p75NTR. Pro-NGF-induced apoptosis is postulated to be dependent upon membrane expression of the sortilin receptor, which interacts with p75NTR to promote a high-affinity binding site for pro-NGF. Here, we explore the expression of pro-NGF, sortilin and p75NTR in the mouse lumbar dorsal root ganglion (DRG) to understand the potential for this trimeric signaling complex to function in injury-induced neuronal death of DRG neurons. Our results reveal the expression of all 3 components within the DRG and that a subpopulation of neurons coexpresses sortilin and p75NTR. Following sciatic nerve transection, the expression of these proteins appears insensitive to injury; however, the majority of small p75NTR-sortilin coexpressing neurons are lost 25 days after sciatic nerve transection. These results propose pro-NGF-induced, p75NTR-sortilin-mediated neuronal death as a critical aspect of nerve injury-induced death in the DRG.

Diabetes-induced chemogenic hypoalgesia is paralleled by attenuated stimulus-induced fos expression in the spinal cord of diabetic mice.

UNLABELLED: Chronic hyperglycemia in diabetes induces abnormal nerve pathologies, resulting in diabetic neuropathy (DN). Sensory symptoms of DN can manifest as positive (painful), negative (insensate), or both. Streptozotocin (STZ)-induced diabetic C57Bl/6 mice have reduced cutaneous innervation and display reduced behavioral responses to noxious stimuli, reflecting the insensate aspect of the human syndrome. Current studies were undertaken to determine whether the diabetes-induced deficits in pain responses are reflected by changes in spinal activation in this model of DN. Nocifensive responses of nondiabetic and diabetic mice to formalin injection were measured 1, 3, 5, and 7 weeks after STZ, and at each time point formalin-induced spinal Fos expression was quantified. Responses of diabetic mice were significantly reduced during the second phase of the formalin test beginning 3 weeks after STZ and during Phase 1 beginning 5 weeks after STZ. Consistent with the behavioral responses, the number of Fos-positive cells in the dorsal horn of diabetic animals was significantly reduced beginning 3 weeks after STZ and continuing 5 and 7 weeks after STZ. The deficits at 5 weeks after STZ were restored by 2-week treatments with insulin or neurotrophins. These results demonstrate that the reduced sensation occurring from progressive peripheral axon loss results in functional deficits in spinal cord activation. PERSPECTIVE: The reduced expression of the immediate early gene Fos as an indicator of pain transmission supports the diabetes-induced loss of sensation in this Type 1 model of diabetes. This murine model may be better suited to understanding the insensate symptoms of diabetic patients in the absence of chronic pain.

Selective changes in nocifensive behavior despite normal cutaneous axon innervation in leptin receptor-null mutant (db/db) mice.

Much of our understanding of the effects of diabetes on the peripheral nervous system is derived from models induced by streptozotocin in which hyperglycemia is rapidly caused by pancreatic beta-cell destruction. Here, we have quantified sensory impairments over time in leptin receptor (lepr)-null mutant -/- mice, a type 2 model of diabetes in which the absence of leptin receptor signaling leads to obesity and chronic hyperglycemia by 4 weeks of age. To assess these mice as a model for peripheral neuropathy, we quantified the responsiveness of lepr -/- mice to mechanical, thermal, and chemogenic stimuli, as well as epidermal and dermal innervation of the hind paw. Compared with wild-type +/+ and heterozygous +/- mice, lepr -/- mice displayed reduced sensitivity to mechanical stimuli by 6 weeks of age, and however, responses to noxious heat were normal. Lepr -/- mice also devoted less activity to their injected paw during the second phase following formalin administration. However, epidermal and dermal innervation of lepr -/- mice was not different from that of lepr +/+ and +/- mice even after 10 weeks of hyperglycemia, suggesting that cutaneous innervation is resistant to chronic hyperglycemia in these mice. These results suggest that certain rodent nocifensive behaviors may be linked to the abundance of cutaneous innervation, while others are not. Finally, these results reveal that the lepr -/- mice may not be useful to study neuropathy associated with distal axonal degeneration but may be better suited for studies of hyperglycemia-induced sensory neuron dysfunction without distal nerve loss.

Increased FNDC5 is associated with insulin resistance in high fat-fed mice.

FNDC5/irisin, has recently been identified as a novel protein that stimulates the "browning" of white adipose by inducing thermogenesis via increased uncoupling protein 1 (UCP1). We tested the hypothesis that high fat diet-induced prediabetic mice would exhibit increased FNDC5 and this effect would be attenuated by chronic exercise. C57BL/6 mice were randomized into three groups for the 4 week intervention: Standard diet (Std, n = 12), High fat diet (HF, n = 14), or High fat diet and free access to a running wheel (HFEX, n = 14). Body weight, glucose, insulin, and the homeostatic model assessment of insulin resistance (HOMA-IR) were greater in HF compared to Std and HFEX after the 4 week intervention. In support of our hypothesis, FNDC5 was higher in HF in both skeletal muscle and adipose compared to Std and was lower in adipose only in HFEX compared to HF mice. Following the same pattern, PGC-1α was significantly higher in HF compared to Std in skeletal muscle and significantly lower in HFEX compared to HF in adipose. UCP1 was significantly lower in HFEX versus Std (in skeletal muscle) and versus HF (in adipose). HOMA-IR was significantly correlated with FNDC5 protein levels in adipose. Increased FNDC5 in adipose and skeletal muscle may be a compensatory mechanism to offset high fat diet-induced weight gain and insulin resistance by increasing energy expenditure.

Rats bred for low and high running capacity display alterations in peripheral tissues and nerves relevant to neuropathy and pain.

INTRODUCTION: Diet and activity are recognized as modulators of nervous system disease, including pain. Studies of exercise consistently reveal a benefit on pain. This study focused on female rats to understand differences related to metabolic status and peripheral nerve function in females. METHODS: Here, we investigated parameters of peripheral nerve function relevant to pain in rats selectively bred for high (high-capacity runners; HCR) or low endurance exercise capacity (low-capacity runners; LCR) resulting in divergent intrinsic aerobic capacities and susceptibility for metabolic conditions. RESULTS: LCR female rats have reduced mechanical sensitivity, higher intraepidermal nerve fiber density and TrkA-positive epidermal axons, increased numbers of Langerhans and mast cells in cutaneous tissues, and a higher fat content despite similar overall body weights compared to female HCR rats. Sensory and motor nerve conduction velocities, thermal sensitivity, and mRNA expression of selected genes relevant to peripheral sensation were not different. CONCLUSIONS: These results suggest that aerobic capacity and metabolic status influence sensory sensitivity and aspects of inflammation in peripheral tissues that could lead to poor responses to tissue damage and painful stimuli. The LCR and HCR rats should prove useful as models to assess how the metabolic status impacts pain.

Urinary bladder hypersensitivity and dysfunction in female mice following early life and adult stress.

Early adverse events have been shown to increase the incidence of interstitial cystitis/painful bladder syndrome in adulthood. Despite high clinical relevance and reports of stress-related symptom exacerbation, animal models investigating the contribution of early life stress to female urological pain are lacking. We examined the impact of neonatal maternal separation (NMS) on bladder sensitivity and visceral neuroimmune status both prior-to, and following, water avoidance stress (WAS) in adult female mice. The visceromotor response to urinary bladder distension was increased at baseline and 8d post-WAS in NMS mice, while colorectal sensitivity was transiently increased 1d post-WAS only in naïve mice. Bladder micturition rate and output, but not fecal output, were also significantly increased following WAS in NMS mice. Changes in gene expression involved in regulating the stress response system were observed at baseline and following WAS in NMS mice, and WAS reduced serum corticosterone levels. Cytokine and growth factor mRNA levels in the bladder, and to a lesser extent in the colon, were significantly impacted by NMS and WAS. Peripheral mRNA levels of stress-responsive receptors were differentially influenced by early life and adult stress in bladder, but not colon, of naïve and NMS mice. Histological evidence of mast cell degranulation was increased in NMS bladder, while protein levels of protease activated receptor 2 (PAR2) and transient receptor potential ankyrin 1 (TRPA1) were increased by WAS. Together, this study provides new insight into mechanisms contributing to stress associated symptom onset or exacerbation in patients exposed to early life stress.

Neurotrophin-3 reverses chronic mechanical hyperalgesia induced by intramuscular acid injection.

Injection of acid into the gastrocnemius muscle results in a persistent, mechanical hyperalgesia of the hindpaw (Sluka et al., 2001). Here, the ability of neurotrophins to alter the development of this secondary hyperalgesia was assessed using transgenic mice and exogenous neurotrophin administration. Acid-induced hyperalgesia was measured in wild-type and transgenic mice that overexpress neurotrophin-3 (NT-3) in muscle (myo/NT-3 mice). Mechanical and thermal sensitivity of the hindpaws were assessed after injections of acidic saline, pH 4, into the right medial gastrocnemius. Wild-type mice exhibited mechanical but not thermal hyperalgesia in both paws 1 d after acid injection. In contrast, myo/NT-3 mice developed a transient mechanical hyperalgesia in both paws that disappeared by 2-3 d. The reversal of hyperalgesia in myo/NT-3 mice could be mimicked by intramuscular administration of exogenous NT-3 to acid-injected mice but not by other neurotrophins. The route of NT-3 administration appears critical, because intrathecal or intraperitoneal delivery were ineffective. The hyperalgesia could only be reversed by NT-3 treatment concurrent with acid injection and not after the emergence of hyperalgesia. The acid-induced hyperalgesia did not redevelop after the termination of NT-3 treatment, suggesting that NT-3 permanently reversed the hyperalgesia. Consistent with the behavioral data, paw palpation of acid-injected mice significantly increased Fos expression in the spinal cord of wild-type but not myo/NT-3 or NT-3-injected mice. The attenuation of hyperalgesia suggests that NT-3 may be a modulator of muscle-derived pain, and NT-3 may suppress events that lead to secondary hyperalgesia triggered by insult to muscle afferents.

Neonatal vaginal irritation results in long-term visceral and somatic hypersensitivity and increased hypothalamic-pituitary-adrenal axis output in female mice.

Experiencing early life stress or injury increases a woman's likelihood of developing vulvodynia and concomitant dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. To investigate the outcome of neonatal vaginal irritation (NVI), female mouse pups were administered intravaginal zymosan on postnatal days 8 and 10 and were assessed as adults for vaginal hypersensitivity by measuring the visceromotor response to vaginal balloon distension (VBD). Western blotting and calcium imaging were performed to measure transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) in the vagina and innervating primary sensory neurons. Serum corticosterone (CORT), mast cell degranulation, and corticotropin-releasing factor receptor 1 (CRF1) expression were measured as indicators of peripheral HPA axis activation. Colorectal and hind paw sensitivity were measured to determine cross-sensitization resulting from NVI. Adult NVI mice had significantly larger visceromotor response during VBD than naive mice. TRPA1 protein expression was significantly elevated in the vagina, and calcium transients evoked by mustard oil (TRPA1 ligand) or capsaicin (TRPV1 ligand) were significantly decreased in dorsal root ganglion from NVI mice, despite displaying increased depolarization-evoked calcium transients. Serum CORT, vaginal mast cell degranulation, and CRF1 protein expression were all significantly increased in NVI mice, as were colorectal and hind paw mechanical and thermal sensitivity. Neonatal treatment with a CRF1 antagonist, NBI 35965, immediately before zymosan administration largely attenuated many of the effects of NVI. These results suggest that NVI produces chronic hypersensitivity of the vagina, as well as of adjacent visceral and distant somatic structures, driven in part by increased HPA axis activation.

Beneficial actions of neurotrophin treatment on diabetes-induced hypoalgesia in mice.

Studies were carried out in streptozotocin-treated diabetic mice to evaluate their behavioral responses to different noxious stimuli. In opposition to rats, streptozotocin-injected diabetic mice display a persistent hypoalgesia to non-noxious mechanical stimulation (von Frey monofilament). Similarly, nocifensive responses of diabetic mice to formalin injection were significantly reduced in both acute and inflammatory phases. However, no overt differences were detected between nondiabetic and diabetic mice in their sensitivity to noxious heat (radiant heat), cold (acetone), or noxious mechanical (pinprick) stimuli applied to the hind paw. To evaluate whether neurotrophin treatment could normalize the sensory deficits, nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) was administered intrathecally to diabetic mice for 3 weeks. Neurotrophin-treated mice were also compared to mice that received insulin for 3 weeks. Both NGF and insulin treatment significantly restored mechanical and chemogenic behavioral responses of diabetic mice. In contrast, GDNF treatment only reversed behavioral responses to chemogenic stimuli during the acute phase of the formalin test. These results demonstrate that diabetic mice develop reduced sensitivity to mechanical and chemical stimuli. Furthermore, these studies show that dorsal root ganglion neurons in diabetic mice are responsive to treatment with either NGF or GDNF; however, these 2 neurotrophins differ in their ability to affect distinct somatosensations.