Loss Of Intraepidermal Nerve Fibers Research Articles

Prevention of chemotherapy-induced peripheral neuropathy by the small-molecule inhibitor pifithrin-μ.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of cancer treatment. It is the most frequent cause of dose reduction or treatment discontinuation in patients treated for cancer with commonly used drugs including taxanes and platinum-based compounds. No FDA-approved treatments for CIPN are available. In rodents, CIPN is represented by peripheral mechanical allodynia in association with retraction of intraepidermal nerve fibers. The mechanism of chemotherapy-induced neurotoxicity is unclear, but it has been established that mitochondrial dysfunction is an important component of the dysregulation in peripheral sensory neurons. We have shown earlier that inhibition of mitochondrial p53 accumulation with the small compound pifithrin-μ (PFT-μ) prevents cerebral neuronal death in a rodent model of hypoxic-ischemic brain damage. We now explore whether PFT-μ is capable of preventing neuronal mitochondrial damage and CIPN in mice. We demonstrate for the first time that PFT-μ prevents both paclitaxel- and cisplatin-induced mechanical allodynia. Electron microscopic analysis of peripheral sensory nerves revealed that PFT-μ secured mitochondrial integrity in paclitaxel-treated mice. In addition, PFT-μ administration protects against chemotherapy-induced loss of intraepidermal nerve fibers in the paw. To determine whether neuroprotective treatment with PFT-μ would interfere with the antitumor effects of chemotherapy, ovarian tumor cells were cultured in vitro with PFT-μ and paclitaxel. Pifithrin-μ does not inhibit tumor cell death but even enhances paclitaxel-induced tumor cell death. These data are the first to identify PFT-μ as a potential therapeutic strategy for prevention of CIPN to combat one of the most devastating side effects of chemotherapy.

Meeting Report: Inaugural Chemotherapy-Induced Peripheral Neuropathy Symposium - Santa Barbara, CA, February 2015.

Chemotherapy-induced peripheral neuropathy is a common, dose-limiting side effect of cancer treatment. This conference was the first of its kind to bring together a wide range of clinicians, researchers, and industry professionals to address the potential causes, preventions, and treatments for this drug toxicity. Intraepidermal nerve fiber loss, axonal degeneration, immune cell infiltration, alterations in tubulin protein expression and microtubule stability, axonal transport, and mitochondrial dysfunction were addressed as possible mechanisms. Problems with animal models of the disease were discussed, as well as the potential of patient-derived induced sensory neurons to serve as a novel in vitro model.

Prevention of chemotherapy-induced peripheral neuropathy by the small-molecule inhibitor Pifithrin-mu in a mouse model

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of cancer treatment and a frequent cause of treatment discontinuation. No FDA-approved treatments for CIPN are available. In rodents CIPN is represented by peripheral mechanical hyperalgesia in association with retraction of intra-epidermal nerve fibers (IENFs). The mechanism of chemotherapy-induced neurotoxicity is unclear but it has been established that mitochondrial dysfunction is an important component of the dysregulation. We have shown earlier that inhibition of mitochondrial p53 accumulation with the small compound pifithrin-mu (PFT-mu) prevents cerebral neuronal death in a rodent model of hypoxic-ischemic brain damage. We now explored whether PFT- mu is capable of preventing neuronal mitochondrial damage and CIPN in mice. We demonstrate for the first time that PFT-mu prevents both paclitaxel- and cisplatin-induced mechanical hyperalgesia. Electron microscopic analysis of peripheral sensory nerves revealed that PFT-mu secured mitochondrial integrity in paclitaxel-treated mice. In addition, PFT-mu administration protected against chemotherapy-induced loss of IENFs in the paw. To determine whether neuroprotective treatment with PFT-mu would interfere with the anti-tumor effects of chemotherapy, ovarian tumor cells were cultured in vitro with PFT-mu and paclitaxel. The results showed that PFT-mu does not inhibit tumor cell death but even enhances paclitaxel-induced tumor cell death. These data are the first to identify PFT-mu as a potential therapeutic strategy for prevention of CIPN to combat the devastating side effects of chemotherapy.

Intraepidermal nerve-fibre density as a biomarker of the course of neuropathy in patients with Type2 diabetes mellitus.

This paper aims to investigate whether intraepidermal nerve-fibre density (IENFD) may be used as a marker of the course of neuropathy in patients with Type 2 diabetes mellitus. Skin biopsies from the distal leg were serially evaluated in a group of 30 patients with Type 2 diabetes mellitus (median age 60 years, 17 men) with a short duration of diabetes (< 3 years) and good glucose control, and in 23 age- and sex-matched controls. The time intervals between biopsies were > 2 years (median 33.8 months). Eighteen patients with Type 2 diabetes mellitus had symptoms or signs of distal symmetrical diabetic polyneuropathy, 12 had no neuropathy. At first skin biopsy, IENFD was normal in all controls and in patients without neuropathy (mean 9.5 and 7.9 fibres/mm, respectively) compared with abnormal IENFD in 77.8% in patients with polyneuropathy (mean 3.4 fibres/mm). The annual rate of intraepidermal nerve-fibre (IENF) loss expressed as a percentage of the first IENFD value in patients with diabetic polyneuropathy was significantly higher [mean (se), 11.95 (3.82)%] compared with controls [1.92 (1.81)%, P < 0.001] and similar to patients without polyneuropathy [12.16 (4.38)%]. The rate of IENF loss did not correlate with degree of glucose control. The annual rate of IENF loss in patients with Type 2 diabetes mellitus was several times higher than that of healthy participants, irrespective of the presence of signs or symptoms of diabetic polyneuropathy at initial evaluation. The change in IENFD is not linear and should be expressed as a proportion of initial IENFD to serve as a marker of the course of diabetic neuropathy.

Mitochondrial transcription factor A regulation of mitochondrial degeneration in experimental diabetic neuropathy.

Oxidative stress-induced mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in peripheral neurons is considered to be important in the development of diabetic neuropathy. Mitochondrial transcription factor A (TFAM) wraps mtDNA and promotes mtDNA replication and transcription. We studied whether overexpression of TFAM reverses experimental peripheral diabetic neuropathy using TFAM transgenic mice (TFAM Tg) that express human TFAM (hTFAM). Levels of mouse mtDNA and the total TFAM (mouse TFAM + hTFAM) in the dorsal root ganglion (DRG) increased by approximately twofold in the TFAM Tg mice compared with control (WT) mice. WT and TFAM Tg mice were made diabetic by the administration of streptozotocin. Neuropathy end points were motor and sensory nerve conduction velocities, mechanical allodynia, thermal nociception, and intraepidermal nerve fiber density (IENFD). In the DRG neurons, mtDNA copy number and damage to mtDNA were quantified by qPCR, and TFAM levels were measured by Western blot. Mice with 16-wk duration of diabetes developed motor and sensory nerve conduction deficits, behavioral deficits, and intraepidermal nerve fiber loss. All of these changes were mostly prevented in diabetic TFAM Tg mice and were independent of changes in blood parameters. Mice with 16 wk of diabetes had a 40% decrease in mtDNA copy number compared with nondiabetic mice (P < 0.01). Importantly, the mtDNA copy number in diabetic TFAM Tg mice reached the same level as that of WT nondiabetic mice. In comparison, there was upregulation of mtDNA and TFAM in 6-wk diabetic mice, suggesting that TFAM activation could be a therapeutic strategy to treat peripheral neuropathy.

Ca2+ toxicity due to reverse Na+/Ca2+ exchange contributes to degeneration of neurites of DRG neurons induced by a neuropathy-associated Nav1.7 mutation.

Gain-of-function missense mutations in voltage-gated sodium channel Nav1.7 have been linked to small-fiber neuropathy, which is characterized by burning pain, dysautonomia and a loss of intraepidermal nerve fibers. However, the mechanistic cascades linking Nav1.7 mutations to axonal degeneration are incompletely understood. The G856D mutation in Nav1.7 produces robust changes in channel biophysical properties, including hyperpolarized activation, depolarized inactivation, and enhanced ramp and persistent currents, which contribute to the hyperexcitability exhibited by neurons containing Nav1.8. We report here that cell bodies and neurites of dorsal root ganglion (DRG) neurons transfected with G856D display increased levels of intracellular Na(+) concentration ([Na(+)]) and intracellular [Ca(2+)] following stimulation with high [K(+)] compared with wild-type (WT) Nav1.7-expressing neurons. Blockade of reverse mode of the sodium/calcium exchanger (NCX) or of sodium channels attenuates [Ca(2+)] transients evoked by high [K(+)] in G856D-expressing DRG cell bodies and neurites. We also show that treatment of WT or G856D-expressing neurites with high [K(+)] or 2-deoxyglucose (2-DG) does not elicit degeneration of these neurites, but that high [K(+)] and 2-DG in combination evokes degeneration of G856D neurites but not WT neurites. Our results also demonstrate that 0 Ca(2+) or blockade of reverse mode of NCX protects G856D-expressing neurites from degeneration when exposed to high [K(+)] and 2-DG. These results point to [Na(+)] overload in DRG neurons expressing mutant G856D Nav1.7, which triggers reverse mode of NCX and contributes to Ca(2+) toxicity, and suggest subtype-specific blockade of Nav1.7 or inhibition of reverse NCX as strategies that might slow or prevent axon degeneration in small-fiber neuropathy.

BTBR ob/ob mice as a novel diabetic neuropathy model: Neurological characterization and gene expression analyses

Given the lack of treatments for diabetic neuropathy (DN), a common diabetic complication, accurate disease models are necessary. Characterization of the leptin-deficient BTBR ob/ob mouse, a type 2 diabetes model, demonstrated that the mice develop robust diabetes coincident with severe neuropathic features, including nerve conduction deficits and intraepidermal nerve fiber loss by 9 and 13weeks of age, respectively, supporting its use as a DN model. To gain insight into DN mechanisms, we performed microarray analysis on sciatic nerve from BTBR ob/ob mice, identifying 1503 and 642 differentially expressed genes associated with diabetes at 5 and 13weeks, respectively. Further analyses identified overrepresentation of inflammation and immune-related functions in BTBR ob/ob mice, which interestingly were more highly represented at 5weeks, an observation that may suggest a contributory role in DN onset. To complement the gene expression analysis, we demonstrated that protein levels of select cytokines were significantly upregulated at 13weeks in BTBR ob/ob mouse sciatic nerve. Furthermore, we compared our array data to that from an established DN model, the C57BKS db/db mouse, which reflected a common dysregulation of inflammatory and immune-related pathways. Together, our data demonstrate that BTBR ob/ob mice develop rapid and robust DN associated with dysregulated inflammation and immune-related processes.

Reduced epidermal thickness, nerve degeneration and increased pain-related behavior in rats with diabetes type 1 and 2

To examine the mechanisms contributing to pain genesis in diabetic neuropathy, we investigated epidermal thickness and number of intraepidermal nerve fibers in rat foot pad of the animal model of diabetes type 1 and type 2 in relation to pain-related behavior. Male Sprague-Dawley rats were used. Diabetes type 1 was induced with intraperitoneal injection of streptozotocin (STZ) and diabetes type 2 was induced with a combination of STZ and high-fat diet. Control group for diabetes type 1 was fed with regular laboratory chow, while control group for diabetes type 2 received high-fat diet. Body weights and blood glucose levels were monitored to confirm induction of diabetes. Pain-related behavior was analyzed using thermal (hot, cold) and mechanical stimuli (von Frey fibers, number of hyperalgesic responses). Two months after induction of diabetes, glabrous skin samples from plantar surface of the both hind paws were collected. Epidermal thickness was evaluated with hematoxylin and eosin staining. Intraepidermal nerve fibers quantification was performed after staining skin with polyclonal antiserum against protein gene product 9.5. We found that induction of diabetes type 1 and type 2 causes significant epidermal thinning and loss of intraepidermal nerve fibers in a rat model, and both changes were more pronounced in diabetes type 1 model. Significant increase of pain-related behavior two months after induction of diabetes was observed only in a model of diabetes type 1. In conclusion, animal models of diabetes type 1 and diabetes type 2 could be used in pharmacological studies, where cutaneous changes could be used as outcome measures for predegenerative markers of neuropathies.

Small Nerve Fiber Pathology in Critical Illness

BackgroundDegeneration of intraepidermal nerve fibers (IENF) is a hallmark of small fiber neuropathy of different etiology, whose clinical picture is dominated by neuropathic pain. It is unknown if critical illness can affect IENF.MethodsWe enrolled 14 adult neurocritical care patients with prolonged intensive care unit (ICU) stay and artificial ventilation (≥ 3 days), and no previous history or risk factors for neuromuscular disease. All patients underwent neurological examination including evaluation of consciousness, sensory functions, muscle strength, nerve conduction study and needle electromyography, autonomic dysfunction using the finger wrinkling test, and skin biopsy for quantification of IENF and sweat gland innervation density during ICU stay and at follow-up visit. Development of infection, sepsis and multiple organ failure was recorded throughout the ICU stay.ResultsOf the 14 patients recruited, 13 (93%) had infections, sepsis or multiple organ failure. All had severe and non-length dependent loss of IENF. Sweat gland innervation was reduced in all except one patient. Of the 7 patients available for follow-up visit, three complained of diffuse sensory loss and burning pain, and another three showed clinical dysautonomia.ConclusionsSmall fiber pathology can develop in the acute phase of critical illness and may explain chronic sensory impairment and pain in neurocritical care survivors. Its impact on long term disability warrants further studies involving also non-neurologic critical care patients.

Na+/H+ exchanger 1 inhibition reverses manifestation of peripheral diabetic neuropathy in type 1 diabetic rats

Evidence for an important role for Na(+)/H(+) exchangers in diabetic complications is emerging. The aim of this study was to evaluate whether Na(+)/H(+) exchanger 1 inhibition reverses experimental peripheral diabetic neuropathy. Control and streptozotocin-diabetic rats were treated with the specific Na(+)/H(+) exchanger 1 inhibitor cariporide for 4 wk after 12 wk without treatment. Neuropathy end points included sciatic motor and sensory nerve conduction velocities, endoneurial nutritive blood flow, vascular reactivity of epineurial arterioles, thermal nociception, tactile allodynia, and intraepidermal nerve fiber density. Advanced glycation end product and markers of oxidative stress, including nitrated protein levels in sciatic nerve, were evaluated by Western blot. Rats with 12-wk duration of diabetes developed motor and sensory nerve conduction deficits, thermal hypoalgesia, tactile allodynia, and intraepidermal nerve fiber loss. All these changes, including impairment of nerve blood flow and vascular reactivity of epineurial arterioles, were partially reversed by 4 wk of cariporide treatment. Na(+)/H(+) exchanger 1 inhibition was also associated with reduction of diabetes-induced accumulation of advanced glycation endproduct, oxidative stress, and nitrated proteins in sciatic nerve. In conclusion, these findings support an important role for Na(+)/H(+) exchanger 1 in functional, structural, and biochemical manifestations of peripheral diabetic neuropathy and provide the rationale for development of Na(+)/H(+) exchanger 1 inhibitors for treatment of diabetic vascular and neural complications.

Induction of Monocyte Chemoattractant Protein-1 (MCP-1) and Its Receptor CCR2 in Primary Sensory Neurons Contributes to Paclitaxel-Induced Peripheral Neuropathy

The use of paclitaxel (Taxol), a microtubule stabilizer, for cancer treatment is often limited by its associated peripheral neuropathy (chemotherapy-induced peripheral neuropathy [CIPN]), which predominantly results in sensory dysfunction, including chronic pain. Here we show that paclitaxel CIPN was associated with induction of chemokine monocyte chemoattractant protein-1 (MCP-1) and its cognate receptor CCR2 in primary sensory neurons of dorsal root ganglia. Immunostaining revealed that MCP-1 was mainly expressed in small nociceptive neurons whereas CCR2 was expressed in large and medium-sized myelinated neurons. Direct application of MCP-1 consistently induced intracellular calcium increases in dorsal root ganglia large and medium-sized neurons but not in small neurons mainly dissociated from paclitaxel-treated but not vehicle-treated animals. Paclitaxel also induced increased expression of MCP-1 in spinal astrocytes, but no CCR2 signal was detected in the spinal cord. Local blockade of MCP-1/CCR2 signaling by anti-MCP-1 antibody or CCR2 antisense oligodeoxynucleotides significantly attenuated paclitaxel CIPN phenotypes including mechanical hypersensitivity and loss of intraepidermal nerve fibers in hindpaw glabrous skin. These results suggest that activation of paracrine MCP-1/CCR2 signaling between dorsal root ganglion neurons plays a critical role in the development of paclitaxel CIPN, and targeting MCP-1/CCR2 signaling could be a novel therapeutic approach. PerspectiveCIPN is a severe side effect accompanying paclitaxel chemotherapy and lacks effective treatments. The current study suggests that blocking MCP-1/CCR2 signaling could be a new therapeutic strategy to prevent or reverse paclitaxel CIPN. This preclinical evidence encourages future clinical evaluation of this strategy.

Transient Receptor Potential Channels in Chemotherapy-Induced Neuropathy

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a common dose-limiting side effect of many chemotherapeuticdrugs, including platinum-based compounds (e.g., cisplatin and oxaliplatin), taxanes (e.g., paclitaxel), vinca alkaloids (e.g., vincristine), and the first-in-class proteasome inhibitor, bortezomib. Among the various sensory symptoms of CIPN, paresthesia, dysesthesia, spontaneous pain, and mechanical and thermal hypersensitivity are prominent. Inflammation, oxidative stress, loss of intraepidermal nerve fibers, modifications of mitochondria, and various ion channels alterations are part of the several mechanisms contributing to CIPN. Because attempts to mitigate chemotherapeutic- induced acute neuronal hyperexcitability and the subsequent peripheral neuropathy have yielded unsatisfactory results, a more in-depth understanding of the mechanism(s) responsible for the neurotoxic action of anticancer drugs is required. Some members of the transient receptor potential (TRP) family of channels, as the TRPV1 and TRPV4 (vanilloid), TRPA1 (ankyrin) and TRPM8 (melastatin) are expressed on the plasma membrane of primary sensory neurons (nociceptors), where they are activated by an unprecedented series of physical and chemical stimuli. There is evidence that TRPV1, TRPV4, TRPA1 and TRPM8 are prominent contributors of mechanical and thermal hypersensitivity in models of CIPN. In particular, in vitro and in vivo studies have pointed out the unique role of TRPA1 and oxidative stress in the mechanism responsible for cold and mechanical hyperalgesia in rodent models of CIPN.

Endoplasmic Reticulum Stress Plays a Key Role in the Pathogenesis of Diabetic Peripheral Neuropathy

Endoplasmic reticulum stress resulting from abnormal folding of newly synthesized proteins impairs metabolism, transcriptional regulation, and gene expression, and it is a key mechanism of cell injury. Endoplasmic reticulum stress plays an important role in cardiovascular and neurodegenerative diseases, cancer, and diabetes. We evaluated the role for this phenomenon in diabetic peripheral neuropathy. Endoplasmic reticulum stress manifest in upregulation of multiple components of unfolded protein response was identified in neural tissues (sciatic nerve, spinal cord) of streptozotocin diabetic rats and mice. A chemical chaperone, trimethylamine oxide, administered for 12 weeks after induction of diabetes (110 mg⋅kg−1⋅d−1, a prevention paradigm) attenuated endoplasmic reticulum stress, peripheral nerve dysfunction, intraepidermal nerve fiber loss, and sciatic nerve and spinal cord oxidative-nitrative stress in streptozotocin diabetic rats. Similar effects on diabetes-induced endoplasmic reticulum stress and peripheral nerve dysfunction were observed with a structurally unrelated chemical chaperone, 4-phenylbutyric acid (100 mg⋅kg−1⋅d−1, intraperitoneal). CCAAT/enhancer-binding protein homologous protein (CHOP)−/− mice made diabetic with streptozotocin displayed less severe sciatic nerve oxidative-nitrative stress and peripheral neuropathy than the wild-type (C57Bl6/J) mice. Neither chemical chaperones nor CHOP gene deficiency reduced diabetic hyperglycemia. Our findings reveal an important role of endoplasmic reticulum stress in the development of diabetic peripheral neuropathy and identify a potential new therapeutic target.

The Protective Effects of DA-9801 (Dioscorea Extract) on the Peripheral Nerves in Streptozotocin-Induced Diabetic Rats

It has been reported that DA-9801, an extract mixture of Dioscorea japonica Thunb and Dioscorea nipponica Makino, produces a neurotrophic activity. Therefore, this study was conducted to examine the neuroprotective effects of DA-9801 in streptozotocin-induced diabetic rats. The experimental rats were divided into six groups: the control group, Group I (non-diabetic rats treated with DA-9801), Group II (diabetic, non-treated rats) and Groups III, IV, and V (diabetic rats treated with DA-9801 at doses of 10, 50 or 100 mg/kg/d). Following a 16-wk course of oral treatment with DA-9801, functional parameters (von Frey filament test, hot plate test), biochemical parameters (nerve growth factor (NGF), tumor necrosis factor (TNF)-α, interleukin (IL)-6) were measured. An immunohistochemical staining was done to assess the neuroprotective effects of DA-9081 in the skin, sciatic nerve, gastric mucosa and renal cortex. In Week 8, pain was evoked by either tactile or thermal stimuli, whose threshold was significantly higher in Group III, IV and V than Group II. Western blot analysis showed a more significant increase in NGF and decrease in TNF-α and IL-6 in Group III, IV and V than in Group II (p<0.05). Moreover, following the treatment with DA-9801, a loss of intraepidermal nerve fibers (IENFs) was inhibited to a significant level in the skin, myelinated axonal fibers of the sciatic nerve and small nerve fibers innervating the gastric mucosa or renal cortex (p<0.05). Our results demonstrated that DA-9801 is a beneficial agent that protects the peripheral nerves in diabetic rats.

12/15-Lipoxygenase inhibition counteracts MAPK phosphorylation in mouse and cell culture models of diabetic peripheral neuropathy

Increased mitogen-activated protein kinase (MAPK) phosphorylation has been detected in peripheral nerve of human subjects and animal models with diabetes as well as high-glucose exposed human Schwann cells, and have been implicated in diabetic peripheral neuropathy. In our recent studies, leukocytetype 12/15-lipoxygenase inhibition or gene deficiency alleviated large and small nerve fiber dysfunction, but not intraepidermal nerve fiber loss in streptozotocin-diabetic mice. To address a mechanism we evaluated the potential for pharmacological 12/15-lipoxygenase inhibition to counteract excessive MAPK phosphorylation in mouse and cell culture models of diabetic neuropathy. C57Bl6/J mice were made diabetic with streptozotocin and maintained with or without the 12/15-lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC). Human Schwann cells were cultured in 5.5 mM or 30 mM glucose with or without CDC. 12(S) HETE concentrations (ELISA), as well as 12/15-lipoxygenase expression and p38 MAPK, ERK, and SAPK/JNK phosphorylation (all by Western blot analysis) were increased in the peripheral nerve and spinal cord of diabetic mice as well as in high glucose-exposed human Schwann cells. CDC counteracted diabetes-induced increase in 12(S)HETE concentrations (a measure of 12/15-lipoxygenase activity), but not 12/15-lipoxygenase overexpression, in sciatic nerve and spinal cord. The inhibitor blunted excessive p38 MAPK and ERK, but not SAPK/ JNK, phosphorylation in sciatic nerve and high glucose exposed human Schwann cells, but did not affect MAPK, ERK, and SAPK/JNK phosphorylation in spinal cord. 12/15-lipoxygenase inhibition counteracts diabetes related MAPK phosphorylation in mouse and cell culture models of diabetic neuropathy and implies that 12/15-lipoxygenase inhibitors may be an effective treatment for diabetic peripheral neuropathy.

Neuropathy‐associated NaV1.7 variant I228M impairs integrity of dorsal root ganglion neuron axons

Small-fiber neuropathy (SFN) is characterized by injury to small-diameter peripheral nerve axons and intraepidermal nerve fibers (IENF). Although mechanisms underlying loss of IENF in SFN are poorly understood, available data suggest that it results from axonal degeneration and reduced regenerative capacity. Gain-of-function variants in sodium channel Na(V)1.7 that increase firing frequency and spontaneous firing of dorsal root ganglion (DRG) neurons have recently been identified in ∼30% of patients with idiopathic SFN. In the present study, to determine whether these channel variants can impair axonal integrity, we developed an in vitro assay of DRG neurite length, and examined the effect of 3 SFN-associated variant Na(V)1.7 channels, I228M, M932L/V991L (ML/VL), and I720K, on DRG neurites in vitro. At 3 days after culturing, DRG neurons transfected with I228M channels exhibited ∼20% reduced neurite length compared to wild-type channels; DRG neurons transfected with ML/VL and I720K variants displayed a trend toward reduced neurite length. I228M-induced reduction in neurite length was ameliorated by the use-dependent sodium channel blocker carbamazepine and by a blocker of reverse Na-Ca exchange. These in vitro observations provide evidence supporting a contribution of the I228M variant Na(V)1.7 channel to impaired regeneration and/or degeneration of sensory axons in idiopathic SFN, and suggest that enhanced sodium channel activity and reverse Na-Ca exchange can contribute to a decrease in length of peripheral sensory axons.

Skin biopsy in painful and immune‐mediated neuropathies

Starting from the original studies of the 19th century, this review covers some of the advances achieved over the last 15 years since skin biopsy has become a diagnostic tool for neurologists. In a relatively short period of time, focused works demonstrated the correlation between the loss of intraepidermal nerve fibers (IENF) and symptoms and signs of small fiber neuropathy (SFN), and provided standardized protocols for nerve morphometry as well as normative reference values to be used in clinical practice. This contributed to the definition of the diagnostic criteria for SFN that is now recognized as a distinct nosologic entity. The relationship between IENF degeneration and neuropathic pain led to the recent discovery that SFN can be caused by mutations in sodium channels, providing evidence for a new diagnostic approach to the etiology of the disease in patients. The presence of myelinated nerve fibers in the dermis prompted studies focused on demyelinating neuropathies of genetic and immune-mediated origin. Specific changes in dermal myelinated nerves have been described suggesting a potential role for skin biopsy also in these fields. Finally, studies on the sequence of events occurring after nerve degeneration in experimental models and patients with chronic neuropathies allowed to understand better the ability of skin nerves to regenerate and the reasons for its failure, providing important hints for the use of skin biopsy as an outcome measure in clinical practice and neuroprotective trials.

Murine Immunodeficiency Virus-Induced Peripheral Neuropathy and the Associated Cytokine Responses

Distal symmetrical polyneuropathy is the most common form of HIV infection-associated peripheral neuropathy and is often associated with pain. C57BL/6 (B6) mice infected with LP-BM5, a murine retroviral isolate, develop a severe immunodeficiency syndrome similar to that in humans infected with HIV-1, hence the term murine AIDS. We investigated the induction of peripheral neuropathy after LP-BM5 infection in B6 mice. Infected B6 mice, like HIV-infected humans, exhibited behavioral (increased sensitivity to mechanical and heat stimuli) and pathological (transient loss of intraepidermal nerve fibers) signs of peripheral neuropathy. The levels of viral gag RNA were significantly increased in all tissues tested, including spleen, paw skin, lumbar dorsal root ganglia, and lumbar spinal cord, postinfection (p.i.). Correlated with the development of peripheral neuropathy, the tissue levels of several cytokines, including IFN-γ, IL-1β, IL-6, and IL-12, were significantly elevated p.i. These increases had cytokine-specific and tissue-specific profiles and kinetics. Further, treatment with the antiretroviral agent zidovudine either significantly reduced or completely reversed the aforementioned behavioral, pathologic, and cytokine changes p.i. These data suggest that LP-BM5 infection is a potential mouse model of HIV-associated distal symmetrical polyneuropathy that can be used for investigating the roles of various cytokines in infection-induced neuropathic pain. Further investigation of this model could give a better understanding of, and lead to more effective treatments for, HIV infection-associated painful peripheral neuropathy.

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

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

Characterization of oxaliplatin-induced chronic painful peripheral neuropathy in the rat and comparison with the neuropathy induced by paclitaxel

Anti-neoplastic agents in the platinum-complex, taxane, vinca alkaloid, and proteasome-inhibitor classes induce a dose-limiting, chronic, distal, symmetrical, sensory peripheral neuropathy that is often accompanied by neuropathic pain. Clinical descriptions suggest that these conditions are very similar, but clinical data are insufficient to determine the degree of similarity and to determine if they share common pathophysiological mechanisms. Animal models do not have the limitations of clinical studies and so we have characterized a rat model of chronic painful peripheral neuropathy induced by a platinum-complex agent, oxaliplatin, in order to compare it with a previously characterized model of chronic painful peripheral neuropathy induced by a taxane agent, paclitaxel. The oxaliplatin model evokes mechano-allodynia, mechano-hyperalgesia, and cold-allodynia that have a delayed onset, gradually increasing severity, a distinct delay to peak severity, and duration of about 2.5 months. There is no effect on heat sensitivity. Electron microscopy (EM) analyses found no evidence for axonal degeneration in peripheral nerve, and there is no upregulation of activating transcription factor-3 in the lumbar dorsal root ganglia. There is a statistically significant loss of intraepidermal nerve fibers in the plantar hind paw skin. Oxaliplatin treatment causes a significant increase in the incidence of swollen and vacuolated mitochondria in peripheral nerve axons, but not in their Schwann cells. Nerve conduction studies found significant slowing of sensory axons, but no change in motor axons. Single fiber recordings found an abnormal incidence of A- and C-fibers with irregular, low-frequency spontaneous discharge. Prophylactic dosing with two drugs that are known to protect mitochondria, acetyl-l-carnitine and olesoxime, significantly reduced the development of pain hypersensitivity. Our results are very similar to those obtained previously with paclitaxel, and support the hypothesis that these two agents, and perhaps other chemotherapeutics, produce very similar conditions because they have a mitotoxic effect on primary afferent neurons.