Elsevier

Neurobiology of Disease

Volume 45, Issue 1, January 2012, Pages 280-287
Neurobiology of Disease

Nerve growth factor/p38 signaling increases intraepidermal nerve fiber densities in painful neuropathy of type 2 diabetes

https://doi.org/10.1016/j.nbd.2011.08.011Get rights and content

Abstract

Painful diabetic neuropathy (PDN) is a common, yet devastating complication of type 2 diabetes. At this time, there is no objective test for diagnosing PDN. In the current study, we measured the peptidergic intraepidermal nerve fiber densities (IENFD) from hind paws of the db/db mouse, an animal model for type 2 diabetes, during the period of mechanical allodynia from 6 to 12 weeks of age. Intraepidermal nerve fibers (IENF) of the hind footpads were identified by protein gene product (PGP) 9.5 immunohistochemistry. The peptidergic IENF were determined by double immunofluorescence using anti-PGP9.5 and antibodies against tropomyosin-receptor-kinase (Trk) A. We observed a significant increase in PGP9.5-positive IENFD at 8 and 10 weeks of age. Similarly, Trk A-positive peptidergic IENF, which also express substance P and calcitonin gene related peptide in db/db mice, were observed to be elevated from 1.5 to 2 fold over controls. This upregulation ended at 16 weeks of age, in accordance with the reduction of mechanical allodynia. Anti-NGF treatment significantly inhibited the upregulation of peptidergic IENFD during the period of mechanical allodynia, suggesting that increased neurotrophism may mediate this phenomenon. In addition, SB203580, an inhibitor of p38, blocked the increase in peptidergic IENFD in db/db mice. The current results suggest that peptidergic IENFD could be a potential diagnostic indicator for PDN in type 2 diabetes. Furthermore, the inhibition of NGF-p38 signaling could be a potential therapeutic strategy for treating this painful condition.

Highlights

► We found increased peptidergic nerve fiber densities in diabetic skin. ► Anti-nerve growth factor inhibited the increased peptidergic nerve fiber densities. ► SB203580 inhibited the upregulation of peptidergic nerve fiber densities. ► Nerve growth factor/p38 signaling mediates increased nerve fibers in diabetic skin.

Introduction

Diabetes mellitus affects over 20 million Americans. About 60% of patients diagnosed with diabetes develop diabetic neuropathy (DN). Painful diabetic neuropathy (PDN) is an early manifestation of DN, which frequently presents in the pre-diabetic states of impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) (Boulton et al., 2005, Feldman et al., 2005, Ziegler et al., 2009). This devastating complication can be found in 40% to 50% of patients with diabetes that have documented neuropathies (Galer et al., 2000). Regrettably, the quality of life for diabetic patients with PDN is significantly diminished (Dworkin et al., 2005, Dworkin et al., 2007, Jensen et al., 2006).

Patients with PDN experience mechanical allodynia and thermal hyperalgesia, which are frequently described as continuously burning, tingling, electric-like, crampy, or achy pain. Allodynia occurs when normally non-painful stimuli become painful, whereas hyperalgesia is an increase in sensitivity to normally painful stimuli. PDN begins in the feet and progresses proximally over time. Symptoms of neuropathic pain are generated by small-calibered Aδ and C fibers that innervate the skin.

At present, there is no available diagnostic tool to objectively determine the severity of PDN. Clinicians must rely on subjective reports from patients concerning their personal experiences of pain, which are frequently influenced by their psychosocial conditions and potential secondary gain from illness. As a result, such measures often lack credibility. In order to objectively test for DN, nerve conduction study and skin biopsy are most commonly used. Unfortunately, nerve conduction study is not a reliable method to diagnose PDN due to its inability to detect small fiber pathology. Skin biopsy can detect small fiber pathology, and thus has been widely used to determine intraepidermal nerve fiber density (IENFD) in DN (Catalan et al., 2007), in addition to the diagnosis of other small-fiber neuropathies. However, its value for determining or predicting the development of PDN is still under debate (Sorensen et al., 2006a, Sorensen et al., 2006b).

The current method of IENFD measurement uses protein gene product (PGP) 9.5 to identify all IENF in the skin (Johnson et al., 2008, Sullivan et al., 2007, Zandecki et al., 2008). Furthermore, PGP9.5 IENFD is considered a standard method to determine small fiber sensory neuropathies including DN (Lauria et al., 2005). Many published animal and human studies have demonstrated a decrease of PGP9.5 IENFD in DN (Kennedy et al., 1996, Shun et al., 2004). Such a decrease of PGP9.5 IENFD is detected early in the course of diabetes and has been found in patients with IGT (Sumner et al., 2003). Nevertheless, the value of PGP9.5 IENFD for the diagnosis of PDN has yet to be determined.

In the present study, our aim was to determine if there is evidence of a correlation between IENFD and mechanical allodynia specific to PDN. Additionally, we intended to further explore our hypothesis that there is enhanced axonal sprouting or regeneration present at the initial stage of PDN to mediate mechanical allodynia in the db/db mouse. Based on previous findings, which report that nerve growth factor (NGF) mediates mechanical allodynia in db/db mice, we focused on subgroups of IENF called peptidergic fibers that express neuropeptides involved in the mediation of nociception. The development of these fibers is dependent on NGF, and these peptidergic fibers are positive for tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for NGF. Two of the major neuropeptides expressed by these peptidergic fibers are substance P (SP) and calcitonin gene related peptide (CGRP). In order to quantify the peptidergic IENFD, we performed double immunofluorescent staining with Trk A and either SP or CGRP on footpads from hind paws of the db/db mouse, an animal model of type 2 diabetes. Additionally, we applied anti-NGF and SB203580, a p38 inhibitor, to db/db mice to gain insight on the molecular mechanisms underlying the changes in IENFD during the early phase of mechanical allodynia.

Previously, we reported that mechanical allodynia is detected in the db/db mouse from 6 to 12 weeks of age. Additionally, we reported an upregulation of SP and NGF at 8 and 10 weeks of age in the db/db mouse (Cheng et al., 2009). Here, we observe a 1.5- to 2-fold elevation in SP and CGRP expression compared to the control values in Trk A-positive peptidergic IENF. Furthermore, we report a cessation of the increase in peptidergic IENFD at 16 weeks of age, corresponding to the reduction of pain behaviors at this time point.

Anti-NGF treatment and SB203580 treatment significantly inhibit the increase in peptidergic IENFD during the period of mechanical allodynia, suggesting that increased neurotrophism may mediate PDN. These results suggest that quantification of peptidergic IENFD could be a potential diagnostic indicator for PDN in type 2 diabetes, and that inhibiting NGF-p38 signaling could be a potential therapeutic strategy for the treatment of PDN.

Section snippets

Animals

Male C57BLKS db/db (stock number 000642) mice were purchased from Jackson Laboratories (Bar Harbor, ME). The homozygous (Leprdb/Leprdb, or db/db) mice were used as a model of type 2 diabetes, while heterozygous mice (Leprdb/+, or db/+) served as nondiabetic controls. Analyses and procedures were performed in compliance with protocols established by the Animal Models of Diabetic Complications Consortium (AMDCC) (http://www.amdcc.org) and were approved by the Use and Care of Animals Committee at

PGP9.5 and Trk A-positive IENFD increase in diabetic animals during the period of mechanical allodynia

Previously, we reported that the db/db mouse, an animal model of type 2 diabetes, develops mechanical allodynia at 6–12 weeks of age (Cheng et al., 2009). In our current study, we examined the temporal correlation between the change of peptidergic IENFD and the development of mechanical allodynia using the hind foot pads from animals characterized in previous studies (Cheng et al., 2009, Cheng et al., 2010). First, we performed immunohistochemistry of a pan-neuronal marker, PGP 9.5, on hind

Discussion

The results from our current study support the use of peptidergic IENFD measurement for the diagnosis of PDN of type 2 diabetes in clinical practice. As was previously mentioned, PGP9.5 has been established as the best pan-neuronal marker across species to localize IENF (Karanth et al., 1991). However, PGP9.5 IENFD measurement has not been proven to be a useful method for the diagnosis of pain. Similar to the current protocol for human IENFD measurement, PGP9.5 immunohistochemistry has been

Conclusions

In conclusion, the upregulation of peptidergic IENFD via NGF-p38 signaling is observed in an animal model of type 2 diabetes during PDN. This phenomenon is associated with and better correlated with the development of mechanical allodynia than the changes in PGP9.5-positive IENFD. These findings not only support the measurement of peptidergic IENFD for the diagnosis of PDN in type 2 diabetes, but also identify the NGF-p38 signaling pathway as a possible pharmacological target for treating this

Acknowledgments

The authors thank Carey Backus and Sang Su Oh for technical assistance, and Jennifer Cheng for her help in the preparation of this manuscript. This work utilized the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center funded by NIH (5P60 DK20572) from the National Institute of Diabetes & Digestive & Kidney Diseases.

This study is supported by National Institutes of Health [UO1-DK60994 (ELF); 1K08NS061039 (HTC)] and the Juvenile Diabetes Research Foundation

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