Elsevier

Experimental Neurology

Volume 179, Issue 2, February 2003, Pages 188-199
Experimental Neurology

Regular article
Restorative effects of neurotrophin treatment on diabetes-induced cutaneous axon loss in mice

https://doi.org/10.1016/S0014-4886(02)00017-1Get rights and content

Abstract

Chronic hyperglycemia in diabetes causes a variety of somatosensory deficits, including reduced cutaneous innervation of distal extremities. Deficient neurotrophin support has been proposed to contribute to the development of diabetic neuropathy. Here, studies were carried out in streptozotocin (STZ)-treated mice to determine whether (1) cutaneous innervation deficits develop in response to hyperglycemia, (2) neurotrophin production is altered in the skin, and (3) neurotrophin treatment improves cutaneous innervation deficits. Cutaneous innervation was quantified in the hindlimb skin using antibodies that label nerve growth factor- (NGF) responsive (CGRP), glial cell line-derived neurotrophic factor (GDNF)/neurturin (NTN) -responsive (P2X3), or all cutaneous axons (PGP 9.5). Diabetic mice displayed severely reduced cutaneous innervation for all three antibodies in both flank and footpad skin regions, similar to reports of cutaneous innervation loss in human diabetic patients. Qualitative assessment of mRNAs for NGF, GDNF, and NTN demonstrated that these mRNAs were expressed in hindlimb flank and footpad skin from diabetic mice. Next, diabetic mice were then treated intrathecally for 2 weeks with NGF, GDNF, or NTN. NGF treatment failed to improve cutaneous innervation, but stimulated axon branching. In comparison, GDNF and NTN treatment increased cutaneous innervation and axon branching. Our results reveal that similar to human diabetic patients, STZ-induced diabetes significantly reduces hindlimb cutaneous innervation in mice. Importantly, intrathecal treatment using GDNF or NTN strongly stimulated axon growth and branching, suggesting that administration of these trophic factors can improve cutaneous innervation deficits caused by diabetes.

Introduction

Peripheral neuropathy is a serious complication of diabetes that affects nerve fibers innervating the distal regions of the limbs. Small cutaneous sensory fibers that respond to mechanical, thermal, and/or chemical stimuli are most commonly affected. Diabetes-induced sensory problems include loss of sensation in the feet and hands, paresthesia, or chronic pain. Neuropathic peripheral nerves commonly display segmental demyelination or axon atrophy and loss, suggesting that the peripheral processes of sensory axons are vulnerable to chronic diabetes. Quantification of axon numbers in skin biopsies has confirmed the loss of cutaneous axons in humans with symptoms of diabetic neuropathy (DN) 1, 23, 28, 36.

Neurotrophic factors play a vital role in the support and regulation of cutaneous afferents. Nerve growth factor (NGF) supports the survival of skin afferents during embryonic development (reviewed in Snider and McMahon, 1998). Postnatally, 50% of cutaneous afferents become unresponsive to NGF, leaving only about 40% of dorsal root ganglion (DRG) neurons that are sensitive to NGF in adulthood 5, 6, 37, 38. NGF-responsive cutaneous afferents respond to noxious stimuli and express neuropeptides associated with nociception, including calcitonin gene-related peptide (CGRP) and substance P 25, 37, 39. Cutaneous CGRP-positive axons are commonly varicose and adhere closely to vascular patterns in the skin 41, 43.

The unmyelinated neurons that become unresponsive to NGF during postnatal periods will subsequently express receptors for glial cell line-derived neurotrophic factor (GDNF) 8, 39. GDNF is a member of the TGFβ superfamily and is closely related to neurturin (NTN), artemin, and persephin. GDNF-related ligands utilize a two-receptor system consisting of a tyrosine kinase receptor, Ret, and one of four GPI-linked ligand-binding receptors. Each of the identified GPI-linked receptors (GFRα1-4) preferentially binds a different GDNF-related factor. NTN is also believed to have biological effects on unmyelinated nociceptive neurons (reviewed in Airaksinen et al., 1999).

Unmyelinated GDNF-responsive neurons comprise up to 35% of lumbar DRG neurons and generally do not express CGRP or substance P 5, 39. Most GDNF-responsive neurons bind the plant-derived isolectin B4 (IB4) and express the enzyme thiamine monophosphatase (TMP) and the purinoreceptor subtype, P2X3 9, 31, 39, 46. It is suggested that GDNF-responsive neurons preferentially innervate cutaneous regions since a greater proportion of IB4-positive neurons project to the skin compared to CGRP-expressing neurons 6, 30. P2X3-immunoreactive cutaneous axons are small, fine fibers that terminate predominantly in the epidermis (Cockayne et al., 2000).

Recent findings have demonstrated that cutaneous innervation of the skin is substantially reduced in human patients with DN and these reductions correlate with electrophysiological and somatosensory deficits. Peripheral axon loss has been quantified in humans by measuring intraepidermal nerve fiber densities from calf biopsies and these measurements have been used as a clinical indicator of neuropathy in diabetic humans 1, 23, 34. We previously demonstrated in streptozotocin (STZ)-treated mice that diabetes results in significant deficits in the central terminals of GDNF-responsive sensory neurons in the spinal dorsal horn. Furthermore, GDNF administration reversed these diabetes-induced deficits in the spinal cord (Akkina et al., 2001). Here, we report that similar to human DN, diabetic mice develop severe reductions in the abundance and branching of peripheral sensory afferents in hindlimb skin. Moreover, GDNF or NTN treatment both increased the abundance and branching of cutaneous axons, suggesting that GDNF and/or NTN may be effective in improving the poor cutaneous innervation that develops in diabetic patients.

Section snippets

Diabetic mice

Fifty-one male C57BL/6 mice (Charles River, Wilmington, MA) were used in this study and housed in the animal facilities at the University of Kansas Medical Center under pathogen-free conditions and received water and mouse chow ad libitum. Once animals reached 8 weeks of age, diabetes was induced by a single intraperitoneal injection of STZ (n = 35; 180 mg/kg, Sigma, St. Louis, MO) dissolved in 0.4 ml sodium citrate buffer, pH 4.5 (Wang et al., 1993). Control mice (n = 11) were injected with

STZ-induced diabetes in mice

Within 2 days after injection of STZ, mice showed typical signs of STZ-induced diabetes including weight loss, polyuria, and polydipsia. At the time of sacrifice, 88% of all STZ-injected mice (35 of 40) had developed at least a 2.5-fold increase in blood glucose levels compared to sham-injected control mice. Only STZ-injected mice with greater than a 10% reduction in body weight and blood glucose levels greater than 300 mg/dL were included in the diabetic group (Wang et al., 1993). Control mice

Discussion

A growing body of work suggests that compromised neurotrophic support contributes to sensory deficits in diabetes (Apfel, 1999). Although all fiber classes are affected in diabetic neuropathy, small-caliber nociceptors are often the earliest affected and neuropathies associated with these axons provide the most discomfort for patients. Using a mouse model of diabetic neuropathy, we have examined the effects of diabetes on innervation of the hindlimb skin and tested the ability of NGF, GDNF, and

Conclusion

We have shown that peripheral axons are lost in the hindlimb skin of a murine model of experimentally induced diabetes. These results are unique since similar observations have not been reported in diabetic rats (Karanth et al., 1990). The diabetes-induced reduction in cutaneous axons is due to the loss of both NGF- and GDNF-responsive axons. The impact of reduced skin innervation in diabetic patients is unclear, but recent studies have begun to focus on the relationship of reduced epidermal

Acknowledgements

This work was supported by NIH Grants R21NS38844 and PO1DE07734 (D.E.W.). The authors thank Colleen Patterson and Janelle Ryals for technical assistance and Dr. Cheryl Stucky for helpful comments during the preparation of the manuscript.

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