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