Diabetic Neuropathy & Nerve Regeneration

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Progress in Neurobiology 69 (2003) 229285

Diabetic neuropathy and nerve regeneration

Hitoshi Yasuda, Masahiko Terada, Kengo Maeda, Shuro Kogawa, Mitsuru Sanada,Masakazu Haneda, Atsunori Kashiwagi, Ryuichi Kikkawa

Division of Neurology, Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan

Received 20 June 2001; accepted 20 February 2003

Abstract

Diabetic neuropathy is the most common peripheral neuropathy in western countries. Although every effort has been made to clarify the

pathogenic mechanism of diabetic neuropathy, thereby devising its ideal therapeutic drugs, neither convinced hypotheses nor unequivocally

effective drugs have been established. In view of the pathologic basis for the treatment of diabetic neuropathy, it is important to enhance

nerve regeneration as well as prevent nerve degeneration. Nerve regeneration or sprouting in diabetes may occur not only in the nerve

trunk but also in the dermis and around dorsal root ganglion neurons, thereby being implicated in the generation of pain sensation. Thus,

inadequate nerve regeneration unequivocally contributes to the pathophysiologic mechanism of diabetic neuropathy. In this context, the

research on nerve regeneration in diabetes should be more accelerated. Indeed, nerve regenerative capacity has been shown to be decreased

in diabetic patients as well as in diabetic animals. Disturbed nerve regeneration in diabetes has been ascribed at least in part to allor some of

decreased levels of neurotrophic factors, decreased expressionof their receptors, alteredcellular signal pathways and/or abnormal expression

of cell adhesion molecules, although the mechanisms of their changes remain almost unclear. In addition to their steady-state changes

in diabetes, nerve injury induces injury-specific changes in individual neurotrophic factors, their receptors and their intracellular signal

pathways, which are closely linked with altered neuronal function, varying from neuronal survival and neurite extension/nerve regeneration

to apoptosis. Although it is essential to clarify those changes for understanding the mechanism of disturbed nerve regeneration in diabetes,

very few data are now available. Rationally accepted replacement therapy with neurotrophic factors has not provided any success in treating

diabetic neuropathy. Aside from adverse effects of those factors, more rigorous consideration for their delivery system may be needed forany possible success. Although conventional therapeutic drugs like aldose reductase (AR) inhibitors and vasodilators have been shown

to enhance nerve regeneration, their efficacy should be strictly evaluated with respect to nerve regenerative capacity. For this purpose,

especially clinically, skin biopsy, by which cutaneous nerve pathology including nerve regeneration can be morphometrically evaluated,

might be a safe and useful examination.

2003 Elsevier Science Ltd. All rights reserved.

Abbreviations: ABC, avidin-biotinylated enzyme complex; ALS, amyotrophic lateral sclerosis; AP-1, activator protein-1; AR, aldose reductase; ARI,

aldose reductase inhibitor; ATF-2, activating transcription factor-2; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; Cdk5,

cyclin-dependent kinase 5; CGRP, calcitonin gene-related protein; CMAP, compound muscle action potential; CNS, central nervous system; CNTF,

ciliary neurotrophic factor; CRE, cAMP responsive element; CSF, cerebrospinal fluid; DRG, dorsal root ganglion; ELISA, enzyme-linked immunosorbent

assay; ERK1, extracellular signal-related kinase 1 (42 kDa); ERK2, extracellular signal-related kinase 2 (44 kDa); GAP-43, growth-associated protein-43;GDC, granular disintegration of the cytoskeleton; GDNF, glial cell-derived neurotrophic factor; GFR, GDNF family receptor component; GPI,

glycosylphosphatidylinositol; GSK-3, glycogen synthase-3; ICAM, intercellular cell adhesion molecule; IDDM, insulin-dependent diabetes mellitus;

IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; JNK, c-Jun N-terminal kinase; L1, L1-CAM (L1 cell adhesion

molecule); LIF, leukemia inhibitory factor; MAG, myelin-associated glycoprotein; MAP, mitogen-activated protein; MAPK, MAP kinase; MAPKK, MAP

kinase kinase; MMPs, matrix metalloproteinases; MNF, myelinated nerve fiber; NCAM, neural cell adhesion molecule; NF, neurofilament; NF-H, high

molecular weight mass (200 kDa) neurofilament; NF-L, light molecular weight mass (68kDa) neurofilament; NF-M, medium molecular weight mass

(145 kDa) neurofilament; NGF, nerve growth factor; NIDDM, non-insulin-dependent diabetes mellitus; NPY, neuropeptide Y; NT-3, neurotrophin-3; NT-4/5,

neurotrophin-4/5; p75NTR, p75 neurotrophin receptor; pak, p21-activated kinase; PGE1, prostaglandin E1; PGI2, prostaglandin I2; PGP 9.5, protein

gene-product 9.5; PKC, protein kinase C; PNS, peripheral nervous system; RET, glial cell-derived neurotrophic factor receptor tyrosine kinase; rhNGF,

recombinant human nerve growth factor; SAPK, stress-activated protein kinase; SCa, slow component a; SCb, slow component b; STZ, streptozocin;

TNF-, tumor necrosis factor-; TrkA, tyrosine-receptor kinase A; TUNNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling;

UMNF, unmyelinated nerve fiber; VEGF, vascular endothelial growth factor; VIP, vosoactive intestinal polypeptide Corresponding author. Tel.: +81-77-548-2222; fax: +81-77-543-3858.

E-mail address:[email protected] (H. Yasuda).

0301-0082/03/$ see front matter 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0301-0082(03)00034-0


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Contents

1. Introduction: aims and scope of review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

2. Intrinsic and extrinsic factors associated with nerve regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

2.1. Intrinsic neuronal regenerating activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

2.1.1. Growth-associated protein-43 (GAP-43) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

2.1.2. Tubulin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

2.2. Growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2342.2.1. Neurotrophins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

2.2.1.1. Nerve growth factor (NGF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

2.2.1.2. Brain-derived neurotrophic factor (BDNF). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

2.2.1.3. Neurotrophin-3 (NT-3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

2.2.1.4. Neurotrophin-4/5 (NT-4/5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

2.2.2. Insulin-like growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

2.2.2.1. Insulin-like growth factor-I and -II (IGF-I/II). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

2.2.2.2. Insulin-like growth factor binding protein (IGFBP) . . . . . . . . . . . . . . . . . . . . . . . . 238

2.2.3. Hematopoietic cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

2.2.3.1. Ciliary neurotrophic factor (CNTF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

2.2.3.2. Tumor necrosis factor-(TNF-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

2.2.3.3. Interleukin-6 (IL-6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

2.2.4. Glial cell line-derived neurotrophic factor (GDNF). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

2.3. Extracellular matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

2.3.1. Laminin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

2.3.2. Fibronectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

2.3.3. Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

2.3.4. Matrix metalloproteinases (MMPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

2.4. Cell adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

2.4.1. Neural cell adhesion molecule (NCAM). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

2.4.2. L1 cell adhesion molecule (L1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

2.4.3. Myelin-associated glycoprotein (MAG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

2.4.4. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

2.5. Cell signal messengers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

2.5.1. cAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

2.5.2. Protein kinase C (PKC). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

2.5.3. Mitogen-activated protein kinases (MAPKs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2452.5.3.1. c-Jun N-terminal protein kinase (JNK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

2.5.3.2. Extracellular signal-related kinase 1/2 (ERK1/2) . . . . . . . . . . . . . . . . . . . . . . . . . . 247

2.5.4. Cyclin-dependent kinases and small GTPases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

2.6. Immediate early genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

2.6.1. c-Jun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

2.6.2. c-Fos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

2.6.3. Activating transcription factor-2 (ATF-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

2.7. Endoneurial microenvironment including vascularization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

3. Experimental studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

3.1. Relevance of examining nerve regeneration in experimental diabetic models . . . . . . . . . . . . . . . 250

3.2. Evaluation of nerve regeneration in animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

3.3. Degeneration and regeneration of the peripheral nerve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

3.3.1. Cellular events in Wallerian degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

3.3.2. Early axonal changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2513.3.3. Schwann cell responses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

3.3.4. Macrophage responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

3.3.5. Nerve regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

3.3.6. Axonal sprouting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

3.3.7. Growth cone and axonal elongation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

3.3.8. Cell body reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

3.3.9. Maturation of regenerating nerve fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

3.4. Wallerian degeneration in experimental diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

3.4.1. Axonal degeneration and pathway clearance in diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . . 253

3.4.2. Degradation of cytoskeletal proteins in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

3.4.3. Macrophage responses in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

3.4.4. Schwann cell response in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256


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3.5. Nerve regeneration in experimental diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

3.5.1. Axonal sprouting, elongation and maturation in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

3.5.2. Cell body reaction in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

3.5.3. Partial denervation in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

4. Clinical observation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

4.1. Pathological findings suggesting nerve regeneration in diabetic nerves. . . . . . . . . . . . . . . . . . . . 258

4.2. Rationale for using neurotrophic factors for diabetic patients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2594.3. Clinical trials: current state. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

4.3.1. Lessens from other trials: amyotrophic lateral sclerosis and toxic neuropathies . . . . . . 260

4.3.2. Nerve growth factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

4.3.3. Other neurotrophic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

4.3.4. Aldose reductase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

4.4. Specific problems for the use of neurotrophic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

5. Regenerating nerve fibers with special reference to pain generation . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

6. Evaluation of nerve pathology including regeneration by skin biopsy . . . . . . . . . . . . . . . . . . . . . . . . . 264

6.1. Morphometric analysis of cutaneous nerves by immunohistochemistry . . . . . . . . . . . . . . . . . . . . 265

6.2. Morphometric analysis of cutaneous nerves by ultrastructural examination . . . . . . . . . . . . . . . . 267

6.3. Neurotrophins and cutaneous innervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

6.4. Evaluation of therapeutic compounds for diabetic neuropathy by skin biopsy . . . . . . . . . . . . . . 268

7. Neuronal cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

7.1. Apoptosis induced by the sera from diabetic patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2697.2. Apoptosis is induced under high glucose or hyperglycemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

7.3. Apoptosis and neurotrophins/MAP kinase/cAMP in hyperglycemia . . . . . . . . . . . . . . . . . . . . . . . 270

7.3.1. Neurotrophins and their receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

7.3.2. MAP kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

7.3.3. cAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

8. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

1. Introduction: aims and scope of review

Diabetic polyneuropathy, the most common of the pe-ripheral neuropathies, occurs widely in western countries. It

most often develops in the midst of complications observed

in diabetes. The putative pathogenesis of diabetic neuropa-

thy includes increased polyol pathway activity leading to

the accumulation of sorbitol and fructose (Gabby et al.,

1966; Gabby and OSullivan, 1968) and imbalances in

nicotinamide adenine dinucleotide phosphate/nicotinamide

adenine dinucleotide, reduced form (Williamson et al.,

1993); auto-oxidation of glucose leading to the formation

of reactive oxygen species (Low et al., 1997); advanced

glycation end-products produced by non-enzymatic gly-

cation of proteins (Brownlee et al., 1988); inappropriate

activation of protein kinase C (PKC) (Greene et al., 1985,

1999a,b; Koya and King, 1998); and a deficit of neu-

rotrophic supports (Tomlinson et al., 1997)(Fig. 1).Based

on these observations, several therapeutic drugs including

aldose reductase inhibitors (ARIs) (Kikkawa et al., 1984;

Sima et al., 1988a,b),anti-oxidant drugs (Low et al., 1997),

aminoguanidine (Yagihashi et al., 1992), a selective PKC

inhibitor (Nakamura et al., 1999),and neurotrophic factors

(Tomlinson et al., 1996, 1997) have been used to treat dia-

betic neuropathy and have been reported to ameliorate nerve

dysfunction and/or morphologic abnormalities in diabetic

animals and/or patients.

Although some of these compounds have had significant

effects on morphological abnormalities of nerve fibers as

well as nerve dysfunction, usually the magnitude of the ef-fects has been smaller in clinical trials than in experimen-

tal studies. The discrepancy may be due in part to the dif-

ference in the amount of nerve fiber lesions: patients with

diabetic neuropathy usually show nerve fiber loss to some

degree, whereas diabetic animals, especially streptozocin

(STZ)-induced ones, do not have any significant degree of

nerve fiber loss (Yasuda et al., 1989a,b; Zochodne et al.,

2001). Moreover, pancreas transplantation has not produced

the expected beneficial effects on nerve dysfunction; com-

plete normalization of blood glucose has resulted in a no-

table but still mild degree of improvement in nerve function

even 10 years after transplantation (Navarro et al., 1997).

Patients who received pancreas transplantation usually had

moderate to severe neuropathy; thus, it may follow that the

ineffectiveness of glycemic control for diabetic neuropathy

may be due to the difficulty for nerve fibers to regenerate

once degenerated. This tendency toward irreversibility in the

peripheral nerve in diabetes has also been supported in many

clinical trials on the therapeutic drugs for diabetic neuropa-

thy in which nerve function has been improved only by a few

meter/s in nerve conduction velocity during 1 year (Boulton

et al., 1990; Goto et al., 1995;Greene et al., 1999a,b).

These results may suggest that peripheral nerve tissue

damage cannot be measurably reversed once pathological


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Fig. 1. Schematic hypothetical mechanisms of diabetic neuropathy. In this figure, possible causative factors are simply presented, although each factor is

closely associated with other factors. In addition, there are other putative factors which may be involved in its pathogenesis. One of the points which

should be emphasized in this schema is that nerve regeneration is closely involved in nerve degeneration such that the balance between nerve degeneration

and regeneration may be directed toward nerve degeneration in diabetic condition. PKC, protein kinase C.

changes even mildly occur. Thus, early diagnosis of dia-

betic neuropathy followed by drug therapy combined with

glycemic control may be warranted to prevent the pro-

gression of diabetic neuropathy, i.e. arrest the degenerative

changes of nerve fiber pathology. However, even with these

early efforts, it may be nearly impossible to normalize

glycemic level.

The pathology of diabetic polyneuropathy includes ax-

onal atrophy, demyelination, loss of nerve fibers, and the

blunted regeneration of nerve fibers (Sima et al., 1988a,b;

Dyck and Giannini, 1996). Thus, nerve regeneration coex-ists with degeneration and contributes to nerve function as

well as nerve pathology especially at the later stage of di-

abetic neuropathy. The goal in treating diabetic neuropathy

is not only to prevent the progression of neuropathic symp-

toms and nerve dysfunction and degeneration (Kennedy

et al., 1990; The Diabetes Control and Complications Trial

Research Group, 1993) but also to promote regeneration of

degenerated nerve fibers. Thus, in addition to prevention

of nerve degenerative process, augmentation of nerve fiber

regenerative capacity may be important and useful for the

treatment of diabetic neuropathy (Fig. 1).For this purpose,

the fundamental understanding about nerve regenerative

mechanisms especially with respect to the pathogenesis of

diabetic neuropathy should be rigorously studied.

The decreased nerve regenerative capacity in diabetes

has been associated with impaired neurotrophic tone, which

could reflect diminished synthesis, secretion or responsive-

ness of neurotrophic factors such as nerve growth factor

(NGF) in sensory and autonomic nerve fibers. Although neu-

rotrophic factors including NGF are required not only for the

development but also for the maintenance of NGF-sensitive

sensory and autonomic neurons and their axons, most of their

serum and nerve levels have been reported to be decreased

in the diabetic condition. Retrograde axonal transport of

peripherally synthesized neurotrophic factors including NGF

from target organs to neuronal cell bodies, which is required

for normal maintenance and regeneration of the peripheral

nervous system (PNS), is also disturbed in the diabetic state

(Schmidt et al., 1985; Fernyhough et al., 1998a,b). In ad-

dition, novel neurotrophic factors are still being discovered

and some of these may be deficient in the diabetic condi-

tion. Furthermore, the signal transduction of neurotrophic

factors has not been fully uncovered although it is consid-

erably changed in diabetes, and its abnormality is closely

implicated in either disrupted neuronal survival or disturbednerve regeneration.

Most diabetic animals have not shown nerve fiber loss in

steady state, and it is not satisfactory to use their uninjured

nerves for nerve regeneration research in diabetic condition.

However, examining nerve fiber regeneration in animal mod-

els of nerve injuries, e.g. crush, freezing, or transection, may

be a useful alternative method.

In this manuscript, we focus on the pathophysiology of

nerve regeneration with special reference to diabetic sensory

polyneuropathy and the roles of its associated neurotrophic

factors, their receptors, extracellular matrices, etc. in the

pathogenesis and treatment of diabetic polyneuropathy. Our

work with respect to nerve regeneration in diabetic state is

described.

2. Intrinsic and extrinsic factors associated with

nerve regeneration

2.1. Intrinsic neuronal regenerating activity

2.1.1. Growth-associated protein-43 (GAP-43)

The synthesis and axonal transport of GAP-43/B-50 are

induced in the process of axonal elongation. GAP-43 is a


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Fig. 2. Molecular biological changes during nerve regeneration after nerve injury. See Abbreviations for details.

major constituent of the axonal growth cone (Fig. 2),where

it is localized exclusively in the membrane skeleton. GAP-43

is never induced in injured neurons of the central nervous

system (CNS), where nerve regeneration does not occur un-

der physiological conditions. By contrast, the protein is dra-matically induced after nerve injury in the PNS (Vanselow

et al., 1994). In steady state, GAP-43 is expressed only in

small dorsal root ganglion (DRG) neurons under normal con-

dition, whereas it is expressed by all sizes of DRG neurons

after nerve injury. However, the induction depends on the

level of the axotomized site along the entire length of axons;

central axotomy does not induce GAP-43 mRNA in DRG

(Chong et al., 1994). This observation suggests that unknown

substrates from the periphery might regulate GAP-43 ex-

pression. The substrates may include NGF, which increases

GAP-43 mRNA by increasing its half-life. This effect of

NGF was mimicked by phorbol ester, PKC agonist, and was

inhibited by PKC inhibitor or down-regulation of PKC in

PC12 cells, suggesting the possible PKC-dependent stabi-

lization of mRNA (Perrone-Bizzozero et al., 1993).Sensory

neurons from mice lacking GAP-43, however, can extend

neurites and form filopodia in culture condition, suggesting

that GAP-43 is not required to create growth cones. In cer-

tain decision points, such as optic chiasm, GAP-43 is nec-

essary for pathfinding (Strittmatter et al., 1995). Apart from

neuronal cells of the PNS, unmyelinating Schwann cells also

express GAP-43, although its function is unknown.

In diabetic rats, the mRNA level of GAP-43 has been

reported to be reduced in uninjured DRGs. After nerve

crush, the level is consistently up-regulated in DRG neu-

rons, although the magnitude of its increase by quantitative

analysis was different between reports;Maeda et al. (1996)

reported a lower increase of GAP-43 mRNA in DRGs of

diabetic rats than in those of control rats, whereas Pekineret al. (1996)reported its unchanged expression in DRGs of

diabetic rats compared with those of non-diabetic rats. A

decrease in GAP-43 mRNA after nerve crush in diabetic

rats was reported by others (Mohiuddin and Tomlinson,

1997). The protein level of GAP-43 in injured DRG neurons

was reduced in the sciatic nerve of diabetic rats (Pekiner

et al., 1996).

In the autonomic nervous system, there was no difference

in immunohistochemical localization of GAP-43 in sympa-

thetic ganglions between control and long-term diabetic rats

(Schmidt et al., 1991).

2.1.2. Tubulin

Tubulin protein is a cytoskeleton that is up-regulated after

nerve injury (Hoffman and Cleveland, 1988; Miller et al.,

1989; Oblinger et al., 1989; Moskowitz et al., 1993). Mi-

crotubules which are the major cytoskeletal component of

regenerating axons are formed by heterodimers of- and

-tubulin.

In uninjured diabetic DRG, T1-tubulin mRNA is

decreased (Mohiuddin et al., 1995a; Scott et al., 1999).

Recently, class III -tubulin mRNA has been shown to be

increased in diabetic DRG by in situ hybridization (Liuzzi

et al., 1998). It is difficult to explain the discrepancy


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between-and-tubulin mRNA in diabetic DRG. In addi-

tion, it remains to be clarified whether these alterations of

tubulin synthesis are associated with regeneration failure in

diabetic nerves. Tubulin protein is modified posttranslation-

ally. Non-enzymatic glycation of tubulin has been observed

as early as 2 weeks after induction of diabetes ( Cullum et al.,

1991; McLean et al., 1992). Posttranslational modificationmight have some effects on tubulin assembly. However,

if any, its direct association with regeneration failure in

diabetes is also uncertain.

2.2. Growth factors

Although many neurotrophic factors are known (Table 1),

few are studied in the field of the pathogenesis and treatment

of diabetic neuropathy; most of them are associated with

nerve growth factor (NGF). Although the mechanism of

action is as yet unknown, the current knowledge of growth

factors and their relationship to diabetic neuropathy sug-

gest a pathophysiological role of reduced levels of growthfactors in the development of diabetic neuropathy; neuronal

function may be compromised by their deficits (Table 2).

Furthermore, atrophy of neurons or nerves and even neu-

ronal death may be induced due to growth factor reduction

in diabetic neuropathy. It also remains to be established

whether growth factor deficiency is due to a decrease in its

synthesis, an inability of the factor to bind to its receptor, or

disturbances in retrograde axonal transport or intraneuronal

processing. Further studies aimed at understanding the dis-

turbances in expression of the genes and proteins involved

in diabetic neuropathy, as well as their receptor binding

and subsequent transport from sites of synthesis to sites ofaction, may clarify the relationship between growth factors

and diabetic neuropathy.

2.2.1. Neurotrophins

2.2.1.1. Nerve growth factor (NGF). NGF shows trophic

effects on a subpopulation of sensory neurons in DRG and

sympathetic postganglionic neurons (Smeyne et al., 1994;

Crowley et al., 1994). NGF is produced by the target tissues

including skeletal muscle (Amano et al., 1991) and skin.

NGF released by the target tissue is incorporated with its

high affinity receptor, TrkA (Figs. 24)at the nerve ending.

NGFphosphorylated TrkA complex is retrogradely trans-

ported to the neuronal body and transduces its signal to the

nucleus. The function of another NGF receptor, p75NTR,

which is also bound to other neurotrophins with low affinity,

is controversial. The DRG neurons supported by NGF are

small in size and mediate nociception. During development,

NGF is essential for the survival of DRG small neurons.

However, in adult, NGF is not necessary for the survival but

maintains neuropeptide levels such as substance P. When

the nerve is injured, the delivery of NGF from the target

is decreased. Interleukin (IL)-1 released by macrophages

infiltrating the endoneurium increases NGF expression in

Table 1

List of neurotrophic factors

Neurotrophins (NT)

Nerve growth factor (NGF)

Brain-derived neurotrophic factor (BDNF)

NT-3

NT-4/5

NT-6

Hematopoietic cytokines

Ciliary neurotrophic factor (CNTF)

Leukemia inhibitory factor (LIF)

Oncogene M

Interleukin (IL)-1

IL-3

IL-6

IL-7

IL-9

IL-11

Granulocyte colony-stimulating factor

Insulin-like growth factors (IGF)

Insulin

IGF-IIGF-II

Heparin-binding family

Acidic-fibroblast growth factor (FGF)

Basic FGF

int-2 onc

hst/k-fgf onc

FGF-4

FGF-5

FGF-6

Keratinocyte growth factor

Epidermal growth factor (EGF) family

EGF

Transforming growth factor (TGF)-

TGF- family

TGF-1

TGF-2

TGF-3

Glial-derived neurotrophic factor (GDNF)

Neurturin

Persephin

Activin A

Bone morphogenetic proteins

Tyrosine kinase-associated cytokines

Platelet-derived growth factor (PDGF)

Colony-stimulating factor-1

Stem cell factor

OthersACTH analogues

Gangliosides

Amyloid precursor protein

-Interferon

Galectin-1

fibroblasts in the distal stump (Heumann et al., 1987a,b;

Lindholm et al., 1987, 1988; Brown et al., 1991; Rotshenker

et al., 1992). Schwann cells, which do not respond to IL-,

also increase NGF expression (Matsuoka et al., 1991). NGF

mRNA is also increased in the denervated skin (Mearow


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Table 2

Neurotrophins and their receptors in experimental diabetes

Neurotrophins Receptors

Nerve growth factor (NGF) TrkA

Serum DRG

Skin

Nerve Heart

Brain-derived neurotrophic factor (BDNF) TrkB

Muscle Not reported

Nerve

DRG

Neurotrophin-3 TrkC

Muscle DRG

Skin

Nerve

Neurotrophin-4/5 TrkB

Nerve Not reported

et al., 1993). The expression of both trkA and p75NTRmRNA in DRGs is decreased after nerve injury.

What effect NGF has on nerve regeneration is a contro-

versial issue. Although the early studies showed that NGF

improved nerve regeneration (Chen et al., 1989; Rich et al.,

1989; Gold et al., 1991),it has been clearly shown that NGF

stimulates collateral sprouting from uninjured neurons but

not regeneration from injured neurons (Diamond et al., 1992;

Mearow et al., 1994). This fact may not be disappointing

in terms of the treatment of diabetic peripheral neuropathy,

Fig. 3. Distribution of neurotrophic factors in the peripheral nerve under normal and injured conditions. Solid circles imply neurotrophic factors which

are secreted from various cells including Schwann cells, captured and taken up from the axons and transported to the neuronal body. The mode of

production and secretion are extraordinarily different either among involved factors or among involved conditions.

since the goal of treatment is reinnervation of the target tis-

sues. NGF increases tubulin mRNA in sympathetic neurons

(Mathew and Miller, 1990)and DRG neurite outgrowth in

three-dimensional extracellular matrix via the up-regulation

of matrix metalloproteinase (MMP)-2 (Muir, 1994).

In diabetic patients, serum (Faradji and Soleto, 1990)and

skin NGF levels were reported to be decreased (Anand et al.,1996), comparable to the data from experimental diabetes

studies (Table 2). However, discrepant results have been re-

ported; skin NGF mRNA was increased in diabetic patients

(Diemel et al., 1999), and the serum NGF level in IDDM

patients was increased compared with age-matched control

subjects and NIDDM patients (Azar et al., 1999).

In experimental models, tissue NGF levels were decreased

in diabetic mice, and this decrease was ameliorated by trans-

plantation of islet cells (Hellweg et al., 1991) (Table 2).

Others also found a decrease in NGF content in the sciatic

nerves of diabetic rats (Hanaoka et al., 1992). Insulin treat-

ment was shown to improve decreased NGF mRNA in the

skin of diabetic rats (Fernyhough et al., 1994).These reportssuggest that hyperglycemia and/or hypoinsulinemia induce

a deficit of NGF synthesis. This deficit might also be caused

by altered corticosterone and 1,25-(OH)2 D3 (Neveu et al.,

1992). Corticosterone decreases NGF, whereas 25-(OH)2D3

increases NGF; in diabetes, serum concentration of corti-

costerone is increased, whereas that of 1,25-(OH)2 D3 is

decreased. A Vitamin D3 derivative not only induces NGF

but also improves neuropeptide content (Riaz et al., 1999).

Accumulation of polyol could decrease NGF synthesis; ARI


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Fig. 4. The receptors for neurotrophins. TrkA, TrkB and TrkC are high affinity receptors specific for NGF, BDNF and NT-4/5 and NT-3, respectively, all

of which have intracellular kinase domain. Both TrkA and TrkB also show an affinity for other neurotrophins, although relatively weak. All neurotrophins

are able to bind to low affinity receptor p75 which does not have intracellular kinase domain.

epalrestat corrects decreases in both NGF content in diabetic

nerves and NGF mRNA in cultured Schwann cells under

high glucose condition (Ohi et al., 1998). The anti-oxidant

-lipoic acid, which is known to be effective for diabetic

neuropathy, was also reported to reverse nerve NGF content

in diabetic rats (Garrett et al., 1997; Hounsom et al., 1998).

The uptake of exogenous NGF into DRG or superior

mesenteric ganglion and retrograde axonal transport of NGF

have been reported to be decreased in diabetic rats (Jakobsen

et al., 1981; Schmidt et al., 1983; Hellweg et al., 1994).

NGF ameliorates decreases in mRNAs and proteins of sub-

stance P and CGRP in peripheral nerves, DRGs or dorsal

horns of the spinal cord (Diemel et al., 1994;Fernyhough

et al., 1995a,b; Schmidt et al., 1995; Unger et al., 1998),

nociceptive threshold (Apfel et al., 1994) and amplitude of

electrically evoked C-fiber response (Elias et al., 1998). In

addition, neurogenic cutaneous vasodilatation and plasma

extravasation (Bennett et al., 1998a,b) and myelinated nerve

fiber (MNF) morphology in the sural nerve (Unger et al.,

1998) are also restored by NGF, whereas it does not im-

prove nerve blood flow or motor nerve conduction velocity

in diabetic rats (Maeda et al., 1997). Decreased NGF pro-

duction may lead to up-regulation of trkA mRNA in the

keratinocytes (Terenghi et al., 1997).

In the autonomic nervous system, NGF mRNA (Kanki

et al., 1999)and protein (Hellweg and Hartung, 1990)were

increased in cardiac muscle at the early stage of diabetes

and decreased in long-term diabetes (Schmid et al., 1999).

In the iris, NGF mRNA was also increased in untreated di-

abetic rats (Brewster et al., 1995). Although the regenera-

tion of postganglionic noradrenergic nerves after chemical

denervation by 6-hydroxydopamine was well preserved in

diabetic rats, a response to exogenous NGF was defective

when assessed by the content of noradrenaline, suggesting

loss of TrkA function (Vo and Tomlinson, 1999).

It was reported that the expression of p75NTR was de-

creased in DRGs of STZ-induced diabetic rats compared

with those of control rats (Delcroix et al., 1997), whereas that

of TrkA was unchanged (Delcroix et al., 1997) or decreased

in DRGs of diabetic rats (Mohiuddin and Tomlinson, 1997).

In addition, anterograde and retrograde axonal transport of

p75NTR was also decreased, while that of TrkA was unaf-

fected in diabetic rats (Delcroix et al., 1998). NGF treatment

reversed changes in transcripts and protein of p75NTR but


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did not reverse those of TrkA (Delcroix et al., 1998).Thus,

the ability to capture and retrogradely transport NGF may

be impaired in diabetic state because of suboptimal produc-

tion of p75NTR, and NGF therapy may overcome this de-

ficiency. It was reported that extracellular cleavage product

of NGF receptor (p75NTR) was increased in the urine of

diabetic patients with neuropathy (Hurska et al., 1993), al-though the significance is not clear.

Decreased NGF effects in diabetes were also observed

after nerve injury. The expression of NGF mRNA in the

distal stump was unexpectedly increased in diabetic rats

compared with non-diabetic rats. By contrast, the expres-

sion of trkA mRNA in DRG was decreased in diabetic rats.

Messenger RNAs of substance P and GAP-43, which are

regulated by NGF, were also decreased in diabetes (Maeda

et al., 1996). Combined with the early observation that the

uptake of exogenous NGF into DRG was decreased in di-

abetic rats (Jakobsen et al., 1981), this experiment suggests

that both deficits in the synthesis of NGF and in the cou-

pling of NGF and trkA may contribute to a decrease in theeffects of NGF on diabetic nerves.

NGF that was administered at the proximal stumps of

transected nerves corrected the molecular changes in DRGs

caused by axotomy; both an increase in GAP-43 mRNA

in DRG and a decrease in -preprotachykinin mRNA were

reversed by NGF therapy (Mohiuddin et al., 1999). In this

situation, interestingly, NGF supernormalized decreased

p75NTR mRNA but did not affect decreased trkA mRNA

in DRGs. The decreased ratio of TrkA/p75NTR was ob-

served in axotomized DRG in diabetes. It was reported that

excessive NGF might cause apoptosis via p75NTR (Frade

et al., 1996). In the neuroblastoma cell line, expressingp75NTR, NGF, but neither BDNF nor NT-3, induced apop-

tosis (Kuner and Hertel, 1998). Thus, DRG neurons may

undergo apoptotic changes when nerves are injured in dia-

betic condition. This phenomenon is discussed inSection 7.

2.2.1.2. Brain-derived neurotrophic factor (BDNF).

BDNF has trophic effects on DRG sensory neurons (Ernfors

et al., 1994a,b; Klein et al., 1993). Although BDNF is a

member of NGF family, it has a distinct aspect different

from NGF. BDNF is produced not only by target tissues

but also by the neuron itself and transported anterogradely

(Zhou et al., 1996)(Figs. 2 and 3). This neuronal produc-

tion suggests its local neurotrophic effect via paracrine or

autocrine action. BDNF supports medium-sized DRG neu-

rons and motoneurons at the spinal anterior horn via its

high affinity receptor TrkB (Fig. 4).

Peripheral axotomy increases BDNF mRNA linearly in

the distal stump of axotomized nerves (Meyer et al., 1992)

and denervated muscles (Funakoshi et al., 1993). The tran-

sient increase in NGF synthesis in the distal stump of in-

jured nerves may serve to stimulate the expression of BDNF

(Apfel et al., 1996). BDNF mRNA is also increased in ax-

otomized DRG neurons (Ernfors et al., 1993). Its receptor

trkB mRNA was reported to be increased (Ernfors et al.,

1993)or unchanged in rat DRGs after nerve injury (Sebert

and Shooter, 1993).

Reports on BDNF in diabetes are less available than those

on NGF. Messenger RNAs of BDNF in soleus muscle and

DRG were increased in diabetic rats, and this increase was

reversed with insulin treatment (Fernyhough et al., 1995a,b).

However, endogenous BDNF protein in the sciatic nerve andantero- and retrograde axonal transport of BDNF are de-

creased in STZ-induced diabetic rats. The observation that

transport of radio-labeled BDNF is not affected by diabetes

suggests that reduced BDNF transport in diabetes is not a

result of impaired capacity for receptor-mediated transport

(Mizisin et al., 1999). The trkB mRNA in both full-length

and truncated forms was also decreased in the sciatic nerve

of 6-week diabetic rats but returned to the control level at

12-week diabetes (Rodiguez-Pena et al., 1995). Decreased

protein and increased mRNA levels of BDNF may indicate

compensatory up-regulation of BDNF production. These al-

terations may be due, in part, to the osmotic effect of hyper-

glycemia since similar results are reported in galactose-fedrats (Mizisin et al., 1999).

BDNF did not ameliorate a decrease of substance P or

CGRP in the sciatic nerve of diabetic rats (Diemel et al.,

1994). In galactose-fed rats, which develop a neuropathy

characterized by nerve conduction deficits and axonal at-

rophy, BDNF improved motor nerve conduction velocity

deficit in the sciatic nerve and ameliorated the diminution of

the caliber of dorsal root sensory axons but did not improve

sensory nerve conduction velocity deficit (Mizisin et al.,

1997a).During nerve regeneration, muscular BDNF mRNA

is regulated differently in soleus and gastrocnemius muscles

in diabetic rats (Fernyhough et al., 1996).

2.2.1.3. Neurotrophin-3 (NT-3). NT-3 is the third mem-

ber of the NGF family. This neurotrophin supports large

neurons of DRGs that mediate proprioception (Klein et al.,

1994;Ernfors et al., 1994a,b;Tessarollo et al., 1994). NT-3

binds to its high affinity receptor TrkC (Fig. 4) and promotes

peripheral nerve regeneration (Sterne et al., 1997). After

peripheral axotomy, NT-3 mRNA was decreased in the dis-

tal stump of the transected nerve and spinal cord but was

unchanged in the gastrocnemius (Funakoshi et al., 1993)

(Fig. 2).NT-3 enhances neurite outgrowth and up-regulates

mRNAs for GAP-43 and T1-tubulin in culture condition

(Mohiuddin et al., 1995a,b).

In uninjured diabetic rats, NT-3 mRNA in the hindlimb

skeletal muscle and sciatic nerve was decreased compared

with control rats (Rodiguez-Pena et al., 1995; Ihara et al.,

1996; Fernyhough et al., 1998a,b), while that in the dor-

sal root and sural nerve was increased (Cai et al., 1999)

(Table 2). The expression of trkC mRNA in the sciatic nerve

was also decreased in diabetic rats (Rodiguez-Pena et al.,

1995). NT-3 mRNA in the skin was not altered in diabetic

rats (Cai et al., 1999). NT-3 protein (Kennedy et al., 1998)

and trkC mRNA (Terenghi et al., 1997) were increased in

human diabetic skin. Together with the results of decreased


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axonal transport of NT-3 and decreased trkC mRNA ex-

pression in DRGs in diabetes (Fernyhough et al., 1998a,b),

this suggests that diabetic DRG neurons receive less support

by NT-3, which can be ameliorated with insulin treatment

(Fernyhough et al., 1998a,b).

2.2.1.4. Neurotrophin-4/5 (NT-4/5). NT-4 and NT-5 wereseparately discovered, but it became apparent that these two

factors were likely to be inter-species variants of the same

protein. That is why these proteins are commonly referred

to as NT4/5. NT-4/5 shows trophic effects via binding to

trkB. Thus, this protein has a spectrum of activity similar

to that of BDNF with respect to neuronal populations. In

the diabetic uninjured nerve, NT-4/5 mRNA is decreased at

12-week diabetes (Rodiguez-Pena et al., 1995).

2.2.2. Insulin-like growth factors

2.2.2.1. Insulin-like growth factor-I and -II (IGF-I/II).

Insulin-like growth factors (IGFs) are a family of struc-turally and functionally related proteins that include insulin,

IGF-I and IGF-II. IGFs have been implicated in the growth

and differentiation of neurons; both IGFs promote neu-

rite outgrowth of neuroblastoma cells (Recio-Pinto et al.,

1984a,b). IGFs bind to their own receptors, type-I and

type-II IGF receptors, although there is considerable cross

reactivity. The liver is the main source of serum IGFs in

rats, although neural tissue also produces IGFs. The major

sites of production and secretion of circulating IGF-II in the

postnatal animal are the choroid plexus and leptomeninges,

and its mRNA also exists in the nervous tissue in the

adult.IGFs also accelerate regeneration of sensory (Fernyhough

et al., 1993) and motor nerves (Near et al., 1992). Following

nerve injury, the expression of IGFs is increased in Schwann

cells in the distal stump (Glazner et al., 1994) (Fig. 2).

IGF receptors exist in neuronal cell bodies, axons and nerve

terminals. Serum IGFs are able to gain access to neurons

through fenestrated capillaries in DRG.

Serum IGF-I level is lower in diabetic patients than in

non-diabetic subjects, while serum IGF-II level is not dif-

ferent between the two groups. In comparing STZ-induced

diabetic rats with control rats, there is a decrease of mRNAs

of both IGF-I and IGF-II in the nerve of the STZ-induced

diabetic rats. The mRNA and protein expression of IGF-I

receptor is also decreased in the superior cervical ganglia of

STZ-diabetic rats (Bitar et al., 1997). This is also the case

with the model of NIDDM, obese Zucker rats, in which

IGF-II mRNA was decreased in the sciatic nerve, spinal cord,

and brain, while IGF-I mRNA was decreased in the liver

but not in the nervous system (Zhuang et al., 1997). Insulin

treatment partially improves nerve IGFs mRNA (Wuarin

et al., 1994). Systemic IGF-I or IGF-II treatment and local

application of IGF-I improved the regenerating rate of sen-

sory nerves in diabetic rats without affecting plasma glucose

levels (Ishii, 1995).

2.2.2.2. Insulin-like growth factor binding protein (IGFBP).

IGFBPs comprise a family of several proteins that bind

IGFs with high affinity and specificity and thereby regulate

IGF-dependent actions. Thus, IGFs exist not as free peptides

but bound to one of their binding proteins; six IGF-binding

proteins (IGFBPs) have been cloned, sequenced and char-

acterized (Shimasaki et al., 1991). IGFBP-3 is the ma-jor form in plasma and is responsible for regulating the

half-life of IGFs. In circulation, most IGFs are found in a

ternary complex with IGFBP-3 and an acid-labile subunit

that does not bind IGFs (Baxter, 1988). The ternary com-

plex (150 kDa) is unable to leave circulation because of

its size, leading to a prolonged half-life in IGFs within the

complex (Binoux and Hossenlopp, 1988). Therefore, this

complex acts as a circulating reservoir for IGFs. Small com-

plexes of IGFs and other IGFBPs (50 kDa) than IGFBP-3

are able to cross the capillary wall and serve to transport

IGFs to the tissues. Thus, IGFBPs are closely implicated

in the turnover of IGFs. The binary complexes, includ-

ing IGFs and the binding protein, have increased affinityfor the acid-labile unit, thereby forming the ternary com-

plex. In this state, IGF-I has reduced bioavailability and

prolonged half-life. By contrast, when IGF-I is bound to

any of the binding proteins without the acid-labile subunit,

IGF-I has a shorter turnover and increased bioavailabil-

ity. IGFBPs are synthesized in all tissues, including the

nervous system, and act as local regulators of IGF ac-

tions. IGFBPs have several functions including transport-

ing the IGFs in the circulation, mediating IGF transport

out of the vascular compartment, localizing the IGFs to

specific cell types and modulating both IGF binding to

receptors and growth-promoting actions. The functions ofIGFBPs appear to be altered through the reduction of the

binding affinity of IGFBPs for IGFs due to proteolysis

or posttranslational modifications including glycosylation

and phosphorylation. Proteolysis of the IGFBP decreases

its affinity for IGF, promoting its release to the receptor.

Phosphorylation alters the binding affinity of IGFBP-1 for

IGFs and also may alter its ability to bind to cell sur-

faces. The role of the oligosaccharide chains of IGFBPs

is unknown.

Circulating levels of IGFBP-1 are increased in IDDM

patients (Suikkari et al., 1988) and IDDM patients with

neuropathy (Crosby et al., 1992). An inverse relationship

between IGFBP-1 and insulin level was demonstrated in

adolescents with IDDM (Batch et al., 1991). In addition,

IGFBP-1 levels are correlated with mean 12-month HbA1c

levels, and improvement of glycemic control with contin-

uous subcutaneous insulin infusion for 2 months results

in normalization of IGFBP-1 levels in adolescents with

IDDM (Batch et al., 1991). These findings may well ex-

plain the regulation of IGFBP-1 by insulin. However, it was

also reported that diabetic patients had markedly elevated

plasma IGFBP-1 levels and lower plasma IGF-I levels even

though these patients were hyperinsulinemic. Thus, overall,

poor glycemic control in type 1 diabetes is associated with


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elevated serum IGFBP-1 levels and reduced IGF-I levels.

Increasing age is accompanied by a further decrease in

serum IGF-I levels as well as an increase in IGFBP-1 levels

in adult diabetic type 1 and type 2 subjects. IGFBP-3 levels

are decreased in poorly controlled IDDM subjects (Baxter

and Martin, 1986). The puberty-related rise in IGFBP-3

levels is also blunted by diabetes (Batch et al., 1991).Theseobservations may be explained by the decreased levels of

IGF-I in patients with IDDM (Bach and Rechler, 1992).

As in IDDM patients, STZ-diabetic rats showed increased

IGFBP-1 (Unterman et al., 1990) and decreased IGFBP-3

serum level (Zapf et al., 1989).

2.2.3. Hematopoietic cytokines

Peripheral nerve production of cytokines originates from

resident and recruited macrophages, lymphocytes, masto-

cytes, Schwann cells and probably neurons. Cytokines are

involved in nerve lesions and repair. Tumor necrosis factor-

(TNF-) injected into nerve induces Wallerian degeneration,

whereas interleukin-1 (IL-1) production promotes detersionby scavenger macrophages and synthesis of neurotrophic

factors (NGF and leukemia inhibitory factor (LIF)). After

experimental axotomy, other neurotrophic factors, including

IL-6, LIF and transforming growth factor-1 (TGF-1), are

overexpressed in the nerve and promote axonal growth until

axonSchwann cell contact.

2.2.3.1. Ciliary neurotrophic factor (CNTF). CNTF is

produced by myelinating Schwann cells in the intact nerve,

although it is not released under normal condition. Once

the nerve is injured, CNTF is released and incorporated

into DRG neurons (Sendtner et al., 1992). Although DRGneurons express CNTF receptor complex consisting of

CNTFR-, gp130 and LIFR-, they do not increase the ex-

pression of CNTF receptor complex after axotomy (Curtis

et al., 1993). An increase in the uptake of CNTF after

axotomy might be associated with increased release of

CNTFR-from the denervated muscles (Davis et al., 1993).

In the spinal cord, CNTFR mRNA is increased after nerve

injury (Mata et al., 1993). The treatment with CNTF after

axotomy resulted in an increase in the number of MNFs

and myelination in rats. CNTF receptor is more important

for the nervous system than CNTF itself; mice lacking

CNTFR- showed significant motor neuron loss unlike

CNTF knockout mice (De Chiara et al., 1995).

Immunohistochemical staining showed a decrease in

CNTF expression in the sciatic nerve of patients with mo-

tor neuron disease but not in patients with diabetic motor

neuropathy (Lee et al., 1996).In diabetic rats, nerve CNTF

mRNA and bioactivity (Calcutt et al., 1992) are reported

to be decreased or to be unchanged (Ohi et al., 1998).

The production failure seen in experimental models might

be due to metabolic changes caused by an accelerated

polyol pathway, since ARI treatment increased CNTF in the

galactose-fed rat, which is the model of the accumulation

of polyol (Mizisin et al., 1997b).

2.2.3.2. Tumor necrosis factor- (TNF-). TNF- is one

of the major cytokines and is implicated in a variety of ac-

tions, including regulation of immune response and con-

trol of cell growth and differentiation through paracrine

and autocrine networks in a variety of tissues, including

the nervous system (Vassalli, 1992). There is growing cir-

cumstantial evidence that TNF- plays a role in the patho-genesis of inflammatory demyelinating disorders, including

Gullain-Barre syndrome (Tsukada et al., 1991) and multi-

ple sclerosis (Hofman et al., 1989). TNF- is expressed in

macrophages, Schwann cells or fibroblasts within the en-

doneurium in healthy subjects, and its immunoreactivity has

been reported to be enhanced in neuropathies of various eti-

ologies (Deprez et al., 2001).Indeed, TNF- exerts delete-

rious effects including demyelination on nerve fibers either

in vitro (Selmaj and Raine, 1988)or in vivo (Redford et al.,

1995).

Although by immunohistochemistry the expression of

TNF- has not been shown to be increased in the PNS in

diabetic condition, its serum level has been reported to be el-evated in diabetic patients (Katsuki et al., 1998; Lechleitner

et al., 2000) as well as in diabetic animals. Interestingly,

inhibitors of TNF- including N-acetylcysteine (Sagara

et al., 1996), troglitazone (Qiang et al., 1998a,b) and gli-

clazide (Qiang et al., 1998a,b) have been shown to inhibit

the development of peripheral neuropathy in STZ-induced

diabetic rats. Since these compounds also have the feature

of free radical scavenger, it is unclear whether the effect of

those compounds on nerve dysfunction is mediated through

inhibiting TNF-activity and whether the increased level of

serum TNF-contributes to nerve dysfunction or disturbed

nerve regeneration in diabetes.

2.2.3.3. Interleukin-6 (IL-6). IL-6 belongs to the neu-

ropoietic cytokine superfamily, which includes various cy-

tokines presented inTable 1.All of these cytokines use the

common signal-transducing receptor compound gp130 (Ip

et al., 1992).The activation of this receptor is triggered by

several types of receptorligand interactions. IL-6 is synthe-

sized in a subpopulation of developing peripheral sensory

and sympathetic neurons (Murphy et al., 1995; Gadient and

Otton, 1996). In the adult nervous system, IL-6 level is

hardly detectable, but IL-6 synthesis appears to be strongly

increased during pathological situations. The level of IL-6

mRNA was elevated in the non-neuronal cells surrounding

the motor fibers of the facial nucleus after motoneuron ax-

otomy (Kiefer et al., 1993). An increase in IL-6 synthesis

was found either in the sciatic nerve at sites undergoing

Wallerian degeneration (Bolin et al., 1995; Bourde et al.,

1996) or in the DRG neurons within 1 day after sciatic

nerve injury (Murphy et al., 1995). After sciatic nerve crush,

its functional recovery was delayed in IL-6 gene knockout

mice as analyzed from a behavioral footprint assay. Com-

pound action potentials after crush lesion showed that there

was a very low level of recovery of the sensory but not of

the motor branch of the mice. Thus, sensory functions were


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impaired in the intact adult animals and regeneration of the

lesioned sensory axons was delayed in IL-6 gene knockout

mice (Zhong et al., 1999).The magnitude of increased IL-6

level after sciatic nerve transection was smaller in diabetic

nerves than in control nerves, although its significance

remains unclear (Takagi et al., 2001).

2.2.4. Glial cell line-derived neurotrophic factor (GDNF)

GDNF is a neurotrophic polypeptide, distantly related to

transforming growth factor- (TGF-). Although GDNF

was originally reported to support dopaminergic neurons

(Lin et al., 1993, 1994), it has more recently been shown

to be a potent neurotrophic factor for peripheral neu-

rons, including enteric, sympathetic, motor and sensory

ones.

GDNF is produced in the skin, kidney, stomach and

testis. Low levels of GDNF mRNAs have also been

found in the skeletal muscle, ovary, lung, adrenal gland,

spinal cord, superior cervical ganglion and DRG (Trupp

et al., 1995). The receptor tyrosine kinase RET is thesignaling receptor (Trupp et al., 1996) and acts in con-

cert with one or several glycosylphosphatidylinositol

(GPI)-linked proteins: GDNF family receptor compo-

nents (GFR) 1 - 4 (Airaksinen et al., 1999). In DRG,

non-peptidergic small neurons postnatally switch their

dependency from NGF to GDNF (Silos-Santiago et al.,

1995; Bennett et al., 1996; Molliver and Snider, 1997).

These neurons bind the isolectin IB4 and express thi-

amine monophosphate (TMP). IB4-positive central axons

terminate in the inner layer of lamina II in the dorsal

horn (Averill et al., 1995). IB4-positive neurons synthe-

size RET, GFR 1 and GFR 2 (Molliver et al., 1997;Bennett et al., 1998a,b). The function of these neurons is

unclear. Although IB4-positive neurons have a high density

of voltage-gated tetrodotoxin-resistant Na+ channels com-

pared to peptidergic neurons (Stucky and Lewin, 1999),

noxious heat causes a great currents in peptidergic neurons,

but much smaller currents in IB4 neurons (Stucky et al.,

1999). IB4 neurons might be associated with neuropathic

pain but not inflammatory pain (Snider and McMahon,

1998).

Two days after transection of the sciatic nerve, GDNF

mRNA is increased dramatically (400500 times) (Trupp

et al., 1995).In DRG, RET mRNA is increased only slightly.

However, GFR1 and 2 are highly increased after axotomy

(Kashiba et al., 1998; Bennett et al., 2000). The treatment

with GDNF restores IB4 binding/TMP expression (Bennett

et al., 2000) and stimulates the regeneration of P2X3-positive

axons (Ramer et al., 2000). In addition, GDNF treatment

improved the recovery of the sensitivity to noxious heat and

pressure.

Although any change in either GDNF synthesis or its re-

ceptor expression has not been reported in diabetes, intrathe-

cal GDNF treatment restored TMP labeling in the inner layer

of lamina II in STZ-induced diabetic mice (Akkina et al.,

2001).

2.3. Extracellular matrix

The extracellular matrix of the peripheral nerve mechani-

cally supports the cells that it surrounds, but it also regulates

their behavior through specific interactions mediated via

molecules on the cell surface, such as integrin receptors

and cell surface proteoglycans. Thus, changes in the struc-ture and composition of the extracellular matrix may alter

cellular functions in multiple ways. At the ultrastructural

level, these changes include thickening of vascular, per-

ineurial and Schwann cell associated basement membranes;

accumulation of microfibrillar material in the vicinity of

perineurial cells; and increased diameter of endoneurial

collagen fibrils. At the molecular level, the changes may

be associated with altered metabolism of various collagen

types, such as type IVI collagens, laminin and fibronectin.

2.3.1. Laminin

Laminin is an 800 kDa heterotrimeric glycoprotein con-

sisting of three subunits, a large A chain and two smallerchains, B1 and B2, and constitutes one of the major extra-

cellular matrices consisting of basement membrane (Lander,

1987). Laminin readily binds to itself, type VI collagen,

proteoglycans, entactin and perhaps other extracellular ma-

trix constituents. In the PNS, the A chain is replaced by the

merosin M chain, a major component of basal lamina. Nerve

elongation is modified by neurite outgrowth domain peptide

(p20) of B2 chain (Liesi et al., 1989) via31 integrin re-

ceptor (Tomaselli et al., 1993). Antibodies against laminin

(Wang et al., 1992) or its receptor (Toyota et al., 1990) inhibit

peripheral nerve regeneration. Although the significance is

unknown, neuronal cells, as well as non-neuronal cells, pro-duce laminin B2 chain (Le Beau et al., 1994) and up-regulate

its mRNA during nerve regeneration (Le Beau et al., 1995).

Attachment of neurons to extracellular substrate is an im-

portant phase for neurite outgrowth in culture. Diabetic DRG

neurons are impaired to adhere to laminin, type I and IV

collagens, and fibronectin (Sango et al., 1995).This defect

was prevented by treatment with aldose reductase inhibitor

(ARI) ONO-2235 (Sango et al., 1999). The mechanism of

this therapeutic effect of ARI remains unclear, although

the function of receptors for extracellular matrix might be

ameliorated.

Extracellular matrix proteins are glycated under long-term

hyperglycemia. Indeed, the presence of advanced glycosy-

lation end products was shown in the nerve of STZ-induced

diabetic rats (Yagihashi et al., 1992) and diabetic patients

(Sugimoto et al., 1997). Glycation may prevent binding of

laminin to members of the integrin family and thus com-

promise its metabolic activity. It was reported that non-

enzymatic glycosylation of laminin and the laminin peptide

IKVAV inhibited neurite outgrowth by cultured neuroblas-

toma cells (Federoff et al., 1993). Immunohistochemical

study showed the labeling for laminin in the basal laminae

of blood vessels and Schwann cells. In the perineurium,

it was restricted to the innermost layer, where the amount


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of laminin appeared to be increased in the diabetic nerve

compared with the control nerve (Bradley et al., 2000).

2.3.2. Fibronectin

Fibronectin, a dimer of 440 kDa, is a major constituent of

extracellular matrix in the developing PNS and contributes to

nerve regeneration. It is also found in soluble form in plasma,which leaks into the endoneurium from endoneurial mi-

crovessels immediately after nerve injury (Salonen, 1987).

There are various related isoforms because of RNA splic-

ing and posttranslational modifications. Its neuronal recep-

tors are molecules in the integrin family (Reichardt et al.,

1989).During nerve development and regeneration, axonal

integrin 51, which is localized in the filopodia and central

region of growth cones (Yanagida et al., 1999),is thought to

bind to fibronectin and interact with actin filament (Lefcort

et al., 1992).Non-neuronal cells from the sciatic nerve ex-

press increased levels of mRNAs for fibronectin and type I

and IV collagens under high glucose concentration (Muona

et al., 1991). By imunohistochemistry, the labeling for fi-bronectin was noted in the basal laminae of blood vessels and

Schwann cells and in those of the perineurial cells through-

out all layers in the sural nerve. The distribution and stain-

ing of fibronectin was almost similar among normal nerves,

the nerves from diabetic neuropathy and those from other

neuropathies (Bradley et al., 2000).

2.3.3. Collagen

Type IV collagen is another major constituent of the base-

ment membrane and also supports nerve regeneration. Under

high glucose condition, mRNAs of pro-1 and pro-2 type

IV collagen and pro-1 type I collagen chains have been re-ported to be increased in Schwann cells and perineurial cells

(Muona et al., 1991). Indeed, increased synthesis of type IV

collagen might make a massive deposit of microfibrils be-

tween perineurial cell layers (Muona et al., 1993; Bradley

et al., 2000).

Circular basal laminal tubes after axonal degeneration are

characteristically observed in diabetic polyneuropathy (King

et al., 1989).In normal condition, these Schwann cell basal

laminal tubes collapse following the removal of the axonal

and myelin debris. By contrast, these tubes are filled with

densely packed collagen fibrils in diabetes, forming circular

basal laminal tubes. These fibrils may impede nerve fiber

regeneration. The diameter of endoneurial collagen fibrils is

increased in diabetic BB Wistar rats (Muona et al., 1989). Al-

though the significance of this finding remains unclear, this

is also found in patients with hereditary motor and sensory

neuropathy as well as in diabetic patients, suggesting that

the increase in the diameter of collagen fibrils is not specific

for diabetic neuropathy but merely a part of nerve degener-

ation (Bradley et al., 2000). Among various types of colla-

gen, type IV collagen was increased in the endoneurium in

diabetic patients (Muona et al., 1993; Bradley et al., 2000).

The deposit of collagen fibrils per se might prevent nerve re-

generation. In addition, glycated collagen, which is expected

to be increased in diabetic condition, is resistant to protease

digestion, which could be enhanced in regenerating axons

(Lubec and Pollak, 1980), possibly leading to impaired nerve

regeneration in diabetic neuropathy.

2.3.4. Matrix metalloproteinases (MMPs)

Both tissue repair and elongation of axons play importantroles in the process of nerve regeneration. Proteases partici-

pate in the former event; matrix metalloproteinases (MMPs)

are one group of proteases involved in wound healing.

MMPs are expressed within various cells in inflammatory

lesions. The substrates for MMPs include extracellular ma-

trixes, including type IV collagen and fibronectin. In the

PNS, both MMP-2 and MMP-9 are known to be expressed

during nerve regeneration after crush injury (La Fleur et al.,

1996). Sensory neurons responsive to NGF in DRG express

MMP-2 (Muir, 1994). Schwann cells and perineurial cells

also express MMP-2 (Kherif et al., 1998). Remodeling of

the extracellular matrix by proteolytic activity is known

to be crucial for growth-cone motility. Neurite outgrowthof DRG neurons was reported to be suppressed by MMP

inhibitor and inversely activated by NGF treatment accom-

panied with increased expression of MMP-2 (Muir, 1994).

However, in vivo study showed the discrepant results that

MMP inhibitor BB-1101 had no effects on compound

muscle action potential recorded from denervated exten-

sor digitorum or morphometric results on the diameter of

regenerating axons (Demestre et al., 1999).

Extracellular matrix is glycated in diabetic condition. Gly-

cated type IV collagen is resistant to proteolysis by MMPs

(Mott et al., 1997). This may be true for the basement mem-

brane of the epidermis. Our skin biopsy studies suggest thateither epidermal nerve fiber number or length, which are

decreased in diabetic patients, is unable to be elongated by

treatment with aldose reductase inhibitor, whereas dermal

nerve fiber length is able to be elongated (Hirai et al., 2000;

Yasuda et al., 2000). Since epidermal reinnervation is ob-

tained mainly by the reentry of dermal nerves into the epi-

dermis through the basement membrane of epidermal basal

cells, it is likely that glycated extracellular matrix prevents

the penetration of regenerating or sprouting nerve fibers into

the epidermis, although there is no clear evidence.

2.4. Cell adhesion molecules

Interactions of cell membranes with adhesion molecules

expressed either on other cell membranes or on extracel-

lular matrices may play a potentially important role in the

morphogenesis of the nervous tissue. Among cell adhesion

molecules (CAMs), neural cell adhesion molecule (NCAM),

L1 cell adhesion molecule (L1) and myelin-associated

glycoprotein (MAG) all belong to the immunoglobu-

lin superfamily and are best characterized. The former

two molecules and N-cadherin are expressed on axons

and Schwann cells. These molecules are involved in the

axon-to-axon and axon-to-Schwann cell attachments using


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homophilic interactions. Axonal integrins such as 11 and

61, which function as receptors, mediate the attachment

between axons (integrin) and Schwann cell basal laminae

(laminin) using heterophilic interactions. Growing cultured

neurites are slowed by the presence of antibodies against

these proteins. The level of CAMs in Schwann cells are

decreased with myelination and re-expressed at high lev-els during Wallerian degeneration after nerve transection.

These observations suggest that CAMs contribute to mem-

brane interactions with other membranes or matrix during

development and axonal regeneration after injury.

2.4.1. Neural cell adhesion molecule (NCAM)

NCAM is the first adhesion molecule that was identified

in the nervous system, and it is known that NCAM has an

important role in axon guidance and projection to targets.

Therefore, changes in its expression or nature may provide

a significant influence on the function of nerve fibers. It was

already reported that NCAM, tenascin and N-cadherin were

significantly up-regulated, whereas polysialic acid was sig-nificantly decreased with direct and indirect enzyme-linked

immunosorbent assays (ELISAs) in diabetic rats, although

no differences were detected by immunohistochemistry be-

tween diabetic (6-month BB/W) and control rats. In view

of the fact that impaired nodal Na+ currents are associ-

ated with displacement of nodal Na+ channels across the

damaged paranodal barrier, which is made up of adhesion

molecules, these data may suggest that imbalances between

highly interactive molecules responsible for the adhesive-

ness between the terminal Schwann cell loop and paranodal

axolemma in diabetes may underlie the critical paranodal

barrier defect in diabetic neuropathy (Merry et al., 1998).In addition, since glycating hexoses, including glucose and

fructose, bind to the lysine residue of peptides, which is

the amino acid residue to which polysialic acid associates

with NCAM, it may be possible that increasing glycation of

NCAM may prevent the interaction between polysialic acid

and NCAM, thereby diminishing the adhesiveness of junc-

tional complexes.

2.4.2. L1 cell adhesion molecule (L1)

L1 is a cell adhesion molecule that is expressed on the sur-

face of developing axons, growth cones, and Schwann cells

of unmyelinated fibers (Martini and Schachner, 1986)and is

involved in neurite outgrowth, migration and fasciculation.

These actions are elicited by its homophilic and heterophilic

interactions (Lagenaur and Lemmon, 1987; Lemmon et al.,

1989). The homophilic interactions on growth cones activate

the fibroblast growth factor (FGF) receptor, which initiates

arachidonic acid production, Ca2+ influx, cytoskeletal rear-

rangements and neurite outgrowth (Williams et al., 1994).

The cytoplasmic domain of L1 can bind ankyrin (Davis,

1994) and may provide a mechanism for transducing

extracellular signals and dynamic cytoskeletal rearrange-

ments required for cell migration (Burden-Gulley et al.,

1997). L1 heterophilic interactions with TAG-1/axonin-1,

DM1-GRASP and 13 integrin have also been impli-

cated in neurite outgrowth (Kuhn et al., 1991; DeBernardo

and Chang, 1996), although the downstream mechanisms

are not well understood. An analysis of L1-deficient mice

showed that axonal-L1 maintains Schwann cell ensheath-

ment of adult sensory unmyelinated axons by heterophilic

binding mechanisms and that loss of axonal-L1 resulted inaxonal degeneration (Haney et al., 1999).

Although L1 is a membrane glycoprotein expressed on

neural cells, the soluble form of L1 is generated in vivo by

proteolysis. The soluble form of L1 without cytoplasmic

and membrane spanning domains, which is secreted from a

stable transfectant of CHO cells, induced neurite outgrowth

of explants from embryonic chick brain stem comparable

with that with substrate-bound L1 (Sugawa et al., 1997).

It was also reported that cerebellar neurons responded to

a soluble recombinant L1-Fc chimera by extending longer

neurites than controls. The response was inhibited by pre-

treating neurons with antibodies to L1 (Doherty et al.,

1995).These data suggest that the ability of CAMs to stim-ulate neurite outgrowth can be dissociated from their ability

as substrate-associated adhesion molecules and point to the

potential of using the soluble form of L1 to promote nerve

regeneration.

In the PNS, in uninjured animals, L1 and its close ho-

mologue CHL1 mRNAs were expressed at moderate levels

by small- to medium-sized sensory neurons, and L1 mRNA

was expressed at moderate levels by motor neurons (Zhang

et al., 2000).Many large sensory neurons expressed neither

L1 nor CHL1 mRNAs, and motor neurons expressed little

or no CHL1 mRNA. Neither up-regulation of L1 mRNA in

all neurons nor that of CHL1 mRNA was found after axo-tomy. CHL1 mRNA was transiently increased following sci-

atic nerve crush and declined to control levels. CHL1 mRNA

was also up-regulated by many presumptive Schwann cells

in injured sciatic nerves.

There has been neither data on the expression of L1 nor

reports on its contribution to impaired nerve regeneration in

diabetes.

2.4.3. Myelin-associated glycoprotein (MAG)

MAG, a well-characterized myelin protein, is a bi-

functional molecule: (1) inhibiting neurite outgrowth

from both developing cerebellar and adult DRGs in vitro

(Mukhopadhyay et al., 1994) and axonal regeneration (Tang

et al., 1997) and sprouting (Shen et al., 1998);or (2) promot-

ing neurite outgrowth from newborn DRG (Mukhopadhyay

et al., 1994). MAG knockout mice reveal that MAG is not

essential for the initiation of myelination, although it plays

an important role in maintaining a stable interaction between

axon and myelin (Bartsch et al., 1997). The distal segment

of the crush-injured sciatic nerve showed a decrease in the

level of MAG mRNA 2 days after crush injury, which was

followed by its progressive increase between 7 and 21 days

after injury. By Western blot, the level of MAG protein was

shown to be substantially decreased between 7 and 21 days


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after injury (Gupta et al., 1990). By contrast, both mRNA

and protein of MAG were undetectable in the distal segment

of the sciatic nerve 35 days after permanent transection,

suggesting distinct down-regulation of MAG gene expres-

sion after permanent transection of the peripheral nerve

(Gupta et al., 1990). Thus, the expression of MAG may

play a significant role in the process of nerve regeneration.Myelin is synthesized about the time of birth. The

Src-family tyrosine kinase Fyn, which is present in

myelin-forming cells, is activated through stimulation of

cell surface receptors such as large myelin-associated gly-

coprotein (L-MAG) at the initial events of myelination

(Umenomori et al., 1994). Myelin basic protein (MBP),

which is a major myelin-specific protein and plays special

roles in the initial stages of myelinogenesis, is significantly

reduced in Fyn-deficient mice. In addition, Fyn has been

shown to stimulate the promoter activity of the MBP gene,

suggesting an important role of Fyn in myelination through

transactivation of the MBP gene (Umenomori et al., 1999);

although, the possibility that MAG and Fyn act indepen-dently to initiate myelination has been proposed in that

possible compensatory mechanisms other than MAGFyn

signaling pathway may well explain a slight hypomyelina-

tion in MAG-deficient mice (Biffiger et al., 2000).

Although the expression of MAG in diabetic nerves does

not differ from that in control nerves, the expression of MAG

during Wallerian degeneration after nerve crush is larger

in diabetic than control nerves (unpublished observations).

This increase may contribute to impaired nerve regeneration

in diabetic state.

2.4.4. OthersNa+/K+-ATPase plays an important role in peripheral

nerve function. Three isoforms of catalytic subunits and

two isoforms of glycosylated subunits of the enzyme

have been identified (Shull et al., 1986), and all distribute

in a cell- and tissue-specific manner. Nerve injury ex-

periments using immunohistochemical and Western blot

analysis have revealed that 3 and 1 isoforms are ex-

clusive for axons and 2 and 2 isoforms are exclusive

for Schwann cells, although axonal contact regulates 2

and 2 isoform expressions; after sciatic nerve injury, 3

and 1 isoforms completely disappeared from the dis-

tal segment, whereas 2 and 2 isoform expressions are

markedly increased in Schwann cells in the distal segment

of the injured sciatic nerve, followed by a return to the

baseline with nerve regeneration (Kawai et al., 1997). Be-

cause the 2 isoform is known as an adhesion molecule

on glia (AMOG) (Gloor et al., 1990), increased expres-

sion of AMOG/2 on Schwann cells in the segment dis-

tal to sciatic nerve injury suggests that AMOG/2 may

act as an adhesion molecule in peripheral nerve regen-

eration. Although possibly altered AMOG/2 expression

in diabetes may contribute to disturbed nerve regenera-

tion, there have been no data on its expression in diabetic

nerves.

The serum levels of soluble forms of CAMs including

intercellular adhesion molecule-1 (sICAM-1) have been

reported to be elevated under poor glycemic control and to

be reversed by intensive insulin treatment. It was reported

that plasma CAMs might be a predictor of the develop-

ment of diabetic neuropathy (Jude et al., 1998):in a 5-year

follow-up study of 28 diabetic patients, they found thatboth P-selectin and ICAM-1 were increased at baseline

in patients with neuropathy compared to non-neuropathic

patients. P-selectin and E-selectin were also found to be

significantly higher at baseline in patients who at follow-up

showed deterioration in peroneal nerve conduction velocity

of more than 3 m/s. P-selectin and ICAM strongly correlated

with the velocity. Univariate and multivariate regression

analyses showed a significant inverse association between

increasing log P-selectin, log E-selectin and log ICAM-1

with decreasing velocity. This was true even after adjust-

ment for glycemic control. P-selectin and E-selectin were

significantly associated with the risk of deterioration of the

conduction velocity after 5 years. These results suggest animportant role of CAMs in the development and progression

of peripheral neuropathy in diabetes mellitus. However, it

remains unclear how important they are and whether they

are associated with the process of nerve regeneration in

diabetic condition.

2.5. Cell signal messengers

2.5.1. cAMP

Cyclic AMP, synthesized from ATP by adenylate cyclase,

is a second messenger of various cellular signals. In neu-

ronal cells, adenylate cyclase is associated with receptors ofvarious molecules, such as neurotransmitters, neurotrophic

factors, prostaglandins, and others. The activated adenylate

cyclase promotes synthesis and accumulation of cAMP in

neuronal cells, and the increased cAMP activates protein

kinase A.

There is increasing evidence that the activation of

cAMP-signal promotes neuronal survival and axonal elon-

gation in neuronal cells. In a series of experiments,Roisen

et al. (1972) reported that dibutyryl cAMP, a cAMP ana-

logue, promoted the in vitro elongation of neurites from

chick sensory ganglia. In addition to the in vitro effect,

intramuscular injections of this compound enhanced sig-

nificantly the regenerative rate of sensorimotor nerves in

either crushed or hemisected rat sciatic nerves (Pichichero

et al., 1973). Furthermore, the compound accelerated the

initial process of Wallerian degeneration and enhanced the

rate of increase in number and diameter of myelinated

nerve fibers (Gershenbaum and Roisen, 1980). However,

discrepant results

Diabetic Neuropathy & Nerve Regeneration

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