diabetic neuropathy & nerve regeneration
TRANSCRIPT
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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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232 H. Yasuda et al. / Progress in Neurobiology 69 (2003) 229285
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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234 H. Yasuda et al. / Progress in Neurobiology 69 (2003) 229285
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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236 H. Yasuda et al. / Progress in Neurobiology 69 (2003) 229285
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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H. Yasuda et al. / Progress in Neurobiology 69 (2003) 229285 237
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