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Amelioration of diabetic peripheral neuropathy by implantation of hematopoietic mononuclear cells in streptozotocin-induced diabetic rats

https://doi.org/10.1016/j.expneurol.2005.11.001Get rights and content

Abstract

This study was performed in order to evaluate the angiogenic effect of implantation of either peripheral blood mononuclear cells (PBMNCs) or bone marrow mononuclear cells (BMMNCs) on diabetic peripheral neuropathy. Streptozotocin (50 mg/kg) was injected intravenously into 6-week-old male Lewis rats. Four weeks after the induction of diabetes, 6 × 107 of PBMNCs or 1 × 108 of BMMNCs were implanted into the left hindlimb muscle. Motor nerve conduction velocity (MNCV) was monitored before and after implantation. At the end of the experiment, bilateral nerve blood flow (NBF) was measured by laser Doppler and the number of vessels in the sciatic nerves quantified by Factor VIII staining of the sections. Diabetes resulted in an approximately 20% reduction (P < 0.01) in sciatic MNCV. Four weeks after implantation, MNCV was improved by 54% with PBMNCs and by 67% with BMMNCs (both P < 0.01). Moreover, the effects of implantation were almost abolished by administration of VEGF-neutralizing antibody. Sciatic NBF was reduced by approximately 50% by diabetes (P < 0.05). This reduction in perfusion was improved by 74% by implantation of PBMNCs and by 62% by implantation of BMMNCs (P < 0.05 and P < 0.01, respectively). These effects were observed only in the implanted limb. Immunohistochemical staining of sciatic nerve sections for Factor VIII showed no significant increase in the number of vessels in the sciatic nerve following implantation of either PBMNCs or BMMNCs. These data suggest that implantation of hematopoietic mononuclear cell fractions is associated with an improvement in MNCV as a result of arteriogenic effects in the sciatic nerve, and that VEGF may contribute to this effect. This improvement occurred in the absence of angiogenesis. Implantation of these cell fractions may therefore be a potential new therapeutic method for treating diabetic peripheral neuropathy.

Introduction

Peripheral neuropathy is a common complication in patients with both type 1 and type 2 diabetes mellitus. Axonal degeneration and disappearance, segmental demyelination and disordered repair are all observed in the nerves of patients with diabetes. In addition, during hyperglycemia, nerve maturation secondary to nerve fiber loss becomes disturbed, with abnormal excitement of these immaturely regenerated nerve fibers causing spontaneous pain, numbness and paresthesia. In the majority of patients, these characteristic changes of diabetic peripheral neuropathy decrease the quality of life considerably, with progression of neuropathy leading to skin ulceration, gangrene and ultimately amputation of the foot.

It has been proposed that diabetic peripheral neuropathy is accompanied by metabolic abnormalities caused by hyperglycemia per sé and also by ischemia and hypoxia in nutrient vessels in the nerves (Cameron et al., 2001). The precise pathogenesis of diabetic neuropathy, however, remains to be established. A number of mechanisms have been proposed to account for the metabolic derangements and include nonenzymatic glycation products, activated protein kinase Cβ, altered neural polyol metabolism, oxidative stress and reduced availability of neurotrophic factors. Several basic and clinical trials have been carried out to investigate these mechanisms using compounds such as aminoguanidine, aldose reductase inhibitors, a protein kinase Cβ inhibitor, the antioxidant, dl-α-lipoic acid, nerve growth factor (NGF) and neurotrophin 3 (NT-3) (Chen et al., 2004, Elias et al., 1998, Goss et al., 2002, Hotta et al., 2001, Kishi et al., 1999, Monnier, 1996, Nakamura et al., 1999, Pradat et al., 2001, Shimoshige et al., 2000, Steele et al., 1993, Yagihashi et al., 2001). However, all these trials were unable to restore neural function completely after the onset of subjective symptoms.

Ischemia is considered to be another factor that contributes to the development of diabetic neuropathy (Cameron et al., 2001). Because the microcirculation is regulated by humoral, endothelial and neural factors, a potential pathogenic vicious cycle may develop in which microcirculatory dysfunction results in peripheral nerve dysfunction, that in turn results in abnormal regulation of the microcirculation leading to further nerve dysfunction. The reduction in endoneural blood flow has been shown to be ameliorated by treatment with various vasodilatory agents such as prostaglandin E1 analogues, alpha-adrenergic receptor blockers, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists and endothelin receptor antagonists (Cameron et al., 1991, Cameron et al., 1992, Jarvis et al., 2000, Maxfield et al., 1995, Obrosova et al., 2000, Yasuda et al., 1989). These treatments were also accompanied by beneficial effects on neural function in animal models of diabetes.

We reported previously that blood flow was improved by implantation of hematopoietic mononuclear cells in ischemic myocardium and hindlimb muscles (Iba et al., 2002, Kamihata et al., 2001, Kamihata et al., 2002, Tateishi-Yuyama et al., 2002). Therefore, we postulate that therapeutic angiogenesis resulting from implantation of blood cells may also ameliorate neural dysfunction in diabetic animals. In the present study, we evaluated the angiogenic effect of implantation of peripheral blood mononuclear cells (PBMNCs) and bone marrow mononuclear cells (BMMNCs) on diabetic peripheral neuropathy.

Section snippets

Animal and experimental protocol

The procedures used in the study were approved by the Animal Experimentation Committee of Kansai Medical University and were carried out in accordance with the “Guidelines for the Care and Use of Experimental Animals” of the Japanese Society of Laboratory Animals. Six-week-old, male Lewis rats (180–200 g) were housed two per plastic cage on a 12–12 h light–dark cycle with food and water available ad libitum. The rats were assigned randomly to either diabetic or control group and then equally to

Results

In order to evaluate peripheral nerve function, we measured sciatic MNCV (Fig. 2). Streptozotocin-induced diabetes caused a 20% reduction in sciatic MNCV before implantation. PBMNCs (∼ 6 × 107 cells) or BMMNCs (∼ 1 × 108 cells) were then implanted into the left hindlimb muscle of the STZ rats. Two weeks later, the reduction in MNCV was ameliorated significantly by implantation of both cell types. Four weeks after implantation, the reduction in MNCV was improved by 54% with PBMNCs and by 67% with

Discussion

The main finding of this study was that implantation of PBMNCs and BMMNCs into the hindlimb muscle of streptozotocin-induced diabetic rats (STZ rats) effectively ameliorated diabetic peripheral neuropathy. STZ rats are a model of type 1 diabetes and develop diabetic neuropathy characterized by a reduction in nerve conduction velocity, a decrease in myelinated fiber size and several metabolic derangements. As these changes are recognized as classical features of diabetic neuropathy, the clinical

Acknowledgments

This study was supported in part by grants-in-aid to A. K. from the Ministry of Education, Science, and Culture and from the Ministry of Health Labor and Welfare, Japan.

We thank Ms. Yoshimi Togawa, Ms. Midori Nakata and Dr. Kazuta Yasui for the technical assistance and Yumiko Izuo for the secretarial aid.

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