Effects of Basic Fibroblast Growth Factor on Experimental Diabetic Neuropathy in Rats
http://www.100md.com
糖尿病学杂志 2006年第5期
1 Division of Metabolic Diseases, Department of Internal Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan
2 Department of Ophthalmology, Fujita Health University School of Medicine, Aichi, Japan
3 Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan
4 Institute for Frontier Medical Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan
bFGF, basic fibroblast growth factor; CGH, cross-linked gelatin hydrogel; HGF, hepatocyte growth factor; MCT, mean circulation time; MNCV, motor nerve conduction velocity; NBF, nerve blood flow; NGF, nerve growth factor; RBF, retinal blood flow; SNBF, sciatic NBF; STZ, streptozotocin; VEGF, vascular endothelial growth factor; VFA, video fluorescein angiography
ABSTRACT
Basic fibroblast growth factor (bFGF) stimulates angiogenesis and induces neural cell regeneration. We investigated the effects of bFGF on diabetic neuropathy in streptozotocin-induced diabetic rats. Diabetic rats were treated with human recombinant bFGF as follows: 1) intravenous administration, 2) intramuscular injection into thigh and soleus muscles with cross-linked gelatin hydrogel (CGH), and 3) intramuscular injection with saline. Ten or 30 days later, the motor nerve conduction velocity (MNCV) of the sciatic-tibial and caudal nerves, sensitivity to mechanical stimuli, sciatic nerve blood flow (SNBF), and retinal blood flow (RBF) were measured. Delayed MNCV in the sciatic-tibial and caudal nerves, hypoalgesia, and reduced SNBF in diabetic rats were all ameliorated by intravenous administration of bFGF after 10, but not 30, days. Intramuscular injection of bFGF with CGH also improved sciatic-tibial MNCV, hypoalgesia, and SNBF after 10 and 30 days, but caudal MNCV was not improved. However, intramuscular injection of bFGF with saline had no significant effects. bFGF did not significantly alter RBF in either normal or diabetic rats. These observations suggest that bFGF could have therapeutic value for diabetic neuropathy and that CGH could play important roles as a carrier of bFGF.
Diabetic neuropathy is one of the most common and important complications in diabetic patients. About one-half of patients with diabetes have some degree of diabetic neuropathy, and the progression of diabetic neuropathy causes various problems in the daily life and may affect the prognosis of diabetic patients (1). Therefore, it is important to prevent the development of diabetic neuropathy and to treat it at an early stage. Although strict glycemic control can prevent the onset and progression of diabetic neuropathy (2), the effectiveness has not been satisfactory. Then, additional treatment based on the pathogenic mechanisms becomes necessary.
The effects of various agents on diabetic neuropathy based on the pathogenic hypotheses, including increased polyol pathway activities (3,4), enhanced nonenzymatic glycation (5), altered protein kinase C activities (6), and increased oxidative stress (7,8) have been experimentally and clinically investigated. Most of these agents have demonstrated promising results in animal studies but have failed to deliver convincing data in clinical trials. In terms of prevention or cessation of diabetic neuropathy, there are some promising data showing efficacy in some clinical trials with aldose reductase inhibitors or -lipoic acid; however, therapeutic or reparative effects on advanced diabetic neuropathy could not be exerted by these agents. Neural cell degeneration and decreased nerve blood flow (NBF) (6,8) have been recognized as pathophysiologically characteristic features of diabetic neuropathy. Therefore, agents that can act as both a neurotrophic and an angiogenic factor may be useful for treatment of diabetic neuropathy, even at an advanced stage.
Basic fibroblast growth factor (bFGF) is a single-chain polypeptide composed of 146 amino acids. It was originally isolated from bovine brain and pituitary gland and found to have stimulatory actions on fibroblast proliferation (9eC11). With recent advances in molecular biology, bFGF has been recognized as a multifunctional growth factor that stimulates angiogenesis, acts as a vasodilatator (12,13), has antiapoptotic effects (14,15), and induces proliferation in various kinds of cells (10,11,16). Actually, the effects of bFGF have been investigated in the field of wound healing (17), bone regeneration (18,19), acute ischemic models (20,21), and myocardial infarction (22,23), both experimentally and clinically. The half-life time of bFGF is relatively short (24), and cross-linked gelatin hydrogel (CGH) has been used to maintain the bioactivity of locally administered bFGF for an extended period (25,26). Furthermore, it has been reported that bFGF has effects on the central nervous system (27,28) and peripheral nervous system (29,30). However, the effects of bFGF on diabetic neuropathy have not been investigated.
This study was conducted to investigate the effects of human recombinant bFGF administered intravenously or intramuscularly with CGH or saline on diabetic neuropathy in streptozotocin (STZ)-induced diabetic rats. The effects of bFGF on retinae in normal and diabetic rats were also evaluated. This is the first report to demonstrate therapeutic effects of bFGF on diabetic neuropathy.
RESEARCH DESIGN AND METHODS
Human recombinant bFGF and CGH were kind gifts from Kaken Pharmaceutical (Tokyo, Japan). CGH was made of glutaraldehyde cross-linking of acidic gelatin with an isoelectric point of 5.0, as previously reported (25,26,31).
Eight-week-old male Wistar rats (Chubu Kagakushizai, Nagoya, Japan) with an initial body weight of 210eC240 g were allowed to adapt to the experimental animal facility for 7 days. They were housed in an aseptic animal room at a temperature of 20eC24°C and a humidity of 40eC70%, with a 12-h light cycle and 12 fresh air changes per hour, and were allowed free access to rat chow and water. Diabetes was induced by intraperitoneal injection of STZ (60 mg/kg) (Sigma, St. Louis, MO). Control rats received an equal volume of citric acid buffer. One week after STZ administration, rats with plasma glucose concentrations of >16 mmol/l were selected as the diabetic group. Control and diabetic rats had free access to rat chow and water. After 8 weeks, normal and diabetic rats were randomly divided into experimental groups and treated with bFGF as described below. The Nagoya University Institutional Animal Care and Use Committee approved all protocols.
In study 1, to evaluate the effects of intravenous administration of bFGF on diabetic neuropathy and to determine the appropriate dose of bFGF, saline alone, 2 e蘥/100 g body wt bFGF, or 20 e蘥/100 g body wt bFGF diluted with saline (total volume 400 e蘬) were injected into the tail veins of normal and diabetic rats for 3 consective days. Ten or 30 days later, the parameters described below were measured.
In study 2, to determine whether local treatment with bFGF has therapeutic effects on diabetic neuropathy, 20 e蘥/100 g body wt bFGF in 1.25 ml CGH or saline were injected into the right thigh and soleus muscles and 1.25 ml CGH or saline alone injected into the left thigh and soleus muscles of normal and diabetic rats. Ten or 30 days later, the following parameters were bilaterally measured.
Measurement of motor nerve conduction velocity.
Rats were placed on a heated pad in a room maintained at 25°C to ensure a constant rectal temperature of 37°C. After intraperitoneal injection of sodium pentobarbital (5 mg/100 g), the sciatic-tibial motor nerve conduction velocity (MNCV) between the ankle and sciatic notch and the caudal MNCV were determined with a Neuropak NEM-3102 instrument (Nihon-Koden, Osaka, Japan), as previously described (5,6,32).
Measurement of sensitivity to mechanical stimuli.
During the testing, rats were standing on a metal grid and the paw of the hindlimb stimulated with a series of calibrated monofilaments (Stoelting, Wood Dale, IL). A series of monofilaments of ascending force (0.5eC21 g) were applied to the middle plantar surface of the hind paw. Monofilaments were applied in ascending order for a duration of 1 s. The response threshold was noted as the lowest force that elicited a 50% withdrawal response (5 of 10 applications).
Measurement of sciatic endoneurial nutritive blood flow.
After anesthesia with sodium pentobarbital (5 mg/100 g), rats were placed on a heated pad in a room maintained at 25°C to ensure a constant rectal temperature of 37°C. Sciatic nerve blood flow (SNBF) was measured by the hydrogen clearance technique with an analog recorder (BW-4; Biochemical Science, Kanazawa, Japan) and an electrolysis tissue blood flow meter (RBA-2; Biochemical Science), as previously described (6,32), and calculated with the equation of Koshu et al. (33).
Measurements of retinal vessel diameters, mean circulation time (MCT), and retinal blood flow.
Video fluorescein angiography (VFA) was performed to measure these parameters, as previously described (34,35). Twenty-four hours before the measurements, the animals underwent catheterization with a polyvinyl catheter inserted into the right jugular vein under anesthesia with sodium pentobarbital (5 mg/100 g). The catheter was flushed with 0.1 ml of 1,000 units/ml sodium heparin before and after implantation. It was positioned subcutaneously along the shoulder, and the distal end was externalized to the back of the neck. On the day of measurement, after the left eye was dilated with 1% tropicamide under anesthesia, a 100-e蘬 syringe containing 10% sodium fluorescein was connected to the catheter and positioned on a platform attached to the imaging camera. Rats were maintained on a heated pad during the course of the measurements. The optic disc was centered and focused in the field of view, the VFA recording sequence initiated, and a 5-e蘬 bolus of fluorescein dye rapidly injected into the jugular vein catheter. The injection time was marked on the video recording. The recorded fluorescein angiograms were digitized on a frame-by-frame basis and analyzed densitometrically to determine the retinal vessel diameters and MCT. Sample sites were chosen using primary retinal vessels at a fixed (one optic disc diameter) radical distance from the center of the optic disc. Vessel diameters in units of pixels were determined during peak fluorescein arterial and venous filling times at the defined vessel sample sites using a boundary-crossing algorithm. The average diameter for each vessel was measured for each sample site. The average vessel diameters for each eye represent the average of the individual vessel diameters for that eye. At the fixed-vessel sites, the average vessel fluorescence within a sample area defined by the vessel width was measured on a frame-by-frame basis to generate temporal fluorescence intensity or dye dilution curves. The resultant artery and vein fluorescence data were fit to a logeCnormal distribution function, from which average arterial appearance time of the dye bolus, defined as the time between dye injection and the first detectable appearance (vessel fluorescence intensity greater than background level by two times the SD of the average background intensity) of dye in the retinal artery, represents an assessment of systemic circulation times. The average MCT was calculated as the difference between the average retinal mean arterial and venous filling times for all primary arteries and veins. Retinal blood flow (RBF) was calculated by dividing the sum of the squares of the arterial and venous diameters by the MCT. Data establishing the sensitivity of this technique have been previously reported (36).
Statistical analyses.
Results are presented as means ± SD. Differences among experimental groups were detected by ANOVA, and the significance of differences between groups was evaluated by Scheffe’s S test. Significance was defined as a P value <0.05.
RESULTS
Body weight and plasma glucose concentration.
Diabetic rats demonstrated no body weight gain and remarkable hyperglycemia compared with normal rats. Neither treatment with intravenous (Table 1) nor intramuscular (Table 2) administration of bFGF with or without CGH or saline altered the body weights or plasma glucose concentrations in normal and diabetic rats.
Effects of intravenous administration of bFGF on MNCV of sciatic-tibial nerves and tail nerves and sensitivity to mechanical stimuli.
The MNCV of the sciatic-tibial nerves and tail nerves in the diabetic rats treated with saline alone (37.7 ± 3.4 and 27.1 ± 3.4 m/s, respectively) was significantly delayed compared with that in normal rats (54.0 ± 4.5 and 35.4 ± 4.0 m/s, respectively). Intravenous administration of bFGF did not significantly ameliorate these delays at a dose of 2 e蘥/100 g body wt but almost normalized the MNCV at a dose of 20 e蘥/100 g body wt after 10 days (sciatic-tibial 47.7 ± 6.9, tail 32.6 ± 3.8 m/s) (Fig. 1A and B). However, intravenous administration of bFGF at a dose of either 2 or 20 e蘥/100 g body wt did not alter the MNCV of the sciatic-tibial nerves and tail nerves in the normal rats. On the other hand, beneficial effects of intravenous administration of bFGF on the MNCV of sciatic tibial nerves were not observed after 30 days (untreated normal 56.2 ± 3.5, untreated diabetic 43.5 ± 1.4, and 20 e蘥 bFGFeCtreated diabetic 45.1 ± 1.7 m/s) (Fig. 1C). Effects of intravenous administration of bFGF on sensitivity to mechanical stimuli were similar to those on MNCV. Untreated diabetic rats (13.0 ± 2.7 g) required a significantly greater force to elicit a 50% withdrawal response compared with untreated normal rats (8.4 ± 0.9 g). This reduced sensitivity to mechanical stimuli was ameliorated by 20 e蘥 bFGF (9.4 ± 0.5 g) after 10 days (Fig. 2A). However, this effect was not maintained for 30 days (Fig. 2B).
Effects of intravenous administration of bFGF on SNBF.
There were no differences in the SNBF between any of the groups of normal rats. The SNBF in diabetic rats treated with saline alone (6.5 ± 1.1 ml · mineC1 · 100 g body wteC1) was significantly reduced compared with that in normal rats (16.1 ± 2.1 ml · mineC1 · 100 g body wteC1). Intravenous administration of bFGF at a dose of 2 e蘥/100 g body wt (11.6 ± 2.3 ml · mineC1 · 100 g body wteC1) partially but significantly improved the decreased SNBF in diabetic rats, and this effect was more prominent at the high dose of 20 e蘥/100 g body wt (14.1 ± 2.5 ml · mineC1 · 100 g body wteC1) after 10 days (Fig. 3A). After 30 days, however, the effects of intravenous administration of bFGF on the SNBF were not observed (untreated normal 16.2 ± 1.8, untreated diabetic 8.3 ± 2.3, and 20 e蘥 bFGFeCtreated diabetic 11.3 ± 1.7 ml · mineC1 · 100 g body wteC1) (Fig. 3B).
Effects of intravenous administration of bFGF on retinal vessel diameters, MCT, and RBF.
There were no significant differences in the arterial or venous diameters between the normal and diabetic rats with or without intravenous administration of bFGF. The prolonged MCT and reduced RBF in the diabetic rats were not significantly altered by the intravenous administration of bFGF (Table 3). Moreover, fluorescein angiography showed no abnormality, including aneurism, occlusion, or neovascularization in retinal arteries, veins, or capillaries.
Effects of intramuscular administration of bFGF with saline or CGH on MNCV of sciatic-tibial nerves and tail nerves, as well as sensitivity to mechanical stimuli.
Intramuscular administration of CGH or saline alone did not affect the MNCV of the sciatic-tibial nerves in normal or diabetic rats, and bFGF with CGH or saline had no effects on the MNCV of the sciatic-tibial nerves in normal rats. The decreased MNCV of the sciatic-tibial nerves in the untreated diabetic rats (untreated normal 55.1 ± 4.5, untreated diabetic 40.2 ± 3.1 m/s) was significantly ameliorated by intramuscular administration of bFGF with CGH (49.3 ± 4.6 m/s) after 10 days but not by that of bFGF with saline (Fig. 4A). On the other hand, the delayed MNCV of the tail nerves in diabetic rats was not improved by local injection of bFGF with CGH or saline into the thigh and soleus muscles (Fig. 4B). In addition, the effect of intramuscular administration of bFGF with CGH on the MNCV of the sciatic-tibial nerves was maintained for 30 days (Fig. 6A). The reduced sensitivity to mechanical stimuli in untreated diabetic rats (12.9 ± 2.7 g) compared with that in untreated normal rats (8.4 ± 1.6 g) was significantly ameliorated by intramuscular administration of bFGF with CGH (9.3 ± 1.0 g) after 10 days (Fig. 4C), and this effect was maintained for 30 days (Fig. 6B). However, intramuscular administration of bFGF with saline showed no significant effects.
Effects of intramuscular administration of bFGF with saline or CGH on SNBF.
The effects of intramuscular injection of bFGF with saline or CGH on the SNBF were similar to those on the MNCV of the sciatic-tibial nerves. No significant differences were found in the SNBF between any of the groups of normal rats. The reduced SNBF in the diabetic rats (7.9 ± 0.8 ml · mineC1 · 100 g body wteC1) was almost normalized by the intramuscular administration of bFGF with CGH (14.7 ± 1.9 ml · mineC1 · 100 g body wteC1) to the level of the untreated normal rats after 10 days (15.7 ± 1.5 ml · mineC1 · 100 g body wteC1) (Fig. 5), but bFGF with saline did not significantly improve the reduction (Fig. 4). The beneficial effect of bFGF with CGH on SNBF was also observed after 30 days (Fig. 6C).
Effects of intramuscular administration of bFGF with saline or CGH on retinal vessel diameters, MCT, and RBF.
No significant differences in the arterial and venous diameters between any of the experimental groups were observed after 10 days. The delayed MCT and decreased RBF in the diabetic rats were not altered by the local injection of bFGF with CGH or saline into the thigh and soleus muscles (data not shown). Because of the development of severe cataracts, the VFA measurements were not performed after 30 days.
DISCUSSION
The present study demonstrated that intravenous administration of bFGF has therapeutic effects on diabetic neuropathy and does not affect the retinal circulation and that local treatment by intramuscular injection of bFGF with CGH, a unique drug-delivery system, ameliorated hypoalgesia and the decreased MNCV and SNBF in diabetic rats without demonstrating systemic actions.
In the present study, sensitivity to mechanical stimuli was measured to evaluate a sensory nerve function. Our preliminary experiment demonstrated that diabetic rats develop hyperalgesia at 2eC4 weeks after induction of diabetes and hypoalgesia at 7eC8 weeks, which is consistent with a previous report by Calcutt et al. (37). However, deficits in MNCV and SNBF of diabetic rats are clearly demonstrated in 8 weeks. Therefore, therapeutic intervention with bFGF was performed 8 weeks after induction of diabetes.
Previous studies have shown that bFGF has various functions both in vitro and in vivo. In in vitro studies, the induction of proliferation activities by bFGF in various types of cells, including vascular cells (10,11,16), and the antiapoptotic action of bFGF on neural cells (14) have been reported. In in vivo studies, the effects of bFGF have been investigated in the fields of central and peripheral nervous systems (27eC30), skin (17), bone (18,19), heart (22,23), and vasculatures (20,21), and beneficial effects have been reported both experimentally and clinically. In addition to these effects, bFGF induces NO production through endothelial NO synthase activation, resulting in vasodilation (12,13,38).
Several neurotrophic factors and growth factors such as nerve growth factor (NGF) (39), vascular endothelial growth factor (VEGF) (40), and hepatocyte growth factor (HGF) (41) have been recognized to ameliorate diabetic neuropathy in animal models. Although the effects of NGF on diabetic neuropathy were most extensively investigated experimentally, a clinical trial failed to establish the usefulness of NGF for diabetic neuropathy (42,43). The therapeutic effects of VEGF (40) and HGF (41) through their angiogenic actions on diabetic neuropathy have been reported. On the other hand, the induction of HGF and VEGF by treatment with bFGF in vitro and in vivo has been demonstrated (44eC46). Interestingly, according to these studies (44eC46), under ischemic conditions, bFGF exerts its angiogenic effects by not only inducing but also harmonizing with these endogenous angiogenic factors. Moreover, it has been confirmed that the vasculatures induced by bFGF consist of both endothelial cells and smooth muscle cells, which act as functional vessels. Considering these reports, bFGF may be a plausible angiogenic growth factor and therapeutic agent for diabetic neuropathy.
In this study, we compared the effects of bFGF administered through three different delivery systems on diabetic neuropathy. Despite its short half-life, intravenous administration of bFGF dose dependently ameliorated the reduced SNBF and delayed MNCV of both the sciatic-tibial and tail nerves in diabetic rats after 10 days (24). These effects may not be mediated through direct actions of bFGF on nerve tissues, but through systemic actions on other tissues, and bFGF might trigger the activation of a cytokine cascade, causing its effect to continue even after its inactivation. However, further long-term effects of bFGF cannot be expected by intravenous injection. In fact, beneficial effects of intravenous administration of bFGF on hypoalgesia, sciatic-tibial MNCV, and SNBF after 10 days were not observed after 30 days in this study. In addition, systemic administration of bFGF by intravenous injection might cause cytotoxicity in various tissues, including retinae and kidneys, through its angiogenic and cell-proliferative actions. In the present study, no beneficial or adverse effects of intravenously administered bFGF on the retinae were observed. The reason for this result remains unclear. Differences may exist in the distribution of exogenously administered bFGF among tissues, which might cause the inconsistent effects on nerve tissues and retinal tissues. To clarify the action mechanisms and establish long-term safety, histological analyses, including angiogenesis and measurement of the distribution of exogenously administered bFGF, would be required. Although the effects on renal functions were not investigated in this study, previous studies have in fact demonstrated toxic actions of bFGF on kidneys, such as podocyte injury (47), epithelial injury (48), and mesangial cell proliferation and matrix accumulation (49). Therefore, delivery systems that can be locally administered and retain the biological activity of bFGF for a long period would be required for the clinical application of bFGF.
Recently, local injection of VEGF gene (40) or HGF gene (41) and transplantation of endothelial progenitor cells (32) to muscles around peripheral nerves have been reported to improve experimental diabetic neuropathy through the induction of local angiogenesis. Therefore, we explored the usefulness of local treatment of bFGF using two drug delivery systems, CGH and saline. CGH is considered an excellent and unique carrier of bFGF (25,26). CGH was prepared through cross-linking of acidic gelatin with an isoelectric point (IEP) of 5.0 and bFGF with an IEP of 9.0. This difference in the IEP enabled bFGF to maintain its bioactivity for an extended period. bFGF administered with CGH can function when released by the biodegradation of the hydrogel (25,26,31). In this study, bFGF administered with saline did not improve the decreased MNCV and SNBF in diabetic rats, but bFGF injected with CGH brought about a remarkable amelioration of the sciatic-tibial MNCV and SNBF in the diabetic rats, and its effects continued for at least 30 days. In addition, this local treatment did not improve the MNCV or SNBF of the opposite side, the tail nerve MNCV, or the retinal circulation in diabetic rats. These findings support the efficiency and safety of local treatment of bFGF with CGH.
Intravenous administration of bFGF at a low dose significantly improved SNBF but not MNCV, and long-term effects of bFGF were not observed by intravenous injection. In addition, intramuscular injection of bFGF with saline tended to increase SNBF without changing MNCV. These observations suggest that the primary action sites of bFGF would be the endoneurial microvasculature, that partial correction of SNBF would be insufficient to improve MNCV, and that the nonvascular actions of bFGF may account for little of its therapeutic efficacy.
In clinical practice, it is recognized that treatment of advanced symptomatic diabetic neuropathy patients with intravenous administration of vasodilatory agents, such as prostaglandin E1, ameliorates their symptoms. However, this effect cannot be maintained for a long period. Long-term efficacy of intramuscular administration of bFGF with CGH was clearly demonstrated in this study, suggesting a clinical benefit of this treatment.
In summary, intravenous administration of bFGF demonstrated a systemic action on the nerve functions of diabetic rats, including amelioration of hypoalgesia and delayed MNCV in both the sciatic-tibial and tail nerves. On the other hand, intramuscular injection of bFGF with CGH, but not that with saline, improved hypoalgesia, sciatic-tibial MNCV, and SNBF only in the treated side, and these effects were maintained for at least 30 days. Although the direct effects of bFGF on neural cells remain unclear and further studies, including histological analyses, will be required, these results strongly suggest that bFGF could have therapeutic value for diabetic neuropathy through the improvement of microvascular blood flow. Given its apparent safety and long-term efficiency, intramuscular administration of bFGF with CGH could be suitable for clinical application.
ACKNOWLEDGMENTS
This work was supported in part by a diabetes research grant from the Ministry of Health and Welfare of Japan.
The authors thank Kaken Pharmaceutical (Tokyo, Japan) for kindly providing bFGF and CGH and thank Yuko Maehata for technical assistance.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Vinik AI, Park TS, Stansberry KB, Pittenger GL: Diabetic neuropathies. Diabetologia 43:957eC973, 2000
The DCCT Research Group: The effect of intensive treatment of diabetes on the development and progression of lomg-term complications in insulin-dependent diabetes mellitus. N Engl J Med 329:977eC986, 1993
Tomlinson DR, Willars GB, Carrington AL: Aldose reductase inhibitors and diabetic complications. Pharmacol Ther 54:151eC194, 1992
Yagihashi S, Kamijo M, Ido Y, Mirrless DJ: Effect of long-term aldose reductase inhibition in development of experimental diabetic neuropathy: ultrastructural and morphometric studies of sural nerve in streptozotocin-induced diabetic rats. Diabetes 39:690eC696, 1990
Yagihashi S, Kamijo M, Baba M, Yagihashi N, Nagai K: Effect of aminoguanidine on functional and structural abnormalities in peripheral nerve of STZ-induced diabetic rats. Diabetes 41:47eC52, 1992
Nakamura J, Kato K, Hamada Y, Nakayama M, Chaya S, Nakashima E, Naruse K, Kasuya Y, Mizubayashi R, Miwa K, Yasuda Y, Kamiya H, Ienaga K, Sakakibara F, Koh N, Hotta N: A protein kinase C--selective inhibitor ameliorates neural dysfunction in streptozotocin-induced diabetic rats. Diabetes 48:2090eC2095, 1999
Vincent AM, Russell JW, Low P, Feldman EL: Oxidative stress in the pathogenesis of diabetic neuropathy. Endocr Rev 25:612eC628, 2004
Cameron NE, Cotter MA: Neurovascular dysfunction in diabetic rats: potential contribution of autooxidation and free radicals examined using transition metal chelating agents. J Clin Invest 96:1159eC1163, 1995
Abraham JA, Mergia A, Whang JL, Tumolo A, Friedman J, Hjerrild KA, Gospodarowicz D, Fiddes JC: Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science 233:545eC548, 1986
Schweigerer L, Neufeld G, Mergia A, Abraham JA, Fiddes JC, Gospodarowicz D: Basic fibroblast growth factor in human rhabdomyosarcoma cells: implications for the proliferation and neovascularization of myoblast-derived tumors. Proc Natl Acad Sci U S A 84:842eC846, 1987
Esch F, Baird A, Ling N, Ueno N, Hill F, Denoroy L, Klepper R, Gospodarowicz D, Bohlen P, Guillemin R: Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci U S A 82:6507eC6511, 1985
Yang HT, Yan Z, Abraham JA, Terjung RL: VEGF121- and bFGF-induced increase in collateral blood flow requires normal nitric oxide production. Am J Physiol Heart Circ Physiol 280:H1097eCH1104, 2001
Cuevas P, Carceller F, Ortega S, Zazo M, Nieto I, Gimenez-Gallego G: Hypotensive activity of fibroblast growth factor. Science 254:1208eC1210, 1991
Shaw R, Cianchetti R, Pleasure D, Kreider B: Basic fibroblast growth factor prevents cAMP-induced apoptosis in cultured Schwann cells. J Neurosci Res 47:400eC404, 1997
Iwai-Kanai E, Hasegawa K, Fujita M, Araki M, Yanazume T, Adachi S, Sasayama S: Basic fibroblast growth factor protects cardiac myocytes from iNOS-mediated apoptosis. J Cell Physiol 190:54eC62, 2002
Boilly B, Vercoutter-Edouart AS, Hondermarck H, Nurcombe V, Le Bourhis X: FGF signals for cell proliferation and migration through different pathways. Cytokine Growth Factor Rev 11:295eC302, 2000
Tanaka E, Ase K, Okuda T, Okumura M, Nogimori K: Mechanism of acceleration of wound healing by basic fibroblast growth factor in genetically diabetic mice. Biol Pharm Bull 19:1141eC1148, 1996
Kawaguchi H, Kurokawa T, Hanada K, Hiyama Y, Tamura M, Ogata E, Matsumoto T: Stimulation of fracture repair by recombinant human basic fibroblast growth factor in normal and streptozotocin-diabetic rats. Endocrinology 135:774eC781, 1994
Nakamura T, Hanada K, Tamura M, Shibanushi T, Nigi H, Tagawa M, Fukumoto S, Matsumoto T: Stimulation of endosteal bone formation by systemic injections of recombinant basic fibroblast growth factor in rats. Endocrinology 136:1276eC1284, 1995
Stark J, Baffour R, Garb JL, Kaufman J, Berman J, Rhee S, Norris MA, Friedmann P: Basic fibroblast growth factor stimulates angiogenesis in the hindlimb of hyperglycemic rats. J Surg Res 79:8eC12, 1998
Baffour R, Berman J, Garb JL, Rhee SW, Kaufman J, Friedmann P: Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor. J Vasc Surg 16:181eC191, 1992
Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA: Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation 105:788eC793, 2002
Yanagisawa-Miwa A, Uchida Y, Nakamura F, Tomaru T, Kido H, Kamijo T, Sugimoto T, Kaji K, Utsuyama M, Kurashima C, Ito H: Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science 257:1401eC1403, 1992
Yuge T, Furukawa A, Nakamura K, Nagashima Y, Shinozaki K, Nakamura T, Kimura R: Metabolism of the intravenously administered recombinant human basic fibroblast growth factor, trafermin, in liver and kidney: degradation implicated in its selective localization to the fenestrated type microvasculatures. Biol Pharm Bull 20:786eC793, 1997
Tabata Y, Nagano A, Ikada Y: Biodegradation of hydrogel carrier incorporating fibroblast growth factor. Tissue Eng 5:127eC138, 1999
Tabata Y, Ikada Y: Vascularization effect of basic fibroblast growth factor released from gelatin hydrogels with different biodegradabilities. Biomaterials 20:2169eC2175, 1999
Abe K, Saito H: Effects of basic fibroblast growth factor on central nervous system functions. Pharmacol Res 43:307eC312, 2001
Katsuki H, Itsukaichi Y, Matsuki N: Distinct signaling pathways involved in multiple effects of basic fibroblast growth factor on cultured rat hippocampal neurons. Brain Res 885:240eC250, 2000
Grothe C, Nikkhah G: The role of basic fibroblast growth factor in peripheral nerve regeneration. Anat Embryol 204:171eC177, 2001
Fujimoto E, Mizoguchi A, Hanada K, Yajima M, Ide C: Basic fibroblast growth factor promotes extension of regenerating axons of peripheral nerve: in vivo experiments using a Schwann cell basal lamina tube model. J Neurocytol 26:511eC528, 1997
Kawaguchi H, Nakamura K, Tabata Y, Ikada Y, Aoyama I, Anzai J, Nakamura T, Hiyama Y, Tamura M: Acceleration of fracture healing in nonhuman primates by fibroblast growth factor-2. J Clin Endocrinol Metab 86:875eC880, 2001
Naruse K, Hamada Y, Nakashima E, Kato K, Mizubayashi R, Kamiya H, Yuzawa Y, Matsuo S, Murohara T, Matsubara T, Oiso Y, Nakamura J: Therapeutic neovascularization using cord blood-derived endothelial progenitor cells for diabetic neuropathy. Diabetes 54:1823eC1828, 2005
Koshu K, Kamiyama K, Oka N, Endo S, Takaku A, Saito T: Measurement of regional blood flow using hydrogen gas generated by electrolysis. Stroke 13:483eC487, 1982
Abiko T, Abiko A, Clermont AC, Shoelson B, Horio N, Takahashi J, Adamis AP, King GL, Bursell SE: Characterization of retinal leukostasis and hemodynamics in insulin resistance and diabetes: role of oxidants and protein kinase-C activation. Diabetes 52:829eC837, 2003
Horio N, Clermont AC, Abiko A, Abiko T, Shoelson BD, Bursell SE, Feener EP: Angiotensin AT(1) receptor antagonism normalizes retinal blood flow and acetylcholine-induced vasodilatation in normotensive diabetic rats. Diabetologia 47:113eC123, 2004
Takagi C, King GL, Clermont AC, Cummins DR, Takagi H, Bursell SE: Reversal of abnormal retinal hemodynamics in diabetic rats by acarbose, an alpha-glucosidase inhibitor. Curr Eye Res 14:741eC749, 1995
Calcutt NA, Freshwater JD, Mizisin AP: Prevention of sensory disorders in diabetic Sprague-Dawley rats by aldose reductase inhibition or treatment with ciliary neurotrophic factor. Diabetologia 47:718eC724, 2004
Kostyk SK, Kourembanas S, Wheeler EL, Medeiros D, McQuillan LP, D’Amore PA, Braunhut SJ: Basic fibroblast growth factor increases nitric oxide synthase production in bovine endothelial cells. Am J Physiol 269:H1583eCH1589, 1995
Goss JR, Goins WF, Lacomis D, Mata M, Glorioso JC, Fink DJ: Herpes simplex-mediated gene transfer of nerve growth factor protects against peripheral neuropathy in streptozotocin-induced diabetes in the mouse. Diabetes 51:2227eC2232, 2002
Schratzberger P, Walter DH, Rittig K, Bahlmann FH, Pola R, Curry C, Silver M, Krainin JG, Weinberg DH, Ropper AH, Isner JM: Reversal of experimental diabetic neuropathy by VEGF gene transfer. J Clin Invest 107:1083eC1092, 2001
Kato N, Nemoto K, Nakanishi K, Morishita R, Kaneda Y, Uenoyama M, Ikeda T, Fujikawa K: Nonviral gene transfer of human hepatocyte growth factor improves streptozotocin-induced diabetic neuropathy in rats. Diabetes 54:846eC854, 2005
Apfel SC: Nerve growth factor for the treatment of diabetic neuropathy: what went wrong, what went right, and what does the future hold Int Rev Neurobiol 50:393eC413, 2002
Pittenger G, Vinik A: Nerve growth factor and diabetic neuropathy. Exp Diabesity Res 4:271eC285, 2003
Masaki I, Yonemitsu Y, Yamashita A, Sata S, Tanii M, Komori K, Nakagawa K, Hou X, Nagai Y, Hasegawa M, Sugimachi K, Sueishi K: Angiogenic gene therapy for experimental critical limb ischemia: acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2. Circ Res 90:966eC973, 2002
Onimaru M, Yonemitsu Y, Tanii M, Nakagawa K, Masaki I, Okano S, Ishibashi H, Shirasuna K, Hasegawa M, Sueishi K: Fibroblast growth factor-2 gene transfer can stimulate hepatocyte growth factor expression irrespective of hypoxia-mediated downregulation in ischemic limbs. Circ Res 91:923eC930, 2002
Tsutsumi N, Yonemitsu Y, Shikada Y, Onimaru M, Tanii M, Okano S, Kaneko K, Hasegawa M, Hashizume M, Maehara Y, Sueishi K: Essential role of PDGFRalpha-p70S6K signaling in mesenchymal cells during therapeutic and tumor angiogenesis in vivo: role of PDGFRalpha during angiogenesis. Circ Res 94:1186eC1194, 2004
Sasaki T, Hatta H, Osawa G: Cytokines and podocyte injury: the mechanism of fibroblast growth factor 2-induced podocyte injury. Nephrol Dial Transplant 14 (Suppl. 1):33eC34, 1999
Sasaki T, Jyo Y, Tanda N, Tamai H, Osawa G: The role of basic fibroblast growth factor (FGF2) in glomerular epithelial cell injury. Contrib Nephrol 118:68eC77, 1996
Floege J, Eng E, Young BA, Alpers CE, Barrett TB, Bowen-Pope DF, Johnson RJ: Infusion of platelet-derived growth factor or basic fibroblast growth factor induces selective glomerular mesangial cell proliferation and matrix accumulation in rats. J Clin Invest 92:2952eC2962, 1993(Mika Nakae, Hideki Kamiya)
2 Department of Ophthalmology, Fujita Health University School of Medicine, Aichi, Japan
3 Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan
4 Institute for Frontier Medical Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan
bFGF, basic fibroblast growth factor; CGH, cross-linked gelatin hydrogel; HGF, hepatocyte growth factor; MCT, mean circulation time; MNCV, motor nerve conduction velocity; NBF, nerve blood flow; NGF, nerve growth factor; RBF, retinal blood flow; SNBF, sciatic NBF; STZ, streptozotocin; VEGF, vascular endothelial growth factor; VFA, video fluorescein angiography
ABSTRACT
Basic fibroblast growth factor (bFGF) stimulates angiogenesis and induces neural cell regeneration. We investigated the effects of bFGF on diabetic neuropathy in streptozotocin-induced diabetic rats. Diabetic rats were treated with human recombinant bFGF as follows: 1) intravenous administration, 2) intramuscular injection into thigh and soleus muscles with cross-linked gelatin hydrogel (CGH), and 3) intramuscular injection with saline. Ten or 30 days later, the motor nerve conduction velocity (MNCV) of the sciatic-tibial and caudal nerves, sensitivity to mechanical stimuli, sciatic nerve blood flow (SNBF), and retinal blood flow (RBF) were measured. Delayed MNCV in the sciatic-tibial and caudal nerves, hypoalgesia, and reduced SNBF in diabetic rats were all ameliorated by intravenous administration of bFGF after 10, but not 30, days. Intramuscular injection of bFGF with CGH also improved sciatic-tibial MNCV, hypoalgesia, and SNBF after 10 and 30 days, but caudal MNCV was not improved. However, intramuscular injection of bFGF with saline had no significant effects. bFGF did not significantly alter RBF in either normal or diabetic rats. These observations suggest that bFGF could have therapeutic value for diabetic neuropathy and that CGH could play important roles as a carrier of bFGF.
Diabetic neuropathy is one of the most common and important complications in diabetic patients. About one-half of patients with diabetes have some degree of diabetic neuropathy, and the progression of diabetic neuropathy causes various problems in the daily life and may affect the prognosis of diabetic patients (1). Therefore, it is important to prevent the development of diabetic neuropathy and to treat it at an early stage. Although strict glycemic control can prevent the onset and progression of diabetic neuropathy (2), the effectiveness has not been satisfactory. Then, additional treatment based on the pathogenic mechanisms becomes necessary.
The effects of various agents on diabetic neuropathy based on the pathogenic hypotheses, including increased polyol pathway activities (3,4), enhanced nonenzymatic glycation (5), altered protein kinase C activities (6), and increased oxidative stress (7,8) have been experimentally and clinically investigated. Most of these agents have demonstrated promising results in animal studies but have failed to deliver convincing data in clinical trials. In terms of prevention or cessation of diabetic neuropathy, there are some promising data showing efficacy in some clinical trials with aldose reductase inhibitors or -lipoic acid; however, therapeutic or reparative effects on advanced diabetic neuropathy could not be exerted by these agents. Neural cell degeneration and decreased nerve blood flow (NBF) (6,8) have been recognized as pathophysiologically characteristic features of diabetic neuropathy. Therefore, agents that can act as both a neurotrophic and an angiogenic factor may be useful for treatment of diabetic neuropathy, even at an advanced stage.
Basic fibroblast growth factor (bFGF) is a single-chain polypeptide composed of 146 amino acids. It was originally isolated from bovine brain and pituitary gland and found to have stimulatory actions on fibroblast proliferation (9eC11). With recent advances in molecular biology, bFGF has been recognized as a multifunctional growth factor that stimulates angiogenesis, acts as a vasodilatator (12,13), has antiapoptotic effects (14,15), and induces proliferation in various kinds of cells (10,11,16). Actually, the effects of bFGF have been investigated in the field of wound healing (17), bone regeneration (18,19), acute ischemic models (20,21), and myocardial infarction (22,23), both experimentally and clinically. The half-life time of bFGF is relatively short (24), and cross-linked gelatin hydrogel (CGH) has been used to maintain the bioactivity of locally administered bFGF for an extended period (25,26). Furthermore, it has been reported that bFGF has effects on the central nervous system (27,28) and peripheral nervous system (29,30). However, the effects of bFGF on diabetic neuropathy have not been investigated.
This study was conducted to investigate the effects of human recombinant bFGF administered intravenously or intramuscularly with CGH or saline on diabetic neuropathy in streptozotocin (STZ)-induced diabetic rats. The effects of bFGF on retinae in normal and diabetic rats were also evaluated. This is the first report to demonstrate therapeutic effects of bFGF on diabetic neuropathy.
RESEARCH DESIGN AND METHODS
Human recombinant bFGF and CGH were kind gifts from Kaken Pharmaceutical (Tokyo, Japan). CGH was made of glutaraldehyde cross-linking of acidic gelatin with an isoelectric point of 5.0, as previously reported (25,26,31).
Eight-week-old male Wistar rats (Chubu Kagakushizai, Nagoya, Japan) with an initial body weight of 210eC240 g were allowed to adapt to the experimental animal facility for 7 days. They were housed in an aseptic animal room at a temperature of 20eC24°C and a humidity of 40eC70%, with a 12-h light cycle and 12 fresh air changes per hour, and were allowed free access to rat chow and water. Diabetes was induced by intraperitoneal injection of STZ (60 mg/kg) (Sigma, St. Louis, MO). Control rats received an equal volume of citric acid buffer. One week after STZ administration, rats with plasma glucose concentrations of >16 mmol/l were selected as the diabetic group. Control and diabetic rats had free access to rat chow and water. After 8 weeks, normal and diabetic rats were randomly divided into experimental groups and treated with bFGF as described below. The Nagoya University Institutional Animal Care and Use Committee approved all protocols.
In study 1, to evaluate the effects of intravenous administration of bFGF on diabetic neuropathy and to determine the appropriate dose of bFGF, saline alone, 2 e蘥/100 g body wt bFGF, or 20 e蘥/100 g body wt bFGF diluted with saline (total volume 400 e蘬) were injected into the tail veins of normal and diabetic rats for 3 consective days. Ten or 30 days later, the parameters described below were measured.
In study 2, to determine whether local treatment with bFGF has therapeutic effects on diabetic neuropathy, 20 e蘥/100 g body wt bFGF in 1.25 ml CGH or saline were injected into the right thigh and soleus muscles and 1.25 ml CGH or saline alone injected into the left thigh and soleus muscles of normal and diabetic rats. Ten or 30 days later, the following parameters were bilaterally measured.
Measurement of motor nerve conduction velocity.
Rats were placed on a heated pad in a room maintained at 25°C to ensure a constant rectal temperature of 37°C. After intraperitoneal injection of sodium pentobarbital (5 mg/100 g), the sciatic-tibial motor nerve conduction velocity (MNCV) between the ankle and sciatic notch and the caudal MNCV were determined with a Neuropak NEM-3102 instrument (Nihon-Koden, Osaka, Japan), as previously described (5,6,32).
Measurement of sensitivity to mechanical stimuli.
During the testing, rats were standing on a metal grid and the paw of the hindlimb stimulated with a series of calibrated monofilaments (Stoelting, Wood Dale, IL). A series of monofilaments of ascending force (0.5eC21 g) were applied to the middle plantar surface of the hind paw. Monofilaments were applied in ascending order for a duration of 1 s. The response threshold was noted as the lowest force that elicited a 50% withdrawal response (5 of 10 applications).
Measurement of sciatic endoneurial nutritive blood flow.
After anesthesia with sodium pentobarbital (5 mg/100 g), rats were placed on a heated pad in a room maintained at 25°C to ensure a constant rectal temperature of 37°C. Sciatic nerve blood flow (SNBF) was measured by the hydrogen clearance technique with an analog recorder (BW-4; Biochemical Science, Kanazawa, Japan) and an electrolysis tissue blood flow meter (RBA-2; Biochemical Science), as previously described (6,32), and calculated with the equation of Koshu et al. (33).
Measurements of retinal vessel diameters, mean circulation time (MCT), and retinal blood flow.
Video fluorescein angiography (VFA) was performed to measure these parameters, as previously described (34,35). Twenty-four hours before the measurements, the animals underwent catheterization with a polyvinyl catheter inserted into the right jugular vein under anesthesia with sodium pentobarbital (5 mg/100 g). The catheter was flushed with 0.1 ml of 1,000 units/ml sodium heparin before and after implantation. It was positioned subcutaneously along the shoulder, and the distal end was externalized to the back of the neck. On the day of measurement, after the left eye was dilated with 1% tropicamide under anesthesia, a 100-e蘬 syringe containing 10% sodium fluorescein was connected to the catheter and positioned on a platform attached to the imaging camera. Rats were maintained on a heated pad during the course of the measurements. The optic disc was centered and focused in the field of view, the VFA recording sequence initiated, and a 5-e蘬 bolus of fluorescein dye rapidly injected into the jugular vein catheter. The injection time was marked on the video recording. The recorded fluorescein angiograms were digitized on a frame-by-frame basis and analyzed densitometrically to determine the retinal vessel diameters and MCT. Sample sites were chosen using primary retinal vessels at a fixed (one optic disc diameter) radical distance from the center of the optic disc. Vessel diameters in units of pixels were determined during peak fluorescein arterial and venous filling times at the defined vessel sample sites using a boundary-crossing algorithm. The average diameter for each vessel was measured for each sample site. The average vessel diameters for each eye represent the average of the individual vessel diameters for that eye. At the fixed-vessel sites, the average vessel fluorescence within a sample area defined by the vessel width was measured on a frame-by-frame basis to generate temporal fluorescence intensity or dye dilution curves. The resultant artery and vein fluorescence data were fit to a logeCnormal distribution function, from which average arterial appearance time of the dye bolus, defined as the time between dye injection and the first detectable appearance (vessel fluorescence intensity greater than background level by two times the SD of the average background intensity) of dye in the retinal artery, represents an assessment of systemic circulation times. The average MCT was calculated as the difference between the average retinal mean arterial and venous filling times for all primary arteries and veins. Retinal blood flow (RBF) was calculated by dividing the sum of the squares of the arterial and venous diameters by the MCT. Data establishing the sensitivity of this technique have been previously reported (36).
Statistical analyses.
Results are presented as means ± SD. Differences among experimental groups were detected by ANOVA, and the significance of differences between groups was evaluated by Scheffe’s S test. Significance was defined as a P value <0.05.
RESULTS
Body weight and plasma glucose concentration.
Diabetic rats demonstrated no body weight gain and remarkable hyperglycemia compared with normal rats. Neither treatment with intravenous (Table 1) nor intramuscular (Table 2) administration of bFGF with or without CGH or saline altered the body weights or plasma glucose concentrations in normal and diabetic rats.
Effects of intravenous administration of bFGF on MNCV of sciatic-tibial nerves and tail nerves and sensitivity to mechanical stimuli.
The MNCV of the sciatic-tibial nerves and tail nerves in the diabetic rats treated with saline alone (37.7 ± 3.4 and 27.1 ± 3.4 m/s, respectively) was significantly delayed compared with that in normal rats (54.0 ± 4.5 and 35.4 ± 4.0 m/s, respectively). Intravenous administration of bFGF did not significantly ameliorate these delays at a dose of 2 e蘥/100 g body wt but almost normalized the MNCV at a dose of 20 e蘥/100 g body wt after 10 days (sciatic-tibial 47.7 ± 6.9, tail 32.6 ± 3.8 m/s) (Fig. 1A and B). However, intravenous administration of bFGF at a dose of either 2 or 20 e蘥/100 g body wt did not alter the MNCV of the sciatic-tibial nerves and tail nerves in the normal rats. On the other hand, beneficial effects of intravenous administration of bFGF on the MNCV of sciatic tibial nerves were not observed after 30 days (untreated normal 56.2 ± 3.5, untreated diabetic 43.5 ± 1.4, and 20 e蘥 bFGFeCtreated diabetic 45.1 ± 1.7 m/s) (Fig. 1C). Effects of intravenous administration of bFGF on sensitivity to mechanical stimuli were similar to those on MNCV. Untreated diabetic rats (13.0 ± 2.7 g) required a significantly greater force to elicit a 50% withdrawal response compared with untreated normal rats (8.4 ± 0.9 g). This reduced sensitivity to mechanical stimuli was ameliorated by 20 e蘥 bFGF (9.4 ± 0.5 g) after 10 days (Fig. 2A). However, this effect was not maintained for 30 days (Fig. 2B).
Effects of intravenous administration of bFGF on SNBF.
There were no differences in the SNBF between any of the groups of normal rats. The SNBF in diabetic rats treated with saline alone (6.5 ± 1.1 ml · mineC1 · 100 g body wteC1) was significantly reduced compared with that in normal rats (16.1 ± 2.1 ml · mineC1 · 100 g body wteC1). Intravenous administration of bFGF at a dose of 2 e蘥/100 g body wt (11.6 ± 2.3 ml · mineC1 · 100 g body wteC1) partially but significantly improved the decreased SNBF in diabetic rats, and this effect was more prominent at the high dose of 20 e蘥/100 g body wt (14.1 ± 2.5 ml · mineC1 · 100 g body wteC1) after 10 days (Fig. 3A). After 30 days, however, the effects of intravenous administration of bFGF on the SNBF were not observed (untreated normal 16.2 ± 1.8, untreated diabetic 8.3 ± 2.3, and 20 e蘥 bFGFeCtreated diabetic 11.3 ± 1.7 ml · mineC1 · 100 g body wteC1) (Fig. 3B).
Effects of intravenous administration of bFGF on retinal vessel diameters, MCT, and RBF.
There were no significant differences in the arterial or venous diameters between the normal and diabetic rats with or without intravenous administration of bFGF. The prolonged MCT and reduced RBF in the diabetic rats were not significantly altered by the intravenous administration of bFGF (Table 3). Moreover, fluorescein angiography showed no abnormality, including aneurism, occlusion, or neovascularization in retinal arteries, veins, or capillaries.
Effects of intramuscular administration of bFGF with saline or CGH on MNCV of sciatic-tibial nerves and tail nerves, as well as sensitivity to mechanical stimuli.
Intramuscular administration of CGH or saline alone did not affect the MNCV of the sciatic-tibial nerves in normal or diabetic rats, and bFGF with CGH or saline had no effects on the MNCV of the sciatic-tibial nerves in normal rats. The decreased MNCV of the sciatic-tibial nerves in the untreated diabetic rats (untreated normal 55.1 ± 4.5, untreated diabetic 40.2 ± 3.1 m/s) was significantly ameliorated by intramuscular administration of bFGF with CGH (49.3 ± 4.6 m/s) after 10 days but not by that of bFGF with saline (Fig. 4A). On the other hand, the delayed MNCV of the tail nerves in diabetic rats was not improved by local injection of bFGF with CGH or saline into the thigh and soleus muscles (Fig. 4B). In addition, the effect of intramuscular administration of bFGF with CGH on the MNCV of the sciatic-tibial nerves was maintained for 30 days (Fig. 6A). The reduced sensitivity to mechanical stimuli in untreated diabetic rats (12.9 ± 2.7 g) compared with that in untreated normal rats (8.4 ± 1.6 g) was significantly ameliorated by intramuscular administration of bFGF with CGH (9.3 ± 1.0 g) after 10 days (Fig. 4C), and this effect was maintained for 30 days (Fig. 6B). However, intramuscular administration of bFGF with saline showed no significant effects.
Effects of intramuscular administration of bFGF with saline or CGH on SNBF.
The effects of intramuscular injection of bFGF with saline or CGH on the SNBF were similar to those on the MNCV of the sciatic-tibial nerves. No significant differences were found in the SNBF between any of the groups of normal rats. The reduced SNBF in the diabetic rats (7.9 ± 0.8 ml · mineC1 · 100 g body wteC1) was almost normalized by the intramuscular administration of bFGF with CGH (14.7 ± 1.9 ml · mineC1 · 100 g body wteC1) to the level of the untreated normal rats after 10 days (15.7 ± 1.5 ml · mineC1 · 100 g body wteC1) (Fig. 5), but bFGF with saline did not significantly improve the reduction (Fig. 4). The beneficial effect of bFGF with CGH on SNBF was also observed after 30 days (Fig. 6C).
Effects of intramuscular administration of bFGF with saline or CGH on retinal vessel diameters, MCT, and RBF.
No significant differences in the arterial and venous diameters between any of the experimental groups were observed after 10 days. The delayed MCT and decreased RBF in the diabetic rats were not altered by the local injection of bFGF with CGH or saline into the thigh and soleus muscles (data not shown). Because of the development of severe cataracts, the VFA measurements were not performed after 30 days.
DISCUSSION
The present study demonstrated that intravenous administration of bFGF has therapeutic effects on diabetic neuropathy and does not affect the retinal circulation and that local treatment by intramuscular injection of bFGF with CGH, a unique drug-delivery system, ameliorated hypoalgesia and the decreased MNCV and SNBF in diabetic rats without demonstrating systemic actions.
In the present study, sensitivity to mechanical stimuli was measured to evaluate a sensory nerve function. Our preliminary experiment demonstrated that diabetic rats develop hyperalgesia at 2eC4 weeks after induction of diabetes and hypoalgesia at 7eC8 weeks, which is consistent with a previous report by Calcutt et al. (37). However, deficits in MNCV and SNBF of diabetic rats are clearly demonstrated in 8 weeks. Therefore, therapeutic intervention with bFGF was performed 8 weeks after induction of diabetes.
Previous studies have shown that bFGF has various functions both in vitro and in vivo. In in vitro studies, the induction of proliferation activities by bFGF in various types of cells, including vascular cells (10,11,16), and the antiapoptotic action of bFGF on neural cells (14) have been reported. In in vivo studies, the effects of bFGF have been investigated in the fields of central and peripheral nervous systems (27eC30), skin (17), bone (18,19), heart (22,23), and vasculatures (20,21), and beneficial effects have been reported both experimentally and clinically. In addition to these effects, bFGF induces NO production through endothelial NO synthase activation, resulting in vasodilation (12,13,38).
Several neurotrophic factors and growth factors such as nerve growth factor (NGF) (39), vascular endothelial growth factor (VEGF) (40), and hepatocyte growth factor (HGF) (41) have been recognized to ameliorate diabetic neuropathy in animal models. Although the effects of NGF on diabetic neuropathy were most extensively investigated experimentally, a clinical trial failed to establish the usefulness of NGF for diabetic neuropathy (42,43). The therapeutic effects of VEGF (40) and HGF (41) through their angiogenic actions on diabetic neuropathy have been reported. On the other hand, the induction of HGF and VEGF by treatment with bFGF in vitro and in vivo has been demonstrated (44eC46). Interestingly, according to these studies (44eC46), under ischemic conditions, bFGF exerts its angiogenic effects by not only inducing but also harmonizing with these endogenous angiogenic factors. Moreover, it has been confirmed that the vasculatures induced by bFGF consist of both endothelial cells and smooth muscle cells, which act as functional vessels. Considering these reports, bFGF may be a plausible angiogenic growth factor and therapeutic agent for diabetic neuropathy.
In this study, we compared the effects of bFGF administered through three different delivery systems on diabetic neuropathy. Despite its short half-life, intravenous administration of bFGF dose dependently ameliorated the reduced SNBF and delayed MNCV of both the sciatic-tibial and tail nerves in diabetic rats after 10 days (24). These effects may not be mediated through direct actions of bFGF on nerve tissues, but through systemic actions on other tissues, and bFGF might trigger the activation of a cytokine cascade, causing its effect to continue even after its inactivation. However, further long-term effects of bFGF cannot be expected by intravenous injection. In fact, beneficial effects of intravenous administration of bFGF on hypoalgesia, sciatic-tibial MNCV, and SNBF after 10 days were not observed after 30 days in this study. In addition, systemic administration of bFGF by intravenous injection might cause cytotoxicity in various tissues, including retinae and kidneys, through its angiogenic and cell-proliferative actions. In the present study, no beneficial or adverse effects of intravenously administered bFGF on the retinae were observed. The reason for this result remains unclear. Differences may exist in the distribution of exogenously administered bFGF among tissues, which might cause the inconsistent effects on nerve tissues and retinal tissues. To clarify the action mechanisms and establish long-term safety, histological analyses, including angiogenesis and measurement of the distribution of exogenously administered bFGF, would be required. Although the effects on renal functions were not investigated in this study, previous studies have in fact demonstrated toxic actions of bFGF on kidneys, such as podocyte injury (47), epithelial injury (48), and mesangial cell proliferation and matrix accumulation (49). Therefore, delivery systems that can be locally administered and retain the biological activity of bFGF for a long period would be required for the clinical application of bFGF.
Recently, local injection of VEGF gene (40) or HGF gene (41) and transplantation of endothelial progenitor cells (32) to muscles around peripheral nerves have been reported to improve experimental diabetic neuropathy through the induction of local angiogenesis. Therefore, we explored the usefulness of local treatment of bFGF using two drug delivery systems, CGH and saline. CGH is considered an excellent and unique carrier of bFGF (25,26). CGH was prepared through cross-linking of acidic gelatin with an isoelectric point (IEP) of 5.0 and bFGF with an IEP of 9.0. This difference in the IEP enabled bFGF to maintain its bioactivity for an extended period. bFGF administered with CGH can function when released by the biodegradation of the hydrogel (25,26,31). In this study, bFGF administered with saline did not improve the decreased MNCV and SNBF in diabetic rats, but bFGF injected with CGH brought about a remarkable amelioration of the sciatic-tibial MNCV and SNBF in the diabetic rats, and its effects continued for at least 30 days. In addition, this local treatment did not improve the MNCV or SNBF of the opposite side, the tail nerve MNCV, or the retinal circulation in diabetic rats. These findings support the efficiency and safety of local treatment of bFGF with CGH.
Intravenous administration of bFGF at a low dose significantly improved SNBF but not MNCV, and long-term effects of bFGF were not observed by intravenous injection. In addition, intramuscular injection of bFGF with saline tended to increase SNBF without changing MNCV. These observations suggest that the primary action sites of bFGF would be the endoneurial microvasculature, that partial correction of SNBF would be insufficient to improve MNCV, and that the nonvascular actions of bFGF may account for little of its therapeutic efficacy.
In clinical practice, it is recognized that treatment of advanced symptomatic diabetic neuropathy patients with intravenous administration of vasodilatory agents, such as prostaglandin E1, ameliorates their symptoms. However, this effect cannot be maintained for a long period. Long-term efficacy of intramuscular administration of bFGF with CGH was clearly demonstrated in this study, suggesting a clinical benefit of this treatment.
In summary, intravenous administration of bFGF demonstrated a systemic action on the nerve functions of diabetic rats, including amelioration of hypoalgesia and delayed MNCV in both the sciatic-tibial and tail nerves. On the other hand, intramuscular injection of bFGF with CGH, but not that with saline, improved hypoalgesia, sciatic-tibial MNCV, and SNBF only in the treated side, and these effects were maintained for at least 30 days. Although the direct effects of bFGF on neural cells remain unclear and further studies, including histological analyses, will be required, these results strongly suggest that bFGF could have therapeutic value for diabetic neuropathy through the improvement of microvascular blood flow. Given its apparent safety and long-term efficiency, intramuscular administration of bFGF with CGH could be suitable for clinical application.
ACKNOWLEDGMENTS
This work was supported in part by a diabetes research grant from the Ministry of Health and Welfare of Japan.
The authors thank Kaken Pharmaceutical (Tokyo, Japan) for kindly providing bFGF and CGH and thank Yuko Maehata for technical assistance.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Vinik AI, Park TS, Stansberry KB, Pittenger GL: Diabetic neuropathies. Diabetologia 43:957eC973, 2000
The DCCT Research Group: The effect of intensive treatment of diabetes on the development and progression of lomg-term complications in insulin-dependent diabetes mellitus. N Engl J Med 329:977eC986, 1993
Tomlinson DR, Willars GB, Carrington AL: Aldose reductase inhibitors and diabetic complications. Pharmacol Ther 54:151eC194, 1992
Yagihashi S, Kamijo M, Ido Y, Mirrless DJ: Effect of long-term aldose reductase inhibition in development of experimental diabetic neuropathy: ultrastructural and morphometric studies of sural nerve in streptozotocin-induced diabetic rats. Diabetes 39:690eC696, 1990
Yagihashi S, Kamijo M, Baba M, Yagihashi N, Nagai K: Effect of aminoguanidine on functional and structural abnormalities in peripheral nerve of STZ-induced diabetic rats. Diabetes 41:47eC52, 1992
Nakamura J, Kato K, Hamada Y, Nakayama M, Chaya S, Nakashima E, Naruse K, Kasuya Y, Mizubayashi R, Miwa K, Yasuda Y, Kamiya H, Ienaga K, Sakakibara F, Koh N, Hotta N: A protein kinase C--selective inhibitor ameliorates neural dysfunction in streptozotocin-induced diabetic rats. Diabetes 48:2090eC2095, 1999
Vincent AM, Russell JW, Low P, Feldman EL: Oxidative stress in the pathogenesis of diabetic neuropathy. Endocr Rev 25:612eC628, 2004
Cameron NE, Cotter MA: Neurovascular dysfunction in diabetic rats: potential contribution of autooxidation and free radicals examined using transition metal chelating agents. J Clin Invest 96:1159eC1163, 1995
Abraham JA, Mergia A, Whang JL, Tumolo A, Friedman J, Hjerrild KA, Gospodarowicz D, Fiddes JC: Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science 233:545eC548, 1986
Schweigerer L, Neufeld G, Mergia A, Abraham JA, Fiddes JC, Gospodarowicz D: Basic fibroblast growth factor in human rhabdomyosarcoma cells: implications for the proliferation and neovascularization of myoblast-derived tumors. Proc Natl Acad Sci U S A 84:842eC846, 1987
Esch F, Baird A, Ling N, Ueno N, Hill F, Denoroy L, Klepper R, Gospodarowicz D, Bohlen P, Guillemin R: Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci U S A 82:6507eC6511, 1985
Yang HT, Yan Z, Abraham JA, Terjung RL: VEGF121- and bFGF-induced increase in collateral blood flow requires normal nitric oxide production. Am J Physiol Heart Circ Physiol 280:H1097eCH1104, 2001
Cuevas P, Carceller F, Ortega S, Zazo M, Nieto I, Gimenez-Gallego G: Hypotensive activity of fibroblast growth factor. Science 254:1208eC1210, 1991
Shaw R, Cianchetti R, Pleasure D, Kreider B: Basic fibroblast growth factor prevents cAMP-induced apoptosis in cultured Schwann cells. J Neurosci Res 47:400eC404, 1997
Iwai-Kanai E, Hasegawa K, Fujita M, Araki M, Yanazume T, Adachi S, Sasayama S: Basic fibroblast growth factor protects cardiac myocytes from iNOS-mediated apoptosis. J Cell Physiol 190:54eC62, 2002
Boilly B, Vercoutter-Edouart AS, Hondermarck H, Nurcombe V, Le Bourhis X: FGF signals for cell proliferation and migration through different pathways. Cytokine Growth Factor Rev 11:295eC302, 2000
Tanaka E, Ase K, Okuda T, Okumura M, Nogimori K: Mechanism of acceleration of wound healing by basic fibroblast growth factor in genetically diabetic mice. Biol Pharm Bull 19:1141eC1148, 1996
Kawaguchi H, Kurokawa T, Hanada K, Hiyama Y, Tamura M, Ogata E, Matsumoto T: Stimulation of fracture repair by recombinant human basic fibroblast growth factor in normal and streptozotocin-diabetic rats. Endocrinology 135:774eC781, 1994
Nakamura T, Hanada K, Tamura M, Shibanushi T, Nigi H, Tagawa M, Fukumoto S, Matsumoto T: Stimulation of endosteal bone formation by systemic injections of recombinant basic fibroblast growth factor in rats. Endocrinology 136:1276eC1284, 1995
Stark J, Baffour R, Garb JL, Kaufman J, Berman J, Rhee S, Norris MA, Friedmann P: Basic fibroblast growth factor stimulates angiogenesis in the hindlimb of hyperglycemic rats. J Surg Res 79:8eC12, 1998
Baffour R, Berman J, Garb JL, Rhee SW, Kaufman J, Friedmann P: Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor. J Vasc Surg 16:181eC191, 1992
Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA: Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation 105:788eC793, 2002
Yanagisawa-Miwa A, Uchida Y, Nakamura F, Tomaru T, Kido H, Kamijo T, Sugimoto T, Kaji K, Utsuyama M, Kurashima C, Ito H: Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science 257:1401eC1403, 1992
Yuge T, Furukawa A, Nakamura K, Nagashima Y, Shinozaki K, Nakamura T, Kimura R: Metabolism of the intravenously administered recombinant human basic fibroblast growth factor, trafermin, in liver and kidney: degradation implicated in its selective localization to the fenestrated type microvasculatures. Biol Pharm Bull 20:786eC793, 1997
Tabata Y, Nagano A, Ikada Y: Biodegradation of hydrogel carrier incorporating fibroblast growth factor. Tissue Eng 5:127eC138, 1999
Tabata Y, Ikada Y: Vascularization effect of basic fibroblast growth factor released from gelatin hydrogels with different biodegradabilities. Biomaterials 20:2169eC2175, 1999
Abe K, Saito H: Effects of basic fibroblast growth factor on central nervous system functions. Pharmacol Res 43:307eC312, 2001
Katsuki H, Itsukaichi Y, Matsuki N: Distinct signaling pathways involved in multiple effects of basic fibroblast growth factor on cultured rat hippocampal neurons. Brain Res 885:240eC250, 2000
Grothe C, Nikkhah G: The role of basic fibroblast growth factor in peripheral nerve regeneration. Anat Embryol 204:171eC177, 2001
Fujimoto E, Mizoguchi A, Hanada K, Yajima M, Ide C: Basic fibroblast growth factor promotes extension of regenerating axons of peripheral nerve: in vivo experiments using a Schwann cell basal lamina tube model. J Neurocytol 26:511eC528, 1997
Kawaguchi H, Nakamura K, Tabata Y, Ikada Y, Aoyama I, Anzai J, Nakamura T, Hiyama Y, Tamura M: Acceleration of fracture healing in nonhuman primates by fibroblast growth factor-2. J Clin Endocrinol Metab 86:875eC880, 2001
Naruse K, Hamada Y, Nakashima E, Kato K, Mizubayashi R, Kamiya H, Yuzawa Y, Matsuo S, Murohara T, Matsubara T, Oiso Y, Nakamura J: Therapeutic neovascularization using cord blood-derived endothelial progenitor cells for diabetic neuropathy. Diabetes 54:1823eC1828, 2005
Koshu K, Kamiyama K, Oka N, Endo S, Takaku A, Saito T: Measurement of regional blood flow using hydrogen gas generated by electrolysis. Stroke 13:483eC487, 1982
Abiko T, Abiko A, Clermont AC, Shoelson B, Horio N, Takahashi J, Adamis AP, King GL, Bursell SE: Characterization of retinal leukostasis and hemodynamics in insulin resistance and diabetes: role of oxidants and protein kinase-C activation. Diabetes 52:829eC837, 2003
Horio N, Clermont AC, Abiko A, Abiko T, Shoelson BD, Bursell SE, Feener EP: Angiotensin AT(1) receptor antagonism normalizes retinal blood flow and acetylcholine-induced vasodilatation in normotensive diabetic rats. Diabetologia 47:113eC123, 2004
Takagi C, King GL, Clermont AC, Cummins DR, Takagi H, Bursell SE: Reversal of abnormal retinal hemodynamics in diabetic rats by acarbose, an alpha-glucosidase inhibitor. Curr Eye Res 14:741eC749, 1995
Calcutt NA, Freshwater JD, Mizisin AP: Prevention of sensory disorders in diabetic Sprague-Dawley rats by aldose reductase inhibition or treatment with ciliary neurotrophic factor. Diabetologia 47:718eC724, 2004
Kostyk SK, Kourembanas S, Wheeler EL, Medeiros D, McQuillan LP, D’Amore PA, Braunhut SJ: Basic fibroblast growth factor increases nitric oxide synthase production in bovine endothelial cells. Am J Physiol 269:H1583eCH1589, 1995
Goss JR, Goins WF, Lacomis D, Mata M, Glorioso JC, Fink DJ: Herpes simplex-mediated gene transfer of nerve growth factor protects against peripheral neuropathy in streptozotocin-induced diabetes in the mouse. Diabetes 51:2227eC2232, 2002
Schratzberger P, Walter DH, Rittig K, Bahlmann FH, Pola R, Curry C, Silver M, Krainin JG, Weinberg DH, Ropper AH, Isner JM: Reversal of experimental diabetic neuropathy by VEGF gene transfer. J Clin Invest 107:1083eC1092, 2001
Kato N, Nemoto K, Nakanishi K, Morishita R, Kaneda Y, Uenoyama M, Ikeda T, Fujikawa K: Nonviral gene transfer of human hepatocyte growth factor improves streptozotocin-induced diabetic neuropathy in rats. Diabetes 54:846eC854, 2005
Apfel SC: Nerve growth factor for the treatment of diabetic neuropathy: what went wrong, what went right, and what does the future hold Int Rev Neurobiol 50:393eC413, 2002
Pittenger G, Vinik A: Nerve growth factor and diabetic neuropathy. Exp Diabesity Res 4:271eC285, 2003
Masaki I, Yonemitsu Y, Yamashita A, Sata S, Tanii M, Komori K, Nakagawa K, Hou X, Nagai Y, Hasegawa M, Sugimachi K, Sueishi K: Angiogenic gene therapy for experimental critical limb ischemia: acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2. Circ Res 90:966eC973, 2002
Onimaru M, Yonemitsu Y, Tanii M, Nakagawa K, Masaki I, Okano S, Ishibashi H, Shirasuna K, Hasegawa M, Sueishi K: Fibroblast growth factor-2 gene transfer can stimulate hepatocyte growth factor expression irrespective of hypoxia-mediated downregulation in ischemic limbs. Circ Res 91:923eC930, 2002
Tsutsumi N, Yonemitsu Y, Shikada Y, Onimaru M, Tanii M, Okano S, Kaneko K, Hasegawa M, Hashizume M, Maehara Y, Sueishi K: Essential role of PDGFRalpha-p70S6K signaling in mesenchymal cells during therapeutic and tumor angiogenesis in vivo: role of PDGFRalpha during angiogenesis. Circ Res 94:1186eC1194, 2004
Sasaki T, Hatta H, Osawa G: Cytokines and podocyte injury: the mechanism of fibroblast growth factor 2-induced podocyte injury. Nephrol Dial Transplant 14 (Suppl. 1):33eC34, 1999
Sasaki T, Jyo Y, Tanda N, Tamai H, Osawa G: The role of basic fibroblast growth factor (FGF2) in glomerular epithelial cell injury. Contrib Nephrol 118:68eC77, 1996
Floege J, Eng E, Young BA, Alpers CE, Barrett TB, Bowen-Pope DF, Johnson RJ: Infusion of platelet-derived growth factor or basic fibroblast growth factor induces selective glomerular mesangial cell proliferation and matrix accumulation in rats. J Clin Invest 92:2952eC2962, 1993(Mika Nakae, Hideki Kamiya)