THE INFLUENCE OF MOMORDICA CHARANTIA FRUIT JUICE ON PERIPHERAL NERVE STRUCTURE IN EXPERIMENTAL DIABETES
Ahmed I., Sharma A.K., Samad P.A. Faculty of Med., United Arab Emirates University, Al Ain, UAE
Momordica charantia fruit is traditionally used as a vegetable and is known to have hypoglycemic effect in human and experimental diabetes. Ultrastructural observations were made on myelinated fibers in the tibial nerve of streptozotocin-diabetic rats. Four groups of male Wistar rats, aged 12 weeks were studied over a period of four months: untreated diabetic rats, diabetic rats treated with M charantia, controls treated with M charantia and age-matched controls. Blood glucose concentrations were significantly reduced in M charantia treated diabetic animals when compared with untreated diabetic rats (p< 0.05), but were still significantly greater (p< 0.05) than those of age-matched controls. The cross-sectional myelinated fiber area (p< 0.03), axonal area (p< 0.02) and myelin area (p< 0.04) including the mean maximum myelinated fiber area (p< 0.03) were significantly less in untreated diabetics when compared with age-matched controls. In the M charantia treated diabetic animals, these parameters were intermediate between untreated diabetics and age-matched controls without showing significant difference when compared with either group. The axon to fiber ratio ('g'ratio) did not differ between any of the experimental groups. In conclusion, the administration of M charantia fruit juice reduced blood glucose levels and partially corrected the structural abnormalities of peripheral nerves in experimental diabetes.
INCREASED GLYCATION AND NERVE DYSFUNCTION IN EXPERMENTAL DIABETES
Carrington A., Jacot J., Orlowski L., Cannan S., Letsinger J., Litchfield J. Institute for Diabetes Discovery, Branford, CT, USA
The impaired nerve function in diabetic neuropathy is associated with non-enzymatic glycation of nerve proteins. This study examined conduction velocity (CV) and detection of early glycation products in peripheral nerve from streptozotocin (STZ)-induced diabetic rats. Study 1: a monoclonal mouse IgM primary antibody raised against glucose derived Amadori-modified lysine residues (1 - deoxyfructosyl lysine) was used for Western blot (WB) analysis of sciatic nerve glycated proteins. Study 2: rats were anesthetized and motor nerve CV was measured in the sciatic nerve-interosseus muscle system. In addition, the sciatic nerves were processed for immunohistochemistry (IHC) of glycated proteins (5m thick-paraffin sections) using the same Amadori antibody and avidin-biotin-peroxidase-DAB-NiCo substrate. All diabetic rats were hyperglycemic (>300mg/dl). WB demonstrated a two fold increased glycation of protein bands in the 37kD to 90kD range, at 1, 2, 3 and 4 weeks post STZ. MNCV was significantly decreased at 1, 2 and 3 months of diabetes compared to controls, p< 0.001. IHC results complemented the WB data showing an apparent increase in glycation of diabetic nerve compared to controls. This is suggestive of a link between nerve dysfunction and glycation of nerve proteins and consistent with previous studies demonstrating increased glycation of nerve proteins in clinical and experimental diabetes.
ELECTRICAL AND TACTILE SENSORY CONDUCTION VELOCITY IN PERIPHERAL NERVES OF DIABETIC PATIENTS
Caruso G., Nolano M., Crisci C., Lanzillo B., Di Lorenzo N., Lullo F., D'Addio G. Department of Neurological Sciences, University of Naples Federico II, Naples, Italy, Fondazione Salvatore Maugeri, IRCCS, Telese, Benevento, Italy
Orthodromic sensory conduction velocity induced by electrical and mechanical stimulation was studied along the median nerve of a group of young subjects affected by diabetes mellitus for different periods of time. Some of these patients underwent a cutaneous punch biopsy of the tip of digit III, which served for the study of Meissner's corpuscles and epidermal nerve fibers. Compared with control values, in patients there was a significant slowing of conduction velocity associated with a marked decrease of sensory evoked potential amplitude. However, in each individual patient tactile conduction velocity was almost constantly more affected than electrically stimulated conduction velocity. In addition, changes in tactile conduction appeared significantly earlier in the course of the disease, to such an extent that the study of tactile conduction velocity could be considered a reliable procedure for the detection of early involvement of peripheral nerve fibers in diabetic patients. In patients for whom cutaneous biopsy was available, there was a marked reduction of the density of the epidermal nerve fibers and diffuse involvement of Meissner's corpuscles.
EVIDENCE OF INFLAMMATORY CELL INFILTRATION AND DEATH IN DIABETIC NERVES
Conti G., Scarpini E., Baron P.L., Erba M.(1), Bianchi R.(2), De Riz M., Livraghi S., Scarlato G. Dept. Of Neurology, Dino Ferrari Center, (1)Dept of Vascular Surgery, IRCCS Osp. Maggiore Policlinico, University of Milan, (2)Mario Negri Institute, Milan, Italy
Activated monocytes/macrophages and lymphocytes have been recently found in diabetic nerves, with a possible role in chemotaxis and regeneration. We studied the presence of inflammatory cells within nerves of diabetic patients affected by distal symmetrical polyneuropathy and in rats that become diabetic after the intraperitoneal injection of streptozotocin. Using antibodies specific for Schwann cells, T-lymphocytes and macrophages on teased nerve fibers, we observed that macrophages and T-cells were infiltrating the endoneurium in both human and experimental diabetic nerves. By TUNEL and DAPI staining we observed in the teased fibers from the same nerves that 20% of these inflammatory cells undergo apoptosis. The endoneurial infiltration and death by apoptosis of inflammatory cells in diabetic nerves may be crucial in the regulation of nerve damage, and suggest a potential effect of anti-inflammatory and/or anti-immune drugs in the treatment of diabetic neuropathies.
THE PATTERN OF SENSATION ABNORMALITIES IN THE DIABETIC FOOT - A CONTROLLED STUDY OF TWO COHORTS FROM ROCHESTER, MN
Dyck P.J., Gillen D., Larson T., Velosa J. Mayo Clinic, Rochester, MN 55905 USA
OBJECTIVE: To characterize the patterns of sensation abnormality in the foot of a representative cohort of community diabetics in Rochester, MN (RDNS) (n=380) and in Pancreas-renal or Pancreas (PR-P) transplant (n=80) cohorts. There is limited information on the prevalence of hyperesthesia, hyperalgesia, hypesthesia, or hypoalgesia in representative diabetic cohorts and whether these phenomena precede or accompany diabetic polyneuropathy. METHODS: CASE IV was used to assess vibration (VDT) and cooling (CDT) detection thresholds and heat-pain 5 and 0.5 thresholds of foot in the two cohorts. Percentile values and normal deviate values specific for site, age, and applicable anthropometric variables were available from a previous study (HS-RDNS) (n=~500). Conceptually threshold values were classified as hyperesthetic or hyperalgesic (£ 5th percentile) or hypoesthetic or hypoalgesic (> 95th percentile). RESULTS: In the RDNS, hypesthesia and hypoalgesia were much commoner than hyperesthesia and hyperalgesia. However, mean threshold values were significantly higher than in controls. 2) In the PR-P cohort, unequivocal hypesthesia and hypoalgesia occurred in most patients and protective sensation was often abnormal. 3) Hyperesthesia and hyperalgesia were uncommon in both cohorts. CONCLUSION: In two cohorts, the one characteristic of community diabetes (RDNS) and the other (PR-P) typical of type 1 patients with severe complications, hypofunction due to loss of peripheral sensory units is much commoner than is hyperesthesia and hyperalgesia as evaluated by QST. These facts have clinical and research implications.
NEURAL AND ENDOTHELIAL CONTROL OF THE MICROCIRCULATION IN DIABETES MELLITUS
Kilo S., Berghoff M., Hilz M., Freeman R. Beth Israel Deaconess Medical Center, Boston, MA USA
Complications of diabetes include microvascular and neural dysfunction. The respective roles of these abnormalities and their interaction in the causation of diabetic polyneuropathy is unknown. We assessed neural and endothelial control of the microcirculation in 16 type 1 and 17 type 2 diabetic patients and their respective age- and gender-matched controls. Seven incrementing doses of the endothelium-dependent vasodilator acetylcholine (Ach) and the NO-donor sodium nitroprusside (SNP) were applied to the forearm using iontophoresis. Direct and axon reflex mediated vasodilation evoked by Ach and SNP were recorded using laser Doppler flowmetry. NP iontophoresis resulted in a ~4 fold increase in blood flow (p<0.05). There was no difference in baseline or SNP evoked blood flow in diabetic subjects compared to controls. There was a significant delay in the onset of the endothelium-mediated blood flow increase with a preserved maximal response in type 1 diabetics (p<0.05). In contrast, the Ach-induced, endothelium-mediated bloodflow increase was reduced in type 2 diabetes (p<0.05). The Ach induced axon-reflex was reduced in both diabetic groups (p<0.05). These data indicate that early microcirculatory changes in diabetes have a neural and endothelial but no smooth muscle mechanism. The endothelial mechanisms include a decreased sensitivity of the endothelium to Ach or alteration in NO synthesis or release. Furthermore, these data suggest that microcirculatory changes seen in type 1 and type 2 diabetes mellitus may have a different pathogenesis. These abnormalities may be secondary to and play a role in the causation of diabetic polyneuropathy
INSULIN TREATMENT ENHANCES INSULIN LIKE GROWTH FACTOR I (IGF-I) EXPRESSION IN SURAL NERVES OF DIABETIC PATIENTS
Grandis M., Nobbio L., Barreca A.(1), Minuto F.(1), Banchi L., Gherardi G.F., Abbruzzese M., Mancardi G.L., Schenone A. Department of Neurological Sciences , (1)Department of Metabolic and Endocrinologic Sciences, University of Genoa, Italy
IGF-I regulation may be impaired in diabetic neuropathy. IGF-I deficiency has been demonstrated only in experimental models of IDDM. Moreover, insulin therapy, which prevents nerve damage due to diabetes, may influence IGF-I expression. We studied IGF-I and IGF-I receptor expression, by semiquantitative RT-PCR and immunohistochemistry, in sural nerves from 8 diabetic subjects with peripheral neuropathy, 5 axonal neuropathies and 4 normal controls. To analyze the effects of insulin therapy on IGF-I expression, we divided insulin treated (IT) and non-insulin treated (NIT) patients, irrespective of the type of diabetes. IGF-I and IGF-I receptor mRNA was found in sural nerves from all cases. IGF-I mRNA levels were not different in diabetic subjects compared to axonal neuropathies or normal controls (1.18±0.8 vs 1.45±0.47 vs 0.36±0.18); only axonal neuropathies were significantly higher than normal controls (p<0.05). In IT diabetic patients IGF-I expression was higher than in NIT subjects (1.75± 0.65 vs 0.6±0.2, p<0.05). IGF-I receptor behaved as its ligand. IGF-I immunoreactivity was observed in all the examined nerves, particularly in Schwann cells, axons and endothelial cells. A higher immunoreactivity was observed in sural nerves undergoing axonal degeneration and in IT than in NIT diabetic patients and normal controls. In conclusion insulin may exert its protective action on peripheral nerves also increasing expression of IGF-I. Like in animal models of NIDDM, IGF-I is not reduced in sural nerves of our NIT patients; therefore, IGF-I may be claimed as a cofactor more in the pathogenesis of neuropathy during IDDM than NIDDM. Finally, failure of diabetic nerves to upregulate IGF-I mRNA, in absence of insulin treatment, may account for the impairment of nerve regeneration due to diabetes.
CORRELATION BETWEEN BLOOD HbAl LEVELS AND DECREASE IN NERVE CONDUCTION VELOCITY IN DIABETICS
Hasegawa O., Mori I., Matsumoto S., Iino M., Arita T. Department of Neurology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
We investigated the relationship between blood levels of HbAl, an indicator of diabetic control, and polyneuropathy index (PNI), an indicator of diabetic polyneuropathy. A total of 155 patients (mean age at the last study; 65.0 years) with non-insulin-dependent diabetes mellitus were followed for more than 5 years and were subjected to a series of motor conduction studies every 1 to 3 years. Their mean HbAl level during the period was 9.3%. The PNI was calculated as a mean percentage of normal value for 12 indices derived from the conduction velocity and long-distance latency in motor conduction studies of 4 nerves. The initial PNI was 86.6% and the last PNI decreased to 82.7% on the mean. The mean follow-up period was 8.1 years, and during that time the mean decrease in the PNI was 4.0%. This value corresponds to 0.49% of reduction per year. There was a significant negative correlation between mean HbAl levels and the percent changes in PNI per year. The higher the HbA1 level during the period, the larger the decline in PNI was. In patients with low initial PNI levels the change in PNI tended to be small in spite of high HbAl levels. Likewise, in patients with high initial PNI levels the decrease in PNI tended to be large despite relatively good control of their HbAl levels. Careful control of diabetes mellitus is of importance to prevent the progression of neuropathies, especially in the initial stage.
THREE DIMENSIONAL NETWORK OF ENDONEURIAL MICROVESSELS IN CHRONIC DIABETIC RATS
Honma H., Katoh J., Yasaki S., Saito N. 3rd Department of Internal Medicine, St.Marianna University School of Medicine, Kawasaki, Japan
The abnormalities of endoneurial microvessels have been considered to be implicated in the pathogenesis of experimental diabetic neuropathy. But the precise vascular structure of endoneurial microvessels in chronic diabetic animals has not been studied. To study the endoneurial vascular structure in chronic diabetic rats, we infused a watery soluble dye (Microfil) into abdominal aortas in chronic diabetic rats (12 months) induced by streptozotocin. Sciatic nerve was taken after 12 months and histological studies were done. Three dimensional structure of the network of endoneurial microvessels were estimated quantitatively by stereoscopic microscope and electron microscope. Motor nerve conduction velocity (MCV) was also measured. In diabetic rats, MCV reduced from that in controls and the networks of endoneurial microvessels were coarse. Intramicrovascular areas were increased in diabetic rats. These results suggest that an ischemic change of endoneurial microvessels play the role of inducing neuropathy.
PROXIMO-DISTAL DISTRIBUTION OF NERVE CONDUCTION DEFICITS IN STREPTOZOTOCIN-DIABETIC AND GALACTOSE-INTOXICATED RATS
Kalichman M.W., Dines K.C., Mizisin A.P. University of California, San Diego, La Jolla, CA USA
Human diabetic neuropathy generally develops first in distal nerves; however, this pattern of deficits has not been verified in experimental models of diabetic neuropathy. The two most widely-studied animal models of Type I and Type II diabetic neuropathy are streptozotocin diabetes and galactose intoxication, respectively. To better understand the mechanisms of neuropathic complications in diabetes, motor nerve conduction measurements were made at various locations in the peripheral nervous system of rats after 3 days, 1 week, 4 weeks, and 8 weeks of either streptozotocin diabetes or intoxication with a diet of 40% galactose. After 3 days of streptozotocin diabetes, motor nerve conduction velocity (MNCV) was significantly reduced at distal (sciatic nerve) and intermediate (between 4th lumbar vertebra and sciatic notch), but not proximal (ventral roots), locations at 3 days. After one week of streptozotocin diabetes, MNCV was no longer reduced at any of the three locations; however, significant and similar reductions were recorded at all three locations at 28 days (-20.3 to -21.1%) and at 56 days (-23.8 to -30.5%). Galactose intoxication had no significant impact after 3 days; however, at 7 days, MNCV was significantly reduced proximally, but not more distally, by 20.2%. MNCV reductions with galactose, similar to those seen with streptozotocin, were -13.2 to -21.2% at 28 days and -19.4 to -29.2% at 56 days. Other than an early (3 days) reversible effect of streptozotocin diabetes on MNCV in distal locations, streptozotocin resulted in parallel reductions across proximal and distal locations of the peripheral nervous system at 28 and 56 days. In contrast, galactose resulted in early proximal reductions in MNCV at 7 days, followed by more distal reductions at 28 - 56 days. These data are not consistent with the pattern of proximo-distal changes observed in human diabetic neuropathy.
ALTERED VASOREACTIVE TO ANGIOTENSIN II IN EXPERIMENTAL DIABETIC NEUROPATHY - ROLE OF NITRIC OXIDE
Kihara M., Takahashi M. Department of Neurology, Kinki University, School of Medicine, Osaka, Japan
OBJECTIVE: We evaluated the effects of Angiotensin II and angiotensin converting enzyme inhibitor (cilazapril) on nerve blood flow (NBF) and electrophysiology in control and diabetic rats. METHODS: We measured NBF in sciatic nerve by microelectrodes H2 polarography following the applied locally special agents. We also performed nerve conduction studies. RESULTS: When applied locally to the sciatic nerve, dose-response curve of angiotensin II provide to be more potent in EDN than control rats. However there was not difference of angiotensin II in serum between EDN and controls. The rats were given typical rat pellets or pellets treated with 10mg/Kg/day cilazapril for 4 weeks. Diabetes caused a significant reduction in NBF, nerve conduction velocity and compound muscle action potential amplitudes (CMAP). NBF was significantly increased in diabetic rats supplemented with cilazapril diet and nerve conduction velocity and amplitudes of the CMAP were also improved after 4 weeks on this diet. Direct application 10(exp-3)M cilazapril on sciatic nerve did not increase NBF in normal and diabetic neuropathy (EDN). We topically applied the nitric oxide syntheses inhibitor NG-nitro-L-argine and observed reduced inhibition of NBF in EDN, correctable with cilazapril diet. CONCLUSION: These results suggested that diabetic neuropathy may have the increasing of vasopressor action with Angiotensin II and it is likely to be the mechanism of NOS inhibition. Angiotensin II converting enzyme inhibitors may have potential in treatment of diabetic neuropathy.
AXONAL TRANPORT OF BDNF IN EXPERIMENTAL DIABETES
Mizisin A.P., DiStefano P.S., Liu X., Garrett D., Tonra J.R. University of California, San Diego, La Jolla CA and Regeneron Pharmaceuticals, Inc, Tarrytown NY, USA
Recent demonstrations of anterograde transport of BDNF and its dramatic increase after axotomy point to a role for altered anterograde movement of this neurotrophin in neuropathic conditions. Taken together with reports of increased expression of BDNF mRNA in DRG and skeletal muscle of streptozotocin-diabetic rats, these observations suggest that axonal transport of BDNF may be increased in experimental diabetes. Given this, we investigated the impact of streptozotocin diabetes or galactose feeding on axonal transport of BDNF. Female Sprague-Dawley rats (200-250 g) were either made diabetic (60 mg/kg streptozotocin ip) or fed a diet containing 40% D-galactose while control animals were either vehicle-injected or fed a diet containing 40% solka floc, a nonnutritive fiber. After 8 weeks of experimental diabetes, the capacity to transport endogenous BDNF was evaluated by measuring, with a commercial ELISA kit (Promega), the accumulation of neurotrophin against two sciatic nerve ligatures placed 1 cm apart and in place for 18-20 hours. In nerves from control, streptozotocin-diabetic and galactose-fed rats (11-12/group), anterograde accumulation of BDNF was greater than retrograde accumulation. Anterograde BDNF accumulation against the proximal ligature was significantly decreased in both streptozotocin-diabetic: (P<0.03) and galactose-fed (P<0.02) rats compared to controls, consistent with a reduction in anterograde transport. While there was a significant decrease in BDNF accumulation against the distal ligature in diabetic rats compared to controls (P<0.05), indicating a reduction in retrograde transport, this decrease was not significant in galactose-fed rats. There was also a significant reduction in the amount of BDNF trapped between the ligatures in streptozotocin-diabetic rats (P<0.001). In addition, the BDNF content of unligated contralateral nerves was significantly decreased in both streptozotocin-diabetic (P< 0.0001) and galactose-fed (P < 0.01) rats compared to controls. Therefore, the reported increase in BDNF mRNA in DRG and skeletal muscle may reflect a compensatory attempt to improve neurotrophic support in experimental diabetes. Supported by NIH Grant NS32339.
EFFECTS OF OPB-9195, A NEW ANTI-GLYCATION AGENT, ON STZ-INDUCED EXPERIMENTAL DIABETIC NEUROPATHY
Nishizawa Y., Wada R., Ishikawa S., Yasumura K., Yagihashi S. Hirosaki. University School of Medicine, Otsuka Fujii Memorial Research Institute, Hirosaki and Otsu, Japan
Non-enzymatic glycation of neural proteins has been implicated in the pathogenesis of diabetic neuropathy. We examined the effects of OPB-9195 (OPB), a new anti-glycation agent, on the peripheral nerve structure and function in STZ-induced diabetic rats. Eight-week old Wistar rats were made diabetic by STZ (60mg/kg, iv) and OPB (60mg/kg/day) was given by gavage for 24 weeks. Age- and sex-matched normal Wistar rats were used for comparison. Diabetic rats showed marked hyperglycemia and less body weight and significant delay of tibial motor nerve conduction velocity (MCV) (75% of control levels) during the whole experimental period. OPB treatment did not affect the body weight, elevated levels of blood glucose and glycated hemoglobin. Serum AGE levels measured by ELISA were significantly elevated in diabetic rats and this increase was significantly inhibited by OPB treatment. Delayed MCV was significantly improved in OPB-treated diabetic rats by 60%. Sciatic nerve Na+,K+-ATPase activity was reduced in diabetic rats and OPB treatment restored this activity. Teased nerve fiber analysis on the sural nerve demonstrated an increased incidence of fibers with paranodal demyelination and swelling which tended to decrease in OPB-treated diabetic rats. Morphometric analysis demonstrated significant decrease in average myelinated fiber area in diabetic rats but this decrease was not significantly influenced by OPB treatment. The current study thus indicated that increased glycation has a pathogenetic role in diabetic neuropathy and 0PB is partially but significantly beneficial for the prevention of diabetic neuropathy.
EVALUATION OF A SORBITOL DEHYDROGENASE INHIBITOR ON DIABETES-INDUCED CHANGES IN PERIPHERAL NERVE METABOLISM AND ANTIOXIDATIVE DEFENSE: A PREVENTION STUDY
Obrosova I.G., Fathallah L., Lang H.J., Greene D.A. University of Michigan, Ann Arbor, MI, USA, Hoechst Marion Roussel, Frankfurt/Main, Germany
Studies of the role for sorbitol dehydrogenase (SDH) in diabetes-induced
nerve conduction slowing, using SDH inhibitors (SDIs) as well
as SDH/- vs. SDH/+ mice, reported contradictory results. As metabolic
effects of SDIs in the diabetic nerve have never been studied
other than in non-steady-state in vitro incubations, we
evaluated an SDI on sorbitol pathway intermediates, mitochondrial
and cytosolic NAD-redox and energy status, as well as parameters
of oxidative stress in vivo using a prevention approach
with SDI administration in the drinking water (i.e. conditions
which, according to some reports, are essential for demonstration
of beneficial effects of SDIs). Control (C) and STZ-diabetic (D)
rats were treated with/without SDI-157 (100 mg/kg/d for 3 wks).
Sciatic nerve free mitochondrial (cristae and matrix) and cytosolic
NAD+/NADH ratios were calculated from metabolites of the b-hydroxy-butyrate, glutamate and lactate
dehydrogenase systems. Phosphocreatine (PCr) creatine (Cr) and
ATP were measured enzymatically, total MDA plus 4-hydroxyalkenals
(4-HA) with N-methyl-2-phenylindole. The increased flux through
SDH (manifested by fructose levels) was 92% inhibited in D+SDI
vs. D. Free NAD+/NADH ratios in both mitochondrial cristae and
matrix were similarly decreased in D and D+SDI vs. C (consistent
with endoneurial hypoxia). Free cytosolic NAD+/ NADH were decreased
as well (680±113 vs 1372±300 in C) and this decrease
was not prevented by SDI (829±124). Decrease in PCr levels
(2.42±0.38 vs 3.14±0.46 mmol/g
in C) and PCr/Cr (0.41±0.08 vs 0.69±0.17 in C) as
well as total MDA plus 4-HA accumulation and GSH depletion were
further exacerbated by SDI. In conclusion, the results do not
support the concept of "diabetic pseudohypoxia", and
are consistent with the idea that minor flux through SDH can not
account for the changes in such a dynamic system as the redox
state of nicotinamide dinucleotides, in the diabetic nerve. Moreover,
exacerbation of energy deficiency and oxidative stress in D+SDI
indicates that inhibition of SDH in diabetes is adverse rather
than beneficial, and is in agreement with the findings of a dramatic
increase in neuroaxonal dystrophy (Schmidt et al, 1998)
rather than with reports of salutatary effects of SDIs.
ELECTROPHYSIOLOGICAL STUDIES OF EXPERIMENTAL HYPOGLYCEMIC NEUROPATHY IN RATS
Ohshima J., Nukada H. Department of Medicine, University of Otago Medical School, Dunedin, New Zealand
Reduction of nerve blood flow may play an important role in the underlying mechanism of the development of acute hypoglycemic neuropathy. Insulin induced hypoglycemic rats have been used extensively in the investigation of hypoglycemic neuropathy. However electrophysiological studies have been little studied. We measured sciatic nerve blood flows (NBFs) and nerve vascular resistances (NVRs) using a laser Doppler flowmeter, and nerve conduction velocities (NCVs) at sciatic and caudal nerves during 3 hours of insulin-induced hypoglycemia in rats. Systematic blood pressure and arterial blood gas were also monitored. NBFs were significantly decreased and NVRs were significantly increased at 1 hour after the insulin injection at mid and lower thigh levels of sciatic nerves. Sciatic NCVs became slower during 3 hours of hypoglycemia and were significantly reduced at 3 hours after the insulin injection. Caudal NCVs bad also slowed, but not significant statistically. Reduced NBFs and increased NVRs were found at thigh levels in the sciatic nerves after 1 hour insulin injection. These phys-iological changes suggest that ischemia is implicated in the underlying mechanism of acute hypoglycemic neuropathy.
POSSIBLE NEW PATHOGENIC ASSOCIATIONS IN DIABETIC NEUROPATHY
Reddy Iska, A.K., Chichili R. Diabetes Research Centre, Pogathota, Nellore, A.P., South India.
The purpose of the present study is to estimate the prevalence of Diabetic Neuropathy diagnosed through the evaluation of vibration perception threshold and its possible association with various other risk factors such as Hypertension, cigarette smoking, dislipidimea, microvascular-like retinopathy and Neuropathy or macrovascular-like peripheral arteriopathy lesions and other clinical and biochemical parameters. For this purpose 1892 diabetic patients who attended to our clinic from 1996 July to 1998 June were randomly incorporated in a cross sectional study using a common protocol. The average prevalence of Neuropathy is 38%. The univariate analysis showed that Neuropathy is significantly associated with Hypertension, coronary artery disease, cigarette smoking, dislipidemia, retinopathy, Nephropathy and duration of diabetes. The prevalence of Neuropathy is more so in males (51%). The relative importance of the variables, determined by standardized Co-efficient (SD) is, Retinopathy (0.69), age (0.42), Vasculopathy (0.31), Neuropathy (0.21), Sex (0.24), duration of diabetes (0.26), Dislipidemia (0.16), Cigarette smoking (0.31), Hypertension (0.21). These results suggest that the vascular component (micro and macroangiopathy) Hypertension, dislipidemia, cigarette smoking play an important role in the pathogenesis of Diabetic Neuropathy.
EFFECT OF a-TOCOPHEROL SUPPLEMENTATION ON PERIPHERAL NERVE STRUCTURE IN EXPERIMENTAL DIABETES
Sharma A.K., Lawrence P.A., Samad P.A., Ahmed I., Dhanaseker S., Sheen R. Faculty of Med., United Arab Emirates University, Al Ain, UAE
The ultrastructure of myelinated fibers in the tibial nerves of streptozotocin-diabetic rats was examined in order to investigate the beneficial effects of a-tocopherol. Male Wistar rats, aged 12 weeks were used over a period of three months: untreated diabetic animals, diabetics treated with a-tocopherol and age-matched controls. In the diabetic animal, administration of a-tocopherol resulted in a significant increase (p< 0.02) in total plasma vitamin E levels when compared with untreated diabetics and age-matched controls. Myelinated fiber cross-sectional area (p< 0.03), axonal area (p< 0.02) and myelin area (p< 0.05) were significantly less in the tibial nerve of diabetic animals as compared with age matched control, but not different from those of onset controls. In the a-tocopherol treated diabetic animals, the values for myelinated fiber area, axonal area and myelin area were intermediate between untreated diabetics and age matched controls without showing significant difference when compared with either group. The 'g' ratio (axon to fiber ratio) did not differ between any of the experimental groups. The mean maximum myelinated fiber area in the a-tocopherol treated diabetic animals was found to be similar to that of age-matched controls. In conclusion, this ultrastructural study reiterated that structural abnormalities of myelinated fibers occur in experimental diabetes and that the a-tocopherol administration may be useful in preventing the development of these abnormalities.
C-PEPTIDE TREATMENT HAS A LONG-TERM AMELIORATING EFFECT ON TYPE-1 DIABETIC NEUROPATHY
Sima A.A.F., Zhang W., Xu G., Wahren J.1, Sugimoto K. Wayne State University, Detroit MI U.S.A., 1Karolinska Institute, Stockholm, Sweden
C-peptide has an beneficial effect on neural and renal Na+/K+-ATPase activity and activates the high affinity insulin receptor (IR) which in peripheral nerve is located to the node of Ranvier and endoneurial vessels. In this study diabetic type-1, C-peptidopenic BB/W-rats were treated with C-peptide (75nmol/kg/d) (BB/W+C) (n=7) via osmopumps and compared to non-treated diabetic BB/W-rats (BB/W+O) (n=8) and non-diabetic control rats (C-BB/W) (n=8). C-peptide had no effect on blood glucose levels (19.3 ±0.1; 20.3 ± 3.8 and 5.0 ± 0.4 mmol/l, resp.) or body weight (345 ± 15; 344 ± 22 and 384 ± 58 g resp.). Motor NCV:s showed a progressive recovery in BB/W+C starting after initiation of C-peptide therapy at 1 wk and was not significantly different from BB/W+O after 5 mo of treatment (60.8 ± 1.1, 52.4 ± 4.1 and 62.9 ± 1.7 m/sec, resp.). These effects corresponded to only a partial but significant (p< 0.05) increase in evoked potential amplitudes (8.5 ± 1.4; 5.8 ± 2.2 and 16.6 ± 2.7 mV resp.). The latencies of thermal plantar tests did not differ between groups at 5 mo. We conclude that C-peptide treatment has an ameliorating effect on the function of IR-bearing myelinated fibers, but less so on unmyelinated fibers. Supported by JDFI and Thomas Foundation
AUTONOMIC NEUROPATHY IN CHRONIC HYPERGLYCEMIC VERSUS HYPOGLYCEMIC RATS
Yagihashi S., Yamagishi S-I, Wada R., Tanaka M., Aze Y. Hirosaki University School of Medicine, Hirosaki and Ono Safety Research Institute, Fukui, Japan
Autonomic neuropathy is a common and serious complication of diabetic patients. It is not known, however, whether repeated episodes of hypoglycemia affects the development of autonomic nerve lesions. We performed detailed structural studies on the sympathetic nervous systems with morphometric analysis in STZ-induced hyperglycemic (22-33mmol/l) rats and non-diabetic hypoglycemic rats (2.5-3.9mmol/1) with daily overdose insulin injection after 10 month duration. Effects of meticulous blood glucose control by insulin injection were also examined in STZ-induced hyperglycemic rats. During the experiment, tibial motor nerve conduction velocity (MN) and CV-RR were monitored. Significant delay of MCV and decreased CV-RR were detected in hyperglycemic rats but not in hypoglycemic rats. These changes in hyperglycemic rats were nearly normalized in insulin-treated hyperglycemic rats. Structurally, significant increase in the incidence of axonal dystrophy in the celiac ganglion and postganglionic mesenteric nerve was detected in hyperglycemic rats compared to controls and insulin-treatment completely prevented the increase in dystrophy. By contrast, hypoglycemic rats also exhibited similar but much less dystrophic changes compared to hyperglycemic rats. Morphometric analysis on postganglionic fibers in the mesenteric nerves did not reveal any differences in fiber area, density and occupancy between all groups. These findings indicate that axonal dystrophic changes detected in both human and rodent diabetic autonomic nerves may mainly be ascribed to continuous hyperglycemia and the effects of hypoglycemia appeared less evident.
BIOSYNTHESIS OF PERIPHERAL NERVE GLYCOLIPIDS IN EXPERIMENTAL DIABETIC NEUROPATHY
Yao, J.K., Schmelzer, J.D. and Low, P.A. VA Pittsburgh Healthcare System (Highland Dr.), Pittsburgh, PA 15206 and Mayo Clinic, Rochester, MN 55905, USA
The biosynthesis of peripheral nerve monogalactosyl lipids (MGL) was studied by in vitro incorporation of [3H]galactose (Gal), [3H]glucose (Glc), or [35S]sulfate into the endoneurium of normal and diabetic rat sciatic nerve. Six (D6) and 16 (D16) weeks after the onset of streptozotocin-induced diabetes, the desheathed sciatic nerves were harvested for incorporation studies. In normal rat nerve, MGL biosynthesis decreased rapidly as myelination proceeded. After 3 months, the decrease continued but at a much slower rate. [3H]Gal was primarily incorporated into monogalactosyl diacylglycerol (MGDG), galactocerebroside (GalCe), and phosphatidylcholine, while [3H]Glc was mainly incorporated into phospholipids with much lower levels of glycolipids. The incorporation of [3H]Gal or [3H]Glc into endoneurial MGL of D6 group was reduced by 20% and 60%, respectively. In addition, the percent of 3H-labeling in MGDG was found substantially higher (~2 folds) in diabetic than in normal control nerves. Such an increase continued in diabetic nerves from D16 group, albeit no significant differences of total incorporation were shown between D16 and normal control group. These acylated glycolipids have long been considered as a marker for myelination in rat brain as well as peripheral nerves. Thus, a decreased synthesis of GalCe and a selectively increased biosynthesis of MGDG may reflect some biochemical alterations of myelin synthesis and maintenance in experimental diabetic neuropathy. On the other hand, biosynthesis of galactosulfatides from [35S] sulfate remains intact in diabetic nerve.