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Immunohistochemical detection of advanced glycation endproducts on peripheral nerves in NIDDM patients with proximal diabetic neuropathy
Track 4. Basic Research
P1430 Changes in Calcium Signalling in Spinal Dorsal Horn Neurons in Rats with Streptozotocin-Induced Diabetes E.P. KOSTYUK, N.V. Voitenko, LA. Kmglikov, PG. Kostyuk, AS. Efimov. Bogomolerz Institute of Physiology NAS, and Instiriute of Endocrinology
and Metabolism AMS, Kiev, Ukraine
Intracellular Ca*+ transients were studied in spinal cord dorsal horn neurons transmitting nociceptive signals in rats with streptozotocin (STZ) induced diabetes mellitus versus control animals. Neurons were loaded with fluorescent Ca*+ indicator Fura- AM and recorded in 300 pm thick slices. The resting level of cytosolic Ca*’ ([Ca*‘]i) did not shown significant difference in neurons of the two groups (68+15 nM versus 82fl7 nM). The amplitudes of [Ca”]i transients in response to membrane depolarization were 719f132 nM in normal and 694f155 nM in diabetic conditions. The major difference in diabetic case was a marked deceleration of Ca*’ removal from the cytoplasm. In all cells, in addition to a fast component of recovery, a second very slow exponential recovery became evident with time course of 23f5 sec. In some cases large Ca*’ load damaged diabetic neurons and elevated [Ca*‘]i did not recover to the basal level for up to 20 min washout with Tyrode solution. To analyze possible nature of such changes, the Ca**-accumulating function of the endoplasmic reticulum was investigated. The amplitudes of caffeine-induced [Ca”]i transients were substantially reduced in diabetic neurons (4lf9 nM versus 268f29 nM in control, P
PM31 Protective Effects of Captopril and Taurine on Structure and Function in Sciatic Nerve of Diabetic Rats YANHU DONG, Guoliang Sui, GuoQing Liu. Depart of Endocrinology, Qingdao Health Second People’s Hospital, Qingdao, China
Objective: To investigate the protective effect of captopril and taurine on the structure and function of the sciatic nerve of diabetic rats. Methods: Forty Male Sprague-Dawley rats were randomly divided into nondiabetic group, untreated diabetic group, captopril treated diabetic group, taurine treated diabetic group. The diabetic Rats were made by single injection of streptozocin (STZ). After 12 weeks treatment, motor nerve conduction velocity (MNCV) of sciatic nerve were measured. Blood malondialdehyde(MDA), the content of the sciatic nerve cyclic adenosine monophosphate(cAMP), and advanced glycosylation end products(AGEs) were also measured. The morphometric quantitative analysis of sural nerve were also investigated in all experimental rats.
S363
Results: Compared to the nondiabetic group, blood MDA and the content of sciatic nerve AGES were significant elevated, the content of sciatic nerve CAMP was markedly decreased in untreated diabetic rats. Sciatic nerve MNCV were markedly slowed in untreated diabetic group. The morphometric quantitative analysis of sural nerve showed the reduced total transverse section dimension and nerve fiber size. In captopril and taurine groups, blood MDA and sciatic nerve AGES content were markedly reduced, while the content of sciatic nerve CAMP in captopril group was increased (cAMp:O. 202 fO.046 vs 0.079fO.021 pmol/mg wet weight, ptO.01) compared with those in untreated diabetic rats. Blood MDA and the content of sciatic nerve AGES in diabetic rats treated with taurine were significant lower than untreated diabetic rats. MNCV in captopril and taurine group was markedly improved (40. 3 f 3. 5 vs 33. 0 f 2. lm/s; 41.4 f 3.2 vs 33.0 f 2.lm/s; all p
P1432 Immunohistochemical Detection of Advanced Glycation Endproducts on Peripheral Nerves in NIDDM Patients with Proximal Diabetic Neumpathy IRENA MIS, Zdenka Turk, Zvonko Milicevic, Ante Barada. The Croatian Group for the Study of Proximal Diabetic Neuropathy; University Clinic Vuk Vrhovac, Zagreb, Croatia
Background and Aims. AGE formation in peripheral nerves of diabetic animal model is well documented. However, there are less data on humans due to the unavailability of tissues for analysis. The aim of this study was to examine localisation of AGE-immunoreactivity in situ in sural or/and femoral nerve obtained by biopsy from NIDDM patients with proximal diabetic neuropathy (PDN). Methods. Twelve NIDDM patients with classic, abrupt-onset, sensorimotor form of PDN were included in the study (age 65.0f6.6 yrs, male/female 7/5, diabetes duration 12.5f5.6 yrs, PDN duration 4.2f1.7 mo, HbAlc 7.7%). The specimens were collected by biopsy of a small sensory branch of the femoral (n=6) and sural nerve (n=8). Five- to 8 pm thin tissue sections were cut on a cryostat and used for detection of AGE deposits, by an indirect immunofluorescence technique with fluoresceine and rhodamine as markers. The primary antibody was polyclonal anti-AGE raised in rabbit. Results. Immunohistochemical examination under fluorescence microscope demonstrated that AGE deposit was located in the perineurium, focally in the endoneurium, and in the mijelin sheath area. In femoral nerve sections, AGES were mainly found in the mijelin sheath (83%), while in sural nerve all the patterns were AGE positive (87%). Negative results were obtained for 1 femoral (17%) and 1 sural section (13%). Summary. The AGE modified proteins were found in almost 85% of the tested femoral and sural nerves of diabetic patients with abrupt-onset form of PDN. Conclusion. The glycation process leading to AGE accumulation on neural cytoskeletal components could be one of the mechanisms involved in the pathogenesis of peripheral nervous system dysfunction.
P1430 Changes in Calcium Signalling in Spinal Dorsal Horn Neurons in Rats with Streptozotocin-Induced Diabetes E.P. KOSTYUK, N.V. Voitenko, LA. Kmglikov, PG. Kostyuk, AS. Efimov. Bogomolerz Institute of Physiology NAS, and Instiriute of Endocrinology
and Metabolism AMS, Kiev, Ukraine
Intracellular Ca*+ transients were studied in spinal cord dorsal horn neurons transmitting nociceptive signals in rats with streptozotocin (STZ) induced diabetes mellitus versus control animals. Neurons were loaded with fluorescent Ca*+ indicator Fura- AM and recorded in 300 pm thick slices. The resting level of cytosolic Ca*’ ([Ca*‘]i) did not shown significant difference in neurons of the two groups (68+15 nM versus 82fl7 nM). The amplitudes of [Ca”]i transients in response to membrane depolarization were 719f132 nM in normal and 694f155 nM in diabetic conditions. The major difference in diabetic case was a marked deceleration of Ca*’ removal from the cytoplasm. In all cells, in addition to a fast component of recovery, a second very slow exponential recovery became evident with time course of 23f5 sec. In some cases large Ca*’ load damaged diabetic neurons and elevated [Ca*‘]i did not recover to the basal level for up to 20 min washout with Tyrode solution. To analyze possible nature of such changes, the Ca**-accumulating function of the endoplasmic reticulum was investigated. The amplitudes of caffeine-induced [Ca”]i transients were substantially reduced in diabetic neurons (4lf9 nM versus 268f29 nM in control, P
PM31 Protective Effects of Captopril and Taurine on Structure and Function in Sciatic Nerve of Diabetic Rats YANHU DONG, Guoliang Sui, GuoQing Liu. Depart of Endocrinology, Qingdao Health Second People’s Hospital, Qingdao, China
Objective: To investigate the protective effect of captopril and taurine on the structure and function of the sciatic nerve of diabetic rats. Methods: Forty Male Sprague-Dawley rats were randomly divided into nondiabetic group, untreated diabetic group, captopril treated diabetic group, taurine treated diabetic group. The diabetic Rats were made by single injection of streptozocin (STZ). After 12 weeks treatment, motor nerve conduction velocity (MNCV) of sciatic nerve were measured. Blood malondialdehyde(MDA), the content of the sciatic nerve cyclic adenosine monophosphate(cAMP), and advanced glycosylation end products(AGEs) were also measured. The morphometric quantitative analysis of sural nerve were also investigated in all experimental rats.
S363
Results: Compared to the nondiabetic group, blood MDA and the content of sciatic nerve AGES were significant elevated, the content of sciatic nerve CAMP was markedly decreased in untreated diabetic rats. Sciatic nerve MNCV were markedly slowed in untreated diabetic group. The morphometric quantitative analysis of sural nerve showed the reduced total transverse section dimension and nerve fiber size. In captopril and taurine groups, blood MDA and sciatic nerve AGES content were markedly reduced, while the content of sciatic nerve CAMP in captopril group was increased (cAMp:O. 202 fO.046 vs 0.079fO.021 pmol/mg wet weight, ptO.01) compared with those in untreated diabetic rats. Blood MDA and the content of sciatic nerve AGES in diabetic rats treated with taurine were significant lower than untreated diabetic rats. MNCV in captopril and taurine group was markedly improved (40. 3 f 3. 5 vs 33. 0 f 2. lm/s; 41.4 f 3.2 vs 33.0 f 2.lm/s; all p
P1432 Immunohistochemical Detection of Advanced Glycation Endproducts on Peripheral Nerves in NIDDM Patients with Proximal Diabetic Neumpathy IRENA MIS, Zdenka Turk, Zvonko Milicevic, Ante Barada. The Croatian Group for the Study of Proximal Diabetic Neuropathy; University Clinic Vuk Vrhovac, Zagreb, Croatia
Background and Aims. AGE formation in peripheral nerves of diabetic animal model is well documented. However, there are less data on humans due to the unavailability of tissues for analysis. The aim of this study was to examine localisation of AGE-immunoreactivity in situ in sural or/and femoral nerve obtained by biopsy from NIDDM patients with proximal diabetic neuropathy (PDN). Methods. Twelve NIDDM patients with classic, abrupt-onset, sensorimotor form of PDN were included in the study (age 65.0f6.6 yrs, male/female 7/5, diabetes duration 12.5f5.6 yrs, PDN duration 4.2f1.7 mo, HbAlc 7.7%). The specimens were collected by biopsy of a small sensory branch of the femoral (n=6) and sural nerve (n=8). Five- to 8 pm thin tissue sections were cut on a cryostat and used for detection of AGE deposits, by an indirect immunofluorescence technique with fluoresceine and rhodamine as markers. The primary antibody was polyclonal anti-AGE raised in rabbit. Results. Immunohistochemical examination under fluorescence microscope demonstrated that AGE deposit was located in the perineurium, focally in the endoneurium, and in the mijelin sheath area. In femoral nerve sections, AGES were mainly found in the mijelin sheath (83%), while in sural nerve all the patterns were AGE positive (87%). Negative results were obtained for 1 femoral (17%) and 1 sural section (13%). Summary. The AGE modified proteins were found in almost 85% of the tested femoral and sural nerves of diabetic patients with abrupt-onset form of PDN. Conclusion. The glycation process leading to AGE accumulation on neural cytoskeletal components could be one of the mechanisms involved in the pathogenesis of peripheral nervous system dysfunction.