Study On Pathogenesis And Protective Mechanism Of Diabetic Peripheral Neuropathy In Rats

Recently, the incidence of diabetes mellitus (DM) rises in the world. In the present, there are 170 million DM patients all over the world. It is estimated that it will be doubled in 2003 and the new patients mainly will come from the Asian developing countries, such as the People's Republic of China and India. Diabetic peripheral neuropathy (DPN) is one of the most common microangiopathy complications of DM. The incidence of DPN is about 5%-50%. In American, DPN incidence is up to 32.7% among the DM patients aged over 40 and it will rise up with the advanced course. The pathogenesis of DPN is not very clear. Hence, the studies on the pathogenesis and treatment strategy of DPN have become a very important work of medical researchers.DPN is induced by multiple causes and complicated mechanisms, which are not clear so far. Presently, there are several theories of DPN's pathogenesis: metabolism theory, microvascular dysfunction theory, nerve nutrition factors deficiency theory, immunity theory, and genetic theory. Recently, it is believed that microvascular and metabolic dysfunction play key roles in DPN development and new researches are mainly focused on them. However, the initial factor of DPN development and correlation among related factors are unknown. Hence, our study is designed to observe the simultaneous and consecutive changes on streptozocin-injection rats systematically and all-sidely, which includes electrophysiology, microcirculation blood flow in low extremities, pathological morphology, oxidative stress related factors (VEGF, TNF-α, IL-1, Il-6, NF-κB, p38MAPK, Caspase-3, Bax, Bcl-2), and ET/NO system (ET-1,ETA receptor, ETB receptor, iNOS, eNOS, nNOS). Prostaglandin E1 (PGE1) is used to observe its effects on the DPN rats. The effects and relationships between these factors and DPN and the protective effects of PGE1 are studied from multiple views and in many ways. The purpose of this study is to make further investigation of DPN's pathogenesis and try to find more accurate target point of treatment for the clinical practice.Main study methods: 1. Experimental DPN model is made by streptozocin-injection rats. 2. Study groups: Experimental DPN rats are divided into two groups. One is normal control group (NC group) and another is diabetes mellitus group (DM group). DM group is divided into five subgroups: DM 4W group, DM8W group, DM 12W group, Treatment 1 group (T1 group), and Treatment 2 group (T2 group). 3. Motor nerve conduction velocity (MNCV), sensory nerve conduction (SNCV), and compound muscle action potential (CMAP), sensory nerve action potential (SNAP) of sciatic nerve are measured on experimental DPN rats by Meditronic keypoint-4 workstation electromyography. 4. Microcirculation blood flow in lower extremities is measured on experimental DPN rats by Laser Doppler flowmetry (LDF). 5. Morphological changes of nerve inner membrane capillary and sciatic nerve are observed by light microscope with HE dye and by electronmicroscope. 6. Expression of oxidative stress related factors (VEGF,TNF-α,IL-1,Il-6,NF-κB,p38MAPK,Caspase-3,Bax,Bcl-2) and ET/NO system related factors (ET-1,ETA receptor,ETB receptor,iNOS,eNOS,nNOS) are observed by immunohistochemistry methods.Main study results: 1. Body weight and blood glucose level changes of each group rats: Body weights of DM group rats obviously decrease compared to itself before streptozocin injection or to the NC group rats. Blood glucose level is kept on a high level (>16.7mmol/L) and without spontaneous relief phenomenon. 2. Electrophysiological changes of each group rats: Compared with itself before streptozocin injection or to the NC group rats, MNCV, SNCV and CMAP, SNAP of DM group rats'sciatic nerve begin to decrease markedly since 4W after streptozocin injection. They progress with the advanced DPN course. They can be distinctly improved by PGE1. 3. Microcirculation blood flow changes in low extremities of each group rats: Compared with itself before streptozocin injection or to the NC group rats, microcirculation blood flow of DM group rats starts to decrease sharply since 2W after streptozocin injection. It progresses with the advanced DPN course. Its decrease are up to over 50% when 12W. It is obviously improved by PGE1. 4. Morphological changes of each group rats'sciatic nerve: (1) Since 4W, the vessel walls of nerve inner membrane capillary in DM group rats begins to thick progressively by HE dye method. It also shows irregular and narrowed vessel lumen and swelling, out-of-shape endothelium cells. It progresses with the advanced DPN course. Simultaneously, myelinated and unmyelinated sciatic nerve fiber of DM group rats also start to line loosely. As the course advanced, they gradually show thinner and separated myelin sheath. Vacuolus formation and axon atrophy also can be seen. (2) Through electronmicroscope, it is found in DM groups rats'sciatic nerve with thick capillary endothelium, unclear basement membrane, inflammatory cells infiltration, unequal or separated or drop-off myelin sheath, decreased nerve filament inside axon, swollen mitochondria, expanded rough endoplasmic reticulum. It progresses with the advanced DPN course. All these manifestations can be obviously improved by PGE1 and early treatment is better than late. 5. Oxidative stress related factors expression changes: Compared with itself before streptozocin injection or to the NC group rats, expressions of VEGF,TNF-α,IL-1,NF-κB,p38MAPK,Caspase-3,Bax increase and expressions of Il-6,Bcl-2 decrease in DM group rats sciatic nerve. These expression changes progress with the advanced DPN course. Expression of VEGF,TNF-α,IL-1,IL-6,NF-κB,p38MAPK,Caspase-3,Bax can be distinctly improved by PGE1 and early treatment is better than late. But Bcl-2 expression is not effected by PGE1. 6. ET/NO system related factors expressions changes: NOS mainly expresses inside axon. Compared with itself before streptozocin injection or to the NC group rats, expressions of eNOS,iNOS,nNOS decrease in DM group rats'sciatic nerve. These expression changes progress with the advanced DPN course. Normally, ET-1 and its receptors express little in the rats'sciatic nerve. However, their expressions obviously increase under pathological status. Compared with itself before streptozocin injection or to the NC group rats, expressions of ET-1,ETA receptor,ETB receptor increase. These expression changes progress with the advanced DPN course. These changes can be improved by PGE1 and early treatment is better than late.Conclusion: 1. Experimental DPN rat model can be successfully established by streptozocin injection. 2. Microcirculation blood flow changes in the lower extremities are earlier than the NCV changes and morphological changes of nerve inner membrane capillary and sciatic nerve. So it is possible that microcirculation blood flow decrease may be the initial factor of DPN development. Mircrocirculation dysfunction is an important pathogenesis of DPN. 3. Oxidative stress related factors (VEGF,TNF-α,IL-1,Il-6,NF-κB,p38MAPK,Caspase-3,Bax,Bcl-2) take part in the pathophysiological changes of DPN. On the one hand, microcirculation blood flow changes in the lower extremities are earlier than the oxidative stress related factors changes. On the other hand, activations of oxidative stress related factors aggravate microcirculation dysfunction. 4. In DPN rats, microcirculation blood flow changes in the lower extremities are earlier than the ET/NO system related factors changes. In DM, through up-regulated ET-1 and its receptors expression and down-regulated NOS expression, ET/NO system is activated to effect microcirculation and to take part in the DPN development.Through the observations of effects and relationships of oxidative stress related factors, ET-1 and its receptors, and NOS during the DPN development and progress, our study makes further investigations of DPN's pathogenesis. Our study makes it clear that microcirculation is the initial factor of DPN. Hence, our results provide more accurate target point of treatment in the clinical practice.