Experimental Study On Inhibition Of Nitric Oxide Synthase Enhancing Sciatic Nerve Regeneration In Rats

Nitric oxide (NO) is a widespread mediator of physiological and pathophysiological processes, acts as a messenger or modulator molecule in many biological systems, plays an important role in a wide range of physiologic processes. The major physiologic function of NO are :①v asodilatation;②Act as a message material of PNS and CNS;③Act as the effector molecule of WBC to execute the function of antisepticize and anti-tumour. Two of the three functions have close relation to neural regeneration. For the nervous system,NO participates the controlling of the growth in early days and the regeneration process of nerve fiber after nerve injury,it has a positive effect on the regeneration of the nerve. At the same time, the NO has the nerve toxicity under the certain condition of,it can performance for repressing the regeneration of nerve and the toxicity function to the regenerating nerve.It seem that NO has dual function for the regeneration nerve.The NO has the positive meaning in nerve regeneration: The NO can cause the growth cone fall sink through the flyback acts function, stop axon growth cone continuously extension, and adjust the axon growth cone .The NO carries on the modifier to the quantity, appearance and functions for the early growth cone, carry on the fine concocting of the regeneration nerve.The NO also can lead to a serieses of nerve toxicity and nerve physiologic derangement. NO can mediate some matter(such as ammonia, ONOO-, and PARS) to effect the nomal physiological process of the injury sever, lead it to damage.The formation of the contrary effection is because of the different region concentration. And the concentration is determined by NOS. The half-life of NO is short, so the speed of birth is important to its region concentration. NOS is the key element to change the speed of th birth speed.The focus that we pay attention to is the effection of NO to the PNS injury. The experiment is to proving what effection the inhabitor of NOS(L-NAME) can bring to PNS and to study the mechanism of action of it. 50 SD rat weighing 100–130 g were raised in mouse cages(2-4 /cage) with free access to food and water were studied. Under anaesthesia with 1% ketamine(40mg/kg)injected i.p., the right sciatic nerve was exposed and completely transected with fine surgical scissors at a level just 1cm distal to the sciatic notch. After nerve section, resewed with 7-0 atraumatic needle and thread, the muscles and skin were resewn and the animal allowed to recover.The rat were randomly divided to 5 groups(L8, D8, L12, D12, S12) assigned L8 , L12 to receive a 10 day course of Nω-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg in a volume of 0.1 ml); 0.1 ml; given once on the day of surgery then twice daily for 10 days) And D8, D12 received an identical course of its inactive enantiomer Nω-nitro-D -arginine methyl ester (D-NAME).S12 received the identical course of saline. Multifiber motor conduction studies of sciatic-tibial fibers were carried out under anaesthesia with 1% ketamine(40mg/kg)injected i.p. by stimulating at the sciatic notch and knee and recording the M (compound muscle action) potential with subcutaneous platinum electrodes muscle belly of soleus muscle. The amplitude of the M potential reflects the number of innervating motor axons in the absence of primary muscle pathology. Near nerve temperature during the recordings was maintained at 37°C with a subcutaneous thermistor probe and a radiant heating lamp. Bilateral sciatic- tibial recordings were made at 8 and 12 weeks later. Then, the rats were put to death and following that, the sciatic nerve and its tibial branches were removed for histological study. The nerves were stained with HE and toluidine blue.Morphometric studies of myelinated fibers were observed using a video-microscopy image analysis setup. Results of the electrophysiological and morphological measurements in L-NAME and D-NAME treated rats were calculated as means with Microsoft Excel.M potential amplitudes and sciatic-tibial conduction velocity measurements were comparable in right and left sciatic-tibial territories at 8 weeks and 12 weeks, the amplitudes of the M potential in the L-NAME treated rats were substantially higher than those of the D-NAME treated rats, indicating greater reinnervation of soleus muscle. The amplitudes of M potentials were higher in L-NAME treated rats irrespective of whether the potentials were measured from sciatic notch stimulation or knee stimulation. Also, there was a significantly higher percentage recovery of the M potential as a function of the initial M potential in the L-NAME treated rats . There was a non-significant trend toward higher conduction velocity in the regenerated L-NAME motor fibers.We obtained the data in these aspects:Triceps surae muscle cuadros index(TSCI): TSCI=weight of triceps surae muscle /total weight×100.Results of the electrophysiological: Conduction velocity(CV) and the amplitudes of sciatic-tibial territories comparable .The diameter of medullated nerve fibers axonal(NFD), width of medullated nerve fibers myelin(NFW) and count of medullated nerve fibers ( NFC)in stained with toluidine blue.Comparisons between the two groups were made using a two-tailed Student's t-test.The amplitudes of the M potential in the L-NAME treated rats were substantially higher than those of the D-NAME treated rats, indicating greater reinnervation of triceps surae muscle at 8 and 12 weks after operation. The amplitudes of M potentials were higher in L-NAME treated rats irrespective of whether the potentials were measured from sciatic notch stimulation or knee stimulation. Also, there was a significantly higher percentage recovery of the M potential as a function of the initial M potential in the L-NAME treated rats. There also was a significant trend toward higher conduction velocity in the regenerated L-NAME motor fibers.The morphometric studies identified a larger mean NFD in L-NAME treated rats and a shift in the axon size histogram toward higher values in this group. Mean axonal area, the number of large myelinated fibers, and mean fiber diameter all tended toward larger values in the L-NAME treated rats. The NFCs were non-significant trends toward larger numbers of all myelinated fibers and a greater density of large myelinated fibers in the L-NAME treated rats. NFW was different between the two groups. The L-NAME treated rats had a trend toward a more mature regenerating medullated nerve fibers. The findings in this work indicate that inhibition of NOS with L-NAME is associated with substantially better myelinated fiber regeneration than that associated with administration of its inactive enantiomer, D-NAME. The better outcome in the L-NAME group was indicated by more complete electrophysiological recovery, evidence of more mature larger myelinated fibers distal to the injury site. We think that this difference must be based on the agents were given for only the first 10 days after operation. These certificate our hypothesis that NOS inhibition would enhance fiber regeneration by reducing the NO generation to the regenerative milieu.Since reinnervation was faster and more complete in the L-NAME group and regenerating myelinated fibers larger, it may be that an'NO-free' microenvironment at the tip of a regenerating peripheral nerve stump is more permissive to earlier and more extensive axonal sprouting. Excessive local elaboration of NO has been implicated as a mediator of cellular and neurite toxicity in several neurological disorders. There is evidence that NO might reversibly inhibit growth cones by inhibiting thioester-linked long-chain fatty acylation of neuronal proteins. NO reacts with the superoxide anion to produce the oxidant peroxynitrite, with actions that could include lipid oxidation and nitration of proteins such as neurofilaments or Trk-receptors for neurotrophins. Most of the neurotoxic actions of NO have been studied at the level of the cell body, where NO may act to cause DNA strand breaks, and activate poly (ADP-ribose) synthetase in turn, depleting cellular energy stores.NO also can effect PN axons regeneration by this means: a mechanism implicated to result in acute axonal damage of fibers in inflammatory neuropathies. Since the degenerating myelinated profiles were large, this finding might suggest degeneration of large or mature fibers. later stage of regeneration. It may be more feasible to speculate that myelinated fibers in the L-NAME treated rats more effectively penetrated bands of Bungner and somehow promoted faster clearance of fiber and myelin debris including degenerating myelinated fiber profiles from the original transection, and it can reduced hyperaemia at this site.Immunohistochemistry identified increased staining of all three isoforms of NOS following injury. The work has suggested that there is local generation of NO at the distal tip of proximal nerve stumps of transected nerves where regenerative sprouting occurs. we use the non-specific inhibition of NOS(-NAME) to enhance the regeneration of axon.Now, the exact dose-effect relationship of L-NAME and the side-effect of other systems are to be definited. Further time course studies will help sort out this problem.