Study On The Ischemia-reperfusion Injury Of The Sino-atrial Node And The Effects Of L-Arginine In Rabbit

Acute and chronic ischemia of the sinoatrial node may result in its tissue pathological chemistry and its impulse handicap. This is one of the important and well-known factors about the exceptional electrophysiologic action and the higher incidence of sinus sinoatrial node syndrome. SAN function is affected easily by the single accommodating coronary artery because of its small volume, a few of function units, simple cellar connection and abundant blood demand. Once its intramural parenchyma was occurred pathological changes, its function will be resumed dificultly. Now, there were wide studies of ischemia-reperfusion about the ordinary cardiac muscle, and some of high-efficiencious and high-selective nitric oxide carrier medication has been used for preventing and curing much of the cardiovascular diseases. But a little of research is very impercipient on ischemia-reperfusion about the conductive system of the heart. So it is important to explore the changes of the structure and function and the pathologic mechanism about the injury on the ischemia -reperfusion SAN.This study will design some contents with the morphologic and electrophysi -ologic aspects in vivo or in vitro as follows:Part one: Sino-atrial node ischemia-reperfusion injury and effect of L-Arginine in vivo.Chaptert one. Establishment and confirmation of sino-node ischemia-reperfusion model in rabbitObjective: To establish the animal model of the ischemia-reperfusion sino-atrial node in the rabbit.Methods: 10 rabbits were permeated by emulsion, the right coronary and its branches were observed, I/R models of ischemia were tested by occluding or loosening the root of right coronary artery in 12 rabbits and their heart rhythm were logged in the computer, Sinoatrial nodes were observed under the light and electron mirror.Results: The right coronary was branched away from the root of aorta. There were 8 cases with typical right coronary preponderant in 10 cases. About (1.9+0.3) mm was the distance between the first branch of right coronary and the root of aorta. In experiment rabbits, sinus and atrial arrhythmia were found in 8 cases, atrio-ventricular block was found in 10 cases. All of rabbits rhythm were aggravated in the forepart of the reperfusion period and recovered in the afternoon of the reperfusion period gradually: Under the light or electron mirror, the borderline of P cells were illegibility, fuscous karyon was dyed and chromatin was parted or disappeared.Conclusion: Opening up the thorax in the middle of breastbone, ligating the root of the right coronary and consulting the ECG should be combined for ischemia and ischemia-reperfusion model of SAN.Chaptert two: Roles of L-Arginine on arrhythmia induced by ischemia reperfusion of right coronary artery in rabbitObjective: To explore the role of L-arginine on the arrhythmia induced by I/R of the right coronary artery in rabbit.Metheds: 4 groups randomly were set up: I30R120 group (120 min reperfusion after 30 min ischemia); I120R120 group (120 min reperfusion after 120 min ischemia); l3oRi2o+L-Arg group; Ii2oRi2o+L-Arg group. I/R model were established by occluding and loosening the root of the right coronary artery in the rabbit. The changes of ECG and arrhythmia were recorded and graded.Results: ?The longer time of IR was, the score of the arrhythmia was higher; The incidence of atrio-ventricular block (AVB), sinus-atrial block (SAB), even sinus arres were detected and aggravated gradually; ?The incidence of AVB was decreased and III ° -* II ° — I markedly, some of sinus and atrial arrhythmia were transformed into sinus rhythm gradually, and all of the arrhythmia scores of I30R120 group were decreased significantly as compared with the same time phases of I120R120 group (P<0.01); (3)A11 of the arrhythmia scores of l3oRi2o+L-Arg and Ii2oRi2o+L-Arg groups were decreased dramatically as compared with the same time phases of I30R120 and I120R120 groups (P<0.01); @A11 of the arrhythmia scores of l3oRi2o+L-Arg group were low when compared with InoRno+L-Arg group (P<0.0l).Conclusion: Supplying appropriate L-Arginine of the tissue was benefit for inhibiting arrhythmia during ischemia and reperfusion, and the longer time of ischemia was, the role of L-Arginine on the arrhythmia was worse during the period of reperfusion.Chaptert three: Effects of L-arginine on functions of sinus node by ischemia-reperfusion of right coronary artery in rabbitsObjective: To study the influence of L-arginine on functions of SAN by ischemia- reperfusion of right coronary artery in rabbits.Methods: 4 groups: control group; I90R90+NS group; LjoRgo+L-arginine group and Ic>oR9o+L-NG-arginine group. At the different time phases after ligating or loosening of the artery, sinus atrial conduction time (S ACT), sino-atrial node recoverytime (SNRT).Results: Compared with the control group, all of SACT and SNRT of every group were prolonged significantly (P<0.01). Compared with I90R90+NS group of every same time point, SACT and SNRT of I^Rw+L-Arg group were descended during ischemia and forepart of reperfusion period, and raised during afternoon of reperfusion period, but the results of I90R90+L-NAME group were contrary to that of l9oR9o+L-Arg group.Conclusion: The longer time of ischemia was, the functions of SAN as well as AVN were worse; supplying appropriate L-Arginine of tissue during ischemia and forepart period, it was benefit for them, but there was no effective treatment in rabbits during the afternoon of reperfusion period, and the functions of SAN were aggravated contrarily.Chaptert four: Effects of L-Arginine on apoptosis and protein expression of bcl-2, bax and iNOS in sinoatrial node cells during ischemia-reperfusion and the effect of L-ArginineObjective: To explore poptosis and expression of bcl-2, bax and iNOS protein and remedial effects of L-Arginine.Motheds: 4 groups were set up: control group; I90R90+NS group; IwRgo+L-Arg group; I90R90 group. Apoptotic rate was calculated by TUNEL-staining method and protein expression about bcl-2, bax and iNOS were observed by means of immuno-histochemistry and its optical densities were analysed by image analysis system.Results: Apoptotic rate of the SAN was the lowest (P<0.01); ischemia can induce apoptosis and reperfusion can aggravate it and increase expression of iNOS protein, the changes of AI was congruous with iNOS-OD; increasing production of endogenous NO inhibit apoptosis (P<0.01); AI and bcl-2-OD were increased, but bax-OD was reduced in IgoRw+L-Arg group significantly.Conclusion: It is showed that expression of iNOS probably has relation to apoptosis; L-Arginine can degrease apoptosis induced by IR and this action is mediated by the reduction of bcl-2 and increase of bax expression.Chaptert five: Morphologic reconstruction of collagen netmush in sinoatrial node following ischemia-reperfusionObjective: To observe the morphologic changes of collagen netmush in SAN following ischemia-reperfusion.Methods: Adapting SET and immunohistochemistry technique on ischemia reperfusion SAN tissue to observe the ultrastructer change of collagen framework and the protein expression of collagen I and collagen III.Results: Thick collagen bundles were distorted; thin collagen fiberes were tangled and incompact; expression of collagen I and collagen III were decreasd (P<0.05) and lost it's balance.Conclusion: SAN ischemia-reperfusion result in damage on collagen network, this must affect the inpulse producting and conducting of SAN and it may be one of factors about sinus sinoatrial node syndrome.Part two: Injury on Sino-atrial node cultured cells by simulating ischemia- reperfusion and effect of L-Arginine in vitroChaptert one: Orientation and morphologic observation of the neonatal rabbit Objective: To offer plenary morphologic information for exsecting SAN tissueexactly.Methods: Taking out the whole hearts from 8 neonatal rabbits with its big bloodvesseles. The tissues were embedded in paraffin and sliced up for H-E and Massionstaining, The micrometric rule was employed for measuring the size of SAN andprotracting its sketch map.Results: The area of SAN was stained lightly, cells were small and glomerate, the borderline of cell was slur, karyon was bigger and its round appeared pale area. No obvious myocardial cells but a small quantity of fibroblasts were oberved. Cell composing was simple and puerile under the transmission electron microscope. There were more collagen fibers in the region of SAN than that of the atrium. The sinoatrial node localized in the anterior wall of the superior cava vena and the posterior-lateral atrial wall. Its appearance was like "inversion triangle" and its size is about 0.68 mm X 1.51 mm X 0.49 mm.Conclusion: Reserving enough superior cava vena when we take out the neonatal rabbit SAN for it's locution was more especial; Cell construct is simple and puerile, so it is fit for purifying cultivation.Chaptert two: Exsecting and purifying cultivation of sino-atrial node cells isolated from new-born rabbitsObjective: To offer sufficient SAN simplex celles for studying its morphologic characteristics and pacing mechanism.Methods: Setting up SAN cells culture system in vitro: The SAN of the neonatal rabbits were removed by microsurgical technique. The tissues were then isolated by digesting method with trypsin and collagenase. Cells were cultured in DMEM culture medium added with 10% TBS and BrdU (0.1 mmol /L). The growth and morphologic characteristics were observed by the inverted phase contract microscope.Results: Three distinctly different types of cells were observed among the cultured cells of SAN: spindle, araneiform and polygon. The spindle cells covered the greatest proportion of the cultured cells of SAN (59.6 %±7.3 %). The frequency of spontaneous contraction of spindle cells was the highest among the constrcting cells (145 ±9/ min). The results of ultrastructure observation showed that myofibrils and other organelles in spindle cells were poorly organized and significantly decreased innumber compared to araneiform cells. There was no significant difference between araneiform cells isolated from SAN and from atrial muscle.Conclusion: Among the cultured cells from neonatal rabbit SAN tissue, the spindle cells are the pacemaker cells of SAN.Chaptert three: Effect of L-Arginine on damnification of simulating ischemia-reperfusion sinoatrial node cultured cells.Objective: To observe SAN celles damnification on simulating I/R and the role of L-Arginine.Methods: Simulating ischemia-reprefusion for cultured celles with low sweet and hypoxygenation. Setting up 7 groups: control group; I120; I120R120 group; I120+ L-Arg group; Ii2oRi2o+L-Arg group; Ino+L-Arg+L-NAME group; Ii2oRi2o+L-Arg+ L-NAME group.Results: In group I120 and group Ii2o+L-Arg+L-NAME, karyon was concentrated, cytoplasm was stained amaranth; AO-fluorescence staining showed that the apoptotic cells was dwindled, karyon was kelly and fractured into many small shiveres. It was enwraped and heaved on cells surface; chromatin of the apoptotic karyon was concentrated and conglomerated below the karyotheca, even was decompounded into mass or crescent-shaped pieces and dissociated among the cytoplast. Those damages were easily obseved, so much as necromancy in group I120R120 and group InoRno+L-Arg+L-NAME, but a few of those damages were occurred in group I120+ L-Arg and group I120R120+ L-Arg.Conclusion: The degree of damnification on SAN celles undergoing simulating ischemia reprefusion injury increase with time of I/R; L-Arginine can inhibt apoptosis in this pathological procession.Chaptert four: Protective mechanism of L-Arginine on damnification of simulating ischemia-reperfusion sinoatrial node cultured cells.Objective: To discuss that the possible mechanism of L-Arginine on the simulating ischemia-reperfusion cultured SAN cells.Methods: Setting up 7 groups: control group; I120; I120R120 group; Ino+L-Arg group; InoRuo+L-Arg group; Ino+L-Arg+L-NAME group; InoRm+L-Arg+L-NAME group. To examine contents of SOD, MDA, iNOS and NO from the different groups SAN celles.Results: SOD activity decreased and MDA content increased obviously (/xO.Ol), as well as production of NOS and NO ascended (P<0.01 or P<0.05) in group I120 and group I120R120; but SOD activity decreased and NOS, NO and MDA contents increased (P<0.01 orP<0.05) .Conclusion: Degree of damage of reperfusion is worse than that of ischemia, it may be relate with much oxygen free radicals during reperfusion period. Supplying appropriate L-Arginine can clean out and decrease the excrescent oxygen free radicals, improve SOD activity for interdicting the apoptotic process by oxygen free radicals.