| Background and objectsAccording to the investigation documents of the Department of Health in 2006, the morbidity rate of diabetes of our country is about 3%, and the diabetic populations are 23 million, but the populations of impaired blood sugar modulation are 33 billion. Diabetes, as one of the most popular chronic diseases, brings huge burdens on the population health and society development of our country. Diabetic cardiac autonomic neuropathy (DCAN) is one of the common complications of diabetes. The prevalence rates of DCAN are 1% to 90% because of the differences in the diagnostic criteria. DCAN aggravates the prognosis of the diabetic badly. Toyry et al reported that the mortality of the type 2 diabetics with autonomic neuropathy clinical manifestation in 5 years is 50%, and 28% of them died of sudden cardiac deaths (SCDs). Sivieri et al also reported that DCAN could prolong the QT intervals to induce fatal arrhythmias, which might lead to SCDs in the type 1 diabetics. In clinical cases, the basal mechanism of most SCDs is ventricular fibrillation (Vf), and the analyses of electrocardiograms before sudden deaths reveal that 75% to 90% of sudden deaths are induced by ventricular fibrillation, so the incidence rate of ventricular arrhythmias (VAs) in diabetics may reflect their prognoses. Additional researches prompted that DCAN was one of the reasons to induce ventricular arrhythmias in the diabetics and the severity of ventricular arrhythmias could also reflect the severity of DCAN. However, the influences of diabetes on the cardiac sympathetic nerve and the relationships between the influences and ventricular arrhythmias are still needed to be researched and illuminated.The main objects of this study are: 1) Diabetic models were erected in rabbits, and immunohistochemistry and real time fluorescent quantitation reverse transcription polymerase chain reaction (RT-PCR) were used to observe the influences of diabetes on the cardiac autonomic nerve (sympathetic nerve). 2) Programmed electrical stimulation (PES) was used to induce ventricular arrhythmias, and the relationships were approached between the changes of cardiac autonomic nerve (sympathetic nerve) in diabetes and the incidences of inducible ventricular arrhythmias. The final object is to provide theory foundations for the prevention and cure of ventricular arrhythmias induced by DCAN.MethodsTwenty-six male New Zealand white rabbits were randomly assigned to two groups after fasting blood glucoses (FBS) were measured. Diabetes mellitus group rabbits (DM group, n-16) were induced into diabetic models. This group of animals was fed with the high-fat/high-sucrose diet in the first two months, which was made from 10% lard, 37% sucrose and 53% regular rabbit chow, and then every rabbit was injected with alloxan (80mg/kg body weight) via the ear vein. At 72 hours and 7 days after the injection, rabbits who demonstrated fasting blood glucose levels of 14mmol/L or above were accepted as diabetic rabbits. If the fasting blood glucose of rabbit was lower than 14mmol/L, another injection of alloxan was given. The rabbit which fasting blood glucose was still lower than 14mmol/L was rejected. Then the diabetic rabbits were fed with the regular rabbit chow for 6 months until the end of the experiment. The control group rabbits (C group, n=10) were always fed with the regular rabbit chow at the same time. They were injected with normal saline via the ear vein when the DM group rabbits were injected with alloxan. 8 months after the beginning, programmed electrical stimulation was applied to induce ventricular arrhythmias. Then immunohistochemistry and real time fluorescent quantitation RT-PCR were applied to detect tyrosine hydroxylase (TH) protein and gene expression, as the marker of sympathetic nerve, to reveal the changes of sympathetic nerve innervation densities and TH mRNA gene expression levels in different cardiac segments of the two groups of rabbits.Results3 of the DM group rabbits did not catch the standard of diabetes, 2 died of severe high blood glucoses, and 11 were accepted as diabetic rabbits and survived to the end of the experiment. The C group rabbits all survived to the end of the experiment.Ventricular arrhythmias induced by programmed electrical stimulation occurred in 2/10 (20%) rabbits in the C group, which are all ventricular fibrillation. In the DM group, 27.3% (3/11) of the rabbits had ventricular tachycardia (VT), 9.1% (1/11) had ventricular flutter (VF), 27.3% (3/11) had ventricular fibrillation, and the total ventricular arrhythmias incidence was 63.6% (7/11). There are statistical differences between the two groups (P<0.05).In the C group, the TH positive nerve fibers mainly distribute at the perivascular regions and go along with the vessels, some in the connective tissue between the myocardial bundles. A lot of thick nerve fiber stems can be seen in the proximal segments of left ventricles, and most nerve fibers are slender in the distal segments of left ventricles, while both slender nerve fibers and slightly slender nerve fiber stems coexist in the middle segments of left ventricles. The densities of TH positive nerve fibers were higher in the proximal and the middle segments of left ventricles, lower in the distal segments of left ventricles, but there are not statistical differences among all the five parts of the C group (P>0.05). The distribution of TH positive nerve fibers in the DM group are similar to that in the C group, but the densities of the nerve fibers in the proximal, middle and distal segments of left ventricles and in the middle segments of right ventricles and interventricular septa all decrease compared with the corresponding segments in the C group (P<0.05, P=0.035 between the proximal segments of left ventricles in the two groups, P<0.01 between all the other corresponding segments in the two groups). In the DM group, the nerve fibers densities decrease obviously in the distal segments of left ventricles, the nerve fibers densities in which are much lower than those in the proximal segments and the middle segments of left ventricles (P<0.01, respectively). The nerve fibers densities in the middle segments of left ventricles are much lower than those in the proximal segments of left ventricles, too (P<0.01). The nerve fibers densities in the middle segments of left ventricles are lower than those in the middle segments of right ventricles (P<0.05). But there are not statistical differences in the nerve fibers densities between the interventricular septa and the middle segments of left ventricles or right ventricles (P>0.05, respectively). There are not statistical differences in the expression levels of TH mRNA among all the five parts in the C group (P>0.05). The expression levels of TH mRNA in all the five parts in the DM group decrease compared with the corresponding segments in the C group, too (P<0.05, P=0.043 between the proximal segments of left ventricles in the two groups, P=0.019 between the middle segments of right ventricles in the two groups, P<0.01 between the other corresponding segments in the two groups). In the DM group, the expression levels of TH mRNA in the distal segments of left ventricles are much lower than those in the proximal segments and the middle segments of left ventricles (P<0.01, respectively). The expression levels of TH mRNA in the middle segments of left ventricles are also much lower than those in the proximal segments of left ventricles (P<0.01). The expression levels of TH mRNA in the middle segments of left ventricles are lower than those in the middle segments of right ventricles (P<0.05). But there are not statistical differences in the expression levels of TH mRNA between the interventricular septa and the middle segments of left ventricles or right ventricles (P >0.05, respectively). The expression levels of TH mRNA detected by real time fluorescent quantitation RT-PCR are coincident to the TH positive nerve fibers densities revealed through immunohistochemistry in every segment of the two groups.Conclusion1 Diabetes damages the cardiac sympathetic nerve of rabbits conspicuously. The damages are displayed by the decline of TH positive nerve fibers densities and expression levels of TH mRNA in the DM group compared with in the C group, and the decreasing degrees among the five cardiac segments in the DM group are different, which aggravate gradually from the proximal segments to the distal segments of left ventricles and are heavier in the middle segments of left ventricles than in the middle segments of right ventricles. There are disequilibrium of sympathetic innervation among the distal segments, the proximal segments and the middle segments of left ventricles and between the middle segments of left ventricles and right ventricles.2 The influence of diabetes on the cardiac sympathetic nerve of rabbits increases the affectabilities of ventricular arrhythmias.
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