| Background and objectives: Neonate hyperbilirubinemia is a common disease in neonate period, erupting simultaneously bilirubin encephalopathy will often cause neonate death in acute stage, even if survive also frequently leave behind the neuronal sequelae, especially with hearing damage is the most common one of which. In neonate period, because the bilirubin is produced more, absorbing and transforming ability of the liver to the bilirubin relatively low, in addition bilirubin intestines liver circulation, easy to occur neonate hyperbilirubinemia; Under the pathological state, because the combined degree of bilirubin and albumin is decreased, the excessive bilirubin can deposit in the brain tissue through the open blood brain barrier , cause nerve cell toxicity to damage .The harm to hearing of hyperbilirubinemia is a part of bilirubin neuronal toxicity. Through measure ABR of hyperbilirubinemia neonate , find the change of I - II wave interphase and wave amplitude of II wave appearing most obvious and first, considering wave origin , clue to that brain stem cochlear nucleus is very sensitive to bilirubin toxicity, namely the damage of cochlear nucleus is earlier than the system of sense of hearing around. The toxicity mechanism of bilirubin to nerve cell is not clear so far. In previous studying , it isverified that bilirubin depositing brain tissue would suppress the neuron energy metabolism, then cause the brain cell hydroncus. Research in recent years indicate central nervous system excitatory aminoacids(EAAs) neurotransmitter and N-methyl-D-aspartate-receptor(NMDAR) activate excessively to lead bilirubin encephalopathy take place. When brain energy metabolism obstacle , ATP repertory exhausted , The obstacle of transports and metabolism of glutaminic acid neurotransmitter happens, and glutaminic acid assembles in the nerve fiber . The NMDA receptor activate excessively and causes nerve cell Na+ , Ca2+ overload .It is the important mechanism of nerve cell damage. And cell Ca2+ form Ca2+ - CaM compound thing , can start target enzyme causing a series of pathology react just to combine usually to adjust with calcium to overload, producing many kinds of harms function on the cell. In case of pathology, NMDAR and CaM activation change and with relation of hearing damage in auditory brainstem nuclei, have not seen yet both at home and abroad at present that there are reports. For probe into hearing damage mechanism in hyperbilirubinemia , NMDAR and CaM function of here at the disease, this subject adopts the technology of immunohistochemistry on the basis of making animal's model of hyperbilirubinemia, observing the activity change of NMDAR and CaM in hyperbilirubinemia newborn rats cochlear nucleus tissue , and analyse its reason, trying to offer theoretical foundation for the prevention and therapy of hyperbilirubinemia hearing damage in clinical, and probe into the feasible way . Materials and Methods:1. 60 healthy SD rats of 7 days old, there are no limitations on male and female, the weight is in about 9-20g, not used ototoxicity drug. Divide for contrast group (C group ) at random, experiment 1 group (Ml group ) and experiment 2 group (M2 group ), every group 20. Inject bilirubin solution to rat abdominal cavity , group of Ml and group of M2 , inject dosage lOOug/g weight respectively (0. 171umol/g weight) and 200ug/g weight (0. 342umol/g weight), it gives to be constant wet and photophobic environment feed 24h naturally on constant temperature after the medicine. The rats of group C replaced by 0. 5ml physiological saline outside bilirubin solution, other conditions are the same as group Ml and group M2.2. Every group animal test Auditory brainstem response(ABR) before putting to death separately, write down ABR I ,11,111 delitescence, and I - II, II -III, I -III incubation period .3. Every group animal is fetch 0.5ml blood after using medicine 24h, mensurating the density of serum bilirubin with the heavy nitrogen; the brain organization of SD rat is fetched, and is cut along centre, one side react with heavy nitrogen to mensurate the content of bilirubin of brain tissue, and mensurate brain tissue Na+-K+-ATP enzyme vigor another side.4. Every group animal do endocardial perfusion , turn on cranium and fetch brain stem entering 4% polyformaldehyde phosphate buffer(PH7. 4) after ABR testing fixing for one week, the paraffin wax is chartered and buried, cut into slices successively. Each rat selects the specific level namely cochlear nucleus to cut into slices, fetches four sets slices altogether. One were studied with HE dye , one were studied with NMDAR immunohistochemistry dye , one were studied with CaM immunohistochemistry dye, doing negative contrast another set.5. Using HPIAS-1000 image-quantative analysis system to exam the average gray value of the positive reactivity production of NMDAR and CaM in rat's cochlear nucleus of every group .6. Statistical analysis of the experimental data was performed with SPSS 11.5 software, adopting one-way ANOVA and correlation analysis, all data show bycounting X ±s , With P <0. 05 has statistics meanings for the difference. Results:1. Density of serum bilirubin of group Ml group M2 are obviously higher than group C, the difference having statistics meanings (P<0. 05); Group M2 is obviously higher than group Ml, the difference having statistics meanings (P<0. 05). The content of bilirubin of brain tissue of group Ml and group M2 obviously increase, and group M2 is obviously higher than group Ml, the difference having statistics meanings (P<0. 05).2. ABR reflecting threshold obviously increases of group Ml and group M2 afterusing medicine, K IK III wave delitescence and I - II, II-IIL I-III incubation period obvious extend, especially with II wave delitescence and I - II incubation period lengthen most remarkable, difference compared with group C having statistics meaning (P <0. 05).3. Group Ml and group M2 brain tissue Na+-K+-ATP enzyme vigor reduced obviously after using medicine , difference compared with group C having statistics meaning (P <0. 05); M2 group animals brain tissue Na+-K+-ATP enzyme vigor is obviously lower than group Ml, the difference having statistics meaning (P <0. 05).4. Group Ml and group M2 rats cochlea nucleus can be seen bilirubin deposited , nerve cell figure obviously reduced, glue cell hyperplasia, nerve cell swelling, the nuclei and somata shrinked. The quantity of Nissl bodies decreased.There were lipofuscin and hollow bubbles in the cells. The membranes of the nuclei were irregular. The capillary cavities increased.The endotheliums proliferated and hypertrophied.The walls of the capillaries became thick.5. The positive reactivity production of NMDAR and CaM in rat's cochlear nucleus assume pale brown under the optics microscope. The average gray value of cochlear nucleus NMDAR and CaM in group Ml and group M2 are obviously lower than group C, namely NMDAR and CaM activation are obviously strengthened, the difference having statistics meaning (P <0. 05). The average gray value of cochlear nucleus NMDAR and CaM in group M2 are obviously lower than group Ml, the difference having statistics meaning (P <0. 05).6. The content of bilirubin of group Ml, group M2 brain tissue to increase as the density of serum bilirubin increases, but is respectively without obvious correlation. Brain tissue Na+-K+-ATP enzyme vigor , The average gray value of cochlear nucleus NMDAR and CaM and the content of bilirubin of brain tissue presented negative correlation. ABR reflecting threshold of group Ml and group M2 increase as the content of brain tissue bilirubin increases, presented remarkable positive correlation (P <0. 05). The average gray value of cochlear nucleus NMDAR and CaM and ABR reflecting threshold presented remarkable negative correlation (P <0. 05).Conclusions:1. The experimental newborn rats have behavior of hearing damages in hyperbilirubinemia.2. The experimental newborn rats brain tissue Na+-K+-ATP enzyme vigor drops in hyperbilirubinemia.3. The experimental newborn rats cochlear nucleus NMDAR activate excessivly in hyperbilirubinemia.4. The experimental newborn rats cochlear nucleus CaM activation obviously rises in hyperbilirubinemia.5. The experimental newborn rat's hearing damage degree and NMDAR and CaM activation present obvious positive correlation, which point out that NMDAR and CaM activation rising play an important role in hearing damage in hyperbilirubinemia.
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