| Objective(1)What’s the first part puepose was to establish a kind of reasonable animal model for the further research work.(2)The main aim of the second part was to explore the effects of Electroacupuncture on the dregree of the hydrops in the cochlea.Moreover the ion channel protein about the sodium would be our main molecule to be discussed.Methods(1)The albino guinea pigs were randomly divided into normal group and group treated with arginine vasopressin via intraperitoneal injection with for seven days.What we next did was using the ABR to test the auditory threshold,using the HE staining to observe the degree of the hydrops.(2)The albino guinea pigs were divided into normal group,the ELH group and ELH+EA group with seven days and fourteen days after the intraperitoneal injection respectively.The acoupoints we selected was baihui(GV20)and tinggong(SI 19).HE staining was used to reveal the morphological change including the degree of the hydrops and the cell density of Reissner’s membranes;ELISA was used to test the concentration of vasopressin.The experiment was about the expression of Na+-K+-ATPaseal subunit and epithelial sodium channel(ENaC)a subunit in the cochlea.Immunohistochemistry and western-blot and RT-PCR were applied to exam the localization,quantitation of the protein and mRNA respectively.Results(1)compared with the normal group,there was no significant difference between the normal group and group treated with AVP in auditory threshold(P>0.05).(2)The success rate of the model could reach 100%.(3)HE staining:besides the normal group,the groups with AVP including ELH groups and EA groups showed hydrops with a significant difference(P<0.05).However,the degree of ELH was much lower in the EA groups than that in the groups treated with AVP at the same time point(P<0.05).The hydrops would not vanish as the time extension but even had a trend of increase.The hydrops in EA groups was lower,but the development trend was also same with the ELH group.(4)The cell density is a sign of hydrops degree,because the width of RM is the main influence infector.Compared with the normal group,the cell density did not change much in the early stage meaning the groups treated for seven days without significant difference(P>0.05).However,the density decreased in the groups treated for fourteen days,the EA could increase the density.The P value had a significant meaning between them(P<0.05).(5)The concentration of p-AVP:The concentration of p-AVP in the ELH groups and EA groups was much higher than that in the normal group(P>0.05);the concentration of p-AVP in the EA group was much lower,compared with the ELH group in the same time point.The concentration of p-AVP decrease with the time extension in the ELH groups as well as the EA groups.(6)The location of Na+-K+-ATPaseaisubunit was tested via immunohistochemistry with specific antibody.The Positive expression of this protein can be found in the RM,lateral wall area containing spiral ligament and vascular stria,and spiral ganglion.The RM and lateral wall area is crucial for the water metabolism,so we only focused on the protein expression in RM and lateral wall.The IOD of Na+-K+-ATPaseα1 subunit in RM revealed a decrease when comparing the normal group with groups treated with AVP(P<0.05).The IOD value had an increase in the EA groups when compared with the ELH group in both time points,which could be seen form the P value that was less than 0.05.The expression of this protein seemed to have a decrease trend in the extent of observed time.(7)The positive expression of ENaC a subunit could be seen in the RM.Compared the normal group,the expression of this protein increased with a significant meaning.Compared with the Ml group,the expression of this protein increased(P<0.05).Compared with the M2group,the results did not show a significant difference.The expression of this protein increased with the time.However,the EA group had the opposite trend.(8)The positive expression of Na+-K+-ATPaseα1 subunit also could be seen in the latter wall.The IOD value in the ELH and EA groups had a significant decrease when compared with the normal group.The comparison results between ELH and EA groups yield a favor to the EA groups at the same time points,which mean the IOD value was greater than that in the ELH group.The development trend was the same as the trend in the RM.(10)the positive expression of ENaC a subunit could be seen in the latter wall.Compared with the normal group,the IOD value in ELH and EA group increased,with statistical difference(P<0.05).Compared with the M1 group,the expression of this protein increased(P<0.05).Compared with the M2group,the results did not show a significant difference.The expression of this protein increased with the time.However,the EA group had a opposite trend.(10)Compared with the normal group,the relative amount of each cochlear Na+-K+-ATP enzyme alpha 1 subunit mRNA decreased,at the same time,the expression of the EA group compared with the model group,with statistical significance(P<0.05),the model group and the electroacupuncture group showed the expression decreased with time;Compared with the normal group of cochlear epithelial sodium channel alpha subunit mRNA expression increased(P<0.05),EA1 group and M1 group after compared expression was statistically significant(P<0.05);EA1 group and M1 group after compared expression was statistically significant(P<0.05).The ELH group showed a gradual upward trend,the expression trend of EA group was on the contrary.(11)Western-Blot results showed that:compared with the normal group cochlear Na+-K+-ATP enzyme alpha 1 subunit expression decreased(P<0.05),at the same time,the electro acupuncture group compared with model group,the protein expression increased(P<0.05),model group and EA group decreased with time.;Compared with the normal group of cochlear epithelial sodium channel alpha subunit expression was increased(P<0.05),Compared with the M2group,the results did not show a significant difference.The expression of this protein increased with the time.However,the EA group had a opposite trend.Conclusions(1)Intraperitoneal injection of arginine vasopressin can establish stable endolymphatic hydrops,the success rate can reach 100%.(2)EA can relieve the degree of endolymphatic hydrops in guinea pigs and Baihui and Tinggong acupoints are the important point’s selection for treatment of ELH and Meniere’s disease.(3)EA could reduce the concentration of vasopressin in plasma of guinea pig,which might be the reason that EA could reduce the degree of hrdrops.(4)The EA could suppress the decease of Na+-K+-ATP enzyme alpha 1 subunit expression,and benignly regulate the expression of ENaC,which might be an underlying mechanism that acupuncture reduce the degree of ELH. |
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