| Objective:To observe the difference of PEBP expression in hippocampus between the fimbria-fornix-transected rats and normal ones. Methods:(1) Real-time PCR:Forty-two SD rats were randomly divided into 7 groups,6 rats in each group. One group served as normal control group and the others served as fimbria-fornix transected 1st,3rd,7th,14th,21st and 28th day group respectively. Then hippocampi were isolated and total RNA was extracted and then the first strand cDNA was synthesized, the method of Real-time PCR was used to observe the changes of PEBP mRNA expression in hippocampus on different time points. CT value of PEBP and GAPDH in normal group and transected groups were detected by the method of 2-△△CT T respectively. The relative expression level of PEBP mRNA was indicated by the result of the method of 2-△△CT. (2) In Situ Hybridization:Six SD rats'right fimbria-fornix were transected. On the 7th day after transection, the rats were perfused and fixed, then cryostat sections of hippocampus were prepared. DIG-labeled PEBP RNA probe was prepared and used in hybridization in order to observe the expression change of PEBP mRNA in hippocampus of both sides. One section was taken from anterior, middle and posterior location respectively of each rat. The number and the mean gray value of PEBP mRNA positive cells were measured in transected and normal sides of pyramidal layer, granular layer of dentate gyrus and on 0.03mm2 area of hilus, subgranular of dentate gyrus respectively. (3) Western Blot:Forty-two SD rats were randomly divided into 7 groups,6 rats in each group. One group served as normal control, and fimbria-fornix of the other groups rats were transected on the 1st,3rd,7th,14th,21st and 28th day respectively. Then hippocampi were isolated and the total protein was acquired from the fimbria-fornix transected and the normal hippocampus, then detected the concentration of each group, the protein sample was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the membrane. The expression was detected by PEBP polyclonal antibody immunoblot. The mean gray value of PEBP and B-actin positive strips was then detected, the relative expression level of PEBP protein was indicated by the reciprocal of the mean gray value of PEBP to B-actin. (4) Immunohistochemistry:Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group, and served as right fimbria-fornix transected 1st,3rd,7th,14th,21st and 28th day group respectively. The rats were perfused and fixed, then cryostat sections of hippocampus were prepared for PEBP immunohistochemistry. One section was taken from anterior, middle and posterior location respectively of each rat. The number and gray value of PEBP positive cells in pyramidal layer and granular layer of dentate gyrus, in hilus of dentate gyrus and subgranular layer of 0.06mm2 area on both transected and normal sides were measured respectively. (5) Analysis of variance and mutual comparision of each group was analyzed by SPSS 11.5 software, and the both sides'results of in situ hybridization and immuohistochemisty were conducted by the method of paired t test. Results:(1) Real-time PCR:In normal group, the relative expression level of PEBP mRNA was 1.16±0.10. It started to increase on 3rd day (1.51±0.08) after transection, and the peak appeared on 7th day(1.64±0.07), then decreased slowly to pre-transection level on 21st (1.14±0.13). One-factor analysis of variance and comparison between each two groups of the PEBP mRNA relative expression manifested that there were significant difference between the fimbria-fornix transected 3rd,7th day groups and other groups (P<0.05). The difference was not significant between 3rd and 7th day group, and not significance existed among other groups (P>0.05). (2) In Situ Hybridization:PEBP mRNA was expressed in CA1～CA3 pyramidal layer of hippocampus and granular layer of dentate gyrus on both sides. The number of PEBP mRNA positive cells manifested no discrepancy between two sides. But the mean gray value of PEBP mRNA positive cells in transected side (78.47±4.45) was lower than that in normal side (102.58±15.99), the signal was higer in transected side than normal side. PEBP mRNA positive cells was also seen in hilus and subgranular layer of dentate gyrus, in transected side, the number of PEBP mRNA positive cells was 60.67±6.71, the mean gray value was 67.16±9.28. In normal side, there was less PEBP mRNA positive cells (50.33±8.52), and the mean gray value was 99.62±13.44. The positive cell numbers and mean gray value of two sides existed significant difference (P<0.05). (3) Western Blot:Western Blot showed that the expression of PEBP protein was 0.0551±0.0093 in control group. It started to increase on 3rd day (0.077±0.0083) and the peak appeared on 7th day (0.0965±0.0089), then decreased slowly to pre-transection level on 28th day (0.0505±0.0066). When transected groups compared with the normal group, statistic results showed no significant difference between the 1st,28th day group and the normal group (P<0.05), while significant difference existed between the normal group and the other five transected groups (P<0.05). When transected groups compared mutually, the difference between the 7th day and other transected groups was significant (P<0.05), the significant difference also existed between the 3rd,14th,21st day group and 1st,28th day groups, while the difference among 3rd,14th,21st day groups were not significant (P>0.05), the difference between the 1st and 28th day was not significan either. (P>0.05). (4) Immunohistochemistry:The result of PEBP immunohistochemistry showed that PEBP was majorly expressed in the kytoplasm of cells which located in the pyramidal layer of CA1～CA3 areas and granular layer, hilus, subgranular layer of dentate gyrus. The number of PEBP protein positive cells has no discrepancy between two sides in the pyramidal layer of CA1～CA3 areas and (P>0.05). But the stain of PEBP positive cells in transected side was deeper than normal side, especially on 7th day, the mean gray value was 68.41±10.53, while the mean gray value was only 91.52±7.77 in the normal side, the significant discrepancy existed between the two sides (P<0.01). More deeper stained PEBP positive cells could be seen in the hilus and subgranular of dentate gyrus, especially on the 7th day after transection, the number of positive cells was 53.33±4.27, the mean gray value was 65.34±15.84, while the number and mean gray value of positive cells in the normal side was 37.5±4.04 and 97.16±11.62 respectively, the significant discrepancy existed between the two sides (P<0.05). The expression of PEBP in pyramidal layer, hilus and subgranular of dentate gyrus was decreased on the 14th day after transection and reached pre-transection level on the 28th day. Conclusion:(1) The expression of PEBP mRNA and protein obviously increased after fimbria-fornix transection, the expression changed with time, it increased at first and then decreased to normal level, the ascending and descending tendency of PEBP mRNA and protein was generally coincident, it reached peak on the 7th day after transection. (2) The PEBP mRNA and protein majorly expressed in the kytoplasm which located in the CA1～CA3 areas of pyramidal layer, granular layer, hilus and subgranular layer of dentate gyrus. (3) Cells in the CA1～CA3 areas of pyramidal layer, granular layer of dentate gyrus were stained deeper in the transection side than the normal side. However, compared with the normal side, cells in the hilus and subgranular layer of dentate gyrus were not only stained deeper but also possessed more numbers of positive cells. The results indicated that the higher expression of PEBP mRNA and protein in the fimbria-fornix transection side hippocampus may involve the hippocampal neural regeneration which induce the neural stem cells differentiating into neurons or cholinergic neurons.
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