The Effects Of Progesterone On The Expression Of ZO-1 In The Intracalvarium Of Rats After Diffuse Axonal Injury

Introduction: Diffuse axonal injury is a common serious craniocerebral injury under the effect of shearing force which causes the white matter to form the multiple brain injury presenting the major characteristics of axonal swelling and rupture. This kind of primary injury often results in the serious damage of blood brain barrier and the increase of its permeability secondarily, which in turn causes the diffuse brain edema. This is the main reason for the comparatively bad effects of its prognosis. Zonula occludens-1 (ZO-1) is one of important components in tight injection of blood brain barrier with other components, and to which the structure and function of ZO-1 are closely related. So the expression level of ZO-1 should be able to reflect the state of the blood brain barrier. Progesterone (PROG) is a natural pregnancy hormone, the research in recent years show that it can be formed in nervous system and play a role in alleviating the brain edema. Its mechanism might be that progesterone makes the effects of antioxidant through its reduction products 5α-dihydro progesterone (5α-DHP) and 3α, 5α-allopregnanolone (3α, 5α-THP) to reduce the damage of the blood brain barrier. However, whether there exists a certain effect on the ZO-1 expression hasn't been reported at home and abroad so far.Objective: The expression level of ZO-1 in brain tissues of SD rats after DAI was detected, and the permeability rate of the blood-brain barrier for evens blue was also observed to investigate weather there is a relationship between them. Furthermore, the expression level of ZO-1 would be detected after treatment with progesterone to investigate weather there is an effect after the treatment.Methods: Randomly dividing 85 healthy adults male SD rats weighing from 350 to 375g (provided by the experiment animal centre of Hebei medical university) into the following groups: sham operated group (10), DAI group (50), progesterone treatment group (25). DAI group and progesterone treatment group were divided into 5 subgroups respectively according to the observation time: 6 h, 24 h, 48 h, 3 days, 7 days. For the sham operated group, the scalp would be slit exposing the cranium and periosteum, and then it would be directly stitched. For the DAI group and progesterone treatment group, the rats were made DAI model according to that Marmarou's method, and the striking height was 150cm with striking weight 450g. For the progesterone group, the progesterone (8mg/kg) would be immediately injected into rats' abdomen after DAI and this injection will be given every 12h. Randomly choosing 5 rats from the 10 rats of sham operated group and every subgroup of DAI group, according to that Belayey's method to detect the permeability of BBB, the BE was used as a tracer. The rest rats ' heads would be quickly cut and the brains will be stripped after the perfusion of 4% paraformaldehyde solution according to the observation time. All samples would be put into 4% paraformaldehyde solution to be fixed and kept in icebox at 4℃for use.Through HE staining to the samples, appearance of typical pathological changes of Axonal retraction ball (Arb) would be observed after the DAI.By using the Immunohistochemical staining method(SP), the ZO-1 protein expression of the samples in the DAI group and progesterone treatment group would be detected. The immunohistochemical staining positive reaction would be analyzed through observing the average integral optical density value of the positive reaction.SPSS13.0 statistics software was adopted. Results of analyzing the immunohistochemistry would be shown in the form of mean±standard deviation ( x±S). Two sample T-test would be adopted for comparison between two groups, while single-factor analysis of variance would be adopted for comparing more than two groups. When the analysis of variance shows presents the significant difference, q-test would be adopted to do further comparison between each two groups, and the directly relativity analysis would be conducted based on the requirement of the experiment. P<0.05 was taken as the standard to judge whether the difference has the statistic significance.Results: 1 Observation results by using optical microscopeFor the DAI group and Progesterone group, there was a wide range of subarachnoid hemorrhage on the brain surface. RBC oozed from some samples' ventricle. Deep neuronal nuclei, shallow cytoplasm and shrinkage of neural cells around the transparent gap could be seen from neural cells and the surrounding area of small blood vessels. Axis becomes thicker and swelling, the typical Arb appeared. The pathology changes of the progesterone treatment group were less than that of DAI group.2 The changes of permeability of the blood-brain barrier for EBPermeability rate of the blood-brain barrier for EB appeared to rise in the DAI 6h subgroup, continued to rise in 24h subgroup, increased to the peak in 48h subgroup, and then began to decline gradually. However it was still higher in 7d subgroup than in the sham operated group. Comparing each subgroup of DAI group with the sham operated group, there was the statistical difference (p <0.05).3 The expression of ZO-1 in sham operated groupThe positive expression of ZO-1 in sham operated group stained on the inner membrane and cytoplasm of cerebral micro-vascular endothelial cells the expression.4 The changes of ZO-1 expression in DAI groupZO-1 in DAI group stained on the inner membrane and cytoplasm of cerebral micro-vascular endothelial cells and each subgroup had the expression. The expression level of ZO-1 in sham operated group was higher than each subgroup of DAI group (p<0.05). The expression of ZO-1 in DAI 6h subgroup began to decline, fell to the lowest level in 48h subgroup, and started to rise gradually, and the expression in 7d subgroup was lower than that of sham operated group. The change of ZO-1 expression was negatively related with the changes of the permeability rate of blood brain barrier for EB (r=-0.843, p<0.01).5 The changes of ZO-1 expression in PROG treatment groupZO-1 in PROG treatment group still stained on the inner membrane and cytoplasm of cerebral micro-vascular endothelial cells after PROG treatment. There was no obvious difference comparing the ZO-1 in PROG treatment 6h subgroup with DAI 6h subgroup(p>0.05); comparing the 24h ,48h, 3d and 7d subgroup of PROG treatment group with the corresponding subgroup of DAI group, the expression of the former was higher than that of the latter(p<0.0). There was no obvious difference comparing ZO-1 expression in PROG treatment 7d subgroup with that of the sham operated group (p>0.05). While there was the obvious difference comparing ZO-1 expression in the 6h, 24h, 48h, 3d subgroup of PROG treatment group with that of sham operated group(p<0.05).Conclusions: 1 The permeability rate of blood brain barrier for EB increased significantly after DAI and reach to the peak at 48h, which indicated that the blood brain barrier is seriously damaged after DAI.2 The damage of the blood brain barrier accompanied with the degression of ZO-1 after DAI.3 The changes of ZO-1 expression in the brain tissues was negatively related with the permeability of the blood and brain for EB, which indicated that the decline of the ZO-1 expression could be regarded as a symbol for the measuring the damage degree of the blood brain barrier.4 The expression of ZO-1 in brain tissues in PROG treatment group was higher than that of the DAI group, which indicated that the treatment of PROG inhibited the degression of ZO-1 after DAI and it might has some protective function for the blood brain barrier.