| Objective: Pirfenidone is a recently discovered anti-fibrosis drug. Itsanti-fibrotic effect is clearly recognized. Thus it has great applicationprospects in the treatment of the brain chronic hydrocephalus which is causedby mechanisms of fibrosis. But there is no proof of its safety on the brain.Therefore, this experimental study is intended to observe the effect ofpirfenidone on the rat nerve injury by determination of serum andcerebrospinal fluid neuron-specific enolase after the intragastric administrationof pirfenidone.Methods：1Rat grouping and processing: A total of50healthy adult clean SD malerats, weighting220±10g was chosen. Randomly divided the rats into10groups, each group had five rats.The rats were divided into control group,50mg/kg group,100mg/kg group,300mg/kg group,500mg/kg group,700mg/kg group,900mg/kg group,1100mg/kg group,1300mg/kg group,1500mg/kg group. Collect the blood sample from rat orbital sinus at the timeof one day before intragastric administration,1hour,12hours,24hours afterthe intragastric administration. Collect the CSF sample at the time of24hoursafter the intragastric administration. When the collection of CSF samplefinished, rats in each group were killed and the brains were collected.2Intragastric administration: Before the intragastric administration, therats of each group were fasted for12hours. Using the rat intragastric deviceextracted the well prepared PFD suspension, then fed the rats.3Behavioral tests in rats: To score the rats behavior ability by the beamwalking test and the rotarod test at the day before, after, and the day of theintragastric administration.4Collection of the serum samples: The rats blood samples were collected from the orbital venous at a predetermined time and tested with the ELISAmethod to obtain serum concentrations of NSE.5Collection of the CSF samples: The rats CSF samples were collectedfrom the cisterna magna at a predetermined time and tested with the ELISAmethod to obtain CSF concentrations of NSE.6Brain specimens drawn: Rats in each group were killed at the presettime, and the brains were collected after the perfusion. The brain samples werekept in paraffin wax waiting for test.7Dyeing treatment: Observing the histopathological changes in thecerebral cortex of rats with and silver staining.8Data analysis: The results were calculated by statistical treatment withIBM SPSS Statistics version21software.Results：1General state: During the experiment the control group rats weregenerally in good condition, the spirit were good, eating well, the coat wereshiny, activities were flexible, they showed no abnormal condition and nosignificant changes in body weight. In the experimental group, the rats oflow-dose group behaved general state similar situation with the control groupof rats. As the doses increasing the rats performed worse, the general stateworse, apathetic, reducing dietary water intake, reduced activity. Withextended time, the state gradually recovered, but still not as good as before theexperiment. There were no death in any groups.2Compare the weight of the rats: Rats of any group were losing someweight after the experiment, but before and after the experiment each groupshowed no significant difference in weight of the rats (P>0.05). The weightchanges were considered experimental stress-induced stimulation.3The beam walking test: The rats were tested with non-parametric ranksum test, there were no significant differences of each group the day beforeintragastric administration (P>0.05). Significant differences were detectedbetween the groups at the intragastric administration day (P<0.05), and sowere the day after the intragastric administration (P<0.05). A comparison between the groups of rats at different time points were taken, the results werethat there were no significant differences at different time points in the blankgroup,50mg/kg group and100mg/kg group (P>0.05), there were significantdifferences at different time points in the300mg/kg group,500mg/kg group,700mg/kg group,1100mg/kg group,1300mg/kg group and1500mg/kg group(P<0.05).4The rotarod test: The rats were tested with non-parametric rank sum test,there were no significant differences of each group the day before intragastricadministration (P>0.05). Significant differences were detected between thegroups at the intragastric administration day and the day before it (P<0.05).There were no significant differences at different time points in the blankgroup,50mg/kg group,100mg/kg group,300mg/kg group and500mg/kg group(P>0.05), there were significant differences at different time points in the700mg/kg group,900mg/kg group,1100mg/kg group,1300mg/kg group and1500mg/kg group (P<0.05).5Comparison of weight and density of brain tissue: The rats brain tissueweight (g) of any two groups were compared, the brain tissue of the smalldoses group,50mg/kg group,100mg/kg group,300mg/kg group andcontrolled group, were smaller than the bigger doses group,1100mg/kg group,1300mg/kg group,1500mg/kg group, and the difference was statisticallysignificant (P<0.05). The rats brain tissue volume (ml) of any two groups werecompared, the brain tissue of the big doses group,1100mg/kg group,1300mg/kg group and1500mg/kg group, were smaller than the small dosesgroup,300mg/kg group and the controlled group, and the differences weresurely statistically significant (P<0.05), but there were no significantdifferences between the other groups (P>0.05). No significant differenceswere found between any groups of the brain tissue density (P>0.05).6Comparison of serum NSE: There were no significant differences of theserum NSE between every other groups the day before gavage (P>0.05). Theserum NSE contents of the rats1hour late of the gavage were compared. Thecontrolled group，50mg/kg group,100mg/kg group and300mg/kg group were sharing the same contents of the serum NSE, and there were no significantdifferences (P>0.05). And so were the500mg/kg group and700mg/kg group,no significant differences were found between them (P>0.05). The900mg/kggroup and the1100mg/kg group had no significant differences either (P>0.05).But the1500mg/kg group compared with any other groups, there weresignificant differences (P<0.05). The serum NSE contents of the rats6hourlate of the gavage were compared. The controlled group，50mg/kg group,100mg/kg group and300mg/kg group were sharing the same contents of theserum NSE, and there were no significant differences (P>0.05). But any of theother groups compared with each other, there were always significantdifferences (P<0.05). The serum NSE contents of the rats12hour late of thegavage were compared. The controlled group，50mg/kg group,100mg/kggroup and300mg/kg group were sharing the same contents of the serum NSE,and there were no significant differences (P>0.05). The900mg/kg group andthe1100mg/kg group were sharing the same content of serum NSE (P>0.05).But the other groups were different with each other, and there were significantdifferences (P<0.05). The serum NSE contents of the rats24hour late of thegavage were compared. There were significant differences between any othergroups (P<0.05).There were no significant differences of the serum NSE contant betweendifferent time point in the small dose groups, controlled group,50mg/kg group,100mg/kg group,300mg/kg group (P>0.05). The comparison of the serumNSE content at different time point in the500mg/kg group,700mg/kg groupand1100mg/kg group, showed that at the6hours and12hours after thegavage was the highest, the1hour and24hours followed by, the day beforethe gavage was the lowest, and the difference was statistically significant(P<0.05). In the900mg/kg group, the order was that12hours was the highest,6hours came to the second,1hour and24hours followed by, and also the daybefore gavage was the lowest, and the difference was statistically significant(P<0.05). In the1300mg/kg group any of the time point was different with theothers, and the difference was statistically significant (P<0.05). Same in the 1500mg/kg group, any of the time point was different with the others, and thedifference was statistically significant (P<0.05), and the ascending order wasthe day before gavage,1hour after gavage,24hours after,6hours after,12hours after.7Comparison of CSF NSE: There were significant differences of theCSF NSE between the groups (P<0.05). Compared with the serum NSE24hours after the gavage, the CSF NSE content were higher, and the differenceswere significant (P<0.05).8Silver staining: The number of nerve cells in the brain tissuedistribution and morphology were normal in the controlled group, thepyramidal cells were oval, the nucleolus were clear. The small dose groups,50mg/kg group,100mg/kg group,300mg/kg group, shared the samecircumstances, the number of nerve cells in the brain tissue distribution andmorphology were almost the same with the rats in the controlled group. Therewere rat brain tissue necrosis in the larger dose groups (>500mg/kg), with thedose increased, increased necrosis.Conclusion：1The neuron-specific enolase can be used as an important test of ratdamage index, and correlated with the content and damage degree.2The neuron-specific enolase has its distribution after brain injury, itscontent levels in CSF is higher than in the serum.3The pirfenidone do has some injury on rat brain.4The pirfenidone on brain injury in rats has its dose law.50mg/kg,100mg/kg,300mg/kg have no obvious effect on the nerve of rats.500mg/kgand larger doses having damage in rats nerve function is a positive correlation. |
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