An Experimental Study On The Damage To Baby Brain Induced By Prednisone Or Corticotrophin

PART I A RAT MODEL SET UP WITH THERAPEUTIC-DOSE RANGE FOR PREDNISONE OR CORTICOTROPHINObjective: To set up a rat model with therapeutic-dose range for prednisone or corticotrophin.Methods: The dose of prednisone or corticotrophin in rats was determined according to their efficacy on HPAA.①Dose determination for prednisone:10 morning serum samples were collected from pediatric patients treated with therapeutic-dose prednisone lasting more than 5 days and cortisol concentration was detected by chemiluminescence. According to calculation by the conversion formula based on body surface area,24 7-day-old male rats were divided into 4 groups (n=6) and orally administrated 2 mg/kg/d,3 mg/kg/d and 4mg/kg/d prednisone and 0.5%CMC(control group) respectively at 8am for 7 consecutive days. Serum samples were collected at 8am of the 8th day and corticosterone concentration was detected by EIA.②Dose determine for corticotrophin: 60 14-day-old male rats were divided into 9 experimental groups and one control group(n=6).Rats of the experimental groups were intraperitoneal injected with corticotrophin in 3 different dose (3.8U/m~2/d,38 U /m~2/d,150 U /m~2/d) at 3 different time(2 pm,3pm,3: 30pm) respectively after anesthesia.Control group was anesthetized simultaneously without any other intervention. Serum samples were collected at 4pm and corticosterone concentration was detected as before.Results:①The mean cortisol contration of 10 in-patient children's serum was 87.18±39.71 nmol/L ,which was 15.79% of the normal value.②Serum corticosterone concentration of rats treated with prednisone (4mg/kg/d) was 15.17% of the control group,which was coincidental with clinical data.③A peak concentration of corticosterone was reached 30 minutes after intraperitoneal injection with corticotrophin in all groups and began to fall down at 60 minutes,then returned to the basal level at 120 minutes after injection.Conclusions:①The depression effect on cortisol of prednisone in clinic can be successfully mimic by oral administration with 4mg/kg/d prednisone for 7 consecutive days. So the dose of 4mg/kg/d should be taken as the experimental dose in present study.②There was no difference in serum corticosterone concentration 30 minutes after corticotrophin administration in different dose groups, which indicated that the corticotrophin dosage for present research had to be calculated by conversion formula based on body surface area according to clinic dosage for infantile spasm treatment.PART II HISTOPATHOLOGICAL OBSERVATION ON BRAIN DAMAGE IN BABY RATS INDUCED BY PREDNISONE OR CORTICOTROPHINObjective: To investigate the histopathologic abnormality induced by prednisone or corticotrophin on immature brain of rats.Methods: 192 baby (P7d) and 192 adul(taged 2mo)male SD rats were selected and treated with prednisone or corticotrophin as follows:(1) 96 baby and 96 adult rats,were divided into 3 groups(n=32) as therapeutic-dose group(4mg/kg/d),low-dose group(2mg/kg/d)and control group(0.5%CMC) respectively. Prednisone was given by oral administration at 8am once a day for either short-term(10 days,n=16) or long-term(3 weeks,n=16) observation.(2)Another 96 baby and adult rats were divided into 3 groups(n=32) and treated with corticotrophin (150U/m~2/d or 38U/m~2/d) or saline(control group) by intraperitoneal injection twice a day for either 10 days or 3 weeks.For each group after prednisone or corticotrophin withdrawal for 24 hours(n=8) or 4 weeks(n=8),rats were sacrificed and body and brain weight was recorded. Serum corticosterone concentration was quantitated by EIA .Morphology in the frontal cortex and hippocampus was observed by HE staining and Nissl staining. Ultrastructural changes of the hippocampus were observed by the tranmission electron microscopy.Results:①A suppression level of 10.75% of corticosterone was identified in rat serum after administration with prednisone (4mg/kg/d) for short term and was coincidental with clinical data. Also an increased corticosterone level of 1.83 times of control was identified in the rats treated with corticotrophin (150U/m~2/d) for short term and can mimic their effect on glucocorticoid in clinic.②Compared with the control, 7.9~17.5% reduction of body weight was found in short-term prednisone or corticotrophin treated groups in a dose-dependent way. 4 weeks after withdrawal,thouth recovered with various degrees,body weight of both baby and adult groups was still lower than control.Body weight loss was more serious after long-term treatment, as high as 20.6~37.7% in baby groups.4 weeks after withdrawal,a reduction of 9.8~23.5% body weight still existed in either baby or adult groups.③The brain weight of baby rats was reduced particularly after treatment. Compared to control, reduction of brain weight was 5.3~10.7% in a dose-dependent way in baby rats treated with prednisone or corticotrophin for short term. 4 weeks after withdrawal,the brain weight of prednisone groups recorverd nearly to normal level,but that of ACTH groups was still nearly 20% lower than control.In the baby rats treated with prednisone for long term,the decrease degree of brain weight was similar to short-term groups,but as high as 11.7~15.9% in ACTH groups.4 weeks after withdrawal,brain weight was 4.1% and 13.2% lower than control in prednisone and ACTH therapeutic-dose group respectively. 2.2~4.8% brain weight loss was also found in adult rats treated with either drug for long term,which recovered after 4 weeks withdrawal.④Significant neurodegeneration and neuronal necrosis was found by HE staining in all prednisone or corticotrophin treated baby rats(except short-term low-dose groups) and long-term therapeutic-dose adult groups.However,no obvious histopathologic changes was observed 4 weeks after withdrawal.⑤Loss of neurocyte,degradation of nissl body and disorganization of brain were found by nissl staining in all prednisone or corticotrophin treated baby groups(except short-term low-dose groups).Cell count at the frontal cortex was less than their controls of 25.9~32.14 wee(prednisone)%,24.4~29.1%(ACTH).4 weeks after withdrawal, obvious hispathologic abnormality was no longer observed except for cell count was still 15.8~20.3% less than control at frontal cortex of corticotrophin therapeutic-dose groups.Degradation of nissl body was also found in the hippocampus of long-term therapeutic-dose adult groups but recovered completely 4 weeks after withdrawal.⑥By electron microscopy, numerous chromatin clumps, swelling of rough endoplasmic reticulum and mitochondia, myelin sheath delamination were observed in hippocampus neurocyte of baby and adult rats taken therapeutic-dose prednisone or corticotrophin for long term,which disappeared 4 weeks after withdrawal.Conclusion:①Prednisone and corticotrophin both could reduce the body weight gain of baby or adult rats.②Both prednisone and corticotrophin could induce brain damage and be more sensitive to immature brain. After 4 weeks withdrawal, the neurocyte number in frontal cortex of baby rats was still significantly less than control even no obvious histology abnormality was observed which indicated a persistent damage in the brain.③Since the reduction of brain weight gain was not parallel to the reduction of neurocyte number,there might be some other mechanism involved in the loss of brain weight.PART III A PRELIMINARY STUDY ON THE PATHOGENESIS OF BRAIN DAMAGE IN BABY RATS INDUCED BY PREDNISONE OR CORTICOTROPHIN Objective: To explore the pathogenesis of brain damage in baby rats induced by prednisone or corticotrophin initially.Methods: Animals and sample preparation were as Part II. NSE level in serum was quantitated by ELISA. Expression of apoptosis-related proteins Bcl-2 and Bax was detected by immunohistochemistry. Neuronal apoptosis was detected by TUNEL.Results:①Compared with the controls, NSE increased by 50.62% and 57.34% (P﹤0.05 or﹤0.01)in baby rats treated with therapeutic-dose prednisone or ACTH for short term, 103.23% and 100.04%(P﹤0.01)in those for long term. No difference of NSE level was found in adult rats treated in short course, but 66.81% and 60.40% enhancement was found in groups treated with long-term therapeutic-dose prednisone or ACTH respectively(P﹤0.05)②Compared with the controls, no overexpression of Bcl-2 in frontal cortex and hippocampus was found in all adult or baby rats treated with prednisone or corticotrophin for short or long term either. In contrast, significant increase of Bax expression was found in baby but not adult rats in the short-term therapeutic-dose groups of both drugs, and the ratio of Bax/Bcl-2 also increased to 1.91～2.29 times of that in controls. Overexpression of Bax was found in frontal cortex and hippocampus of the baby rats treated with both drugs with therapeutic or low dose, and the ratio of Bax/Bcl-2 became 1.92～2.70 times of that in controls, which also found in hippocampus of therapeutic-dose adult groups after long-term treatment. All those changes almost recovered after 4 weeks withdrawl.③Remarkable TUNEL-positive cells was found in frontal cortex and hippocampus of baby rats treated with therapeutic-dose in short- or long-term,as well treated with low-dose but long-term. Significant TUNEL-positive cells were also shown in the hippocampus of adlut rats exposed to long-term therapeutic-dose of either drug.Conclusion:①Necrosis process of cells could be one of the main pathogenesis on the brain damage induced by prednisone or corticotrophin except for apoptosis.②Overexpression of Bax might be the important promoter in the development of neurocyte apotosis.③Both prednisone and corticotrophin can induce brain damage which mainly occurred in baby age in a dose-dependent way but significantly recovery 4 weeks after withdrawal, which also happened in adult rats treated long-term therapeutic dose.