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The Effects of Methylprednisolone on Acute Critical Illness-Related Corticosteroid Insufficiency Associated with Traumatic Brain Injury in Rats

Author: ZhangBin
Tutor: ZhangJianNing
School: Tianjin Medical University
Course: Surgery
Keywords: Traumatic brain injury Corticosteroid insufficiency Apoptosis Methylprednisolone Critical Illness Related Corticosteroid Insufficiency HPA axis
CLC: R651.15
Type: Master's thesis
Year: 2011
Downloads: 15
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Abstract

Objective:About two thirds of patients with traumatic brain injury suffer from neuroendocrine dysfunction, and the deficiency in the hypothalamic-pituitary-adrenal (HPA) axis have significant effects on the mortality and prognosis of the patients. Compared with other critical illness, the incidence and mechanism of CRICI after TBI are different. The purpose of this study was to test the hypothesis that methylprednisolone (MP), a synthetic glucocorticoid, promotes post-traumatic apoptosis in both the hypothalamus and pituitary, resulting in acute CIRCI and increased mortality in the acute phase of traumatic brain injury (TBI).Materials and methods:220 adult male wistar rats (300-350g,8 weeks age, from Academy of Military Medical of China) were randomly divided into four groups:naive group (n=16), injury control (NS, n=55), low-dose MP treatment (n=55) and high-dose MP treatment (n=94). Rats were anesthetized with 10% chloral hydrate (0.3ml/kg) administered intraperitoneally and then placed in a stereotaxic frame. A 4.0 mm craniotomy was performed over the right parietal skull to expose the dura (4.4 mm posterior from bregma and 2.4 mm lateral to the sagittal suture). The pressure pulse of the fluid percussion device between 2.0 to 2.5 atm. After the FPI, rats were treated with MP (5 and 30 mg/kg designated as low and high dose, respectively) intraperitoneally once a day for 4 days after FPI. Control rats received an equal volume of 0.9% saline in the same manner.15 rats from each of three groups (Injury Control, Low-dose MP Treatment, High-dose MP Treatment) were examined for corticosteroids response. Rats were consecutively subjected to electrical stimulation (ES) on three test days:pre-injury day 7, post-injury day 7, and post-injury day 14. During each test day, blood was collected from the rats pre-ES,30 min,90 min and 24hr post-ES in order to assay the dynamic changes of serum corticosterone (CORT), the primary form of GCs in rats. Whole blood samples were collected from the orbital sinus of each rat under inhaled light anesthesia, and An ELISA kit was used to measure the serum concentration of CORT. At 7 and 14 days after FPI,10 rats at each time point were anesthetized with chloride hydrate (30 ml/kg, i.p.) and sacrificed. Brains and pituitary were removed and fixed in 4% paraformaldehyde for 24 h. After fixation, the tissue was paraffin embedded and processed for immunohistological examinations. Two successive brain and three successive pituitary sections from each rat were used for hematoxylin and eosin (H&E), terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) and immunohistochemical staining. Tissue (1 mm×1 mm) was obtained from the PVN of each animal and fixed in a mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M PBS (pH 7.4) overnight at 4℃for 1h. Serial sections which were cut on an ultratome and double stained with uranyl acetate and lead citrate were examined in a TEM at 80 kV.Results:1. The mortality rates of rats in different group are 10%-53.3%. The mortality rate of rats receiving high-dose MP treatment (53.3%) was significantly higher than that of the injury control group(16.7%)(P<0.05)and the low-dose MP treatment group(10%)(P<0.05).2. There was no difference of CII among all the groups before TBI.7 days after FPI, the CII of the high does MP treating group was significantly worse than that of the low-does MP treatment(p<0.05) and injury control group(p<0.05), consequently, the incidence of CIRCI was significantly higher than other groups. But there was no difference between the low-does MP treat group and injury control group.14 days post TBI, there was no difference of stress ability among all the groups. The value of CII in dead rats was significantly lower than the survival ones on post-injury day 7(P<0.05).3. TUNEL-positive cells were observed to be significantly increased in the hypothalamus of injured compared to non-injured rats (p<0.05). Moreover, on postinjury day 7, the number of TUNEL-positive cells was significantly higher in injured rats that had received high-dose MP compared to injury control rats (p<0.05). However TUNEL-positive cells were not detected in the pituitary across the experimental groups at either 7 or 14 days after FPI in rats that survived injury. However, autopsies performed on rats that did not survive after injury and treatment did reveal TUNEL-positive cells in the adenohypophysis of the pituitary.4. At 7 days after FPI, the number of CRH cells were significantly decreased in injured rats receiving high-dose MP(compared with injury control group, P<0.01; compared with low-dose MP treatment group, P<0.01)5. Two kinds of cell death-necrosis and apoptosis were detected by transmission electronic microscopy.Conclusion:1. TUNEL-positive cells were observed in P VN of hypothalamus and pituitary at 7 days after FPI in the survival and death rats respectly, and the use of high-dose MP increase the number of TUNEL-positive cell.2. The injury of the hypothalamus and pituitary after FPI lead to a decrease of CII after electronic stimulation, which causes the incidence of CIRCI. The use of high-dose MP exacerbate the injury of hypothalamus and pituitary and increase the incidence of CIRCI, consequently, increase the mortality.3. High-dose MP exacerbates the injury of hypothalamus and pituitary and increases the incidence of CIRCI and the mortality, so high-dose MP replacement to CIRCI after TBI was not recommended, but moderate or low-dose MP were necessary.

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