Changs Of MRNA And Protein Level Of S-100 And Neuron Specific Enolase In Normal And Hypoxic-ischemic Brain Damage Rats Following Pneumoperitoneun With Carbon Dioxide

Objective With the rapid development and renovation of laparoscopic technology, many experimental and clinical researches concerned on the function changes of normal organs under carbon dioxide pneumoperitoneun which was largely used expend manipulative working space during laparoscopic procedure. With the rapid development of minimally invasiving surgery, the spectrum of laparoscopic operation have been growing wider and the difficulty of them also have been the focus in the field. Some studies have indicated that the brain ischemic and reperfusive injury developed during pneumoperitoneun and the function of Central nerous system were impaired after pneumoperitoneun. The reason for the abdominal compartment syndrome is an important factor for the injury of parenchymatous organ. The researches about the influence of pneumoperitoneun on the respiration, circulation, immunology and the vital abdominal organs are spring up. Howerver, the studies on the influence of pneumoperitoneun on the vital parenchymal organs in the pathological situation were very few and superficial. In healthy patients, carbon dioxide pneumoperitoneun effect on the body is transient and without any apparent sequelae. however, few articles in the literature were reported about the influence of CO₂ PP on the body with dysfunctional organ , such as heart failure,chronic liver dysfunction and so on. The purpose of this study was to evaluate the changes of blood ,cerebrospinal fluid(CSF) and brain tissue S-100 protein (S-100),neuron specific enolse (NSE) levels and the expression of the mRNA and protein level for S-100 and NSE following pneumoperitoneun with carbon dioxide (CO₂ PP) at different of time and pressuer,and to explore whether CO₂ PP produces effect on central nerous system(CNS) in normal and hypoxic-ischemic brain damage rats. Methods Healthy adult Wistar rats were used to establish models of CO₂ PP and of hypoxic-ischemic brain damage .80 model rats were randomly divided into group s.The abdominal cavity of these rats were gradually insufflated CO₂ gas by closeing method and maintained at the pressure of 5mmHg (0.67Kp) for 1hour and 10mmHg (1.33Kp) for 2 hours.animals in each group were sacrificed at 48 hours individually after deflation .Astrocytes and neurons in the hippocamps of brian tissue of rat were observed by light microscope and electron microscope. Their serial blood and CSF S-100 and NSE were measured by ELISA way. By using the RT-PCR technique the expression of mRNA for S-100 and NSE in the brain tissue were tested .Immunohistochemical assay was used to investigate the changes of the expression of S-100 and NSE at the protein level. Detected by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick and labeling (TUNEL),apoptosis of the astrocytes and neurons in the hippocampus of brian tissue of rat was counted on each slices.Results (1) There were no any histologic different between A_5-1 group and control group after pneumoperitoneun with carbon dioxide. The astrocytes and neurons in the hippocampus of brian tissue of rat in A_10-2 group showed local hydropic degeneration under light microscope , mitochondrion swelling under electron microscope. There were significantly histologic difference between B group and control group . The astrocytes and neurons in the hippocampus of brian tissue of rat in B group showed hydropic degeneration, no nuclear necrosis under light microscope , mitochondrion swelling under electron microscope. Astrocytes in the hippocampus of the B_10-2 group showed diffuse hydropic degeneration, nuclear pyknosis, and cell shrinkage under light microscope.mitochondrion and glycogen swelling , fragmentation of ridge myelinogenesis in the astrocytes .Neuron in the B_10-2 group showed diffuse hydropic degeneration, cytoplasmic hyperosinophilia , local nuclear pyknosis and cell shrinkage under light microscope .Nuclear membrane disruption under electron microscope. (2) The values of S-100, NSE were (0.415±0.096)ug/L and (0.511±0.022) ng/ml respectively in A_5-1 group and (0.445±0.086)ug/L, (0.531±0.04)ng/ml respectively in A_10-2 group in the blood, were (0.440±0.082) ug/L and (0.538±0.059) ng/ml respectively in A_5-1 group and (0.458±0.127)ug/L, (0.574±0.048)ng/ml respectively in A_10-2 group in the CSF after CO₂ PP. The values of S-100 ,NSE were increased in both blood and CSF samples in A_5-1 group and A_10-2 group after CO₂ PP than the values of control group (0.393±0.100ug/L,0.493±0.045 ng/ml and 0.431±0.124ug/L,0.531±0.048ng/ml) respectively. Nevertheless there were no significance between A_5-1 group and control group or A_10-2 group and control group ( P>0.05). The findings showed a significantly increased levels of S-100 and NSE in the B group compared with the control group (P<0.05) or the B_10-2 group compared with the B group (P<0.05)respectively. The values of S-100, NSE in the blood were found to be positively correlated with the values in the CSF (P<0.05) )respectively.(3) The values of expression of mRNA for S-100 and NSE were (64.22±10.40) and (8.98±2.38) in A_5-1 group and (67.02±13.34), (9.18±3.37) respectively in A_10-2 group after CO₂ PP,were (59.34±11.38) and (8.50±2.94) in control group .The expressions of S-100 and NSE increased with the increase of pressure and time of CO₂ PP. There were no significance between them ( P>0.05). The protein s of mRNA for S-100 and NSE increased progressively in B group ,B_5-1 group and B_10-2 group than the expressions of control group. There were significance between B group and control group (P<0.05) or B_10-2 group and B group (P<0.05).(4) The positive areas of the expression of S-100,NSE in the brian tissue increased progressively with the increase of pressure and time of CO₂ PP. There were no significance between A_5-1 group and control group ( P>0.05) or A_10-2 group and control group ( P>0.05). There were significance between B group and control group ( P<0.05) or B_10-2 group and Bgroup ( P<0.05).(5) Apoptosis of astrocytes and neurons in the hippocampus of brian tissue of rat had the similar amount to the control group at low IAP. Dramatical increase of apoptosis of astrocytes and neurons in per high visual field were demonstrated increased significantly after CO₂ PP pressure at 10mmHg (1.33Kp) for 2 hours .There were significance between B_10-2 group and control group. Apoptosis cells in astrocytes and neurons in the hippocampus of brian tissue had slightly but not significantly enhanced in normal rat, significantly enhanced in hypoxic-ischemic brain damage rats at 10mmHg (1.33Kp) for 2 hours.Conclusion (1) CO₂ PP at pressure of 5 mmHg and within 1 hour does not produce any effect on histology, biochemical marker, protein transcriptase level, protein expression level and apoptosis of astrocyte and neuron of central nervous system in normal rat. CO₂ PP is used during this extent is safe to central nervous system.(2) CO₂ PP at pressure of 10mmHg and within 2 hours does not produce significantly effect on histology, biochemical marker, protein transcriptase level, protein expression level and apoptosis of astrocyte and neuron of central nervous system in normal rat. CO₂ PP is used during this extent is safe to central nervous system . (3) CO₂ PP at pressure of 5 mmHg and within 1 hour produce a some effect on histology, biochemical marker, protein transcriptase level, protein expression level and apoptosis of astrocyte and neuron of central nervous system in hypoxic-ischemic brain damage rats. CO₂ PP is used during this extent is safe to central nervous system (4) CO₂ PP at pressure of 10 mmHg and within 2 hours produce significantly effect on histology, biochemical marker, protein transcriptase level, protein expression level and apoptosis of astrocyte and neuron of central nervous system in hypoxic-ischemic brain damage rats. CO₂ PP is used during the extent is injurious to central nervous system, and trying to short PP time or use control way.