Alterations Of Enterocyte Mitochondrial Function Following Traumatic Brain Injury

Traumatic brain injury (TBI) is an increasing problem worldwide because of its highmortality and disability.Many patients with severe TBI often die of multiple organdysfunction syndrome (MODS), but not of the injury itself. Many studies have shown thatthe intestine may play an important role in the development and progression of SIRS,sepsis and MODS. Thus, it is believed that the intestine is not only the target organ, butalso the promoter of MODS. For these reasons, gastrointestinal dysfunction following TBIis an increasingly recognized phenomenon and is currently an important research hotspotin the bio-medical field.There are a number of complications after TBI, one of which is gastrointestinaldysfunction. Gastrointestinal dysfunction is shown by the decrease of gastrointestinalmotility, intestinal nutrient absorption barriers and mucosal damage. Most previous studiesfocused on microstructure of gut mucosal, mucosal barriers fracture, mucosal blood flow,inflammatory mediators and cytokines. Ischemia or hypoperfusion of intestinal mucosaoften takes place after TBI in order to perfuse vital organs such as heart and brain.Unfortunately, the energy store of intestinal mucosa is too short to cope with ischemia orhypoperfusion and intestinal epithelial cells are very sensitive to ischemia and hypoxia.Once TBI happens, intestinal mucosa would be in the face of decreased blood supply andintestinal mucosal epithelial cells are under high pressure of oxidative stress.Mitochondria are important plant of oxidative phosphorylation and energy supply. Inaddition, they are also main place where generate reactive oxygen species (ROS) and thebulk of mitochondrial ROS is generated at the electron transport chain. For that reason,mitochondria are thought to be the primary target of oxidative damage and play animportant role in oxidative stress. However, it is regrettable that alterations of ratenterocyte mitochondrial function following traumatic brain injury have not been describedpreviously． Part I Two step preparation of intestinal epithelial cell mitochondriabased high quality in ratsObjective：To develop a two-step method to isolate high guality mitochondria fromrat intestinal epithelial cells. Methods：Firstly， enzyme digestion with collagenase andhyaluronidase was used to extract cells from rat small intestinalepithelial.scecondly,mitochondria were isolated from prepared rat small intestinal epitheliacells according to standard extraction procedures of mitochondria from cultured tissue cells.BCA protein assay kit was used to measure protein concentrations of preparedmitochondria. Pax-5,β-actin and Cox IV level in our prepared sample and referencesamples were evaluated by Western blotting to reveal their purity and contamination. Theintegrity, stability and biological activity of prepared mitochondria were also determined.Result：maximum mitochondrial protein concentration among20prepared samples is4.4516μg/μl, minimum value is2.2108μg/μl and mean value is3.1932μg/μl. The levels ofPax-5and β-actin are very low while the expression of Cox IV is high in preparedmitochondrial sample. Using cytochrome c oxidase as biomaker, the integrity ofmitochondria is still more than97%after an incubation time of two hours at0-4℃.Cytochrome c oxidase in prepared mitochondria can carry out oxidation reaction withNADH and malic acid, its activity can also be inhibited by KCN(1mmol/L). The ATPenzyme content of prepared mitochondria is not lower than content of the control group(mitochondria from the cultured IEC-6cell line). Conclusion：It is suggested that ourtwo-step method is simple and rapid for preparing high guality mitochondria from ratintestinal epithelial cells. The purity and protein of prepared mitochondria is high. Theintegrity, stability and biological activity of prepared mitochondria is good. Our preparedmitochondria are high guality for experimental studies of mitochondrial function.Part II Alterations in enterocyte mitochondrial respiratory functionand enzyme activities following brain injuryObjective：To determine the alterations in rat enterocyte mitochondrial respiratoryfunction and enzyme activities following traumatic brain injury (TBI). Methods：Fifty-six male SD rats were randomly divided into seven groups (8rats in each group)：controlgroup (rats with sham operation) and traumatic brain injury groups at6,12,24h, day2,day3and day7after operation．TBI models were induced by Feendy’s free-falling method.Mitochondrial respiratory function (respiratory control ratio and ADP/O ratio) wasmeasured with a Clark oxygen electrode. The activities of respiratory chain complex I-IVand related enzymes were determined by spectrophotometry．Results：Compared with thecontrol group, the mitochondrial respiratory control ratio (RCR) declined at6h andremained at a low level until day7after TBI (control,5.42±0.46;6h,5.20±0.18;12h,4.55±0.35;24h,3.75±0.22;2d,4.12±0.53;3d,3.45±0.41;7d,5.23±0.24; P＜0.01). Thevalue of phosphate-to-oxygen (P/O) significantly decreased at12,24h, day2and day3,respectively (12h,3.30±0.10;24h,2.61±0.21;2d,2.95±0.18;3d,2.76±0.09; P＜0.01)compared with the control group (3.46±0.12). Two troughs of mitochondrial respiratoryfunction were seen at24h and day3after TBI．The activities of mitochondrial complex I(6h:110±10,12h:115±12,24h:85±9,2d:80±15,3d:65±16; P＜0.01) and complex II(6h:105±8,12h:110±92,24h:80±10,2d:76±8,3d:68±12; P＜0.01) were increased at6h and12h following TBI, and then significantly decreased at24h, day2and day3,respectively．However, there were no differences in complex III and IV activities betweenthe control and TBI groups．Furthermore, pyruvate dehydrogenase (PDH) activity wassignificantly decreased at6h and continued up to7days after TBI compared with thecontrol group (6h:90±8,12h:85±10,24h:65±12,2d:60±9,3d:55±6,7d:88±11; P＜0.01). The changes in α-ketoglutaric dehydrogenase (KGDH) activity were similar to PDH,except that the decrease in KGDH activity began at12h after TBI (12h:90±12,24h:80±9,2d:76±15,3d:68±7,7d:90±13; P＜0.01). No significant change in malatedehydrogenase (MDH) activity was observed. Conclusion：Rat enterocyte mitochondrialrespiratory function and enzyme activities are inhibited following TBI. Mitochondrialdysfunction may play an important role in TBI-induced gastrointestinal dysfunction.Part III Alterations of enterocyte mitochondrial membrane potentialand permeability transition following traumatic brain injury in ratsObjective To investigate the alterations of rat enterocyte mitochondrial membrane potential and permeability transition following traumatic brain injury． MethodsNinety-six male SD rats were randomly divided into two groups (48rats each group)：control group (rats with sham operation) and traumatic brain injury group. Each group wasdivided into six subgroups(n=8) as6，12，24h，day2，day3and day7after operation．TBImodels were induced by Feendy’s free-falling. The apoptotic index of intestinal mucosalepithelial cells was determined by flow cytometry. Mitochondrial membrane potential andthe activities of permeability transition pore were measured by flow cytometry andspectrophotometry．Results Compared with control group，the apoptotic index ofintestinal mucosal epithelial cells was significantly increased at6h and kept a low leveluntil day7after operation (P＜0.05）．Enterocyte mitochondrial membrane potential wasdecreased (P＜0.05）and the activities of permeability transition pore were increased at3hand kept changes until day7after operation (P＜0.05）. Conclusion The alterations ofenterocyte mitochondrial membrane potential and permeability transition might contributeto the TBI-related increased early apoptosis rate of intestinal mucosal epithelial cellsfollowing traumatic brain injury in rats.