Effects Of Brain Iron On Secondary Brain Injury After Experimental TBI And Study Of ICP,CPP And P_btO₂Monitoring In Moderate And Severe TBI

Part ⅠEffects and mechanisms of brain iron on secondary brain injury afterexperimental traumatic brain injury （TBI）, and the protective effects of deferoxamineBackgroundTraumatic brain injury （TBI） is one of the critical problems affected the health andeconomics in the world. TBI is the first killer of the people younger than45years-old, andthe first reason of disability and mortality. Although the methods of treatment for TBI haveimproved greatly within several decades, the mobility and mortality are still very high.Usually, some of the neurological functions of patients suffered from TBI recovered quicklywithin months, but some impairment, such as the deteriorations of congnition and memory,and the brain atrophy would last for several years. It was indicated there would be somelasting mechanisms of secondary brain injury after TBI. However, some medicines, such asagents for anti-apoptosis, obstructing glutamate-induced neurotoxicity, anti-inflammation,and inhibiting oxidative stress were failed to take advantages for the recovery ofneurological functions. There were more than30drugs which were proved useful for TBI inanimals but were failed in the Ⅲ stage clinical trials. There would other factors whichinduced the secondary brain injury.It was reported that iron over-loading would induce neurotoxicity in experimentalintracerebral hemorrhage （ICH）, neurodegenerative diseases and experimental chronichydrocephalus. The abnormal iron accumulation played critical roles on the brain atrophyand neurological injuries in ICH and neurodegenerative diseases. In experimental ICH,administration of deferoxamine （DFO） could reduce the brain atrophy and neurologicalinjuries. In our previous study, injection of FeCl3to the ventricle could induce chronic hydrocephalus, and administration of DFO would decrease the the morbility of chronichydrocephalus. Which suggested that iron over-loading would induce neurotoxicity, andadministration of DFO can lower the possibility of brain atrophy and the neurologicalinjuries. Hemorrhage is also one of the most frequent pathological changes in TBI，especially in contusion and laceration of brain and intracerebral hematoma. Theneurotoxicity caused by iron over-loading might be happened in TBI.The purpose of this study was to disclose the effects of iron over-loading on TBI andthe therapeutic effects and the safety of DFO. Furthermore, the roles of iron metabolismproteins in the TBI were demonstrated.Materials and methods1. One hundred and four healthy adult male SD rats were divided randomly intothree groups: sham group, injury group, and DFO-treated group. and the last group dividedinto Early DFO-treated subgroups: E50subgroup （50mg/Kg）, E100subgroup （100mg/Kg）,E150subgroup （150mg/Kg）, and Delayed DFO-treated subgroups: D50subgroup（50mg/Kg）, D100subgroup （100mg/Kg）, and D150subgroup （150mg/Kg）. EarlyDFO-treated subgroups were administrated DFO2h after TBI，12h interval for28days,and delayed DFO-treated subgroups were administrated DFO3days after TBI，12h intervalfor25days. The sham group and injury group were administrated saline.2. Morris water maze was done at the endpoint of the experiment. The time andstrategy of searching platform were calculated to evaluate the ability of learning andmemory.3. The volume of brain lose was calculated by Coniglobus formula after the animalswere perfused by4%Paraformaldehyde and the brains were taken.4. The content of the peri-injury brain iron was calculated by spectrophotometer.5. The perl’s staining、HE staining and Nissl staining（brain tissue） were performed inthe tissues of peri-injury brain, femur, sternum, spleen, and liver.6. The expression of ferritin light chain （FL）, ferritin heavy chain （FH）, transferring（Tf） and TRPC6in the neurons, astrocytes, and oligodendroglias were detected withimmunofluorescence.7. The expression of Beclin1was detected with the immunohistochemisty. 8. The expressions of FL, FH, Tf and TRPC6were also detected by western blot.Results1. The volume of brain lose was （209.99±16.7mm³）. And it could be decreasedsignificantly （115.35±13.7mm³，P＜0.05） by early100mg/Kg DFO-treated.2. Time of platform searching was14.52±1.86s, and strategy of platform searchingwas3.63±0.52with Morris water maze in nomal rats. While time of platform searchingextended to110±47.34s （P＜0.05）, and strategy of platform searching lowered to2.13±0.64（P＜0.05） in injury group. The early （2h） DFO administration （100mg/Kg） coulddecrease the time of platform searching to36.15±26.63s （P＜0.05）, and improve thestrategy of platform searching to3.13±0.35（P＜0.05）.3. Compared with sham group, the content of brain iron in injury group increasesgreatly （P＜0.01）. However, the administration of DFO failed to decrease the brain iron （P＞0.05）.4. There were no statistical differences of sexuality, rectal temperature, glucose andhemoglobin in each group.5. Compared with sham group, the expressions of FL, FH, Tf and TRPC6wereup-regulated （P＜0.05） after TBI and could be down-regulated by early （2h after TBI）administration of100mg/Kg DFO. In injury group, there were no neurons and astrocytes inthe peri-injury zones after TBI, while many shrinkage neurons and astrocytes with TRPC6expressing were detected in group with early （2h after TBI） administration of100mg/KgDFO.6. The up-regulation of Beclin1was detected after injury. And the expression wasinhibited by early （2h after TBI） administration of100mg/Kg DFO.7. The best therapeutic effect of DFO was appeared in the group which the DFO wasadministrated in2hours after TBI with dosage of100mg/Kg.8. There were no affects of motality, hemoglobin and iron metabolism caused by DFOadministration after TBI.Conclusions1. Over-load brain iron plays the role of neurotoxicity at28thday after experimental TBI.2. DFO is the effective drug in experimental TBI, which can significantly reduce thevolume of brain defect, and significantly improve the ability of spatial learning and memoryin experimental animals.3. Taken the time-effect relationship into account, the best treatment time of DFO is2h after TBI, and as the dose-effect relationship, the optical dose is100mg/Kg.4. There is few intact neurons and astrocytes peri-injury after TBI,while DFOadministered at2h after TBI, more neurons and astrocytes are detected though the cellshrinkage, which suggests that the protective effect of neurons and astrocytes in“penumbra” at the cell level, and TRPC6may play important role in the procedure.5. The expression of Beclin1，the marker of autophagy, upregulates significantlyperi-injury after TBI,and down regulation after DFO administered, which suggests thatautophagy injury exists after TBI, and DFO administered can reduce autophagy, and ironmay play important role..6. In this experiment, the time of DFO administered extends to28d, and the doseincreases to150mg/Kg, which has no significant changes in mortality, hemoglobin and ironmetabolisms. In other words, DFO is a safe drug of TBI treatment.Part Ⅱ The application of ICP,CPP and P_btO₂monitoring in moderate andsevere traumatic brain injuryBackgroundThe characteristics of traumatic brain injury （TBI） are high morbility, disability andmortality. The rate of disability and mortality caused by TBI were not decreasedsignificantly for decades. Lacking of guidelines and controversies on the strategies of thetreatment of contusion and laceration of brain limited the treatment based on theexperiences of the individual neurosurgeons. So ICP, CPP and P_btO₂monitoring mightprovide more evidence for guiding the treatment of TBI.Materials and methodsSelected standard Patients of moderate and severe traumatic brain injuries with contusion and lacerationof brain and/or subdural hematoma, GCS from3to12were selected.Exclusion criteriaSome cases were eliminated from the experiment in order to keep the reliability,including （1） patients with instability vital signs and exhaustion of brainstem functions;（2）hemorrhagic diathesis or hemorrhagic diathesis before TBI;（3） combining with chronicdisease of important organ or fail to tolerance operation;（4） pure epidural hematoma anddiffuse axonal injury with CT scan.Clinical dataFrom May2009to Mar2011,43moderate to severe TBI cases from our hospital,Fuling center hospital, Sanxia center hospital and people’s hospital of Banan district wereperformed ICP and P_btO₂monitoring. Within the43patients, there were31male and12female, aged from14to76years old.There were4cases with GCS12,3cases with GCS9,14cases with GCS8,1case with GCS7,5cases with GCS6,6cases with GCS5,2caseswith GCS4, and8cases with GCS3.When discharged from hospital, there were19caseswith GOS5,9cases with GOS4,1case with GOS3,4cases with GOS2, and10caseswith GOS1.The ratio of good outcome （GOS from4to5） was65.1%, the motality was23.3%as discharged, and30.2%after6months.Results1. The blood glucose increased in both the group of good outcome and the group ofbad outcome. Compared with the patients with good outcome, the concentration of bloodglucose was higher in patients with bad outcome at pre-operation,1day and5days afteroperation.2. The volumes of hemorrhage were usually huge in the bad outcome group,4casesabove100ml,6from70ml to100ml. While in the good outcome group, there were mostfrom30ml to50ml.3. The values of ICP were detected pre-operation in the bad outcome group higher thanthese in the good group, and decreased after operation, but still increased significant abovethe normal level, and then increased quickly. While the values of ICP were still detectedincreasing pre-operation, but decreased significant after operation, lower to normal lever, though certain increased at3d.Compared with the bad outcome group, the values of ICPdecreasing were significant at pre-operation,1d and5d（P＜0.01）,even at3d（P＜0.05）.4. The values of CPP were little lower compared with the normal lever in the goodoutcome group and increased soon after operation. These were decreased in the badoutcome group. Compared with in the bad outcome group, those in the good outcome groupincreased significantly （P＜0.01）.5. The values of P_btO₂were little lower compared with the normal lever in the goodoutcome group and then recovered to normal after operation. However, these in the badgroup lowered significantly pre-operation. Eventhough, this condition couldn’t be improvedafter operation. Compared with the bad outcome group, the levers increased significantly atpre-operation,1d,5d（P＜0.01） amd3d（P＜0.05）.6. Standard large trauma craniotomy was performed in the experiment, the lever of ICPdecreased significantly both in the bad outcome group （29.45±3.30mmHg） and the goodoutcome group （31.22±2.73mmHg）. However, it failed to improve the lever of CPP andP_btO₂in the bad outcome group, while it did in the good outcome group. Compared with thebad outcome group, Standard large trauma craniotomy was performed to improve CPP andP_btO₂（P＜0.05）, but there was no difference in decreasing ICP in both groups.Conclusions1. It is very necessary to monitor ICP and CPP after moderate to severe TBI, which candecide the case to operate or not early, and the secondary hemorrhage and outcome can bealso judged early.2. The monitoring of P_btO₂is a safe and effective method in neurosurgery intensivecare unit, and more sensitive and specific than the monitoring of ICP and CPP, which isearlier in reflecting the changes of patient’s condition than the later. It is important tohypoxia, homeostasis and energy metabolism of brain with monitoring of P_btO₂, and theoutcome also can be judged early.3. Multi-monitoring of ICP,CPP and P_btO₂should be performed in neurosurgeryintensive care unit, which can provide more information to neurosurgeon early, and makethe treatment reliable, and can be as the guideline for the clinical medication and outcome.4. Only taken effect of decompression into account, the standard large trauma craniotomy is supported to be the preference approach in our experiment, which not onlycan decrease ICP significantly, but also improve CPP and P_btO₂.