The Expression And Role Of Serum GFAP And UCH-L1 As Biomarkers After Traumatic Brain Injury

Background Traumatic brain injury(TBI)is a serious medical and socioeconomic problem for its high incidence,the devastating effect of disease and the huge mortality and morbidity.TBI induces complex pathological responses in the patient brain including ischemia,edema,inflammation,and impaired blood-brain barrier(BBB),which lead to degeneration or death of glial cells and neurons.Following the death of brain cells,a wide range of brain-specific proteins are released into interstitial fluid or cerebrospinal fluid and eventually enter the blood via the impaired BBB or other pathways,which could be used as biomarkers of injury.Glial fibrillary acidic protein(GFAP)and ubiquitin carboxyl-terminal hydrolase L1(UCH-L1)have been studied as novel markers of glial and neuronal cell damage in the recent years.It was reported that serum GFAP and UCH-L1 were increased after TBI and could reflect the severity of brain injury and disease progression.Further,the reliability,sensitivity,and specificity of GFAP and UCH-L1 were said to be better than the traditionally studied biomarkers such as neuron specific enolase(NSE),or S100 B protein.Thus,in our current research,we investigated the blood levels of GFAP and UCH-L1 after TBI and their utilities in diagnosis and prognosis of brain injury.Additionally,with the use of hypothermia therapy,we assessed the value of serum GFAP and UCH-L1 to monitor the treatment effect.Our study were divided into FOUR sections: 1)the establishment of fluid percussion injury(PFI)model in rats and treatment of hypothermia or normothermia;2)the ability of serum GFAP and UCH-L1 to monitor the treatment effect of hypothermia;3)the utility of early serum GFAP and UCH-L1 to predict outcome of brain-injured patients;4)the preliminary use of serum GFAP and UCH-L1 in the LTH-1 trial.Section 1 The establishment of fluid percussion injury model in rats and treatment of hypothermia or normothermia Objective To establish stable and repetitive rat models of FPI and hypothermia or normothermia therapy and assess the quality of animal models.Methods 72 adult male SD rats weighted from 348 to 385 g were randomly assigned to four groups: injury with normothermia,injury plus hypothermia,sham injury and sham injury plus hypothermia(n=18 for each).FPI was induced with a specific instrument under the pressure of 1.8-2.1 ATM.Moderate hypothermia(33.0±0.5?)was achieved via ice bath cooling,maintained for 4 h and discontinued with slow rewarming in 2 h.Rats in the groups of sham injury underwent all surgical,anesthetic and hypothermia produces except for PFI.At 24 h post-injury,6 rats in each group were sacrificed and gross appearance of the brain(n=3)and histopathological observation with HE staining(n=3)were performed.12 rats in each group were used for beam walking test(n=6)and Morris water maze test(n=6).Results The body temperature of rats in the hypothermia groups decreased rapidly to the target(33.0±0.5?).Rats from two sham injury groups showed no obvious pathological change in the brain.However,in the normothermia group,the most severe injury with subdural hematoma and subarachnoid hemorrhage in the brain was observed,which was less in the rats receiving hypothermia.For the neurobehavioral tests,the injured rats from hypothermia group had better performance than that from normothermia group.Conclusions The models of FPI and treatment with hypothermia or normothermia were successfully established.The histopathological and neurobehavioral findings supported the protective effect of hypothermia.Section 2 The ability of serum GFAP and UCH-L1 to monitor treatment effect of hypothermiaObjective To assess the effect of hypothermia on serum levels of GFAP and UCH-L1 at 6h?24h and the numbers of intact astrocytes and degenerating neurons at 24 h post injury.Methods 64 male rats were assigned into four groups and treated as described in Section 1.Whole blood was obtained at baseline(prior to injury),6h and 24 h post injury and assessed for the levels of serum GFAP and UCH-L1.Brain sections were collected at 24 h post injury and stained with GFAP immunohistochemistry or Fluoro-Jade B histofluorescence.Correlations between serum levels of GFAP and UCH-L1 and the number of intact astrocytes and degenerating neurons respectively were evaluated with Pearson correlation test.Results Hypothermia therapy caused no change in the serum levels of GFAP and UCH-L1 in the groups of sham injury.For the two injury groups,hypothermia reduced the serum GFAP level at 6h(P=0.0017)and at 24h(P=0.043)post injury and serum UCH-L1 level at 6h(P=0.0013)but not at 24h(P=0.162).Histological findings showed a reduction of injured astrocytes and degenerating neurons in the cortex and the CA2/3 regions of the hippocampus after injury.Further analysis revealed strong correlations between serum levels of GFAP at 6h?24h and number of intact astrocytes(6h time-point: R=-0.616,P=0.0002;24h time-point: R=-0.313,P=0.0132).A correlation between serum level of UCH-L1 at 6h post injury and numbers of degenerating neurons was observed(R=0.365,P=0.0399),but not for the serum UCH-L1 at 24 h post injury.Conclusions Hypothermia therapy could reduce serum levels of GFAP and UCH-L1 and injury of glial and neuronal cells.At earlier time points serum biomarkers may be used to detect the effect of hypothermia.Section 3 The utility of early serum GFAP and UCH-L1 to predict outcome of brain-injured patientsObjective To evaluate the predictive utility of early(4h post injury)serum levels of GFAP and UCH-L1 in 6-month outcome of traumatically brain-injured patients.Methods From March 2012 to September 2014,adult severe TBI patients admitted to Shanghai Renji Hospital were recruited in this study.Inclusion criteria were older than 18 years without gender restriction,admitted to hospital within 4h post injury,and with a GCS score of 3-8.In addition,a total of 135 healthy volunteers with no prior neurological diseases were enrolled as the control group.Serum specimens from patients and healthy subjects were collected on admission and the levels of GFAP and UCH-L1 were measured.Patient outcome was assessed at 6 months post injury with the GOS scale and further grouped into death versus survival and unfavorable versus favorable.The accuracy of serum GFAP and UCH-L1 for outcome prediction was assessed with ROC analysis.Results A total of 67 patients were enrolled during the study period.The mean time from injury to admission was 2.6 h with the median admission GCS score of 6.Compared with healthy subjects,patients with severe TBI had increased serum levels of GFAP and UCH-L1(P<0.001 for both).Results of ROC analysis showed that both serum GFAP and UCH-L1 could predict neurological outcome at 6 months.The AUC values for GFAP were 0.761 for death and 0.823 for unfavorable outcome and for UCH-L1 were 0.722 for death and 0.728 for unfavorable outcome,respectively.The predictive ability of biomarkers was better than clinical variables such as age,GCS score,and pupil reactions.Conclusions Serum GFAP and UCH-L1 on admission were increased in patients with severe TBI and were predictive of neurological outcome at 6 months.Section 4 The preliminary use of serum GFAP and UCH-L1 in the LTH-1 trialObjective To evaluate the feasibility of serum GFAP and UCH-L1 as surrogate markers in the LTH-1 trial.Methods The LTH-1 trial is a prospective,nationwide multicenter,randomized,controlled clinical trial to examine the efficacy of safety of long-term mild hypothermia in adult patients after severe TBI.The current study is the single-center results of prospectively collected data from 14 patients admitted to Shanghai Renji Hospital.Serum specimens were collected on admission and over the first 6 days and assessed for levels of GFAP and UCH-L1.Patient outcome was determined at 6 months post injury.Results From November 12,2013 to September 2,2015,a total of 14 patients were enrolled and completed the 6-month follow-up.The age,gender,admission GCS score,cause of injury,and pupil reactions demonstrated no statistical differences between long-term hypothermia group and control group.During the 6-day period,the serum levels of both GFAP and UCH-L1 decreased in both groups.Patients in long-term hypothermia group showed more reductions in serum levels of GFAP and UCH-L1 compared to that of control group.The 6-month follow-up results demonstrated a trend of hypothermia to reduce the risk of death and unfavorable outcome;however,with a very limited sample size,the differences did not reach statistical significance.Conclusions Hypothermia could reduce the blood levels of GFAP and UCH-L1.Biomarkers may be helpful in TBI trials as surrogate biomarkers.