Combination of Neutrophil-to-Lymphocyte Ratio and Admission Glasgow Coma Scale Score Is Independent Predictor of Clinical Outcome in Diffuse Axonal Injury.

BACKGROUND: The neutrophil-to-lymphocyte ratio (NLR) is an independent predictor of clinical outcome of different diseases, such as acute ischemic stroke, intracerebral hemorrhage, malignant tumor, and traumatic brain injury. However, the prognostic value of NLR plus admission Glasgow Coma Scale score (NLR-GCS) is still unclear in patients with diffuse axonal injury (DAI). Therefore this study assessed the relationship between the NLR-GCS and 6-month outcome of DAI patients. METHODS: The clinical characteristics of DAI patients admitted to our department between January 2014 and January 2020 were retrospectively analyzed. The candidate risk factors were screened by using univariate analysis, and the independence of resultant risk factors was evaluated by the binary logistic regression analysis and least absolute shrinkage and selection operator regression analysis. The predictive value of NLR-GCS in an unfavorable outcome was assessed by the receiver operating characteristics curve analysis. RESULTS: A total of 93 DAI patients were included. Binary logistic regression analysis and least absolute shrinkage and selection operator regression analysis showed the level of NLR on admission was an independent risk factor of unfavorable outcomes in DAI patients. The ROC curve analysis showed that the predictive capacity of the combination of NLR and admission GCS score and combination of NLR and coma duration outperformed NLR, admission GCS score, and coma duration alone. CONCLUSIONS: The higher NLR level on admission is independently associated with unfavorable outcomes of DAI patients at 6 months. Furthermore, the combination of NLR and admission GCS score provides the superior predictive capacity to either NLR or GCS alone.

Serum levels of tau protein increase according to the severity of the injury in DAI rat model.

Traumatic brain injury (TBI) in the form of diffuse axonal injury (DAI) is difficult to diagnose in the early phase of the injury. Early diagnosis of DAI may provide opportunity for developing treatment and management strategies. Tau protein has been demonstrated to increase in the early phase of TBI with high diagnostic accuracy in patients with DAI. We tested the biological plausibility of tau protein using a rat DAI model by evaluating the association between serum tau levels and the severity of brain injury. DAI was induced in animals using the Marmarou model. After a survival of 60 minutes, rats were anesthetized and sacrificed after obtaining blood samples (5ml) from the heart. Eighteen rats were employed in the present study and were randomly subjected to sham-operated control (n=4), mild DAI (n=7), and severe DAI (n=7). Of seven severe DAI rats, two rats that had focal injury caused by skull fracture were excluded in the measurement of tau protein level. The serum levels of tau protein in the rat DAI model were found to increase significantly and consistently according to the severity of the injury. Rats with DAI showed significantly higher serum levels of tau protein compared to sham rats; the severe DAI rats had higher levels of tau than moderate DAI and sham rats (sham vs. mild,  P=0.02; mild vs. severe,  P=0.02). In conclusion, serum tau protein levels may be useful as a biomarker for diagnosing and estimating the severity of DAI in the early phase.

Neurofilament light as a biomarker in traumatic brain injury.

OBJECTIVE: To determine whether serum neurofilament light (NfL) correlates with CSF NfL, traumatic brain injury (TBI) diagnosis, injury severity, brain volume, and diffusion tensor imaging (DTI) estimates of traumatic axonal injury (TAI). METHODS: Participants were prospectively enrolled in Sweden and the United States between 2011 and 2019. The Swedish cohort included 45 hockey players with acute concussion sampled at 6 days, 31 with repetitive concussion with persistent postconcussive symptoms (PCS) assessed with paired CSF and serum (median 1.3 years after concussion), 28 preseason controls, and 14 nonathletic controls. Our second cohort included 230 clinic-based participants (162 with TBI and 68 controls). Patients with TBI also underwent serum, functional outcome, and imaging assessments at 30 (n = 30), 90 (n = 48), and 180 (n = 59) days and 1 (n = 84), 2 (n = 57), 3 (n = 46), 4 (n = 38), and 5 (n = 29) years after injury. RESULTS: In athletes with paired specimens, CSF NfL and serum NfL were correlated (r = 0.71, p < 0.0001). CSF and serum NfL distinguished players with PCS >1 year from PCS ≤1 year (area under the receiver operating characteristic curve [AUROC] 0.81 and 0.80). The AUROC for PCS >1 year vs preseason controls was 0.97. In the clinic-based cohort, NfL at enrollment distinguished patients with mild from those with moderate and severe TBI (p < 0.001 and p = 0.048). Serum NfL decreased over the course of 5 years (ß = -0.09 log pg/mL, p < 0.0001) but remained significantly elevated compared to controls. Serum NfL correlated with measures of functional outcome, MRI brain atrophy, and DTI estimates of TAI. CONCLUSIONS: Serum NfL shows promise as a biomarker for acute and repetitive sports-related concussion and patients with subacute and chronic TBI. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that increased concentrations of NfL distinguish patients with TBI from controls.

Time course and diagnostic utility of NfL, tau, GFAP, and UCH-L1 in subacute and chronic TBI.

OBJECTIVE: To determine whether neurofilament light (NfL), glial fibrillary acidic protein (GFAP), tau, and ubiquitin C-terminal hydrolase-L1 (UCH-L1) measured in serum relate to traumatic brain injury (TBI) diagnosis, injury severity, brain volume, and diffusion tensor imaging (DTI) measures of traumatic axonal injury (TAI) in patients with TBI. METHODS: Patients with TBI (n = 162) and controls (n = 68) were prospectively enrolled between 2011 and 2019. Patients with TBI also underwent serum, functional outcome, and imaging assessments at 30 (n = 30), 90 (n = 48), and 180 (n = 59) days, and 1 (n = 84), 2 (n = 57), 3 (n = 46), 4 (n = 38), and 5 (n = 29) years after injury. RESULTS: At enrollment, patients with TBI had increased serum NfL compared to controls (p < 0.0001). Serum NfL decreased over the course of 5 years but remained significantly elevated compared to controls. Serum NfL at 30 days distinguished patients with mild, moderate, and severe TBI from controls with an area under the receiver-operating characteristic curve (AUROC) of 0.84, 0.92, and 0.92, respectively. At enrollment, serum GFAP was elevated in patients with TBI compared to controls (p < 0.001). GFAP showed a biphasic release in serum, with levels decreasing during the first 6 months of injury but increasing over the subsequent study visits. The highest AUROC for GFAP was measured at 30 days, distinguishing patients with moderate and severe TBI from controls (both 0.89). Serum tau and UCH-L1 showed weak associations with TBI severity and neuroimaging measures. Longitudinally, serum NfL was the only biomarker that was associated with the likely rate of MRI brain atrophy and DTI measures of progression of TAI. CONCLUSIONS: Serum NfL shows greater diagnostic and prognostic utility than GFAP, tau, and UCH-L1 for subacute and chronic TBI. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that serum NfL distinguishes patients with mild TBI from healthy controls.

Tau protein as a diagnostic marker for diffuse axonal injury.

BACKGROUND: Diffuse axonal injury (DAI) is difficult to identify in the early phase of traumatic brain injury (TBI) using common diagnostic methods. Tau protein is localized specifically in nerve axons. We hypothesized that serum level of tau can be a useful biomarker to diagnose DAI in the early phase of TBI. METHODS & RESULTS: We measured serum tau levels in 40 TBI patients who were suspected of DAI within 6 hours after TBI to evaluate the accuracy of the tau level as a diagnostic marker for DAI. Diagnosis of DAI was confirmed according to magnetic resonance imaging (MRI) findings. The serum tau level in the DAI group (n = 13) was significantly higher than that in the non-DAI group (n = 27) (DAI vs. non-DAI, 25.3 [0 to 99.1] pg/mL vs. 0 [0 to 44.4] pg/mL, P = 0.03)). A receiver-operating characteristic curve to evaluate the diagnostic ability of serum tau level within 6 hours for DAI showed an area under the curve of 0.690 with 74.1% for sensitivity and 69.2% for specificity. Serum tau level was not significantly higher in unfavorable outcome group (Glasgow Outcome scale [GOS] score = 1-3 at hospital discharge) compared with favorable outcome group (GOS score = 4-5) (P = 0.19). CONCLUSIONS: Tau protein may be a useful biomarker for diagnosis of DAI in the early phase of TBI.

Integrated Proteomics and Metabolomic Analyses of Plasma Injury Biomarkers in a Serious Brain Trauma Model in Rats.

Diffuse axonal injury (DAI) is a prevalent and serious brain injury with significant morbidity and disability. However, the underlying pathogenesis of DAI remains largely unclear, and there are still no objective laboratory-based tests available for clinicians to make an early diagnosis of DAI. An integrated analysis of metabolomic data and proteomic data may be useful to identify all of the molecular mechanisms of DAI and novel potential biomarkers. Therefore, we established a rat model of DAI, and applied an integrated UPLC-Q-TOF/MS-based metabolomics and isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis to obtain unbiased profiling data. Differential analysis identified 34 metabolites and 43 proteins in rat plasma of the injury group. Two metabolites (acetone and 4-Hydroxybenzaldehyde) and two proteins (Alpha-1-antiproteinase and Alpha-1-acid glycoprotein) were identified as potential biomarkers for DAI, and all may play important roles in the pathogenesis of DAI. Our study demonstrated the feasibility of integrated metabolomics and proteomics method to uncover the underlying molecular mechanisms of DAI, and may help provide clinicians with some novel diagnostic biomarkers and therapeutic targets.

Identification of plasma biomarkers for diffuse axonal injury in rats by iTRAQ-coupled LC-MS/MS and bioinformatics analysis.

DAI is a serious and complex brain injury associated with significant morbidity and mortality. The lack of reliable objective diagnostic modalities for DAI delays administration of therapeutic interventions. Hence, identifying reliable biomarkers is urgently needed to enable early DAI diagnosis in the clinic. Herein, we established a rat model of DAI and applied an isobaric tags for a relative and absolute quantification (iTRAQ) coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) proteomics approach to screen differentially expressed plasma proteins associated with DAI. A total of 58 proteins were found to be significantly modulated in blood plasma samples of the injury group in at least one time point compared to controls. Bioinformatics analysis of the differentially expressed proteins revealed that the pathogenesis of axonal injury underlying DAI is multi-stage biological process involved. Two significantly changed proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hemopexin (Hpx), were identified as potential diagnostic biomarkers for DAI, and were successfully confirmed by further western blot analysis. This proteomic profiling study not only identified novel plasma biomarkers that may facilitate the development of clinically diagnostic for DAI, but also provided enhanced understanding of the molecular mechanisms underlying DAI.

Integration of 1H NMR- and UPLC-Q-TOF/MS-based plasma metabonomics study to identify diffuse axonal injury biomarkers in rat.

Diffuse axonal injury (DAI) is much common during traumatic brain injury (TBI) and is associated with high mortality and poor neurological outcome. Although many studies have been examined, there are still no reliable objective diagnostic modalities available for clinicians to make an early diagnosis of DAI. Therefore, we established a rat model of DAI, applying an integrated 1H NMR- and UPLC-Q-TOF/MS-based metabonomics approach to identify differentially changed metabolites in plasma. A total of twenty-two metabolites in the injury group were identified as differentially changed. Among them, four metabolites, glutamine, pyruvate, glycerol and phosphocholine, were identified as candidate biomarkers based on their high fold-changes and biological functions, and may play important roles in axonal injury progression in DAI. Our study not only identified several novel biomarkers that improved our understanding of the metabolic events underlying DAI, but also may provide some potential novel therapeutic targets for preventing axonal injury in DAI.

[Application of Susceptibility Weighted Imaging in the Identification of Hemorrhagic Diffuse Axonal Injury].

OBJECTIVES: To explore the application value of susceptibility weighted imaging （SWI） in the forensic identification of hemorrhagic diffuse axonal injury （DAI）. METHODS: The forensic identification materials of 20 DAI cases were analyzed retrospectively. The sequence image materials of T1WI, T2WI, diffusion weighted imaging （DWI）, fluid attenuated inversion recovery （FLAIR） and susceptibility weighted imaging （SWI） were analyzed statistically. RESULTS: DAI related hemorrhage focus commonly located in the superficial area of brain. The detection rate of hemorrhage focus by SWI was the highest, which showing significant difference （P<0.05） compared with other sequence images. CONCLUSIONS: The SWI plays an important role in the forensic identification of DAI.

Serum Neurofilament Light Protein as a Marker for Diffuse Axonal Injury: Results from a Case Series Study.

Diffuse axonal injury (DAI) is an important cause of morbidity in patients with traumatic brain injury (TBI). There is currently no simple and reliable technique for early identification of patients with DAI, or to prognosticate long-term outcome in this patient group. In the present study, we examined acute serum concentrations of neurofilament light (NFL) in nine patients with severe TBI and DAI using a novel ultrasensitive single molecule array (Simoa) assay. The relationships between the NFL concentrations and MRI in the acute stage as well as clinical outcome and magnetic resonance diffusion tensor imaging (MR-DTI) parameters at 12 months were analyzed. We found that the mean NFL concentrations among the patients displayed a 30-fold increase compared with controls, and that NFL completely discriminated between the patients and controls. We also found a relationship between serum NFL and MR-DTI parameters, with higher NFL concentrations in patients with higher trace (R2 = 0.79) and lower fractional anisotropy (FA) (R 2 = 0.83). These results suggest that serum NFL may be a valuable blood biomarker for TBI, reflecting the severity of DAI.

[Proteomic Analysis of Rat Brain Stem with DAI by MALDI-TOF-MS].

OBJECTIVE: To establish a diagnostic model for diffuse axonal injury (DAI) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To screen the proteins or peptides associated with DAI for providing the biomarkers with theoretic foundation. METHODS: Fifteen male Sprague-Dawley rats were randomly divided into DAI group (n = 10) and control group (n = 5). The protein or peptide expression profiles of rat brain stem were detected by MALDI-TOF-MS. ClinProTools 2.2 software was used to find specific peaks, and a diagnostic model was established by the genetic algorithm. RESULTS: There were significant differences in 61 peaks of DAI group (P < 0.05), 9 peaks were down-regulated and 52 up-regulated. The diagnostic model was established based on 5 different peaks. The specificity and sensitivity of cross validation was 96.14% and 95.98%; while the specificity and sensitivity of blind validation showed was 73.33% and 70.00%, respectively. CONCLUSION: A specific and sensitive diagnostic model of DAI can be established by MALDI-TOF-MS to provide a potential value for determining DAI in forensic practice.

What are the progesterone-induced changes of the outcome and the serum markers of injury, oxidant activity and inflammation in diffuse axonal injury patients?

To permit appropriate targeted therapy, the present clinical study was aimed to investigate the effects of progesterone on the outcome and the serum markers of injury, oxidant activity and inflammation in diffuse axonal injury (DAI). Forty-eight male DAI patients were divided into two groups (control and progesterone). Progesterone group received progesterone in dose of 1mg/kg per 12h for five days. The outcome was investigated using Extended Glasgow Outcome Scale (GOS-E) and functional independence measure (FIM). The markers of inflammation [interleukin-1ß (IL-1ß), IL-6, transforming growth factor-ß1 (TGF-ß1)], injury (brain protein of S-100B), and oxidant activity [malondialdehyde (MDA)] were evaluated in the serum of the patients. Higher GOS-E and FIM scores were observed in progesterone group at the six-month follow-up (P<0.05 and P<0.01, respectively). Meanwhile, a reduction in the serum levels of IL-1ß, MDA and S-100B was noticed in progesterone group 24h after injury (P<0.05, P<0.001 and P<0.05, respectively), and there was an increase in serum levels of IL-6 and TGF-ß1 (P<0.01 and P<0.05, respectively). Also, lower levels of MDA and S-100B, and higher levels of TGF-ß1 were observed in progesterone group six days after injury (P<0.05). According to these findings, progesterone may improve the outcome in DAI patients probably through modulation in the levels of cytokines, and reduction in the injury and oxidant activity.

Serum SNTF Increases in Concussed Professional Ice Hockey Players and Relates to the Severity of Postconcussion Symptoms.

Biomarkers for diffuse axonal injury could have utilities for the acute diagnosis and clinical care of concussion, including those related to sports. The calpain-derived αII-spectrin N-terminal fragment (SNTF) accumulates in axons after traumatic injury and increases in human blood after mild traumatic brain injury (mTBI) in relation to white matter abnormalities and persistent cognitive dysfunction. However, SNTF has never been evaluated as a biomarker for sports-related concussion. Here, we conducted longitudinal analysis of serum SNTF in professional ice hockey players, 28 of whom had a concussion, along with 45 players evaluated during the preseason, 17 of whom were also tested after a concussion-free training game. Compared with preseason levels, serum SNTF increased at 1 h after concussion and remained significantly elevated from 12 h to 6 days, before declining to preseason baseline. In contrast, serum SNTF levels were unchanged after training. In 8 players, postconcussion symptoms resolved within a few days, and in these cases serum SNTF levels were at baseline. On the other hand, for the 20 players withheld from play for 6 days or longer, serum SNTF levels rose from 1 h to 6 days postconcussion, and at 12-36 h differed significantly from the less-severe concussions (p=0.004). Serum SNTF exhibited diagnostic accuracy for concussion, especially so with delayed return to play (area under the curve=0.87). Multi-variate analyses of serum SNTF and tau improved the diagnostic accuracy, the relationship with the delay in return to play, and the temporal window beyond tau alone. These results provide evidence that blood SNTF, a biomarker for axonal injury after mTBI, may be useful for diagnosis and prognosis of sports-related concussion, as well as for guiding neurobiologically informed decisions on return to play.

Evaluation of TGF ß1, IL-8 and nitric oxide in the serum of diffuse axonal injury patients and its association with clinical status and outcome.

AIM: The aim was to evaluate the level of interleukin 8 (IL-8), transforming growth factor ß1 (TGF ß1) and Nitric oxide (NO) in diffuse axonal injury (DAI) and its association to the outcome and clinical status. MATERIAL AND METHODS: This cross-sectional study was conducted on 20 patients with DAI and 20 patients with multiple traumas without head injury and 20 healthy subjects as controls. Blood levels of IL-8, TGF ß1 and nitric oxide in the 1st, 2nd, 3rd and 7th days of injury were measured. Glasgow coma scale (GCS) of patients was recorded. The patients' outcome was evaluated by Glasgow Outcome Scale (GOS). RESULTS: The level of TGF ß1 was increasing during the admission and had the maximum level at the 7th day. In the DAI group, there was significant correlation between GOS score and serum IL-8 at 7th day of admission (r=-0.68, p= 0.002). In this group the GCS was found to be significantly correlated with the IL-8 concentration at 7th day of admission (p= 0.026, r=-0.55). CONCLUSION: IL-8 has negative correlation with GCS and GOS. TGF ß1 could protect the brain from cytotoxics, hypoxia and acidosis so its level comes down in brain injuries as a result of its overuse.

Dyskalaemia following diffuse axonal injury: case report and review of the literature.

Traumatic brain injury, and its management, commonly causes derangements in potassium balance. There are a number of recognised causative factors including head trauma, hypothermia and iatrogenic factors such as pharmacological agents and permissive cooling. We describe a case of a 19-year-old man with a severe traumatic brain injury. In a 36-h period, his intracranial pressure increased despite maximal medical therapy and he developed refractory hypokalaemia. Immediately following a decompressive craniectomy, the patient was noted to be profoundly hyperkalaemic; this led to the development of ventricular tachycardia and cardiac arrest, from which the patient did not recover. The effects of brain injury on potassium balance are not well appreciated; the effect of decompressive craniectomy on potassium (K(+)) balance has not been described previously. We would like to emphasise the potential effect of diffuse axonal injury, a severe form of brain injury and decompressive craniectomy on potassium balance.

Cerebral endothelial injury in severe head injury: the significance of measurements of serum thrombomodulin and the von Willebrand factor.

Thrombomodulin (TM), which is located in the surface of the endothelium in the arteries, veins, and capillaries of major organs such as the brain, lungs, liver, kidneys, skeletal muscles, and gastrointestinal tract, is one of several indicators of endothelial injury. Von Willebrand factor (vWf), which is synthesized by endothelial cells, is also an endothelial specific glycoprotein. The serum level of vWf increases in response to various stimuli without endothelial injury. An elevated serum level of vWf may suggest endothelial activation in severe head injury. We hypothesize that the degree of cerebral endothelial activation or injury depends on the type of head injury and that measuring the TM and vWf is useful for predicting delayed traumatic intracerebral hematoma (DTICH), produced by weakness of the vessel wall, occuring either as a direct or indirect effect of head injury. The values of vWf in focal brain injury (ranging from 332.5 +/- 52.8% to 361.7 +/- 86.2%) were significantly higher than those in diffuse axonal injury from 2 h to 7 days after the injury occurred (ranging from 201.6 +/- 59.5% to 242.5 +/- 51.7%). The serum level of TM in focal brain injury (ranging from 3.84 +/- 1.54 to 4.12 +/- 1.46 U/mL) was higher than that in diffuse axonal injury (ranging from 2.96 +/- 0.63 to 3.67 +/- 1.70 U/mL), but these differences were not statistically significant. In patients with DTICH, TM was significantly higher than in patients without DTICH (p < 0.01). The results of our study demonstrate that the degree of endothelial activation in focal brain injury was significantly higher than in diffuse brain injury. In addition, the serum level of TM in patients with DTICH was significantly higher than in patients without DTICH. These findings suggest that cerebral tissue injury is often accompanied by cerebral endothelial activation, and that these two phenomena should be distinguished from each other. The levels of serum TM and vWf appear to be good indicators of the cerebral endothelial injury and of endothelial activation in severe head injury.

Thyroid hormones in comatose patients with traumatic brain injury.

The objective was to study if thyroid hormones, cortisol, prolactin and brain injury marker levels were changed in traumatic brain injury (TBI) patients with changing levels of consciousness. We estimated the above named parameters in 32 patients (27 men and 5 women aged 11-55). Admission Glasgow Coma Score was < 8. Follow-up period--30 days. The length of coma was 3 to 25 days. There were significant decreases in TSH, TBG, FT3 and F_levels (p < 0.05, for each) and a T3 increase (as compared to very low preceding values) on day 1 before emergence from coma and considerable post-coma increase in TBG, FT3, TSH and F levels (p < 0.001 each) on days 1-3 in patients with diffuse axonal injury (DAI). In patients with contusions and epidural and subdural hematomas (CH) T3 and T4 levels continued to fall until 4-6 postcoma days. TSH values significantly increased up to average normal ranges (p < 0.05) on days "-" 2 and "-" 1 before emergence from coma and remained so. Significantly lower levels of TSH, F and PRL were found in patients with CH in the mostly remote period (on days "-" 12-"-" 8) before emergence from coma in comparison with DAI patients. In blood the following correlations of examined parameters were established: between NSE and T3 (r = -0.39), NSE and FT3 (r = -0.59), TNF alpha and TBG (r = -0.64), TNF alpha and T3 (r = -0.3) and S-100 and T3 (r = -0.3) (p < 0.05, for each). The results obtained confirmed a low T3 syndrome in comatose TBI patients. We demonstrated an objective and informative interdependence: the turning-point moment of the emergence from coma was accompanied by significant changes of examined hormone levels and brain injury marker levels. The results may serve as a base for recommending monitoring FT3 and T3 levels simultaneously with that of other injury markers and adequate T3 replacement therapy in the early posttraumatic period.