Expression And Prognostic Value Of The Inflammasome Proteins In Traumatic Brain Injury

Background:Traumatic brain injury (TBI) constitutes a major health and socioeconomic problem throughout the world, which is the leading cause of mortality and disability among young individuals. As the development of diagnosis and management of TBI, the mortality of patients has reduced, whereas the rate of disability is still high. In the meantime, the global incidence of TBI is rising sharply, mainly in low and middle-income countries. Early diagnosis of severity in patients with TBI is crucial for making management decisions and predicting patient’s outcome. Currently, the Glasgow Coma Scale (GCS) and CT scan are the best available clinical predictor of injury severity and functional outcome. However, they are subjective and insufficiently sensitive for the demand of modern medicine. In recent years there have been some new developed methods to assess the severity and outcome of TBI, including neuroimaging techniques, such as diffusion tension imaging (DTI), magnetic resonance spectrum (MRS) and functional MRI (fMRI), electrophysiology techniques, such as electroencephalography (EEG), somato-sensitive evoked potentials (SSEPs) and event related potentials (ERPs), as well as the use of biochemical markers for TBI. It is the focus of research and social responsibility of modern medicine to screen and find a series of accurate and practical prognostic methods and build a comprehensive evaluation system of TBI. Inflammasomes are molecular complexes, which generally have three main components:the sensor NOD like receptors (NLRs), the enzyme caspase1and the adaptor protein apoptosis-associated speck-like protein containing a caspase-activating recruitment domain protein (ASC). Several members of the NLR family proteins, such as NLRP1and NLRP3can assemble the inflammasome. Through the sensing of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) on infectious agents or disease-associated host molecules by NLRs, inflammasomes trigger the maturation and release of proinflammatory cytokines such as interleukin-lb (IL-1b) and IL-18to engage innate immune inflammatory response, as well as pyroptotic cell death. In the CNS, inflammasomes exist in microglia, astrocytes, and neurons, which can induce inflammatory response at the local site of infection or injury (that is, proliferation and activation of glia and infiltration of blood leukocytes.) The robust and poorly controlled neuroinflammation response may cause secondary brain injury. IL-1b, the main product of inflammasome signaling pathways, is a relatively definite marker of the neuroinflammation and brain injury. It had been found that IL-1b is elevated in the blood or Cerebrospinal Fluid (CSF) of TBI patients and related with the injury severity and clinical outcome. As the upstream regulatory proteins of IL-lb, inflammasomes play crucial role in the production of IL-lb and the initiation of neuroinflammation, which involve the pathogenesis and pathophysiology of neurological diseases, including meningitis, stroke and Alzheimer’s disease. It has also been found that NLRP1and NLRP3inflammasomes were associated with TBI. However, the expression and role of inflammasomes after brain trauma are still not clear. Therefore, our first purpose in this research is to build TBI model in mice and observe the expression of NLRP1and NLRP3inflammasomes in cerebral cortex after TBI. Second, we performe immunological analysis of inflammasome proteins NLRP1and NLRP3in the CSF samples from TBI patients and investigate the value of inflammasome proteins as biomarkers to assesse TBI severity and functional outcome.Methods:1, The expression of inflammasome proteins NLRP1and NLRP3in cerebral cortex after TBI in mice model.1.1, The Establishment of TBI model in mice. Male, pathogen-free C57BL/6mice (10～12weeks, weighing28～40g) were used in this study. The mice were randomly divided into6groups:normal group (n=5), sham-operated group (n=5), four TBI groups (n=8) at6h,1day,3day, and7day. The modified Shohami model was used to induce experimental TBI in mice. Specifically, used self-made device to set a metal rod (weight333g)2.5cm above the mouse head and allowed a free fall of the rod hits the exposed skull. Then, assessed initial neurological impairment by Neurological Severity Score (NSS)1h after TBI, which is the best indicator for the severity of TBI. The severe injury mice whit NSS7-8were included. They were sacrificed6h,1d,3d and7d after trauma and the pericontusional tissues were collected and frozen until use.1.2, Western blot analysis for inflammasome proteins in the brain tissue of mice. Extracted total proteins from the pericontusional brain tissues of experimental mice. Protein concentrations were estimated by Bradford method. Then perform sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) based on the protein concentrations. For imunobotting, the following antibodies were used:anti-b-actin, anti-NLRP1antibody, anti-NLRP3antidoby and horseradish peroxidase (HRP)-linked secondary antibody. The results were analyzed by chemiluminescence (ECL) detection system and AlphaEase FC softwere. b-actin was used as loading control.1.3, Statistical analysis. All data in this study were analyzed in SPSS13.0softwere. Single factor factor variance analysis was used to analyze the data among multi-groups. Statistic difference was considered to be significant if P<0.05.2, The prognostic value of inflammasome proteins NLRP1and NLRP3in patients with TBI.2.1, Enroll and management of TBI petients. Twenty patients with severe TBI (sTBI) admitted to our hospital between May and July in2013were recruited with the following inclusion criteria:hospital admission within72hours after the accident; severe brain trauma (GCS≤8); age>12years. The exclusion criteria were no CSF sample taken; no informed consent; obvious CNS infection; serious extracranial trauma. Seven moderate TBI patients (8<GCS<13) and eight non-trauma patients without CNS infection were recuited as control. TBI Patients had an immediate CT scan taken after resuscitation. The CT findings were classified into six categories according to the Marshall’s criteria. In category Ⅰ, no visible intracranial pathology is present; in Ⅱ, cisterns are present with midline shift0to5mm or lesion densities with a volume not exceeding25ml; in category Ⅲ, cisterns are compressed or absent, with a midline shift0to5mm, no high or mixed density lesions≥25ml; category Ⅳ is as Ⅲ but with a midline shif t≥5mm; category Ⅴ is presence of any mass lesion that is surgically evacuated; category Ⅵ denotes the presence of any mass lesion≥25ml that is not surgically evacuated. Outcome was assessed using the Glasgow Outcome Scale (GOS) at6months post injury. GOS were dichotomized into favorable (GOS-4or5) and unfavorable (GOS-3,2, or1) outcome. The following data were recorded:age, GCS, pupillary reactions, CT classification, and GOS.2.2, Enzyme-linked immuno sorbent assay (ELISA) for inflammasome proteins in the CSF of TBI patients. CSF samples from sTBI patients were taken at24h and72h after injury through ventriculostomy or lumbar puncture. The mean concentration of inflammasome proteins were collected for statistics. The control CSF samples were taken for once by lumbar puncture. NLRP1and NLRP3were analyzed according to the manufacturer’s instructions by using commercially available ELISA kits.2.3, Statistical analysis. Statistical comparisons were made using nonparametric tests. Mann-Whitney Test was used to analyze the data between two groups. The significance among multi-group were detected with Kruskal-Wallis test, and LSD’s post-hoc tests controlled for multiple comparisons. Correlations between expression levels of NLRP3and patients age, GCS, CT classification, and GOS were analyzed by Spearman rank correlation, linear regression was utilized to analyze the Correlations between expression levels of NLRP3and patients prognosis assessd by GOS. Statistic difference was considered to be significant if P<0.05.Results:1, The expression of inflammasome proteins NLRP1and NLRP3in cerebral cortex after TBI in mice mode.1.1, The Establishment of TBI model in mice. After brain trauma induced by a weight of333g and a distance of2.5cm,24mice in totol of32experienced obvious neurological disorder with the mean NSS (7.5±0.3). In the meanwhile, Cortical contusion could been saw in the brain of mice. In the other hand, we found3mice with NSS below7and5mice died within1hour post injury. The mortality was15.6%and the total model success rate is75%.1.2, The expression of inflammasome proteins in cerebral cortex in mice. The results of western blot showed that inflammasome protains NLRP1and NLRP3exist in the brain of mice. The NLRP3protein level was rise as early as6hour after injury then gradually decreased. There was a statistically significant difference between TBI groups in each time point post injury and the control group (P<0.05). However, no significant changes in the level of NALP1were observed at any time point examined.2, The prognostic value of inflammasome proteins NLRP1and NLRP3in patients with TBI.2.1, The expression of Inflammasome Proteins in the CSF of patients with TBI. Patients with sTBI exhibited significantly higher CSF levels of NLRP3([33.8±11.6] ng/ml) compared to moderate TBI patients ([11.4±2.9] ng/ml, Z=-3.873, P<0.001) or non-trauma controls ([1.1±0.3] ng/ml, Z=-4.068, P<0.001). There was a significant difference among the sTBI patients with abnormal pupillary reactions in both sides (A), one side (B), and normal pupillary reactions (C)(x2=7.484, P=0.024). LSD’s post-hoc tests showed that difference between (A) and (C) as well as (B) and (C) were significant, indicating that NLRP3were significantly higher in sTBI patients with mydriasis([42.3±4.6] ng/ml) than that with nomal pupillary reactions ([31.1±12.8] ng/ml, P=0.024). Spearman rank correlation showed that expression of NLRP3correlated significantly with CT classification (r=0.601, P=0.005), but not age and GCS. unfavorable outcomes, including death, vegetative state and severe disability (P=0.048).2.2, The relationship of the CSF level of Inflammasome Proteins NLRP3in patients with sTBI and their prognosis. NLRP3were significantly higher in sTBI patients with unfavorable outcomes, including death, vegetative state and severe disability ([42.8±14.4] ng/ml) than that with favorable ontcome ([29.9±7.9] ng/ml, Z=-1.98, P=0.048). Linear regression analysis showed that expression of NLRP3correlated significantly with GOS at six months post injury (r=0.665, P=0.001).Conclusions:1, A weight of333g and a distance of2.5cm was found suitable for establishment of TBI model in mice whit good repeatability. Inflammasome protains NLRP1and NLRP3both express in the brain of mice. The level of NLRP3wes significantly improved after brain injury, indicating that NLRP3inflammasome involve the neurological inflammatory responses induced by TBI, especially in the early postinjury period.2, CSF level of inflammasome protein NLRP1in patients with sTBI was greatly low and showed no significant difference to non-trauma controls. NLRP1has poor sensitivity to be the biomarker of TBI. On the contrary, NLRP3in CSF reflects the extend of brain injury, which is potential biomarker to assess TBI severity and functional outcome, serving as adjunct to clinical predictor.