iTRAQ-Based Quantitative Proteomics Reveals the New Evidence Base for Traumatic Brain Injury Treated with Targeted Temperature Management
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This study aimed to investigate the effects of targeted temperature management (TTM) modulation on traumatic brain injury (TBI) and the involved mechanisms using quantitative proteomics technology. SH-SY5Y and HT-22 cells were subjected to moderate stretch injury using the cell injury controller (CIC), followed by incubation at TTM (mild hypothermia, 32°C), or normothermia (37°C). The real-time morphological changes, cell cycle phase distribution, death, and cell viability were evaluated. Moderate TBI was produced by the controlled cortical impactor (CCI), and the effects of TTM on the neurological damage, neurodegeneration, cerebrovascular histopathology, and behavioral outcome were determined in vivo. Results showed that TTM treatment prevented TBI-induced neuronal necrosis in the brain, achieved a substantial reduction in neuronal death both in vitro and in vivo, reduced cortical lesion volume and neuronal loss, attenuated cerebrovascular histopathological damage, brain edema, and improved behavioral outcome. Using an iTRAQ proteomics approach, proteins that were significantly associated with TTM in experimental TBI were identified. Importantly, changes in four candidate molecules (plasminogen [PLG], antithrombin III [AT III], fibrinogen gamma chain [FGG], transthyretin [TTR]) were verified using TBI rat brain tissues and TBI human cerebrospinal fluid (CSF) samples. This study is one of the first to investigate the neuroprotective effects of TTM on the proteome of human and experimental models of TBI, providing an overall landscape of the TBI brain proteome and a scientific foundation for further assessment of candidate molecules associated with TTM for the promotion of reparative strategies post-TBI.
KeywordsTargeted temperature management Traumatic brain injury Proteomics Isobaric tags for relative and absolute quantitation Cerebrospinal fluid
Traumatic brain injury (TBI) remains a major health-care problem worldwide, which is accompanied with high fatality and disability for children and adults . In China, the mortality rate of severe TBI patients is 21.8% according to the Chinese Head Trauma Databank (CHTDB) , but these studies of morbidity, mortality, and disability do not take the large number of mild TBI events into consideration for which patients are not hospitalized but only suffer from non-specific symptoms such as headache . For the common and potential devastating effects of TBI, improved early diagnostic methods and treatment strategies to prevent the diverse and complex nature of the pathological processes activated by TBI are critical in the clinical setting, posing major challenges for neurological research.
In 2011, five international critical care societies recommended replacing the term “therapeutic hypothermia” with “targeted temperature management (TTM)” to emphasize the importance of defining a complete temperature profile . Growing evidence suggests that there are multiple pathways in the cascade of brain injury after TBI, while TTM acts via several mechanisms that have a protective effect against brain injury by promoting neuronal sprouting , alleviating inflammation , inhibiting necroptosis [7, 8], reducing brain oxygen consumption, excitotoxicity, blood–brain barrier (BBB) disruption and chemical-induced seizures, and inhibiting oxidative stress and cell death [9, 10, 11].
There have been various proposals of the mechanisms by which TTM attenuates TBI, including reduction of cerebral metabolic rate of oxygen, cerebral glucose demand, and excitotoxicity in the acute phase, inhibition of inflammation, BBB disruption in the sub-acute phase, and modulation of axonal injury in the chronic phase post-TBI , but the translation of these positive data into clinical applications has proven to be controversial. Several large multi-center clinical trials did not show a significant cerebroprotective effect [13, 14], most likely due to the poor understanding of TBI, which reflects the complex, multifactorial nature of cellular responses. Therefore, there is an urgent need for consistent support by key physiological parameters to improve the selection of the candidates during TTM .
Recently, neuroproteomics has become a prominent method to assess post-TBI conditions, and plays an increasing role in TBI biomarker discovery . Furthermore, there has been a rapid development of quantitative protein expression technologies, including iTRAQ combined with liquid chromatography (LC) and tandem mass spectrometry (MS/MS), which can construct data-driven systems biology models of TBI by high-throughput data sets, thereby increasing the possibility of identifying less abundant proteins . In the present study, we applied iTRAQ followed by LTQ Orbitrap Velos protein identification and bioinformatics analysis to determine the changes of differentially expressed proteins, identify potential biomarkers associated with TTM, and elucidate the possible role of TTM in the molecular pathways following TBI.
Materials and Methods
Cell Culture, Stretch Injury, and Temperature Modulation
Human neuroblastoma cells (SH-SY5Y) and mouse hippocampal cells (HT-22) were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Cells were randomly divided into 4 groups: 1) injury + normothermia; 2) injury + TTM; 3) uninjured + normothermia; and 4) uninjured + TTM. Cells were seeded at 1 × 105/ml in Bioflex culture plates (FlexCell International, Burlington, NC, USA). For the injury groups, cells were subjected to moderate stretch injury with the cell injury controller II (CIC; Custom Design & Fabrication, Richmond, VA, USA), which uses a controlled pulse of compressed nitrogen gas to transiently deform the silastic membrane and to achieve a predetermined degree of stretch . The parameters for the moderate stretch injury were set at 28 psi with a 50-ms duration, resulting in 10.2-psi peak pressure at the well. Uninjured cells served as controls. Temperature was changed immediately following injury by placing the Bioflex plates in temperature-controlled incubators that were maintained at 32°C (TTM) or 37°C (normothermia) for 6 h. The cells were then routinely harvested. The temperature of 32°C was selected based on previous studies [18, 19, 20].
Flow Cytometry and Digital Holographic Microscopy (DHM) Assays
Cells were collected by trypsinization, washed with ice-cold phosphate-buffered saline (PBS), and centrifuged at 200 g for 5 min at 4°C. After vortexing, cells were re-suspended in 70% ethanol, and then stored at 4°C overnight. Cell suspensions were centrifuged at 200 g for 5 min, washed once in ice-cold PBS, and then incubated for 1 h at room temperature in 1 ml of 50 μg/ml of propidium iodine (PI) solution containing 250 μg/ml of DNase-free RNase. The DNA content of cells was immediately measured using a FACSCalibur (BD Biosciences, San Jose, CA, USA) and the proportion of cells within the sub-G1, G0/G1, S, and G2/M phases were analyzed with the FCS Express software (Version 3; De Novo Software, Los Angeles, CA, USA).
The digital holographic microscope (HoloMonitor M4; Phase Holographic Imaging, Lund, Sweden), placed in an incubator, was used to record 3-dimensional information of injured cells after exposure to hypothermia or normothermia. Digital photos were taken at 12-min intervals for 6 h and time-lapse movies were constructed.
Cell Viability and Cell Death Assays
For cell viability assay, cells were harvested with 0.05% trypsin and re-suspended in PBS. The cell suspensions were assayed using 0.4% trypan blue (Sigma-Aldrich, St. Louis, MO, USA) with the automated Countess System (Life Technologies, Waltham, MA, USA), according to the manufacturer’s instructions.
For cell death assay, PI (Sigma-Aldrich) was added at a final concentration of 1 μg/ml and cells were incubated for 15 min at room temperature. On each slide, 5 fields were chosen randomly, and the mean number of PI-positive cells was calculated. Additionally, Annexin V/PI staining was performed using the Annexin V/PI Detection Kit (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, the cell suspension was incubated with 5 μl Annexin V–fluorescein isothiocyanate and 10 μl PI for 30 min at room temperature in the dark, followed by cytometric analysis. All experiments were performed in triplicate.
Study Design of Animal Models
A total of 160 adult male Sprague Dawley rats (mean body weight 300–350 g) were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China) and housed at 20°C to 25°C and 50 ± 5% humidity with ad libitum access to food and water and a 12-h light/dark cycle. All surgical and behavioral procedures were done in accordance with the rules and regulations of the Institutional Animal Care and Use Committee of Logistics University of PAP, and complied with the Guide for the Care and Use of Laboratory Animals. Cortical contusion impact (CCI)-induced rate mortality of moderate TBI is usually 10% or less, whereas the mortality rate of this experiment was about 4%. All the deaths of all groups were filled up. Animal groups included: 1) sham + TTM; 2) sham + normothermia; 3) CCI + TTM; and 4) CCI + normothermia (n = 40 per group). The study design is presented in Fig. S1. The study consisted of 2 stages: 1) 6 rats/group, regional cerebral blood flow (rCBF) test, modified neurological severity score (mNSS; for 3 days), and magnetic resonance imaging (MRI; for 4 days) were performed 1 d after CCI, and MWM was performed 11 d after CCI (for continuous 7 d); and 2) 1 day after CCI, the rats were sacrificed for the measurement of the following parameters, with 4 rats/group: lesion volume, brain water content, BBB integrity, histology, Fluoro-Jade C (FJC) staining, immunofluorescence, and Western blotting. In addition, 6 rats per group were subjected to the iTRAQ assay.
Cortical Contusion Impact Injury
The experimental TBI method used in this study was performed using an electric cortical contusion impactor device (eCCI-6.3; Custom Design & Fabrication), as described previously with minor modifications . This model is a predominately focal TBI model . Rats were anesthetized with a nitrous oxide/oxygen mixture (70%/30%) containing 1.4% isoflurane delivered by nose cone. A midline scalp incision was made to expose the skull under sterile conditions. A craniotomy (4 mm in diameter) was performed over the right parietal–temporal cortex at 4.0 mm posterior to the bregma and 3.0 mm lateral from the midline. A 3-mm flat-tipped impactor was placed on the dural surface at a 20° angle, and a brain injury of moderate severity was performed with an impact velocity of 5 m/s, impact depth of 2 mm, and impact dwell time of 200 ms. After impact, the bone was replaced and fixed using bone wax, and the incision was closed with absorbable sutures. Sham-operated animals underwent all surgical craniotomy procedures except for the CCI injury, in order to serve as uninjured controls.
Temperature manipulation was initiated 30 min after CCI or sham surgery. Core body temperature was continuously monitored with rectal temperature probes. TTM was induced with a hypothermia therapy apparatus (HEMA, Shenzhen, China) by applying an ice blanket over the dorsum of prone rats until a body temperature of 32 ± 0.5°C was achieved. The temperature of 32°C for TTM was chosen according to previous studies in rats [23, 24]. A similar degree of hypothermia is recommended by the American Association of Neurological Surgeons Guidelines for the management of severe TBI . After 6 h of TTM, the rats were allowed to gradually rewarm back to their baseline temperature (37 ± 0.5°C) during a 1-h period using heating lamps. Normothermic animals were maintained at normal baseline temperature following CCI induction. Sham-operated animals received normothermia treatment identical to the normothermia CCI animals.
T2-weighted MRI scanning was performed using a clinical 3.0-Tesla MRI scanner (Siemens, Erlanger, Germany) equipped with a rat RF coil (50 mm; Chengguang Medical Technologies, Shanghai, China), which was used to visualize lesions following CCI. Briefly, at 1 to 4 days post-CCI, rats (n = 6 in each group) were anesthetized as described above and placed in a prone position with their heads in the rat RF coil. T2-weighted images were acquired using a 2-dimensional rapid acquisition with relaxation enhancement (RARE) sequence with the following parameters: repetition time (TR) = 4000 ms; echo time (TE) = 75 ms; (RARE) factor = 10; field of view (FOV)= 19.2 × 19.2 mm; matrix size = 320 × 320; number of slices = 20; slice thickness = 2 mm.
The rCBF in the cerebral cortex of animals was measured by laser Doppler flowmetry (Perimed Periflux 5010; Perimed, Järfälla, Sweden). After 24 h of CCI, rats (n = 6 in each group) were anesthetized as described above and laid in a prone position. The laser probe was placed over the skull in the right middle cerebral artery distribution (centered at 4.0 mm posterior to the bregma and 3.0 mm lateral from the midline). The region to be scanned was 4 × 4 mm.
Lesion Volume Assessment
After 24 h of CCI, animals (n = 4 in each group) were anesthetized as described above and sacrificed by cervical dislocation. Brains were removed for the following lesion volume assessment. For each animal, six 500-μm-thick coronal sections that spanned across a total distance of 3 mm were obtained and stained with cresyl violet (Sigma-Aldrich) for analysis. Contralateral (left hemisphere) and ipsilateral (right hemisphere) substructure volumes were calculated based on stereological methods, as previously described . Volume of tissue loss for each substructure was calculated by subtracting the ipsilateral volume from the contralateral volume.
Determination of Brain Water Content
Cerebral edema was evaluated by water content of the brain tissue at 24 h after CCI (n = 4 in each group), using wet-to-dry weight ratio . Following sacrifice, the brain parenchyma (after discarding the olfactory bulb and cerebellum) was removed and divided into 2 cerebral hemispheres along the midline: ipsilateral (right) and contralateral (left). The cerebrum samples were immediately weighted to obtain the wet weight (WW), and then dried for 24 h at 110°C using a drying oven to obtain the dry weight (DW). The water content was calculated based on the following formula: water content (%) = (WW – DW) / WW × 100%.
Evaluation of BBB Integrity
The integrity of the BBB was determined by assessing the Evans blue (EB) dye extravasation, as described previously . At 23 h post-CCI, rats were anesthetized with an overdose of isoflurane (5%), then injected with 2% solution of EB dye (Sigma-Aldrich) at a dose of 4 ml/kg via the caudal vein, and then allowed to circulate for 1 h prior to sacrifice. Rats were transcardially perfused with saline through the left ventricle until colorless perfusion was achieved. Following sacrifice, the brain was removed, weighted, homogenized in formamide (3 μl/mg), and incubated at room temperature for 48 h. After centrifugation, the supernatant was collected and the optical density was measured at 625 nm, which was used to determine the relative amount of EB dye.
Specimen Preparation and Histological Analysis
After 24 h of CCI, animals (n = 4 in each group) were anesthetized as described above. Cardiac regions exposing by thoracotomy, and left ventricular cannulation of the ascending aorta was performed. Rats were perfused with 200 ml buffered saline solution to flush the blood followed by 700 ml of 4% paraformaldehyde in a (pH 7.2–7.4) for perfusion fixation. Following complete perfusion, rats were decapitated and whole brains were extracted. Whole-brain tissues were fixed overnight in fresh 4% paraformaldehyde at 4°C and then embedded in paraffin. Sections were dried overnight at room temperature, stained using hematoxylin and eosin, and observed by bright-field microscopy (Leica Microsystems, Erlangen, Germany).
FJC (Chemicon, Temecula, CA, USA) was used as a marker to specially stain degenerating neurons following TBI, as previously described . Briefly, the slides were rinsed in distilled water for 2 min, incubated in descending concentrations of ethanol (5 min in 100%, and 2 min each in 70% and 30%), and then immersed in 0.06% KMnO4 for 10 min. After rinsing in water, sections were exposed to 0.001% FJC in the dark for 30 min, and 4’,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Subsequently, slides were rinsed 3 times in water and dried at 37°C. The mean number of FJC-positive cells was counted by a blinded observer in 3 coronal sections in 5 random, non-repeated visual fields covering the injured cortex site using the inverted Leica DMI 4000B fluorescence microscope.
Immunofluorescence Histochemistry and Confocal Imaging
Specimens were dewaxed and hydrated, treated with 2% rabbit serum (Epitomics, Burlingame, CA, USA), and incubated with the primary antibody overnight at 4°C. Then, slides were washed 3 times with a washing buffer for 5 min each and stained with the secondary antibody for 30 min in darkness at 37°C. After washing with 10 μM PBS 3 times for a total of 15 min, DAPI was used to stain the nuclei. Finally, sections were sealed and imaged on a TCS SP8 laser scanning confocol microscope (Leica Microsystems). In this regard, the following primary antibodies produced in rabbit and obtained from Abcam (Cambridge, MA, USA), including anti-antithrombin III (AT III, ab126598), anti-plasminogen (Plg, ab154560), anti-fibrinogen γ chain (Fgg, ab96532), and anti-transthyretin (TTR, ab78548), were diluted 1:100 in PBS. The secondary antibody was fluorescein isothiocyanate-labeled goat antirabbit antibody (1:400; KPL Gaithersburg, MD, USA).
mNSS was used to evaluate neurological function deficits in rats (n = 20 in each group) at 2 days after CCI, which is a composite of motor, sensory, balance, and reflex tests. The maximum points are 6, 2, 6, and 4, respectively. Thus, a higher score indicates more severe neurological dysfunction (13–18 designates severe, 7–12 moderate, and 1–6 mild) .
Morris Water Maze
Starting on post-injury day (PID) 11, the Morris water maze (MWM) test was performed by a blinded observer to test the spatial learning ability of rats, as previously described . Briefly, the rats (n = 20 in each group) were placed in a circular tank containing opaque water and were trained to find a submerged platform. Acquisition training consisted of a total of 28 trials, given 4 trials per day for consecutive days [postinjury day (PID) 11–17]. For each trial, the rat was randomly placed at 1 of 4 starting positions. The escape latency time to reach the platform and the swimming path were recorded by the automatic tracking system (Zhenghua Biologic Apparatus, Huaibei, China).
Protein Digestion and Isobaric Tag Labeling
The brain tissues just covering the injury or the corresponding part of the sham rats were snap frozen in liquid nitrogen and homogenized in urea solution (8 M urea, 50 mM ammonium bicarbonate, and 5 mM iodoacetamide). Equal amount of protein (n = 6 in each group) were pooled. Samples were diluted 4 times before digested with trypsin at a 1:50 ratio overnight. The digested peptides were filtered with a microcon (Cat. 42410; Millipore, Billerica, MA, USA), desalted, and vacuum dried. Dried peptides were labeled with 4-plex iTRAQ reagent kits (Applied Biosystems, Foster City, CA, USA) in triethylammonium bicarbonate buffer (pH 8.5) at room temperature for 1 h. Samples of sham + 37°C, sham + 32°C, CCI + 37°C, and CCI + 32°C were labeled as 114, 115, 116, and 117 iTRAQ tags, respectively. The labeled peptides were desalted by stagetips and then dried .
The labeled peptides were dissolved in phase A (3% acetonitrile (ACN), pH was adjusted to 10.0 by NH3•H2O) and mixed equally. Then the mixed peptides were loaded onto an L-3000 HPLC System (Rigol, Beijing, China) and separated with a Durashell RP column (3 μm, 700 Å, 250 mm × 4.6 mm i.d.; Agela, Wilmington, DE, USA) using the following gradient: 1% to 6% B (98% ACN, pH 10.0), 8 min; 6% to 21% B, 40 min; 21% to 35% B, 28 min; 35% to 95% B, 8 min; 95% B, 6 min. The flow rate was 800 μl/min and 35 fractions were collected. All the samples were dried under vacuum.
The separated peptides were re-suspended in 1% acetonitrile, 0.5 (v/v) formic acid (FA) and loaded onto a nanoflow HPLC system (NannoAcquity LC; Waters, Milford, MA, USA) equipped with a 15-cm C18-reversed phase column (75 mm inner diameter, 3 μm, Dr Maisch GmbH Reprosil-Pur 120 C18-AQ resin) with a 60 min gradient from 2% to 60% acetonitrile in 0.5% acetic acid at a flow of 250 nl/min. The separated peptides were injected into an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, San Jose, CA, USA) in data-dependent mode. The MS parameters were as follows: ion transfer tube was at 250°C and the voltage of the ion source at 1.8 kV. The MS1 scan range was m/z 300 to 1600 with resolution of 30,000. The top-20 intense peaks charged from 2 to 7 were acquired with high energy collision dissociation (HCD). Normalized collision energy was 45%. The automatic gain control (AGC) was 3 × 104 with a maximum ion accumulation time of 250 ms and a signal intensity threshold of 500. Dynamic exclusion time was 30 s. HCD fragmentation ions were detected in the orbitrap at a resolution setting of 7.5 × 103.
Biological Function Analysis
The raw files were analyzed with MaxQuant (version 22.214.171.124) and MS/MS spectra were searched against the Swiss-Prot Rattus norvegicus reference protein database Uniprot (release 2015_08, 7928 query number) and quantified by iTRAQ 4-plex report ions. The target–decoy based strategy was used to control the peptide and protein false discovery rate (FDR). Fully tryptic digestion and up to 2 missed cleavages were selected. Precursor ion peaks were searched with an initial mass tolerance of 20 ppm and fragmented ion peaks were searched with an initial mass tolerance of 0.1 Da. The fixed modifications of carbamidomethylation (+57.021465), iTRAQ 4-plex (K), and iTRAQ 4-plex (N-term) were specified and the variable modification was methionine oxidation (+15.99492). The FDR of peptides and proteins was set to 0.01.
Gene Ontology (GO) annotation analysis was performed using DAVID (https://david.ncifcrf.gov/) [33, 34], and the Rattus norvegicus was selected as the background and species. The significance was determined with slight modifications as recommended by the authors of DAVID according to the Benajmini correction of p < 0.05. Ingenuity Pathway Analysis software (IPA, version 14400082; Ingenuity Systems, Redwood City, CA, USA) was used to assign the signaling pathway analysis for these altering proteins. The protein–protein interaction (PPI) network was generated using the CluePedia plugin of the Cytoscape version 3.2.1.
Human Subject Characteristics and Cerebrospinal Fluid Collection
Twenty people with TBI (7 mild TBI: Glasgow Coma Scale [GCS] score 13–15; 5 moderate TBI: GCS score 9–12; 8 severe TBI: GCS score 3–8) were prospectively enrolled at the neuro-intensive care unit of the Neurosurgery and Neurology Hospital of PAP, Tianjin, China between 2015 and 2016. The study was approved by the Institutional Review Board at the Logistics University of PAP (PJHEC-2016-A8) and informed consent was obtained for collection of the cerebrospinal fluid (CSF). The inclusion criteria were age between 18 and 65 years, a history of confirmed closed TBI within 6 h before admission, and abnormal images confirmed by computed tomography scan of the brain, including skull fracture, contusion, or hematoma. Patients with severely combined injury, pre-existing medical conditions, for instance cardiac arrest or penetrating TBI, and pregnant or breast-feeding women were excluded.
All treatment procedures were standardized, step-wise, based on current guidelines [20, 25], for instance head elevation, ventilation control, sedation and analgesia, hyperosmolar therapy, and extraventricular drainage (EVD) catheter for intracranial pressure monitoring and management, as previously described , to maintain internal environment and intracranial pressure. In this study, patients with severe TBI who were randomized to the TTM group were cooled to 32°C with the use of cooling blankets and maintained for 24 h. Thereafter, the patients were rewarmed slowly at no greater than 0.5°C every 4 h to prevent rebound intracranial hypertension. Patients with severe TBI randomized to normothermia and other patients with moderate or mild TBI were maintained at 37°C during the entire study period.
For patients with moderate or severe TBI, CSF was obtained directly from EVD catheters, which were placed as a standard of care. For patients with mild TBI, CSF was collected via lumbar puncture. CSF was acquired at separate time points: mild TBI (PID 1), moderate TBI (PID 1), and severe TBI (PID 1 and 3). A volume of 2 ml per sample was collected, centrifuged at 4°C for 10 min, and stored at –80°C until analysis.
Western Blotting and Enzyme-Linked Immunosorbent Assay
CSF samples (10 μl) were separated by 10% sodium dodecyl sulfate polyacrylamid gel electrophoresis and transferred to nitrocellulose (NC) membranes. The membranes were blocked by 10% non-fat milk and incubated with the primary antibodies (PLG, AT III, FGG, and TTR; Abcam, Cambridge, MA, USA) at a dilution of 1:1000 overnight at 4°C. After washing 3 times with PBS/0.1% Tween-20, membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibody (IgG at 1:10,000 dilution; KPL). The membranes were washed as mentioned above.
Enzyme-Linked Immunosorbent Assays
CSF samples were diluted and measured by enzyme-linked immunosorbent assay (ELISA) kits [Cloud-Clone (USCN), Houston, TX, USA] for quantitative analysis of AT III (SEC313Hu), PLG (SEB236Hu), FGG (SEC477Hu), and TTR (SEA726Hu) levels according to the manufacturer’s protocols. Briefly, the plates were coated with detecting antibody and washed with a buffered solution. Samples and known control solutions were then applied to individual wells. Absorbance was quantified using a microplate spectrophotometer (Multiskan GO; Thermo Scientific), and concentrations of samples were calculated based on standard curves generated from known solutions.
Small Interfering RNA and Cell Viability
For the knockdown of PLG, AT III, and F GG genes, PLG (103869), AT III (118569), and FGG (121886) sequences were obtained from Thermo Fisher. Knockdown was then performed in HT-22 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. The cell-damage assessment and TTM assay were conducted at 24 h after cell transfection. The effectiveness of gene knockdown was verified by Western blot. Cell viability was measured 12 h after cell damage, using the CellTiter 96 AQueous One solution Cell Proliferation Assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Each experiment was repeated 3 times. Non-targeting small interfering RNA (siRNA) (5’-UUCUCCGAACGUGUCACGU-3’) was used for the controls in the siRNA knock-down experiment.
The Gaussian distribution for the quantitative proteomics data was fit and the SD was calculated. Differences of 2 times the SD between the sham + 32°C and sham + 37°C groups were set as thresholds for significantly differentially expressed proteins (p < 0.05) . Other data are presented as the mean ± SD. SPSS 13.0 (IBM, Armonk, NY, USA) was used for analysis. Differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls or Tukey’s post-hoc analysis. All tests were 2-tailed and a p-value < 0.05 was considered to be statistically significant.
TTM Overcomes TBI-Induced Necrosis of Neurons in vitro
We also examined the effect of TTM on neuronal cell death and viability, as assessed by PI positivity (Fig. 1d) and cell counting (Fig. 1e) assays, respectively. Strikingly, stretch injury induced extensive SH-SY5Y cell death (p < 0.01) and resulted in a reduction in the number of viable cells (p < 0.001) compared with uninjured control cells, whereas the sensitivity of cells to stretch injury exposed to TTM was significantly lower than that of 37°C, as evidenced by PI positivity (p < 0.01) and cell viability (p < 0.001), as well as HT-22 cells (data not shown).
Cell-cycle phase distribution was then detected by flow cytometry to monitor the efficacy of TTM treatment. As demonstrated in Figure 1f, exposure to 37°C caused a significant accumulation of the injured SH-SY5Y cells in sub-G1 phase (69.26%) compared with that of the uninjured cells (0.45%). However, TTM treatment considerably decreased sub-G1 populations (from 69.26% to 4.17%). Further investigation into the TBI-induced cell death was performed through necrosis that occurs in the early phase, or apoptosis that occurs in the late phase after injury. Necrosis and apoptosis of SH-SY5Y and HT-22 cells postinjury were quantified using Annexin V/PI (Fig. 1g). To our surprise, stretch injury can induce necrosis in the early phase of SH-SY5Y cells as detected by PI, in which the percentage of necrotic cells increased significantly (from 0.00 ± 0.00% to 21.48 ± 0.55%). After TTM treatment, cells showed a sharp reduction in proportion of necrosis (9.15 ± 1.30%) compared with that of normothermia (p < 0.05; Fig. 1g, left). Similar results were obtained with HT-22 cells (Fig. 1g, right). Altogether, these in vitro results suggest that TTM overcomes TBI-induced damage of neurons.
TTM Decreases Neurological Damage and Attenuates Neurodegeneration Post-TBI in vivo
TTM Attenuates Cerebrovascular Histopathology and Improves Behavioral Outcome after TBI in vivo
To test whether TTM improved functional performance of rats after TBI, the mNSS and MWM tests were performed in the different groups. Significantly lower mNSS scores were found to be markedly improved in TTM animals compared with those in the normothermia groups on days 1, 2, and 3 after CCI (all p < 0.01; Fig. 3d). Furthermore, MWM was used to detect the spatial learning and memory for 7 consecutive days, starting on PID day 11 after TBI. Surprisingly, the CCI + 32oC group showed significantly shortened escape latencies (p < 0.05 or p < 0.01; Fig. 3e) from day 12 to day 17 post-CCI and increased time spent in the target quadrant (p < 0.01; Fig. 3f) relative to the CCI + 37oC group. Altogether, these in vivo results confirmed that behavioral outcome is negatively affected by TBI, such as neurological dysfunction or cognitive deficits, and can be improved by TTM.
Protein Identification, Quantification, and Bioinformatics Analysis of Candidate Molecules Associated with TTM Post-TBI
The results showed that the distribution of log2 (sham + 32°C/sham + 37°C) ratio could be fit to a Gaussian curve with a SD of 0.22 (n = 1526), that of log2 (CCI + 37°C/sham + 37°C) ratio with a SD of 0.28 (n = 1523), and 1529 proteins were quantified with a SD of 0.30 for the distribution of log2 (CCI + 32°C/sham + 37°C) ratio (Fig. 4b). In the sham + 32°C/sham + 37°C groups, 163 proteins were changed, with 127 proteins being upregulated and 36 proteins being downregulated. After TBI, 71 proteins were changed, with 42 proteins being upregulated and 29 proteins being downregulated. TTM changed 41 proteins after TBI, with 16 proteins being upregulated and 25 being downregulated. Interestingly, we noticed that the amount of altered proteins in the CCI + 32°C group was larger than that in the CCI + 37oC group. The results of cluster analysis for differentially expressed proteins showed that CCI + 32°C treatment could significantly reverse many protein expression trends compared with the CCI + 37oC group (Fig. 4c). The quantification of differentially expressed proteins is shown in Table S1.
To explore the global possible PPIs using the Cytoscape, we found that the fibrinogen gamma chain (FGG), fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), apolipoprotein A1 (APOA1), glutathione S-transferase P (GSTP1), serphin family C member 1 (SERPINC1), plasminogen (PLG), apolipoprotein H (APOH), and histidine-rich glycoprotein (HRG) were responsible for hemostasis, platelet aggregation, blood coagulation, and many other processes in the CCI + 37°C group (Fig. 5e). Meanwhile, TTM could alleviate the TBI-induced neurotrauma through regulation of hemostasis, platelet aggregation, blood coagulation, and many other processes (Fig. 5f).
Confirmation of iTRAQ Data in Rat Brain Tissue and Human CSF Post-TBI
Owing to the limitations of the rat database, the number of proteins validated in the quantitative proteomics analysis was limited, and not every differentially expressed protein could be classified when analyzed with DAVID, IPA, and Cytoscape. Moreover, in order to screen for the potential diagnostic biomarkers of TBI severity, this study focused on secretory proteins. In the purpose of hunting for proper potential biomarkers, the top-20 proteins for each upregulated and downregulated side after TBI intervention or after reversion of TBI effect with TTM were analyzed, and 11 molecules were sorted out to be the shared proteins. For screening biomarkers, this study selected upregulated secretory proteins after TBI intervention (downregulated proteins could be undetectable by ELISA). The results of the GO and IPA analyses for MS data showed that the expression levels of coagulation system-related molecules in the TTM groups, PLG, AT III, albumin, and FGG, were significantly decreased compared with those of the TBI groups (p < 0.05), indicating that TTM treatment can be an effective strategy applied for patients with TBI . Considering that albumin, the most abundant protein in serum, could be influenced by liver function, infection, nutrition, and a number of other factors, it was excluded from the list for validation. Thus, 4 proteins were rendered for validation: PLG, ATI II, FGG, and TTR.
Meanwhile, the expression level of TTR in the TBI groups was significantly increased compared with that of the control group, and that of TTM groups was significantly decreased compared with that of the TBI group (Tables S2 and S3). A recent study showed that the accumulation or mutation of TTR, which is a potent scavenger of amyloid peptides in the central nervous system, would result in different organization of amyloidosis and neuronal degeneration . Hence, we verified whether TTM was associated with significant change in the expression of a variety of factors involved in TBI among PLG, AT III, Fgg, and TTR in vivo using immunofluorescence.
Next, we addressed whether there were altered levels by using quantitative ELISA on patient CSF. The temporal profiles of CSF PLG, AT III, FGG, and TTR acquired from patients with severe TBI (n = 4, GCS 3–8), moderate TBI (n = 5, GCS 9–12), and mild TBI (n = 7, GCS 13–15) at 1 day after trauma are shown in Figure 6(b). The mean CSF PLG levels in patients with severe and moderate TBI (1336.83 ± 558.08 and 1456.38 ± 808.25 ng/ml) were sharply increased compared with that in patients with mild TBI (176.96 ± 182.55 ng/ml; p = 0.0014 and p = 0.0042, respectively). Additionally, as with PLG, the mean AT III, FGG, and TTR levels in patients with severe and moderate TBI were higher than those in patients with mild TBI [AT III: 127.81 ± 69.28 and 60.97 ± 37.95 vs 16.23 ± 10.99 ng/ml (p = 0.0044 and p = 0.0225); FGG: 78.49 ± 57.44 and 44.82 ± 6.37 vs 15.49 ± 7.30 ng/ml (p = 0.0293 and p < 0.0001); TTR: 4.52 ± 2.70 and 4.62 ± 2.22 vs 1.24 ± 1.39 ng/ml (p = 0.0396 and p = 0.0147)].
In addition, the temporal profiles of CSF proteins mentioned above acquired from patients with severe TBI with standard therapy or TTM therapy at separate time points after injury are shown in Figure 6(c). The results suggest that CSF PLG, AT III, and FGG did not change over time when examined 1 and 3 days after severe TBI with normothermia therapy (p > 0.05), but the mean TTR levels were significantly increased over time (4.52 ± 2.70 vs 19.29 ± 7.59 ng/ml; p = 0.0191). Additionally, the mean CSF PLG, AT III, and FGG levels were sharply lower in patients with TTM therapy at 3 days versus 1 day post-TBI [PLG: 1189.04 ± 497.11 vs 606.31 ± 109.00 ng/ml (p = 0.0127); AT III: 117.02 ± 3.43 vs 54.60 ± 18.03 ng/ml (p = 0.0011); FGG: 78.96 ± 10.91 vs 51.94 ± 7.19 ng/ml (p = 0.0038)], but CSF TTR did not change over time (p > 0.05). The results of Western blot and ELISA for these proteins were consistent with the proteomic data. Taken together, these data suggested that PLG, AT III, FGG, and TTR were present in human CSF after TBI, and TTM treatment showed the inverse change in expression pattern, supporting the notion that these molecules may be useful as future biomarkers associated with TTM. Nevertheless, these results should be taken with caution because the exact source of the CSF (EVD vs LP) could affect the results. Additional studies are necessary to examine this point.
siRNA and Cell Viability
Further investigation on the expression levels of FGG, AT III, and PLG (Fig. 7b, c, d) showed that the levels of these proteins were increased after cell damage, whereas TTM could reduce the expression of FGG, AT III, and PLG in damaged cells, but the application of TTM did not significantly change the levels of FGG, AT III, and PLG in undamaged cells, and the effect of FGG was more marginal than that of PLG and AT III. These findings suggest that the changes of FGG, AT III, and PLG by TTM occurred after cell damage. For AT III and PLG, TTM and gene knockdown showed synergistic effects. TTM further reduced the protein expression after knockdown. These findings suggest that the combined use of TTM in addition to ATIII and PLG knockdown could more effectively improve cell repair. In contrast, TTM increased FGG protein levels in the FGG gene knockdown group of damaged cells. We measured proliferating cell nuclear antigen (PCNA) and found that PCNA was decreased significantly after cell damage, whereas TTM treatment decreased the levels of PCNA (Fig. 7d). We speculated that TTM could not increase the proliferation of the cells.
Previous in vivo and in vitro experimental studies showed the beneficial effects of TTM in TBI in fluid percussion injury (FPI) animal and scratch injury cell models [40, 41, 42]. In our previous studies, we found that post-traumatic TTM significantly improved survival of neurons and reduced β-amyloid and other indicators of abnormal protein accumulation . Nevertheless, the pathology of TBI is so diverse that precise mechanisms underlying this phenomenon are still unclear, mainly because the phenotypic effects of TTM emerge through a currently intractable, not well-understood, multicellular process involving hundreds of interacting molecular components .
In the current study, we exposed neuron-like SH-SY5Y cells and mouse hippocampal HT-22 cells to 32oC following CIC, which is widely used as a cell model to investigate TBI in vitro [17, 43], and observed the accumulation of injured cells in sub-G1 phase, PI-positive cells, and cell death, whereas TTM decreased sub-G1 populations and the sensitivity of cells to stretch injury as evidenced by PI positivity and cell viability compared with normothermia, suggesting that TTM inhibition overcomes TBI-induced damage of neurons. Similar to our previous studies and other reports, TBI produces behavioral abnormalities and neurodegenerative changes resulting from regional patterns of cell death and circuit dysfunction [21, 44]. As supported by previous studies of TTM on TBI, we also demonstrated that using CCI rats as an in vivo model that TTM attenuated several phenotypes of animal post-TBI, markedly reduced cell death [7, 8, 11], BBB leakage [45, 46, 47], cortical lesion volume , cerebrovascular histopathology [49, 50], and improved behavioral and pathological outcomes . These results may help determine both the safety and efficacy for postinjury TTM, and such laboratory models (in vitro or in vivo) may present an ideal opportunity for the application of proteomic technological approach to biomarker discovery after TBI.
Systems biology is a crucial approach to investigate the complex, multifactorial nature of secondary cellular interactions to TBI, from which a testable hypothesis about the underlying TBI-related molecular mechanisms that can be experimentally validated is generated by the analysis of data-driven, high-throughput genomics or proteomics data sets of biomolecules and their interactions rather than traditional research approaches [52, 53, 54]. In the present study we applied isobaric peptide tags (iTRAQ) and multidimensional LC-MS/MS to identify proteins that were differentially expressed in 32oC versus 37oC injured brain specimens derived from a CCI model of TBI. Of the altered proteins after TBI, regulation of fibrinolysis and blood coagulation, which were the most important processes, were reported in terms of vascular injury and hemorrhage . Besides, another important process—response to wounding—is another natural response to injury, as shown by altered mitotic cell cycle, such as revealed by neural stem cell proliferation after TBI. Response to metal ions correspond to Ca2+ stimuli, and cytokine-mediated signaling pathways correspond to the tumor necrosis factor-α and interleukin-1β stimuli. TTM seems to reduce these dysfunctions. Above all, the present study is only the first systematic study of TBI and TTM, and additional studies are still needed to confirm the TTM effects comprehensively.
TBI is a complicated process because of various injury causes, locations, and severities. Biomarkers for function outcome and prognosis are urgently needed. In the past decades, a few biomarkers have been identified. For example, S100B is a widely used protein for the assessment and prediction of TBI [18, 56, 57, 58]. Recently, MIF was found to change along with inflammation factors and could be a prognosis biomarker for good clinical outcome . Sharma et al.  tested 87 potential serum biomarkers from 110 patients with mild TBI, and found a biomarker panel that identifies complicated mild TBI from uncomplicated mild TBI. Another systematic biomarker screening study—also based on antibody-based proteomics—was conducted on normoxic or hypoxic TBI models, and potential biomarkers for TBI were identified but failed to separate normoxic or hypoxic TBI models .
In the present study, we also focused on screening a series of altering expression molecules in CCI with TTM treatment, including Fgg, fibrinogen alpha chain, fibrinogen beta chain, apolipoprotein A1, glutathione S-transferase P, serphin family C member 1, PLG, apolipoprotein H, and histidine-rich glycoprotein, which are involved in vital biological processes, such as hemostasis, platelet aggregation, and blood coagulation. In particular, PLG, AT III, Fgg, and TTR could be used as assessment criteria of TBI healing after TTM treatment, and the expression pattern was confirmed by immunostaining on tissue from CCI rats treated with TTM, as well as by Western blot and ELISA assay on CSF samples acquired from patients with TBI followed by TTM.
In conclusion, based on iTRAQ quantitative proteomics profiling, we have generated an extensive and unique data set of protein expression profiles that recapitulates the change of proteins in TBI treated with TTM. The validation of the iTRAQ result for the PLG, AT III, FGG, and TTR proteins suggests that the combined proteomics and bioinformatics analysis applied has generated valid candidates that may be used for diagnostic and prognostic counseling of patients with TBI. These results may provide more accurate information about TBI status and clinical outcome, and may be further evaluated for their role associated with TTM in the progression from TBI.
This work was supported by grants from the National Natural Science Foundation of China (31200809), Science & Technology Program of Tianjin, China (15ZXLCSY00040), and Technology Research Projects (AWS15J001).
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