Setting and design
We conducted a prospective longitudinal case–control study (STROBEs criteria followed), in a level 1 trauma care center, for the duration of 30 months (December 2010–May 2013). 70 isolated traumatic brain injury patients (age group 16–65 years), were included in the study and categorized into four groups (n = 15) i.e. (i) severe head injury (SHI) (GCS ≤ 8) who died within 5 days of injury, (ii) SHI who survived beyond 5 days of injury, (iii) moderate and mild head injury (MHI) (GCS > 8–14) who were discharged within 5 days of injury, (iv) MHI who were hospitalized for more than 5 days of injury, following the assessment for injury using tools like Glasgow coma scale, and computed tomography (CT) findings.
Patients with isolated skull fracture, also patients who are immune-compromised or having pre-existing medical problem (diabetes/hypertension/hepatitis) were excluded from the study. Patients admitted after ≥24 h of injury and referred from other institutes were also excluded (Fig. 1).
Age and gender matched 15 healthy controls (HC) were included in the study.
Parameters such as hospital length of stay (HLOS), ICU length of stay (ILOS) and Glasgow outcome score (GOS) at discharge, and development of sepsis (blood culture positivity) and cerebral meningitis (CSF culture positive) throughout hospital stay was recorded.
Peripheral blood was drawn on day 1 and day 5 of injury subsequently for measurement of chemokine RANTES using standard laboratory techniques.
Five contused brain tissue samples were also collected for chemokine analysis, from SHI who survived beyond 5 days of injury, at the time of surgery (within 24 h of injury) from the site of evacuation.
Also, 10 cerebrospinal fluid samples were taken only when clinically indicated from 10 separate patients with SHI the patients, as per the standard scheme of neurosurgical management.
Diagnosis of traumatic brain injury
TBI was diagnosed based on admission head CT findings .
Sample size calculation
Assuming a common mean ± SD (standard deviation) of 82,840 ± 400 (ρg/ml), one way ANOVA would have 90% power to detect 5% level, a difference in mean RANTES levels, with a sample size in each of three groups (viz. no TBI, mild and moderate, severe TBI) of 10 . Therefore we proposed 15 subjects per group.
Similarly samples for day 5 were studied but only among moderate and severe who had hospital stay for more than 5 days.
Sample collection and processing
3 ml of intravenous blood samples was collected in EDTA (ethylenediaminetetraacetic acid) vial (1:4) as part of routine blood analysis. Plasma was separated by centrifugation and stored at −20 °C until analysis.
500 μl of cerebrospinal fluid was, collected. Samples were centrifuged at 3000 rpm for 10 min to remove cellular debris and supernatants was decanted and stored at −20 °C until analysis.
Contused brain tissue
Tissue were removed and placed in cold (2–4 °C) phosphate buffered saline (PBS) and stored at −80 °C until analysis. Tissue was homogenized as previously described by Hulse et al. .
Briefly, the tissue sample was rinsed with cell wash buffer, taken from the Bio-Plex™ Cell Lysis Kit (catalog #171-304012 Bio-Rad; Hercules, CA) once. Tissue was cut into 3 × 3 mm pieces. 500 mM Phenylmethylsulfonyl Fluoride (PMSF) was prepared by adding 0.436 g PMSF (#P-7626 Sigma, St. Louis, MO, USA) to 5 ml dimethyl sulphoxide (#D2650 Sigma, St. Louis, MO, USA) [DMSO], stored in 0.5 ml aliquots at −20 °C. Lysing solution (10 ml) was prepared by mixing the other contents of the Cell Lysis Kit (#171-304012 Bio-Rad) as per manufacturer’s instructions, vortexed gently and set aside on ice, and 40 μl of 500 mM PMSF was added afterwards. To 500 μl of lysing solution, tissue sample was added, tissue disruption was accomplished by drawing the samples up and down through a 1 ml pipette tip (cut back to a 2 mm opening) 20 times, subsequently centrifuged at 4500g for 15 min at 4 °C, supernatant was collected.
RANTES concentrations were measured in duplicates by ELISA (catalog #BMS287/2INST, eBiosciences, Vienna, Austria), following the manufacturer’s instructions.
Statistical analysis was performed for the comparison of RANTES levels between the groups. Quantitative variables were summarized as mean ± SD or as median (range). Categorical data was expressed as frequency (%) and analyzed using Pearson chi square test. One-way analysis of variance (ANOVA) was applied for comparison between three groups. A p value of ≤0.05 was considered to be statistically significant.