Table 1.
Summary of investigations on Bcl-2 and apoptosis after TBI in human subjects.
| Author and Year | Study Type | N | Sample Characteristics | Description | Methodology | Results |
|---|---|---|---|---|---|---|
| Pericontusional Tissue Analysis | ||||||
| Clark et al., 1999 [12] | Prospective cohort | 8 TBI adults (6 male, 2 female), 6 non-TBI controls (2 male, 4 female) | Pericontusional tissue of adults who underwent decompressive craniectomy and surgical resection | PCD cascade activation and neuronal expression of Bcl-2 | Immunohistochemistry and expression of Bcl-2, Bcl-xL and Bax, and cleavage of caspase-1 and caspase-3. Detection of TUNEL-positive apoptotic cells. | Compared to non-TBI controls, TBI patients had increased Bcl-2 expression (17-fold, p = 0.020). Pro-caspase-1 showed 2-fold reduction, while the p10 fragment of caspase-1 was increased (78-fold increase, p < 0.001). Caspase-3 is upregulated 14-fold (p = 0.020) to permit formation of active enzyme complexes. TUNEL-positive cells were detected in most TBI samples and in 2/6 non-TBI controls. |
| Ng et al., 2000 [14] | Prospective cohort | 11 severe TBI adults (8 male, 3 female) | Pericontusional tissue (7 frontal, 4 temporal, 4 parietal) of adults who underwent craniotomy and surgical resection for mass effect | PCD cascade activation and Bcl-2 expression after severe TBI | Immunohistochemistry and expression of Bcl-2, Bax, and p53. Detection of TUNEL-positive apoptotic cells. | Bcl-2 was detected in 6/11 (55%) samples. Notably, 4/5 (80%) of patients without Bcl-2 expression had time of trauma to surgery 8 h, versus 2/6 (33%) of patients positive for Bcl-2 expression. ICPs were 13.5 3.72 mm Hg in Bcl-2-positive patients versus 40.8 30.28 mm Hg in Bcl-2-negative patients (p = 0.057). 6/6 Bcl-2-positive patients were alive at 6 months compared to 1/5 Bcl-2-negative patients (p = 0.01). TUNEL-positive cells were detected in 8 (73%) of the 11 patients. |
| Nathoo et al., 2004 [16] | Prospective cohort | 29 moderate and severe TBI (27 male, 2 female), 3 epilepsy-surgery controls | Pericontusional tissue of adults who required emergency craniotomy for supratentorial pathology | Identify evidence of apoptosis associated with traumatic cerebral contusions and correlation with clinical outcomes |
Immunohistochemistry of apoptosis-related cell proteins Bcl-2, p53, Bax, and caspase-3 | There were increases of Bax (18-fold; p < 0.005) and caspase-3 (20-fold; p < 0.005), whereas Bcl-2 was upregulated in only 14 patients (48.3%; 2.9-fold increase) compared with control tissue. Bcl-2-positive patients experienced improved outcome on GOS when compared with the Bcl-2-negative patients at 18 months of follow up (p = 0.03), despite having a higher mean age and lower admission GCS scores. Regression analysis found Bcl-2-negative status (p < 0.04, OR 5.5; 95% CI 1.1–28.4) and caspase-3-positive status (p < 0.01, OR 1.4, 95% CI 1.1–1.8) as independent predictors of poor outcome. |
| Miñambres et al., 2008 [15] | Prospective cohort and in vitro | 11 severe TBI adults (8 male, 3 female), 5 non-TBI controls (4 male, 1 female) | Pericontusional tissue obtained from surgical resection (4) or post-mortem via autopsy (4) | PCD cascade activation, Bcl-2 expression, and in vitro neuronal apoptosis (PC12 cells) | Immunohistochemistry and expression of Bcl-2, Bcl-xL, Bim, Bax, and Fas. Detection of TUNEL-positive apoptotic cells. In vitro apoptosis induced by TBI patients’ serum | Bcl-2 (4/11 versus 0/5) and Fas (6/11 versus 0/5) were only immunoreactive in TBI patients. Compared to controls, Bcl-2 expression was higher in craniotomy group (p = 0.027), Fas was higher in both craniotomy (p = 0.09) and post-mortem (p = 0.007) groups, and Bcl-xL was lower in post-mortem group (p = 0.014). Anti-apoptotic Bcl-2 (p = 0.027) and Bcl-xL (p = 0.014) were higher in the emergency craniotomy cohort relative to post-mortem TBI patients. TUNEL-positive cells were detected in 4/4 samples of craniotomy cohort, 4/7 (57%) of post-mortem cohort, and 0/5 of controls (p = 0.026). There was greater early apoptosis in the cultures of PC12 induced by the serum of patients with low Bcl-2 and Bcl-xL levels (median 64.4% versus 73.8%), and with non-survivors. |
| Dai et al., 2018 [17] | Prospective and in vitro | 30 patients with glioma and 25 TBI patients of unknown severity | Glioma tissues from biopsy/resection and cerebral tissues from TBI patients were collected | Determine the mechanism by which Nur77 and Bcl-2 protein expression influence apoptosis after TBI | Nur77 and Bcl-2 expression by IHC assay and immunofluorescence. Detection of TUNEL-positive apoptotic cells. Nur77 inhibitor via injection with 1 mL/kg CsA, and Bcl-2 inhibitor using 1 mL/kg APG- 1252 | Apoptotic cells are increased in TBI cohort compared to glioma group (p < 0.001). Nur77 and Bcl-2 expression is upregulated after TBI (p < 0.001), and there was a positive correlation between Nur77 and Bcl-2 in TBI tissues (r = 1.051, p < 0.001). Nur77 play a promoting factor in nerve cell apoptosis-induced TBI via Bcl-2/Cyto C/Caspase 3 in vitro and vivo. Bcl-2 may promote apoptosis in some cases, acting as a pro-apoptotic protein. |
| CSF Biomarker Analysis | ||||||
| Clark et al., 2000 [13] | Prospective cohort | 23 severe TBI pediatric patients (14 male, 9 female), 19 non-TBI controls (11 male, 8 female) | CSF samples collected on days 1, 2, and 3 after TBI, and brain tissue of 2 patients who needed decompressive craniectomy and surgical resection | PCD cascade activation, Bcl-2 expression, and DNA degradation in infants and children | Levels of Bcl-2 and oligonucleosomes in CSF. Detection of TUNEL-positive apoptotic cells. | Mean CSF Bcl-2 concentrations were increased in patients after TBI compared with control (9.70 ± 1.43 versus 2.68 ± 0.85 U/mL, p = 0.01). Increased CSF Bcl-2 was independently associated with patient survival on multivariate analysis (p = 0.018). CSF oligonucleosome concentration increased after TBI compared with control (428 ± 77 versus 168 ± 52 mU/mL, p = 0.08) and did not correlate with CSF Bcl-2 (r = –0.015, p = 0.905). |
| Uzan et al., 2006 [19] | Prospective cohort | 14 patients with severe TBI (11 male, 3 female), 14 controls without TBI or spinal pathology | CSF samples drained on days 1, 2, 3, 5, 7, and 10 from pediatric (5) and adult (9) patients | Determine if soluble Bcl-2, Fas and caspase-3 would be increased in CSF after severe head injury | Bcl-2, sFas, and caspase-3 were measured in drained CSF samples after severe TBI. The concentrations of Bcl-2 were analyzed via ELISA | No Bcl-2, Fas, or caspase-3 were detected in CSFof controls, while levels were higher in CSF of patients at all time points post-trauma (p < 0.01). Peak Bcl-2 levels varied by individual, but frequently on days 3 and 4 (7 patients). Mean peak bcl-2 concentration was noted on day 3 (119 28.5 ng/mL) and declined after day 5. Bcl-2 levels in CSF did not correlateto ICP (p = 0.9), CPP (p = 0.7) and initial CTfindings (p = 0.4). |
| Hoh et al., 2010 [23] | Prospective cohort | 205 subjects (163 male, 42 female) with severe TBI aged 16–75 years old | DNA was extracted from CSF or blood specimens for genotyping of regions within and around the Bcl-2 gene. | Investigate if variation in the Bcl-2 gene contributes to variability in the outcomes attained after severe TBI | All of the genetic variability associated with the Bcl-2 gene were characterized utilizing 17 tSNPs. The GOS, DRS, and NRS-R scores were conducted at 3, 6, 12, and 24 months post-TBI. | The variant allele of rs17759659 was associated with poorer outcomes (GOS, p = 0.001; DRS, p = 0.002), higher mortality (p = 0.02; OR = 4.23; CI 1.31–13.61), and worse NRS-R scores (p = 0.05). The variant allele for rs1801018 was associated with poorer outcomes (GOS, p = 0.02; DRS, p = 0.009), and mortality (p = 0.03; OR = 3.86; CI 1.18–12.59). Other polymorphisms including rs7236090 and rs949037 were associated with variable outcomes on the NRS-R, and DRS, although the only finding that stood up to Bonferroni correction was rs17759659 for GOS. |
| Wagner et al., 2011 [11] | Prospective cohort | 76 severe TBI patients (61 male, 15 female) aged 16–65 years old, 10 healthy adult control subjects | CSF samples for biomarker analysis from EVDs were collected for up to 6 days after initial trauma. Control subjects’ CSF was obtained via lumbar puncture | Bcl-2 and cytochrome C levels over time may reflect cellular injury response and predict long-term outcomes after TBI | CSF Bcl-2 and CytoC concentrations were measured daily from day 1 to 6 after injury via ELISA. GOS score and Disability Rating Scale (DRS) at 6- and 12-month follow-up were the primary outcome measures | Bcl-2 levels are significantly higher than controls on days 1, 2, and 4 (p < 0.05 all comparisons) and trended toward significance on day 3 (p = 0.051). CytoC levels peaked 24 h after injury and were higher than controls for days 1 to 4 (p < 0.043 all comparisons) and trended toward significance on day 0 (p = 0.074). Bcl-2 temporal profiles were categorized as riser (11%), low (31%), and high (58%). The combined low and riser Bcl-2 groups were significantly associated with better GOS-6 and GOS-12 scores (p = 0.009 and p = 0.002, respectively) when compared with the high Bcl-2 group. Patients in the low and riser Bcl-2 groups were 9 times more likely to have better outcome 12 months after injury. |
| Other | ||||||
| Indharty et al., 2013 [22] | Prospective randomized trial | 40 moderate TBI adults aged 18–29 years old | Standard therapy versus standard therapy plus intranasal ACTH in patients with contusion on CT scan without the indication for surgery | ACTH4-10Pro8-Gly9-Pro10 as a synthetic peptide constituting a short fragment of ACTH to potentially inhibit apoptosis by increasing Bcl-2 while minimizing hormonal side effects | 5-day course of intranasal ACTH4-10Pro8-Gly9-Pro10 (Semax®): 9 mg/day 1, 6 mg/day 2, and 3 mg daily for the remaining 3 days. Blood draws on day 1 and day 5 to quantify Bcl-2 via ELISA | In the control group, mean Bcl-2 on day 1 was 1.68 ± 1.34 ng/mL and at day 5 was 1.66 ± 1.06 ng/mL. In the intervention arm, serum Bcl-2 level increased from day 1 1.93 ± 1.35 ng/mL to 3.81 ± 1.00 ng/mL on day 5 (p < 0.05), which was also significant on intergroup comparison (p < 0.05). There was no difference in clinical outcomes by the Barthel Index or MMSE, but there was a trend toward shorter hospital stay within the ACTH4-10Pro8-Gly9-Pro10 intervention cohort. |
TBI = traumatic brain injury; PCD = programmed cell death; Bcl-2 = b-cell lymphoma 2; TUNEL = terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick-end labeling; GOS = Glasgow Outcome Score; GCS = Glasgow Coma Scale; CI = confidence interval; CSF = cerebrospinal fluid; DNA = deoxyribonucleic acid; tSNP = tagging single nucleotide polymorphism; DRS = Disability Rating Scale; Neurobehavioral Rating Scale-Revised; ACTH = adrenocorticotropic hormone; MMSA = Mini-Mental State Exam; ELISA = enzyme-linked immunosorbent assay.