B-Cell Lymphoma 2 (Bcl-2) and Regulation of Apoptosis after Traumatic Brain Injury: A Clinical Perspective.

Background and Objectives: The injury burden after head trauma is exacerbated by secondary sequelae, which leads to further neuronal loss. B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein and a key modulator of the programmed cell death (PCD) pathways. The current study evaluates the clinical evidence on Bcl-2 and neurological recovery in patients after traumatic brain injury (TBI). Materials and Methods: All studies in English were queried from the National Library of Medicine PubMed database using the following search terms: (B-cell lymphoma 2/Bcl-2/Bcl2) AND (brain injury/head injury/head trauma/traumatic brain injury) AND (human/patient/subject). There were 10 investigations conducted on Bcl-2 and apoptosis in TBI patients, of which 5 analyzed the pericontutional brain tissue obtained from surgical decompression, 4 studied Bcl-2 expression as a biomarker in the cerebrospinal fluid (CSF), and 1 was a prospective randomized trial. Results: Immunohistochemistry (IHC) in 94 adults with severe TBI showed upregulation of Bcl-2 in the pericontusional tissue. Bcl-2 was detected in 36-75% of TBI patients, while it was generally absent in the non-TBI controls, with Bcl-2 expression increased 2.9- to 17-fold in TBI patients. Terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick-end labeling (TUNEL) positivity for cell death was detected in 33-73% of TBI patients. CSF analysis in 113 TBI subjects (90 adults, 23 pediatric patients) showed upregulation of Bcl-2 that peaked on post-injury day 3 and subsequently declined after day 5. Increased Bcl-2 in the peritraumatic tissue, rising CSF Bcl-2 levels, and the variant allele of rs17759659 are associated with improved mortality and better outcomes on the Glasgow Outcome Score (GOS). Conclusions: Bcl-2 is upregulated in the pericontusional brain and CSF in the acute period after TBI. Bcl-2 has a neuroprotective role as a pro-survival protein in experimental models, and increased expression in patients can contribute to improvement in clinical outcomes. Its utility as a biomarker and therapeutic target to block neuronal apoptosis after TBI warrants further evaluation.

CSF Bcl-2 and cytochrome C temporal profiles in outcome prediction for adults with severe TBI.

The biochemical cascades associated with cell death after traumatic brain injury (TBI) involve both pro-survival and pro-apoptotic proteins. We hypothesized that elevated cerebrospinal fluid (CSF) Bcl-2 and cytochrome C (CytoC) levels over time would reflect cellular injury response and predict long-term outcomes after TBI. Cerebrospinal fluid Bcl-2 and CytoC levels were measured for 6 days after injury for adults with severe TBI (N=76 subjects; N=277 samples). Group-based trajectory analysis was used to generate distinct temporal biomarker profiles that were compared with Glasgow Outcome Scale (GOS) and Disability Rating Scale (DRS) scores at 6 and 12 months after TBI. Subjects with persistently elevated temporal Bcl-2 and CytoC profiles compared with healthy controls had the worst outcomes at 6 and 12 months (P≤0.027). Those with CytoC profiles near controls had better long-term outcomes, and those with declining CytoC levels over time had intermediate outcomes. Subjects with Bcl-2 profiles that remained near controls had better outcomes than those with consistently elevated Bcl-2 profiles. However, subjects with Bcl-2 values that started near controls and steadily rose over time had 100% good outcomes by 12 months after TBI. These results show the prognostic value of Bcl-2 and CytoC profiles and suggest a dynamic apoptotic and pro-survival response to TBI.

Evaluation of apoptosis in cerebrospinal fluid of patients with severe head injury.

OBJECTIVE: To determine whether sFas, caspase-3, proteins which propagate apoptosis, and bcl-2, a protein which inhibits apoptosis, would be increased in cerebrospinal fluid (CSF) in patients with severe traumatic brain injury (TBI) and to examine the correlation of sFas, caspase-3, and bcl-2 with each other and with clinical variables. METHODS: sFas, caspase-3, and bcl-2 were measured in CSF of 14 patients with severe TBI on days 1, 2, 3, 5, 7, and 10 post-trauma. The results were compared with CSF samples from control patients who had no brain and spinal pathology and had undergone spinal anesthesia for some other reason. Soluble Fas and bcl-2 were measured by ELISA while caspase-3 was measured enzymatically. RESULTS: No sFas, caspase-3, and bcl-2 activities were found in CSF of controls, but activities significantly increased in CSF of patients at all time points post-trauma (p < 0.01). Caspase-3 significantly correlated to intracranial pressure (p = 0.01) and cerebral perfusion pressure (p = 0.04). Soluble Fas and caspase-3 peaks coincided on day 5 post-trauma and there was significant association between sFas and caspase-3 increase (p = 0.01). CONCLUSION: This study indicates a prolonged activation of pro-apoptotic (sFas, caspase-3) and anti-apoptotic (bcl-2) proteins after severe TBI in humans. The degree of activation of particularly caspase-3 may be related to the severity of the injury. Parallel increases of these three molecules may indicate a pivotal role of apoptosis in the pathophysiology of post-traumatic brain oedema, secondary cell destruction and chronic cell loss following severe TBI and may open new targets for post-traumatic therapeutic interventions.

Increases in bcl-2 protein in cerebrospinal fluid and evidence for programmed cell death in infants and children after severe traumatic brain injury.

OBJECTIVES: To determine whether bcl-2, a protein that inhibits apoptosis, would be increased in cerebrospinal fluid (CSF) in infants and children after traumatic brain injury (TBI) and to examine the association of bcl-2 concentration with clinical variables. STUDY DESIGN: Bcl-2 was measured in CSF from 23 children (aged 2 months-16 years) with severe TBI and from 19 children without TBI or meningitis (control subjects) by enzyme-linked immunosorbent assay. CSF oligonucleosome concentration was also determined as a marker of DNA degradation. Brain samples from 2 patients undergoing emergent decompressive craniectomies were analyzed for bcl-2 with Western blot and for DNA fragmentation with TUNEL (terminal deoxynucleotidyl-transferase mediated biotin-dUTP nick-end labeling). RESULTS: CSF bcl-2 concentrations were increased in patients with TBI versus control subjects (P =.01). Bcl-2 was increased in patients with TBI who survived versus those who died (P =.02). CSF oligonucleosome concentration tended to be increased after TBI (P =.07) and was not associated with bcl-2. Brain tissue samples showed an increase in bcl-2 in patients with TBI versus adult brain bank control samples and evidence of DNA fragmentation within cells with apoptotic morphology. CONCLUSIONS: Bcl-2 may participate in the regulation of cell death after TBI in infants and children. The increase in bcl-2 seen in patients who survived is consistent with a protective role for this anti-apoptotic protein after TBI.

Intracerebral production of tumor necrosis factor-alpha, a local neuroprotective agent, in Alzheimer disease and vascular dementia.

The local pattern of proinflammatory cytokine release was studied in Alzheimer disease (AD) and vascular dementia (VAD), by measuring intrathecal levels of IL-1 beta, IL-6, TNF-alpha, and its naturally occurring antagonists, soluble TNF receptors I and II. The cytokine levels were related to neuronal damage, as measured by the intrathecal tau concentration, to cerebral apoptosis assessed by levels of Fas/APO-1 and bcl-2, and to clinical variables. In vitro analysis was performed to study the effect of TNF-alpha on the production of bcl-2, an antiapoptotic factor, by human neuronal cells. Patients with both AD and VAD displayed significantly higher intrathecal levels of TNF-alpha compared to controls. In addition, patients with AD showed significantly negative correlations between the intrathecal levels of TNF-alpha and the levels of Fas/APO-1 as well as of tau protein. The level of bcl-2 in supernatants of TNF-alpha-exposed cultures of human neuronal cells was up to three times higher than in control supernatants. Our study demonstrates intrathecal production of TNF-alpha in patients with dementias, suggesting that this cytokine may have a neuroprotective role in these neurodegenerative conditions as evidenced by negative correlations between this cytokine and (i) levels of intrathecal Fas/APO-1 and (ii) levels of tau protein, both parameters closely related to brain damage. Our in vitro data suggest that TNF-alpha exerts its neuroprotective effect by stimulating neuronal cells to express bcl-2, a molecule which downregulates apoptosis.

Intrathecal expression of proteins regulating apoptosis in acute stroke.

BACKGROUND AND PURPOSE: The neuronal death that accompanies an ischemic stroke has previously been attributed to a necrotic process. However, numerous studies in experimental models of ischemia have recently indicated that programmed cell death, also called apoptosis, may contribute to neuronal death. The aim of the present study was to investigate the intrathecal levels of proteins regulating apoptosis in acute stroke and to relate these levels to brain damage and to production of proinflammatory and anti-inflammatory cytokines. METHODS: Thirty stroke patients were studied prospectively on days 0 to 4, 7 to 9, 21 to 26, and after day 90 with clinical evaluation, radiological assessment, and analysis of cerebrospinal fluid (CSF) levels of soluble (s) Fas/APO-1 and sbcl-2, 2 proteins that regulate apoptosis. In addition, analysis of the intrathecal levels of cytokines interleukin (IL)-1beta, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha was performed. Nineteen CSF samples from healthy subjects were used for control purposes. The patients were examined with MRI 1 to 3 months after stroke onset for measurement of infarct volume RESULTS: Significantly decreased CSF levels of sFas/APO-1 were observed during the entire observation period, with a maximal decrease on day 21 after the onset of stroke. The intrathecal levels of sFas/APO-1 were significantly negatively correlated with the volume of brain infarct and with the neurological deficit 3 weeks and 3 months after the onset of the stroke. In addition, the intrathecal levels of sFas/APO-1 were significantly correlated with the levels of IL-1beta, IL-6, IL-10, and GM-CSF 3 weeks after the onset of the disease. The intrathecal levels of sbcl-2 were significantly decreased during the first 3 days after stroke onset and at the same time were positively correlated with the levels of IL-6 and tumor necrosis factor-alpha. CONCLUSIONS: Our study demonstrates decreased intrathecal levels of proteins with antiapoptotic properties, suggesting that patients with acute stroke display a propensity toward apoptosis. Control of factors regulating apoptosis may lead to decreased delayed brain damage in stroke.