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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 8, 2003 as doi:10.1096/fj.02-1067fje. |
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,
Departments of
* Critical Care Medicine,
Neurology,
¶ Pediatrics,
Neurological Surgery, and
Cell Biology and Physiology, The Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh; and
Geriatric Research Educational and Clinical Center, VA Pittsburgh Health System, Pittsburgh, Pennsylvania
2Correspondence: Safar Center for Resuscitation Research, 3434 Fifth Ave., Pittsburgh, PA 15260, USA. E-mail: clarkrs{at}ccm.upmc.edu
SPECIFIC AIM
To determine whether the death receptor pathway of programmed cell death is initiated in humans after severe traumatic brain injury (TBI).
PRINCIPAL FINDINGS
1. Caspase-8 mRNA is increased after TBI
Caspase-8 mRNA was examined by digoxigenin-labeled reverse transcription polymerase chain reaction (RT-PCR) in brain tissue samples removed from adult patients during surgical management of severe intracranial hypertension (n=17), and compared with postmortem control brain tissue samples (n=6). Caspase-8/actin mRNA is increased in TBI patients compared with controls (10.6 [0–62.2] vs. 0 [0–4.7], respectively; P<0.05). Predicted PCR products of 211 and 294 bp for caspase-8 and actin, respectively, were detected.
2. Caspase-8 is increased and cleaved after TBI
Western blot analysis detected the 
48 kDa precursor form of caspase-8 (pro-caspase-8) in control brain tissue samples as well as in those of TBI patients (Fig 1
). The relative abundance of pro-caspase-8 was increased 1.5-fold in TBI patients compared with controls (P<0.05; Fig 1B
). Proteolytic processing of caspase-8 was seen in 15/16 TBI patients but in only 1/6 control patients. Peptides ranged from 20 to 40 kDa. The antibody used for these studies was generated against an epitope within the p20 fragment. The p20 fragment was detected in the majority of TBI patients but not in controls (P<0.01; Fig 1B
). Caspase-8 levels positively correlated with caspase-8/actin mRNA levels (p20, rs=0.564, P=0.018; p48, rs=0.436, P=0.078), supporting induction of caspase-8.
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3. Fas, but not FasL, is increased after TBI
Since caspase-8, Fas, and FasL are components of the DISC, relative protein levels of Fas and FasL were also determined using Western blot. The relative abundance of Fas was increased 7.5-fold in TBI patients compared with controls (P<0.02; Fig 1C
). In contrast, the relative abundance of FasL did not differ between TBI patients and controls (P=0.98; Fig 1D
). Consistent with DISC formation, relative Fas levels were associated with relative levels of pro-caspase-8 (rs=0.576, P=0.015) and the p20 cleaved fragment of caspase-8 (rs=0.703, P=0.001); TBI and control patients were both included in this analysis.
4. Relative levels of Fas protein are associated with death and patient age after TBI
Relative levels of caspase-8 (p48 and p20), Fas, and FasL were compared with the clinical variables age, gender, initial GCS, latest GOS, survival, time of resection post-TBI, and whether or not the patients received therapeutic hypothermia as part of the clinical management. Relative levels of Fas were increased by 71% in TBI patients that died vs. those that survived (P=0.05). Relative levels of Fas were positively associated with increasing patient age (rs=0.707, P=0.006). There was no association between caspase-8, Fas, or FasL and any other clinical variables analyzed.
5. Caspase-8 is expressed primarily in neurons after TBI
Multi-label immunocytochemical analysis using neuronal (NeuN) and glial (GFAP) specific markers demonstrated that the majority of cells with increased caspase-8 immunoreactivity were neurons (Fig 2
). Peri-contusional areas without frank necrosis were examined from the TBI patients (1, 5, 6, 8, and 11). Sections from patients 1 (Fig 2D-F
) and 8 (Fig 2A-C
) are shown and are representative of all sections examined, where cellular caspase-8 immunoreactivity was seen almost exclusively in NeuN-positive cells in gray matter and was not detected in GFAP-positive cells.
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CONCLUSIONS AND SIGNIFICANCE
The novel findings of this study include 1) caspase-8 mRNA is increased in injured brain; 2) caspase-8 protein is increased and proteolytically processed in injured brain, an event important for activation of the enzyme; 3) caspase-8 mRNA levels correlate with protein levels; 4) Fas, but not FasL, is increased in injured brain; 5) Fas levels are associated with pro-caspase-8, the p20 cleaved fragment of caspase-8, increasing patient age, and death after TBI; and 6) caspase-8 appears to be expressed predominately in neurons after TBI in humans. These data demonstrate the involvement of the death receptor or extrinsic pathway of programmed cell death involving Fas and caspase-8 in human brain after TBI.
Experimental models have established that the death receptor pathway of programmed cell death is operative in mammalian brain after TBI. After controlled cortical impact injury in rats, increases in Fas and FasL in neurons and astrocytes with morphological changes consistent with programmed cell death are seen. In the present study, an increase in Fas, but not FasL, was seen in injured human brain. The latter pattern is also seen in mice after controlled cortical impact, perhaps implying species differences in the composition of the DISC after TBI. The capacity for TBI to initiate formation of the DISC is now evident in mice and patients. Further downstream, caspase-8 proteolysis occurs after fluid percussion injury and controlled cortical impact in rats and is localized to neurons, astrocytes, and oligodendrocytes. DISC formation and activation of caspase-8 could lead to subsequent proteolysis and activation of caspase-3 culminating in programmed cell death, an event that has been well documented in multiple models of experimental TBI. In addition to Fas-FasL/FADD/caspase-8, there is a role for TNF-TNFRI/TRADD/caspase-2 and -10 in death receptor-mediated sequelae after TBI in rodents and humans.
The results of this study confirm that similar to experimental models, TBI can initiate multiple programmed cell death pathways in human brain. Neuronal cell death with apoptotic phenotypes has been reported in multiple studies. Evidence for participation of the mitochondrial or intrinsic pathway includes reports showing alterations in bcl-2 family proteins in injured brain after trauma in adult and pediatric patients. In terms of the extrinsic pathway, others have demonstrated DISC formation in injured brain from five patients with severe TBI. Additional evidence includes the finding that CSF levels of soluble Fas ligand are increased and positively associated with intracranial pressure after TBI, implying a relationship between Fas and cerebral edema. These and the present study showing induction and proteolysis of caspase-8 and increases in brain tissue Fas levels in 17 patients with severe TBI clearly demonstrate the participation of the death receptor or extrinsic pathway of programmed cell death after TBI. Activation of caspase-3, positioned at the confluence of the two pathways, has also been reported after TBI in humans.
Is activation of the death receptor pathway after TBI beneficial, detrimental, both, or neither after TBI? Studies using transgenic mice suggest that the presence of TNF is detrimental early after TBI related to acute edema formation, but may be necessary later after TBI as part of the regenerative response. In experimental models of ischemia, epilepsy, and neurodegenerative diseases the extrinsic pathway is thought to participate in neuronal cell death. In humans after TBI, activation of the Fas death receptor pathway appears primarily detrimental, as CSF levels of soluble FasL correlate with increasing intracranial pressure and early CSF levels of soluble Fas correlate with CSF neuron specific enolase, a biochemical marker of neuronal cell death. Consistent with these findings, in the present study injured brain tissue levels of Fas were increased in patients that died vs. those that survived.
Like the intrinsic or mitochondrial programmed cell death pathway, activation of the extrinsic pathway is important for normal development and cell turnover, particularly in inflammatory cells. Therefore, a homeostatic role for Fas and caspase-8 after TBI similar to that observed for TNF cannot be ruled out. In mammalian brain, Fas is essential for normal embryologic development, with expression diminishing after birth. The finding in the present study that brain tissue levels of Fas are positively associated with patient age suggests that Fas reactivation may occur more rapidly with increasing age. An alternative possibility is that the elderly patients had a greater severity of injury, although there was no association between Fas and initial GCS.
In conclusion, these data demonstrate that induction, up-regulation, and proteolysis of caspase-8 occurs in human brain after trauma and that relative caspase-8 levels are associated with levels of Fas. An association between relative Fas levels and patient death suggest that the participation of the extrinsic programmed cell death pathway after TBI may be detrimental, but further investigation in experimental models and humans is necessary.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1067fje; doi: 10.1096/fj.02-1067fje 
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