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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 21, 2005 as doi:10.1096/fj.05-4024fje. |
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* School of Biomedical Engineering, Science, and Health Systems, DrexelUniversity, Philadelphia, Pennsylvania, USA;
Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA; and
Department of Radiation Oncology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA
1 Correspondence: School of Biomedical Engineering, 3141 Chestnut St., Philadelphia, PA 19104, USA. E-mail: barbee{at}drexel.edu
SPECIFIC AIMS
The mechanism of cell death and the progressive degeneration of neural tissue after traumatic brain injury (TBI) have come under intense investigation. However, the complex interactions among the evolving pathologies in multiple cell types obscure the causal relationships between the initial effects of the mechanical trauma at cellular level and the long-term dysfunction and cell death. Our aims are to 1) determine the molecular mechanisms of neuronal cell death in an in vitro model of mechanical trauma and 2) evaluate the cellular mechanism of neuroprotection by Poloxamer 188 (P188) treatment in mechanically damaged cells.
PRINCIPAL FINDINGS
1. Mechanical injury induced both necrosis and apoptosis in neuronal cells mimicking in vivo findings
To determine the acute and delayed cellular response to mechanical injury, PC2 cells (at DIV7) were subjected to fluid shear stress. Examination of the cell death mechanism was performed with TUNEL assay, which is based on the analysis of DNA fragmentation in injured cells. Mechanical injury caused a marked increase in both apoptotic (Fig. 1
) and necrotic cells (Fig. 2
) at 24 h postinjury. To assess the activation of different MAPKs (ERK1/2, JNK1/2, and p38) at 15 min, 3 h, 6 h, and either 16 h or 24 h postinjury, Western blot analysis was used with antibodies that recognize both total and phosphorylated forms of the proteins. ERK1/2, JNK1/2, and p38 were rapidly phosphorylated at 15 min postinjury. ERK1/2 phosphorylation increased 1.44 ± 0.07-fold 15 min postinjury, then decreased gradually with time, eventually falling below the baseline compared with sham controls at 16 h. JNK1/2 phosphorylation was increased 1.35 ± 0.15-fold at 15 min, but returned to control levels by 3 h and remained similar to sham controls at later time points. Phosphorylation of p38 showed the largest increase compared with ERK1/2 and JNK1/2 with a peak of 2.31 ± 0.21-fold at 15 min.
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2. Acute repair of injured plasma membrane prevents both necrosis and apoptosis in injured cells
To examine the effect of plasma membrane repair on the cellular protection from cell death, we applied the nonionic surfactant P188 to the cells within 10 min after mechanical injury. In the presence of P188, the rate of apoptosis and the rate of necrosis were dramatically reduced to levels that were not significantly different from sham controls (Figs. 1
, 2)
.
3. The cellular response to mechanical injury involves multiple signaling pathways, and the targeting of acute membrane repair appears to be an effective approach for neuronal protection
Membrane repair interfered with one of the signaling pathways, p38, which is related to apoptosis. Western blot analysis of injured cells showed that both SB203580 (10 µM), a pharmacological inhibitor of the p38 pathway, and P188 (100 µM) significantly reduced transient p38 phosphorylation 15 min after injury. At both 15 min and 24 h, p38 activation was not significantly different from control cells for SB203580- or P188-treated cells. Inhibition of p38 activity with SB203580 in injured cells provided a partial inhibition in apoptosis from 33.1 ± 2.9% to 24.5 ± 2.9% (Fig. 1)
. In contrast, P188 treatment reduced the rate of apoptosis in injured cells from 33.1 ± 2.9% to 8.7 ± 0.7% (Fig. 1)
. No significant reduction in necrosis was observed in injured cells with SB203580 treatment (22.4±10.7% of SB203580-treated cells compared with 30±14.14% of untreated injured cells; Fig. 2
). In injured cells, P188 treatment significantly reduced the number of necrotic cells from 30 ± 14.1% to 4.2 ± 4.5% (Fig. 2)
. Microscopic evaluation of the cells treated with P188 at 24 h showed a remarkable recovery of the morphology characteristic of uninjured cells and sham controls.
CONCLUSION AND SIGNIFICANCE
This work shows that mechanical injury leads not only to necrosis, but also to apoptosis in neuronal cells with the concert and transient activation of MAPK pathways. To our knowledge, this is the first demonstration of apoptosis in response to mechanical trauma in a neuronal cell culture model.
Previous in vitro injury models that involve acute destruction of a subpopulation of cells have given rise to primarily necrotic cell death via an excitotoxic mechanism. P188 has been shown to protect both neuronal and non-neuronal cells from cell death by resealing the injured cell membrane and enhancing the functional recovery of the cells injured by electric shock, heat (thermal) shock, and excitotoxic and oxidative agents. However, in the excitoxicity case the membrane damage follows the excitotoxic insult and represents a final pathway to necrotic cell death. In contrast, in mechanical injury we demonstrated that the membrane damage is the precipitating event initiating the signaling cascades leading to apoptosis. Among the signaling pathways we investigated, p38 activation was the most prominent. However, the direct inhibition of the p38 pathway provided only partial protection from apoptosis and did not protect from necrosis. In contrast, P188 treatment provided protection from necrosis and apoptosis in mechanically injured cells. These data suggest that the stimulation of apoptosis by trauma may involve other signaling pathways in addition to the p38 pathway.
The production of acute sublethal membrane injury in our in vitro model mimics the in vivo condition for most forms of closed head injury. In this study, we have demonstrated the importance of preserving membrane integrity in developing treatment strategies for TBI. The complexity of the cellular mechanisms and the existence of redundant signaling pathways initiated by traumatic injury present many challenges to the development of new treatment strategies. However, our novel approach of targeting the membrane integrity appears to affect multiple pathways by repairing the structural damage that triggers the secondary events after traumatic injury. As there are currently no drugs available for the treatment of TBI, P188 alone or in combination with other strategies may hold great promise for the treatment of traumatic injury induced diseases and dysfunctions. Furthermore, the identification of plasma membrane integrity as therapeutic target for mitigation of cell injury may have broad applications beyond TBI including mechanical injury of other tissues.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4024fje;
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