Optimal Treatment Timing to Rescue Neuronal Cells from Spinal Cord Injury via Bcl-2 Gene Transfer In Vitro and In Vivo

Washington University, St. Louis, MO, USA

INTRODUCTION: Following spinal cord injury (SCI), there is both a primary response of necrotic cell death from the external mechanical injury, and importantly a secondary injury cascade. The secondary injury is actively mediated by cellular and molecular processes including edema, inflammation, growth factors, ischemia, superoxide radicals and, importantly, apoptosis. Apoptosis is a distinct form of programmed cell death that occurs based on information from the cellular environment, metabolism, and its genome. Recently, apoptotic cell death has been documented to occur in the secondary injury cascade of SCI and thus provides an opportunity for treatment with anti-apoptotic factors (eg. Bcl-2 protein) that can attenuate apoptosis and potentially improve cell survival following SCI.

PURPOSE: To investigate the optimum treatment time to deliver the anti-apoptotic protein Bcl-2 via gene transfer with recombinant adenovirus to rescue cells following neural insult both in vitro and in vivo.

METHODS: Bcl-2 gene transfer was mediated by a recombinant adenovirus using human Bcl-2 oncogene (Adv-Bcl-2). The adenovirus carrying b-galactosidase gene (Adv-Bgal) served as a control. A serum withdrawal model was used to induce apoptosis in vitro. At various time points before or after serum withdrawal (-24, 0, 2,4,8, 24 H), the motor neuron cells (NSC-19 cells) were infected with either Adv-Bcl-2 or Adv-Bgal. 72 hours after serum withdrawal, the number of apoptotic cells was counted using Hoechst 33342 staining to obtain the apoptotic index. For the in vivo animal experiments, a weight drop injury model (10g x 5.0cm) was used on 81 rats. At various time points before or after experimental spinal injury (-24, 0, 2, 4, 8, 24 H), .1ml virus solution including 2.3 x 10⁸pfu of Adv-Bcl-2 or Adv-Bgal was injected at the epicenter of the injured spinal cord. The degree of cord injury was measured 72 hours after trauma and quantified with NIH software as the injury index. TUNEL staining was performed to count apoptotic cells per section in the cases injected at time 0 H.

RESULTS: In vitro: Adv-Bcl-2 infection reduced the apoptotic index (cell death) significantly between 24 hours before (3.5%) and 4 hours after (5.5%) serum withdrawal (P<.01), but not at the two later timepoints 8 H (7.5%) and 24 H (9.2%) following serum withdrawal In vivo: Adv-Bcl-2 injection at 0 H (immediately after injury) significantly decreased the injury index by 24% (P ‹ .01) and significantly reduced the number of TUNEL positive apoptotic cells by 28.5% vs. 34.2% in controls (P ‹ .01).

CONCLUSION: Early initiation of Bcl-2 gene transfer (-24 H before to 4 H after in vitro, and at time 0 H in vivo) produces improved cell and tissue recovery following neural insults by limiting apoptotic cell death. These results stress important temporal considerations in future gene therapy strategies for SCI.