Cell viability in three ex vivo rat models of spinal cord injury

Abstract

Spinal cord injury (SCI) is a devastating disorder that has a poor prognosis of recovery. Animal models of SCI are useful to understand the pathophysiology of SCI and the potential use of therapeutic strategies for human SCI. Ex vivo models of central nervous system (CNS) trauma, particularly mechanical trauma, have become important tools to complement in vivo models of injury in order to reproduce the sequelae of human CNS injury. Ex vivo organotypic slice cultures (OSCs) provide a reliable model platform for the study of cell dynamics and therapeutic intervention following SCI. In addition, these ex vivo models support the 3R concept of animal use in SCI research – replacement, reduction and refinement. Ex vivo models cannot be used to monitor functional recovery, nor do they have the intact blood supply of the in vivo model systems. However, the ex vivo models appear to reproduce many of the post traumatic events including acute and secondary injury mechanisms. Several well‐established OSC models have been developed over the past few years for experimental spinal injuries ex vivo in order to understand the biological response to injury. In this study, we investigated cell viability in three ex vivo OSC models of SCI: stab injury, transection injury and contusion injury. Injury was inflicted in postnatal day 4 rat spinal cord slices. Stab injury was performed using a needle on transverse slices of spinal cord. Transection injury was performed on longitudinal slices of spinal cord using a double blade technique. Contusion injury was performed on longitudinal slices of spinal cord using an Infinite Horizon impactor device. At days 3 and 10 post‐injury, viability was measured using dual staining for propidium iodide and fluorescein diacetate. In all ex vivo SCI models, the slices showed more live cells than dead cells over 10 days in culture, with higher cell viability in control slices compared with injured slices. Although no change in cell viability was observed between time‐points in stab‐ and contusion‐injured OSCs, a reduction in cell viability was observed over time in transection‐injured OSCs. Taken together, ex vivo SCI models are a useful and reliable research tool that reduces the cost and time involved in carrying out animal studies. The use of OSC models provides a simple way to study the cellular consequences following SCI, and they can also be used to investigate potential therapeutics regimes for the treatment of SCI.

Ex Vivo Rat Transected Spinal Cord Slices as a Model to Assess Lentiviral Vector Delivery of Neurotrophin-3 and Short Hairpin RNA against NG2

“…The levels of NT-3 measured at day 7 were lower than those observed at day 3 for slices transduced with NT-3 or with NT-3/NG2; however, they remained significantly higher than the control slices ( Figure 6). Previous studies in our group have shown that there is not a significant decrease in cell viability between day 3 and day 10 post-injury [53]. As lentiviral transduction leads to integration in the host Immunocytochemistry was performed to confirm the reduction in NG2 protein in Neu7 cells via measurement of total cell fluorescence intensity (Figure 4).…”

“…Each spinal cord was cut into pieces 1cm in length and transferred to sterile tissue chopper discs. Longitudinal sections of spinal cord were prepared and cultured as described previously [5,53]. In brief, spinal cord sections were cut to 350µm thickness on the McIIwain tissue chopper (Mickle Laboratory Engineering, Surrey, UK).…”

An In Vitro and Ex Vivo Analysis of the Potential of GelMA Hydrogels as a Therapeutic Platform for Preclinical Spinal Cord Injury

“…Historically, the SCI preclinical landscape has used in vitro, ex vivo, and in vivo models to unravel the complex multiphasic injury pathophysiology. [7][8][9] Lasting for up to 24 h, the "primary injury" phase is initiated by mechanical damage to the spinal cord and results in death of local neural and glial cells. [10,11] This triggers the "secondary injury" phase that propagates damage away from the primary injury site and lasts from weeks to months, and as a result is therapeutically targetable.…”