Engraftment, migration and differentiation of neural stem cells in the rat spinal cord following contusion injury.

BACKGROUND AIMS: Spinal cord injury is a devastating injury that impacts drastically on the victim's quality of life. Stem cells have been proposed as a therapeutic strategy. Neural stem (NS) cells have been harvested from embryonic mouse forebrain and cultured as adherent cells. These NS cells express markers of neurogenic radial glia. METHODS: Mouse NS cells expressing green fluorescent protein (GFP) were transplanted into immunosupressed rat spinal cords following moderate contusion injury at T9. Animals were left for 2 and 6 weeks then spinal cords were fixed, cryosectioned and analyzed. Stereologic methods were used to estimate the volume and cellular environment of the lesions. Engraftment, migration and differentiation of NS cells were also examined. RESULTS: NS cells integrated well into host tissue and appeared to migrate toward the lesion site. They expressed markers of neurons, astrocytes and oligodendrocytes at 2 weeks post-transplantation and markers of neurons and astrocytes at the 6-week time-point. NS cells appeared to have a similar morphologic phenotype to radial glia, in particular at the pial surface. CONCLUSIONS: Although no functional recovery was observed using the Basso Beattie Bresnahan (BBB) locomotor rating scale, NS cells are a potential cellular therapy for treatment of injured spinal cord. They may be used as delivery vehicles for therapeutic proteins because they show an ability to migrate toward the site of a lesion. They may also be used to replace lost or damaged neurons and oligodendrocytes.

Effect of cyclosporin A on functional recovery in the spinal cord following contusion injury.

Considerable evidence has shown that the immunosuppressant drug cyclosporin A (CsA) may have neuroprotective properties which can be exploited in the treatment of spinal cord injury. The aim of this study was to investigate the cellular environment within the spinal cord following injury and determine whether CsA has an effect on altering cellular interactions to promote a growth-permissive environment. CsA was administered to a group of rats 4 days after they endured a moderate contusion injury. Functional recovery was assessed using the Basso Beattie Bresnahan (BBB) locomotor rating scale at 3, 5 and 7 weeks post-injury. The rats were sacrificed 3 and 7 weeks post-injury and the spinal cords were sectioned, stained using histological and immunohistochemical methods and analysed. Using stereology, the lesion size and cellular environment in the CsA-treated and control groups was examined. Little difference in lesion volume was observed between the two groups. An improvement in functional recovery was observed within CsA-treated animals at 3 weeks. Although we did not see significant reduction in tissue damage, there were some notable differences in the proportion of individual cells contributing to the lesion. CsA administration may be used as a technique to control the cell population of the lesion, making it more permissive to neuronal regeneration once the ideal environment for regeneration and the effects of CsA administration at different time points post-injury have been identified.

Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations.

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 µm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).