The effect of grafted neuroectodermal stem cells on injured spinal motoneurons following ventral root avulsion and reimplantation: insights into the molecular mechanism Ph.D. thesis

signaling by grafted neuroectodermal stem cells rescues motoneurons destined to die. Summary Avulsion of one or more ventral roots from the spinal cord leads to the death of the majority of affected motoneurons. This process is due to a cascade of events involving activation of astrocytes and microglial cells and the excessive amounts of excitotoxic glutamate release in the injured cord. The aim of the present study was to analyse and compare the therapeutic potential of transplanted NE-GFP-4C murine neuroectodermal cells applied in topically different transplantation paradigms and determine the factors responsible for the motoneuron-rescuing effect. The lumbar 4 (L4) ventral root of Sprague-Dawley rats was avulsed and reimplanted ventrolaterally into the injured cord. Neuroectodermal stem cells were injected immediately following avulsion injury into the L4 segment, into the reimplanted ventral root or were placed in fibrin clot around the reimplanted root. Three months after the primary surgery the L4 motoneuron pool was retrogradely labelled with Fast Blue and the numbers of reinnervating motoneurons were determined. Expression of various factors expected to prevent motoneuron death in the grafted cord was determined by PCR and immunohistochemistry in short term experiments. Animals that received intraspinal stem cell grafts have 70% of their L4 motoneurons regenerated into the vacated endoneural sheaths of the reimplanted root. Morphological reinnervation was accompanied by significant functional recovery. Intraradicular neural stem cell grafting (transplantation into the reimplanted root) resulted in good morphological and functional reinnervation, while both negative controls and animals with perineural stem cell treatment showed poor motor recovery. Stem cell grafts produced the modulatory cytokines IL-1-alpha, IL-6, IL-10, TNF-alpha and MIP-1-alpha, but no neurotrophic factors. The neurons and astrocytes in the ventral horn of grafted animals also produced IL-6 and MIP-1-alpha. The infusion of function-blocking antibodies against all cytokines into the grafted cords completely abolished their motoneuron-rescuing effect, while neutralization of only IL-10 suggested its strong effectivity as concerns motoneuron survival and a milder effect on reinnervation. In this study we have provided evidence that significant numbers of motoneurons can be rescued both by intraradicular and intraspinal stem cell grafting. The pro-inflammatory and anti-inflammatory cytokines selectively secreted by grafted stem cells act in concert to save motoneurons and to promote reinnervation of the target muscles.