On CNS injury and olfactory ensheathing cell engraftment strategies

The intrinsic regenerative capacity of the adult mammalian central nervous system (CNS) is limited because of lack of nerve-growth stimulatory factors and presence of an insurmountable molecular environment for injured axons. This thesis aims to study neural regeneration following adult CNS injury, using rodent models of spinal cord injury, dorsal root avulsion, and Parkinson s disease. A focus is cell therapy with olfactory ensheathing cells (OEC), a unique type of glial cells assisting regeneration of olfactory axons from the periphery into the otherwise non-permissive CNS throughout adulthood.

To elucidate roles of intercellular communication via gap junctions after spinal cord injury, we investigated the expression patterns of principle gap junctional genes and proteins, connexin 43 (Cx43), Cx36, and Cx32. After a transection of the adult spinal cord, the levels of Cx43 mRNA and protein were upregulated within hours and lasted over 4 weeks post-injury primarily in astrocytes. In contrast, Cx36 and Cx32 mRNA and proteins were relatively sparse and mainly unchanged after spinal cord injury along the entire axis of the spinal cord. We suggest that long-term up-regulation of Cx43 may be one critical component in the rearrangement of the local astroglial network following spinal cord injury.

A growing number of repair strategies have shown neuroprotection/regeneration potential in CNS injury, including the use of various cell transplants, neurotrophic factors, blockade of inhibitory constituents, and stimulation of intrinsic growth capacity. Nevertheless, the degree of functional recovery is generally modest when individual approaches have been applied. Combinatorial treatments targeting multiple independent mechanisms may have additive effects. We evaluated the effects of OEC transplants alone and in combination with a cocktail treatment including acidic fibroblast growth factor (aFGF), chondroitinase ABC, and rolipram after spinal cord injury. Improvements of locomotor and sensory recovery were observed in behavioral tests and functional magnetic resonance imaging (MRI) in OEC-transplanted, cocktail-treated, and combination-treated rats. However, there was no robust evidence that the combination of OEC and the cocktail led to additive effects.

A lack of specific markers of OEC has hindered studies assessing survival and function of OEC following transplantation. We examined the usefulness of superparamagnetic iron oxide nanoparticles (SPION) as a cell label to allow in vivo tracking of grafted OEC by MRI. We found that labeled OEC were readily detectable in vivo for at least 2 months with extensive migration in normal spinal cord as shown by MRI and histological markers. However, OEC showed limited migration in injured spinal cord and were not able to cross a glial scar region. In a similar experiment, we tracked grafted neural s tem cells labeled by gold-coated SPION in the rat spinal cord. Gold-coated SPION exert powerful contrast-enhancing properties making it possible to detect cellular clusters of as few as about 20 cells.

In the model of dorsal root avulsion, we investigated whether peripheral nerve grafts combined with aFGF could improve reconnection of transected dorsal roots with the spinal cord as evaluated by somatosensory evoked potentials (SEPs). Four to twenty weeks after rhizotomy, most rats receiving such treatment had recovery of SEPs while none of the controls showed such recovery. The reappearing SEPs were eliminated by re-transection of the repaired roots, verifying their source as the regenerated roots.

In the unilateral 6OHDA denervation model of Parkinson's disease fetal ventral mesencephalic tissue (VM) grafts can partly restore dopamine innervation and counteract functional deficits, but the approach is hampered by limited graft survival and restricted dopaminergic reinnervation of striatum. We co-grafted VM with OEC to examine if OEC might promote survival and/or growth of grafted VM. The presence of OEC co-grafts improved dopamine cell survival, striatal reinnervation and functional recovery compared to VM graft alone, and caused a strikingly long-distance growth of graft-derived dopamine nerve fibers. Comparable results were observed in a co-culture system where OEC promoted dopamine cell survival and neurite elongation through mechanisms involving both releasable factors and direct cell contacts.

List of scientific papers

I. Lee IH, Lindqvist E, Kiehn O, Widenfalk J, Olson L (2005). Glial and neuronal connexin expression patterns in the rat spinal cord during development and following injury. J Comp Neurol. 489(1): 1-10.
https://pubmed.ncbi.nlm.nih.gov/15977163

II. Lee LM, Huang MC, Chuang TY, Lee LS, Cheng H, Lee IH (2004). Acidic FGF enhances functional regeneration of adult dorsal roots. Life Sci. 74(15): 1937-43.
https://pubmed.ncbi.nlm.nih.gov/14761674

III. Lee IH, Bulte JW, Schweinhardt P, Douglas T, Trifunovski A, Hofstetter C, Olson L, Spenger C (2004). In vivo magnetic resonance tracking of olfactory ensheathing glia grafted into the rat spinal cord. Exp Neurol. 187(2): 509-16.
https://pubmed.ncbi.nlm.nih.gov/15144877

IV. Wang FH, Lee IH, Holmstrom N, Yoshitake T, Kim DK, Muhammed M, Frisen J, Olson L, Spenger C, Kehr J (2005). Magnetic resonance tracking of nanoparticle-labelled neural stem cells in the rat spinal cord. [Manuscript]

V. Johansson S, Lee IH, Olson L, Spenger C (2005). Olfactory ensheathing glial co-grafts improve functional recovery in rats with 6-OHDA lesions. Brain. Oct 26: Epub ahead of print.
https://pubmed.ncbi.nlm.nih.gov/16251218

VI. Lee IH, Endo T, Nissen J, Dominguez C, Lilja J, Xu X, Cao YH, Wiesenfeld-Hallin Z, Spenger C, Olson L (2005). Olfactory ensheathing glia alone or combined with aFGF, chondroitinase and rolipram promote functional recovery after severe spinal cord contusion. [Manuscript]