Glial restricted precursor cell transplant with cyclic adenosine monophosphate improved some autonomic functions but resulted in a reduced graft size after spinal cord contusion injury in rats

Abstract

Transplantation of glial restricted precursor (GRP) cells has been shown to reduce glial scarring after spinal cord injury (SCI) and, in combination with neuronal restricted precursor (NRP) cells or enhanced expression of neurotrophins, to improve recovery of function after SCI. We hypothesized that combining GRP transplants with rolipram and cAMP would improve functional recovery, similar to that seen after combining Schwann cell transplants with increasing cAMP. A short term study, (1) uninjured control, (2) SCI + vehicle, and (3) SCI + cAMP, showed that spinal cord [cAMP] was increased 14 days after SCI. We used 51 male rats subjected to a thoracic SCI for a 12-week survival study: (1) SCI + vehicle, (2) SCI + GRP, (3) SCI + cAMP, (4) SCI + GRP + cAMP, and (5) uninjured endpoint age-matched control (AM). Rolipram was administered for 2 weeks after SCI. At 9 days after SCI, GRP transplantation and injection of dibutyryl-cAMP into the spinal cord were performed. GRP cells survived, differentiated, and formed extensive transplants that were well integrated with host tissue. Presence of GRP cells increased the amount of tissue in the lesion; however, cAMP reduced the graft size. White matter sparing at the lesion epicenter was not affected. Serotonergic input to the lumbosacral spinal cord was not affected by treatment, but the amount of serotonin immediately caudal to the lesion was reduced in the cAMP groups. Using telemetric monitoring of corpus spongiosum penis pressure we show that the cAMP groups regained the same number of micturitions per 24 hours when compared to the AM group, however, the frequency of peak pressures was increased in these groups compared to the AM group. In contrast, the GRP groups had similar frequency of peak pressures compared to baseline and the AM group. Animals that received GRP cells regained the same number of erectile events per 24 hours compared to baseline and the AM group. Since cAMP reduced the GRP transplant graft, and some modest positive effects were seen that could be attributable to both GRP or cAMP, future research is required to determine how cAMP affects survival, proliferation, and/or function of progenitor cells and how this is related to function. cAMP may not always be a desirable addition to a progenitor cell transplantation strategy after SCI.

Introduction

Endogenous repair has been shown to occur following contusion spinal cord injury (SCI; (Beattie et al., 1997, Mothe and Tator, 2005, Namiki and Tator, 1999, Vaquero et al., 1981, Wallace et al., 1987), however, the response of the endogenous progenitor cell population is insufficient and does not lead to adequate recovery (Beattie et al., 1997, Horner et al., 2000, Mothe and Tator, 2005, Namiki and Tator, 1999). This insufficient endogenous response may be due to insufficient numbers of progenitor cells, the hostile milieu of the lesion environment, lack of neurotrophic stimulation, absence of permissive substrates, and presence of inhibitory factors (Cao et al., 2002, Horner and Gage, 2000, Popovich et al., 1999, Schmidt and Leach, 2003). Our laboratory has previously shown that when glial restricted precursor (GRP) cells, derived from transgenic embryonic rats harboring the heat-stable human placental alkaline phosphatase (hPLAP) gene (Rao et al., 1998), are transplanted into acute thoracic contusion SCI, the genetically-identified donor cells survived 6 weeks and differentiated into cells with glial markers, including CC-1 and glial fibrillary acidic protein (GFAP). In addition, these cells appeared to integrate into the white matter, produced some apparent remyelination, and modulated the inflammatory response and the formation of a glial scar (Hill et al., 2004). Furthermore, GRP cell transplants in combination with neuronal restricted precursor (NRP) cells from the same embryonic stage, and genetically modified multineurotrophin expressing GRP cells, have been shown to improve functional recovery after spinal cord lesions (Cao et al., 2005, Mitsui et al., 2005b). Transplantation of astrocytes derived from bone morphogenetic protein-4 (BMP-4) treated GRP cells have also been shown to result in significant histopathological and functional improvement (Davies et al., 2006, Davies et al., 2008). Also, combining transplantation of neural precursor cells with growth factors and anti-inflammatory and immunosuppressive treatment, enhanced survival of donor cells and improved functional recovery (Karimi-Abdolrezaee et al., 2006). More recently, a group has shown positive effects of GRP/NRP and bone marrow stromal cell transplantations on bladder and sexual function after SCI (Temeltas et al., 2009a, Temeltas et al., 2009b).

cAMP has been shown to play an important role in axon regeneration in the adult central nervous system, largely in part through downstream effects of cAMP-dependent protein kinase (PKA), that ultimately help overcome the inhibitory cues of the injured spinal cord milieu (Bregman et al., 1998, Cai et al., 1999, Gao et al., 2004, Hannila and Filbin, 2008, Spencer and Filbin, 2004). The phosphodiesterase (PDE)-IV inhibitor rolipram increases intracellular cAMP and cGMP concentrations in various tissues, and mainly in monocytes, macrophages, granulocytes, T-lymphocytes, and dopaminergic and adrenergic nerve cells. Furthermore, rolipram has the ability to suppress pro-inflammatory cytokines (predominantly TNF-α) and chemokines (Zhu et al., 2001). Recent evidence suggests that rolipram not only enhances axonal regeneration, but also protects against apoptotic insults, for example by reducing caspase-3 activity (Chen et al., 2007). In addition rolipram reduces oligodendrocyte death at 24–72 hours after SCI (Whitaker et al., 2008).

Pearse et al. (2004) demonstrated in vivo that SCI results in a reduction of spinal cord cAMP concentrations, and that this could be prevented by exogenous administration of rolipram and dibutyryl (db) -cAMP. Additionally, the increase of TNF-α that occurred within the first 6 hours following SCI was retarded in animals that received rolipram. Moreover, he showed that the combination of Schwann cell transplants and elevation of cAMP after SCI resulted in significant improvement of locomotor function and promoted supraspinal and propriospinal axon sparing and myelination, as well as serotonergic fiber growth into and beyond the graft (Pearse et al., 2004). Another study showed similar beneficial effects after transplanting embryonic spinal tissue and administering rolipram in a hemisection model of SCI (Nikulina et al., 2004). These findings suggest a beneficial role for elevating cAMP in the injured spinal cord.

In the present work, we sought to use the Pearse et al. (2004) strategy aimed at elevating cAMP concentrations to determine whether we could enhance the therapeutic potential of GRP cells transplanted into subacute lesions, 9 days after mid-thoracic SCI. In order to monitor a wider variety of functional outcomes, we included in-cage monitoring of bladder and sexual function to the functional testing database. Autonomically mediated functions, such as urogenital tract function, following SCI and its subsequent complications are highly prevalent and clinically very important (Anderson, 2004, Hicken et al., 2001, Noreau et al., 2000). Recently, we developed a method to assess recovery of micturition and erectile function in conscious freely moving rats by monitoring corpus spongiosum penis (CSP) pressure using telemetry (Nout et al., 2007, Nout et al., 2005). In the following study we determine recovery of both micturition and erectile events following delayed transplantation of GRP cells and administration of local db-cAMP and systemic rolipram after SCI. Furthermore, detailed histopathology allows assessment of graft characteristics and fate of transplanted cells.