In the majority of rats (28 of 40) the lesion consisted of a single large cavity that gradually enlarged to only be surrounded by a thin rim of spared white matter at the level of the lesion center. At this level, a large number of DAPI positive cell bodies were present, suggestive of scar tissue that appeared to have entered the lesion cavity from the surface of the spinal cord. Concomitant with this infiltration of cells was the development of multiple cavities separated by trabeculae, spared white matter, and tissue containing graft cells. Moving caudad the lesion was gradually filled in until it was no longer visible. In the other animals, the lesion consisted of more than 1 cavity that gradually was filled in until no longer visible ( top panel).
Figure 1 Lesion region analysis. In A the top panel shows a horizontal section through the lesion in which the extent of the hPLAP positive transplant can be seen; cranial to the left. The bottom panel shows representative images of 9 of the 10 animals that received (more ...)
In all animals that received GRP cell transplants, extensive areas consisting of hPLAP positive cells were identified in the injured spinal cord at 84 days after SCI (Figures and ). In the bottom panel of , representative images are shown of the transplants of 9 of the 10 animals from which the spinal cord was sectioned transversally. Images are shown from an area in the lesion region, adjacent but not at lesion center. Grafts were present throughout the lesion region and were well integrated with the spared tissue of the host. Graft cells bordered the lesion cavity and were present in the spared tissue and trabeculae within the lesion cavity. The density of hPLAP positive cells was high in the center of the graft and gradually reduced as the graft integrated with the host tissue. The three injection sites and their respective cell deposits were not individually recognizable, as the hPLAP positive cells formed a continuous graft in 9 out of 11 animals (sectioned transversally). Needle tracts from the injection were present in 5 out of 11 animals and were limited to either the cranial (3) and/or caudal (4) injection sites.
Figure 2 Lesion center analysis. Representative lesion centers show in A = OP control, B = GRP control, C = cAMP control, and D = GRP cAMP. Sections are 20 μm thick and stained for GFAP, hPLAP, and DAPI. Green represents astrocytes (GFAP), red / yellow (more ...)
The area occupied by tissue (GFAP positive) and GRP cells (hPLAP positive) was determined from the transversally cut spinal cords from 10 animals, 35 sections were imaged per animal. The distance between consecutively imaged sections was approximately 200μm. shows that there was significantly more tissue present throughout the lesion region in animals that received a GRP cell transplant (p=0.003; F=6.9). In the volumes of the lesion region and transplants are shown. The expected cord volume not accounted for tissue loss of the lesion region is shown as well as the actual volume of the remaining tissue and the volume of the GRP transplant. demonstrates that the volume occupied by GRP cells in the lesion region in the GRP control group was significantly (p=0.047; F=5.5) larger than that in the GRP cAMP group. At the lesion center, there was no residual gray matter, but a thin rim of white matter remained (). The percentage white matter sparing was not different between groups and was 8.1 ± 1.5 % for OP control, 6.1 ± 1.4% for GRP control, 9.6 ± 1.1% for cAMP control, and 7.1 ± 2.0% for GRP camp (). The hPLAP positive area at the lesion center was significantly larger in the GRP control group compared to the GRP cAMP group (p<0.001; F=15.6; ).
At the time of sacrifice, 75 days after transplantation GRP cells expressed nestin, GFAP, CC-1, and NG2 (). Our quantification method allowed determination of cell differentiation of a total of 22,684 hPLAP positive cells (12,291 GRP control and 10,393 GRP cAMP); 5,356 were examined for nestin expression, 5,018 for GFAP expression, 5,672 for CC-1 expression, and 6,638 for NG2 expression. The proportion of hPLAP positive cells that expressed nestin was 12 ± 4% for GRP control and 10 ± 2% for GRP cAMP. The proportion of hPLAP positive cells that expressed GFAP was 60 ± 7% for GRP control and 42 ± 6% for GRP cAMP (p=0.07; F=4.4). The proportion of hPLAP positive cells that expressed CC-1 was 33 ± 5% for GRP control and 41 ± 5% for GRP cAMP. The proportion of hPLAP positive cells that expressed NG2 was 8.8 ± 1.8% for GRP control and 11.2 ± 1.4% for GRP cAMP. Examination of sections immunostained for hPLAP and PDGFRα demonstrated few cells expressing both markers, consistent with the results of immunostaining for hPLAP and NG2.
Figure 3 Differentiation of hPLAP positive GRP cells. Each confocal image (A-H) represents one optical slice from a 20 μm section. In A and B GRP cells are shown that express both hPLAP (red) and nestin (green). In C and D GRP cells are shown that express (more ...)
In all groups a significant reduction in serotonin caudal to the lesion was seen when compared to the amount cranial to the lesion (). The percentage of serotonin found caudal to the lesion compared to cranial to the lesion was 33 ± 6% for OP control, 18 ± 7% for GRP control, 3 ± 1% for cAMP control, and 9 ± 4% for GRP cAMP. In both groups that received cAMP, this reduction was significantly more (p=0.004; F=6.8) than the reduction seen in the OP control group. For 44 animals 20 cells from the DM and DL nuclei in the lumbosacral spinal cord were imaged. The location of the DM and DL nuclei is shown in , respectively. Quantification of the amount of serotonin in close proximity to these motor neurons () was significantly less following SCI when compared to the AM control group (p<0.001; F=5.4), however, no differences between groups were found ().
Figure 4 Location of dorsomedial (A) and dorsolateral nuclei (B) in the lumbosacral spinal cord, examples of motor neurons located in the dorsomedial or dorsolateral nuclei (C and D), and quantification of serotonin in close proximity to these motor neurons ( (more ...) 3.2. Functional outcomes
In the short-term study, rats were approximately 80 days and weighed 325 ± 3 g at the time of SCI. At the time of transplant, animals in the OP control group weighed significantly more than animals in the cAMP control group (304 ± 6 g and 285 ± 2 g, respectively). At the time of sacrifice (14 days post SCI) the UI control animals weighed 363 ± 4 g, which was significantly more (p<0.001) than the OP control (291 ± 6 g) and cAMP control (279 ± 1 g) animals. In the long-term study, rats were 71 ± 2 days and weighed 293 ± 1 g for transducer implantation. Rats were 77 ± 2 days and weighed 318 ± 2 g at the time of SCI. At the time of transplant and sacrifice, rats weighed 289 ± 3 g and 385 ± 6, respectively. Average weights of animals at the time of sacrifice were similar in all groups (OP control: 392 ± 11 g; GRP control: 383 ± 14 g; cAMP control: 380 ± 13 g; GRP cAMP: 391 ± 12 g); however, when grouping animals together the animals in the combined OP and GRP control groups weighed significantly more than animals in the combined GRP cAMP and cAMP control groups (295 ± 4 and 284 ± 3; p=0.05). Five animals died unexpectedly during the course of this study; 1 animal from the OP control group died on day 9 following SCI, 1 animal in the cAMP control group died on day 11 following SCI, 1 animal in the GRP control group died on day 22 following SCI, and 2 animals in the GRP cAMP group died on days 53 and 79 following SCI. Three animals (1 GRP control, 2 GRP cAMP) had episodes of autophagia that resolved spontaneously.
The BL average number of micturition events per 24 hours per rat was 22 ± 2 (OP control), 20 ± 2 (GRP control), 29 ± 2 (cAMP control), and 27 ± 1 (GRP cAMP). First spontaneous micturition events were detected by telemetry 2 – 4 days following SCI. Although in most animals recovery of micturition was sufficiently effective that manual bladder expression was no longer required, the number of micturitions per 24 hours at the endpoint of the study remained significantly lower than that at BL for all groups (OP control: p=0.03; F=12; GRP control: p=0.05; F=11; cAMP control: p=0.02; F=11; GRP cAMP: p=0.008; F=18). However, the number of micturitions at the endpoint of the study in the cAMP control (15 ± 3) and GRP cAMP (19 ± 1) groups was not lower than that recorded from the AM control group (23 ± 2; ). In contrast, the OP control (12 ± 3) and GRP control (10 ± 2) groups did have significantly fewer micturitions per 24 hours at endpoint compared to the AM control group (p=0.008; F=4.6; ). Detailed pressure waveform analysis revealed increased duration of micturition events and increased frequency of micturition pressure peaks over time (). Micturition peak frequency is a robust parameter as shown by small standard errors and little variability between groups. Also, the micturition peak frequency recorded in the AM control group was not significantly different from BL data of the other 4 groups. Comparing day 84 to BL revealed a significant increase of micturition peak frequency for the OP control and cAMP control groups (p=0.03; F=11.6 and p=0.01; F=12.7, respectively), whereas in the animals that received GRP cells there was no significant difference in this parameter. At day 84 the micturition peak frequency of the cAMP control and the GRP cAMP groups were significantly larger when compared to the AM control group (p=0.02; F=3.6).
Figure 5 Recovery of micturition function shown for all groups. A shows the estimated marginal means of the number of micturitions per 24 hours determined by ANCOVA set with the coveriate baseline (BL) at 25.3 micturitions/24h for BL and days 2, 5, 14, and 84 (more ...)
Number of full erectile events per 24 hours per rat at BL was 52 ± 3 (OP control), 63 ± 4 (GRP control), 47 ± 6 (cAMP control), and 63 ± 9 (GRP cAMP; ). At 84 days after SCI the total number of full erectile events was significantly reduced from BL in the OP control (32 ± 2; p=0.006; F=28) and cAMP control groups (30 ± 4; p=0.03; F=8.7). In contrast the reduction of events in the GRP control and GRP cAMP groups was not significant (40 ± 10 and 37 ± 7, respectively). No significant differences were present between groups or between the injured groups and the AM control group (45 ± 5 erectile events per 24h). The number of partial erectile events per 24 hours per rat at BL was 8 ± 2 (OP control), 8 ± 2 (GRP control), 7 ± 2 (cAMP control), and 13 ± 3 (GRP cAMP; ). The total number of partial erectile events on day 84 following SCI was reduced in all groups, but only in the GRP control group (3 ± 2) was this reduction not significant from BL. In the other groups there were significantly fewer partial erectile events (OP control: 3 ± 1, p=0.002, F=54; cAMP control: 3 ± 1, p=0.005, F=19; GRP cAMP: 3 ± 1, p=0.007, F=19). In all groups the number of partial erectile events per 24 hours at 84 days after SCI was not significantly lower than in the AM group (6 ± 3).
Figure 6 Recovery of erectile events shown for all groups. The estimated marginal means determined by ANCOVA with baseline being the covariate is shown for baseline (BL) and days 2, 5, 14, and 84 after SCI. In A full erectile events are shown set at BL=54 erectile (more ...)
Following SCI, latency (time of first erectile event following sheath retraction) decreased at all time points (). Although partial recovery occurred, latency remained significantly lower than BL for all groups (OP control: p<0.001; F=28; GRP control: p<0.001; F=29; cAMP control: p=0.002; F=16; GRP cAMP: p<0.001; F=61). Furthermore, latency recorded in the AM group was significantly lower than latency measured at BL in the injured groups (p<0.001; F=14). At day 86 after SCI, latency in the GRP control and GRP cAMP groups remained significantly lower than that of the AM group (p<0.001; F=14). The number of suprasystolic pressure peaks that occurred during erectile events increased from BL at all time points following SCI (). At day 86 after SCI the number of pressure peaks per event was significantly higher than at BL for the GRP control (p=0.002; F=28) and cAMP control (p=0.016; F=92) groups. Furthermore, the number of pressure peaks at day 86 following SCI remained significantly greater than that recorded in the AM control group for all groups (p<0.001; F=7.6).
Ex copulatory reflex erection test results. For all groups of rats the latency (time to first erection after sheath retraction) (A) and the number of peaks per event (B) are shown for baseline (BL) and for the duration of the study.
The 25 g-cm SCI resulted in an initial profound paraplegia in all rats characterized by BBB locomotor scores of 0.6 ± 0.3 (OP control) 0.5 ± 0.3 (GRP control) 0.4 ± 0.3 (cAMP control), and 0.6 ± 0.1 (GRP cAMP) at 24 hours after SCI. Gradually, rats showed some recovery of hind limb locomotor function (), however, no significant differences were found between groups. BBB locomotor scores were 8.2 ± 0.4 (OP control) 8.0 ± 0.6 (GRP control) 8.0 ± 0.5 (cAMP control), and 8.6 ± 0.4 (GRP cAMP) at the time of sacrifice. This remained significantly different from BL (p<0.001; F=404). In the short-term survival study, BBB locomotor scores at 14 days after SCI for the OP control (6.6 ± 1.0) and cAMP control (7.1 ± 1.1) animals were not significantly different from those in the long-term study at 16 days after SCI. The level of activity of the rats when housed in their cages, determined by telemetry, was reduced immediately after SCI (). This was followed by a steady recovery; however, the transplantation surgery at day 9 resulted in a second drop in level of activity. The level of activity reached a plateau between 3 – 5 weeks following SCI, and at the endpoint of this study was similar to that of the AM control group, which was significantly less than activity recorded for rats at BL for the OP control (p=0.03; F=9), cAMP control (p<0.001; F=36), and GRP cAMP (p=0.009; F=14) groups.
Figure 8 Recovery of locomotion function. For the 4 groups of rats the BBB locomotor score (A) and level of activity in home cage (B; AVG ± SE) are shown for baseline (BL) and for the duration of the study. The open arrow indicates time of transplantation (more ...) 3.3 cAMP concentrations
Concentrations of cAMP were determined in cranial spinal cord, lesion center, caudal spinal cord, serum, and CSF 14 days after SCI (). Lesion center [cAMP] were not significantly different between the groups, however, in the cranial sections significantly more cAMP was found in the cAMP control group vs. the UI control group (p=0.035). In addition, in the caudal sections significantly more cAMP was found in both the OP control and cAMP control groups vs. the UI control group (p=0.04 and p=0.02, respectively; ). Concentrations of cAMP measured in serum and CSF were not different between groups ().
Figure 9 Quantification of cAMP concentrations at 14 days after injury. A shows cAMP concentrations determined in a cranial section, lesion center, and caudal section of spinal cord. B shows cAMP concentrations determined in cranial spinal cord, serum, and cerebrospinal (more ...)
Here we show that hPLAP positive GRP cells transplanted at 9 days after contusion SCI exhibited robust survival and integration into host tissue for up to 85 days, and expressed markers for both oligodendrocytes (CC1) and astrocytes (GFAP). As GRP cells are immunoreactive negative for CC1 and GFAP prior to transplantation, the expression of these makers suggest in vivo differentiation of the GRP cells along the astrocytic and oligodendrocytic pathways. Extensive analysis of histopathology and neurological outcome showed some desirable effects attributable to the GRP transplant such as the increased tissue volume throughout the injured cord suggesting, again, that these transplants are neuroprotective. In addition, animals with GRP cell transplants did not have significantly increased micturition peak frequencies and they did not have a significant reduction of erectile events per 24 hours compared to baseline data at the end point of the study. On the down side, animals with GRP cells had significantly shorter latency times in reflex erection tests. The only positive effect we could attribute to cAMP treatment was that by the end of the study these animals had a similar number of micturitions per 24 hours as the age-matched control animals. Adverse effects associated with administration of rolipram and db-cAMP were plentiful and included increased body-weight loss in the acute and chronic phase after SCI and increased micturition peak frequencies compared to age-matched control animals. Perhaps surprisingly, analysis of the 12-week survival transplants also showed that in rats that were administered db-cAMP and rolipram for two weeks after SCI, a smaller area of the injured cord was occupied by hPLAP-positive cells from the donor, suggesting that this treatment reduced survival or proliferation of GRP cells. In addition, in animals that received db-cAMP and rolipram, the amount of serotonin immediately caudal to the lesion was reduced and there was a trend towards less differentiation of precursor cells into astrocytes. Although rolipram + cAMP and GRP cells affected some of the functional outcome parameters, there was no consistent beneficial effect across outcomes from either treatment or from the combination of both.
The present results are consistent with recent reports suggesting that undifferentiated GRP cells may not be therapeutically effective without further in vitro
or in vivo
treatments to promote astrocyte differentiation (Davies, et al., 2006
, Davies, et al., 2008
). Further, while treatments with db-cAMP and rolipram appear to be anti-inflammatory and may enhance neurite outgrowth and/or attenuate oligodendrocyte death in a number of in vitro
and in vivo
assays (Bregman, et al., 1998
, Cai, et al., 1999
, Pearse, et al., 2004
, Spencer and Filbin, 2004
, Whitaker, et al., 2008
), the protocol applied here in combination with GRP cells was not therapeutically effective. In addition, it may have reduced the size of the 12-week survival transplant and reduced serotonergic fibers immediately caudal to the lesion. A recent study reported beneficial effects of a one-time administration of rolipram on histopathological and functional outcome measurements after SCI only when it was administered in combination with thalidomide (Koopmans, et al., 2009
Our moderate contusion injury produced a very consistent lesion with the area of spared white matter at the lesion center in the OP control group (8.1 ± 1.5%) in the same range as described previously for this level of injury (9.9 ± 4.8%; Basso et al., 1996
). There were no group differences with respect to spared white matter or functional outcomes, consistent with the idea that these two parameters are highly correlated (Basso, et al., 1996
). Furthermore, in this study, cAMP concentrations in the spinal cord appeared to be increased 14 days after SCI, contrary to what others have shown previously (Pearse, et al., 2004
Pearse et al. (2004)
demonstrated in vivo
that spinal cord [cAMP] are reduced after SCI (up till 14 days after SCI), and showed that administration of rolipram and db-cAMP, using the same methodology as we did in our study, prevented this SCI-induced reduction of spinal cord [cAMP]. They demonstrated that the combination of Schwann cell transplants and elevation of [cAMP] following a 12.5g-cm SCI resulted in significant improvement of the BBB score at 8 weeks after transplant (injured-only group: 10.4; treated group: 15.0). At 1 week after transplant the injured-only group had a BBB score of 7 vs 10.5 in the treated group but no BBB scores were reported prior to 1 week after transplant. This treatment strategy promoted supraspinal and propriospinal axon sparing and myelination, as well as serotonergic fiber growth into and beyond the graft. The extent of the transplant grafts within the injury lesion region and whether or not this was affected by cAMP were not quantified. Noting that Schwann cells are very different from the progenitor cells we used here, the results still suggest that the effects of cAMP and rolipram on progenitor cell proliferation and survival warrant further careful study if this approach is to be used in other combination strategies for repairing SCI. Another study by this group showed that treatment with rolipram improved functional recovery, promoted axonal growth, and attenuated astrogliosis (Nikulina, et al., 2004
). This is particularly interesting in light of our findings of slightly reduced differentiation of GRP cells into astrocytes. Perhaps reduced activation of endogenous astrocytes and reduced glial scarring is produced through similar mechanisms of cAMP that alter lineage differentiation of GRP cells. In our study less hPLAP-GFAP positive cells were seen in the animals that received db-cAMP and rolipram, and although more hPLAP-CC-1 positive cells were seen in the GRP cAMP group, this was not statistically significant and there was no beneficial effect on functional outcome. With increased availability of oligodendrocytes, one would expect to see enhanced re-myelination and potentially improved functional outcome. However, it is possible that even if the proportion of hPLAP-CC-1 positive cells was larger, the total number of hPLAP-positive oligodendrocytes would not have been increased in our GRP cAMP group since cAMP appeared to have a negative effect on GRP cell transplant size. In addition, we identified very few hPLAP-NG2-positive and hPLAP-PDGFRα-positive cells, suggesting that few donor cells were differentiating into oligodendrocyte precursor cells.
As far as the authors are aware, our study is the only one that has quantified extent of transplant within the injured spinal cord. Certainly there appear not to be any other reports on the effect of cAMP on survival of transplanted cells within the injured spinal cord. Our study suggests that administering db-cAMP and rolipram is not necessarily beneficial to survival of GRP cells. This is unlikely to be due to the altered immune response that is caused by cAMP, since it has been well established that cAMP has predominantly anti-inflammatory effects. Macrophages failed to differentiate into activated macrophages when exogenous cAMP was added in an in vitro
model (Peters, et al., 1990
) and phagocyte function of macrophages was inhibited by cAMP (Aronoff, et al., 2005
, Peters, et al., 1990
). However, numerous reports have shown that cAMP is involved in regulation of cell survival, in particular of neoplastic and progenitor cells.
In B-cell chronic lymphocytic leukemia (B-CLL) PDE-IV is the predominant PDE expressed in the neoplastic cells. Rolipram induces apoptosis in B-CLL cells through a cAMP dependent and caspase dependent manner. Rolipram increases the efficacy of glucocorticoid-mediated apoptosis in B-CLL cells by modulating glucocorticoid receptor signal transduction, a process that requires PKA (Tiwari, et al., 2005
). Interestingly, this up-regulation of glucocorticoid receptor transcript levels does not take place in normal circulating hematopoietic cells, or in cells of T-cell chronic lymphocytic leukemia (T-CLL) (Meyers, et al., 2009
, Meyers, et al., 2007
). Also, rolipram was found to suppress the anti-apoptotic members of the Bcl-2 family and induce the pro-apoptotic protein Bax. Combining these mechanisms results in a shift of the balance between pro- and anti-apoptotic members of the Bcl-2 family towards a pro-apoptotic direction (Siegmund, et al., 2001
). Both acute and chronic lymphoblastic leukemia cells undergo apoptosis following administration of rolipram. Contributing mechanisms include G1 and G2/M cell cycle arrest (Ogawa, et al., 2002
), mitochondrial depolarization, release of cytochrome c into the cytosol, and caspase-9 and -3 activation (Moon and Lerner, 2003
). Elevation of cAMP concentration has also been shown to inhibit cell growth and induce apoptosis in other cancer cell lines such as retinoblastoma cells (Fassina, et al., 1997
), papilloma cells (Marko, et al., 1998
), glioma cells (Chen, et al., 2002
), neuroblastoma cells (Kumar, et al., 2004
), and esophageal cancer cells (Wang, et al., 2005
). In cancer cell lines this effect of PDE-IV inhibition is an obvious area of interest since halting growth of these cells is a much-desired therapeutic goal. Furthermore, in the treatment of bronchial asthma, PDE inhibition is thought to be an important mechanism of the anti-inflammatory actions of theophylline. Theophylline and rolipram have been shown to increase eosinophil intracellular cAMP concentrations and inhibit eosinophil survival (Momose, et al., 1998
, Takeuchi, et al., 2002
, Wang, et al., 2005
). Recently it has been demonstrated that cAMP elevation resulted in significant inhibition of colony growth and induced apoptosis of progenitor cells in asthmatics, but not in normal subjects. These effects were not limited to the eosinophil lineage alone (Wang, et al., 2003
). A number of possible mechanisms for this are internucleosomal DNA cleavage, G1 cell arrest, and effects through CREB gene regulation. Another potential mechanism for the pro-apoptotic effect of cAMP is given in a recent study of pulmonary hypertension. Pulmonary arterial hypertension is a proliferative vascular disease characterized by aberrant regulation of smooth muscle cell proliferation and apoptosis in distal pulmonary arteries. Elevating cAMP concentrations through administration of PDE inhibitors suppressed proliferation and matrix metalloproteinase activity and promoted apoptosis in these cells. One of the important mechanisms appeared to be attenuation of DNA synthesis (Growcott, et al., 2006
). These studies, and the results of our study, suggest that (progenitor) cell survival should be carefully examined in treatment strategies that include the use of elevating cAMP.
In the present study, GRP cells alone did not improve locomotor and only modestly improved some autonomic behavioral outcomes. Similarly, Davies et al (2006)
showed no beneficial effects from transplantation of undifferentiated GRP cells, however transplantation of GRP-derived astrocytes (GDAs) resulted in improvement of histopathological and locomotor outcomes. Other studies examining the effects of different glial progenitor cells such as the human embryonic oligodendrocyte precursor cells (OPCs) (Keirstead, et al., 2005
) and oligodendrocyte precursors (Bambakidis and Miller, 2004
) have demonstrated positive effects on locomotor outcome, but these studies were conducted in less severe SCI models. One elegantly performed study in male rats of similar weight as ours, demonstrated a positive effect of transplanting oligodendrocyte-type 2 astrocyte progenitors (O-2A) into the lesion center on locomotor recovery after a 25 g-cm SCI (Lee, et al., 2005
). Although a significant difference was found in BBB score at 6 weeks after transplantation (9.5 in operated control animals vs. 12.3 in treated animals), few significant effects of transplantation were seen during electrophysiological assessment of these animals. The O-2A cells used in that study differentiated into oligodendrocytes that had the ability to promote remyelination and regeneration of axons. Differences between GRP cells and O-2A cells include different developmental age of isolation, different chemokine response patterns, different responses to inducers of differentiation, and different behaviors of the GRP cells versus O-2As following transplantation (Rao, et al., 1998
). The latter is most striking and perhaps reflects the embryonic nature of the GRP cells that have the potential to differentiate into two distinct types of astrocytes and O-2A cells (Gregori, et al., 2002
). In contrast, the postnatally generated O-2A cells predominantly differentiated into oligodendrocytes while their ability to generate astrocytes is still controversial (Noble, et al., 2004
). Differences in characteristics of these progenitor cells undoubtedly affect eventual functional outcome and need to be studied in detail in future experiments. In addition to finding the best progenitor cell characteristics to use in the development of transplantation strategies, investigators also need to consider the occurrence of harmful side effects. Davies et al (2008)
saw evidence of allodynia with GRP cells and GDAs and, although we did not formally test for this as we have done in other studies (Lindsey, et al., 2000
), we did not see obvious features of allodynia or hyperreflexia related to GRP cell transplants in this study. We note, however, that the effects reported in Davies et al (2008)
are quite modest, and may not have been severe enough to affect sensation to the degree that we would notice it in the normal handling of the animals. The reports from Davies et al (2006
) strongly suggest that future studies should employ these tests.
Recovery of autonomic function after transplantation strategies has been studied by Mitsui et al.
using fibroblasts engineered to secrete brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3 (Mitsui, et al., 2005
) and a combined application of NRP and GRP cells (Mitsui, et al., 2005
). In these studies, rats were observed in metabolic cages and recovery of micturition from the period of bladder areflexia appeared accelerated in the treatment groups; however treatment differences were no longer apparent by 4 weeks after transplantation. Both of these treatments appeared to reduce signs of detrusor sphincter dyssynergia and detrusor hyperreflexia as determined by cystometry and determination of bladder weights. In the combined NRP GRP transplant study, rats underwent a protocol similar to ours in which SCI was followed by transplantation of precursor cells in 3 sites of the lesion at 9 days after injury. Transplant injections in the dorsal midline of the spinal cord were performed using a Hamilton syringe with a tip of 310μm which is significantly larger than what we used (180μm) and transplanting a combination of NRP and GRP cells resulted in improvement of the BBB locomotor score from 7.1 (operated control group) to 9.4 (NRP GRP group). The comparatively low BBB score seen in the operated control group was attributed to the modified-moderate contusion injury that is used, in which the impactor rod is left on the spinal cord for an additional 5 sec following a 25 g-cm contusion (Mitsui, et al., 2005
). Interestingly, in the previous study that demonstrated a positive effect of transplanting fibroblasts expressing BDNF and NT-3 on recovery of locomotor function (BBB from 3.5 to 8.2), (Mitsui, et al., 2005
), this modified-moderate contusion model resulted in BBB scores of 3.5 in the operated control group at 8 weeks after transplant. The fibroblast study was performed using a similar experimental paradigm as the NRP GRP study. Considering the difference in BBB locomotor scores between the 2 groups (3.5 vs 8.2) after transplant of genetically modified fibroblasts, it is perhaps not surprising other behavioral outcomes improved as well. Development of thermal hypersensitivity was not affected by this treatment. In addition, both of these studies reported increased serotonergic input to the DL nucleus in the lumbosacral spinal cord; however from the data shown it is unclear what nucleus was examined for serotonin (Mitsui, et al., 2005
). Although in our study the number of micturitions per 24 hours, micturition duration, and erectile event duration appeared to have recovered more in the GRP cAMP treatment groups, none of these outcomes were unequivocally positively affected by our intervention. We did not find significant beneficial effects of our transplantation strategy on micturition or sexual outcome measurements. Another recent study, demonstrated only very slight improvement of some urodynamic bladder function outcomes following transplantation of either a combination of NRP and GRP cells or bone marrow stromal cells (Temeltas, et al., 2009
). Locomotor outcomes and histopathological quantification of transplant size and white matter sparing were not provided. The same group also showed improvement in erectile function outcomes using the same treatment (Temeltas, et al., 2009
). GRP cells have been shown to have beneficial effects on locomotor recovery when they are genetically modified to express multineurotrophins (Cao, et al., 2005
). The experimental paradigm in this study was similar to our study and the studies conducted by Mitsui et al.
(2000a and 2000b), in that transplantation was performed 9 days after SCI. However, the level of injury (150 kdyn; Infinite Horizon device) was much less severe and resulted in BBB scores of 10 – 12 prior to transplant. This strategy resulted in BBB scores of 12.2 – 15.6 (operated control and multineurotrophin-expressing GRP group, respectively) at 6 weeks after SCI.
In this study we used telemetry to monitor micturition and erectile events in rats as a measurement of autonomic function. We believe this technique is very useful because anesthesia, animal handling, or animal restraint does not affect the results. Although we found some significant differences in some of the measurements, there was no unequivocal positive outcome of the applied treatment, similar to the results we showed for the locomotor function outcome. Telemetry also provided us with a measurement of activity in the home-cage, which appears a useful indicator of gross voluntary movement. This measurement was the only one that showed clear deterioration of the recovery curve at the time of the transplantation surgery. Telemetry is a valuable system to obtain data on recovery of spontaneous functions, in particular autonomically mediated outcomes.
In conclusion, our study showed modest beneficial effects on some functional outcome measurements of transplanting GRP cells and administering cAMP after SCI. Although elevation of cAMP has been reported to have beneficial effects in neural regeneration and may have beneficial effects on lineage differentiation of GRP cells, from our study it appears that cAMP is not always reduced after SCI and that administering rolipram and db-cAMP may reduce survival or proliferation of GRP cells following transplantation into the injured spinal cord. Continued research should be undertaken to specifically study the effects of cAMP on endogenous and exogenously administered progenitor cells and future studies should examine effects of combined treatment strategies, such as this one, for SCI. Transplant strategies appear promising; however it seems appropriate to use combinatorial strategies in which cell transplants are combined with other therapies such as treatments that induce differentiation of progenitor cells into populations with desired functional characteristics, treatments that affect regeneration, and/or treatments that alter the spinal cord milieu. It is important that investigators critically examine effects and interactions of all these treatments, not only on functional outcome measurements but also on histopathological features. Furthermore, effects of the complimentary treatments such as administered neurotrophins or cAMP administration may have different effects in normal adult cells vs. in progenitor cells such as used in current transplantation strategies.
- GRP cells survived, differentiated, and formed extensive transplants.
- GRP cells and cAMP had modest positive effects on micturitions and erections.
- cAMP reduced the graft size throughout the lesion region and at the lesion center.
- Serotonin immediately caudal to the lesion was reduced in the cAMP groups.