This study was designed to investigate the effects of a neonatal mNPC intervention on the cognitive and neuroanatomical changes that occur in DS, compared to sham saline and untreated controls. The implantation of mNPC may not be a viable early intervention for DS at this time. Despite evidence of some long-term survival, there were no reliable improvements in cognitive function.
Both the trisomic and disomic neural environments allowed for the survival of implanted mNPC throughout the changes associated with neonatal development, adolescence, and adulthood. While the rate of survival was low, the proportion of animals with surviving implanted cells was higher than those previously reported 
. Further, a greater proportion of trisomic brains had surviving cells than disomic brains, even though the disomic brains had greater total numbers surviving and greater migration. Although the brain is a partially immunologically privileged site, the limited survival of implanted cells remains a significant challenge in the field of cell transplantation research. We had hypothesized that by implanting at PND2, we might overcome the low post-implantation survival rate of NPC reported in many adult implant studies 
. At this time point, we were able to prevent the formation of a glial scar, another limitation to effective transplantation, but did not increase the number of surviving implanted cells compared to these previous studies.
Earlier studies suggest that implanted NPC migrate to sites of damage in the injured brain by following chemokine signals 
. The presence of implanted mNPC in certain areas of the brain may indicate a need for support in those areas. In the current study, we found that the implanted mNPC had migrated throughout the brain in both karyotypes. However, trisomic brains contained more mNPC at the site of implantation, the hippocampus, than did disomic brains. This may indicate that the hippocampus in trisomic mice is especially compromised, but not enough mNPC survived to affect cognition. Alternatively, the low migration rate may suggest that implanted mNPC did not or were unable to migrate readily in the trisomic host environment.
One mechanism we investigated for producing long-term change in the trisomic brain was the potential for mNPC to decrease the DG hypocellularity reported in Ts65Dn mice 
. A karyotype specific hypocellularity was not found in this study. This could result from differences in quantification methods or, more likely, the postnatal handling during DM assessment, which can increase the retention of immature and mature DG neurons 
. Despite the lack of karyotype controlled changes, implantation with mNPC, but not saline, resulted in an increased density of dentate granule cells. We did not find evidence that implanted mNPC differentiated into granule cells, as the total number of mNPC found in this area could not account for the 33% increase in granule density measured in implanted animals. This would suggest that the effects of the mNPC are indirect, facilitating an environment that promotes endogenous DG neurogenesis, a phenomenon that has been previously reported in the aged hippocampus implanted with NPC 
. This ability to increase the density of the DG may have beneficial effects in older Ts65Dn mice, which also have fewer neurons in the DG 
No significant effects of mNPC implantation on cognition were found. It is possible that the low number of surviving cells was not enough to affect cognition. While some individual animals that received implantations of either saline or mNPC had impressive performances in some behavior tasks, the effects were not consistent within karyotypes, treatments, or across tasks. The limited survival of implanted cells precluded statistically meaningful correlations with behavior data, and the degree to which survival corresponded with improved performance on behavioral tasks was inconsistent.
The current study confirmed the cognitive deficits of trisomic Ts65Dn mice compared to disomic mice in Developmental Milestones, Plus Maze, MWM, and CTA measures of cognition. The treatment with mNPC did not enhance the cognitive function of either trisomic or disomic mice. Low surviving numbers of mNPC and modest changes in granule cell density may not have been sufficient to produce changes in behavior. However, while the implanted mNPC did not affect the cognitive abilities of the trisomic mice in adulthood, it is possible they will offer some protection against the secondary cognitive decline of Alzheimer's disease that emerges in DS 
. The modest improvement in granule cell density and novel alternations in the Plus maze could be suggestive of differences that will be significant with time. These results contribute to a growing foundation of information on developmental delays and cognitive impairments in the Ts65Dn mouse, confirming the fidelity of this model with previous studies and providing insight into possible alternative treatment innovations in the future.