The results of the present study indicated that the transplantation of NSC and MSC decreased the number of Aβ plaques in the hippocampus of APP/PS1 mice, although cell transplantation was not sufficient to diminish the size of plaques as no difference was observed between APP/PS1 groups when the plaque area was analyzed. In addition, NSC induced an increase in microglial cells in the hippocampus compared to the APP/PS1 group. Also, NSC were able to reverse the hyperlocomotion present in APP/PS1 animals, although both NSC and MSC cells did not affect the object recognition memory.
Even showing different characteristics, both cell types, NSC and MSC, were able to decelerate the buildup of new plaques reducing the amount of Aβ plaques. Considering that MSC were not found in the hippocampus six weeks after surgery (data not shown), it is plausible that the cell infusion produced an immunomodulatory effect by paracrine action, reducing neuroinflammation that, in turn, could inhibit the formation of new Aβ plaques. Reduced numbers of plaques caused by NSC transplantation has other underlying mechanisms, however. NSC express BACE2, a degrading enzyme of Aβ peptide able to cleave the existing Aβ proteins, destroying the molecule (25). In addition, NSC has been proposed to act as CNS immunomodulator, allowing homeostasis, maintaining and repairing tissue, not only structurally but also functionally (26). NSC transplantation in AD helps preparing the microenvironment, releasing immunoregulatory factors capable of modulating the progression of neuroinflammation (27).
Neuroinflammation was examined by the quantitation of microglial cells expressing Iba-1 protein. This marker belongs to a family of proteins with Ca²+ binding domains, with significant activity in cell signaling in the CNS. Iba-1 is found specifically in microglial cells, and in the case of inflammation there is an increase in expression due to cell activation. In fact, APP/PS1 + NSC group, together with the reduction in Aβ plaques, also showed a significant increase in hippocampal microglia. As the transplanted cells did not differentiate into microglia, it is reasonable to think that the number of microglia increased in NSC-treated mice as a consequence of an intensification of Aβ hippocampal levels, causing them to have a positive effect on the number but not on the size of the plaques in the hippocampus. Previous results corroborate our findings that microglial clusters around Aβ plaques may be benefic, suggesting that microglia could phagocyte the plaques, not allowing formation of new ones or effectively reducing their size (28, 29, 30). However, once the disease progresses, microglial function is reduced and, therefore, Aβ plaques continue to increase. Studies suggest that the phagocytic products of the plaques are not metabolized by microglia, which in turn enters an apoptotic process and releases the amyloid load back to the extracellular matrix, possibly forming new plaques (31). These high concentrations of microglia were preferentially found surrounding the Aβ plaques after NSC transplantation, which is in accordance with the suggestion that microglia are activated by the presence of Aβ proteins, clustering around it to secrete cytokines, neurotoxins and to phagocyte the plaques (32).
Different results were observed after MSC transplantation. Six weeks after intrahippocampal transplantation of MSC no alterations were observed in the number of microglial clusters (Iba-1-positive cells). It seems that MSC could attenuate Aβ deposition by modulating inflammatory response through a mechanism other than the microglial activation pathway and, considering that APP/PS1 animals have Aβ plaques as a permanent condition, the plausible explanation is that microglia activation should be continuous due to the continuous formation of new plaques, even with lower numbers of Aβ plaques in transplanted groups. Interestingly, after cell transplantation into the brain a local neuroinflammation is supposed to occur due to the injection site lesion in brain tissue (33). However, even with no evident increase in microglia, MSC were capable of inhibiting the local inflammation caused in the injection site after the invasive procedure, possibly promoting tissue recovery and probably inducing the microglial phagocytic activity of Aβ plaques. Studies demonstrated that MSC can reduce the deposition of Aβ peptides, inhibiting the expression of APP-cleaving protein in the β site (BACE1), though immunomodulation and cytokine secretion (26, 34). Then, it is likely that the two cell types use different mechanisms of action for the regulation of Aβ plaques formation.
Seven-months old APP/PS1 mice showed hyperlocomotion in the Open Field test when compared to WT animals, and only NSC transplantation could reverse such behavior. APP/PS1 mice in the same age showed an increase in peripheric locomotion in the same test. Clinic observations already describe agitation and increase in locomotor activity in AD patients (35). Studies also describe the same behavior in 7 to 8-months old APP/PS1 mice both in Open Field and bright-dark tests.
Transplanted NSC are able to differentiate into neurons. Literature data show that newly formed neurons can increase connectivity and synaptic density (36, 37, 38, 39). Even though MSC are also capable of releasing neurotrophic factors and microglial and astrocytic modulators, increasing the tissue’s ability to eliminate Aβ plaques in the hippocampus (40, 32, 41), these cells are not able to differentiate into neurons. These data may explain the NSC capability of reverting hyperlocomotion in the Open Field test (42).
APP/PS1 mice do not show impairment in recognition memory, as revealed by the NOR, being in agreement with previous reports (43, 44). Published studies already demonstrated that 9-months old APP/PS1 animals showed no neuronal loss in the hippocampus, since the amount of Aβ plaques were not yet capable of leading to neuronal death (45, 46). Therefore, our study was not able to evaluate whether stem cell transplantation could protect APP/PS1 animals from neuronal loss in later years, as demonstrated by (47), (48) and (49). Future research is needed to evaluate the same parameters in older animals, to identify whether NSC and MSC transplantation could protect recognition memory. The reduction of hyperlocomotion observed in APP/PS1 + NSC animals plays an important role, since it approaches APP/PS1 mice in their behavior to their WT counterparts. The fact that, 6 weeks after transplantation, MSC were not found in the hippocampus may account for the absence of significant differences observed in the hyperlocomotion between APP/PS1 and APP/PS1 + MSC animals.