Our results showed for the first time that a therapeutic effect of intramyocardial hADSC injection on the brain and heart simultaneously through engagement of the ROS pathway. Previous studies have shown that when cell therapy is administered peripherally, the cells migrate to many organs. Obviously, our results were not consistent with the results. We did not find any hADSCs migrating into the hippocampus. Thus, direct cell implantation can likely not contribute to the alleviation of cognitive impairment. Mechanistically, the structural recovery and cognitive enhancements elicited by exposure to hADSCs were at least partially mediated by ROS, rather than hADSC engraftment. Taken together, our novel findings suggest that early treatment with hADSCs may decrease the risk of cognitive impairment after MI. The present study was consistent with the notion that functional recovery mediated by transplanted cells could occur without the grafted cells entering the brain if neuroprotective molecules secreted by the cells could reach the injured brain site by crossing the blood–brain barrier.
Our results were consistent with the beneficial effects of intramyocardial administration of hADSCs on cognitive function, as documented structurally by DHE stain, Nissl stain, Golgi stain and immunofluorescent stain; molecularly by hippocampal synaptophysin and PSD95 proteins; biochemically by plasma superoxide levels, and functionally by passive avoidance test. The beneficial effects of hADSCs on MI-induced cognitive impairment are supported by 4 lines of evidence.
1) ROS in hippocampus was increased after MI. The present study demonstrates the hippocampal ROS levels were increased after MI, in a correlation with systemic ROS activation. A closer link between the brain and the heart after cardiac damage has been reported than with other nonhematopoietic organs . Damaged cardiomyocytes at the site of MI can increase superoxide anions that rapidly enter the bloodstream. Brain has very low levels of catalase and moderated levels of superoxide dismutase . Thus, even low-grade ROS also substantially affects the blood-brain barrier . Thus, on reaching brain, superoxide regulates acute cytokine response and mediates trafficking of hippocampus to injured hearts. ROS are increasingly being shown to be intrinsic mediators of communication between the brain and heart.
2) hADSC administration was associated with improved hippocampus-dependent cognitive function after MI. In the brain, the hippocampus is responsible for learning and memory, and dysfunction of the hippocampus has been shown to play a major role in altering cognitive ability. In this study, we used the passive avoidance test to analyze the cognitive and learning ability of the rats to elucidate whether MI could induce cognitive dysfunction. The passive avoidance test has been widely validated as a measure of hippocampus-dependent associative memory function in rodents . A fundamental role in fear memory to plasticity in the hippocampus includes the dentate gyrus . The latency in refraining from entering the compartment in which the rats are punished has been shown to serve as an index of avoidance ability, through which memory can be assessed. The time required to re-enter the compartment in which the rats are punished is mostly influenced by learning ability . Thus, the latency time in this study predominantly reflected the memory function of the rats. Our findings indicate that there were memory impairments after MI, but that hADSCs could significantly reverse these memory impairments. Our results support these clinical observations and demonstrate mechanistic links between ROS and post-MI cognitive decline.
3) hADSC administration ameliorated synaptic plasticity via the superoxide radical pathway. The causative effect of ROS on synaptic plasticity has been demonstrated in experimental studies . ROS overproduction suppresses hippocampal neuroplasticity.35 Many studies have shown that a reduction of synaptic plasticity was associated with dysfunction in learning and memory performance [37, 38]. A reduction in synaptophysin immunoreactivity has been shown to be accompanied by a decrease in connectivity, which in turn leads to functional deficits in synaptic transmission and eventually cell loss [16, 17]. Since synaptophysin and PSD95 regulated neuronal synaptic activity , increases in synapse number assessed by the intensity of synaptophysin puncta colocalized with PSD95 can lead to cognitive improvement. In our experiment, 3 days after MI, we found that the neuron numbers (Nissl stain-positive cells, Fig. 4A), the densities of dendritic spines (Golgi stain-positive cells, Fig. 4B), and the synaptic numbers (co-localization of synaptophysin and PSD95, Fig. 5A) were lower in the vehicle-treated infarcted rats than those in the sham. The impairment can be significantly reversed after adding hADSCs. Furthermore, the addition of SIN-1, a peroxynitrite donor, abolished benefits compared with hADSCs alone, implying that hADSCs may mediate the effects through a superoxide-dependent pathway. Our results were consistent with the findings of Knapp et al , showing that exogenous superoxide administration resulted in transient reduction in postsynaptic response, followed by a late form of long-term potentiation, which can be inhibited by superoxide dismutase application.
4) ROS, not cell engraftment, play a role in mediating neuroprotection.
The therapeutic effect of hADSCs is through remote mechanisms. In this study, we used species-specific competitive PCR-based assays to investigate the mechanisms behind the strong behavioral and neurological effects of the hADSCs, as this could yield information regarding how gene families are involved in stem cell migration. Our results showed no amplification in PCR reactions with human β-2-microglobulin primer sets at the hippocampus. We proposed the mechanisms through which the remotely distributed hADSCs exert their therapeutic effect by direct antioxidant effect on the hippocampus. As anticipated, reduction in superoxide anion was prevented, and the behavioral and histological protective effects were completely blocked by co-treatment of hADSCs with SIN-1. Our results are consistent with previous studies which showed that stem cells administered intravenously did not reach brain tissue , indicating that the benefits of treatment observed for this route of administration are not due to brain stem cell engrafting. Our results were consistent with the paracrine hypothesis of stem cells. Stem cells secrete a variety of extracellular vesicles (EVs) that serve as a cell-free therapeutic agent. Mesenchymal stem cells-derived EVs which can cross the blood-brain barrier  were enriched in antioxidant miRNAs and exhibited remarkable antioxidant activity evident .
Previous studies have shown an increase in blood-brain barrier permeability at 3 hours of reperfusion post middle cerebral artery occlusion, subsequently reaching a maximum at 48 hours and then decreasing from 4 days post stroke . To determine whether hADSCs may have entered at an earlier period and then died during disease progression, MI was induced in additional animals (n = 4 per group) followed by intramyocardial injection of hADSCs. These animals were randomly euthanized at 3 and 48 hours after MI, and the analysis still revealed no detectable hADSCs in the brain (data not shown).
Our results demonstrated that the intramyocardial administration of hADSCs could ameliorate cognitive impairment and synaptic plasticity, and this may be used to develop interventions targeting cognitive decline. More importantly, we hope that by using this approach to alter basic processes, it may become possible to counteract the cellular abnormalities that lead to neurodegeneration after MI. These findings emphasize the importance of an appropriate intervention at the cerebral level to prevent hippocampus injury after MI. Diverse and multifaceted decrements involving co-mobility and medications have been described that impact multiple cognitive domains that persist long after MI in humans. In addition, animal studies conducted in the absence of confounding diseases have shed considerable light on many factors contributing to cognitive impairment. Clinicians commonly encounter patients with heart-brain syndrome, and it has been shown to affect their prognosis, morbidity, and mortality. Consequently, more attention should be paid to cardiac dysfunction-associated brain damage. Besides, treatment with hADSCs overcome the limitations associated with drug-based therapies and offer additional advantages after intramyocardial administration.
First, in the present study, we explored the potential clinical efficacy of hADSCs in the treatment of patients after MI. However, our results using human ADSCs cannot be necessarily extrapolated to rodent ADSCs. There were differential effects between species. Previous studies have shown that the transplantation of human glial progenitor cells into the frontal cortices of immune-deficient neonatal mice has significantly enhanced the cognitive function of adult and aged mice . In contrast, the transplantation of murine glial progenitor cells had no such effects . Such species-specific differences may be explained by the propagation of calcium waves, during which astrocytes can communicate. Human progenitor calcium waves propagate at least three times faster than mouse calcium waves, possible because they are much larger and have a more complex structure. These differences may also contribute to enhanced long-term potentiation. Second, although it appears that the large accumulation of ROS is necessary for pathological processes to prevail over physiological processes, the levels of tolerance and toxicity have yet to be clarified. Few studies have investigated whether there is a dose-dependent relationship between ROS and plasticity, and future investigations are warranted to investigate this issue. Finally, although our experiments do not exclude the possibility of hADSC-derived EVs playing a role in antioxidation. Comparative analyses of ADSCs and their EVs demonstrated diverse genetic cargo including mRNA and miRNA, and protein contents that play role in angiogenesis, adipogenesis, and regulation of inflammation . Moreover, protein levels and surface markers also differ between EVs and their parent cells . Thus, our results can not be explored into the EVs.