This study demonstrates that rTMS significantly reduces mitochondrial damage, apoptotic neuronal death, and glial activation and supports functional recovery in a rat model of remote damage after focal cerebellar injury.
Here, we showed that rTMS significantly reduced HCb-induced cell death of precerebellar neurons by blocking cyt-c-associated apoptosis. These findings are consistent with earlier reports on the anti-apoptotic effects of rTMS in the perilesional area after TBI [
17] and after transient cerebral ischemia [
18]. Although remote mechanisms differ substantially from those in perilesional areas after traumatic or ischemic insults [
2,
12], our data demonstrate the effectiveness of rTMS in counteracting apoptotic cell death in areas that are distant from the site of damage. Although the efficacy of rTMS in reducing apoptotic cell death in our model is quite specific, further mechanistic studies are required to identify signaling pathways of rTMS effects on precerebellar neurons. In addition to the effects on neurons, our data also showed that glial cells, specifically astrocytes and microglia, responded to rTMS stimulation. In fact, in our model rTMS significantly reduces HCb-induced inflammatory responses, which have been shown to contribute to remote degeneration [
2]. At present, there is limited information regarding the response of astrocytes and microglia to rTMS in health and disease [
19]. Present data demonstrating the rTMS effects on neuroinflammation, although pointing to a direct effect of TMS on glial cells, do not allow to rule out a direct effect on neurons and their survival. Overall, taking into account the key role of neuron-glia crosstalk in CNS physiology and pathophysiology, the influence of TMS on glial cells is critical to open up novel therapeutic options. However, further studies are needed to clarify the specific effect of TMS on neuron and glial cells as well as on their crosstalk mechanisms to being able to develop TMS approaches for modulating specific cellular responses. In this line, it is worth considering that in our model, as well as in many brain pathologies, plastic responses to injury are not limited to mitochondrial damage or glial activation. We cannot exclude that other factors, in addition to those mentioned, are also sensitive to rTMS. On the other hand, as the clinical significance and positive therapeutic effects of rTMS in a great variety of CNS disorders suggest that they are determined by a combination of multiple factors, we can speculate that, also in our model, rTMS-mediated neuroprotection is a multifactorial process in which many elements play a role. Future research on these mechanisms and factors will be critical for the development of more powerful and reliable TMS therapeutic protocols. In particular, interactions between neurophysiological and cellular/molecular effects of TMS represent a new and intriguing field, which is opening up new lines of research to address neuronal survival and plasticity after CNS insults.
We are aware that the use of a commercial human-sized coil with high-intensity field strengths (≥1 T) might be a limitation of our study [
20,
21], rendering dose efficacy or target selectivity requirements unable to be evaluated. However, the selectivity of the effects of rTMS on lesion-induced changes and the patent differences between sham and rTMS treatments support the reliability of our findings. Furthermore, the high sensitivity of the damaged tissue to rTMS is also notable. No changes in any of our parameters were observed in the unlesioned group.
Establishing the link between the sparing of neuronal death in a given population and improvements in functional recovery is always challenging. We cannot exclude that rTMS, especially using so large a coil, might influence outcomes by acting on neural centers that differ from those that we have considered. Plasticity-related changes after rTMS can occur in regions that are functionally connected to the stimulated area and thus contribute to the efficacy of rTMS [
22‐
25]. Despite these cautions, the demonstration of cellular and molecular changes in a key node of the cerebro-cerebellar loop—i.e., the Pn—supports the importance of Pn survival in the recovery.