The current work provides the first randomized, triple-blind, placebo-controlled trial combining MRS and TMS techniques, in the same NF1 group, to investigate whether outcome measures based on cortical inhibitory dysfunction may be rescued by lovastatin. Previous statin trials have failed at definitively establishing the effect of treatment in this population (11–17). This may be due to the previous choice of available outcome measures. Here, we proposed a feasibility study testing outcomes at the neural system level by investigating effects of treatment from the neurochemical and physiological points-of-view.
Considering physiological outcomes, we did observe a significant increase in the absolute and relative CSP:MEP amplitude ratios, consistent with the hypothesis of increased intrahemispheric inhibition following lovastatin. The corrected measure allowed us to address CSP intrinsic variability, when studying corticospinal pathway’s net excitability (22). Importantly, the muscle contraction applied during this paradigm did not influence CSP results as attested by the absence of significant correlations between these measures (23). The origin of the CSP is still under debate. Although some authors claim that spinal processes may also play an important part, the literature points out a major role of cortical mechanisms through the recruitment of inhibitory interneurons in the cortex (22, 24). The CSP paradigm has been demonstrated to be valuable in studying inhibition of the motor cortex, particularly GABAB-mediated alterations (22–24).
Regarding the paired-pulse protocol, we observed a trend towards a decrease in peak-to-peak amplitude of motor-evoked potentials ratio for an ISI of 50 ms, suggesting superior long-interval intracortical inhibition after lovastatin intake. Interestingly, although at baseline NF1 patients did not show an inhibition for LICI 50 ms interval, unlike healthy individuals, possibly resulting from counteracting facilitatory mechanisms (25), after lovastatin the expected inhibition was observed. This effect at a neurophysiological transition zone between excitation and inhibition might also reinforce a tendency for lovastatin to modulate the excitatory-inhibitory imbalance. This was corroborated by the only study we found including TMS outcome measures to address statin intervention in NF1 (13), wherein modulation of inhibition by lovastatin intake was also detected in another paired-pulse inhibitory protocol, particularly in SICI intervals. In our study, however, SICI and ICF were not altered, suggesting an effect of lovastatin in paired-pulse GABAB-mediated measures, namely LICI.
In fact, our main findings included LICI and CSP measures, both being associated to GABAB receptor, with CSP measuring the duration and LICI assessing the magnitude of cortical inhibition (26). After lovastatin, we found a significant moderate negative correlation between the duration of absolute and relative silent periods and the MEP ratio in LICI (mean of 50 and 100 ms ISIs), that was not observed in placebo. A longer silent period correlated with an inferior peak-to-peak amplitude of MEPs in the LICI paradigm, both indicating an increase of inhibition.
Regarding our MRS findings, a trend towards a decrease in absolute Glx in motor cortex was observed after lovastatin intake, whereas GABA+ concentrations were not altered. The Placebo condition revealed no effect. The Glx findings are in line with those reported in a trial, in NF1 young children, showing reduced gray nuclei Glx after treatment with simvastatin (15) and support preliminary evidence that statins may introduce changes in the excitatory-inhibitory imbalance described in NF1 (5, 7).
Taken together, our findings suggest that some measures of TMS, namely CSP and, into a lesser extent paired-pulse stimulation, demonstrated higher sensitivity than MRS to detect changes in the GABAergic system induced by lovastatin. It is worth mentioning that a reliable biomarker should be able to detect statistically significant differences while minimizing the presence of dropouts. Taking this into account, we do not recommend future studies to rely on LICI measures since the lower signal-to-noise ratio highly impacts the amount of good-quality data for analysis.
An important purpose of this study was to investigate potentially more sensitive outcome measures, trying to overcome the lack of prior efficient detection of lovastatin effects in NF1 excitation-inhibition imbalance as well as in behavioral measures. Although the effectiveness of lovastatin in rescuing NF1 symptoms can only be assessed in larger trials, our study sheds some light on the selection of more suitable outcome measures for future Phase 2b/III clinical trials. Specifically, we highlight the potential of cortical silent period TMS as a promising tool to investigate the potential benefits of therapeutic interventions in NF1.
Methodological demands associated with this challenging feasibility study design require caution when interpreting the results. In this work, we studied cortical inhibition in the motor cortex, which has been barely explored in NF1 (23) and, therefore, we cannot draw conclusions about the effects of lovastatin in other areas of the brain. It is possible that only larger studies will have enough statistical power to capture subtle effects of lovastatin in rescuing altered GABA concentrations in NF1. The amount of data eligible for analyses was impacted by the rigorous quality criteria employed. Additionally, the lovastatin dosage was established based on safety criteria from common use in other pathologies such as hypercholesterolemia. We also selected a shorter duration treatment, when comparing with other studies (14–17), targeting earlier neurochemical and physiological changes in the GABAergic system, while enhancing patients’ compliance.
This study was performed with the aim of investigating alternative outcome measures based on impaired synaptic inhibition changes elicited by lovastatin intervention. Using objective physiological approach provides an alternative to behavioral outcome measures. We observed alterations in the excitatory-inhibitory push-pull mechanism in NF1 patients, induced by lovastatin as measured by CSP stimulation protocol. These altered mechanisms should be further explored in future large-scale studies in which TMS based cortical silent period measures seem to represent an important asset to evaluate the lovastatin-induced effects in the treatment of neurological conditions, such as NF1.