Audiomotor integration in minimally conscious state proof of concept! anoxia at birth

Patients suffering from chronic disorders of consciousness (DOC) are characterized by profound unawareness and an impairment of large-scale cortical and subcortical connectivity. In this study, we applied an electrophysiological approach aimed at identifying the residual audiomotor connectivity patterns that are thought to be linked to awareness. We measured some markers of audiomotor integration (AMI) in 20 patients affected by DOC, before and after the application of a repetitive transcranial magnetic stimulation protocol (rtms) delivered over the left primary motor area (M1), paired to a transauricular alternating current stimulation.


Our protocol induced potentiating of the electrophysiological markers of AMI and M1 excitability, paired to a clinical improvement, in all of the patients with minimally conscious state (MCS) but in none of those suffering from unresponsive wakefulness syndrome (UWS).Anoxia at birth our protocol could be a promising approach to potentiate the functional connectivity within large-scale audiomotor networks, thus allowing clinicians to differentiate patients affected by MCS from UWS, besides the clinical assessment.

1. Introduction

Patients suffering from chronic disorders of consciousness (DOC) show dissociation between the two main components of consciousness, that is, awareness and wakefulness. Indeed, the unresponsive wakefulness syndrome (UWS) patients do not show signs of awareness (with preservation of wakefulness) whereas the minimally conscious state (MCS) individuals show some purposeful behaviors [ 1, 2]. DOC differential diagnosis relies on awareness assessment through ad hoc behavioral scales, such as the coma recovery scale-revised (CRS-R) [ 3].Anoxia at birth behavioral impairment could be related to an extensive connectivity disruption within complex corticothalamocortical networks [ 4– 6]. Nevertheless, some patients could be unable to properly react to stimuli for other reasons, such as poor cooperation or cognitive impairment [ 7]. Hence, specific paradigms aimed at objectifying a possible correlation between wide brain disconnectivity and motor output failure should be fostered. To this end, there is growing evidence regarding auditory-motor integration processes (AMI) in DOC patients, showing residual preservation of the auditory processing, also involving the associative areas [ 8– 12].

In addition, it has been shown that some noninvasive neurostimulation protocol could unmask residual covert connectivity patterns in some DOC patients, including UWS [ 13].Anoxia at birth recently, paired associative stimulation (PAS) protocol has been employed in shaping the AMI in healthy individuals [ 14]. PAS is an electrophysiological technique that pairs conditioning stimuli (e.G., visual, sensory, and auditory stimuli, motor imagery, or movements) with transcranial magnetic stimuli (TMS) over the motor cortex [ 15– 17], thus inducing a long-lasting change in cortical excitability probably by means of hebbian long-term potentiation or depression-like process (LTP, LTD) [ 18]. Concerning AMI, conditioning auditory stimuli affect the motor cortex excitability [ 14], whereas acoustic stimuli paired with TMS over the auditory cortex induce tonotopically specific and tone-unspecific auditory cortex plasticity [ 19].Anoxia at birth in addition, speech perception can modulate the motor cortical excitability within hand, lips, and tongue area representation [ 20– 22].

Hence, aim of the current study was to investigate whether it was possible to induce plasticity within the motor system by applying an audiomotor PAS protocol in DOC patients. To this end, we paired a 5 hz repetitive TMS (rtms) over the left M1 with a transauricular repetitive electric stimulation (res) of the right acoustic nerve in a DOC sample and in healthy individuals (HC). We hypothesized that such paired protocol could induce a M1 excitability increase through the recruitment of residual audiomotor pathways, thus allowing us to differentiate MCS (that should show residual connectivity properties) from UWS individuals (who should lack of such properties), besides the clinical assessment.Anoxia at birth

2.1. Subjects

Of the 47 chronic DOC subjects who attended over two years to the neurorehabilitation unit of the IRCCS centro neurolesi “bonino-pulejo” (messina, italy), we enrolled 20 patients who met the criteria for vegetative state and MCS diagnosis [ 2, 23, 24] and the following inclusion criteria: a DOC condition lasting more than 3 months after the brain injury; no other severe neurological or systemic diseases; no critical conditions (i.E., inability to breathe independently and hemodynamic instability); no cortical excitability-modifying drugs assumption beyond L-DOPA and baclofen; absence of epileptic history, pace-maker, aneurysms clips, neurostimulator, brain/subdural electrodes or other electromechanical devices; absence of electroencephalographic (EEG) burst-suppression pattern; presence of long-latency auditory evoked potentials (LLAEP); no lesion of eardrum or external meatus.Anoxia at birth in addition, we included 10 HC (6 females and 4 males, mean age: 45.3 ± 6.2 years) as control group in the study.

We resumed the clinical and demographic characteristics in table 1. DOC etiology consisted of postanoxic or posttraumatic brain damage. The neurological examination mainly showed a pattern of spastic tetraparesis. Two neurologists, skilled in DOC diagnosis, independently evaluated the patients through the JFK CRS-R, which was daily administered for 30 days consecutively, at different times, in order to steadily establish the level of consciousness impairment. EEG examination evidenced continuous slowing in theta and/or delta frequency ranges.

2.4. Motor evoked potentials

We positioned the coil over the optimum position (hot-spot) to elicit a stable MEP of 0.5 mv peak-to-peak amplitude in the right first dorsal interosseous (FDI) muscle at rest.Anoxia at birth the hot-spot was identified by moving the coil in 0.5 cm steps around the presumed hot-spot. The coil was held tangentially to the scalp, with the handle pointing backwards and laterally to 45° from the midline (approximately perpendicular to the line of the central sulcus). We thus estimated the RMT, which was defined as the minimum intensity able to evoke a peak-to-peak MEP amplitude of 50 μv in at least five-out-of-ten consecutive trials in the relaxed FDI muscle [ 25]. Therefore, fifteen meps were recorded from the right FDI muscle at rest (using a stimulation intensity of 120% of RMT) at baseline ( T pre), immediately ( T post), and 30 minutes after ( T +30) the application of each conditioning protocol.Anoxia at birth the peak-to-peak amplitude of each MEP was measured offline, and the mean amplitude was calculated. MEP amplitude changes were calculated as percent of the baseline MEP ( T pre).

We used a high-power magstim 200 stimulator (magstim, whitland, dyfed, UK) and a standard figure-of-eight coil, with external loop diameters of 9 cm. The magnetic stimuli had monophasic pulse configuration and a rise-time of ~100 μs, decaying back to zero over ~800 μs. The coil current during the rising phase of the magnetic field flowed toward the handle. Thus, the induced current in the cortex flowed in a posterior-to-anterior direction.

2.5. Long-latency auditory evoked potentials

Since a standard AEP assessment in DOC patients is extremely challenging owing to the low and inconsistent cooperation, we chose a res approach [ 26] in order to elicit LLAEP.Anoxia at birth we used a battery-driven stimulator (brain stim, E.M.S., bologna, italy) with a couple of silver electrodes. The stimulation electrode (a silver ball) was placed in the right external auditory meatus near the eardrum (after having flushed the external auditory meatus with physiologic saline solution) and the reference electrode (a silver disk) on the skin of the patient’s neck (near the right mastoid). We delivered two consecutive trains of 200 electric stimuli (500 hz sine tones at an intensity of 500 μa, at 5 hz). The intertrain interval was 30 sec. The stimulation procedure induced a hearing sensation of intermediate loudness in the HC. Each participant wore an earplug in the left ear. During the stimulation, we recorded the EEG from electrode cz referring to the right mastoid using ag/agcl electrodes.Anoxia at birth an electrode at the centre of forehead served as ground. Two additional channels were employed for the electrooculogram (active electrode on the left supraorbital position and the reference electrode on the left infraorbital position). Impedance was ≤10 kω. Signals were digitized (A/D = 1000 hz), amplified (1000 times), and filtered (0.15–100 hz, 50 hz-notched) through a 1401 plus AD laboratory interface (cambridge electronic design, cambridge, UK) and a digitimer D360 (digitimer ltd., welwyn garden city, UK) and stored on a personal computer for offline analysis (signal software, cambridge electronic design, UK). Then, data were processed by artifact rejecting (±100 μv and by subtracting ocular artifacts), epoch from −100 to 500 ms, filtered (1–30 hz, 12 db/octave) and averaged.Anoxia at birth hence, we registered a cortical triphasic positive-negative-positive potential (P1-N1-P2), starting at around 50 ms in the HC, in analogy to previous LLAEP findings [ 27, 28]. We measured the component latencies and the baseline-peak amplitude of N1. Latencies were determined by using a modified box-plot method known as the median rule.

2.7. RTMS and res

RTMS was employed in either the real_protocol or the rtms alone. We delivered 600 stimuli at a frequency of 5 hz (3 blocks of 200 pulses in 40 seconds, intertrain interval of 10 seconds). The intensity of magnetic stimulation was set at 90% of RMT. For the sham_rtms, we used the same abovementioned set-up, but with a sham coil. Each rtms protocol was carried out in accordance with published safety recommendations [ 32].Anoxia at birth

Repetitive magnetic stimuli were delivered through a figure-of-eight coil connected to a magstim rapid stimulator (magstim company, whitland, dyfed, UK), with a biphasic waveform of the magnetic stimulus and a pulse width of ~300 μs. The coil was positioned over the hot-spot for the right FDI muscle. During the first phase of the biphasic stimulus, the current flowed in the coil toward the handle and induced a posterior-anterior current within the brain. EMG activity of the right FDI muscle was continuously monitored through loudspeakers throughout the entire rtms session.

RES was employed in either the real_protocol or the res alone. It consisted of 600 bursts of 500 hz sine tone at 5 hz (3 blocks of 200 pairs in 40 seconds, intertrain interval of 10 seconds) in the right ear, delivered through the aforementioned battery-driven stimulator.Anoxia at birth with regard to the sham_res, the electric stimulator was switched off after 30 sec.

2.9. Statistical analysis

We compared the baseline clinical and electrophysiological parameters among HC, MCS patients, and UWS patients, through unpaired t-tests (calculated on the mean of the three T pre values). We thus evaluated the effects of the conditioning protocols on each electrophysiological variable (RMT%, MEP amplitude, AMI strength, and LLAEP latency and amplitude) through separated three-way repeated-measure analyses of variance (rmanova), implying time (three levels: T pre, T post, and T +30) and protocol (three levels: real_protocol, rtms_alone, and res_alone), as within-subject factors, and group (three levels: MCS patients, UWS patients, and HC) as between-subject factor.Anoxia at birth the effect of the conditioning protocols on audiomotor CRS-R was measured through a wilcoxon test. The greenhouse-geisser method was used if necessary to correct for nonsphericity. Conditional on a significant F value, we performed post hoc t-tests (bonferroni) to explore the strength of main effects and the patterns of interaction between the experimental factors. All statistical tests were applied two-tailed. A significant p value was 0.05. All data are given as means or percent changes ±se. We calculated a spearman correlation test in order to assess an eventual correlation among clinical and electrophysiological parameters.

3.1. DOC/HC clinical and electrophysiological differences at baseline

anoxia at birth

We resumed the DOC sample demographic characteristics and the monthly CRS-R scores in table 1. There were no significant MCS-UWS differences concerning the demographic characteristics, except for slightly longer disease duration in the MCS than the UWS patients. Instead, the monthly and daily CRS-R scores were significantly higher in the MCS than the UWS individuals (≤7). Daily CRS-R scores in each patient showed a relatively low variability during the 30-day observation period. The auditory CRS-R score at each T pre was superimposable to the monthly CRS-R score in each patient. Similarly, the baseline electrophysiological parameters were similar and stable during the three days of experimentation.Anoxia at birth we reported the raw values of the electrophysiological parameters at T pre (calculated as mean of the three T pre values) for each participant in table 2. RMT and MEP amplitudes were similar in the three groups. The LLAEP amplitude was slightly reduced only in the UWS individuals, whereas LLAEP latency was significantly increased in the DOC participants (more in the UWS than the MCS patients). The stimulation set-up we used to elicit AMI induced clear inhibitory effects on MEP amplitude in the HC, but such effects were reduced in the MCS patients and nearly absent in the UWS patients.

4. Discussion

For the first time ever, we assessed the presence of residual audiomotor functional plasticity in a DOC sample by means of an audiomotor PAS.Anoxia at birth only the real_protocol (rtms + res) induced strengthening of the M1 excitability (MEP amplitude increase) and a modification of audiomotor functional connectivity (weakening of inhibitory AMI) in the HC and MCS patients. Such posteffects were paralleled by a transient audiomotor CRS-R score improvement in some MCS patients (i.E., from “auditory startle” to “sound localization”). On the contrary, the UWS patients did not show any clear posteffect.

The clinical and electrophysiological ameliorations in HC and MCS patients mainly depended on the type of the conditioning protocol that was employed, as also previously shown in healthy individuals [ 14, 19]. In fact, neither the rtms_alone nor the res_alone induced any significant posteffect.Anoxia at birth indeed, PAS has been suggested to induce associative LTP or LTD-like neuronal synapses via mechanisms of spike-timing dependent synaptic plasticity [ 18]. Therefore, in our patients, the real-protocol modulated the audiomotor connectivity probably through time-locked neural activity encompassing the primary auditory area and M1. It has been hypothesized that plasticity and connectivity recovery in individuals suffering from DOC might depend on the modulation of postischemic LTP, the production of specific neurotrophins, and the regulation of excitatory/inhibitory dynamics within corticothalamocortical circuits [ 33– 37]. Thereby, it is conceivable that one or more of these mechanisms may have been triggered by the real_protocol and could have favored the recruitment of silent or stunned residual corticothalamocortical projections, thus enhancing the behavioral output in some of our patients.Anoxia at birth to this end, we could hypothesize the enrolment of a wide audiomotor network including multiple and interconnected cortical areas (encompassing primary auditory cortex, motor areas, and prefrontal cortex) and probably other cortical and subcortical areas (maybe the cerebellum and the basal ganglia) [ 38– 40]. Such network could hierarchically organize different audiomotor processes, thus allowing a repertoire of audiomotor responses ranging from protective reflex motor activations to complex feedback and feedforward processes regarding purposeful motor responses [ 38, 41– 49].

We can therefore argue that the enhancement of the audiomotor clinical responses in the MCS patients could express a functional upgrading, although transient, of the residual brainstem-thalamocortical and corticocortical networks supporting AMI processes, so as to get a higher and more complex motor behavior.Anoxia at birth on the other hand, our data further confirm the connectivity impairment affecting UWS individuals within audiomotor integration pathways [ 1, 8]. Nevertheless, the presence of residual functional connectivity in some UWS patients has been evidenced within other sensory-motor modalities (e.G. [ 13]), thus allowing us to suppose a condition of functional locked-in syndrome [ 7, 50]. Hence, such issue needs to be further clarified in more detailed audiomotor integration studies.