This observational study aims to integrate anamnestic, clinical, and innovative neurophysiological data to assess their impact on the diagnosis and prognosis of patients with Disorders of Consciousness. The main questions it aims to answer are:

• Is it possible to identify biomarkers with high diagnostic relevance for distinguishing subcategories of patients with Disorders of Consciousness?
• Can biomarkers with potential medium- and long-term prognostic value be identified for each category of patients with Disorders of Consciousness?

Participants will undergo an EEG examination during near-nerve stimulation of the median nerve. Clinical and anamnestic data will also be recorded and collected at follow-up.

At the enrollment of each patient with Disorders of Consciousness, demographic, anamnestic, and clinical data are prospectively collected. The etiology is categorized as traumatic, anoxic, hemorrhagic, or ischemic. The lesion side is defined according to the latest CT scans from the acute phase and is grouped as left, right, and bilateral. All patients undergo a standard neurophysiological evaluation, and the level of consciousness is assessed using the CRS-R scale following international guidelines. It is repeated to confirm the clinical status of the patient, and a reduction of sedation is performed if deemed safe by the physician. Two months after admission to the IRU, the neurophysiological and consciousness assessments are repeated.

In addition to clinical and standard neurophysiological evaluations, two types of SSEPs are acquired during continuous EEG recording through both Surface Cutaneous Stimulation (SCS) and Near-Nerve Stimulation (NNS) of the median nerve. EEG signals are collected using a 61-channel cap with a sampling rate of 512 Hz.

The electrical stimulation is applied over the median nerve proximally to the wrist, and the stimulation side is chosen contralaterally to the less affected hemisphere. Superficial bipolar electrodes are used for SCS, whereas a microneedle with 1 MΩ of impedance is inserted under the skin for NNS. The electrical pulse of 0.2 ms is delivered at a rate of 1 Hz in two sets of 50 repetitions. The stimulation amplitude is chosen as the minimum value able to generate a response only in sensory or sensory-motor fibers for NNS and SCS, respectively. The only sensory activation in NNS is preferred to minimize the influence of motor twitches on brain responses. For SCS, due to the difficulty of exclusively stimulating sensory fibers, the amplitude is set as the minimal value required to elicit both sensory and motor activation.

To monitor the stimulated fibers, the peripheral pathway of the median nerve is recorded. In particular, surface electrodes are positioned on the three fingers innervated by the median nerve for the acquisition of Sensory Nerve Action Potential (SNAP) and on the palm proximally to the motor twitch for the acquisition of Compound Motor Action Potential (CMAP). This information is essential in patients with DoC for different reasons:

i) Identifying the proximity to the median nerve, a crucial step due to the unresponsiveness and lack of cooperation of this population.ii) Identifying the type of active fibers.iii) Monitoring feedback to confirm the accurate placement of the microelectrode.

Whenever the minimum amplitude evokes a CMAP/SNAP, the related amplitude is selected for SCS/NNS. The device automatically extracts the mean values of amplitude and latency of CMAP/SNAP during the experimental session.

Inclusion Criteria:

• Age > 18 years, both sexes;
• Severe acquired brain injury (regardless of etiology);
• Patients with severe alteration of consciousness state due to severe acquired brain injury of various etiologies (Cardiac Arrest, Traumatic Brain Injury, Cerebral Ischemia or Hemorrhage);
• Informed consent signature by family member/caregiver/legal guardian.

Exclusion Criteria:

• Time since the acute event greater than 3 months;
• Previous neurological diseases;
• Previous acute brain injuries, psychiatric disorders, neurodegenerative diseases, neoplasms, severe systemic diseases;
• Modified Barthel Index pre-event < 50;
• Unstable medical conditions.

Patients who were admitted with a Disorder of Consciousness to the Intensive Rehabilitation Unit (IRU) of Fondazione Don Carlo Gnocchi (Florence).

Upon publication of results, anonymized data and analysis code will be shared to ensure reproducibility. Zenodo and GitHub repositories will be created.

Upon publication of results

Data and code will be available for research purposes upon request to authors.