Introduction

Improving the ability to detect conscious processing in non communicating patients remains a main goal of clinical and cognitive neuroscience (Balconi 2006; Gawryluk et al. 2010). A diagnosis of DOC (disorder of consciousness) is difficult to provide, because clinical examination is limited in these unresponsive patients (Giacino et al. 2002, 2009; Giacino and Whyte 2005).

Appropriate diagnostic tools that can take into account these barriers and that ensure a more accurate assessment are needed to provide an exhaustive examination of the cognitive status of these patients. Available standardized tools, specifically developed for the evaluation of patients with severe impairment, are very limited and mostly relatively insensitive to minor changes in level of consciousness. Also they do not consider the obstacles induced by the pathological condition (due to the presence of motor, sensory or cognitive deficits) that may affect the detection of responses, increasing the risk of mistakenly attributing the patient to a state of unresponsiveness or not to highlight improvements.

These disorders were classified in terms of awareness and wakefulness (Gawryluk et al. 2010). Awareness refers to a state in which people have complete experiences such as thoughts, memories and emotions. Wakefulness refers to the state in which people can open their eyes and presents motor responsiveness. Three different typologies of distinct disorders of consciousness (DOC) can be defined based on these two states: coma (C), where patients show absence of both awareness and wakefulness; persistent vegetative state (VS), where there is wakefulness without awareness; minimally conscious state (MCS), where both awareness and wakefulness are partially preserved. In contrast to patients in VS they also reveal discernible evidence of awareness: for example they may speak a word or make a gesture in response to a command. Thus it is commonly agreed that the two states patients in VS and MCS are frequently misdiagnosed (Giacino et al. 2002).

As suggested by Giacino et al., the behavioral measures remain the “goal standard” for routine assessment of consciousness, but it relies exclusively on observable responses (2009). Clinical tests rely on a patient demonstrating awareness by means of overt behaviors, as does the Coma Near Coma (CNC), the Glasgow Coma Scale (GCS) and the Disability Rating Scale (DRS) (Rappaport 2005; Rappaport et al. 1982; Teasdale and Jennett 1974). Also recently CRS-R (Coma Recovery Scale-Revised) (Giacino et al. 2004) was developed to assess differences between levels of consciousness (Coma, Vs and MCS), whereas a critical point remains the low reliability levels for patients with significant cognitive syndromes (such as aphasia or neglect). Also, other scales were used, as the Sensory Stimulation Assessment Measure (SSAM, Rader and Ellis 1994), the Sensory Modality Assessment and Rehabilitation Techniques (Gill-Thwaite and Mundy 2004), and the Wessex Head Injury Matrix (Shiel et al. 2000). However, the ability to perform these overt behaviors is often decoupled from consciousness as a direct outcome of the traumatic brain injury.

Specifically, the DRS has the goal to furnish a quantitative measure of the disability outcomes for patients with severe head injury, documenting evolution from initial coma to various stages of impaired levels of consciousness (Rappaport 2005; Rappaport et al. 1982; Teasdale and Jennett 1974). However, it allows the monitoring of patient progress and it is predictive about the long-term outcomes. Thus this measure can offer the evolution through various stages of impaired level of consciousness. It is composed by 8 items divided into four categories and the scoring values are reversed (high scores correspond to more deterioration). The patient’s behavior is evaluated through observation and by analysis of the responses to various stimuli. It is considered a good-level scale and suitable instrument when used in association with other scales such as the CNC. Moreover, it was documented also a high correlation between DRS and some measures of brain function (such as ERPs, event-related potentials).

These ERP measures allow to describe the potential variations on the scalp and are directly related to some cognitive and emotive functions. Specific peak deflections are responsive of different processes, as shown by a vast amount of literature (Balconi and Pozzoli 2005). For this reason it is considered a relevant marker of the state of consciousness in association with psychophysiological measures (Rohaut et al. 2015; Sculthorpe-Petley et al. 2015; Sozzi and Inzaghi 2011).

The CNC scale was used to evaluate levels of responsiveness of patients with severe brain injury and it analyzes the patient’s response to stimulation with different sensory modalities (Rappaport 2005; Scozzafava et al. 2010). Compared to the DRS it has a higher sensitivity in detecting small changes that define the degree of patient’s responsiveness. It was shown the CNC correlates with changes of physical and mental patient’s condition. Moreover it is useful to predict the outcome even when the evaluation is made after a long period from the brain injury. The final score can be referred to a class level and each level has its own definition in term of variation of “coma” (from “extreme coma” to “no coma”). Both the DRS and CNC measures were used to distinguish VS from MCS, based on two different cut-off: scores more than 2 and more than 21 respectively to the CNC (range = 1–4) and DRS (range = 0–30) scales were considered an index of VS. The final score may be attributed to class level and each level has its own definition in terms of variation of “coma” (from extreme coma to near coma). However, due to the frequent misdiagnosis, it is was debated the role these measures may have to describe the patients’ responsiveness to some cognitive tasks, such as linguistic tasks (Balconi 2011; Sozzi and Inzaghi 2011).

Thus, some approach considered the physiological measures to evaluate DOC profile in order to avoid the significant limitations of some behavioural measures. There is a critical need to improve the clinical evaluation of consciousness state using alternative measures such as electrophysiological indices which may introduce important evidences on the functional responsiveness of the injured-brain (Balconi 2011; Balconi and Mazza 2009; Cavinato et al. 2011). Based on the evidence to-date, electroencephalographic assessments of consciousness provide valuable information for evaluation of residual functions, formation of differential diagnoses, and estimation of prognosis.

Specifically, ERPs might be used to assess consciousness, providing an on-line monitoring of information processing in the brain. Based on previous evidences, it was shown that the brain of many patients with disorder of consciousness can perform very complex information processing operations. The first ERP study indicated electrophysiological signs of profound stimulus processing, such as P300 effect to rare stimuli. Indeed an increased P300 peak amplitude is observable, within a sequence of stimuli, in the case of a rare stimulus compared to a frequent stimulus (Reuter et al. 1989). Nevertheless, only recently it was evaluated the relevance of different ERP components in large sample of patients with DOC. In fact it was found a significant contribution by these electrophysiological measures to explore the preserved cognitive functions in patients with DOC (Amantini et al. 2011; Kotchoubey 2005).

It is useful to distinguish different planes of analysis related to ERP profile. Sensory ERPs, generally appearing in response to perceptual features of a stimulus (exogenous potentials), are used for clinical assessment of basic sensory functions. Contrarily, cognitive ERPs are applied to evaluate higher level functions like attention, memory, and language. These long-latency potentials are considered well-suited to assess functions of consciousness, and four specific components were considered focusing on the analysis of conscious awareness, that is N100, the mismatch negative (MMN) (Qin et al. 2008), the P300 (Faugeras et al. 2011; Fischer et al. 2010), and the N400 (Schoenle and Witzke 2004). These different ERP correlates were used to assess consciousness state across the spectrum of pathologies associated with DOC (for a review about these ERP correlates and their function see Vanhaudenhuyse et al. 2008).

The N100 indices perceptual functions during visual, auditory and somatosensory processing. The MMN, a negative deflection occurring around 150–250 ms. post-stimulus, was linked to processing of deviant auditory stimuli and it occurs below the level of consciousness. It is regarded as an automatic response of the brain to stimulus deviation from preceding repetitive auditory stimuli and requires fully processed physical features of the auditory stimuli (Näätänen 1990). Specifically, in case of patients in VS, it was shown larger MMN components were associated with increased signs of consciousness (Fischer et al. 2010). The P300 generally occurs to deviant or oddball stimuli (i.e. infrequent compared to more frequent stimuli within a sequence) generally appearing when subjects attend to the stimulus, and is thought to reflect higher level processing. It was considered the most appropriate candidate as an indicator of conscious awareness (Kotchoubey 2005). Recent research showed a significant P300 increasing related to the appearance of highly significant stimuli in patients with DOC, using a series of equiprobable first names among which is included the subject’s own name (Perrin et al. 2006). Using a similar paradigm it was shown that patients with DOC (both MCS and VS) displayed a significant brain oscillation synchronization (theta-synchronization) in response to their own proper name in comparison with other names (Fellinger et al. 2011). However, in other cases, significant differences were observed in ERP profile between an active and a passive task for the MCS, whereas the VS did not show the same differences (Schnakers et al. 2008). In general, these results suggest that partially preserved semantic processing could be observed in noncommunicative brain-damaged patients, notably for the detection of salient stimuli, such as the subject’s own name.

Finally, the N400 is observed following sentences that end with semantically inappropriate words or, more generally, significant stimuli (Balconi and Pozzoli 2003, 2005). Its amplitude was found to be related to the semantic congruence, semantic relatedness or proximity, meaning probability and contextual constraint. Thus the semantic violation paradigm supported by the N400 may be used to assess high linguistic and conceptual skills in subjects. This linguistic marker may be used to test the preserved abilities to respond to linguistic stimuli. About the MCS a significant amount of data confirmed the presence of some cognitive functions, as it was shown by analysis of N400 (Balconi 2011; Fischer et al. 2010). Some previous research focalized on semantic processing by using classical linguistic task (Rämä et al. 2010; Schoenle and Witzke 2004; Vanhaudenhuyse et al. 2008). Preserved abilities were found in DOC in response to linguistic stimuli (Steppacher et al. 2013). Functional skills to process semantic meaning were observed in some conditions, that is in MCS and sometimes in single-case reports of VS (Balconi et al. 2013; Schoenle and Witzke 2004). The N400 highlights the ability to evaluate intent, which is a critical component of awareness (Overgaard 2009). At this regard, in some cases misdiagnosis occurs for patients who are considered in VS and who instead could be more correctly classified as minimally conscious, by adopting some ERP measures such as N400.

The present research aimed to associate the behavioral measures (the CNC and DRS scales) with ERP (N400) modulation in response to a semantic task, to test the diagnostic significance of these two levels of measure. Thus the main goal of the present research was to verify the relationship between traditional assessment measures and ERP indices. Indeed, previous study did not directly compare the psychometric measures with the ERP approach (Balconi et al. 2013). Within an experimental task, some patients with DOC were required to implicitly process congruous versus incongruous linguistic semantic associations by auditory stimulation. Due to the experimental paradigm which was used in the present research, for the first time a specific preserved ability to evaluate semantic associations based on auditory word meaning was tested in the MCS and VS. The auditory stimulation may make easier to comprehend the semantic meaning, due to the difficulty in many patients with DOC to process visual words.

This task in healthy people generally induces an increased N400 amplitude (higher peak deflection) as a consequence of the semantic processing of an incongruous condition (Balconi and Pozzoli 2004, 2005). We intended to explore the presence of residual semantic comprehension process in patients with DOC and its relationship with the clinical diagnosis as stated by behavioral measures. Specifically patients with DOC were tested for their ability to comprehend congruous versus incongruous semantic associations between pair of words. A significant N400 higher amplitude was expected in response to incongruous patterns in comparison with congruous patterns for patients able to semantically process the linguistic anomaly. About the cortical localization of the N400, a more frontal distribution was expected based on the fact that the present study used an auditory stimulation. Indeed, previous studies revealed a more significant frontal distribution of the N400 for auditory stimuli in comparison to visual stimuli, which generally induce a more parietal cortical distribution (Balconi and Pozzoli 2004).

Also significant differences were expected for ERP profile as a function of the CNC/DRS scores. Specifically, we intended to comprehend the diagnostic value of the two measures in relationship with the semantic processing, as it was tested by the ERP marker. We expected that lower scores in the CNC and DRS measure could support different degree of semantic preserved ability, as shown by an increasing of the N400. Possible differences were also supposed between the two behavioral measures, since the DRS was found to be more sensitive to the cognitive changes in patients with DOC than the CNC.

Methods

Patients

The sample included 22 patients (10 men and 12 women) aged between 25 and 64 (M age 53; SD = 8.98). The patients with DOC followed a coma due to anoxia (10), traumatic brain injury (7), stroke (5). The time between coma onset and the experiment ranged between 6 and 63 months (mean 48 months). The patients had no history of neurologic disorder prior to coma. The two scales (CNC and DRS) were administered to the patients by three expert neuropsychologists following the standard guidelines (see details in Rappaport et al. 1982; Rappaport 2005). They rated each patient in blind condition (without having any previous clinical indications about the patients’ classification in terms of MCS/VS), with independent evaluations that were successively compared. However, due to the presence of significant eye- or motor-artifacts that make difficult to analyze the ERP profiles, four patients were not considered for the successive analysis and only 18 were used for the statistical analysis.

Based on these scorings patient group was sub-divided into two subgroups for the successive statistical analysis: patients in VS and MCS. The first one included seven patients who scored more than 2.00 on the CNC; more than 22 (range 22–24) to the DRS; the second one 11 patients who scored between 0.00 and 2.00 on the CNC; less than 22 (range 12–20) on the DRS. This classification was based on previous cut-off, as suggested by literature to classify MCS and VS (see Rappaport et al. 1982, 2005 for the cut-off parameters). These two measures showed to be highly correlated (as reported in Table 3) and they are able to furnish a clear cut-off between high and low impaired patients with DOC. The study was approved by the Ethic Committee of the Catholic University of Milan. The demographic and clinical data were reported in Table 1. Patients were in rehabilitation center supported by medical assistance and rehabilitation for all the hospitalization.

Table 1 Demographic and clinical data of DOC patients
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Stimulus

Each auditory word sequence was composed of four words that were presented to the subjects, and it had a congruous (semantic related words) or an incongruous (semantic unrelated words) final word based on its semantic content. The semantically unrelated final words were chosen on the basis of belonging to distant semantic categories and a lack of association according to the association norms. For example subjects were presented the following incongruous words sequence: [cherry, apple, melon, cuttle]; or congruous words sequence: [cherry, apple, melon, grapes]. Specifically, the association norms were drawn from the standard psycholinguistics database (Peressotti et al. 2002). Prior to the experimental phase, nine judges evaluated the semantic proximity and relatedness (semantic associates) of each pair with a nine-point Likert scale. The judges were required to consider the degree of the semantic association (“Do you think the final word is directly semantically related to the other preceding words?”). They independently evaluated the stimuli with a blind procedure. Subsequently their scores were averaged. The congruous and incongruous word categories obtained the following scores for semantic association: semantically related words M = 8.43, SD = 0.12; semantically unrelated words M = 1.98, SD = 0.30.

Words used to create semantic associations were Italian nouns (from four to seven letters) of moderate frequency, a stated by De Mauro (1993). The familiarity of the words was assessed for the whole stimulus material before the experimental task by a group of ten judges. Familiarity was evaluated using a five-point Likert scale, and all of the words in the study showed similar high familiarity rates (M = 4.87; SD = 0.22). All of the words that were included were counterbalanced relative to the word length and their abstract versus concrete contents (De Mauro et al. 1993).

Procedure

The task was performed in a quiet room (the hospital bedroom) in which patients were tested one at a time. They were trained with the task in a preliminary session, with the same associative implicit task (four examples, two for congruous and two for incongruous condition, these examples were not used also for the successive experimental session). The sequences (30 congruous and 30 incongruous sequences) were presented orally. To make the cognitive task more simple and to reduce the cognitive effort, the entire battery (60 trials) was subdivided into six sub-sequences of 10 trials (five congruous and five incongruous sequences) each. Two loudspeakers were placed behind the participant, to the right and left of participant’s head at a distance of 30 cm. The volume was distinctly audible (about 60 dB). They were introduced in a word-by-word presentation (duration range 2–3 s.) with an ISI interval of 3 s and an inter-trial interval of 15 s. The order of the sequences was counterbalanced across participants.

EEG Data Reduction

The EEG was recorded using a 64-channel DC amplifier (SYNAMPS system, Compumedics Neuroscan, Charlotte, USA) and acquisition software (NEUROSCAN 4.2, Compumedics Neuroscan, Charlotte, USA). An ElectroCap with Ag/AgCl electrodes was used to record the EEG from active scalp sites (which were used to calculate the ERP profile) that were referred to the earlobes (10/20 system of electrode placement, Jaspers 1958). The data were recorded using a sampling rate of 500 Hz, with a frequency band of 0.01–50 Hz. An off-line common average reference was successively computed to limit the signal-to-noise ratio (for this aspect see Teplan 2002). In addition, two EOG electrodes were placed on the outer side of the eyes. The impedance remained below 5 kΩ. After the EOG correction and preliminary visual inspection to check the morphological profile of each wave, only the artefact-free trials were examined (rejected epochs 6.69 %). The signal was visually checked, with the artefacts removed. A successive regression-based eye movement correction was used and data were corrected by an ICA-based algorithm (Jung et al. 2000). The computerised artefact rejection criterion excluded the peak-to-peak amplitude when it exceeded 40 μV. An averaged waveform (off-line) was obtained for each condition, each electrode and each participant (not less than 23 epochs were averaged).

The peak amplitude measurement was quantified relative to the 100-ms pre-stimulus, and it considered the most negative peak value within the temporal window of the 300–500 ms post-stimulus. The time onset was coincident with the appearance of the final word. The peak deflection appeared with a mean latency of 420 ms post-stimulus.

Results

The morphological analysis of ERPs showed a significant negative deflection for all the patients. In order to test the presence of a significant N400 effect in each subject, a preliminary statistical calculation was performed on averaged traces from individuals: amplitudes and latencies of the N400 deflection were calculated for individual averages. Subsequently, individual waveforms were analyzed on a point-by-point basis using serial t scores, which take into account the variance of the individual ERP trials composing the grand-averaged ERP. t scores were computed for all subjects in a temporal window of 100 ms. around the peak latency of the N400. Bonferroni corrections for multiple comparison was applied. The analysis was applied to three specific regions of interest (see the following analysis for details) and two conditions. Results were considered significant at P < .05, and patients were inserted in the successive data analysis when at least one significant difference was found between regions of interest or condition. Therefore, only significant differences were considered to apply the second set of analysis (see the following ANOVAs). Thus, the 18 selected patients showed significant differences and they were used for the successive steps (see Table 1).

Successively, two sets of analysis were conducted: the first (repeated measure ANOVA) was conducted to compare the two categories of DOC patients (VS and MCS) with respect of ERP profile modulation in response to the congruous versus incongruous condition. The second (Pearson’s correlation analysis) was aimed to correlate the ERP measure with the behavioral assessment measures (respectively the CNC and DRS ratings).

Two dependent measures were used, respectively the N400 peak amplitude and the peak latency. The ERPs were entered into a three-way repeated measure ANOVA. Independent, repeated factors were Congruence (2), DOC Category (2), and Region (3, fronto-central, temporo-parietal and occipital). Indeed, to obtain specific regions of interest, the data were averaged for the fronto-central (Fz, F3, F4, Cz, C3, C4), temporo-parietal (T7, T8, Pz, P3, P4) and occipital (O1, Oz, O2) electrode location.

With respect to the peak amplitude measure, significant main effects were found for Congruence (F(1,17) = 9.45, P = 0.001, η2 = .39) and Congruence × Region (F(2,17) = 9.12, P = 0.001, η2 = .38) (Fig. 1a–d). No other effect was significant (for all P > .10). A more negative deflection was found in response to the incongruous condition, as compared to the congruous condition (Table 2).

Fig. 1
figure 1

N400 peak amplitude (grand average) for: a patients in MCS, b patients in VS; and N400 peak latency for c patients in MCS, d patients in VS

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Table 2 Mean peak amplitude (mVolt) and latency (msec.) of N400 effect as a function of congruence and region of interest
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Secondly, as shown by contrast analysis (Bonferroni alpha corrections for multiple comparisons), a higher fronto-central deflection was found in comparison with temporo-parietal (F(1,17) = 11.46, P = 0.001, η2 = .44) and occipital (F(1,17) = 10.13, P = 0.001, η2 = .40) sites in case of incongruence Condition (Figs. 2, 3, 4).

Fig. 2
figure 2

Cortical maps of congruous (a) and incongruous (b) condition for DOC patients. An increased peak amplitude (increased red colour) was reported in response to incongruous condition at the fronto-central areas (Color figure online)

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Fig. 3
figure 3

N400 profile (grand average) as a function of regions of interest and patients in MCS/VS

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Fig. 4
figure 4

Scatterplots for: a N400 peak amplitude and CNC scores; b peak amplitude and DRS scores; c N400 peak latency and DRS scores

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About the peak latency variable, significant main effects were found for Congruence (F(1,17) = 7.12, P = 0.01, η2 = .33) Congruence × Region (F(2,17) = 8.78, P = 0.001, η2 = .39), and Congruence × Category × Region (F(2,17) = 8.70, P = 0.001, η2 = .39). As revealed by the post hoc analysis, a delayed peak was found in response to incongruous Condition (F(1,17) = 8.96, P = 0.001, η2 = .40) (Table 2). Secondly, a more delayed fronto-central deflection was found in incongruous than congruous Condition (F(1,17) = 7.10, P = 0.01, η2 = .32). About the three-way interaction, post hoc comparisons revealed a significant difference between congruous and incongruous Condition within the fronto-central sites for both VS (F(1,17) = 7.60, P = 0.01, η2 = .33) and MCS (F(1,17) = 9.03, P = 0.001, η2 = .43), who showed delayed peak deflections in case of incongruous condition. Moreover, VS and MCS patients differed in incongruous Condition within the fronto-central sites (F(1,17) = 6.77, P = 0.01, η2 = .30), with delayed peak for VS. No other comparison was significant.

Pearson Correlational Analysis

A specific correlational analysis (Pearson coefficient) was conducted between the behavioral measures (CNC and DRS ratings) and respectively the peak amplitude and latency measure.

A significant inverse correlation was found between the peak amplitude for incongruous condition and the two behavioral scales: thus a decreased impairment (a lower CNC and DRS scoring) was related to an increased peak amplitude for incongruous condition. Moreover significant correlations were found between the DRS and the peak latency measure, since increased latency values were significantly correlated with increased DRS scores (Table 3).

Table 3 Pearson’s correlational values between N400 peak amplitude and behaviorual scales (CNC and DRS)
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Discussion

Four main results were found in the present research: (1) a first main result was that a N400, peaking at about 420 ms. post-stimulus, more frontally distributed and higher for incongruous condition, was found for patients with DOC, taking into account both patient in VS and MCS category. (2) This effect was similar in amplitude between the two patients’ categories. However, the comparison between the peak latencies in the two conditions (congruence and incongruence) revealed significant differences, with a delayed peak in incongruous in comparison with congruous condition. Moreover patients in VS showed a higher increased peak latency of the N400 in incongruous condition than patients in MCS. (3) In addition, the correlational analysis showed a significant relationship between the behavioral measure (the CNC and DRS) and the ERP modulation for the semantic processing. In other words, a decreased impairment shown by CNC/DRS ratings was related to an increased N400 amplitude in response to incongruous patterns. (4) Finally, the DRS scale showed to be more sensitive to N400 latency modulation, with an increased latency for higher DRS scores.

About the functional significance of the N400, it was found to be particularly useful for indexing some preservation of awareness, such as comprehension of meaning or associative semantic relationship (Cavinato et al. 2011). Previous research found that some patients diagnosed as VS showed some semantic capabilities (Rämä et al. 2010; Schoenle and Witzke 2004). However, no consistent results were obtained comparing different research. About the MCS a significant amount of data confirmed the presence of some cognitive functions, such as it was shown by analysis on P300 and N400 ERPs (Fellinger et al. 2011; Fischer et al. 2010; Qin et al. 2008; Perrin et al. 2006).

The present contribution explicitly explored the semantic processing that includes the evaluation of conceptual relationship between words. We found that final words semantically unrelated to previous words elicited a larger N400 than semantically related words in all patients with DOC, as revealed by morphological, individual and group analysis of ERP. Moreover, the N400 appeared to be morphologically similar to the classical peak deflection observed for auditory stimulation. In fact, whereas a more posterior (parietal) localization was generally found for N400 in response to visual stimuli, a more anterior (fronto-central) cortical distribution was found for auditory stimuli (Balconi and Pozzoli 2005; Kutas and Federmeier 2011).

Since the N400 was supposed to reflect some aspects of how words are related to their semantic context, we may hypothesize that DOC patients showed N400 amplitude variation depending on the difficulty of implicitly retrieving knowledge associated with context (Gerrig and McKoon 1998). Based on previous research on N400, it was suggested that the N400 amplitude may be determined by the difficulty of restoring linguistic material. This hypothesis was referred to as “memory retrieval account” and it was used to explain the variations due to the degree of relevance and general “coherence” of information within a context. Thus the amplitude of the N400 is modulated by the degree of plausibility of a conceptual dependency and the strength of the underlying associations: indirect dependencies or conceptual independencies result in an increased negativity (Kutas and Federmeier 2000). Therefore, we may suppose that patients with DOC preserved the ability to execute some high level linguistic functions and that these functions can be remarked by electrophysiological measures (Fischer et al. 2008).

This relevant result partially goes against the concept of patients in VS and the general supposition on their poor conceptual skills. The question was raised of which ERP marker may reflect the semantic preserved abilities in DOC states taking into account the different level of impaired consciousness (Sörös 2011). The present research elucidated the role N400 may have at this regard. Moreover, it showed as a critical point the supposition that patients with DOC, and specifically patients diagnosed as in VS, lack of significant conceptual and semantic competencies. Contrarily, we found that also VS may preserve some important cognitive functions, such as some semantic associative processes.

About the delayed N400 we found in response to incongruous associations it could be explained taking into account the increased difficulty for patients with DOC to restore the appropriate semantic context due to the unattended associations. In fact, it was found that potentially unrelated information is processed with increased cognitive costs, as a function of how this information has to be mentally discarded as not relevant and the conceptual context has to be restored (Balconi and Pozzoli 2008). This fact may reflect the implication of higher cognitive resources that involve semantic processes and language comprehension, which are less preserved in the case of patients with DOC. In other words, in case of DOC, conceptual processes could take longer time to be completed. Related to this result, an important difference was observed between patients in VS and MCS, since patients in VS took more time to produce the N400 response within the fronto-central sites. This fact may be explained suggesting an increased difficulty by VS to activate the coherent cognitive process related to violations of semantic expectations, as revealed by the higher N400 amplitude for incongruous pairs. Thus, whereas the two patient categories did not differ in terms of the qualitative profile and the amplitude of the N400, the latency variable marked the gap between these two clinical profiles, with higher cognitive costs for the VS more than the MCS.

The other main point of the present research was related to the correlation between behavioral (CNC and DRS) and ERP measures. As shown by the correlational analysis, the N400 appeared to have a an important role when it was related to the classical assessment measure, since the N400 appears to vary as a function of the increasing/decreasing of the patients’ impairment, as stated by CNC and DRS scales. Specifically, both CNC and DRS scores were associated with the semantic skills in patients diagnosed with DOC as characterized by N400. Moreover, the present results are mainly relevant taking into account the concomitance of variations in N400 and both the behavioral measures. Thus we can state that, whereas the EEG data are based on physiological behavior and it is responsive to the cognitive processes (for the long-latency ERPs), firstly it may be directly related to the clinical assessment and secondly that it allows to increase the diagnostic power of the classical measures. Moreover, although CNC and DRS considered different descriptive aspects of patients’ with DOC behavior, respectively the responsiveness of DOC to different sensory stimulation (CNC) and the behavioral disability by quantitative scales (DRS), they show to have a clear and consistent relationship with the N400, as a function of the semantic processing complexity.

However, DRS was found to be more sensitive than CNC to the cognitive performance in patients with DOC, since a significant relationship was also found between DRS scores and latency measures. As it was reported, the increased latency of the N400 effect in response to the incongruous associations was explained taking into account the higher cognitive costs related to an unattended semantic relationship. Also, VS patients showed to increase their peak latency due to this higher cognitive complexity. Thus in general we may suppose DRS might be more able to underline this increased difficulty in processing incongruence, and, thus, to distinguish the two clinical profiles (VS and MCS) based on pshychophysiological variations.

To conclude, ERP index appears as a dynamic measure which was able to characterize good competences and covert preserved skills (such as intact semantic processing) also in presence of an overt deficit (for example related to a motor behavior). Patients previously classified as VS maintain a consistent ability to respond to the semantic task, by showing a significant preserved comprehension of incongruous associations, although they need more time to process the semantic incongruence. This consideration may help to elucidate the question whether VS/MCS patients suffer only from performance problems or whether they have lost their inner knowledge and processing capabilities in the various mental and cognitive domains. More generally, since patients with DOC were able to process language, this is strong evidence for preserved higher cognitive functions and an argument against the assumption that these patients only respond at a basic perceptual level (Amantini et al. 2011).

Due to the present results we have to consider the hypothesis that patients with DOC retain some specific semantic and preserved cognitive high level functions. In addition, the current diagnostic criteria for DOC, mainly based on observing patients’ behavior, may prevent to deeply comprehend the real cognitive potentialities of this patient group and to correctly classify different consciousness profiles. An adjunctive open question remains to be answered, whether in some cases misdiagnosis occurs for patients who are considered in VS and that instead could be more correctly classified as minimally conscious, by adopting ERP measures (and specifically N400). Future research should discuss the critical point of direct comparison of patient group previously classified on the basis of other behavioral measures (by other psychometric scales, such as GCS), to check the correlation between different clinical and psychophysiological markers.

However, due to the sample size limitation and the number of patients included in the MCS and VS category, in the future the present data should be integrated. Secondly, the systematic analysis of the aetiology of DOC patients may help to better comprehend possible within-category (MCS, VS, or C) differences. An exhaustive analysis about the relationship between different clinical profiles, psychometric measures and ERPs might furnish a more complete overview.