How somatosensory evoked potentials improve the diagnosis of the disturbance of consciousness: A retrospective analysis

ABSTRACT The interpeak latency is a crucial characteristic of upper limb somatosensory evoked potentials (USEPs). However, the existing research on the correlation between interpeak latency and consciousness disorders is currently limited. We aimed to investigate how USEPs can contribute to the diagnosis of consciousness disorders. A retrospective analysis was conducted on 10 patients who underwent repetitive transcranial magnetic stimulation (rTMS) for consciousness disorders. The interpeak latency N13-N20, Glasgow coma scale (GCS), and Chinese Nanjing persistent vegetative state scale (CNPVSS) were evaluated before and after rTMS treatment, and the linear correlation between N13-N20, GCS, and CNPVSS was analysed. The scores of CNPVSS and GCS significantly increased in the first, second, and third months after rTMS. The N13-N20 was shorter in the second and third months after rTMS compared to before treatment. rTMS was found to shorten the N13-N20 latency, and there was a negative correlation between N13-N20 and the score of consciousness disorders. N13-N20 can serve as an objective index for evaluating consciousness disorders. This research provides potential insights for doctors in diagnosing patients with consciousness disorders.


Introduction
Cerebral haemorrhage is usually divided into traumatic cerebral haemorrhage and spontaneous cerebral haemorrhage.Compared with spontaneous cerebral haemorrhage, traumatic cerebral haemorrhage is more likely to have different degrees of impaired consciousness (Tan et al. 2010).Disorders of consciousness (DOC) are classified as coma, vegetative state (VS), and minimally conscious state (MCS) based on differences in symptoms and timing.The main methods of clinical assessment of DOC include behavioural scales, neurophysiology and neuroimaging (Giacino et al. 2013).Among the widely used behavioural scales are the consciousness recovery scale-revised (CRS-R), the Glasgow coma scale (GCS), and the Chinese Nanjing persistent vegetative state scale (CNPVSS).Several studies have shown that the reliability and validity of CNPVSS and CRS-R scores are consistent, especially in identifying VS and MCS impairment of consciousness (Wang et al. 2020;Xingming et al. 2022).
Repetitive transcranial magnetic stimulation (rTMS) is recommended by the National Institutes of Health and Human Services for the treatment of traumatic brain injury-induced DOC, central pain, depression, and many other conditions (Hosseinian et al. 2021;Pink et al. 2021).Patients in a minimally conscious state (MCS) following brain injury may potentially experience benefits from rTMS (Liu et al. 2018).The combination of rTMS with electroencephalography (EEG), known as rTMS-EEG, could serve as an effective assessment tool to evaluate the therapeutic efficacy of rTMS protocols in DOC (Bai et al. 2016).rTMS produces excitatory or inhibitory potentials in cortical neurons and alters cortical functions, which is a noninvasive, safe and effective neurophysiological treatment technique (Chen et al. 2022).Different frequencies of transcranial magnetic stimulation produce different effects.Low-frequency rTMS (0.5 Hz, 1 Hz) reduces the excitability of cortical neurons and decreases cerebral blood flow and neurotransmitter expression (Qiu et al. 2020;Liu et al. 2022).In contrast, high-frequency rTMS (5 Hz, 10 Hz, 20 Hz) elevates neuronal excitability and increases cerebral blood flow and neurotransmitter production (Zhu et al. 2021).Multiple studies have demonstrated that high-frequency rTMS can enhance the level of awareness in patients with consciousness disorders and have a stimulating effect on wakefulness (Liu et al. 2016;He et al. 2018;Fan et al. 2022).
How to accurately evaluate the level of consciousness impairment in a patient remains a challenge in modern medicine.Behavioural scales, though commonly used, can be subjective and may not provide clear criteria for classifying DOC.Therefore, there is a need for an objective indicator that can accurately assess the severity and state of consciousness disorders.N13 and N20 are key indicators of upper limb somatosensory evoked potentials (USEPs; Inoue et al. 2004).N13 represented the posterior horn synaptic potential of the cervical spinal cord, N20 represented the central posterior cortical potential (Reisecker et al. 1985;Tinazzi et al. 1997).N13-N20 represented the central conduction time and was used to analyse the conduction velocity between the posterior horn of the spinal cord and the sensory cortex (Edwards et al. 2010;Kalina et al. 2023).These potentials can be used to assess nerve conduction velocity and the status of the central nervous system, and they hold significant importance in the fields of neuroscience research and clinical diagnosis (Ganji et al. 1988;Moncho et al. 2015).Previous studies have shown that patients with impaired consciousness have prolonged N13 and N20 wave latency and reduced amplitude (Logi et al. 2003).However, peak latency and amplitude are disturbed by peripheral nerve conduction velocity, and the degree of impaired consciousness cannot be assessed with complete accuracy.The interpeak latency N13-N20 refers to the conduction time between the two wave peaks N13 and N20, which indicates the central conduction time.The interpeak latency is more representative than the wave crest latency in indicating the excitability of the central nervous system (Shevchenko et al. 2020).
In this study, we retrospectively analysed the neurophysiological and clinical responses of 10 patients with consciousness impairment after cerebral haemorrhage, both before and after rTMS treatment.We focused on investigating the linear regression between the interpeak latency N13-N20 in somatosensory evoked potentials and the level of consciousness impairment.

Clinical data
Research subjects.The study sample consisted of 15 patients with impaired consciousness after cerebral haemorrhage.These patients were admitted to the rehabilitation Center of Nanjing Zijin Hospital between June 2020 and June 2021.All patients received conventional medical wake-up therapy and rehabilitation.In the study, five patients were unable to complete the rTMS treatment course due to being discharged from the hospital.Consequently, a total of 10 patients with impaired consciousness resulting from cerebral haemorrhage were included for rTMS treatment.The patients were all male, with an average age of 45.7 ± 5.51 years and an average course of 21.2 ± 1.41 weeks (Table 1 for details).Prior to the treatment, all patients underwent assessment of clinical impairment of consciousness scores and USEPs.These assessments were repeated monthly for a total follow-up period of three months after the treatment.

Inclusion criteria
(1) Patients who are in a coma following a traumatic brain injury for more than 1 month.(2) Patients with no concurrent severe brainstem lesions.
(3) Patients with stable disease, without hydrocephalus or severe brain atrophy.(4) Patients with all haemorrhagic lesions that have been absorbed.
(5) Patients who have signed informed consent.

Exclusion criteria
(1) Patient with a previous history of epilepsy or a history of idiopathic epilepsy in a first-degree relative.

Treatment methods
All patients were treated with rTMS on the basis of conventional drug treatment and rehabilitation treatment.The rTMS (Magneuro 100, Weiss Medical, Nanjing, China) and a matching figure-of-eight coil were used for treatment.The patients were positioned supine, and the coil was placed tangentially at a 45°angle to the target area of the scalp.Using the international 10-20 system for electroencephalogram localization, the stimulation sites were determined to be 1 cm away from the anterior, posterior, upper, and lower points of F3 or F4 on the affected side.The stimulation target was confirmed when motor evoked potentials of at least 50 μv were detected at the abductor pollex brevis of the affected hand in at least 5 out of 10 attempts.The rTMS treatment involved delivering 2,000 pulses at a frequency of 10 Hz, with the stimulation intensity set at 90% of the resting motor threshold (RMT).Each stimulation lasted for 5 s, followed by a 10 s interval, resulting in a total treatment time of 10 minutes.The treatment was administered once a day, five times a week, for a duration of four weeks, with an additional eight-week follow-up at the end of the treatment course (Xie and Zhang 2012).

Assessment of clinical impairment of consciousness
We performed impairment of consciousness ratings using the CNPVSS and GCS for all enrolled patients with post-cerebral haemorrhage impairment before and after rTMS treatment, which was assessed and recorded monthly by a regular therapist.The CNPVSS score consists of five sub-items, namely auditory function, eye movement, limb movement, feeding function, and emotional response, which tests different dimensions of neurological function related to the level of consciousness.Each sub-item is scored on a 5-point scale, with a score of 0 for complete unresponsiveness and a score of 4 for the ability to perform the function at will.Patients with no spontaneous eye opening or stimulus-induced eyeopening, and with scores equal to or less than 1 for all items, were rated as comatose; patients with spontaneous eye opening or stimulus-induced eyeopening, and with scores equal to or less than 1 for all items, were rated as VS; those with scores equal to 2 for any one item and not 3 or more for other items were rated as MCS; those with scores of 3 for any one item and not 4 for other items were rated as MCS+, and those with scores of 4 for any one item were rated as out of microcosm.The GCS score includes an eye-opening response, verbal response, and body movement, with a maximum score of 15 for consciousness, 3-8 for coma, and less than 3 for severe coma with no vocalization due to intubation.Prognosis: mild coma (13-14 points), moderate coma (9-12 points), severe coma (3-8 points) (Nik et al. 2018).

USEPs
USEPs were examined using a myoelectric evoked potential instrument (SIERRA SUMMIT, Cadwell, USA).Scalp electrodes were placed in the international standard 10-20 system lead method (Bagnato et al. 2021),with the electrode at cervical 7, Erb's point, scalp C3 and C4, and a reference electrode on the forehead.We applied pulses with a strength of 8-15 mA, frequency of 3 Hz, and duration of 0.1 ms to the median nerve at the wrist.The intensity was gradually increased until slight movement of the thumb was observed, and the time of nerve conduction was recorded.We utilized Sierra Summit software to process the experimental parameters.The sensitivity of the USEPs was set at 2.6 μv/div, with the scanning speed at 5 ms/div.To minimize the impact of noise and enhance signal legibility, we employed low-pass and high-pass filters configured to allow frequencies ranging from 20 Hz to 3 kHz.The threshold for determining real peak values was established based on the signal-to-noise ratio and peak characteristics.Subsequent analysis focused on potential peaks identified, comparing various features such as amplitude, width, and shape to ascertain the authenticity of each peak and its corresponding position.Multiple repeated measurements were conducted, and the results were averaged to mitigate the influence of noise and improve measurement accuracy.N13 (recorded at the cervical 7 electrode) represented the posterior horn synaptic potential of the cervical spinal cord, N20 (recorded at the C3 or C4 electrode) represented the central posterior cortical potential, and N13-N20 represented the peak-to-peak latency between N13 and N20, which reflected the central conduction time and was used to analyse the conduction velocity between the posterior horn of the spinal cord and the sensory cortex.

Numerical analysis and Pearson's correlation coefficient
Statistical data were analysed using SPSS 23.0 software, and measures that met the conditions of normal distribution and chi-square were described as mean ± standard deviation (SD).When the data did not conform to a normal distribution, they were described as median (P25 to P75).Multiple time point measurements before and after treatment were compared using repeated measures ANOVA with Bonferroni correction, and P < 0.05 indicated statistical significance.Pearson's correlation coefficient method was used to analyse the correlation between N13-N20 and disorders of consciousness score (CNPVSS and GCS).R value was used to represent the strength of correlation.According to Cohen's classification, r = 0 was no correlation, r ≥ 0.20 was a weak correlation, r ≥ 0.50 was a moderate correlation, r ≥ 0.80 was a strong correlation (Lopes et al. 2017).

Representative figures of USEPs before and after rTMS in patients with cerebral hemorrhage
We conducted USEPs tests in cerebral haemorrhage patients experiencing consciousness disorders before and at regular intervals following rTMS treatment, as illustrated in Figure 1.N9 indicates the potential of the brachial plexus, N13 represents the postsynaptic potential in the posterior horn of the spinal cord, and N20 signifies the cortical potential in the postcentral gyrus.The interpeak latency between N13 and N20 reflects the central conduction time.

Impaired consciousness scores in patients with cerebral hemorrhage with impaired consciousness before and after rTMS treatment
All patients with cerebral haemorrhage with impaired consciousness received the same course of rTMS, and the pre-treatment CNPVSS scores and GCS scores did not conform to a normal distribution by the Shapiro-Wilk test and were transformed to conform to a normal distribution by the normal score method.Multiple time point measurements before treatment were compared with those at the first, second, and third months after treatment by repeated measures ANOVA.As shown in Table 2, CNPVSS scores and GCS scores were significantly higher in the post-treatment compared with pre-treatment (P< 0.05).

rTMS shortens interpeak latency N13-N20 in patients with impaired consciousness after cerebral hemorrhage
The values of N13-N20 before and after rTMS treatment conformed to a normal distribution and were expressed as mean ± SD.Multiple time point measurements before and after treatment were compared using repeated measures ANOVA.As indicated in Table 3, there was no substantial disparity in N13-N20 between the first and second months after treatment compared to baseline.However, there was a decrease in N13-N20 at the third month after treatment when compared to baseline (P = 0.002).

Linear regression of interpeak latency N13-N20 with CNPVSS and GCS scores
We conducted an analysis to examine the correlation between the interpeak latency of N13-N20 and the CNPVSS and GCS scores.The results, which are shown in Figure 2 and Table 4, revealed the following findings.Prior to the rTMS treatment, the correlation coefficient (R value) between N13-N20 and the GCS scores was 0.300 (P = 0.399).
Additionally, the correlation between N13-N20 and CNPVSS scores was 0.098 (P = 0.788), indicating a weak positive correlation.During the first month after treatment, the correlation between N13-N20 and GCS scores decreased slightly to −0.184 (P = 0.610), implying a weak negative correlation.Similarly, the correlation between N13-N20 and CNPVSS scores decreased to −0.227 (P = 0.529), indicating a weak negative correlation.At the second month after treatment, there was a stronger negative correlation between N13-N20 and GCS scores, with an R value of −0.721 (P = 0.019).Furthermore, the correlation between N13-N20 and CNPVSS scores was −0.646 (P = 0.043), showing a moderate negative correlation.By the third month after treatment, the negative correlation between N13-N20 and GCS scores became even stronger, with an R value of −0.845 (P = 0.002).Similarly, the correlation between N13-N20 and CNPVSS scores was −0.672 (P = 0.033), suggesting a moderate negative correlation.

The analyze of R-value between N13-N20 and CNPVSS and GCS scores
To further investigate the correlation between the interpeak latency N13-N20 and CNPVSS and GCS scores, we conducted an analysis of the R-values.A heat map depicting the correlation coefficient R values before and after treatment was generated.The heat maps in Figure 3 illustrate the correlation coefficients of the interpeak latency N13-N20 with CNPVSS and GCS scores.Prior to rTMS treatment, the R values showed a positive correlation, indicating that N13-N20 was positively associated with CNPVSS and GCS scores.However, after rTMS treatment, the R values became negative and gradually approached −1, suggesting a strong negative correlation between N13-N20 and CNPVSS and GCS scores at the second and third month.

Discussion
In our study, we retrospectively analysed 10 patients who experienced DOC following cerebral haemorrhage.We followed up with these patients for three months, utilizing SEPs, CNPVSS and GCS assessment scales.Our findings revealed that, through the use of rTMS and comprehensive rehabilitation  treatment, the degree of consciousness impairment gradually improved.Notably, the N13-N20 also displayed a gradual decrease, particularly during the third month, showing a significant decrease compared to the pretreatment period.Furthermore, we discovered a negative correlation between N13-N20 and CNPVSS and GCS scores during the second and third months after treatment.As the treatment duration increased, the R-value approached −1, indicating an increased correlation between N13-N20 and the degree of consciousness impairment.Hence, N13-N20 may serve as a valuable indicator for measuring recovery following an event that induces DOC.As a non-invasive brain stimulation physiotherapy, rTMS can effectively improve the consciousness of patients with DOC, to promote wakefulness and restore consciousness (Zhang et al. 2021).The mechanism of rTMS may be related to the activation of brain neuronal excitability (Kasten and Herrmann 2022).While EEG can capture superficial changes in the electrical activity of the brain, it necessitates the patient to maintain a relatively motionless state during a coma.However, coma patients frequently experience symptoms like tics and muscle spasms, which can disrupt the gathering and examination of EEG signals (Auksztulewicz et al. 2012;Cho et al. 2021).Researchers have further demonstrated that the SEPs can serve as a surrogate index of cortical excitability (Hermann et al. 2022).Prolonged interpeak latency suggests a slower nerve conduction velocity, indicating potential issues with the transmission of signals along the nerves (Peng et al. 2014).In our study, we primarily observed  a correlation between the interpeak latency N13-N20 and the clinical impairment of consciousness scores (CNPVSS, GCS).The shorter the N13-N20, the higher the CNPVSS and GCS scores, indicating that the central conduction time gradually approached normalcy with the course of rTMS treatment and the central neuronal excitability gradually recovered and increased.Activation of the cerebral cortex and the superior reticular nervous system is the key to treatment in the rehabilitation process of recovery and arousal of impaired consciousness.Numerous studies have shown that rTMS improves brain metabolism and brain function by stimulating bilateral dorsolateral prefrontal lobes and regulating the balance between excitatory and inhibitory neurons (Zhong et al. 2021),thereby improving patients' impaired consciousness (He et al. 2020;Wu et al. 2022).In the present study, we also observed a significant increase in CNPVSS and GCS scores in patients with impaired consciousness after rTMS treatment compared with those before treatment.We followed up for three months and found that patients recovered best from impaired consciousness in the 3rd month after rTMS treatment, which may be related to the time of repairing damaged neurological functions in the brain.In addition, we found that the higher the CNPVSS and GCS scores, the closer the N13-N20 values were to normal.These results indicate that patients with impaired consciousness tend to improve after rTMS treatment because the central conduction time is shortened, the function of damaged neurons is gradually restored, and the balance between excitatory and inhibitory neurons is re-established.rTMS can improve the neurobehavioral function of patients with impaired consciousness, and the longer the treatment course, the better the improvement of impaired consciousness (Arnts et al. 2022).This is closely related to the changes in cortical electrophysiology, which explains why the interpeak latency N13-N20 correlates with CNPVSS and GCS, and the longer the time, the better the recovery of consciousness.
The findings of this study should be interpreted with caution due to several limitations.Firstly, it is important to note that this study had a small sample size and was conducted over a short-term period.Furthermore, the participants included patients with cerebral haemorrhage who exhibited varying degrees of impaired consciousness, ranging from coma to vegetative state or minimally conscious state.Additionally, a 3-month follow-up study was conducted to assess the wake-promoting efficacy of rTMS and its correlation with interpeak latency N13-N20.Finally, It is important to acknowledge that the absence of a control group and randomization in this study prevents us from completely ruling out the possible influence of a placebo effect on patients' reported results.It is worth mentioning that the recovery process for consciousness disorders is known to be a lengthy and gradual journey.Therefore, future research should aim to explore the wake-promoting effect and mechanism of rTMS in patients with consciousness disorders.This could involve extending the duration of follow-up, increasing the sample size of the study, and investigating the correlation between the frequency and amplitude of EEG waveforms in patients with different degrees of consciousness disorders.These efforts will contribute to establishing a scientific and theoretical foundation for the use of rTMS in the treatment of consciousness disorders.

Conclusion
This study provided evidence that rTMS can effectively promote wakefulness in individuals with impaired consciousness by analysing a limited clinical dataset using linear correlation.Additionally, our findings indicated that N13-N20 can serve as an objective measure to evaluate the severity of impaired consciousness, offering a theoretical basis for quantifying central nervous system excitability.These findings hold promise for potential clinical applications.

Disclosure statement
No potential conflict of interest was reported by the author(s).
(2) Patient with epileptiform discharge signals observed on electroencephalogram. (3) Patients with implanted brain shunt, implantable pacemaker, cardiac catheter, or electronic heart pump.(4) Patients with metal aneurysm clips, metal staples, vascular sutures, subdural electrodes, or the use of metal plates to close cranial defects.(5) Patients with cranial defects.(6) Patients with complications such as deep vein thrombosis, pulmonary embolism, acute infection, sepsis, or severe anaemia.(7) Patients whose family members voluntarily withdrew from the clinical trial midway.

Figure 1 .
Figure 1.Representative figures of upper limb sensory evoked potentials (USEPs) in patients with intracerebral hemorrhage undergoing repetitive transcranial magnetic stimulation (rTMS) treatment.A: pre-rTMS treatment.B: the first month after rTMS treatment.C: Second month after rTMS treatment.D: Third month after rTMS treatment.N20 represent potentials recorded in the C3 or C4 region, N13 represents potentials recorded in the C7 region, and N9 represents potentials recorded in the Erb's region.

Figure 2 .
Figure2.Linear correlation analysis of N13-N20 with CNPVSS and GCS scores.Using Pearson's correlation coefficient method, CNPVSS and GCS scores were negatively correlated with N13-N20 at the 2nd and 3rd months after treatment, *P < 0.05.

Figure 3 .
Figure 3.The plot of the trend of correlation coefficient R values over time.The darker the color, the larger the value; the lighter the color, the smaller the value.

Table 4 .
Linear correlation analysis of N13-N20 with CNPVSS and GCS scores.