Neuron-specific enolase, representing 1.5% of total soluble brain protein, is a ~ 78 kDa enzyme found predominantly in neurons and neuroendocrine cells [24, 25]. Enolases (2-phospho-D-glycerate hydrolases) are catabolic glycolytic enzymes that convert 2-phosphoglycerate to phosphoenolpyruvate as part of the cellular mechanism for ATP production . Functional enzymes are formed by homo- and heterodimerization of α, β, and γ subunits differentially expressed in every cell type, with the neuron-specific form of enolase comprised of a γ-γ homodimer [26, 27]. Under normal conditions NSE levels in serum should be zero. However, ELISA-based methods for measuring NSE rely upon antibody recognition of the γ subunit, which is also found in platelets and erythrocytes, predominantly as an α-γ heterodimer . As a result, baseline levels of γ-enolase in serum are approximately 10 ng/mL ; in our study, healthy control values ranged from 3 to 22 ng/mL. During neurologic disease states, increased serum NSE is predictive of outcome and correlated to injury severity. For example, in closed-head traumatic brain injury (TBI), ~ 80 ng/mL NSE correlated with severe TBI, ~ 55 ng/mL correlated with moderate injury, and ~ 20 ng/mL was associated with mild head trauma . Moreover, in this same study the level of serum NSE was 87% sensitive and 82% specific in predicting poor outcome. For the majority of trauma-related studies, including extracorporeal circulation-induced injury associated with cardiac surgery, the peak level of NSE was measured within 6–12 h of the inciting event, slowly decaying with an apparent half-life of 24–48 h . This pattern suggests an accumulative building of NSE in the serum over the first few hours after injury followed by a gradual decline that is the sum of ongoing injury-dependent release and catabolic degradation of the enzyme in circulation. However, this pattern is at odds with our observations, in which large increases in NSE were detected within the space of 3 h and large decreases occurred over similar time frames. Our findings suggest acute but transient neuronal injury events that result in a rapid spike of serum NSE followed by rapid decay of the existing NSE without ongoing replacement by continuous neuronal injury.
Assessment of NSE levels at multiple timepoints over the course of several days provided an unbiased dataset that upon post hoc analysis revealed a correlation between seizure and spike events and concomitant rises in serum NSE levels. By comparison to simultaneous measurement of S100β in the same subject along with similar temporal profiling in healthy control subjects, we identified statistically significant NSE signal changes in the epilepsy patients in our study. These findings are strengthened by the general stability of the S100β measurements through time, which rules out sample quality variability as an explanation for the NSE changes. An important caveat, however, is that S100β exhibits a much shorter half-life than NSE, potentially obscuring rapid changes in this molecule due to the sampling window employed. Nonetheless, while all four control subjects exhibited signal variation values indicative of no change (1.0 or less), all 7 epilepsy patients had values above 1.0 (Fig. 4b). Comparison of the 3 patients with low values (< 1.5) versus the 4 patients with high values (> 1.5) revealed no effect of age ([25–49 y] vs [31–45 y]) or disease duration ([5 mo - 29 y] vs [4–41 y]). The low variation in at least one patient (E007) is likely the result of an algorithmic false negative caused by the presence of two spikes in NSE level separated by a time window that masks the sample entropy difference (Fig. 2g). Likewise, the low variation score in E003 may arise from the relative “noisiness” of the NSE measurements in this individual (Fig. 2c), while the lower variation value measured in E002 may arise from the narrow dynamic range of the change in this patient (Fig. 2b). Alternatively, these individuals may have different underlying etiologies or seizure foci/semiologies that preclude neuronal injury or there may be masking effects associated with different drug regimens or comorbidities. Overall, we are unable to determine whether all patients with temporal lobe epilepsy experience ongoing neuronal injury associated with seizures, but our findings support the presence of such injury in at least some patients.
Others have measured NSE and S100β in epilepsy patients, though none of these studies employed the same longitudinal profiling strategy in both patients and healthy controls. A study from Palmio and colleagues showed a statistically significant increase in both NSE and S100β at around 6 h after a seizure and provided evidence that this change occurred in patients with temporal lobe epilepsy but not in individuals with extra-temporal lobe epilepsy . While this supports our findings, it is notable that the change in NSE following seizures in this study was from 8.4 pg/mL to only 13.5 pg/mL, averaged across all of the patients with temporal lobe epilepsy, and the maximum NSE value measured in the study was around 22 pg/mL. In contrast, our averaged measurements ranged from 7.6 pg/mL to 35.0 pg/mL and the maximum NSE level we measured was 117 pg/mL. Whether this difference reflects aspects of the patient cohort, the unbiased sampling strategy employed in our study, or variations in sample processing is unknown. Nonetheless, the Palmio findings along with a number of other published studies [15, 32, 33] support the contention that at least some patients with epilepsy experience ongoing neurodegeneration triggered by individual seizures. This concept is nicely reviewed by Pitkanen and Sutula .
S100β is a glial injury marker and the absence of variation in this protein in the serum is a good indicator of the reliability of NSE as a primary biomarker for the neuronal injury. S100β is a calcium binding protein that at low levels behaves as a trophic factor, but at μM concentrations engages the receptor for advanced glycation endproducts (RAGE) system and causes cell apoptosis . In our multiple serum sampling experiments we did not observe significant changes in S100β. We think that this observation, in contrast to the observed changes in NSE, point toward either low levels of glial cell injury relative to neuronal injury during seizures or a lack of sustained release of S100β . In fact the significantly lower S100β levels in most EMU patients compared to healthy controls is noteworthy. While the reasoning for this apparent suppression of S100β is still unclear and beyond the scope of this paper, we can speculate that less glial cell trophic activity in patients with epilepsy may be the underlying cause . Moreover, low and unchanging levels of S100β indicate that blood brain barrier (BBB) changes do not underlie the NSE elevation observed in association with seizures .
While this study was strengthened by the direct comparison of epilepsy patient measurements to repeated samples collected from healthy control subjects under similar conditions (e.g. intravenous line placement rather than repeated venipuncture, collection under in-patient-like conditions), a number of potential limitations require cautious interpretation of the findings. One of the most significant limitations is the absence of overnight serum samples. This precludes continuous evaluation of the changes in NSE, especially in patients with clinical seizure events that occurred outside of the 6 AM to 6 PM collection window. Likewise, the absence of overnight serum samples may alter the correlation of spike frequency to NSE level. Obviously, these experiments are logistically quite challenging and expensive to perform. In addition to the demands on clinical personnel required for continuous sampling every 3 h for up to 72 h or more, the need to prepare each serum sample immediately after collection requires a concerted round-the-clock laboratory effort. In the absence of some sort of indwelling NSE sensor, however, all such studies will be limited by the sampling frequency and the difficulty of comparing a continuous measurement (EEG) to a discontinuous measurement (serum factors). Since IEDs are sub-clinical events, these have been often overlooked and rarely sought as a measurement of severity of epilepsy . We believe that the spikes in NSE levels prove that IEDs, despite being incapable of causing clinical changes, are capable of damage. Indeed, transient cognitive impairment has been attributed to IEDs situated outside of the seizure onset zone and the frequency of spikes usually depends on seizure frequency in TLE . In addition, another potential issue in this study was the collection of clinical quality EEG rather than research quality data. While we were able to perform automated spike frequency analysis in four of the seven EMU subjects, it is possible that the lower quality EEG restricted the sensitivity of the analysis. This suggests that future studies may benefit from either higher quality EEG, better algorithms for analysis of noisy EEG, or serum sampling in patients with intracranial electrodes. Likewise, the methods employed for measuring NSE and S100β signal variation are challenged by the small number of samples and by sampling gaps. While our strategy for measuring sample entropy and signal variation accounts for the small sample size, this metric would benefit from more measurements and finer temporal resolution. A key example of the difficulties presented by a small sample size is the apparent false negative finding in E007, as discussed above. This patient exhibits a clear spike in NSE at the beginning of the study, but the second, albeit smaller, spike that occurs during the second day of measurements resulted in a low sample entropy score. Presumably, the availability of overnight serum samples would have filled the gap between these two spikes and enhanced the accuracy of entropy analysis. However, this problem at least suggests that the identification of high sample entropies and large signal variation metrics in the other patients are not false positives and were made despite a tendency of the algorithm and the gapped data to underestimate information content. The early NSE spike in patient E007 also reduced our ability to assess the impact of preceding seizures and EEG spiking events on changes in NSE levels, as we had less than 3 h of EEG data collected before the NSE spike. Due to the post hoc nature of the serum analyses we were also unable to ascertain whether the subject had any relevant clinical seizures over the 24 h preceding their enrollment into our study. The issue of sample collection timing also impacted the temporal association between repeated seizure events and changes in NSE levels. Due to the standard clinical practice of terminating the EMU stay after sufficient data are collected to permit identification of seizure foci, the number of samples collected in our study after the third seizure was small compared to the first event. Therefore, the absence of an association between elevated NSE levels and the third clinical event may reflect data insufficiency rather than biology; particularly since this outcome is counter-intuitive (one would predict that more seizures would result in even more detectable NSE). Future studies will require prolonged monitoring after multiple seizures to determine whether the NSE response decays with repeated events. Finally, our study ultimately provides pilot data, rather than comprehensive evidence of neural injury in patients with epilepsy. However, our findings may support a larger, perhaps multi-center investigation into the relationship between EEG and serum biomarkers of neural injury that will provide additional insight into the need for neuroprotective strategies in patients with drug-refractory seizures.
Despite these issues we did obtain several compelling associations. Increased levels of serum NSE were associated with increased time after the first seizure at P = 0.0064. The coefficient of determination for this linear regression is 0.143, indicating that the relationship between time after seizure and increasing NSE levels is noisy. However, 100 iterations of 20% k-fold crossvalidation confirmed that this R2 value was significantly different from zero (95% confidence interval of the k-fold R2: 0.07 to 0.14; P < 0.0001 by Wilcoxon signed rank test against a null hypothesis that R2 = 0; power = 0.999). Due to the discrete nature of both the seizure events and the serum measurements it is difficult to identify a specific post-ictal time domain for the increase in NSE. However, simple inspection of the plot in Fig. 5a suggests that the NSE levels trend upward at around 20 h after the first seizure. This time domain also appears to be relevant to the detection of increased NSE levels following increased spiking on the EEG. Visual inspection of Fig. 5d suggests a broad, albeit low significance, trend toward increased serum NSE from about 15 to 21 h after an increase in spike frequency. Statistically, the strongest association between a preceding increase in EEG spiking and detection of increased serum NSE occurs at 24 h. This time domain exhibited a strong coefficient of determination (R2 = 0.595), high statistical significance (P = 0.0003), and high statistical power (0.9922), suggesting that despite the limitations of our current data we revealed a strong association between an electrophysiological disturbance and a concomitant rise in a neuronal injury marker in the serum after about 24 h. Unfortunately, our ability to determine the length of time over which this rise in serum NSE persists after 24 h is limited by the length and variability of the EEG recording session for the EMU patients. Analysis of Fig. 5d shows that by 27 h after an increase in spike frequency our data are too sparse to draw interpretable conclusions (indicated by the broad 95% confidence interval bands (light red) around the regression fit (red line)). This suggests that future studies will need to retain the EMU subjects for longer EEG recording. This would also permit more serum measurements, further strengthening our ability to detect significant associations. Nonetheless, our current data support the strong, biologically relevant conclusion that an increase in serum levels of the neuronal injury marker NSE is detected approximately 24 h after an electrophysiological event consistent with neuronal hyperactivity. If our interpretation of these findings is correct, then post-ictal assessment of serum NSE may serve as a surrogate biomarker for measuring the efficacy of acute neuroprotective therapies aimed at preserving neurons in patients with epilepsy .
Cognitive impairment may be due to both circuit abnormalities and neuron loss, recurrent seizures often result in cell death and concomitant synaptic reorganization, a process that is apparent in hippocampal sclerosis. Due to the multitude of changes that occur in association with seizures, it is difficult to tease out a specific relationship between cognitive impairment and neuron loss . However, several studies in animal models of epilepsy have shown increased calcium flux -dependent excitotoxicity and neuronal death . In addition, epilepsy patients over 50 years of age have a greater risk of dementia and Alzheimer-type pathology as a resultant of neuronal loss in the hippocampus .
We recently reported that treatment of mice with an oral calpain inhibitor after the start of behavioral seizures induced by the neuroinflammatory response to acute viral infection resulted in preservation of hippocampal CA1 pyramidal neurons, preservation of cognitive performance, and abrogation of further seizure events . Likewise, calpain inhibitor therapy started after onset of status epilepticus reduced seizure burden in the rat pilocarpine model  and preserved CA1 neurons in the kainic acid model . Because loss of hippocampal neurons, whether excitatory or inhibitory, may underlie the transition from spontaneous seizures to epilepsy as well as the persistence or spread of epileptic foci , neuroprotective drugs may block epileptogenesis, prevent cognitive sequelae associated with seizures and epilepsy, and facilitate maintenance of seizure-free outcomes following brain resection surgery. However, directly measuring the efficacy of such neuroprotective drugs is challenged by time-to-effect and by the difficulty of correlating the absence of subsequent seizures, etc., to drug efficacy. We therefore propose that measurement of serum NSE will provide causal evidence of drug efficacy, particularly during acute post-ictal windows and perhaps especially in the context of a trial involving calpain inhibitor therapy delivered immediately after a seizure.