Cognitive impairment is a common symptom following mild traumatic brain injury (mTBI or concussion) and can persist for years in some individuals. Hippocampal slice preparations following closed-head, rotational acceleration injury in swine have previously demonstrated reduced axonal function and hippocampal circuitry disruption. However, electrophysiological changes in hippocampal neurons and their subtypes in a large animal mTBI model have not been examined. Using in vivo electrophysiology techniques, we examined laminar oscillatory field potentials and single unit activity in the hippocampal network 7 days post-injury in anesthetized minipigs. Concussion altered the electrophysiological properties of pyramidal cells and interneurons differently in area CA1. While the firing rate, spike width and amplitude of CA1 interneurons were significantly decreased post-mTBI, these parameters were unchanged in CA1 pyramidal neurons. In addition, CA1 pyramidal neurons in TBI animals were less entrained to hippocampal gamma (40–80 Hz) oscillations. Stimulation of the Schaffer collaterals also revealed hyperexcitability across the CA1 lamina post-mTBI. Computational simulations suggest that reported changes in interneuronal physiology may be due to alterations in voltage-gated sodium channels. These data demonstrate that a single concussion can lead to significant neuronal and circuit level changes in the hippocampus, which may contribute to cognitive dysfunction following mTBI. In vivo extracellular recordings in pigs reveal that concussion preferentially disrupts the activity of hippocampal interneurons leading to network hyperexcitability, with in silico models implicating altered voltage-gated sodium channel kinetics. Cognitive impairment is common following concussion and deficits in episodic memory, learning, and delayed recall have been described4. Importantly, a growing body of data suggests that neurocognitive dysfunction observed post-concussion can persist for years after initial insult in some people9. Given the important role of the hippocampus in learning and memory, it is a central region of interest in diseases affecting cognitive processes10. While the underlying mechanisms of cognitive dysfunction following mTBI remain largely unknown, structural changes in the human hippocampus up to 1-year post-concussion have been reported using imaging techniques12. Recent reports also link concussion to hyperexcitability14. Moreover, recent reports based on the epidemiological data may indicate a 2-times increased risk for post-traumatic epilepsy in adult population with mTBI17.Concussion, or mild TBI (mTBI), is a prevalent form of brain injury, affecting approximately 1.3 million people every year in the US alone20. While studies utilizing rodent models of TBI can provide some mechanistic insight into electrophysiological changes following TBI, brains of large animals such as pigs, with their gyrencephalic structure and appropriate white-to-grey matter ratios, more closely resemble human architecture and are important for an accurate biomechanical modeling of all aspects of human TBI21. Using a biomechanically relevant and gyrencephalic model of concussion in swine, we demonstrated injury-related compensatory mechanisms and a reduction in axonal function in the ex vivo hippocampus following TBI in the apparent absence of intra-hippocampal neuronal or axonal degeneration22. Deafferentation of temporal lobe structures (such as the hippocampus) via axonal pathology has been proposed as a driver of hyperexcitability in this model and may disrupt the precise timing of hippocampal oscillations and associated neuronal activity23. Diffuse axonal injury is the predominant pathology observed in this preclinical model, however direct evidence that concussion disrupts neuronal network activity, specific neuronal subtypes, or laminar oscillations in the hippocampus of the intact swine has yet to be demonstrated.Rodent studies of TBI have shown changes in neuronal firing and local hippocampal oscillations, potentially suggesting abnormalities in temporal coding, a crucial element of episodic memory formation and encoding25. Laminar oscillatory field potentials and single unit activity were examined and compared between injured and control animals under isoflurane anesthesia. Our data suggest that even a single concussion may alter both individual neuronal function and circuit-level activity in the hippocampus and may provide insight into aspects of hyperexcitability and cognitive dysfunction following concussion.We utilized an established porcine model of closed-head, rotational acceleration to induce concussion, and then applied high-density in vivo electrophysiology techniques to examine neuronal and oscillatory changes in the intact hippocampus at 7 days post-injury. Notably, the function of hippocampal circuits was assessed using laminar, multichannel silicon probes specifically designed for recording from deep brain structures of large animals26. Electrophysiological properties of CA1 neurons such as firing rate, spike width and amplitude  and post-mTBI  animals at 7 days post-concussion . The spike width of 0.358 ± 0.015 m in the control group was significantly reduced to 0.270 ± 0.008 ms in the post-mTBI group (p < 0.0001), and the spike amplitude was significantly reduced from 300 ± 27 µV in the control group to 187 ± 27 µV in the post-mTBI group of animals (p = 0.0003). These findings suggest that concussion may alter the intrinsic properties of CA1 neurons, which could affect their synchronization with hippocampal oscillations.To examine changes in hippocampal circuitry induced by concussion, in vivo electrophysiological recordings were obtained using 32-channel silicon probes that spanned the laminar structure of CA1 in the dorsal hippocampus Fig. . These pude Fig.  were calion Fig. . While t27. Previously published criteria for rodent hippocampal cells were used for classification28, with several modifications accounting for species differences. Briefly, the probe’s anatomical location coupled with electrophysiological characteristics were utilized to exclude cells recorded at the bottom portion of the probe, as these cells could be identified as putative dentate granule cells, mossy cells, or interneurons in the dentate . Additionally, unclassifiable cells located throughout the length of the probe  were omitted from the analysis. Next, electrophysiological parameters .To further characterize the effects of mTBI on the hippocampal circuitry and to assess changes in specific subpopulations of cells in CA1, recorded cells were separated into putative CA1 pyramidal cells or putative CA1 interneuronsp = 0.0555), spike width , and spike amplitude  were not significantly different between the control (n = 8) and post-mTBI (n = 9) groups of animals at 7 days following concussion  and in the 103–117 Hz frequency band . Additionally, we found a significant reduction in gamma oscillation (35–75 Hz) power  detected locally to pyramidal CA1 cells . These results indicate that concussion disrupts the generation of gamma oscillations in the pyramidal cell layer due to the changes in afferent inputs coming into CA1 and/or alterations in local CA1 pyramidal-interneuron or interneuron-interneuron interactions.Electrophysiological properties of putative CA1 pyramidal cells in the post-mTBI group such as firing rate , spike width , and spike amplitude  at 7 days post-concussion  to 3.78 ± 0.93 Hz in animals injured at ~190 rad/sec , and further to 2.33 ± 0.51 Hz in animals injured at ~260 rad/sec  . These results indicate that concussion alters intrinsic and/or synaptic properties of CA1 interneurons, which affects local gamma and high-frequency oscillations presumably generated by local CA1 interneurons. These changes likely alter the way CA1 pyramidal cells interact with local hippocampal oscillations.Analysis of putative CA1 interneurons revealed that their electrophysiological properties were significantly altered. In contrast to CA1 pyramidal cells, CA1 interneurons had a significantly reduced firing rate  to assess changes in the excitability levels in the intact brain25. Representative examples of the evoked laminar field potentials in dorsal CA1 using Schaffer collateral stimulation are shown as averaged responses over a 300 ms time period  for a control  animals injured at ~260 rad/sec  basket cells provide peri-somatic, feedforward inhibition in the hippocampal CA1 region38. These cells stabilize hippocampal circuitry during changes in input and control the selection of cell ensembles by allowing only certain number of pyramidal cells to fire in response to the stimulus40. Interestingly, pathological changes in PV+ interneurons in CA1 have been also shown to play a crucial role in the development of epilepsy42. To elucidate potential mechanisms underlying the electrophysiological changes observed in CA1 interneurons in our animal model of concussion, we used the NEURON simulation environment44, specifically a model of hippocampal PV+ basket cells described previously45, and adjusted parameters to fit the observed decrease in spike size and firing rate observed in interneurons of mTBI animals (Table inf) in the model , spike width , and spike amplitude   and inactivation (hinf) curves , is decreased by ~30% following inertial injury. The model output using the best fit parameters is shown in Fig. + interneuron dysfunction post-mTBI.As detailed in Table  mV Fig. , arrow. 47. Specifically, concussion is known to lead to acute and chronic cognitive deficits in a subset of mTBI patients, which manifest as poor performance in tests of memory, learning, and delayed recall up to several weeks post injury4. The hippocampus is involved in learning and memory processes10, and its role in integrating information to support these processes may be particularly sensitive to temporal coding disruptions associated with axonal injury or disruptions in oscillatory activity23. Temporal coding in the CA1 region of the hippocampus supports the acquisition, consolidation, and retrieval of spatial and episodic memories by integrating information from entorhinal and CA3 inputs in a temporally precise manner before routing it to downstream structures49. Changes in neuronal firing and local hippocampal oscillations have been reported in rodent models of mTBI, suggesting abnormalities in temporal coding20. However, studies investigating electrophysiological changes following clinical concussion are sparse due to the lack of data from human patients50.Concussion (or mild TBI) is a prevalent type of brain injury with the potential for long-term consequences51, and these models may be the most relevant for understanding the effects of concussion on cognitive processing and memory. We have previously reported changes in hippocampal circuitry following mTBI using an ex vivo slice preparation22. In the current study, we advanced our investigation of electrophysiological changes in CA1 physiology following rotational acceleration injury by performing high-density laminar electrophysiology in the intact anesthetized swine at 7 days post-mTBI. We found that CA1 interneurons had a significantly decreased firing rate, spike amplitude, and spike width, in the injured compared to the control animals. Moreover, we found that concussion resulted in hippocampal hyperexcitability in vivo, which confirms findings from prior work utilizing this model22. Because CA1 activity is known to support learning and memory processes, our combined observations suggest that mTBI-associated changes in CA1 physiology may underlie some aspects of cognitive disruption following mTBI.Large animal gyrencephalic models offer several advantages in modeling the complex biomechanics and resultant pathophysiology of inertial injury+ channels. While other groups have proposed a coupled left-shift of activation/availability of sodium channels using an in vitro model of stretch injury52, our computational model did not predict this negative shift in sodium channel activation but instead predicted a negative shift in sodium channel inactivation. Notably, pathological changes affecting the inactivation of voltage-gated Na+ channels have been previously reported following injury54. Using an in vitro model of axonal stretch injury, we previously demonstrated that traumatic deformation of axons induced abnormal sodium influx through TTX-sensitive Na+ channels, leading to an increase in the intracellular Ca2+ concentration in axons54. Furthermore, axonal stretch injury induced structural changes in the alpha-subunit of voltage-gated sodium channels and led to changes in sodium channel inactivation, followed by a long-term increase in the intracellular Ca2+ and subsequent proteolysis of the sodium channel alpha subunit53. Most recently, widespread and progressive loss of nodal axonal sodium channels have been reported in our pig model55. These results continue to implicate voltage-gated Na+ channel dysfunction as a consequence of TBI, and our translational model of mTBI further suggests that this dysfunction may preferentially affect interneurons in the CA1 region of the hippocampus.We found that concussion-induced changes in neuronal firing in CA1 were cell-type specific and preferentially affected CA1 interneurons. We showed that concussion, induced by inertial rotation, led to changes in the firing rate and spike shape of CA1 interneurons but not pyramidal cells. Specifically, CA1 interneurons had decreased firing rates at 7 days following concussion, potentially contributing to a decrease in local inhibition resulting in increased excitation and hyperexcitability in the CA1 pyramidal cell layer. Our NEURON modeling results indicated that the decreased firing rate and a reduction in spike amplitude and width observed in CA1 interneurons of mTBI animals may be due to a negative shift in the inactivation of voltage-gated Na57. PV+ CA1 interneurons  are directly involved in feedforward inhibition and have been proposed to stabilize the frequency of local gamma oscillations and maintain a sparsity of neuronal ensembles in the presence of changes in the excitatory drive40. This observation may further support TBI associated disruptions in feedforward inhibition provided by PV+ interneurons in CA1. This disruption may also impair behavioral tasks requiring network sparsity , that have been previously reported to be disrupted in a rodent model of mTBI58. That concussion may lead to interneuronal changes underscores the importance of cognitive tasks that rely on spatiotemporal precision for clinical diagnosis. However, rigorously correlating these changes requires awake behaving electrophysiology and the development of behavioral tasks sensitive to this injury24.The changes in oscillatory power and entrainment of CA1 neurons that we observed at 7 days following concussion may also suggest a potential shift in the recruitment/activation of CA1 neuronal subpopulations28. Electrophysiological characteristics that define spike shape and firing properties of pig hippocampal neurons may vary from those defining rodent CA1 neurons due to the differences in the size of neurons as well as variability in the voltage-gated channels responsible for the generation of action potentials59. Also, anesthesia has been known to affect neuronal firing rate as well as the pattern and strength of inputs arriving at the dendritic arbor of the CA1 pyramidal cells60. These caveats notwithstanding, the relative firing properties and spike shapes of pyramidal cells vs interneurons in CA1 follow the same patterns in swine as in rats with pyramidal cells typically firing in bursts  power detected locally to CA1 pyramidal cells post-concussion. The reduction in low gamma oscillations (35–55 Hz) could be due to a decrease in the fiber volleys of Schaffer collaterals (inputs from the CA3 layer) previously reported in this model22. Taking into account the fact that networks of interneurons in CA1 support the generation of local high gamma oscillations (55–75 Hz) and entrain CA1 pyramidal cells to these oscillations61, these results support our findings above that CA1 interneurons are preferentially affected by inertial injury and that they have not fully recovered by 7 days following concussion. Due to the hypothesized role of theta-gamma coupling in memory and recall, loss of entrainment of CA1 pyramidal cells to gamma oscillations may underlie aspects of post-concussive syndrome23. However, oscillatory activity in this study is influenced by anesthesia, suggesting that these results need to be confirmed in the awake behaving animal.Although we found no alterations in intrinsic properties, we demonstrated that CA1 pyramidal cells lose significant synchronization with local gamma oscillations at 7 days following concussion. Previously, we characterized the laminar profile of oscillations in the pig hippocampus34. In TBI, spontaneously generated paroxysmal depolarizations have been only detected in organotypic hippocampal cultures following long-term activity deprivation, potentially due to a homeostatic plasticity mechanism62. Therefore, the prolonged depolarizing shift observed using our model of concussion is the first such epileptogenic event described in the in vivo preparation to the best of our knowledge. A long-lasting depolarization shift and accompanying repetitive action potentials following paired-pulse stimulation of Schaffer collaterals could be due to changes in fiber volleys, given the differential response to varying stimulation intensities in slice previously reported between control and injured animals using this model22. Additionally, we also observed recurring paroxysmal oscillatory events in the form of arcuate spikes occurring in a synchronized manner across all hippocampal layers in all post-mTBI but not control animals. Similar epileptogenic spikes have been previously reported after experimental TBI in rodent models and are thought to reflect repeated population spikes associated with hypersynchronous multi-unit discharges63. Both findings could be substantially influenced by the loss of activity of hippocampal interneurons. Therefore, there are potentially multiple pathological mechanisms involved in the generation of the prolonged depolarizations since we are observing different inputs to the CA1 dendritic arbor simultaneously with these laminar probes.In this study, we observed a prolong depolarization in a subset of injured animals (2 out of 9), followed by a loss of synaptic responses to stimulation. In epilepsy, paroxysmal depolarizing shifts and spreading depolarization have been observed in the hippocampus and cortex using animal models as well as in human patients64. However, mechanisms underlying the development of post-traumatic epileptogenesis following concussion have not yet been identified. Here, we reported a significant reduction in the power of high frequency oscillations (80–300 Hz) detected locally to interneurons  CA1 cells, which may be due to the observed reduction in the firing rate of CA1 interneurons or desynchronization of the CA1 network. A reduction in the number of hippocampal interneurons is a prominent pathology associated with the development of unprovoked seizures both in experimental models and in human temporal lobe epilepsy65. While interneuronal cell loss in our porcine inertial injury model has not been histologically assessed, the number of electrophysiologically active cells recorded in our model was not significantly different between the control and injured animals. Nevertheless, changes in the electrophysiological properties of interneurons may affect circuit level hippocampal interactions and increase the risk for hyperexcitability, and therefore potentially epileptogenesis.Concussion has been previously identified as a risk factor for the development of epilepsy+ interneurons have been previously reported66. While histological and electrophysiological studies have previously identified PV+ interneurons to be selectively altered in experimental models of rodent TBI70, we could not identify a specific sodium channel responsible for the observed changes in CA1 interneurons following concussion. Using NEURON modeling of a CA1 PV+ interneuron , we observed a reduction in the sodium channel window current which in turn may also affect resurgent and/or persistent currents generated by voltage-gated sodium channels71. Interestingly, changes in the window current of voltage-gated sodium channels have been previously reported in epilepsy73. Potential mechanisms for the negative shift of sodium channel inactivation include altered properties of sodium channel auxiliary subunits (beta74 and gamma subunits) as well as the involvement of slow inactivation caused by prolonged depolarization75, both of which were proposed to play a crucial role in sodium channel associated epilepsies72. In addition, multifocal disruption of the blood brain barrier (BBB) has been demonstrated in this model up to at least 72 h post-mTBI as evidenced by extravasation of serum proteins, fibrinogen, and immunoglobulin G, and could potentially contribute to observed electrophysiological changes76.Hippocampal CA1 hyperexcitability and epileptogenesis caused by changes in sodium channels expressed in inhibitory PV78. However, we have no evidence to suggest that anesthesia would differentially affect intrinsic properties or synaptic release probabilities of injured interneurons vs. pyramidal neurons post-injury80. Differences in metabolic states may also contribute to the observed electrophysiological changes, along with potential injury-induced vascular remodeling or changes in neurovascular coupling81. We selectively focused on hippocampal area CA1 due to our prior results demonstrating profound changes in this region in a hippocampal slice preparation with this model22, as well as the known role of the hippocampus in various aspects of cognition and its selective vulnerability in TBI and post-traumatic epilepsy. Verification of these data in awake, chronically implanted animals and expansion to other limbic areas involved in these processes is important future work82.There are several limitations to our electrophysiological approach, including the anesthetized preparation. The predominant effects of isoflurane are known to be on GABA, glycine, and NMDA receptors, and we cannot rule out differential effects on interneurons vs. pyramidal cells+ basket cells and may underlie the exhibited decrease in local gamma power and the loss of entrainment of pyramidal cells to local gamma oscillations. We also observed hippocampal hyperexcitability in the local field potentials, predominantly following stimulation. These changes may underlie aspects of cognitive dysfunction following concussion, as well as increase the risk for post-traumatic epilepsy16. Future studies are needed to investigate if the changes in sodium channel function predicted by our model are observed experimentally. Further assessing these changes over time and determining the differential involvement of specific interneuron populations may reveal therapeutic targets for improving concussion induced memory dysfunction and/or epileptogenesis. Restoring GABAergic interneuronal function before hyperexcitability proceeds to epileptogenesis may improve cognitive outcomes and decrease seizure frequency, as has been previously demonstrated in a mouse model of post-traumatic epilepsy83. While it is unknown whether the observed changes in interneuronal function persist at time points greater than one-week post-injury, pharmacological interventions centered on restoring sodium channel inactivation may be a potential target for improving cognitive function following concussion.In summary, using a translational, large animal model of concussion we have demonstrated that a single inertial injury alters hippocampal circuitry at 7 days post-injury by affecting the electrophysiological properties of CA1 neurons. In particular, the physiological properties of interneurons in CA1 are substantially altered following concussion. These interneuronal alterations may be due to a change in the inactivation of voltage-gated sodium channels expressed in the somata of PVn = 17) were purchased from Sinclair and underwent the current studies at the age of 6 months and a mean weight of 29.8 ± 1.3 kg (mean ± SEM). The animals were fed standard pig chow and water ad libitum. All animals were pair housed when possible and were always in a shared room with other pigs in a research facility certified by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC facility). All animal experiments were approved by and conducted according to the ethical guidelines set by the Institutional Animal Care and Use Committee of the University of Pennsylvania and adhered to the guidelines set forth in the NIH Public Health Service Policy on Humane Care and Use of Laboratory Animals (2015).Yucatan miniature castrated male pigs  and 0.5 mg/kg of midazolam , anesthesia was provided with using 2–2.5% isoflurane  via a snout mask and glycopyrrolate was given subcutaneously to curb secretions . The animals were intubated with a size 6.0 mm endotracheal tube and anesthesia was maintained with 2–2.5% isoflurane per 2 liters O84. Briefly, the injury was performed under isoflurane anesthesia, while physiological parameters of the animals were being closely monitored. Closed-head rotational acceleration was performed using the HYGE pneumatic actuator, a device capable of producing non-impact head rotation (up to 110 degrees in less than 20 ms) with a controlled relationship between maximum rotational acceleration and injury severity. Angular velocity was recorded using a magneto-hydrodynamic sensor  connected to a National Instruments DAQ, controlled by LabVIEW. The animal’s head was secured to a padded bite plate under anesthesia and attached to the HYGE device. A single rapid head rotation was performed within a range of rotational velocities (168–269 rad/s), with the mean angular peak velocity being 226 ± 13 rad/s . For a subset of analyses, the injured animals were divided into two subgroups, those injured at ~190 rad/s  versus those injured at ~260 rad/s . The injury model has been extensively characterized in previously published studies86. Control animals  included naïve animals (n = 4). Another subset (n = 3) received an additional craniectomy anterior to the recording site as control for another study . A single sham animal underwent all injury procedures 7 days prior to the recording, including attachment to the HYGE device without firing it, and was kept under anesthesia for the same amount of time as injured animals.An injury was induced according to the previously described protocolsn = 17 total) according to the previously described protocols25. Briefly, pigs undergoing electrophysiological recordings  were placed in a stereotaxic frame prior to electrode placement. Large animal stereotaxis combined with single-unit electrophysiological mapping via a tungsten monopolar electrode  was used to precisely place a 32-channel laminar silicon probe into the CA1 region of the dorsal hippocampus. For laminar multichannel recordings, either a NN32/LIN linear 32-channel silicon probe  or a NN32/TET custom-design 32-channel silicon probe  was used for electrophysiological recordings (see ref. 24 for more details on multichannel silicon probes used in this study). NN32/LIN probes were used in 9 animals (control = 5 vs. post-mTBI = 4), and NN32/TET probes were used in 8 animals (control = 3 vs. post-mTBI = 5). A skull screw was placed over the contralateral cortex as a reference signal.Electrode implantation was performed in all control and post-mTBI animals . Anesthesia Neurophysiological signals were amplified and acquired continuously at 32 kHz on a 64-channel Digital Lynx 4SX acquisition system with Cheetah recording and acquisition software . The final location of the silicon probes was confirmed electrophysiologically as described previously25. Stimulation amplitude was not significantly different between the control and post-mTBI groups of animals .In a subset of animals , electrical stimulation was performed in the Schaffer Collaterals using custom-designed concentric bipolar electrodes (Cat# CBBPC75(AU1), FHC) to investigate CA1 responses to the inputs coming in from the hippocampal CA3 layer. A-M Systems Model 3800 stimulator with MultiStim software installed was used to generate electrical pulses, while A-M Systems Model 3820 Stimulus Isolation Unit was used to deliver the current. Input-output stimulation in the range of 100–1000 μA was performed prior to the experimental stimulation for each animal, and the final stimulation amplitude (range 200–500 μA) was selected at the half maximum response to stimulationAt the end of electrode insertion procedure, all pigs underwent transcardial perfusion under anesthesia using 0.9% heparinized saline followed by 10% neutral buffered formalin (NBF). Brains were extracted and inspected for gross pathology. Minimal subdural and/or subarachnoid blood regionally associated with electrode insertion was appreciated in all animals. In three animals , minimal subarachnoid blood was also observed ventral to the brainstem. No gross evidence of brain swelling was identified, consistent with prior descriptions of the model.http://klusta-team.github.io/klustakwik/), and clusters were manually refined with KlustaViewa software (https://github.com/klusta-team/klustaviewa)87. The number of single units per animal recorded with either type of multichannel silicon probes (NN32/TET vs. NN32/LIN) was not significantly different  and post-TBI (ncells = 93) groups used for comparison between the groups. The number of recorded cells per animal was not different between the groups . There were no significant differences detected between a number of single units recorded with two types of electrodes  or between a number of single units recorded with each type of electrode for control vs. post-mTBI groups of animals . Resulting single-unit clusters were then imported into Matlab (version R2023a) for visualization and further analyses using custom and built-in routines (https://www.mathworks.com/products/matlab.html). In order to minimize waveform shape distortion for visualization, the wideband signal was high pass filtered using a wavelet multi-level decomposition and reconstruction filter 88. Waveforms were then extracted from this filtered signal and averaged for display. The freely available Matlab packages FMAToolbox (http://fmatoolbox.sourceforge.net48) and Chronux (http://chronux.org89) were used for analysis of neuronal bursting properties.Off-line spike detection and sorting was performed on the wideband signals using the Klusta software packages (Firing rate (in Hz) was calculated as the average number of spikes per second. All firing rates reported here are of spontaneously active neurons recorded under anesthesia. For each unit, the autocorrelogram was calculated by binning spike times into 1 ms bins and computing the correlation of the binned spike times at lags from 1 ms to 50 ms. The counts in each bin were divided by the total number of counts to create a probability distribution that is used for plotting. The first moment of the autocorrelogram (in ms) was defined as the mean value of the autocorrelogram in time. Spike amplitude (in μV) was calculated as a distance from baseline to the highest peak. Spike width (in ms) was calculated at the half point of spike amplitude.27 following previously described protocols28. Briefly, the following criteria were used: 1) anatomical location: only single units from the top portion of the multichannel silicon probe were selected, which anatomically corresponded to the pyramidal CA1 cell layer; 2) firing rate threshold: putative pyramidal CA1 cells had firing rate of 7 Hz or below, while CA1 interneurons had firing rate above 7 Hz (under anesthesia); and 3) spike waveform and autocorrelogram: first moment of autocorrelogram and symmetry of spike waveforms were used to manually place cells into putative CA1 pyramidal cells vs. CA1 interneurons category similarly to previously published criteria for rodent studies28. We also performed the automated clustering of pyramidal cells and interneurons using Matlab K-means clustering function (with k = 2 clusters) as previously described in the rodent literature28 and compared the results of automated vs. manual clustering , Chronux (http://chronux.org89), EEGLAB (http://sccn.ucsd.edu/eeglab/index.html90), and CSDplotter, version 0.1.191. Entrainment strength was determined by computing the modulation vector length (MVL) each single unit with oscillations ranging from 1 to 300 Hz on the channel that contained the maximal spike amplitude. Entrainment strength for all single units was then averaged and compared between the control and post-mTBI groups. Power spectrum density analysis of hippocampal oscillations was performed for all channels with detectable single units as previously described25. If more than one single unit was recorded on the same channel, the duplicate power spectrum was excluded from the analysis. The calculated power spectrum density was averaged across all animals then z-score normalized for comparative analysis between the control and post-mTBI groups of animals.Acquired wideband local field potentials recorded with the 32-channel silicon probe were downsampled to 2 kHz for further analysis. Signals were imported into Matlab software, version R2023a and processed using a combination of custom and modified routines from the freely available Matlab packages FMAToolbox  basket cells, fast-spiking CA1 interneurons that synapse on the somata and proximal dendrites of CA1 pyramidal cells, was used as described previously92. The individual parameters of the model  were altered either alone or in a combination with other parameters in a blinded manner to fit the experimental data  and post-TBI (ncells = 93) groups were used for comparison. The data were analyzed using Matlab  as well as Graphpad Prism (version 10) software. Non-parametric Mann-Whitney test was used for comparative analyses of neuronal properties  between the control and post-mTBI groups of animals. Data were displayed as mean ± SEM. For a subset of analyses of neuronal properties, post-mTBI animals were divided into two subgroups, injured at ~190 rad/sec (n = 4) vs. at ~260 rad/sec (n = 5). Ordinary one-way ANOVA was used for comparative analysis between the control, ~190 rad/sec, and ~260 rad/sec groups of animals. For power analyses, two sample t-test was used for comparison, and data were presented as mean ± STD. For entrainment analyses, non-parametric Mann-Whitney test was used for comparison, and data were presented as mean ± SEM. The threshold for significance was used with p-value of less than 0.05.Electrophysiological data were collected in 17 animals total . Total number of single units in control (nFurther information on research design is available in the Peer Review FileSupplementary Figure 1Reporting Summary


In this Perspective, we present the recent advances inatomic frictionmeasured of two-dimensional materials obtained by friction forcemicroscopy. Starting with the atomic-scale stick–slip behavior,a beautiful highly nonequilibrium process, we discuss the main factorsthat contribute to determine sliding friction between single asperityand a two-dimensional sheet including chemical identity of material,thickness, external load, sliding direction, velocity/temperature,and contact size. In particular, we focus on the latest progress ofthe more complex friction behavior of moiré systems involving2D layered materials. The underlying mechanisms of these frictionalcharacteristics observed during the sliding process by theoreticaland computational studies are also discussed. Finally, a discussionand outlook on the perspective of this field are provided. I1 A fundamental understanding oforigin of friction on the nanoscale is of essential importance tofind solutions to reduce energy dissipation and achieve wearless motionin microscopic and nanoscopic electromechanical devices.2 With the advent of atomic force microscopy3 and its adaption5 to study friction of single asperitieson the nanoscale, significant advances in detecting forces on thesub-nanonewton level resisting relative motion between probe and substratehave been witnessed in the past three decades. This leads to a profoundunderstanding of the physical processes underlying friction at theatomistic level.Friction is a universalphenomenon existing in our daily life spanninglength scales from earthquakes and mechanical bearings down to nanoscalemotors.7 to datean ever-growing number of atomic friction studies have been demonstratedon various surfaces and external conditions in the field of nanotribology.Atomic stick–slip instabilities, a fascinating highly nonequilibriumprocess, have been observed on numerous material surfaces includingmetals,10 semiconductors,11 andinsulators.12 In FFM experiments, the effectsof normal load, sliding velocity, temperature, and scanning directionon atomic friction have been investigated. The role of the normalload in atomic friction processes was studied by Socoliuc et al. inFFM measurements between a silicon tip and NaCl crystal surface.13 They observed a transition from dissipativestick–slip motion to smooth sliding which is reversible onlydepending on the applying load.13 Thisis a universal physical phenomenon confirmed by subsequent experimentson other surfaces such as graphite14 andgold.9 Furthermore, they developed a methodto switch friction on and off by applying external mechanical excitationsof the sliding system perpendicular to the contact plane.15 The velocity dependence of atomic friction betweenthe AFM tip and crystal surface has been experimentally revealed byGnecco et al.,12 representing a logarithmicdependence of friction force. Several measurements based on frictionforce microscope confirmed this typical logarithmic regime validatespanning many decades of velocity.18 The observed velocitydependence of friction can be well described under the frame of thermallyactivated Prandtl–Tomlinson (PTT) model20 based on reaction rate theory. The PTT model also successfully interpretedthe relevant atomic friction measurements with varying temperaturesshowing an increase in friction with decreasing temperature.21 To investigate the origin of friction at theatomic scale, both noncontact and contact friction measurements23 are performed on superconductors across the critical temperature.Thus, the electronic and photonic contributions could be determinedseparately. In fact, the contact is not always a single atom as describedby the PTT model but hundreds of atoms within the contacting region.Thus, a sharp tip and a blunt tip lead to different friction forcesat the single cleavage step edge.24 Thecertain degree of commensurability between the tip and sample raisesthe issue of anisotropic orientational dependence of atomic friction.25 Another anisotropy effect relying on the slidingdirection of AFM tip is also observed,27 which originatedfrom the structural anisotropy of the sample surface. Further studiesbased on molecular dynamics were performed to obtain a better understandingof atomic friction process in the buried contacting interface.28In friction force microscope experiments, asharp tip is draggedvia a spring along the crystalline surface at a constant normal loadand sliding velocity. When the cantilever starts moving relativelyto the substrate, the spring extends, which leads to an increase oflateral force and flattening the energy barrier for the tip to overcome.Instabilities are involved during sliding. Since the first atomicstick–slip process represented by Mate et al. using a tungstentip sliding on graphite surface,29 Its tribological performance is of both fundamentaland practical importance for massive applications emerging in microscopicdevices. The stacking order between the 2D layer and subsurface givesrise to the moiré superstructure,30 a novel degree of freedom to effectively modulate the frictionalproperties. In this Perspective, we focus on two aspects of atomicfriction processes on 2D materials: atomically flat 2D surfaces andmoiré corrugated superlattice induced by surface reconstruction materialssuch as graphene, h-BN, and transition metal dichalcogenides (TMDs),where atomically flat nanocontact can be investigated. By way of comparison,such thin surfaces consisting of one or several layers of atoms onone hand exhibit excellent friction reduction properties39 For such applications,the tribological properties of 2D material surfaces are of particularimportance from scientific and technological perspectives. SeveralAFM measurements report the reduction of friction on graphene patchesin contrast to a variety of surrounding substrate surfaces includingmetal,31 semiconductor,40 and insulator.32 More interestingly,friction on suspended graphene is investigated as well showing similarcharacteristics as on graphene supported by a rough substrate.32These layered materials are predictedto show ultralow frictionand wearless motion when it is scratched by the AFM tip based on thefact of their high in-plane elastic modulus due to the intralayercovalent bonding, weak van der Waals interaction between probe andsurface, and high chemical inertness. These unique features providea new promising solution in solid lubrication by coating the substratewith 2D material layers.41 Single-layer graphene exhibitsa higher average friction than bilayers. They suggested the differencein electron–phonon couplings for monolayer and bilayer graphenegives rise to the revealed friction contrast. The evolution of nanofrictionfrom single layer to few layers has been studied by Lee et al. fora variety of mechanically exfoliated 2D materials under ambient conditions.32 They found a negative layer dependence on frictionin all measured surfaces and distortion of the regular stick–slipmotion. Out-of-plane deformation of 2D layered materials, also referredto as the puckering effect where a typical hexagonal stick–slip motion has been observedin both monolayer and bilayer graphene under ultrahigh vacuum (UHV)conditions.2 substrate. Choi et al. observedsignificant anisotropic ratios of friction up to 215% with a periodicityof 180° on each frictional domain.33 This directional dependence on friction arises from the ripple structureresulting from interfacial inhomogeneous interaction during the samplepreparation process. Another recent example of anisotropic frictionis related to transition metal dichalcogenides (TMDs). Vazirisereshket al. studied the atomic friction acting on single asperity slidingon wrinkle-free MoS2 surface by means of FFM and MD simulations.43 A 2-fold symmetry has been obtained in bothexperiments and simulations due to the distorted, low-symmetry tip–samplepotential energy surface. Another factor that should be taken intoaccount is the self-assembly of adsorption of contaminants on surfacesof 2D materials in ambient conditions leading to the frictional anisotropyin FFM measurements45 as well.For mechanically exfoliated 2D material films,the anisotropy offriction has been reported in atomic friction experiments and simulations.An impressive example of friction anisotropy is given by graphenetransferred onto a SiO2 The load dependence of friction could be tunedby applying mechanical strain in the suspended single-layer graphenewhich is attributed to the change of contact quality between slidingAFM probe and surface.38 As discussed above,the contact region in realistic AFM experimental measurements wasinterpreted to be hundreds of atoms, where the degree of commensurabilitybetween the tip and substrate plays an important role in the atomicfriction features. The combination of the quantity of atoms in contactingarea and quality of contacting interface determines the frictionalresponse of 2D materials surface such as strengthening effect.46 Actually, high contact pressure can lead tothe intermittent formation of covalent bonds which limits the superlow friction at tip–sample interface.47 As for the relative scanning speed, the friction of monolayer 2Dlayer as a function of sliding velocity shows a logarithmic dependence48 similar to the observed results from the bulksample. Zhao et al. studied the dependence of friction on temperatureobserving the exponential increase with decreasing temperature inthe case of MoS2.11The dependenceof friction on 2D materials on the external appliedload is of fundamental scientific interest where nonlinear dependenceof friction on normal load could be fitted with DMT or JKR relations.2 with the same probe have demonstrated that graphene exhibits a lowerfriction than MoS2 depending on the difference in energybarriers for the tip to overcome.40 InTMDs systems, lattice constants of the chalcogen also give rise tofriction contrast, where the larger lattice constants lead to lowersliding friction.49 Furthermore, Zhou etal. found that the vertical interlayer force constant of various TMDs,changing transition metal, dominates the nanoscale friction behaviorof MoS2 vs WS2 and MoSe2 vs WSe2.50Asdiscussed above, within the family of 2D layered materials,graphene, h-BN, and TMDs have shown similar frictional performancesuch as extremely low friction, layer dependence, and friction anisotropy;however, the fundamental understanding of the role of the chemicalidentity of these 2D materials surface remains unclear. To this end,in situ AFM measurements of monolayer graphene and MoS35 oxidation,34 andfluorination.36 Fessler et al. studiedthe atomic friction on pristine and hydrogenated graphene exfoliatedon SiO2 substrate in a single measurement showing thatthe frictional behavior is the practically same on both surfaces oncecontamination adhering to hydrogenated regions is cleaned.35 However, a 6-fold enhancement of atomic frictionmeasured on fluorinated graphene has been revealed by Kwon et al.under UHV conditions36 compared to theintrinsic layer. They attribute this observation to the enhancementof the out-of-plane bending stiffness of fluorinated graphene. A systematicstudy of friction on hydrogenated, fluorinated, and oxidized graphene,compared to pristine graphene, shows 2-, 6-, and 7-fold enhanced nanoscalefriction on their surfaces, respectively.34 Relevant density-functional theory calculations indicate the maindissipation route of the out-of-plane vibrations. Corresponding functionalgroup removal from 2D materials has been revealed as well. Felts etal. reported the bond scission technique induced by mechanical stresswhich enables the cleavage of chemical groups such as oxygen, fluorine,and hydrogen from graphene.51 Zambudioperformed friction force measurement of atomic monovacancies on defectivegraphene prepared by means of Ar+ irradiation.37 A 5-fold enhancement of effective friction coefficientwas observed with low density of atomic vacancies due to the chemicalreactivity of dangling bonds and long-range strain distribution inducedby the defect.Chemical modificationis a powerful tool to modulate the chemicalcomposition of 2D layered materials surface, for example, hydrogenation,IIISo far, we have discussed the atomic friction process on a 2D materialssurface with atomic flatness. However, with the growing progress inthe field of moiré materials, the influence of periodic long-rangereconstruction induced by lattice mismatch between 2D layer and subsurfaceon the atomic friction process has opened a new nanofriction researchfrontier. In this section, we focus on the atomic friction behaviorson 2D materials with a moiré superlattice.52 finding the modulation effectsof friction force by long-range superstructure due to the latticemismatch between these two materials. A tiny out-of-plane surfacecorrugation accounts for the modulation of the amplitude of the potentialcorrugation owing to the moiré superlattice. Filleter et al.used the AFM tip to slide graphene film grown on SiC(0001) with long-periodsuperstructure.53 Although the lateralforces acting on the tip are modulated with reconstruction features,there is no variation in the energy dissipated during the slidingprocess. In the framework of the Tomlinson model, the influence ofadsorbed molecules on friction was investigated by Tshiprut et al.by introducing local perturbations,54 anda second harmonic in the tip–surface interaction potentialto mimic the disordered surface was taken into account by Fajardoand co-workers.55 After reviewing atomicfriction measurements on superstructures, Steiner et al. extend thePT model to the ordered superlattice by modifying the substrate potentialdescribing the interaction between the AFM tip and surface.56 They found two types of potential modulationsfor interpreting the corresponding observed atomic friction process:56 (i) amplitude modulation, multiplication ofatomic potential by the superstructure potential; (ii) centerlinemodulation, superposition of atomic sinusoidal potential and long-periodpotential. These, in turn, give rise to the modulations of lateralforce in both cases, as observed above. In contrast to amplitude modulation,the centerline modulation does not affect friction dissipation, asthe trace and retrace of friction are consistently changed.56 The origin of the difference in these two typesof modulations might be attributed to the unique physical and chemicalproperties of two-dimensional layered materials. The weak interfacialvan der Waals interactions between tip and surface in combinationwith high chemical inertness strong intralayer covalent bonding of2D materials may be the main reasons leading to the superpositionof the short-range and moiré-level potentials. Another specificexample of the surface reconstruction is the Au(111) herringbone whichis imaged successfully by contact friction force microscopy underUHV conditions.57 As for noncontact friction, charge-densitywaves where a superstructure is formed by a charge redistributionmake a key contribution to energy dissipation compared to metal phasein the case of NbSe2.58Variationof atomic friction due to superstructure was investigatedexperimentally and theoretically about one decade ago. Maier et al.experimentally studied the atomic friction process on reconstructedsurface by depositing KBr film on NaCl(001) substrate for the firsttime,1 represented by fixed periodicpotentials. Thus, the classic PT model fails to describe the realscenario during the sliding process considering the in-plane and out-of-planedeformations of the moiré supercell. This local straining inboth directions induced by scanning probe was confirmed by STM measurementof graphene on an h-BN substrate.59 Furthermore,the effects of periodic dimensions and mechanical and electrical propertiesof moiré superlattice on the frictional properties of 2D materialshould not be ignored.These examples are the first observations of modulated lateralforce induced by moiré superlattice at the atomic level. However,the origin of the modulation effect is not fully understood, as itcould be attributed to geometrical effect or other factors such asinternal degree of freedom of the supercell. In fact, the Tomlinsonmodel developed above assumes AFM tip sliding over a rigid crystallinesurface53 themoiré superstructure involving 2D atomic layers offer a uniqueplatform to gain fundamental understanding of friction on a varietyof ordered superstructures, where the topographic, mechanical, andelectrical properties could be tuned widely.30 Chan et al. measured friction vs the relative orientation of graphenefilm grown on Pt(111) surface in UHV.61 They observed that in addition to atomic stick–slip motion,lateral force modulation critically depends on the moiré patternoriginating from the natural lattice misfit between the graphene filmand Pt(111) surface. Similar lateral force modulation induced by themoiré supercell was revealed by means of molecular dynamics(MD) simulations which is attributed to the geometric corrugationof graphene.62 Long-range stick–slipdynamics was observed by Shi et al. in the case of graphene on Ru(0001)depending on the interfacial interaction of the heterogeneous structure.63 Zheng et al. represent similar experimentalobservations of graphene on Ge(111) such as long-range centerlinemodulation of lateral force along the moiré pattern.64 Furthermore, they proved that this nanoscaleconfinement induced by moiré superlattice leads to a strongsuppression of out-of-plane of the 2D atomic layer even when the surfacewas chemically modified by fluorination or oxidation where normallysignificant enhancement of friction emerges. Because of a similarreason, graphene confined with the moiré pattern on Pt(111)substrate gives rise to substantial enhancement of the load carryingcapacity during tip sliding.39 Furtherstudy shows the capability to tune the moiré level lateralforce by applying external bias voltage introducing current transferfrom the conductive AFM probe to graphene on Ru(0001).65As indicated by Filleter et al.,60 Two distinct regimes of friction with slidingvelocities have been revealed: (i) friction force remains ultralowand nearly constant below some threshold; (ii) at high velocities,the logarithmic frictional dependence on speed appears, as shown in 66 To bridge the gap of sliding velocities between MD simulations andexperiments, a phenomenological model is derived, showing the abilityto calculate the relevant friction force in excellent agreement withexperimental results.It is of great interest and importancein both technological applicationsand scientific research to find the fundamental mechanism of moiréfriction in addition to atomic stick–slip motions. To thisend, Song et al. conducted the friction measurements on superlatticeof graphene-coated Pt(111) surfaces 3a with s6 Thistransition is influenced by moiré size as well, showing thecapability of tuning frictional dissipation widely in a simple andreversible manner. These findings could be explained very well inthe framework of the dissipative moiré-scale stick–slipmechanism. Further studies that investigated the atomic friction ongraphene/h-BN superlattices with larger periodicities67 confirm that moiré-level stick slip behaviors attributedto the in-plane elastic deformation and release of the graphene superstructureexist up to the moiré pattern with a period of ∼15 nm.67 Based on MD simulations, Huang et al. revealthe underlying mechanism that moiré level stick–sliporiginated from the coupling between in-plane strain and out-of-planedeformation of the graphene layer on an h-BN substrate.68In addition to the velocity dependence, the loaddependence ofatomic friction was also studied with the same graphene/Pt(111) system. 69 The influence of the strength of interfacialinteractions on moiré friction should be investigated. Songet al. prepared MoS2 film on Au(111) surface under UHVconditions70 to exclude the contaminants.45 Relatively strong adhesion has been predictedby previous DFT calculations for the MoS2/Au(111) system.71 It was confirmed by another FFM experiment revealingpronounced adhesion and higher sliding friction between MoS2 and the Au-coated tip, in contrast to the silicon tip.72 The friction force measurements of the MoS2/Au(111) system were carried out, revealing apparent independenceof friction on load with a relatively large moiré size of 3.3nm,70 in contrast to the previously reportedmoiré level friction at the ridge leading to a highly dissipatedregime. The puckering effect is not observable when analyzing thecorresponding lateral force trace and retrace (2 and the gold surface. The disappearance ofthe “strengthening” effect (2 layer scanned by an AFM probe.70 Thepreparation and friction measurement of the MoS2 surfaceunder UHV conditions are important for the experimental findings toexclude potential contaminations. This paves the way to control moiréfriction by varying the strength of interaction of 2D atomic layerwith the underlying substrate.All the cases discussed above are relatedto the moiré systemswith relatively weak van der Waals interlayer interaction, such asfor the case of graphene/h-BN heterostructure microscale robust superlubricsliding has been uncovered between these two surfaces. retrace 4. It is ” effect 4c confirIVDetermination of the friction dissipation of unideal2D layered materials surface with various types of defects such aszero-dimensional point defects and line defects. More experiments,particularly in UHV, are needed to reveal the mechanism in the beginning.Subsequent systematic studies on defect friction in various environmentscould lead to further understanding of the corresponding atomic frictionalbehaviors for more realistic application scenarios.As discussed above, the moiré superstructuregives rise to various frictional characteristics due to its uniquemechanical properties. Phase transition of 2D layered materials providesanother new degree of freedom for varying not only electronic propertiesbut also frictional response of atomic surfaces. Further AFM studieson corrugated moiré surfaces in combination with phase transitionwould be useful to investigate atomic-scale and moiré-scalefrictional behaviors of 2D material over phase transitions.Over the course of thisPerspective, we have emphasized the varietyof atomic friction processes on 2D materials surfaces which has beeninvestigated by friction force microscopy. Material identity, thickness,sliding direction, load, velocity, and chemical modifications havesignificant effects on frictional behaviors of 2D layers. More importantly,we have represented that moiré superstructures involving a2D atomic layer provide a new dominant energy dissipation route inaddition to atomic stick–slip dynamics. We want to emphasizethe important role of the elastic deformations, which extend overa larger length scale related to the moiré pattern. Becauseof the large extension of the elastic deformations during the stickphase, a larger energy can be dissipated in the slip phase, whichexplains the dominance of moiré friction compared to atomicfriction. This behavior is observed not only experimentally but alsoby MD simulations and is well represented by the phenomenologicaltwo-state model. Despite being very successful in atomic frictionstudies on 2D materials, there is still much work to be done. Herewe emphasize a few directions that we feel are important and needto be further addressed:


O6-methylguanine-DNA-methyltransferase (MGMT) gene has emerged as a clinically relevant prognostic marker in glioblastoma (GBM). Methylation of the MGMT promoter has been shown to increase chemotherapy efficacy. While traditionally reported as a binary marker, recent methodological advancements have led to quantitative methods of measuring promoter methylation, providing clearer insight into its functional relationship with survival.Epigenetic inhibition of the MGMT promoter methylation index. GBMs of 240 newly diagnosed patients were sequenced and risk for mortality was assessed. Nonlinearities were captured by fitting splines to Cox proportional hazard models and plotting smoothed residuals. Covariates included age, Karnofsky performance status, IDH1 mutation, and extent of resection.A CLIA assay and bisulfite sequencing was utilized to develop a quantitative, 17-point, P < .005). GBM patients with low levels of promoter methylation (1-6 CpG sites) fared markedly worse  than individuals who were unmethylated. Subjects with medium levels of promoter methylation (7–12 sites) had the greatest reduction in hazard , followed by individuals in the highest promoter methylation tertile .Median follow-up time and progression-free survival were 16 and 9 months, respectively. A total of 176 subjects experienced death. A one-unit increase in promoter CpG methylation resulted in a 4% reduction in hazard (95% CI 0.93–0.99, MGMT promoter methylation and survival in GBM may be nonlinear. These findings challenge the current understanding of MGMT and underlines the clinical importance of determining its prognostic utility. Potential limitations include censoring, sample size, and extraneous mutations.Our findings suggest that the relationship between the extent of  MGMT promoter methylation suggests nonlinear prognostic effect.- Analysis of quantitative MGMT promoter methylation may be insufficient for prognostication.- Binary representation of MGMT biomarker in glioblastoma. The results of this study challenge our current understanding of how promoter methylation of the MGMT gene effects prognosis and demonstrates the potential downside of binary marker representation. While previous literature suggests that any amount of MGMT promoter methylation is beneficial and that the prognostic benefit increases linearly, the results of this analysis bring this current understanding into question. The analysis carried out in this study indicates low-level promoter methylation to prognostically disadvantageous and the highest levels of promoter methylation to be less beneficial than medium levels of promoter methylation. MGMT promoter methylation is commonly assessed in glioblastoma patients and treatment regiments are often modulated in response to the results of this marker. For this reason, a clear and quantitative understanding of how the marker shapes prognosis is imperative.The following manuscript details a novel laboratory and statistical approach to modeling the nonlinearities of the 2 Prognostic outlook for GBM is quite poor as it is the most aggressive primary malignant tumor of the central nervous system. Common prognostic indicators of glioblastoma include Karnofsky performance status (KPS), age, extent of tumor resection, radiotherapy, chemotherapy, tumor size, tumor location, and molecular profiling for biomarkers such as IDH1, EGFR, PTEN, and MGMT.4 Since the introduction of the 5th edition of the WHO CNS Tumor Classification, IDH-mutant glioblastomas are now referred to as ‘Astrocytoma, IDH-mutant, CNS WHO grade 4.Glioblastoma (GBM) currently represents approximately 50% of high-grade gliomas diagnosed in individuals.O6-methylguanine DNA methyltransferase (MGMT) sequence is located at the 10q26.3 position of the 10th chromosome. MGMT encodes for the DNA repair protein, O6-alkylguanine-DNA-alkyltransferase (AGT),6 which repairs alkylation at the O6 position on guanine.7AGT is instrumental in genomic stability and functions by removing alkylating lesions to repair DNA and prevent errors during DNA replication and transcription.6 This repair process occurs with either open or condensed chromatin, indicating that AGT acts independent of target site chromatin remodeling, apart from when its promoter is methylated.10 Furthermore, AGT does not associate or work in concert with other transcription-coupled repair proteins, indicating AGT’s independence as a repair mechanism.8Highly conserved, the AGT.5 The activity of this repair enzyme correlates inversely with sensitivity to agents such as temozolomide, that form O6-alkylguanine DNA adducts.11 Expression of the MGMT gene is known to play a role in carcinogenesis as a number of malignancies, including gliomas, have increased MGMT expression due to lower levels of promoter methylation.5 When the MGMT promoter is unmethylated, the MGMT gene is expressed and, therefore, likely to be more effective in repairing DNA that has been damaged by alkylating chemotherapy,12 rendering treatment less effective. This finding is consistent with studies that have shown that tumors with unmethylated MGMT promoters have less favorable disease progression and survival outcomes when compared to their methylated counterparts.9,10,13–18Cells, tissues, and individuals vary greatly in the expression of MGMT promoter methylation is thought to be an independent favorable prognostic marker for improved response to radiation and chemotherapies. Tumors with unmethylated MGMT promoters have a less favorable response to alkylating chemotherapeutic agents19 due to AGT being actively expressed and repairing the lesions in the DNA caused by treatment.20–22 Epigenetic silencing of the MGMT gene has also been shown to be associated with decreased mortality and increased progression-free survival (PFS) and overall survival (OS) when compared to glioblastoma patients with unmethylated MGMT promoters.13MGMT promoter methylation, methylation specific PCR (MSP), does not provide quantitative information on the extent of methylation, reporting promoter methylation status as a binary outcome (methylated/unmethylated).23 MSP is commonly utilized due to its efficacy with small Bx samples and relatively low cost when compared to other methods. One of the largest downsides to utilizing MSP is that some weakly or partially methylated tumors are unable to be unequivocally assigned to either a methylated or unmethylated category, rendering the marker uninterperable.24 To circumnavigate this issue, UCSF currently employs a CLIA-based assay and bisulfite sequencing technique that interrogates 17 distinct CpG sites, generating a quantitative, 17-point index.To date, the most prevalent method of testing for 25 To bypass these pitfalls, alternative methods of analyzing MGMT promoter methylation have emerged, including pyrosequencing and high-resolution melt analysis, which have provided promising results and improved discrimination between methylated and unmethylated tumors when compared to MSP.26–29The majority of research utilizing such methods, especially MSP, model promoter methylation’s protective effect as a linear relationship and seek to define an optimal cutoff point.31 Conversely, a recent study utilizing pyrosequencing detailed that MGMT promoter methylation had a nonlinear prognostic relationship where low levels of promoter methylation were inversely associated with survival.32 Conflicting descriptions of the association between MGMT promoter methylation and survival highlight the importance of further elucidating its protective effect’s functional form. The present study aims to determine how MGMT promoter methylation’s advantages in treatment efficacy transform as the extent of promoter CpG silencing increases. By utilizing a novel, methylation assay and various statistical techniques to capture nonlinearities, we further illuminate the functional relationship between MGMT promoter methylation and GBM survival.While other quantitative methylation techniques are available, methods involving protein expression or activity-based assays run a nonnegligible risk of generating false positives due to nontumor tissue contamination.oC for 15 s, 54oC for 30 s, and 72oC for 30 s. In the second PCR round, a 1 μl aliquot from the first PCR is used in each of 2 amplification reactions where either the forward or reverse PCR primer is biotinylated. After the second PCR, the reactions are purified with streptavidin magnetic beads and subjected to Sanger Sequencing on an Applied Biosystems 3130 instrument. To control for PCR contamination, each of the 2 rounds of PCR utilizes an FFPE DNA extraction blank control. Prior to sequencing, these blank controls are run on an agarose gel along with the amplification reactions to ensure appropriate amplification of the targeted 191 bp amplicon without any amplification in the blank controls. The DNA sequencing reactions from each biotinylated primer were performed in duplicate, yielding a total of 4 overlapping DNA sequencing reactions. The sequences were assembled and visualized using CodonCode Aligner . All 31 cytosine control sites are required to be converted to thymines before a methylation index, ranging from 0 to 17, is determined. Our Sanger bisulfite sequencing assay does not quantify the extent of promoter methylation at individual CpG sites. Rather, it detects the methylation of a CpG site as denoted by the presence of a cytosine nucleotide, within the sensitivity of Sanger DNA sequencing (approximately 10%). The total number of methylated CpG sites is reported as the methylation index. An index score of 0 corresponds to an unmethylated MGMT promoter, whereas an index score ranging from 1 to 17 corresponds to a methylated MGMT promoter. A one-unit increase in promoter methylation index score corresponds to an additional CpG site being methylated.DNA was extracted following macro-dissection of tumor tissue from formalin-fixed paraffin-embedded (FFPE) sections that contained at least 70% tumor cells. DNA was treated with a bisulfite reagent  followed by two PCR amplification rounds aimed at the synthesis of a 191 bp amplicon  with the following forward and reverse PCR primers 5ʹ-ATTATTTTTGTGATAGGAAAAGGTA-3ʹ and 5ʹ-AAACAATCTACGCATCCT-3ʹ. The 191 bp amplicon spans 17 CpG sites and 31 nonCpG cytosines that serve as internal controls for the efficiency of bisulfite treatment. The 191 bp amplicon includes 17 CpG sites with the first CpG site located 228 bp upstream of rs2782888. CpGs 1-5 are located 85 bp upstream of DMR1 and CpGs 6-17 extend 106 bp within DMR1. Each PCR round consisted of 35 cycles at 95Prior to surgery, all subjects signed consent for the use of their tissue, demographic information, and clinical information obtained through query of electronic medical records. This tissue consent was approved by the UCSF institutional Review Board. The inclusion criteria defined for this study were adult patients (≥18 years of age) with glioblastoma (WHO grade IV) centered in the cerebral hemispheres, who underwent surgical intervention at UCSF, including biopsy (Bx), subtotal resection (STR), or gross total resection (GTR) and received standard adjuvant radiation/chemotherapy. Tumor samples acquired from UCSF patients with newly diagnosed GBM were analyzed using the previously described assay. Promoter methylation results from 300 patients’ FFPE tumor samples collected during initial surgical resection between 2009 and 2017 were evaluated and followed for survival until 2020. Data cleaning procedures where subjects with lower grade gliomas, nonprimary GBM cases, and cases with no follow-up data (less than 2 weeks) were excluded, yielded a final cohort of 240 subjects. Median follow-up time for OS was 16 months (IQR 21.9).isocitrate dehydrogenase 1 mutation status (IDH1-R132H), and methylation index score were retrospectively collected. Extent of resection was defined by neuroradiology in conjunction with the UCSF tumor board and based on a comparison of pre and postoperative contrast imaging.Data on age, gender, KPS, extent of resection, location of tumor, time to recurrence, time to death (or last follow-up), IDH1 mutation. The statistical significance level was 0.05. Multivariate Cox models were then utilized to create survival plots. To capture nonlinearities, basis splines, P-splines, and restricted cubic splines were fit to the aforementioned Cox models. To test model fit and to further assess the functional form of MGMT promoter methylation, we plotted the smoothed Martingale residuals of an unadjusted, Cox proportional hazard model. The assumption of proportionality was tested by plotting Schoenfeld residuals against the transformed time.Data cleaning, wrangling, and analysis were performed using R software (version 4.0.2). Cox Proportional Hazard models were constructed with methylation index results of patients in continuous and ordinal categorical (methylation tertiles and quintiles) formats. Models were adjusted for age, KPS, extent of resection, and MGMT promoter methylation. The median number of methylated CpG sites (index score) was 2 (IQR 0–12). Of the patients with methylated tumors, the median index score was 8. IDH1-R123H mutation data was available for 92% of patients with 15 patients (6%) testing positive.Patient baseline characteristics parsed out by methylation tertile are listed in MGMT promoter methylation increased. Despite median OS increasing in a uniform fashion as promoter methylation increases  for every one-unit increase in promoter methylation index. Modeling promoter methylation status as a binary marker resulted in methylated subjects experiencing a 40% reduction of risk for mortality . Moreover, GBM patients with low levels of promoter methylation fared markedly worse  than individuals who were unmethylated. Subjects with medium levels of promoter methylation had the greatest reduction in hazard , followed by individuals in the highest methylation tertile . Our Cox proportional hazard model had a Concordance index of 0.66 (SE = 0.025).Total median follow-up time was 16 months, during which 176 subjects experienced death. Median OS for unmethylated subjects was 14 months. OS increased linearly with extent of promoter methylation where low-level promoter methylation (1–6 CPG sites) had a median OS of 15.4 months, medium-level methylated subjects (7–12 CpG sites) had a median OS of 25.7 months, and high-level promoter methylation (13–17 CpG sites) had a median OS of 27.5 months. Summarized in ncreases , hazard P-value was 0.15 (Chi-square = 2.06 on 1 df). In this case, a significant P-value indicates that the proportional hazards assumption is violated.To test the assumption of proportionality, we plotted scaled Schoenfeld residuals corresponding to different variables included in the Cox model to determine if the residuals increased or decreased over time . We determined that there was no significant interaction effect between time and promoter methylation  as the PP = 2e−06), a Wald Chi-square statistic of 33.89 on 6 df (P = 7e−06), and a (Logrank) test = 35.29 on 6 df (P = 4e−06). There is a significant difference between different promoter methylation groups, index scores, and status (methylated/unmethylated). The difference between the ordinal categorical levels of promoter methylation is visually apparent in the survival curves illustrated in IDH-1 mutation status.We reject the null hypothesis in favor of the full as our model reported an estimated likelihood ratio of 36.8 on 6 df (P < 2e−16) and approximates the form of both the survival curves and ordinal categorical Cox model. Alternate spline models including P-splines and B-splines demonstrated the same shape or functional form of the relationship between MGMT promoter methylation and OS. All spline models contained the same covariates as the previously mentioned survival plot in When modeling promoter methylation index score as a continuous variable we utilized splines to capture nonlinearities in its relationship with OS. As indicated in IDH-1 mutation, and extent of resection. Subjects with low levels of promoter methylation (1–6) showed no significant change in risk  when compared to subjects with unmethylated tumors. Subjects with medium levels of promoter methylation (7–12) showed a 73% reduction in risk  whereas subjects with highly methylated tumors (13–17) had a 54% reduction in risk for mortality . Differences in time to radiographic disease progression between levels of promoter methylation are visualized in the survival plot in IDH1 mutation status. Additional illustrations for the PFS endpoint can be found in the supplemental materials section.The median time to radiographic disease progression was 9 months. Unmethylated subjects had a median PFS time of 8 months, low-level methylated subjects (1–6 CpG sites) had a median PFS of 9.7 months, medium levels of promoter methylation (7–12 CpG sites) had a median PFS of 19.9 months, and highly methylated subjects (13–17 CpG sites) had a median PFS of 17.5 months. The Cox PH forest plot in MGMT promoter methylation index to determine how the extent of promoter methylation effects OS and time to disease progression. While MGMT promoter methylation’s relationship with survival has traditionally been described as linear,34 our data contrasts this, indicating that the prognostic effect of MGMT promoter methylation follows a pattern of nonlinear conformation. We found that low-level promoter methylation carried a significantly higher risk for mortality than being unmethylated. Furthermore, our data indicates that MGMT promoter methylation offers the greatest prognostic benefits when 9–13 CpG sites exhibit methylation, as subjects with medium levels of promoter methylation had a lower risk for mortality when compared to subjects who were highly methylated. These findings suggest that a cutoff point or threshold may not be optimal in delineating how a patient will perform based on their level of promoter methylation.In this study, we utilize a novel P < .00001).13 In our study we also found that low levels of promoter methylation were associated with increased risk for mortality. When stratifying our 17-point methylation index into quintiles, subjects who had 1–3 CpG sites methylated had a 100% increase in hazard. . We expected that the benefits of MGMT promoter methylation would follow a linear trend where the highest levels of promoter methylation offered the greatest prognostic benefits. Surprisingly, our data exhibited a pattern of conformation where subjects with medium levels of promoter methylation had a greater reduction in risk than those with the highest levels of promoter methylation. This pattern of nonlinear conformation was also present for the PFS outcome; however, we did not find a statistically significant difference between lower levels of promoter methylation and the unmethylated reference group.A recent study utilizing a quantitative pyrosequencing approach conducted by Giuseppe et al. concluded that methylation at less than 15% of the CpG sites they interrogated was associated with impaired survival  that model nonlinearity. While it is possible this trend in hazard ratio may be due in part to sample size, these findings are consistent with those of Giuseppe et al., which had a total enrollment of 681 patients. Our OS Cox model did not include the extent of resection as a covariate, as including extent of resection did not change the shape of how the estimated hazard ratio transformed over an increase in promoter methylation index score. Extent of resection was left out of this model as it increased the estimated The analysis presented in this study includes two important measurements: median OS and hazard ratio. Median OS is a measure of central tendency, reflecting the time point at which half of the population has died. In contrast, HRs represent the adjusted relative risk of an event occurring in one promoter methylation group compared to another group. It is important to note that the trends between these two measurements differ as they measure different aspects of survival.12 Several ideas have been proposed to better explain this “gray zone,” including heterogeneous methylation patterns across the MGMT CpG islands, as well as a biochemical challenge in adequately distinguishing between methylated and unmethylated in tumor tissue with variable contamination of benign tissue.12 In several instances, subjects who progressed and went on to have subsequent surgeries would send out tissue for central review as part of the inclusion/exclusion criterium of a clinical trial. We found that in some cases, subjects with low promoter methylation index scores (1–3) would be considered unmethylated by a central lab’s MS-PCR analysis, affecting the patient’s eligibility for enrollment in an investigational therapy.It remains unclear as to what may be responsible for causing the low-level promoter methylation group’s large increase in hazard. It is possible that subjects who had lower levels of promoter methylation were barred from or wrongly permitted to participate in certain investigation interventions upon recurrence, affecting survival. This may be due in part to a potential “gray zone.” As initially discussed by Wick et al., the “gray zone” represents weakly or partly methylated tumors that often cannot be unequivocally assigned to the methylated or unmethylated categories.The apparent increase in risk from medium to high-level promoter methylation may be due in part to small sample sizes. Outlying long-term survivors (LTS) may skew the medium level of the promoter methylation group’s estimated hazard ratio. Eighteen patients had a time to last follow-up greater than 3 years. Of these 18 long term survivors, 2 were unmethylated, 2 had low levels of promoter methylation (1 CpG site), 8 exhibited medium levels of promoter methylation (9–10 sites), and 6 were highly methylated (13–16 sites). A follow-up study with a larger sample size would be necessary to verify whether the presence of LTS outliers is affecting the shape of our data.35 Upon recurrence, these TMZ-treated hypermutated tumors harbored driver mutations in key tumor suppressors and oncogenes.36 It is quite likely that temozolomide increases tumor mutational load in primary GBM as well37, affecting OS and time to disease progression. MGMT promoter methylation increases temozolomide efficacy, thus having high levels of promoter methylation may correlate with increased intertumoral mutation.35 Hypermutated gliomas are more resistant to alkylating chemotherapies and harbor unique molecular vulnerabilities such as greater expression of mutant neoantigens on their cell surfaces.36 The conformation of promoter methylation’s prognostic effect found in our data may represent an optimal level of AGT inhibition where the repair enzyme is able to mitigate some of temozolomide’s cytotoxic effects by repairing mutagenic DNA lesions while still rendering the chemo alkylating agent more effective than it would be under lower levels of promoter methylation.Recent research has found that temozolomide induces somatic hypermutation, leading to transformation of grades.36 To this extent, MGMT promoter methylation may vary within samples of a single lesion, different multi-focal lesions, or between reoccurrences.38 This variability may be a source of sampling bias that is unaccounted for. In addition to tumor heterogeneity, left and right censoring may affect model estimates and survival curves. We have no clear way of inferring how long a patient was living with GBM before time of diagnosis, and only 176 out of 240 subjects experienced death, several were lost to follow-up. It also remains unclear how important a predictor MGMT promoter methylation is as a stand-alone prognostic marker. Despite including IDH-1 mutated patients (n = 15) in our models, we adjusted for IDH-1 status as a covariate. We found that excluding IDH-1 patients did not significantly affect model estimates or the functional form of our regressor’s relationship with OS.There are a number of additional factors to take into consideration when interpreting the estimates from our models. For one, glioblastoma tumors are cellularly and molecularly heterogeneous, both within and between tumors.MGMT promoter methylation assays is the mixed extent of methylation at each individual CpG sites. Each CpG island varies in how heavily methylated the site is. For this reason, we did not calculate the methylation percent of a methylated CpG site, but simply scored it as methylated when a cytosine nucleotide was detected on a background of fully converted 31 CpG control sites. We controlled for the integrity of the bisulfite treatment by ensuring the conversion of the 31 control sites before calculating the methylation index score. Finally, the possibility of point mutations introduced during the amplification process by DNA polymerase errors was controlled by the use of a Taq DNA polymerase with a proofreading function, as well as subsequent monitoring of the aberrant nucleotides noted by the analysis software.A potential confounding factor in all MGMT promoter methylation in GBM has been found to be both a prognostic and predictive marker. While it is well-established that MGMT promoter methylation leads to improved response to radiation and chemotherapy, it is also possible that MGMT silencing may be a pretumorigenic mechanism. Studies have shown that MGMT silencing is linked to mutations in other tumor-related genes, such as p53, k-ras, and CDKN1A/2A, which are markers of poor prognosis.8 Although the relationship between methylation of genes in carcinogenesis is not entirely clear, these findings suggest that MGMT promoter methylation likely has an impact on GBM tumor biology beyond treatment resistance.MGMT gene affect the marker’s prognostic performance. Research of this nature will assist to further characterizing the inherent qualities of the relationship between MGMT promoter methylation and survival in glioblastoma.Our findings suggest that making treatment decisions guided by an “optimal methylation cut-off point,” as suggested by other studies, may carry inherent risks and negative clinical implications. When interpreting quantitative promoter methylation testing results, it is imperative that clinicians consider that the functional form of the relationship between promoter methylation and OS may not be linear. To validate and build upon the findings of this study, we intend to conduct research utilizing next-generation sequencing data from a larger sample in order to derive how the extent of methylation of each individual CpG site and their locations within the promoter region of the vdad115_suppl_Supplementary_Figure_S1Click here for additional data file.vdad115_suppl_Supplementary_Figure_S2Click here for additional data file.vdad115_suppl_Supplementary_TableClick here for additional data file.