Epilepsy phenotype and its reproducibility after lateral fluid percussion-induced traumatic brain injury in rats: Multicenter EpiBioS4Rx study project 1.

OBJECTIVE: This study was undertaken to assess reproducibility of the epilepsy outcome and phenotype in a lateral fluid percussion model of posttraumatic epilepsy (PTE) across three study sites. METHODS: A total of 525 adult male Sprague Dawley rats were randomized to lateral fluid percussion-induced brain injury (FPI) or sham operation. Of these, 264 were assigned to magnetic resonance imaging (MRI cohort, 43 sham, 221 traumatic brain injury [TBI]) and 261 to electrophysiological follow-up (EEG cohort, 41 sham, 220 TBI). A major effort was made to harmonize the rats, materials, equipment, procedures, and monitoring systems. On the 7th post-TBI month, rats were video-EEG monitored for epilepsy diagnosis. RESULTS: A total of 245 rats were video-EEG phenotyped for epilepsy on the 7th postinjury month (121 in MRI cohort, 124 in EEG cohort). In the whole cohort (n = 245), the prevalence of PTE in rats with TBI was 22%, being 27% in the MRI and 18% in the EEG cohort (p > .05). Prevalence of PTE did not differ between the three study sites (p > .05). The average seizure frequency was .317 ± .725 seizures/day at University of Eastern Finland (UEF; Finland), .085 ± .067 at Monash University (Monash; Australia), and .299 ± .266 at University of California, Los Angeles (UCLA; USA; p < .01 as compared to Monash). The average seizure duration did not differ between UEF (104 ± 48 s), Monash (90 ± 33 s), and UCLA (105 ± 473 s; p > .05). Of the 219 seizures, 53% occurred as part of a seizure cluster (≥3 seizures/24 h; p >.05 between the study sites). Of the 209 seizures, 56% occurred during lights-on period and 44% during lights-off period (p > .05 between the study sites). SIGNIFICANCE: The PTE phenotype induced by lateral FPI is reproducible in a multicenter design. Our study supports the feasibility of performing preclinical multicenter trials in PTE to increase statistical power and experimental rigor to produce clinically translatable data to combat epileptogenesis after TBI.

Successful harmonization in EpiBioS4Rx biomarker study on post-traumatic epilepsy paves the way towards powered preclinical multicenter studies.

OBJECTIVE: Project 1 of the Preclinical Multicenter Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) consortium aims to identify preclinical biomarkers for antiepileptogenic therapies following traumatic brain injury (TBI). The international participating centers in Finland, Australia, and the United States have made a concerted effort to ensure protocol harmonization. Here, we evaluate the success of harmonization process by assessing the timing, coverage, and performance between the study sites. METHOD: We collected data on animal housing conditions, lateral fluid-percussion injury model production, postoperative care, mortality, post-TBI physiological monitoring, timing of blood sampling and quality, MR imaging timing and protocols, and duration of video-electroencephalography (EEG) follow-up using common data elements. Learning effect in harmonization was assessed by comparing procedural accuracy between the early and late stages of the project. RESULTS: The animal housing conditions were comparable between the study sites but the postoperative care procedures varied. Impact pressure, duration of apnea, righting reflex, and acute mortality differed between the study sites (p < 0.001). The severity of TBI on D2 post TBI assessed using the composite neuroscore test was similar between the sites, but recovery of acute somato-motor deficits varied (p < 0.001). A total of 99% of rats included in the final cohort in UEF, 100% in Monash, and 79% in UCLA had blood samples taken at all time points. The timing of sampling differed on day (D)2 (p < 0.05) but not D9 (p > 0.05). Plasma quality was poor in 4% of the samples in UEF, 1% in Monash and 14% in UCLA. More than 97% of the final cohort were MR imaged at all timepoints in all study sites. The timing of imaging did not differ on D2 and D9 (p > 0.05), but varied at D30, 5 months, and ex vivo timepoints (p < 0.001). The percentage of rats that completed the monthly high-density video-EEG follow-up and the duration of video-EEG recording on the 7th post-injury month used for seizure detection for diagnosis of post-traumatic epilepsy differed between the sites (p < 0.001), yet the prevalence of PTE (UEF 21%, Monash 22%, UCLA 23%) was comparable between the sites (p > 0.05). A decrease in acute mortality and increase in plasma quality across time reflected a learning effect in the TBI production and blood sampling protocols. SIGNIFICANCE: Our study is the first demonstration of the feasibility of protocol harmonization for performing powered preclinical multi-center trials for biomarker and therapy discovery of post-traumatic epilepsy.

Image data harmonization tools for the analysis of post-traumatic epilepsy development in preclinical multisite MRI studies.

Preclinical MRI studies have been utilized for the discovery of biomarkers that predict post-traumatic epilepsy (PTE). However, these single site studies often lack statistical power due to limited and homogeneous datasets. Therefore, multisite studies, such as the Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx), are developed to create large, heterogeneous datasets that can lead to more statistically significant results. EpiBioS4Rx collects preclinical data internationally across sites, including the United States, Finland, and Australia. However, in doing so, there are robust normalization and harmonization processes that are required to obtain statistically significant and generalizable results. This work describes the tools and procedures used to harmonize multisite, multimodal preclinical imaging data acquired by EpiBioS4Rx. There were four main harmonization processes that were utilized, including file format harmonization, naming convention harmonization, image coordinate system harmonization, and diffusion tensor imaging (DTI) metrics harmonization. By using Python tools and bash scripts, the file formats, file names, and image coordinate systems are harmonized across all the sites. To harmonize DTI metrics, values are estimated for each voxel in an image to generate a histogram representing the whole image. Then, the Quantitative Imaging Toolkit (QIT) modules are utilized to scale the mode to a value of one and depict the subsequent harmonized histogram. The standardization of file formats, naming conventions, coordinate systems, and DTI metrics are qualitatively assessed. The histograms of the DTI metrics were generated for all the individual rodents per site. For inter-site analysis, an average of the individual scans was calculated to create a histogram that represents each site. In order to ensure the analysis can be run at the level of individual animals, the sham and TBI cohort were analyzed separately, which depicted the same harmonization factor. The results demonstrate that these processes qualitatively standardize the file formats, naming conventions, coordinate systems, and DTI metrics of the data. This assists in the ability to share data across the study, as well as disseminate tools that can help other researchers to strengthen the statistical power of their studies and analyze data more cohesively.

MRI-Guided Electrode Implantation for Chronic Intracerebral Recordings in a Rat Model of Post-Traumatic Epilepsy-Challenges and Gains.

Brain atrophy induced by traumatic brain injury (TBI) progresses in parallel with epileptogenesis over time, and thus accurate placement of intracerebral electrodes to monitor seizure initiation and spread at the chronic postinjury phase is challenging. We evaluated in adult male Sprague Dawley rats whether adjusting atlas-based electrode coordinates on the basis of magnetic resonance imaging (MRI) increases electrode placement accuracy and the effect of chronic electrode implantations on TBI-induced brain atrophy. One group of rats (EEG cohort) was implanted with two intracortical (anterior and posterior) and a hippocampal electrode right after TBI to target coordinates calculated using a rat brain atlas. Another group (MRI cohort) was implanted with the same electrodes, but using T2-weighted MRI to adjust the planned atlas-based 3D coordinates of each electrode. Histological analysis revealed that the anterior cortical electrode was in the cortex in 83% (25% in targeted layer V) of the EEG cohort and 76% (31%) of the MRI cohort. The posterior cortical electrode was in the cortex in 40% of the EEG cohort and 60% of the MRI cohort. Without MRI-guided adjustment of electrode tip coordinates, 58% of the posterior cortical electrodes in the MRI cohort will be in the lesion cavity, as revealed by simulated electrode placement on histological images. The hippocampal electrode was accurately placed in 82% of the EEG cohort and 86% of the MRI cohort. Misplacement of intracortical electrodes related to their rostral shift due to TBI-induced cortical and hippocampal atrophy and caudal retraction of the brain, and was more severe ipsilaterally than contralaterally (p < 0.001). Total lesion area in cortical subfields targeted by the electrodes (primary somatosensory cortex, visual cortex) was similar between cohorts (p > 0.05). MRI-guided adjustment of coordinates for electrodes improved the success rate of intracortical electrode tip placement nearly to that at the acute postinjury phase (68% vs. 62%), particularly in the posterior brain, which exhibited the most severe postinjury atrophy. Overall, MRI-guided electrode implantation improved the quality and interpretation of the origin of EEG-recorded signals.

Seizure Susceptibility and Sleep Disturbance as Biomarkers of Epileptogenesis after Experimental TBI.

Objectives: We investigated whether seizure susceptibility increases over weeks−months after experimental traumatic brain injury (TBI), and whether seizure susceptibility in rats predicts the development of post-traumatic epilepsy (PTE) or epileptiform activity. We further investigated whether rats develop chronic sleep disturbance after TBI, and whether sleep disturbance parameters­alone or in combination with pentylenetetrazol (PTZ) test parameters­could serve as novel biomarkers for the development of post-traumatic epileptogenesis. Methods: TBI was induced in adult male Sprague-Dawley rats with lateral fluid-percussion injury. Sham-operated experimental controls underwent craniectomy without exposure to an impact force. Seizure susceptibility was tested with a PTZ test (30 mg/kg, intraperitoneally) on day (D) 30, D60, D90, and D180 after TBI (n = 28) or sham operation (n = 16) under video electroencephalogram (vEEG). In the 7th post-injury month, rats underwent continuous vEEG monitoring to detect spontaneous seizures and assess sleep disturbances. At the end of the experiments, rats were perfused for brain histology. Results: In the TBI group, the percentage of rats with PTZ-induced seizures increased over time (adjusted p < 0.05 compared with D30). Combinations of three PTZ test parameters (latency to the first epileptiform discharge (ED), number of EDs, and number of PTZ-induced seizures) survived the leave-one-out validation for differentiating rats with or without epileptiform activity, indicating an area under the receiver operating curve (AUC) of 0.743 (95% CI 0.472−0.992, p = 0.05) with a misclassification rate of 36% on D90, and an AUC of 0.752 (95% CI 0.483−0.929, p < 0.05) with a misclassification rate of 32% on D180. Sleep analysis revealed that the number of transitions to N3 or rapid eye movement (REM) sleep, along with the total number of transitions, was increased in the TBI group during the lights-on period (all p < 0.05). The sleep fragmentation index during the lights-on period was greater in the TBI rats than in sham-operated rats (p < 0.05). A combination of sleep parameters showed promise as diagnostic biomarkers of prior TBI, with an AUC of 0.792 (95% CI 0.549−0.934, p < 0.01) and a misclassification rate of 28%. Rats with epilepsy or any epileptiform activity had more transitions from N3 to the awake stage (p < 0.05), and the number of N3−awake transitions differentiated rats with or without epileptiform activity, with an AUC of 0.857 (95% CI 0.651−1.063, p < 0.01). Combining sleep parameters with PTZ parameters did not improve the biomarker performance. Significance: This is the first attempt to monitor the evolution of seizure susceptibility over months in a well-described rat model of PTE. Our data suggest that assessment of seizure susceptibility and sleep disturbance can provide diagnostic biomarkers of prior TBI and prognostic biomarkers of post-traumatic epileptogenesis.

Hippocampal position and orientation as prognostic biomarkers for posttraumatic epileptogenesis: An experimental study in a rat lateral fluid percussion model.

OBJECTIVE: This study was undertaken to identify prognostic biomarkers for posttraumatic epileptogenesis derived from parameters related to the hippocampal position and orientation. METHODS: Data were derived from two preclinical magnetic resonance imaging (MRI) follow-up studies: EPITARGET (156 rats) and Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx; University of Eastern Finland cohort, 43 rats). Epileptogenesis was induced with lateral fluid percussion-induced traumatic brain injury (TBI) in adult male Sprague Dawley rats. In the EPITARGET cohort, T 2 ∗ -weighted MRI was performed at 2, 7, and 21 days and in the EpiBioS4Rx cohort at 2, 9, and 30 days and 5 months post-TBI. Both hippocampi were segmented using convolutional neural networks. The extracted segmentation mask was used for a geometric construction, extracting 39 parameters that described the position and orientation of the left and right hippocampus. In each cohort, we assessed the parameters as prognostic biomarkers for posttraumatic epilepsy (PTE) both individually, using repeated measures analysis of variance, and in combination, using random forest classifiers. RESULTS: The extracted parameters were highly effective in discriminating between sham-operated and TBI rats in both the EPITARGET and EpiBioS4Rx cohorts at all timepoints (t; balanced accuracy > .9). The most discriminating parameter was the inclination of the hippocampus ipsilateral to the lesion at t = 2 days and the volumes at t ≥ 7 days after TBI. Furthermore, in the EpiBioS4Rx cohort, we could effectively discriminate epileptogenic from nonepileptogenic animals with a longer MRI follow-up, at t = 150 days (area under the curve = .78, balanced accuracy = .80, p = .0050), based on the orientation of both hippocampi. We found that the ipsilateral hippocampus rotated outward on the horizontal plane, whereas the contralateral hippocampus rotated away from the vertical direction. SIGNIFICANCE: We demonstrate that assessment of TBI-induced hippocampal deformation by clinically translatable MRI methodologies detects subjects with prior TBI as well as those at high risk of PTE, paving the way toward subject stratification for antiepileptogenesis studies.

Convolutional Neural Networks Enable Robust Automatic Segmentation of the Rat Hippocampus in MRI After Traumatic Brain Injury.

Registration-based methods are commonly used in the automatic segmentation of magnetic resonance (MR) brain images. However, these methods are not robust to the presence of gross pathologies that can alter the brain anatomy and affect the alignment of the atlas image with the target image. In this work, we develop a robust algorithm, MU-Net-R, for automatic segmentation of the normal and injured rat hippocampus based on an ensemble of U-net-like Convolutional Neural Networks (CNNs). MU-Net-R was trained on manually segmented MR images of sham-operated rats and rats with traumatic brain injury (TBI) by lateral fluid percussion. The performance of MU-Net-R was quantitatively compared with methods based on single and multi-atlas registration using MR images from two large preclinical cohorts. Automatic segmentations using MU-Net-R and multi-atlas registration were of excellent quality, achieving cross-validated Dice scores above 0.90 despite the presence of brain lesions, atrophy, and ventricular enlargement. In contrast, the performance of single-atlas segmentation was unsatisfactory (cross-validated Dice scores below 0.85). Interestingly, the registration-based methods were better at segmenting the contralateral than the ipsilateral hippocampus, whereas MU-Net-R segmented the contralateral and ipsilateral hippocampus equally well. We assessed the progression of hippocampal damage after TBI by using our automatic segmentation tool. Our data show that the presence of TBI, time after TBI, and whether the hippocampus was ipsilateral or contralateral to the injury were the parameters that explained hippocampal volume.

Regulation of Parvalbumin Interactome in the Perilesional Cortex after Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) causes 10-20% of structural epilepsy, with seizures typically originating in the cortex. Alterations in the neuronal microcircuits in the cortical epileptogenic zone, however, are poorly understood. Here, we assessed TBI-induced changes in perisomatic gamma aminobutyric acid (GABA)-ergic innervation in the perilesional cortex. We hypothesized that TBI will damage parvalbumin (PV)-immunoreactive inhibitory neurons and induce regulation of the associated GABAergic molecular interactome. TBI was induced in adult male Sprague-Dawley rats by lateral fluid-percussion injury. At 1-month post-TBI, the number of PV-positive somata was plotted on unfolded cortical maps and the distribution and density of immunopositive terminals analyzed. Qualitative analysis revealed either patchy microlesions of several hundred micrometers in diameter or diffuse neuronal loss. Quantitative analysis demonstrated a reduction in the number of PV-positive interneurons in patches down to 0% of that in sham-operated controls in the perilesional cortex. In the majority of patches, the cell numbers ranged from 71% to 90% that of the controls. The loss of PV-positive somata was accompanied by decreased axonal labeling. In situ hybridization revealed downregulated PV mRNA expression in the perilesional cortex. Gene Set Enrichment Analysis indicated a robustly downregulated expression profile of PV-related genes, which was confirmed by quantitative reverse transcriptase polymerase chain reaction. Specifically, we found that genes encoding postsynaptic GABA-A receptor genes, Gabrg2 and Gabrd, were downregulated in TBI animals compared with controls. Our data suggests that patchy reduction in PV-positive perisomatic inhibitory innervation contributes to the development of focal cortical inhibitory deficit after TBI.

Reorganization of Thalamic Inputs to Lesioned Cortex Following Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) disrupts thalamic and cortical integrity. The effect of post-injury reorganization and plasticity in thalamocortical pathways on the functional outcome remains unclear. We evaluated whether TBI causes structural changes in the thalamocortical axonal projection terminals in the primary somatosensory cortex (S1) that lead to hyperexcitability. TBI was induced in adult male Sprague Dawley rats with lateral fluid-percussion injury. A virus carrying the fluorescent-tagged opsin channel rhodopsin 2 transgene was injected into the ventroposterior thalamus. We then traced the thalamocortical pathways and analyzed the reorganization of their axonal terminals in S1. Next, we optogenetically stimulated the thalamocortical relays from the ventral posterior lateral and medial nuclei to assess the post-TBI functionality of the pathway. Immunohistochemical analysis revealed that TBI did not alter the spatial distribution or lamina-specific targeting of projection terminals in S1. TBI reduced the axon terminal density in the motor cortex by 44% and in S1 by 30%. A nematic tensor-based analysis revealed that in control rats, the axon terminals in layer V were orientated perpendicular to the pial surface (60.3°). In TBI rats their orientation was more parallel to the pial surface (5.43°, difference between the groups p < 0.05). Moreover, the level of anisotropy of the axon terminals was high in controls (0.063) compared with TBI rats (0.045, p < 0.05). Optical stimulation of the sensory thalamus increased alpha activity in electroencephalography by 312% in controls (p > 0.05) and 237% (p > 0.05) in TBI rats compared with the baseline. However, only TBI rats showed increased beta activity (33%) with harmonics at 5 Hz. Our findings indicate that TBI induces reorganization of thalamocortical axonal terminals in the perilesional cortex, which alters responses to thalamic stimulation.

Long-lasting blood-brain barrier dysfunction and neuroinflammation after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) causes 10-20% of acquired epilepsy, which typically develops within 2 years post-injury with poorly understood mechanisms. We investigated the location, severity, evolution and persistence of blood-brain barrier (BBB) dysfunction and associated neuroinflammation after TBI, and their contribution to post-traumatic seizure susceptibility. METHODS: TBI was induced with lateral fluid-percussion in adult male Sprague-Dawley rats (6 sham, 12 TBI). Permeability of the BBB was assessed using T1-weighted magnetic resonance imaging (MRI) with gadobutrol (Gd) contrast enhancement at 4 days, 2 weeks, 2 months, and 10 months post-injury and with intravenously administered fluorescein at 11 months post-TBI. Continuous (24/7) video-EEG monitoring was performed for 3 weeks at 11 months post-injury followed by the pentylenetetrazol (PTZ) seizure-susceptibility test. In the end, rats were perfused for histology to assess albumin extravasation, iron deposits, calcifications, reactive astrocytes, microglia and monocytes. To investigate the translational value of the data obtained, BBB dysfunction and neuroinflammation were investigated immunohistochemically in autopsy brain tissue from patients with TBI and PTE. RESULTS: MRI indicated persistent Gd leakage in the impacted cortex and thalamus of variable severity in all rats with TBI which correlated with fluorescein extravasation. In the impacted cortex BBB dysfunction was evident from 4 days post-injury onwards to the end of the 10-months follow-up. In the ipsilateral thalamus, leakage was evident at 2 and 10 months post-injury. The greater the BBB leakage in the perilesional cortex at 10 months after the injury, the greater the expression of the endothelial cell antigen RECA-1 (r = 0.734, p < 0.01) and the activated macrophages/monocytes/microglia marker CD68 (r = 0.699, p < 0.05) at 11 months post-injury. Seven of the 12 rats with TBI showed increased seizure susceptibility in the PTZ-test. Unlike expected, we did not find any association between increased Gd-leakage or neuroinflammation with seizure susceptibility at 11 months post-TBI. Analysis of human autopsy tissue indicated that similar to the animal model, chronic BBB dysfunction was also evident in the perilesional cortex and thalamus of patients with PTE, characterized by presence of albumin, iron deposits and calcifications as well as markers of neuroinflammation, including reactive astrocytes, microglia and monocytes. CONCLUSIONS: Rats and humans with TBI have long-lasting cortical BBB dysfunction and neuroinflammation. Focal Gd-enhancement matched with loci of neuroinflammation, particularly in the thalamus. Although BBB leakage did not associate with increased seizure susceptibility after TBI, our data suggest that for treatments aimed to mitigate BBB damage and its secondary pathologies like chronic neuroinflammation, there is a region-specific, long-lasting therapeutic time window.

Droplet digital polymerase chain reaction-based quantification of circulating microRNAs using small RNA concentration normalization.

Quantification of plasma microRNAs (miRNAs) as non-invasive disease biomarkers is subject to multiple technical variabilities. This study aimed to develop an optimized protocol for miRNA quantification from rodent plasma. We hypothesized that a fixed small RNA concentration input for reverse transcription (RT) reaction will provide better miRNA quantification than a fixed RNA volume input. For this, tail-vein plasma was collected from 30 naïve, adult male Sprague-Dawley rats. Plasma hemolysis was measured with NanoDrop-1000 and Denovix DS-11 spectrophotometers. Plasma was then pooled, and RNA was extracted from 50-µl, 100-µl or 200-µl pool aliquots. Small RNA concentration was measured with Qubit miRNA assay. A fixed RNA volume (un-normalized) or a fixed small RNA concentration was used for RT (concentration-normalized). The method was setup with miR-23a-3p and validated with miR-103a-3p and miR-451a. Hemolysis measurements from Denovix and NanoDrop strongly correlated. Qubit revealed increased small RNA concentrations with increasing starting plasma volumes. With concentration-normalization, miRNA levels from 100-µl and 200-µl plasma volume groups mostly normalized to the level of the 50-µl in ddPCR. Our results indicate that miRNA quantification with ddPCR should be performed with small RNA concentration-normalization to minimize variations in eluted RNA concentrations occuring during RNA extraction.

Web Application for Quantification of Traumatic Brain Injury-Induced Cortical Lesions in Adult Mice.

Disabilities resulting from traumatic brain injury (TBI) strongly correlate with the cytoarchitectonic part of the brain damaged, lesion area, and type of lesion. We developed a Web application to estimate the location of the lesion on mouse cerebral cortex caused by TBI induced by lateral fluid-percussion injury. The application unfolds user-determined TBI lesion measurements, e.g., from histologic sections to a reference template, and estimates the total lesion area, including the percentage of cortex damaged in different cytoarchitectural cortical regions. The resulting lesion can be visualized on a two-dimensional map of mouse cerebral cortex. The application also visualizes the development of the lesion over time when measurements from multiple time points are available. The web application was validated by comparing its performance to the manual method. The total area of the cortical lesion was similar between the manual (9.19 ± 0.66 mm2, range 4.25-14.93 mm2) and the automated analysis (9.27 ± 0.66 mm2, range 4.50-15.10 mm2) (p = 0.938). The results of the manual and automated analyses were strongly correlated (r = 0.999, p < 0.0001, Pearson correlation). The lesion localized in the same cytoarchitectonic regions when the unfolded map from the automated method was superimposed onto the map obtained using the manual method. The Web application-automated method is faster than the manual method in generating unfolded cortical lesion maps. The accuracy of the presented automated method in determining the anteroposterior level and outlining the lesion is equal to or greater than that of the manual method. Our application provides a novel tool for accurately quantifying and visualizing TBI lesions on mouse cerebral cortex.

Acute Downregulation of Novel Hypothalamic Protein Sushi Repeat-Containing Protein X-Linked 2 after Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) causes damage to the hypothalamo-hypophyseal axis, leading to endocrine dysregulation in up to 40% of TBI patients. Hence, there is an urgent need to identify non-invasive biomarkers for TBI-associated hypothalamo-hypophyseal pathology. Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel hypothalamic protein expressed in both rat and human brain. Our objective was to investigate the effect of acquired brain injury on plasma SRPX2 protein levels and SRPX2 expression in the brain. We induced severe lateral fluid-percussion injury in adult male rats and investigated changes in SRPX2 expression at 2 h, 6 h, 24 h, 48 h, 72 h, 5 days, 7 days, 14 days, 1 month, and 3 months post-injury. The plasma SRPX2 level was assessed by Western blot analysis. Hypothalamic SRPX2-immunoreactive neuronal numbers were estimated from immunostained preparations. At 2 h post-TBI, plasma SRPX2 levels were markedly decreased compared with the naïve group (area under the curve = 1.00, p < 0.05). Severe TBI caused a reduction in the number of hypothalamic SRPX2-immunoreactive neurons bilaterally at 2 h post-TBI compared with naïve group (5032 ± 527 vs. 9440 ± 351, p < 0.05). At 1 month after severe TBI, however, the brain and plasma SRPX2 levels were comparable between the TBI and naïve groups (p > 0.05). Unsupervised hierarchical clustering using SRPX2 expression differentiated animals into injured and uninjured clusters. Our findings indicate that TBI leads to an acute reduction in SRPX2 protein expression and reduced plasma SRPX2 level may serve as a candidate biomarker of hypothalamic injury.

Lateral fluid-percussion injury leads to pituitary atrophy in rats.

Traumatic brain injury (TBI) causes neuroendocrine dysregulation in up to 40% of humans, which is related to impaired function of the hypothalamo-hypophyseal axis and contributes to TBI-related co-morbidities. Our objective was to investigate whether hypophyseal atrophy can be recapitulated in rat lateral fluid-percussion injury model of human TBI. High-resolution structural magnetic resonance images (MRI) were acquired from rats at 2 days and 5 months post-TBI. To measure the lobe-specific volumetric changes, manganese-enhanced MRI (MEMRI) scans were acquired from rats at 8 months post-TBI, which also underwent the pentylenetetrazol (PTZ) seizure susceptibility and Morris water-maze spatial memory tests. MRI revealed no differences in the total hypophyseal volume between TBI and controls at 2 days, 5 months or 8 months post-TBI. Surprisingly, MEMRI at 8 months post-TBI indicated a 17% reduction in neurohypophyseal volume in the TBI group as compared to controls (1.04 ± 0.05 mm3 vs 1.25 ± 0.05 mm3, p < 0.05). Moreover, neurohypophyseal volume inversely correlated with the number of PTZ-induced epileptiform discharges and the mean latency to platform in the Morris water-maze test. Our data demonstrate that TBI leads to neurohypophyseal lobe-specific atrophy and may serve as a prognostic biomarker for post-TBI outcome.

Genotype and Injury Effect on the Expression of a Novel Hypothalamic Protein Sushi Repeat-Containing Protein X-Linked 2 (SRPX2).

Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel hypothalamic protein and a ligand of the urokinase-type plasminogen activator receptor (uPAR), which is essential for proteolysis of extracellular matrix and tissue remodeling after an insult to the brain. However, little is known about regulation of SRPX2. Our objective was to investigate if SRPX2 expression is altered by (i) the deficiency of uPAR or uPA (urokinase-type plasminogen activator), and (ii) traumatic brain injury (TBI). SRPX2 expression was assessed in wild type (Wt), Plaur-/- (uPAR-deficient), and Plau-/- (uPA-deficient) mice, with and without controlled cortical impact injury (CCI). The number of SRPX2+ neurons in hypothalamus was comparable to that in Wt littermates in Plaur-/- (2985 ±â€¯138 vs. 2890 ±â€¯92, p > 0.05) and Plau-/- mice (2180 ±â€¯232 vs. 2027 ±â€¯77, p > 0.05). The number of hypothalamic SRPX2+ neurons in the Wt-CCI group was comparable to that in controls (3645 ±â€¯288 vs. 3385 ±â€¯192, p > 0.05). Hypothalamic, hippocampal and thalamic Srpx2 gene expression was unaltered after TBI. However, at 4 days post-TBI Srpx2 gene expression was upregulated in the perilesional cortex of Plau-/--CCI mice up to 123% of that in the sham group (p < 0.05). Unsupervised hierarchical clustering using SRPX2 expression did not identify genotype or injury-specific clusters. Our data demonstrate that SRPX2 expression in the hypothalamus is resistant to genetic deficiencies in the urokinase-system or to the hypothalamus-affecting TBI. The contribution of elevated Srpx2 gene expression in perilesional cortex to post-TBI recovery process, however, requires further exploration.

Harmonization of the pipeline for seizure detection to phenotype post-traumatic epilepsy in a preclinical multicenter study on post-traumatic epileptogenesis.

RATIONALE: The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) Centre without walls is an NIH funded multicenter consortium. One of EpiBioS4Rx projects is a preclinical post-traumatic epileptogenesis biomarker study that involves three study sites: The University of Eastern Finland, Monash University (Melbourne) and the University of California Los Angeles. Our objective is to create a platform for evaluating biomarkers and testing new antiepileptogenic treatments for post-traumatic epilepsy (PTE) using the lateral fluid percussion injury (FPI) model in rats. As only 30-50% of rats with severe lateral FPI develop PTE by 6 months post-injury, prolonged video-EEG monitoring is crucial to identify animals with PTE. Our objective is to harmonize the surgical and data collection procedures, equipment, and data analysis for chronic EEG recording in order to phenotype PTE in this rat model across the three study sites. METHODS: Traumatic brain injury (TBI) was induced using lateral FPI in adult male Sprague-Dawley rats aged 11-12 weeks. Animals were divided into two cohorts: a) the long-term video-EEG follow-up cohort (Specific Aim 1), which was implanted with EEG electrodes within 24 h after the injury; and b) the magnetic resonance imaging (MRI) follow-up cohort (Specific Aim 2), at 5 months after lateral FPI. Four cortical epidural screw electrodes (2 ipsilateral, 2 contralateral) and three intracerebral bipolar electrodes were implanted (septal CA1 and the dentate gyrus, layers II and VI of the perilesional cortex both anterior and posterior to the injury site). During the 7th post-TBI month, animals underwent 4 weeks of continuous video-EEG recordings to diagnose of PTE. RESULTS: All centers harmonized the induction of TBI and surgical procedures for the implantation of EEG recordings, utilizing 4 or more EEG recording channels to cover areas ipsilateral and contralateral to the brain injury, perilesional cortex and the hippocampus and dentate gyrus. Ground and reference screw electrodes were implanted. At all sites the minimum sampling rate was 512 Hz, utilizing a finite impulse response (FIR) and impedance below 10 KΩ through the entire recording. As part of the quality control criteria we avoided electrical noise, and monitoring changes in impedance over time and the appearance of noise on the recordings. To reduce electrical noise, we regularly checked the integrity of the cables, stability of the EEG recording cap and the appropriate connection of the electrodes with the cables. Following the pipeline presented in this article and after applying the quality control criteria to our EEG recordings all of the sites were successful to phenotype seizure in chronic EEG recordings of animals after TBI. DISCUSSION: Despite differences in video-EEG acquisition equipment used, the three centers were able to consistently phenotype seizures in the lateral fluid-percussion model applying the pipeline presented here. The harmonization of methodology will help to improve the rigor of preclinical research, improving reproducibility of pre-clinical research in the search of biomarkers and therapies to prevent antiepileptogenesis.

Harmonization of pipeline for detection of HFOs in a rat model of post-traumatic epilepsy in preclinical multicenter study on post-traumatic epileptogenesis.

Studies of chronic epilepsy show pathological high frequency oscillations (HFOs) are associated with brain areas capable of generating epileptic seizures. Only a few of these studies have focused on HFOs during the development of epilepsy, but results suggest pathological HFOs could be a biomarker of epileptogenesis. The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy" (EpiBioS4Rx) is a multi-center project designed to identify biomarkers of epileptogenesis after a traumatic brain injury (TBI) and evaluate treatments that could modify or prevent the development of post-traumatic epilepsy. One goal of the EpiBioS4Rx project is to assess whether HFOs could be a biomarker of post-traumatic epileptogenesis. The current study describes the work towards this goal, including the development of common surgical procedures and EEG protocols, an interim analysis of the EEG for HFOs, and identifying issues that need to be addressed for a robust biomarker analysis. At three participating sites - University of Eastern Finland (UEF), Monash University in Melbourne (Melbourne) and University of California, Los Angeles (UCLA) - TBI was induced in adult male Sprague-Dawley rats by lateral fluid-percussion injury. After injury and in sham-operated controls, rats were implanted with screw and microwire electrodes positioned in neocortex and hippocampus to record EEG. A separate group of rats had serial magnetic resonance imaging after injury and then implanted with electrodes at 6 months. Recordings 28 days post-injury were available from UEF and UCLA, but not Melbourne due to technical issues with their EEG files. Analysis of recordings from 4 rats - UEF and UCLA each had one TBI and one sham-operated control - showed EEG contained evidence of HFOs. Computer-automated algorithms detected a total of 1,819 putative HFOs and of these only 40 events (2%) were detected by all three sites. Manual review of all events verified 130 events as HFO and the remainder as false positives. Review of the 40 events detected by all three sites was associated with 88% agreement. This initial report from the EpiBioS4Rx Consortium demonstrates the standardization of EEG electrode placements, recording protocol and long-term EEG monitoring, and differences in detection algorithm HFO results between sites. Additional work on detection strategy, detection algorithm performance, and training in HFO review will be performed to establish a robust, preclinical evaluation of HFOs as a biomarker of post-traumatic epileptogenesis.

Harmonization of lateral fluid-percussion injury model production and post-injury monitoring in a preclinical multicenter biomarker discovery study on post-traumatic epileptogenesis.

Multi-center preclinical studies can facilitate the discovery of biomarkers of antiepileptogenesis and thus facilitate the diagnosis and treatment development of patients at risk of developing post-traumatic epilepsy. However, these studies are often limited by the difficulty in harmonizing experimental protocols between laboratories. Here, we assess whether the production of traumatic brain injury (TBI) using the lateral fluid-percussion injury (FPI) in adult male Sprague-Dawley rats (12 weeks at the time of injury) was harmonized between three laboratories - located in the University of Eastern Finland (UEF), Monash University in Melbourne, Australia (Melbourne) and The University of California, Los Angeles, USA (UCLA). These laboratories are part of the international multicenter-based project, the Epilepsy Bioinformatics Study for Antiepileptogenesis Therapy (EpiBioS4Rx). Lateral FPI was induced in adult male Sprague-Dawley rats. The success of methodological harmonization was assessed by performing inter-site comparison of injury parameters including duration of anesthesia during surgery, impact pressure, post-impact transient apnea, post-impact seizure-like behavior, acute mortality (<72 h post-injury), time to self-right after the impact, and severity of the injury (assessed with the neuroscore). The data was collected using Common Data Elements and Case Report Forms. The acute mortality was 15% (UEF), 50% (Melbourne) and 57% (UCLA) (p < 0.001). The sites differed in the duration of anesthesia, the shortest being at UEF < Melbourne < UCLA (p < 0.001). The impact pressure used also differed between the sites, the highest being in UEF > Melbourne > UCLA (p < 0.001). The impact pressure associated with the severity of the functional deficits (low neuroscore) (P < 0.05) only at UEF, but not at any of the other sites. Additionally, the sites differed in the duration of post-impact transient apnea (p < 0.001) and time to self-right (P < 0.001), the highest values in both parameters was registered in Melbourne. Post-impact seizure-like behavior was observed in 51% (UEF), 25% (Melbourne) and 2% (UCLA) of rats (p < 0.001). Despite the differences in means when all sites were compared there was significant overlap in injury parameters between the sites. The data reflects the technical difficulties in the production of lateral FPI across multiple sites. On the other hand, the data can be used to model the heterogeneity in human cohorts with closed-head injury. Our animal cohort will provide a good starting point to investigate the factors associated with epileptogenesis after lateral FPI.

Harmonization of pipeline for preclinical multicenter MRI biomarker discovery in a rat model of post-traumatic epileptogenesis.

Preclinical imaging studies of posttraumatic epileptogenesis (PTE) have largely been proof-of-concept studies with limited animal numbers, and thus lack the statistical power for biomarker discovery. Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is a pioneering multicenter trial investigating preclinical imaging biomarkers of PTE. EpiBios4Rx faced the issue of harmonizing the magnetic resonance imaging (MRI) procedures and imaging data metrics prior to its execution. We present here the harmonization process between three preclinical MRI facilities at the University of Eastern Finland (UEF), the University of Melbourne (Melbourne), and the University of California, Los Angeles (UCLA), and evaluate the uniformity of the obtained MRI data. Adult, male rats underwent a lateral fluid percussion injury (FPI) and were followed by MRI 2 days, 9 days, 1 month, and 5 months post-injury. Ex vivo scans of fixed brains were conducted 7 months post-injury as an end point follow-up. Four MRI modalities were used: T2-weighted imaging, multi-gradient-echo imaging, diffusion-weighted imaging, and magnetization transfer imaging, and acquisition parameters for each modality were tailored to account for the different field strengths (4.7 T and 7 T) and different MR hardwares used at the three participating centers. Pilot data collection resulted in comparable image quality across sites. In interim analysis (of data obtained by April 30, 2018), the within-site variation of the quantified signal properties was low, while some differences between sites remained. In T2-weighted images the signal-to-noise ratios were high at each site, being 35 at UEF, 48 at Melbourne, and 32 at UCLA (p < 0.05). The contrast-to-noise ratios were similar between the sites (9, 10, and 8, respectively). Magnetization transfer ratio maps had identical white matter/ gray matter contrast between the sites, with white matter showing 15% higher MTR than gray matter despite different absolute MTR values (MTR both in white and gray matter was 3% lower in Melbourne than at UEF, p < 0.05). Diffusion-weighting yielded different degrees of signal attenuation across sites, being 83% at UEF, 76% in Melbourne, and 80% at UCLA (p < 0.05). Fractional anisotropy values differed as well, being 0.81 at UEF, 0.73 in Melbourne, and 0.84 at UCLA (p < 0.05). The obtained values in sham animals showed low variation within each site and no change over time, suggesting high repeatability of the measurements. Quality control scans with phantoms demonstrated stable hardware performance over time. Timing of post-TBI scans was designed to target specific phases of the dynamic pathology, and the execution at different centers was highly accurate. Besides a few outliers, the 2-day scans were done within an hour from the target time point. At day 9, most animals were scanned within an hour from the target time point, and all but 2 outliers within 24 h from the target. The 1-month post-TBI scans were done within 31 ± 3 days. MRI procedures and animal physiology during scans were similar between the sites. Taken together, the 10% inter-site difference in FA and 3% difference in MTR values should be included into analysis as a covariate or balanced out in post-processing in order to detect disease-related effects on brain structure at the same scale. However, for a MRI biomarker for post-traumatic epileptogenesis to have realistic chance of being successfully translated to validation in clinical trials, it would need to be a robust TBI-induced structural change which tolerates the inter-site methodological variability described here.

Informatics tools to assess the success of procedural harmonization in preclinical multicenter biomarker discovery study on post-traumatic epileptogenesis.

The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is a National Institutes for Neurological Diseases and Stoke funded Centers-Without-Walls international multidisciplinary study aimed at preventing epileptogenesis. The preclinical biomarker discovery in EpiBios4Rx applies a multicenter study design to allow the number of animals that are required for adequate statistical power for the analysis to be studied in an efficient manner. Further, the use of multiple centers mimics the clinical trial situation, and therefore potentially the chance of successful clinical translation of the outcomes of the study. Its successful implementation requires harmonization of procedures and data analyses between the three contributing centers in Finland, Australia, and USA. The objective of the present analysis was to develop metrics for analysis of the success of harmonization of procedures to guide further data analyses and plan the future multicenter preclinical studies. The interim analysis of data is based on the analysis of data from 212 rats with lateral fluid-percussion injury or sham-operation included in the biomarker discovery by April 30, 2018. The details of protocols, including production of injury, post-injury follow-up, blood sampling, electroencephalogram recording, and magnetic resonance imaging have been presented in the accompanying manuscripts in this Supplement. Implementation of protocols in EpiBios4Rx project participant centers was visualized in 2D using t-distributed stochastic neighborhood embedding (t-SNE). The protocols applied to each rat were presented as feature vectors of procedure related variables (e.g., impact pressure, anesthesia time). The total number of protocol features linked to each rat was 112. The missing data was accounted in visualization by utilizing imputation and adding the number of missing values as a third dimension to 2D t-SNE plot, resulting in a 3D overview of protocol data. Intraclass correlation coefficient (ICC) using Euclidean distances and area under receiver operating characteristic curve (AUC) of k-nearest neighbor classifier (KNN) were utilized to quantify the degree of clustering by center. Both subsets of data with incomplete protocol vectors omitted and missing protocol data imputed were assessed. Our data show that a visible clustering by center was observed in all t-SNE plots, except for day 7 neuroscores. Both ICC and AUC indicated clustering by center in all protocol variable subsets, excluding unimputed day 7 neuroscores (ICC 0.04 and AUC 0.6). ICC for imputed set of all protocol related variables was 0.1 and KNN AUC 0.92. In conclusion, both ICC and AUC indicated differences in protocol between EpiBios4Rx participating centers, which needs to be taken into account in data analysis. Importantly, the majority of observed differences are recoverable as they relate to insufficient updates in record keeping. While AUC score of KNN is a more sensitive measure for protocol harmonization than ICC for data that displays complex splintered clustering, ICC and AUC provide complementary measures to assess the degree of procedural harmonization. This experience should be helpful for other groups planning such multicenter post-traumatic epileptogenesis studies in the future.