Diffusion tensor imaging (DTI) reveals microstructural features of grey and white matter non-invasively. The contrast produced by DTI, however, is not fully understood and requires further validation. We used serial block-face scanning electron microscopy (SBEM) to acquire tissue metrics, i.e., anisotropy and orientation, using three-dimensional Fourier transform-based (3D-FT) analysis, to correlate with fractional anisotropy and orientation in DTI. SBEM produces high-resolution 3D data at the mesoscopic scale with good contrast of cellular membranes. We analysed selected samples from cingulum, corpus callosum, and perilesional cortex of sham-operated and traumatic brain injury (TBI) rats. Principal orientations produced by DTI and 3D-FT in all samples were in good agreement. Anisotropy values showed similar patterns of change in corresponding DTI and 3D-FT parameters in sham-operated and TBI rats. While DTI and 3D-FT anisotropy values were similar in grey matter, 3D-FT anisotropy values were consistently lower than fractional anisotropy values from DTI in white matter. We also evaluated the effect of resolution in 3D-FT analysis. Despite small angular differences in grey matter samples, lower resolution datasets provided reliable results, allowing for analysis of larger fields of view. Overall, 3D SBEM allows for more sophisticated validation studies of diffusion imaging contrast from a tissue microstructural perspective.

Objective: To identify magnetic resonance imaging (MRI) biomarkers for post‐traumatic epilepsy.Methods: The EPITARGET (Targets and biomarkers for antiepileptogenesis, epitarget.eu) animal cohort completing T₂ relaxation and diffusion tensor MRI follow‐up and 1‐month‐long video‐electroencephalography monitoring included 98 male Sprague‐Dawley rats with traumatic brain injury and 18 controls. T₂ imaging was performed on day (D) 2, D7, and D21 and diffusion tensor imaging (DTI) on D7 and D21 using a 7‐Tesla Bruker PharmaScan MRI scanner. The mean and standard deviation (SD) of the T₂ relaxation rate, multiple diffusivity measures, and diffusion anisotropy at each time‐point within the ventroposterolateral and ventroposteromedial thalamus were used as predictor variables in multi‐variable logistic regression models to distinguish rats with and without epilepsy.Results: Twenty‐nine percent (28/98) of the rats with traumatic brain injury (TBI) developed epilepsy. The best‐performing logistic regression model utilized the D2 and D7 T₂ relaxation time as well as the D7 diffusion tensor data. The model distinguished rats with and without epilepsy (Bonferroni‐corrected p‐value < .001) with a cross‐validated concordance statistic of 0.74 (95% confidence interval [CI] 0.60–0.84). In a cross‐validated classification test, the model exhibited 54% sensitivity and 91% specificity, enriching the epilepsy rate within the study population from the expected 29% to 71%. A model using the D2 T₂ data only resulted in a 73% enriched epilepsy rate (regression p‐value .007, cross‐validated concordance 0.70, 95% CI 0.56–0.80, sensitivity 29%, specificity 96%).Significance: An MRI parameter set reporting on acute and subacute neuropathologic changes common to experimental and human TBI presents a diagnostic biomarker for post‐traumatic epileptogenesis. Significant enrichment of the study population was achieved even when using a single time‐point measurement, producing an expected epilepsy rate of 73%.

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 parameters—alone or in combination with pentylenetetrazol (PTZ) test parameters—could 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.

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.