Diffusion MRI approaches for investigating microstructural complexity in a rat model of traumatic brain injury.

Our study explores the potential of conventional and advanced diffusion MRI techniques including diffusion tensor imaging (DTI), and single-shell 3-tissue constrained spherical deconvolution (SS3T-CSD) to investigate complex microstructural changes following severe traumatic brain injury in rats at a chronic phase. Rat brains after sham-operation or lateral fluid percussion (LFP) injury were scanned ex vivo in a 9.4 T scanner. Our region-of-interest-based approach of tensor-, and SS3T-CSD derived fixel-, 3-tissue signal fraction maps were sensitive to changes in both white matter (WM) and grey matter (GM) areas. Tensor-based measures, such as fractional anisotropy (FA) and radial diffusivity (RD), detected more changes in WM and GM areas as compared to fixel-based measures including apparent fiber density (AFD), peak FOD amplitude and primary fiber bundle density, while 3-tissue signal fraction maps revealed distinct changes in WM, GM, and phosphate-buffered saline (PBS) fractions highlighting the complex tissue microstructural alterations post-trauma. Track-weighted imaging demonstrated changes in track morphology including reduced curvature and average pathlength distal from the primary lesion in severe TBI rats. In histological analysis, changes in the diffusion MRI measures could be associated to decreased myelin density, loss of myelinated axons, and increased cellularity, revealing progressive microstructural alterations in these brain areas five months after injury. Overall, this study highlights the use of combined conventional and advanced diffusion MRI measures to obtain more precise insights into the complex tissue microstructural alterations in chronic phase of severe brain injury.

Acute Hippocampal Damage as a Prognostic Biomarker for Cognitive Decline but Not for Epileptogenesis after Experimental Traumatic Brain Injury.

It is necessary to develop reliable biomarkers for epileptogenesis and cognitive impairment after traumatic brain injury when searching for novel antiepileptogenic and cognition-enhancing treatments. We hypothesized that a multiparametric magnetic resonance imaging (MRI) analysis along the septotemporal hippocampal axis could predict the development of post-traumatic epilepsy and cognitive impairment. We performed quantitative T2 and T2* MRIs at 2, 7 and 21 days, and diffusion tensor imaging at 7 and 21 days after lateral fluid-percussion injury in male rats. Morris water maze tests conducted between 35-39 days post-injury were used to diagnose cognitive impairment. One-month-long continuous video-electroencephalography monitoring during the 6th post-injury month was used to diagnose epilepsy. Single-parameter and regularized multiple linear regression models were able to differentiate between sham-operated and brain-injured rats. In the ipsilateral hippocampus, differentiation between the groups was achieved at most septotemporal locations (cross-validated area under the receiver operating characteristic curve (AUC) 1.0, 95% confidence interval 1.0-1.0). In the contralateral hippocampus, the highest differentiation was evident in the septal pole (AUC 0.92, 95% confidence interval 0.82-0.97). Logistic regression analysis of parameters imaged at 3.4 mm from the contralateral hippocampus's temporal end differentiated between the cognitively impaired rats and normal rats (AUC 0.72, 95% confidence interval 0.55-0.84). Neither single nor multiparametric approaches could identify the rats that would develop post-traumatic epilepsy. Multiparametric MRI analysis of the hippocampus can be used to identify cognitive impairment after an experimental traumatic brain injury. This information can be used to select subjects for preclinical trials of cognition-improving interventions.

The effect of magnetic resonance imaging on mercury release from dental amalgam at 3T and 7T.

OBJECTIVES: To measure mercury release from standardised hydroxyapatite/amalgam constructs during MRI scanning and investigate the impact of static field strength and radiofrequency (RF) power on mercury release. METHODS: Amalgam was placed into 140 hydroxyapatite disks and matured for 14-days in artificial saliva. The solution was replaced, and samples split into five groups of 28 immediately prior to MRI. One group had no exposure, and the remainder were exposed to either a 3T or 7T MRI scanner, each at high and low RF power. Mercury concentration was measured by inductively coupled plasma mass spectrometry. Groups were compared using one-way ANOVA, and two-way ANOVA for main effects/ interaction of field strength/ RF power. RESULTS: Mercury concentration was increased in the 7T groups (high/ low: 15.43/ 11.33 ng mL-1) and 3T high group (3.59) compared to control (2.44). MRI field strength significantly increased mercury release (p < .001) as did RF power (p = .030). At 3T, mercury release was 20.3 times lower than during maturation of dental amalgam, and for the average person an estimated 1.50 ng kg-1 of mercury might be released during one 3T investigation; this is substantially lower than the tolerable weekly intake of 4,000 ng kg-1. CONCLUSION: Mercury release from amalgam shows a measurable increase following MRI, and the magnitude changes with magnetic field strength and RF power. The amount of mercury released is small compared to release during amalgam maturation. Amalgam mercury release during MRI is unlikely to be clinically meaningful and highly likely to remain below safe levels.

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.

Microstructural Tissue Changes in a Rat Model of Mild Traumatic Brain Injury.

Our study investigates the potential of diffusion MRI (dMRI), including diffusion tensor imaging (DTI), fixel-based analysis (FBA) and neurite orientation dispersion and density imaging (NODDI), to detect microstructural tissue abnormalities in rats after mild traumatic brain injury (mTBI). The brains of sham-operated and mTBI rats 35 days after lateral fluid percussion injury were imaged ex vivo in a 11.7-T scanner. Voxel-based analyses of DTI-, fixel- and NODDI-based metrics detected extensive tissue changes in directly affected brain areas close to the primary injury, and more importantly, also in distal areas connected to primary injury and indirectly affected by the secondary injury mechanisms. Histology revealed ongoing axonal abnormalities and inflammation, 35 days after the injury, in the brain areas highlighted in the group analyses. Fractional anisotropy (FA), fiber density (FD) and fiber density and fiber bundle cross-section (FDC) showed similar pattern of significant areas throughout the brain; however, FA showed more significant voxels in gray matter areas, while FD and FDC in white matter areas, and orientation dispersion index (ODI) in areas most damage based on histology. Region-of-interest (ROI)-based analyses on dMRI maps and histology in selected brain regions revealed that the changes in MRI parameters could be attributed to both alterations in myelinated fiber bundles and increased cellularity. This study demonstrates that the combination of dMRI methods can provide a more complete insight into the microstructural alterations in white and gray matter after mTBI, which may aid diagnosis and prognosis following a mild brain injury.

Inflammatory reaction in the retina after focal non-convulsive status epilepticus in mice investigated with high resolution magnetic resonance and diffusion tensor imaging.

Pathophysiological consequences of focal non-convulsive status epilepticus (fNCSE) have been difficult to demonstrate in humans. In rats fNCSE pathology has been identified in the eyes. Here we evaluated the use of high-resolution 7 T structural T1-weighted magnetic resonance imaging (MRI) and 9.4 T diffusion tensor imaging (DTI) for detecting hippocampal fNCSE-induced retinal pathology ex vivo in mice. Seven weeks post-fNCSE, increased number of Iba1+ microglia were evident in the retina ipsilateral to the hemisphere with fNCSE, and morphologically more activated microglia were found in both ipsi- and contralateral retina compared to non-stimulated control mice. T1-weighted intensity measurements of the contralateral retina showed a minor increase within the outer nuclear and plexiform layers of the lateral retina. T1-weighted measurements were not performed in the ipsilateral retina due to technical difficulties. DTI fractional anisotropy(FA) values were discretely altered in the lateral part of the ipsilateral retina and unaltered in the contralateral retina. No changes were observed in the distal part of the optic nerve. The sensitivity of both imaging techniques for identifying larger retinal alteration was confirmed ex vivo in retinitis pigmentosa mice where a substantial neurodegeneration of the outer retinal layers is evident. With MR imaging a 50 % decrease in DTI FA values and significantly thinner retina in T1-weighted images were detected. We conclude that retinal pathology after fNCSE in mice is subtle and present bilaterally. High-resolution T1-weighted MRI and DTI independently did not detect the entire pathological retinal changes after fNCSE, but the combination of the two techniques indicated minor patchy structural changes.

Acute thalamic damage as a prognostic biomarker for post-traumatic epileptogenesis.

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%.

Quantitative susceptibility mapping of the rat brain after traumatic brain injury.

The primary lesion arising from the initial insult after traumatic brain injury (TBI) triggers a cascade of secondary tissue damage, which may also progress to connected brain areas in the chronic phase. The aim of this study was, therefore, to investigate variations in the susceptibility distribution related to these secondary tissue changes in a rat model after severe lateral fluid percussion injury. We compared quantitative susceptibility mapping (QSM) and R2 * measurements with histological analyses in white and grey matter areas outside the primary lesion but connected to the lesion site. We demonstrate that susceptibility variations in white and grey matter areas could be attributed to reduction in myelin, accumulation of iron and calcium, and gliosis. QSM showed quantitative changes attributed to secondary damage in areas located rostral to the lesion site that appeared normal in R2 * maps. However, combination of QSM and R2 * was informative in disentangling the underlying tissue changes such as iron accumulation, demyelination, or calcifications. Therefore, combining QSM with R2 * measurement can provide a more detailed assessment of tissue changes and may pave the way for improved diagnosis of TBI, and several other complex neurodegenerative diseases.

Early Increase in Cortical T2 Relaxation Is a Prognostic Biomarker for the Evolution of Severe Cortical Damage, but Not for Epileptogenesis, after Experimental Traumatic Brain Injury.

Prognostic biomarkers for post-injury outcome are necessary for the development of neuroprotective and antiepileptogenic treatments for traumatic brain injury (TBI). We hypothesized that T2 relaxation magnetic resonance imaging (MRI) predicts the progression of perilesional cortical pathology and epileptogenesis. The EPITARGET animal cohort used for MRI analysis included 120 adult male Sprague-Dawley rats with TBI induced by lateral fluid-percussion injury and 24 sham-operated controls. T2 MRI was performed at days 2, 7, and 21 post-TBI. The lesioned cortex was outlined, and the T2 value of each imaging voxel within the lesion area was scored using a five-grade pathology classification. Analysis of 1-month video-electroencephalography recordings initiated 5 months post-TBI indicated that 27% (31 of 114) of the animals with TBI developed epilepsy. Multiple linear regression analysis indicated that T2-based classification of lesion volume at day 2 and day 7 post-TBI explained the necrotic lesion volume with greatly increased T2 (>102 ms) at day 21 post-TBI (F(13,103) = 52.5; p < 0.001; R2 = 0.87; adjusted R2 = 0.85). The volume of moderately increased (78-102 ms) T2 at day 7 post-TBI predicted the evolution of large (>12 mm3) cortical lesions (area under the curve, 0.92; p < 0.001; cutoff, 1.9 mm3; false positive rate, 0.10; true positive rate, 0.62). Logistic regression analysis, however, showed that the different severities of T2 lesion volumes at days 2, 7, and 21 post-TBI did not explain the development of epilepsy (χ2(18,95) = 18.4; p = 0.427). In addition, the location of the T2 abnormality within the cortex did not correlate with epileptogenesis. A single measurement of T2 relaxation MRI in the acute post-TBI phase is useful for identifying post-TBI subjects at highest risk of developing large cortical lesions, and thus, in the greatest need of neuroprotective therapies after TBI, but not the development of post-traumatic epilepsy.

Nonconvulsive status epilepticus in rats leads to brain pathology.

OBJECTIVE: Status epilepticus (SE) is an abnormally prolonged epileptic seizure that if associated with convulsive motor symptoms is potentially life threatening for a patient. However, 20%-40% of patients with SE lack convulsive events and instead present with more subtle semiology such as altered consciousness and less motor activity. Today, there is no general consensus regarding to what extent nonconvulsive SE (NCSE) is harmful to the brain, which adds uncertainty to stringent treatment regimes. METHODS: Here, we evaluated brain pathology in an experimental rat and mouse model of complex partial NCSE originating in the temporal lobes with Western blot analysis, immunohistochemistry, and ex vivo diffusion tensor imaging (DTI). The NCSE was induced by electrical stimulation with intrahippocampal electrodes and terminated with pentobarbital anesthesia. Video-electroencephalographic recordings were performed throughout the experiment. RESULTS: DTI of mice 7 weeks post-NCSE showed no robust long-lasting changes in fractional anisotropy within the hippocampal epileptic focus. Instead, we found pathophysiological changes developing over time when measuring protein levels and cell counts in extracted brain tissue. At 6 and 24 hours post-NCSE in rats, few changes were observed within the hippocampus and cortical or subcortical structures in Western blot analyses of key components of the cellular immune response and synaptic protein expression, while neurodegeneration had started. However, 1 week post-NCSE, both excitatory and inhibitory synaptic protein levels were decreased in hippocampus, concomitant with an excessive microglial and astrocytic activation. At 4 weeks, a continuous immune response in the hippocampus was accompanied with neuronal loss. Levels of the excitatory synaptic adhesion molecule N-cadherin were decreased specifically in rats that developed unprovoked spontaneous seizures (epileptogenesis) within 1 month following NCSE, compared to rats only exhibiting acute symptomatic seizures within 1 week post-NCSE. SIGNIFICANCE: These findings provide evidence for a significant brain pathology following NCSE in an experimental rodent model.

Behavioral and stereological characterization of Hdc KO mice: Relation to Tourette syndrome.

A premature termination codon in the human histidine decarboxylase (Hdc) gene has been identified in a family suffering from Guilles de la Tourette syndrome (GTS). In the current study we investigated if mice lacking the histamine producing enzyme HDC share the morphological and cytological phenotype with GTS patients by using magnetic resonance (MRI) and diffusion tensor imaging (DTI), unbiased stereology and immunohistochemistry. Behavior of Hdc knock-out (Hdc KO) mice was assessed in an open field test. The results of stereological, volumetric and DTI analysis measurements showed no significant differences between control and Hdc KO mice. The numbers and distribution of GABAergic parvalbumin or nitric oxide-expressing and cholinergic interneurons were normal in Hdc KO mice. Cortical morphology and layering in adult Hdc KO mice were also preserved. In open field test Hdc KO mice showed impaired exploratory activity and habituation when introduced to novel environment. Our results indicate that Hdc deficiency in mice does not disturb the development of striatal and cortical interneurons and does not lead to the morphological and cytological phenotypes characterized by humans with GTS. Nevertheless, histamine deficiency leads to behavioral alterations probably due to neurotransmitter dysbalance on the level of the striatum.

Luminometric Nanoparticle-Based Assay for High Sensitivity Detection of ß-Amyloid Aggregation.

A nanoparticle-based assay utilizing time-resolved luminescence resonance energy transfer (TR-LRET) was developed for the detection of ß-amyloid aggregation. The assay is based on the competitive adsorption of the sample and the acceptor-labeled protein to donor europium(III) polystyrene nanoparticles. The performance of the assay was demonstrated by following the fibrillization of ß-amyloid peptide 1-42 (Aß42) as a function of time and by comparing to the reference methods atomic force microscopy (AFM) and thioflavin T (ThT) assay. The fibrillization leads to reduced adsorption of Aß42 to the nanoparticles increasing the TR-LRET signal. The investigated methods detected fibril formation with equal sensitivities. Eight potential fibrillization inhibitor compounds reported in the literature were tested and the results obtained with each method were compared. It was shown with AFM imaging that the inhibition of fibril formation was not complete with any of the compounds. The developed TR-LRET nanoparticle assay gave corresponding results with the AFM imaging. However, the ThT assay led to contradictory results, as low fluorescence signal was measured in the presence of all tested compounds suggesting inhibition of fibrillization. Our results suggest that the developed TR-LRET nanoparticle assay can be exploited for screening of potential ß-amyloid aggregation inhibitors, whereas some of the tested compounds may be measured as false positive inhibitors with the much-utilized ThT assay.