RESUMEN

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.

Asunto(s)

Lesiones Traumáticas del Encéfalo , Epilepsia Postraumática , Epilepsia , Animales , Ratas , Biomarcadores , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/diagnóstico por imagen , Modelos Animales de Enfermedad , Epilepsia/etiología , Epilepsia/diagnóstico , Epilepsia Postraumática/etiología , Epilepsia Postraumática/tratamiento farmacológico , Convulsiones , Estudios Multicéntricos como Asunto

RESUMEN

We report on a case study of a Wistar rat that was investigated in detail because it exhibited no N3 sleep in electroencephalography (EEG) after lateral fluid-percussion injury (FPI)-induced traumatic brain injury (TBI). The rat (#112) belonged to a cohort of 28 adult Wistar rats exposed to lateral FPI. Rats were monitored by continuous video EEG for 30 days to follow-up on the evolution of sleep disturbances. The beam walking test was used to measure post-TBI functional recovery. Severity of the cortical lesion area, total brain volume, and cortical volume were measured from histological brain sections. Rat #112 had a normal body and skull appearance. Its baseline body weight did not differ from that of the rest of the cohort. At baseline, rat #112 crossed the beam in 6.3 sec (score range for the rest of the cohort, 4.7-44.3) and showed no evident slipping of the paws, scoring a 5.3 (score range for the rest of cohort, 4.3-6.0). On day 30 post-TBI, however, rat #112 was the only rat with a score of 0 on the beam. Histological analysis at 30 days post-TBI revealed a small 0.6-mm2 post-TBI lesion in the somatosensory cortex (lesion size range for the rest of the cohort, 1.2-10.9). The brain volume of rat #112 was 2-fold larger than the mean volume of the rest of the cohort (1592 vs. 758 mm3), the ventricles were remarkably enlarged, and the layered cerebral cortex was very thin. Analysis of the sleep EEG revealed that rat #112 had rapid eye movement sleep and wakefulness, but no N3 sleep, during the 72-h EEG epoch analyzed. This case report demonstrates that brain abnormalities presumably unrelated to the impact-induced cortical lesion, such as presumed pre-existing hydrocephalus, may worsen TBI-induced behavioral and electrographical outcome measures and complicate the assessment of the cause of the abnormalities.

RESUMEN

OBJECTIVE: We used the lateral fluid percussion injury (LFPI) model of moderate-to-severe traumatic brain injury (TBI) to identify early plasma biomarkers predicting injury, early post-traumatic seizures or neuromotor functional recovery (neuroscores), considering the effect of levetiracetam, which is commonly given after severe TBI. METHODS: Adult male Sprague-Dawley rats underwent left parietal LFPI, received levetiracetam (200 mg/kg bolus, 200 mg/kg/day subcutaneously for 7 days [7d]) or vehicle post-LFPI, and were continuously video-EEG recorded (n = 14/group). Sham (craniotomy only, n = 6), and naïve controls (n = 10) were also used. Neuroscores and plasma collection were done at 2d or 7d post-LFPI or equivalent timepoints in sham/naïve. Plasma protein biomarker levels were determined by reverse phase protein microarray and classified according to injury severity (LFPI vs. sham/control), levetiracetam treatment, early seizures, and 2d-to-7d neuroscore recovery, using machine learning. RESULTS: Low 2d plasma levels of Thr231 -phosphorylated tau protein (pTAU-Thr231 ) and S100B combined (ROC AUC = 0.7790) predicted prior craniotomy surgery (diagnostic biomarker). Levetiracetam-treated LFPI rats were differentiated from vehicle treated by the 2d-HMGB1, 2d-pTAU-Thr231 , and 2d-UCHL1 plasma levels combined (ROC AUC = 0.9394) (pharmacodynamic biomarker). Levetiracetam prevented the seizure effects on two biomarkers that predicted early seizures only among vehicle-treated LFPI rats: pTAU-Thr231 (ROC AUC = 1) and UCHL1 (ROC AUC = 0.8333) (prognostic biomarker of early seizures among vehicle-treated LFPI rats). Levetiracetam-resistant early seizures were predicted by high 2d-IFNγ plasma levels (ROC AUC = 0.8750) (response biomarker). 2d-to-7d neuroscore recovery was best predicted by higher 2d-S100B, lower 2d-HMGB1, and 2d-to-7d increase in HMGB1 or decrease in TNF (P < 0.05) (prognostic biomarkers). SIGNIFICANCE: Antiseizure medications and early seizures need to be considered in the interpretation of early post-traumatic biomarkers.

Asunto(s)

Lesiones Traumáticas del Encéfalo , Proteína HMGB1 , Ratas , Masculino , Animales , Levetiracetam/farmacología , Ratas Sprague-Dawley , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Convulsiones/tratamiento farmacológico , Biomarcadores , Proteínas Sanguíneas

RESUMEN

As traumatic brain injury (TBI) is one of the major causes of permanent disability, there is increasing interest in the long-term outcome of TBI. While motor deficits, cognitive impairment and longer-term risks of neurodegenerative disease are well-established consequences in animal models of TBI, pain is discussed less often despite its high prevalence. The current study addresses the need to characterize the extent of chronic pain and long-term behavioral impairments induced by moderate lateral fluid percussion injury (latFPI) in mice up to 12 months post-TBI and evaluates the validity of the model. Adult male BALB/c mice were subjected to latFPI, and the results were compared with outcomes in sham-operated mice. Mouse behavior was assessed at 1 and 7 days and 1, 3, 6, 9, and 12 months post-injury using sensory-motor (neurological severity score, NSS), cold (acetone) and mechanical sensitivity (von Frey), depressive-like behavior (tail suspension), locomotor (open field), motor coordination (rotarod) and cognitive (Morris water maze, y-maze, passive avoidance) tests. Animals with TBI demonstrated significantly higher NSS than the sham-operated group for up to 9 months after the injury. Cold sensitization was significantly increased in the contralateral hind paw in the TBI group compared to that of the sham group at 3, 6, and 9 months after TBI. In the von Frey test, the withdrawal threshold of the contralateral and ipsilateral hind paws was reduced at 6 months after TBI and lasted for up to 12 months post-injury. latFPI induced progressive depressive-like behavior starting at 6 months post-injury. No significant deficits were observed in memory, motor coordination or locomotion over the 12-month assessment period. The present study demonstrates that moderate TBI in mice elicits long-lasting impairment of sensory-motor function, results in progressive depression and potentiates peripheral pain. Hence, the latFPI model provides a relevant preclinical setting for the study of the link between brain injury and chronic sequelae such as depression and peripheral pain.

RESUMEN

Neuroprotective effects of Sigma-1 receptor (S1R) ligands have been observed in multiple animal models of neurodegenerative diseases. Traumatic brain injury (TBI)-related neurodegeneration can induce long-lasting physical, cognitive, and behavioral disabilities. The aim of our study was to evaluate the role of S1R in the development of neurological deficits after TBI. Adult male wild-type CD-1 (WT) and S1R knockout (S1R-/-) mice were subjected to lateral fluid percussion injury, and behavioral and histological outcomes were assessed for up to 12 months postinjury. Neurological deficits and motor coordination impairment were less pronounced in S1R-/- mice with TBI than in WT mice with TBI 24 h after injury. TBI-induced short-term memory impairments were present in WT but not S1R-/- mice 7 months after injury. Compared to WT animals, S1R-/- mice exhibited better motor coordination and less pronounced despair behavior for up to 12 months postinjury. TBI induced astrocyte activation in the cortex of WT but not S1R-/- mice. S1R-/- mice presented a significantly reduced GFAP expression in Bergmann glial cells in the molecular layer of the cerebellum compared to WT mice. Our findings suggest that S1R deficiency reduces TBI-induced motor coordination impairments by reducing GFAP expression in Bergmann glial cells in the cerebellum.

Asunto(s)

Lesiones Traumáticas del Encéfalo/metabolismo , Cerebelo/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Neuroglía/metabolismo , Receptores sigma/metabolismo , Animales , Astrocitos , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Masculino , Ratones , Ratones Noqueados , Enfermedades Neurodegenerativas/metabolismo , Fármacos Neuroprotectores/metabolismo , Receptor Sigma-1

RESUMEN

We assessed the effect of antioxidant therapy using the Food and Drug Administration-approved respiratory drug N-acetylcysteine (NAC) or sulforaphane (SFN) as monotherapies or duotherapy in vitro in neuron-BV2 microglial co-cultures and validated the results in a lateral fluid-percussion model of TBI in rats. As in vitro measures, we assessed neuronal viability by microtubule-associated-protein 2 immunostaining, neuroinflammation by monitoring tumor necrosis factor (TNF) levels, and neurotoxicity by measuring nitrite levels. In vitro, duotherapy with NAC and SFN reduced nitrite levels to 40% (p < 0.001) and neuroinflammation to -29% (p < 0.001) compared with untreated culture. The treatment also improved neuronal viability up to 72% of that in a positive control (p < 0.001). The effect of NAC was negligible, however, compared with SFN. In vivo, antioxidant duotherapy slightly improved performance in the beam walking test. Interestingly, duotherapy treatment decreased the plasma interleukin-6 and TNF levels in sham-operated controls (p < 0.05). After TBI, no treatment effect on HMGB1 or plasma cytokine levels was detected. Also, no treatment effects on the composite neuroscore or cortical lesion area were detected. The robust favorable effect of duotherapy on neuroprotection, neuroinflammation, and oxidative stress in neuron-BV2 microglial co-cultures translated to modest favorable in vivo effects in a severe TBI model.

Asunto(s)

Acetilcisteína/farmacología , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Isotiocianatos/farmacología , Microglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Sulfóxidos/farmacología , Animales , Antioxidantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Masculino , Ratones Endogámicos C57BL , Microglía/citología , Microglía/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Neuronas/citología , Neuronas/metabolismo , Ratas Sprague-Dawley

RESUMEN

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.

Asunto(s)

Lesiones Traumáticas del Encéfalo/patología , Corteza Motora/patología , Corteza Somatosensorial/patología , Tálamo/patología , Animales , Anisotropía , Ritmo beta/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Masculino , Optogenética , Estimulación Luminosa , Ratas Sprague-Dawley

RESUMEN

Binding of urokinase-type plasminogen activator receptor (uPAR) to its ligand uPA or to its plasma membrane partner, platelet-derived growth factor receptor ß (PDGFRß), promotes neuroprotection, cell proliferation, and angiogenesis. Following injury, single deficiency in uPA or uPAR leads in increased tissue loss and compromised vascular remodeling. We hypothesized that double-deficiency of uPAR (Plaur) and uPA (Plau) would result in increased lesion area and poor vascular integrity after traumatic brain injury (TBI). TBI was induced by lateral fluid-percussion injury in Plau/Plaur double-knockout (dKO) and wild-type (Wt) mice. The cortical lesion area was quantified in unfolded cortical maps prepared from thionin-stained sections at 4 d or 30 d post-TBI. The density of PDGFRß+ pericytes and blood vessels was calculated from immunostained sections. Blood-brain barrier leakage was analyzed using ImageJ® from IgG-immunostained sections. Genotype had no effect on the total area of the cortical lesion at 4 d or 30 d post-TBI (p > 0.05) or its progression as the overall lesion area was comparable at 4 d and 30 d post-TBI in both genotypes (p > 0.05). Subfield analysis, however, indicated that damage to the visual cortex at 4 d post-TBI in dKO-TBI mice was 53 % of that in Wt-TBI mice (p < 0.05). Both genotypes had a higher density of PDGFRß-positive pericytes at 4 d than at 30 d post-TBI (p < 0.05), but no genotype effect was detected between these time-points (p > 0.05). TBI-induced increase in the density of PDGFRß+ blood vessels at the region adjacent to the lesion core was comparable in both genotypes (p > 0.05). Genotype had no effect on TBI-induced IgG leakage into the perilesional cortical parenchyma (p > 0.05). Contrary to our expectations, Plau/Plaur double-deficiency did not aggravate TBI-related structural outcome.

Asunto(s)

Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/patología , Encéfalo/irrigación sanguínea , Receptores del Activador de Plasminógeno Tipo Uroquinasa/deficiencia , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones Noqueados , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética

RESUMEN

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.

Asunto(s)

Lesiones Traumáticas del Encéfalo/patología , Corteza Cerebral/patología , Epilepsia Postraumática/patología , Imagen por Resonancia Magnética/métodos , Animales , Modelos Animales de Enfermedad , Masculino , Pronóstico , Ratas , Ratas Sprague-Dawley

RESUMEN

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.

Asunto(s)

Lesiones Traumáticas del Encéfalo/metabolismo , Regulación hacia Abajo/fisiología , Hipotálamo/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Biomarcadores/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Hipotálamo/patología , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is a NINDS funded Center-Without-Walls international study aimed at preventing epileptogenesis after traumatic brain injury (TBI). One objective of EpiBioS4Rx relates to preclinical biomarker discovery for post-traumatic epilepsy. In order to perform a statistically appropriately powered biomarker discovery study, EpiBioS4Rx has made a rigorous attempt to harmonize the preclinical procedures performed at the three EpiBioS4Rx centers, located in Finland, Australia, and the USA. Moreover, we have also performed a rigorous interim analysis of the success of procedural harmonization, which is reported in this virtual special issue. The analysis included harmonization of the production of animal model, blood sampling, electroencephalogram analyses (seizures, high-frequency oscillations) and magnetic resonance imaging analysis. Based on lessons learned, we propose a 3-stage protocol to facilitate the success of preclinical multicenter studies: preparation ⇨ testing ⇨ multicenter study. The need of funding for preparation and testing phases, which precede the actual multicenter study and are necessary for its success, should be taken into account in the design of funding schemes.

Asunto(s)

Biomarcadores/sangre , Investigación Biomédica , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Epilepsia Postraumática/tratamiento farmacológico , Animales , Biología Computacional/métodos , Modelos Animales de Enfermedad , Humanos

RESUMEN

Severe traumatic brain injury (TBI) induces seizures or status epilepticus (SE) in 20-30% of patients during the acute phase. We hypothesized that severe TBI induced with lateral fluid-percussion injury (FPI) triggers post-impact SE. Adult Sprague-Dawley male rats were anesthetized with isoflurane and randomized into the sham-operated experimental control or lateral FPI-induced severe TBI groups. Electrodes were implanted right after impact or sham-operation, then video-electroencephalogram (EEG) monitoring was started. In addition, video-EEG was recorded from naïve rats. During the first 72 h post-TBI, injured rats had seizures that were intermingled with other epileptiform EEG patterns typical to non-convulsive SE, including occipital intermittent rhythmic delta activity, lateralized or generalized periodic discharges, spike-and-wave complexes, poly-spikes, poly-spike-and-wave complexes, generalized continuous spiking, burst suppression, or suppression. Almost all (98%) of the electrographic seizures were recorded during 0-72 h post-TBI (23.2 ± 17.4 seizures/rat). Mean latency from the impact to the first electrographic seizure was 18.4 ± 15.1 h. Mean seizure duration was 86 ± 57 sec. Analysis of high-resolution videos indicated that only 41% of electrographic seizures associated with behavioral abnormalities, which were typically subtle (Racine scale 1-2). Fifty-nine percent of electrographic seizures did not show any behavioral manifestations. In most of the rats, epileptiform EEG patterns began to decay spontaneously on Days 5-6 after TBI. Interestingly, also a few sham-operated and naïve rats had post-operation seizures, which were not associated with EEG background patterns typical to non-convulsive SE seen in TBI rats. To summarize, our data show that lateral FPI-induced TBI results in non-convulsive SE with subtle behavioral manifestations; this explains why it has remained undiagnosed until now. The lateral FPI model provides a novel platform for assessing the mechanisms of acute symptomatic non-convulsive SE and for testing treatments to prevent post-injury SE in a clinically relevant context.

Asunto(s)

Lesiones Traumáticas del Encéfalo/complicaciones , Estado Epiléptico/etiología , Animales , Modelos Animales de Enfermedad , Electroencefalografía , Masculino , Ratas , Ratas Sprague-Dawley , Estado Epiléptico/fisiopatología

RESUMEN

BACKGROUND: Both the type and severity of functional impairments caused by damage to the cerebral cortex depend on the functional cortical areas and networks affected, as well as the lesion extent and type. NEW METHOD: To accelerate the laborious quantitative analysis of cortical lesion location and size, we created user-friendly software that generates two-dimensional unfolded cortical maps of the lesions in adult rats. The program imports and superimposes simple user-made measurements, e.g., from histologic sections, on a template. The software then quantifies the total lesion area and the area of damage in each cortical cytoarchitechtonic region. To validate the accuracy of the software, we compared computer-generated maps with manually created unfolded maps from 32 rats with lateral fluid-percussion-induced traumatic brain injury. RESULTS: The total area of the cortical lesions varied from 7.37 to 38.45 mm2 in the automated analysis and from 7.26 to 38.97 mm2 in the manual analysis (p > 0.05). The Pearson correlation coefficient between the automated and manual analyses was 0.998 (p < 0.001). The mean difference between the automated and manual measurements was 0.50 ± 0.69 mm2 (range 0.00-3.30 mm2). The slight differences between the analyses related to human error in positioning the measurements to the anteroposterior coordinates on the template map. COMPARISON WITH EXISTING METHODS: Compared with the manual method, the automated method accurately and quickly generates unfolded cortical maps of lesioned cortical areas. CONCLUSIONS: Application provides a novel tool for accurately positioning the lesioned area on the cortical mantle and quantifying the lesion area in histologic sections or magnetic resonance images.

Asunto(s)

Lesiones Traumáticas del Encéfalo/diagnóstico por imagen , Lesiones Traumáticas del Encéfalo/patología , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/patología , Procesamiento de Imagen Asistido por Computador/métodos , Animales , Internet , Masculino , Reconocimiento de Normas Patrones Automatizadas/métodos , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Programas Informáticos

RESUMEN

Diagnosis of ongoing epileptogenesis and associated hyperexcitability after brain injury is a major challenge. Given that increased neuronal activity in the brain triggers a blood oxygenation level-dependent (BOLD) response in functional magnetic resonance imaging (fMRI), we hypothesized that fMRI could be used to identify the brain area(s) with hyperexcitability during post-injury epileptogenesis. We applied fMRI to detect onset and spread of BOLD activation after pentylenetetrazol (PTZ)-induced seizures (PTZ, 30 mg/kg, intraperitoneally) in 16 adult male rats at 2 months after lateral fluid percussion (FPI)-induced traumatic brain injury (TBI). In sham-operated controls, onset of the PTZ-induced BOLD response was bilateral and first appeared in the cortex. After TBI, 5 of 9 (56%) rats exhibited ipsilateral perilesional cortical BOLD activation, followed by activation of the contralateral cortex. In 4 of 9 (44%) rats, onset of BOLD response was bilateral. Interestingly, latency from the PTZ injection to onset of the BOLD response increased in the following order: sham-operated controls (ipsilateral 132 ± 57 sec, contralateral 132 ± 57 sec; p > 0.05) < TBI with bilateral BOLD onset (ipsilateral 176 ± 54 sec, contralateral 178 ± 52 sec; p > 0.05) < TBI with ipsilateral BOLD onset (ipsilateral 406 ± 178 sec, contralateral 509 ± 140 sec; p < 0.05). Cortical lesion area did not differ between rats with ipsilateral versus bilateral BOLD onset (p > 0.05). In the group of rats with ipsilateral onset of PTZ-induced BOLD activation, none of the rats showed a robust bilateral thalamic BOLD response, only 1 of 5 rats had robust ipsilateral thalamic calcifications, and 4 of 5 rats had perilesional astrocytosis. These findings suggest the evolution of the epileptogenic zone in the perilesional cortex after TBI, which is sensitive to PTZ-induced hyperexcitability. Further studies are warranted to explore the evolution of thalamo-cortical pathology as a driver of epileptogenesis after lateral FPI.

Asunto(s)

Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/fisiopatología , Convulsiones/etiología , Convulsiones/fisiopatología , Animales , Imagen por Resonancia Magnética/métodos , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

Although rodent models of traumatic brain injury (TBI) reliably produce cognitive and motor disturbances, behavioral characterization resulting from left and right hemisphere injuries remains unexplored. Here we examined the functional consequences of targeting the left versus right parietal cortex in lateral fluid percussion injury, on Morris water maze (MWM) spatial memory tasks (fixed platform and reversal) and neurological motor deficits (neurological severity score and rotarod). In the MWM fixed platform task, right lateral injury produced a small delay in acquisition rate compared to left. However, injury to either hemisphere resulted in probe trial deficits. In the MWM reversal task, left-right performance deficits were not evident, though left lateral injury produced mild acquisition and probe trial deficits compared to sham controls. Additionally, left and right injury produced similar neurological motor task deficits, impaired righting times, and lesion volumes. Injury to either hemisphere also produced robust ipsilateral, and modest contralateral, morphological changes in reactive microglia and astrocytes. In conclusion, left and right lateral TBI impaired MWM performance, with mild fixed platform acquisition rate differences, despite similar motor deficits, histological damage, and glial cell reactivity. Thus, while both left and right lateral TBI produce cognitive deficits, laterality in mouse MWM learning and memory merits consideration in the investigation of TBI-induced cognitive consequences.

Asunto(s)

Conducta Animal , Lesiones Traumáticas del Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Lóbulo Parietal/lesiones , Animales , Lesiones Traumáticas del Encéfalo/psicología , Lateralidad Funcional , Hipocampo/fisiopatología , Masculino , Aprendizaje por Laberinto , Ratones Endogámicos C57BL , Neuroglía/fisiología , Prueba de Desempeño de Rotación con Aceleración Constante

RESUMEN

OBJECTIVE: Posttraumatic epilepsy (PTE) accounts for 20% of acquired epilepsies. Experimental models are important for studying epileptogenesis. We previously reported that repetitive high-frequency oscillations with spikes (rHFOSs) occur early after lateral fluid percussion injury (FPI) and may be a biomarker for PTE. The objective of this study was to use multiple electrodes in rat hippocampal and neocortical regions to describe the long-term electroencephalographic and behavioral evolution of rHFOSs and epileptic seizures after traumatic brain injury (TBI). METHODS: Adult male rats underwent mild, moderate, or severe FPI or sham injury followed by video-electroencephalography (EEG) recordings with a combination of 16 neocortical and hippocampal electrodes at an early, intermediate, or late time-point after injury, up to 52 weeks. Recordings were analyzed for the presence of rHFOSs and seizures. RESULTS: Analysis was done on 28 rats with FPI and 7 shams. Perilesional rHFOSs were recorded in significantly more rats after severe (70.3%) than mild (20%) injury or shams (14.3%). Frequency of occurrence was significantly highest in the early (10.8/h) versus late group (3.2/h). Late focal seizures originating from the same electrodes were recorded in significantly more rats in the late (87.5%) versus early period (22.2%), occurring almost exclusively in injured rats. Seizure duration increased significantly over time, averaging 19 s at the beginning of the early period and 27 s at the end of the late period. Seizure frequency also increased significantly over time, from 4.4 per week in the early group to 26.4 per week in the late group. Rarely, rats displayed early seizures or generalized seizures. SIGNIFICANCE: FPI results in early rHFOSs and later spontaneous focal seizures arising from peri-lesional neocortex, supporting its use as a model for PTE. Epilepsy severity increased over time and was related to injury severity. The association between early rHFOSs and later focal seizures suggests that rHFOSs may be a potential noninvasive biomarker of PTE.

Asunto(s)

Lesiones Traumáticas del Encéfalo/complicaciones , Ondas Encefálicas/fisiología , Progresión de la Enfermedad , Epilepsia Postraumática/etiología , Animales , Lesiones Traumáticas del Encéfalo/etiología , Mapeo Encefálico , Modelos Animales de Enfermedad , Electrodos Implantados , Electroencefalografía , Lateralidad Funcional , Masculino , Percusión/efectos adversos , Ratas , Ratas Sprague-Dawley , Grabación en Video

RESUMEN

More than 2.5 million Americans suffer a traumatic brain injury (TBI) each year. Even mild to moderate TBI causes long-lasting neurological effects. Despite its prevalence, no therapy currently exists to treat the underlying cause of cognitive impairment suffered by TBI patients. Following lateral fluid percussion injury (LFPI), the most widely used experimental model of TBI, we investigated alterations in working memory and excitatory/inhibitory synaptic balance in the prefrontal cortex. LFPI impaired working memory as assessed with a T-maze behavioral task. Field excitatory postsynaptic potentials recorded in the prefrontal cortex were reduced in slices derived from brain-injured mice. Spontaneous and miniature excitatory postsynaptic currents onto layer 2/3 neurons were more frequent in slices derived from LFPI mice, while inhibitory currents onto layer 2/3 neurons were smaller after LFPI. Additionally, an increase in action potential threshold and concomitant decrease in firing rate was observed in layer 2/3 neurons in slices from injured animals. Conversely, no differences in excitatory or inhibitory synaptic transmission onto layer 5 neurons were observed; however, layer 5 neurons demonstrated a decrease in input resistance and action potential duration after LFPI. These results demonstrate synaptic and intrinsic alterations in prefrontal circuitry that may underlie working memory impairment caused by TBI.

RESUMEN

Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, ß2, ß3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, ß2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei.

Asunto(s)

Lesiones Encefálicas/metabolismo , Hipocampo/metabolismo , Receptores de GABA-A/metabolismo , Tálamo/metabolismo , Animales , Autorradiografía , Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/patología , Modelos Animales de Enfermedad , Lateralidad Funcional , Expresión Génica , Hipocampo/patología , Inmunohistoquímica , Hibridación in Situ , Captura por Microdisección con Láser , Masculino , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Tálamo/patología

RESUMEN

Traumatic brain injury (TBI) causes 10-20% of acquired epilepsy in humans, resulting in an ictogenic region that is often located in the cerebral cortex. The thalamus provides heavy projections to the cortex and the activity of thalamocortical pathways is controlled by GABAergic afferents from the reticular nucleus of the thalamus (RT). As rats with TBI induced by lateral fluid-percussion injury (FPI) undergo epileptogenesis, we hypothesized that damage to the parvalbumin (PARV)-immunoreactive (ir) neurons in the RT is associated with seizure susceptibility after lateral FPI. To address this hypothesis, adult Sprague-Dawley rats (n=13) were injured with lateral FPI. At 6months post-TBI, each animal underwent a pentylenetetrazol (PTZ) seizure susceptibility test and 2weeks of continuous video-electroencephalography (EEG) monitoring for detection of the occurrence of spontaneous seizures. Thereafter, the brain was processed for PARV immunohistochemistry. We (a) estimated the total number of PARV-ir neurons in the RT using unbiased stereology, (b) measured the volume of the ventroposteromedial (VPM) and ventroposterolateral (VPL) nuclei of the thalamus, which receive PARV-ir inputs from the RT and project to the perilesional cortex, (c) quantified the density of PARV-ir terminals in the VPM-VPL, and (d) studied the expression of GABAA receptor subunits in a separate group of rats using laser-dissection of the thalamus followed by Real-Time polymerase chain reaction (RT-PCR) array studies. At 6months post-TBI, only 64% of PARV-ir neurons were remaining in the RT ipsilaterally (p<0.001 as compared to controls) and 84% contralaterally (p<0.05). Accordingly, the volume of the ipsilateral RT was 58% of that in controls ipsilaterally (p<0.001) and 90% contralaterally (p>0.05). Also, the volume of the VPM-VPL was only 51% of that in controls ipsilaterally (p<0.001) and 91% contralaterally (p<0.05). The density of PARV-ir axonal labeling was remarkably increased in the lateral aspects of the VPM and VPL (both p<0.001). Expression of the ε- and θ-subunits of the GABAA receptor was down-regulated (0.152, p<0.01 and 0.302, p<0.05, respectively), which could relate to the inclusion of the hypothalamus into the tissue analyzed with RT-PCR arrays. In controls, the lower the number of PARV-ir neurons in the RT, the higher the seizure susceptibility in the PTZ test. Rats with TBI showed seizure susceptibility comparable to that in controls with the lowest number of PARV-ir neurons in the RT. Our data show that the RT and VPM-VPL undergo remarkable degeneration after lateral-FPI which results in reorganization of PARV-ir terminals in the VPM-VPL. The contribution of RT damage to seizure susceptibility and post-traumatic epileptogenesis deserves further studies.

Asunto(s)

Lesiones Encefálicas/patología , Núcleos Talámicos Laterales/metabolismo , Parvalbúminas/metabolismo , Receptores de GABA-A/metabolismo , Núcleos Talámicos Ventrales/metabolismo , Animales , Lesiones Encefálicas/complicaciones , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia/inducido químicamente , Lateralidad Funcional , Antagonistas del GABA/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Captura por Microdisección con Láser , Núcleos Talámicos Laterales/patología , Masculino , Enfermedades Neurodegenerativas/etiología , Neuronas/metabolismo , Parvalbúminas/genética , Pentilenotetrazol/toxicidad , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Núcleos Talámicos Ventrales/patología