Alejandra Sierra

Finding a better drug for epilepsy: Antiepileptogenesis targets

For several decades, both in vitro and in vivo models of seizures and epilepsy have been employed to unravel the molecular and cellular mechanisms underlying the occurrence of spontaneous recurrent seizures (SRS)—the defining hallmark of the epileptic brain. However, despite great advances in our understanding of seizure genesis, investigators have yet to develop reliable biomarkers and surrogate markers of the epileptogenic process. Sadly, the pathogenic mechanisms that produce the epileptic condition, especially after precipitating events such as head trauma, inflammation, or prolonged febrile convulsions, are poorly understood. A major challenge has been the inherent complexity and heterogeneity of known epileptic syndromes and the differential genetic susceptibilities exhibited by patients at risk. Therefore, it is unlikely that there is only one fundamental pathophysiologic mechanism shared by all the epilepsies. Identification of antiepileptogenesis targets has been an overarching goal over the last decade, as current anticonvulsant medications appear to influence only the acute process of ictogenesis. Clearly, there is an urgent need to develop novel therapeutic interventions that are disease modifying—therapies that either completely or partially prevent the emergence of SRS. An important secondary goal is to develop new treatments that can also lessen the burden of epilepsy comorbidities (e.g., cognitive impairment, mood disorders) by preventing or reducing the deleterious changes during the epileptogenic process. This review summarizes novel antiepileptogenesis targets that were critically discussed at the XIth Workshop on the Neurobiology of Epilepsy (WONOEP XI) meeting in Grottaferrata, Italy. Further, emerging neurometabolic links among several target mechanisms and highlights of the panel discussion are presented.

Manganese enhanced MRI detects mossy fiber sprouting rather than neurodegeneration, gliosis or seizure-activity in the epileptic rat hippocampus

We tested a hypothesis that manganese enhanced magnetic resonance imaging (MEMRI) after systemic injection of MnCl(2) could detect axonal sprouting in the hippocampus following kainate (KA) induced status epilepticus (SE). MEMRI was performed at 3 h, 25 h, 4 days, and 2 months post-SE. To assess the contribution of various cellular alterations that occur in parallel with sprouting to the MEMRI signal, we sacrificed animals for histology at 4 days and 2 months post-SE. Neurodegeneration was assessed from thionin and Fluoro-Jade B stained preparations, astrogliosis from GFAP (glial fibrillary acidic protein) and microgliosis from Ox-42 immunostained preparations. Sprouting of granule cells axons (mossy fibers) in the dentate gyrus was analyzed from Timm stained sections. Occurrence of spontaneous epileptic seizures was analyzed at 2 months post-SE using continuous video-EEG monitoring. Integrity of the blood-brain barrier (BBB) was studied using Gd-enhanced MRI. We found abnormal MEMRI hyperintensity in the CA1 and the dentate gyrus at 2 months post-SE but not at earlier time points. Based on histologic analysis of individual animals with MEMRI hyperintensity, hippocampal MEMRI changes could be attributed to increasing axonal density rather than to neurodegeneration, astrogliosis, or microgliosis. Moreover, MEMRI contrast was not affected by seizure activity, and we could not detect any leakage of the BBB that could have explained the observed MEMRI hyperintensity. Present data show that systemic MEMRI can reveal axonal sprouting, and thus, can potentially serve as a marker for neuroplasticity in preclinical studies.

Diffusion tensor MRI of axonal plasticity in the rat hippocampus

The aim of this study was to explore non-invasive imaging methods to detect post-injury structural axonal plasticity. Brain injury was launched by status epilepticus induced by intraperitoneal injection of either kainic acid or pilocarpine. Several months later, ex vivo diffusion tensor magnetic resonance imaging (DTI) showed increased FA in the dentate gyrus of both kainic acid (p<0.01) and pilocarpine animals (p<0.01). Importantly, FA changes correlated (p<0.01) with histologically verified axonal plasticity of myelinated and non-myelinated neuronal fibers. The changes observed in DTI parameters ex vivo in the septal dentate gyrus were also seen by in vivo DTI. As DTI is completely a non-invasive imaging method, these results may pave the way for non-invasive in vivo imaging of axonal plasticity after brain insults.

Diffusion tensor MRI with tract-based spatial statistics and histology reveals undiscovered lesioned areas in kainate model of epilepsy in rat

In this study, we used tract-based spatial statistics (TBSS) to analyze diffusion tensor MR imaging (DTI) data acquired from the rat brain, ex vivo, for the first time. The aim was to highlight potential changes in the whole brain anatomy in the kainic acid model of epilepsy, and further characterize the changes with histology. Increased FA was observed in dorsal endopiriform nucleus, external capsule, corpus callosum, dentate gyrus, thalamus, and optic tract. A decrease in FA was seen in the horizontal limb of the diagonal band, stria medullaris, habenula, entorhinal cortex, and superior colliculus. Some of the areas have been described in kainic acid model before. However, we also found regions that to our knowledge have not been previously reported to undergo structural changes, in this model, including stria medullaris, nucleus of diagonal band, habenula, superior colliculus, external capsule, corpus callosum, and optic tract. Four of the areas highlighted in TBSS (dentate gyrus, entorhinal cortex, thalamus and stria medullaris) were analyzed in more detail with Nissl, Timm, and myelin-stained histological sections, and with polarized light microscopy. TBSS together with targeted histology confirmed that DTI changes were associated with altered myelination, neurodegeneration, and/or calcification of the tissue. Our data demonstrate that DTI in combination with TBSS has a great potential to facilitate the discovery of previously undetected anatomical changes in animal models of brain diseases.

Quantitative T2 mapping as a potential marker for the initial assessment of the severity of damage after traumatic brain injury in rat

Severity of traumatic brain injury (TBI) positively correlates with the risk of post-traumatic epilepsy (PTE). Studies on post-traumatic epileptogenesis would greatly benefit from markers that at acute phase would reliably predict the extent and severity of histologic brain damage caused by TBI in individual subjects. Currently in experimental models, severity of TBI is determined by the pressure of applied load that does not directly reflect the extent of inflicted brain injury, mortality within experimental population, or impairment in behavioral tests that are laborious to perform. We aimed to compare MRI markers measured at acute post-injury phase to previously used indicators of injury severity in the ability to predict the extent of histologically determined post-traumatic tissue damage. We used lateral fluid-percussion injury model in rat that is a clinically relevant model of closed head injury in humans, and results in PTE in severe cases. Rats (48 injured, 12 controls) were divided into moderate (mTBI) and severe (sTBI) groups according to impact strength. MRI data (T2, T2*, lesion volume) were acquired 3 days post-injury. Motor deficits were analysed using neuroscore (NS) and beam balance (BB) tests 2 and 3 days post-injury, respectively. Histological evaluation of lesion volume (Fluoro-Jade B) was used as the reference outcome measure, and was performed 2 weeks after TBI. From MRI parameters studied, quantitative T2 values of cortical lesion not only correlated with histologic lesion volume (P<0.001, r=0.6, N=34), as well as NS (P<0.01, r=-0.5, N=34) and BB (P<0.01, r=-0.5, N=34) results, but also successfully differentiated animals with mTBI from those with sTBI 70.6 +/- 6.2 6.2 ms vs. 75.9 +/- 2.6 ms, P<0.001). Quantitative T2 of the lesion early after TBI can serve as an indicator of the severity of post-traumatic cortical damage and neuro-motor impairment, and has a potential as a clinical marker for identification of individuals with elevated risk of PTE.

Water spin dynamics during apoptotic cell death in glioma gene therapy probed by T1ρ and T2ρ

Longitudinal and transverse relaxations in the rotating frame, with characteristic time constants T1ρ and T2ρ, respectively, have potential to provide unique MRI contrast in vivo. On‐resonance spin‐lock T1ρ with different spin‐lock field strengths and adiabatic T2ρ with different radiofrequency‐modulation functions were measured in BT4C gliomas treated with Herpes Simplex Virus thymidine kinase (HVS‐tk) gene therapy causing apoptotic cell death. These NMR tools were able to discriminate different treatment responses in tumor tissue from day 4 onward. An equilibrium two‐site exchange model was used to calculate intrinsic parameters describing changes in water dynamics. Observed changes included increased correlation time of water associated with macromolecules and a decreased fractional population of this pool. These results are consistent with destructive intracellular processes associated with cell death and the increase of extracellular space during the treatment. Furthermore, association between longer exchange correlation time and decreased pH during apoptosis is discussed. In this study, we demonstrated that T1ρ and T2ρ MR imaging are useful tools to quantify early changes in water dynamics reflecting treatment response during gene therapy. Magn Reson Med 59:1311–1319, 2008.

Early Maternal Alcohol Consumption Alters Hippocampal DNA Methylation, Gene Expression and Volume in a Mouse Model

The adverse effects of alcohol consumption during pregnancy are known, but the molecular events that lead to the phenotypic characteristics are unclear. To unravel the molecular mechanisms, we have used a mouse model of gestational ethanol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first 8 days of gestation (GD 0.5-8.5). Early neurulation takes place by the end of this period, which is equivalent to the developmental stage early in the fourth week post-fertilization in human. During this exposure period, dynamic epigenetic reprogramming takes place and the embryo is vulnerable to the effects of environmental factors. Thus, we hypothesize that early ethanol exposure disrupts the epigenetic reprogramming of the embryo, which leads to alterations in gene regulation and life-long changes in brain structure and function. Genome-wide analysis of gene expression in the mouse hippocampus revealed altered expression of 23 genes and three miRNAs in ethanol-exposed, adolescent offspring at postnatal day (P) 28. We confirmed this result by using two other tissues, where three candidate genes are known to express actively. Interestingly, we found a similar trend of upregulated gene expression in bone marrow and main olfactory epithelium. In addition, we observed altered DNA methylation in the CpG islands upstream of the candidate genes in the hippocampus. Our MRI study revealed asymmetry of brain structures in ethanol-exposed adult offspring (P60): we detected ethanol-induced enlargement of the left hippocampus and decreased volume of the left olfactory bulb. Our study indicates that ethanol exposure in early gestation can cause changes in DNA methylation, gene expression, and brain structure of offspring. Furthermore, the results support our hypothesis of early epigenetic origin of alcohol-induced disorders: changes in gene regulation may have already taken place in embryonic stem cells and therefore can be seen in different tissue types later in life.

Detection of calcifications in vivo and ex vivo after brain injury in rat using SWIFT

Calcifications represent one component of pathology in many brain diseases. With MRI, they are most often detected by exploiting negative contrast in magnitude images. Calcifications are more diamagnetic than tissue, leading to a magnetic field disturbance that can be seen in phase MR images. Most phase imaging studies use gradient recalled echo based pulse sequences. Here, the phase component of SWIFT, a virtually zero acquisition delay sequence, was used to detect calcifications ex vivo and in vivo in rat models of status epilepticus and traumatic brain injury. Calcifications were detected in phase and imaginary SWIFT images based on their dipole like magnetic field disturbances. In magnitude SWIFT images, calcifications were distinguished as hypointense and hyperintense. Hypointense calcifications showed large crystallized granules with few surrounding inflammatory cells, while hyperintense calcifications contained small granules with the presence of more inflammatory cells. The size of the calcifications in SWIFT magnitude images correlated with that in Alizarin stained histological sections. Our data indicate that SWIFT is likely to better preserve signal in the proximity of a calcification or other field perturber in comparison to gradient echo due to its short acquisition delay and broad excitation bandwidth. Furthermore, a quantitative description for the phase contrast near dipole magnetic field inhomogeneities for the SWIFT pulse sequence is given. In vivo detection of calcifications provides a tool to probe the progression of pathology in neurodegenerative diseases. In particular, it appears to provide a surrogate marker for inflammatory cells around the calcifications after brain injury.

Diffusion tensor imaging detects chronic microstructural changes in white and gray matter after traumatic brain injury in rat

Traumatic brain injury (TBI) is a major cause of disability and death in people of all ages worldwide. An initial brain injury caused by external mechanical forces triggers a cascade of tissue changes that lead to a wide spectrum of symptoms and disabilities, such as cognitive deficits, mood or anxiety disorders, motor impairments, chronic pain, and epilepsy. We investigated the detectability of secondary injury at a chronic time-point using ex vivo diffusion tensor imaging (DTI) in a rat model of TBI, lateral fluid percussion (LFP) injury. Our analysis of ex vivo DTI data revealed persistent microstructural tissue changes in white matter tracts, such as the splenium of the corpus callosum, angular bundle, and internal capsule. Histologic examination revealed mainly loss of myelinated axons and/or iron accumulation. Gray matter areas in the thalamus exhibited an increase in fractional anisotropy associated with neurodegeneration, myelinated fiber loss, and/or calcifications at the chronic phase. In addition, we examined whether these changes could also be detected with in vivo settings at the same chronic time-point. Our results provide insight into DTI detection of microstructural changes in the chronic phase of TBI, and elucidate how these changes correlate with cellular level alterations.

Posttraumatic epilepsy — Disease or comorbidity?

Traumatic brain injury (TBI) can cause a myriad of sequelae depending on its type, severity, and location of injured structures. These can include mood disorders, posttraumatic stress disorder and other anxiety disorders, personality disorders, aggressive disorders, cognitive changes, chronic pain, sleep problems, motor or sensory impairments, endocrine dysfunction, gastrointestinal disturbances, increased risk of infections, pulmonary disturbances, parkinsonism, posttraumatic epilepsy, or their combinations. The progression of individual pathologies leading to a given phenotype is variable, and some progress for months. Consequently, the different post-TBI phenotypes appear within different time windows. In parallel with morbidogenesis, spontaneous recovery occurs both in experimental models and in human TBI. A great challenge remains; how can we dissect the specific mechanisms that lead to the different endophenotypes, such as posttraumatic epileptogenesis, in order to identify treatment approaches that would not compromise recovery?