Beatriz M. Longo

Blockade of pilocarpine- or kainate-induced mossy fiber sprouting by cycloheximide does not prevent subsequent epileptogenesis in rats

Post-injury sprouting of hippocampal mossy fibers has been suggested to be a causal mechanism underlying the development of temporal lobe epilepsy. However, this hypothesis rests entirely on indirect correlational evidence. Here we demonstrate that cycloheximide, a protein synthesis inhibitor, blocked pilocarpine- and kainate-induced mossy fiber sprouting in rats, but did not prevent the subsequent development of spontaneous seizures or affect their frequency. These results provide direct evidence against a causal role for mossy fiber sprouting in temporal lobe epileptogenesis.

Cell damage and neurogenesis in the dentate granule cell layer of adult rats after pilocarpine- or kainate-induced status epilepticus.

Dentate granule cells are generally considered to be relatively resistant to excitotoxicity and have been associated with robust synaptogenesis after neuronal damage. Synaptic reorganization of dentate granule cell axons, the mossy fibers, has been suggested to be relevant for hyperexcitability in human temporal lobe epilepsy and animal models. A recent hypothesis suggested that mossy‐fiber sprouting is dependent on newly formed dentate granule cells. However, we recently demonstrated that cycloheximide (CHX) can block the mossy‐fiber sprouting that would otherwise be induced by different epileptogenic agents and does not interfere with epileptogenesis in those models. Here, we investigated cell damage and neurogenesis in the dentate gyrus of pilocarpine‐ or kainate‐treated animals with or without coadministration of CHX. Dentate granule cells were highly vulnerable to pilocarpine induced‐status epilepticus (SE), but were hardly damaged by kainate‐induced SE. CHX pretreatment markedly reduced the number of injured neurons after pilocarpine‐induced SE. Induction of SE dramatically increased the mitotic rate of KA‐ and KA + CHX‐treated animals. Induction of SE in animals injected with pilocarpine alone led to 2–7‐fold increases in the mitotic rate of dentate granule cells as compared to 5‐ and 30‐fold increases for pilocarpine + CHX animals. We suggest that such increased mitotic rates might be associated with a protection of a vulnerable precursor cell population that would otherwise degenerate after pilocarpine‐induced SE. We further suggest that mossy‐fiber sprouting and neurogenesis of granule cells are not necessarily linked to one another. Hippocampus 10:169–180, 2000

Behavioral changes resulting from the administration of cycloheximide in the pilocarpine model of epilepsy

Cycloheximide influences synaptic reorganization resulting from pilocarpine-induced status epilepticus (SE). To investigate the possible behavioral consequences of this effect, we subjected animals to pilocarpine-induced SE either in the absence (Pilo group) or presence of cycloheximide (Chx group). Animals were further divided regarding the occurrence of spontaneous recurrent seizures (SRS). Two months after SE induction animals were exposed to different behavioral tests. Age-matched naïve animals were used as controls. All epileptic groups showed a significantly diminished freezing time in contextual and tone fear conditioning, performed poorly in the Morris water maze and present less seconds in immobility position as compared to controls. Only Pilo animals explored more extensively the open arms of the elevated plus maze and showed increased in horizontal exploratory activity in the open field as compared to controls. With the exception of Pilo animals without recorded SRS, all other groups had extensive tissue shrinkage in central nucleus of the amygdala as compared to controls. Cycloheximide-treated animals differed from Pilo animals in the extent of hilar loss and supragranular mossy fiber sprouting as well as tissue shrinkage in the dorsal hippocampus. Despite the histological differences seen in the dorsal hippocampus between experimental groups, no differences were encountered in the cognitive tests used to evaluate dorsal hippocampal function. The encountered histological differences between Chx and Pilo animals, however, might underlie the different emotional responses between the two groups.

Effect of long-term spontaneous recurrent seizures or reinduction of status epilepticus on the development of supragranular mossy fiber sprouting

In a recent report we have shown that a protein synthesis inhibitor, cycloheximide (CHX), is able to block the mossy fiber sprouting (MFS) that would otherwise be triggered by pilocarpine (Pilo)-induced status epilepticus (SE), and also gives relative protection against hippocampal neuronal death. Under this condition animals still showed spontaneous recurrent seizures (SRS) which led us to question the role played by sprouted mossy fibers in generating those seizures. In both patients and animal models of epilepsy the relative contribution of SE (when present) and/or SRS for the development of MFS is not known. In the present study we investigated the relationship between MFS, SE and SRS, and evaluated whether the CHX-induced blockade of MFS was transient or permanent in nature. We performed a chronic study which included animals subject to Pilo-induced SE in the presence of CHX and sacrificed between 8 and 10 months later, and animals that were subject to Pilo-induced SE in the presence of CHX and underwent a reinduction of SE with Pilo, 45 days after the first induction, but this time in the absence of CHX. Re-induction of SE or a long period of chronic seizures, were able to trigger supragranular MFS even in animals where the first (or only) SE event was triggered in the presence of CHX. MFS did not show any association with the frequency of SRS, and thus seemed to depend more critically on time. Our current findings allow us to suggest that MFS are neither the cause nor the consequence of SRS in the pilocarpine model.

Therapeutic effects of the transplantation of VEGF overexpressing bone marrow mesenchymal stem cells in the hippocampus of murine model of Alzheimer's disease

Alzheimer’s disease (AD) is clinically characterized by progressive memory loss, behavioral and learning dysfunction and cognitive deficits, such as alterations in social interactions. The major pathological features of AD are the formation of senile plaques and neurofibrillary tangles together with neuronal and vascular damage. The double transgenic mouse model of AD (2xTg-AD) with the APPswe/PS1dE9 mutations shows characteristics that are similar to those observed in AD patients, including social memory impairment, senile plaque formation and vascular deficits. Mesenchymal stem cells (MSCs), when transplanted into the brain, produce positive effects by reducing amyloid-beta (Aβ) deposition in transgenic amyloid precursor protein (APP)/presenilins1 (PS1) mice. Vascular endothelial growth factor (VEGF), exhibits neuroprotective effects against the excitotoxicity implicated in the AD neurodegeneration. The present study investigates the effects of MSCs overexpressing VEGF in hippocampal neovascularization, cognitive dysfunction and senile plaques present in 2xTg-AD transgenic mice. MSC were transfected with vascular endothelial growth factor cloned in uP vector under control of modified CMV promoter (uP-VEGF) vector, by electroporation and expanded at the 14th passage. 2xTg-AD animals at 6, 9 and 12 months old were transplanted with MSC-VEGF or MSC. The animals were tested for behavioral tasks to access locomotion, novelty exploration, learning and memory, and their brains were analyzed by immunohistochemistry (IHC) for vascularization and Aβ plaques. MSC-VEGF treatment favored the neovascularization and diminished senile plaques in hippocampal specific layers. Consequently, the treatment was able to provide behavioral benefits and reduce cognitive deficits by recovering the innate interest to novelty and counteracting memory deficits present in these AD transgenic animals. Therefore, this study has important therapeutic implications for the vascular damage in the neurodegeneration promoted by AD.

Effect of neuronal precursor cells derived from medial ganglionic eminence in an acute epileptic seizure model

Most of the γ‐aminobutyric acid (GABA)ergic interneurons in the cerebral cortex originate from restricted regions of the ventral telencephalon known as the caudal and medial ganglionic eminence (MGE) and from the preoptic area. It is well established that dysfunction of GABAergic interneurons can lead to epilepsy. During the last decade new approaches to prevent, reduce, or reverse the epileptic condition have been studied, including cell‐based therapy from different sources. Recent studies have shown that transplanted neuronal precursor cells derived from MGE have the ability to migrate, differentiate into inhibitory GABAergic interneurons, and integrate into cortical and hippocampal networks, modifying the inhibitory tone in the host brain. Therefore, transplantation of neuronal precursors derived from MGE into the postnatal central nervous system (CNS) could modify the neuronal circuitry in neurologic diseases in which inhibitory synaptic function is altered, such as in epilepsy. Here, we evaluated the seizure susceptibility of mice transplanted with MGE‐derived cells in the maximum electroconvulsive shock (MES) model and we review some data from different studies using GABAergic precursor or GABA‐releasing cell grafts in animal models of seizure and epilepsy.

Effects of rimonabant on the development of single dose-induced behavioral sensitization to ethanol, morphine and cocaine in mice.

RATIONALE The endocannabinoid system has been implicated in the neurobiological mechanism underlying drug addiction, especially the primary rewarding dopamine-dependent processes. Therefore, endocannabinoid receptor antagonists, such as the CB1 cannabinoid antagonist rimonabant, have been proposed as candidates for preventive addiction therapies. OBJECTIVES Investigate the possible involvement of CB1 receptors in the development of behavioral sensitization to ethanol, morphine and cocaine in mice. METHODS We compared the effects of different doses of rimonabant (0.3, 1, 3 and 10mg/kg) on spontaneous locomotor activity in the open-field, hyperlocomotion induced by acute administration of ethanol (1.8g/kg), morphine (20mg/kg) or cocaine (10mg/kg) and on subsequent drug-induced locomotor sensitization using a two-injection protocol in mice. We also investigated a possible depressive-like effect of an acute rimonabant challenge at the highest dose and its potential anxiogenic property. RESULTS At the highest dose, rimonabant abolished ethanol- and cocaine-induced hyperlocomotion and behavioral sensitization without modifying spontaneous and central locomotor activity or inducing depressive-like behavior on the forced swim test in mice. The other doses of rimonabant also selectively blocked acute ethanol-induced central hyperlocomotion. Although rimonabant at 0.3 and 1mg/kg potentiated the central hyperlocomotion induced by acute morphine injection, it was effective in attenuating morphine-induced behavioral sensitization at all doses. CONCLUSIONS Because the neural basis of behavioral sensitization has been proposed to correspond to some components of addiction, our findings indicate that the endocannabinoid system might be involved in ethanol, cocaine and morphine abuse.

The Anti-Tumor Effects of Adipose Tissue Mesenchymal Stem Cell Transduced with HSV-Tk Gene on U-87-Driven Brain Tumor

Glioblastoma (GBM) is an infiltrative tumor that is difficult to eradicate. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. To use this strategy, abundantly present adipose-tissue-derived mesenchymal stem cells (AT-MSCs) were evaluated for the treatment of GBM in mice. AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal protein and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 μM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection, combined with GCV treatment, drastically reduced tumors to smaller than 0.5 mm2. Immunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, showing chemoattraction between them. The abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.

Changes in Hippocampal Volume are Correlated with Cell Loss but Not with Seizure Frequency in Two Chronic Models of Temporal Lobe Epilepsy

Kainic acid (KA) or pilocarpine (PILO) have been used in rats to model human temporal lobe epilepsy (TLE) but the distribution and severity of structural lesions between these two models may differ. Magnetic resonance imaging (MRI) studies have used quantitative measurements of hippocampal T2 (T2HP) relaxation time and volume, but simultaneous comparative results have not been reported yet. The aim of this study was to compare the MRI T2HP and volume with histological data and frequency of seizures in both models. KA- and PILO-treated rats were imaged with a 2 T MRI scanner. T2HP and volume values were correlated with the number of cells, mossy fiber sprouting, and spontaneous recurrent seizures (SRS) frequency over the 9 months following status epilepticus (SE). Compared to controls, KA-treated rats had unaltered T2HP, pronounced reduction in hippocampal volume and concomitant cell reduction in granule cell layer, CA1 and CA3 at 3 months post SE. In contrast, hippocampal volume was unchanged in PILO-treated animals despite detectable increased T2HP and cell loss in granule cell layer, CA1 and CA3. In the following 6 months, MRI hippocampal volume remained stable with increase of T2HP signal in the KA-treated group. The number of CA1 and CA3 cells was smaller than age-matched CTL group. In contrast, PILO group had MRI volumetric reduction accompanied by reduction in the number of CA1 and CA3 cells. In this group, T2HP signal was unaltered at 6 or 9 months after status. Reductions in the number of cells were not progressive in both models. Notably, the SRS frequency was higher in PILO than in the KA model. The volumetry data correlated well with tissue damage in the epileptic brain, suggesting that MRI may be useful for tracking longitudinal hippocampal changes, allowing the assessment of individual variability and disease progression. Our results indicate that the temporal changes in hippocampal morphology are distinct for both models of TLE and that these are not significantly correlated to the frequency of SRS.

Manganese-enhanced magnetic resonance imaging detects mossy fiber sprouting in the pilocarpine model of epilepsy

Purpose:  Mossy fiber sprouting (MFS) is a frequent finding following status epilepticus (SE). The present study aimed to test the feasibility of using manganese‐enhanced magnetic resonance imaging (MEMRI) to detect MFS in the chronic phase of the well‐established pilocarpine (Pilo) rat model of temporal lobe epilepsy (TLE).