White Matter Integrity in a Rat Model of Epileptogenesis: Structural Connectomics and Fixel-Based Analysis.

Introduction: Electrophysiological and neuroimaging studies have demonstrated that large-scale brain networks are affected during the development of epilepsy. These networks can be investigated by using diffusion magnetic resonance imaging (dMRI). The most commonly used model to analyze dMRI is diffusion tensor imaging (DTI). However, DTI metrics are not specific to microstructure or pathology and the DTI model does not take into account crossing fibers, which may lead to erroneous results. To overcome these limitations, a more advanced model based on multi-shell multi-tissue constrained spherical deconvolution was used in this study to perform tractography with more precise fiber orientation estimates and to assess changes in intra-axonal volume by using fixel-based analysis. Methods: dMRI images were acquired before and at several time points after induction of status epilepticus in the intraperitoneal kainic acid (IPKA) rat model of temporal lobe epilepsy. Tractography was performed, and fixel metrics were calculated in several white matter tracts. The tractogram was analyzed by using the graph theory. Results: Global degree, global and local efficiency were decreased in IPKA animals compared with controls during epileptogenesis. Nodal degree was decreased in the limbic system and default-mode network, mainly during early epileptogenesis. Further, fiber density (FD) and fiber-density-and-cross-section (FDC) were decreased in several white matter tracts. Discussion: These results indicate a decrease in overall structural connectivity, integration, and segregation and decreased structural connectivity in the limbic system and default-mode network. Decreased FD and FDC point to a decrease in intra-axonal volume fraction during epileptogenesis, which may be related to neuronal degeneration and gliosis. Impact statement To the best of our knowledge, this is the first longitudinal multi-shell diffusion magnetic resonance imaging study that combines whole-brain tractography and fixel-based analysis to investigate changes in structural brain connectivity and white matter integrity during epileptogenesis in a rat model of temporal lobe epilepsy. Our findings present better insights into how the topology of the structural brain network changes during epileptogenesis and how these changes are related to white matter integrity. This could improve the understanding of the basic mechanisms of epilepsy and aid the rational development of imaging biomarkers and epilepsy therapies.

Chemogenetic Seizure Control with Clozapine and the Novel Ligand JHU37160 Outperforms the Effects of Levetiracetam in the Intrahippocampal Kainic Acid Mouse Model.

Expression of inhibitory designer receptors exclusively activated by designer drugs (DREADDs) on excitatory hippocampal neurons in the hippocampus represents a potential new therapeutic strategy for drug-resistant epilepsy. To overcome the limitations of the commonly used DREADD agonist clozapine, we investigated the efficacy of the novel DREADD ligand JHU37160 in chemogenetic seizure suppression in the intrahippocampal kainic acid (IHKA) mouse model for temporal lobe epilepsy (TLE). In addition, seizure-suppressing effects of chemogenetics were compared to the commonly used anti-epileptic drug (AED), levetiracetam (LEV). Therefore, an adeno-associated viral vector was injected in the sclerotic hippocampus of IHKA mice to induce expression of a tagged inhibitory DREADD hM4Di or only a tag (control) specifically in excitatory neurons using the CamKIIα promoter. Subsequently, animals were treated with LEV (800 mg/kg), clozapine (0.1 mg/kg), and DREADD ligand JHU37160 (0.1 mg/kg) and the effect on spontaneous seizures was investigated. Clozapine and JHU37160-mediated chemogenetic treatment both suppressed seizures in DREADD-expressing IHKA mice. Clozapine treatment suppressed seizures up to 34 h after treatment, and JHU37160 effects lasted for 26 h after injection. Moreover, both compounds reduced the length of seizures that did occur after treatment up to 28 h and 18 h after clozapine and JHU37160, respectively. No seizure-suppressing effects were found in control animals using these ligands. Chemogenetic seizure treatment suppressed seizures during the first 30 min after injection, and seizures remained suppressed during 8 h following treatment. Chemogenetics thus outperformed effects of levetiracetam (p < 0.001), which suppressed seizure frequency with a maximum of 55 ± 9% for up to 1.5 h (p < 0.05). Only chemogenetic and not levetiracetam treatment affected the length of seizures after treatment (p < 0.001). These results show that the chemogenetic therapeutic strategy with either clozapine or JHU37160 effectively suppresses spontaneous seizures in the IHKA mouse model, confirming JHU37160 as an effective DREADD ligand. Moreover, chemogenetic therapy outperforms the effects of levetiracetam, indicating its potential to suppress drug-resistant seizures.

Level of hM4D(Gi) DREADD Expression Determines Inhibitory and Neurotoxic Effects in the Hippocampus.

Selective neuromodulation using designer receptors exclusively activated by designer drugs (DREADDs) has become an increasingly important research tool, as well as an emerging therapeutic approach. However, the safety profile of DREADD expression is unknown. Here, different titers of adeno-associated viral (AAV) vector were administered in an attempt to vary total expression levels of the inhibitory DREADD hM4D(Gi) in excitatory hippocampal neurons. Male Sprague Dawley rats were injected with AAV2/7 encoding DREADD-mCherry, DREADD, or mCherry. Pronounced neuronal loss and neuroinflammatory reactions were observed after transduction with the high titer DREADD AAV, which also resulted in the highest DREADD expression levels. No such effects were observed in the mCherry control group, despite an equally high titer, nor in conditions where lower viral vector titers were injected. In the high titer DREADD conditions, dentate gyrus (DG) evoked potentials were inhibited on clozapine-induced activation of hM4D(Gi), while in low titer conditions DG evoked potentials were enhanced. Recordings of single neuronal activity nevertheless indicated a reduction in spontaneous firing of granule cell layer neurons. Our results indicate that prolonged, high levels of DREADD expression can have neurotoxic effects and that chemogenetic suppression of excitatory hippocampal neurons can paradoxically enhance DG evoked potentials.

Long-term chemogenetic suppression of seizures in a multifocal rat model of temporal lobe epilepsy.

OBJECTIVE: One third of epilepsy patients do not become seizure-free using conventional medication. Therefore, there is a need for alternative treatments. Preclinical research using designer receptors exclusively activated by designer drugs (DREADDs) has demonstrated initial success in suppressing epileptic activity. Here, we evaluated whether long-term chemogenetic seizure suppression could be obtained in the intraperitoneal kainic acid rat model of temporal lobe epilepsy, when DREADDs were selectively expressed in excitatory hippocampal neurons. METHODS: Epileptic male Sprague Dawley rats received unilateral hippocampal injections of adeno-associated viral vector encoding the inhibitory DREADD hM4D(Gi), preceded by a cell-specific promotor targeting excitatory neurons. The effect of clozapine-mediated DREADD activation on dentate gyrus evoked potentials and spontaneous electrographic seizures was evaluated. Animals were systemically treated with single (.1 mg/kg/24 h) or repeated (.1 mg/kg/6 h) injections of clozapine. In addition, long-term continuous release of clozapine and olanzapine (2.8 mg/kg/7 days) using implantable minipumps was evaluated. All treatments were administered during the chronic epileptic phase and between 1.5 and 13.5 months after viral transduction. RESULTS: In the DREADD group, dentate gyrus evoked potentials were inhibited after clozapine treatment. Only in DREADD-expressing animals, clozapine reduced seizure frequency during the first 6 h postinjection. When administered repeatedly, seizures were suppressed during the entire day. Long-term treatment with clozapine and olanzapine both resulted in significant seizure-suppressing effects for multiple days. Histological analysis revealed DREADD expression in both hippocampi and some cortical regions. However, lesions were also detected at the site of vector injection. SIGNIFICANCE: This study shows that inhibition of the hippocampus using chemogenetics results in potent seizure-suppressing effects in the intraperitoneal kainic acid rat model, even 1 year after viral transduction. Despite a need for further optimization, chemogenetic neuromodulation represents a promising treatment prospect for temporal lobe epilepsy.

Dynamic functional connectivity and graph theory metrics in a rat model of temporal lobe epilepsy reveal a preference for brain states with a lower functional connectivity, segregation and integration.

Epilepsy is a neurological disorder characterized by recurrent epileptic seizures. The involvement of abnormal functional brain networks in the development of epilepsy and its comorbidities has been demonstrated by electrophysiological and neuroimaging studies in patients with epilepsy. This longitudinal study investigated changes in dynamic functional connectivity (dFC) and network topology during the development of epilepsy using the intraperitoneal kainic acid (IPKA) rat model of temporal lobe epilepsy (TLE). Resting state functional magnetic resonance images (rsfMRI) of 20 IPKA animals and 7 healthy control animals were acquired before and 1, 3, 6, 10 and 16 weeks after status epilepticus (SE) under medetomidine anaesthesia using a 7 T MRI system. Starting from 17 weeks post-SE, hippocampal EEG was recorded to determine the mean daily seizure frequency of each animal. Dynamic FC was assessed by calculating the correlation matrices between fMRI time series of predefined regions of interest within a sliding window of 50 s using a step length of 2 s. The matrices were classified into 6 FC states, each characterized by a correlation matrix, using k-means clustering. In addition, several time-variable graph theoretical network metrics were calculated from the time-varying correlation matrices and classified into 6 states of functional network topology, each characterized by a combination of network metrics. Our results showed that FC states with a lower mean functional connectivity, lower segregation and integration occurred more often in IPKA animals compared to control animals. Functional connectivity also became less variable during epileptogenesis. In addition, average daily seizure frequency was positively correlated with percentage dwell time (i.e. how often a state occurs) in states with high mean functional connectivity, high segregation and integration, and with the number of transitions between states, while negatively correlated with percentage dwell time in states with a low mean functional connectivity, low segregation and low integration. This indicates that animals that dwell in states of higher functional connectivity, higher segregation and higher integration, and that switch more often between states, have more seizures.

Long-term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy.

OBJECTIVE: More than one-third of patients with temporal lobe epilepsy (TLE) continue to have seizures despite treatment with antiepileptic drugs, and many experience severe drug-related side effects, illustrating the need for novel therapies. Selective expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allows cell-type-specific reduction of neuronal excitability. In this study, we evaluated the effect of chemogenetic suppression of excitatory pyramidal and granule cell neurons of the sclerotic hippocampus in the intrahippocampal mouse model (IHKA) for temporal lobe epilepsy. METHODS: Intrahippocampal IHKA mice were injected with an adeno-associated viral vector carrying the genes for an inhibitory DREADD hM4Di in the sclerotic hippocampus or control vector. Next, animals were treated systemically with different single doses of clozapine-N-oxide (CNO) (1, 3, and 10 mg/kg) and clozapine (0.03 and 0.1 mg/kg) and the effect on spontaneous hippocampal seizures, hippocampal electroencephalography (EEG) power, fast ripples (FRs) and behavior in the open field test was evaluated. Finally, animals received prolonged treatment with clozapine for 3 days and the effect on seizures was monitored. RESULTS: Treatment with both CNO and clozapine resulted in a robust suppression of hippocampal seizures for at least 15 hours only in DREADD-expressing animals. Moreover, total EEG power and the number of FRs were significantly reduced. CNO and/or clozapine had no effects on interictal hippocampal EEG, seizures, or locomotion/anxiety in the open field test in non-DREADD epileptic IHKA mice. Repeated clozapine treatment every 8 hours for 3 days resulted in almost complete seizure suppression in DREADD animals. SIGNIFICANCE: This study shows the potency of chemogenetics to robustly and sustainably suppress spontaneous epileptic seizures and pave the way for an epilepsy therapy in which a systemically administered exogenous drug selectively modulates specific cell types in a seizure network, leading to a potent seizure suppression devoid of the typical drug-related side effects.

Alterations in the functional brain network in a rat model of epileptogenesis: A longitudinal resting state fMRI study.

Epilepsy is a neurological disorder characterized by recurrent epileptic seizures. Electrophysiological and neuroimaging studies in patients with epilepsy suggest that abnormal functional brain networks play a role in the development of epilepsy, i.e. epileptogenesis, resulting in the generation of spontaneous seizures and cognitive impairment. In this longitudinal study, we investigated changes in functional brain networks during epileptogenesis in the intraperitoneal kainic acid (IPKA) rat model of temporal lobe epilepsy (TLE) using resting state functional magnetic resonance imaging (rsfMRI) and graph theory. Additionally, we investigated whether these changes are related to the frequency of occurrence of spontaneous epileptic seizures in the chronic phase of epilepsy. Using a 7T MRI system, rsfMRI images were acquired under medetomidine anaesthesia before and 1, 3, 6, 10 and 16 weeks after status epilepticus (SE) induction in 20 IPKA animals and 7 healthy control animals. To obtain a functional network, correlation between fMRI time series of 38 regions of interest (ROIs) was calculated. Then, several graph theoretical network measures were calculated to describe and quantify the network changes. At least 17 weeks post-SE, IPKA animals were implanted with electrodes in the left and right dorsal hippocampus, EEG was measured for 7 consecutive days and spontaneous seizures were counted. Our results show that correlation coefficients of fMRI time series shift to lower values during epileptogenesis, indicating weaker whole brain network connections. Segregation and integration in the functional brain network also decrease, indicating a lower local interconnectivity and a lower overall communication efficiency. Secondly, this study demonstrates that the largest decrease in functional connectivity is observed for the retrosplenial cortex. Finally, post-SE changes in functional connectivity, segregation and integration are correlated with seizure frequency in the IPKA rat model.