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Pericardial Injection of Kainic Acid Induces a Chronic Epileptic State in Larval Zebrafish

ABSTRACT: Epilepsy is a common disorder of the brain characterized by spontaneous recurrent seizures, which develop gradually during a process called epileptogenesis. The mechanistic processes underlying the changes of brain tissue and networks toward increased seizure susceptibility are not fully understood. In rodents, injection of kainic acid (KA) ultimately leads to the development of spontaneous epileptic seizures, reflecting similar neuropathological characteristics as seen in patients with temporal lobe epilepsy (TLE). Although this model has significantly contributed to increased knowledge of epileptogenesis, it is technically demanding, costly to operate and hence not suitable for high-throughput screening of anti-epileptic drugs (AEDs). Zebrafish, a vertebrate with complementary advantages to rodents, is an established animal model for epilepsy research. Here, we generated a novel KA-induced epilepsy model in zebrafish larvae that we functionally and pharmacologically validated. KA was administered by pericardial injection at an early zebrafish larval stage. The epileptic phenotype induced was examined by quantification of seizure-like behavior using automated video recording, and of epileptiform brain activity measured via local field potential (LFP) recordings. We also assessed GFP-labeled GABAergic and RFP-labeled glutamatergic neurons in double transgenic KA-injected zebrafish larvae, and examined the GABA and glutamate levels in the larval heads by liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). Finally, KA-injected larvae were exposed to five commonly used AEDs by immersion for pharmacological characterization of the model. Shortly after injection, KA induced a massive damage and inflammation in the zebrafish brain and seizure-like locomotor behavior. An abnormal reorganization of brain circuits was observed, a decrease in both GABAergic and glutamatergic neuronal population and their associated neurotransmitters. Importantly, these changes were accompanied by spontaneous and continuous epileptiform brain discharges starting after a short latency period, as seen in KA rodent models and reminiscent of human pathology. Three out of five AEDs tested rescued LFP abnormalities but did not affect the seizure-like behavior. Taken together, for the first time we describe a chemically-induced larval zebrafish epilepsy model offering unique insights into studying epileptogenic processes in vivo and suitable for high-throughput AED screening purposes and rapid genetic investigations.

SUBMITTER: Heylen L

PROVIDER: S-EPMC8551382 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveIn order to better investigate the cause/effect relationships of human mesial temporal lobe epilepsy (mTLE), we hereby describe a new non-human primate model of mTLE.MethodsTen macaques were studied and divided into 2 groups: saline control group (n?=?4) and kainic acid (KA) injection group (n?=?6). All macaques were implanted bilaterally with subdural electrodes over temporal cortex and depth electrodes in CA3 hippocampal region. KA was stereotaxically injected into the right hippocampus of macaques. All animals were monitored by video and electrocorticography (ECoG) to assess status epilepticus (SE) and subsequent spontaneous recurrent seizures (SRS). Additionally, in order to evaluate brain injury produced by SE or SRS, we used both neuroimaging, including magnetic resonance image (MRI) & magnetic resonance spectroscopy (MRS), and histological pathology, including Nissl stainning and glial fibrillary acid protein (GFAP) immunostaining.ResultsThe typical seizures were observed in the KA-injected animal model. Hippocampal sclerosis could be found by MRI & MRS. Hematoxylin and eosin (H&E) staining and GFAP immunostaining showed neuronal loss, proliferation of glial cells, formation of glial scars, and hippocampal atrophy. Electron microscopic analysis of hippocampal tissues revealed neuronal pyknosis, partial ribosome depolymerization, an abnormal reduction in rough endoplasmic reticulum size, expansion of Golgi vesicles and swollen star-shaped cells. Furthermore, we reported that KA was able to induce SE followed by SRS after a variable period of time. Similar to human mTLE, brain damage is confined to the hippocampus. Accordingly, hippocampal volume is in positive correlations with the neuronal cells count in the CA3, especially the ratio of neuron/glial cell.ConclusionsThe results suggest that a model of mTLE can be developed in macaques by intra-hippocampal injection of KA. Brain damage is confined to the hippocampus which is similar to the human mTLE. The hippocampal volume correlates with the extension of the hippocampal damage.

Project description:Objective: Our study aimed to explore whether gasdermin D (GSDMD)-mediated pyroptosis is involved in the mechanism of kainic acid-induced seizures. Methods: C57BL/6 mice were randomly divided into sham and epilepsy groups. The epilepsy group was intrahippocampally injected with kainic acid to induce status epilepticus (SE), and the sham group was injected with an equal volume of saline. Dimethyl fumarate (DMF) was used as the GSDMD N-terminal fragments (GSDMD-N) inhibitor and suspended in 0.5% sodium carboxymethyl cellulose (CMC) for orally administration. The epilepsy group was divided into SE + CMC and SE + DMF groups. In the SE + DMF group, DMF was orally administered for 1 week before SE induction and was continued until the end of the experiment. An equal volume of CMC was administered to the sham and SE + CMC groups. Recurrent spontaneous seizures (SRSs) were monitored for 21 days after SE. Western blot analysis and immunofluorescent staining was performed. Results: The expression of GSDMD increased at 7-21 days post-SE, and GSDMD-N expression was significantly elevated 7 days after SE in both ipsilateral and contralateral hippocampus. GSDMD-positive cells were co-labeled with astrocytes, but not neurons or microglia. Astroglial damage occurs following status epilepticus (SE). Damaged astrocytes showed typical clasmatodendrosis in the CA1 region containing strong GSDMD expression at 7-21 days post-SE, accompanied by activated microglia. In the SE + DMF group, the expression of GSDMD-N was significantly inhibited compared to that in the SE + CMC group. After administration of DMF, SRSs at 7-21 days after SE were significantly decreased, and the number of clasmatodendritic astrocytes, microglia, and the expression of inflammatory factors such as IL-1β and IL-18 were significantly attenuated. Conclusion: GSDMD-mediated pyroptosis is involved in the mechanism of kainic acid-induced seizures. Our study provides a new potential therapeutic target for seizure control.

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Pericardial Injection of Kainic Acid Induces a Chronic Epileptic State in Larval Zebrafish

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