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Scientific Reports Dec 2023Transient brain insults including status epilepticus (SE) can initiate a process termed 'epileptogenesis' that results in chronic temporal lobe epilepsy. As a...
Transient brain insults including status epilepticus (SE) can initiate a process termed 'epileptogenesis' that results in chronic temporal lobe epilepsy. As a consequence, the entire tri-synaptic circuit of the hippocampus is fundamentally impaired. A key role in epileptogenesis has been attributed to the CA1 region as the last relay station in the hippocampal circuit and as site of aberrant plasticity, e.g. mediated by acquired channelopathies. The transcriptional profiles of the distinct hippocampal neurons are highly dynamic during epileptogenesis. Here, we aimed to elucidate the early SE-elicited mRNA signature changes and the respective upstream regulatory cascades in CA1. RNA sequencing of CA1 was performed in the mouse pilocarpine-induced SE model at multiple time points ranging from 6 to 72 h after the initial insult. Bioinformatics was used to decipher altered gene expression, signalling cascades and their corresponding cell type profiles. Robust transcriptomic changes were detected at 6 h after SE and at subsequent time points during early epileptogenesis. Major differentially expressed mRNAs encoded primarily immediate early and excitability-related gene products, as well as genes encoding immune signalling factors. Binding sites for the transcription factors Nfkb1, Spi1, Irf8, and two Runx family members, were enriched within promoters of differentially expressed genes related to major inflammatory processes, whereas the transcriptional repressors Suz12, Nfe2l2 and Rest were associated with hyperexcitability and GABA / glutamate receptor activity. CA1 quickly responds to SE by inducing transcription of genes linked to inflammation and excitation stress. Transcription factors mediating this transcriptomic switch represent targets for new highly selected, cell type and time window-specific anti-epileptogenic strategies.
Topics: Mice; Animals; Hippocampus; Status Epilepticus; Epilepsy, Temporal Lobe; Neurons; Pilocarpine; Transcription Factors; Disease Models, Animal
PubMed: 38092829
DOI: 10.1038/s41598-023-49310-y -
JCI Insight Jan 2024Prolonged seizures can disrupt stem cell behavior in the adult hippocampus, an important brain structure for spatial memory. Here, using a mouse model of...
Prolonged seizures can disrupt stem cell behavior in the adult hippocampus, an important brain structure for spatial memory. Here, using a mouse model of pilocarpine-induced status epilepticus (SE), we characterized spatiotemporal expression of Lin28a mRNA and proteins after SE. Unlike Lin28a transcripts, induction of LIN28A protein after SE was detected mainly in the subgranular zone, where immunoreactivity was found in progenitors, neuroblasts, and immature and mature granule neurons. To investigate roles of LIN28A in epilepsy, we generated Nestin-Cre:Lin28aloxP/loxP (conditional KO [cKO]) and Nestin-Cre:Lin28a+/+ (WT) mice to block LIN28A upregulation in all neuronal lineages after acute seizure. Adult-generated neuron- and hippocampus-associated cognitive impairments were absent in epileptic LIN28A-cKO mice, as evaluated by pattern separation and contextual fear conditioning tests, respectively, while sham-manipulated WT and cKO animals showed comparable memory function. Moreover, numbers of hilar PROX1-expressing ectopic granule cells (EGCs), together with PROX1+/NEUN+ mature EGCs, were significantly reduced in epileptic cKO mice. Transcriptomics analysis and IHC validation at 3 days after pilocarpine administration provided potential LIN28A downstream targets such as serotonin receptor 4. Collectively, our findings indicate that LIN28A is a potentially novel target for regulation of newborn neuron-associated memory dysfunction in epilepsy by modulating seizure-induced aberrant neurogenesis.
Topics: Animals; Nestin; Pilocarpine; Seizures; Status Epilepticus; Hippocampus; Neurogenesis; Epilepsy
PubMed: 38193536
DOI: 10.1172/jci.insight.175627 -
Epilepsy Research Nov 2023The use of zebrafish as a model organism is gaining evidence in the field of epilepsy as it may help to understand the mechanisms underlying epileptic seizures. As...
The use of zebrafish as a model organism is gaining evidence in the field of epilepsy as it may help to understand the mechanisms underlying epileptic seizures. As zebrafish assays became popular, the heterogeneity between protocols increased, making it hard to choose a standard protocol to conduct research while also impairing the comparison of results between studies. We conducted a systematic review to comprehensively profile the chemically-induced seizure models in zebrafish. Literature searches were performed in PubMed, Scopus, and Web of Science, followed by a two-step screening process based on inclusion/exclusion criteria. Qualitative data were extracted, and a sample of 100 studies was randomly selected for risk of bias assessment. Out of the 1058 studies identified after removing duplicates, 201 met the inclusion criteria. We found that the most common chemoconvulsants used in the reviewed studies were pentylenetetrazole (n = 180), kainic acid (n = 11), and pilocarpine (n = 10), which increase seizure severity in a dose-dependent manner. The main outcomes assessed were seizure scores and locomotion. Significant variability between the protocols was observed for administration route, duration of exposure, and dose/concentration. Of the studies subjected to risk of bias assessment, most were rated as low risk of bias for selective reporting (94%), baseline characteristics of the animals (67%), and blinded outcome assessment (54%). Randomization procedures and incomplete data were rated unclear in 81% and 68% of the studies, respectively. None of the studies reported the sample size calculation. Overall, these findings underscore the need for improved methodological and reporting practices to enhance the reproducibility and reliability of zebrafish models for studying epilepsy. Our study offers a comprehensive overview of the current state of chemically-induced seizure models in zebrafish, highlighting the common chemoconvulsants used and the variability in protocol parameters. This may be particularly valuable to researchers interested in understanding the underlying mechanisms of epileptic seizures and screening potential drug candidates in zebrafish models.
Topics: Animals; Zebrafish; Reproducibility of Results; Anticonvulsants; Seizures; Epilepsy; Pentylenetetrazole
PubMed: 37801749
DOI: 10.1016/j.eplepsyres.2023.107236 -
Diagnostics (Basel, Switzerland) Dec 2023In this study, we evaluated the effectiveness of a single-dose oral pilocarpine administration on tear film (TF), as well as dry eye and dry mouth symptoms, in 53 eyes...
In this study, we evaluated the effectiveness of a single-dose oral pilocarpine administration on tear film (TF), as well as dry eye and dry mouth symptoms, in 53 eyes of 27 Sjögren syndrome (SS) patients who were experiencing dry mouth. To evaluate the changes in tear volume, a digital video-meniscometer was used to measure the radius of the lower central tear meniscus curvature (R, mm) of each eye at prior to the administration of 5 mg oral pilocarpine, and at 15 (R:(15)), 30 (R:(30)), and 60 (R:(60)) minutes after administration. The fluorescein breakup time (FBUT, seconds) and ocular and oral dryness symptoms were evaluated before and at 60 min after administration using a visual analogue scale (VAS, mm). A significant increase in R was observed at 15 and 30 min after administration compared to that at prior to administration. FBUT showed significant improvement at 60 min after administration, and the VAS score for ocular and oral dryness symptoms was found to have decreased significantly at 60 min after administration. A single-dose administration of 5 mg oral pilocarpine had a beneficial effect on TF, as well as on ocular and oral dryness symptoms, in patients with SS.
PubMed: 38201400
DOI: 10.3390/diagnostics14010091 -
Neurochemical Research Feb 2024Endoplasmic reticulum (ER) dysfunction caused by excessive ER stress is a crucial mechanism underlying seizures-induced neuronal injury. Studies have shown that...
Endoplasmic reticulum (ER) dysfunction caused by excessive ER stress is a crucial mechanism underlying seizures-induced neuronal injury. Studies have shown that mitochondrial reactive oxygen species (ROS) are closely related to ER stress, and our previous study showed that activating transcription factor 5 (ATF5)-regulated mitochondrial unfolded protein response (mtUPR) modulated mitochondrial ROS generation in a hippocampal neuronal culture model of seizures. However, the effects of ATF5-regulated mtUPR on ER stress and the underlying mechanisms remain uncertain in epilepsy. In this study, ATF5 upregulation by lentivirus infection attenuated seizures-induced neuronal damage and apoptosis in a rat model of pilocarpine-induced epilepsy, whereas ATF5 downregulation by lentivirus infection had the opposite effects. ATF5 upregulation potentiated mtUPR by increasing the expression of mitochondrial chaperone heat shock protein 60 (HSP60) and caseinolytic protease proteolytic subunit (ClpP) and reducing mitochondrial ROS generation in pilocarpine-induced seizures in rats. Additionally, upregulation of ATF5 reduced the expression of glucose-regulated protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), suggesting suppression of ER stress; Moreover, ATF5 upregulation attenuated apoptosis-related proteins such as B-cell lymphoma-2 (BCL2) downregulation, BCL2-associated X (BAX) and cleaved-caspase-3 upregulation. However, ATF5 downregulation exerted the opposite effects. Furthermore, pretreatment with the mitochondria-targeted antioxidant mito-TEMPO attenuated the harmful effects of ATF5 downregulation on ER stress and neuronal apoptosis by reducing mitochondrial ROS generation. Overall, our study suggested that ATF5-regulated mtUPR exerted neuroprotective effects against pilocarpine-induced seizures in rats and the underlying mechanisms might involve mitochondrial ROS-mediated ER stress.
Topics: Rats; Animals; Reactive Oxygen Species; Pilocarpine; Endoplasmic Reticulum Stress; Unfolded Protein Response; Apoptosis; Mitochondria; Apoptosis Regulatory Proteins; Epilepsy; Proto-Oncogene Proteins c-bcl-2; Seizures; Neurons; Lentivirus Infections
PubMed: 37847329
DOI: 10.1007/s11064-023-04042-3 -
The Journal of Neuroscience : the... Oct 2023A significant proportion of temporal lobe epilepsy (TLE) patients experience drug-resistant seizures associated with mesial temporal sclerosis, in which there is...
A significant proportion of temporal lobe epilepsy (TLE) patients experience drug-resistant seizures associated with mesial temporal sclerosis, in which there is extensive cell loss in the hippocampal CA1 and CA3 subfields, with a relative sparing of dentate gyrus granule cells and CA2 pyramidal neurons (PNs). A role for CA2 in seizure generation was suggested based on findings of a reduction in CA2 synaptic inhibition (Williamson and Spencer, 1994) and the presence of interictal-like spike activity in CA2 in resected hippocampal tissue from TLE patients (Wittner et al., 2009). We recently found that in the pilocarpine-induced status epilepticus (PILO-SE) mouse model of TLE there was an increase in CA2 intrinsic excitability associated with a loss of CA2 synaptic inhibition. Furthermore, chemogenetic silencing of CA2 significantly reduced seizure frequency, consistent with a role of CA2 in promoting seizure generation and/or propagation (Whitebirch et al., 2022). In the present study, we explored the cellular basis of this inhibitory deficit using immunohistochemical and electrophysiological approaches in PILO-SE male and female mice. We report a widespread decrease in the density of pro-cholecystokinin-immunopositive (CCK) interneurons and a functional impairment of CCK interneuron-mediated inhibition of CA2 PNs. We also found a disruption in the perisomatic perineuronal net in the CA2 stratum pyramidale. Such pathologic alterations may contribute to an enhanced excitation of CA2 PNs and CA2-dependent seizure activity in the PILO-SE mouse model. Impaired synaptic inhibition in hippocampal circuits has been identified as a key feature that contributes to the emergence and propagation of seizure activity in human patients and animal models of temporal lobe epilepsy (TLE). Among the hippocampal subfields, the CA2 region is particularly resilient to seizure-associated neurodegeneration and has been suggested to play a key role in seizure activity in TLE. Here we report that perisomatic inhibition of CA2 pyramidal neurons mediated by cholecystokinin-expressing interneurons is selectively reduced in acute hippocampal slices from epileptic mice. Parvalbumin-expressing interneurons, in contrast, appear relatively conserved in epileptic mice. These findings advance our understanding of the cellular mechanisms underlying inhibitory disruption in hippocampal circuits in a mouse model of spontaneous recurring seizures.
Topics: Humans; Male; Female; Mice; Animals; CA2 Region, Hippocampal; Epilepsy, Temporal Lobe; Cholecystokinin; Hippocampus; Interneurons; Seizures; Pilocarpine; Status Epilepticus; Disease Models, Animal
PubMed: 37643861
DOI: 10.1523/JNEUROSCI.2091-22.2023 -
Journal of Ethnopharmacology Jan 2024Traditional Chinese medicine (TCM) uses Chaihu-Longgu-Muli decoction (CLMD) to alleviate disease, clear away heat, calm the mind, and temper excitation. It has been...
ETHNOPHARMACOLOGICAL RELEVANCE
Traditional Chinese medicine (TCM) uses Chaihu-Longgu-Muli decoction (CLMD) to alleviate disease, clear away heat, calm the mind, and temper excitation. It has been widely used for the therapy of neuropsychiatric disorders including epilepsy, dementia, anxiety, insomnia, and depression for several centuries in China.
AIM OF THE STUDY
This study aims to analyze differentially expressed proteins (DEPs) in the plasma exosomes of patients with temporal lobe epilepsy (TLE) and after the Chaihu-Longgu-Muli Decoction (CLMD) therapy and to explore the biomarkers of TLE and the potential targets of CLMD in treating TLE.
MATERIALS AND METHODS
The plasma exosomes of normal people and patients with TLE before the treatment of oxcarbazepine (OXC) and combined treatment of OXC and CLMD (OXC.CLMD) were harvested. The exosomes were separated from plasma through ultracentrifugation and then identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. The DEPs were analyzed by proteomics and then subjected to gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The protein level of key genes was detected using Western blot. A lithium chloride-pilocarpine-induced epilepsy rat model was established and treated with OXC alone, OXC. CLMD, and CLMD alone (low dose and high dose). Neuronal injury in the hippocampal dentate gyrus and ribosomal protein L6 (RPL6) expression in the brain tissues were detected using H&E staining, Nissl staining, and Western blot.
RESULTS
The proteomic analysis showed several DEPs were present among plasma exosomes in the four groups; DEPs were enriched in epilepsy-related function and pathway. Four key proteins were screened, including RPL6, Nucleolin (NCL), Apolipoprotein A1 (APOA1), and Lactate Dehydrogenase A (LDHA). Among them, RPL6, NCL, and LDHA protein levels were downregulated and APOA1 protein level was upregulated in the plasma exosomes of TLE patients. After OXC and OXC. CLMD treatment, the protein level of RPL6, NCL, and LDHA was increased, and the APOA1 protein level was decreased. Moreover, the RPL6 protein level was further elevated after OXC. CLMD treatment than that after OXC treatment. In the TLE rat model, neuronal degeneration and necrosis in the hippocampal dentate gyrus increased and RPL6 expression level decreased. After the treatment with OXC, OXC. CLMD, and CLMD alone, the degeneration and necrosis of neurons decreased, and the RPL6 expression level was increased; RPL6 upregulation was remarkably obvious after CLMD treatment.
CONCLUSIONS
RPL6, NCL, LDHA and APOA1 are the DEPs in the plasma exosomes of patients with TLE before and after therapy. RPL6 might be a potential biomarker of CLMD in treating TLE.
Topics: Rats; Animals; Epilepsy, Temporal Lobe; Proteome; Exosomes; Proteomics; Epilepsy; Necrosis; Hippocampus
PubMed: 37479071
DOI: 10.1016/j.jep.2023.116928 -
Journal of Cystic Fibrosis : Official... Jan 2024The sweat test using pilocarpine iontophoresis remains the gold standard for diagnosing cystic fibrosis, but access and reliability are limited by specialized equipment...
BACKGROUND
The sweat test using pilocarpine iontophoresis remains the gold standard for diagnosing cystic fibrosis, but access and reliability are limited by specialized equipment and insufficient sweat volume collected from infants and young children. These shortcomings lead to delayed diagnosis, limited point-of-care applications, and inadequate monitoring capabilities.
METHODS
We created a skin patch with dissolvable microneedles (MNs) containing pilocarpine that eliminates the equipment and complexity of iontophoresis. Upon pressing the patch to skin, the MNs dissolve in skin to release pilocarpine for sweat induction. We conducted a non-randomized pilot trial among healthy adults (clinicaltrials.gov, NCT04732195) with pilocarpine and placebo MN patches on one forearm and iontophoresis on the other forearm, followed by sweat collection using Macroduct collectors. Sweat output and sweat chloride concentration were measured. Subjects were monitored for discomfort and skin erythema.
RESULTS
Fifty paired sweat tests were conducted in 16 male and 34 female healthy adults. MN patches delivered similar amounts of pilocarpine into skin (1.1 ± 0.4 mg) and induced equivalent sweat output (41.2 ± 25.0 mg) compared to iontophoresis (1.2 ± 0.7 mg and 43.8 ± 32.3 mg respectively). Subjects tolerated the procedure well, with little or no pain, and only mild transient erythema. Sweat chloride concentration measurements in sweat induced by MN patches (31.2 ± 13.4 mmol/L) were higher compared to iontophoresis (24.0 ± 13.2 mmol/L). Possible physiological, methodological, and artifactual causes of this difference are discussed.
CONCLUSIONS
Pilocarpine MN patches present a promising alternative to iontophoresis to enable increased access to sweat testing for in-clinic and point-of-care applications.
Topics: Adult; Child; Child, Preschool; Female; Humans; Infant; Male; Chlorides; Cystic Fibrosis; Erythema; Pilocarpine; Reproducibility of Results; Sweat
PubMed: 37236899
DOI: 10.1016/j.jcf.2023.04.014 -
Epilepsy Research May 2024The hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) is predominantly located in key regions associated with epilepsy, such as the neocortex and...
BACKGROUND
The hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) is predominantly located in key regions associated with epilepsy, such as the neocortex and hippocampus. Under normal physiological conditions, HCN1 plays a crucial role in the excitatory and inhibitory regulation of neuronal networks. In temporal lobe epilepsy, the expression of HCN1 is decreased in the hippocampi of both animal models and patients. However, whether HCN1 expression changes during epileptogenesis preceding spontaneous seizures remains unclear.
OBJECTIVE
The aim of this study was to determine whether the expression of HCN1 is altered during the epileptic prodromal phase, thereby providing evidence for its role in epileptogenesis.
METHODS
We utilized a cobalt wire-induced rat epilepsy model to observe changes in HCN1 during epileptogenesis and epilepsy. Additionally, we also compared HCN1 alterations in epileptogenic tissues between cobalt wire- and pilocarpine-induced epilepsy rat models. Long-term video EEG recordings were used to confirm seizures development. Transcriptional changes, translation, and distribution of HCN1 were assessed using high-throughput transcriptome sequencing, total protein extraction, membrane and cytoplasmic protein fractionation, western blotting, immunohistochemistry, and immunofluorescence techniques.
RESULTS
In the cobalt wire-induced rat epilepsy model during the epileptogenesis phase, total HCN1 mRNA and protein levels were downregulated. Specifically, the membrane expression of HCN1 was decreased, whereas cytoplasmic HCN1 expression showed no significant change. The distribution of HCN1 in the distal dendrites of neurons decreased. During the epilepsy period, similar HCN1 alterations were observed in the neocortex of rats with cobalt wire-induced epilepsy and hippocampus of rats with lithium pilocarpine-induced epilepsy, including downregulation of mRNA levels, decreased total protein expression, decreased membrane expression, and decreased distal dendrite expression.
CONCLUSIONS
Alterations in HCN1 expression and distribution are involved in epileptogenesis beyond their association with seizure occurrence. Similarities in HCN1 alterations observed in epileptogenesis-related tissues from different models suggest a shared pathophysiological pathway in epileptogenesis involving HCN1 dysregulation. Therefore, the upregulation of HCN1 expression in neurons, maintenance of the HCN1 membrane, and distal dendrite distribution in neurons may represent promising disease-modifying strategies in epilepsy.
Topics: Animals; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Male; Epilepsy; Rats, Sprague-Dawley; Rats; Hippocampus; Disease Models, Animal; Potassium Channels; Pilocarpine; Cobalt; Electroencephalography; Neurons; Neocortex
PubMed: 38555654
DOI: 10.1016/j.eplepsyres.2024.107355 -
Hippocampus Jul 2024Olfactory oscillations may enhance cognitive processing through coupling with beta (β, 15-30 Hz) and gamma (γ, 30-160 Hz) activity in the hippocampus (HPC). We...
Olfactory oscillations may enhance cognitive processing through coupling with beta (β, 15-30 Hz) and gamma (γ, 30-160 Hz) activity in the hippocampus (HPC). We hypothesize that coupling between olfactory bulb (OB) and HPC oscillations is increased by cholinergic activation in control rats and is reduced in kainic-acid-treated epileptic rats, a model of temporal lobe epilepsy. OB γ2 (63-100 Hz) power was higher during walking and immobility-awake (IMM) compared to sleep, while γ1 (30-57 Hz) power was higher during grooming than other behavioral states. Muscarinic cholinergic agonist pilocarpine (25 mg/kg ip) with peripheral muscarinic blockade increased OB power and OB-HPC coherence at β and γ1 frequency bands. A similar effect was found after physostigmine (0.5 mg/kg ip) but not scopolamine (10 mg/kg ip). Pilocarpine increased bicoherence and cross-frequency coherence (CFC) between OB slow waves (SW, 1-5 Hz) and hippocampal β, γ1 and γ2 waves, with stronger coherence at CA1 alveus and CA3c than CA1 stratum radiatum. Bicoherence further revealed a nonlinear interaction of β waves in OB with β waves at the CA1-alveus. Beta and γ1 waves in OB or HPC were segregated at one phase of the OB-SW, opposite to the phase of γ2 and γ3 (100-160 Hz) waves, suggesting independent temporal processing of β/γ1 versus γ2/γ3 waves. At CA1 radiatum, kainic-acid-treated epileptic rats compared to control rats showed decreased theta power, theta-β and theta-γ2 CFC during baseline walking, decreased CFC of HPC SW with γ2 and γ3 waves during baseline IMM, and decreased coupling of OB SW with β and γ2 waves at CA1 alveus after pilocarpine. It is concluded that β and γ waves in the OB and HPC are modulated by a slow respiratory rhythm, in a cholinergic and behavior-dependent manner, and OB-HPC functional connectivity at β and γ frequencies may enhance cognitive functions.
PubMed: 38949057
DOI: 10.1002/hipo.23622