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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 -
International Journal of Molecular... May 2023Epilepsy is a challenging brain disorder that is often difficult to treat with conventional therapies. The gut microbiota has been shown to play an important role in the...
Epilepsy is a challenging brain disorder that is often difficult to treat with conventional therapies. The gut microbiota has been shown to play an important role in the development of neuropsychiatric disorders, including epilepsy. In this study, the effects of , a probiotic, on inflammation, neuronal degeneration, and behavior are evaluated in a lithium-pilocarpine model of temporal lobe epilepsy (TLE) induced in young adult rats. was administered orally at a dose of 10 CFU/rat for 30 days after pilocarpine injection. The results show that treatment has beneficial effects on the TLE-induced changes in anxiety levels, neuronal death in the amygdala, and body weight recovery. In addition, increased the expression of anti-inflammatory and neuroprotective genes, such as and . However, the probiotic had little effect on TLE-induced astrogliosis and microgliosis and did not reduce neuronal death in the hippocampus and temporal cortex. The study suggests that may have a beneficial effect on TLE and may provide valuable insights into the role of gut bacteria in epileptogenesis. In addition, the results show that may be a promising drug for the comprehensive treatment of epilepsy.
Topics: Rats; Animals; Epilepsy, Temporal Lobe; Pilocarpine; Lithium; Bifidobacterium longum; Hippocampus; Epilepsy; Probiotics; Disease Models, Animal
PubMed: 37176158
DOI: 10.3390/ijms24098451 -
International Journal of Molecular... Apr 2020Epilepsy is a devastating neurological condition exhibited by repeated spontaneous and unpredictable seizures afflicting around 70 million people globally. The basic...
Epilepsy is a devastating neurological condition exhibited by repeated spontaneous and unpredictable seizures afflicting around 70 million people globally. The basic pathophysiology of epileptic seizures is still elusive, reflecting an extensive need for further research. Developing a novel animal model is crucial in understanding disease mechanisms as well as in assessing the therapeutic target. Most of the pre-clinical epilepsy research has been focused on rodents. Nevertheless, zebrafish disease models are relevant to human disease pathophysiology hence are gaining increased attention nowadays. The current study for the very first time developed a pilocarpine-induced chronic seizure-like condition in adult zebrafish and investigated the modulation in several neuroinflammatory genes and neurotransmitters after pilocarpine exposures. Seizure score analysis suggests that compared to a single dose, repeated dose pilocarpine produces chronic seizure-like effects maintaining an average seizure score of above 2 each day for a minimum of 10 days. Compared to the single dose pilocarpine treated group, there was increased mRNA expression of HMGB1, TLR4, TNF-α, IL-1, BDNF, CREB-1, and NPY; whereas decreased expression of NF-κB was upon the repeated dose of pilocarpine administration. In addition, the epileptic group demonstrates modulation in neurotransmitters levels such as GABA, Glutamate, and Acetylcholine. Moreover, proteomic profiling of the zebrafish brain from the normal and epileptic groups from LCMS/MS quantification detected 77 and 13 proteins in the normal and epileptic group respectively. Summing up, the current investigation depicted that chemically induced seizures in zebrafish demonstrated behavioral and molecular alterations similar to classical rodent seizure models suggesting the usability of adult zebrafish as a robust model to investigate epileptic seizures.
Topics: Animals; Chromatography, Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Gene Expression Regulation; Gene Regulatory Networks; Male; NF-kappa B; Neurotransmitter Agents; Pilocarpine; Proteomics; Seizures; Tandem Mass Spectrometry; Zebrafish; Zebrafish Proteins
PubMed: 32260203
DOI: 10.3390/ijms21072492 -
Optometry and Vision Science : Official... Jun 2020This is a proof-of-concept study showing the possibility of pharmacological control for choroidal thickness using pilocarpine as an agent that causes 2 to 5% choroidal...
SIGNIFICANCE
This is a proof-of-concept study showing the possibility of pharmacological control for choroidal thickness using pilocarpine as an agent that causes 2 to 5% choroidal thinning in healthy eyes after the instillation.
PURPOSE
The purpose of this article was to study the effect of instillation of 1% pilocarpine on choroidal thickness in healthy subjects.
METHODS
Sixteen healthy individuals (seven males and nine females; mean ± standard deviation age, 25.8 ± 3.3 years) were included. All participants received optical coherence tomography to evaluate subfoveal choroidal thickness (SCT) and choroidal area on cross-sectional scan within 4-mm central area. Axial length was measured using optical biometry. Optical coherence tomography was performed before and after pilocarpine was instilled six times for a 75-minute period in one eye; the fellow eye was used as the control. Subfoveal choroidal thickness and choroidal area were measured by two masked graders in random fashion and averaged for analysis.
RESULTS
After instillation of 1% pilocarpine, percentage SCT change in study and control eye was -3.3 ± 3.8% and 0.4 ± 3.2%, respectively (P = .03). Percentage change choroidal area in study and control eye was -2.3 ± 2.5% and 0.8 ± 3.3%, respectively (P < .001). There was a correlation between percentage SCT change and axial length (r = -0.56, P < .001), as well as between percentage SCT change and baseline SCT (r = 0.72, P < .001).
CONCLUSIONS
Instillation of 1% pilocarpine causes a decrease of choroidal thickness, which is more substantial in eyes with short axial length and thick choroid.
Topics: Administration, Ophthalmic; Adult; Choroid; Cross-Sectional Studies; Female; Healthy Volunteers; Humans; Male; Miotics; Ophthalmic Solutions; Organ Size; Pilocarpine; Tomography, Optical Coherence; Young Adult
PubMed: 32511168
DOI: 10.1097/OPX.0000000000001521 -
Environmental Science and Pollution... Jan 2022Epilepsy is characterized by recurrent epileptic seizures, and its effective management continues to be a therapeutic challenge. Oxidative stress and local inflammatory...
Epilepsy is characterized by recurrent epileptic seizures, and its effective management continues to be a therapeutic challenge. Oxidative stress and local inflammatory response accompany the status epilepticus (SE). This study evaluated the effect of Melissa officinalis extract (MOE) on oxidative stress, inflammation, and neurotransmitters in the hippocampus of pilocarpine (PILO)-administered rats, pointing to the involvement of Nrf2/HO-1 signaling. Rats received PILO via intraperitoneal administration and were treated with MOE for 2 weeks. MOE prevented neuronal loss; decreased lipid peroxidation, Cox-2, PGE2, and BDNF; and downregulated glial fibrillary acidic protein in the hippocampus of PILO-treated rats. In addition, MOE enhanced GSH and antioxidant enzymes, upregulated Nrf2 and HO-1 mRNA abundance, and increased the nuclear translocation of Nrf2 in the hippocampus of epileptic rats. Na/K-ATPase activity and GABA were increased, and glutamate and acetylcholine were decreased in the hippocampus of epileptic rats treated with MOE. In conclusion, MOE attenuated neuronal loss, oxidative stress, and inflammation; activated Nrf2/HO-1 signaling; and modulated neurotransmitters, GFAP, and Na/K-ATPase in the hippocampus of epileptic rats. These findings suggest that M. officinalis can mitigate epileptogenesis, pending further studies to explore the exact underlying mechanisms.
Topics: Animals; Rats; Heme Oxygenase (Decyclizing); Hippocampus; Inflammation; Melissa; NF-E2-Related Factor 2; Oxidative Stress; Pilocarpine; Plant Extracts; Rats, Wistar; Signal Transduction
PubMed: 34363578
DOI: 10.1007/s11356-021-15825-y -
Cellular, molecular, and therapeutic characterization of pilocarpine-induced temporal lobe epilepsy.Scientific Reports Sep 2021Animal models have expanded our understanding of temporal lobe epilepsy (TLE). However, translating these to cell-specific druggable hypotheses is not explored. Herein,...
Animal models have expanded our understanding of temporal lobe epilepsy (TLE). However, translating these to cell-specific druggable hypotheses is not explored. Herein, we conducted an integrative insilico-analysis of an available transcriptomics dataset obtained from animals with pilocarpine-induced-TLE. A set of 119 genes with subtle-to-moderate impact predicted most forms of epilepsy with ~ 97% accuracy and characteristically mapped to upregulated homeostatic and downregulated synaptic pathways. The deconvolution of cellular proportions revealed opposing changes in diverse cell types. The proportion of nonneuronal cells increased whereas that of interneurons, except for those expressing vasoactive intestinal peptide (Vip), decreased, and pyramidal neurons of the cornu-ammonis (CA) subfields showed the highest variation in proportion. A probabilistic Bayesian-network demonstrated an aberrant and oscillating physiological interaction between nonneuronal cells involved in the blood-brain-barrier and Vip interneurons in driving seizures, and their role was evaluated insilico using transcriptomic changes induced by valproic-acid, which showed opposing effects in the two cell-types. Additionally, we revealed novel epileptic and antiepileptic mechanisms and predicted drugs using causal inference, outperforming the present drug repurposing approaches. These well-powered findings not only expand the understanding of TLE and seizure oscillation, but also provide predictive biomarkers of epilepsy, cellular and causal micro-circuitry changes associated with it, and a drug-discovery method focusing on these events.
Topics: Animals; Anticonvulsants; Biomarkers; Datasets as Topic; Disease Models, Animal; Drug Discovery; Epilepsy, Temporal Lobe; Gene Expression Regulation; Hippocampus; Humans; Interneurons; Male; Mice; Pilocarpine; Pyramidal Cells; RNA-Seq; Single-Cell Analysis; Temporal Lobe
PubMed: 34580351
DOI: 10.1038/s41598-021-98534-3 -
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 -
International Journal of Developmental... Feb 2023This study aimed to elucidate the effects of Gentiopicroside (Gent) on epileptogenesis and underlying mechanisms.
OBJECTIVES
This study aimed to elucidate the effects of Gentiopicroside (Gent) on epileptogenesis and underlying mechanisms.
METHODS
The status epilepticus (SE) model was established by intraperitoneal (i.p.) injection of lithium chloride (127 mg/kg) and pilocarpine (50 mg/kg) in immature rats. HAPI microglial cellular inflammation model was induced by lipopolysaccharide (LPS, 1 μg/ml) and adenosine triphosphate (ATP, 5 mM). The differential concentrations of Gent were used to pretreat animal (200, 400, and 800 mg/kg) and model cells (50, 100, and 200 μM). Epileptic discharges were assessed by electroencephalography (EEG) and Racine scale. Changes in spatial memory function were measured using the Morris water maze task test. Nissl and FJB staining were employed to assess the damage to hippocampus tissues. ELISA was used to detect the production of IL-1β, IL-18, and TNF-α. The expressions of P2X7R and NLRP3 were detected by q-PCR, immunofluorescence staining, and Western blot, and cell viability was determined by cell counting kit-8 (CCK-8).
RESULTS
Lithium chloride and pilocarpine (LICL-PILO) induced abnormal EEG activities, behavioral alterations, brain damage, and inflammatory responses in immature rats. However, Gent pretreatment significantly reduced the neuronal damage and spatial memory dysfunction induced by LICL-PILO. Additionally, Gent suppressed the production of inflammatory cytokines and inhibited the expression of P2X7R, NLRP3, ASC, and Caspase-1 in LPS/ATP-induced HAPI microglial cells.
DISCUSSION
Gent intervention could improve epileptogenesis in immature rats partially due to suppressing P2X7R and NLRP3 inflammasome.
Topics: Rats; Animals; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Rats, Sprague-Dawley; Lipopolysaccharides; Pilocarpine; Lithium Chloride; Adenosine Triphosphate
PubMed: 36342791
DOI: 10.1002/jdn.10237 -
Scientific Reports May 2021We have compared the diagnostic ability of different concentrations of 0.125% and 0.0625% dilute pilocarpine for detecting denervation supersensitivity in unilateral... (Observational Study)
Observational Study
We have compared the diagnostic ability of different concentrations of 0.125% and 0.0625% dilute pilocarpine for detecting denervation supersensitivity in unilateral Adie's tonic pupil. This retrospective, observational, case-control study involved 117 subjects, consisting of 56 patients with unilateral Adie's tonic pupil and 61 controls with other causes of unilateral dilated pupils. Subjects underwent the dilute pilocarpine test with one of the two concentrations, 0.125% or 0.0625%. Pupillary light reflex was recorded with a dynamic pupillometer at baseline and at 30-40 min after instilling one of the two concentrations of dilute pilocarpine. Diagnostic accuracy of two different concentrations of the dilute pilocarpine test, 0.125% group versus 0.0625% group, were compared by area under the receiver operating characteristic curve (AUC). Diagnostic ability of the dilute pilocarpine test for detecting denervation supersensitivity in unilateral Adie's tonic pupil was significantly better in the 0.0625% group than in the 0.125% group (AUC = 0.954 vs. 0.840, respectively, P = 0.047). In the 0.0625% group, the change in maximal pupil diameter of ≥ 0.5 mm after topical pilocarpine instillation showed 100% sensitivity and 82.8% specificity for detecting Adie's tonic pupil. This study confirmed that pupillary constriction with 0.0625% pilocarpine is better than 0.125% pilocarpine for detecting denervation supersensitivity in Adie's tonic pupil. Digital pupillometry is a reliable method for assessing denervation supersensitivity in Adie's tonic pupil.
Topics: Adie Syndrome; Adult; Aged; Case-Control Studies; Female; Humans; Male; Middle Aged; Pilocarpine; Reflex, Pupillary; Retrospective Studies
PubMed: 33980910
DOI: 10.1038/s41598-021-89148-w