RESUMO
Although several adult rat models of medial temporal lobe epilepsy (mTLE) have been described in detail, our knowledge of mTLE epileptogenesis in infant rats is limited. Here, we present a novel infant rat model of mTLE (InfRPil-mTLE) based on a repetitive, triphasic injection regimen consisting of low-dose pilocarpine administrations (180 mg/kg. i.p.) on days 9, 11, and 15 post partum (pp). The model had a survival rate of >80% and exhibited characteristic spontaneous recurrent electrographic seizures (SRES) in both the hippocampus and cortex that persisted into adulthood. Using implantable video-EEG radiotelemetry, we quantified a complex set of seizure parameters that demonstrated the induction of chronic electroencephalographic seizure activity in our InfRPil-mTLE model, which predominated during the dark cycle. We further analyzed selected candidate genes potentially relevant to epileptogenesis using a RT-qPCR approach. Several candidates, such as the low-voltage-activated Ca2+ channel Cav3.2 and the auxiliary subunits ß 1 and ß 2, which were previously reported to be upregulated in the hippocampus of the adult pilocarpine mTLE model, were found to be downregulated (together with Cav2.1, Cav2.3, M1, and M3) in the hippocampus and cortex of our InfRPil-mTLE model. From a translational point of view, our model could serve as a blueprint for childhood epileptic disorders and further contribute to antiepileptic drug research and development in the future.
Assuntos
Modelos Animais de Doenças , Epilepsia do Lobo Temporal , Pilocarpina , Animais , Epilepsia do Lobo Temporal/fisiopatologia , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/induzido quimicamente , Ratos , Eletroencefalografia , Hipocampo/metabolismo , Animais Recém-Nascidos , Encéfalo/metabolismo , Ratos Sprague-Dawley , Masculino , FemininoRESUMO
BACKGROUND: Hyposalivation is treated using oral cholinergic drugs; however, systemic side effects occasionally lead to discontinuation of treatment. We aimed to investigate the effects of transdermal pilocarpine on the salivary gland skin on saliva secretion and safety in rats. METHODS: Pilocarpine was administered to rats orally (0.5 mg/kg) or topically on the salivary gland skin (5 mg/body). Saliva volume, the number of sweat dots, and fecal weight were measured along with pilocarpine concentration in plasma and submandibular gland tissues. RESULTS: Saliva volume significantly increased 0.5 h after oral administration and 0.5, 3, and 12 h after topical administration. Fecal weight and sweat dots increased significantly 1 h after oral administration; however, no changes were observed after topical application. The pilocarpine concentration in the submandibular gland tissues of the topical group was higher than that in the oral group at 0.5, 3, and 12 h of administration. CONCLUSIONS: Pilocarpine application to salivary gland skin persistently increased salivary volume in rats without inducing sweating or diarrhea. Transdermal pilocarpine applied to the skin over the salivary glands may be an effective and safe treatment option for hyposalivation.
Assuntos
Administração Cutânea , Pilocarpina , Glândulas Salivares , Salivação , Xerostomia , Pilocarpina/administração & dosagem , Pilocarpina/farmacologia , Animais , Salivação/efeitos dos fármacos , Ratos , Masculino , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/metabolismo , Xerostomia/induzido quimicamente , Xerostomia/tratamento farmacológico , Agonistas Muscarínicos/administração & dosagem , Agonistas Muscarínicos/farmacologia , Saliva/metabolismo , Saliva/química , Administração Oral , Glândula Submandibular/efeitos dos fármacos , Glândula Submandibular/metabolismo , Ratos Sprague-DawleyRESUMO
The ciliary muscle constitutes a crucial element in refractive regulation. Investigating the pathophysiological mechanisms within the ciliary muscle during excessive contraction holds significance in treating ciliary muscle dysfunction. A guinea pig model of excessive contraction of the ciliary muscle induced by drops pilocarpine was employed, alongside the primary ciliary muscle cells was employed in in vitro experiments. The results of the ophthalmic examination showed that pilocarpine did not significantly change refraction and axial length during the experiment, but had adverse effects on the regulatory power of the ciliary muscle. The current data reveal notable alterations in the expression profiles of hypoxia inducible factor 1 (HIF-1α), ATP2A2, P53, α-SMA, Caspase-3, and BAX within the ciliary muscle of animals subjected to pilocarpine exposure, alongside corresponding changes observed in cultured cells treated with pilocarpine. Augmented levels of ROS were detected in both tissue specimens and cells, culminating in a significant increase in cell apoptosis in in vivo and in vitro experiments. Further examination revealed that pilocarpine induced an increase in intracellular Ca2+ levels and disrupted MMP, as evidenced by mitochondrial swelling and diminished cristae density compared to control conditions, concomitant with a noteworthy decline in antioxidant enzyme activity. However, subsequent blockade of Ca2+ channels in cells resulted in downregulation of HIF-1α, ATP2A2, P53, α-SMA, Caspase-3, and BAX expression, alongside ameliorated mitochondrial function and morphology. The inhibition of Ca2+ channels presents a viable approach to mitigate ciliary cells damage and sustain proper ciliary muscle function by curtailing the mitochondrial damage induced by excessive contractions.
Assuntos
Apoptose , Cálcio , Senescência Celular , Pilocarpina , Animais , Pilocarpina/farmacologia , Cobaias , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Senescência Celular/efeitos dos fármacos , Corpo Ciliar/metabolismo , Masculino , Células Cultivadas , Espécies Reativas de Oxigênio/metabolismoRESUMO
To present the expression of calsyntenin-1 (Clstn1) in the brain and investigate the potential mechanism of Clstn1 in lithium-pilocarpine rat seizure models. Thirty-five male SD adult rats were induced to have seizures by intraperitoneal injection of lithium chloride pilocarpine. Rats exhibiting spontaneous seizures were divided into the epilepsy (EP) group (n = 15), whereas those without seizures were divided into the control group (n = 14). Evaluate the expression of Clstn1 in the temporal lobe of two groups using Western blotting, immunohistochemistry, and immunofluorescence. Additionally, 55 male SD rats were subjected to status epilepticus (SE) using the same induction method. Rats experiencing seizures exceeding Racine's level 4 (n = 48) were randomly divided into three groups: SE, SE + control lentivirus (lentiviral vector expressing green fluorescent protein [LV-GFP]), and SE + Clstn1-targeted RNA interference lentivirus (LV-Clstn1-RNAi). The LV-GFP group served as a control for the lentiviral vector, whereas the LV-Clstn1-RNAi group received a lentivirus designed to silence Clstn1 expression. These lentiviral treatments were administered via hippocampal stereotactic injection 2 days after SE induction. Seven days after SE, Western blot analysis was performed to evaluate the expression of Clstn1 in the hippocampus and temporal lobe. Meanwhile, we observed the latency of spontaneous seizures and the frequency of spontaneous seizures within 8 weeks among the three groups. The expression of Clstn1 in the cortex and hippocampus of the EP group was significantly increased compared to the control group (p < .05). Immunohistochemistry and immunofluorescence showed that Clstn1 was widely distributed in the cerebral cortex and hippocampus of rats, and colocalization analysis revealed that it was mainly co expressed with neurons in the cytoplasm. Compared with the SE group (11.80 ± 2.17 days) and the SE + GFP group (12.40 ± 1.67 days), there was a statistically significant difference (p < .05) in the latency period of spontaneous seizures (15.14 ± 2.41 days) in the SE + Clstn1 + RNAi group rats. Compared with the SE group (4.60 ± 1.67 times) and the SE + GFP group (4.80 ± 2.05 times), the SE + Clstn1 + RNAi group (2.0 ± .89 times) showed a significant reduction in the frequency of spontaneous seizures within 2 weeks of chronic phase in rats (p < .05). Elevated Clstn1 expression in EP group suggests its role in EP onset. Targeting Clstn1 may be a potential therapeutic approach for EP management.
Assuntos
Modelos Animais de Doenças , Pilocarpina , Ratos Sprague-Dawley , Convulsões , Animais , Masculino , Pilocarpina/toxicidade , Ratos , Convulsões/metabolismo , Convulsões/induzido quimicamente , Convulsões/genética , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/metabolismo , Neurocalcina/metabolismo , Neurocalcina/genética , Hipocampo/metabolismo , Cloreto de Lítio , Lobo Temporal/metabolismo , Encéfalo/metabolismoRESUMO
Epilepsy, a neurological disorder characterized by excessive neuronal activity and synchronized electrical discharges, ranks among the most prevalent global neurological conditions. Despite common use, antiepileptic drugs often result in adverse effects and lack effectiveness in controlling seizures in temporal lobe epilepsy (TLE) patients. Recent research explored the potential of occidentalin-1202, a peptide inspired by Polybia occidentalis venom, in safeguarding Wistar rats from chemically induced seizures. The present study evaluated the new analog from occidentalin-1202 named NOR-1202 using acute and chronic pilocarpine-induced models and an acute kainic acid (KA) male mice model. NOR-1202 was administered through the intracerebroventricular (i.c.v.), subcutaneous, or intraperitoneal routes, with stereotaxic procedures for the i.c.v. injection. In the acute pilocarpine-induced model, NOR-1202 (i.c.v.) protected against generalized seizures and mortality but lacked systemic antiepileptic activity. In the KA model, it did not prevent generalized seizures but improved survival. In the chronic TLE model, NOR-1202's ED50 did not differ significantly from the epileptic or healthy groups regarding time spent in spontaneous recurrent seizures during the five-day treatment. However, the NOR-1202 group exhibited more seizures than the healthy group on the second day of treatment. In summary, NOR-1202 exhibits antiepileptic effects against chemoconvulsant-induced seizures, but no effect was observed when administered systemically.
Assuntos
Anticonvulsivantes , Modelos Animais de Doenças , Convulsões , Animais , Masculino , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Camundongos , Convulsões/tratamento farmacológico , Convulsões/induzido quimicamente , Pilocarpina , Ácido Caínico/análogos & derivados , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Peptídeos/administração & dosagem , Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/induzido quimicamenteRESUMO
BACKGROUND: The present study elucidates a common significant postoperative complication of micropulse transscleral laser treatment (mTLT) and explores its potential management strategies for younger patients with good central vision. CASE PRESENTATION: Three younger Chinese glaucoma patients with good central vision maintained high intraocular pressures (IOPs) (36, 25, and 30 mmHg) on maximally tolerated topical anti-glaucoma medications. All patients were treated with mTLT because of a higher risk of complications with filtering surgery. After the procedure, their best-corrected visual acuities were not significantly changed, IOPs were significantly decreased, and the number of topical anti-glaucoma medicines was gradually decreased. However, all patients complained about reduced near visual acuity (NVA) for 1-5 months. Slit-lamp examination revealed pupillary dilation, and binocular accommodative function examination indicated accommodation loss. After treatment with 2% topical pilocarpine, all patients reported an improvement in NVA. Among them, we could observe pupillary constriction, recovery of accommodation function, and improved NVA, even discontinuation of pilocarpine in Patient 2. CONCLUSION: In younger patients with good central vision, topical pilocarpine might ameliorate accommodation loss and pupillary dilation after mTLT.
Assuntos
Acomodação Ocular , Pressão Intraocular , Pilocarpina , Humanos , Pilocarpina/uso terapêutico , Pilocarpina/administração & dosagem , Masculino , Feminino , Adulto , Pressão Intraocular/fisiologia , Acomodação Ocular/fisiologia , Acuidade Visual , Mióticos/administração & dosagem , Mióticos/uso terapêutico , Pupila/efeitos dos fármacos , Esclera/cirurgia , Glaucoma/cirurgia , Glaucoma/fisiopatologia , Terapia a Laser/métodos , Soluções Oftálmicas , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Administração TópicaRESUMO
Epilepsy is a common neurological disease. Increasing evidence has highlighted the role of miRNAs in the molecular mechanisms underlying the development of neurological diseases such as epilepsy. In this study, we established a lithium chloride-pilocarpine epilepsy mouse model, performed miRNA sequencing of hippocampal tissue samples, and compared the obtained miRNA expression profile with that of normal control mice to determine differences in expression levels. We found that 55 miRNAs were differentially expressed in status epilepticus mice compared with normal control mice, with 38 upregulated and 17 downregulated miRNAs. Through subsequent analysis of the five downregulated miRNAs (mmu-let-7a-1-3p, mmu-let-7a-2-3p, mmu-let-7c-5p, mmu-let-7d-5p, and mmu-let-7e-5p) with the most significant differences in expression, the key pathways involved included the MAPK signaling pathway and focal adhesion, among others. Therefore, we believe that let-7 family miRNAs may be potential therapeutic targets for epilepsy.
Assuntos
Modelos Animais de Doenças , Epilepsia , Hipocampo , MicroRNAs , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Hipocampo/metabolismo , Camundongos , Epilepsia/genética , Epilepsia/metabolismo , Perfilação da Expressão Gênica/métodos , Masculino , Pilocarpina , Regulação da Expressão Gênica , Estado Epiléptico/genética , Estado Epiléptico/metabolismo , Estado Epiléptico/induzido quimicamente , Cloreto de Lítio/farmacologiaRESUMO
The interplay between circadian rhythms and epilepsy has gained increasing attention. The suprachiasmatic nucleus (SCN), which acts as the master circadian pacemaker, regulates physiological and behavioral rhythms through its complex neural networks. However, the exact role of the SCN and its Bmal1 gene in the development of epilepsy remains unclear. In this study, we utilized a lithium-pilocarpine model to induce epilepsy in mice and simulated circadian disturbances by creating lesions in the SCN and specifically knocking out the Bmal1 gene in the SCN neurons. We observed that the pilocarpine-induced epileptic mice experienced increased daytime seizure frequency, irregular oscillations in core body temperature, and circadian gene alterations in both the SCN and the hippocampus. Additionally, there was enhanced activation of GABAergic projections from the SCN to the hippocampus. Notably, SCN lesions intensified seizure activity, concomitant with hippocampal neuronal damage and GABAergic signaling impairment. Further analyses using the Gene Expression Omnibus database and gene set enrichment analysis indicated reduced Bmal1 expression in patients with medial temporal lobe epilepsy, potentially affecting GABA receptor pathways. Targeted deletion of Bmal1 in SCN neurons exacerbated seizures and pathology in epilepsy, as well as diminished hippocampal GABAergic efficacy. These results underscore the crucial role of the SCN in modulating circadian rhythms and GABAergic function in the hippocampus, aggravating the severity of seizures. This study provides significant insights into how circadian rhythm disturbances can influence neuronal dysfunction and epilepsy, highlighting the therapeutic potential of targeting SCN and the Bmal1 gene within it in epilepsy management.
Assuntos
Ritmo Circadiano , Hipocampo , Camundongos Endogâmicos C57BL , Núcleo Supraquiasmático , Animais , Núcleo Supraquiasmático/metabolismo , Camundongos , Hipocampo/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Masculino , Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Epilepsia/genética , Pilocarpina , Convulsões/metabolismo , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/fisiopatologia , Camundongos Knockout , Neurônios GABAérgicos/metabolismoRESUMO
In order to investigate the effectiveness and safety of miR-23b-3p in anti-seizure activity and to elucidate the regulatory relationship between miR-23b-3p and Cx43 in the nervous system, we have established a lithium chloride-pilocarpine (PILO) status epilepticus (SE) model. Rats were randomly divided into the following groups: seizure control (PILO), valproate sodium (VPA+PILO), recombinant miR-23b-3p overexpression (miR+PILO), miR-23b-3p sponges (Sponges+PILO), and scramble sequence negative control (Scramble+PILO) (n = 6/group). After experiments, we got the following results. In the acute phase, the time required for rats to reach stage IV after PILO injection was significantly longer in VPA+PILO and miR+PILO. In the chronic phase after SE, the frequency of spontaneous recurrent seizures (SRSs) in VPA+PILO and miR+PILO was significantly reduced. At 10 min before seizure cessation, the average energy expression of fast ripples (FRs) in VPA+PILO and miR+PILO was significantly lower than in PILO. After 28 days of seizure, Cx43 expression in PILO was significantly increased, and Beclin1expression in all groups was significantly increased. After 28 days of SE,the number of synapses in the CA1 region of the hippocampus was significantly higher in the VPA+PILO and miR+PILO groups compared to that in the PILO group. After 28 days of SE ,hippocampal necrotic cells in the CA3 region were significantly lower in the VPA+PILO and miR+PILO groups compared to those in the PILO group. There were no significant differences in biochemical indicators among the experimental group rats 28 days after SE compared to the seizure control group. Based on the previous facts, we can reach the conclusion that MiR-23b-3p targets and blocks the expression of hippocampal Cx43 which can reduce the formation of pathological FRs, thereby alleviating the severity of seizures, improving seizure-induced brain damage.
Assuntos
Conexina 43 , Hipocampo , MicroRNAs , Ratos Sprague-Dawley , Estado Epiléptico , Animais , Masculino , Ratos , Lesões Encefálicas/metabolismo , Conexina 43/metabolismo , Conexina 43/genética , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , MicroRNAs/metabolismo , MicroRNAs/genética , Pilocarpina/toxicidade , Convulsões/metabolismo , Convulsões/induzido quimicamente , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/metabolismoRESUMO
Status epilepticus (SE) is a medical emergency associated with high mortality and morbidity. Na+, K+-ATPase, is a promising therapeutic target for SE, given its critical role in regulation of neuron excitability and cellular homeostasis. We investigated the effects of a Na+, K+-ATPase-activating antibody (DRRSAb) on short-term electrophysiological and behavioral consequences of pilocarpine-induced SE. Rats were submitted to pilocarpine-induced SE, followed by intranasal administration (2 µg/nostril). The antibody increased EEG activity following SE, namely, EEG power in theta, beta, and gamma frequency bands, assessed by quantitative analysis of EEG power spectra. One week later, DRRSAb-treated animals displayed less behavioral hyperreactivity in pick-up tests and better performance in novel object recognition tests, indicating that the intranasal administration of this Na+, K+-ATPase activator immediately after SE improves behavioral outcomes at a later time point. These results suggest that Na+, K+-ATPase activation warrants further investigation as an adjunctive therapeutic strategy for SE.
Assuntos
Administração Intranasal , Eletroencefalografia , Pilocarpina , ATPase Trocadora de Sódio-Potássio , Estado Epiléptico , Animais , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/tratamento farmacológico , ATPase Trocadora de Sódio-Potássio/metabolismo , Masculino , Pilocarpina/farmacologia , Eletroencefalografia/métodos , Eletroencefalografia/efeitos dos fármacos , Ratos , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Ratos Wistar , Anticorpos/farmacologia , Anticorpos/administração & dosagemRESUMO
This in vivo mouse model study was conducted to investigate the temporal alteration of the function of CD36 in salivary secretion. CD36 was highly expressed in the parotid gland of BALB/c mice. No significant variations were shown in the CD36 levels in the 8-, 48-, and 72-week-old animals. However, pilocarpine-induced salivary secretion was reduced in an age-dependent manner, showing a significantly low level at the age of 72 weeks. Pilocarpine-induced salivary secretion was significantly reduced by pretreatment with a CD36 inhibitor at 8 and 48 weeks, but not at 72 weeks. In senescence-accelerated mice (SAM), the pilocarpine-induced salivary secretion was significantly reduced at the age of 56 weeks, and a significantly lower amount of CD36 was demonstrated in the parotid gland, compared with the control. These results suggest that the involvement of parotid CD36 in mouse salivary secretion is altered with age.
Assuntos
Envelhecimento , Antígenos CD36 , Camundongos Endogâmicos BALB C , Glândula Parótida , Saliva , Animais , Glândula Parótida/metabolismo , Antígenos CD36/metabolismo , Camundongos , Masculino , Saliva/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Pilocarpina/farmacologia , Salivação/efeitos dos fármacosRESUMO
Epilepsy is known to cause alterations in neural networks. However, many details of these changes remain poorly understood. The objective of this study was to investigate changes in the properties of hippocampal CA1 pyramidal neurons and their synaptic inputs in a rat lithium-pilocarpine model of epilepsy. In the chronic phase of the model, we found a marked loss of pyramidal neurons in the CA1 area. However, the membrane properties of the neurons remained essentially unaltered. The results of the electrophysiological and morphological studies indicate that the direct pathway from the entorhinal cortex to CA1 neurons is reinforced in epileptic animals, whereas the inputs to them from CA3 are either unaltered or even diminished. In particular, the dendritic spine density in the str. lacunosum moleculare, where the direct pathway from the entorhinal cortex terminates, was found to be 2.5 times higher in epileptic rats than in control rats. Furthermore, the summation of responses upon stimulation of the temporoammonic pathway was enhanced by approximately twofold in epileptic rats. This enhancement is believed to be a significant contributing factor to the heightened epileptic activity observed in the entorhinal cortex of epileptic rats using an ex vivo 4-aminopyridine model.
Assuntos
Região CA1 Hipocampal , Modelos Animais de Doenças , Epilepsia , Lítio , Pilocarpina , Células Piramidais , Animais , Células Piramidais/patologia , Células Piramidais/metabolismo , Ratos , Epilepsia/induzido quimicamente , Epilepsia/patologia , Epilepsia/fisiopatologia , Masculino , Região CA1 Hipocampal/patologia , Lítio/toxicidade , Lítio/farmacologia , Córtex Entorrinal/patologia , Ratos WistarRESUMO
BACKGROUND: Status epilepticus (SE) as a severe neurodegenerative disease, greatly negatively affects people's health, and there is an urgent need for innovative treatments. The valuable neuroprotective effects of glucagon-like peptide-1 (GLP-1) in several neurodegenerative diseases have raised motivation to investigate the dipeptidyl peptidase-4 (DPP-4) inhibitor; alogliptin (ALO), an oral antidiabetic drug as a potential treatment for SE. ALO has shown promising neuroprotective effects in Alzheimer's and Parkinson's diseases, but its impact on SE has not yet been studied. AIM: The present study aimed to explore the repurposing potential for ALO in a lithium/pilocarpine (Li/Pil)-induced SE model in rats. MAIN METHODS: ALO (30 mg/kg/day) was administered via gavage for 14 days, and SE was subsequently induced in the rats using a single dose of Li/Pil (127/60 mg/kg), while levetiracetam was used as a standard antiepileptic drug. KEY FINDINGS: The results showed that ALO reduced seizure severity and associated hippocampal neurodegeneration. ALO also increased γ-aminobutyric acid (GABA) levels, diminished glutamate spikes, and corrected glial fibrillary acidic protein (GFAP) changes. At the molecular level, ALO increased GLP-1 levels and activated its downstream signaling pathway, AMP-activated protein kinase (AMPK)/sirtuin-1 (SIRT1). ALO also dampened the brain's pro-oxidant response, curbed neuroinflammation, and counteracted hippocampal apoptosis affording neuroprotection. In addition, it activated autophagy as indicated by Beclin1 elevation. SIGNIFICANCE: This study suggested that the neuroprotective properties and autophagy-enhancing effects of ALO make it a promising treatment for SE and can potentially be used as a management approach for this condition.
Assuntos
Autofagia , Fator 2 Relacionado a NF-E2 , Fármacos Neuroprotetores , Pilocarpina , Piperidinas , Convulsões , Sirtuína 1 , Animais , Ratos , Sirtuína 1/metabolismo , Fármacos Neuroprotetores/farmacologia , Masculino , Autofagia/efeitos dos fármacos , Piperidinas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Lítio/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Uracila/análogos & derivados , Uracila/farmacologia , Ratos Sprague-Dawley , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Mesial temporal lobe epilepsy (MTLE) is characterized by recurring focal seizures that arise from limbic areas and are often refractory to pharmacological interventions. We have reported that optogenetic stimulation of PV-positive cells in the medial septum at 0.5 Hz exerts seizure-suppressive effects. Therefore, we compared here these results with those obtained by optogenetic stimulation of medial septum PV-positive neurons at 8 Hz in male PV-ChR2 mice (P60-P100) undergoing an initial, pilocarpine-induced status epilepticus (SE). Optogenetic stimulation (5 min ON, 10 min OFF) was performed from day 8 to day 12 after SE at a frequency of 8 Hz (n = 6 animals) or 0.5 Hz (n = 8 animals). Surprisingly, in both groups, no effects were observed on the occurrence of interictal spikes and interictal high frequency oscillations (HFOs). However, 0.5 Hz stimulation induced a significant decrease of seizure occurrence (p < 0.05). Such anti-ictogenic effect was not observed in the 8 Hz protocol that instead triggered seizures (p < 0.05); these seizures were significantly longer under optogenetic stimulation compared to when optogenetic stimulation was not implemented (p < 0.05). Analysis of ictal HFOs revealed that in the 0.5 Hz group, but not in the 8 Hz group, seizures occurring under optogenetic stimulation were associated with significantly lower rates of fast ripples compared to when optogenetic stimulation was not performed (p < 0.05). Our results indicate that activation of GABAergic PV-positive neurons in the medial septum exerts seizure-suppressing effects that are frequency-dependent and associated with low rates of fast ripples. Optogenetic activation of medial septum PV-positive neurons at 0.5 Hz is efficient in blocking seizures in the pilocarpine model of MTLE, an effect that did not occur with 8 Hz stimulation.
Assuntos
Epilepsia do Lobo Temporal , Optogenética , Convulsões , Animais , Optogenética/métodos , Epilepsia do Lobo Temporal/fisiopatologia , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/terapia , Masculino , Convulsões/fisiopatologia , Camundongos , Pilocarpina/toxicidade , Camundongos Transgênicos , Modelos Animais de Doenças , Septo do Cérebro , Núcleos Septais/fisiopatologia , Camundongos Endogâmicos C57BLRESUMO
Epilepsy affects 1% of the general population and 30% of patients are resistant to antiepileptic drugs. Although optogenetics is an efficient antiepileptic strategy, the difficulty of illuminating deep brain areas poses translational challenges. Thus, the search of alternative light sources is strongly needed. Here, we develop pH-sensitive inhibitory luminopsin (pHIL), a closed-loop chemo-optogenetic nanomachine composed of a luciferase-based light generator, a fluorescent sensor of intracellular pH (E2GFP), and an optogenetic actuator (halorhodopsin) for silencing neuronal activity. Stimulated by coelenterazine, pHIL experiences bioluminescence resonance energy transfer between luciferase and E2GFP which, under conditions of acidic pH, activates halorhodopsin. In primary neurons, pHIL senses the intracellular pH drop associated with hyperactivity and optogenetically aborts paroxysmal activity elicited by the administration of convulsants. The expression of pHIL in hippocampal pyramidal neurons is effective in decreasing duration and increasing latency of pilocarpine-induced tonic-clonic seizures upon in vivo coelenterazine administration, without affecting higher brain functions. The same treatment is effective in markedly decreasing seizure manifestations in a murine model of genetic epilepsy. The results indicate that pHIL represents a potentially promising closed-loop chemo-optogenetic strategy to treat drug-refractory epilepsy.
Assuntos
Epilepsia , Neurônios , Optogenética , Animais , Concentração de Íons de Hidrogênio , Camundongos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Epilepsia/fisiopatologia , Epilepsia/metabolismo , Epilepsia/tratamento farmacológico , Humanos , Convulsões/tratamento farmacológico , Convulsões/fisiopatologia , Convulsões/metabolismo , Halorrodopsinas/metabolismo , Halorrodopsinas/genética , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Masculino , Luciferases/metabolismo , Luciferases/genética , Células Piramidais/metabolismo , Células Piramidais/efeitos dos fármacos , Imidazóis/farmacologia , Pilocarpina/farmacologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Células HEK293 , PirazinasRESUMO
INTRODUCTION: Our previous work reveals a critical role of activation of neuronal Alox5 in exacerbating brain injury post seizures. However, whether neuronal Alox5 impacts the pathological process of epilepsy remains unknown. OBJECTIVES: To prove the feasibility of neuron-specific deletion of Alox5 via CRISPR-Cas9 in the blockade of seizure onset and epileptic progression. METHODS: Here, we employed a Clustered regularly interspaced short-palindromic repeat-associated proteins 9 system (CRISPR/Cas9) system delivered by adeno-associated virus (AAV) to specifically delete neuronal Alox5 gene in the hippocampus to explore its therapeutic potential in various epilepsy mouse models and possible mechanisms. RESULTS: Neuronal depletion of Alox5 was successfully achieved in the brain. AAV delivery of single guide RNA of Alox5 in hippocampus resulted in reducing seizure severity, delaying epileptic progression and improving epilepsy-associated neuropsychiatric comorbidities especially anxiety, cognitive deficit and autistic-like behaviors in pilocarpine- and kainic acid-induced temporal lobe epilepsy (TLE) models. In addition, neuronal Alox5 deletion also reversed neuron loss, neurodegeneration, astrogliosis and mossy fiber sprouting in TLE model. Moreover, a battery of tests including analysis of routine blood test, hepatic function, renal function, routine urine test and inflammatory factors demonstrated no noticeable toxic effect, suggesting that Alox5 deletion possesses the satisfactory biosafety. Mechanistically, the anti-epileptic effect of Alox5 deletion might be associated with reduction of glutamate level to restore excitatory/inhibitory balance by reducing CAMKII-mediated phosphorylation of Syn ISer603. CONCLUSION: Our findings showed the translational potential of AAV-mediated delivery of CRISPR-Cas9 system including neuronal Alox5 gene for an alternative promising therapeutic approach to treat epilepsy.
Assuntos
Araquidonato 5-Lipoxigenase , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Epilepsia , Hipocampo , Neurônios , Animais , Camundongos , Araquidonato 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/genética , Neurônios/metabolismo , Hipocampo/metabolismo , Epilepsia/genética , Masculino , Camundongos Endogâmicos C57BL , Deleção de Genes , Epilepsia do Lobo Temporal/genética , Dependovirus/genética , PilocarpinaRESUMO
Epilepsy, a chronic neurological disorder characterized by recurrent unprovoked seizures, presents a substantial challenge in approximately one-third of cases exhibiting resistance to conventional pharmacological treatments. This study investigated the effect of 4-allyl-2,6-dimethoxyphenol, a phenolic compound derived from various natural sources, in different models of induced seizures and its impact on animal electroencephalographic (EEG) recordings. Adult male Swiss albino mice were pre-treated (i.p.) with a dose curve of 4-allyl-2,6-dimethoxyphenol (50, 100, or 200â¯mg/kg), its vehicle (Tween), or standard antiepileptic drug (Diazepam; or Phenytoin). Subsequently, the mice were subjected to different seizure-inducing models - pentylenetetrazole (PTZ), 3-mercaptopropionic acid (3-MPA), pilocarpine (PILO), or maximal electroshock seizure (MES). EEG analysis was performed on other animals surgically implanted with electrodes to evaluate brain activity. Significant results revealed that animals treated with 4-allyl-2,6-dimethoxyphenol exhibited increased latency to the first myoclonic jerk in the PTZ and PILO models; prolonged latency to the first tonic-clonic seizure in the PTZ, 3-MPA, and PILO models; reduced total duration of tonic-clonic seizures in the PTZ and PILO models; decreased intensity of convulsive seizures in the PTZ and 3-MPA models; and diminished mortality in the 3-MPA, PILO, and MES models. EEG analysis indicated an increase in the percentage of total power attributed to beta waves following 4-allyl-2,6-dimethoxyphenol administration. Notably, the substance protected from behavioral and electrographic seizures in the PTZ model, preventing increases in the average amplitude of recording signals while also inducing an increase in the participation of theta and gamma waves. These findings suggest promising outcomes for the tested phenolic compound across diverse pre-clinical seizure models, highlighting the need for further comprehensive studies to elucidate its underlying mechanisms and validate its clinical relevance in epilepsy management.
Assuntos
Anticonvulsivantes , Modelos Animais de Doenças , Eletroencefalografia , Eletrochoque , Pentilenotetrazol , Convulsões , Animais , Masculino , Convulsões/tratamento farmacológico , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Camundongos , Anticonvulsivantes/farmacologia , Pentilenotetrazol/toxicidade , Eletroencefalografia/efeitos dos fármacos , Anisóis/farmacologia , Relação Dose-Resposta a Droga , Pilocarpina/toxicidade , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Ácido 3-Mercaptopropiônico/farmacologia , Convulsivantes/toxicidadeRESUMO
Epilepsy is a neurological disorder characterized by recurring seizures. It is necessary to further understand the mechanisms of epilepsy in order to develop novel strategies for its prevention and treatment. Abnormal endoplasmic reticulum stress (ERS) activation is related to the pathogenesis of epilepsy. Nuclear protein 1, transcriptional regulator (NUPR1) is involved in ERS and it might play a role in epilepsy progression. In the present study, we generated an epileptic mouse model using pilocarpine induction. After 72 h of pilocarpine treatment, the expression of NUPR1 was increased in epileptic mice. Furthermore, NUPR1 knockdown reduced the number of spontaneous recurrent seizures and alleviated hippocampal damage in these mice. Interestingly, NUPR1 knockdown also reduced the protein expression levels of LC3, PINK1, and Parkin in the mitochondria, and decreased the PINK1 expression in hippocampus. Additionally, the expression of ERS-related proteins-cleaved caspase-12, ATF4, and CHOP-decreased in epileptic mice following NUPR1 knockdown. In vitro experiments showed that the absence of NUPR1 reduced the expression of ATF4, CHOP, and cleaved caspase-12 in hippocampal neurons and inhibited the neuron apoptosis. In all, our study suggested that NUPR1 maybe a potential molecular target for epilepsy therapy.
Assuntos
Estresse do Retículo Endoplasmático , Epilepsia , Hipocampo , Animais , Epilepsia/metabolismo , Epilepsia/genética , Epilepsia/patologia , Camundongos , Masculino , Hipocampo/metabolismo , Hipocampo/patologia , Progressão da Doença , Neurônios/metabolismo , Neurônios/patologia , Apoptose/genética , Técnicas de Silenciamento de Genes , Pilocarpina , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Proteínas de Ligação a DNA , Proteínas de NeoplasiasRESUMO
The development of an effective transdermal drug delivery protocol to eccrine sweat glands is important for the advancement of research on the human sweating response. We investigated whether microneedle treatment prior to the application of pilocarpine, a hydrophilic and sudorific agent that does not induce sweating due to a limited percutaneous passive diffusion by skin application alone, augments sweat production. We applied three microneedle arrays to forearm skin sites simultaneously (n = 20). Upon removal of the microneedles, 1 % pilocarpine was applied to each site for 5-, 15-, and 30-min for the assessment of sweat gland function. In parallel, pilocarpine was administered by transdermal iontophoresis (5-min) at a separate site. Sweat rate was assessed continuously via the ventilated capsule technique. Pilocarpine augmented sweat rate at the 15- and 30-min periods as compared to the application at 5-min. The sweating responses induced by the 15- and 30-min application of pilocarpine were equivalent to â¼ 80 % of that measured at the iontophoretically treated sites. Notably, we observed a correlation in sweat rate between these two transdermal drug delivery methods. Altogether, our findings show that pre-treatment of microneedle arrays can enhance transdermal delivery efficiency of pilocarpine to human eccrine sweat glands.
Assuntos
Administração Cutânea , Iontoforese , Agulhas , Pilocarpina , Sudorese , Pilocarpina/administração & dosagem , Humanos , Sudorese/efeitos dos fármacos , Masculino , Adulto , Iontoforese/métodos , Feminino , Adulto Jovem , Sistemas de Liberação de Medicamentos/instrumentação , Agonistas Muscarínicos/administração & dosagem , Suor , Pele/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Dingxian Pill (DXP), a famous traditional Chinese medicine prescription, and has been widely proven to have positive therapeutic effects on "Xianzheng" (the name of epilepsy in ancient China). However, the anti-epileptic molecular mechanisms of DXP are not yet fully understood and remain to be further investigated. AIM OF THE STUDY: To elucidate the molecular mechanism of DXP's improvement in epileptic neuronal loss, damage and apoptosis by regulating TNF-α/TNFR1 signaling pathway. MATERIALS AND METHODS: Sixty Kunming mice were randomly divided in 6 groups: control group (equal volume of normal saline), model group (180 mg kg-1 pilocarpine hydrochloride - used to establish the epilepsy animal model), carbamazepine group (30 mg kg-1), and low, medium, and high-dose Dingxian Pill groups (4.08, 8.16, and 16.32 g kg-1, respectively - oral administration once daily for 2 weeks). Successful establishment of the epileptic mouse model was monitored with electroencephalography. Pathological changes in hippocampal tissue were analyzed with hematoxylin-eosin staining. Hippocampal neuronal apoptosis was analyzed with TUNEL staining. TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein expression levels in hippocampal tissue were analyzed with real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot, respectively. Cleaved caspase-8 protein levels in hippocampal tissue were measured with immunohistochemistry and Western blot. RESULTS: Compared to control, the model group showed an increase in continuous epileptic discharge waves on EEG, a damaged hippocampal neuron morphological structure, increased hippocampal neuronal apoptosis, and significantly increased TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein levels, and increased caspase-8 cleavage (P < 0.05). Compared to the model group, the carbamazepine group as well as the low-, medium-, and high-dose Dingxian Pill groups showed decreased epileptic discharges on EEG, an obvious hippocampal neuron morphological structure restoration, varying degrees of attenuated hippocampal neuronal apoptosis, and significantly decreased TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein levels as well as decreased caspase-8 cleavage (P < 0.05). CONCLUSIONS: Dingxian Pill exerts an anti-epileptic effect through inhibition of TNF-α/TNFR1 signaling pathway-mediated apoptosis in hippocampal neurons.