Electroencephalographic and Behavioral Effects of Intranasal Administration of a Na+, K+-ATPase-Activating Antibody after Status Epilepticus.

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.

Non-Status Epilepticus female rats show seizure-like behaviors in the chronic phase of Pilocarpine experimental model / Ratas sem Status Epilepticus apresentam comportamento do tipo crise epiléptica na fase crônica do modelo experimental da Pilocarpina

Only few studies have focus on animals that received Pilocarpine (Pilo) and did not develop behavioral status epilepticus (SE) and, whether they may become epileptic in the model's chronic phase. Previews works observed mossy fiber sprouting in the hippocampus of Non-SE (NSE) rats, while others observed spontaneous and recurrent seizures (SRS) 6 - 8 months after animals received Pilo. It is known that neuronal excitability is influenced by female hormones, as well as, the occurrence of SE in castrated and non-castrated female rats. However, it is not known whether females that received Pilo and did not show SE, may have SRS. The aim of this work was to investigate whether castrated and non-castrated female rats that did not show behavioral SE after Pilo, will develop SRS in the following one-year. For that, animals received 360 mg/kg of Pilo and were video monitored for 12 months. SE females from castrated and non-castrated groups became epileptic since the first month after drug injection. Epileptic behaviors were identified watching video monitoring recordings in the fast speed. Castrated and Non castrated NSE animals showed behaviors resembling seizures described by Racine Scale stages 1 - 3. Motor alterations showed by NSE groups could be observed only when recordings were analyzed in slow speed. In addition, behavioral manifestations as, rhythmic head movements, sudden head movements, whole body movements and immobility were also observed in both, SE and NSE groups. We concluded that NSE female rats may have become epileptic. Adding to it, slow speed analysis of motor alterations was essential for the observation of NSE findings, which suggests that possibly many motor alterations have been underestimated in epilepsy experimental research.

Poucos são os estudos com foco em animais que receberam Pilocarpina (Pilo) e não desenvolveram status epilepticus (SE) comportamental e, se os mesmos se tornarão epilépticos na fase crônica do modelo. Autores observaram o brotamento das fibras musgosas no hipocampo de ratos Não-SE (NSE), enquanto outros observaram crises espontâneas e recorrentes (CER) 6 - 8 meses após receberam a droga. A excitabilidade neuronal é influenciada pelos hormônios femininos e, da mesma forma, a ocorrência de SE em ratas castradas e não-castradas. Entretanto, não é sabido se as fêmeas que não apresentam SE terão CER. O objetivo deste trabalho foi investigar se fêmeas castradas e não castradas que não tiveram SE comportamental após a injeção de Pilo desenvolverão CER dentro de um ano. Para isto, os animais receberam 360 mg/kg de Pilo e foram videomonitorados por 12 meses. As fêmeas SE castradas e não-castradas se tornaram epilépticas desde o primeiro mês pós Pilo. O comportamento epiléptico foi identificado assistindo as gravações na velocidade rápida. As fêmeas NSE castradas e não-castradas apresentaram comportamentos similares aos estágios 1 - 3 da Escala de Racine. As alterações motoras nestes grupos (NSE) foram observadas apenas quando as videomonitoração foi analisada na velocidade lenta. Além destas, manifestações comportamentais como movimentos rítmicos da cabeça, movimentos súbitos da cabeça, movimentos de todo o corpo e imobilidade também foram observadas em ambos grupos, SE e NSE. Concluímos que as fêmeas NE podem ter se tornado epilépticas. Adicionado a isto, a análise das alterações motoras na velocidade lenta foi essencial para a observação dos achados das fêmeas NSE, o que sugere que possivelmente muitas alterações motoras têm sido subestimados na pesquisa em epilepsia experimental.

Non-Status Epilepticus female rats show seizure-like behaviors in the chronic phase of Pilocarpine experimental model / Ratas sem Status Epilepticus apresentam comportamento do tipo crise epiléptica na fase crônica do modelo experimental da Pilocarpina

Only few studies have focus on animals that received Pilocarpine (Pilo) and did not develop behavioral status epilepticus (SE) and, whether they may become epileptic in the model's chronic phase. Previews works observed mossy fiber sprouting in the hippocampus of Non-SE (NSE) rats, while others observed spontaneous and recurrent seizures (SRS) 6 - 8 months after animals received Pilo. It is known that neuronal excitability is influenced by female hormones, as well as, the occurrence of SE in castrated and non-castrated female rats. However, it is not known whether females that received Pilo and did not show SE, may have SRS. The aim of this work was to investigate whether castrated and non-castrated female rats that did not show behavioral SE after Pilo, will develop SRS in the following one-year. For that, animals received 360 mg/kg of Pilo and were video monitored for 12 months. SE females from castrated and non-castrated groups became epileptic since the first month after drug injection. Epileptic behaviors were identified watching video monitoring recordings in the fast speed. Castrated and Non castrated NSE animals showed behaviors resembling seizures described by Racine Scale stages 1 - 3. Motor alterations showed by NSE groups could be observed only when recordings were analyzed in slow speed. In addition, behavioral manifestations as, rhythmic head movements, sudden head movements, whole body movements and immobility were also observed in both, SE and NSE groups. We concluded that NSE female rats may have become epileptic. Adding to it, slow speed analysis of motor alterations was essential for the observation of NSE findings, which suggests that possibly many motor alterations have been underestimated in epilepsy experimental research.(AU)

Poucos são os estudos com foco em animais que receberam Pilocarpina (Pilo) e não desenvolveram status epilepticus (SE) comportamental e, se os mesmos se tornarão epilépticos na fase crônica do modelo. Autores observaram o brotamento das fibras musgosas no hipocampo de ratos Não-SE (NSE), enquanto outros observaram crises espontâneas e recorrentes (CER) 6 - 8 meses após receberam a droga. A excitabilidade neuronal é influenciada pelos hormônios femininos e, da mesma forma, a ocorrência de SE em ratas castradas e não-castradas. Entretanto, não é sabido se as fêmeas que não apresentam SE terão CER. O objetivo deste trabalho foi investigar se fêmeas castradas e não castradas que não tiveram SE comportamental após a injeção de Pilo desenvolverão CER dentro de um ano. Para isto, os animais receberam 360 mg/kg de Pilo e foram videomonitorados por 12 meses. As fêmeas SE castradas e não-castradas se tornaram epilépticas desde o primeiro mês pós Pilo. O comportamento epiléptico foi identificado assistindo as gravações na velocidade rápida. As fêmeas NSE castradas e não-castradas apresentaram comportamentos similares aos estágios 1 - 3 da Escala de Racine. As alterações motoras nestes grupos (NSE) foram observadas apenas quando as videomonitoração foi analisada na velocidade lenta. Além destas, manifestações comportamentais como movimentos rítmicos da cabeça, movimentos súbitos da cabeça, movimentos de todo o corpo e imobilidade também foram observadas em ambos grupos, SE e NSE. Concluímos que as fêmeas NE podem ter se tornado epilépticas. Adicionado a isto, a análise das alterações motoras na velocidade lenta foi essencial para a observação dos achados das fêmeas NSE, o que sugere que possivelmente muitas alterações motoras têm sido subestimados na pesquisa em epilepsia experimental.(AU)

N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes.

Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 µg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.

Ellagic acid and allopurinol decrease H2O2 concentrations, epileptiform activity and astrogliosis after status epilepticus in the hippocampus of adult rats.

Status epilepticus (SE) can result in an overproduction of hydrogen peroxide (H2O2), which contributes to oxidative stress and brain injury during different phases of epileptogenesis and seizures. The purpose of this study was to evaluate the effects of ellagic acid and allopurinol administered after SE on H2O2 concentrations, electrical activity and GFAP immunoreactivity in the hippocampus of rats evaluated on Day 18 after SE. H2O2 levels were measured using an online technique with high temporal resolution and simultaneous electrical activity recording. For this purpose, the lateral ventricles of male Wistar rats (200-250 g) were injected with pilocarpine (2.4 mg/2 µl) to induce SE. After SE, rats were injected with ellagic acid (50 mg/kg i.p., and two additional doses at 24 and 48 h) or allopurinol (50 mg/kg i.p., single dose). Administration of ellagic acid or allopurinol after SE significantly reduced the H2O2 concentrations and decreased the presence of epileptiform activity and GFAP immunoreactivity in the hippocampus 18 days after SE. In conclusion, the administration of antioxidants potentially reduces oxidative stress, which indicates the possible attenuation of the neurobiological consequences after SE.

Lactation in large litters influences anxiety, memory, and spreading depression in adult male rats that were chronically subjected to a non-convulsive pilocarpine dose.

Objectives: Unfavorable lactation influences brain excitability and behavioral reactions in adults. Administration early in life of the cholinergic agonist, pilocarpine, even at non-convulsive doses, alters the brain excitability-related phenomenon known as cortical spreading depression (CSD), and produce anxiogenic-like behavior. However, the influence of unfavorable lactation on the CSD- and memory-effects of pilocarpine administration late in life has not been investigated. Herein, we analyzed the ponderal, electrophysiological (CSD), and behavioral effects of chronic treatment with a non-convulsive dose of pilocarpine, in adult rats suckled under favorable and unfavorable conditions.Methods: Wistar rats were suckled in litters with 9 or 15 pups (groups L9 and L15, respectively). A very low dose of pilocarpine (45/mg/kg/day) was chronically administered in mature rats from postnatal day (PND) 69-90. Behavioral tests occurred at PND91 [elevated plus maze (EPM)], PND93 [open field (OF)], and PND94-95 [object recognition memory (ORM)]. CSD was recorded between PND96-120.Results: Pilocarpine-treated rats performed worse in the anxiety and memory tests, and displayed lower CSD propagation velocity when compared with saline-treated controls. In addition, L15 rats showed an increase in the distance traveled and a decrease in the immobility time in the EPM, impaired ORM, and accelerated CSD propagation when compared with L9 rats (p ≤ 0.05).Discussion: These data suggest that sub-convulsive pilocarpine treatment in adult rats can affect behavioral and excitability-related reactions. In addition, unfavorable lactation increases the ambulatory effects of pilocarpine. Further studies should investigate the possible cholinergic molecular mechanisms involved in these effects.

The effects of pharmacological accommodation and cycloplegia on axial length and choroidal thickness / Efeitos da acomodação farmacológica e da cicloplegia no comprimento axial e na espessura da coroide

ABSTRACT Purpose: To investigate the effects of pharmacological accommodation and cycloplegia on ocular measurements. Methods: Thirty-three healthy subjects [mean (±SD) age, 32.97 (±5.21) years] volunteered to participate in the study. Measurement of the axial length, macular and choroidal thickness, refractive error, and corneal topography, as well as anterior segment imaging, were performed. After these procedures, pharmacological accommodation was induced by applying pilocarpine eye drops (pilocarpine hydrochloride 2%), and the measurements were repeated. The measurements were repeated again after full cycloplegia was induced using cyclopentolate eye drops (cyclopentolate hydrochloride 1%). The correlations between the measurements were evaluated. Results: A significant increase in subfoveal choroidal thickness after applying 2% pilocarpine was identified (without the drops, 319.36 ± 90.08 µm; with pilocarpine instillation, 341.60 ± 99.19 µm; with cyclopentolate instillation, 318.36 ± 103.0 µm; p<0.001). A significant increase in the axial length was also detected (without the drops, 23.26 ± 0.83 mm; with pilocarpine instillation, 23.29 ± 0.84 mm; with cyclopentolate instillation, 23.27 ± 0.84 mm; p=0.003). Comparing pharmacological accommodation and cycloplegia revealed a significant difference in central macular thickness (with pilocarpine instillation, 262.27 ± 19.34 µm; with cyclopentolate instillation, 265.93 ± 17.91 µm; p=0.016). Pilocarpine-related miosis (p<0.001) and myopic shift (p<0.001) were more severe in blue eyes vs. brown eyes. Conclusion: Pharmacological accommodation may change ocular measurements, such as choroidal thickness and axial length. This condition should be considered when performing ocular measurements, such as intraocular lens power calculations.(AU)

RESUMO Objetivo: Investigar os efeitos da acomodação farmacológica e da cicloplegia nas medições oculares. Métodos: participaram do estudo 33 voluntários saudáveis (média de idade [± DP], 32,97 anos [± 5,21 anos]). Foram medidos o comprimento axial, a espessura macular e coroidal e o erro refrativo, bem como realizados exames de imagem da topografia corneana e do segmento anterior. Em seguida, foi induzida a acomodação farmacológica aplicando-se colírio de pilocarpina (cloridrato de pilocarpina a 2%) e as medições foram repetidas nos participantes. As mesmas medições foram repetidas depois de induzir a cicloplegia completa com colírio de ciclopentolato (cloridrato de ciclopentolato a 1%) e foram avaliadas as correlações entre as medidas. Resultados: Identificou-se aumento significativo da espessura coroidal subfoveal com o uso da pilocarpina a 2% (sem colírio, 319,36 ± 90,08 µm; com a instilação de pilocarpina, 341,60 ± 99,19 µm; com a instilação de ciclopentolato, 318,36 ± 103,0 µm; p<0,001). Detectou-se também aumento significativo do comprimento axial (sem colírio, 23,26 ± 0,83 mm; com a instilação de pilocarpina, 23,29 ± 0,84 mm; com a instilação de ciclopentolato, 23,27 ± 0,84 mm; p=0,003). Ao se comparar a acomodação farmacológica e a cicloplegia, houve diferença significativa na espessura macular central (com a instilação de pilocarpina, 262,27 ± 19,34 µm; com a instilação de ciclopentolato, 265,93 ± 17,91 µm; p=0,016). Observou-se que a miose associada à pilocarpina (p<0,001) e o desvio miópico (p<0,001) foram mais severos nos olhos azuis que nos castanhos. Conclusão: A acomodação farmacológica pode alterar medidas oculares como a espessura da coroide e o comprimento axial. Essa possibilidade deve ser levada em consideração ao se efetuarem medições oculares, tais como cálculos de potência de lentes intraoculares.(AU)

Changes in Physiological and Pathological Behaviours Produced by Deep Microelectrode Implantation Surgery in Rats: A Temporal Analysis.

Physiological behaviours such as the sleep-wake cycle and exploratory behaviours are important parameters in intact and sham-operated animals and are usually thought to be unaffected by experimental protocols in which neurosurgery is performed. However, there is insufficient evidence in the literature on the behavioural and cognitive effects observed after deep microelectrode implantation surgery in animal models of neurological diseases. Similarly, in studies that utilize animal models of neurological diseases, the impact of surgery on the pathological phenomena being studied is often minimized. Based on these considerations, we performed a temporal analysis of the effects of deep microelectrode implantation surgery in the hippocampus of rats on quiet wakefulness, sleep, and exploratory activity and the pathological behaviours such as convulsive seizures according to the Racine scale. Male Wistar rats (210-300 g) were used and grouped in sham and epileptic animals. Single doses of pilocarpine hydrochloride (2.4 mg/2 µl; i.c.v.) were administered to the animals to generate spontaneous and recurrent seizures. Deep microelectrode implantation surgeries in both groups and analysis of Fast ripples were performed. Physiological and pathological behaviours were recorded through direct video monitoring of animals (24/7). Our principal findings showed that in epileptic animals, one of the main behaviours affected by surgery is sleep; as a consequence of this behavioural change, a decrease in exploratory activity was also found as well as the mean time spent daily in seizures of scale 4 and the number of seizure events of scales 4 and 5 was increased after surgery. No significant correlations between the occurrence of FR and seizure events of scale 4 (rho 0.63, p value 0.25) or 5 (rho -0.7, p value 0.18) were observed. In conclusion, microelectrode implantation surgeries modified some physiological and pathological behaviours; therefore, it is important to consider this fact when it is working with animal models.

Proconvulsant effects of sildenafil citrate on pilocarpine-induced seizures: Involvement of cholinergic, nitrergic and pro-oxidant mechanisms.

Sildenafil is a phosphodiesterase 5 inhibitor used for the treatment of erectile dysfunction and pulmonary hypertension. Proconvulsant effect is a serious adverse event associated with sildenafil use. Here, we investigated the possible proconvulsant effects of sildenafil in pilocarpine (PILO)-induced seizures model, which mimics some aspects of temporal lobe epilepsy. We also evaluated sildenafil's effects on hippocampal markers related to PILO-induced seizure, for instance, acetylcholinesterase (AChE) activity, oxidative stress and nitric oxide (NO) markers, namely nitrite, inducible NO synthase (iNOS) and neuronal NOS (nNOS). The influences of muscarinic receptors blockade on sildenafil proconvulsant effects and brain nitrite levels were also evaluated. Male mice were submitted to single or repeated (7 days) sildenafil administration (2.5, 5, 10 and 20 mg/kg). Thirty minutes later, PILO was injected and mice were further evaluated for 1 h for seizure activity. Sildenafil induced a dose- and time-progressive proconvulsant effect in PILO-induced seizures. Sildenafil also potentiated the inhibitory effect of PILO in AChE activity and induced a further increase in nitrite levels and pro-oxidative markers, mainly in the hippocampus. Repeated sildenafil treatment also increased the hippocampal expression of iNOS and nNOS isoforms, while the blockade of muscarinic receptors attenuated both sildenafil-induced proconvulsant effect and brain nitrite changes. Our data firstly demonstrated the proconvulsant effect of sildenafil in PILO-model of seizures. This effect seems to be related to an increased cholinergic-nitrergic tone and pro-oxidative brain changes. Also, our findings advert to caution in using sildenafil for patients suffering from neurological conditions that reduces seizure threshold, such as epilepsy.

Enduring effects of muscarinic receptor activation on adult hippocampal neurogenesis, microRNA expression and behaviour.

The cholinergic system is one of the most important neurotransmitter systems in the brain with key roles in autonomic control and the regulation of cognitive and emotional responses. However, the precise mechanism by which the cholinergic system influences behaviour is unclear. Adult hippocampal neurogenesis (AHN) is a potential mediator in this context based on evidence, which has identified this process as putative mechanism of antidepressant action. More recently, post-transcriptional regulation by microRNAs is another candidate mechanism based on its involvement in the regulation of AHN and neurotransmission. Taking into account this background, we evaluated the behavioural effects of a non-convulsant dose of pilocarpine - a non-selective muscarinic receptor (mAChR) agonist - in adult Wistar rats. Furthermore, we quantified the expression of different microRNAs implicated in the regulation of AHN. Our results suggests that pilocarpine treatment increases AHN in the granular cell layer but also induced ectopic neurogenesis. Pilocarpine treatment reduced immobility time in forced swimming test but did not affect fear conditioning response, sucrose preference or novelty supressed feeding behaviour. In addition, treatment with pilocarpine down-regulated the expression of 6 microRNAs implicated in the regulation of neurotrophin signalling and axon guidance pathways. Therefore, we suggest that the low-dose stimulation of the cholinergic system is sufficient to alter AHN of rats through post-transcriptional mechanisms, which might contribute to long-lasting behavioural effects.

Coronary vasodilation impairment in pilocarpine model of epilepsy.

We investigated the coronary arteries reactivity alterations in rats with epilepsy induced by pilocarpine. To do so, male Wistar rats weighing between 250 g and 300 g were used. Status epilepticus (SE) was induced in rats using 385 mg/kg (i.p.) of pilocarpine. After 60 days from the first spontaneous seizure, rats were submitted to heart rate measurements and then, one day after, euthanized, and the heart was dissected and submitted to constant flow Langendorff approaches to evaluate coronary reactivity. Rats with epilepsy showed higher resting heart rate and impairment of coronary vasodilation induced by bradykinin. Endothelial nitric oxide synthase (eNOS) and superoxide dismutase (SOD) presented a reduced staining in coronary arteries, and eNOS expression was also reduced in the left ventricle of rats with epilepsy. Our findings demonstrated, for the first time, that epilepsy can cause impairment of coronary arteries reactivity, probably because of an endothelial dependent mechanism.

Central muscarinic and LPBN mechanisms on sodium intake.

Central cholinergic activation stimulates water intake, but also NaCl intake when the inhibitory mechanisms are blocked with injections of moxonidine (α2 adrenergic/imidazoline agonist) into the lateral parabrachial nucleus (LPBN). In the present study, we investigated the involvement of central M1 and M2 muscarinic receptors on NaCl intake induced by pilocarpine (non-selective muscarinic agonist) intraperitoneally combined with moxonidine into the LPBN or by muscimol (GABAA agonist) into the LPBN. Male Holtzman rats with stainless steel cannulas implanted bilaterally in the LPBN and in the lateral ventricle were used. Pirenzepine (M1 muscarinic antagonist, 1 nmol/1 µl) or methoctramine (M2 muscarinic antagonist, 50 nmol/1 µL) injected intracerebroventricularly (i.c.v.) reduced 0.3 M NaCl and water intake in rats treated with pilocarpine (0.1 mg/100 g of body weight) injected intraperitoneally combined with moxonidine (0.5 nmol/0.2 µL) into the LPBN. In rats treated with muscimol (0.5 nmol/0.2 µL) into the LPBN, methoctramine i.c.v. also reduced 0.3 M NaCl and water intake, however, pirenzepine produced no effect. The results suggest that M1 and M2 muscarinic receptors activate central pathways involved in the control of water and sodium intake that are under the influence of the LPBN inhibitory mechanisms.

A visão em pseudofácicos com ametropia residual após instilação de pilocarpina a 2% / Pseudophakic Vision with residual ametropia after pilocarpine 2% instillation

Resumo Objetivo: Avaliar modificações de acuidade visual, refração, campo visual e diâmetro pupilar, em pacientes pseudofácicos, após a instilação de pilocarpina a 2%. Métodos: Ensaio clínico, controlado, mascarado e randomizado realizado entre maio de 2015 e setembro de 2016 no Hospital Universitário Gaffrée e Guinle, RJ, Brasil. Quarenta pacientes divididos em 2 grupos foram acompanhados em pós-operatório de facectomia com implante de LIO. No grupo de casos houve aplicação de uma gota de pilocarpina a 2%, no grupo controle, uma gota de lubrificante no olho operado. Foram avaliadas antes e 1 hora após a instilação do colirio: a acuidade visual para longe e perto com e sem correção; a refração; o diâmetro pupilar e o campo visual. Resultados: No grupo de casos, a acuidade visual s/c para longe aumentou de 0,33 para 0,57 (p = 0,0001) e para perto melhorou também, 13 pacientes (59,09%) possuíam acuidade visual de J1 ou J2 antes da instilação e depois o número aumentou para 18 ou 81,81% (p = 0,0054). O diâmetro pupilar reduziu de 2,00mm para 1,85mm (p < 0,0001). Não houve alteração do campo visual central. No grupo controle, não houve variação estatisticamente ou clinicamente significativa de qualquer um dos parâmetros medidos. Conclusão: A administração tópica de uma gota de pilocarpina a 2% melhorou a visão de pacientes pseudofácicos com ametropia residual para longe e para perto. Estudos de dose-efetividade adicionais podem indicar melhores concentrações e posologias para alcançar maiores melhoras de acuidade visual.

Abstract Objective: Evaluate the visual acuity, refraction, visual field changes and pupillary diameter in pseudophakic patients after instillation of 2% pilocarpine eye drops. Methods: Controlled, masked and randomized clinical trial carried out between May, 2015 and September, 2016 at the Gaffrée and Guinle University Hospital, RJ, Brazil. Forty patients, divided into 2 groups, were followed up in the postoperative period of a facectomy with intraocular lens implant. The patients in the group of cases were submitted to a drop of 2% pilocarpine and those of the control group to a drop of lubricant in the operated eye. Before eye drop instillation nd one hour after it, the authors evaluated: visual acuity for distance and near; refraction; pupillary diameter and visual field. Results: In case group visual acuity increased from 0.33 to 0.57 for far (p = 0.0001)and also increased for near, 13 patients (59.09%) had visual acuity of J1 or J2 before instillation and 18 or 81.81% after it (p = 0.0054). The median pupillary diameters raised from 2.00 mms to 1.85 mm(p <0.0001). Central visual fields did not have significant alteration. In the control group, there were no statistically or clinically significant changes in any of the measured parameters. Conclusion: Topical administration of a 2% pilocarpine eye drop was effective to improve pseudophakic patients vision with residual ametropia for far and near. Additional dose-effectiveness studies may indicate better concentrations and dosages to achieve greater improvements in visual acuity.

Neuroprotective effects of levetiracetam, both alone and combined with propylparaben, in the long-term consequences induced by lithium-pilocarpine status epilepticus.

Status epilepticus (SE) is a neurological condition that frequently induces severe neuronal injury in the hippocampus, subsequent epileptogenesis and pharmacoresistant spontaneous recurrent seizures (SRS). The repeated administration of LEV (a broad-spectrum antiepileptic drug) during the post-SE period does not prevent the subsequent development of SRS. However, this treatment reduces SE-induced neurodegeneration in the hippocampus. Conversely, propylparaben (PPB) is a widely used antimicrobial that blocks voltage-dependent Na+ channels, induces neuroprotection and reduces epileptiform activity in vitro. The present study attempted to determine if the neuroprotective effects induced by LEV are augmented when combined with a sub-effective dose of PPB. Long-term SE-induced consequences (hyperexcitability, high glutamate release, neuronal injury and volume loss) were evaluated in the hippocampus of rats. LEV alone, as well as combined with PPB, did not prevent the occurrence of SRS. However, animals treated with LEV plus PPB showed high prevalence of low frequency oscillations (0.1-4 Hz and 8-90 bands, p < 0.001) and low prevalence of high frequency activity (90-250 bands, p < 0.001) during the interictal period. In addition, these animals presented lower extracellular levels of glutamate, decreased rate of neurodegeneration and a similar hippocampal volume compared to the control conditions. This study's results suggest that LEV associated with PPB could represent a new therapeutic strategy to reduce long-term consequences induced by SE that facilitate pharmacoresistant SRS.

Pilocarpine/ascorbic acid interaction in the immature brain: Electrophysiological and oxidative effects in well-nourished and malnourished rats.

Ascorbic acid (AA) administration has been associated with neuroprotection against oxidative stress, although at high doses it can facilitate oxidation and acts like a proconvulsing drug. The pilocarpine-induced epilepsy model has been widely studied. However, less is known about the effects of sub-convulsive doses of pilocarpine on brain activity in immature animals under normal or deficient nutritional conditions. Herein, we investigated the effects of chronic pilocarpine administration in a sub-convulsive dose, with or without AA, on the excitability-related phenomenon denominated as cortical spreading depression (CSD) and levels of lipid peroxidation-induced malondialdehyde in well-nourished and malnourished rats. At postnatal days 7-28, rats received no gavage treatment (naïve group), saline (vehicle group), 45 mg/kg/d of pilocarpine and/or 120 mg/kg/d of AA. CSD propagation and malondialdehyde levels were analyzed at 34-40 days. The pilocarpine group presented with lower CSD velocities, while AA groups exhibited higher CSD velocities and augmented malondialdehyde levels compared with controls. The co-administration of AA partially antagonized the pilocarpine CSD effects, but did not revert it to control levels. Malnutrition increased CSD amplitude and velocity in comparison to the well-nourished condition. The electrocorticogram (ECoG) amplitude increased after CSD (ECoG potentiation) when compared with the baseline amplitude before CSD. However, no intergroup difference was observed in this CSD-related ECoG potentiation. The results support the hypothesis of a pilocarpine/ascorbic acid interaction in the immature rat brain and might help further the understanding of this interaction on neuronal electrical activity and oxidative stress.

Pupillometry in Chagas disease.

PURPOSE: We investigated parasympathetic innervation abnormalities of the iris sphincter and ciliary muscles in chronic Chagas disease by measuring pupillary diameter and intraocular pressure. METHODS: A group of 80 patients with Chagas disease was compared with 76 healthy individuals without chagasic infection. The following procedures were performed: pupillometry, hypersensitivity test to pilocarpine 0.125%, intraocular pressure measurement (IOP), basal pupil diameter (BPD), absolute pupillary constriction amplitude (ACA), relative pupillary constriction amplitude (RCA) and the presence of anisocoria. RESULTS: The prevalence of anisocoria was higher in chagasic patients (p<0.01). These patients had mean basal pupillary diameter, mean photopic pupillary diameter and mean value of absolute pupillary constriction amplitude significantly lower than non-chagasic ones (p<0.01, mean difference -0.50mm), (p=0.02, mean difference -0.20mm), (p<0.01, mean difference -0.29mm), respectively. The relative pupillary constriction amplitude did not differ between the two groups (p=0.39, mean difference -1.15%). There was hypersensitivity to dilute pilocarpine in 8 (10%) of the chagasic patients in the right eye and in 2 (2.5%) in the left eye and in 1 (1.25%) in both eyes. The mean value of intraocular pressure had a marginal statistical significance between the two groups (p=0.06, mean difference -0.91mmHg). CONCLUSIONS: Patients with chagasic infection may exhibit ocular parasympathetic dysfunction, demonstrable by pupillometry and the dilute pilocarpine hypersensitivity test.

Pupillometry in Chagas disease / Pupilometria na doença de Chagas

ABSTRACT Purpose: We investigated parasympathetic innervation abnormalities of the iris sphincter and ciliary muscles in chronic Chagas disease by measuring pupillary diameter and intraocular pressure. Methods: A group of 80 patients with Chagas disease was compared with 76 healthy individuals without chagasic infection. The following procedures were performed: pupillometry, hypersensitivity test to pilocarpine 0.125%, intraocular pressure measurement (IOP), basal pupil diameter (BPD), absolute pupillary constriction amplitude (ACA), relative pupillary constriction amplitude (RCA) and the presence of anisocoria. Results: The prevalence of anisocoria was higher in chagasic patients (p<0.01). These patients had mean basal pupillary diameter, mean photopic pupillary diameter and mean value of absolute pupillary constriction amplitude significantly lower than non-chagasic ones (p<0.01, mean difference -0.50mm), (p=0.02, mean difference -0.20mm), (p<0.01, mean difference -0.29mm), respectively. The relative pupillary constriction amplitude did not differ between the two groups (p=0.39, mean difference -1.15%). There was hypersensitivity to dilute pilocarpine in 8 (10%) of the chagasic patients in the right eye and in 2 (2.5%) in the left eye and in 1 (1.25%) in both eyes. The mean value of intraocular pressure had a marginal statistical significance between the two groups (p=0.06, mean difference -0.91mmHg). Conclusions: Patients with chagasic infection may exhibit ocular parasympathetic dysfunction, demonstrable by pupillometry and the dilute pilocarpine hypersensitivity test.

RESUMO Introdução: Investigaram-se anormalidades da inervação parassimpática dos músculos esfíncter da íris e ciliar na doença de Chagas crônica, através de medidas pupilares e da pressão intraocular. Métodos: Foram estudados dois grupos, um com 80 chagásicos e outro com 76 indivíduos saudáveis sem infecção chagásica. Foram realizados os seguintes procedimentos: pupilometria, teste de hipersensibilidade à pilocarpina a 0,125%, medida da pressão intraocular (PIO), diâmetro basal da pupila (DBP), amplitude de constrição pupilar absoluta (ACA), amplitude de constrição pupilar relativa (ACR), e presença de anisocoria. Resultados: A prevalência de anisocoria foi maior nos chagásicos (p<0,01). Estes pacientes apresentaram diâmetro basal pupilar médio, diâmetro fotópico médio e valor médio da amplitude de constrição pupilar absoluta, significativamente menores que os não chagásicos, (p<0,01, diferença de média -0,50mm), (p=0.02, diferença de média -0,20mm), (p<0,01, diferença de média -0,29mm), respectivamente. A amplitude de constrição pupilar relativa não diferiu entre os dois grupos (p=0,39, diferença de média -1,15%). Houve hipersensibilidade à pilocarpina diluída em 8 (10%) chagásicos no olho direito em 2 (2,5%) no olho esquerdo e em 1 (1,25%) em ambos os olhos. O valor médio da pressão intraocular teve significância marginal entre os dois grupos (p=0,06, diferença de média -0,91mmHg). Conclusões: Pacientes com infecção chagásica podem apresentar disfunção parassimpática ocular, demonstrável pela pupilometria e pelo teste de hipersensibilidade à pilocarpina diluída.

The apoptotic effect of midazolam on the salivary gland of rats and the reversal effect by pilocarpine.

To evaluate the apoptosis in parotid glands of rats treated with midazolam associated or not with pilocarpine, 60 Wistar rats were assigned to 6 groups: control groups received saline solution for 30 days (S30) and 60 days (S60) and the other groups received pilocarpine for 60 days (P60), midazolam for 30 days (M30), midazolam for 30 days and 30 days of saline (M30 + S30), and finally midazolam for 30 days and 30 days of midazolam and pilocarpine (M30 + MP30). Histological sections were subjected to the TdT-mediated dUTP-biotin nick and labeling technique. The number of positive and negative cells was quantified, calculating the apoptotic index. ANOVA at 2 criteria and Tukey's test were used. A greater apoptotic index was observed in the M30 (52.79 ± 9.01) and M30 + S30 (62.43 ± 8.52) groups when compared with the S30 (37.94 ± 5.94) and S60 (31.85 ± 9.18) groups, respectively (p < 0.05). There was no difference between M30 + MP30 (30.98 ± 6.19) and S60 (31.85 ± 9.18) groups regarding apoptotic index. Chronic administration of midazolam has been shown to increase the number of apoptotic cells in the parotid glands of rats. However, pilocarpine inhibited this effect, thus inhibiting the apoptosis.

Behavioral and biochemical effects of ethanol withdrawal in zebrafish.

Chronic alcohol use induces adaptations and toxicity that can induce symptoms of anxiety, autonomic hyperarousal, and epileptic seizures when alcohol is removed (withdrawal syndrome). Zebrafish has recently gained wide attention as a behavioral model to study the neurobehavioral effects of acute and chronic alcohol use, including withdrawal. The literature, however, is very contradictory on findings regarding withdrawal effects, with some studies reporting increased anxiety, while others report no effect. A meta-analytic approach was taken to find the sources of this heterogeneity, and ethanol concentration during exposure and exposure duration were found to be the main sources of variation. A conceptual replication was also made using continuous exposure for 16 days in waterborne ethanol (0.5%) and assessing anxiety-like behavior in the light/dark test after 60 min withdrawal. Withdrawal was shown to reduce preference for darkness, consistent with decreased anxiety, but to increase risk assessment, consistent with increased anxiety. Animals were also subjected to the withdrawal protocol and injected with pilocarpine in a sub-convulsive dose to assess susceptibility to epileptic seizure-like behavior. The protocol was sufficient to increase susceptibility to epileptic seizure-like behavior in animals exposed to ethanol. Finally, withdrawal also decreased catalase activity in the brain, but not in the head kidney, suggesting mechanisms associated with the behavioral effects of ethanol withdrawal.

DNA damage and oxidative stress induced by seizures are decreased by anticonvulsant and neuroprotective effects of lobeline, a candidate to treat alcoholism.

The alkaloid lobeline (Lob) has been studied due to its potential use in treatment of drug abuse. This study evaluates the possible anticonvulsant and neuroprotective activities of Lob to obtain new information on its properties that could confirm it as a candidate in the treatment of alcohol addiction. The anticonvulsant effect of Lob was evaluated using a pilocarpine-induced seizure model. In addition, possible neuroprotective effects were investigated measuring DNA damage using the comet assay, assessing free radical levels by dichlorofluorescein diacetate (DCF) oxidation, and measuring the antioxidant potential using the α, α-diphenyl-ß-picrylhydrazyl (DPPH) scavenging assay, besides measuring superoxide dismutase (SOD) and catalase (CAT) enzyme activities in brain tissues. Lobeline increased the latency to the first seizure and decreased the percentage of seizures in a similar way as diazepam, used as control. DNA damage induced by Pil and hydrogen peroxide were decreased in hippocampus and cerebral cortex from mice treated with Lob. The levels of free radicals and CAT activity increased in cortex and hippocampus, respectively, in mice treated with Pil. Lobeline decreased CAT in hippocampus, leading to similar values as in the saline negative control. In conclusion, Lob has anticonvulsant and neuroprotective actions that may be mediated by antioxidant-like mechanisms, indicating its potential as candidate drug in alcoholism therapy.