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Febrile seizures are childhood convulsions resulting from an infection that leads to an inflammatory response and subsequent convulsions. Prenatal stress has been shown to heighten the progression and intensity of febrile seizures. Current medications are costly and have adverse effects associated with prolonged use. Quercetin flavonoid exhibits anti-inflammatory, anti-convulsant, and anti-stress effects. This study was aimed to investigate the therapeutic effect of quercetin in a prenatally stressed rat model of febrile seizures. We hypothesized that quercetin will alleviate the effects of prenatal stress in a febrile seizure rat model. On gestational day 13, Sprague-Dawley rat dams were subjected to restraint stress for 1 hour/d for 7 days. Febrile seizures were induced on postnatal day 14 on rat pups by intraperitoneally injecting lipopolysaccharide followed by kainic acid and quercetin on seizure onset. Hippocampal tissue was harvested to profile cytokine concentrations. Our results show that quercetin suppresses prenatal stress-induced pro-inflammatory marker (interleukin 1 beta) levels, subsequently attenuating febrile seizures. This shows that quercetin can be therapeutic for febrile seizures in prenatally stressed individuals.
In total, 10 female and 5 male Sprague-Dawley rats were used for mating; these rats were acquired from the Biomedical Resource Centre of the University of KwaZulu-Natal where they were accommodated under standard laboratory conditions: room temperature of ±22°C, humidity of 70%, and a 12-hour light/dark cycle. Animals had free access to food and water ad libitum, and all experimental procedures performed were approved by the Animal Ethics Research Committee of the University of KwaZulu-Natal (ethics number AREC/057/015) in accordance with guidelines of the National Institutes of Health, USA. To prepare the female rats for mating, their oestrus cycle was synchronized 4 days before mating by putting 2 females in 1 cage. Studies show that ovarian cycles of female rats that live together become synchronized and this is mediated by pheromones.13,14 The oestrus cycle is a reproductive cycle of female rats lasting for 4 to 5 days and is observed by morphological modifications in the uterus, ovaries, and vagina.15 It is divided into 4 phases as follows: pro-oestrus, oestrus, metoestrus, and dioestrus.16 These phases are distinguished based on cell types observed in vaginal smears.16 The pro-oestrus and oestrus phases each last for 12 hours, whereas metoestrus takes 21 hours and dioestrus persists for 57 hours. The oestrus cycle was assessed daily by examination of a vaginal smear, and when a female was at pro-oestrus phase, a male was placed into the cage and a vaginal smear was obtained the following morning. Vaginal smears were collected as described by Marcondes et al,15 and the presence of sperm in the vaginal smear indicated that mating had taken place and that day was marked as gestational day 0 (GND0). The male rat was then taken out of the cage, and the pregnant dams were left undisturbed until gestational day 14 (GND14). On GND14, the pregnant dams were divided into 2 main groups: stressed group and non-stressed. The stress protocol was conducted from GND14 to GND20 as described by Cassim et al.17 Stress was introduced on GND14 because this is when gross neural structures of the CNS start specializing and also because the placental barrier becomes less active, thus the foetus is exposed to maternal stress hormones.18,19 Following birth, the pups remained with their dams until postnatal day (PND) 14. A total number of 24 pups (n = 3) were used in the study. Each group was divided into the following subgroups: non-stressed saline (NS-S), who received saline only; non-stressed febrile seizure (NS-FS), who received lipopolysaccharide (LPS) + kainic acid (KA); non-stressed quercetin, who received saline + quercetin (Q); and NS-FS treated with quercetin (NS-FSQ), who received LPS + KA + Q. Stressed saline (S-S), who received saline only; stressed febrile seizure (S-FS), who received LPS + KA; stressed quercetin, who received saline + quercetin; and S-FS treated with quercetin (S-FSQ), who received LPS + KA + Q. On PND 14, pups were separated from their dams and moved to the experimental room 1 hour before inducing febrile seizures for acclimatization. The control animals were injected intraperitoneally with 1-mL saline solution (0.9% NaCl; Adcock Ingram, Mid-rand, South Africa). The febrile seizure group was injected with a gram-negative bacterial endotoxin LPS (200 µg/kg, 0.2 mL, intraperitoneal; Sigma-Aldrich, St. Louis, MO, USA) and 2.5 hours later injected with a sublethal dose of the glutamate analogue KA (1.75 mg/kg, 0.2 mL, intraperitoneal; Sigma-Aldrich) to trigger convulsions.17 The treatment group was also injected with quercetin (10 mg/kg, 0.2 mL, intraperitoneal; Sigma-Aldrich) once there were noticeable convulsions in all the rats injected with LPS and KA.20 All the drugs were dissolved in 0.9% normal saline. The assessment of seizure intensity is schematically represented in Figure 1. Assessment of seizure intensity21,22 All the animals were moved to the autopsy room 1 hour post-seizure to be sacrificed. The animals were decapitated using a guillotine; the brains were harvested and dissected to collect hippocampal tissue. The tissue was then placed in 2-mL Eppendorf tubes, snap frozen in liquid nitrogen, and stored at −80°C bio-freezer for later analysis of cytokine levels. The total protein content of the hippocampal samples was determined using an assay that was a reducing agent compatible as well as a detergent compatible (Bio-Rad Laboratories, Hercules, CA, USA). The samples were prepared for analysis by adding ice-cold phosphate-buffered saline (0.01 M, pH = 7.4) and volume added based on the sample weight. The samples were then placed in ice and homogenized to disrupt the cell wall and release the cell contents using an ultrasonic sonicator (Qsonica, Newtown, CT, USA). The homogenates were then centrifuged for 5 minutes at 5000g to obtain the supernate. The supernate was transferred to new Eppendorf tubes. The concentration of IL-1ra was measured using a commercially available Rat IL-1ra enzyme-linked immunosorbent assay kit (Elabscience Biotechnology, Wuhan, China). The concentration of IL-1β, TNF-α, IL-6, and IL-10 was measured using a commercially available Bio-Plex Multiplex Rat Cytokine immunoassay (Bio-Rad Laboratories). The data were analysed with GraphPad Software Inc. La Jolla, CA, USA (version 5) software. Analysis of variance was performed on the rat hippocampal concentration of the following cytokines: IL-1β, TNF-α, IL-6, IL-1ra, and IL-10 with stress and febrile seizure as factors between rats. Significant main effects were followed by Bonferroni post hoc test. Differences were considered significant when P ⩽ .05.
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