| BackgroundEpilepsy, a common chronic neurologic disorder characterized by recurrent seizures, is caused by abnormal, paroxysmal changes in the electrical activity of the brain. It has high morbidity in children and adults. Convulsion, characterized by an abnormal, involuntary contraction of the muscles, is most typically seen with seizure disorders and is always accompanied by conscious disturbance. Because a convulsion is often a symptom of an epileptic seizure, the term convulsion is sometimes used as a synonym for seizure. Frequent and serious seizures can lead to further brain injury as well as persistent mental and behavioral disorders, bringing troubles to the patients, their families and society. Epileptogenesis is defined as the process of developing epilepsy. The mechanisms of epileptogenesis are complicated, which involve neurotransmitter imbalance, ion channel and genetic abnormalities, and glial dysfunction. Although great progress has been made in the etiology, pathogenesis and treatment of epilepsy, the exact mechanisms of epileptogenesis still remain unclear.Astrocyte and microglia, the important component cells of the central nervous system (CNS), play an essential role in maintaining brain homeostasis, development and pathologies and participate actively in the process of neuronal injury and repair. Neuroinflammation is the term used to describe CNS inflammatory responses characterized by activation and proliferation of glia (especially astrocytes and microglia) and the associated up-regulation of proinflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)a, and IL-6, and anti-inflammatory cytokines such as IL-10. In recent years, accumulated data have indicated that neuroinflammation participates in the pathogenesis of epilepsy. A recognized response to seizures and a potential contributor to mechanisms of epileptogenesis is excessive or prolonged glial activation and the associated increase in proinflammatory cytokine production. Appropriate neuroinflammation occurs as protective responses to CNS insults. However, excessive neuroinflammation can cause damage to the blood-brain-barrier (BBB), leading to increased BBB permeability and exaggerated leukocyte infiltration and enhance neuronal excitability and exacerbate seizure-induced brain injury. Thus, the relationship between neuroinflammation and epileptogenesis is represented as an amplifying feedback loop.There is an increasing body of evidence suggesting that infection occurring within the intrauterine or perinatal environment can not only produce acute brain injury but affect brain as well as psychological and behavioral development in the long term. Insults to the fetus during critical times of development may alter fetal brain structure and function thereby acting as risk factors for mental, psychiatric and behavioral disorders later in life. Maternal infections of both viral and bacterial origin have been linked with later development in the offspring of autism, schizophrenia and cerebral palsy. For instance, prospective epidemiological studies indicate that maternal influenza, toxoplasmosis, and genital/reproductive infection are associated with schizophrenia. Moreover, inflammation in the neonatal period can also have long-term functional effects on metabolic, immune, behavioral and neuroendocrine system that persist into adulthood, leading to increased susceptibility to neuropsychiatric and behavioral disorders. Rodent models have been developed to determine the behavioral and biological effects of maternal immune activation (MIA) on offspring. In these studies, lipopolysaccharide (LPS) or the synthetic, double-stranded RNA polyriboinosinic-polyribocytidilic acid (polyI：C) are administered to pregnant dams to mimic the immune-stimulating actions of live bacterial or viral infections, respectively. LPS is a large lipid-polysaccharide complex released from the outer cell wall of Gram-negative bacteria that binds to the toll-like receptor4(TLR4) and TLR2expressed on the surface of certain cell types resulting in the activation of nuclear factor-KB (NF-kB) which induces proinflammatory cytokine expression. In the MIA model, maternal LPS increases proinflammatory cytokine mRNA expression and/or proteins in the maternal serum, amniotic fluid, and placenta. Increased cytokine gene expression in fetal brain has been reported in some studies that used a much higher dose of LPS. Recently, accumulating data from animal studies have indicated that prenatal or neonatal LPS can induce long-term hippocampal microglial and astrocytic activation that persisted into adulthood. Since excessive neuroinflammation can cause increased seizure susceptibility, we hypothesize that early life immune challenge may predispose the maturing brain more vulnerable to later-life seizures.In accordance with our hypothesis, recent epidemiological studies have indicated that maternal infection during pregnancy, such as cystitis, vaginal yeast infection, consistent diarrhea lasting4days and coughs lasting1week was associated with an increased risk for childhood epilepsy. Moreover, intrapartum infection is associated with a higher risk for newborn seizures. A recent animal study has shown that neonatal immune challenge can increase seizure susceptibility in adult rats. However, whether early life infection causes long-lasting brain injury and heightened susceptibility to later seizures and the possible mechanisms underlying this association remain largely unexplored experimentally. To make clealy these questions will help to clarify the mechanisms of epileptogenesis, and will be possible to find new methods to treat this disease.Chapter I Effect of maternal immune activation during late gestation on seizure susceptibility in juvenile rat offspringObjectivesThe study was designed to investigate whether maternal immune challenge during late gestation could influence juvenile seizure susceptibility and the associated brain injury as well as the possible mechanisms. Methods1. The construction of maternal immune activation modelLipopolysaccharide (LPS) diluted in saline was injected intraperitoneally into pregnant Sprague-Dawley (SD) rats on gestational day (GD)19and20at a dose of300ug/kg. Control dams were injected with a corresponding volume of normal saline (NS) at the same GDs. Pregnant females were monitored for the parturition date. In addition, litter size and birth weight of pups were recorded.2. Kainic acid (KA)-induced status epilepticus (SE) model in juvenile rat offspringJuvenile offspring whose mothers received NS or LPS during late pregnancy received7.5mg/kg KA intraperitoneally at postnatal day (P)21. Instead of KA administration, control rats received comparable volume of NS as given to KA-injected rats. A seizure severity grade was assigned according to a modified Racine scale as follows:0—no response; Ⅰ—motionless staring, and slight facial and mouth movements; Ⅱ—pawing, head nobbing, and wet-dog shake; Ⅲ—unilaternal forelimb clonus; Ⅳ—bilaternal forelimb clonus with rearing; Ⅴ—rearing, generalized tonic-clonic seizures and transient loss of postural control. Animals were closely observed and seizure severity and latency to the first sign of seizure were recorded. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)2h after onset of SE. Only animals with grade IV or V seizures were included in this study. Animals were returned to their dams after behavioral monitoring.Thus, four experimental groups were studied, including normal control (NS-NS), prenatal inflammation (LPS-NS), juvenile seizure (NS-KA), and "two-hit"(LPS-KA) groups.3. KA seizure susceptibility testingThe latency to the first behavioral seizure after KA administration, referred to as seizure onset time (SOT), was defined by the occurrence of forelimb clonus, rearing and loss of balance, and was recorded to the nearest second for each animal by an individual blind to the prenatal treatment of the animal. SOT is a commonly used measure to describe seizure susceptibility to convulsant compounds in rats. 4. At acute stage, rats were killed6h after SE, and examined for production of cytokines, including IL-1β, TNFa, IL-6and IL-10using real-time polymerase chain reaction (RT-PCR).5. Glial activation state was analyzed by western blotting of glial markers, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule1(Ibal)24h after SE.6. Three days after SE, rats were sacrificed for further examination of glial activation by immunohistological methods.7. Three days after SE, rats were sacrificed for examination of neuronal injury by NeuN, Nissl and Fluoro-Jade B (FJB) staining.8. At P70, water maze task was used to evaluate the long-term impact of maternal inflammation on spatial learning and memory ability of adult rat offspring.9. After water maze test, locomotor activity and exploratory ability were tested using an open-field apparatus.Results:1. The effect of maternal LPS exposure on gestational length, litter size and birth weight of pupsWe did not observe any significant difference in gestation length or litter size between LPS-exposed dams and controls, though some LPS-exposed pregnant rats died after LPS application, suffered from miscarriage or had stillborn pups. The offspring of LPS-treated dams displayed significantly reduced birth weight and weight gain at P9compared to controls. Pup body weight did not differ between groups at P21.2. Prenatal LPS increases juvenile seizure susceptibility to KARats exposed to LPS prenatally showed faster mean SOT compared with controls, indicating that maternal LPS exposure increased juvenile seizure susceptibility.3. Cytokine expression in the hippocampus of P21rats after KA-induced SEThere was no difference in the mRNA expression of the four cytokines, including IL-1β, TNF-a, IL-6and IL-10between LPS-NS and NS-NS groups. The expression of the four cytokines was higher in NS-KA group compared with NS-NS group. Compared with NS-KA group, the expression of IL-1β mRNA and IL-6mRNA was higher in LPS-KA group. However, no difference in the expression of IL-10mRNA and TNFa mRNA was observed between LPS-KA and NS-KA rats.4. Hippocampal microgilal activation after KA-induced SEWestern blot analysis of Iba1expression and immunohistochemical staining for Ibal showed similar results. Compared with NS-NS group, LPS-NS and NS-KA rats had higher hippocampal Ibal expression. Compared with NS-KA group, LPS-KA group had higher hippocampal Ibal expression. These results demonstrated that prenatal inflammation not only caused prolonged microglial response but also enhanced KA-induced microglial activation.5. Hippocampal astrocyte activation after KA-induced SEWestern blot analysis of GFAP expression and immunohistochemical staining for GFAP showed similar results. Compared with NS-NS group, LPS-NS and NS-KA rats had higher hippocampal GFAP expression. Compared with NS-KA group, LPS-KA group had higher hippocampal GFAP expression. These results demonstrated that prenatal inflammation not only caused prolonged astrocytic response but also enhanced KA-induced astrocytic activation.6. Neuronal damage in the hippocampus3days after KA-induced SENissl staining showed that there was no neuronal damage in NS-NS rats. Compared to NS-NS group, Nissl-positive cell counts were significantly reduced in LPS-NS, NS-KA and LPS-KA groups. Fewer Nissl-staining cells were detected in the LPS-KA group compared with NS-KA group.The results for NeuN and FJB staining were consistent with those of Nissl staining.7. The performance of adult rat offspring in the Morris water maze after KA-induced SEPerformance improved in all groups over the four training days in the place navigation test. Compared to NS-NS group, escape latency was significantly increased in LPS-NS, NS-KA and LPS-KA groups. Escape latency in LPS-KA rats was greater than that in NS-KA rats. On the day of probe testing, NS-NS rats spent significantly more time in the (target quadrant) TQ than animals from the other three groups."Two-hit" rats spent significantly less time in the TQ than animals in the NS-KA group.8. The performance of adult rat offspring in the open field after KA-induced SENo difference in the number of squares crossed and rearing frequency was observed among NS-NS, LPS-NS, NS-KA and LPS-KA groups.No difference in the number of head dippings was observed between NS-NS and LPS-NS groups. NS-KA and LPS-KA groups demonstrated smaller number of head dippings compared with that of NS-NS control."Two-hit" rats showed smaller number of head dippings compared with NS-KA group.Conclusins1. Maternal infection increases seizure susceptibility to KA in juvinile rat offspring;2. Maternal infection, without perturbation of cytokine expression, causes long-lasting neuronal injury, glial alteration and cognitive deficit in juvenile offspring;3. Maternal infection during late pregnancy enhances neuronal injury, exaggerates neuroinflammatory responses, and exacerbates long-term neurobehavioral impairment associated with a second adolescent KA insult.In sum, primed glia by prenatal infection may underlie, at least in part, long-term "perinatal programming" of later-life vulnerability to seizure insult. Chapter II Prenatal immune challenge in rats increases susceptibility to seizure-induced brain injury in adulthoodObjectivesThe study was designed to investigate whether maternal immune challenge during gestation could influence adult seizure susceptibility and the associated brain injury. Methods1. The construction of maternal immune activation modelLipopolysaccharide (LPS, Escherichia coli, serotype055:B5) diluted in saline was injected intraperitoneally into pregnant Wistar rats on gestational day (GD)15and16at a dose of200ug/kg. Control dams were injected with a corresponding volume of normal saline (NS) at the same GDs.2. Lithium-pilocarpine (LiPC)-induced status epilepticus (SE) model in adult rat offspringAt P45, male offspring from LPS-or NS-treated mothers received intraperitoneal injections of lithium chloride (127mg/kg) followed16h later by pilocarpine (36mg/kg, i.p.)(LiPC). Instead of pilocarpine application, control rats received corresponding volumes of NS as given to the pilocarpine-injected rats. A seizure severity grade was assigned according to a modified Racine scale (detailed in part1). Animals were closely observed and seizure severity and latency to the first sign of seizure were recorded. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)1h after the onset of SE. Only animals with grade IV or V seizures were included in this study.Four experimental groups were studied, including normal controls (NS-NS), prenatal inflammations (LPS-NS), adult seizures (NS-LiPC), and "two-hit"(LPS-LiPC) animals.3. LiPC seizure susceptibility testingSOT was used to assess seizure susceptibility (detailed in part1).4. Three days after SE, rats were sacrificed for examination of neuronal injury by Nissl staining.5. At P61, locomotor activity and exploratory ability were tested using an open-field apparatus.6. At P68, anxiety was examined using the elevated plus maze.7. Begining at P75, water maze task was used to evaluate long-term impact of maternal inflammation on spatial learning and memory ability of adult rat offspring. Results:1. Prenatal LPS increases adult seizure susceptibility to LiPCRats exposed to LPS prenatally showed faster mean SOT compared with controls, indicating that maternal LPS exposure increased adult seizure susceptibility.2. Neuronal damage in the hippocampus3days after KA-induced SENissl staining showed that there was no hippocampal neuronal damage in NS-NS rats. Compared to NS-NS group, Nissl-positive cell counts were significantly reduced in LPS-NS and NS-LiPC groups. Fewer Nissl-staining cells were detected in the LPS-LiPC group compared with NS-LiPC group. These results demonstrated that prenatal inflammation not only caused neuronal injury but also enhanced LiPC-induced neuronal damage.3. The performance of adult rat offspring in the open field after LiPC-induced SENo difference in the number of squares crossed and rearing frequency was observed among NS-NS, LPS-NS, NS-LiPC and LPS-LiPC groups.No difference in the number of head dippings was observed between NS-NS and LPS-NS groups. In addition, no difference in the number of head dippings was observed between NS-NS and NS-LiPC groups."Two-hit" rats showed smaller number of head dippings compared with NS-LiPC group.4. The performance of adult rat offspring in the elevated plus maze after LiPC-induced SEThe offspring of LPS-treated mothers (LPS-NS) had significantly lower anxiety score than NS-NS controls, indicating that maternal immune challenge caused increased anxiety in offspring. No significant difference in anxiety scores was found in LPS-LiPC rats compared with NS-LiPC rats. The "single hit" NS-LiPC animals had higher anxiety scores than NS-NS controls.5. The performance of adult rat offspring in the Morris water maze after KA-induced SEPerformance improved in all groups over the four training days in the place navigation test. Compared to NS-NS group, escape latency was significantly increased in LPS-NS and NS-LiPC groups. Escape latency in LPS-LiPC rats was greater than that in NS-LiPC rats.On the day of probe testing, NS-NS rats spent significantly more time in the TQ than animals from the other three groups. No significant difference existed between NS-LiPC and LPS-LiPC groups. Conclusins1. Maternal infection increases seizure susceptibility to LiPC in the adult rat offspring;2. Maternal infection alone causes long-lasting neuronal injury, increased anxiety level and cognitive deficit in the offspring rats;3. Maternal infection during late pregnancy enhances neuronal injury and exacerbates long-term neurobehavioral impairment associated with a second adult LiPC insult.In sum, prenatal infection can increase adult seizure susceptibility and exacerbates the associated brain injury. Chapter Ⅲ Neonatal inflammation exacerbates seizure-induced hippocampus-dependent memory impairment in adult ratsObjectivesThe study was designed to examine whether neonatal lipopolysaccharide (LPS) exposure is associated with changes in microglia and whether these alternations could influence later seizure-induced neurobehavioral outcomes.Methods1. The construction of neonatal immune activation modelLPS diluted in saline was injected intraperitoneally (i.p.) into male Sprague-Dawley pups at postnatal (P) day3and P5at a dose of50ug/kg. Control pups were injected with a corresponding volume of normal saline (NS).2. To study the effect of neonatal LPS on hippocampal microglia using immunofluorescence staining, male pups were sacrificed4,16, or40days after the last administration of NS or LPS on P5. Immunofluorescence staining of Ibal, a marker of microglia, was used to study the time course of LPS-induced microglia alternation.3. To study the inhibitory effect of minocycline on neonatal LPS-induced microglia alternation, pups administered saline or LPS neonatally were treated with phosphate buffered saline (PBS) or minocycline for6consecutive days. Three groups of pups were studied, including normal controls treated with PBS (SS), neonatally LPS-exposed pups treated with PBS (LS), and neonatally LPS-exposed pups treated with minocycline (LM). On P9(4days after administration of NS or LPS on P5) and P21, rats were sacrificed and evaluated for hippocampal microglial activation using immunofluorescence staining of Ibal.4. To investigate the effect of early-life LPS exposure on seizure-induced neurobehavioral impairment and to further explore whether minocycline would prevent changes in susceptibility to later seizure-induced brain injury, a separate study was conducted. Rat pups injected with saline or LPS neonatally were administered kainic acid (KA) at P45. Four groups were studied, including SSS (normal controls), SSK.(adult seizures treated with PBS neonatally), LSK ("two-hit" animals treated with PBS at the time of neonatal LPS exposure), and LMK ("two-hit" animals treated with minocycline at the time of neonatal LPS exposure).(1) KA-induced status epilepticus (SE) model in adult ratsAt P45, KA dissolved in saline (15mg/kg) was administered i.p. to rats that were injected with saline or LPS neonatally. Controls received equal volumes of NS. A seizure severity grade was assigned according to a modified Racine scale (detailed in part1). Animals were closely observed and seizure severity and latency to the first sign of seizure were recorded. Seizure onset time (SOT) was used to assess seizure susceptibility. Seizures were terminated with chloral hydrate (400mg/kg, i.p.)2h after the onset of SE. Only animals with grade IV or V seizures were included in this study;(2) At acute stage (6h after SE), animals were killed for quantification of hippocampal IL-1β and TNFa production using real-time polymerase chain reaction (RT-PCR); (3) Three days after SE, rats were sacrificed for further examination of microglial activation by immunofluorescence staining of Ibal;(4) From P46to P55, Y maze task was used to evaluate hippocampus-dependent spatial learning ability of adult rats;(5) From P60to P65, water maze task was used to evaluate hippocampus-dependent spatial learning and memory ability of adult rats;(6) At P70, inhibitory avoidance task was used to evaluate hippocampus-dependent nonspatial memory ability of adult rats.Results:1. Neonatal LPS has no effect on KA-induced seizure susceptibility in adult rats.There were no significant difference in SOTs among SSK, LSK, and LMK groups, indicating that neonatal LPS has no effect on KA-induced seizure susceptibility in adulthood.2. Transient microglia activation after neonatal LPS exposure.The analysis of immunostaining of Ibal revealed that LPS-treated pups exhibited significantly greater hippocampal expression of Ibal than NS-treated pups at P9and P21. However, we observed no significant difference in the expression of Ibal at P45. In short, dual exposure to LPS on P3and P5caused persistent activation of hippocampal microglial cells. This activation, however, was not permanent and subsided within40days.3. Minocyclne inhibits neonatal LPS-induced microglial activationCompared with SS, LS showed higher expression of Ibal at P9and P21. Minocycline administration significantly reduced LPS-induced microglia activation in LM pups compared with LS pups at both P9and P21. There was no difference in the expression of Ibal between SS controls and LM pups at both P9and P21.4. Cytokine expression in the hippocampus of adult rats after KA-induced SECompared with SSS group, SSK, LSK and LMK rats showed higher expression of IL-1βmRNA. Compared with SSK group, the expression of IL-1β mRNA was higher in LSK group. There was no difference in the expression of IL-1(3mRNA between SSK and LMK groups. Compared with SSS group, SSK, LSK and LMK rats showed higher expression of TNFa mRNA. Compared with SSK group, the expression of TNFa mRNA was higher in LSK group. There was no difference in the expression of TNFamRNA between SSK and LMK groups.5. Hippocampal microgilal activation after KA-induced SECompared with SSS group, SSK, LSK and LMK rats had higher hippocampal Ibal expression. Compared with SSK and LMK groups, LSK group had higher hippocampal Ibal expression. There was no difference in the expression of Ibal between SSK and LMK groups.6. The performance of adult rats in the Y maze after KA-induced SEAnimals in SSK group, LSK group, and LMK group all demonstrated lower spontaneous alternation scores when compared with SSS controls. Compared with SSK and LMK rats, the percent of alternation in "two-hit" rats (LSK) was significantly reduced. There was no difference in the percent alternation between SSK and LMK groups.7. The performance of adult rats in the water maze after KA-induced SEAll groups of rats have comparable rate of acquisition. On the day of probe testing, SSS rats spent significantly more time in the TQ than animals from the other three groups. LSK rats spent significantly less time in the TQ than animals in SSK and LMK groups. SSK and LMK rats spent comparable time in the TQ.8. The performance of adult rats in the inhibitory avoidance task after KA-induced SESSS, SSK, and LMK rats demonstrated comparable retention time at1h after training. However, the latency to step into the dark compartment was significantly decreased in LSK rats compared with SSS group.At24h, the latency to step into the dark compartment was significantly increased in SSS rats compared with SSK, LSK and LMK groups. Compared with LSK group, SSK and LMK groups spent longer time to step into the dark compartment. No difference in escape latency was observed between SSK and LMK groups. Conclusins1. Neonatal inflammation caused persistent activation of hippocampal microglial cells;2. Minocycline inhibited neonatal LPS-induced microglial activation;3. Neonatal inflammation predisposed the immatured brain to exacerbated neuroinflammatory response and worse hippocampus-dependent behavioral deficit following seizures in adulthood, possibly by priming microglia. |
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