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Keywords:

  • Brain development;
  • Interleukins;
  • Neurodegeneration;
  • Hippocampus;
  • Seizures;
  • Rat

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Summary: Purpose: We investigated the activation of microglia and astrocytes, induction of cytokines, and hippocampal neuronal damage, 4 and 24 h after kainic acid–induced status epilepticus (SE) in postnatal day (PN) 9, 15, and 21 rats.

Methods: Limbic seizures were induced by systemic injection of kainic acid. Glia activation and neuronal cell loss were studied by using immunocytochemistry and Western blot. Cytokine expression was analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR) followed by Southern blot quantification.

Results: After SE onset, hippocampal glia activation, cytokine expression, and neuronal damage are all age-dependent phenomena. In the hippocampus, neuronal injury occurs only when cytokines are induced in glia, and cytokine synthesis precedes the appearance of degenerating neurons. Neuronal injury is more pronounced when interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are produced in addition to IL-1β.

Conclusions: This study shows that cytokine induction in rat brain after sustained seizures is age dependent, and it is associated with the appearance of cell injury.

Proinflammatory cytokines and related inflammatory and antiinflammatory molecules are rapidly overexpressed by glia in adult rodent hippocampus in various models of limbic seizures (1–3). In the adult brain, cytokines are expressed to a larger extent when seizures are associated with neuronal damage, suggesting a link between cytokine production and the occurrence of neuronal injury (1,4). Thus it has been shown that in mature rat brain, proinflammatory cytokines, and in particular interleukin (IL)-1β, act as modulators of various forms of neurodegeneration (5–7).

In humans and in experimental models of epilepsy, seizure susceptibility and the associated neuronal damage are age-dependent phenomena, changing dramatically during postnatal development. In the first 2 postnatal weeks, the brain is more prone to seizure activity, but it is relatively resistant to irreversibile seizure-induced damage as compared with adult brain (8–12).

The factors implicated in the occurrence of age-dependent seizure-related injury are still unclear. We tested the hypothesis that glia activation and the subsequent cytokine production may be involved in this process. We used rats at distinct postnatal (PN) ages (PN9, PN15, PN21), known to have age-specific seizure susceptibility and neuronal injury after kainic acid–induced seizures.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Experimental animals

Male Sprague–Dawley rats (Charles River, Calco, Italy) of PN 9, 15, and 21 (with PN0 defined as the day of birth) were used. All animals were used before weaning. Pups were housed with their dams at constant temperature (23°C) and relative humidity (60%), with a fixed 12-h light–dark cycle and free access to food and water. Procedures involving animals and their care were conducted in conformity with institutional guidelines that are in compliance with national and international policies.

Status epilepticus (SE) induction

Kainic acid (1.3, 5, and 7.5 mg/kg) was intraperitoneally injected in PN9, PN15, and PN21 pups, respectively, because of the age-specific seizure susceptibility to kainate (9). Control age-matched rats were injected with an equivalent volume of phosphate-buffered saline (PBS, pH 7.4). Rats were monitored for 3 h after kainic acid injection for behavioral SE manifestation.

Kainic acid induced SE at all ages, with an onset of 30 min, lasting for 3 h. In PN9 and PN15 pups, SE was characterized by bilateral clonic seizures of all extremities, whereas in PN21 rats, limbic motor seizures were characterized by continuous forelimb clonus (13). After the experiment, rats were then housed with their lactating mother until they were killed.

Immunohistochemistry

Experimental pups and their controls (n = 5–10 in each group) were killed 4 and 24 h after SE onset. These times were chosen because of the significant glia activation by SE in adult rat brain (1,3). Rats were deeply anesthetized with Equithesin (1% pentobarbital/4% (vol/vol) chloral hydrate; 3.5 ml/kg, i.p.) and trascardially perfused with 0.05 M PBS (pH 7.4) followed by 4% paraformaldehyde in PBS. Brains were postfixed in fixative for 4 h, and then cryoprotected in 20% sucrose in PBS and rapidly frozen at –25°C.

Serial cryostat horizontal 40-μm forebrain sections were cut throughout the whole septotemporal aspect of the hippocampus. Adjacent sections were collected for detection of microglia (complement receptor type 3, OX-42; Serotec Ltd, Oxford, U.K.; 1:100), astrocytes (anti–glial fibrillary acidic protein, GFAP; Chemicon International, Temecula, CA, U.S.A.; 1:2500) and neuronal injury (Fluoro-Jade; Histo-Chem, Jefferson, AR, U.S.A.) (14). Immunostaining was done as previously described (1,14,15).

RNA extraction and RT-PCR analysis

Kainic acid–treated pups and their age-matched controls (n = 6–10 for each group) were killed at various times after SE onset (2–24 h). During this interval, we previously found maximal cytokine overexpression by SE in adult rat brain (3). Rats were decapitated, and their hippocampi were dissected out, immediately frozen on dry ice, and kept at –80°C until use. Total messenger RNA (mRNA) was extracted from the hippocampi, and reverse transcriptase–polymerase chain reaction (RT-PCR) analysis was performed as previously described in detail (16). An amount of PCR mixture (7 μl) was electrophoresed in 1.5% agarose gel, stained with ethidium bromide, photographed, and transferred to Gene Screen Plus membrane (NEN, Boston, MA, U.S.A.). Membranes were prehybridized in sodium chloride, 5× SSC, containing 1% sodium dodecylsulfate (SDS), 0.5% nonfat milk, and 100 μg/ml of denatured salmon sperm DNA at 37°C for 4 h. Oligonucleotide probes internal to PCR primers were radiolabeled with [32P]-adenosine triphosphate (ATP) by T4 polynucleotide kinase (Promega, Madison, WI, U.S.A.). All labeled probes were purified through G-50 Sephadex Quick Spin Columns. Membranes were hybridized overnight at 37°C, washed first in 6× SSC at room temperature (RT), and then in 3M tetraethyl ammonium chloride, 50 mM Tris-HCl (pH 8), and 0.1% SDS at 58°C. Subsequently, membranes were washed in 2× SSC and dried before autoradiography.

Western blot

Pups (n = 4 each group) were decapitated 4 h after SE onset. Hippocampi were dissected out at 4°C, pooled, and homogenized in 20 mM Tris-HCl (pH 7.4), containing 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM EGTA, 1 mM Na-vanadate, 2 μg/μl aprotinin, 1 μg/μl pepstatin, 1 μg/μl leupeptin (30 mg tissue/100 μl homogenization buffer). Twenty-five micrograms of total proteins each lane were analyzed with SDS-polyacrylamide gel electrophoresis (PAGE) and 10% acrylamide, and each sample was run in duplicate in two different gels. Proteins were transferred to Hybond nitrocellulose membrane by electroblotting. For immunoblotting, we used an antibody that recognizes a specific band of ∼50 kDa corresponding to GFAP (1:5000). Immunoreactivity was visualized with enhanced chemiluminescence (ECL), and densitometry was used to quantify the changes in protein levels in the immunoblots (AIS image analyzer).

Statistical analysis of data

Mann–Whitney one-way analysis of variance (ANOVA) was used to evaluate the effect of SE on cytokine mRNA and GFAP levels.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Glia activation induced by SE

SE enhanced microglia (OX-42) and astrocyte (GFAP) immunoreactivity (IR) mainly in the hippocampus. These changes were age dependent.

Figure 1 shows microglia activation in the dentate gyrus of the hippocampus from representative PN9 and PN21 rats in control conditions and 24 h after SE onset. Weak IR was found in control sections at all postnatal ages (PN15 not shown; see PN9 and PN21 in Fig. 1). SE induced little microglia activation in PN9 rats both 4 and 24 h after seizure onset (see 24 h after SE onset in Fig. 1). In PN15 and PN21 rats, the IR was enhanced after seizures at both times analyzed. Strongly immunoreactive cells resembling reactive microglia (i.e., round to oval shape and developed processes) were observed at both postnatal ages (PN15 not shown; see for comparison PN21 in Fig. 1). In PN15 and PN21 rats, microglia immunostaining was enhanced also in extrahippocampal areas (not shown).

image

Figure 1. Photomicrographs showing OX-42 immunoreactivity in the dentate gyrus of PN9 and PN21 rats 24 h after status epilepticus (SE) onset. In control sections, faint immunostaining was found at both postnatal ages. SE induced little activation of microglia in PN9 rats, whereas OX-42 staining was strongly increased by seizures in PN21 rats. GC, granule cell layer; sm, stratum molecolare; h, hilus. Scale bar, 100 μm.

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GFAP IR was low in control pups at all times tested (PN15 not shown; see PN9 and PN21 in Fig. 2). Little increase in GFAP staining was observed after seizures at PN9 at both times (see 24 h after SE onset in Fig. 2). In PN15 and PN21 rats, a pronounced increase in GFAP IR was found 4 and 24 h after SE onset (see PN21, 24 h after SE onset in Fig. 2).

image

Figure 2. Photomicrographs showing glial fibrillary acidic protein (GFAP)-positive astrocytes in the dentate gyrus of PN9 and PN21 rats 24 h after status epilepticus (SE) onset. Faint GFAP staining was observed in controls at different ages. Little increase of staining was found in PN9 rats after SE induction, whereas the GFAP signal was strongly enhanced by seizures in PN21 rats. GC, granule cell layer; sm, stratum molecolare; h, hilus. Scale bar, 100 μm.

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To have a semiquantitative analysis of astrocytosis, we ran a Western blot of GFAP, by using hippocampi of PN9, PN15 and PN21 rats 4 h after SE onset, versus their age-matched controls. Densitometric analysis showed that GFAP levels were enhanced in control rats in an age-dependent manner. In PN15 rats, GFAP concentration was 26 ± 1.5% higher than in PN9 rats (p < 0.05; Fig. 3). In PN21 rats, GFAP concentration was 34 ± 1.5% higher than that in PN15 rats (p < 0.05; Fig. 3). Four hours after SE onset, GFAP levels did not significantly change in PN9 rats, but they increased by 35% on average in both PN15 and PN21 rats (p < 0.05 in PN15 and in PN21; Fig. 3).

image

Figure 3. Western blot analysis of glial fibrillary acidic protein (GFAP) in the hippocampus 4 h after status epilepticus (SE) onset in PN9, PN15, and PN21 rats. Bar graphs depict mean ± SEM of the optical density (OD) of the ∼50-kDa band corresponding to GFAP, expressed as a percentage of respective control values. In each age group, the white bar graph shows GFAP level in the control condition, whereas the black bar graph shows the increase in GFAP concentration induced by seizures. *p < 0.05; **p < 0.001 vs. age-matched control by Mann–Whitney test; within the control groups, #p < 0.05 vs. the younger control age by Mann–Whitney test.

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Because the experimental denaturized conditions required for Western blot were not compatible for microglia analysis with OX-42 antibody, we cannot provide any semiquantitative measurements of microglia activation.

Inflammatory response: cytokine mRNA expression

We measured the level of cytokine mRNAs in the hippocampi of PN9, PN15, and PN21 rats at various times after SE onset (2–24 h). Cytokine mRNA expression was not increased in kainic acid–treated PN9 rats as compared with that in control animals (not shown). A detailed time course showed that in PN15 rats, IL-1β mRNA expression was not modified 2 h after SE onset, but it was enhanced by 2.2-fold (p < 0.05) 4 h after seizure induction and returned to control levels by 18 h (Fig. 4). The levels of other cytokines [IL-1Ra, IL-6, and tumor necrosis factor (TNF)-α] were not modified at any time studied (Fig. 4). At PN21, all cytokines were increased: IL-1β and IL-6 mRNA was enhanced by 3.6-fold (p < 0.05); TNF-α and IL-1Ra, by 1.5-fold versus control level (p < 0.05).

image

Figure 4. Densitometric analysis of cytokine messenger RNA (mRNA) level in the hippocampus of PN15 rats at different times after status epilepticus (SE) onset. Data are expressed as optical density (OD) values (mean ± SEM, n = 6–10) of cytokine mRNA bands normalized to the respective cyclophilin mRNA. **p < 0.05 vs. control by Mann–Whitney test.

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Neuronal injury

Neuronal cell injury was assessed by using Fluoro-Jade staining. No evidence of Fluoro-Jade–positive neurons was found in PN9 rats both 4 and 24 h after SE. In PN15 and PN21 rats, damaged neurons were observed 24 h after SE onset only.

In PN15 rats, we found scattered positive neurons in the CA3 pyramidal sector and in the dorsal part of the subiculum (not shown). At PN21, various degenerating neurons were observed in both the dorsal and ventral CA1 (not shown) and CA3 sector (Fig. 5A1 and B). At this age, we found Fluoro-Jade–positive cells in the basolateral amygdala (not shown), in entorhinal and perirhinal (Fig. 5C) cortices, and in the periventricular nuclei in 40% of rats.

image

Figure 5. Fluoro-Jade labeling of forebrain neurons 24 h after status epilepticus (SE) onset in PN21 rats. At PN9, no degenerating neurons were observed in the hippocampus and in other forebrain areas (not shown); at PN15, few scattered damaged neurons were noted in the CA3 pyramidal cell layer and in the subiculum (not shown); at PN21, we observed abundant injured neurons in the CA3 area both in the dorsal (A1) and in the ventral hippocampus (B) and in other forebrain areas (here shown damage in the perirhinal cortex, C). Scale bar, 100 μm.

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In PN15 and PN21, we observed glia activation in the same brain areas showing damaged neurons (not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

These data show an age-dependent activation of microglia and astrocytes and induction of cytokines in the rat hippocampus after SE. In PN9 rats, little activation of both glia populations was found, and we did not observe any cytokine neosynthesis. In PN15 and PN21 rats, glia were markedly activated. IL-1β is the only cytokine induced in PN15 rats, whereas in PN21 rats, all cytokines were produced, similar to adult rat brain (3). The limited activation of microglia and astrocytes and the lack of cytokine expression after SE in PN9 rats are not due to the inability of glia to react to injurious stimuli at this age. Thus after hypoxic or ischemic events in PN9 rats, microglia, astrocytes, and cytokines are activated (17–19).

The analysis of neuronal cell loss showed that in immature brain, neuronal injury occurs only when cytokines (and in particular IL-1β) are induced, and their synthesis precedes the appearance of neuronal damage. In PN15 and PN21 rats, IL-1β mRNA was enhanced 4 h after SE onset, thus before neurodegenerating neurons appear (i.e., 24 h after seizures). Neuronal injury was larger in PN21 rats, when all proinflammatory cytokines were produced. These data suggest that IL-1β must act synergistically with other cytokines to induce significant neuronal injury. Accordingly, in neuronal cell cultures, although IL-1β and TNF-α alone were not toxic, their combination caused pronounced neuronal injury (20). If the induction of inflammatory processes by seizures plays a role in age-dependent neuronal death, at least two pathways related to cytokine-activated signal transduction may be involved: the induction of iNOS and cyclooxygenase (COX)-2. Thus these pathways are developmentally regulated, as IL-1β induction by seizures. After kainic acid-induced seizures, no COX-2 mRNA induction was found before PN14 (21). Despite prolonged seizures, immature rats do not show increase in reactive oxygen species as in adults (22).

The mechanism by which IL-1β may contribute to neuronal injury also may be related to the functional interaction between IL-1β–receptor type I and N-methyl-d-aspartate (NMDA) receptors. We recently found that the exposure of hippocampal neurons to IL-1β enhances the phosphorylation of the NR2A/2B subunit of the NMDA receptor (23). This receptor modification upregulates NMDA activity, thus increasing its channel-gating properties (24). The age-dependent induction of IL-1β during seizures may contribute to the occurrence of neuronal damage by inducing posttranslational modification in NR2 subunit.

Experiments are in progress to validate these hypotheses. Further insights into this field will be of value for better understanding molecular pathways critically involved in seizure-associated neuronal damage.

Acknowledgments

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Acknowledgment:  This work was supported by CURE, Fondazione Mariani Onlus, the Heffer Family Foundation (A.V.), and grants NS-36238 (J.V.) and NS-2023 (S.L.M.) from the NINDS. S.L.M. is the recipient of the Martin A. and Emily L. Fisher fellowship in Neurology and Pediatrics.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES
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