Research reportKindling modulates the IL-1β system, TNF-α, TGF-β1, and neuropeptide mRNAs in specific brain regions
Introduction
Epilepsy is characterized by abnormal patterns of highly synchronized recurrent neuronal discharges in one or more brain areas, which often recruit anatomically related structures. Generally, seizures are relatively brief, as in the complex partial seizures of temporal lobe epilepsy, but occasionally may persist for minutes or hours, as in the case of status epilepticus (SE), and may culminate in neuronal damage within several limbic and cortical areas. To study complex partial epilepsy in animals, the model most often used is kindling, whereas systemically applied drugs (e.g., pilocarpine preceded by lithium treatment) and neurotoxins (e.g., kainic acid) have typically been used to model SE.
In an effort to better understand the relationship between discrete seizures and SE-induced brain damage, we assessed the effects of systemically applied kainic acid in hippocampal-kindled rats [20]. The earlier kindling experience sensitized the rats, such that the kainic acid-induced SE had a faster onset and longer duration relative to that of the non-kindled rats. However, despite the increased seizure activity during SE, previous kindling was associated with a surprising and marked neuroprotection of multiple brain structures that are ordinarily damaged/destroyed by SE, including the piriform and parietal cortices, the amygdala and hippocampus [20].
There are various mechanisms that could provide neuroprotection during kindling including an up-regulation of growth factors and cytokines. Neurotrophic factors, such as brain-derived neurotrophic factor, fibroblast growth factor and nerve growth factor, are up-regulated by seizures, particularly by kindling, and could provide a degree of neuroprotection 43, 48. Likewise, cytokines are up-regulated in a variety of pathophysiological conditions and could potentially act as growth factors and neuroprotectants, although they may also promote deleterious and neurotoxic effects 28, 41. The data concerning cytokine involvement in brain processes are unclear and have included demonstrations of damage, neuroregeneration, neurotoxicity, and neuroprotection 28, 29, 41, 54. Among other factors, the functional effects of cytokines will depend on their concentrations [29]and the overall cytokine profile, that is, the balance between pro-inflammatory (stimulatory) and anti-inflammatory (inhibitory) cytokines [30].
Consistent with a role of cytokines in the pathophysiology of epilepsy and/or brain damage following seizure activity, it has been reported that cells immunoreactive for IL-1β are increased in tissue samples obtained from patients with temporal lobe epilepsy [42]. Moreover, experimentally triggered SE with either hippocampal kainic acid injections or electrical stimulation increases mRNA expression for hippocampal IL-1β, TNF-α and other cytokines [50]. Because SE induces relatively widespread brain damage, evaluation of local brain cytokine production during and following SE is problematic. After all, the damage induced by SE may significantly up-regulate the cytokine network independent of seizure activity per se. Thus, the analysis of regional cytokine changes (using mRNA as index of local production) following discrete seizures that are not associated with gross neurodegeneration (e.g., after kindling) could aid in dissociating cytokine activation following brief acute seizures from that associated with protracted seizures and neurodegeneration.
In the present study, using amygdala-kindled rats, we determined the expression of mRNA for several cytokine components in distinct brain regions known to be important for the development and manifestation of kindling-induced convulsions. Cytokine and cytokine receptor components mRNA expression was determined in the parietal, prefrontal and piriform cortices, amygdala, hippocampus and hypothalamus 2 h and 3 weeks following the last generalized convulsive seizure triggered from the ipsilateral kindled amygdala. Both of these time windows are associated with neuroprotection upon challenge with kainic acid-induced SE [20].
Using sensitive and specific RNase protection assays, we analyzed mRNA for the following cytokine components: two stimulatory cytokines (IL-1β and TNF-α), two inhibitory cytokines (IL-1 receptor antagonist and transforming growth factor-β1), IL-1β system receptor-associated components (IL-1 receptor type I and IL-1 receptor accessory proteins),1 and glycoprotein 130 (gp 130, a common signal transducer among receptors for members of the IL-6 subfamily). It appears likely that cytokine–cytokine and cytokine–neuropeptide interactions play an important role in cytokine action on the brain. These include interactions between IL-1β and neuropeptide Y (NPY) [46]and IL-1β and endogenous opioids [23]. NPY is an endogenous antiepileptic 2, 18, 22, 47, 48, 51, and endogenous opioids respond to kindling [8]. Thus, we also determined the mRNA levels for NPY, and the opioid peptide precursor, pro-opiomelanocortin (POMC).
Cytokine- and neuropeptide-associated mRNAs were examined in the same brain region samples. This concomitant analysis of cytokine and neuropeptide systems can provide information on potential endogenous chemical (cytokine–cytokine and cytokine–neuropeptide) interactions. The data show that cytokine and neuropeptide mRNA components are differentially modulated in specific brain regions of amygdala-kindled rats.
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Subjects and maintenance
Male, Long–Evans hooded rats (Charles River Laboratories, Canada), 300–350 g, served as experimental subjects. All protocols and procedures met the guidelines of the Canadian Council of Animal Care and were approved by the Institutional's Animal Care and Use Committees. Rats were individually housed in standard polypropylene cages and were maintained ad libitum on rat food and tap water. Room temperature was maintained at 21±2°C, and artificial illumination was from 0700 to 1900 h (12:12 h
Electrophysiological profile
The characteristics of amygdala kindling were typical of this neuroanatomical site. The AD threshold and kindling rate (number of stimulations to develop the first stage-5 seizure) for the 2 h- and 3 week-kindled groups were not different: AD threshold=57±16 and 56±9 μA; kindling rate=14.6±2.4 and 12±1 stimulations, respectively.
Plasma corticosterone levels
Analysis of plasma corticosterone levels indicated that the levels among control rats (3.49±0.26 μg/dl) did not differ from the 2 h- and 3 week-kindled groups
Discussion
The data show that amygdala kindling induces significant mRNA changes for cytokine and neuropeptide components in specific brain regions. The effects of a kindled seizure on cytokine mRNAs were very marked when brain tissue was taken 2 h after the last seizure, but, with few exceptions, 3 weeks following the last seizure, cytokine mRNA levels were not different from controls. At the 2-h time point, IL-1β and TNF-α mRNA levels were up-regulated in all cortical areas, as well as the amygdala and
Acknowledgements
We thank Dr. Ronald P. Hart (Department of Biological Sciences, Rutgers University) for providing the rat IL-1β, IL-1Ra, IL-1RI and IL-1R AcP cDNAs, Dr. Karl Decker (Biochemisches Institut der Albert Ludwigs Universität) for providing the rat TNF-α cDNA, Dr. David Danielpour (National Cancer Institute) for providing the rat TGF-β1 cDNA, Dr. Steven L. Sabol (Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, NIH) for providing the rat neuropeptide Y cDNA, Dr. Gerald
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