Experimental Study Of The Role Of Interleukin-18 In KA-induced Neurodegeneration In Hippocampus Of Mouse

Glutamate excitotoxicity plays a key role in inducing neuronal cell death inmany neurological diseases. Administration of kainic acid (KA), an analog ofthe excitotoxin glutamate, to rodents results in neuron death and seizures, whichprovides a well-characterized model for studies of human neurodegenerativediseases. In the course of KA-induced hippocampal injury, neurons interact withthe surrounding cells, including astrocytes and microglia. The modulation of theactivity of these cells may influence the survival and repair of hippocampalneurons after injury. Interleukin-18 (IL-18), a recently identified pleiotropic cytokine, was firstisolated as interferon-γ (IFN-γ) inducing factor, an inflammatory mediator oftissue damage. More and more accumulating evidences suggested that IL-18plays an important role in the communication between central nervous system(CNS) and immune system. Purpose: In the present study, we investigated the role of IL-18 in excitotoxicneurodegeneration by using the model of KA-induced hippocampal injury inIL-18 knockout (IL-18 KO) mice. In addition, in a separate experiment,wild-type mice were treated with recombinant mouse IL-18 (rmIL-18) beforeKA application to further explore the role of IL-18 in KA-inducedneurodegeneration.Methods:A total of 77 (39 male and 38 female) 6-to 8-week-old IL-18 KO mice,which carry a mutation in the IL-18 gene and are unable to produce biologicallyactive IL-18, and 94 sex-and age-matched wild-type C57BL/6 mice were usedin the present study.KA administration and assessment of clinical signsThe mice were partially anesthetized with Isofluen. KA dissolved indistilled water was slowly and gently dropped by micropipette into the noses ofthe mice at a dose of 40mg KA per kg body weight. Mice were monitoredcontinuously for 5 h to register the onset and extent of seizure activity. Seizureswere rated as follows: 0, normal;1, immobilization;2, rearing and falling;3,seizure for less than 1 h;4, seizure for 1-3 h;5, seizure for more than 3 h;and 6,death.Histopathological analysisThe mice were anesthetized with sodium pentobarbital and transcardiallyperfused with PBS followed by 4% buffered formaldehyde either 1 day or 7days after the administration of KA. The brains were kept in 10% sucrose untilbeing frozen. Coronal sections were stained by Nissl's method to evaluate themorphology of neurons. For assessment of severity and extent ofneurodegeneration in the hippocampus according to Nissl's staining, sectionswere scored using a semiquantitative grading system.Recombinant mouse IL-18 treatmentThe wild-type mice received rmIL-18 intraperitoneally for 5 consecutivedays before KA administration. From these mice also frozen sections of thehippocampi were prepared to evaluate pathological changes as described above.Immunohistochemistry of brain sectionsFrozen hippocampal sections were prepared as described forhistopathological analysis. After washes with Tris buffer, the sections wereblocked by "protein block" for 30 min. Subsequently, they were exposed to ratantibodies to CD11b, rabbit antibodies to GFAP, rat antibodies to IL-12, rabbitantibodies to IL-1β, rat antibodies to IFN-γ and rabbit antibodies to TNF-α,respectively, followed by staining with the avidin-biotin technique and DABsystems.Isolation and flow cytometry analysis of microgliaOne and seven days after KA or water treatments, the mice from differenctgroups were perfused with PBS and sacrificed. The hippocampi were dissectedand dissociated. Microglia were isolated by a series of trypsiniazation, filtrationand centrifugation. Then microglia enriched cell suspensions were incubatedwith the following antibodies: rat anti-mouse CD11b, rat anti-mouse IFN-γ andrat anti-mouse TNF-α. The mean fluorescence intensity (MFI) of each antibodyon the microglia was determined.Open-field test with zone monitoringOpen-field activity was measured one day before and six days after KAadministration. At the beginning each mouse was placed into the center ofopen-field arena and its locomotion as well as rearing were recorded every 5minduring 60 min.Results:1. IL-18 deficiency does not affect KA-induced clinical signsWithin 20-30min after KA administration, most mice displayed continuousseizures, which lasted less than 5h. Severity of seizure did not differsignificantly between the two groups.2. RmIL-18 increases the severity of KA-induced neurodegeneration inwild-type miceAfter KA treatment, both the clinical and pathological signs (seven days) in thehippocampus were markedly aggravated in mice treated with the higher dose ofrmIL-18 compared to the mice without rmIL-18 treatment or mice receiving thelow dose of rm IL-18. Therefore, rmIL-18 increased the severity of KA-inducedneurodegeneration in mice in a dose-dependent manner.3. The locomotion activity of IL-18 KO mice reveals increasedsusceptibility to KA administrationKA administration did not influence obviously the rearing activity in bothIL-18 KO and wild-type mice. However, 6 days after KA treatment, locomotionactivity in IL-18 KO mice was significantly increased compared to wild-typemice, which indicates that the habituation to the novel environment ofKA-treated IL-18 KO was impaired more severely than KA-treated wild-typemice.4. IL-18 KO mice show increased hippocampal neurodegenerationSeven days after KA treatment, IL-18 KO mice exhibited more severeneurodegeneration than wild-type mice, but not on one day after KA treatment.The control group treated with water showed no pathological changes in thehippocampi.5. IL-18 deficiency enhances KA-induced microglia activation andastrogliosis in hippocampiOne day after KA treatment, both IL-18 KO and wild-type mice displayedslightly more activated microglia and stronger astrogliosis. However, there wasno obvious difference between the two groups. Seven days after KA treatment,the intensity of the activated microglia and astrogliosis of IL-18 KO mice wasmuch greater than those of wild-type mice with similar pathological changes.6. KA treatment increases IL-12 and IFN-γ, but not IL-1β and TNF-αexpression in IL-18 KO miceSeven days after KA delivery a stronger IL-12 expression was found inIL-18 KO mice than in wild-type mice and the positive immunostainingaccumulated in the lesioned CA3 area. Double-staining for activated microgliatogether with IL-12 showed that the increased IL-12 expression was restricted tothe activated microglia. Similarly, IFN-γ expression in the hippocampus ofIL-18 KO mice was higher than that of wild-type mice. Although IL-1β andTNF-α expression was also detected in the hippocampi of wild-type and IL-18KO mice, the levels of their expression were similar after KA treatment. Whenanalyzing microglial expression of cytokines by flow cytometry, we found thatin both IL-18 KO and wild-type mice, one day after KA treatment, TNF-αexpression was higher than in the corresponding mice without KA treatment, butno difference between the IL-18 KO and wild-type mice. On the other hand,seven days after KA treatment, in the IL-18 KO mice there was a clear tendencyof increased IFN-γ expression compared to untreated IL-18 KO mice.Conclusions:1. RmIL-18 increases the severity of KA-induced neurodegeneration inwild-type mice in a dose-dependent manner, suggesting that as an inflammatorycytokine, IL-18 involves in KA-induced injury.2. IL-18 KO mice show increased hippocampal neurodegeneration,enhanced microglia activation and astrogliosis, decreased habituation to thenovel environment after KA treatment, suggesting the high susceptibility ofIL-18 KO mice to KA injury.3. KA treatment increases IL-12 and IFN-γ, but not IL-1β and TNF-αexpression in IL-18 KO mice.4. Taken together, although exogenous IL-18 administration can aggravatethe KA-induced neurodegeneration, IL-18 KO mice are more sensitive to theKA administration compared to the animals with normal supply of IL-18. Thedeficiency of a potentially pro-inflammatory cytokine such as IL-18 does notnecessarily reduce the effect of KA-induced excitotoxicity. The roles of IL-18 inexcitotoxic injury in IL-18 deficient mice may be overcompensated by increaseof other microglia-derived disease-promoting factors, of which IL-12 is onecandidate.The traits of present study:1. It is the first time to use IL-18 KO mice to investigate the role of IL-18in KA-induced neurodegeneration.2. It is also the first time to provide the evidences that deficiency of apro-inflammatory cytokine such as IL-18 does not necessarily reduce the effectof KA-induced excitotoxicity.