IL-18 deficiency aggravates kainic acid-induced hippocampal neurodegeneration in C57BL/6 mice due to an overcompensation by IL-12

The role of interleukin-18 (IL-18) in excitotoxic neurodegeneration is largely unknown. To address this issue, we used kainic acid (KA)-induced hippocampal neurodegeneration in IL-18 knockout (KO) mice. One day after KA administration, clinical symptoms and histopathological changes did not differ between IL-18 KO mice and wild-type mice. However, 7 days after KA application the hippocampal neurodegeneration was markedly severe in IL-18 KO mice as demonstrated by increased locomotion and prominent histopathological changes including neuronal cell loss, microglia activation and astrogliosis. Surprisingly, when wild-type mice received recombinant mouse IL-18 (rmIL-18) in advance, after KA treatment both the clinical and pathological signs were dose-dependently aggravated compared to mice without rmIL-18 pre-treatment. To clarify the mechanism behind this, we assessed the expression of the IL-18 associated cytokines IL-12, IL-1beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) in the hippocampus by immunohistochemistry and flow cytometry. IL-12 and IFN-gamma expression was strongly increased in IL-18 KO mice when compared to wild-type mice 7 days after KA treatment in agreement with increased microglia activation. These results suggest that the role of IL-18 in excitotoxic injury in IL-18 deficient mice may be overcompensated by increased IL-12 secretion.     

Gender differences in susceptibility to kainic acid-induced neurodegeneration in aged C57BL/6 mice

Some epidemiological studies concerning gender differences in Alzheimer's disease (AD) support the higher prevalence and incidence of AD in women, while most studies using animal models of aging have included only male subjects. It is still uncommon for aged males and females to be compared in the same study. In the present study, we investigated how age and gender influence the excitotoxic neurodegeneration by treating C57BL/6 mice (aged females and males as well as adult females and males) with kainic acid (KA) intranasally. Clinical signs, behavioural changes, pathological changes and astrocyte proliferation were tested; and the levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured after KA treatment. The results showed that aged female mice were more sensitive to KA-induced excitotoxicity as demonstrated by severer seizure activity, increased locomotion and rearing in open-field test, prominent hippocampal neuronal damage, enhanced astrocyte proliferation compared with aged males, adult females and adult male mice. In addition, higher BDNF level in hippocampus of aged female mice was observed. These results denote the disparity of aging and gender in KA-induced hippocampal neurodegeneration and aged female mice are more sensitive to the excitotoxicity.     

Excitotoxic neurodegeneration induced by intranasal administration of kainic acid in C57BL/6 mice

Glutamate excitotoxicity plays a key role in inducing neuronal cell death in many neurological diseases. In mice, administration of kainic acid, an analogue of the excitotoxin glutamate, results in hippocampal cell death and seizures. Kainic-acid-induced seizures in mice provide a well-characterized model for studies of human neurodegenerative diseases. However, C57BL/6 mice, which are often used for genetic analyses and transgenic and knockout studies, are resistant to excitotoxicity induced by subcutaneous administration of kainic acid. In the present study, kainic acid administered by the intranasal route was shown to result in continuous tonic-clonic seizures in C57BL/6 mice. These seizures continued for 1-5 h and successfully induced selective lesions in area CA3 of the hippocampus. The survival rate was high even after mice experienced severe seizures. The hippocampal lesions were associated with a high level of cyclooxygenase-2 production as well as astrogliosis. Administration of kainic acid also altered behavioral responses, with mice showing a significant increase in locomotion and rearing activity as indicated by an open-field test. This animal model could provide a valuable tool for exploring the role of excitotoxicity in neuropathological conditions and should be further evaluated in gene-targeting studies of neurodegenerative diseases.     

小剂量海人酸导致C57BL/6 小鼠产生慢性神经退行性病变

目的探索应用海人酸在C57BL/6小鼠建立慢性进行性神经退行性病变的新的动物模型。方法小剂量(3mg/kg体重)海人酸经鼻滴入C57BL/6小鼠,每3d给药1次,连续20次。观察临床表现,并应用旷场行为实验检测小鼠的行为学变化;通过Nissl染色方法评估鼠脑病理变化以及免疫组织化学方法分析小胶质细胞活化和星形胶质细胞增生情况。结果应用小剂量海人酸反复多次经鼻给药,小鼠虽无明显临床症状,但引起其皮质和海马发生兴奋性毒性所致的神经细胞退行性病变,小胶质细胞活化和星形胶质细胞增生以及行为学变化。结论此慢性动物模型的神经病理改变与人类神经退行性疾病的变化相似,因此,本文为研究人类神经系统慢性退行性疾病的发病机制及治疗提供了一个有用的动物模型。     

Kainic acid-induced excitotoxic hippocampal neurodegeneration in C57BL/6 mice: B cell and T cell subsets may contribute differently to the pathogenesis

The roles of T cells and B cells in kainic acid (KA)-induced hippocampal lesions were studied in C57BL/6 mice lacking specific T cell populations (CD4, CD8, and CD4/CD8 cells) and B cells [Igh-6(-/-)]. At 48 mg/kg of KA administrated intranasally, KA-induced convulsions were seen in all groups. However, CD4/CD8(-/-) mice exhibited the mildest seizures; the responses of CD8(-/-), Igh-6(-/-) and wild-type mice were intermediate, whereas CD4(-/-) mice displayed much more severe clinical signs and 100% early mortality, indicating that a deficiency of CD4 T cells obviously increased susceptibility to KA-induced brain damage. Histopathological analysis of the mice that survived 7 days after KA administration revealed that CD4/CD8(-/-) mice had the fewest pathologic changes but Igh-6(-/-) mice showed more severe lesions in area CA3 of the hippocampus than CD8(-/-) and wild-type mice. Reactive astrogliosis were prominent in all KA-treated mice. Locomotor activity as assessed by open-field test increased after KA administration in Igh-6(-/-) and wild-type mice only. These results denote the influence of the adaptive immune response on KA-induced hippocampal neurodegeneration and suggest that B cell and T cell subsets may contribute differently to the pathogenesis.     

A deficiency of axonal regeneration in C57BL/6J mice

Axonal regeneration within peripheral nerves and dorsal spinal roots was investigated in inbred strains of mice with known differences in macrophage recruitment and inflammatory functions. During the second week after sciatic nerve crush, counts of regenerating newly myelinated fibres were significantly lower in C57BL/6J mice than in 4 other strains. After dorsal root crush with or without concomitant sciatic nerve transection to enhance regeneration, fibre counts in roots of C57BL/6J were one-fifth of those in A/J mice. Axonal regeneration is subnormal in C57BL/6J mice but this defect appears not to be linked to known deficiencies in macrophage function.     

TNF-alpha receptor 1 deficiency enhances kainic acid-induced hippocampal injury in mice

The exact role of TNF-alpha in excitotoxic neurodegeneration of the brain is unclear. To address this issue, the kainic acid (KA)-induced hippocampal injury model, a well-characterized model of human neurodegenerative diseases, was used in TNF-alpha receptor 1 (TNFR1)-knockout (TNFR1-/-) mice in the present study. After nasal application of a single dose of 40 mg of KA per kilogram body weight, TNFR1-/- mice showed significantly more severe seizures than the wild-type mice. In addition, obvious neurodegeneration, enhanced microglia activation, and astrogliosis in the hippocampus, as well as increased locomotor activity, were found in TNFR1-/- mice compared with the wild-type controls 8 days after KA delivery. Moreover, CC chemokine receptor 3 expression on activated microglia was increased 3 days after KA treatment in TNFR1-/- mice, as measured by flow cytometry. These data suggest that TNF-alpha may play a protective role through TNFR1 signaling.     

Lack of morphine-induced hyperactivity in C57BL/6 mice following striatal kainic acid lesions

Bilateral injection of kainic acid (0.15 micrograms/0.3 microliters) into the striatum (caudatus/putamen) of C57BL/6 mice prevented stimulation of locomotor activity by morphine (20 mg/kg, i.p.). This effect was specific to morphine since mice with the same lesion did not show any impairment of amphetamine (2 mg/kg)-induced locomotor hyperactivity. Histological inspections showed neuron damage also in the nucleus accumbens, while hippocampus was not damaged by kainic acid. Moreover, mice with kainic acid lesions in the hippocampus were more stimulated by morphine, compared with the morphine-injected sham lesion group. The results, which suggest the existence of non-catecholaminergic mediations in the locomotor effects of morphine, are discussed in terms of opioid systems in the brain.     

Enhanced seizures and hippocampal neurodegeneration following kainic acid-induced seizures in metallothionein-I + II-deficient mice

Metallothioneins (MTs) are major zinc binding proteins in the CNS that could be involved in the control of zinc metabolism as well as in protection against oxidative stress. Mice lacking MT-I and MT-II (MT-I + II deficient) because of targeted gene inactivation were injected with kainic acid (KA), a potent convulsive agent, to examine the neurobiological importance of these MT isoforms. At 35 mg/kg KA, MT-I + II deficient male mice showed a higher number of convulsions and a longer convulsion time than control mice. Three days later, KA-injected mice showed gliosis and neuronal injury in the hippocampus. MT-I + II deficiency decreased both astrogliosis and microgliosis and potentiated neuronal injury and apoptosis as shown by terminal deoxynucleotidyl transferase-mediated in situ end labelling (TUNEL), detection of single stranded DNA (ssDNA) and by increased interleukin-1beta-converting enzyme (ICE) and caspase-3 levels. Histochemically reactive zinc in the hippocampus was increased by KA to a greater extent in MT-I + II-deficient compared with control mice. KA-induced seizures also caused increased oxidative stress, as suggested by the malondialdehyde (MDA) and protein tyrosine nitration (NITT) levels and by the expression of MT-I + II, nuclear factor-kappaB (NF-kappaB), and Cu/Zn-superoxide dismutase (Cu/Zn-SOD). MT-I + II deficiency potentiated the oxidative stress caused by KA. Both KA and MT-I + II deficiency significantly affected the expression of MT-III, granulocyte-macrophage colony stimulating factor (GM-CSF) and its receptor (GM-CSFr). The present results indicate MT-I + II as important for neuron survival during KA-induced seizures, and suggest that both impaired zinc regulation and compromised antioxidant activity contribute to the observed neuropathology of the MT-I + II-deficient mice.     

Regional neuropathology following kainic acid intoxication in adult and aged C57BL/6J mice

We evaluated regional neuropathological changes in adult and aged male mice treated systemically with kainic acid (KA) in a strain reported to be resistant to excitotoxic neuronal damage, C57BL/6. KA was administered in a single intraperitoneal injection. Adult animals were dosed with 35 mg/kg KA, while aged animals received a dose of 20 mg/kg in order to prevent excessive mortality. At time-points ranging from 12 h to 7 days post-treatment, animals were sacrificed and prepared for histological evaluation utilizing the cupric-silver neurodegeneration stain, immunohistochemistry for GFAP and IgG, and lectin staining. In animals of both ages, KA produced argyrophilia in neurons throughout cortex, hippocampus, thalamus, and amygdala. Semi-quantitative analysis of neuropathology revealed a similar magnitude of damage in animals of both ages, even though aged animals received less toxicant. Additional animals were evaluated for KA-induced reactive gliosis, assayed by an ELISA for GFAP, which revealed a 2-fold elevation in protein levels in adult mice, and a 2.5-fold elevation in aged animals. Histochemical evaluation of GFAP and lectin staining revealed activation of astrocytes and microglia in regions with corresponding argyrophilia. IgG immunostaining revealed a KA-induced breach of the blood-brain barrier in animals of both ages. Our data indicate widespread neurotoxicity following kainic acid treatment in C57BL/6J mice, and reveal increased sensitivity to this excitotoxicant in aged animals.     

Epileptic seizures and hippocampal damage after cuprizone-induced demyelination in C57BL/6 mice

Epileptic seizures are known to occur in different animal models of demyelination and have also been described in demyelinating diseases of the central nervous system (CNS) such as multiple sclerosis. How myelin deficiency might cause seizures is unknown, but may involve axonal pathology and resultant alterations in neuronal excitability. The cause of seizures occurring in rodent demyelination models is unknown. In the present study, we used EEG/video monitoring to record seizures occurring during chronic demyelination of C57BL/6 mice fed for 12 weeks with 0.2% cuprizone. Furthermore, in the search for a morphological correlate of the seizures, the hippocampal formation was examined histologically. Epileptiform spikes resembling interictal spikes known from chronic epilepsy were recorded in all cuprizone-treated mice, but not in controls. Most cuprizone-treated animals exhibited generalized tonic-clonic seizures upon stress-inducing stimuli. In addition to the known demyelination of the corpus callosum, massive demyelination was found in the hippocampal formation. This was associated with neuronal alterations, including a loss of neurons in the hilus of the dentate gyrus. In view of the role of the dentate gyrus in epileptogenesis, demyelination leading to axonal pathology and thus neuronal damage as observed in the hilus may be causally involved in the paroxysmal alterations observed after prolonged treatment with cuprizone. The present data suggest a potential role of the hippocampal formation for seizures occurring as a consequence of neuronal damage secondary to CNS demyelination.     

Ethanol-induced apoptotic neurodegeneration in the developing C57BL/6 mouse brain

Recent studies have shown that administration of ethanol to infant rats during the synaptogenesis period (first 2 weeks after birth), triggers extensive apoptotic neurodegeneration throughout many regions of the developing brain. While synaptogenesis is largely a postnatal phenomenon in rats, it occurs prenatally (last trimester of pregnancy) in humans. Recent evidence strongly supports the interpretation that ethanol exerts its apoptogenic action by a dual mechanism--blockade of NMDA glutamate receptors and hyperactivation of GABA(A) receptors. These findings in immature rats represent a significant advance in the fetal alcohol research field, in that previous in vivo animal studies had not demonstrated an apoptogenic action of ethanol, had not documented ethanol-induced cell loss from more than a very few brain regions and had not provided penetrating insight into the mechanisms underlying ethanol's neurotoxic action. To add to the mechanistic insights recently gained, it would be desirable to examine gene-regulated aspects of ethanol-induced apoptotic neurodegeneration, using genetically altered strains of mice. The feasibility of such research must first be established by demonstrating that appropriate mouse strains are sensitive to this neurotoxic mechanism. In the present study, we demonstrate that mice of the C57BL/6 strain, a strain frequently used in transgenic and gene deletion research, are exquisitely sensitive to the mechanism by which ethanol induces apoptotic neurodegeneration during the synaptogenesis period of development.     

Reduced susceptibility to kainic acid-induced excitoxicity in T-cell deficient CD4/CD8(-/-) and middle-aged C57BL/6 mice

Kainic acid (KA)-induced hippocampal injury is a good model for studying human neurodegenerative diseases. To investigate the roles of immune cells and age related changes in neurodegeneration, we used this model to assess reactions in young and middle-aged wild-type and CD4/CD8(-/-) mice by intranasal administration of KA. We found that CD4/CD8-deficiency resulted in a significant reduction of the severity of clinical signs and pathological changes in KA-treated young, but not in KA-treated middle-aged mice. Middle-aged wild-type mice had a similar reaction to KA insult as young and middle-aged CD4/CD8(-/-) mice. CD4/CD8(-/-) mice exhibited decreased locomotor and rearing activities as they approached to middle-aged state, which was not seen in wild-type mice. In addition, CD4/CD8-deficiency and increased age prevented KA-induced increase of both locomotor and rearing activities. The results suggest that a decline of immunological function is associated with aging, and both of them may contribute to the relative resistance to KA-induced neurotoxicity.     

B细胞和T细胞亚型在海人藻酸引起的C57BL/6鼠海马神经元损伤过程中不同的作用

目的应用海人藻酸在C57BL/6免疫缺陷小鼠建立了神经退行性病变并观察了免疫活性B细胞和T细胞亚型在病变过程中的作用。方法经鼻滴人海人藻酸观察其临床和病理变化、细胞流式仪检测和分析脾细胞表面标记。结果海人藻酸引起了CD_4基因敲除(CD_4-/-)、CD_8(CD_8-/-)、CD_(48)(CD_(48)-/-)和B细胞(Igh6-/-)基因敲除鼠的临床抽搐症状和海马损伤。其临床症状在CD_4(-/-)鼠最重,CD_8(-/-),Igh6(-/-)以及野生型鼠次之,而CD_4、CD_8双重基因缺陷鼠最轻。病理变化大约和临床症状相平行,脾细胞表面标记的表达也证实了上述发现。结论获得性免疫反应参与了海人藻酸引起的海马损伤。CD_4T细胞和B细胞在病变过程中可能起到了保护作用,而CD8T细胞则加重神经退行性病变。     

Observational learning in C57BL/6j mice

The ability of mice to solve a complex task by observational learning was investigated with C57BL/6j mice. Four female demonstrators were trained to reliably perform a sequence that consisted in pushing a piece of food into a tube attached to the side of a puzzle box, and recovering it by opening a drawer in front of the box. They then performed this sequence in front of naive mice assigned to individual cubicles in a box with a wire mesh front arranged in a row facing the demonstrators. A total of 25 naive mice (13 males and 12 females) were used. Fifteen mice observed 14 demonstrations a day for 5 days; 10 control mice were placed in similar cubicles, but behind a plastic screen which prevented them from observing the demonstrators. The mice were post-tested in the demonstrator situation, and 6 of 15 observers immediately reproduced the complete task successfully, but none of the naive or control mice were able to solve the task. The observers and controls were then subjected to a five level individual learning schedule. Observers learned the individual task significantly faster than the controls. No sex difference was found. These results suggest that observational learning processes at work were based on stimulus enhancement and observational conditioning.     

A comparison of the behavior of C57BL/6 and C57BL/10 mice

Selection of an appropriate animal model is a crucial first step in many research programs. The C57BL/6 (B6) mouse is the most widely used inbred mouse strain in biomedical research; this is particularly so in behavioral studies. However, there are several C57BL substrains, all derived from common ancestors. C57BL/10 (B10) mice are superficially almost identical to B6 mice in appearance and behavior and widely used in inflammation and immunology research, yet rarely in behavioral studies. The present study assessed the comparability of behavioral results from these two strains, to determine whether they could be used interchangeably in future behavioral experiments. The results showed that the behavior of B6 mice clearly differed from that of B10 mice: in tests of cognition, species-typical behaviors, and motor coordination the B6 strain performed better. Consequently, B6 mice will probably remain the preferred choice for behavioral studies. Interpretation of results derived from the B10 strain should take into account its particular behavioral characteristics.     

Sensitive indicators of injury reveal hippocampal damage in C57BL/6J mice treated with kainic acid in the absence of tonic-clonic seizures

Sensitive indices of neural injury were used to evaluate the time course of kainic acid (KA)-induced hippocampal damage in adult C57BL/6J mice (4 months), a strain previously reported to be resistant to kainate-induced neurotoxicity. Mice were injected systemically with saline or kainate, scored for seizure severity (Racine scale), and allowed to survive 12 h, one, three, or seven days following which they were evaluated for neuropathological changes using histological or biochemical endpoints. Most kainate-treated mice exhibited limited seizure activity (stage 1); however, cupric-silver and Fluoro-Jade B stains revealed significant damage by 12 h post-treatment. Immunohistochemistry and immunoassay of glial fibrillary acidic protein and lectin staining revealed a strong treatment-induced reactive gliosis and microglial activation. Immunostaining for immunoglobulin G revealed a kainate-induced breach in the blood-brain barrier. Nissl and hematoxylin stains provided little information regarding neuronal damage, but revealed the identity of non-resident cells which infiltrated the pyramidal layer. Our data suggest sensitive indicators of neural injury evaluated over a time course, both proximal and distal to treatment, are necessary to reveal the full extent of neuropathological changes which may be underestimated by traditional histological stains. The battery of neuropathological indices reported here reveals the C57BL/6J mouse is sensitive to excitotoxic neural damage caused by kainic acid, in the absence of tonic-clonic seizures.     

Neuronal stress and injury in C57/BL mice after systemic kainic acid administration

Kainate-induced seizures are widely studied as a model of human temporal lobe epilepsy due to behavioral and pathological similarities. While kainate-induced neuronal injury is well characterized in rats, relatively little data is available on the use of kainate and its consequences in mice. The growing availability of genetically altered mice has focused attention on the need for well characterized mouse seizure models in which the effects of specific genetic manipulations can be examined. We therefore examined the kainate dose–response relationship and the time-course of specific histopathological changes in C57/BL mice, a commonly used founder strain for transgenic technology. Seizures were induced in male C57/BL mice (kainate 10–40 mg/kg i.p.) and animals were sacrificed at various time-points after injection. Seizures were graded using a behavioral scale developed in our laboratory. Neuronal injury was assayed by examining DNA fragmentation using in situ nick translation histochemistry. In parallel experiments, we examined the expression an inducible member of the heat shock protein family, HSP-72, another putative marker of neuronal injury, using a monoclonal antibody. Seizure severity paralleled kainate dosage. At higher doses DNA fragmentation is seen mainly in hippocampus in area CA3, and variably in CA1, thalamus and amygdala within 24 h, is maximal within 72 h, and is largely gone by 7 days after administration of kainate. HSP-72 expression is also highly selective, occurring in limbic structures, and it evolves over a characteristic time-course. HSP-72 is expressed mainly in structures that also manifest DNA fragmentation. Using double-labeling techniques, however, we find essentially no overlap between neurons expressing HSP-72 and DNA fragmentation. These findings indicate that DNA fragmentation and HSP-72 expression are complementary markers of seizure-induced stress and injury, and support the notion that HSP-72 expression is neuroprotective following kainate-induced seizures.     

p75NTR independent oligodendrocyte death in cuprizone-induced demyelination in C57BL/6 mice

Feeding C57Bl/6 J mice the copper chelator cuprizone leads to selective apoptosis of mature oligodendrocytes and concomitant demyelination predominantly in the corpus callosum. The process of oligodendrocyte apoptosis in this animal model for multiple sclerosis (MS) involves early microglial activation, but no infiltration of T-lymphocytes. Therefore, this model could mimic early stages of oligodendrocyte degeneration Affected oligodendrocytes express the common neurotrophin receptor, p75(NTR), a 'stress-receptor' which under certain circumstances can induce apoptosis. Only affected oligodendrocytes in MS lesions and MS animal models express this receptor. In order to study the significance of p75(NTR) in the fate of oligodendrocytes, we have exposed wild-type as well as p75(NTR)-knockout mice to a 0.2% (w/w) cuprizone diet and performed a comparative immunohistochemical analysis of the corpus callosum at various time points. Surprisingly, our results show that the absence of p75(NTR) did not alter cuprizone-induced oligodendrocyte death (and subsequent de- or remyelination). Apparently, intracellular apoptosis pathways in adult oligodendrocytes do not require p75(NTR) activated signal transduction in the absence of T-lymphocytes and T-lymphocyte derived cytokines.     

MBP-PLP fusion protein-induced EAE in C57BL/6 mice

Gene knock-out and knock-in mice are becoming increasingly indispensable for mechanism-oriented studies of EAE. Most gene-modified mice are on the C57BL/6 background, for which presently there are only two EAE models available, the MOG peptide 35-55 and the PLP 178-191 peptide induced disease. However, because MS is not a single pathogenic entity, different EAE models are required to reproduce and study its various features. Here we are introducing MBP-PLP fusion protein (MP4)-induced EAE for C57BL/6 mice. B cell- and CD8+ T cell-dependence, as well as multi-determinant recognition are among the unique features of this demyelinating EAE.