Swim-induced grooming in mice is mediated by a dopaminergic substrate

Summary Grooming induced in mice after a period of swimming was potently and dose-dependently blocked by neuroleptics. The order of potency of the neuroleptics was spiroperidol>haloperidol>cis-flupenthixol>pimozide>chlorpromazine>thioridazine. The trans isomer of flupenthixol was inactive at 40M/kg. The-adrenergic receptor antagonists, phentolamine and phenoxybenzamine, and the catecholamine synthesis inhibitor,-methyl-p-tyrosine were essentially without effect on the grooming behaviour. Amitriptyline inhibited grooming behaviour only in doses which severely affected the animals motor function. Fluoxetine was without effect. Cisflupenthixol was less active in inhibiting grooming in animals chronically treated with haloperidol than in control animals, indicating the presence of supersensitive dopamine receptors. The data indicate that swim-induced grooming in mice is mediated via dopaminergic systems.     

Lipopolysaccharide-induced depressive-like behavior is mediated by indoleamine 2,3-dioxygenase activation in mice

Although elevated activity of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) has been proposed to mediate comorbid depression in inflammatory disorders, its causative role has never been tested. We report that peripheral administration of lipopolysaccharide (LPS) activates IDO and culminates in a distinct depressive-like behavioral syndrome, measured by increased duration of immobility in both the forced-swim and tail suspension tests. Blockade of IDO activation either indirectly with the anti-inflammatory tetracycline derivative minocycline, that attenuates LPS-induced expression of proinflammatory cytokines, or directly with the IDO antagonist 1-methyltryptophan (1-MT), prevents development of depressive-like behavior. Both minocycline and 1-MT normalize the kynurenine/tryptophan ratio in the plasma and brain of LPS-treated mice without changing the LPS-induced increase in turnover of brain serotonin. Administration of L-kynurenine, a metabolite of tryptophan that is generated by IDO, to naive mice dose dependently induces depressive-like behavior. These results implicate IDO as a critical molecular mediator of inflammation-induced depressive-like behavior, probably through the catabolism of tryptophan along the kynurenine pathway.     

Interferon-gamma-inducing factor (IL-18) and interferon-gamma in inflammatory CNS diseases.

OBJECTIVE: To examine the intrathecal production of a newly identified cytokine, interferon-gamma-inducing factor (IL-18), together with interferon-gamma itself, in inflammatory diseases of the CNS (i.e., bacterial meningitis, viral meningoencephalitis, and MS). RESULTS: IL-18 concentrations in CSF were significantly increased in bacterial meningitis and tended toward increased levels in viral meningoencephalitis. In contrast, IL-18 was detectable only in a few patients with MS and healthy controls. Interestingly, interferon-gamma was significantly increased selectively in CSF of patients with viral meningoencephalitis. CONCLUSION: The observation of an intrathecal release of IL-18 in patients with meningitis argues for a pathophysiologic role of this novel cytokine in immunity against invading microorganisms the CNS.     

Antigen-induced changes in odor attractiveness and reproductive output in male mice

Modulation of social signals by antigen-induced immunoenhancement is a significant component of behavioral and reproductive adaptations of a host population to parasitic pressure. To investigate this concept, we studied odor attractiveness and reproductive output of ICR male mice treated with keyhole limpet hemocyanin (KLH) as an antigenic stimulus. We collected urine samples for olfactory preference tests (control vs. KLH administration) on different days following treatment. We found that the differences in odor attractiveness between control and immunized males, which were observed on the 3rd day, disappeared soon afterwards. Odor attractiveness of male mice positively correlated with their immunoresponsiveness, which was assessed by the sum of anti-KLH IgG1 and anti-KLH IgG2a titers. According to the hypothesis of terminal investment, antigen-treated males had higher reproductive output in comparison with control males and produced more progeny as a result.     

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.     

炎症小体NLRP3相关炎症因子IL-1β、IL-18与脑小血管病相关性研究

目的检测脑小血管病(CSVD)患者血清中炎症小体相关炎症因子IL~(-1)β、IL~(-1)8浓度,建立炎症小体NLRP3与CSVD之间的潜在关联。方法纳入CSVD患者50例和同期具有脑血管病危险因素,但头颅影像学检查正常的患者(主要指头颅磁共振平扫及弥散成像)30例。采集血样标本,使用双抗体夹心法对血清IL~(-1)β、IL~(-1)8浓度进行检测,比较其与CSVD的相关性。结果 CSVD组IL~(-1)β血清浓度(19.21±3.77ng·L~(-1))比对照组(16.56±2.84ng·L~(-1))高(P<0.05);CSVD组IL~(-1)8血清浓度(73.38±19.27ng·L~(-1))比对照组(70.70±2.84ng·L~(-1))高,但差异无统计学意义(P>0.05)。以CSVD为因变量,以年龄、性别、高血压、糖尿病、冠心病、既往卒中史、吸烟史、饮酒史、颈动脉内膜增厚、颈动脉斑块形成、血同型半胱氨酸、糖化血红蛋白、空腹血糖、三酰甘油、低密度脂蛋白、总胆固醇、IL~(-1)β、IL~(-1)8为自变量,进行Logistic回归分析。IL~(-1)β、年龄、吸烟是CSVD的危险因素(B=0.446,OR=1.562,P=0.003;B=0.166,OR=1.180,P=0.007;B=4.107,OR=0.016,P=0.010)。结论血清IL~(-1)β浓度升高与CSVD有相关性;IL~(-1)β、年龄、吸烟是脑小血管病的危险因素。     

Increased blood-brain barrier permeability in LP-BM5 infected mice is mediated by neuroexcitatory mechanisms.

Serum protein levels in LP-BM5 infected mouse brains were investigated to gain insight into the contribution of blood-brain barrier (BBB) patency to the pathogenesis of retroviral encephalopathy. Evans blue uptake by the forebrain and cerebellum was significantly increased between 8-12 weeks post inoculation. Immunohistochemistry revealed foci of albumin, transferrin, alpha(2)-macroglobulin and IgG transudation around blood vessels particularly in the cerebral cortex and cerebellar vermis. These leaks were often associated with astrocytosis and apoptotic cells. Unlike the other serum proteins, IgG immunoreactivity extended from the circumventricular organs and disseminated throughout the brain parenchyma, accumulating on the plasma membranes of hippocampal and cortical neurons. Consistent with the chronic elevation of free glutamate levels in LP-BM5 infected mice, the increase in Evans blue uptake into the forebrain was completely reversed following dizocilpine administration. Thus, the chronic increase in free glutamate levels in LP-BM5 infected mouse brain contributes to BBB disruption. Furthermore, the CNS accumulation of serum proteins, particularly IgG, observed in these mice may increase osmotic load, impair neuronal function, and cause white matter pallor. Administration of NMDA receptor antagonists may prove useful in managing BBB permeability in those neuropathologies, such as HIV-associated dementia/cognitive/motor complex, having a glutamatergic component.     

The adrenal gland is a source of stress-induced circulating IL-18

The present study compared plasma IL-18 levels between sham-operated and adrenalectomized mice following stress to investigate whether the adrenal gland contributes to the elevation of circulating IL-18 during stress. Two hours of stress provoked a robust, stressor-dependent, elevation of IL-18 mRNA and peptide in the adrenal gland in sham-operated mice. Consistently, levels of circulating mature IL-18 increased during stress and remained elevated for up to 6 h after stress. The stress-induced increase in circulating IL-18 was abolished by adrenalectomy. These findings demonstrate that the adrenal gland is required to achieve elevation of circulating IL-18 during stress.     

Increased vulnerability of nigrostriatal terminals in DJ-1-deficient mice is mediated by the dopamine transporter

Mutations in the gene for DJ-1 have been associated with early-onset autosomal recessive parkinsonism. Previous studies of null DJ-1 mice have shown alterations in striatal dopamine (DA) transmission with no DAergic cell loss. Here we characterize a new line of DJ-1-deficient mice. A subtle locomotor deficit was present in the absence of a change in striatal DA levels. However, increased [(3)H]-DA synaptosomal uptake and [(125)I]-RTI-121 binding were measured in null DJ-1 vs. wild-type mice. Western analyses of synaptosomes revealed significantly higher dopamine transporter (DAT) levels in pre-synaptic membrane fractions. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure exacerbated striatal DA depletion in null DJ-1 mice with no difference in DAergic nigral cell loss. Furthermore, increased 1-methyl-4-phenylpyridinium (MPP(+)) synaptosomal uptake and enhanced MPP(+) accumulation were measured in DJ-1-deficient vs. control striatum. Thus, under null DJ-1 conditions, DAT changes likely contribute to altered DA neurotransmission and enhanced sensitivity to toxins that utilize DAT for nigrostriatal entry.     

Effect of spleen-cell-conditioned medium on [3H]-choline uptake by retinal cells in vitro is mediated by IL-2

Cytokines are essential molecules throughout the development of the nervous system and also play an important role during the adult life span. In the present work, we analyzed in vitro the effect of spleen-cell-conditioned medium (SCM) on the survival and [3H]-choline uptake of neonatal rat retinal cells. SCM induced an increase in neuronal survival, glial cell proliferation and neurite outgrowth, as evaluated by biochemical and morphological criteria. These effects were time dependent; after 120 h, SCM induced a 6-fold increase in the total protein level. The effect of SCM was blocked both by the inhibition of protein tyrosine kinase activity (10 microM genistein) and by the inhibition of cell division (20 microM fluorodeoxyuridine). SCM also increased the uptake of [3H]-choline by retinal cells. The effect was time dependent. The maximum effect was obtained after 48 h and was maintained at a high level until 120 h. Treatment by 10 microM genistein and 15 microM bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) (an intracellular calcium chelator) completely blocked this effect. However, 20 microM fluorodeoxyuridine did not abolish it. Conditioned medium obtained from glial cells stimulated with SCM (S-GCM) induced an effect on [3H]-choline uptake earlier than that promoted by SCM. Anti-interleukin-2 (IL-2) antibodies blocked the effect of both SCM and S-GCM on [3H]-choline uptake after 48 and 72 h. IL-2 (50 U/ml) elicited the same effect as that observed when the cells were maintained in the presence of SCM. Taken together, our results suggest that IL-2 plays an important role in controlling the survival and differentiation of retinal cells in vitro.     

IL-6-deficient mice show impaired inflammatory response in a model of myosin-induced experimental myositis

Inflammatory/immune reactions against muscle cells are responsible for the damage in idiopathic inflammatory myopathies. We investigated the role of IL-6, a cytokine known to contribute to local leukocyte accumulation, in a model of myosin-induced experimental myositis. After injection of rabbit myosin in CFA/pertussis toxin, normal mice develop clinically evident muscle deficit and damage, as demonstrated by myofiber necrosis and leukocyte infiltration, while IL-6-deficient mice have no clinical or histological signs of muscle damage. This study evidences that selective deficiency of IL-6 directly or indirectly hinders the local inflammatory response and its harmful effects in this model of muscle damage.     

The PD-1/PD-L pathway is up-regulated during IL-12-induced suppression of EAE mediated by IFN-gamma

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), is mediated by autoantigen-specific T-helper1 (Th1) cells. IL-12, an inducer of Th1 cell development, exerts immunomodulatory effects in EAE. Programmed death-1 (PD-1) and PD-1 ligand (PD-L), new members of the B7 superfamily of costimulatory molecules, play a critical role in regulating EAE. Whether the interaction of IL-12 and the PD-1/PD-L pathway regulates EAE is unclear. We have previously shown that IL-12 suppresses EAE induced by MOG35-55 in C57BL/6 mice, but not in IFN-gamma-deficient mice, suggesting that IFN-gamma is required for the inhibitory effects of IL-12 on EAE. In the current study, PD-L1 expression is up-regulated following IL-12 treatment in wild-type mice, but not in IFN-(-deficient EAE mice. Similarly, IL-12 induces IFN-gamma and PD-L1 expression in cultured MOG-specific T cells from wild-type mice but not from IFN-gamma-deficient mice. Furthermore, PD-L1 expression increased specifically in CD11b+ antigen presenting cells (APCs) after IL-12 administration. These data suggest that one mechanism of IL-12 suppression of EAE is mediated by PD-1/PD-L signaling downstream of IFN-gamma induction in CD11b+ APCs. The regulation of PD-1/PD-L1 may have potential therapeutic effects for EAE and MS.     

Increased blood–brain barrier permeability in LP-BM5 infected mice is mediated by neuroexcitatory mechanisms

Serum protein levels in LP-BM5 infected mouse brains were investigated to gain insight into the contribution of blood–brain barrier (BBB) patency to the pathogenesis of retroviral encephalopathy. Evans blue uptake by the forebrain and cerebellum was significantly increased between 8–12 weeks post inoculation. Immunohistochemistry revealed foci of albumin, transferrin, α₂-macroglobulin and IgG transudation around blood vessels particularly in the cerebral cortex and cerebellar vermis. These leaks were often associated with astrocytosis and apoptotic cells. Unlike the other serum proteins, IgG immunoreactivity extended from the circumventricular organs and disseminated throughout the brain parenchyma, accumulating on the plasma membranes of hippocampal and cortical neurons. Consistent with the chronic elevation of free glutamate levels in LP-BM5 infected mice, the increase in Evans blue uptake into the forebrain was completely reversed following dizocilpine administration. Thus, the chronic increase in free glutamate levels in LP-BM5 infected mouse brain contributes to BBB disruption. Furthermore, the CNS accumulation of serum proteins, particularly IgG, observed in these mice may increase osmotic load, impair neuronal function, and cause white matter pallor. Administration of NMDA receptor antagonists may prove useful in managing BBB permeability in those neuropathologies, such as HIV-associated dementia/cognitive/motor complex, having a glutamatergic component.     

Elevated intracranial IL-18 in humans and mice after traumatic brain injury and evidence of neuroprotective effects of IL-18-binding protein after experimental closed head injury.

Proinflammatory cytokines are important mediators of neuroinflammation after traumatic brain injury. The role of interleukin (IL)-18, a new member of the IL-1 family, in brain trauma has not been reported to date. The authors investigated the posttraumatic release of IL-18 in murine brains following experimental closed head injury (CHI) and in CSF of CHI patients. In the mouse model, intracerebral IL-18 was induced within 24 hours by ether anesthesia and sham operation. Significantly elevated levels of IL-18 were detected at 7 days after CHI and in human CSF up to 10 days after trauma. Published data imply that IL-18 may play a pathophysiological role in inflammatory CNS diseases; therefore its inhibition may ameliorate outcome after CHI. To evaluate the functional aspects of IL-18 in the injured brain, mice were injected systemically with IL-18-binding protein (IL-18BP), a specific inhibitor of IL-18, 1 hour after trauma. IL-18BP-treated mice showed a significantly improved neurological recovery by 7 days, accompanied by attenuated intracerebral IL-18 levels. This demonstrates that inhibition of IL-18 is associated with improved recovery. However, brain edema at 24 hours was not influenced by IL-18BP, suggesting that inflammatory mediators other than IL-18 induce the early detrimental effects of intracerebral inflammation.     

l-Glutamine-induced apoptosis in microglia is mediated by mitochondrial dysfunction

Under physiological conditions, astrocytes take up l ‐glutamate from the synaptic gap, metabolize it to l ‐glutamine and return it to neurons, where l ‐glutamine is metabolized to l ‐glutamate and stored in neurotransmitter vesicles. However, under pathological conditions, such as hepatic failure, l ‐glutamine and ammonium are elevated globally in the brain. The Trojan horse hypothesis of l ‐glutamine toxicity assumes that intramitochondrial hydrolysis of l ‐glutamine enhances ammonium locally and leads to mitochondrial dysfunction. In the present study, we show that exposure of murine primary microglia as well as of the microglial cell‐line BV‐2 to l ‐glutamine promotes chromatin condensation and formation of crescent‐like structures in the nucleus. Furthermore, l ‐glutamine induced an increase in annexin‐V labelling, cell shrinkage (apoptotic volume decrease), cell fragmentation and formation of apoptotic bodies. Inhibition of the phosphate‐activated glutaminase with 6‐diazo‐5‐oxo‐l ‐norleucine suppressed chromatin condensation and annexin‐V labelling in l ‐glutamine‐exposed cells. In addition, inhibition of the glutamine synthetase with l ‐methionine sulfoximine suppressed chromatin condensation and annexin‐V labelling in ammonium‐exposed cells. l ‐Glutamine and ammonium enhanced production of reactive oxygen species, as detected with CM‐H₂DCFDA. Apoptosis, induced by l ‐glutamine, was inhibited either by the radical scavenger α‐tocopherol or by the nitric oxide synthase blocker N ^G‐methyl‐l ‐arginine. Cyclosporin A, a ligand of the permeability transition pore complex component cyclophilin D, prevented l ‐glutamine‐triggered apoptosis. Furthermore, blockade of caspase‐9 activity with Z‐LEHD‐FMK prevented l ‐glutamine‐triggered apoptosis. Taken together, our results indicate that hydrolysis of l ‐glutamine and, accordingly, accumulation of ammonium in mitochondria induce the intrinsic pathway of apoptosis, characterized by mitochondrial dysfunction and activation of caspase‐9, which activates caspase‐3.