Parkinson's disease (PD) is an age-related progressive neurodegenerative disorder caused by selective loss of dopaminergic neurons from the substantia nigra (SN) to the striatum. The initial factor ...that triggers neurodegeneration is unknown; however, inflammation has been demonstrated to be significantly involved in the progression of PD. The present study was designed to investigate the role of the pro-inflammatory cytokine interleukin-1 (IL-1) in the activation of microglia and the decline of motor function using IL-1 knockout (KO) mice.
Lipopolysaccharide (LPS) was stereotaxically injected into the SN of mice brains as a single dose or a daily dose for 5 days (5 mg/2 ml/injection, bilaterally). Animal behavior was assessed with the rotarod test at 2 hr and 8, 15 and 22 days after the final LPS injection.
LPS treatment induced the activation of microglia, as demonstrated by production of IL-1β and tumor necrosis factor (TNF) α as well as a change in microglial morphology. The number of cells immunoreactive for 4-hydroxynonenal (4HNE) and nitrotyrosine (NT), which are markers for oxidative insults, increased in the SN, and impairment of motor function was observed after the subacute LPS treatment. Cell death and aggregation of α-synuclein were observed 21 and 30 days after the final LPS injection, respectively. Behavioral deficits were observed in wild-type and TNFα KO mice, but IL-1 KO mice behaved normally. Tyrosine hydroxylase (TH) gene expression was attenuated by LPS treatment in wild-type and TNFα KO mice but not in IL-1 KO mice.
The subacute injection of LPS into the SN induces PD-like pathogenesis and symptoms in mice that mimic the progressive changes of PD including the aggregation of α-synuclein. LPS-induced dysfunction of motor performance was accompanied by the reduced gene expression of TH. These findings suggest that activation of microglia by LPS causes functional changes such as dopaminergic neuron attenuation in an IL-1-dependent manner, resulting in PD-like behavioral impairment.
We hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of ...gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.
Unilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNgamma or IL-4.
Gp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNgamma, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.
Classical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.
Microglial cells account for approximately 12-15 % of the cells in the central nervous system (CNS). Microglial cells are polarized by pathological stimuli such as cytokines, chemokines, and growth ...factors, and play important roles in the deterioration and repair of the CNS. Here, we established cultures of primary microglial cells isolated from the brains of adult C57/BL6 mice using Percoll density gradients. The cells were cultured and stained with antibodies against CD11b, glial fibrillary acidic protein, myelin basic protein and NeuN to determine microglial, astroglial, oligodendroglial, and neuronal cells respectively. Moreover, the cells were exposed to interferon-γ (IFNγ) plus interleukin-1β (IL-1β) or IL-4 for 24 h to demonstrate the activating phenotypes with inducible nitric oxide synthase (iNOS), Ym1, and Iba-1 immunoblotting. At least 95 % of the cultured cells were CD11b-positive and -negative for astroglial, neuronal, and oligodendrocyte markers. IFNγ plus IL-1β treatment resulted in classical activation, which was represented by an increase in iNOS. The cells also displayed alternative activation, which increased Ym1 when treated with IL-4. The present study indicates that the microglial cells isolated as described here are a useful tool for elucidating adult microglial function.
Because rheumatoid arthritis (RA), an autoimmune disease, the patients often recognize side-effects due to the medication, alternative therapeutic strategies might potentially offer a clinical ...advantage. We evaluated the effect of nucleoprotein from salmon soft roe on animal model of arthritis. Mice transgenic for human T-cell leukemia virus type I (HTLV-1 Tg) were divided into three experimental groups and supplemented on either nucleoprotein-free (nonNP), or 0.6% or 1.2% nucleoprotein mixed (NP0.6 or NP1.2) diet for 3 months. The mice were evaluated arthritis by morphology, and measured with rheumatoid factor (RF). Moreover, macrophages and oxidative metabolites were assessed in the ankle and/or serum. Anti-oxidative potentials in nucleoprotein were determined with biological anti-oxidative potential (BAP) test, and electron spin resonance (ESR) analysis. NonNP-diet HTLV-1 Tg mice increased an arthritis symptoms and RF. The symptoms were ameliorated in NP-diet groups. Macrophages detected by F4/80 staining, and oxidative metabolites in the serum and/or joints were clearly decreased in 1.2% NP-diet HTLV-1 Tg mice. Nucleoprotein and DNA-nucleotide, but less protamine, had direct anti-oxidative potency with BAP test and/or ESR in vitro. These observations suggest that dietary nucleoprotein ameliorates arthritis symptoms in HTLV-1 Tg mice and offers hope as an alternative treatment for this debilitating medical condition.
Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. ...Previous studies showed that calpain-an intracellular modulatory protease-interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells.
This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice.
Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 (T helper type1/2) subsets. This occurs via the proteolytic degradation of MEKK1 (mitogen-activated protein kinase kinase kinase 1) and the subsequent inhibition of TGF (transforming growth factor)-β1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-β type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice.
Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.
We have demonstrated that ischemic neuronal death (apoptosis) of rat CA1 region of the hippocampus was prevented by infusing pituitary adenylate cyclase-activating polypeptide (PACAP) either ...intracerebroventricularly or intravenously. We have also demonstrated that the activity of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38, was increased in the hippocampus within 1–6 h after brain ischemia. The molecular mechanisms underlying the PACAP anti-apoptotic effect were demonstrated in this study. Ischemic stress had a strong influence on MAP kinase family, especially on JNK/SAPK and p38. PACAP inhibited the activation of JNK/SAPK and p38 after ischemic stress, while ERK is not suppressed. These findings suggest that PACAP inhibits the JNK/SAPK and p38 signaling pathways, thereby protecting neurons against apoptosis.
Interleukin-1 (IL-1) contributes to ischemic neurodegeneration. However, the mechanisms regulating action of IL-1 are still poorly understood. In order to clear this central issue, mice that were ...gene deficient in IL-1α and β (IL-1 KO) and wild-type mice were subjected to 1-h transient middle cerebral artery occlusion (tMCAO). Expression levels of IL-1β and IL-1 receptor I (IL-1RI) were then examined. Generation of peroxynitrite and the expression of mRNAs for nitric oxide synthase (NOS) subtypes were also determined. Immunostaining for IL-1β was increased from 6 h and peaked at 24 h after tMCAO in the microglia and macrophage. The immunoreactivities of IL-1RI were increased progressively in the microvasculature and neuron-like cells of the ipsilateral hemisphere. Infarct volumes were significantly lower in IL-1 KO mice compared with wild-type mice 48 h after tMCAO (
P<0.01). The immunoreactivities of 3-nitro-
l-tyrosine were determined in the neurons and microvasculature 24 h after tMCAO and were significantly decreased in the IL-1 KO mice compared to wild-type mice. In addition, expression levels of NOS mRNA in IL-1 KO mice were lower than that measured in wild-type mice. These results indicate that IL-1 is up-regulated and may play a role in neurodegeneration by peroxynitrite production during ischemia.
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