The Synergy of Aging and LPS Exposure in a Mouse Model of Parkinson's Disease.

Aging is an inevitable physiological challenge occurring in organisms over time, and is also the most important risk factor of neurodegenerative diseases. In this study, we observed cellular and molecular changes of different age mice and LPS-induced Parkinson disease (PD) model. The results showed that behavioral performance and dopaminergic (DA) neurons were declined, accompanied by increased expression of pro-inflammatory factors (TLR2, p-NF-kB-p65, IL-1ß and TNF-α), as well as pro-oxidative stress factor gp91phox in aged mice compared with young mice. Aging exaggerated inflammatory M1 microglia, and destroyed the balance between oxidation and anti-oxidation. The intranasal LPS instillation induced PD model in both young and aged mice. The poor behavioral performance and the loss of DA neurons as well as TLR2, p-NF-kB-p65, IL-1ß, TNF-α, iNOS and gp91phox were further aggravated in LPS-aged mice. Interestingly, the expression of Nrf2 and HO-1 was up-regulated by LPS only in young LPS-PD mice, but not in aged mice. The results indicate that the synergy of aging process and LPS exposure may prominently aggravate the DA neurons loss caused by more serious neuroinflammation and oxidative stress in the brain.

The inhibition of CB1 receptor accelerates the onset and development of EAE possibly by regulating microglia/macrophages polarization.

Cannabinoid 1 receptor (CB1R) regulates the neuro-inflammatory and neurodegenerative damages of experimental autoimmune encephalomyelitis (EAE) and of multiple sclerosis (MS). The mechanism by which CB1R inhibition exerts inflammatory effects is still unclear. Here, we explored the cellular and molecular mechanisms of CB1R in the treatment of EAE by using a specific and selective CB1R antagonist SR141716A. Our study demonstrated that SR141716A accelerated the clinical onset and development of EAE, accompanied by body weight loss. SR141716A significantly up-regulated the expression of toll like receptor-4 (TLR-4) and nuclear factor-kappaB/p65 (NF-κB/p65) on microglia/macrophages of EAE mice as well as levels of inflammatory factors (TNF-α, IL-1ß, IL-6) and chemokines (MCP-1, CX3CL1), accompanied by the shifts of cytokines from Th2 (IL-4, IL-10) to Th1 (IFN-γ)/Th17 (IL-17) in the spinal cords of EAE mice. Similar changes happened on splenic mononuclear cells (MNCs) except chemokine CX3CL1. Consistently, SR141716A promoted BV-2 microglia to release inflammatory factors (TNF-α, IL-1ß, IL-6) while inhibited the production of IL-10 and chemokines (MCP-1, CX3CL1). Furthermore, when splenic CD4+ T cells co-cultured with SR141716A-administered BV-2 microglia, the levels of IL-4 and IL-10 were decreased while production of IL-17 and IFN-γ increased significantly. Our research indicated that inhibition of CB1R induced M1 phenotype-Th17 axis changed of microglia/macrophages through TLR-4 and NF-κB/p65 which accelerated the onset and development of EAE. Therefore, CB1R may be a promising target for the treatment of MS/EAE, but its complexity remains to be carefully considered and studied in further clinical application.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Neuroprotective Effect Is Driven Through the Upregulation of CB1 Receptor in Experimental Autoimmune Encephalomyelitis.

During immuno-mediated demyelinating lesions, endocannabinoid system participates in both inflammatory and neurodegenerative damage through several mechanisms that involve neuronal and immune cells. Here, we constructed lentiviral vector to upregulate CB1 receptor (CB1R) in the lumbar spinal cord 5-6 region and observe the effect of clinical score and possible mechanism on the occurrence and development of experimental autoimmune encephalomyelitis (EAE). The results show that overexpression of CB1R delayed the onset of clinical signs and ameliorated the severity of disease. Overexpression of CB1R significantly inhibited the expression of NF-kB/p65 and TLR-4 as well as levels of IL-1ß, IL-6, and TNF-α, followed by a decrease of IL-17 and an increase of IL-10 in the spinal cord of mice. The percentage of M1 marker CD11b(+)CD16/32(+) cells was decreased, while the percentage of M2 marker CD11b(+)CD206(+) and CD11b(+)IL-10(+) cells was elevated in splenic mononuclear cells (MNCs) of mice with overexpression of CB1R. Interestingly, overexpression of CB1R dramatically enhanced the expression of neurotrophic NT-3, BDNF, and GDNF in the spinal cord. These results indicate that local overexpression of CB1R in the spinal cord exhibited neuroprotective effects in EAE, mainly suppressing inflammatory microenvironment and elevating neurotrophic factors, slightly declining IL-1ß and IL-17 in the spleen, and increased IL-10 in the brain. Its complexity remains to be carefully considered and further studied in further investigation.

Immunoregulation of experimental autoimmune encephalomyelitis by the selective CB1 receptor antagonist.

During immune-mediated demyelinating lesions, the endocannabinoid system is involved in the pathogenesis of both neuroinflammation and neurodegeneration through different mechanisms. Here we explored the cellular distribution of the CB1 receptor (CB1R) in the central nervous system (CNS) and detected the level of CB1R expression during experimental autoimmune encephalomyelitis (EAE) by RT-qPCR, Western blotting, and immunostaining. Expression of CB1R was observed in neurons and microglia/macrophages but was barely detected in astrocytes. During EAE, the expression of CB1R in spinal cords was reduced at days 9, 17, and 28 postimmunization (p.i.), but the level of CB1R expression in spleens did not show a significant difference compared with complete Freund's adjuvant (CFA)-immunized mice. A selective CB1R antagonist (SR141716A) increased EAE clinical score, accompanied by weight loss. Unexpectedly, SR141716A inhibited the expression of CB1R but increased the expression of CB2R in brains, spinal cords, and spleens simultaneously. The administration of SR141716A increased interferon-γ, interleukin-17 (IL-17), and inflammatory cytokines such as IL-1ß, IL-6, and tumor necrosis factor-α in brains and/or spinal cords. A similar increase was observed in spontaneous and specific antigen-stimulated splenic mononuclear cells compared with vehicle controls. Interestingly, the expression of CX3CL1 was increased in brains and spinal cords but declined in spleens of EAE mice treated with SR141716A. These results indicate that manipulation of the CB1R may have therapeutic value in MS, but its complexity remains to be carefully considered and studied in further clinical application.

Targeting CB(2) receptor as a neuroinflammatory modulator in experimental autoimmune encephalomyelitis.

During immune mediated demyelinating lesions, the endocannabinoid system is involved in the pathogenesis of both neuroinflammation and neurodegeneration through different mechanisms. Here, we explored the cellular distribution of cannabinoid 2 receptor (CB(2)R) in the central nervous system (CNS) and detected the level of CB(2)R expression during experimental autoimmune encephalomyelitis (EAE) by RT-PCR, Western blot and immunostaining. Our results show that CB(2)R was expressed in neurons, microglia and astrocytes. During EAE, the expression of CB(2)R in spinal cord rose slowly at days 9 and 17 post immunization (p.i.), and elevated rapidly at day 28 p.i., while the expression of CB(2)R in spleen elevated rapidly and got a plateau at days 17 and 28 p.i. Only the increase of CB(2)R expression in spinal cord demonstrated a significant difference when compared to control mice immunized with complete Freund's adjuvant (CFA). The selective CB(2)R antagonist (SR144528) exacerbated EAE clinical severity accompanied by weight loss. SR144528 inhibited the expression of CB(2)R, but increased the expression of CB(1)R in brain, spinal cord and spleen. The administration of SR144528 declined interferon-γ, IL-17, IL-4, IL-10, IL-1ß, IL-6 and tumor necrosis factor-α, but increased CX3CL1 in brain and/or spinal cord. In contrast, IL-17 and MCP-1 were increased, while CX3CL1 was decreased in splenic mononuclear cells as compared to vehicle controls. These results indicate that manipulation of CB(2)R may have therapeutic value in MS, but its complexity remains to be considered and studied for further clinical application.

Translocation and neuroprotective properties of transactivator-of-transcription protein-transduction domain-neuroglobin fusion protein in primary cultured cortical neurons.

Ngb (neuroglobin) is a newly discovered hexaco-ordinate globin that is expressed in vertebrate brain and peripheral nervous systems. Expression of Ngb increases in response to oxygen deprivation and protects neurons from hypoxia in vitro and in vivo. However, the lack of its transduction ability into cells resulted in limited neuroprotection. To educe its neuroprotection under hypoxia, a cell-permeable Ngb fusion protein was generated. A rat brain Ngb gene was cloned and fused with a gene fragment encoding the nine-amino-acid TAT PTD (transactivator-of-transcription protein-transduction domain; RKKRRQRRR) of HIV-1 in a prokaryotic expression vector to generate a genetic in-frame N-terminal hexahistidine-tagged) TAT PTD-Ngb fusion protein. It was expressed in soluble form in Escherichia coli BL21(DE3)plysS and purified with Ni(2+)-affinity chromatography. The results showed that the purified fusion protein TAT PTD-Ngb can enter into the primary cultured cortical neurons in a dose-dependent manner when added exogenously to the culture media and can be detected in cells within 48 h. The cell viability under hypoxia was increased and apoptosis induced by hypoxia was decreased after TAT PTD-Ngb was transduced into cortical neurons. The results provide a clue for the research of Ngb and suggest that transduction of TAT PTD-Ngb may be one of the ways for the therapy of CNS (central nervous system) diseases, especially cerebrovascular diseases and neurodegenerative diseases.