Abstract
Although therapeutic hypothermia (TH) provides neuroprotection, the cellular mechanism underlying the neuroprotective effect of TH has not yet been fully elucidated. In the present study, we investigated the effect of TH on microglial activation to determine whether hypothermia attenuates neuronal damage via microglial activation. After lipopolysaccharide (LPS) stimulation, BV-2 microglia cells were cultured under normothermic (37 °C) or hypothermic (33.5 °C) conditions. Under hypothermic conditions, expression of pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) was suppressed. In addition, phagocytosis of latex beads was significantly suppressed in BV-2 cells under hypothermic conditions. Moreover, nuclear factor-κB signaling was inhibited under hypothermic conditions. Finally, neuronal damage was attenuated following LPS stimulation in neurons co-cultured with BV-2 cells under hypothermic conditions. In conclusion, hypothermia attenuates neuronal damage via inhibition of microglial activation, including microglial iNOS and pro-inflammatory cytokine expression and phagocytic activity. Investigating the mechanism of microglial activation regulation under hypothermic conditions could contribute to the development of novel neuroprotective therapies.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Bhalala US, Koehler RC, Kannnan S (2014) Neuroinflammation and neuroimmune dysregulation after acute hypoxic-ischemic injury of developing brain. Front Pediatr 2:144. https://doi.org/10.3389/fped.2014.00144
Ciesielski-Treska J, Grant NJ, Ulrich G, Corrotte M, Bailly Y, Haeberle AM, Chasserot-Golaz S, Bader MF (2004) Fibrillar prion peptide (106–126) and Scrapie prion protein hamper phagocytosis in microglia. Glia 46:101–115. https://doi.org/10.1002/glia.10363
Chung H, Brazil MI, Soe TT, Maxfield FR (1999) Uptake, degradation, and release of fibrillar and soluble forms of Alzheimer's amyloid beta-peptide by microglial cells. J Biol Chem 274:32301–32308. https://doi.org/10.1074/jbc.274.45.32301
Cunha C, Gomes C, Vaz AR, Brites D (2016) Exploring new inflammatory biomarkers and pathways during LPS-induced M1 polarization. Mediators Inflamm 2016:6986715. https://doi.org/10.1155/2016/6986175
Derecki NC, Cronk JC, Lu Z, Xu E, Abbot SB, Guyenet PG, Kipnis J (2012) Wild-type microglia arrest pathology in a mouse model of Rett syndrome. Nature 484:105–109. https://doi.org/10.1038/nature10907
Dixon BJ, Reis C, Ho WM, Tang J, Zhang JH (2015) Neuroprotective strategies after neonatal hypoxic ischemia. Int J Mol Sci 16:22368–22401. https://doi.org/10.3390/ijms160922368
Fu R, Shen Q, Xu P, Luo JJ, Tang Y (2014) Phagocytosis of microglia in the central nervous system disease. Mol Neurobiol 49:1422–1434. https://doi.org/10.1007/s12035-013-8620-6
Gibbons H, Sato TA, Dragunow M (2003) Hypothermia suppresses inducible nitric oxide synthase and stimulates cyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cells. Mol Brain Res 110:63–75. https://doi.org/10.1016/s0169-328x(02)00585-5
Hayden MS, Ghosh S (2008) Shared principles in NF-κB signaling. Cell 132:344–362. https://doi.org/10.1016/j.cell.2008.01.020
Kakita H, Aoyama M, Nagaya Y, Asai H, Hussein MH, Suzuki M, Kato S, Saitoh S, Asai K (2013) Diclofenac enhances proinflammatory cytokine-induced phagocytosis of cultured microglia via nitric oxide production. Toxicol Appl Pharmacol 268:99–105. https://doi.org/10.1016/j.taap.2013.01.024
Kanda Y (2013) Investigation of the freely available easy-to-use software “EZR” for medical statistics. Bone Marrow Transplant 48:452–458. https://doi.org/10.1038/bmt.2012.244
Kozuka N, Itofusa R, Kudo Y, Morita M (2005) Lipopolysaccharide and proinflammatory cytokines require different states to induce nitric oxide production. J Neuro Res 82:717–728. https://doi.org/10.1002/jnr.20671
Kozuka N, Kudo Y, Morita M (2007) Multiple inhibitory pathways for lipopolysaccharide and pro-inflammatory cytokine-induced nitric oxide production in cultured astrocytes. Neuroscience 144:911–919. https://doi.org/10.1016/j.neuroscience.2006.10.040
Lenz K, Nelason LH (2018) Microglia and beyond innate immune cells as regulators of brain development and behavioral function. Front Immunol 9:698. https://doi.org/10.3389/fimmu.2018.00698
Liu L, Liu X, Wang R, Yan F, Luo Y, Cahndra A, Ding Y, Ji X (2018) Mild focal hypothermia regulates the dynamic polarozation of microglial after ischemic stroke in mice. Neurol Res 40:508–515. https://doi.org/10.1080/01616412.2018.1454090
Loane DJ, Byrnes KR (2010) Role of microglia in neurotrauma. Neurotherapeutics 7:366–377. https://doi.org/10.1016/j.nurt.2010.07.002
Long-Smith CM, Sullivan AM, Nolan YM (2009) The influence of microglia on the pathogenesis of Parkinson's disease. Prog Neurobiol 89:277–287. https://doi.org/10.1016/j.pneurobio.2009.08.001
Magire O, O'Loughlin K, Minderman H (2015) Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry. J Immunol Methods 423:3–11. https://doi.org/10.1016/j.jim.2015.03.018
Maksoud MJE, Tellios V, An D, Xiang YY, Lu WY (2019a) Nitric oxide upregulates microglia phagocytosis and increases transient receptor potential vanilloid type 2 channel expression on the plasma membrane. Glia 67:2294–2311. https://doi.org/10.1002/glia.23685
Maksoud MJE, Tellios V, Xiang YY, Lu WY (2019b) Nitric oxide signaling inhibits microglia proliferation by activation of protein kinase-G. Nitric Oxide 94:125–134. https://doi.org/10.1016/j.niox.2019.11.005
Nagaya Y, Aoyama M, Tamura T, Kakita H, Kato S, Hida H, Saitoh S, Asai K (2014) Inflammatory cytokine tumor necrosis factor αsuppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia-inducible factor-2α. Eur J Neurosci 173:1541–1544. https://doi.org/10.1111/ejn.12747
Napoli I, Nerumann H (2010) Protective effects of microglia in multiple sclerosis. Exp Neurol 225:24–28. https://doi.org/10.1016/j.expneurol.2009.04.024
Park JY, Paik SR, Jou I, Park SM (2008) Microglial phagocytosis is enhanced by monomeric alpha-synuclein, not aggregated alpha-synuclein: implications for Parkinson's disease. Glia 56:1215–1223. https://doi.org/10.1002/glia.20691
Perego C, Fumagalli S, De Simori MG (2013) Temporal characterization of microglia/macrophage phenotype in a mouse model of neonatal hypoxic-ischemic brain injury. J Vis Exp 79:50605. https://doi.org/10.3791/50605
Prokop S, Miller KR, Heppner FL (2013) Microglia activation in Alzheimer's disease. Acta Neuropathol 126:461–477. https://doi.org/10.1007/s00401-013-1182-x
Seo JW, Kim JH, Kim JH, Seo M, Han HS, Park J, Suk K (2012) Time-dependent effects of hypothermia on microglial activation and migration. J Neuroinflamm 9:164. https://doi.org/10.1186/1742-2094-9-164
Serdar M, Kempe K, Rizazad M, Herz J, Bendix I, Felderhoff-Müser U, Sabir H (2019) Early pro-inflammatory microglia activation after inflammation-sensitized hypoxic-ischemic brain injury in neonatal rats. Front Cell Neurosci 13:237. https://doi.org/10.3389/fncel.2019.00237
Tamura T, Aoyama M, Ukai S, Kakita H, Sobue K, Asai K (2017) Neuroprotective erythropoietin attenuates microglial activation, including morphological changes, phagocytosis, and cytokine production. Brain Res 1662:65–74. https://doi.org/10.1016/j.brainres.2017.02.023
Valles SL, Iradi A, Aldasoro M, Vila JM, Aldasoro C, de la Torre J, Campos-Campos J, Jorda A (2019) Function of glia in aging and the brain diseases. Int J Med Sci 16:1473–1479. https://doi.org/10.7150/ijms.37769
Viatour P, Merville MP, Bours V, Chariot A (2005) Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation. Trends Biochem Sci 30:43–52. https://doi.org/10.1016/j.tibs.2004.11.009
Wang N, Liang H, Zen K (2014) Molecular mechanisms that influence the macrophage m1–m2 polarization. Front Immunol 5:614. https://doi.org/10.1056/NEJMcps050929
Yamamoto Y, Gaynor RB (2004) IkappaB kinases: key regulators of the NF-kappaB pathway. Trends Biochem Sci 29:72–79. https://doi.org/10.1016/j.tibs.2003.12.003
Yenari MA, Han HS (2012) Neuroprotective mechanisms of hypothermia in brain ischaemia. Nature Rev Neurosci 13:267–278. https://doi.org/10.1038/nrn3174
Yenari MA, Kauppinen TM, Swanson RA (2010) Microglia activation in stroke: therapeutic targets. Neurotherapeutics 7:378–391. https://doi.org/10.1016/j.nurt.2010.07.005
Zhang F, Dong H, Lv T, Jin K, Jin Y, Zhang X, Jiang J (2018) Moderate hypothermia inhibits microglial activation after traumatic brain injury by modulating autophagy/apoptosis and the MyD88-dependent TL4 signaling pathway. J Neuroinflammation 15:273. https://doi.org/10.1186/s12974-018-1315-1
Acknowledgements
We acknowledge the assistance of the Research Equipment Sharing Center at Nagoya City University.
Funding
This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, KAKEN Grant Numbers 16K10101, 17K10197, and 18K07832.
Author information
Authors and Affiliations
Contributions
Conceptualization: TK, KT, HK, TT, ST, YY, and MA; methodology: TK, KT, and ST; formal analysis and investigation: TK, KT, HK, TT, ST, and MA; writing: original draft preparation: TK and KT; writing: review and editing: HK, MA; funding acquisition: HK, TT, ST, YY, and MA; resources: HK, TT, ST, YY, and MA; supervision: YY and MA; final manuscript approval: TK, KT, HK, TT, ST, YY, and MA.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical Approval
The present study was approved by the Animal Care and Use Committee of Nagoya City University Graduate School of Pharmaceutical Sciences (Protocol Number, H27-P-03), and all experiments were performed in accordance with institutional and US National Institutes of Health guidelines for the care and use of laboratory animals.
Informed Consent
Not applicable.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Tomoka Kimura and Kohki Toriuchi have contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
10571_2020_860_MOESM1_ESM.tiff
Supplementary file1 Supplemental Fig. 1 Time course of inducible nitric oxide synthase (iNOS) and inflammatory cytokine gene expression in lipopolysaccharide (LPS)-stimulated BV-2 cells. (A) Illustration of the time course of the experiment. Expression of the (B) iNOS, (C) tumor necrosis factor-α (TNFα), and (D) interleukin-1β (IL-1β) genes was determined in BV-2 cells stimulated with LPS. Data are the mean ± SEM (n = 3 in each group). *P < 0.05 compared with 0-h expression level. (TIFF 5574 kb)
10571_2020_860_MOESM2_ESM.tiff
Supplementary file2 Supplemental Fig. 2 Photographs of whole Western blot membranes. (A) Whole membrane of the results shown in Figure 3A. (B) Whole membrane of the results shown in Figure 4A. (C) Whole membrane of the results shown in Figure 4C. (TIFF 5574 kb)
Rights and permissions
About this article
Cite this article
Kimura, T., Toriuchi, K., Kakita, H. et al. Hypothermia Attenuates Neuronal Damage via Inhibition of Microglial Activation, Including Suppression of Microglial Cytokine Production and Phagocytosis. Cell Mol Neurobiol 41, 459–468 (2021). https://doi.org/10.1007/s10571-020-00860-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10571-020-00860-z