Mediators of Inflammation

Volume 2013 (2013), Article ID 436263, 7 pages

http://dx.doi.org/10.1155/2013/436263

Research Article

Hypothermia Reduces Toll-Like Receptor 3-Activated Microglial Interferon-β and Nitric Oxide Production

Tomohiro Matsui,¹ Yukari Motoki,¹ and Yusuke Yoshida²

¹Department of Laboratory Sciences, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-kogushi, Ube, Yamaguchi 755-8505, Japan

²ACEL, Inc., SIC1 1201, 5-4-21 Nishihashimoto, Midori-ku, Sagamihara, Kanagawa 252-0131, Japan

Received 8 January 2013; Revised 18 February 2013; Accepted 18 February 2013

Academic Editor: Fulvio D'Acquisto

Copyright © 2013 Tomohiro Matsui et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Therapeutic hypothermia protects neurons after injury to the central nervous system (CNS). Microglia express toll-like receptors (TLRs) that play significant roles in the pathogenesis of sterile CNS injury. To elucidate the possible mechanisms involved in the neuroprotective effect of therapeutic hypothermia, we examined the effects of hypothermic culture on TLR3-activated microglial release of interferon (IFN)-β and nitric oxide (NO), which are known to be associated with neuronal cell death. When rat or mouse microglia were cultured under conditions of hypothermia (33°C) and normothermia (37°C) with a TLR3 agonist, polyinosinic-polycytidylic acid, the production of IFN-β and NO in TLR3-activated microglia at 48 h was decreased by hypothermia compared with that by normothermia. In addition, exposure to recombinant IFN-β and sodium nitroprusside, an NO donor, caused death of rat neuronal pheochromocytoma PC12 cells in a concentration-dependent manner after 24 h. Taken together, these results suggest that the attenuation of microglial production of IFN-β and NO by therapeutic hypothermia leads to the inhibition of neuronal cell death.

1. Introduction

Toll-like receptors (TLRs) are major sensors of pathogen-associated molecular patterns (PAMPs) that mediate innate immunity and are involved in adaptive immune responses [1]. Production and release by damaged cells of molecules that are abnormally expressed or whose structures are altered can stimulate the activity of TLRs [2, 3]. Under these conditions, these molecules are recognized as damage- or danger-associated molecular patterns that trigger immediate responses or enhance reactions to tissue injury and inflammation [3–5].

Microglia express TLRs and are principal immune cells in the central nervous system (CNS). Their functional characteristics have received much attention because these cells represent the major source of immune mediators in the brain [6]. Although stimulation of TLRs in microglia activates functions that are important for the elimination of pathogens [7], microglial TLRs, particularly TLR2 and TLR4, mediate stroke-induced injury to the CNS [8, 9], neuroinflammation, and neuronal damage [4, 5, 10, 11] by responding to endogenous compounds. Lehnardt et al. investigated in detail the role of endogenous mechanisms that trigger activation of microglial TLRs [4]. They found that the molecular chaperon, heat shock protein 60 (HSP60), serves as a signal of CNS injury by activating microglial signal pathways mediated by TLR4 and the TLR adapter protein called myeloid differentiation factor 88 (MyD88). Dying CNS cells release HSP60 that binds to microglia, which in turn secrete neurotoxic nitric oxide (). These data provided the first evidence for an endogenous pathway that may be common to many forms of neuronal injury and that bidirectionally links CNS inflammation with neurodegeneration. Lehnardt et al. [4] characterized these events as a “vicious cycle of neurodegeneration,” in which the initial cause of CNS cell death, irrespective of its nature, leads to the release of endogenous molecules from dying cells, which activate microglia via TLRs. This leads to the release of neurotoxic molecules that cause further injury to neighboring neurons. Consistent with this hypothesis, necrotic neurons activate an MyD88-dependent pathway in microglia, leading to the release of not only , but also proinflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α [5], which are associated with neuronal injury [12, 13].

Therapeutic hypothermia can potentially protect neurons after severe brain damage, such as that occurring after traumatic brain injury (TBI) and cardiac arrest [14, 15]. To elucidate the possible mechanisms responsible for this neuroprotective effect, we and others have examined whether decreasing temperature affects in vitro microglial release of inflammatory factors through activation of TLR2 and TLR4 and have demonstrated that the production of microglial TNF-α, IL-6, and is in fact reduced under hypothermic culture conditions [16–20].

In the present study, we focused on examining the effects of hypothermic culture on the production of interferon (IFN)-β and by microglia, in which TLR3 was activated, to better understand the relationship between therapeutic hypothermia and microglial responses. TLR3 is a major mediator of cellular responses to viral infection, even in the CNS [21] because it responds to double-strand RNA (dsRNA), a common intermediate of viral replication [22]. This antiviral response is characterized by high expression of type I IFNs, predominantly IFN-α and IFN-β, which is induced by the stimulation of TLR3 [23]. During inflammation, TLR3 also recognizes RNA released from necrotic cells as an endogenous ligand [24–26], leading to the release of type I IFNs [24, 25]. Thus, host-derived nucleic acids are likely to act as endogenous ligands that activate TLR3, particularly in microglia, which may further amplify inflammation in the CNS.

In the present study, we used a synthetic analog of dsRNA, polyinosinic-polycytidylic acid (poly(I:C)), to stimulate TLR3 signaling. This compound activates microglia in vitro [27, 28] and in vivo [29–31], and the latter leads to neurodegeneration [29]. Therefore, poly(I:C) can be used to study TLR3-driven neuroinflammation mediated by microglia in the CNS. Poly(I:C) induces type I IFNs [32], IFN-β more so than IFN-α, in microglia [27]. Despite some anti-inflammatory effects in the CNS, for example, IFN-β reduces the expression of proinflammatory cytokines [33, 34] and inhibits the infiltration of T cells [35], it directly induces neuronal cell death [36] and as found for other cytokines, excessive levels or inappropriate activity of type I IFNs can cause toxicity and even death (neurodegeneration) [37, 38]. Thus, the function of IFN-β in the CNS is somewhat controversial.

To determine their involvement in neuronal protection induced by hypothermia, we investigated whether IFN-β and directly induced death of a neuronal pheochromocytoma cell line (PC12).

2. Materials and Methods

The Animal Care Committee of Yamaguchi University School of Medicine reviewed and approved all protocols used in this study.

2.1. Isolation of Microglia

Microglia were isolated from primary cultures of the brains of 1- to 3-day-old Wistar rats or C57BL/6N mice (purchased from Japan SLC, Hamamatsu, Japan) as described in our previous reports, including removal of the meninges [20, 39]. Cell purity was >95% as determined by flow cytometric analysis and immunocytochemistry staining of the microglial markers, Mac-1 (CD11b) and Iba1, respectively. Both markers are reliable markers for microglia in this conventional method for isolation of these cells with similar purities (90%–99.5%) [5, 40, 41]. We used anti-Mac-1 (Immunotech, Marseille, France) and anti-Iba1 (Wako Pure Chemical Industries, Osaka, Japan) antibodies, respectively, for this purpose. In addition, we confirmed that the culture did not contain astrocytes using an antibody against glial fibrillary acidic protein by immunocytochemistry staining, in accordance with the result of another study [40].

2.2. Microglial Cell Culture

Rat or mouse microglia ( cells/well in untreated 96-well plates) were incubated with or without poly(I:C) (100 μg/mL; Imgenex, San Diego, CA, USA) in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco). Cells were incubated under hypothermia (33°C) and normothermia (37°C) for 48 h to measure the production of IFN-β and . On the basis of our preliminary investigations on the optimal responses to each variable, the dose of poly(I:C) and incubation period were determined. Cell-free supernatants were stored at −80°C.

2.3. IFN-β Assay

Concentrations of mouse IFN-β present in microglial culture supernatants were measured in duplicate using an enzyme-linked immunosorbent assay (ELISA) kit (PBL Interferon Source, Piscataway, NJ, USA), according to the manufacturer’s instructions. We performed IFN-β assay on mice because ELISA kit for only mouse IFN-β was commercially available.

2.4. NO Assay

production in rat and mouse microglia was detected and quantified as nitrite (), a relatively stable metabolite of that accumulates in the culture medium. A colorimetric assay using Griess reagent (Sigma-Aldrich, St. Louis, OH, USA) was performed as previously described [20, 39].

2.5. PC12 Cell Culture

The rat PC12 cell line was obtained from the RIKEN BioResource Center (RIKEN, Ibaraki, Japan). The undifferentiated cells were grown at 37°C in DMEM (Gibco) supplemented with 10% FBS (Nichirei Bioscience, Tokyo, Japan) and 10% horse serum (HS) (Gibco).

2.6. Cytotoxicity Assay

PC12 cells ( cells/well) were placed on type I collagen-coated 96-well plates containing culture medium and incubated for 24 h at 37°C. Thereafter, the culture supernatants were substituted for the conditioned medium by DMEM supplemented with 0.1% FBS, 0.1% HS, and 10 ng/mL mouse nerve growth factor 2.5S (Almone Labs, Jerusalem, Israel), including various concentrations of rat recombinant IFN-β (Sigma-Aldrich) or sodium nitroprusside dehydrate (an donor) (SNP; Wako Pure Chemical Industries), and the cells were cultured at 37°C for 24 h. The viability of the cultures was determined colorimetrically using WST-8 reagent (Nacalai Tesque, Kyoto, Japan) in a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay [42]. In brief, culture supernatants were replaced by the conditioned medium including 10% WST-8 reagent and incubated at 37°C for 1 h. The absorbance was measured at 450 nm using a microplate reader. Cell viabilities are presented as values relative to those obtained when cells were treated with vehicle (0.04% sterile distilled water) to control for variation between experiments.

2.7. Statistical Analysis

Data are expressed as mean ± standard  error  of  the  mean  (SEM). Differences in values for two groups or among groups were analyzed using the paired -test or one-way analysis of variance followed by the Newman-Keuls multiple comparison method (StatFlex Ver5.0, Artech, Osaka, Japan). The value of was considered to indicate a significant difference.

3. Results

3.1. Effect of Hypothermic Culture on the Production of IFN-β

IFN-β was virtually undetectable in unstimulated mouse microglia after 48 h of culture. Application of poly(I:C) to mouse microglia cultured at either 33°C or 37°C induced IFN-β production at 48 h (Figure 1). There was a significant reduction in the level of IFN-β at 33°C (hypothermia) compared with that at 37°C (normothermia) (Figure 1).

Figure 1: Effect of hypothermic culture on IFN-β production by poly(I:C)-stimulated mouse microglia. Mouse microglia ( cells/well) were cultured with 100 μg/mL poly(I:C) under hypothermic (33°C) and normothermic (37°C) conditions for 48 h. IFN-β levels in culture supernatants were measured by ELISA. Data are expressed as (). * compared with 37°C.

3.2. Effects of Hypothermic Culture on the Production of NO

was detected at low levels in an unstimulated rat and mouse microglia after 48 h of culture, and in both cases poly(I:C) increased production (Figures 2(a) and 2(b), resp.). Production of by untreated or treated rat and mouse microglia was reduced by hypothermia compared with normothermia (Figures 2(a) and 2(b), resp.).

(a)

(b)

Figure 2: Effects of hypothermic culture on production by poly(I:C)-stimulated rat and mouse microglia. Rat (a) and mouse (b) microglia ( cells/well) were cultured with or without 100 μg/mL poly(I:C) under hypothermic (33°C) and normothermic (37°C) conditions for 48 h. levels in culture supernatants were measured using a colorimetric assay with Griess reagent. Data are expressed as ( for rat microglia (a) and for mouse microglia (b)). ** compared with 37°C.

3.3. Effects of IFN-β and SNP, an NO Donor, on the Viability of Neuronal PC12 Cells

IFN-β and SNP induced death of neuronal PC12 cells in a concentration-dependent manner 24 h after exposure (Figures 3(a) and 3(b), resp.). These decreases in cell survival were statistically significant at 12–300 U/mL IFN-β (80%–69% reduction) and at 0.08–10 μM SNP (79%–63% reduction), compared with vehicle (Figures 3(a) and 3(b), resp.).

(a)

(b)

Figure 3: Effects of IFN-β and SNP, an donor, on the viability of neuronal PC12 cells. Neuronal PC12 cells ( cells/well) were treated with or without recombinant IFN-β (a) or SNP, an donor (b), for 24 h at 37°C. Cell viability was determined using a colorimetric assay with WST-8 reagent as described in the Methods section. Data are presented as values relative to those obtained in cells treated with vehicle and are expressed as (). *, ** compared with vehicle.

4. Discussion

TLR signaling can be induced by recognition of either PAMPs or endogenous components. Under pathophysiological conditions, these endogenous agonists are produced or released at unusual concentrations or are present in nonphysiological appearance [43, 44] and trigger immediate responses or enhance reactions to tissue injury and inflammation in microglia [4, 5, 8–11]. Therefore, understanding TLR-driven neuroinflammation in microglia seems to be of particular significance for elucidating the possible mechanisms behind the neuroprotective effects of therapeutic hypothermia. We previously examined the effects of hypothermic culture on the release of inflammatory factors by microglia through activation of TLR2 and TLR4 [19, 20]. Here we focused on the stimulation of TLR3 and showed that, in the TLR3-activated microglia, hypothermia (33°C) reduced the production of IFN-β and at 48 h of culture. To the best of our knowledge, this is the first paper describing microglial responses to hypothermia using poly(I:C), an activator of TLR3. Reduction of levels under hypothermic cultures is consistent with reports regarding activators of TLR2 and TLR4 [16–20].

Increased levels of several proinflammatory cytokines, such as IL-1 and IL-6, and are present in cerebrospinal fluid (CSF) after severe head injury in humans [14, 45, 46]. These potentially neurotoxic factors are produced by activated microglia when neurons are destroyed after ischemia or trauma [6, 47], and they are associated with secondary brain damage [13, 48]. Thus, evidence indicates that suppression of the release of these factors by microglia contributes to the neuroprotective effects of therapeutic hypothermia after severe brain damage [14, 15, 49, 50]. In fact, therapeutic hypothermia attenuates the increase in levels of proinflammatory cytokines and in the CNS after brain injury [14, 49, 51], and this is associated with a favorable outcome compared with normothermia [14, 49]. Further, hypothermia during severe perinatal asphyxia prevents increases in 3′,5′-cyclic monophosphate (as a marker of ) in the rat brain. In this study, 100% of the hypothermic rats survived, whereas 70% mortality was observed in the normothermic group [52]. We are not aware of any reports showing that levels of IFN-β increase in the CSF after brain injury in vivo; however, one animal study indicates production of increased levels of IFN-α and IFN-β after sterile CNS injury [53]. Despite certain anti-inflammatory effects in the CNS [33–35], IFN-β may still be deleterious. The mechanisms responsible for neurotoxic effects of type I IFNs remain unclear; however, some investigators have postulated that the indirect effects of cytokines are mediated by their actions on either peripheral organs or glial cells, for example, type I IFNs induce proinflammatory mediators release from microglial cells [54, 55]. Another possibility is that type I IFNs may exert toxic effects directly on neuronal cells. Gene chip analysis of RNA from a culture of brain cells treated with IFN-α indicates that neurons are very responsive target cells for IFNs [56]. Consistent with these findings, IFN-β induces death of neuronal cells [36]. Here we found that hypothermia reduced the production of IFN-β by microglia expressing activated TLR3. Taken together, our findings suggest that the neuroprotective effects of therapeutic hypothermia are related to the attenuation of the production of and IFN-β by microglia, although the clinical significance of these findings remains to be determined.

To determine a possible pathophysiological involvement of the decreased production of IFN-β and by microglia for hypothermic neuronal protection, we examined whether IFN-β and/or directly induced neuronal PC12 cell death. We were able to demonstrate that IFN-β and independently decrease cell survival in a concentration-dependent manner, in agreement with a previous study on the effects of [57]. To the best of our knowledge, the present study is the first to demonstrate that IFN-β induces this effect, although we are aware that it induces apoptosis in a human neuroblastoma cell line [36]. The concentration-dependent IFN-β- and -induced neuronal cell death and in vivo findings of their elevated levels in the CNS after CNS injury [46, 53] support the conclusion that a decrease in their levels during hypothermia contributes toward protection of neurons. The conditioned media from TLR3-activated microglia may have yielded similar direct effects in such experiments; however, we were unsuccessful in our preliminary attempts to answer this question.

Because we used poly(I:C), a synthetic analog of dsRNA, to stimulate TLR3 in microglia, it is possible that activation of this receptor in our present study differs from that in sterile CNS injury. However, poly(I:C) has been used to induce TLR3 signaling to examine its contribution to the pathophysiology of certain noninfectious conditions [29–31]. Nonetheless, it would be of interest to utilize RNAs that act as endogenous TLR3 ligands [24–26] from injured CNS cells [4] in such experiments. It would also be interesting to study other CNS-derived endogenous ligand(s) that colocalize with TLR3 in microglia, such as stathmin, a regulator of microtubules [58], which is present in myelin sheaths and upregulated during neuroinflammation [59]. Further research using these models to confirm our present findings may yield much clinically relevant data. Our study shows that the production of IFN-β and by microglia was reduced when TLR3 signaling was activated by poly(I:C) under hypothermic culture conditions. Inactivating TLR3 using methods such as RNA interference and/or the use of TLR3-knockout mice would further support the role of TLR3 signaling.

5. Conclusions

Here, we demonstrated that hypothermia reduced the production of IFN-β and by microglia expressing activated TLR3 and that these factors induced neuronal cell death. Our results suggest that the attenuation of the production of IFN-β and by microglia induced by therapeutic hypothermia leads to the inhibition of neuronal cell death. Studies of stroke using animal models suggest that activation of TLRs by the release of endogenous ligands contributes to tissue injury and indicates that TLR2 and TLR4 [8, 9, 60, 61], but not TLR3, contribute to this pathological process [62]. Although studies of mice lacking TLR3 support these findings, the activation of TLR3 in microglia by poly(I:C) leads to neurodegeneration [29]. This indicates that expression of TLR3 by microglia plays a role in CNS injury under certain conditions. Our studies here focused on TLR3 signaling in microglia and are the first to show the effects of hypothermia on TLR3-driven neuroinflammation. They reveal a possible mechanism responsible for the neuroprotective effects of therapeutic hypothermia.

Acknowledgments

This research was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Grant-in-Aid for Young Scientists (B), no. 22791435 to T. Matsui. The authors would like to thank Enago (http://www.enago.jp/) for the English language review.

References

S. Akira, S. Uematsu, and O. Takeuchi, “Pathogen recognition and innate immunity,” Cell, vol. 124, no. 4, pp. 783–801, 2006.

M. F. Tsan and B. Gao, “Endogenous ligands of toll-like receptors,” Journal of Leukocyte Biology, vol. 76, no. 3, pp. 514–519, 2004.

G. B. Johnson, G. J. Brunn, and J. L. Platt, “Activation of mammalian toll-like receptors by endogenous agonists,” Critical Reviews in Immunology, vol. 23, no. 1-2, pp. 15–44, 2003.

S. Lehnardt, E. Schott, T. Trimbuch et al., “A vicious cycle involving release of heat shock protein 60 from injured cells and activation of toll-like receptor 4 mediates neurodegeneration in the CNS,” The Journal of Neuroscience, vol. 28, no. 10, pp. 2320–2331, 2008.

T. F. Pais, C. Figueiredo, R. Peixoto, M. H. Braz, and S. Chatterjee, “Necrotic neurons enhance microglial neurotoxicity through induction of glutaminase by a MyD88-dependent pathway,” Journal of Neuroinflammation, vol. 5, article 43, 2008.

L. Minghetti and G. Levi, “Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide,” Progress in Neurobiology, vol. 54, no. 1, pp. 99–125, 1998.

R. B. Rock, G. Gekker, S. Hu, et al., “Role of microglia in central nervous system infections,” Clinical Microbiology Reviews, vol. 17, no. 4, pp. 942–964, 2004.

S. Lehnardt, S. Lehmann, D. Kaul, et al., “Toll-like receptor 2 mediates CNS injury in focal cerebral ischemia,” Journal of Neuroimmunology, vol. 190, no. 1-2, pp. 28–33, 2007.

G. Ziegler, D. Harhausen, C. Schepers et al., “TLR2 has a detrimental role in mouse transient focal cerebral ischemia,” Biochemical and Biophysical Research Communications, vol. 359, no. 3, pp. 574–579, 2007.

O. Hoffmann, J. S. Braun, D. Becker, et al., “TLR2 mediates neuroinflammation and neuronal damage,” The Journal of Immunology, vol. 178, no. 10, pp. 6476–6481, 2007.

A. A. Babcock, M. Wirenfeldt, T. Holm, et al., “Toll-like receptor 2 signaling in response to brain injury: an innate bridge to neuroinflammation,” The Journal of Neuroscience, vol. 26, no. 49, pp. 12826–12837, 2006.

S. M. Allan and N. J. Rothwell, “Cytokines and acute neurodegeneration,” Nature Reviews Neuroscience, vol. 2, no. 10, pp. 734–744, 2001.

S. M. Allan, P. J. Tyrrell, and N. J. Rothwell, “Interleukin-1 and neuronal injury,” Nature Reviews Immunology, vol. 5, no. 8, pp. 629–640, 2005.

D. W. Marion, L. E. Penrod, S. F. Kelsey et al., “Treatment of traumatic brain injury with moderate hypothermia,” The New England Journal of Medicine, vol. 336, no. 8, pp. 540–546, 1997.

S. A. Bernard, T. W. Gray, M. D. Buist et al., “Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia,” The New England Journal of Medicine, vol. 346, no. 8, pp. 557–563, 2002.

Q. S. Si, Y. Nakamura, and K. Kataoka, “Hypothermic suppression of microglial activation in culture: inhibition of cell proliferation and production of nitric oxide and superoxide,” Neuroscience, vol. 81, no. 1, pp. 223–229, 1997.

S. Maekawa, M. Aibiki, Q. S. Si, Y. Nakamura, Y. Shirakawa, and K. Kataoka, “Differential effects of lowering culture temperature on mediator release from lipopolysaccharide-stimulated neonatal rat microglia,” Critical Care Medicine, vol. 30, no. 12, pp. 2700–2704, 2002.

H. Gibbons, T. A. Sato, and M. Dragunow, “Hypothermia suppresses inducible nitric oxide synthase and stimulates cyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cells,” Molecular Brain Research, vol. 110, no. 1, pp. 63–75, 2003.

T. Matsui and T. Kakeda, “IL-10 production is reduced by hypothermia but augmented by hyperthermia in rat microglia,” Journal of Neurotrauma, vol. 25, no. 6, pp. 709–715, 2008.

T. Matsui, M. Tasaki, T. Yoshioka, Y. Motoki, H. Tsuneoka, and J. Nojima, “Temperature- and time-dependent changes in TLR2-activated microglial NF-κB activity and concentrations of inflammatory and anti-inflammatory factors,” Intensive Care Medicine, vol. 38, no. 8, pp. 1392–1399, 2012.

P. A. Carpentier, D. S. Duncan, and S. D. Miller, “Glial toll-like receptor signaling in central nervous system infection and autoimmunity,” Brain, Behavior, and Immunity, vol. 22, no. 2, pp. 140–147, 2008.

L. Alexopoulou, A. C. Holt, R. Medzhitov, and R. A. Flavell, “Recognition of double-stranded RNA and activation of NF-κB by toll-like receptor 3,” Nature, vol. 413, no. 6857, pp. 732–738, 2001.

A. N. Theofilopoulos, R. Baccala, B. Beutler, and D. H. Kono, “Type I interferons (α/β) in immunity and autoimmunity,” Annual Review of Immunology, vol. 23, pp. 307–336, 2005.

K. Karikó, H. Ni, J. Capodici, M. Lamphier, and D. Weissman, “mRNA is an endogenous ligand for toll-like receptor 3,” The Journal of Biological Chemistry, vol. 279, no. 13, pp. 12542–12550, 2004.

F. Brentano, O. Schorr, R. E. Gay, S. Gay, and D. Kyburz, “RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via toll-like receptor 3,” Arthritis and Rheumatism, vol. 52, no. 9, pp. 2656–2665, 2005.

K. A. Cavassani, M. Ishii, H. Wen et al., “TLR3 is an endogenous sensor of tissue necrosis during acute inflammatory events,” The Journal of Experimental Medicine, vol. 205, no. 11, pp. 2609–2621, 2008.

J. K. Olson and S. D. Miller, “Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs,” The Journal of Immunology, vol. 173, no. 6, pp. 3916–3924, 2004.

T. Town, D. Jeng, L. Alexopoulou, J. Tan, and R. A. Flavell, “Microglia recognize double-stranded RNA via TLR3,” The Journal of Immunology, vol. 176, no. 6, pp. 3804–3812, 2006.

L. M. Melton, A. B. Keith, S. Davis, A. E. Oakley, J. A. Edwardson, and C. M. Morris, “Chronic glial activation, neurodegeneration, and APP immunoreactive deposits following acute administration of double-stranded RNA,” Glia, vol. 44, no. 1, pp. 1–12, 2003.

Z. Zhang, K. Trautmann, and H. J. Schluesener, “Microglia activation in rat spinal cord by systemic injection of TLR3 and TLR7/8 agonists,” Journal of Neuroimmunology, vol. 164, no. 1-2, pp. 154–160, 2005.

I. K. Patro, Amit, M. Shrivastava, S. Bhumika, and N. Patro, “Poly I:C induced microglial activation impairs motor activity in adult rats,” Indian Journal of Experimental Biology, vol. 48, no. 2, pp. 104–109, 2010.

M. Matsumoto and T. Seya, “TLR3: interferon induction by double-stranded RNA including poly(I:C),” Advanced Drug Delivery Reviews, vol. 60, no. 7, pp. 805–812, 2008.

T. K. Makar, D. Trisler, C. T. Bever, et al., “Stem cell based delivery of IFN-beta reduces relapses in experimental autoimmune encephalomyelitis,” Journal of Neuroimmunology, vol. 196, no. 1-2, pp. 67–81, 2008.

M. Chen, G. Chen, H. Nie, et al., “Regulatory effects of IFN-beta on production of osteopontin and IL-17 by CD4⁺ T cells in MS,” European Journal of Immunology, vol. 39, no. 9, pp. 2525–2536, 2009.

O. Stüve, N. P. Dooley, J. H. Uhm, et al., “Interferon beta-1b decreases the migration of T lymphocytes in vitro: effects on matrix metalloproteinase-9,” Annals of Neurology, vol. 40, no. 6, pp. 853–863, 1996.

S. Dedoni, M. C. Olianas, and P. Onali, “Interferon-β induces apoptosis in human SH-SY5Y neuroblastoma cells through activation of JAK-STAT signaling and down-regulation of PI3K/Akt pathway,” Journal of Neurochemistry, vol. 115, no. 6, pp. 1421–1433, 2010.

Y. Akwa, D. E. Hassett, M. L. Eloranta et al., “Transgenic expression of IFN-α in the central nervous system of mice protects against lethal neurotropic viral infection but induces inflammation and neurodegeneration,” The Journal of Immunology, vol. 161, no. 9, pp. 5016–5026, 1998.

C. Reyes-Vázquez, B. Prieto-Gómez, and N. Dafny, “Interferon modulates central nervous system function,” Brain Research, vol. 1442, pp. 76–89, 2012.

T. Matsui, Y. Motoki, T. Inomoto, et al., “Temperature-related effects of adenosine triphosphate-activated microglia on pro-inflammatory factors,” Neurocritical Care, vol. 17, no. 2, pp. 293–300, 2012.

A. Bal-Price and G. C. Brown, “Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity,” The Journal of Neuroscience, vol. 21, no. 17, pp. 6480–6491, 2001.

L. Zhang, S. Zhao, X. Wang, C. Wu, and J. Yang, “A self-propelling cycle mediated by reactive oxide species and nitric oxide exists in LPS-activated microglia,” Neurochemistry International, vol. 61, no. 7, pp. 1220–1230, 2012.

T. Mosmann, “Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays,” Journal of Immunological Methods, vol. 65, no. 1-2, pp. 55–63, 1983.

U. K. Hanisch, T. V. Johnson, and J. Kipnis, “Toll-like receptors: roles in neuroprotection?” Trends in Neurosciences, vol. 31, no. 4, pp. 176–182, 2008.

K. Takeda, T. Kaisho, and S. Akira, “Toll-like receptors,” Annual Review of Immunology, vol. 21, pp. 335–376, 2003.

M. J. Bell, P. M. Kochanek, L. A. Doughty et al., “Interleukin-6 and interleukin-10 in cerebrospinal fluid after severe traumatic brain injury in children,” Journal of Neurotrauma, vol. 14, no. 7, pp. 451–457, 1997.

R. S. B. Clark, P. M. Kochanek, W. D. Obrist et al., “Cerebrospinal fluid and plasma nitrite and nitrate concentrations after head injury in humans,” Critical Care Medicine, vol. 24, no. 7, pp. 1243–1251, 1996.

M. N. Woodroofe, G. S. Sarna, M. Wadhwa et al., “Detection of interleukin-1 and interleukin-6 in adult rat brain, following mechanical injury, by in vivo microdialysis: evidence of a role for microglia in cytokine production,” Journal of Neuroimmunology, vol. 33, no. 3, pp. 227–236, 1991.

L. Ott, C. J. McClain, M. Gillespie, and B. Young, “Cytokines and metabolic dysfunction after severe head injury,” Journal of Neurotrauma, vol. 11, no. 5, pp. 447–472, 1994.

M. Aibiki, S. Maekawa, S. Ogura, Y. Kinoshita, N. Kawai, and S. Yokono, “Effect of moderate hypothermia on systemic and internal jugular plasma IL-6 levels after traumatic brain injury in humans,” Journal of Neurotrauma, vol. 16, no. 3, pp. 225–232, 1999.

Hypothermia after Cardiac Arrest Study Group, “Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest,” The New England Journal of Medicine, vol. 346, no. 8, pp. 549–556, 2002.

S. Yamaguchi, K. Nakahara, T. Miyagi, T. Tokutomi, and M. Shigemori, “Neurochemical monitoring in the management of severe head-injured patients with hypothermia,” Neurological Research, vol. 22, no. 7, pp. 657–664, 2000.

C. F. Loidl, J. De Vente, M. M. van Ittersum et al., “Hypothermia during or after severe perinatal asphyxia prevents increase in cyclic GMP-related nitric oxide levels in the newborn rat striatum,” Brain Research, vol. 791, no. 1-2, pp. 303–307, 1998.

R. Khorooshi and T. Owens, “Injury-induced type I IFN signaling regulates inflammatory responses in the central nervous system,” The Journal of Immunology, vol. 185, no. 2, pp. 1258–1264, 2010.

M. O. Kim, Q. Si, and J. N. Zhou, “Interferon-beta activates multiple signaling cascades in primary human microglia,” Journal of Neurochemistry, vol. 81, no. 6, pp. 1361–1371, 2002.

L. L. Hua and S. C. Lee, “Distinct patterns of stimulus-inducible chemokine mRNA accumulation in human fetal astrocytes and microglia,” Glia, vol. 30, no. 1, pp. 74–81, 2000.

J. Wang and I. L. Campbell, “Innate STAT1-dependent genomic response of neurons to the antiviral cytokine alpha interferon,” Journal of Virology, vol. 79, no. 13, pp. 8295–8302, 2005.

K. Wada, N. Okada, T. Yamamura, and S. Koizumi, “Nerve growth factor induces resistance of PC12 cells to nitric oxide cytotoxicity,” Neurochemistry International, vol. 29, no. 5, pp. 461–467, 1996.

M. Bsibsi, J. J. Bajramovic, M. H. J. Vogt et al., “The microtubule regulator stathmin is an endogenous protein agonist for TLR3,” The Journal of Immunology, vol. 184, no. 12, pp. 6929–6937, 2010.

A. Liu, C. Stadelmann, M. Moscarello et al., “Expression of stathmin, a developmentally controlled cytoskeleton-regulating molecule, in demyelinating disorders,” The Journal of Neuroscience, vol. 25, no. 3, pp. 737–747, 2005.

C. X. Cao, Q. W. Yang, F. L. Lv, J. Cui, H. B. Fu, and J. Z. Wang, “Reduced cerebral ischemia-reperfusion injury in toll-like receptor 4 deficient mice,” Biochemical and Biophysical Research Communications, vol. 353, no. 2, pp. 509–514, 2007.

F. Hua, J. Ma, T. Ha et al., “Activation of toll-like receptor 4 signaling contributes to hippocampal neuronal death following global cerebral ischemia/reperfusion,” Journal of Neuroimmunology, vol. 190, no. 1-2, pp. 101–111, 2007.

K. Hyakkoku, J. Hamanaka, K. Tsuruma et al., “Toll-like receptor 4 (TLR4), but not TLR3 or TLR9, knock-out mice have neuroprotective effects against focal cerebral ischemia,” Neuroscience, vol. 171, no. 1, pp. 258–267, 2010.