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Strain-dependent variation in the early transcriptional response to CNS injury using a cortical explant system
Journal of Neuroinflammationvolume 8, Article number: 122 (2011)
While it is clear that inbred strains of mice have variations in immunological responsiveness, the influence of genetic background following tissue damage in the central nervous system is not fully understood. A cortical explant system was employed as a model for injury to determine whether the immediate transcriptional response to tissue resection revealed differences among three mouse strains.
Immunological mRNAs were measured in cerebral cortex from SJL/J, C57BL/6J, and BALB/cJ mice using real time RT-PCR. Freshly isolated cortical tissue and cortical sections incubated in explant medium were examined. Levels of mRNA, normalized to β-actin, were compared using one way analysis of variance with pooled samples from each mouse strain.
In freshly isolated cerebral cortex, transcript levels of many pro-inflammatory mediators were not significantly different among the strains or too low for comparison. Constitutive, baseline amounts of CD74 and antisecretory factor (ASF) mRNAs, however, were higher in SJL/J and C57BL/6J, respectively. When sections of cortical tissue were incubated in explant medium, increased message for a number of pro-inflammatory cytokines and chemokines occurred within five hours. Message for chemokines, IL-1α, and COX-2 transcripts were higher in C57BL/6J cortical explants relative to SJL/J and BALB/cJ. IL-1β, IL-12/23 p40, and TNF-α were lower in BALB/cJ explants relative to SJL/J and C57BL/6J. Similar to observations in freshly isolated cortex, CD74 mRNA remained higher in SJL/J explants. The ASF mRNA in SJL/J explants, however, was now lower than levels in both C57BL/6J and BALB/cJ explants.
The short-term cortical explant model employed in this study provides a basic approach to evaluate an early transcriptional response to neurological damage, and can identify expression differences in genes that are influenced by genetic background.
Inbred strains of mice with identical or nearly identical genotypes have been developed and used extensively in experimental research. They provide a valuable means to study genetic influence on various biological determinants. Susceptibility to, or the severity of, experimental models of neurological disease and injury is often strain-dependent [1–3]. In such systems, inflammatory mediators and immunological activation are recognized as key factors. Gene linkage analysis of hybrids from two strains of mice and rats have implicated many immunologically relevant genes that may regulate in clinical susceptibility or severity [2, 4–15].
SJL/J mice are a strain commonly used in animal models of neurological disease. Variations in immune responsiveness have also been well defined between C57BL/6J and BALB/cJ mice in non-CNS tissues. It is not fully known whether the immediate response to injury in CNS tissue differs among these strains. A simple ex vivo system was devised to address this. The transcriptional response of inflammatory-related genes was measured in cerebral cortical tissue that was incubated in explant medium for less than five hours from resection. The inflammation-related transcriptional targets selected for analysis were based on their previously documented involvement in models of neurological disease and injury [16–18]. This included pro-inflammatory cytokines, chemokines, CD74, and antisecretory factor. CD74 is differentially regulated among inbred strains following CNS injury [4, 19]. Antisecretory factor is an understudied molecule with anti-inflammatory activity that has been implicated in severity of experimental autoimmune encephalomyelitis , a model system known to exhibit well established strain-dependent variability [21, 22]. In this study, the levels of mRNAs were compared in freshly isolated cerebral cortex and cortical explants among three mouse strains. A classic injury response of pro-inflammatory mediators was observed in cortical explants, yet differences based on genetic background were also observed.
The Institutional Animal Care and Use Committee at Dartmouth College approved all experimental protocols. All mice were obtained from Jackson Laboratory (Bar Harbor, ME). SJL/J (n = 11), C57BL/6J (7), and BALB/cJ (11) strains were housed at Borwell Animal Facility for several weeks before use in cortical explant experiments. All mice were female with an average age of 3.9 ± 0.6 months. Only female mice were used in this study to avoid gender differences that are well documented in the SJL/J strain, for which a polymorphism on the Y chromosome has been implicated .
Mice were euthanized via halothane over-exposure and then decapitated. Brains were removed and set in an acrylic brain matrices (Braintree Scientific, Braintree, MA) where two 1-mm-thick coronal sections positioned within 2 mm from either side of bregma were cut using a razor blade. Cortex was dissected at the corpus callosum and the midline producing four sections per mouse brain. One section of cortex was processed for RNA isolation immediately to determine basal mRNA levels. Other sections of cortical tissue was placed in individual wells of a 48-well Falcon tissue culture plate containing 0.5 ml of pre-warmed DMEM/High Glucose medium (Thermo Scientific HyClone, Rockford, IL ) supplemented with fetal bovine serum (FBS; 10%; Thermo Scientific HyClone), L-glutamine (2 mM), and penicillin (100 units/ml)/streptomycin (100 ug/ml). Explants were placed in a humidified incubator at 37°C with 5% CO2 for designated times. The elapsed time from euthanasia to commencement of incubation of cortical explants was less than ten minutes.
Quantitative real time reverse transcription (RT)-PCR
Cortical explants were stored immediately in RNAlater solution (Invitrogen, Carlsbad, CA) for one day at 4°C and then stored at -80°C until RNA isolation. RNA was extracted using TRIzol Reagent (Invitrogen). Eluted RNA was quantified by spectrophotometry and 1 ug was reverse-transcribed using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). Quantitative real-time PCR was performed using PerfeCTa SYBR Green FastMix with low ROX (Quanta Biosciences), 4 ng sample cDNA, and 300 nM of a RT-PCR primer set (IDT, San Jose, CA) listed in Table 1. Settings for analysis using an ABI 7500 machine were as follows: initial denaturation (95°C/3 min) was followed by 50 cycles of denaturation (95°C/15 s) and primer annealing (60°C/45 s). A melt curve was performed on all samples for quality control. The relative quantity of gene expression was analyzed by the 2(-ΔΔCt) method with normalization to the endogenous control β-actin.
Baseline levels of immunological mRNA in SJL/J cerebral cortex
Freshly isolated sections of cortical tissue from SJL/J mice were immediately processed for RNA to determine baseline levels of fourteen immunological transcripts. β-actin mRNA tissue levels served as a reference amount. All were lower with messages for chemokines, IL-1α and β, IL-6, and IL-23 less than 0.1% of β-actin levels (Table 2).
Comparison of constitutive mRNA levels in cerebral cortex from three mouse strains
Levels of immunological transcripts in freshly isolated SJL/J cerebral cortex were compared to baseline levels in freshly isolated C57BL/6J and BALB/cJ cortices. Amounts of CD74 and antisecretory factor (ASF) mRNA differed (Figure 1). CD74 in C57BL/6J and BALB/cJ were lower than 50% of that in SJL/J tissue. ASF was 30% higher in C57BL/6 relative to SJL/J and BALB/cJ tissues. No significant differences in constitutive expression of COX-2 (one-way analysis of variance; P = 0.7), TNF-α (0.1), IL-12 p35 (0.08), or CiiTA (0.6) were found. Baseline levels of chemokines, IL-1α and β, IL-6, and IL-23 were negligible (see Table 2) and considered too low to reliably compare among strains based on the detection limits for real-time RT-PCR.
Immunological transcriptional response in SJL/J cortical explants
Tissue levels of mRNA in sections of cortical tissue from SJL/J mice incubated in explant medium were expressed as a fold difference relative to baseline levels in freshly isolated SJL/J cerebral cortex. The quantity of eleven transcripts changed within five hours of incubation (Table 3). Chemokines and pro-inflammatory cytokines increased in cortical explants with the exception to the p35 subunit of IL-12. Message for ASF decreased in a time-dependent manner (Figure 2).
Comparison of mRNA levels in cortical explant from three mouse strains
Sections of cortical tissue from SJL/J, C57BL/6J, and BALB/cJ mice were incubated in explant medium for 4.5 hours. Immunological transcripts in cortical explants from these strains were determined and expressed as a percent of the amount in SJL/J - i.e., for this interstrain comparison the transcript amount for the moieties studied were calculated using the levels found in SJL/J explants as the standard. Transcripts for many pro-inflammatory mediators and antisecretory factor revealed differential tissue levels among strains (Figure 3). C57BL/6J explants had higher mRNA amounts for chemokines and IL-1α relative SJL/J and BALB/cJ explants. IL-1β, TNF-α, IL-12/23 p40, and COX-2 revealed a similar profile with SJL/J and C57BL/6J having similar amounts that were higher than in BALB/cJ explants. No significant difference in abundance of IL-6 (one-way analysis of variance; P = 0.6), IL-12 p35 (0.07), IL-23 p19 (0.4), CiiTA (0.1) mRNA were observed among these strains. Since strain differences in CD74 and ASF mRNA were found in freshly isolated cortex and in cortical explants, fold differences within each strain was evaluated. CD74 mRNA was down-regulated in C57BL/6J and BALB/cJ, but not in SJL/J explants relative to baseline levels, while ASF was down-regulated by varying degrees in all three strains (Figure 4).
The data reported in this study establish that differences in the immediate gene response to damage of central nervous system (CNS) tissue occur among mouse strains. Message for several genes involved in CNS injury and neurological diseases with autoimmunity or chronic innate immune activation were strain-dependently altered. Short-term explants of cortical sections provided a reliable system for defining immunologically relevant transcriptional changes in CNS tissue and the model may serve as a cost-effective method to test novel immunomodulating pharmaceuticals.
One millimeter-thick explants of adult cerebral cortex were used in this study. Acute CNS explants of this thickness have been previously demonstrated to induce pro-inflammatory mRNA expression consistent to the temporal profile observed following injury in vivo [24, 25]. The production of immunologically relevant mRNAs is likely caused by a combination of tissue damage at the periphery of the explant due to tissue sectioning and axotomy of projection neurons throughout the explant, and some undefined amount of ischemia in the center of the explant. Pro-inflammatory mRNA increase occurs in cortex within hours following lesioning  or ischemia [27, 28]in vivo. Since there were no established foci of inflammation in the tissue prior to sectioning in our explant model, the influence of the miniscule number of leukocytes in the vasculature would contribute only marginally and is limited to the residual cells in the vasculature at the time of sectioning. Therefore, the extent of the measured pro-inflammatory response is predominantly by resident CNS cells.
Pro-inflammatory cytokines and chemokines expression are activated in cells of the monocyte/macrophage lineage in the innate response to injury or infections. Microglia are considered the primary cell type within the CNS parenchyma that carry out this function. Pattern recognition receptors recognize specific molecules released from damaged host cells or foreign microbes leading to the activation of transcription factors that induce transcription of certain inflammatory genes. Sections of cerebral cortex incubated ex vivo in explant medium were demonstrated to increase mRNA for pro-inflammatory cytokines and chemokines within five hours. The constitutive amounts in uninjured cortical tissue were mostly very low and transcripts that could be confidently quantified did not differ significantly among mouse strains. However the amount measured in cortical explants from these strains was different for several of the transcripts suggesting strain-related alterations in their induction. The chemokines evaluated were CCL2 (MCP-1), CCL3 (MIP-1α), and CCL4 (MIP-1β). These transcripts were found in increasing abundance in BALB/cJ, SJL/J, and C57BL/6J, respectively. Interestingly, the abundance of IL-1α and COX-2 mRNA revealed a similar pattern to the chemokines suggesting these inflammatory mediators could have a common regulatory mechanism that is distinct among these mouse strains. Messages for IL-1β and TNF-α were similar in SJL/J and C57BL/6 explants, but higher than BALB/cJ. Taken together, BALB/cJ appears to have a dampened immunological response to tissue damage in cerebral cortex.
Expression of genes associated with autoimmunity was also examined in this study. Experimental autoimmune encephalomyelitis is a widely studied autoimmune model, and involves infiltration of CD4+ lymphocytes and immunological activation of microglia within the CNS [21, 22, 29]. Although MHC class II haplotype and its binding to specific myelin autoantigen play a pivotal role in this model, non-MHC class II effectors are also implicated . BALB/cJ is EAE resistant while SJL/J and C57BL/6J are susceptible [2, 3, 31]. Cytokines IL-12 and IL-23 are implicated in the pathogenesis of autoimmune diseases including EAE . The p35 subunit of IL-12 and p19 subunit of IL-23 form respective cytokines with a common p40 subunit. Blocking the IL-12/23 p40 subunit with inhibiting antibodies is effective in non-CNS autoimmune diseases such as psoriasis . In cortical explants, the p40 mRNA was upregulated. Its levels were considerably lower in the resistant BALB/cJ explants. This suggests that inherent difference within the CNS tissues may contribute to strain susceptibility to autoimmunity.
Antisecretory factor (ASF) has been shown to affect the severity of EAE. Blocking its activity with an inhibiting antibody increases clinical severity implying it has an anti-inflammatory property . Our results showed that differences in ASF mRNA expression occurred in normal cortical tissue and in cultured cortical explants. C57BL/6J mice had high constitutive ASF mRNA. Its levels decreased after injury in cortical explants in each strain, but to a lesser degree in BALB/cJ. This supports the hypothesis that higher amounts of ASF due to genetic background may contribute to EAE resistance.
Antigen presentation by MHC class II is critical for many autoimmune diseases. CD74 (invariant chain, Ii) acts as an MHC class II chaperone [33, 34]. CD74 mRNA was higher in SJL/J cortex relative to BALB/cJ and C57BL/6. A similar trend among these strains was reported in facial nucleus two weeks following facial nerve axotomy . Our study revealed that higher levels were found in uninjured cortex and that these differences were increased further in explants due to down-regulation in C57BL/6J and BALB/cJ tissues. CD74 also mediates transcription by NF-κB [35, 36] and regulates dendritic cell migration . This suggests its altered expression among strains could influence a wide range of effects.
Now, identifying the inherent genetic polymorphisms that control the variations in transcriptional response to an injury stimulus among strains is important for understanding genetically variable responses to a spectrum of neurological disorders. Approaches such as quantitative trait locus analysis and/or haplotype-based computational genetic mapping can be utilized with the cortical explant model. Haplotype-based computational genetic mapping has recently pinpointed genetic variation of Nalp1 as a contributor to interstrain differences in the inflammatory response to injured skin . It is important to recognize that the involvement of multiple genes may be required and that such analyses will likely require data from additional strains and transcripts.
The genes expressed differentially in cortical explants derived from disparate strains of mice reveal that genetic background can influence immediate response to neurological damage within the CNS. The straightforward approach described in this study may help uncover the inherent regulatory mechanism that control altered immunological responsiveness and perhaps neurological disease susceptibility in future studies.
CC chemokine ligand
class II transactivator
central nervous system
fetal bovine serum
reverse transcription- polymerase chain reaction
tumor necrosis factor
Heiman-Patterson TD, Deitch JS, Blankenhorn EP, Erwin KL, Perreault MJ, Alexander BK, Byers N, Toman I, Alexander GM: Background and gender effects on survival in the TgN(SOD1-G93A)1Gur mouse model of ALS. J Neurol Sci. 2005, 236: 1-7. 10.1016/j.jns.2005.02.006.
Encinas JA, Lees MB, Sobel RA, Symonowicz C, Greer JM, Shovlin CL, Weiner HL, Seidman CE, Seidman JG, Kuchroo VK: Genetic analysis of susceptibility to experimental autoimmune encephalomyelitis in a cross between SJL/J and B10. S mice. J Immunol. 1996, 157: 2186-92.
Royle SJ, Collins FC, Rupniak HT, Barnes JC, Anderson R: Behavioural analysis and susceptibility to CNS injury of four inbred strains of mice. Brain Res. 1999, 816: 337-49. 10.1016/S0006-8993(98)01122-6.
Diez M, Abdelmagid N, Harnesk K, Ström M, Lidman O, Swanberg M, Lindblom R, Al-Nimer F, Jagodic M, Olsson T: Identification of gene regions regulating inflammatory microglial response in the rat CNS after nerve injury. J Neuroimmunol. 2009, 212: 82-92. 10.1016/j.jneuroim.2009.05.004.
Piehl F, Swanberg M, Lidman O: The axon reaction: Identifying the genes that make a difference. Physiol Behav. 2007, 92: 67-74. 10.1016/j.physbeh.2007.05.030.
Swanberg M, Harnesk K, Ström M, Diez M, Lidman O, Piehl F, Linden R: Fine mapping of gene regions regulating neurodegeneration. PLoS One. 2009, 4: 689-700.
Butterfield RJ, Sudweeks JD, Blankenhorn EP, Korngold R, Marini JC, Todd JA, Roper RJ, Teuscher C: New genetic loci that control susceptibility and symptoms of experimental allergic encephalomyelitis in inbred mice. J Immunol. 1998, 161: 1860-7.
Baker D, Rosenwasser OA, O'Neill JK, Turk JL: Genetic analysis of experimental allergic encephalomyelitis in mice. J Immunol. 1995, 155: 4046-51.
Sundvall M, Jirholt J, Yang HT, Jansson L, Engström Å, Pettersson U, Holmdahl R: Identification of murine loci associated with susceptibility to chronic experimental autoimmune encephalomyelitis. Nat Genet. 1995, 10: 313-7. 10.1038/ng0795-313.
Croxford JL, O'Neill JK, Baker D: Polygenic control of experimental allergic encephalomyelitis in Biozzi ABH and BALB/c mice. J Neuroimmunol. 1997, 74: 205-11. 10.1016/S0165-5728(96)00219-6.
Teuscher C, Butterfield RJ, Ma RZ, Zachary JF, Doerge RW, Blankenhorn EP: Sequence polymorphisms in the chemokines Scya1 (TCA-3), Scya2 (monocyte chemoattractant protein (MCP)-1), and Scya12 (MCP-5) are candidates for eae7, a locus controlling susceptibility to monophasic remitting/nonrelapsing experimental allergic encephalomyelitis. J Immunol. 1999, 163: 2262-6.
Marta M, Stridh P, Becanovic K, Gillett A, Öckinger J, Lorentzen JC, Jagodic M, Olsson T: Multiple loci comprising immune-related genes regulate experimental neuroinflammation. Genes Immun. 2009, 11: 21-36.
Teuscher C, Doerge RW, Fillmore PD, Blankenhorn EP: eae36, a locus on mouse chromosome 4, controls susceptibility to experimental allergic encephalomyelitis in older mice and mice immunized in the winter. Genetics. 2006, 172: 1147-53.
Butterfield RJ, Blankenhorn EP, Roper RJ, Zachary JF, Doerge RW, Teuscher C: Identification of genetic loci controlling the characteristics and severity of brain and spinal cord lesions in experimental allergic encephalomyelitis. Am J Pathol. 2000, 157: 637-45. 10.1016/S0002-9440(10)64574-9.
Spach KM, Case LK, Noubade R, Petersen CB, McElvany B, Zalik N, Hickey WF, Blankenhorn EP, Teuscher C: Multiple linked quantitative trait loci within the Tmevd2/Eae3 interval control the severity of experimental allergic encephalomyelitis in DBA/2J mice. Genes Immun. 2010, 11: 649-59. 10.1038/gene.2010.40.
Wang CX, Shuaib A: Involvement of inflammatory cytokines in central nervous system injury. Prog Neurobiol. 2002, 67: 161-72. 10.1016/S0301-0082(02)00010-2.
Kroenke MA, Carlson TJ, Andjelkovic AV, Segal BM: IL-12-and IL-23-modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition. J Exp Med. 2008, 205: 1535-41. 10.1084/jem.20080159.
Fischer FR, Santambrogio L, Luo Y, Berman MA, Hancock WW, Dorf ME: Modulation of experimental autoimmune encephalomyelitis: effect of altered peptide ligand on chemokine and chemokine receptor expression. J Neuroimmunol. 2000, 110: 195-208. 10.1016/S0165-5728(00)00351-9.
Harnesk K, Swanberg M, Diez M, Olsson T, Piehl F, Lidman O: Differential nerve injury-induced expression of MHC class II in the mouse correlates to genetic variability in the type I promoter of C2ta. J Neuroimmunol. 2009, 212: 44-52. 10.1016/j.jneuroim.2009.04.019.
Davidson TS, Hickey WF: Antisecretory factor expression is regulated by inflammatory mediators and influences the severity of experimental autoimmune encephalomyelitis. J Leukoc Biol. 2004, 76: 835-44. 10.1189/jlb.0204085.
Furlan R, Cuomo C, Martino G: Animal models of multiple sclerosis. Methods Mol Biol. 2009, 549: 157-73. 10.1007/978-1-60327-931-4_11.
Gold R, Linington C, Lassmann H: Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research. Brain. 2006, 129: 1953-71. 10.1093/brain/awl075.
Spach KM, Blake M, Bunn JY, McElvany B, Noubade R, Blankenhorn EP, Teuscher C: Cutting edge: the Y chromosome controls the age-dependent experimental allergic encephalomyelitis sexual dimorphism in SJL/J mice. J Immunol. 2009, 182: 1789-93. 10.4049/jimmunol.0803200.
Pan JZ, Ni L, Sodhi A, Aguanno A, Young W, Hart RP: Cytokine activity contributes to induction of inflammatory cytokine mRNAs in spinal cord following contusion. J Neurosci Res. 2002, 68.
Rice T, Larsen J, Rivest S, Yong VW: Characterization of the early neuroinflammation after spinal cord injury in mice. J Neuropathol Exp Neurol. 2007, 66: 184-10.1097/01.jnen.0000248552.07338.7f.
Rostworowski M, Balasingam V, Chabot S, Owens T, Yong VW: Astrogliosis in the neonatal and adult murine brain post-trauma: elevation of inflammatory cytokines and the lack of requirement for endogenous interferon-γ. J Neurosci. 1997, 17: 3664.
Wang X, Yue TL, Barone FC, White RF, Gagnon RC, Feuerstein GZ: Concomitant cortical expression of TNF-α and IL-1β mRNAs follows early response gene expression in transient focal ischemia. Mol Chem Neuropathol. 1994, 23: 103-14. 10.1007/BF02815404.
Berti R, Williams AJ, Moffett JR, Hale SL, Velarde LC, Elliott PJ, Yao C, Dave JR, Tortella FC: Quantitative real-time RT-PCR analysis of inflammatory gene expression associated with ischemia-reperfusion brain injury. J Cereb Blood Flow Metab. 2002, 22: 1068-79.
Murphy ÁC, Lalor SJ, Lynch MA, Mills KHG: Infiltration of Th1 and Th17 cells and activation of microglia in the CNS during the course of experimental autoimmune encephalomyelitis. Brain Behav Immun. 2010, 24: 641-51. 10.1016/j.bbi.2010.01.014.
Happ M, Wettstein P, Dietzschold B, Heber-Katz E: Genetic control of the development of experimental allergic encephalomyelitis in rats. Separation of MHC and non-MHC gene effects. J Immunol. 1988, 141: 1489-94.
Teuscher C, Hickey WF, Grafer CM, Tung KSK: A common immunoregulatory locus controls susceptibility to actively induced experimental allergic encephalomyelitis and experimental allergic orchitis in BALB/c mice. J Immunol. 1998, 160: 2751-9.
Leonardi CL, Kimball AB, Papp KA, Yeilding N, Guzzo C, Wang Y, Li S, Dooley LT, Gordon KB: Efficacy and safety of ustekinumab, a human interleukin-12/23 monoclonal antibody, in patients with psoriasis: 76-week results from a randomised, double-blind, placebo-controlled trial (PHOENIX 1). The Lancet. 2008, 371: 1665-74. 10.1016/S0140-6736(08)60725-4.
Roche PA, Teletski CL, Stang E, Bakke O, Long EO: Cell surface HLA-DR-invariant chain complexes are targeted to endosomes by rapid internalization. Proc Natl Acad Sci USA. 1993, 90: 8581-5. 10.1073/pnas.90.18.8581.
Cresswell P: Assembly, transport, and function of MHC class II molecules. Annu Rev Immunol. 1994, 12: 259-91. 10.1146/annurev.iy.12.040194.001355.
Teo BHD, Wong SH: MHC class II-associated invariant chain (Ii) modulates dendritic cells-derived microvesicles (DCMV)-mediated activation of microglia. Biochem Biophys Res Commun. 2010, 400: 673-8. 10.1016/j.bbrc.2010.08.126.
Matza D, Kerem A, Medvedovsky H, Lantner F, Shachar I: Invariant chain-induced B cell differentiation requires intramembrane proteolytic release of the cytosolic domain. Immunity. 2002, 17: 549-60. 10.1016/S1074-7613(02)00455-7.
Faure-Andre G, Vargas P, Yuseff MI, Heuze M, Diaz J, Lankar D, Steri V, Manry J, Hugues S, Vascotto F: Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain. Science. 2008, 322: 1705-10. 10.1126/science.1159894.
Hu Y, Liang D, Li X, Liu HH, Zhang X, Zheng M, Dill D, Shi X, Qiao Y, Yeomans D, Carvalho B, Angst MS, Clark JD, Peltz G: The Role of Interleukin-1 in Wound Biology. Part I: Murine In Silico and In Vitro Experimental Analysis. Anesth Analg. 2010, 111: 1525-33. 10.1213/ANE.0b013e3181f5ef5a.
DJG and WFH acknowledge support from the Department of Pathology, Dartmouth Medical School. DJG and BTH were supported in part by grants from Reata Pharmaceuticals and the ALS Center of Dartmouth-Hitchcock Medical Center.
The authors declare that they have no competing interests.
DJG carried out the experiments and evaluated the data. DJG, BTH, and WFH participated in the design and assisted with the preparation of the manuscript. All authors have read and approved the final version of the manuscript.