Toll-Like Receptor Agonists as Antecedent Therapy for Ischemic Brain Injury: Advancing Preclinical Studies to the Nonhuman Primate

  • Frances Rena Bahjat
  • Keri B. Vartanian
  • G. Alexander West
  • Mary P. Stenzel-PooreEmail author
Part of the Springer Series in Translational Stroke Research book series (SSTSR)


Antecedent therapy for ischemic brain injury has the potential to protect a large, high-risk patient population from the devastating effects of cerebral ischemia associated with cardiac surgery. Substantial evidence has shown that preconditioning with a modestly damaging stimulus induces powerful endogenous neuroprotection. Pharmacological agents that stimulate toll-like receptors (TLRs) induce robust neuroprotective effects as preconditioning stimuli against cerebral ischemia in mouse and nonhuman primate models of stroke. Here we describe the progress of our preclinical development of TLR agonists as antecedent therapy against cerebral ischemic injury. The objective was to discuss studies that begin with in vitro validation in cell cultures to in vivo efficacy studies using mouse and nonhuman primate models of stroke, with particular emphasis on the TLR9 agonist CpG oligonucleotide. We provide an in-depth discussion of our novel rhesus macaque stroke model and cover the progress we have made in therapeutic testing and evaluation in these animals. These studies represent a logical path for the development of TLR agonists as antecedent therapy for the prevention of the damaging neurological complications resulting from cerebral ischemic injury.


Cerebral Ischemia Middle Cerebral Artery Infarct Volume Rhesus Macaque Ischemic Brain Injury 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Barber PA, Darby DG, Desmond PM, Gerraty RP, Yang Q, Li T, et al. Identification of major ischemic change. Diffusion-weighted imaging versus computed tomography. Stroke. 1999;30(10):2059–65.PubMedCrossRefGoogle Scholar
  2. 2.
    McKhann GM, Grega MA, Borowicz Jr LM, Baumgartner WA, Selnes OA. Stroke and encephalopathy after cardiac surgery: an update. Stroke. 2006;37(2):562–71.PubMedCrossRefGoogle Scholar
  3. 3.
    Johnston SC, Gress DR, Browner WS, Sidney S. Short-term prognosis after emergency department diagnosis of TIA. JAMA. 2000;284(22):2901–6.PubMedCrossRefGoogle Scholar
  4. 4.
    Simon RP, Niiro M, Gwinn R. Prior ischemic stress protects against experimental stroke. Neurosci Lett. 1993;163:135–7.PubMedCrossRefGoogle Scholar
  5. 5.
    Virca GD, Kim SY, Glaser KB, Ulevitch RJ. Lipopolysaccharide induced hyporesponsiveness to its own action in RAW 264.7 cells. J Biol Chem. 1989;264(36):21951–6.PubMedGoogle Scholar
  6. 6.
    Tasaki K, Ruetzler CA, Ohtsuki T, Martin D, Nawashiro H, Hallenbeck JM. Lipopolysaccharide pre-treatment induces resistance against subsequent focal cerebral ischemic damage in spontaneously hypertensive rats. Brain Res. 1997;748(1–2):267–70.PubMedCrossRefGoogle Scholar
  7. 7.
    Heemann U, Szabo A, Hamar P, Muller V, Witzke O, Lutz J, et al. Lipopolysaccharide pretreatment protects from renal ischemia/reperfusion injury: possible connection to an interleukin-6-dependent pathway. Am J Pathol. 2000;156(1):287–93.PubMedCrossRefGoogle Scholar
  8. 8.
    Vartanian K, Stenzel-Poore M. Toll-like receptor tolerance as a mechanism for neuroprotection. Transl Stroke Res. 2010;1(4):252–60.PubMedCrossRefGoogle Scholar
  9. 9.
    Broad A, Kirby JA, Jones DE. Toll-like receptor interactions: tolerance of MyD88-dependent cytokines but enhancement of MyD88-independent interferon-beta production. Immunology. 2007;120(1):103–11.PubMedCrossRefGoogle Scholar
  10. 10.
    Biswas SK, Lopez-Collazo E. Endotoxin tolerance: new mechanisms, molecules and clinical significance. Trends Immunol. 2009;30(10):475–87.PubMedCrossRefGoogle Scholar
  11. 11.
    Marsh B, Stevens SL, Packard AE, Gopalan B, Hunter B, Leung PY, et al. Systemic lipopolysaccharide protects the brain from ischemic injury by reprogramming the response of the brain to stroke: a critical role for IRF3. J Neurosci. 2009;29(31):9839–49.PubMedCrossRefGoogle Scholar
  12. 12.
    Caso JR, Pradillo JM, Hurtado O, Lorenzo P, Moro MA, Lizasoain I. Toll-like receptor 4 is involved in brain damage and inflammation after experimental stroke. Circulation. 2007;115(12):1599–608.PubMedCrossRefGoogle Scholar
  13. 13.
    Lehnardt S, Lehmann S, Kaul D, Tschimmel K, Hoffmann O, Cho S, et al. Toll-like receptor 2 mediates CNS injury in focal cerebral ischemia. J Neuroimmunol. 2007;190(1–2):28–33.PubMedCrossRefGoogle Scholar
  14. 14.
    Ziegler G, Harhausen D, Schepers C, Hoffmann O, Rohr C, Prinz V, et al. TLR2 has a detrimental role in mouse transient focal cerebral ischemia. Biochem Biophys Res Commun. 2007;359(3):574–9.PubMedCrossRefGoogle Scholar
  15. 15.
    Brea D, Blanco M, Ramos-Cabrer P, Moldes O, Arias S, Perez-Mato M, et al. Toll-like receptors 2 and 4 in ischemic stroke: outcome and therapeutic values. J Cereb Blood Flow Metab. 2011;31(6):1424–31.PubMedCrossRefGoogle Scholar
  16. 16.
    Hua F, Ma J, Ha T, Kelley J, Williams DL, Kao RL, et al. Preconditioning with a TLR2 specific ligand increases resistance to cerebral ischemia/reperfusion injury. J Neuroimmunol. 2008;199(1–2):75–82.PubMedCrossRefGoogle Scholar
  17. 17.
    Stevens SL, Ciesielski TM, Marsh BJ, Yang T, Homen DS, Boule JL, et al. Toll-like receptor 9: a new target of ischemic preconditioning in the brain. J Cereb Blood Flow Metab. 2008;28(5):1040–7.PubMedCrossRefGoogle Scholar
  18. 18.
    Stroke Therapy Academic Industry Round Table (Fisher M. Chair). Enhancing the development and approval of acute stroke therapies: stroke therapy academic industry roundtable. Stroke. 2005;36(8):1808–13.CrossRefGoogle Scholar
  19. 19.
    Fukuda S, del Zoppo GJ. Models of focal cerebral ischemia in the nonhuman primate. ILAR J. 2003;44(2):96–104.PubMedGoogle Scholar
  20. 20.
    Karpiak SE, Tagliavia A, Wakade CG. Animal models for the study of drugs in ischemic stroke. Annu Rev Pharmacol Toxicol. 1989;29:403–14.PubMedCrossRefGoogle Scholar
  21. 21.
    Carmichael ST. Rodent models of focal stroke: size, mechanism, and purpose. NeuroRx. 2005;2(3):396–409.PubMedCrossRefGoogle Scholar
  22. 22.
    American Academy of Pediatrics Committee on Drugs. Alternative routes of drug administration—advantages and disadvantages (subject review). Pediatrics. 1997;100(1):143–52.CrossRefGoogle Scholar
  23. 23.
    DeGraba T, Pettigrew L. Why do neuroprotective drugs work in animals but not humans? Neurol Clin. 2000;18:475–93.PubMedCrossRefGoogle Scholar
  24. 24.
    Kapoor K, Kak VK, Singh B. Morphology and comparative anatomy of circulus arteriosus cerebri in mammals. Anat Histol Embryol. 2003;32(6):347–55.PubMedCrossRefGoogle Scholar
  25. 25.
    Ge Y, Grossman RI, Babb JS, Rabin ML, Mannon LJ, Kolson DL. Age-related total gray matter and white matter changes in normal adult brain. Part II: quantitative magnetization transfer ratio histogram analysis. AJNR Am J Neuroradiol. 2002;23(8):1334–41.PubMedGoogle Scholar
  26. 26.
    Arakawa S, Wright PM, Koga M, Phan TG, Reutens DC, Lim I, et al. Ischemic thresholds for gray and white matter: a diffusion and perfusion magnetic resonance study. Stroke. 2006;37(5):1211–6.PubMedCrossRefGoogle Scholar
  27. 27.
    Maeda M, Takamatsu H, Furuichi Y, Noda A, Awaga Y, Tatsumi M, et al. Characterization of a novel thrombotic middle cerebral artery occlusion model in monkeys that exhibits progressive hypoperfusion and robust cortical infarction. J Neurosci Methods. 2005;146(1):106–15.PubMedCrossRefGoogle Scholar
  28. 28.
    Hirouchi Y, Suzuki E, Mitsuoka C, Jin H, Kitajima S, Kohjimoto Y, et al. Neuroimaging and histopathological evaluation of delayed neurological damage produced by artificial occlusion of the middle cerebral artery in Cynomolgus monkeys: establishment of a monkey model for delayed cerebral ischemia. Exp Toxicol Pathol. 2007;59(1):9–16.PubMedCrossRefGoogle Scholar
  29. 29.
    Huang J, Mocco J, Choudhri TF, Poisik A, Popilskis SJ, Emerson R, et al. A modified transorbital baboon model of reperfused stroke. Stroke. 2000;31(12):3054–63.PubMedCrossRefGoogle Scholar
  30. 30.
    Mack WJ, Komotar RJ, Mocco J, Coon AL, Hoh DJ, King RG, et al. Serial magnetic resonance imaging in experimental primate stroke: validation of MRI for pre-clinical cerebroprotective trials. Neurol Res. 2003;25(8):846–52.PubMedCrossRefGoogle Scholar
  31. 31.
    West GA, Golshani KJ, Doyle K, Lessov NS, Hobbs TR, Kohama SG, et al. A new model of cortical stroke in the rhesus macaque. J Cereb Blood Flow Metab. 2009;29(6):1175–86.PubMedCrossRefGoogle Scholar
  32. 32.
    Burrows AM, Waller BM, Parr LA. Facial musculature in the rhesus macaque (Macaca mulatta): evolutionary and functional contexts with comparisons to chimpanzees and humans. J Anat. 2009;215(3):320–34.PubMedCrossRefGoogle Scholar
  33. 33.
    Iredale SK, Nevill CH, Lutz CK. The influence of observer presence on baboon (Papio spp.) and rhesus macaque (Macaca mulatta) behavior. Appl Anim Behav Sci. 2010;122(1):53–7.PubMedCrossRefGoogle Scholar
  34. 34.
    Spetzler RF, Selman WR, Weinstein P, Townsend J, Mehdorn M, Telles D, et al. Chronic reversible cerebral ischemia: evaluation of a new baboon model. Neurosurgery. 1980;7(3):257–61.PubMedCrossRefGoogle Scholar
  35. 35.
    Mori E, Ember JA, Copeland BR, Thomas WS, Koziol JA, del Zoppo GJ. Effect of tirilazad mesylate on middle cerebral artery occlusion/reperfusion in nonhuman primates. Cerebrovasc Dis. 1995;5(5):342–9.CrossRefGoogle Scholar
  36. 36.
    De Haan R, Horn J, Limburg M, Van Der Meulen J, Bossuyt P. A comparison of five stroke scales with measures of disability, handicap, and quality of life. Stroke. 1993;24(8):1178–81.PubMedCrossRefGoogle Scholar
  37. 37.
    De Vries SI, Van Hirtum HW, Bakker I, Hopman-Rock M, Hirasing RA, Van Mechelen W. Validity and reproducibility of motion sensors in youth: a systematic update. Med Sci Sports Exerc. 2009;41(4):818–27.PubMedCrossRefGoogle Scholar
  38. 38.
    Papailiou A, Sullivan E, Cameron JL. Behaviors in rhesus monkeys (Macaca mulatta) associated with activity counts measured by accelerometer. Am J Primatol. 2008;70(2):185–90.PubMedCrossRefGoogle Scholar
  39. 39.
    Mann TM, Williams KE, Pearce PC, Scott EA. A novel method for activity monitoring in small non-human primates. Lab Anim. 2005;39(2):169–77.PubMedCrossRefGoogle Scholar
  40. 40.
    Krieg AM. Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov. 2006;5(6):471–84.PubMedCrossRefGoogle Scholar
  41. 41.
    Leifer CA, Verthelyi D, Klinman DM. Heterogeneity in the human response to immunostimulatory CpG oligodeoxynucleotides. J Immunother. 2003;26(4):313–9.PubMedCrossRefGoogle Scholar
  42. 42.
    Gursel M, Verthelyi D, Gursel I, Ishii KJ, Klinman DM. Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide. J Leukoc Biol. 2002;71(5):813–20.PubMedGoogle Scholar
  43. 43.
    Verthelyi D, Kenney RT, Seder RA, Gam AA, Friedag B, Klinman DM. CpG oligodeoxynucleotides as vaccine adjuvants in primates. J Immunol. 2002;168(4):1659–63.PubMedGoogle Scholar
  44. 44.
    Bahjat FR, Williams-Karnesky RL, Kohama SG, West GA, Doyle KP, Spector MD, et al. Proof of concept: pharmacological preconditioning with a Toll-like receptor agonist protects against cerebrovascular injury in a primate model of stroke. J Cereb Blood Flow Metab. 2011;31(5):1229–42.PubMedCrossRefGoogle Scholar
  45. 45.
    Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, et al. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke. 2009;40(6):2244–50.PubMedCrossRefGoogle Scholar
  46. 46.
    Viray J, Rolfs B, Smith DG. Comparison of the frequencies of major histocompatibility (MHC) class-II DQA1 and DQB1 alleles in Indian and Chinese rhesus macaques (Macaca mulatta). Comp Med. 2001;51(6):555–61.PubMedGoogle Scholar
  47. 47.
    Ferguson B, Street SL, Wright H, Pearson C, Jia Y, Thompson SL, et al. Single nucleotide polymorphisms (SNPs) distinguish Indian-origin and Chinese-origin rhesus macaques (Macaca mulatta). BMC Genomics. 2007;8:43.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Frances Rena Bahjat
    • 1
  • Keri B. Vartanian
    • 1
  • G. Alexander West
    • 2
  • Mary P. Stenzel-Poore
    • 1
    • 3
    Email author
  1. 1.Department of Molecular Microbiology and ImmunologyOregon Health and Science UniversityPortlandUSA
  2. 2.Colorado Brain and Spine Institute, Neurotrauma Research LaboratorySwedish Medical CenterEnglewoodUSA
  3. 3.Division of NeuroscienceOregon National Primate Research CenterBeavertonUSA

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