Skip to main content

Complexities, Confounders, and Challenges in Experimental Stroke Research: A Checklist for Researchers and Reviewers

  • Protocol
  • First Online:
Book cover Rodent Models of Stroke

Part of the book series: Neuromethods ((NM,volume 47))

Abstract

The quest for internal and external validity in experimental stroke research is fraught with pitfalls and confounders. This article, written as a checklist from the perspective of an editor and reviewer of articles on rodent stroke models and an active bench side stroke researcher, presents a compilation of the common pitfalls and quality issues in experimental stroke research. These include selecting controls for genetically modified animals, effects of stroke on systemic parameters (immunodepression, infection, cachexia, etc.), cerebral blood flow measurement, brain edema correction, study design, statistics and interpretation.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wolfer DP, Crusio WE, Lipp HP (2002) Knockout mice: simple solutions to the problems of genetic background and flanking genes. Trends Neurosci 25:336–340

    Article  PubMed  CAS  Google Scholar 

  2. Gerlai R (1996) Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype? Trends Neurosci 19:177–181

    Article  PubMed  CAS  Google Scholar 

  3. Barone FC, Knudsen DJ, Nelson AH, Feuerstein GZ, Willette RN (1993) Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J Cereb Blood Flow Metab 13:683–692

    Article  PubMed  CAS  Google Scholar 

  4. Beckmann N (2000) High resolution magnetic resonance angiography non-invasively reveals mouse strain differences in the cerebrovascular anatomy in vivo. Magn Reson Med 44:252–258

    Article  PubMed  CAS  Google Scholar 

  5. Fujii M, Hara H, Meng W, Vonsattel JP, Huang Z, Moskowitz MA (1997) Strain-related differences in susceptibility to transient forebrain ischemia in SV-129 and C57black/6 mice. Stroke 28:1805–1810, discussion 181

    Article  PubMed  CAS  Google Scholar 

  6. Majid A, He YY, Gidday JM, Kaplan SS, Gonzales ER, Park TS, Fenstermacher JD, Wei L, Choi DW, Hsu CY (2000) Differences in vulnerability to permanent focal cerebral ischemia among 3 common mouse strains. Stroke 31:2707–2714

    Article  PubMed  CAS  Google Scholar 

  7. (1997) Mutant mice and neuroscience: recommendations concerning genetic background. Banbury Conference on genetic background in mice. Neuron 19:755–759

    Google Scholar 

  8. Conner, D. A. (2005) Transgenic mouse colony management. Curr Protoc Mol Biol Chapter 23, Unit 23.10

    Google Scholar 

  9. Matsui T, Mori T, Tateishi N, Kagamiishi Y, Satoh S, Katsube N, Morikawa E, Morimoto T, Ikuta F, Asano T (2002) Astrocytic activation and delayed infarct expansion after permanent focal ischemia in rats. Part I: enhanced astrocytic synthesis of s-100beta in the periinfarct area precedes delayed infarct expansion. J Cereb Blood Flow Metab 22:711–722

    Article  PubMed  CAS  Google Scholar 

  10. Pantano P, Caramia F, Bozzao L, Dieler C, von Kummer R (1999) Delayed increase in infarct volume after cerebral ischemia: correlations with thrombolytic treatment and clinical outcome. Stroke 30:502–507

    Article  PubMed  CAS  Google Scholar 

  11. Henrich-Noack P, Baldauf K, Reiser G, Reymann KG (2008) Pattern of time-dependent reduction of histologically determined infarct volume after focal ischaemia in mice. Neurosci Lett 432:141–145

    Article  PubMed  CAS  Google Scholar 

  12. Katchanov J, Waeber C, Gertz K, Gietz A, Winter B, Bruck W, Dirnagl U, Veh RW, Endres M (2003) Selective neuronal vulnerability following mild focal brain ischemia in the mouse. Brain Pathol 13:452–464

    Article  PubMed  Google Scholar 

  13. Fujie W, Kirino T, Tomukai N, Iwasawa T, Tamura A (1990) Progressive shrinkage of the thalamus following middle cerebral artery occlusion in rats. Stroke 21:1485–1488

    Article  PubMed  CAS  Google Scholar 

  14. Dihne M, Grommes C, Lutzenburg M, Witte OW, Block F (2002) Different mechanisms of secondary neuronal damage in thalamic nuclei after focal cerebral ischemia in rats. Stroke 33:3006–3011

    Article  PubMed  Google Scholar 

  15. Coimbra C, Drake M, Boris-Moller F, Wieloch T (1996) Long-lasting neuroprotective effect of postischemic hypothermia and treatment with an anti-inflammatory/antipyretic drug. Evidence for chronic encephalopathic processes following ischemia. Stroke 27:1578–1585

    Article  PubMed  CAS  Google Scholar 

  16. Li F, Irie K, Anwer MS, Fisher M (1997) Delayed triphenyltetrazolium chloride staining remains useful for evaluating cerebral infarct volume in a rat stroke model. J Cereb Blood Flow Metab 17:1132–1135

    Article  PubMed  CAS  Google Scholar 

  17. Vogel J, Mobius C, Kuschinsky W (1999) Early delineation of ischemic tissue in rat brain cryosections by high-contrast staining. Stroke 30:1134–1141

    Article  PubMed  CAS  Google Scholar 

  18. Liszczak TM, Hedley-Whyte ET, Adams JF, Han DH, Kolluri VS, Vacanti FX, Heros RC, Zervas NT (1984) Limitations of tetrazolium salts in delineating infarcted brain. Acta Neuropathol 65:150–157

    Article  PubMed  CAS  Google Scholar 

  19. Hatfield RH, Mendelow AD, Perry RH, Alvarez LM, Modha P (1991) Triphenyltetrazolium chloride (TTC) as a marker for ischaemic changes in rat brain following permanent middle cerebral artery occlusion. Neuropathol Appl Neurobiol 17:61–67

    Article  PubMed  CAS  Google Scholar 

  20. Bednar MM, Fanburg JC, Anderson ML, Raymond SJ, Dooley RH, Gross CE (1994) Comparison of triphenyltetrazolium dye with light microscopic evaluation in a rabbit model of acute cerebral ischaemia. Neurol Res 16:129–132

    PubMed  CAS  Google Scholar 

  21. Klohs J, Grafe M, Graf K, Steinbrink J, Dietrich T, Stibenz D, Bahmani P, Kronenberg G, Harms C, Endres M, Lindauer U, Greger K, Stelzer EH, Dirnagl U, Wunder A (2008) In vivo imaging of the inflammatory receptor CD40 after cerebral ischemia using a fluorescent antibody. Stroke 39:2845–2852

    Article  PubMed  CAS  Google Scholar 

  22. Stern MD, Lappe DL, Bowen PD, Chimosky JE, Holloway GA Jr, Keiser HR, Bowman RL (1977) Continuous measurement of tissue blood flow by laser-Doppler spectroscopy. Am J Physiol 232:H441–H448

    PubMed  CAS  Google Scholar 

  23. Dirnagl U, Kaplan B, Jacewicz M, Pulsinelli W (1989) Continuous measurement of cerebral cortical blood flow by laser-Doppler flowmetry in a rat stroke model. J Cereb Blood Flow Metab 9:589–596

    Article  PubMed  CAS  Google Scholar 

  24. Endres M, Biniszkiewicz D, Sobol RW, Harms C, Ahmadi M, Lipski A, Katchanov J, Mergenthaler P, Dirnagl U, Wilson SH, Meisel A, Jaenisch R (2004) Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase. J Clin Invest 113:1711–1721

    PubMed  CAS  Google Scholar 

  25. Kitagawa K, Matsumoto M, Yang G, Mabuchi T, Yagita Y, Hori M, Yanagihara T (1998) Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery. J Cereb Blood Flow Metab 18:570–579

    Article  PubMed  CAS  Google Scholar 

  26. Endres M, Laufs U, Huang Z, Nakamura T, Huang P, Moskowitz MA, Liao JK (1998) Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci USA 95:8880–8885

    Article  PubMed  CAS  Google Scholar 

  27. Hoehn M, Kruger K, Busch E, Franke C (1999) Validation of arterial spin tagging perfusion MR imaging: correlation with autoradiographic CBF data. In: ISMRM 7th annual meeting, Philadelphia, p 1843

    Google Scholar 

  28. Leithner C, Gertz K, Schrock H, Priller J, Prass K, Steinbrink J, Villringer A, Endres M, Lindauer U, Dirnagl U, Royl G (2008) A flow sensitive alternating inversion recovery (FAIR)-MRI protocol to measure hemispheric cerebral blood flow in a mouse stroke model. Exp Neurol 210:118–127

    Article  PubMed  CAS  Google Scholar 

  29. Gerriets T, Stolz E, Walberer M, Muller C, Kluge A, Bachmann A, Fisher M, Kaps M, Bachmann G (2004) Noninvasive quantification of brain edema and the space-occupying effect in rat stroke models using magnetic resonance imaging. Stroke 35:566–571

    Article  PubMed  CAS  Google Scholar 

  30. Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR (1990) A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 10:290–293

    Article  PubMed  CAS  Google Scholar 

  31. Elliott KA, Jasper H (1949) Measurement of experimentally induced brain swelling and shrinkage. Am J Physiol 157:122–129

    PubMed  CAS  Google Scholar 

  32. Meisel C, Prass K, Braun J, Victorov I, Wolf T, Megow D, Halle E, Volk HD, Dirnagl U, Meisel A (2004) Preventive antibacterial treatment improves the general medical and neurological outcome in a mouse model of stroke. Stroke 35:2–6

    Article  PubMed  CAS  Google Scholar 

  33. Liesz A, Suri-Payer E, Veltkamp C, Doerr H, Sommer C, Rivest S, Giese T, Veltkamp R (2009) Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat Med 15:192–199

    Article  PubMed  CAS  Google Scholar 

  34. Harms H, Prass K, Meisel C, Klehmet J, Rogge W, Drenckhahn C, Gohler J, Bereswill S, Gobel U, Wernecke KD, Wolf T, Arnold G, Halle E, Volk HD, Dirnagl U, Meisel A (2008) Preventive antibacterial therapy in acute ischemic stroke: a randomized controlled trial. PLoS One 3:e2158

    Article  PubMed  Google Scholar 

  35. Klehmet J, Harms H, Richter M, Prass K, Volk HD, Dirnagl U, Meisel A, Meisel C (2009) Stroke-induced immunodepression and post-stroke infections: lessons from the preventive antibacterial therapy in stroke trial. Neuroscience 158:1184–1193

    Article  PubMed  CAS  Google Scholar 

  36. Prass K, Meisel C, Hoflich C, Braun J, Halle E, Wolf T, Ruscher K, Victorov IV, Priller J, Dirnagl U, Volk HD, Meisel A (2003) Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1-like immunostimulation. J Exp Med 198:725–736

    Article  PubMed  CAS  Google Scholar 

  37. Meisel C, Schwab JM, Prass K, Meisel A, Dirnagl U (2005) Central nervous system injury-induced immune deficiency syndrome. Nat Rev Neurosci 6:775–786

    Article  PubMed  CAS  Google Scholar 

  38. McColl BW, Allan SM, Rothwell NJ (2009) Systemic infection, inflammation and acute ischemic stroke. Neuroscience 158:1049–1061

    Article  PubMed  CAS  Google Scholar 

  39. Ozdemir YG, Bolay H, Erdem E, Dalkara T (1999) Occlusion of the MCA by an intraluminal filament may cause disturbances in the hippocampal blood flow due to anomalies of circle of Willis and filament thickness. Brain Res 822:260–264

    Article  PubMed  CAS  Google Scholar 

  40. Furuya K, Kawahara N, Kawai K, Toyoda T, Maeda K, Kirino T (2004) Proximal occlusion of the middle cerebral artery in C57Black6 mice: relationship of patency of the posterior communicating artery, infarct evolution, and animal survival. J Neurosurg 100:97–105

    Article  PubMed  Google Scholar 

  41. Buchan A, Pulsinelli WA (1990) Hypothermia but not the N-methyl-D-aspartate antagonist, MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia. J Neurosci 10:311–316

    PubMed  CAS  Google Scholar 

  42. Busto R, Dietrich WD, Globus MY, Valdes I, Scheinberg P, Ginsberg MD (1987) Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury. J Cereb Blood Flow Metab 7:729–738

    Article  PubMed  CAS  Google Scholar 

  43. Miyazawa T, Hossmann KA (1992) Methodological requirements for accurate measurements of brain and body temperature during global forebrain ischemia of rat. J Cereb Blood Flow Metab 12:817–822

    Article  PubMed  CAS  Google Scholar 

  44. Jonsson AC, Lindgren I, Norrving B, Lindgren A (2008) Weight loss after stroke: a population-based study from the Lund Stroke Register. Stroke 39:918–923

    Article  PubMed  Google Scholar 

  45. Roe SY, Rothwell NJ (1997) Whole body metabolic responses to brain trauma in the rat. J Neurotrauma 14:399–408

    Article  PubMed  CAS  Google Scholar 

  46. Crossley NA, Sena E, Goehler J, Horn J, van der Worp B, Bath PM, Macleod M, Dirnagl U (2008) Empirical evidence of bias in the design of experimental stroke studies: a metaepidemiologic approach. Stroke 39:929–934

    Article  PubMed  Google Scholar 

  47. Macleod MR, Fisher M, O’Collins V, Sena ES, Dirnagl U, Bath PM, Buchan A, van der Worp HB, Traystman RJ, Minematsu K, Donnan GA, Howells DW (2009) Reprint: good laboratory practice: preventing introduction of bias at the bench. J Cereb Blood Flow Metab 29:221–223

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Humana Press

About this protocol

Cite this protocol

Dirnagl, U. (2010). Complexities, Confounders, and Challenges in Experimental Stroke Research: A Checklist for Researchers and Reviewers. In: Dirnagl, U. (eds) Rodent Models of Stroke. Neuromethods, vol 47. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-750-1_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-750-1_19

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-749-5

  • Online ISBN: 978-1-60761-750-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics