- Open Access
Outcome of experimental stroke in C57Bl/6 and Sv/129 mice assessed by multimodal ultra-high field MRI
© Pham et al; licensee BioMed Central Ltd. 2010
- Received: 14 February 2010
- Accepted: 15 March 2010
- Published: 15 March 2010
Transgenic mice bred on C57Bl/6 or Sv/129 genetic background are frequently used in stroke research. It is well established that variations in cerebrovascular anatomy and hemodynamics can influence stroke outcome in different inbred mouse lines. We compared stroke development in C57Bl/6 and Sv/129 mice in the widely used model of transient middle cerebral artery occlusion (tMCAO) by multimodal ultra-high field magnetic resonance imaging (MRI).
C57Bl/6 and Sv/129 mice underwent 60 min of tMCAO and were analyzed by MRI 2 h and 24 h afterwards. Structural and functional images were registered to a standard anatomical template. Probability maps of infarction were rendered by automated segmentation from quantitative T2-relaxometric images. Whole-brain segmentation of infarction was accomplished manually on high-resolution T2-weighted (T2-w) RARE images. Cerebral perfusion (cerebral blood flow, CBF) was measured quantitatively by modified continuous arterial-spin-labeling (CASL) and apparent diffusion coefficients (ADC) by spin-echo diffusion-weighted imaging (DWI).
Probabilities of cortical (95.1% ± 3.1 vs. 92.1% ± 2.5; p > 0.05) and subcortical (100% vs. 100%; p > 0.05) infarctions at 24 h were similar in both groups as was the whole-brain volumetric extent of cerebral infarction. In addition, CBF and ADC values did not differ between C57Bl/6 and Sv/129 mice at any time point or region of interest.
The C57Bl/6 and Sv/129 genetic background is no major confounding factor of infarct size and cerebral perfusion in the tMCAO model.
- Apparent Diffusion Coefficient
- Cerebral Perfusion
- Middle Cerebral Artery Territory
- Transient Middle Cerebral Artery Occlusion
- Bilateral Common Carotid Artery Occlusion
The implementation of transgenic mice has also revolutionized the field of experimental stroke research in that the effects of distinct genes on stroke outcome can be easily assessed. Most of transgenic mice originate from C57Bl/6 or Sv/129 inbred strains. Moreover, these strains are also commonly used as "wild-type controls". However, there are considerable strain-related differences in the susceptibility to cerebral ischemia. Variations in cerebrovascular anatomy and hemodynamics as well as sensitivity to excitotoxicity have been identified as underlying reasons [1–7]. This may become of particular relevance when transgenic mice with mixed genetic background are compared to purebred controls. C57Bl/6 mice have been shown to be more susceptible to ischemic injury compared to Sv/129 mice in models of global (forebrain) ischemia  and to develop larger brain infarctions in permanent middle cerebral artery occlusion (pMCAO) [2, 9]. Whether these findings also extend to the most frequently used murine stroke model, i.e. transient middle cerebral artery occlusion (tMCAO) with an intraluminal thread, is not known. Furthermore, important functional parameters of acute ischemic brain damage such as cerebral perfusion or cytotoxic edema formation have not been systematically compared between these two popular strains until now.
We recently introduced multimodal magnetic resonance imaging (MRI) at high-field strength of 17.6 Tesla as a powerful tool to non-invasively analyze the early phase of cerebral ischemia and the evolution of infarctions in individual mice over time . In the present study, C57Bl/6 and Sv/129 mice were subjected to 60 min tMCAO. MRI outcome measures were chosen to characterize cerebral perfusion by continuous arterial spin labeling (CASL), hypoxic diffusion restriction by diffusion-weighted imaging (DWI), and irreversible infarction by lesion extent on T2-w and quantitative T2 relaxometric images.
Experimental design and animal stroke model
All procedures and animal studies were approved by the appropriate local authorities (Regierung von Unterfranken, Wuerzburg, Germany) and conducted in accordance with recommendations for the performance of basic experimental stroke studies . The experimental groups consisted of 8 6-8-weeks-old male C57Bl/6 mice (Charles River, Sulzfeld, Germany) and 8 6-8-weeks-old male Sv/129 mice (Harlan-Winkelmann, Borchen, Germany) weighing 20-25 g. Animals of each group underwent tMCAO with 60 min occlusion time.
tMCAO was performed as described in detail previously [12–14]. Briefly, a standardized suture coated with silicon rubber (6021PK10; Doccol Corporation, Redlands, CA, USA) was introduced into the right common carotid artery and advanced via the internal carotid artery to the origin of the middle cerebral artery (MCA). The suture was fixed and left in situ and animals were allowed to recover. Operation time per animal did not exceed 15 min. After 60 min, animals were re-anesthetized and the suture was withdrawn to allow tissue reperfusion (tMCAO). The operations were performed under inhalation anesthesia (2.0% isoflurane in a 70%/30% N2O/O2 mixture) and the body temperature was maintained at 37°C using a servo-controlled heating pad. All animals were subsequently followed in-vivo by serial multimodal ultra-high field MRI at 2 h and 24 h.
Multimodal ultra-high field MRI of experimental cerebral ischemia in-vivo
The detailed description of the imaging protocol can be found elsewhere . A short summary of relevant parameters of the employed pulse sequences is given here. Cerebral perfusion was measured using a modified arterial spin labeling (CASL) method [15–17]. To benefit especially from increased longitudinal magnetization and the elevation of the T1 relaxation time for detailed anatomical mapping of CBF and group analysis, all measurements were performed at ultra-high field strength (17.6 T, 750 Hz, Biospin, Bruker BioSpin GmbH, Ettlingen, Germany). Image maps of cerebral perfusion were calculated on a pixel-by-pixel basis according to Detre et al. . The degree of the inversion efficiency was assumed to be alpha = 0.7 [18, 19], and the brain-blood partition coefficient value for water lambda = 0.95 mL/g . Parameters for the fast spin-echo imaging sequence (RARE) were: echo train length (ETL) = 8, effective echo time TEeff = 17.2 ms, repetition time TR = 1 s, slice thickness 1.5 cm, FOV 2.5 × 2.5 cm, matrix of 64 × 64 voxels. The signal was averaged over 12 repetitions resulting in a total acquisition time of 9.5 min.
DWI was performed with a pulsed-field gradient Setjskal-Tanner-like multislice spin echo sequence with diffusion sensitization along the slice direction . Images with different b-values, 0 and 800 s/mm2, were acquired to allow for the calculation of apparent diffusion coefficient (ADC) maps of brain water. Whole brain coverage was achieved by thirteen coronal slices acquired with a matrix size of 64 × 64, FOV 2.5 × 2.5 cm, in plane resolution 282 × 282 μm, slice thickness = 0.5 mm, interslice distance = 1 mm, TE/TR = 22.3/2000 ms. Repeated measurement of the b = 800 s/mm2 DWI experiments (number of repetition NR = 3) resulted at an overall acquisition time for diffusion weighted experiments of 8 min. ADC maps were calculated by applying the common equation ADC = -0.00125 × ln (SIB800/SIB0).
T2 relaxometric mapping was performed for the in-vivo delineation of infarcted brain tissue at 24 h. Single slice T2-w imaging was performed using a Carr-Purcell-Meiboom-Gill (CPMG) multi-spin echo sequence collecting 32 echoes at TR/TE = 4.2/2000 ms. T2 relaxation times constants were calculated voxel-wise by fitting the intensities of the 20 first echoes to a monoexponential model. In addition, a strongly T2 weighted 2D turbo spin-echo sequence was acquired for high resolution anatomical imaging (RARE factor 16, TR = 8 s, effective TE = 56.44 ms, 2 averages, 13 coronal slices with an image matrix of 128 × 128 were acquired, FOV = 2.5 cm × 2.5 cm, slice thickness = 0.5 mm, interslice distance = 1 mm, overall acquisition time of 1 min). The center position of the 1.5 mm slab for measurement of CBF, T1 and T2 relaxometric maps each with the exact same geometry was centered at the bregma as the operational definition of the central MCA territory.
At the host console measurements and data processing were performed with the ParaVision software (version 3.02, Bruker BioSpin GmbH, Ettlingen, Germany). Further image calculation and fitting procedures were done using MATLAB® (The Mathworks Inc., Natick, MA, USA).
During MRI measurements, mice were anesthesized by 2% isoflurane in medical air (21%). The respiratory rate was monitored using an air-balloon positioned ventrally underneath the mouse body. The body temperature was constantly measured on the body surface and actively maintained at 37°C.
Statistical and image analysis
The extraction of brain tissue from the scalp and skull was done by manual segmentation for each subject and time point. Packages from the FMRIB Software Library FSL (version 4.1)  were used for motion correction, registration (FLIRT) , and statistical image analysis. Intra-subject linear alignment and registration to a common standard template  was achieved by a step-wise affine procedure with six degrees of freedom.
Manual whole-brain segmentation of infarction
Probability maps of infarction by quantitative T2 thresholds
Group means of infarct probabilities (%).
53.3 ± 10.1
92.1 ± 2.5
60.7 ± 10.4
95.1 ± 3.1
67.8 ± 12.8
79.1 ± 11.3
Analysis of functional ischemic outcome parameters
Pairwise comparisons between group means of quantitative CBF values were not significant for any time point or region of interest. Two-sided p-values for matched pairwise comparisons between C57BL/6 and Sv/129 mice were: subcortical ROI at 2 h p = 0.7 and p = 0.53 at 24 h; cortical ROI at 2 h p = 0.64 and p = 0.86 at 24 h.
Group means of functional ischemic outcome measures: CBF and ADC.
2 h vs. 24 h
34.1 ± 4.7 vs. 22.3 ± 2.9
38.7 ± 4.8 vs. 25.2 ± 3.7
30.4 ± 4.5 vs.20.8 ± 2.7
32.8 ± 4.6 vs. 23.4 ± 3.5
2 h vs. 24 h
7.8 ± 1.0 vs. 5.8 ± 0.5
7.8 ± 1.0 vs. 5.7 ± 0.6
7.3 ± 1.0 vs. 5.4 ± 0.5
7.2 ± 1.1 vs. 5.3 ± 0.4
As principle finding, we here show that C57Bl/6 and Sv/129 mice, the two most frequently utilized strains in experimental stroke research, behave similar in the tMCAO model regarding critical functional and structural parameters of infarct development. Volumetric extents and probability of cerebral infarctions did not significantly differ between the two groups as assessed by multimodal ultra-high field MRI as did cerebral perfusion and diffusion restriction of free water.
Strain-related differences between C57Bl/6 and Sv/129 mice have already been investigated in models of global and permanent focal cerebral ischemia. C57Bl/6 mice challenged by transient bilateral common carotid artery occlusion in the presence or absence of systemic hypotension developed more severe global forebrain ischemia than Sv/129 mice [7, 8]. Results after permanent MCAO have been inconclusive. While some reports described larger infarctions in C57Bl/6 mice in direct comparison to the Sv/129 strain [2, 9], others could not confirm this observation . We further extend these findings to the most frequently used model of focal cerebral ischemia, i.e. tMCAO with an intaluminal thread. Here, 60 min of focal ischemia had no differential effect on definite infarct volumes on day 1. Importantly, diffusion restriction of free water as an early marker of ischemic cell damage and cytoxic edema likewise occurred similar in both strains. This implies that the C57Bl/6 or Sv/129 genetic background is no major confounding factor of infarct evolution in the acute phase after tMCAO. Whether this also applies to later stages of infarct development or different MCA occlusion times, however, still needs to be assessed.
Besides infarct volume, regional cerebral blood flow (rCBF) is frequently chosen as readout parameter in experimental stroke studies. Both critically depend on the composition of the cerebral vasculature which is known to vary considerably between different mouse strains [1–3]. The posterior communicating artery is absent or hypoplastic in more than 90% of C57/Bl6 mice, but in less than 50% of Sv/129 mice, which is also supported by in-vivo MRI data [4, 25]. Moreover, the MCA territory seems to be larger in C57/Bl6 as compared to Sv/129 mice . In contrast, pial anastomoses between the anterior and middle cerebral artery territories are of similar number and diameter . PWI produced comparable results in C57/Bl6 and Sv/129 mice subjected to tMCAO indicating that the reported anatomical variations in the Circle of Willis do not translate into relevant functional differences in cerebral blood supply. Interestingly, infarction of subcortical areas (e.g. basal ganglia) was present as early as 2 h after tMCAO in both groups as assessed by various structural and functional MRI parameters. Early completed infarction in this subcortical territory of the MCA corresponds to the anatomically limited collateral blood supply of this region . In contrast, severe hypoperfusion was observed in the cortical grey matter of the MCA territory at the same time point (2 h). Irreversible infarction followed only at 24 h determining this region as tissue-at-risk with a prolonged capacity for survival because of a richer collateral network through pial anastomoses. These observations emphasize the need for fast therapeutic interventions in ischemic stroke.
In summary, the C57/Bl6 and Sv/129 genetic background is no major confounding factors of stroke size and cerebral perfusion in the tMCAO model. Multimodal ultra-high field MRI is a valuable tool to non-invasively assess important structural and functional parameters of brain infarction in mice and can help to increase the validity of experimental stroke studies.
This study was supported by the Deutsche Forschungsgemeinschaft, Bonn (Sonderforschungsbereich 688 B1, A13, and Z-02) and a postdoctoral-fellowship granted to MP by the Medical Faculty of the University of Heidelberg.
- Barone FC, Knudsen DJ, Nelson AH, Feuerstein GZ, Willette RN: Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J Cereb Blood Flow Metab 1993, 13: 683–692.PubMedView ArticleGoogle Scholar
- Connolly ES Jr, Winfree CJ, Springer TA, Naka Y, Liao H, Yan SD, Stern DM, Solomon RA, Gutierrez-Ramos JC, Pinsky DJ: Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. Role of neutrophil adhesion in the pathogenesis of stroke. J Clin Invest 1996, 97: 209–216. 10.1172/JCI118392PubMed CentralPubMedView ArticleGoogle Scholar
- Kitagawa K, Matsumoto M, Yang G, Mabuchi T, Yagita Y, Hori M, Yanagihara T: 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 1998, 18: 570–579. 10.1097/00004647-199805000-00012PubMedView ArticleGoogle Scholar
- Majid A, He YY, Gidday JM, Kaplan SS, Gonzales ER, Park TS, Fenstermacher JD, Wei L, Choi DW, Hsu CY: Differences in vulnerability to permanent focal cerebral ischemia among 3 common mouse strains. Stroke 2000, 31: 2707–2714.PubMedView ArticleGoogle Scholar
- Rivera J, Tessarollo L: Genetic background and the dilemma of translating mouse studies to humans. Immunity 2008, 28: 1–4. 10.1016/j.immuni.2007.12.008PubMedView ArticleGoogle Scholar
- Schauwecker PE, Steward O: Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches. Proc Natl Acad Sci USA 1997, 94: 4103–4108. 10.1073/pnas.94.8.4103PubMed CentralPubMedView ArticleGoogle Scholar
- Wellons JC III, Sheng H, Laskowitz DT, Burkhard Mackensen G, Pearlstein RD, Warner DS: A comparison of strain-related susceptibility in two murine recovery models of global cerebral ischemia. Brain Res 2000, 868: 14–21. 10.1016/S0006-8993(00)02216-2PubMedView ArticleGoogle Scholar
- Fujii M, Hara H, Meng W, Vonsattel JP, Huang Z, Moskowitz MA: Strain-related differences in susceptibility to transient forebrain ischemia in SV-129 and C57black/6 mice. Stroke 1997, 28: 1805–1810. discussion 1811PubMedView ArticleGoogle Scholar
- Maeda K, Hata R, Hossmann KA: Regional metabolic disturbances and cerebrovascular anatomy after permanent middle cerebral artery occlusion in C57black/6 and SV129 mice. Neurobiol Dis 1999, 6: 101–108. 10.1006/nbdi.1998.0235PubMedView ArticleGoogle Scholar
- Pham M, Kleinschnitz C, Helluy X, Bartsch AJ, Austinat M, Behr VC, Renne T, Nieswandt B, Stoll G, Bendszus M: Enhanced cortical reperfusion protects coagulation factor XII-deficient mice from ischemic stroke as revealed by high-field MRI. Neuroimage 2010, 49: 2907–2914. 10.1016/j.neuroimage.2009.11.061PubMedView ArticleGoogle Scholar
- Dirnagl U: Bench to bedside: the quest for quality in experimental stroke research. J Cereb Blood Flow Metab 2006, 26: 1465–1478. 10.1038/sj.jcbfm.9600298PubMedView ArticleGoogle Scholar
- Kleinschnitz C, Pozgajova M, Pham M, Bendszus M, Nieswandt B, Stoll G: Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarct size, functional outcome, and intracranial bleeding. Circulation 2007, 115: 2323–2330. 10.1161/CIRCULATIONAHA.107.691279PubMedView ArticleGoogle Scholar
- Kleinschnitz C, Stoll G, Bendszus M, Schuh K, Pauer HU, Burfeind P, Renne C, Gailani D, Nieswandt B, Renne T: Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis. J Exp Med 2006, 203: 513–518. 10.1084/jem.20052458PubMed CentralPubMedView ArticleGoogle Scholar
- Varga-Szabo D, Braun A, Kleinschnitz C, Bender M, Pleines I, Pham M, Renne T, Stoll G, Nieswandt B: The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction. J Exp Med 2008, 205: 1583–1591. 10.1084/jem.20080302PubMed CentralPubMedView ArticleGoogle Scholar
- Detre JA, Leigh JS, Williams DS, Koretsky AP: Perfusion imaging. Magn Reson Med 1992, 23: 37–45. 10.1002/mrm.1910230106PubMedView ArticleGoogle Scholar
- Williams DS, Detre JA, Leigh JS, Koretsky AP: Magnetic resonance imaging of perfusion using spin inversion of arterial water. Proc Natl Acad Sci USA 1992, 89: 212–216. 10.1073/pnas.89.1.212PubMed CentralPubMedView ArticleGoogle Scholar
- Wong EC, Buxton RB, Frank LR: A theoretical and experimental comparison of continuous and pulsed arterial spin labeling techniques for quantitative perfusion imaging. Magn Reson Med 1998, 40: 348–355. 10.1002/mrm.1910400303PubMedView ArticleGoogle Scholar
- Maccotta L, Detre JA, Alsop DC: The efficiency of adiabatic inversion for perfusion imaging by arterial spin labeling. NMR Biomed 1997, 10: 216–221. 10.1002/(SICI)1099-1492(199706/08)10:4/5<216::AID-NBM468>3.0.CO;2-UPubMedView ArticleGoogle Scholar
- Zhang W, Williams DS, Koretsky AP: Measurement of rat brain perfusion by NMR using spin labeling of arterial water: in vivo determination of the degree of spin labeling. Magn Reson Med 1993, 29: 416–421. 10.1002/mrm.1910290323PubMedView ArticleGoogle Scholar
- Herscovitch P, Raichle ME: What is the correct value for the brain--blood partition coefficient for water? J Cereb Blood Flow Metab 1985, 5: 65–69.PubMedView ArticleGoogle Scholar
- Setjskal EO, Tanner JE: Spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient. J Chem Phys 1965, 42: 288–292. 10.1063/1.1695690View ArticleGoogle Scholar
- Smith SM, Jenkinson M, Woolrich MW, Beckmann CF, Behrens TE, Johansen-Berg H, Bannister PR, De Luca M, Drobnjak I, Flitney DE, Niazy RK, Saunders J, Vickers J, Zhang Y, De Stefano N, Brady JM, Matthews PM: Advances in functional and structural MR image analysis and implementation as FSL. Neuroimage 2004,23(Suppl 1):S208–219. 10.1016/j.neuroimage.2004.07.051PubMedView ArticleGoogle Scholar
- Jenkinson M, Bannister P, Brady M, Smith S: Improved optimization for the robust and accurate linear registration and motion correction of brain images. Neuroimage 2002, 17: 825–841. 10.1016/S1053-8119(02)91132-8PubMedView ArticleGoogle Scholar
- MacKenzie-Graham A, Lee EF, Dinov ID, Bota M, Shattuck DW, Ruffins S, Yuan H, Konstantinidis F, Pitiot A, Ding Y, et al.: A multimodal, multidimensional atlas of the C57BL/6J mouse brain. J Anat 2004, 204: 93–102. 10.1111/j.1469-7580.2004.00264.xPubMed CentralPubMedView ArticleGoogle Scholar
- Beckmann N: High resolution magnetic resonance angiography non-invasively reveals mouse strain differences in the cerebrovascular anatomy in vivo. Magn Reson Med 2000, 44: 252–258. 10.1002/1522-2594(200008)44:2<252::AID-MRM12>3.0.CO;2-GPubMedView ArticleGoogle Scholar
- Maeda K, Hata R, Hossmann KA: Differences in the cerebrovascular anatomy of C57black/6 and SV129 mice. Neuroreport 1998, 9: 1317–1319. 10.1097/00001756-199805110-00012PubMedView ArticleGoogle Scholar
- del Zoppo GJ, Mabuchi T: Cerebral microvessel responses to focal ischemia. J Cereb Blood Flow Metab 2003, 23: 879–894. 10.1097/01.WCB.0000078322.96027.78PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.