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Contrast-enhanced MR microangiography of cortical vascular remodeling after unilateral internal carotid artery occlusion in the mouse
Philipp Boehm-Sturm1,2, Till de Bortoli3,4, Stefan Paul Koch1,2, Melina Nieminen3, Susanne Mueller1,2, Giovanna Diletta Ielacqua5, Jan Klohs5, Ulrich Dirnagl1, Peter Vajkoczy3, and Nils Hecht3

1Department of Experimental Neurology and Center for Stroke Research Berlin, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany, 2Cluster of Excellence NeuroCure and Charité Core Facility 7T Experimental MRIs, Charité - Universitätsmedizin Berlin, Berlin, Germany, 3Department of Neurosurgery and Center for Stroke Research Berlin, Charité - Universitätsmedizin Berlin, Berlin, Germany, 4Charité Comprehensive Cancer Center, Charité – Universitätsmedizin Berlin, Berlin, Germany, 5Institute for Biomedical Engineering, University of Zurich & ETH Zurich, Zurich, Switzerland

Synopsis

Collateral flow is an important, yet poorly understood compensatory mechanism in response to brain hypoperfusion. Unilateral internal carotid artery occlusion in the mouse is a model to study collateral growth of penetrating arterioles. We hypothesized that this process could be assessed in vivo by high resolution MR microangiography with iron oxide nanoparticles. Since our MR vessel density measurements contradicted previous histological findings we established atlas tools to validate angiograms with microscopy on vessel-stained tissue slices or with whole brain serial two photon microscopy.

Introduction

Collateral outgrowth with flow augmentation remains the most important mechanism for prevention of hemodynamic ischemic stroke. In these cases, hemodynamic rescue is mainly provided by collateral arterioles in the range of ~20-50 µm diameter1. However, the mechanism of cerebral collateralization (arteriogenesis) remains poorly understood and reliable tools for in vivo assessment of its degree, morphology and dynamics are lacking2,3. Due to the blooming effect of ultra-small superparamagnetic iron oxide nanoparticles (USPIO) we hypothesized that these vessels could directly be visualized and quantified using contrast-enhanced MR microangiography with a cryoprobe4. The goal of this study was to develop noninvasive and quantitative MR readouts in order to characterize the collateral vasculature during cerebral arteriogenesis.

Methods

For simulation of chronic cerebral hypoperfusion, male C57/Bl6 mice were randomized to undergo right side unilateral internal carotid artery occlusion (ICAO, n=8) or a sham procedure (n=8) as described previously2. 21 d after surgery, animals underwent MRI at 7T using a mouse head cryoprobe (Bruker, Ettlingen, Germany). The protocol consisted of T2w MRI for atlas registration (2D RARE, 39 contiguous 0.4 mm thick slices, (80 µm)2 in plane resolution, TR/TEeff=4.2 s/33 ms, RARE factor 8, 2 averages, TA=4:03 min), contrast-free angiography (3D TOF, (80 µm)3 resolution, TR/TE=14 ms/2.1 ms, TA=6:02 min), contrast-enhanced MR microangiography of the cortex (5.2 mm slab) by acquiring images (3D FLASH, (65 µm)3 resolution, TR/TE=54 ms/3.9 ms, TA=29:57 min per image) pre and post injection of the USPIO Ferumoxytol (300 µmol Fe/kg, AMAG Pharmaceuticals, Waltham, MA/USA) through the femoral vein, and DSC MRI of cerebral perfusion during injection (GE-EPI, 15 0.4 mm slices, (293 µm)2 resolution, TR/TE=400 ms/7.2 ms, 200 repetitions. TA=1:20 min). Finally, animals received 100 µL FITC-lectin (Merck, Darmstadt, Germany) to stain cerebral vasculature and were transcardially perfused for confocal fluorescence microscopy (Leica TCS SP8, Leica Microsystems, Wetzlar/Germany) on tissue slices. A subset of animals (n=2 ICAO, n=1 sham) was processed for 3D serial two photon tomography (STPT, (1.2 µm)2 in plane, 25 µm sections, TissueVision, Somerville, MA/USA). MR microangiograms were generated by subtraction of image intensities Spre-Spost. From these, vessel densities in left/right/whole cortex were calculated by a vessel tracing algorithm by an independent lab as described previously4. The relative cerebral blood volume (rCBV) was mapped via change in transverse relaxation rate ΔR2*=1/TE*ln(Spre/Spost)5. MR and histology images were mapped in common spaces using custom MATLAB tools6. All data analyses were performed blind to the group assignment of animals.

Results

One animal (sham) was excluded due to unsuccessful i.v. injection of the contrast agent, one (ICAO) died during MRI leading to a final n=7 in each group. Qualitative inspection of MR microangiograms revealed that penetrating arterioles and venules could be visualized and that image intensity was apparently higher in the ICAO group (Fig. 1). However, quantitative analysis using the vessel tracing algorithm showed a puzzling decrease in vessel density independent of parameter settings whereas rCBV confirmed the increase in vasculature seen in previous studies (Fig. 2). Registration of MRI on FITC-lectin stained tissue sections showed that vessels as small as ~25 µm were detectable by MRI (Fig. 3). Whole brain 3D STPT and MR microangiograms were mapped into Allen brain atlas space for qualitative comparison (Fig. 4). However, a voxel-wise comparison has not yet been possible due to the resolution gap (3D histology <25 µm, MRI 65 µm plus blooming effect). A biophysical model to quantitatively compare the two modalities is still work in progress.

Discussion

Contrast-enhanced MR microangiography visualizes changes in the collateral vasculature. MR/histology image registration can help to assess the scales for which the technique is valid. rCBV seems to be a more robust marker of arteriogenesis but this remains to be confirmed by voxel-wise comparison to 3D histology. Since vessel dilation has been shown to be absent 21 d post ICAO, it is likely that the observed increase in rCBV paired with the apparent decrease in vascular density is an artefact of the vessel tracing algorithm, e.g. that two vessels that are too close to each other will be identified as a single object. Our atlas registration tools to gold standard histology will thus allow development of better vessel tracing algorithms.

Conclusion

In the future, the quantitative tools developed in this study will help to investigate mechanisms of cerebral arteriogenesis in experimental models exploring novel strategies for therapeutic stimulation of cerebral collateralization.

Acknowledgements

Work was supported by the Deutsche Forschungsgemeinschaft Cluster of Excellence NeuroCure (Exc 257) and the German Federal Ministry of Education and Research (BMBF; 01EO0801, Center for Stroke Research Berlin).

References

  1. Faber JE, Chilian WM, Deindl E, van Royen N, Simons M. A Brief Etymology of the Collateral Circulation. Arterioscler Thromb Vasc Biol 2014; 34: 1854–1859.
  2. Hecht N, He J, Kremenetskaia I, Nieminen M, Vajkoczy P, Woitzik J. Cerebral Hemodynamic Reserve and Vascular Remodeling in C57/BL6 Mice Are Influenced by Age. Stroke 2012; 43: 3052–3062.
  3. Hecht N, Marushima A, Nieminen M, Kremenetskaia I, von Degenfeld G, Woitzik J et al. Myoblast-Mediated Gene Therapy Improves Functional Collateralization in Chronic Cerebral Hypoperfusion. Stroke 2015; 46: 203–211.
  4. Klohs J, Baltes C, Princz-Kranz F, Ratering D, Nitsch RM, Knuesel I et al. Contrast-Enhanced Magnetic Resonance Microangiography Reveals Remodeling of the Cerebral Microvasculature in Transgenic ArcA Mice. J Neurosci 2012; 32: 1705–1713.
  5. Lemasson B, Bouchet A, Maisin C, Christen T, Le Duc G, Rémy C et al. Multiparametric MRI as an early biomarker of individual therapy effects during concomitant treatment of brain tumours. NMR Biomed 2015; 28: 1163–1173.
  6. Koch S, Mueller S, Foddis M, Bienert T, von Elverfeldt D, Knab F et al. Atlas registration for edema-corrected MRI lesion volume in mouse stroke models. J Cereb Blood Flow Metab 2017; : 0271678X17726635.

Figures

Fig. 1: MR microangiograms in ICAO and sham animals. Maximum intensity projection through 5 horizontal, cortical slices.

Fig. 2: Vessel density (left+right hemisphere) calculated from MR microangiograms for three different settings (connectivity threshold 3, 6, 9) of the previously published vessel tracing algorithm and rCBV in the same volume of interest in ICAO and sham animals. Comparsion using two sample t-test (*p<0.05).

Fig. 3: Example of cortical MR microangiogram (red, Frangi filtered) registered to FITC-lectin stained vasculature on a single coronal tissue slice (green). Registration artefacts and false negatives are still visible on high resolution microscopy images but circles denote areas with a qualitative match of histology and MRI. Zoomed overlay (left) shows MR detection of a single penetrating cortical vessel of 20-30 µm diameter.

Fig. 4: Example of Allen atlas registration of a cortical MR microangiogram and whole brain STPT (downsampled to 25 µm isotropic resolution) for 3D histological validation.

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)
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