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Research ArticleResearch Article: Confirmation, Novel Tools and Methods

Localizing Microemboli within the Rodent Brain through Block-Face Imaging and Atlas Registration

Matthew W. McDonald, Matthew S. Jeffers, Melissa Filadelfi, Andrea Vicencio, Gavin Heidenreich, Junzheng Wu and Gergely Silasi
eNeuro 16 July 2021, 8 (4) ENEURO.0216-21.2021; DOI: https://doi.org/10.1523/ENEURO.0216-21.2021
Matthew W. McDonald
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
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Matthew S. Jeffers
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
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Melissa Filadelfi
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
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Andrea Vicencio
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
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Gavin Heidenreich
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
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Junzheng Wu
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
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Gergely Silasi
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
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Figures

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  • Figure 1.
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    Figure 1.

    Block-face imaging setup. A, Brains were blocked coronally at the level of the colliculus. B, To ensure a black background during block-face imaging, brains were embedded in 2% agarose darkened with India Ink, using a 45-well plate as a mold. Once solidified, the block was extracted from the well plate and glued to the vibratome chuck for sectioning. C, Alternatively, the brain can be directly glued to the vibratome chuck and black ink can be applied directly to the brain surface. D, Visual representation of the block-face imaging setup. E, Representative block-face images of mouse brain with fluorescent microspheres (scale bar: 1 mm).

  • Figure 2.
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    Figure 2.

    Microsphere detection. A, Validation of image thresholding to produce binary segmentation masks of individual microspheres. Left, Block-face image with visible fluorescent microspheres (green). Fluorescent microspheres are visible ∼300–400 μm into the tissue block. Following image thresholding to distinguish microspheres, serial images are subtracted from each other to determine the microsphere’s true location. Middle left, Microspheres (red) detected with image threshold after subtraction of serial images. Middle right, Microspheres manually counted by an experimenter after subtraction of serial images (green). Right, Overlay of raw block-face image with microspheres manually selected and thresholded (yellow, agreement between manual and image threshold); scale bar: 1 mm. B, Correlation between microspheres identified by experimenter and microspheres that were detected from thresholded image. Each data point represents the number of microspheres identified manually or detected from thresholded image of each coronal image (N = 327; n = 64–67 per brain). C, Total number of microspheres manually identified by an experimenter or from thresholded image for each brain. D, The number of microspheres identified by an experimenter or thresholded image in each block-face image (N = 327). E, Number of microspheres manually counted from block-face images compared with the number of microspheres manually counted in histologic sections. F, Percent of microspheres lost in histologic sections when compared with the equivalent block-face image. Data are mean ± SD. Asterisk = significance based on paired t-test (p < 0.05).

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    Figure 3.

    Block-face imaging workflow. A, Block-face image with three surface microspheres identified. B, Microspheres detected from block-face image registered to AMB Nissl image using QuickNII. Microspheres detected with image threshold (green). C, Block-face image “anchored” to Nissl image of AMB atlas. D, AMB atlas segmentation map of block-face image with overlay of microspheres detected with image threshold (green). Each shade of gray is a unique brain region within the AMB (2017). E, Block-face image anchored to AMB atlas segmentation map. F, Slide-mounted brain section collected after acquiring block-face image. DAPI (blue) and microspheres (green). G, Visual representation of the steps of our block-face imaging workflow and atlas registration of microspheres. For example, using this workflow and Nutil, three surface microspheres were identified to be in the (1) hippocampal region, (2) medial hypothalamic zone, and (3) the lateral hypothalamic zone (scale bar: 1 mm).

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    Figure 4.

    A, Representative image of the widespread distribution of microspheres. Data represent the mouse with the mean number of microspheres (301). Each microsphere color represents target brain regions. B, Total number of microspheres in the brain. C, Microsphere density within the total brain volume sampled in each mouse. D, Heat map representing microsphere density (per mm3) in target brain regions across all mice (n = 29). The largest density of microspheres was found in the thalamus. The A-P coordinate relative to the anterior commissure is provided below each atlas image. Data are mean ± SD.

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    Figure 5.

    Microsphere location until level 7 AMB hierarchical tree across all animals (n = 29). Data are means of the number of microspheres in each anatomic tree location and the proportion relative to total microspheres (in parentheses). Target brain regions (i.e., terminal branches) are in gray.

Tables

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    Table 1

    Agreement between manual allocation of individual microspheres from block-face images and our block-face imaging workflow

    Brain region
    CategoryTotal countsOlfactory
    aeas
    IsocortexCorpus
    callosum
    Hippocampal
    region
    StriatumThalamus and
    midbrain
    Other
    Agreement995 (93.9%)68 (88.3%)383 (94.6%)33 (75%)129 (96.3%96 (92.3%)261 (97%)25 (92.6%)
    Disagreement65 (6.1%)9 (11.7%)22 (4.4%)11 (25%)5 (3.7%)8 (7.5%)8 (3%)2 (7.4%)
    Total106177 (7.3%)405 (38.2%)44 (4.2%)134 (12.6%)104 (9.8%)269 (25.4%)27 (2.5%)
    • Agreement represents the same allocation between our workflow and manual rating. Overall, there is 93.9% agreement between our workflow and a human rater (N = 1061). Microspheres were predominantly found in isocortex and thalamus/midbrain. Data are presented as number of microspheres found in a given region, with the percentage relative to the total counts column found in parenthesis.

    • View popup
    Table 2

    At each hierarchal level (Lvl) of the AMB, the agreement between a blinded rater allocating microspheres from histologic images and our block-face imaging workflow (upper) or compared with registering histologic sections with QuickNII (lower)

    CategoryLvl 10Lvl 9Lvl 8Lvl 7Lvl 6Lvl 5Lvl 3/4Lvl 1/2
    Manual histology compared
    with block-face imaging
    workflow
    Agreement18 (34.6%)29 (55.8%)39 (75%)45 (86.5%)50 (96.2%)50 (96.2%)50 (96.2%)51 (98.1%)
    Microsphere not
    at this level
    27 (51.9%)16 (30.8%)8 (15.4%)4 (7.7%)0 (0%)0 (0%)0 (0%)0 (0%)
    Sum of agreement45 (86.5%)45 (86.5%)47 (90.4%)49 (94.2%)50 (96.2%)50 (96.2%)50 (96.2%)51 (98.1%)
    Disagreement7 (13.5%)7 (13.5%)5 (9.6%)3 (5.8%)2 (3.8%)2 (3.8%)2 (3.8%)1 (1.9%)
    Total5252525252525252
    Manual histology compared
    with registering Histologic
    sections with QuickNII
    Agreement22 (42.3%)30 (57.7%)40 (76.9%)45 (86.5%)48 (92.3%)49 (94.2%)50 (96.2%)51 (98.1%)
    Microsphere not
    at this level
    20 (38.5%)14 (26.9%)4 (7.7%)1 (1.9%)0 (0%)0 (0%)0 (0%)0 (0%)
    Sum of agreement42 (80.7%)44 (84.6%)44 (84.6%)46 (88.5%)48 (92.3%)49 (94.2%)50 (96.2%)51 (98.1%)
    Disagreement10 (19.2%)8 (15.4%)8 (15.4%)6 (11.5%)4 (7.7%)3 (5.8%)2 (3.8%)1 (1.9%)
    Total5252525252525252
    • Agreement represents the number of microsphere given the same allocation between the rater and either workflow. Microsphere not at this level, represents the number of microspheres not located at a given level of the hierarchal tree. Some microspheres are not located at specific levels of the hierarchal tree, since every subregion of the AMB hierarchal tree does not end at the same level. Sum of agreement, the number of microspheres correctly allocated up to the specific hierarchal level. Disagreement, represents the number of microspheres incorrectly allocated at the given level. With 94.2% agreement, we are confident we can allocate microspheres from block-face imaging to level 7 of the hierarchal tree of the AMB atlas (e.g., somatomotor, somatosensory, visual, piriform areas, etc.). In contrast, registering histologic sections with QuickNII, rather than block-face images, results in 88.5% agreement at level 7.

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    Table 3

    Number, proportion, and density of microspheres located in targeted brain regions

    Microsphere
    number (#)
    Microsphere
    proportion (%)
    Microsphere
    density (#/mm3)
    Atlas region of interestNo perfusionPerfusionp valueNo perfusionPerfusionp valueNo perfusionPerfusionp value
    Ventricular systems4.5 (2.9)3.3 (1.7)0.1951.2 (0.9)1.5 (1.0)0.4870.75 (0.49)0.56 (0.29)0.195
    Lateral forebrain bundle system8.4 (5.8)8.8 (3.2)0.8042.7 (1.4)3.7 (1.6)0.0850.60 (0.42)0.63 (0.23)0.804
    Medial forebrain bundle system4.2 (3.1)4.5 (3.3)0.7891.2 (0.7)1.5 (0.9)0.2280.60 (0.44)0.64 (0.46)0.789
    Other fiber tracts3.5 (2.6)3.3 (2.2)0.8521.1 (1.0)1.4 (1.0)0.4390.24 (0.18)0.23 (0.15)0.852
    Midbrain, motor-related areas18.2 (11.7)13.7 (11.8)0.3154.8 (2.3)4.5 (2.7)0.7190.86 (0.55)0.64 (0.56)0.315
    Other midbrain areas5.3 (4.6)4.3 (4.4)0.5761.5 (1.1)1.4 (1.1)0.8200.61 (0.53)0.50 (0.51)0.576
    Hypothalamus12.9 (7.2)12.2 (7.7)0.7954.3 (1.9)4.3 (1.8)0.9910.85 (0.48)0.80 (0.51)0.795
    Pallidum6.1 (4.1)6.1 (4.1)0.9951.6 (1.0)2.1 (1.1)0.2470.66 (0.43)0.65 (0.44)0.995
    Cortical subplate11.1 (5.3)7.9 (6.1)0.1353.1 (1.4)2.6 (1.8)0.4131.25 (0.60)0.88 (0.68)0.135
    Thalamus, polymodal
    association cortex related
    21.3 (8.9)19.1 (12.7)0.6046.7 (2.5)6.9 (3.2)0.8371.68 (0.71)1.51 (1.00)0.604
    Thalamus, sensory-motor
    cortex related
    10.6 (5.2)10.7 (5.9)0.9913.7 (2.7)4.9 (3.9)0.3611.59 (0.77)1.6 (0.89)0.991
    Striatum, dorsal region30.2 (15)21.9 (11)0.0998.8 (2.0)7.9 (1.8)0.2131.16 (0.57)0.84 (0.42)0.099
    Striatum, ventral region8.3 (4.9)5.6 (3.4)0.0942.5 (0.9)2.3 (1.2)0.6230.96 (0.57)0.65 (0.39)0.094
    Other striatum regions4.1 (2.9)3.7 (2.6)0.6421.3 (1.0)1.4 (0.7)0.8490.54 (0.37)0.48 (0.34)0.642
    Olfactory areas25.9 (16.9)18.1 (10.4)0.1467.4 (2.9)7.1 (2.9)0.7490.55 (0.36)0.39 (0.22)0.146
    Agranular insular area8.4 (3.9)7.2 (4.3)0.4312.8 (1.0)2.6 (1.3)0.6521.07 (0.49)0.91 (0.55)0.431
    Auditory areas8.4 (6.6)4.9 (3.2)0.0892.2 (1.6)1.8 (1.0)0.4031.45 (1.13)0.85 (0.56)0.089
    Retrosplenial area8.0 (6.0)5.4 (3.6)0.1642.0 (1.3)2.1 (1.5)0.8150.76 (0.57)0.51 (0.34)0.164
    Somatosensory areas33.5 (19.2)28.7 (14.8)0.4539.3 (3.7)10.6 (3.6)0.3201.01 (0.58)0.86 (0.44)0.453
    Somatomotor areas22.9 (14.4)20.8 (11.7)0.6656.5 (3.3)7.8 (2.7)0.2630.94 (0.59)0.85 (0.48)0.665
    Visual areas12.5 (7.8)7.4 (6.2)0.0604.0 (3.0)2.4 (1.8)0.0980.93 (0.58)0.55 (0.46)0.060
    Orbital area8.1 (6.0)4.8 (4.3)0.1002.5 (2.3)1.7 (1.5)0.2771.37 (1.01)0.81 (0.73)0.100
    Other isocortex areas20.9 (12.3)15.7 (10.1)0.2245.7 (2.4)5.9 (2.5)0.8700.95 (0.56)0.72 (0.46)0.224
    Retrohippocampal area8.8 (6.4)6.8 (7.4)0.4502.4 (1.4)2.2 (2.2)0.7770.48 (0.35)0.37 (0.40)0.450
    Hippocampal region28.3 (13.2)23.7 (11.7)0.3329.7 (5.5)8.6 (2.8)0.5071.19 (0.55)1.00 (0.49)0.332
    Unaccounted microspheres3.0 (2.6)1.9 (1.6)0.1930.8 (0.6)0.7 (0.5)0.517
    Total, all regions combined337.6 (153.6)270.8 (117.3)0.1981001000.922 (0.408)0.738 (0.319)0.186
    • The total row for microsphere number and microsphere proportion are the sum of all regions within their respective columns. The total row for microsphere density uses the mean of all regions within its column. Data within a given column are means with SDs in parentheses.

Extended Data

  • Figures
  • Tables
  • Extended Data 1

    Detailed description of the block-face imaging workflow and Python script for image acquisition. Download Extended Data 1, ZIP file.

  • Extended Data 2

    ImageJ script for image processing. Download Extended Data 2, ZIP file.

  • Extended Data 3

    Python script to facilitate compiling all microsphere location data. Download Extended Data 3, ZIP file.

  • Extended Data 4

    Detailed statistics comparing the distribution of microspheres across identified brain regions. Download Extended Data 4, ZIP file.

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Localizing Microemboli within the Rodent Brain through Block-Face Imaging and Atlas Registration
Matthew W. McDonald, Matthew S. Jeffers, Melissa Filadelfi, Andrea Vicencio, Gavin Heidenreich, Junzheng Wu, Gergely Silasi
eNeuro 16 July 2021, 8 (4) ENEURO.0216-21.2021; DOI: 10.1523/ENEURO.0216-21.2021

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Localizing Microemboli within the Rodent Brain through Block-Face Imaging and Atlas Registration
Matthew W. McDonald, Matthew S. Jeffers, Melissa Filadelfi, Andrea Vicencio, Gavin Heidenreich, Junzheng Wu, Gergely Silasi
eNeuro 16 July 2021, 8 (4) ENEURO.0216-21.2021; DOI: 10.1523/ENEURO.0216-21.2021
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Keywords

  • block-face
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