Figure 1.
Global and 3D imaging of intact mouse CNS.
(A). Spinal cords from C57BL/6 mice were stained with ASMA (red) and were further imaged by OPT. OPT scanned spinal cords were cryosectioned and imaged under a fluorescence microscope with 4× zoom (white frames). Selected regions were also imaged under a confocal microscope with 40× zoom (inlets). Nucleated cells were stained with 4,6-diamidino-2-phenylindole (DAPI). Spinal cord (B) and optic nerve (C) from B10RII mice were stained with MBP (red), and imaged by OPT. Cryosectioning was performed after OPT imaging and images were captured using a fluorescence microscope at 4× zoom. Spinal cords (D) and optic nerves (E) from C57BL/6 mice with a clinical score of 3.0 were stained with CD3 specific antibodies. OPT scanned tissues were cryosectioned and subsequently imaged under a fluorescence microscope at 4× zoom (white frames) or selected regions displaying evidence of CD3+ T cell infiltration were imaged using a confocal microscope at 63× zoom (inlets). CD3+ T cell staining is shown in red and the anatomy reconstructed from the CNS outline on the basis of the autofluorescence signal is shown in green. Scale bar: (A) Whole organ: 2 mm, 4× zoom images: 100 µm; 40× zoom image: 50 µm; (B, C) 4× zoom image: 100 µm; (D) whole organ: 2 mm, 4× zoom image: 100 µm, 40× zoom images: 50 µm, 63× zoom image: 50 µm; (E) whole organ: 2 mm, 4× zoom image: 100 µm, 63× zoom image: 50 µm.
Figure 2.
Spatial assessment of neuroinflammation in B6 EAE.
C57BL/6 mice were immunized with MOG35-55/complete Freunds adjuvants (CFA)/pertussis toxin (PTX) and were observed for EAE. CNS tissue was obtained at different clinical scores and was stained for CD3+ T cells and imaged using OPT. The iso-surface rendered OPT images of spinal cord and optic nerve sections at different clinical scores during the progression of EAE were obtained. Infiltrating CD3+ T cells (red) due to the signal from the CD3 specific antibody. The reconstruction of the CNS outline is based on the autofluorescence signal (green), where the upper part of spinal cord is the cervical and thoracic region while the bottom parts are the lumbar and sacral regions. Images were generated for score 1 (n = 2), score 2 (n = 3), score 3 (n = 2). Scale bar = 2 mm.
Figure 3.
Spatial assessment of neuroinflammation in B10.RIII EAE and DA Rat EAE model.
(A) B10.RIII mice were immunized with MBP89-101/CFA/PTX and were EAE was allowed to progress. At different clinical scores CNS tissue was obtained and was stained for CD3+ T cells and imaged using OPT. The iso-surface rendered OPT images of spinal cord and optic nerve sections at different clinical scores during the progression of EAE were obtained. Infiltrating CD3+ T cells (red) due to the signal from the CD3 specific antibody. The reconstruction of the CNS outline is based on the autofluorescence signal (green), where the upper parts of spinal cord are the cervical and thoracic region while the bottom parts are the lumbar and sacral region. Images were generated for score 1 (n = 4), score 2 (n = 5), score 3 (n = 3) score 4 (n = 4). (B) Intact spinal cord and optic nerves were removed from Dark Agouti rats with a clinical score of 4, and then stained with anti-CD3 antibody. Global imaging was performed using OPT. Iso-surface rendered OPT images of representative spinal cords and optic nerves were obtained. Infiltrating CD3+ T cell lymphocytes staining (red) are based on the signal from the CD3 specific antibody. The reconstruction of the CNS outline is based on the autofluorescence signal (green). Scale bar = 2 mm.
Figure 4.
Progression of inflammation and demyelination during clinical course of EAE in B10.RIII EAE model.
OPT scanned samples were subjected to cryosectioning and subsequently sections were stained with anti-MBP (green) and anti-F480 (cyan). CD3 staining visualizing T cells came from the pre-stained OPT samples. Spinal cord sections obtained from mice with different clinical scores were stained and imaged to record inflammation and demyelination during the progression of EAE. No inflammation was observed in the control spinal cord section (A). Increase in loss of myelin, F480+ cells infiltration and CD3 + T cell infiltration was observed with increase in clinical score severity as shown for score 2 (B) score 3 (C) and score 4 (D). Images were captured at 20× magnification. Sections roughly come from the indicated areas of the intact spinal cord. Data was generated mice/group (n = 2). Scale bar = 500 µm.
Figure 5.
Global quantification of the CD3+T cell inflammatory foci in EAE.
B6/MOG (A) and B10RIII/MBP (B) models were analyzed. The total volume estimates of CD3+ T cell infiltration was measured by OPT and calculated using Volocity software. Each bar indicates the results from an individual spinal cord. The Y-axis indicates the infiltrated CD3+ T cell volumes/spinal cord, and the X-axis indicates the clinical score. Correlation analysis between the volumes of CD3+ T cell foci and corresponding clinical scores in B6 mice r = 0.90513) (Fig. 5A) and B10.RIII r = 0.898869 (Fig. 5B). Each dot in the correlation plot represents one mouse. For B6 EAE quantification mice numbers were: score 1 (n = 2), score 2 (n = 3), score 3 (n = 2) while for B10.RIII quantification mice numbers were: score 1 (n = 4), score 2 (n = 5), score 3 (n = 3) score 4 (n = 4).