Figure 1.
Mouse spinal cord window chamber (SCWC) device and surgical implantation procedures.
(A) The SCWC device design and dimensions are shown. An 8 mm diameter glass coverslip was inserted into the SCWC device and held in place by a metal ring clamp once the device is surgically implanted into the mouse (shown in panel “F” and “G”). Four radial extension arms have been built into the device in order to immobilize the animal during imaging sessions. (B-E) Photographs showing step-by-step surgical procedures for implanting the SCWC in the mouse exposing the spinal cord at the L2–L3 vertebrae. (E) Artificial dura was placed on the dorsal surface of exposed spinal cord below the coverslip to prevent scar tissue formation. Two separate devices were manufactured from either durable (F) polycarbonate or (G) light-weight surgical steel. Polycarbonate was used to allow photoacoustic imaging in vivo. (H) X-ray images were taken following SCWC implantation to confirm the device had been placed over L2–L3 and demonstrate that the spinal cord and vertebrae remain structurally sound after implantation of the device. Scale bars = 1 cm.
Figure 2.
Visual and histological confirmation that SCWC implantation does not damage the spinal cord structure or cause significant inflammation or infection.
(A) White light images following SCWC implantation at 0, 1, 3 and 7 days showed no signs of local infection, excessive bleeding around the installation site, or device rejection. SCWC remained optically clear for 29 days, permitting long-term high-resolution imaging of cord and vascular structures. Yellow arrows indicate the location of the spinal cord. (B–E) Histological analysis and quantification of spinal cord tissue cross-sections cut directly below the caudal edge of the implanted SCWC. (B) H&E staining confirmed tissue morphology was intact after WC implantation. (C) Representative Iba-1 immunohistochemistry images from spinal cords 24, 48 and 72 hours following SCWC implantation. Sham and spinal cord injury (SCI) (Iba-1 positive control) animals are also shown for comparison. SCI positive control animals showed a significant increase in Iba-1 expression, * p<0.001. No notable changes in Iba-1 expression in ex vivo spinal cord were observed between the SCWC implanted groups). (D) Western blot for Iba-1 prior to SCWC implant (sham), and at 24, 48, and 72 hours post-implantation in athymic nude mice. (E) Bar graph representing the fluorescent intensity quantification of Iba-1, and no significant increase in Iba-1 was observed; n = 3 per group (p-values >0.212 for comparisons between all groups). (F) Bar graph showing quantification of Western blot data. Increases in Iba-1 protein were observed in animals receiving SCWC implantation, although these increases were insignificant (p = 0.15); n = 3 per group. (G) Western blot for Iba-1 prior to SCWC implant (sham) (n = 3), and at 24 hours (n = 4), 3 days (n = 3), 10 days (n = 3) and 28 days (n = 3) post-implantation in C57BL6 mice. (H) Bar graph showing quantification of Western blot Iba-1 data (C57BL6 mice). No significant increases in Iba-1 protein were observed (p = 0.405). It is anticipated that the slight increases in Iba-1 in the athymic nude mice may be due to the laminectomy and surgical procedures performed. β-Actin was used as a protein loading control. Scale bars = 400 µm. Data was transformed (square-root transformation) and analyzed using a one-way ANOVA; Tukey post-hoc analysis. SCWC = spinal cord window chamber.
Figure 3.
SCWC model permits X-ray microirradiation of the spinal cord in situ.
(A) Anaesthetized mice were placed directly under the micro-irradiation collimator of the small animal irradiator for delivery of X-rays to the spinal cord through the coverglass of the window chamber. (B, C) In situ fluoroscopic imaging was used for image-guided delivery of the X-ray beam (centered on the crosshairs) to the spinal cord. (D) Custom fit radiochromic film was used to confirm the location of the irradiation beam which had a 3 mm diameter (as seen by the dark blue circle). Scale bar = 1 cm. SCWC = spinal cord window chamber.
Figure 4.
Longitudinal optical imaging of radiation response of the normal spinal cord and its vasculature through the SCWC.
White light, wide-field fluorescence, and svOCT images were taken at (A) 1 day before, (B) 1 hour, (C) 24 hours, and (D) 48 hours following a single 30 Gy radiation dose to the cord. White light images revealed significant radiation-induced hematoma in the spinal cord two days after irradiation (arrows). FITC-dextran was injected intravenously prior to acquiring the fluorescence images at each time point. Fiducial markers consisting of India ink (black dots shown by arrows) on the white light images were used to as spatial landmarks to allow identification and long-term imaging of vascular structures. Compared with vascular function, corresponding en-face projected svOCT images revealed that the posterior spinal cord vein and other vasculature did not suffer significant short-term radiation-induced structural damage. Scale bar = 1 mm. SCWC = spinal cord window chamber. svOCT = speckle variance optical coherence tomography.
Figure 5.
SCWC permits structural, functional and oxygenation imaging of the intact spinal cord vasculature in situ.
(A) Power Doppler ultrasound (color) overlaid on a B-mode structural ultrasound (gray-scale) image obtained through the polycarbonate SCWC along a longitudinal section of the normal spinal cord in vivo (device is shown in Figure 1F). The power Doppler depicts vascular architecture in several vessels of the spinal cord. The color bar represents the signal intensity. (B) Corresponding multispectral photoacoustic imaging of the same cross section of normal spinal cord permitted in situ measurement of hemoglobin oxygen saturation in the anterior spinal artery and posterior spinal vein. It demonstrated that the cord is well oxygenated. The color bar represents the relative hemoglobin oxygen saturation level. (C) Cross-sectional Doppler OCT image demonstrated significant blood flow in the posterior spinal vein. The color bar represents the phase-shift of the backscattered light in radians which is proportional to the velocity of the red blood cells in the axial direction. (D) Corresponding structural OCT image of the spinal cord permitted visualization of key spinal cord features, including the glass coverslip (1), anterior spinal vein (2), white matter (3), and grey matter (4) of the intact cord. Scale Bars = 500 µm (A–D). SCWC = spinal cord window chamber. OCT = optical coherence tomography.
Figure 6.
Hemoglobin oxygen saturation (sO2) measurement made using in vivo photoacoustic imaging.
Baseline vascular sO2 was measured in situ for 1 minute while the animal breathed 100% oxygen mixed with 2% isoflurane. The animal was shifted to breathing 7% oxygen mixed with 2% isoflurane for an additional 1 minute. The oxygen concentration was then returned to 100%.
Figure 7.
Intravital multispectral fluorescence microscopic imaging of medulloblastoma tumor metastasis to the spinal cord.
(A) In vivo bioluminescence images of mice 7 days following intracranial tumor implantation of human WW426 medulloblastoma tumor cells, demonstrating local tumor growth. (B) SCWC was implanted 27 days after tumor implantation, when metastatic GFP+ tumor cells to the spinal cord could be seen using both BLI and intravital two-photon images (color bar indicates bioluminescence signal intensity; BLI units are photons/s/cm2/Sr). The head of the mouse was covered in “B” to reduce the bioluminescence signal from the brain in order to detect lower bioluminescence from the tumor micrometastases. (C) Wide-field fluorescence imaging and (D) confocal fluorescence microscopy of the SCWC-bearing mouse 28 days after initial tumor implantation (1 day post-SCWC installation). The outline of the spinal cord is highlighted with the orange dotted line in “C”. TRITC-dextran shows the posterior spinal cord vein. The arrows in “C” and “D” indicate the location of multiple tumor micrometastases in close proximity to the spinal cord vasculature. Scale bars = 1 mm. SCWC = spinal cord window chamber. BLI = bioluminescence imaging.