Chronic Cranial Windows for Long Term Multimodal Neurovascular Imaging in Mice

Abstract

Chronic cranial windows allow for longitudinal brain imaging experiments in awake, behaving mice. Different imaging technologies have their unique advantages and combining multiple imaging modalities offers measurements of a wide spectrum of neuronal, glial, vascular, and metabolic parameters needed for comprehensive investigation of physiological and pathophysiological mechanisms. Here, we detail a suite of surgical techniques for installation of different cranial windows targeted for specific imaging technologies and their combination. Following these techniques and practices will yield higher experimental success and reproducibility of results.

Keywords: awake; chronic; imaging; mice; multimodal; neural; vascular.

Figure 1

(A) Schematics of the borosilicate glass window implant. (B) Schematic illustration of the window implant over the whisker representation within the primary somatosensory cortex (SI) and the headpost fixed to the skull overlaying the other (contralateral) hemisphere. (C) Images of the brain vasculature through the glass window implant obtained by 2-photon imaging of fluorescein isothiocyanate (FITC)-labeled dextran injected intravenously. The images illustrate preserved integrity of the vasculature between days 1 (left) and 28 (right) following surgical implantation. (D) Two-photon image stack obtained with Alexa 680 labeled dextran injected intravenously illustrating the capability of deep imaging. The resolution for presented images was ~1 μm/pixel. (E) Top: Image of the surface vasculature calculated as a maximum intensity projection (MIP) of an image stack 0–300 μm in depth using a 4 × objective. Individual images were acquired every 10 μm (top, left). A zoomed-in view of the region within the red square acquired with a 20× objective (top, middle, left). A plane 250 μm below the surface corresponding to the region outlined in blue (top, middle, right). The yellow circle indicates a small diving arteriole. An example temporal diameter change profile acquired from the arteriole outlined by the yellow circle imaged 250 μm below the surface (top, right). The vessel diameter was captured by repeated line-scans across the vessel. These line-scans form a space-time image when stacked sequentially, from left to right. White arrowheads indicate the onset of stimulus trials (air puffs to the whisker pad); four trials are shown. Graphs: Single-vessel dilation time-courses extracted from data. Time-courses for individual trials are overlaid for sensory stimuli (left, n = 160 trials) and OG stimuli (right, n = 19 trials); the thick lines show the average. The stimulus onset is indicated by the black arrowhead and the blue vertical line for the sensory and OG panels, respectively. (F) Concurrent IOSI and LSCI in the same subject. A CCD reflectance image of the surface vasculature (left). The corresponding LS contrast image (middle, left). Ratio images of HbO (extracted from the OIS data, see Methods) and LS contrast showing the region of activation following OG stimulation (middle, right, and right). The location of optical fiber is indicated on all images (black dotted line). The same arteriole is outlined by yellow circles. The black parallelogram indicates the region of interest (ROI) used for extraction of time-courses. The resolution for presented images was 5.5 μm/pixel. Graphs: Time-courses of HbO and HbR (shown in red and blue, respectively), and LS contrast (shown in black) in response to sensory and OG stimulation. These time-courses were extracted from the polygonal ROI shown in top images. (G) Corrected GE EPI image (left, top) and a corresponding structural image (TurboRARE, left, bottom). The resolution for EPI and TurboRARE is 200 μm/pixel 100 × 50 and ~75 μm/pixel, slice thickness = 1 mm. Red arrows point to the peripheral edges of the implant, i.e., the glass/bone boundary. The red line indicates the bottom of the glass implant, i.e., the glass/brain boundary. The BOLD signal in response to sensory stimuli in a fully awake mouse (top, middle). Spatiotemporal evolution of the BOLD signal change from a single slice cutting through the center of the evoked response, presented as trial-averaged ratio maps, in response to a 20-s train of 100-ms light pulses delivered at 1 Hz (“blocked” OG stimulus) in a single Emx1-Cre/Ai32 subject. EPI images were thresholded to reflect the sensitivity of the surface RF coil (for display purposes only). The ratio images are overlaid on the structural (TurboRARE) image of the same slice. BOLD response time-courses extracted from the active ROI (top, right). Fifty seven stimulus trials are superimposed. The average is overlaid in thick black. The BOLD signal in response to OG stimuli in a fully awake mouse (bottom, middle). Spatiotemporal evolution of the BOLD signal change from a single slice cutting through the center of the evoked response, presented as trial-averaged ratio maps, in response to a 20-s train of 100-ms light pulses delivered at 1 Hz (“blocked” OG stimulus) in a single Emx1-Cre/Ai32 subject. EPI images were thresholded to reflect the sensitivity of the surface RF coil (for display purposes only). The ratio images are overlaid on the structural (TurboRARE) image of the same slice. BOLD response time-courses extracted from the active ROI (bottom, right). Twenty eight stimulus trials are superimposed. The average is overlaid in thick black. Modified from Desjardins et al. (2019).

Figure 2

(A) Representative image of cranial window immediately after surgery. In this preparation, animal was implanted with a one-point secured flat head bar and half Crystal Skull glass (please see Supplementary Material for details). (B) Intrinsic optical signal imaging of change in total hemoglobin concentration during air puff stimulation of the contralateral forelimb. Black circle indicates the vessel targeted for photothrombotic occlusion. The resolution for presented images was 5.5 μm/pixel. (C) Relative CBF detected by LSCI at 1 h after photothrombosis. The resolution for presented images was 5.5 μm/pixel. (D) OCT angiograms of flowing vessel before stroke (top) and 1-h after photothrombotic stroke (bottom). Transverse and axial resolutions of the OCT system using a 10× objective (Mitutoyo) were 3.5 and 3.5 μm. (E) Representative images showing collateral occlusion (circled in black). Top panel shows laser speckle contrast images as visualized in real time. Bottom panel shows relative blood flow changes associated with the occlusion. (F) Two-photon maximum intensity projections (left) and volumes (right) of 400 μm stack 5 days before photothrombosis (top) and 4 weeks after photothrombosis. Red circle indicates vessel targeted for photothrombosis. Red square indicates regions chosen for volume projections. The resolution for presented images was ~1.5 μm/pixel. Modified from Sunil et al. (2020).

Figure 3

(A) Representative images of US compatible craniotomy (top) and the position of an awake mouse for training and imaging. In this preparation, animal was implanted with a two-point secured machined head bar and a PMP strip (please see Supplementary Material for details). (B) 2-P vascular stack taken with i.v. FITC injection. The resolution for presented images was ~1.5 μm/pixel. (C) LSCI (top) and IOSI (bottom) of both hemispheres during whisker stimulation. The resolution for presented images was 5.5 μm/pixel. (D) Full field OCT angiography. Transverse and axial resolutions of the OCT system using a 5× objective (Mitutoyo) were 7 and 3.5 μm. (E) ULM images acquired by US. The resolution for presented images was ~10 μm/pixel. (F) Experimental paradigm for fUS imaging (top). Trial average of fUS images acquired during whisker stimuli (middle). The resolution for presented images was ~100 μm/pixel. Time series of average velocity values calculated for marked vessels (middle) during whisker stimuli. Note that contralateral cortical and subcortical activation is visible while ipsilateral cortex is not activated. Modified from Tang et al. (2020).