2.1.

In Vitro Activation of the MMP-Activatable Probe

The MMP-activatable probe (MMPSense™ 680, Perkin Elmer) is predominantly activated by MMP-9, -2 and -13, but to a lesser extent also by other MMPs.²⁵ The MMP-activatable probe ($0.2\mu \mathrm{mol}/\mathrm{L}$) was incubated with $4.5\text{-}\mu \mathrm{g}$ human recombinant MMP-9 (Oncogene Research Products, California, USA) at 37°C in a buffer containing $50\mathrm{mmol}/\mathrm{L}$ Tris HCl pH 7.5, $10\mathrm{mmol}/\mathrm{L}$ ${\mathrm{CaCl}}_2$, $100\mathrm{mmol}/\mathrm{L}$ NaCl, and 0.005% Brij-35 (Sigma-Aldrich). Tubes (3 mm diameter, 4 to 5 cm lengths) were filled with either $200\mu \mathrm{l}$ activated probe or nonactivated probe for NIRF imaging. For obtaining MSOT absorbance spectra and standard curve of activated MMP, $1\mu \mathrm{M}$ MMP-activatable probe and $100\mu \mathrm{M}$ of trypsin solution from porcine pancreas (Sigma-Aldrich, Switzerland) in a $1:2$ dilution series were used. The solutions were filled in tubes of the same type used (3 mm diameter, 4 to 5 cm lengths) in NIRF imaging with the Maestro 500 multispectral imaging system (Cambridge Research & Instruments Inc.) and placed in 2-cm-diameter cylindrical phantoms made of 2% agarose (solidifying at 20°C; Sigma-Aldrich) mixed with 5% intralipid.

2.2.

Animal Model

All procedures conformed to the national guidelines of the Swiss Federal Act on Animal Protection and were approved by the Cantonal Veterinary Office Zurich (Permit Number: 18-2014). Thirty-nine male C57BL/6J mice (Janvier, France), weighing 20 to 25 g, 8 to 10 weeks of age, were used including 19 sham-operated mice (2 died during surgery) and 20 tMCAO mice. Data for one tMCAO was excluded due to strong pigment on the mouse head interfering with MSOT and NIRF data acquisition. To assess cerebral oxygenation during the hyperacute phase (i.e., vessel occlusion in tMCAO group), tMCAO ($n=9$) and sham-operated mice ($n=9$) underwent in vivo MSOT and DW MRI and triphenyltetrazolium chloride (TTC) for validation after in vivo imaging. Considering that MSOT measurement and depilating gel on the skin might influence the inflammation status of the mice, another batch of tMCAO ($n=10$) and sham-operated mice ($n=8$) was used to assess for cerebral oxygenation in the acute stage at 48 h after reperfusion: in vivo MSOT, ${\mathrm{T}}_2$-weighted MRI, ex vivo NIRF, and histology staining sequentially. Additionally, six tMCAO mice and four sham-operated mice were assessed for tissue oxygenation at 48 h after surgery and were also injected intravenously with $300\mu \mathrm{l}$ 4 nM of MMPSense™ 680 after 1 h tMCAO. Animals were assessed with MSOT for detecting MMP activity after reperfusion or sham-operation. Three tMCAO mice without MMP probe injection were assessed with MSOT and served as controls.

2.3.

Transient Middle Cerebral Artery Occlusion

Surgeries for tMCAO and sham were performed as described before.²⁶ Anaesthesia was initiated using 3% isoflurane (Abbott, Cham, Switzerland) in a $1:4$ oxygen/air mixture and maintained at 2%. Before the surgical procedure, a local analgesic (Lidocaine, 0.5%, $7\mathrm{mg}/\mathrm{kg}$, Sintectica S.A., Switzerland) was administered subcutaneously (s.c.). Temperature was kept constant at $36.5°\mathrm{C}\pm 0.5°\mathrm{C}$ with a feedback controlled warming pad system. All surgical procedures were performed in less than 15 min. After surgery, buprenorphine was administered as s.c. injection (Temgesic, $0.1\mathrm{mg}/\mathrm{kg}$ b.w.) and then twice s.c. every 6 to 8 h on the day of surgery and was supplied thereafter via the drinking water ($1\mathrm{mg}/\mathrm{kg}$) until 48 h.

2.4.

Multispectral Optoacoustic Tomography

A MSOT inVision 128 imaging system (iThera Medical, Germany) was used as described previously.²⁶ Briefly, a tunable (680 to 980 nm) optical parametric oscillator pumped by an Nd:YAG laser provides 9-ns excitation pulses at a framerate of 10 Hz with a wavelength tuning speed of 10 ms and a peak pulse energy of 100 mJ at 730 nm. Ten arms, each containing an optical fiber bundle, provide even illumination of a ring-shaped light strip with a width of $\sim 8\mathrm{mm}$. For ultrasound detection, 128 cylindrically focused ultrasound transducers with a center frequency of 5 MHz (60% bandwidth), organized in a concave array of 270-deg angular coverage and a curvature radius of 4 cm, were used.

For a phantom MSOT measurement, the phantom was fixed into the supplied rigid phantom holder and placed into the imaging chamber of the MSOT system, filled with 36.5°C warm water. Phantom measurements using different concentrations of trypsin and MMP activatable probe were used to generate MMP MSOT absorbance spectra. After equilibrating of the phantom temperature for 5 min, imaging was performed at 45 wavelengths from 680 to 900 nm with 5-nm increment. Principal component analysis based on Ref. 27 was used for calculating the absorbance spectra of MMP and used in the unmixing and multispectral analysis of the phantom and tMCAO-/sham-operated mice. For the phantom activated and unactivated human recombinant MMP-9 with MMP activatable probe, imaging was performed at 10 wavelengths (i.e., 680, 685, 690, 695, 700, 715, 730, 760, 800, and 850 nm) at 35 to 40 consecutive slices with a step size of 0.1 mm and 10 averages. For in vivo MSOT measurement, animals were anesthetized with 4% and maintained at 1.5% isoflurane in a $1:4$ oxygen/air mixture supplied via a nose cone. Mice were depilated around the head and placed in a mouse holder in the supine position. Preheated ultrasound gel (Diagramma, Switzerland) was applied to the mouse head for ultrasonic coupling and the animals were wrapped in a polyethylene membrane. The mouse holder was placed in an imaging chamber filled with water and kept at 36.5°C. Images were acquired at 10 wavelengths (i.e., 680, 685, 690, 695, 700, 715, 730, 760, 800, and 850 nm), for 35 to 40 consecutive slices with a step size of 0.3 mm and 10 averages, lasting $\sim 5\min$.

2.5.

Near-Infrared Fluorescence Imaging

NIRF imaging was performed with the Maestro 500 multispectral imaging system (Cambridge Research & Instruments Inc.), as previously described.²⁸ The system was equipped with a band-pass filter (615 to 665 nm) for excitation. The fluorescence was detected by a CCD camera fitted with a long-pass filter ($>700\mathrm{nm}$). Fluorescence emission images were acquired by automatically increasing the emission filter wavelengths at 10 nm steps. For in vivo NIRF imaging of MMP, four tMCAO and three sham-operated mice were anesthetized with 3% and maintained at 1.5% isoflurane in a $1:4$ oxygen/air mixture. For ex vivo NIRF imaging, brains were subsequently removed under deep anesthesia without prior perfusion. Coronal brain slices of 1-mm thickness were cut in a brain matrix using a razor blade. Sections were placed on object holders wrapped with black tape. Acquired image cubes were spectrally unmixed using Maestro software (Cambridge Research & Instruments Inc.). Regions of interest (RoIs) were selected over each section on the ex vivo brain slices (Fig. 4). Fluorescence intensity (F.I.) ratios were calculated by dividing the RoI values of the left ischemic (ipsilateral) hemisphere by that of the right (contralateral) hemisphere.

2.6.

Magnetic Resonance Imaging

Data were acquired on a 7 T Bruker Pharmascan (Bruker BioSpin GmbH, Germany), equipped with a volume resonator operating in quadrature mode for excitation and a four element phased array surface coil for signal reception. During MRI acquisition, mice were kept under 1.5% isoflurane anesthesia in a $1:4$ oxygen/air mixture. Body temperature was monitored with a rectal temperature probe (MLT415, ADInstruments) and kept at $36.5°\mathrm{C}\pm 0.5°\mathrm{C}$ using a warm water circuit integrated into the animal support (Bruker BioSpin GmbH, Germany). ${\mathrm{T}}_2$-weighted MR images were obtained using a spin echo sequence (TurboRARE) with echo time 3 ms, repetition time 6 ms, 100 averages, slice thickness 1 mm, field-of-view $2.56\times 1.28{\mathrm{cm}}^2$, matrix size $256\times 128$, giving an in-plane resolution of $100\mu \mathrm{m}\times 100\mu \mathrm{m}$. For DWI, a four-shot spin echo–echo planar imaging sequence with $\text{echo time}=28\mathrm{ms}$, $\text{repetition time}=3000\mathrm{ms}$ was used. Twelve axial slices with a slice thickness of 1 mm and slice gap of 0.3 mm were acquired with a field-of-view of $3.3\times 2{\mathrm{cm}}^2$ and a matrix size of $128\times 128$, resulting in a nominal voxel size of $258\mu \mathrm{m}\times 156\mu \mathrm{m}$. Diffusion-encoding was applied in the $x$-, $y$-, and $z$-directions with $b$-values of 100, 200, 400, 600, 800, and $1000\mathrm{s}/{\mathrm{mm}}^2$, respectively, acquisition time 3 min 48 s.

2.7.

Magnetic Resonance Image Reconstruction

Maps of the apparent diffusion coefficient (ADC) were calculated on a pixel-by-pixel basis by linear regression analysis using the model function in Paravison 6 (Bruker Biospin GmbH, Germany):

2.8.

MSOT Image Reconstruction and Spectral Unmixing

MSOT images were reconstructed using a model-based algorithm, size 20 mm, resolution $100\mu \mathrm{m}$, and filter range from 50.0 kHz to 7 MHz. The model-based reconstruction incorporates a detailed model of detection geometry that allows for more quantifiable images. For the multispectral processing, linear unmixing methods with a non-negative constraint imposed during inversion were applied to resolve signals from the MMP probe, deoxygenated (Hb) and oxygenated hemoglobin (${\mathrm{HbO}}_2$), as described previously.²⁷ The linear regression method uses known spectra for fitting and allows for reproducible results across studies. Tissue oxygen saturation (${\mathrm{SO}}_2$) was calculated by

2.9.

Coregistration of MSOT and MRI Images

Functional MSOT images showing Hb, ${\mathrm{HbO}}_2$, and MMP distribution share the same coordinates with the intrinsic anatomical images. This allows for registering the anatomical images obtained from MSOT to ${\mathrm{T}}_2$-weighted images/ADC maps and for applying the same transformation to all the functional images. Registration of MSOT and MR image was performed using a three-step strategy with MATLAB (R2015a, Mathworks). (The archived version of the code can be freely accessed and executed through Code Ocean: https://codeocean.com/2018/02/20/mri-msot-coregistration-matlab-code-demo.) First, the anatomical image was realigned with the ${\mathrm{T}}_2$-weighted MR image obtained at 48 h after reperfusion (or with the ADC maps at 1 h during occlusion) along the longitudinal axis. The alignment was performed under the assumption that, during measurement, the animal holder in MSOT moved along the $z$-axis, parallel to the central axis of the MRI scanner bore. Anatomical landmarks, such as the eye and brain structures, were selected for alignment. Linear interpolation was applied to MSOT images to generate the same number of frames as MRI for further processing. Second, nonreflective similarity transformation was applied to MSOT images after processing MR images. The transformation matrix was calculated based on the least square algorithm after manually selecting at least six pairs of landmarks in both MSOT and MR images. Rotation, scaling, and transformation were applied, with angles and shapes being preserved during registration. Finally, different MSOT maps (Hb, ${\mathrm{HbO}}_2$, and MMP) were registered with the corresponding MR image. The images were overlaid with multiple channels. RoIs were drawn over the lesion in the cortex, striatum, and the whole lesion in the ipsilateral and contralateral side on the axial overlaid MSOT/MR images using ImageJ (NIH). The focal ischemic lesions were identified on the ${\mathrm{T}}_2$-weighted MR images and ADC maps. The size of RoIs was $\sim 3$ to $10{\mathrm{mm}}^2$ for the cortical-infarct and 5 to $10{\mathrm{mm}}^2$ for the striatal infarct and the corresponding contralateral areas (Fig. 1).

Fig. 1

Registration procedure: (a) coregistration of cross-sectional axial background MSOT and ${\mathrm{T}}_2$-weighted MR images; (b) landmark coregistration; and (c) resulting coregistered MSOT image and RoIs, including ischemic lesion (whole, cortex, and striatum) in the ipsilateral and nonaffected contralateral hemisphere.

For assessing the depth dependence of MSOT signals, five parallel lines, each at two positions perpendicular to the cortex, were drawn on the ${\mathrm{HbO}}_2$ image in the contralateral hemisphere of one tMCAO mouse (illustrated in Fig. 5). The intensity (MSOT. a.u.) in relation to the distance (depth) from the cortex/surface of signal was analyzed.

2.10.

Triphenyltetrazolium Chloride Staining

To assess the ischemic lesion severity in the tMCAO mice and in the sham-operated mice, staining with TTC staining was performed. Brain slices were incubated in a 2.5% TTC solution (Sigma-Aldrich) at 37°C for 3 min. Pictures of stained samples were taken and analyzed by drawing RoIs over the contralateral side and the nonischemic ipsilateral side using ImageJ.

2.11.

Statistics

Unpaired two-tail student’s $t$ test with Welch’s correction was used (Graphpad Prism 6.0, California) for comparing the difference between tMCAO and sham-operated mice. ANOVA with Turkey multiple comparison tested for the difference between sham-injected, tMCAO injected, and noninjected with MMP probe. Paired $t$ test was used for comparison of data between the ipsilateral and contralateral hemisphere. Unpaired two-tail student $t$ test with Welch’s correction was used for comparison of NIRF imaging data between tMCAO and sham-operated mice. All data are present as $\text{mean}\pm \text{standard deviation}$. Significance was set at *, #$p<0.05$ and ***, ###$p<0.001$.

3.1.

In Vivo MSOT of Oxygenation during Occlusion and 48 H After

During occlusion, the lesions were visible on the ADC maps calculated from DWI, but not on the ${\mathrm{T}}_2$-weighted MR images, as expected. Thus, ADC maps were applied in the coregistration with images from MSOT for the hyperacute group. ${\mathrm{T}}_2$-weighted MR images, which gave a better anatomical contrast for the lesions (discernable later than ADC maps), were used for coregistration for the group assessed at 48 h after reperfusion. The same transformations were subsequently applied to Hb, ${\mathrm{HbO}}_2$, and MMP MSOT images. Figure 1 illustrates the MSOT-MRI coregistrated image that we obtained following a three-step strategy.

To assess the influence of acute tMCAO on brain regional tissue oxygenation, we measured the ${\mathrm{HbO}}_2/\mathrm{Hb}$ level in the brain of tMCAO and sham-operated mice during occlusion and at 48 h after reperfusion noninvasively in vivo using MSOT and calculated ${\mathrm{SO}}_2$ (Fig. 2). Lower ${\mathrm{SO}}_2\mathrm{ipsi}/{\mathrm{SO}}_2\mathrm{contra}$ ratios [%] were detected in the lesion of tMCAO ($n=9$) compared with sham-operated mice ($n=9$) ($52.5\pm 23.1\%$, versus $98.6\pm 18.3\%$, $p=0.0003$) and in the cortical lesion ($44.9\pm 28.5\%$ versus $91.6\pm 16.5\%$, $p=0.0006$), indicating severe focal ischemia. ${\mathrm{SO}}_2\mathrm{ipsi}/{\mathrm{SO}}_2\mathrm{contra}$ [%] in the hemisphere and cortical brain region of the tMCAO animals were significantly higher in the 48 h after reperfusion ($99.9\pm 9.4\%$, $n=9$) compared with during occlusion ($93.1\pm 23.5\%$, $n=10$), and similar to the level of sham-operated mice at 48 h after reperfusion ($102.0\pm 10.5\%$, $n=8$).

Fig. 2

In vivo assessment of brain oxygenation with MSOT in tMCAO and sham-operated mice. (a) Hb and ${\mathrm{HbO}}_2$ images of tMCAO and sham-operated mice during occlusion and at 48 h (coronal view, approximately Bregma $-0.58\pm 0.3\mathrm{mm}$); unmixed signal from ${\mathrm{HbO}}_2$ (red) and Hb (blue); (b) reduced tissue ${\mathrm{SO}}_2$ in the whole lesion in the ipsilateral hemisphere and in the cortex compared with contralateral hemisphere in the brain of tMCAO compared with sham-operated mice during occlusion, with no differences between regions of groups at 48 h; results are $\text{mean}\pm \text{standard deviation}$, ***$p<0.001$, tMCAO mice compared with sham-operated mice; ###$p<0.001$, tMCAO mice during occlusion compared with 48 h; one way ANOVA with Turkey multiple comparison; tMCAO, Hb, deoxygenated hemoglobin; ${\mathrm{HbO}}_2$, oxygenated hemoglobin; ${\mathrm{SO}}_2$, oxygen saturation rate; MSOT, multispectral optoacoustic imaging;

3.2.

In Vitro NIRF Imaging and MSOT of MMP Activated and Nonactivated Probe

Analysis of NIRF images of the phantom indicated a threefold increase in the intensity of the MMP-activatable probe after activation with human recombinant MMP-9 compared with that of the nonactivated probe [F.I. average counts over the field, $232.2\pm 31.8$ versus $77.5\pm 10.2$ counts, **$p=0.0086$; Fig. 3(a)]. Similarly, analysis of the MMP probe phantom measured with MSOT showed a three- to fourfold increase in the optoacoustic signal of the MMP activated probe [$19196.0\pm 3692.2$ versus $5546.3\pm 2625.6$ MSOT a.u., $p=0.0004$; Fig. 3(b)].

Fig. 3

In vivo MSOT assessment of MMP activity in phantom and brain of sham-operated and tMCAO mouse at 48 h after reperfusion. (a and b) NIRF imaging and MSOT of activated (human MMP-9) and unactivated MMP probe in a phantom, scale: F.I. 0 to 0.122; (c) sham and tMCAO mouse injected with MMP-activatable probe 48 h after reperfusion (approximately at Bregma $-0.58\pm 0.3\mathrm{mm}$) with unmixed signal from MMP (green) overlaid on ${\mathrm{T}}_2$-weighted MR image, scale 0 to $9\times {10}^4$ MSOT a.u.; (d) quantification of MSOT MMP signal in the ipsi/contralateral in the whole lesion, cortex, and striatum; results are $\text{Mean}\pm \text{standard deviation}$, *$p<0.05$, sham-injected compared with tMCAO-injected; #$p<0.05$, to tMCAO-injected compared with tMCAO noninjected. One-way ANOVA with Turkey multiple comparison. F.I., fluorescence intensity; MMP, matrix metalloproteinase; tMCAO, transient middle cerebral artery occlusion.

3.3.

In Vivo MSOT Imaging of MMP Activity in tMCAO Mice

To reveal MMP activity in vivo, the MMP-activatable probe was injected into tMCAO and sham-operated mice as controls. tMCAO mice without the MMP activatable probe injection were measured as additional controls to assess the potential influence of tissue changes on MMP signal unrelated to the presence of the probe. Mice were assessed with MSOT 48 h after reperfusion with in-house generated MMP spectra and followed by MRI (Fig. 3). Significantly, higher unmixed MMP intensities were observed in the ischemic ipsilateral hemisphere ($4738.7\pm 2867.8$ MSOT a.u., $n=5$) of tMCAO injected, compared with the nonischemic contralateral hemisphere ($1102.7\pm 534.0$ MSOT a.u., $n=5$, $p=0.0309$) and compared with sham-injected ($1138.4\pm 709.7$ MSOT a.u., $n=4$, $p=0.0479$). The MMP signals were higher in the cortical regions compared with that in the striatum and were lower than in all groups: tMCAO injected ($494.2\pm 601.5$, $n=5$) in the striatum similar to that in sham-operated injected mice ($584.9\pm 442.2$, $n=4$, $p=0.9566$). The noninjected tMCAO mice showed low level of signals in both the ipsilateral ($243.6\pm 67.0$, $n=3$) and contralateral hemisphere ($201.1\pm 226.5$, $n=3$), significantly lower than that in the ipsilateral hemisphere of tMCAO injected ($4837.7\pm 2867.8$, $p=0.0258$). Signals were also detected in extracerebral tissue of the mouse head in tMCAO and sham-operated mice injected with the MMP-activatable probe, likely due to MMP activation in muscles due to ligation of the external carotid artery.²³

3.4.

In Vivo and Ex Vivo NIRF Imaging of MMP and TTC Staining in tMCAO Mouse Brains

After MSOT, whole brains were removed from the skull for ex vivo NIRF imaging of the mouse brain and brain slices to assess the distribution of the activated MMP probe (Fig. 4). Higher MMP fluorescence intensities were detected in the ipsilateral lesioned hemisphere compared with contralateral hemisphere of tMCAO mice injected with the MMP-activatable probe (ipsi/contralateral ratio $2.7\pm 0.2$). No difference was observed between the fluorescence intensity in the two hemispheres of sham-operated mice injected with the MMP-activatable probe (ipsi/contralateral ratio $1.0\pm 0.01$, $p<0.0001$ compared with tMCAO). Ex vivo NIRF imaging of brain sections of tMCAO mice validated the high fluorescence intensities to areas of nonviable tissue as demarcated by TTC staining.

Fig. 4

Ex vivo NIRF imaging of MMP signal in the brain of sham-operated and tMCAO mice at 48 h after reperfusion. (a and b) NIRF imaging of the brain, NIRF imaging, and TTC staining on the coronal brain slices, scale 0 to 0.0142; (c) quantification of MMP signals using ex vivo NIRF imaging in tMCAO ($n=4$) and sham ($n=3$), ***$p<0.001$, unpaired two tail student’s $t$ test with Welch’s correction. MMP, matrix metalloproteinase; tMCAO, transient middle cerebral artery occlusion; NIRF, near infrared fluorescence; F.I., fluorescence intensity;

3.4.1.

Depth dependence of MSOT signal

To analyze the depth dependence of MSOT, signal intensity was plotted as a function of depth at two different locations in the right contralateral hemisphere in the brain of a tMCAO mouse. The ${\mathrm{HbO}}_2$ signals were detectable across the cortical layers up to $\sim 3$ to 4 mm at two positions and decreased thereafter with increasing depth (Fig. 5).

Fig. 5

Depth analysis of MSOT signal in a representative tMCAO mouse brain. (a) Coronal view of MSOT image of ${\mathrm{HbO}}_2$ in a tMCAO mouse brain at 48 h after reperfusion and illustration of RoI used for depth analysis, scale 0 to 20 MSOT a.u. (b and c) Plotting of mean signal intensity in relation to depth (mm) in two RoIs as illustrated in (a). ${\mathrm{HbO}}_2$, oxygenated hemoglobin; tMCAO, transient middle cerebral artery occlusion; MSOT, multispectral optoacoustic imaging;