Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

Animal care, preparation, and experimental protocols were approved by the Animal Research Committee of Tokyo University of Agriculture and Technology. For this methodology, the rat is housed in a controlled environment (24 °C, 12 h light/dark cycle), with food and water available ad libitum.

1. Construction of a Conventional Multispectral Diffuse Reflectance Imaging System

1. Mount nine narrowband optical interference filters with center wavelengths of 500, 520, 540, 560, 570, 580, 600, 730, and 760 nm to the filter holes of the motorized filter wheel.
2. Construct a multispectral imaging system using a broadband white light source, a motorized filter wheel with the above set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic CCD camera. The layout of optical components, shown in Figure 2, can be referred to for this construction procedure.
   NOTE: The angle of illumination is approximately 45° with respect to the sample surface.
3. Turn on the halogen lamp light source to illuminate the surface of the sample via an interference filter, the light guide, and the collecting lens.
4. Open the operating software of the CCD camera.

2. Animal Preparation

NOTE: In this protocol, the rat was not used for the future experiments and it was sacrificed immediately after the measurements of multispectral images. 

1. Connect the inlet port of an induction chamber to the outlet port of an anesthesia machine with a tube. Connect the outlet port of the induction chamber to the inlet port of the anesthesia machine with a second tube.
2. Place the rat into the induction chamber and induce anesthesia with 5.0% isoflurane. Maintain anesthesia at a depth such that the rat does not respond to toe pinches. Lower to 2.0% isoflurane using a rotary knob on the anesthesia machine.
3. Fix the rat head in a stereotaxic frame. Attach a mouthpiece for anesthesia to the stereotaxic frame.
4. Connect the inlet port of the mouthpiece to the outlet port of the anesthesia machine with a tube. Connect the outlet port of the mouthpiece to the inlet port of the anesthesia machine with a tube.
5. Shave the head region beyond the prospective incision site using hair clippers until the skin surface appears.
6. Make a longitudinal incision approximately 20 mm long along the midline of the head using a surgical scalpel (Figure 1(a)) and expose the subcutaneous connective tissues (Figure 1(b)).
7. Remove the subcutaneous connective tissues using a sharp curette or a pincer and pull it to both sides of the head to expose the skull bone (Figure 1(c)).
8. Dig an ellipsoidal ditch on the skull bone inside the cranial sutures (coronal suture, sagittal suture, and lambdoid suture) using a high-speed drill (Figure 1(d)).
9. Slowly and homogenously excavate the skull bone inside the ditch using the high-speed drill.
10. Press lightly on the surface of the thinned skull with the tip of the pincer to estimate the bone thickness and strength after a cerebral blood vessel appears. If the thinned skull region depresses easily, terminate the reduction of the skull bone with the high-speed drill.
11. Cut the ellipsoidal border line of the thinned skull piecemeal using the tip of the pincer or small surgical scissors.
12. Remove the thinned skull from the brain surface slowly and gently using the pincer.
13. Gently bathe the cranial window with physiological saline and cover it with a transparent glass plate approximately 0.1 mm thick.

3. Regulating the Fraction of Inspired Oxygen

NOTE: The respiratory condition can be changed by regulating the fraction of inspired oxygen (FiO₂).

1. Using a tube, connect the first port of a Y-shaped tube connector (connector 1) to the first port of another Y-shaped tube connector (connector 2).
2. Connect the inlet port of the mouthpiece to the second port of tube connector 1.
3. Using a tube, connect the third port of tube connector 1 to an oxygen concentration monitor device.
4. With a tube, connect the second port of tube connector 2 to the outlet port of an anesthesia machine.
5. Using a tube, connect the third port of tube connector 2 to the outlet port of a gas mixture device.
6. Connect one inlet port of the gas mixture device to a high-pressure 95% O₂ – 5% CO₂ gas cylinder using a tube.
7. Connect the other inlet port of the gas mixture device to a high-pressure 95% N₂ – 5% CO₂ gas cylinder using a tube.
8. Change the gas flow rates of O₂ and N₂ using the rotary knobs on the gas mixture device.
9. Check and regulate the FiO₂ using the oxygen concentration monitor device.

4. Acquisition of the Multispectral Diffuse Reflectance Images

1. Acquisition of reference images
   NOTE: The optical components used in this experiment, such as the light source, optical fiber, and detectors have their own spectral characteristics. Therefore, the intensity of light passed through these components should be recorded as a reference image. The reference image is an image taken with a standard white diffuser illuminated with the light from the light source.
   1. Put the standard white diffuser on the stage horizontally.
   2. Focus the camera lens on the surface of the white diffuser by rotating the zoom ring on the barrel.
   3. Adjust the integration time of the camera by selecting the appropriate value from the drop-down list of integration times in the operating software of the camera so that the greatest amount of light produces a signal that is approximately 75% of the maximum counts. While watching the histogram of pixel values, adjust the integration time until the signal intensity level is approximately 75% of the maximum counts.
   4. Select the "save" command from the file menu to save an image to a file.
   5. Change the filter location by rotating the filter wheel.
   6. Save an image at the other wavelengths according to the process described above. The file name should identify the sample and the wavelength used (e.g., W500, W520, W540 … W760).
2. Acquisition of sample images
   Note: Images of diffusely-reflected light intensity of exposed rat brain at nine wavelengths are captured and saved on the hard drive of a personal computer using the same acquisition conditions.
   1. Gently place the rat on the stage and slowly adjust the stage level so that the camera can focus on the surface of the rat brain.
   2. Select the "save" command from the file menu to save an image to a file.
   3. Change the filter location by rotating the filter wheel.
   4. Save an image at the other wavelengths according to the process described above. The file name should identify the sample and the wavelength (e.g., R500, R520, R540 … R760).
3. Acquisition of dark images
   NOTE: The CCD camera can generate a light intensity in response to an electrical signal. However, there is some minor output due to noise in the electric circuits and detectors, even if light does not enter to the detector; this is called dark current noise. To accurately measure the spectral intensity of light, the dark current component should be recorded as a dark image and then subtracted from the measured signal. The dark image is an image taken with the light path blocked.
   1. Turn off the halogen lamp light source.
   2. Block the light path to the CCD camera system using a shielding plate.
   3. Select the "save" command from the file menu to save an image to a file. The file name should identify the sample (e.g., Dark).

5. Visualizing the Hemoglobin Content and the Light Scattering Parameter

NOTE: A set of multispectral diffuse reflectance images is saved to the hard drive of a personal computer and analyzed offline. A multiple regression analysis aided by a Monte Carlo simulation¹⁹ of the multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of oxygenated hemoglobin concentration, deoxygenated hemoglobin concentration, total hemoglobin concentration, regional cerebral oxygen saturation, and scattering power. The detailed algorithm has been published in the literatures^17,18.

1. Subtract the dark image from both the reference image and the sample image at each wavelength.
2. Normalize the sample image by the reference image at each wavelength λ. Treat the normalized image as the diffuse reflectance image R.
3. Calculate the absorbance (or optical density) image A by taking the logarithm of the reciprocal of the diffuse reflectance image R at each wavelength λ:
        (1)
4. Generate a three-dimensional matrix by stacking the absorbance images in the order of their wavelengths, where the x–y plane shows the structural information obtained for the brain surface and the z-axis shows the spectral information.
5. Perform a multiple regression analysis for the absorbance spectrum A(λ) at each x-y coordinate.
6. Use the absorbance spectrum A(λ) as the dependent variable and the molar extinction coefficient spectra of oxygenated hemoglobin ε_HbO (λ) and deoxygenated hemoglobin ε_HbR (λ) as the independent variables for step 5.5 (published values for ε_HbO (λ) and ε_HbR (λ) are provided in Table 1).
7. Check the two-dimensional maps (images) of the three multiple regression coefficients a_HbO, a_HbR, and a₀.
8. Generate a three-dimensional matrix by stacking the images of the multiple regression coefficients in the order a_HbO, a_HbR, and a₀, where the x–y plane shows the structural information obtained for the brain surface and the z-axis shows the multiple regression coefficients.
9. Calculate the oxygenated hemoglobin concentration C_HbO, the deoxygenated hemoglobin concentration C_HbR, and the scattering power b from the set of multiple regression coefficients a_HbO, a_HbR, and a₀ at each x-y coordinate using the following empirical formulae (he values of β_HbO,i, β_HbR,i, and β_0,i (i = 0,1,2,3) are provided in Table 2):
        (2)
       (3)
       (4)
10. Check the two-dimensional maps (images) of the oxygenated hemoglobin concentration C_HbO, the deoxygenated hemoglobin concentration C_HbR, and the scattering power b.
11. Calculate a two-dimensional map of the total hemoglobin concentration C_HbT by summing C_HbO and C_HbR at each x–y coordinate.
12. Calculate a two-dimensional map of the regional cerebral oxygen saturation rSO₂ by dividing the oxygenated hemoglobin concentration C_HbO by the total hemoglobin concentration C_HbT at each x–y coordinate.