Part of this work was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science (25350520, 22500401, 15K06105) and the US-ARMY ITC-PAC Research and Development Project (FA5209-15-P-0175).

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 &#176;C, 12 h light/dark cycle), with food and water available ad libitum.
1. Construction of a Conventional Multispectral Diffuse Reflectance Imaging System
<ol>
	<li>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.</li>
	<li>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&#176; with respect to the sample surface.</li>
	<li>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.</li>
	<li>Open the operating software of the CCD camera.</li>
</ol>
2. Animal Preparation
NOTE:&#160;In this protocol, the rat was not used for the future experiments and it was sacrificed immediately after the measurements of multispectral images.&#160;
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Fix the rat head in a stereotaxic frame. Attach a mouthpiece for anesthesia to the stereotaxic frame.</li>
	<li>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.</li>
	<li>Shave the head region beyond the prospective incision site using hair clippers until the skin surface appears.</li>
	<li>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)).</li>
	<li>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)).</li>
	<li>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)).</li>
	<li>Slowly and homogenously excavate the skull bone inside the ditch using the high-speed drill.</li>
	<li>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.</li>
	<li>Cut the ellipsoidal border line of the thinned skull piecemeal using the tip of the pincer or small surgical scissors.</li>
	<li>Remove the thinned skull from the brain surface slowly and gently using the pincer.</li>
	<li>Gently bathe the cranial window with physiological saline and cover it with a transparent glass plate approximately 0.1 mm thick.</li>
</ol>
3. Regulating the Fraction of Inspired Oxygen
NOTE: The respiratory condition can be changed by regulating the fraction of inspired oxygen (FiO2).
<ol>
	<li>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).</li>
	<li>Connect the inlet port of the mouthpiece to the second port of tube connector 1.</li>
	<li>Using a tube, connect the third port of tube connector 1 to an oxygen concentration monitor device.</li>
	<li>With a tube, connect the second port of tube connector 2 to the outlet port of an anesthesia machine.</li>
	<li>Using a tube, connect the third port of tube connector 2 to the outlet port of a gas mixture device.</li>
	<li>Connect one inlet port of the gas mixture device to a high-pressure 95% O2 - 5% CO2 gas cylinder using a tube.</li>
	<li>Connect the other inlet port of the gas mixture device to a high-pressure 95% N2 - 5% CO2 gas cylinder using a tube.</li>
	<li>Change the gas flow rates of O2 and N2 using the rotary knobs on the gas mixture device.</li>
	<li>Check and regulate the FiO2 using the oxygen concentration monitor device.</li>
</ol>
4. Acquisition of the Multispectral Diffuse Reflectance Images
<ol>
	<li>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.
	<ol>
		<li>Put the standard white diffuser on the stage horizontally.</li>
		<li>Focus the camera lens on the surface of the white diffuser by rotating the zoom ring on the barrel.</li>
		<li>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.</li>
		<li>Select the &#34;save&#34; command from the file menu to save an image to a file.</li>
		<li>Change the filter location by rotating the filter wheel.</li>
		<li>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 &#8230; W760).</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Select the &#34;save&#34; command from the file menu to save an image to a file.</li>
		<li>Change the filter location by rotating the filter wheel.</li>
		<li>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 &#8230; R760).</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Turn off the halogen lamp light source.</li>
		<li>Block the light path to the CCD camera system using a shielding plate.</li>
		<li>Select the &#34;save&#34; command from the file menu to save an image to a file. The file name should identify the sample (e.g., Dark).</li>
	</ol>
	</li>
</ol>
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 literatures17,18.
<ol>
	<li>Subtract the dark image from both the reference image and the sample image at each wavelength.</li>
	<li>Normalize the sample image by the reference image at each wavelength &#955;. Treat the normalized image as the diffuse reflectance image R.</li>
	<li>Calculate the absorbance (or optical density) image A by taking the logarithm of the reciprocal of the diffuse reflectance image R at each wavelength &#955;: 
	<img alt="Equation 1" src="/files/ftp_upload/55399/55399eq1.jpg" />&#160;&#160;&#160;&#160; (1)</li>
	<li>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.</li>
	<li>Perform a multiple regression analysis for the absorbance spectrum A(&#955;) at each x-y coordinate.</li>
	<li>Use the absorbance spectrum A(&#955;) as the dependent variable and the molar extinction coefficient spectra of oxygenated hemoglobin &#949;HbO (&#955;) and deoxygenated hemoglobin &#949;HbR (&#955;) as the independent variables for step 5.5 (published values for &#949;HbO (&#955;) and &#949;HbR (&#955;) are provided in Table 1).</li>
	<li>Check the two-dimensional maps (images) of the three multiple regression coefficients aHbO, aHbR, and a0.</li>
	<li>Generate a three-dimensional matrix by stacking the images of the multiple regression coefficients in the order aHbO, aHbR, 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.</li>
	<li>Calculate the oxygenated hemoglobin concentration CHbO, the deoxygenated hemoglobin concentration CHbR, and the scattering power b from the set of multiple regression coefficients aHbO, aHbR, and a0 at each x-y coordinate using the following empirical formulae (he values of &#946;HbO,i, &#946;HbR,i, and &#946;0,i (i = 0,1,2,3) are provided in Table 2): 
	<img alt="Equation 2" src="/files/ftp_upload/55399/55399eq2.jpg" />&#160;&#160;&#160;&#160; (2) 
	<img alt="Equation 3" src="/files/ftp_upload/55399/55399eq3.jpg" /> &#160;&#160;&#160; (3) 
	<img alt="Equation 4" src="/files/ftp_upload/55399/55399eq4.jpg" /> &#160;&#160;&#160; (4)</li>
	<li>Check the two-dimensional maps (images) of the oxygenated hemoglobin concentration CHbO, the deoxygenated hemoglobin concentration CHbR, and the scattering power b.</li>
	<li>Calculate a two-dimensional map of the total hemoglobin concentration CHbT by summing CHbO and CHbR at each x-y coordinate.</li>
	<li>Calculate a two-dimensional map of the regional cerebral oxygen saturation rSO2 by dividing the oxygenated hemoglobin concentration CHbO by the total hemoglobin concentration CHbT at each x-y coordinate.</li>
</ol>

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The authors have nothing to disclose.

The most critical step in this protocol is the removal of the thinned skull region to make the cranial window; this should be performed carefully to avoid unexpected bleeding. This step is important for obtaining high-quality multispectral diffuse reflectance images with high accuracy. The use of a stereomicroscope is recommended for the surgical procedure if possible. Small pieces of gelatin sponge are useful for hemostasis.
The optical system described in this article passes a monochromatic light through an interference filter located in front of the light source. This can be modified by placing the filter wheel in front of the video camera lens or CCD camera. In this case, however, the focal plane can be variable if interference filters with different thicknesses are used, and this will cause a deterioration of the image quality. It is necessary to remove the glass plate from the cranial window if a recording electrode is inserted into the cortical tissue for electrophysiology measurements, such as measurements of the electrical local field potential. In this case, the imaging system can detect undesirable specular reflection from the cortical surface. This problem can be avoided by using a set of polarization plates with a crossed Nicols alignment.
The conventional multispectral imaging apparatus demonstrated in this article is somewhat time-consuming to use, since the filter positions in the wheel are changed mechanically. This means that the imaging system captures each diffuse reflectance image sequentially at a different wavelength-point. Because of this limitation, this system is inadequate to capture fast IOSs, such as changes in the reflectance spectrum due to neuronal activities20. Although oxygenated hemoglobin and deoxygenated hemoglobin are the main chromophores in the living brain tissue, the other chromophores, such as cytochrome c oxidase, flavin adenine dinucleotide and nicotinamide adenine dinucleotide, also contribute to the absorption coefficient in the visible wavelength region. Therefore, the estimated values of CHbO, CHbR, CHbT, rSO2, and b can be affected by the minor chromophores. Moreover, this approach integrates all information along the depth direction because it relies on diffuse reflection. Therefore, the imaging system does not perform depth-resolved measurements.
It is advantageous that the algorithm used for the present system can also be applied to multispectral diffuse reflectance images captured by other rapid spectral imaging techniques, such as an acousto-optical tunable filter21, a multi-aperture lenslet array with interference filters22, and the spectral reconstruction images from an RGB image17,23. Using the proposed algorithm and rapid spectral techniques together is a promising approach for evaluating fast IOS imaging, as well as for use in clinical situations.
Most multispectral brain imaging techniques to date have mainly focused on cortical hemodynamics and tissue metabolism, such as cerebral blood volume, regional cerebral oxygen saturation, and cerebral metabolic rate of oxygen10,11,12,13,14. Several existing approaches evaluate the scattering amplitude under the assumption that the scattering power is constant15,16. However, morphological alterations of tissues due to pathophysiological changes and a reduction of viability in living cortical tissue can affect the size of biological scatterers4,5,6,7,8,9. Therefore, it is important to estimate the scattering parameter of b quantitatively to evaluate the tissue morphologies of the brain. The significance of the present technique with respect to existing methods is its ability to simultaneously measure the spatiotemporal changes in cerebral hemodynamics and cortical tissue morphology.
In terms of future applications, this algorithm can be used for monitoring brain function, vitals, and viability in the cortical tissue of various brain disorder animal models, such as traumatic brain injury, epileptic seizure, stroke, and ischemia.

Multispectral diffuse reflectance imaging is the most common technique for obtaining a spatial map of intrinsic optical signals (IOSs) in cortical tissue. IOSs observed in the in vivo brain are mainly attributed to three phenomena: variations in light absorption and scattering properties due to cortical hemodynamics, variation in absorption depending on the reduction or oxidization of cytochromes in mitochondria, and variations in light scattering properties induced by morphological alterations1.
Light in the visible (VIS) to near-infrared (NIR) spectral range is effectively absorbed and scattered by biological tissue. The diffuse reflectance spectrum of the in vivo brain is characterized by absorption and scattering spectra. The reduced scattering coefficients &#956;s&#39; of brain tissue in the VIS-to-NIR wavelength range result in a monotonous scattering spectrum exhibiting smaller magnitudes at longer wavelengths. The reduced scattering coefficient spectrum &#956;s&#39;(&#955;) can be approximated to be in the form of the power law function2,3 as &#956;s&#39;(&#955;) = a &#215; &#955;-b. The scattering power b is related to the size of biological scatterers in living tissue2,3. Morphological alterations of the tissue and reduction of the viability of living cortical tissue can affect the size of the biological scatterers4,5,6,7,8,9.
An optical system for multispectral diffuse reflectance imaging can be easily constructed from an incandescent light source, simple optical components, and a monochromatic charged-coupled device (CCD). Therefore, various algorithms and optical systems for multispectral diffuse reflectance imaging have been used to evaluate cortical hemodynamics and/or tissue morphology10,11,12,13,14,15,16,17,18.
The method described in this article is used to visualize both the hemodynamics and light scattering properties of rat cerebral tissue in vivo using a conventional multispectral diffuse reflectance imaging system. The advantages of this method over alternative techniques are the ability to evaluate spatiotemporal changes in both cerebral hemodynamics and cortical tissue morphology, as well as its applicability to various brain dysfunction animal models. Therefore, the method will be appropriate for investigations of traumatic brain injury, epileptic seizure, stroke, and ischemia.

<table border="0" cellpadding="0" cellspacing="0" width="856">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="36">
			<td height="36" style="height:36px;width:289px;">150-W halogen-lamp light source</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td style="width:119px;">LA-150SAE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Light guide</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td>LGC1-5L1000</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Collecting lens</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td>SH-F16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@500nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@520nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@540nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@560nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#67766</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@570nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#67767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@580nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@600nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@730nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@760nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#67777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Motorized filter wheel&#160;</td>
			<td>Andover Corporation, NH, USA</td>
			<td>FW-MOT-12.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16-bit cooled CCD camera</td>
			<td>Bitran, Japan</td>
			<td>BS-40</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Video zoom lens</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>VZMTM300i</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:289px;">Spectralon white standard with 99% diffuse reflectance</td>
			<td>Labsphere Incorporated, North Sutton, NH, USA</td>
			<td>SRS-99-020</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous evaluation of cerebral hemodynamics and light scattering properties of the in vivo rat brain using multispectral diffuse reflectance imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured 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 hemodynamics and the light scattering properties of the in vivo rat brain.

Representative spectral images of diffuse reflectance acquired from in vivo rat brains are shown in Figure 3. The images at 500, 520, 540, 560, 570, and 580 nm clearly visualize a dense network of blood vessels in the cerebral cortex. The deterioration of contrast between blood vessels and the surrounding tissue observed in the images at 600, 730, and 760 nm reflects the lower absorption of light by hemoglobin at longer and NIR wavelengths.
Figure 4 shows representative estimated images of an exposed rat brain for oxygenated hemoglobin concentration, deoxygenated hemoglobin concentration, total hemoglobin concentration, regional cerebral oxygen saturation, and scattering power. As expected from the diffuse reflectance images at shorter wavelengths in Figure 3, the total hemoglobin concentration in the blood vessel region is higher than that in the surrounding tissue region. On the other hand, the oxygenated hemoglobin concentrations in arterioles are higher than those in venules due to the hemoglobin in arterial blood being much more oxygenated than in venous blood. Therefore, the distribution of arterioles and venules can be clearly distinguished in the estimated image of regional oxygen saturation.
Representative estimated images of an exposed rat brain during changes in FiO2 for diffuse reflectance at 500 nm r(500), concentration of oxygenated hemoglobin CHbO, concentration of deoxygenated hemoglobin CHbR, concentration of total hemoglobin CHbT, regional cerebral oxygen saturation rSO2, and scattering power b are shown in Figure 5. The value of rSO2 increased under hyperoxic conditions and decreased remarkably after the induction of anoxic conditions. The value of b was slightly increased during the period from the onset of anoxia until respiratory arrest, whereas it continuously decreased during the period from 5 min to 30 min after the onset of anoxia. These changes in the value of b were indicative of morphological changes, such as the swelling and shrinkage of cellular and subcellular structures, induced by the loss of tissue viability in brain.
<img alt="Figure 1" src="/files/ftp_upload/55399/55399fig1.jpg" /> 
Figure 1: Steps in the Surgical Exposure of the Rat Cerebral Cortex. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55399/55399fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55399/55399fig2.jpg" /> 
Figure 2: Schematic Diagram of the Experimental Apparatus for Administering Anesthesia and Changing the Fraction of Inspired Oxygen. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55399/55399fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55399/55399fig3.jpg" /> 
Figure 3: Representative Multispectral Diffuse Reflectance Images at 500, 520, 540, 560, 570, 580, 600, 730, and 760 nm, Obtained from an In Vivo Rat Brain. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55399/55399fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55399/55399fig4.jpg" /> 
Figure 4: Representative Estimated Images of an Exposed Rat Brain. (a) Concentration of oxygenated hemoglobin CHbO, (b) concentration of deoxygenated hemoglobin CHbR, (c) concentration of total hemoglobin CHbT, (d) regional cerebral oxygen saturation rSO2, and (e) scattering power b. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55399/55399fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55399/55399fig5.jpg" /> 
Figure 5: Representative Results of an Exposed Rat Brain During Changes in FiO2. Images of in vivo rat cortical tissue during changes in FiO2 for diffuse reflectance at 500 nm r(500), concentration of oxygenated hemoglobin CHbO, concentration of deoxygenated hemoglobin CHbR, concentration of total hemoglobin CHbT, regional cerebral oxygen saturation rSO2, and scattering power b. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55399/55399fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Wavelength&#160;&#955; nm</td>
			<td>&#949;HbO (&#955;)</td>
			<td>&#949;HbR (&#955;)</td>
		</tr>
		<tr>
			<td>500</td>
			<td>113.03712</td>
			<td>112.6548</td>
		</tr>
		<tr>
			<td>520</td>
			<td>130.69296</td>
			<td>170.58384</td>
		</tr>
		<tr>
			<td>540</td>
			<td>287.4744</td>
			<td>251.5968</td>
		</tr>
		<tr>
			<td>560</td>
			<td>176.11128</td>
			<td>290.4552</td>
		</tr>
		<tr>
			<td>570</td>
			<td>240.2784</td>
			<td>243.3888</td>
		</tr>
		<tr>
			<td>580</td>
			<td>270.5616</td>
			<td>199.908</td>
		</tr>
		<tr>
			<td>600</td>
			<td>17.28</td>
			<td>79.25688</td>
		</tr>
		<tr>
			<td>730</td>
			<td>2.106</td>
			<td>5.95188</td>
		</tr>
		<tr>
			<td>760</td>
			<td>3.1644</td>
			<td>8.36201</td>
		</tr>
	</tbody>
</table>
Table 1: The Values of &#949;HbO and &#949;HbO used for the Multiple Regression Analysis. The molar extinction coefficients of oxygenated hemoglobin &#949;HbO and deoxygenated hemoglobin &#949;HbR at each wavelength &#955;.
<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>i</td>
			<td>&#946;HbO,i</td>
			<td>&#946;HbR,i</td>
			<td>&#946;b,i</td>
		</tr>
		<tr>
			<td>0</td>
			<td>-8.3302</td>
			<td>-5.85271</td>
			<td>-0.76587</td>
		</tr>
		<tr>
			<td>1</td>
			<td>4405.877</td>
			<td>-143.23</td>
			<td>53.34134</td>
		</tr>
		<tr>
			<td>2</td>
			<td>2740.622</td>
			<td>3798.067</td>
			<td>124.4656</td>
		</tr>
		<tr>
			<td>3</td>
			<td>-4.40454</td>
			<td>-2.81699</td>
			<td>-1.36919</td>
		</tr>
	</tbody>
</table>
Table 2: The Values of &#946;HbO,i, &#946;HbR,i, and &#946;0,i (i = 0,1,2,3) used in the Empirical Formulae for CHbO, CHbR, and b. Note that the units of CHbO and CHbR derived from these empirical formulae are the volume concentration, in which the hemoglobin concentration of whole blood with a hematocrit reading of 44% is taken to be the 100% volume concentration of hemoglobin. The empirical formulae for hemoglobin concentrations can be derived from the diffuse reflectance spectra calculated by the Monte Carlo simulation of light transport19. The detailed process for the derivation of the empirical formulae has been described in the literature17,18.

Watch this Scientific Journal Video about Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging at J

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

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

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

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

simultaneous-evaluation-cerebral-hemodynamics-light-scattering

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.6K Views.  Tokyo University of Agriculture & Technology. The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

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

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the test&#39;s susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...