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...

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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 ...

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.
<p class="jove_con...

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 ...

The overall goal of this procedure is to visualize the hemodynamics and light-scattering property of in vivo brain tissue. This method can help answer key questions in the field of biomedical optics through the use of imaging for intrinsic optical signals in rat brain tissues. The main advantage of this technique is that special temporal changes in post cerebral hemodynamics and cortical tissue morphology can be evaluated.The implications of this technique extend throughout monitoring brain functions and viability for various brain disorder models because experiments with those animal models are often performed through the cranial window. So this method can provide insight into the intrinsic optical signals. It can also be applied to other spectral imaging techniques.Begin by affixing the rat head in a stereotaxic frame. After anesthetizing, shave the head region beyond the prospective incision site using hair clippers until the skin surface appears. Then make a longitudinal incision approximately 20 millimeters long along the midline of the head using a surgical scalpel.And expose the subcutaneous connective tissues. Remove the subcutaneous connective tissues using a sharp curette, and pull it to both sides of the head to expose the skull bone. Next use a high speed drill to dig an ellipsoidal ditch on the skull bone inside the cranial sutures.Then slowly and homogeneously excavate the skull bone inside the ditch. Next 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. Then use the tip of the pincer to cut the ellipsoidal border line of the thinned skull piecemeal.And slowly remove the thinned skull from the brain's surface. Finally gently bathe the cranial window with physiological saline and cover it with a transparent glass plate approximately 0.1 millimeters thick. Begin by using a tube to connect the first port of a Y shaped tube called connector one to the first port of another Y shaped tube called connector two.Then connect the inlet port of the mouthpiece to the second port of tube connector one. Next use a tube to connect the third port of tube connector one to an oxygen concentration monitor device. Similarly, use a tube to connect the second port of tube connector two to the outlet port of an anesthesia machine, and use another tube to connect the third port of tube connector two to the outlet port of a gas mixture device.Next use a tube to connect one inlet port of the gas mixture device to a high pressure 95%o2 5%co2 gas cylinder. Likewise connect the other inlet port of the gas mixture device to a high pressure 95%n2 5%co2 gas cylinder using a tube. Then use the rotary knobs on the gas mixture device to change the gas flow rates of o2 and n2.Finally check and regulate the fraction of inspired oxygen or fio2 using the oxygen concentration monitor device. Begin by gently placing the rat on the stage and slowly adjust the STage level so that the camera can focus on the surface of the rat brain. Within the image acquisition software, select the save command from the file menu to save an image to a file, naming it according to the sample and wavelength.Then change the filter location by rotating the filter wheel. Repeat the image acquisition at each of the nine wavelengths. Next turn off the halogen lamp light source.Block the light path to the monochromatic charge coupled device camera system using a shielding plate. Finally select the save command from the file menu and choose a file name that identifies the sample. The estimated images of hemoglobin concentration, oxygenation state, and scattering power for in vivo exposed rat brain are shown here.The oxygenated hemoglobin concentration and regional oxygen saturation in arteriole are higher than those in venules. Here images of an exposed rat brain during changes in fio2 for the light scattering power B are shown here. 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 five minutes to 30 minutes after the onset of anoxia.Once mastered, this technique can be completed in three hours if it is performed properly. While measuring the spectral images, it's important to remember to maintain the arrangement of the optical components. Following this procedure, other methods like rapid multispectral imaging can be performed in order to answer additional questions about post optical intrinsic signals.After watching this video, you should have a good understanding of how to visualize hemodynamics and light scattering properties of in vivo exposed rat brains. Don't forget that working with isoflurane can be hazardous and anesthesia gas rates should be controlled with an anesthetic gas scavenging theater.

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JoVE Journal

Neuroscience

7.6K ビュー.  Tokyo University of Agriculture & Technology. The overall goal of this procedure is to visualize the hemodynamics and light-scattering property of in vivo brain tissue. This method can help answer key questions in the field of biomedical optics through the use of imaging for intrinsic optical signals in rat brain tissues. The main advantage of this technique is that special temporal changes in post cerebral hemodynamics and cortical tissue morphology can be evaluated.The implications of this technique extend throughout monitoring ...

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