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

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.