縦二光子イメージングに続いてマウスの急性脳外傷

Hello, I'm Michel iv, a postdoctoral researcher at the Neuroscience Center University of Helsinki. Hello, I'm Hel Stein, a postgraduate student at the University of Helsinki. In this video, we'll describe a method for application of a local acute trauma to a mouse brain for subsequent to photo imaging of brain cell behavior and post-traumatic conditions.

Let C, Acute brain injury is a severe pathology affecting a substantial proportion of population in developed countries. Multiphoton microscopy allows studying cells in tissue context in a living rodent brain under physiological and pathological conditions. Here we describe a method of acute brain injury applied as a stereotactic prick with a syringe needle, followed by a two photon imaging of brain cell behavior and the post-traumatic conditions.

The method of stereotactic infliction of local brain injury is a tool to study pathophysiological consequences of acute trauma. In mammalian brain, a particular brain region can be targeted specifically and injured with a sharp needle to produce a trauma of desired width and depth in the brain parenchyma. In this video, we'll use acute brain injury in combination with cranial window or skull thinning procedures, followed by a two photon imaging of neurons and glia in the post-traumatic brain.

All of the experiments presented here are performed according to the University of Helsinki regulations for animal experiments, the mouse is anesthetized by interperitoneal injection of ketamine and zine. A piece of skin is removed to expose the skull and the animal is then fixed using ear bars on the heating table attached to the stereotactic manipulator. A syringe with a steel needle of 0.3 millimeter diameter is fixed on the microinjection setup.

The micro injection setup allows us to apply traumatic prick to a defined area of the brain using a binocular microscope, we locate the breg point on the skull. We identify the region of interest using the stereotactic coordinates. We drill the skull gently and carefully at the marked point using a high speed surgical drill.

We apply the prick to the drilled well and briefly immerse the needle into the brain 0.5 to two millimeters deep starting from the level of the bottom of the well. A remarkable advantage of these method is that we can inject DI or drugs in the same small well and therefore deliver solutions precisely to the injury site. For the delivery of solutions to the cortex, we use micro injector with electronic controller and a 10 microliter syringe attached to a stereotactic manipulator.

When the tip of the glass pipette touches the surface of the brain, we dip it further by 0.5 to one millimeter. Then we use the micro injector controller to inject 0.5 to two microliters of D or drug solution into the brain at the rate of five nanoliters per second. The bone is then gently drilled around the injury site and the round piece of the skull is removed.

The brain surface is rinsed with a cortex buffer and a thin round cover slip is placed on top of the skull opening. The cover slip is sealed with the poly acrylamide glue and the steel holder is glued on top of the cover slip. To ensure strong attachment of the cranial window to the skull, the dental cement is placed all around the holder.

The anesthetized mouse is mounted on the two photon microscope, and cells are imaged at the injury site within the living brain. This image was taken three hours after the brain injury. The injury site is shown in the brain of a transgenic mouse that expresses GFP under the GFAP promoter in astrocytes.

In addition to GFP expressing green astrocytes, many astrocytic cells are stained in red by the sulfur rumine 1 0 1 injection. The white fibers represent the second harmonic generation signal, presumably visualizing the fiber structures in the extracellular matrix. The second harmonic generation turns out to be a valuable tool to delineate the injury site.

The signal is emitted by non-central symmetric structures such as protein fibers and doesn't require any exogenous staining. The second harmonic generation signal is most intense at the brain surface and doesn't spread deep into the brain parenchyma. In this image, you can see accumulation of the activated microglia at the injury site.

The microglial cells are shown in green and the second harmonic generation in gray. In the proximity of the injury site activated microglia exhibit A characteristic morphology with few branching processes while preparing the skull surface. Use stereotactic coordinates to locate the brain area to be imaged and mark it with a pen.

Avoid areas of interest directly located over cranial searchers because the skull is less stable in these areas and because the underlying large vessels in the meninges will compromise imaging quality. Place the skull holder coded with glue over the area of interest on the animal's skull by applying minimal pressure. Then place dental cement around the holder wait for approximately five minutes for the skull holder to become firmly glued to the skull.

Wash the opening of the skull holder several times with PBS to remove the residual non polymerized glue. Under a binocular microscope thin, a circular area of skull typically 0.5 to 1.5 millimeters in diameter over the region of interest. The mouse skull consists of two thin layers of compact bone and a thick layer of spongy bone sandwiched between them.

The spongy bone contains tiny cavities arranged in concentric circles and multiple canaliculi that contain blood vessels while thinning, remove the external layer of compact bone and most of the spongy bone. Using a high speed micro drill, use compressed air during drilling to blow away the bone. Debris carry out drilling intermittently in order to avoid friction induced overheating of the bone.

Cool, the boring bit in the liquid, kept at room temperature and periodically apply the cortex buffer to the thinning area to observe heat. Some bleeding from the vessels running through the spongy bone may occur during the thinning process. This bleeding will usually stop spontaneously within a few minutes.

Use second harmonic generation imaging to make sure that the majority of this pongy bone tissue is removed. At this stage, the skull thickness should still be no less than 50 micrometers. Submerge the skull in a drop of the cortex buffer and continue the skull thinning procedure Using a microsurgical blade or a micro finishing bur to obtain a very thin and smooth preparation of approximately 20 micrometers in thickness and 700 micrometers in diameter during the final stage of the thinning process, repeatedly examine the preparation using second harmonic generation imaging.

The chosen region can be imaged before and after the injury infliction. Several different areas of the thin region should be imaged to control for possible surgery induced artifacts for trauma.Infliction. Position the needle above the thin region.

Make sure that there are no large vessels underlying it. Dip the needle into the brain 0.5 to two millimeters downwards according to the coordinates of the prick application site. Remove the needle and suppress hemorrhage appearing after the acute brain injury.

With a hemostatic tampon wait until the blood clots are formed and pulsation at the injury site. Subsides solutions containing, for example, sulfur Rumine 1 0 1 or other dyes can be injected into the injury opening in the same way as demonstrated above for the cranial window preparation. In contrast to the cranial window in the skull thinning preparation, the injury site can be accessed repeatedly for delivery of drugs and dyes.

Maintaining sterility of all operations helps keeping the thinned region transparent by preventing inflammation. Here you can see the injury site imaged after skull thinning. In the left side image, you can see the injury side surrounded by YFP labeled dendrites of cortical neurons 20 minutes after the trauma.

The right image represents the same area of the brain in the same animal five days after trauma. In this image, you can see another example of the brain two photon microscopy. After skull thinning, the bone can be seen by second harmonic generation signal shown in gray.

In the left panel, the microglia shown in green. In this experimental setup, the microglia can be observed under the bone around the injury site as shown in the right panel. In the present study, we used both the chronic cranial window and the skull thinning preparations to study cell behavior under post-traumatic conditions within the living brain.

Both methods have certain advantages and limitations. The chronic cranial window provides better resolution, deeper penetration into the brain tissue, and the convenience of multiple imaging sessions. On the other hand, the skull thinning procedure is less likely to induce inflammation at the imaging site and allows repetitive applications of drugs and dyes.