In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy (pCLE) of Hippocampal Capillaries in Awake Mice

This protocol allowed us to use the confocal laser-scanning fiber-optic endomicroscope to perform simultaneous imaging and electrophysiological recordings in vivo over several hours from neural structures at any depth of the brain in the awake or relatively anesthetized animal. We developed a painless restraint method that allows for deep brain imaging in awake animals using method like those previously employed for electrophysiological recordings. This method has been used to study functional vascular abnormalities in rodent models of epilepsy and age-related macular degeneration.

It is a viable technique to evaluate the contribution of blood flow pathology in many, if not most organs of the diseases. While this technique was developed for a real time study of blood flow dynamics in the brain, we have used it to study blood flow in the in vivo retina. It is not limited to vascular studies in neural tissue.

Demonstrating the procedure will be Jose Maria Gonzalez Martin, a highly qualified technician from my lab. After confirming a lack of response to petal reflex, place the teeth inside the hole of the bite bar, and stabilize the head of the mouse in a rodent stereotaxic frame. Tighten the nose clamp until it is snug.

The position of the body should be as straight as possible. Use ear bars with jaw holder cuffs to secure the head such that the zygomatic processes of the skull are within the clamp. After cleaning and sterilizing the scalp, make 12 to 15 millimeter incision along the midline from the base of the skull to between the eyes along the sagittal midline of the skull, and use four 28 millimeter Bulldog serrefine clamps to retract the borders of the scalp, exposing the skull, and maximizing the working area.

Scrape the periosteum to the incision's edges, and dry the top of the skull. Measure the height of the skull at any distance posterior to bregma, and two equal distances lateral from the midline on either side of the skull. Identify the target point, the right hippocampus with the aid of a stereotaxic atlas.

On the skull, find the target point coordinates relative to bregma with the stereotax, and use a surgical marker to mark four corners of a 1.4 by two millimeter imaging window centered around the target point directly above the hippocampus. Use the surgical marker to draw an additional mark over the top left parietal bone, and another over the right frontal bone for the bone screws. Use the marker to draw one one millimeter on each side of the midline, and one dot on the midline one millimeter behind lambda to indicate the epidural EEG recording electrode insertion points.

To install the head cap, equip a dental drill with a 0.7 millimeter diameter burr, and carefully drill two small holes in the skull at the bone screw placement points. Next place one 70 to 80%ethanol sterilized four millimeter 0.85 diameter stainless steel slotted bone screw into each hole, taking care that the screw tips do not protrude beyond the bottom of the bone into the skull cavity. Then use an L-shaped custom alignment piece clamped to the microdrive mounted to the stereotaxic device to align the head cap along the midline with the anterior ridge overlying bregma, and insert two Fillister head slotted drive screws over the bregma until the screws lie flat on the skull, taking care not to exert too much pressure on the skull.

To set up the imaging window, drill a 0.7 millimeter diameter burr hole at each of the positions of the craniotomy reference marks for the corners of the imaging window before using the drill to gently delineate the periphery of the 1.4 by two millimeter craniotomy window with iteratively deeper cuts. Cover the open window with bone wax, and use a 0.9 millimeter diameter burr to drill the epidural EEG electrode holes, taking care not to cut through the duramater. Cover the three holes with bone wax, and build a wall of dental cement that encompasses the imaging window and EEG holes to bind the wall to the head cap.

When the cement has dried, use 4.0 or 5.0 black braided silk and simple interrupted sutures to close any loose wound margins, and use a cotton tipped applicator to apply lidocaine and antibacterial ointments to the exposed skin. Then place the mouse in a warm cage with monitoring until full recovery. One day after the head cap implantation survey, secure the head cap to a custom mounting bar attached to the stereotaxic frame to position the awake mouse securely to the frame throughout the imaging session.

Insert the ground epidural EEG recording electrodes into the posterior central hole, and insert the recording electrodes in the anterior left and right holes. The tips of the wires are L-shaped, and positioned between the skull and the duramater. To visualize the blood flow within the hippocampus, secure the tail with two fingers to locate the central vein, and holding an ultra fine insulin syringe equipped with an integrated 30 gauge needle parallel to the tail, insert the needle bevel side up into the vein approximately halfway to two thirds from the base of the tail to deliver 200 microliters of 5%green fluorescein lysine-fixable dextran.

Then use the bent tip of a syringe needle to remove the dura and initiate the confocal laser scanning recording. Directly following the tail vein injection, clamp the 300 micron beveled fiber-optic bundle to the mobile arm of the robotic stereotaxic drive in a downward orientation. The restraint system permits stable blood flow recordings of deep brain microvessels, including capillaries and their proximal mural cells over long hours.

In this representative analysis of entire microvessels, blood flow stoppages occurred at the labeled mural cells. The quantity of the fiber recordings can be determined by comparing high resolution two photon imaging recordings of the blood flow to equivalent vasospasms within cortical tissue. Immunohistochemical analysis at the recording sites illustrates that hippocampal mural cells are correctly targeted using this method, not only constricting in response to the treatment, but also spatially associating with strictures in microvessels far from arterials.

Proficiency in the stereotaxic surgery and lateral tail vein injection is crucial to perform this protocol. Thorough practice is essential to reduce error and improve accuracy. The technique allows for realtime imaging of blood flow dynamics in deep brain structures, making it suitable for studying vascular system pathologies and evaluating the efficacy of treatments that target them.