The overall goal of this procedure is to provide favorable metabolic conditions to living tissue samples during the collection of magnetic resonance microscopy data. This method can help answer key questions in the field of medical imaging by revealing the MR contrast properties of living cells and other microscopic tissue components. The advantages of this technique are that it guarantees precise control over perfusate conditions inside the MR scanner and permits continuous flow of perfusate, even during image acquisition.
Visual demonstration of this method is critical as the assembly of the coil probe and the oxygenator hardware is complicated and difficult to describe. To ready the artificial cerebrospinal fluid, or aCSF perfusate, for experimentation, equilibrate previously prepared aCSF to ambient temperature while gassing with 95%carbogyn via direct bubbling for at least one hour. Once the aCSF reaches the desired temperature and carbogen saturation, make sure that the pH is in the physiological range by placing a pH meter probe into the aCSF fluid.
In order to maintain dissolved oxygen content and pH conditions appropriate for healthy tissue metabolism, continue to bubble gas directly into the perfusate reservoir during the experiment. Before starting the priming procedure, make sure that profusion line placement will not interfere with the assembly of the probe body or insertion of the probe into the bore. To prime the profusion lines, submerge the inlet tube connected to the peristaltic micro pump into the prepared aCSF.
Then, pass the oxygenator device and profusion lines through the magnet bore and gradient coil stack from top to bottom. Set the coils aside until probe assembly. In order to catch aCSF effluent, hang the oxygenator above a beaker.
Select and confirm the intended two milliliter per minute flow rate. Switch on the pump to begin filling the profusion lines. Displace the air inside with aCSF by inverting the empty inline bubble trap.
Next, connect the oxygenator's gas port to a second source of carbogen while setting a flow rate of one 16th liter per minute. To make sure that carbogen is flowing over the oxygenator's gas exchange membrane, dip the exhaust port on top of the oxygenator into water. Once aCSF starts dripping from the profusion chamber, manually agitate the inflow lines to release any visible gas bubbles.
Then, submerge the oxygen electrode in the aCSF flowing from the profusion chamber to confirm that the oxygenator is operating correctly. After removing the brain from the euthanized rat's scull, as described in the text protocol, return it to the prone position. To isolate the hippocampus containing central portion of the brain, use a straight edged razor to make two cuts along the transverse plane to remove all tissues coddled to the transverse fissure and rostral to the fimbria.
Then, use cyanoacrylate glue to affix the central portion's coddlemost plane to the center of a Vibratome cutting bath. Add ice cold, carbogen bubbled aCSF, then place the nylon retention hardware inside the Vibratome cutting bath. Next, cut 300 micrometer thick slices to get three to four usable slices per hemisphere, resulting in six to eight total.
Use a scalpel and micro forceps to isolate hippocampus or cortical sections from one hemisphere, and trim the slice in order to fit within the five millimeter diameter tissue well of the micro coil. To prepare the micro coil, use a transfer pipette to fill its tissue chamber with oxygenated aCSF from the Vibratome's cutting bath. Next, use the transfer pipette to place the trimmed brain sample into the micro coil's tissue well.
Position the region of interest over the micro coil using a dissecting microscope. To retain sample position throughout the imaging experiment, insert the tissue retention device, such as a nylon mesh net affixed to a nylon washer. Visual confirmation of correct sample placement after the retention components have been inserted is critical because this will be the last opportunity to adjust the tissue's position.
Next, secure the modified micro coil assembly into a table clamp and insert the oxygenator's acetol support peg into the hole on top of the micro coil. Then, place the profusion chamber over the micro coil's tissue well and clench them together to seal the profusion system. Use wire cutters to trim the excess cable tie.
When the sealing has been successful, aCSF will begin dripping through the outflow lines into a collection tube. Attach the micro coil and oxygenator assembly to the top of the imaging probe body. Finally, slide the gradient stack over the assembly and seat the gradient on top of the probe.
To insert the assembled probe into the magnet bore, first place the probe body close to the spectrometer bore opening at the bottom of the magnet. Then, retract the excess length of perfusion lines through the bore opening at the top of the magnet. After all the available slack has been taken up from the profusion lines, advance the probe body into the magnet bore at the base.
Simultaneously, remove more slack from the profusion lines from the top of the bore. Then, fasten two securing screws into the corresponding slots of the shim stack at the base of the probe. Before proceeding, secure the outflow line into the waste reservoir and verify aCSF outflow to ensure that perfusion lines were not pinched or kinked during probe insertion.
After connecting the probe body, begin image collection. If the in-bore oxygenator is functioning properly, the percent of dissolved oxygen of aCSF perfusate should match the percent concentration of oxygen in the supply gas, as shown here. When carbogen mixtures with variable concentrations of oxygen were used as a supply gas, the percent of oxygen saturation at the sight of tissue profusion approached 100%of the oxygen concentration within the mixture used.
Diffusion signal stability trials were performed and compared between superfused slices, fixed, and nonperfused controls. Normalized diffusion signal values in four cortex slices subjected to different perfusion conditions remain stable for a period of 15.5 hours following euthanasia, as well as in formaldehyde fixed cortex as the positive control in contrast to nonperfused cortex. Proper placement of the samples is critical for MR microscopy and can be confirmed visually in faster and lower resolution pilot scans.
In this preliminary scan, low diffusion weighting results in low contrast between the pyramidal cell layer and adjacent tissues in the hippocampus. At high diffusion weighting, the contrast between the pyramidal cell layer and adjacent tissues in the hippocampus increases, and the pyramidal cell layer becomes darker than the adjacent tissues, confirming that the sample is correctly centered and has not shifted since being positioned under the dissecting scope. While attempting this procedure, it's important to remember to begin superfusion as soon as possible to maximize slice viability.
After watching this video, you should have a good understanding of how to offer metabolic support to excised brain tissues undergoing MR microscopy studies. With alterations to the perfusate and dissolved gasses, other types of living, excised tissues with different metabolic requirements may be investigated.
