Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Measurement of regional rates of brain protein synthesis can trace the response of the brain to long term changes such that occur during development and neuroplasticity. Our method has the advantages that measurements are fully quantitative, and they are made in the awake behaving animal. The quantitative autoradiographic technique permits measurements in all brain regions simultaneously.

Demonstrating the procedure will be Anita Torossian, a post-baccalaureate fellow in my laboratory, and Tianjian Huang, our animal surgeon. Begin this procedure with preparation for surgery as detailed in the text protocol. Once on the surgery stage, use surgical scissors to make a one centimeter incision from the upper medial portion of the left thigh rostrally towards the midline revealing the femoral artery and vein.

Retract loose skin with surgical skin hooks above and on either side of the incision. Secure the skin hooks by taping them to the surgery stage. Apply sterile 0.9%sodium chloride to the exposed area to maintain adequate moisture.

Use forceps to blunt dissect, separating the connective tissue around a small section of the femoral artery and vein. Carefully separate the artery and vein. Now use forceps to thread one strand of absorbable suture under both the femoral vein and artery at the most lateral point of the incision.

Pull the suture halfway through so the ends are even. At a more proximal point to the groin, use forceps to thread a second suture under only the femoral vein. Gently tie a half-knot that will be used to restrict blood flow.

At a point between strand A and strand B, use forceps to thread a third suture under only the femoral vein. Gently tie a full knot that will be used to restrict blood flow. Be careful not to tear the vein.

Gently tug on strand B to restrict blood flow. Use a hemostat to gently tug strand B to maintain blood restriction. Now, connect the non-cut end of the PE tubing to a 32 gauge needle and a one millimeter syringe filled with heparinized saline.

Flush the catheter to remove air bubbles. Cut a small hole in the restricted area of the femoral vein with micro scissors, and carefully insert the angled end of the flushed PE eight tubing toward strand B.Once inserted, release strand B’s tension and guide the catheter further up the vein. Tighten strand B around the vein containing the catheter.

Using strand C, tie an additional knot around the catheter. Make sure this knot does not capture the femoral artery. Gently pull back on the syringe barrel to partially fill the tubing with blood to ensure that the catheter has been implanted properly before inserting a PE 10 catheter into the left femoral artery using the same procedure.

Once both the femoral vein and artery catheters have been secured, tie strand A into a knot around both catheters. After cutting all excess sutures and removing skin hooks, flush the arterial catheter with heparinized saline to prevent clotting. Cauterize the ends of both catheters to create a seal.

Place the mouse in the prone position and make a small incision at the base of the neck applying saline to the exposed area. Insert a hollow metal rod subdermally from the neck incision to the femoral incision. Snake the catheters through the hollow rod and out of the neck incision.

After removing the hollow rod, close the femoral incision with suture followed by post-surgical analgesia. Snake the catheters through a 30 centimeter flexible hollow tube to make the spring tether before suturing the button of the spring tether under the skin followed by post-surgical analgesia. Move the mouse into a clear cylindrical container with a swivel mount and arm to house the mouse during the recovery period.

Place a hand warmer under the container to keep the mouse warm. Ensure that the mouse is in a normal physiological state at the outset of the experiment by taking samples as detailed in the text protocol. To administer the tracer intravenously, use a Y-connector with a syringe holding the C 14 labeled leucine tracer connected to one arm, and a syringe with 100 to 200 microliters of sterile saline to flush the venous line connected to the other arm.

Connect the Y-connector to the venous line. Initiate the study by simultaneously starting a stop watch, injecting the tracer, and collecting timed arterial blood samples. Flush the venous line with saline immediately following injection.

Collect blood samples one through seven continuously throughout the first two minutes of the experiment in the same manner. After collecting the seven samples, collect 30 microliters of dead space blood before each remaining sample. Samples eight through 14 are collected at three, five, 10, 15, 30, 45, and 60 minutes respectively.

At some point during the experiment, process three tubes for internal standards containing tritiated leucine and norleucine as detailed in the text protocol. To quantify plasma leucine concentrations, use an HPLC system with a sodium cation exchange column and post column derivatization with orthophthalaldehyde and flourometric detection. The area under the leucine curve is proportional to the concentration of leucine in the sample.

Use comparison with standards to quantify leucine concentrations in the samples. Then use a liquid scintillation counter to quantify disintegrations per minute of tritium and C 14 in the plasma samples. Use these concentrations to construct the clearance curve of C 14 labeled leucine from the circulation and the time course of its specific activity in the arterial plasma.

From the graph, calculate the integrated C 14 labeled leucine specific activity in the arterial plasma. To perform quantitative autoradiography, prepare brain sections 20 microns in thickness. Section brain by means of a cryostat at minus 20 degrees celsius.

Thaw mount sections on gelatin coated slides. After fixation of slides, arrange slides in an X-ray film cassette along with a set of previously calibrated C 14 labeled methyl methacrylate standards. In a dark room and under safe light, place a piece of X-ray film, emulsion-side down over the sides and standards.

Seal the cassette and place it in a black changing bag. Develop the films according to the manufacturer’s instructions. Automated film development is not recommended because the background may be uneven and can affect quantification.

Construct a calibration curve of optical density versus tissue C 14 concentration based on the optical density values of the set of calibrated standards on the film. Fit these data to a polynomial equation. Either a second or third degree polynomial equation fits very well.

To analyze specific brain regions, locate the region of interest or ROI in six to eight sections by comparison with a brain atlas. Record the optical density of the pixels within an ROI in all sections. Based on the calibration curve, compute the tissue C 14 concentration in each pixel.

Finally, compute the regional rates of cerebral protein synthesis from the average tissue C 14 concentration in the ROI in the integral of the ratios of arterial plasma concentrations of unlabeled and labeled leucine at times t and lamba. The fraction of leucine in the tissue precursor pool that comes from the plasma. Shown here are representative images from a vehicle treated animal compared with an animal treated with anisomycin, an inhibitor of protein synthesis.

Rates of protein synthesis are proportional to the level of darkness in the image. Anisomycin drastically reduces the measured rates of brain protein synthesis indicating the specificity of this method. Here, digitized autoradiograms are shown from an awake behaving mouse at the level of the hippocampus and hypothalamus.

The darker regions have higher regional rates of cerebral protein synthesis. Shown here is a digitized autoradiogram from an awake behaving control mouse at the level of the dorsal hippocampus. Rates of cerebral protein synthesis are color-coated in the images according to the color bar.

While attempting this procedure, it’s important to be sure that animals are in a normal physiological state during the measurements. Our methodology has already demonstrated disregulation of protein synthesis in neurodevelopmental disorders like Fragile X syndrome. It may also be a useful marker for degenerative changes and conditions like Alzheimer’s disease.

The protein synthesis method can be used in conjunction with immune histochemistry in alternating sections to correlate changes in proteins synthesis with regional changes in specific proteins. In summary, the quantitative autoradiographic L-1 C 14 leucine method is ideal for accurate determination of regional rates of protein synthesis in vivo. It offers considerable advantages in terms of accuracy and its applicability to in vivo conditions.