Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

This protocol allows CSF and blood collection in the quantitative correction of CSF protein levels to measure into a thecal protein synthesis in mouse models of neurological disorders. This procedure provides a baseline, against which the pathophysiological origin of CSF proteins of interest, and the stability and functional significance of the blood CSF favor integrity can be assessed. The analysis of interthecal protein synthesis and barrier integrity can be applied to other animal models and human studies.

For example, to check for diseases of the central nervous system. Collecting significant volumes of clean CSF can be technically challenging in mice, so practicing the technique until large volumes of uncontaminated sample can be obtained is advised. Demonstrating the procedures will be Micheal Linzey, a grad student from the program in experimental and molecular medicine at Dartmouth, in our new immunology research group.

For serum collection via retro orbital bleeding. After confirming a lack of response to pedal reflex in a greater than 15 gram anesthetized mouse, grasp the loose skin behind the ears with the thumb and index finger of the non-dominant hand, and use the index finger to draw back the skin above the eyes. Using the thumb to draw back the skin below the eyes, place the tip of a Pasteur pipette, held at an approximately 45 degree angle, into the eye socket underneath the eyeball, directed toward the middle of the eye socket, while rotating the pipette between the fingers.

Then apply brief, gentle pressure and release to allow blood to enter the pipette. When the blood sample has been collected, gently remove the capillary without injuring the eye, and transfer the blood to a 1.5 milliliter centrifuge tube. After closing the eyelid, apply mild pressure with gauze to prevent further bleeding.

Allow the blood to clot for 30 to 60 minutes at room temperature before spinning down the sample by centrifugation. Using a clean pipette technique, collect the separated serum into a new, labeled 500 microliter vial, and immediately freeze the serum at 80 degrees Celsius. After confirming the lack of response to pedal reflex, remove a large enough area of hair to allow collection of the cerebral spinal fluid immediately at the caudal end of the skull of the anesthetized mouse.

Place the mouse in a prone position on a stereotaxic device under sterile conditions, and steady the head with ear bars. Swab the surgical site with 30%chlorhexidine diacetate, and make a sagittal skin incision inferior to the occiput to expose the muscles overlying the cisterna magna. Using forceps, blunt dissect the subcutaneous tissue and muscles, and use micro retractors to hold the muscles apart to expose the dura mater meningeal layer over the cisterna magna.

Gently wash the tissue with sterile PBS to remove any possible blood contamination, and use a sterile cotton swab to blot the dura mater dry. Positioning the initial puncture at the cisterna magna is essential for obtaining abundant, non-contaminated CSF. Using a 30 gauge needle, gently puncture the membrane covering the cisterna magna, and quickly and gently insert a small glass capillary tube into the puncture.

When five to 12 microliters of CSF have been collected, carefully remove the tube from the membrane and use a piece of polyethylene tubing to connect the tube to a three milliliter syringe. Inject the collected CSF into a labeled 500 microliter tube on ice, and use a disposable needle and varied polydioxanone sutures to close the incision. Clean the area of any dried blood or tissue, and place the mouse in a clean, warm cage with monitoring until full recumbency.

Collect the CSF by centrifugation, and visually inspect the pellet and supernate it for any signs of blood contamination. Then using clean pipette technique, transfer the CSF-containing supernate into a new 200 microliter tube, and dilute the CSF at a one to three ratio with PBS for immediate storage at 80 degrees Celsius. For serum collection by cardiac puncture, immediately after CSF collection, as just demonstrated, place the mouse in the supine position.

Swab the abdominal skin with 70%alcohol. Use scissors to open the thoracic cavity, exposing the heart, and insert a 25 gauge needle attached to a 3 milliliter syringe into the left ventricle. Then gently apply negative pressure to the syringe plunger, withdrawing the needle after the blood has been collected.

Depress the plunger to eject the collected blood into a 1.5 milliliter vial. Now, allow the blood to clot for 30 to 60 minutes at room temperature before separating the serum by centrifugation. Then, using a clean pipette technique, transfer the serum into a new labeled 500 microliter vial for immediate storage at 80 degrees Celsius.

To quantify the target proteins and albumen in matched serum and CSF specimens, use a standard protein quantification assay, using referenced standard proteins to prepare a standard curve for each protein of interest. After the analysis, export the raw data to an appropriate software graphing program, and graph the detection signal fluorescence intensity versus the standard protein concentrations to create a standard curve for each protein of interest. Then, use the standard curves to calculate the concentrations of each analyte of interest in the samples.

Finally, use albumen and target analyte concentrations to calculate Q values and intrathecal index. The actual levels of total IgG are significantly increased in the CSF of two tested rodent models of multiple sclerosis, compared to the corresponding age matched sham controls. R-EAE mice show significantly enhanced albumen quotient values, indicating an increased permeability of the blood brain barrier in these mice.

Nonversely, no differences in albumen quotient exist between TMEV-IDD and sham mice, corroborating previous findings of an intact barrier in TMEV-IDD mice. In addition, in TMEV-IDD animals, significantly higher IgG index values and therefore, intrathecal IgG production are observed. The calculation of a protein index facilitates the identification of novel protein biomarkers useful for early diagnosis, outcome prediction, and disease course monitoring for both neuroinflammatory and neurodegenerative diseases.