This technique increases the Cerebrospinal Fluid or CSF glucose levels in mice without affecting their blood glucose concentration. This helps to investigate the direct effects of high CSF glucose levels on the animal's brain function. This technique can establish animal models to explore the theological role of higher CSF glucose on cognitive dysfunction.
Leading to the discovery of new molecular targets to improve cognitive function. The method will also help determine whether high CSF glucose levels are sufficient to mediate phenotypes of neurodegenerative disorders in mice. Attention must be paid to the proper assembly and priming of the cannula setup and insertion of the cannula in the brain at the correct coordinates.
To begin the surgery for Osmotic Minipump Implantation place the anesthetized mouse on the stereotaxic frame. Fit the animal's head on the incisor bar. Cover the nose with the nose cone to maintain the anesthesia and connect isofluorane flow to the nose cone at a rate of 1.5 to 2%Place a heating pad under the mouse to maintain the body temperature.
After disinfecting the surgical area and making an incision expose the skull surface with a spatula and wipe the blood, if any, with cotton tips. Use a cauterizer if bleeding profusely. Rub with a cotton tip dipped in 3%hydrogen peroxide to expose the cranial sutures.
Working under a microscope, take note of bregma and lambda to navigate the skull coordinates. Set all the coordinates to zero at bregma. Using an electric drill carefully and without rupturing the dura mater make a hole at the coordinates 0.5 millimeters posterior to bregma and 1 millimeter lateral to midline toward the right.
Insert a hemostat and gently open and close it to make a pocket for the mini pump. Holding the pump from the tip of the flow modulator push it through the incised skin into the created pocket. Apply some glue on the underside of the cannula, and fix it in the cannula holder.
Ensure that the cannula is not clogged and the solution is flowing properly. Then lower the cannula slowly through the drilled hole, two millimeters ventral to the skull surface. Leave it there for at least five minutes to allow the glue to solidify.
With the help of scissors, clip the top of the cannula ensuring it doesn't dislodge from the skull surface. Cover the cannula with a layer of dental cement for extra support. For Cerebrospinal Fluid or CSF collection, place the properly anesthetized mouse on the stereotaxic frame.
After fixing the head on the frame as demonstrated previously rotate the knobs to tilt the head so that the nose faces downward. After disinfecting the surgical area and making an incision remove the support from the mouse body and let the mouse rest vertically so that the neck is fully extended dorsally. Using curved blunt forceps, gently bisect the posterior neck muscles from the midline to create a small window.
Then use a wet cotton tip applicator to gently displace the neck muscles from the midline to the periphery. Observe whether the cisterna magna is exposed as a triangular window with a transparent dura membrane. Place the micro manipulator assembly on the stereotaxic frame next to the mouse while ensuring the capillary tip does not touch any surface.
Gently break the capillary tip without disturbing the setup. Rotate the corresponding knobs on the micro manipulator to align slowly and move the capillary tip toward the cisterna magna. Some resistance is felt once the capillary tip touches the cisterna magna membrane.
Very slowly push the tip against the membrane with the help of the knobs being careful not to damage any blood vessels in the membrane. As the membrane is pierced, CSF flows into the capillary at once due to negative capillary pressure. Leave the setup for a few minutes until approximately 10 microliters of CSF collects in the capillary.
Carefully remove the capillary from the syringe and attach it to a microcap bulb dispenser. Press the bulb to transfer the CSF into a sterile micro centrifuge tube. Place the support under the mouse and rotate the stereotaxic knobs to level the head.
Close the wound with nylon sutures. Inject 300 microliters of sterile saline subcutaneously before removing the mouse from the apparatus. Give postoperative care to the animals until it is time to remove the sutures, about 7 to 10 days after surgery.
CSF collected 10 days after the surgery showed about a sixfold increase in glucose level in mice, infused with 50%glucose compared to that in mice infused with CSF. However, the blood glucose levels were not different between the groups. While lowering the cannula in the brain, retract it back and check if CSF comes out of the hole.
It is also vital to correctly identify cisterna magna and prevent contamination of CSF with blood. This technique can also be used to deliver any drug that is unable to cross the blood-brain barrier to test its effect on brain function. There is a very limited number of animal models of diabetes, and they disturb glucose homeostasis in the peripheral environment.
This model, however, helps investigate brain specific changes in glucose regulation on the neural function.
