Free-Hand Intracerebroventricular Injections in Mice

We are interested in the neural control of reproductive function, particularly how the brain regulates the release of hormones that control the gonads, both in healthy conditions and in response to stress. The main advantage of this technique is that it is relatively simple and rapid. Delivering drugs into the lateral ventricle is a great way to cast a wide net in testing a signaling pathway to support more detailed follow-up experiments.

There's still much to be learned about the neuroendocrine control of reproduction, including the molecular mechanisms for luteinizing hormone pulse generation, mechanisms for modulating pulse frequency during stress, and generation of the preovulatory LH surge. In the future, we are particularly interested in integrating disparate findings into models. Begin by preparing For a craniotomy by placing a clean bench pad on the work surface.

Tape the nose cone of an isoflurane vaporizer to the work surface. Next, place silastic tubing on the end of an 18-gauge needle, ensuring that 1 millimeter of the needle tip protrudes. Practice needle movement by marking a sheet of paper with a starting point for bregma, another 0.2 millimeters laterally, and make a final mark, 1 millimeter caudal to the second mark.

Practice moving the needle from the start point to the second mark, then the final mark, ensuring the movement is repeatable without guides. Next, shave the head of the anesthetized animal. Place the mouse on the work surface, keeping the head in the nose cone to maintain anesthesia.

And apply eye lubricant on both eyes. Now, scrub the injection site with sterile gauze dipped in iodine solution. Then wipe the area with an alcohol scrub pad.

Repeating this process three times. Firmly hold the head with the non-dominant hand laying the mouse flat on the work surface. Drag the prepared 18-gauge needle across the head skin along the midline in a rostral-caudal direction to identify bregma.

Now, move the needle two millimeters laterally and one millimeter caudal to the injection site. Holding the needle vertically, firmly push it through the skin and bone until the tubing is flush with the skin. Retract the needle, rotate, and press through the skin and bone again, repeating the process to create a small hole in the bone.

Using the blunt needle, check for a sufficient hole in the bone, large enough to allow the needle to pass through. Remove the mouse from the nose cone. Proceed to clean off any blood from the injection site using sterile gauze.

Place the mouse in a cage, on a cage warmer until awake. Begin by placing a clean bench pad or drape material on the work surface. Tape the nose cone of an isoflurane vaporizer to the surface, ensuring it faces away from the experimenter.

Attach a 10-millimeter long, 27-gauge needle, with a 45 degree bevel onto a 5 microliter glass syringe. Add silastic tubing to the needle, allowing 3.5 millimeters of the tip to protrude. And secure the tubing with laboratory tape to the syringe body.

Next, draw three microliters of injection media into the glass syringe by initially drawing more than needed, then ejecting the excess until only three microliters remain. Start a laboratory timer in count-up mode and position it for easy viewing during injections. After practicing moving a needle laterally and caudally, prepare the anesthetized mouse for injection by first cleaning the injection site with iodine and then with alcohol three times.

Identify the injection site using an 18-gauge needle, passing it through the craniotomy site. To inject the mouse, first, firmly hold the head of the mouse with the non-dominant hand. Position the head flat on the work surface.

Next, place the needle of the glass syringe through the hole in the skull until the tubing rests on the skin of the mouse. Holding the syringe vertically to the coronal and sagittal planes, slowly inject the media over one minute. Hold the syringe and needle for another minute to minimize backflow.

Then slowly retract the needle. Remove the mouse from the nose cone. Clean off any blood from the injection site using sterile gauze, and place the mouse in a cage on a cage warmer until awake.

Brief exposure to gas anesthesia and the injection of three microliters of fluid into the ventricular system alone did not alter the pulsatile luteinizing hormone secretion. The injection tracts were visible in neural tissue and intersected with the lateral ventricle.