In neuroscience research, stable and reliable animal models are the cornerstone for exploring life mechanisms and validating treatment strategies. As a core technology for constructing various neuroscience research models, the standardization and accuracy of mouse craniotomy surgery are crucial. A successful surgery not only minimizes animal damage to the greatest extent possible, but also ensures the accuracy and reproducibility of subsequent experimental data.

Mastering craniotomy surgery proficiently requires a deep understanding of the anatomical structure of the brain and repeated refinement of fine operating techniques. In practical operation, many researchers often face the following typical problems:

✅ How to safely remove the skull and ensure the integrity of brain tissue?

✅ How to balance the fixation strength and the integrity of the imaging field when fixing cranial window slides?

✅ What are the important details to pay attention to during aseptic operation and postoperative animal care?

This article will revolve around the classic process of mouse craniotomy surgery, systematically outlining the core methods of this technique through clear step-by-step demonstrations and analysis of key points. As the first article on "Two Photon Fluorescence Imaging Mouse Preparation Surgery", this article aims to help readers solidify their surgical foundation. In the future, we will continue to explain more detailed surgical procedures such as GRIN Lens implantation, spinal cord fenestration, and the combination of two-photon and EEG electrode implantation, accompanying you in continuously improving your technology on the path of scientific research.

　　Kind Reminder:Considering your viewing experience, we have softened some of the surgical images. Please feel free to watch.

　　

　　PART.01Preoperative preparation

1. Surgical instruments: two fine forceps, one double headed micro scraper, and one spring scissor.

2. Experimental consumables: Prepare the necessary consumables and physiological saline (1% ceftriaxone sodium) for the experiment.

3. Instrument disinfection: All instruments or implants used during surgery need to be disinfected before use, and can be disinfected with 75% ethanol. If conditions permit, regular high-pressure sterilization can be performed.

4. Surgical table disinfection: Before use, the surgical table should be wiped with 75% ethanol for disinfection. The surgical table should be padded with sterile cloth, clean absorbent pads or towels, and these materials must be replaced after each surgery.

　　PART.02Anesthesia and fixation

1. Place the mice in the anesthesia chamber for induction anesthesia, with gas flow rate and anesthesia scale set according to the laboratory's gas anesthesia machine. When a mouse shows a significant decrease in breathing and relaxation of its limbs, it indicates that the mouse has entered an anesthetized state.

2. Fix the anesthetized mice on the stereotaxic device and maintain anesthesia. Pay attention to the mice's breathing during the surgery.

　　PART.03hair removal

1. Apply erythromycin eye ointment evenly to the eyes of mice, and the amount should completely cover the eyes of the mice, so that the eyes are not exposed.

2. Dip a medical cotton swab into hair removal cream and apply it evenly to the hair roots against the direction of hair growth. After about 2 minutes, use a cotton swab to wipe off the hair on the head.

3. Use a medical cotton swab dipped in physiological saline to clean the remaining hair removal cream on the mouse head.

4. Use iodine to wipe and clean the depilated skin.

PART.04Expose and clean the skull

1. Use a spring clipper to remove the scalp.

2. Peel off all excess fat from the exposed mucosa and two muscle junctions on the skull.

3. Use a micro double headed scraper to scrape off residual mucosa, connective tissue, etc. on the surface of the skull.

4. Hold the ear wash ball and blow the surface of the skull until completely dry.

　　PART.05have an operation on the skull

1. Place a circular glass slide of appropriate size on the surface of the skull using fine forceps, with the virus injection hole or positioning site as the center.

2. Adjust the speed of the skull drill to low gear, grind it along the size of the slide once, gently blow away the debris with an ear wash ball, repeat this operation until the grinding range fully matches the size of the slide, then adjust the speed to medium high gear and continue grinding.

When cracks appear during the grinding of the skull, adjust the rotation speed of the skull drill to a low level, continue grinding, and repeat the operation until the entire skull window is completely broken.

4. Use fine tweezers to slowly remove the skull fragment.

　　PART.06Break up bone fragments

Use fine tweezers to needle down from the side of the cranial window, gently remove the surrounding bone fragments, and repeat this operation until there are no fragments around the cranial window.

PART.07Install glass slides

1. Use fine tweezers to gently place the cleaned glass slide onto the exposed brain tissue; Gently press the glass slide with tweezers to tightly adhere to the polished craniofacial surface and embed it underneath the skull; Check for bubbles under the slide under a stereomicroscope. If there are bubbles, remove the slide and add physiological saline dropwise. Repeat this process until there are no more bubbles.

2. Use a moisture absorbing paper tip to absorb excess liquid that seeps out of the glass slide, gently dry the surface of the skull using an ear wash ball, and apply a small amount of biological tissue adhesive to the edge skin, entire skull, and cranial window edges in an outward to inward order using a small brush.

3. Wait for the biological tissue adhesive to dry completely, then dip a dental applicator stick into the 406 adhesive and apply it within the same range as above. Wait for the 406 adhesive to dry.

4. Dip a moisture absorbing paper tip into the mixture of 502 and dental cement, and apply a small amount multiple times to the edge of the skull window. During the entire operation, be sure not to let the glue flow to the center of the slide.

PART.08Install the head piece

1. Dissolve and mix the dental cement with 502 glue, apply a thin and uniform layer to cover the exposed skull, completely seal the exposed skull, and install the head piece before the dental cement dries.

2. Fill the gaps or weak connections between the dental cement and the head piece to enhance its stability.

3. Wait for the dental cement to dry completely, then place the mice in an insulated box and wait for them to wake up.

PART.09Postoperative care

1. To provide good living conditions for animals, it is recommended to keep them in a single cage and sprinkle feed inside the cage to avoid rubbing and colliding with the animal's head during feeding or fighting, which may cause visual field deviation or head drop.

2. 1-7 days after surgery, inject ceftriaxone sodium solution into the animal's abdominal cavity once a day.