Implantation of Optoelectronic Devices in the Rodent Spinal Cord

All procedures were conducted according to the guidelines of the Canadian Council for Animal Care and overseen by the University of British Columbia Animal Care Committee. Female Long-Evans rats, weighing 350-450 g and aged 6-8 months, were group-housed (21 °C; 12 h:12 h light cycle) and given ad libitum access to a standard rodent diet prior to and after the surgery. The details of the reagents and equipment used for this study are listed in the Table of Materials.

1. Pre-operative preparation

1. Sterilize all surgical tools using an autoclave.
2. Anesthetize the animal with isoflurane (5% for induction and 2% for maintenance) delivered in oxygen at a flow rate of 1 L/min.
3. Transfer the animal from the induction chamber to a heating pad and promptly connect the isoflurane nose cone. Verify that the animal is under a surgical plane of anesthesia by ensuring a complete loss of toe-pinch reflex on both legs.
4. Shave the back of the rat starting at the base of the ears, as shown in Figure 2A.
5. Apply a generous amount of sterile eye ointment over the eyes and inject buprenorphine (diluted to 0.03 mg/kg) and 10 mL lactated ringers subcutaneously (warmed to body temperature).
6. Wipe the shaved area with antiseptic surgical scrub (chlorhexidine) followed by isopropyl alcohol in a circular motion starting from the center of the shaved area and widening the circle diameter. Repeat the soap/alcohol process two more times.
7. Secure the animal in a stereotaxic frame, positioning the head using lubricated ear bars for stability (Figure 2A).
   NOTE: Throughout the entire procedure, consistently provide thermal support, verify anesthesia depth via toe-pinch reflex, and monitor vital signs.
8. Place a sterile surgical drape on top of the animal.

2. Cervical spinal cord exposure

1. Using sterile forceps, begin by palpating the base of the skull. Feel for a prominent spinous process that extends rostrocaudally near the base of the skull; this is C2¹⁴ (Figures 1B and Figure 2B).
2. Proceed with palpation caudal to C2 to find a notably sharp and pointed spinous process, identifiable as T2¹⁵ (Figures 1B and Figure 2C).
3. Using a scalpel, create an incision in the skin, starting from C2 and extending caudally for about 1.5 cm (Figure 2D).
   NOTE: The incision size may vary among animals of different sizes. Ensure identification of the anatomical landmarks in advance and proceed accordingly.
4. Carefully cut through the subcutaneous adipose layer with the scalpel to expose the intact underlying dorsal musculature.
5. Once the dorsal muscles are exposed, perform blunt dissection by pulling them apart from the midline using two Adson forceps (Figure 2E).
   NOTE: It is important to perform blunt dissection (pulling the muscle fibers apart) rather than cutting the muscles to minimize bleeding. Adequate exposure of the dorsal musculature should reveal a ball-shaped muscle (Figure 2G). This muscle completely covers C2 and partially covers C3.
6. Using rongeurs and sterile forceps, lift a flap of skin immediately caudal to the incised area. Use rongeurs to create a small subcutaneous pocket-this will be the location for the device (Figure 2F).
   NOTE: The subcutaneous pocket should be larger than the device itself. It is recommended that the surgeon(s) reposition themselves to face the animal anteriorly to improve control and visibility when opening the subcutaneous pocket.
7. Place a retractor to expose the vertebral column (Figure 2G).
8. Using rongeurs, remove any remaining muscles or tissues covering the vertebrae and begin to identify the spinal cord segments. Immediately caudal to the ball-shaped muscle is C4, followed by C5 and C6 (Figure 2G-I). Once complete, rinse the surgical area with sterile saline and dry with sterile gauze.
   NOTE: The ball-shaped muscle fully envelops C2 and partially extends over C3. The spinal cord segment directly caudal to it, with minimal contact, is designated as C4.
9. Perform laminectomies depending on the intended purpose of the probe.
   1. For a probe under the lamina, perform a lateral laminectomy at C5 and C6, creating a lateralized opening in the lamina for future probe placement (Figure 2H, Supplementary Figure 1A).
   2. For a probe over the lamina, perform a medial laminectomy of C5, ensuring not to remove the lateral aspects of the spinous process-merely exposing a medial pathway for the probe placement (Figure 2I, Supplementary Figure 1B).
   3. Following the laminectomy, rinse the region with sterile saline and dry with sterile gauze to remove any bone debris.
      NOTE: When performing the laminectomy, it is critical to push the rongeurs up against the bone and avoid any downward movements to prevent damage to the cord.

3. Epidural placement of the device

1. Hold the sterile device with plastic-tip sterile forceps (Figure 3A) and drive it inside the subcutaneous opening made earlier in step 2.9.
   NOTE: It is critical to avoid touching the device with non-sterile gloves/tools to maintain device sterility. Use designated sterile forceps for device placement and positioning under the skin.
2. Suture or glue the device to the neighboring muscle layer to keep it secure (Figure 3B).
   NOTE: Use non-absorbable sutures if suturing the device to the muscles. Otherwise, the device is prone to movement after suture absorption in the body. If using glue, ensure the long-term stability and biocompatibility of the glue/adhesive. Avoid securing the device to the subcutaneous fat/adipose layer to ensure reliable anchoring points.
   1. Plan the device body position before proceeding with this step. Since the device will be permanently fastened, issues with probe placement (step 3.4) can arise if the device is located too close or too far from the desired level at the spinal cord.
3. Place the retractor around the spinal cord and open a suitable window to place the probe on the spinal cord.
4. Probe placements:
   1. Probe placement under the lamina: Carefully insert the probe using plastic-tip forceps under the (C5 and C6) laminae by sliding it through the lateralized channels made in step 2.9.1 (Figure 3D).
   2. Probe placement over the spinal cord:
      1. Adjust the probe using plastic-tip forceps to align and place the probe tip on top of the medial window created at C5 in step 2.9.2 (Figure 3C).
5. In a sterile, small, and preferably ceramic container, prepare cement by mixing one scoop of dental cement powder, 3 drops of high tech grease, and one drop of catalyst. Mix with sterile toothpicks until a viscous consistency is achieved.
   NOTE: It is highly recommended that one person who is not performing the surgery prepares the cement so that the surgeon can hold the probe in place and dry the desired area immediately before applying the cement. This step is time-sensitive. Ensure that the surgeon positions the probe prior to preparing the cement. Once the catalyst is applied, the cement will thicken too much to effectively bond with the bone if there is a delay. Practice with the cement prior to surgery to ensure achieving the proper consistency before placing it on the probe/bone.
6. Dry the intended cementing area on the vertebrae completely to make a reliable anchor point. Apply 1-2 drops of dental cement at the tip of the probe, which should be placed on top of the intended vertebral level, in this case, C4 (Figure 3C,D).
   NOTE: It is critical for the vertebrae to be as dry as possible prior to cementing. Otherwise, the cement will not adhere, and the probe will not be secured.
7. Pause for 30 s and gently touch the cement to verify it is cured.
8. If the cement is not completely cured, wait an additional 20 s and re-apply freshly made cement until the probe is robustly secured to the bone.
   NOTE: The cement has bonded properly when it becomes hard and stiff to the touch.
   CAUTION: Applying the cement while it is too liquid may cause it to seep into the spinal cord tissue between the vertebrae. If this occurs, allow approximately 10 s for the cement to thicken to a more gum-like consistency before gently using forceps to remove cement that has come into contact with the spinal cord.

4. Post-surgical procedures

1. Once the surgery is complete, suture the incision site using 5-0 vicryl sutures. Gently remove the animal from the stereotaxic apparatus and transfer it to a heated recovery chamber. For the initial 3-5 days post-surgery, provide the animal with soft, moistened chow, treats, and hydrogel water.
2. Thoroughly monitor the animal twice daily during the first-week post-surgery. Administer buprenorphine and warmed lactated Ringer's injections twice daily for the subsequent 2 days, or longer if signs of pain persist. Continue daily checks until the animal shows no clinical health concerns. Thereafter, check on the animal at least once per week.
3. (Optional) Outline the device perimeter on the skin after surgery and incision closure for a visual indication of the device location in the body (Figure 4A).
   NOTE: This step is particularly useful when the device requires external access, such as devices needing to be powered wirelessly through an external transmitter where antenna coupling is critical, or drug delivery pumps that must be refilled through the skin^16,17,18.