Implantation of Optoelectronic Devices in the Rodent Spinal Cord

Abstract

Neuromodulation can provide diagnostic, modulatory, and therapeutic applications. While extensive work has been conducted in the brain, modulation of the spinal cord remains relatively unexplored. The inherently delicate and mobile spinal cord tissue imposes constraints that make the precise implantation of neural probes challenging. Despite recent advances in neuromodulation devices, particularly flexible bioelectronics, opportunities to expand their use in the spinal cord have been limited by the surgical complexities of device implantation. Here, we provide a series of surgical protocols tailored specifically for the implantation of a custom-made optoelectronic device that interfaces with the spinal cord in rodents. The steps to place and anchor an optical shank on a specific segment of the spinal cord via two different surgical implantation methods are detailed here. These methods are optimized for a diverse range of devices and applications, which may or may not require direct contact with the spinal cord for optical stimulation. To elucidate the methodology, the vertebral anatomy is referenced first to identify prominent landmarks before making a skin incision. The surgical steps to secure an optical shank over the cervical spine in rodents are demonstrated. Procedures are then outlined for securing the optoelectronic device connected to the optical shank in a subcutaneous space away from the spinal cord, minimizing unnecessary direct contact. Behavioral studies comparing animals receiving the implants to those undergoing sham surgeries indicate that the optical shanks did not adversely affect hindlimb or forelimb function seven days post-implantation. The present work broadens the neuromodulation toolkit for use in future studies aimed at investigating various spinal cord interventions.

Introduction

The spinal cord facilitates a range of essential central nervous system functions, from coordinating motor behaviors to regulating homeostatic processes such as respiration^1,2. Elucidating the role of the sophisticated network of circuitry across the spinal cord requires interfaces, whether for electrical stimulation, recording, drug delivery, or optical stimulation to targeted areas^3,4,5,6. Although devices have been developed to enable such interrogations^7,8,9,10,11, specialized surgical techniques are required for their chronic implantation in the spinal cord⁴. In particular, the spinal cord and associated vertebrae have increased susceptibility to mechanical deformations caused by natural movements such as extension and bending^8,12,13. These unique characteristics of the spinal cord make it intrinsically challenging to ensure that the implanted probes remain stable, functional, and secured at a specific segment over extended periods of time.

Herein, a surgical protocol is described for inserting and securing an optical shank in a targeted segment of the spinal cord (Figure 1A). Since interfacing with the cervical region in particular has been shown to introduce unique challenges⁹, the implantation steps are specifically demonstrated over the C5 cervical region. It is posited that the complexity of the cervical spine arises from its deeper positioning and the abundance of musculature, a characteristic not as prominent along the rest of the spinal cord. Regardless, the procedures outlined in this protocol are designed to be adaptable for surgeries across various spinal cord regions. Stepwise instructions are provided to locate and identify spinal cord segments using pronounced anatomical "landmarks" identifiable from over the skin (Figure 1B). The protocol then elucidates two techniques for surgical implantation: one tailored for probes that require direct contact with the spinal cord, and another for probes that may not require direct contact. The described steps are designed to be reproduced by any researcher with training in rodent survival surgery.

This protocol encompasses step-by-step instructions for the implantation of an optoelectronic device (18 mm x 13 mm) with an attached flexible optical shank over the C5 cervical level. The implantable device is secured subcutaneously caudal to C5 and consists of a microscale light-emitting diode (µLED) indicator, which illuminates when spinal cord optical stimulation occurs, providing live feedback of device functionality. The effect of the implanted optical shank on the natural motor function was assessed on rodents who had received implants and was compared to rodents with sham surgeries. Results indicate that the probes do not adversely affect the natural hindlimb and forelimb function of the animal seven days post-implantation.

Protocol

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.

Representative Results

An optoelectronic device with its detailed functional diagram shown in Supplementary Figure 2 was implanted in four Long Evans rats. Supplementary Figure 3 shows the final optoelectronic device ready to implant. Three other animals received sham surgeries, which involved a medial laminectomy at C5 without device implantation. The optoelectronic device consisted of a flexible probe with an embedded µLED at the tip which was activated by an integrated LED driver. The LED driver is controlled by a microcontroller with programmable firmware. It also consisted of a device body that was sutured to the muscle layer immediately under the skin. A layer of Parylene-C (~10 µm) is deposited on the whole device using chemical vapor deposition (CVD). A second layer of Polydimethylsiloxane (PDMS) (~800 µm) covered the optoelectronic device body (Supplementary Figure 3) to form a soft interface with the tissue. The probe tip was secured at C4 with the µLED hovering over C5. A µLED indicator was utilized on the device (with its light visible from under the skin) that simultaneously turned on with the µLED of the optical shank for live verification of device functionality. The animals were monitored for a period of 7 days following surgery to confirm the sustained reliability of their performance over time (Figure 4B).

The motor functions of the animals were evaluated using the Martinez open-field locomotor rating scale¹⁹. To assess open-field behavior, two trained observers who were unaware of the treatment groups conducted the tests before the operations as well as on days three, five, and seven post-surgery. Following data collection, the Mann-Whitney U test was conducted to determine differences at each timepoint for both the forelimb and hindlimb scores between the implant and sham groups. Our analysis indicates a similar forelimb function score in implant and sham groups by day seven (Figure 5A). Similarly, there were no statistically significant differences between the groups for the hindlimb scores across all timepoints (Figure 5B).

Post-mortem verification was performed 7 days post-implantation to confirm if the probe and the device body had remained in place. No visible detachment of the suture or the device was found. Furthermore, pulling on the device body did not cause its detachment from the tissue (Supplementary Figure 4A). The previously dissected and sutured muscles were then exposed over the spinal cord, and it was confirmed that the probes remained securely cemented over the spinal cord (Supplementary Figure 4B). Similar to the device body, the probe head was pulled back successively against the cementing point to assess its attachment to the probe-lamina mechanical joint.

Figure 1: Schematic overview of device implantation and anatomical landmarks. (A) Demonstrating the placement of the probe over the spinal cord and subcutaneous placement of the device. (B) 3D model indicating the landmarks used to determine spinal cord levels. The C2, T2, and T10 spinous processes are shown for reference. Darker colors indicate the corresponding level. Please click here to view a larger version of this figure.

Figure 2: Exposing the spinal cord and preparing a subcutaneous pocket. (A) The stereotaxic is positioned on the animal. (B) C2 spinous process and (C) T2 spinous process are identified via palpation. (D) An incision through the skin and subcutaneous adipose layer is made to expose dorsal musculature at the point of interest at the cervical level. (E) Through blunt dissection of the dorsal musculature, the cervical vertebrae are exposed. (F) A subcutaneous pocket is created to secure the implantable device caudal to the incision site. (G) A retractor is placed following adequate dissection to expose cervical vertebrae and the ball-shaped muscle, completely covering C2 and partially masking C3. The dashed line indicates the ball-shaped muscle. Once the cervical spinal cord has been exposed, either (H) two lateral laminectomies at C5 and C6 are done for probe placement under the vertebrae, or (I) medial laminectomy is made at C5 for probe placement over the vertebrae. Asterisks indicate the site of lateral laminectomy. Scale bars = 3 mm. Please click here to view a larger version of this figure.

Figure 3: Device implantation and probe placement. (A) The device is placed in the subcutaneous pocket. (B) The device is sutured to the musculature. (C) The probe is secured on top of the C5 lamina, which had received medial laminectomy. (D) The device is placed under the C5 and C6 lamina which both received lateral laminectomy. In both (C) and (D), the tip of the probe is cemented at an intact C4. Scale bars = 3 mm. Please click here to view a larger version of this figure.

Figure 4: Marking device and verifying functionality post-surgery. (A) The location of the device may be optionally marked over the skin after suturing for the ease of its identification post-surgery. (B) The figure depicts an animal post-implantation. The functionality of the device was validated by observing the indicator µLED visible beneath the skin, confirming the successful operation of the device (the bump on the right side of the animal is where the device body is implanted). Please click here to view a larger version of this figure.

Figure 5: Martinez open field behavioral scores in sham and implant groups for forelimb and hindlimb performance over time. The plots illustrate the mean behavioral scores for (A) forelimb and (B) hindlimb open field assessments across four timepoints: 0 (baseline), 3 days, 5 days, and 7 days post-implantation (DPI). Error bars represent the standard error of the mean (SEM). Significant differences (p < 0.05) between the sham and implant groups are indicated with asterisks (*) at specific timepoints. The figure legend indicates the sham groups displayed by the dotted line, while the implant group is shown by the solid line. The sham sample size was n = 3, and the implant was n = 4. The non-parametric Mann-Whitney U test was used to evaluate the significance of differences between groups at each timepoint. Please click here to view a larger version of this figure.

Supplementary Figure 1: Illustration of laminectomy. Dashed lines indicate the regions to resect for (A) two lateral laminectomies for probe placement under the vertebrae, and (B) medial laminectomy for probe placement over the vertebrae. Please click here to download this File.

Supplementary Figure 2: Schematic of the optoelectronic device. The detailed block diagram of the device is shown. The top left block depicts a wireless power receiver antenna resonant LC tank. The received power is rectified and fed into a low-dropout voltage regulator (LDO). A microcontroller unit automatically activates the device based on the programmed parameters, and an LED driver powers any µLEDs embedded in the probe. Please click here to download this File.

Supplementary Figure 3: Optoelectronic device. The final optoelectronic device with biocompatible encapsulation connected to an optical shank comprising 1 µLED at the tip. The dashed rectangle depicts the location of the µLED. Please click here to download this File.

Supplementary Figure 4: Post-mortem verification of device stability. Seven days after implantation, (A) the device body had remained sutured to the musculature in the same position it was implanted, and (B) the cemented probe remained secured on top of the C4 lamina. Please click here to download this File.

Discussion

Neuromodulation and therapeutic interventions of the spinal cord often require the placement of probes in precise, targeted segments^3,4,7,13. Given the inherent mobility of the spinal cord, the probe must be reliably secured to enable chronic studies. Based on the specific application, it may be important to control whether the probe is in physical contact with the spinal cord, or if the contact can be reduced to lessen the inflammatory tissue response when possible. Therefore, surgical steps for each of the two methods are described. The protocol specifically details how to place a probe in the cervical segment of the spinal cord at C5. Nonetheless, using the described landmark for T2 or T10 of the spinal cord, the probe can be similarly placed in a precise location over the thoracic or the lumbar region by counting down the vertebrae from T2 or T10, respectively, once they are exposed. Furthermore, to minimize spinal cord tissue damage, we secured the device body, which is often larger and more rigid compared to the connected probe, in a subcutaneous space away from the spinal cord.

There are some critical points to implanting the device that is coupled with the probe. First, it is critical to decide on the location of the device body prior to cementing the probe. This ensures the distance between the tip of the probe and the device body is optimized to reduce tension on the probe as well as avoid having extra probe length, which can, for example cause probe twisting or displacement. Essentially, the goal is to ensure that the length of the probe is similar to the distance from the subcutaneous space where the device body is placed to the targeted spinal cord region where the probe is cemented. By conducting terminal surgery procedures in which various probe lengths are tested, the optimum size can be determined for a targeted segment.

To maintain sterility, the device should be handled carefully to prevent contact with the outer layer of the skin during insertion into the subcutaneous pocket. Such contact can compromise the device's sterility, potentially leading to post-operative infection. In addition, it is important to minimize the amount of force applied to the device when holding it with forceps to prevent damaging its coating, which is typically a thin protective, insulative, and sterile layer^20,21. Removing the coating may drastically reduce the lifespan of the device by, for example, shortening the circuit, causing electrical shock to the animal, and/or provoking an inflammatory response in the body. Handling the device with plastic tip forceps may help reduce such complications.

When suturing the device to soft tissue, it is important to avoid suturing to subcutaneous adipose tissue. As observed in preliminary trials, fat layers are not a reliable anchoring point for sutures since they are prone to rupture. Instead, the device body was sutured to an adjacent muscle layer in the subcutaneous space using non-absorbable sutures for the permanent placement of the device in the body. On the other hand, when securing the probe to the spinous processes, it is important to ensure the site to which the probe is being secured is dry before applying the cement. Wet bone/probe prolongs the curing time and may result in the complete failure of the process.

There are some critical considerations associated with an implantable device that need to be carefully addressed prior to implantation surgery. (1) Electrically active parts of the device must be encapsulated by an insulative passivation layer. Any deprivation in the passivating layer might cause device functional failure. (2) The implantable must be thoroughly sterilized according to the facility animal protocol. (3) The junction between the device and the neural probes or the stimulatory shanks must be securely formed. The connection will go through repeatable mechanical stress due to constant animal movements. (4) The neural probes or stimulatory shanks attached to the device must be flexible and stretchable enough to avoid snapping at various points.

The described protocol may be extended to implant devices in animal models of different sizes. After identifying the anatomical landmarks, the described surgical methods can be methodically customized to secure any neural probes or stimulatory shanks at targeted segments of the spinal cord and implant their associated control modules. However, depending on the application, different devices can have varying sizes, materials, and thicknesses from the one implanted in this paper; for example, devices connected to an external control module require additional considerations. Additionally, it must be noted that while this protocol is tailored for optogenetic stimulation, other neuromodulatory applications, such as drug delivery or electrical stimulation/recording, require slightly different surgical procedures. Specifically, these applications need subdural implantation to ensure direct contact with the spinal cord beneath the dura mater⁷. However, for optogenetics, intimate contact with the tissue is typically unnecessary because the rodent dura mater does not significantly impede light penetration, which enables light sources to be placed epidurally¹⁰.

Materials

Name	Company	Catalog Number	Comments
Adson Forceps	Fine Science Tools	11027-12
Alm 3 Point Retractor	Fine Science Tools	17010-10
Buprenorphine / Vetergesic	CDMV	124918	Manufacturer provides at 0.3 mg/mL but must be diluted to 0.03 mg/kg for use in rats
Chlorhexidine 2% Solution	Partnar	PCH-020
Curved Long Hemostat Forceps	KaamKaaj Tools	14.5	Curved Long Hemostat Forceps with A Stainless Steel Ratchet Locking Tweezer
CVD Parylene Machine: SCS Labcoter 2	Specialty Coating Systems	PDS 2010
Dental Cement - Catalyst	Parkell, Inc	S371
Dental Cement - Metabond	Parkell, Inc	S398
Dental Cement - Powder	Parkell, Inc	S396
Forceps with Replaceable Plastic Tips	Fine Science Tools	11980-13
Friedman-Pearson Rongeurs	Fine Science Tools	16121-14
Isoflurane USP	Fresenius Kabi	CP0406V2	Provided at 5% for induction and 2% for mainentance through precision vaporizer
Isopropyl Alcohol 70%	McKesson	350600
Lacri-Lube Sterile Eye Ointment	Refresh
Long Evans Rats	Charles River Laboratories	6
Low temperature solder paste	Chip Quik Inc.	11.38
Magnets	Radial Magnets, Inc.	0.53	Magnet Neodymium Iron Boron (NdFeB) N35 (3.00 mm x 1.00 mm)
Olsen-Hegar Needle Holders with Suture Cutters	Fine Science Tools	12002-12
PDMS: SYLGARD 184	Sigma Aldrich	761036
Scalpel Blades - #15	Fine Science Tools	10015-00
Scalpel Handle - #3	Fine Science Tools	10003-12
Solder flux	Chip Quik Inc.	14.25
Stereotaxic Frame	David Kopf Instruments	Model 900
Sterile Kwik-Sil Adhesive	World Precision Instruments	KWIK-SIL-S
UV Flashlight	Vansky	19.99
Wireless Charger	Nilkin	NKT06
Wireless Charging coil	TDK Corporation	WT202012-15F2-ID