This protocol details a surgical procedure for performing spinal cord surgery and for implanting and securing an optical shank over the spinal cord in rodents.
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.
The spinal cord facilitates a range of essential central nervous system functions, from coordinating motor behaviors to regulating homeostatic processes such as respiration1,2. 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 areas3,4,5,6. Although devices have been developed to enable such interrogations7,8,9,10,11, 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 bending8,12,13. 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.
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
2. Cervical spinal cord exposure
3. Epidural placement of the device
4. Post-surgical procedures
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.
Neuromodulation and therapeutic interventions of the spinal cord often require the placement of probes in precise, targeted segments3,4,7,13. 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 layer20,21. 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.
The authors have nothing to disclose.
S.S. is partially funded by a Four-Year Doctoral Fellowship from the University of British Columbia. A.M. is partially supported by a Canada Graduate Scholarship – Master's from the Canadian Institute of Health Research (CIHR). D.S. acknowledges funding from the Michael Smith Health Research British Columbia Scholar Award. This work was partially funded by the Government of Canada's New Frontiers in Research Fund – Transformation (NFRFT-2020-00238). The schematic in Figure 1 was generated using Biorender.com, and the 3D model was obtained with permission from sketchfab.com.
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 |
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