Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Summary

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

Abstract

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

Introduction

Intracortical microelectrodes were originally used to map the circuitry of the brain, and have developed into a valuable tool to enable the detection of motor intentions which can be used to produce functional outputs¹. Detected functional outputs can offer individuals suffering from spinal cord injuries, cerebral palsy, amyotrophic lateral sclerosis (ALS), or other movement-limiting conditions the control of a computer cursor^2,3 or robotic arm^4,5,6, or restore function to their own disabled limb⁷. Therefore, intracortical microelectrode technology has emerged as a promising and quickly growing field⁸.

Due to the successes seen in the field, clinical studies are underway to improve and better understand the possibilities of BMI technology^5,9,10. By realizing the full potential of communication with neurons in the brain, the rehabilitation applications are perceived as limitless⁸. Although there is great optimism for the future of intracortical microelectrode technology, it is also well-known that microelectrodes eventually fail¹¹, possibly due to an acute neuroinflammatory response following implantation. The implantation of a foreign material in the brain results in immediate damage to the surrounding tissue and leads to further damage caused by the neuroinflammatory response that varies depending on properties of the implant¹². In addition, an implant in the brain can cause a microlesion effect: a reduction in glucose metabolism thought to be caused by acute edema and hemorrhage due to the device insertion¹³. Furthermore, the signal quality and the length of time that useful signals can be recorded are inconsistent, regardless of the animal model^11,14,15,16. Several studies have demonstrated the connection between neuroinflammation and microelectrode performance^17,18,19. Therefore, the consensus of the community is that the inflammatory response of the neural tissue that surrounds the microelectrodes, at least in part, compromises electrode reliability.

Many studies have examined local inflammation^11,20,21,22 or explored methods to reduce the damage to the brain caused by insertion^11,23,24,25, with a goal of improving the recording performance over time^14,26. Additionally, we have recently shown that an iatrogenic injury caused by a microelectrode insertion in the motor cortex of rats causes an immediate and lasting fine motor deficit²⁷. Therefore, the purpose of the protocols presented here is to give researchers a quantitative method to assess possible motor deficits as a result of brain trauma following the implantation and persistent presence of intracortical devices (microelectrodes in the case of this manuscript). The behavior tests described here were designed to tease out both gross and fine motor function impairments, and can be used in many models of brain injury. These methods are straightforward, reproducible, and can easily be implemented in a rodent model. Further, the methods presented here allow for a correlation of motor behavior to histological outcomes, a benefit that until recently, the authors have not seen published in the BMI field. Finally, as these methods were designed to test fine motor function²⁸, the gross motor function²⁹, and stress and anxiety behavior^29,30, the methods presented here can also be implemented into a variety of head injury models where the researchers want to rule out (or in) any motor function deficits.

Protocol

All procedures and animal care practices were approved by and performed in accordance with the Louis Stokes Cleveland Department of Veterans Affairs Medical Center Institutional Animal Care and Use Committees.

NOTE: To educate researchers on the decision about the use of a stab injury model as a control, it is recommended to review the work done by Potter et al.²¹.

1. Microelectrode Implantation Surgical Procedure

1. Pre-surgical animal preparation
   1. Anesthetize the animal in an induction chamber using isoflurane (2 - 4%). While under anesthesia, continuously monitor the animal using a vital measurement system to monitor the heart rate and the blood-oxygen content.
   2. Move the animal to a nose cone to continue the anesthetic. Subcutaneously (SQ) inject a cephalosporin antibiotic, e.g. cefazolin (25 mg/kg) and a non-steroidal anti-inflammatory, e.g. carprofen (5 mg/kg) to prevent infection and manage the pain, respectively.
   3. Liberally apply ophthalmic ointment to the animal's eyes to prevent them from drying.
   4. Using small animal nail clippers, trim the toenails to prevent the animal from scratching the sutures during the wound healing. Ensure that the nails are not clipped too short, as this can lead to pain and bleeding for the animal.
   5. Shave the animal's head thoroughly from behind the ears to between the eyes using an electric razor trimmer.
   6. Provide a local analgesia with an SQ injection of bupivacaine (0.3 mL of 0.125% bupivacaine diluted from stock solution) at the top of the animal's head in the area of the incision.
   7. Mount the animal on a stereotaxic frame, using ear bars to keep the head from moving during the surgery. Place a circulating water heating pad under the animal to maintain the animal's internal temperature. 
   8. Apply a sterile drape, e.g., institutionally-approved sterile plastic wrap, to isolate the surgical field.
   9. Scrub the surgical area using an alternating betadine solution and isopropanol scrubs.
   10. Perform a toe pinch according to the institutional protocol to ensure the animal is under the surgical plane.
2. Prepare animal for implantation
   1. Create an incision of approximately 1 in down midline exposing the skull using a No. 10 scalpel blade. Bluntly remove the periosteum using a cotton-tipped applicator, and stop any bleeding using a gauze pad. Retract the surrounding tissue using alligator clips and clean and dehydrate the skull with hydrogen peroxide.
   2. Place a few drops of cyanoacrylate-based tissue adhesive on the exposed skull to improve the dental cement bonding in later steps.
   3. In the chosen hemisphere, mark the region of the motor cortex corresponding to forepaw movement approximately 3 mm lateral to midline and 2 mm anterior to bregma by creating a nick in the bone.
   4. Remove a portion of the skull using a 1.75-mm rounded tip dental drill, taking special consideration not to drill too quickly or too deeply, and supporting one hand on the stereotaxic frame. The drill should be applied to the skull intermittently to avoid overheating³¹.
   5. Reflect the dura using a fine hooked 45° dura pick.
   6. Clean any bleeding using a cotton-tipped applicator and saline, taking care to not directly touch the brain surface.
3. Insertion of microelectrode in motor cortex
   1. Carefully mount the sterilized microelectrode in the universal holder on the stereotaxic frame, taking caution not to bump the shank of the electrode. Ensure that the headstage interface connector of the electrode is firmly held by the holder.
      NOTE: Here, a non-functional Michigan-style silicon shank electrode measuring 2 mm x 123 µm x 15 µm was used, and the shank was inserted using fine forceps.
   2. Using the micromanipulators on the stereotaxic frame, carefully position the tip of the electrode over the open craniotomy.
   3. Gently lower the electrode approximately 2 mm into the brain using the micromanipulators as a measurement guide (depending on the choice of electrode, an automated insertion at controlled rates may be required.) Take caution to avoid any visible vasculature whenever possible. Once the electrode is in place, carefully release the connector from the universal holder and retract the insertion arm.
   4. Carefully clean any bleeding from around the electrode using a cotton-tipped applicator and saline.
   5. Seal off the craniotomy around the implanted electrode using a silicone elastomer.
   6. Fix the electrode to the skull using dental cement.
   7. Once the cement is completely dry, bring the edges of the incision together at the front and back of the cement headcap and suture them shut.
4. Post-operative care
   1. Allow the animal to recover on a circulating water heating pad while continuing to monitor its vital signs. Avoid using heat lamps as the temperature from lamps is more difficult to control and animals can overheat.
   2. Once the animal is fully awake, move the animal to a clean cage with easy access to food and water.
   3. During post-operative days 1 - 3, provide the animals with SQ cephalosporin antibiotic (25 mg/kg) and a non-steroidal anti-inflammatory (5 mg/kg) to prevent infection and manage their pain.
   4. Monitor the animals daily for the signs of pain or discomfort, bleeding, weight change, or suture issues through at least post-operative day 5.

2. Behavioral Testing

1. For all behavior testing, test the animals 2x per test in the week prior to the electrode implantation surgery to calculate their pre-surgery baseline scores. Following the surgery, allow the animals to rest for 1 week before beginning behavior testing 2x per week on each test. Consistent testing conditions should be used throughout the study for both pre- and post-surgical testing to minimize the effects of stress on the performance, which could result in a measurement of anxiety.
   1. Clean all testing equipment with a chlorine dioxide-based sterilant at the beginning of each testing session and after each animal.
   2. Film the open field grid and ladder testing. These tests require a video camera (1080p, minimum of 15 fps, 78° diagonal field of view), a laptop, and room to store the video data.
   3. At the beginning of each testing day, bring the animals to the testing room and allow them to acclimate for at least 20 min before beginning the testing. The room should be light and temperature-controlled, and the same personnel should complete all testing. Ideally, the same room will be used for all animals throughout the course of the testing with no changes to the room.
   4. Use food rewards to encourage the animals to complete the tasks, especially during the ladder training. Cereals or small pieces of banana chips or crackers make good rewards.
   5. Normalize all weekly testing performances to the pre-surgery scores for each individual animal (Equation 1).
      Equation 1:
2. Open field grid testing
   NOTE: The open field grid test was built in-house and has a running surface of 1 m² with approximately 40-cm high opaque side walls. The bottom running surface of the grid is divided into 9 equal squares from the underside using tape (Figure 1A). The recording camera is permanently mounted above the center of the grid on scaffolding.
   1. To begin open field grid testing, place the animal in the center of the grid facing away from the tester.
   2. Allow the animal to run freely for 3 min while recording a video.
   3. When the animal has completed testing, remove the animal from the grid and return it to the cage. Clean the grid thoroughly with chlorine dioxide-based sterilant.
   4. Test each animal 1x per testing day.
   5. Analyze the number of gridlines crossed, the total distance traveled, and the maximum velocity of the animal as metrics of the gross motor function using a video tracking software.
      NOTE: The data presented here were quantified manually by trained researchers, but it is preferred to use a recently developed in-house tracking algorithm³².
3. Ladder testing
   NOTE: The ladder test was built in-house and consists of 2 clear acrylic side walls, each 1 m in length, connected by 3-mm diameter rungs spaced at 2 cm apart (Figure 2A). Ladder testing is a skilled test, and therefore requires 1 week of training prior to recording the pre-surgery baseline scores. The protocol for the training and testing is the same.
   1. Move the animal to a temporary clean holding cage to begin ladder testing.
   2. Set the ladder up so that it bridges 2 cages. The start end of the ladder rests on a clean cage, and the finish end rests on the animal's home cage to serve as a motivation to complete the run.
   3. Position the same (or similar) video camera on a tripod at the center of the ladder. The position of the camera should be at rung height and allow for the whole ladder to be seen.
   4. With the video camera running, hold the animal to the starting line of the ladder, allowing their front paws to touch the first rung.
   5. Allow the animal to cross the ladder at their own pace. The time elapsed between the moment when the animal's paw touches the first rung and the finish line at the third to last rung will determine the animal's time to cross.
   6. If the animal turns around on the ladder or does not move for a period of 20 s, consider the animal to have failed the run. Assign the animals a penalty score time for each failed run. Determine the penalty time by the slowest performance recorded during pre-surgery testing²⁷.
   7. Allow each animal to cross the ladder 5x per testing day with approximately 1 min rest in between each run.
   8. Average the fastest 3 runs per day as a metric of fine motor function. Additionally, record the number of times each of the front paws slips off the rungs using a video tracking software.
      NOTE: The data presented here were quantified manually by trained researchers, but it is preferred to use a recently developed in-house tracking algorithm using Dona et al.³².
4. Grip strength testing
   1. Calibrate the grip strength meter before each testing session, and measure the strength in grams.
   2. Position the grip strength meter on the edge of a counter with the grip handlebars extended over the floor.
   3. Allow the animal to grab the handlebars with both front paws while holding the animal by the base of the tail (Figure 3A).
   4. Once the animal has a firm grip with each paw, pull the animal away from the meter by the base of the tail with slow and steady force.
   5. Record the maximum grip strength exerted by the animal which is displayed on the digital output by the grip strength meter.
   6. Test each animal 3x per testing day with approximately 2 min rest in between each test.
   7. As a metric of fine motor function, record and average the maximum grip strength output from each of the 3 trials.

3. Post-behavioral Protocol

1. Following all behavioral testing (e.g, 8 - 16 weeks after the implantation), anesthetize the animals deeply using ketamine (160 mg/kg) and xylazine (20 mg/kg), transcardially perfuse them, harvest their brains and cryo-slice them, and stain the tissue using immunohistochemical markers to quantify the cellular response around the site of implantation^{33,34,35,36,37,38}.

4. Statistical Analysis

NOTE: A prospective power analysis is strongly suggested for any studies seeking to answer a particular research question. The power analysis, which informs the number of animals required to achieve a statistical significance for a particular study design, should be based on the particular research hypothesis, the design of the experiment, the estimated effect size and variability of the intended treatments, as well the effect size required to achieve clinical or scientific relevance.

1. Conduct statistical analyses using common statistical software.
2. Tabulate the descriptive statistics and display them as Mean ± Standard Error.
3. Analyze the behavioral performance [in the open field grid (step 2.2), ladder (step 2.3), and grip strength testing (step 2.4)] at each weekly time point to compare the control vs. implanted groups using a two-sample t-test. Consider each weekly time point an independent measure.
4. Quantify the longitudinal performance using a mixed effect linear model. The week and the group are fixed factors and an experimental animal is nested within the group as a random effect. An analysis of variance (ANOVA) is used to determine the factor effect with a significance level of p < 0.05.
5. Compare the ladder performance with immunoglobin G (IgG) intensity using a linear regression analysis. Calculate the correlation coefficient by a Pearson's method.

Results

Using the methods presented here, a microelectrode implantation surgery in the motor cortex is completed following established procedures^39,40,41,42, followed by open field grid testing to assess the gross motor function and ladder and grip strength testing to assess the fine motor function²⁷. Motor function testing was completed 2x per w...

Discussion

The protocol outlined here has been used to effectively and reproducibly measure both fine and gross motor deficit in a model of rodent brain injury. Additionally, it allows for the correlation of fine motor behavior to histological outcomes following a microelectrode implantation in the motor cortex. The methods are easy to follow, inexpensive to set up, and can be modified to fit a researcher's individual needs. Further, the behavior testing does not cause great stress or pain to the animals; rather, the researcher...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sprague Dawley rats, male, 201-225g	Charles River	CD
Compac5 anesthesia system	Vetequip	901812
Electric trimmers	Wahl	9918-6171
Stereotaxic frame	David Kopf Instruments	1760
Gaymar heated water pad and pump	Braintree Scientific Inc	TP-700
Vetbond tissue adhesive	3M	07-805-5031
Dental drill	Pearson Dental	O60-0045
Dura pick	Fine Science Tools	10064-14
Silicon shank microelectrode	Made in-house at Cleveland VA Medical Center	N/A
KwikCast silicone elastomer	World Precision Instruments	KWIK-CAST
Teets dental cement	A-M Systems	525000
Webcam HD Pro c920	Logitec	960-000764
Grip strength meter	Harvard Apparatus	565084
Minitab 17 statistical software	Minitab Inc
Open field grid test	Made in-house at Case Western Reserve University	N/A
Ladder test	Made in-house at Case Western Reserve University	N/A
Rabbit anti rat IgG antibody	Bio-Rad	618501