설치류 쥐 모터 피 질에서 Microelectrode 주입으로 인 한 기능적 적자를 평가 하기 위해 테스트 행동

This method can help answer key questions in the field of neural engineering such as whether device implantation has implications for long-term motor function. The main advantage of this technique is that in contrast to endpoint histology, ongoing behavior provides a real-time and global measurement of motor function throughout the course of the study. To begin this procedure inject the animal with a cephalosporin antibiotic subcutaneously to prevent infection and a nonsteroidal anti-inflammatory for pain management.

Next, apply ophthalmic ointment to the animal's eyes to prevent them from drying. Using small animal nail clippers trim the toenails to prevent the animal from scratching the sutures during wound healing after surgery. Subsequently, provide a local analgesic with a subcutaneous injection of bupivacaine in the area of the incision on the animal's head.

Mount the animal on a stereotaxic frame using ear bars to keep the head from moving during the surgery. Then place a circulating water heating pad under the animal to maintain its internal temperature. Afterwards, scrub the surgical area using an alternating Betadine solution and isopropanol scrubs.

Then perform a toe pinch according to the institutional protocol to ensure the animal is under the surgical plane. To prepare the animal for implantation make an incision of approximately one inch along the midline to expose the skull. Bluntly remove the periosteum using a cotton-tipped applicator and stop any bleeding using a piece of gauze.

Then retract the surrounding tissue using alligator clips or hemostats. After the skull is cleaned, apply a few drops of cyanoacrylate-based tissue adhesive to the exposed skull to improve the dental cement bonding in later steps. In the chosen hemisphere, mark the region of the motor cortex corresponding to the fore paw movement by creating a nick in the bone.

Remove a portion of the skull using a 1.75-millimeter rounded tip dental drill taking special consideration not to drill too quickly or too deeply. The drill should be applied to the skull intermittently to avoid overheating. Using saline and light suction remove any bone debris from the skull.

Be careful to not damage the dura during drilling. Then use fine rongeurs or curved micro forceps to remove the remaining bone. Afterward, reflect the dura using a fine-hooked 45-degree dura pick.

Clean any bleeding using a cotton-tipped applicator and saline taking care not to directly touch the brain surface. To insert the microelectrode in the motor cortex 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 head stage interface connector of the electrode is firmly held by the holder.

Using the micromanipulator on the stereotaxic frame carefully position the tip of the electrode over the open craniotomy. Gently lower the electrode approximately two millimeters into the brain. Take caution to avoid any visible vasculature whenever possible.

For manual/by-hand insertion as demonstrated here microforceps are used to insert the silicon nonfunctional dummy microelectrode. Once the electrode is in place carefully release the connector from the universal holder and retract the insertion arm. Carefully clean any bleeding from around the electrode using a cotton-tipped applicator and saline.

Subsequently seal off the craniotomy around the implanted electrode using a silicone elastomer. Then fix the electrode to the skull using dental cement. Once the cement is completely dry bring the edges of the incision together at the front and back of the cement head cap and suture them shut.

For nonfunctional implants, the entire incision may be closed. To begin open field grid testing place the animal in the center of the grid facing away from the tester. Allow the animal to run freely for three minutes while recording a video.

When the animal has completed testing remove it from the grid and clean the grid thoroughly with chlorine dioxide-based sterilant. Analyze the number of grid lines crossed, the total distance traveled, and the maximum velocity of the animal as metrics of the gross motor function using a video-tracking software. For ladder testing one week of training is required prior to recording the presurgery baseline scores.

Then transfer the animal to a temporary clean holding cage to begin ladder testing. Set the ladder up so that it bridges two cages. The start end of the ladder rests on a clean cage, and the finish end rests in the animal's home cage to serve as a motivation to complete the run.

Position the video camera used to film ladder testing on a tripod at the center of the ladder around the rung height. With a video camera running hold the animal at the starting line of the ladder allowing its front paws to touch the first rung. Allow the rat to cross the ladder at its own pace.

The time elapsed between the moment when the animal's paw touches the first rung and the finish line of the third to last rung will determine the animal's time to cross. If the animal turns around on the ladder or does not move for a period of 20 seconds, consider the animal fails. Assign it a penalty score time for each failed run and determine the penalty time by the slowest performance recorded during presurgery testing.

Allow each animal to cross the ladder five times per testing day with approximately one minute rest in between each run. Average the fastest three runs per day as a metric of fine motor function. Additionally, record the number of times each of the front paws slip off the rungs using a video-tracking software.

For grip strength testing, position the grip strength meter on the edge of the counter with the grip handlebars extended over the floor. Then allow the animal to grab the handle bars with both front paws while holding the animal by the base of the tail. Once the animal has a firm grip with each paw pull it away from the meter by the base of the tail with slow and steady force.

Record the maximum grip strength exerted by the animal by the grip strength meter. Test each animal three times per testing day with approximately two minutes rest in between each test. As a metric of fine motor function, record and average the maximum grip strength output from each of the three trials.

Significant difference in open field grid performance between control and implanted animals was seen only in the first week of testing following the recovery period. There was no difference in performance for the remainder of the study indicating no long-term gross motor function deficit. Aside from the first week of post-recovery testing control animals cross the ladder significantly faster than implanted animals.

The implanted animals were not able to maintain presurgery ladder crossing speeds indicating possible deficits in fine motor function. This is further supported by the increased occurrence of right paw slips in implanted animals correlating to the region of the brain receiving an implant. Control animals often achieved significantly higher grip strength results compared to implanted animals over the course of the study.

Additionally, implanted animals perform significantly lower than baseline performance immediately following recovery, but improved over time and returned to baseline performance. Shown here is the correlation of ladder performance to immunoglobulin G fluorescence intensity, a marker of blood-brain barrier permeability. A correlation coefficient of 0.901 was found indicating a very strong correlation.

Once mastered this technique can be performed in approximately 30 minutes per animal per testing session. While attempting this procedure it's important to reduce environmental variables as these may distract the animals from the task at hand. Following this procedure, other methods including immunohistochemistry or other light imaging techniques can be performed in order to investigate cellular or tissue response to implanted microelectronics.

After watching this video, you should have a good understanding of how to perform a rapid, simple and repeatable assay for gross and fine motor deficits that may arise from device implantation or injury in the primary motor cortex.