Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

Using the Median nerve of the rat, it is possible to adequately replicate, peripheral nerve lesions in humans. Clinically, most of these lesions occur, in the upper extremity which lends support to the use of this experimental model. In this paper we will show, how to produce different types of Median nerve lesions in the rat.

Additionally, we will illustrate, how to evaluate functional recovery of this nerve using several tests. The Median nerve originates from the spinal cord segments C6 to T1.The Median nerve is highlighted in green. In this video, surgical drapes are not used for demonstration purposes.

Please include a drape while performing the surgery. The skin in the medial aspect of the right arm and pectoral region is incised until the deep fascias plane. Bleeding vessels are carefully ligated.

Next, the Brachial fascia is carefully cut, taking care not to damage the vascular and nerve structures, in the medial aspect of the arm. A pair of scissors is open bluntly beneath, the terminal insertion of the pectoralis major muscle, in order to tease this muscle, away from the underlying Axillary artery and vein, as well as from the terminal branches of the Brachial plexus. The insertion of pectoralis major muscle, is then divided with an electric cautery.

The pectoralis minor muscle is then exposed and cut. The medial nerve is then bluntly dissected from the brachial vessels and from the Ulnar nerve until the elbow region. It is now possible to observe, the different terminal branches of the Brachial plexus, namely the Median, the Ulnar, the Radial, the Axillary, and the Musculocutaneous nerves.

With the Median nerve exposed and isolated different mechanical injuries can be induced. A Sham group can be created, by only dissecting the Median nerve. In the crush group, the Median nerve is compressed, using microsurgery forceps for 15 seconds.

The excision group results from the removal of, a 10 millimeter long segment of the Median nerve. The proximal stump of the Median nerve is ligated with the 8-0 nylon suture to prevent axonal growth. Alternatively, it is possible to rotate the excised segment 180 degrees and use it as an autologous nerve graft.

In this case, the proximal and distal stumps of the section in the media nerve are sutured to the nerve graft using 10-0 nylon stitches. Physiotherapy is part of the standard of care of patients recovering from peripheral nerve injuries. There are multiple strategies to provide post-operative physiotherapy to rats, which have been submitted to Median nerve injuries.

It is important to choose young rats and allow them to have contact with the physiotherapy instruments before surgery. These ensures an easier and faster adaptation to the exercise settings. Moreover, the physiotherapy instruments should allow the rats to have some degree of freedom and ideally enable them to perform fine movements with the forepaws.

There are several ways of performing physiotherapy. For example, individual spheres can be used. Put the rat inside the sphere and then place it in a room with few obstacles.

Let it explore the room for half an hour. Do this once a day. Rats can also be housed in individual cages.

With incorporated running wheels. In this way, rats can exercise, as much as they want and whenever they want it. Another possibility is accommodating, the rats in groups in larger cages.

Personalize the cages using leathers, ropes, running wheels, and other environmental enrichment elements. These enables them ample freedom of movement, in agreement with their natural exploratory behavior and allows the rats to get familiarized, with some of the elements present, in the Functional tests. Before performing the Functional tests, familiarize the rats with the food treats you chose to use as positive reinforcement.

Conduct the tests in the evening, when the rats are naturally more active. Start with a Grasping test. Place the rat on the grid and lift it by its tail letting it grab the grid.

When the rat is able to grab the grid, with similar strength with both forepaws. It means the Median nerve is functioning and the Grasping test is considered positive. If it can't grab the grid with the injured paw, the Grasping test is considered negative.

For the Pin Prick test, place the rat on the platform and wait a few minutes until exploratory and major grooming activities subside. Start when the rat is stationary and standing on the forepaws. With the help of the mirror, insert the Von Frey hair through the mesh to poke the palmar aspect of the forepaw.

Repeat this five times on each forepaw, alternatively, waiting a few seconds after each evaluation. Score the withdraw response as follows, zero for no withdrawal response, if the rat slowly removes the paw from the filament and two if the rat quickly responds to the stimulus and removes the paw or licks the paw. Place the rat at the bottom of the ladder and gently touch the tail of the rat.

Start the timer once the rat starts walking and stop it once its snouts crosses the boxes entrance. Record the time and repeat the test three times. Put the rat at the bottom of the rope and persuade it to climb it by gently touching its tail.

Consider the test valid if the animal does not, hesitate during the task or stops climbing. Start the timer once the animal initiates the climbing and stop it the moment the rat's snout crosses the platform's entrance. Give the rat a snack after performing, the tasks correctly and allow it, some minutes of rest before repeating it.

Place a piece of graph paper on the corridors floor. Then, grab the rat by its tail and let it hold the painting brush, impregnated in methylene blue. Place it at the entrance of the corridor to make him walk inside the box.

Remove the graph paper from the corridors floor and repeat the tests until you obtain a good, representative impression of both forepaws. From the obtained prints, select one with a good print of successive forepaws and measure the following parameters. Print Length, Finger Spread, Intermediate Finger Spread and Stance Factor.

Finally measure the distance between the second finger of two successive prints, the Stride Length. To perform the Thermographic Evaluation, place a piece of double side glue on the sponge. Place the rat on it and glue the forepaws as shown.

Wait a minute to dissipate the heat exchange from handling the rat's paws. Grab the camera at an angle of 90 degrees and approximately 30 centimeter away from the rats. Make two acquisitions.

First, a full body thermogram, then, approximating the camera to the rat's upper body, make the second one. From the obtained thermogram, we find a region of interest corresponding to the palmar area of the forepaws. Measure the mean, minimum and maximum temperature values of that area on both paws and compute the temperature differences.

To perform the following evaluation, expose the Median nerve on both sides as explained before. Start by placing the rat on its back and put the electrode just shown in the skin. The Ground electrodes on the rat's thigh and the Stimulation and Recording electrodes, on the left upper limb.

Put a recording electrode, on the Flexor digitorum sublimis muscle belly, and the Stimulation electrodes, approximately in the Median nerve. Start the acquisition with a Stimulus amplitude of 10 millivolts and gradually increase it, in 10 millivolt steps until reaching two volts. During the acquisition, register the Stimulus amplitude for the first visible fingers flexion.

That's the Motor stimulation threshold. The minimum Stimulus amplitude, required to obtain a Compound Muscle Action Potential, C.M.A.P.is a Neurological stimulation threshold. Finally, select a representative C.M.A.P, in response to a Supramaximal stimulus and measure the following parameters, Latency, Amplitude, and Duration.

To perform the Flexion Strength Evaluation, Firstly pass a five or six silk suture loop through the palmar area of each forepaw and leave it with five centimeters of length. Position the rat on its back. Start with the left forepaw, hook the suture line in the dynamometer hook and align the forepaw with the dynamometer, without putting too much strain on the hook.

Hold the Stimulating electrodes, approximately in the Median nerve. Adjust the Stimulus amplitude to 1.5 volts and record instant force for 30 seconds. From the obtained plot, analyze the maximum, minimum, and mean force values and the area under the curve.

The Flexor carpi radialis, which is innervated by the Median nerve, is removed bilaterally and weighed in a precision scale. Regarding some Representative Results, in the Grasping test, the percentage of rats with a positive response, is highest in the Sham group and it gradually increases over time, in rats from the Nerve Graft group. Pin Prick test results are higher, in the Sham group compared to those of either the Graft or the Excision groups.

Rat's velocity in the Ladder Running test, is highest in the Sham group, than the rat's submitted to medial lab lesion. Among the lighter, the time to run the ladder, tends to decrease over time paring Median nerve recovery. Analogously to what is observed in the Ladder Running test, the time rats take to climb the rope, is inferior in the Sham group, compared to the groups in which the Median nerve is injured.

Rat speed, in this test, increases in the rats, in which a Median nerve is allowed to recover. As shown in these bar graphs, analysis of walking rats, tends to show changes in the morphology of paw prints. These changes are often more pronounced, in crushing injuries than in Segmental nerve lesions.

This graph shows that thermography seems to be, a useful tool to search for temperature differences, between the forepaws in the first 30 days after surgery. Those temperature differences are more noticeable, in rats with the more severely injured Median nerve, such as in those from the Excision group. Rat's flexion strength is directly correlated, with the degree of nerve recovery.

Being closest to normal when recovery is maximal, as can be seen in these bar graphs comparing strength, in both paws in Excision and the Nerve Graft groups. Concerning Electroneuromyography, various patterns can be observed. A normal C.M.A.P.potential, is typical of a rat from the Sham group.

While the Polyphasic C.M.A.P.is associated, with a variable degree of lesion of the Median nerve, as happens in the crush and in the Nerve Graft groups. In the Excision group, there is no C.M.A.P.The wave and morphology of the Flexor carpi radialis are dependent on the Median nerve recovery. These two images show these muscles, in the Sham and Excision groups.

As shown in this paper, the rat Median nerve is a convenient model of peripheral nerve lesion and repair. Multiple standardized strategies are available to assess Motor and sensory recovery. Many of these are non-invasive, allowing for daily assessment.

Finally, experimental physiotherapy settings, can simulate recovery in the clinical context, facilitating stipulation of results to the human species.