This is the first protocol demonstrating the successful co-infection of the corticospinal tract in neonatal rats. It provides an accurate blueprint to investigate how individual subpopulations of cells in young mammals contribute to the recovery from spinal cord injury. Like other double viral vector techniques, this method allows the user to accurately transduce specific subpopulations of neurons, in turn, allowing researchers to identify the effectiveness of various chemogenetic techniques in promoting recovery.
We would expect researchers that are unfamiliar with this technique to struggle with locating the proper spinal anatomy. And so we encourage you to continually count the vertebral segments often and always refer back to your landmarks. After confirming the depth of anesthesia in a neonatal Sprague Dawley pup, identify the area where the incision will be made by pressing the forceps in the midline on the top of the scalp, feeling for the sagittal suture.
Holding the skin taut to ensure a clean, straight, and precise incision, make a one-centimeter incision along the sagittal suture plane starting immediately above the I line. Next, identify the bregma by gently probing the forceps along the surface of the skull, paying close attention to the suture lines and an indentation caused by the forceps running along the parietal and frontal bones. Maintain the opening by applying weighted hooks on the side of interest.
Clear the aponeurosis using a combination of cotton tips and micro scissors to maximize the exposure of the bregma for increased accuracy. Ensure that the coronal suture is in clear view far enough laterally to provide a rough template for subsequent injections. Using the micro scissors, cut out an approximately three-by-two-millimeter section of the left frontal skull bone immediately adjacent to the bregma.
Clear any debris, cerebrospinal fluid, and blood with cotton tips. To inject the virus, place the needle in place and program the injector to inject one microliter at a rate of 250 nanoliters per minute. Upon completion of the injection, allow the needle to rest in the cortex for three minutes.
Repeat the injections at the other coordinates for a total of three injections along the cortex in relation to the bregma, all at a depth of 0.6 millimeters. After completing the injections, suture the scalp and transfer the animal to the other operating table for cervical laminectomy. Palpate the base of the skull using fingers to identify the incision site.
Hold the skin taut by applying tension with the thumb and index finger. Using a number 11 blade, begin the incision at the midline one to two millimeters posterior to the skull base and extend the incision one centimeter posterior to expose the superficial muscle. Using the sharp point of the blade, gently make a series of cuts along the midline of the superficial muscle to expose the spinal cavity and deep spinal muscles.
Then use a pair of forceps to spread open the muscle and visualize the surgical window. Once the deep spinal muscles have been exposed, insert retractors into the surgical window and retract the surgical window to seven to eight millimeters in width, allowing an unobstructed view of the spine. Identify the second cervical vertebra by its prominent spinous process that projects dorsally and encapsulates a large dome-shaped muscle.
Using the flat edge of a bone scraper, gently scrape away the deep spinal muscle to the sides to expose the C3 to C7 vertebrae. Start medial and scrape laterally along the direction parallel to the vertebra laminae to ensure proper exposure, allowing clear distinction of the vertebral laminae. Control any bleeding with cotton tips.
Using C2 as a guide when counting the vertebral levels, carefully cut the lateral edges of the cartilaginous laminae at C6 and C7 using a pair of micro scissors. Using a pair of fine forceps, carefully remove the dissected portion of the lamina to expose the spinal cord, ensuring that the spinal window is large enough to accommodate the desired injection site. Set the animal up in the stereotaxic cranial holder and place a rolled up piece of gauze under the animal's trunk to elevate its hind quarters.
Before beginning injections, clear the spinal window of any blood or cerebrospinal fluid by gently applying cotton tips and Sugi triangles to the area, taking care not to insult the spinal cord. Further, create a barrier around the perimeter of the window by placing a small piece of absorbable cotton in the lateral portions of the spinal window to prevent occlusion of the injection site by continuous bleeding or CSF leakage. For direct injections into the spinal cord, use the tip of the glass injection needle to approximate the midline of the spinal cord by identifying the spinal artery.
Next, place the needle just posterior to the C5 lamina at the approximate midline and use this as the reference point. Then, using the micro manipulator, move the needle laterally to the right by 0.3 millimeters and lower the needle until it just touches the surface of the spinal cord. From this depth, plunge the needle.
0.6 millimeters into the spinal cord. Inject one microliter of the retro adeno-associated virus two carrying the Cre recombinase gene at 250 nanoliters per minute. Then wait for two minutes for the virus to diffuse into the spinal cord before slowly withdrawing the needle.
The labeling of layer five corticospinal tract neurons in the motor cortex of a brain coronal section expressing Cre-dependent DREADDs mCherry co-injected with a contralateral spine injection of retro Cre is shown here. Being able to target specific neuronal populations will help to delineate the nuances driving recovery and provide more accuracy for treating the underlying spinal cord injury in the developing nervous system. This method paved the way for us to investigate the viability of using excitatory DREADDs as a means for treating spinal cord injury in juvenile rats.
