Accessing the Subdural Space of the Rodent Spinal Cord for Treatment Delivery

We're exploring whether delivering treatment directly onto injured spinal tissue via subdural administration can improve recovery and function after spinal cord injury. Current challenges for our research revolve around broadening the scope of our treatment modalities. We want to create a reproducible and reliable treatment paradigm that can be applied in multiple scenarios.

Recently, our group has shown improvements in motor and sensory function in spinal cord injured rodents following electric field stimulation treatment. Our protocol delivers treatments into the intrathecal space, bypassing the blood spinal cord barrier, offering potentially greater efficacy than standard epidural delivery methods. Moving forward, our group plans to utilize this method in larger animal models, as well as examine its effects on bladder function following spinal cord injury.

To begin, position the anesthetized rat on a clean table and shave the fur along the back of the animal, starting at the top of the hips and ending at the base of the neck. After shaving, administer preoperative subcutaneous injections. Using a chlorhexidine scrub, disinfect the shaved area in a circular motion, moving outward from the center.

Then apply chlorhexidine tincture in the same manner. Administer subcutaneous Marcaine and repeat previous steps for disinfection. Apply ophthalmic lubricant to the eyes to prevent drying out.

Now transfer the rat onto a heating pad placed under the microscope and organize all sterile tools. Drape a press and seal film over the shaved area. Cut an opening just large enough to expose the surgical field without revealing unshaved fur.

Insert a rectal temperature probe to monitor the rat's temperature. Adjust the heating pad as needed to maintain a temperature of approximately 37 degrees Celsius and perform animal welfare checks every 5 to 10 minutes. Next, administer a pedal reflex test to confirm the animal has reached a surgical plane of anesthesia.

Once confirmed, begin the surgical procedure. Using a scalpel blade, make a 6 to 8 centimeter long linear incision in a rostral to caudal direction, beginning just below the base of the neck and extending over the thoracic hump to expose vertebrae T9 to T13. Then with gray forceps and micro spring scissors, gently retract the connective tissue beneath the incision to expose the spinal muscle tissue.

Now identify the T13 and T12 spinous processes by locating the two white V-shaped tendons in the caudal half of the incision. The spinous process just rostral to the second V, moving head to tail, is T13. Starting between T13 and T12, moving toward T9, use micro scissors to make small incisions in the connective tissue between each vertebral process to separate them.

Then cut a parallel channel along the side of the spinous processes to expose the lamina of each segment. Repeat this dissection on both sides of the spine. Next, attach Serrefine clamps to the muscle walls to hold the surgical site open.

Place one pair at T13, another at the T11 T12 border, and a third at the T11 T10 border. Once the channels are defined, use micro scissors or rongeurs to remove the connective tissue between each spinous process. Continue clearing until all connective tissue and muscle is dissected from the lamina between rostral T13 and caudal T9.Locate the caudal portion of the T12 lamina that overlaps the rostral portion of T13.

Position the tip of the rongeurs underneath this overlapping area and gently remove small pieces of the lamina. Alternate from one side to the other, continuing to remove portions of the T12 lamina. To enlarge the opening, extend the cut into the midline of the process and expose the spinal cord, forming a hole approximately 5 millimeters across.

Then position the rongeurs under the lamina on both sides and carefully create a uniform channel extending toward the cranial end of T12. Repeat the same procedure to remove the lamina from T11 and T10. Once the laminectomy exposes an adequate area from T10 to T12, gently irrigate the exposed dura with saline.

Perform the durotomy using the dissection microscope to access the subdural space of the spinal cord. Confirm that the chosen site for the durotomy and the region directly in front of it are free of branching blood vessels from the midline vessel. Bend a 27 gauge needle to a 90 degree angle with the bevel facing upward.

At the desired entry point, pierce the dura mater gently with the tip of the bent needle. Once the dura is punctured, carefully lift the needle dorsally to tear a circular hole in the dura no more than 2 millimeters on either side of the midline blood vessel. Look for a slight leakage of cerebrospinal fluid to confirm successful durotomy.

Use the holes made in the dura to deliver treatment or insert a device into the subdural space. For this protocol insert intrathecal catheters either to guide a stimulation device or to inject hydrogel. Confirm that the catheter tips are visible under the dura mater before proceeding.

A successful placement is evident if the catheters glide beneath the dural membrane and over the small spinal blood vessels. Once in place, use the catheters to deliver the desired treatment. After delivering the treatment, insert a piece of sterile surgical gel foam into the laminectomy cavity to promote hemostasis, aid healing, and maintain spinal structure.

Finally, use 4-0 Polydioxanone absorbable suture to close the muscle and skin layers above the spine. A bio electronic device was implanted or a hydrogel was delivered into 6 to 8 week old Sprague-Dawley rats. Following each procedure, all animals maintained normal motor function with no difference in BBB score between groups 7 days post procedure, indicating the safe implementation of the described protocol.