Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

The overall goal of this procedure is to perform a complete spinal cord, transection and brain dissection for the study of Axon guidance after injury. This is accomplished by first transecting, a lampre spinal cord at the level of the fifth gill. Then the brain is dissected and pinned to syl guard.

After fixing the brain, it can be stored in methanol or immediately used for in situ hybridization ultimately changes in the expression of axonal. Guidance receptors can be seen in a regenerating vertebrae after a spinal cord injury. The advantage of the technique we're going to demonstrate today over more conventional paraffin sectioning or cryostat sectioning, is that it allows you to see the whole brain, including neurons that project to the spinal cord that are large and identified.

Demonstrating the procedure is Dr.Kathy Zang, a research scientist in our laboratory From the streams feeding Lake Michigan tributaries to the Delaware River in Pennsylvania, or streams in Maine. Wild type larval sea lampre of the species. Pet mize on Marus were obtained.

Four to 7-year-old larvae are 10 to 14 centimeters in length and large enough for the injury and dissection. Maintain them in groups of 52, a hundred. In 50 gallon fresh water tanks at 16 degrees Celsius.

The tank requires a one inch gravel substrate so the lamprey can borrow All water added to the tank should be conditioned with charcoal filtering and aeration for at least 48 hours before it is added to the tank. Provided the animals are used within one year, there is no need to feed them for this procedure. Have freshly prepared ringer solution at the ready.

First transfer a lampe to saturated aqueous benzocaine solution. Once anesthetized, transfer it to a dish half filled with sil guard. Put the dish on ice and top the dish off with ice.

Cold ringers. Secure the animal with three 0.15 diameter insect pins, one at the snout and two at the fifth gill. Then position it under a stereo microscope for the surgery.

Begin by using a number 11 scalpel to make a longitudinal incision along the dorsal midline at the level of the fifth gill. This will reveal the spinal cord while making this incision. Use two hooks fashioned from insect pins to secure the body walls using Castro reveal number eight, scissors.

Make a perpendicular cut across the exposed spinal cord. Inspect the cut ends of the spinal cord to ensure that the transection is complete. Then using forceps, realign the cut ends and close the wound.

Transfer the lampre lava onto a piece of filter paper soaked in ringer solution. Move the preparation onto a bed of ice for an hour so the wound can dry after an hour. Transfer the lava to its own small tank of ice cold water.

Allow it to heal there for 24 hours. After 24 hours, check for movement cordal to the transection. If there are any movements, the transection was incomplete.

If there aren't, then the transect was good and the animal can be moved to a room temperature tank for recovery while the animal recovers. Change the water every two to three days. Once the transected animal is sufficiently recovered, anesthetize it and prepare it for dissection as previously described.

To dissect the brain, start with a transverse incision between the nostril and the pineal organ. Then with Castro veal, scissors cut the tissue surrounding the brain while securing the animal. With forceps.

The white oval structures on either side of the brain stem are the otic capsules. Once the brain is exposed, use forceps to remove the choroid plexus. Next, use the modified pin hooks to spread the head of the animal and expose the cranial nerves.

Then cut the spinal cord transversely with the scissors while securing the spinal cord with forceps. Cut the cranial nerves to dissect out the brain. Then transfer the brain to a small piece of silicone and secure it there.

Using one insect pin in the spinal cord and one pin through one of the olfactory bulbs. Then using the scissors, cut the inter olfactory bulb commissure the cerebral protector commissure and the oex of the brainstem along the dorsal midline. Once the commissures and oex are cut, deflect the A LR plates laterally and pin them flat to the silicone with a total of seven insect pins, two in the olfactory bulbs, two in the edges of the cut, cerebral protector commissure, two in the extended oex, and one in the midline of the spinal cord.

Now using RNAs free reagents, fix the preparation in 4%para formaldehyde at room temperature for three hours with gentle agitation. The brains can then be analyzed by in situ two hybridization using the method described expression of neogen in transcripts was identified in spinal cord, projecting neurons of control, animals and in animals. Two weeks post lesion seen here is a representative uninjured control.

After the complete spinal cord transection, there were changes in the expression of the neogen receptor. After two weeks, the neogen receptor in the non-control was preferentially expressed In bad regenerators like the M1 I one I two I four, or MTH neurons. The development of this technique paved the way for our ability to explore expression of guidance receptors, receptors for chondroitin sulfate, protio, glycans, and activation of cast bases all in the same brain.