This protocol is significant because it offers the first model of inflammation sensitized premature brain injury in the ferrets that results in significant and sustained injury. This technique involves a prolonged insults that incorporates many of the triggers and biochemical mechanisms thought to contribute to the types of brain injuries seen in premature human infants. Demonstrating the brain measurement procedure will be Kylie Corry, a research scientist in our lab and Olivia White, an undergraduate student.
Thirty minutes before starting surgery, administer three milligrams per kilogram LPS IP to kits in the injury group and an equivalent volume of sterile saline of three microliters per gram to control animals. Before beginning the procedure, use small animal clippers to remove all of the hair on the ventral neck region of the ferret in a rectangular pattern, taking care to avoid nicking the skin or generating heat rash. Administer local anesthesia to the shaved area and disinfect the skin with three alternating swabs of povidone iodine and seventy percent ethanol.
After confirming an appropriate level of sedation by toe pinch, cover the animal with sterile, disposable cut out drapes that expose the neck region. For bilateral carotid artery ligation, use a single use number ten scalpel blade to make a 1.5 centimeter midline incision in the center of the neck and use fine hemostats and curved forceps to bluntly dissect down to the left carotid artery. Dissect the exposed artery away from the associated neurovascular bundle.
And use a pair of curved fine forceps to pass a looped ten centimeter length of sterile 5/0 silk suture under the artery. Cut the suture in half and securely tie both lengths of suture to ligate the artery, leaving at least two millimeters between the knots. Transect the left carotid artery between the sutures, taking care to leave the nerve intact and repeat the dissection on the right.
Reversibly ligate the right carotid artery with a single sterile 1/8 inch umbilical tie. And close the wound with surgical skin clips. Then, allow the animal to recover in a temperature controlled water bath for at least thirty minutes with monitoring before hypoxia.
For sequential hypoxia, hyperoxia and hypoxia treatment, place the animals in the injury group in an airtight chamber within a water bath. Continuously monitor the oxygen concentration within the chamber, as well as the rectal temperature in at least one sentinel animal. Flush the chamber with humidified nine percent oxygen and 91 percent nitrogen and maintain a flow rate of three to five liters per minute, depending on chamber size.
Once the concentration of oxygen in the chamber has reached nine percent, continue the delivery for thirty minutes. At the end of the hypoxia treatment, switch the gas supply to 80 percent humidified oxygen and 20 percent nitrogen. After thirty minutes of hyperoxia, open the chamber to allow it to more rapidly reach normoxia, by equilibrating with room air, before sealing the chamber and flushing with nine percent humidified oxygen again.
Continuously monitor all of the animals during this second round of hypoxia, taking note of any animals that display bradypnea. Once the concentration of oxygen in the chamber has reached nine percent, continue the hypoxia treatment for thirty minutes. At the end of the treatment, used curved forceps to identify and untie the umbilical tape from the right carotid artery.
Reclose the wound with surgical skin clips. Then return all of the kits to the jills for 60 minutes for nursing and recovery. At the end of the recovery period, return the injured animals to the water baths at 37 to 40 degrees Celsius for six hours, adjusting water temperature as needed, to maintain rectal temperatures of 36 to 37 degrees Celsius.
Then, return kits to their jills again. After brain harvest and fixation at the appropriate experimental endpoint, place the tips of an electronic caliper on the dorsal and ventral aspects of each brain to measure the height of each injured and non-injured brain. Place the tips of the caliper at the most lateral portions of the temporal lobes to measure the width of each brain and weigh each brain.
Use the caliper to measure the longitudinal fissure, all of the sulci from the beginning and end of the most distinct portion of the corresponding sulcus, and all of the gyri from the widest aspect of each corresponding gyrus. Then, place one tip of the caliper at the most posterior point of the longitudinal fissure and the other tip of the caliper at the most posterior part of the cerebellum to measure the amount of exposed cerebellum. Of the 34 animals from six litters exposed to the insult in this study, five animals had moderate injury and four animals had severe injury.
With increasing injury, narrowing of the gyri in the temporal and/or occipital lobes is observed, with an associated sulcul shortening and widening of the longitudinal fissure and larger areas of cystic tissue loss in the most severely injured animals. Over the reflex testing period, the injured animals display a slower time to rotate in the negative geotaxis task. A slower time to rotate away from the edge in the Cliff Aversion task and a slower time to right.
In the catwalk, injured animals demonstrate a similar average speed to controls but display a significantly greater degree of speed variation during each run. The weight adjusted distance between the paws and hind paws is significantly greater in injured animals, with less pressure exerted per unit paw area, through the four paws. In the open field, the injured animals cover less total distance, stop more frequently and spend significantly more time in the center of the field and less time in the corners.
Here, representative heat maps of control and injured animals can be observed. Take care to dissect away the artery from the accompanying nerve and allow the maximum possible hypoxia time. These steps will ensure that the majority of animals survive, whilst also sustaining an injury.
Following this procedure, any biochemical, pathological or behavioral outcomes can be assessed, including assessments that are normally restricted to rodent models of brain injury.
