A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

The overall goal of this procedure is to implant a transmitter to record cardiovascular parameters and a spinal cord injured rat transplanted with embryonic neural stem cells. This is accomplished by first completely transecting the spinal cord at the T four level in an adult rat. The next step taking place two weeks later is to graft brainstem derived neural stem cells from embryonic day 14 rats into the lesion site.

The third step eight weeks later is to implant a transmitter into the femoral artery of the rat. The final steps are to take vital measurements and to induce autonomic dysreflexia by colorectal distension. Ultimately, the results can show partially recovered cardiovascular function in rats grafted with embryonic neural stem cells through telemetric.Recording.

Visual demonstration of this method is critical as transmitter implantation and the colorectal distinction are difficult to learn because they need a professional expertise to be done Eight weeks prior to implanting transmitters. Rats must undergo a spinal cord injury with or without cell grafting. Details on this protocol are provided in the text.

Begin with re anesthetizing these rats or naive controls. Once confirming that the rat is fully anesthetized, shave the abdominal area and hind limbs. Clean the exposed skin with Betadine, then transfer the rat to the surgical site and put it in a supine position.

Make the first incision through the ventral abdomen and right side inner thigh using scissors. Cut through the subcutaneous tissues to expose the femoral nerve and vessels. Now separate the femoral artery vein and nerve using curved tipped forceps.

Then place three sutures under the artery and tie each loosely vasodilate the artery with 0.1 milliliters of 2%Lidocaine. Then secure the vessel distally with a permanent silk knot. Block the artery proximally temporarily by tightening the sutures.

Now puncture the artery with a 20 gauge curved needle into the hole. Insert the tip of an eight centimeter long sterilized telemeter catheter using a catheter insertion tool. Push the catheter in, roly up to four centimeters to place the tip in the thoracic aorta.

To anchor the catheter, tie two more silk sutures around the artery. Now using blunt scissors, make a subcutaneous pocket on the flank between the coddle edge of the rib cage and the most cranial extension of the knees range into this pocket. Insert the body of the transmitter.

Suture the surrounding tissue to better secure the transmitter. Close the skin using six to zero silk suture. Immediately after the surgery, inject five milliliters of lactated, ringers, subcutaneous, and buprenorphine with ampicillin subcutaneously.

Keep the rats in a warm incubator until it wakes up the day after transmitter implantation. Put an animal on the receiver pad and turn the transmitter on. Let the animal habituate for 10 to 15 minutes.

The rat's cardiovascular parameters should stabilize, then measure the resting mean arterial pressure. This is derived from the pulse arterial pressure collected every five seconds over an hour. During this hour, monitor the animals and remove data points that are collected from visible spasms to induce autonomic dysreflexia in a rat.

Begin with restraining it in a towel. Place a rat over a receiver to collect data from an implanted transmitter. Next, insert a balloon tipped catheter into its rectum.

Push it in about two centimeters deep and tape it to the tail. Turn on the transmitter and wait 10 to 15 minutes for the animal's blood pressure to return to pre catheter baseline levels at the baseline. Record the mean arterial pressure rate every three seconds for one minute.

Then inflate the balloon catheter slowly over 10 seconds with 1.4 milliliters of air and hold the pressure for one minute. The resulting pressure is about 30 tour. Continue taking recordings from the transmitter for a minute.

After the inflation, repeat this process two to three times with a minimum of 15 minutes Between trials. Then euthanize the animal with an overdose of anesthetic, followed by perfusion spinal cord. Injured rats were implanted with transmitters as described.

Animals that were given a graft of brain stem derived neural stem cells at the injury had basal mean arterial pressure and heart rates similar to naive animals. 10 weeks after injury, colorectal distension identified dysreflexia rats that showed increases in arterial pressure and a decrease in heart rate. Inevitably, all the rats experience episodic hypertension spasms and barrow reflex IC mediated bradycardia, but arterial pressure was much lower in animals with the cell graft.

The heart rate didn't significantly change between groups, which may be due to reduced sensitivity of barrow receptors after the spinal cord injury. Histological analysis of the animals grafted with cells expressing GFP showed that eight weeks after injury, the neural stem cells filled the gap in the spinal cord. Within this population of grafted cells, many are positive for tyrosine hydroxylase and many are positive for serotonin.

The axons of these cells extended and innervated coddle sympathetic pre ganglionic neurons, which were labeled with fluoro gold in the intermed lateral cell column After its development. This method paved the way for researchers in a field of spinal cord injury to explore cardiovascular dysfunction in rats.