This method can help answer key question in the neurodegenerative disease field, such as Alzheimer's disease. The main advantage of this technique is it mimics Alzheimer's disease in the rat animal model by intracerebroventricular injection of A-beta 25 to 35, combined with aluminum trichloride and recombinant human transforming growth factor beta one. It also provides a fast and relatively simple experimental protocol with a high animal survive rate, high model successful rate, as well as a high rate of duplication, which has been shown to be more economic.
The implications of this technique extend towards screening efficacy of drug against AD because this animal model possess memory impairments and neuronal disturbance close to the states of AD patients. Demonstrating the procedure will be Cheng Jianjun, a technician from my laboratory. On an anesthetized rat, begin by snipping the fur on the vertex of the head with surgical scissors, and disinfect with iodophor.
Next, make an incision on the head skin along the median longitudinal calvaria with surgical bistouries and scissors. Separate the subcutaneous tissue and fascia, wipe the skull calvarium with 0.3%hydrogen peroxide, and mark the bregma with a marker pen. Next, mark three points, the anterodorsal thalamic nucleus for injecting RHTGF beta one and fixing one screw, the lateral ventricle, or LV, area for injecting A-beta 25-35 and AICI3, and lastly the second screw fixing place.
Gently drill three one-millimeter diameter holes with a flexible bone drill at the three marked points on the skull. Stop the bleeding and clean the skull surface repeatedly with sterile dry cotton. Next, insert a needle linked to the microinjection pump to the brain at 4.2 millimeters depth, and gently inject one microliter RHTGF beta one into the ad area.
Keep the microinjection two minutes after injection. Then slowly pull it out. Fix the two screws into the skull, designated in the previous marked points, with a small screwdriver.
Following screw placement, assemble the cannula implantation system by first inserting the dummy cannula into the guide cannula after disinfection with 75%alcohol immersion for 24 hours. Then, insert the stainless-steel tubing guide cannula to the brain at 4.6 millimeters into LV area through the skull hole, with the help of a cannula holder on the rat's stereotaxic apparatus. Next, mix the denture base material with denture base water at a ratio of 1.5 grams per one milliliters.
Put the paste to cover the guide cannula plastic pedestal and two screws for immobilizing the guide cannula. Cover the whole skin incision to avoid skin infection. The next day, draw out the dummy cannula, and insert the internal cannula into the guide cannula.
Screw the fixing screw to immobilize the internal cannula. Set the polyethylene pipe that links the microinjection pump to the internal cannula, and regulate the injection speed to one microliter per minute. Microinject the A-beta 25-35 to the LV.Wait five minutes after finishing the injection, and gently draw out the internal cannula.
Then, insert the dummy cannula again into the guide cannula. On day 15 post-surgery, dismantle the cannula implantation system by removing the denture base material solid with surgical scissors and forceps and disinfecting the wound with iodophor. Finally, fill in the hole of the skull with bone cement, and suture the skin with a simple interrupted suture method.
Begin by blackening the pool water with some black food coloring. Maintain the depth of water at 31.5 centimeters and the temperature at 23 plus or minus one degree Celsius. Then, set a 1.5-centimeter, circular, transparent plexiglass platform below the water surface.
Next, divide the pool into four equal quadrants by imaginary lines for descriptive data collection. Place the hidden platform in the first quadrant, or Q one, of the water maze. Finally, capture the rat swimming behaviors through a video camera, over the water maze linked to a computer-based graphics analytic software.
These results indicate that the sham-operated group of rats always swam freely, while the composited A-beta-treated group rats always swam around the pool perimeter in adaptive swimming in the Morris water maze. Over the four days of screening for memory impairment model rats, all rats had progressively declining times to find the hidden platform. Further, the latencies of the composited A-beta-treated group for finding the hidden platform were significantly longer than those of the sham-operated group, demonstrating that the composited A-beta can elevate the memory re-learning impairment in rats.
In the one-day memory retention trial, the composited A-beta-treated group took less swimming time, swimming distance, and crossing number within 60 seconds, compared to those of the sham-operated group. Lastly, in addition to pathological changes, the neuron number was also significantly reduced in the hippocampus and cerebral cortex in the composited A-beta-treated group compared with the sham-operated group. Once mastered, this technique can be completed in 20 to 30 minute if it is performed properly.
While attempting this procedure, it is important to remember to avoid the guided cannula falling off from the brain screw. Following this procedure, other methods like maze test can be performed in order to answer additional question, like whether the mimicked AD model was successful. After its development, this technique paved the way for researchers in the field of neuroscience to explore neurodegenerative disease in in vivo.
After watching this video, you should have a good understanding of how to set up successful mimic AD animal models through intracerebroventricular injection of A-beta 25 to 35 combined with aluminum trichloride and recombinant human transforming growth factor beta one in rats. Don't forget that working with polymethyl methacrylate can be extremely hazardous, and precaution such as wearing masks and gloves should always be taken while performing this procedure.
