Hi, I'm Fab sva. I'm a graduated student from the lab of at of biomedical science at Federal University of Genic. In this video, I'll be performing a stereotactic surgery to deliver the six side drugs dopamine neurotoxin to induce brain lesion and mouse model this technique crucial to the study of Parkinson's disease.
I'll also show how to evaluate the degree of motor impairment through the upper morphine induced rotation test Materials and instruments. To start the procedure, you must wear gloves and the appropriate protective clothing. Turn on the heating pad and set it to 37 degrees Celsius.
Make sure that all the parts of the stereotactic apparatus, as well as the necessary materials and instruments have been cleaned and sterilized all gels and solutions that may be kept warm, such as anesthetics and sterile saline should be done. So products maintained in an anhydrous form such as six hydroxy dopamine and apo. Morphine should be diluted in sterile injection water and filtered cited lanar flow hood anesthetizing and preparing the animal for surgery.
Weigh the animal and calculate the amount of sedatives necessary to be administered by an intraperitoneal injection while the animal is becoming sedated. Place it on the heating pad to maintain body temperature and remove the fur by shaving. Apply a local anesthetic such as Xylocaine to the ear bars of the Stereotaxic instrument to prevent suffering and post-surgery discomfort.
Confirm that the animal is properly sedated by testing the withdrawal reflex of the hind limb. Only then should you position the animal carefully on the stereotaxic instrument. Insert the superior incisors into the mouthpiece, then clamp it tights.
Next gently insert the ear bars, center the ear bars, and check that the skull has been properly secured. To do so, you may try to move the head up and down and side to side. If the skull has been correctly placed, it should not move.
Apply a drop of ophthalmic solution to the eyes to prevent a corneas from drying out. Stereotactic surgery, disinfect the skin with povidone iodine, followed by 70%ethanol. Repeat this entire process three times.
Confirm again that the animal is well sedated by testing the withdrawal reflex. Use a sterile scalpel to make a 1.5 centimeter sagittal incision, and then use a new blade to scrape away the periosteum. To reveal the sutures, locate the lambda and bgma.
If the animal's positioned correctly, these two reference points should be at the same vertical level and spaced rostral coly by 4.2 millimeters. Otherwise, adjust the angle of the nose piece. Next, adjust the tip of the sterilized Hamilton needle so that it almost touches the bgma.
Note, the anterior, posterior, and the medial lateral coordinate of this reference points. Raise the needle and move it to the desired location at 0.5 millimeters anterior torema and 2.0 millimeters laterally in this case towards the left hemisphere. Mark this point on the skull, then fully raise the Hamilton needle and begin drilling.
It is important to drill with an intermittent action to prevent heating. Once the hole has been drilled, use warm saline and sterile cotton swabs to remove the bone residue. Use a dental curette to carefully retrieve any bone particles, and again, use warm saline and cotton swabs to further clean the area.
Repeat this process of picking and swabbing as necessary to free the area of any loose particles. Check that the six hydroxy dopamine solution is clear. If it has a yellowish brown tin to it, discard it and prepare a fresh one for a total injection of 10 micrograms.
Load the Hamilton with two microliters at a concentration of five micrograms per microliter. Also, keep in mind that once the Hamilton has been loaded with the six hydroxy dopamine, you should cover the syringe with aluminum foil to prevent the solution from oxidizing. Under the observation scope, place the tip of the needle on the dura mater and read the dorsal ventral reference value.
Then slowly insert the Hamilton into the brain tissue. Once you've reached the desired ventral position of 3.0 millimeters below the cortical surface, wait five minutes to allow the tissue to adapt to the needle. Before starting the injection, it is important to inject the six hydroxy dopamine neurotoxin very slowly at a rate of 0.1 microliters per minute.
In other words, this injection process should take no less than 20 minutes. Once the entire volume of two microliters has been injected, wait five minutes and then carefully and slowly withdraw the Hamilton so that the entire three millimeters is only withdrawn in a minimum timeframe of five minutes. Clean the area well with a sterile cotton swab.
Then retract the stereotaxic arm and the scope. Use USP six dash zero size nylon sutures to close the incision. In our case, we'll use 3 2 4 U forma stitches.
A local antibiotics that suggest neomycin may be applied as an extra precaution against infections. Remove the animal from the stereotactic instrument by first loosening the ear bars. Next, undo the mouthpiece and lift the animal up by the scruff to prevent damage to the animal's teeth.
Leave the animal on the heating pad until the sedation has worn off and its internal thermal regulation has been recovered. Hydrate the animal with 500 microliters of warm sterile saline injected subcutaneously for the next two days. After the surgery, add 0.2 milliliters of an ibuprofen suspension to 100 milliliters of water in the drinking bottle to serve as post-surgery analgesia.
Apple morphine induced rotation test. The APO morphine rotation test is generally performed one month after the induction of the lesion, confirm that the APO morphine solution is clear. Load a dose of 0.5 milligrams per kilogram into an insulin syringe and inject subcutaneously in the scruff.
Place the animal in the test container and give it five minutes to house, then continue to monitor its behavior for an additional five minutes. The animal on the left received a six hydroxy dopamine injection while the animal that received a vehicle injection of a 0.2%solution of abic acid diluted in 0.9%Sodium chloride is shown on the right. This graph shows our results from various apomorphine induced rotation tests carried out on a total of 20 Swiss mice, five control, and 15 lesioned one month after having undergone the described stereotactic surgery.
Conclusion, Parkinson's disease is a neurodegenerative disorder of the central nervous system, but in order to study the effectiveness of different therapies, it is crucial to have a well-established model which reproduces the loss of dopaminergic neurons in the midbrain. The lesions induced by this procedure very closely mimics the effects of Parkinson's that's allowing researchers to study, devise, and test new therapies to combat this devastating disease. So taking into consideration the reliable results produced in addition to its straightforward and simplistic design, the appeal of this technique is apparent.
Good luck and we hope that you have found this video helpful in your research.
