There are no words to describe this blog.

Bregma marks the spot. Well, sort of.

I apologize that this post is so long in coming. I typed this post up last week, but unfortunately I accidentally erased it all. Smooth, I know. I did not have time to retype it this weekend, as my parents were visiting me in LA. We had a lot of fun! We did many of the tourist activities in LA which was cool since I hadn’t done many of those things yet even though I have been living in LA for two months! I will post those pictures soon.



Several weeks ago, I was able to assist in my first surgery ever! It was awesome. Before I begin my description on tumor implantation, I want to reiterate that all procedures are done to ensure that the animals are treated in the most humane way possible. The mice are first anesthetized. Then their heads are shaved. Once they are completely under anesthesia, they are placed on the stereotactic frame, as shown above. After the mice are secure, betadine is applied to the incision area and the skin covering the skull is cut. The skull is exposed and the bregma is located. The bregma is actually easier to locate when the skull dries slightly. The bregma is the point of intersection between the coronal suture (horizontal line in the picture to the left) and the sagittal suture (vertical line) where the frontal and parietal bones meet. We use the bregma as the “zero point” or the point of reference. Once we find the bregma, we utilize the stereotactic frame to find the correct point in the right hemisphere where we will inject the tumor cells. A burr hole is created in the skull using a hand drill, a needle is inserted into the hole, and 50 K tumor cells are injected into the hole. After injection, bone wax is applied to cover the burr hole, the skin is clipped together, and pain killers are injected subcutaneously. The mice are observed closely for several hours after surgery and then each day until terminal symptoms become apparent. This particular type of surgery can be performed to derive tumor lines or to observe the effects that different treatment types have on tumor growth and mouse survival time.

To Perfuse or Not to Perfuse... That's Not Even a Question!

As I mentioned before, we do a lot of flow cytometry in the lab where I work. We concentrate most of our efforts on T cells: the molecules expressed on the surface and inside different populations of T cells, the presence or absence of T cells in brains with tumors, how T cells interact with and kill cancer cells, and more. In order to do this, many times we need to remove the brains and/or spleens of mice by perfusion.



I would like to preface my discussion of perfusions by saying that all work that we do on mice is done following a set protocol that has been created in collaboration with veterinarians, research scientists, and healthcare professionals to ensure the most humane methods of euthanasia. Also, all mice studies are only done when other routes of experimentation are unavailable and there is sufficient need for the experiment.


So why is doing a perfusion the definite route to go when you are seeking to resect a brain or spleen for flow cytometry or other types of analyses? I am glad you asked. It is because perfusions allow a scientist to remove the blood from the organ of interest which cuts down on auto fluorescence by RBCs. First, the mouse is anesthetized with a ketamine + xylazine IP injection. The mouse must be completely unconscious before the procedure can begin so that it will not feel any pain, and this can be tested by squeezing its paws and observing the reaction. Next, a dissection is performed to expose the abdominal and thoracic cavities on the ventral side of the mouse. The ribs are removed and the heart is exposed. After exposing the heart, the aorta is cut and a small hole is cut in the left ventricle. A butterfly needle is placed into the left ventricle and PBS is injected into the mouse. The mouse’s heart will carry the PBS throughout the body which will rid the body of RBCs and cause the organs to turn white in color. If the brain tissue needs to be fixed, then PFA can also be injected through the left ventricle to do so. After this procedure is done, the required organs can be removed and properly stored for analysis.


Another important procedure for researchers that are studying cancer is tumor implantation. Tumor implantation is two things: 1. It is a very neat procedure, and 2. It is the topic for my next blog… Stay tuned.