At the heart of the matter: an introduction to transcardial perfusion

WARNING: This post describes a rodent non-survival surgery. Reader discretion advised.

This week, I was able to observe Feng performing transcardial perfusion, a technique used to prepare tissue for immunohistochemistry.

The subject animal (in this case, a mouse) is deeply anesthetized before surgery is performed to expose the heart.

Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. J Vis. Exp. (65), e3564, doi:10.3791/3564 (2012).

A perfusion needle is placed in the left ventricle (the chamber of the heart responsible for pumping blood to the rest of the body), and a saline solution is then pumped at constant pressure through the circulatory system before exiting through an incision made to the right atrium. This clears the subject of blood.

Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. J Vis. Exp. (65), e3564, doi:10.3791/3564 (2012).

A paraformaldehyde (PFA) solution is then accordingly pumped through the cleared circulatory system, quickly and consistently preserving the tissue in a life-like state.

Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. J Vis. Exp. (65), e3564, doi:10.3791/3564 (2012).

The fixed tissue can then be harvested and stored for later experiment. For our experimental needs, whole animal perfusion fixation yields the best possible preservation of the brain for immunohistochemistry.

Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. J Vis. Exp. (65), e3564, doi:10.3791/3564 (2012).

Through my observation, I learned that safety and sterility, for both the animal subject and the human investigator, are of the utmost important in any procedure of this nature. Due to the toxic characteristice of PFA, all work is done under the fume hood, and gloves and a lab coat are donned at all times. And because our lab works with both live tissue and fixed tissue, we have a special set of tools put aside for each, to ensure that no cross-contamination occurs.

It was a very good learning experience, and I enjoyed helping with the set-up and clean-up for the procedure. I'm sure that as Laura begins to undertake her own immunohistochemistry experiments in the future, I'll be assisting with many more perfusions.

Spirit check

In today's post, I'm taking a break from the lab and sharing with you a different facet of my life . . . dancing!

I took my first ballet class when I was three years old, and since then, I've fallen in love with dance both as a sport and as an art form. Dance practice is the space in my busy schedule where I can clear my mind of everything else going on in my life and just focus on my body's movement.

In the spring, I auditioned for UH's Cougar Dolls Dance Team and made it onto their 2013-2014 squad!

The last night of auditions when we received our roses! I'm second from the right in the back row.

This weekend, we travelled to Texas State University in San Marcos to attend UDA (Universal Dance Association) College Camp. We performed as a team for the first time, learned some awesome new choreography to bring back to UH, and got to interact with other college dance teams from all over the country.

Representing the Houston Cougars at fight song evaluations! I'm second from the right in the bottom row.

Overall, it was an amazing experience. We really bonded as a team, and we're now looking forward to football season when we'll get to cheer on our boys at Reliant Stadium!

Keep up with the Dolls at our Facebook page. Go Coogs!

Rabbits and goats and guinea pigs . . . oh my!!

This past week was rather short due to the 4th of July holiday, and my behavioral tests are now postponed indefinitely. :(

But no matter! I am just as happy helping Laura design her future experiment. Last Tuesday, she gave me a crash course in immunohistochemistry (IHC). I also found a neat video that really clarified the technique for me. Watch it here.

We'll be using indirect IHC to visualize a number of inflammation markers in rodent hippocampal brain slices. But instead of using a marker like DAB (as seen in the video), we'll use fluorescent tags on our secondary antibodies that can be imaged with the confocal microscope.

Right now, we're looking at five different primary antibodies - one for each target molecule - from five different host animals: goat, guinea pig, rat, mouse, and rabbit. So many animals!

I'm also having fun playing with Life Technologies' Fluorescence SpectraViewer to map out which fluorophores we want to use with our secondaries. It is important that each tag's excitation and emission wavelengths are unique to prevent crosstalk and to produce a clear image.

Good example, adequate spacing.

Bad example, overlapping signals.

Try it out yourself!

Congrats, Feng!

Today, our senior graduate student Feng defended his dissertation and earned his doctorate degree!

WOOHOO!

For those who are unfamiliar with the terminology, a dissertation is the culmination of a graduate student's research - a lengthy essay that explains the methodology, results, and significance of his work.  The graduate student's "defense" is a presentation of that work in front of a committee that is familiar with his field of study.

My lab mates and I sat in to watch Feng's public presentation before his closed Q&A session with the committee. I thought he did a wonderful job. Feng has done some great work here at UH, and he is definitely an example to me of dedication to your work and how much it pays off in the end!

Afterwards, we threw him a surprise party to celebrate his new Ph.D.

My cupcakes melted a little on the commute in to campus.

Feng will stay with us for two more months, and then he's off to Stanford for his post-doc! Can't wait to hear about all the great work he'll be doing there. I know all of us so are excited for him :)

Flashback Friday

Behavioral testing is temporarily on hold as we wait for our new protocols to be approved. But, I've been keeping busy with lots of reading and writing --- helping Laura with the Autism grant she's writing and helping Feng edit and organize his dissertation!

Today, however, I'd like to travel back in time to Saturday, June 1, to the annual Biology of Behavior Retreat. The Ziburkus lab is part of the Biology of Behavior Institute (BoBI) at UH, so we were all invited to the annual retreat at the Houston Arboretum.

I had a really good time learning about all the interesting projects going on in different labs right now, and I also got to meet several of the undergraduates who are in the SURF program with me this summer.

Feng, Laura, and I in front of Feng's poster presentation!

I had also never been to the arboretum before, despite living in Houston for ten years now! I found a nice little bench surrounded by flowers to sit and have my lunch, and I had some butterfly visitors!

If anyone can identify this pretty butterfly, please comment and let me know!

Overall, it was an enjoyable day, and I was definitely inspired by the passion the speakers had for their respective work.

Happy Friday, everyone!

Being wrong never felt so right

As part of the SURF program, I attend weekly "brown bag" lectures with my fellow undergraduate researchers. Because we all have such diverse interests (from testing cancer treatment to translating Homer!), the sessions are usually designed to address broad topics that can apply to all disciplines.

This week, Dr. Jeremy May, Assistant Professor of Chemistry, talked to us about that great, mysterious entity known as . . . research ethics.

Reading "The Responsible Researcher" before Dr. May's lecture.

For the most part, ethics in the sciences are the same as those in business, politics, education, and just life in general. Don't lie. Don't cheat. Be a conscientious citizen and a good neighbor.

However, because we work on the frontiers of our respective fields, scientists do face unique challenges.

For example, in the 1800s, the French mathematician Urbain Le Verrier argued that Mercury's strange orbit had to be caused by the gravitational force of another celestial object somewhere between Mercury and the Sun. Le Verrier named his hypothetical planet Vulcan and soon received news from other astronomers of Vulcan sightings, seemingly confirming his hypothesis. But, almost 40 years after Le Verrier's death, Albert Einstein's theory of general relativity successfully explained Mercury's orbit and disproved the existence of Vulcan. (more disproven theories here)

Urbain Le Verrier, 1811-1877

Was Le Verrier wrong? Yes. But his science was still good. In his time, he was able to offer the best explanation for Mercury's behavior with the tools at his disposal. He did not fabricate, falsify, or plagiarize; he was simply wrong. And it took 40 years and a revolutionary mind to challenge Le Verrier's hypothesis.

This was the crux of Dr. May's lecture: wrongness is an integral and healthy part of the experimental process. As young researchers, we cannot be afraid to get negative results, to learn something that completely shatters our hypothesis, or to be stumped over and over again by Nature as we attempt to unravel her secrets.

Dr. May's message really spoke to me this week, as I am currently reviewing data from our preliminary behavioral tests, and I am discovering previously unnoticed flaws in our experimental design. In a perfect world I would make no mistakes, but then again, a perfect world would have no need for scientists! Until then, I am embracing my wrongness, learning from my mistakes, and trying, trying again!

An introduction

Welcome!

My name is Cara Riffe, and I am a rising sophomore studying biology here at the University of Houston.

Last semester, thanks to my Tier One scholarship and UH's amazing faculty and staff, I joined Dr. Jokubas Ziburkus' neuroscience lab as an undergraduate research assistant. I was assigned to work with graduate students to design and carry out an experiment to test a new drug treatment for epilepsy.

I learned that although research is often demanding, it is also highly rewarding. So, I have decided to continue my work with UH's Summer Undergraduate Research Fellowship (SURF). As a SURF participant, I will work full time in the lab, as well as attend weekly seminars hosted by SURF in the Honors College. My SURF experience will culminate at Undergraduate Research Day in October, where I will present a poster on my research.

I am excited to have this opportunity to focus exclusively on my project without academic classes competing for my time and attention. This blog will document my summer experiences in the lab, and I hope to include lots of pictures and maybe even some videos!

My Project

Severe myoclonic epilepsy in infancy, commonly known as Dravet syndrome (DS), is a form of childhood epilepsy in which affected children suffer from seizures, loss of motor control, and social and cognitive dysfunctions. The Ziburkus lab has shown promising results in controlling seizures in DS mouse models through the use of a novel pharmaceutical. 

Working in collaboration with and under the supervision of Dr. Ziburkus and graduate students Feng Gu and Laura Montier, I will help test the behavioral effects of this drug on DS. We will run social and learning tests on mice that have either recieved treatment or have been left untreated. The data we collect through this testing will help elucidate the link between the seizures early in life and the cognitive impairment later in life that is observed in those that suffer from DS. Our hope is that, by decreasing the adolescent seizures associated with DS, the novel drug treatment will also save the mice’s cognitive function. Overall, the study is highly translatable and, if successful, could lead towards additional studies in human DS patients.




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