By Jason Glynn, Liberal Studies Major

Here is the second (and hopefully final) installment of my summer experience working in a neuroscience lab at the University of New England, under a Maine-INBRE Fellowship.

I was awarded a National Institutes of Health sponsored fellowship geared towards biomedical research, and I chose to carry it out at UNE’s Biddeford campus for 10 weeks over the summer. To catch up from part one, visit our WordPress site at: https://thesmccbeacon.wordpress.com

Pain, or the perception thereof, is handled by an interconnected web of brain regions and nerves. This interconnected web is commonly referred to as the pain matrix. Pain is not really a “thing,” but rather a perception and/or response to a stimulus, and is designed to keep us safe, or remove that hand from the fire. We had to evolve different ways of perceiving and/or dealing with pain to survive.

There are two-main parts of this pain pathway: the inhibition side, which deals with the incoming signals, or the descending inhibition side, which nulls the pain. The inhibition side has been well studied. This is where things like non-steroidal-anti-inflammatory (NSAIDS like Advil), local anesthesia, and opioids (e.g., Oxycontin) work; but the descending pathway – discovered recently – is relatively unknown. This pathway is believed to be regulated by an endogenous (internal) opioid system, through neurotransmitters such as: serotonin, dopamine, and norepinephrine.

My pilot study was an interesting one, not just topically, but because it was a collaboration of sorts due to my limited time there. So I used rats from other – but related – studies. The rats had already been given osteoarthritis, and most had been subjected to an exercise regimen. I looked into a group of OA-exercised rats, a group of OA-sedentary rats, and a group of non-OA-non-exercised rats as a control.

We hypothesized that exercise was causing this descending inhibition side to be up-regulated, meaning it worked better, and this would be observable through increased levels of tyrosine hydroxylase (TH) – a molecular precursor to dopamine and/or norepinephrine. This up-regulation would be quantifiable by immunohistochemical techniques. We initially wanted to look at three brain regions, but decided to hone in on the rostral anterior cingulate cortex – a prefrontal cortex region implicated in the emotional aspect in the perception of pain.

So now that we had a hypothesis, it was time to get testing it!! I did this by using the well-known Rat Brain Atlas – yes, there really is an atlas dedicated to rat brains; rats are one of science’s most-used models after all. Once located, I used a cryostat to slice and plate numerous rat brains from different cohorts.

Once sliced and plated on charged slides, I began staining for immunohistochemistry. This is a process that uses antibodies to adhere to specific antigens (proteins) if they are present; the antibodies are marked with fluorescent tags to be visible under a fluorescent microscope. We used antigens known to be associated with TH – our target molecule.

After everything was stained, it was time to image them on a fluorescent scope, and analyze the intensity of the fluorescence using FIJI (or ImageJ) software. This software measures intensity through pixel values, and you can compare the values within sections, or bilaterally.

What I found did support our hypothesis; there was a noticeable increase in TH expression in the cohort that was given arthritis and then exercised. What does this mean? Great question, and since this was a pilot study, I have provided other scientists with a new road to explore. Since TH is a precursor molecule, it could mean that dopamine is increased, or norepinephrine. More quantitative research, such as Western Blot Analysis, is currently underway to figure out what this means.

Alas, that is science in the real world; sometimes you don’t find answers to questions, but rather more questions to answer. At the end of the day, it was a great experience; and a life-changing one at that – as I have swapped majors because real science is much more slow-paced and more boring than I had anticipated. But I digress, that’s for the next addition to this story!