.

We killed the mice this morning
harvested their organs for observing
what life is all about
broken down into the molecular cloud

but what is a life in a tube
when a heart was beating gently smooth

just two minutes ago 

before its place in a row

with sixteen other pieces of fur and flesh and bone

they had no way to take flight
from scalpel and knife

into some spectral plane of thought

i  myself have so hungrily sought--

a world of comfort and meaning 

that gives me reason for surviving

Maybe i am a mouse too 

who runs believing time will never end until 

a breeze blows me away

into oblivion. 

Kurzgesagt - In a Nutshell

               Ah, the lovely 96-well polyurethane plate, home of the minuscule samples I gotta deal with. 

So, what is diabetic neuropathy, and what exactly am I researching?

   I'm glad you asked! Let's start with the basics.

   Right now, nearly 30 million Americans suffer from diabetes. The vast majority of them have Type 2 diabetes mellitus (T2 DM)--an acquired form of the disease, which begins with insulin resistance. Insulin (produced by beta cells in the pancreas) regulates blood sugar by promoting glucose absorption in skeletal muscle and fat tissue, storing energy for later use. This happens after you eat, when glucose broken down from carbs and fats enters your bloodstream. But a combination of obesity, lack of exercise, bad diet, and stress can lead to body tissues becoming desensitized--defective--to insulin. (here's how that works) And without regulation, levels of blood sugar in the bloodstream start going bananas, nutty, apples! (Woah, there's a lot of foods that can mean crazy.) Highs and lows and everything in between. And with that comes a kilderkin of complications--neuropathy being a major one of them. Diabetes can more than double an individual's probability of death! So don't take candy from strangers, cause it's high in sugars and you're watching your health!

   Ok, that was my episode of Crash Course for T2 DM. Now let's talk shop about neuropathy.

   You can already figure out a few things about peripheral neuropathy from its name--"peripheral" as in outside, "neuro-" as in nerves, and "pathy-" as in disease. Basically, it's when nerves in your body that conduct signals to the spinal cord and brain become damaged. This damage can cause pain and loss of muscle/sensory function. Typically, it begins in the hands and feet, called a "stocking-glove" pattern. There are several ways that neuropathy can occur, and often it's a result of a combination of factors. In diabetes, there is the potential for high blood sugar to affect the area around nerves, chemically and through inflammation. You know how at Sea World there's a fun splash zone around the pool where all the dolphins, orcas and humans perform? Well, inflammation is like that, only its splash zone is a death zone, where cells have fun by committing suicide en masse. Fantastic. So this may harm other cells around nerves--not to mention that your immune system mounts other responses in the area (inflammation is already an immune response). Here's my more technical (and accurate) explanation of neuropathy:

Diabetic neuropathy encompasses a variety of clinical and subclinical syndromes, and can arise from multiple pathogenic mechanisms. Every case involves focal of diffuse damage to either peripheral somatic or autonomic nerves, which leads to the two main groups of neuropathies: diffuse and focal. Diffuse neuropathies include distal polysymmetric sensorimotor neuropathy (DPN), and diabetic autonomic neuropathy (DAN), while focal neuropathies are rarer, acute, and less long-term. Painful diabetic neuropathy (PDN) of type 2 diabetes causes length-dependent neuropathic pain, increased sensitivity to painful (hyperalgesia) and non-painful (allodynia) stimuli. Symptoms often begin as a stocking-glove combination in patients, with the hands and feet affected first. Large fiber disease impairs proprioception and light touch, and small fiber disease impairs pain and temperature perception, leading to paresthesias, dysthesias, and neuropathic pain. Prolonged development can lead to health complications including significant loss of touch and movement abilities, and corresponds to serious decreases in patient quality of life.  

The pathogenic mechanisms of diabetic neuropathy are varied. There are metabolism-driven pathways: “glucose flux through the polyol pathway, the hexosamine pathway, excess/inappropriate activation of protein kinase C (PKC) isoforms, and accumulation of advanced glycation end-products”. Collectively, the combination of these “lead to excess formation of reactive oxygen species. Increased oxidative stress within the cell leads to activation of the Poly (ADP-ribose) polymerase (PARP) pathway, which regulates the expression of genes involved in promoting inflammatory reactions and neuronal dysfunction” (Edwards 2008).

   Neuropathy is the most common and costly complication that arises from diabetes--more than 50% of patients that have the disease for a prolonged period of time suffer from neuropathy. And it's incurable--it can only be fought through blood-sugar management and alleviating medicines. Diabetic neuropathy sucks.

   My research is primarily concerned with the neuronal markers in neuropathy, and using those to identify tissues. Different cells in the body have different functions and characteristics, and different cell "markers" that distinguish them. I'll be using immunohistochemistry (IHC) to stain samples (footpads from mice that are genetically altered to develop diabetes, more on that later) for specific structures--primarily nerves, to see where inflammation is happening. I'll then look at those samples under a microscope which highlights stained cells (fluorescence). And like any good ol' experiment, there's an experimental group and a control group. I'll then compare the two groups to note differences--more nerve inflammation, presence of macrophages, etc. for finding results. I'm trying to answer these questions: "Is there a difference between high-fat diet mice from wild-type (normal) mice," and "what are those differences, if any?"

  Ultimately though, I'm searching for experience. There's a limit to what conclusive research can be done in the short span of three months, and this project is just a part of a larger experiment (including Alzheimer's research!), part of the even larger effort to help understand and cure diabetic neuropathy. So if there's anything I'm certain of gaining, it'll be the wonderful experience, knowledge, and people I meet here during these three months. And that's an adventure in itself. 

~ Liam



   

Adventure Time!

                            Introductions: Lab bench, blog reader. Blog reader, lab bench.

   Hi there! I'm Liam, a student at BASIS Flagstaff, and this is my senior research project! Let's go explore the magical, wonderful world of diabetes research--so strap on your seat belts; cause this project lifts off now! Whooo!

   I'm conducting my research at Massachusetts General Hospital's Genetics and Aging research unit, headed by Dr. Rudolph Tanzi. I'll be studying peripheral diabetic neuropathy with Dr. Thomas Cheng within the db/db+ mouse model. Preparing samples, confocal microscope imaging, and data crunching are all on my upcoming to-do list titled: "Being a unpaid non-employee researcher whohasnoideahowtodofreakinanything." (This is going to be an interesting experience!) 

   My research is essentially a three-layer chocolate cake: 1.) staining and determining optimal protein concentrations for tissue samples, 2.) quantifying intraepidermal nerve fiber densities (IENFD) via confocal light microscopy, and 3.) calculating mechanical threshold values from Von Frey experiments on high-fat diet mice. This is a lot to unpack, so I'll explain each section in detail within my next few posts. 

I'll leave this here for now, but thanks for coming along on my SRP adventure! I'm excited to see what will happen in the next three months! 

Until next time, 

Liam