Mammoth Approacheth. Pt. 2

                                                         Mouse footpad section with Toluidine Blue 

    At last! The protein concentrations work, the tissue sections work, and now for confocal imaging! I'm posting on the blog too! It's a miracle! Let's get into what I've been doing with my life and my research recently. 

(Ha! Who am I kidding? My research is my life! What a funny joke!)

Ok, seriously. 

   Last week, I did a massive amount of mouse footpad sectioning on the lab microtome. Because peripheral neuropathy starts as a stocking-glove combination in hands and feet, the footpads of high-fat fed mice are the first to show changes in intraepidural nerve fibers (IENF), nerves extending through the layers of skin. IENF detect touch/heat and thus are the primary source of peripheral neuropathic pain. Although researchers currently don't know what really causes neuropathic pain, there are theories about pathways that may contribute to the development of neuropathic pain, including 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 (to name a few). By sectioning these mouse footpads, I can stain them for different proteins and biomarkers to identify the differences between normal mice and high-fat diet mice. Above is a Toluidine Blue staining I did for cell nuclei. It's a quick-and-easy way to see if my sections look good, before using expensive antibodies and extensive IHC staining on them. I'm rather proud of that particular section above. You can see everything so clearly--sweat glands, layers of skin, blood vessels, collagen, connective tissue, all working together in just a tiny footpad of a tiny mouse!

The lovely microtome I work on. The blade cuts through everything like butter, including butter. 

  And with lots of staining, comes lots of tissue sectioning. Sectioning on the microtome is tedious, not just because I have to manage a steely blade less than a foot away from my face, but the mouse footpads are impossible to position just right (I need to cut horizontally across an upright footpad which is, let me reiterate, the footpad of a mouse and therefore tiny), the dry ice is always evaporating, and I have to wash and re-mount the stage for every new footpad I section. I have 64 footpads to section. I have done 14. But science goes on, and my work must too. 

   This experiment isn't just about neuropathy--there's a longitudinal age factor thrown in too. Half of the mice I'm testing were aged 5 weeks, and half aged 36 weeks (Mice live about 1-2 years, or in baby-age speak, 52-104 weeks, so these are fairly young.). Both the control group and the high-fat diet group are split up this way, 16 mice in each, 32 mice total, so the effect of a high-fat diet on neuropathy can be examined from a how-long-have-you-been-eating-cheeseburgers perspective. Never done before, this cheeseburger thing. 

   Here's a more technical look at the causes of high-fat induced neuropathy, and Tumor Necrosis Factor Alpha (TNF-a), one of the proteins I'll be staining the mouse sections with. 

   Research suggests that overall low-grade inflammation in obesity causes insulin resistance (prediabetes and diabetes). Excessive intake of dietary fat then disrupts the homeostasis of cellular metabolism and triggers an inflammatory response in adipose tissue. This obesity-related inflammation is associated with increased numbers of infiltrating proinflammatory macrophages and other inflammatory cells in the fat tissue Circulating fat-derived factors, including C-reactive protein (CRP), TNF-a, and IL-6 contribute to the development of prediabetes and diabetes. Among these proinflammatory mediators, TNF-a is a major cytokine that mediates the development of HF-induced insulin resistance in adipocytes. TNF-a actions directly affect insulin signaling in HF diet-induced obesity. In dorsal root ganglia (DRG) neurons, insulin resistance is detected following chronic insulin treatment and in diabetic animals.

HF diet induces TNF-a/NF-kB signaling in DRG neurons: A HF diet increases TNF-a expression in DRG neurons, which in turn binds to TNFR1 and induces sequential recruitment of adaptors, including TRADD, TRAF2, and RIP. This complex activates the IKK complex which leads to the phosphorylation of IkB and the p65 subunit of NF-kB, causing dissociation of IkB from the NF-kB dimer. The free NF-kB dimer then enters the nucleus to regulate gene expression.

   So far I've stained footpad sections with TNF-alpha, CGRP, Anti-Langerin, CD68, and PGP, with the potential to do Prenselinin and Trk-A as well. Without getting into what each of these proteins are for--because I would die inside and you would fall asleep--I'm doing this so that the cells and nerves in the skin sections can be better identified. Inflammation is caused, and can be seen, in many different cells and structures, many of which may be similar under the microscope. So by staining for many different things, I'm hoping to be able to distinguish between all that's going on. Next week, I'll be finally--finally!--be able to take my finished slides to examine under the confocal microscope here in the lab.

   To be honest, I don't nearly understand everything about neuropathy and its mechanisms. I only work with a small portion of the big picture. Every explanation in science is always a cross-section perspective of the whole process, where many extensive and varied systems and processes are interacting with each other--yet this universal and far-reaching truth can be found in the tiny footpad of a tiny mouse. Life never fails to amaze me with its delicate complexity.  

P.S.:

   You've probably noticed that I finally came up with a pithy title for the blog (Yeah Liam, after like, five weeks). "Near Space Exploration" was a reference to how fluorescent tissue sections looked like galaxies and nebulas through a microscope lens. In retrospect, I should have clarified that. "Cheeseburger Pain" was an alternative title option, but that and "Burger Bliss" just didn't meat my expectations--so I was in a bit of a pickle after they didn't cut the mustard. Hamberger bun puns?

Grill-ty as charged.

Look, a Mammoth! Pt. 1

      The right filter on anything can make it the place where the zombie apocalypse begins.

AHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH

    I'm back! Sorry for the long wait! But I now present, for your personal reading pleasure, the next installment of-- THE BLOG. (Don't you think the entrance to my lab looks inviting? That biohazard sign beats any scratchy welcome mat I know of.) This story comes to you in four chunks, spread over four posts, installed over four days. Today,

-  THE PAST  -

   Let's start with where we left off--the mice.

   The mice live in a separate facility downstairs (that I can't access quite yet.) and are brought up to the lab. When we talked last the mice were being collected --a nicer word for killed, yes, but let me be clear, the mice are put to sleep and then collected as humanly and ethically as possible. Every organ in a mouse is a wealth of information. The brain, brain stem, cerebellum, blood plasma, fat, beige fat, brown fat, lower spinal cord, sciatic nerve (the largest nerve in the body, mice and humans) dorsal root ganglia, the liver, and footpads are all collected in their own little eppendorf tubes for analysis--and with 16 mice, a total of around 240-ish samples are collected for testing. All in all, it took a tag team of three people--Thomas, my mentor/PI, Joe, the lab tech here, and Martin, who researches Alzheimers--two days to get all the samples. Joe dissects the head and removes the brain, Martin makes the first incisions into the rat, and Thomas removes the small samples under a dissecting microscope.

  Stages of labeling 240 tiny tubes--denial, anger, bargaining, depression, and OCD insanity. 

   This study has two groups of mice--control and high-fat. The control are just your ordinary-Joe-schmo mice that you'd meet on the street with an ordinary diet. The high-fat mice are fed with, you guessed it, McDonald's quarter pounders with cheese. Ok, with food pellets that have a high fat content (Which are colored green, so--cool fact--the intestines of the mice look bright green upon dissection). A diet high in fat is associated with weight gain/obesity in humans and mice, which can increase risks of type-2 diabetes and a host of other problems. Although if you're looking at changing your own diet, I recommend you consult your local nutritionist for detailed advice. The high-fat diet can cause symptoms of neuropathy, which leads to my current task involving the staining and sectioning of teeny-tiny mouse footpads. Think "30 micron-thick" teeny-tiny (Human hair is about 50 microns thick). But more on that in Part 2. 

    So the amount of time and money that goes into making a single data point for this study is crazy! Growing the mice for months, then processing the samples for weeks, then analysis of that data--not to mention all the equipment, materials, and manpower--sorry, personpower--in the journey from nothing to published paper. I'm truly grateful for this opportunity to peek into the scientific process, and to play a small part in the inexorable march of science towards discovering the great unknown. Go Science!

A note about "."
  
    In each plastic box a mouse chatters behind metal grates, oblivious to the gloved hand which will drop it into a glass box in a few moments, from which it will never see or smell the sawdust in its enclosure ever again. I'm morbidly fascinated by the entire process by which a living, breathing being is broken down into its component parts in just a matter of moments. So this collection of bones and blood is what sustains life in its infinite beauty and multitudes? I can't help but feel pangs of existential dread, knowing that I am like the mouse, in this fleshy container that I call "me". Alas, poor Yorick! My molecules, aren't alive, they're made up of the same elements found in books and trees and even stars and rocky planets beyond our galaxy. But I believe that consciousness makes the human collection of atoms unique--as Descartes said, "cogito ergo sum." I think, therefore I am. We are bridges that touch the banks of life and death, coalesced into a point in space and time to gaze at the universe all too briefly. One day my dust will return to where it came from--but until then, I will never cease wondering and wandering in the world, learning broadly, and without fear. 

~ Liam 

.

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