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