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.