Spinal ganglion in a five days old mouse - De wereld onder de microscoop

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Spinal ganglion in a five days old mouse

Preparaten > Organen en cellen 2

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Summary

The purpose was to find out if it is histological possible to make a coupe on the microtome of the spinal cord and vertebrates in one section. Primary target in that section was the position of the dorsal root ganglion.


Introduction

A dorsal root ganglion (or spinal ganglion) (also known as a posterior root ganglion), is a cluster of nerve cell bodies (a ganglion) in a dorsal root of a spinal nerve. The dorsal root ganglia contain the cell bodies of sensory neurons (afferent) [1].
The axons of dorsal root ganglion neurons are known as afferents. In the peripheral nervous system, afferents refer to the axons that relay sensory information into the central nervous system (i.e. the brain and the spinal cord). These neurons are of the pseudo-unipolar type, meaning they have an axon with two branches that act as a single axon, often referred to as a distal process and a proximal process [1].

Note: the neuron can consist of three parts:
1.   Dendrite that receives the information and relays it to the Soma, or cell body.
2.   Soma—the cell body of the neuron
3.   Axon: which relays information from the soma.

In a neuron, the dendrite receives information from another neuron's axon at the synapse, and the axon sends information to the next neuron's dendrites, even though the dendrite may be covered with myelin. Unlike the majority of neurons found in the central nervous system, an action potential in posterior root ganglion neuron may initiate in the distal process in the periphery, bypass the cell body, and continue to propagate along the proximal process until reaching the synaptic terminal in the posterior horn of spinal cord [1].
The dorsal root ganglia develop in the embryo from neural crest cells, not neural tube. Hence, the spinal ganglia can be regarded as gray matter of the spinal cord that became translocated to the periphery [1].

Click to enlarge image


Material and Methods

Because the calcification of the bone structures starts immediately after the mouse is born, the samples were taken at five days old mice. No decalcification was taken.
Mice heads were decapitated with a sharp disposable microtome knife and fixed in a mixture of formalin and glutaraldehyde. M.J. Karnovsky developed the mixture and this is still recommended for light and electron microscopy. In this case the mixture ratio was 2% formalin and 2,5% glutaraldehyde in a buffered solution [2].
A 2mm thick section of the neck was cut with a sharp disposable microtome knife and embedded in Technovit 7100. Technovit 7100 is a plastic embedding system based on HEMA (2-hydroxyethyl  methacrylate) [3].
Sections (2µm) were cut on a LKB 2218 Historange rotationmicrotome with a steel disposable knife and dried at 70 degrees Celsius.
Sections were stained with 1% toluidine blue in 1% sodium tetraborate in distilled water [4]. The sections were mounted in Depex, a neutral plastic resin [5].

 

Click to enlarge image


The processing protocol included:

- Fixation in Formalin/Glutaraldehyde (2%/2,5%), 10 days;
- Washing in 3 portions distilled water, 12 hours;
- Specimen dehydration:
   • Ethanol 70%, 12 hours;
   • Ethanol 85%, 12 hours;
   • Ethanol 95%, 12 hours;
   • Ethanol 100%, 12 hours;
- Specimen infiltration:
   • Ethanol 100% + Technovit 7100 (ratio 1:1), 12 hours;
   • Technovit 7100 + harder I (ratio 100ml Technovit + 1gr harder I), 2 portions, every portion 24 hours;
- Embedding:
Mix 15 portions of Technovit 7100 + harder I and 1 portion of harder II;
Place specimen in molds an polymerize.
- Polymerization:
Room temperature, 1 hour;
37 degrees Celsius, 3 hours;
For better cutting results specimen blocks can be hardened at 50 degrees Celsius for 48 hours.


Results

It was pretty easy to make this large coupe and the dorsal root ganglion was well preserved. Because of the calcification of the bone structures this five days old mouse tissue is the maximum time limit to cut on a normal rotation microtome such as the LKB 2218 Historange is.

Pictures of the coupes were taken with a Canon 700D digital camera on a Leitz Orthoplan microscope.




References

[1] Wikipedia, The Free Encyclopedia, https://en.wikipedia.org/wiki/Main_Page

[2] Maria Mulisch and Ulrich Welsch, Romeis Mikroskopische Technik, 18th
edition 2010, ISBN: 978-3-8274-1676-6, Pag 98, index 2.3.2.2.3 Fixierungsgemische mit Glutaraldehyd und Formaldehyd.

[3] Division Kulzer Technique, Kulzer GmbH - Division Technik - Philipp-Reis-Str. 8/13 - 61273 Wehrheim – Germany,  

[4] Prof. Dr. Peter Bock, Romeis Mikroskopische Technik, 17th edition 1989, ISBN: 3-541-11227-1, Pag 248, index 3.2 Schnellfärbung mit Toluidinblau O.


[5] www.ihcworld.com/royellis/ABCSafe/chemicals/depex.htm




 
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