The authors would like to thankthe Jenkins Microscopy Facility at the University of Wyoming for their help, and the members of the Bushman lab, Kelly Roballo, Hayden True, Wupu Osimanjiang and Subash Dhunghana, for assistance in animal care. This publication was made possible by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 2P20GM103432.

Adult Sprague Dawley rats were used in this project and all procedures were approved by the University of Wyoming Institutional Animal Care and Use Committee.
1. Surgery and In Vivo Nerve Fixation
NOTE: Vendor information for all materials and equipment used in this protocol are listed in the Table of Materials.
NOTE: In vivo nerve fixation is used to preserve the tissue and reduce structural degradation that can occur between the time of death and collection of the nerves.&#160;In vivo tissue fixation is a standard practice for preparation of nervous system tissue for histology, where perfusion is often a precursor to this. The placement and size of peripheral nerves allowed for in situ fixation. We do not recommend collection and fixation of tissue after euthanasia due to the possibility of tissue degradation.
<ol>
	<li>Prepare the rat for general anesthesia by placing it in an induction chamber with 2-3% isoflurane, then place the anesthetized rat in the prone position on the top of a dissection mat and maintain at 1-2% of isoflurane via anesthetic nose cone.&#160;To ensure adequate depth of anesthesia, test animals for pedal withdrawal at the hind feet and check for the absence of palpebral reflexes at the eyes by touching the medial canthus of the eye. 
	NOTE: Intraperitoneal injection of ketamine is a commonly used alternative to inhaled isoflurane.</li>
	<li>To expose the sciatic nerve, shave the hair of the hind limb(s) and clean the shaved areas with 70% ethanol. With a scalpel or scissors, make a 2-3 cm incision in the skin along the hind limb from the knee up to the greater trochanter, where palpation of the femur using the sterile scalpel to ensure the proper location of the incision.&#160;The femur is located just proximal to the most accessible segment of the sciatic nerve. Even though this is a non-survival surgery, maintain sterile techniques.</li>
	<li>After making the skin incision, locate the plane between the biceps femoris muscle and the gluteus maximus muscle, and using microdissection scissors separate the underlying fascia and expose 2-3 cm of the underline sciatic nerve. To expose a clear section of the sciatic nerve, use a retractor to widen the gap between the two muscles (Figure 1A). With fine forceps and iris scissors, carefully separate the nerve from surrounding connective tissue, taking great care not to compress or cut the nerve.</li>
	<li>Add sufficient Trump&#8217;s fixative (4% formaldehyde, 1% glutaraldehyde in 1x PBS (phosphate buffered saline) with 1.16 g of NaH2PO4&#183;H2O per 100 mL) into the cavity containing the exposed nerve to cover the nerve and let sit for 10 min. If necessary, place gauze under the hind leg to improve the angle so that more fixative can be added into the cavity. Remove the fixative after 10 min&#160;and repeat this step two more times.</li>
	<li>Using a dissecting microscope, cut the nerve from both sides using fine dissection scissors, making sure not to stretch or pinch the sciatic nerve, and immediately put the nerve sections in 15 mL tubes containing Trump&#39;s fixative at 4 &#176;C for one week, changing the fixative every 48 h.</li>
	<li>Do not leave animals unattended during any part of the surgical procedure. After removal of the nerve, euthanize animals by cervical dislocation while under anesthesia.</li>
</ol>
2. Osmium Tetroxide Treatment and Resin Embedding
<ol>
	<li>Post fixation, carefully remove any remaining fat and connective tissues from the nerve and use a sharp scalpel to cut the nerve into segments approximately 5 mm in length (nerve segments can be separated and labeled in different tubes). Prepare fresh 2% osmium tetroxide diluted in Trump&#39;s fixative solution and keep in a glass tube. Immerse nerve segments in 2% osmium tetroxide for 2 h, which can be in plastic tubes for this short period of time. 
	NOTE: Some plastic tubes react with osmium tetroxide, causing a darkening of the solution, so prolonged storage of osmium tetroxide in plastic tubes is not recommended.</li>
	<li>Using an epoxy embedding medium kit, prepare the final embedding formula:
	<ol>
		<li>Mix the epoxy embedding medium with DDSA solution (dodecenylsuccinic anhydride; mixture A) and mix the epoxy embedding medium with NMA solution (methylnadic anhydride; mixture B). Let both mixtures A and B mix at least 20 min&#160;by stirring with a magnetic stirrer.</li>
		<li>Immediately before use, mix mixture A and B and add the accelerator DPM-30 (2,4,6-tris(dimethylaminomethyl)phenol) in the proportion of 1.5 to 2.0% of the total volume of the mixture. Note that the DPM-30 solution should be measured precisely to prevent the block from becoming dark in color and brittle. All resin chemicals are toxic; take extra care to prevent skin contact or inhalation.</li>
	</ol>
	</li>
	<li>Using 1.5 mL tubes, wash nerve segments with PBS and start the dehydration process by placing the nerve segments in different acetone/distilled water passages:&#160;30%, 60%, 90%, concentrations for 10 min each, then in 100% acetone 3 times for 10 min each.&#160; Note that ethanol can be used in the place of acetone.</li>
	<li>For resin infiltration, place the nerve segments in a mixture of 1 part of the final embedding mixture and 1 part 100% acetone for 30 min, then in a mixture of 2 parts of the final embedding mixture and 1 part 100% acetone for 30 min, and finally place the nerve segments in the final embedding mixture for 30 min.</li>
	<li>Put the nerve segments into silicone rubber embedding molds (we have used 106 mm length x 71 mm width x 7 mm depth; block size 11 mm length x 6 mm width x 3 mm depth). Gently add the resin on top of the nerves, making sure to cover the whole nerve segments and avoid making air bubbles. Leave the mold containing nerve segments with the resin to polymerize at 60 &#176;C overnight.</li>
</ol>
3. Sectioning by Ultramicrotome
<ol>
	<li>Prepare glass knives using a glass knife maker (Figure 1B) and make glass knives with boats(Figure 1C), which are used to collect nerve sections floating on the distilled water.</li>
	<li>Place resin embedded nerve blocks into the ultramicrotome holder with the trapezoidal side facing up. Using the ultramicrotome scope, trim any excess resin surrounding the nerve tissue, and make the block face a trapezoid shape and as close to the longitudinal surface of the nerve as possible using a single-edged blade, such as a razor blade. Do not penetrate the longitudinal surface of the nerve segment, which is recognizable by its dark staining by osmium tetroxide. 
	NOTE: Trimming of excess resin will reduce the area of resin to be sectioned and improve blade performance and cutting in subsequent steps.</li>
	<li>Using the ultramicrotome, make multiple cross sections sufficient to expose a uniform cross section surface of the nerve tissue using a plain glass knife. Once this is done, switch to a glass knife whose boat is filled with distilled water at room temperature and adjust the ultramicrotome to 1-2 &#956;m to cut thin sections.</li>
	<li>Transfer floating thin sections from the glass knife boat to a drop of deionized water on glass slide using metal loop (Figure 1D). Dry slides containing sections by passing several times over a flame for a few seconds, making sure not to overheat the sections. Stain dried sections immediately with toluidine blue or store at room temperature for several days before staining. 
	NOTE: Sections can also be dried using a plate warmer at 60 &#176;C for 15 min. A slow drying process makes the sections smooth.</li>
</ol>
4. Toluidine Blue Staining
<ol>
	<li>Prepare 1% solution of toluidine blue by dissolving 2 g of sodium borate in 100 mL of deionized water, then add 1 g of toluidine blue and stir until dissolved. Filter the solution using filter paper (pore size: 11 &#181;m) and keep the solution in an opaque bottle at room temperature for up to two weeks.</li>
	<li>Using a plastic pipette or micro pipettor, add a drop of toluidine blue solution on the top of the nerve sections and leave for 20-30 s.</li>
	<li>Rinse off all toluidine blue solution excess by gently dipping the slides into deionized water jar and repeat 3-4 times until sections are clear. Dry the slides at least 15 min at 60 &#176;C or overnight at room temperature and then cover the sections with a coverslip using regular mounting medium. Examine the mounted slides immediately or store at room temperature.</li>
	<li>Examine under a light microscope. A 100X oil immersion lens is recommended for detailed images used to calculate g-ratios.</li>
</ol>

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The authors declare that they have no conflicts of interest.

Examinations of the morphological structures of peripheral nerve injury and regeneration are frequent subjects of study13. In this protocol, we describe the steps to obtain high quality images for histological data quantifications using rat sciatic nerve tissue embedded in resin blocks and stained with toluidine blue. This technique provides an image of nerve morphology in which the nerve regeneration can be quantified by measuring the number of axons, degree of myelination, presence of infiltrative fibrotic tissue, and the health nerves. While the images show sectioning and processing of uninjured nerves as samples, the same steps and materials are applicable to injured nerves and nerves regenerated with conduits or tissue grafts, provided that fixation of the tissue is adequate20,21. 
While all steps of the protocol are essential, some of these steps are more likely to lead to poor quality sections or images. Insufficient tissue dehydration may lead to holes in the nerve sections (Figure 5B). One possible explanation is the immiscibility of resin with water, and excess water in the tissue prevents resin from infiltrating the tissue. Therefore, allowing sufficient time and the use of absolute acetone is essential to ensure proper dehydration step. Although we used acetone in this protocol, ethanol can also be used. Many resins, however, are not reactive with ethanol, so the tissue must be treated with propylene oxide to serve as a transition between the ethanol dehydration and resin embedding.
Due to the thinness of the sections, transferring nerve sections from the glass knife to the glass slide should be done with great care (Figure 5C). 1-2 &#956;m sections are very fragile, and improper transfer of the section could cause the section to break. If the section is picked up in the central portion of the section with a needle, the section is prone to folding in on itself and often will be difficult to unfold when transferred to the slide. Different tools can be used for the transfer of the section to the glass slide, including needles and loops, and each should be tested for the specific user to determine which will yield the best transfer results. For our purposes, using a loop that could encompass the entire section greatly reduced the folding of sections when they were transferred from the glass knife to the microscope slide (Figure 1D).
In general, proper handling of the nerves is essential as compression of the nerves can cause cracks to occur in nerve sections (Figure 5A). Nerve tissue compression can happen during transfer of nerve segments in the processes of exposure in the animal, fixation, dehydration, and resin embedding. To avoid nerve tissue compression, nerve segments should be transferred by fine forceps and picked up on one end of the segment ideally by just the epineurial layer, so the entire nerve will not be affected. The presence of cracks and lines (knife marks) in the nerve sections may also occur due to dull or overused glass knives. To guarantee optimal sectioning, we recommend making fresh glass knives and changing the glass knife every 25-30 cuts, fewer if sectioning through nerve encased within a conduit.
Resins can be relatively expensive, are inadequate when longitudinal nerve sections are required, and can require expensive tools such as an ultramicrotome and a glass knife maker. Despite these limitations, toluidine blue staining of resin embedded peripheral nerve sections is still considered the gold standard for visualization of cross sections of peripheral nerve morphometry4,5. Longitudinal sections can also reveal important features of peripheral nerves, such as axonal continuity and nodes of Ranvier, but is most useful with immunohistochemistry. In such cases it is the general practice to differentially process two sections of the same nerve, one for toluidine blue cross sections and the other for longitudinal immunohistochemistry. Due to increased incidences of peripheral nerve trauma and injuries and the needs of a better way of assessing nerve morphological structures, this method can continue to be applied as an essential tool to gain histological data of nerve damage and regeneration.

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons1. Peripheral nerve defects caused by medical disorders and trauma represent a major public health concern and have large economic impacts2,3. Despite the advances in assessing the outcomes of peripheral nerve injuries and understanding nerve regeneration, traditional methods such as nerve histology and staining techniques are essential tools to qualitatively and quantitatively assess nerve health as a terminal outcome measurement in animal models or excised human tissue. This is often paired with electrophysiological measurements of peripheral nerve function, where morphometry can reveal why functional nerve regeneration did or did not occur.
Toluidine blue staining of resin embedded semi-thin peripheral nerve sections is a specialized method for imaging myelinated nerve fibers, providing high quality and clear detailed images of nerve structures4,5,6. Toluidine blue is an acidophilic metachromatic stain, discovered by William Henry Perkin in 18567, and has been used in several medical applications8. Toluidine blue-stained peripheral nerve sections obtained from resin-embedded nerve segments allows for clear visualization of nerve structures. Visualization of myelin sheath structure can be enhanced by the use of osmium tetroxide post-fixation4,9. Osmium tetroxide is a toxic oxidant and lipid fixative agent that interacts with the double bonds in lipids, resulting in starkly defined lipid-rich myelin sheaths10. However, osmium tetroxide is toxic, expensive, requires a longer incubation of nerve segments, and is not always used.
Alternative methods of processing and staining have been developed for visualization of peripheral nerve morphology; Paraffin, cryogenic sectioning, and epoxy resin-embedded nerve sectioning followed by staining with toluidine blue or phenylenediamine solution has been used to quantify morphological changes of peripheral nerve regeneration 11,12. These methods each have their advantages and yield essential data on the number of axons, myelin thickness, axon diameter, and axon diameter to myelinated fiber diameter (g-ratio) 11,13,14,15.
The primary distinction of the resin-embedding in this protocol is that it facilitates obtaining 1-2 &#956;m thickness cross-sections due to the hardness of the resin while maintaining the histological qualities of the nerve. These thin sections, as opposed to the 4-5 &#956;m thickness sections obtained from paraffin embedding, provide peripheral nerve sections with higher resolution, allowing for a more accurate quantification of axon myelination, such as the g-ratio, that cannot be obtained from thicker sections16. While cryogenic sectioning can be used to obtain 1-2 &#181;m sections, it has been our experience that it is more difficult to obtain sections without numerous large cracks. Such cracked sections can cause inaccurate counting of the number of axons and aspects of myelination.
In addition to toluidine blue staining17, a silver staining method18 and Masson&#39;s trichrome staining4 can also be used to show nerve axons. However, using resin embedding of rat median nerve sections stained with either hematoxylin and eosin or Masson&#39;s trichrome showed faint myelin sheaths and unrecognized structures, whereas toluidine blue staining showed clear myelin sheath image and easily can be quantified4. Despite some limitations, toluidine blue staining of resin embedded peripheral nerves is a valuable technique that can be used when high resolution images of nerve morphology are required.
The primary disadvantage for resin embedding is that it is time-consuming and does not allow for immunostaining of the same tissue due to the difficulty of antigen retrieval when compared to paraffin and frozen embedded sections techniques. Thus, it is not generally possible to utilize the same tissue for immunostaining that is processed via resin-embedding for toluidine blue staining. Although not used here, if immunohistochemistry is desired in resin embedded sections, the use of glycol methacrylate embedding resins allows for immunohistochemistry to be performed on tissue sections, but it is relatively expensive19. This can be somewhat mitigated by cutting the peripheral nerve into separate segments, some for resin-embedding and others for immunostaining directly after fixation.
The process of toluidine blue staining of resin embedded peripheral nerves, as with most histopathological analysis, can be broken up into five stages, including fixation, dehydration, embedding, sectioning, and staining20. We aim here to provide a protocol and practical guideline for using resin embedded rat sciatic nerve sections stained with toluidine blue to acquire high quality images.

<table><tbody><tr><td>Dulbecco&#039;s Phosphate Buffer Saline</td><td>Gibco</td><td>14200-075</td><td></td></tr><tr><td>Glutaraldehyde Solution</td><td>Sigma-Aldrich</td><td>G6257</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Sodium Phosphate Monobasic Monohydrate</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>Toluidine Blue O</td><td>Sigma-Aldrich</td><td>T3260</td><td></td></tr><tr><td>Sodium Tetraborate Decahydrate</td><td>Acros Organics</td><td>205950010</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal</td><td>NDC 66794-013-25</td><td></td></tr><tr><td>Epoxy Embedding Medium Kit</td><td>Sigma-Aldrich</td><td>45359</td><td></td></tr><tr><td>Sodium Hydroxide Solution</td><td>Sigma-Aldrich</td><td>72068</td><td>To adjust Trump&#039;s fixative pH</td></tr><tr><td>Acetone</td><td>Fisher Chemical</td><td>170942</td><td></td></tr><tr><td>Osmium Tetroxide Solution</td><td>Sigma-Aldrich</td><td>75632</td><td></td></tr><tr><td>VWR Micro Slides, Superfrost Plus</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific</td><td>12545102</td><td></td></tr><tr><td>Pelco Embedding Cast</td><td>Fisher Scientific</td><td>NC9671811</td><td></td></tr><tr><td>Glass Knife Maker</td><td>RMC Products</td><td>GKM-2</td><td></td></tr><tr><td>Ultramicrotome</td><td>RMC Products</td><td>MT-XL</td><td></td></tr><tr><td>15 mL Conical Tube</td><td>Falcon</td><td>ISO 9001</td><td></td></tr><tr><td>Eppendorf 1.5 mL microcentrifuge tubes</td><td>Sigma-Aldrich</td><td>T9661</td><td></td></tr><tr><td>4 mL Glass Vial</td><td>Sigma-Aldrich</td><td>854190</td><td></td></tr><tr><td>Razor Blades</td><td>VWR</td><td>55411-050</td><td>For trimming resin block</td></tr><tr><td>Perfect Loop</td><td>Electron Microscopy Sciences</td><td>70944</td><td>For picking up thin resin sections</td></tr><tr><td>Ultra Glass Knife Strips 6.4 mm x 25 mm x 400 mm</td><td>Electron Microscopy Sciences</td><td>71012</td><td></td></tr><tr><td>100 Watt Oven</td><td>Millipore&amp;nbsp;</td><td>6350115</td><td></td></tr><tr><td>Whatman Filter Paper</td><td>Sigma-Aldrich</td><td>WHA10010155</td><td></td></tr><tr><td>3 mL plastic pipette</td><td>Sigma-Aldrich</td><td>Z331740</td><td></td></tr><tr><td>Micro-surgical Kit</td><td>World precision instruments</td><td></td><td></td></tr><tr><td>Olympus fluorescence microscope</td><td></td><td></td><td>Dual CCD Color and Monochrome Camera, DP80</td></tr></tbody></table>

toluidine blue staining of resin-embedded sections for evaluation of peripheral nerve morphology

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves are frequently damaged because of trauma or disease. As methods and strategies have been developed to assess peripheral nerve injury in animal models, function and regeneration, analyzing the morphometry of the peripheral nerve has become an essential terminal outcome measurement. Toluidine blue staining of nerve cross sections obtained from resin embedded nerve sections is a reproducible method for qualitative and quantitative assessments of peripheral nerves, enabling visualization of morphology number of axons and degree of myelination. This technique, as with many other histological methods, can be difficult to learn and master using standard written protocols. The intent of this publication is therefore to accentuate written protocols for toluidine blue staining of peripheral nerves with videography of the method, using sciatic nerves harvested from rats. In this protocol, we describe in vivo peripheral nerve fixation and collection of the tissue, and post-fixation with 2% osmium tetroxide, embedding of nerves in epoxy resin, and ultramicrotome sectioning of nerves to 1-2&#956;m thickness. Nerve sections then transferred to a glass slide and stained with toluidine blue, after which they are quantitatively and qualitatively assessed. Examples of the most common problems are shown, as well as steps for mitigating these issues.

Resin embedded peripheral nerve sections stained with toluidine blue allow for accurate histological data quantifications. An overview of the procedure is shown in (Figure 2). Sciatic nerves sections embedded in resin medium and stained with toluidine blue showed clear images with optimal resolution (Figures 3). Nerve damage can cause many changes in nerve morphological structures, for example, changes in nerve fiber, axon diameter, and myelin sheath thickness. This method can preserve nerve structure in its natural form, which facilitates the measurement of several parameters such as the ratio of axon diameter to the total fiber diameter (the g-ratio; Figure 4). A variety of factors can lead to less than optimal peripheral nerve sections including the presence of cracks (Figure 5A) in the section due to improper handling of the nerve. Holes can also occur in the section (Figure 5B) due to insufficient dehydration. Another possible error in the procedure is folding of the sections (Figure 5C), which can be remedied by using inoculating loops for section transfer onto the glass slide.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58031/58031fig1.jpg" /> 
Figure 1: Surgery and sectioning. (A) Rat sciatic nerve during in vivo fixation. (B) Glass knife maker (C) Glass knives with boat. (D)Metal loops used to transfer floated thin sections from the glass knives to a drop of deionized water on glass slide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58031/58031fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58031/58031fig2.jpg" /> 
Figure 2: Schematic of the method used in this protocol. (A) Adult Sprague Dawley rat is anesthetized, followed by in vivo nerve fixation (B) and nerve collection (C). The next step is post-fixation with 2% osmium tetroxide (D) and resin embedding (E). Resin embedded nerve sections are then trimmed (F) and sectioned (G) using an ultramicrotome. Finally, nerve sections on glass slide are stained with toluidine blue (H). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58031/58031fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58031/58031fig3.jpg" /> 
Figure 3: Transverse section of a rat sciatic nerve stained with toluidine blue. Shows sections under different magnifications (10X, 20X, 40X, and 60X) of clear image with optimal resolution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58031/58031fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58031/58031fig4.jpg" /> 
Figure 4: Transverse section of a rat sciatic nerve stained with toluidine blue. This high-resolution image (100X) can be used to measure the ratio of axon diameter to the total fiber diameter (the g-ratio). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58031/58031fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58031/58031fig5.jpg" /> 
Figure 5: Transverse sections of a rat sciatic nerve stained with toluidine blue under different magnifications. Shown aresome of the problems that may be encountered during performance of this protocol. (A) and (B). The presence of cracks and holes inside the nerve section caused by improper handling of the section and insufficient dehydration of the tissue, respectively. (C). Folding of the sections, which may happen during transferring the sections from the glass knives to the slide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58031/58031fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology at JoVE.com

Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology

Here we present a protocol to visualize fine structures of peripheral nerves by obtaining and staining 1-2 &#181;m sections with toluidine blue

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves ...

toluidine-blue-staining-resin-embedded-sections-for-evaluation

Nanyang Technological University

Research

JoVE Journal

Neuroscience

18.7K Views.  University of Wyoming. Here we present a protocol to visualize fine structures of peripheral nerves by obtaining and staining 1-2 µm sections with toluidine blue

Video: Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Developmental Biology

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen films. Favorable clinical outcomes have been achieved when using these tubes for the treatment of damaged inferior alveolar nerve (IAN). A critical factor for the successful nerve regeneration using PGA-C tubes is blood supply to the surrounding tissue. Cervical sympathetic ganglion block (CSGB) creates a sympathetic blockade in the head and neck region thus increasing blood flow in the area. To ensure an adequate effect, the blockade must be administered with local anesthetics one to two times a day for several consecutive weeks; this poses a challenge when creating animal models for investigating this technique. To address this limitation, we developed an ethanol-induced CSGB in a canine model of long-term increase in blood flow in the orofacial region. We examined whether IAN regeneration via PGA-C tube implantation can be enhanced by this model. Fourteen Beagles were each implanted with a PGA-C tube across a 10-mm gap in the left IAN. The IAN is located within the mandibular canal surrounded by bone, therefore we chose piezoelectric surgery, consisting of ultrasonic waves, for bone processing, in order to minimize the risk of nerve and vessel injury. A good surgical outcome was obtained with this approach. A week after surgery, seven of these dogs were subjected to left CSGB by injection of ethanol. Ethanol-induced CSGB resulted in improved nerve regeneration, suggesting that the increased blood flow effectively promotes nerve regeneration in IAN defects. This canine model can contribute to further research on the long-term effects of CSGB.

We evaluated the effect of cervical sympathetic ganglion block on nerve repair using artificial nerve conduits. Male beagle dogs were each implanted with an artificial nerve across a 10-mm gap in the left inferior alveolar nerve; left cervical sympathetic ganglion was blocked by injecting 99.5% ethanol via lateral thoracotomy.

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology

Summary

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Chapters in this video

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve