Duke Histology - Nerve Tissue

In this laboratory we will examine nerves, neuronal cell bodies, and ganglia. Let us start with a few important definitions:

Axons are neuronal processes specialized for electrical impulse conduction. EMs show a cytoskeleton rich in microtubules (neurotubules) and intermediate filaments (neurofilaments). The organelles associated with protein synthesis are rare in axons, but abundant in neuronal cell bodies, where the membrane channels, ion pumps and synaptic machinery needed in axons and dendrites are synthesized. Axons may be long (spinal cord to foot) or short (many interneurons of CNS). Myelin is formed by Schwann cells in the PNS and produces segmental insulation interrupted between successive Schwann cells at the nodes of Ranvier, where ion channels and ion pumps are localized and support saltatory impulse conduction.

I. Peripheral Nerves and the Myelin Sheath

Webslide 0020_O: sciatic nerve, c.s, TB-AF
[DigitalScope]

The sciatic nerve is a mixed nerve, containing sensory axons from neuron cell bodies in dorsal root ganglia and motor axons from neurons in spinal cord gray matter. Like all larger peripheral nerves bundles, it is also mixed in the sense of containing both somatic and autonomic nerve fibers.

Scan the slide at low power to see the fascicular organization, identifying epineurium (dense connective tissue surrounding the nerve and filling in between nerve bundles or fascicles), perineurium (thin layers of flattened cells immediately surrounding and defining each fascicle), and endoneurium (fine connective tissue within each fascicle between nerve fibers--see your text for orientation). Around smaller fascicles, the epineurium becomes very thin, leaving the bundle surface visible as the circumferential wrapping of perineurial cells. Note that the perineurium gives nerve bundles sharply defined boundaries that makes them easy to distinguish from vessels, ducts, CT, and muscle fibers. The nuclei visible within each nerve bundle include crescent-oval Schwann cell nuclei snugly tangent to nerve fibers, and smaller, denser nuclei belonging to endoneurial fibroblasts or the endothelium of capillaries running through the endoneurium, usually parallel to nerve fibers.

At higher magnification you can see the unstained rings representing individual myelin sheaths surrounding axons. Unmyelinated nerve fibers (which typically occupy at least 5% of a nerve's cross-section) can be visualized as axons without surrounding myelin sheaths.

Here you see a nerve lying parallel to 1-2 medium-small blood vessels, cut from a block that presents both l.s. and c.s. views. Here myelin remains only as a pink network of residual protein. Although the endoneurium is very hard to spot, the epineurial CT and the perineurial wrapping are easily seen. Note in c.s., the axons in the center of many nerve fibers. Unmyelinated fibers are impossible to distinguish here, but you should be able to observe the nuclei of Schwann cells, endothelial cells, and endoneurial fibroblasts.

Locate the peripheral nerve in association with other tissues in the bottom right quadrant and the upper right quadrant of the slide. What criteria are useful in identifying a peripheral nerve? Look for both myelinated and non-myelinated fibers. The nervous system, like the vascular system, is very pervasive. You will see it a lot in future slides.

II. Basic Cell Types of the Central Nervous System and Peripheral Nervous System

A. Introduction

In these slides, cell types can be best distinguished in specially stained slides, where a small number of cells and their processes are densely stained. However, the "empty spaces" in this material are deceptive--they contain unstained cell processes whose cell-cell relationships are poorly illustrated. Weblide #19 of this lab, on the other hand, contains practically no "empty space." It enables you to see the region of synaptic contacts (neuropil), but the appreciation of basic cellular shape and orientation is considerably more difficult. It is necessary to combine information from both kinds of preparations to get a feeling for the cellular configurations.

B. Slide Descriptions

This section contains a cross section of the spinal cord. The tiny central canal recalls the origin of the nervous system as an infolding which closes off to form the neural tube. Locate the central canal in the middle of your section and examine the lining layer of ependymal cells. Compare the ependymal nuclei with the neuronal nuclei you observed in previous slides. Ependymal cells also line the ventricles of the brain that are continuous with the central canal of the spinal cord.

During development, newly formed neurons proliferate adjacent to the canal to form a mantle layer, which becomes the gray matter in the central region of the mature spinal cord. This gray matter occupies a butterfly-shaped cross-section in this mature spinal cord. In the gray matter, examine the large motor neuron cell bodies carefully. Depending on how the cells are cut, you may see the nucleus, nucleolus, and axon hillock, as well as the Nissl bodies (rough ER) filling the cytoplasm. Note also the smaller nuclei of neuroglial cells. Neuropil refers to the regions in gray matter that lie between cell bodies, devoid of nuclei but complexly crowded with neuronal cell processes and synapses.

The neurons of the mantle layer produce processes that grow outward to form the fiber tracts of white matter, which carry parallel myelinated axons for long-range interneuronal contacts in CNS. The white matter fills most of this cross-section external to the central butterfly-shaped region of gray matter. This marginal zone is white primarily because of the abundance of myelinated axons, which you can observe in your sections, although the axons are very faint and therefore difficult to see.

Webslide 0019A_O: Spinal cord, mammalian, silver stain
[DigitalScope]

Quickly repeat the observation of the spinal cord as described above, noting the differences that can be readily observed due to the silver staining of the neuropil. Also note the dorsal root ganglia on either side of the spinal cord that contain large neuronal cell bodies similar to those that you will observe in the next slide.

The trigeminal (aka "Gasserian") ganglion (the sensory ganglion of the fifth cranial nerve) is a collection of pseudo-unipolar cells (pseudo because the single cell process arising from the soma splits into two functionally polarized processes). In response to tactile stimuli on the face a nerve impulse travels along the axon, passes through the ganglion containing the cell bodies, and finally reaches a synapse in the sensory nucleus of cranial nerve V in the brain stem. This is the first neuron (primary neuron) in a pathway whose activity will eventually result in your awareness of being touched.

In this slide, you will see the large round cell bodies of these primary neurons, also called ganglion cells. The nuclei are large and stain faintly. The single nucleolus, if it happens to be in the plane of section, stains darkly, giving the nucleus a bull's eye appearance typical of many neurons. Around each neuronal soma are smaller flattened cells, whose darkly stained nuclei form a ring around the ganglion cells. These are specialized astroglial cells called satellite cells (not to be confused with satellite cells of skeletal muscle).

In the same plane of section you will also see collections of light blue disks surrounded by unstained structures that look like white donuts. These are the axons and myelin sheaths of the ganglion cells passing through the plane of section. Look among the myelinated fibers for the wedge-shaped, densely staining nuclei of the Schwann cells, whose membranes form the myelin sheaths.

Among the cells and fibers in the trigeminal ganglion you can observe capillaries, many still containing easily identified red blood cells. Note the connective tissue coverings of the ganglion.

Webslide 4000: Colon, monkey, H&E
[DigitalScope]

In this section of the ileum, you will see autonomic ganglia where axons (whose cell bodies are in the spinal cord) synapse with intrinsic neurons (whose cell bodies are located here in the GI tract). These ganglia (called the Auerbach's or myenteric plexus) are found all along the GI tract between the circular and longitudinal layers of the outer smooth muscle. Like other parts of the peripheral nervous system, these ganglia are covered by a thin connective tissue layer, essentially a perineurium. Like CNS, but unlike other (non-enteric) autonomic ganglia, these enteric ganglia exclude connective tissue, and contain only neurons and glial (supporting) cells. Look for the typical neuronal bull's eye nuclei. Neurons are smaller and glial supporting cells fewer in autonomic ganglia than in sensory ganglia, so they rarely appear as obvious rings of satellite cells like those seen in Webslide #18. A good example of a neuron in an Auerbach’s plexus is shown in this annotated version of the slide (Arrow in 'Region D').

As a review, try and identify the other annotated features:

Arrow in 'Region B' (identify the cell) - ANSWER

Arrow in 'Region C' (identify the tissue) - ANSWER

Pathology Case 005: prostate cancer with perineural invasion
[DigitalScope]

This slide is from an individual who underwent a prostatecotomy for treatment of prostatic adenocarcinoma, or prostate cancer. Note that the term "carcinoma" refers to a cancer derived from epithelial cells and "adeno-" further specifies the epithelium as having been of a "gland." In this case, the tumor came from the epithelium of prostate glands.

First, look at the bottom left portion of the slide to view some typical prostate glands [example]. How would you classify this epithelium? [answer]. Note also the surrounding "fibromusclar stroma" that consists of dense, irregular connective tissue (DICT) mixed in with bundles of smooth muscle. We haven't studied smooth muscle yet, but it consist of densely packed bundles of smooth muscle cells that contain a lot of protein (actin and myosin) and therefore pick up much more of the eosin stain compared to the DICT.

Now, go to the top portion of the slide where there are nests of tumor cells that still form "glandular" structures (i.e. they still have a lumen), but the epithelial cells show many of the characteristics of malignant transfmormation such as pleimorphism (irregular size and shape), more densely packed, and also more proliferative (as evidenced by numerous mitotic figures).

Along the top portion of the slide, there are numerous nerves running through the connective tissue, and you should observe that many of the nerves have tumor cells invested around the perineurium [example]. This is phenomenon known as "perineurial invasion" and is a feature of many prostatic adenocarcinomas as well as pancreatic, gastric, colorectal, and cervical cancers, and many cancers of the head and neck that can metastasize to other places of the body by spreading along the nerve sheaths (as opposed to other tumors that more often spread by invading into lymphatic and/or blood vessels).

While the presence of perineurial invasion here is not necessarily an ominous sign (i.e. many low-grade prostate tumors show some degree of perineurial invasion), it is fair to say that the degree of perineurial invasion observed is highly correlated with poorer outcomes (i.e., the degree of perineurial invasion observed is associated with increased risk of prostate cancer-specific mortality), and this is therefore one of the diagnositc criteria used when evaluating and treating patients for prostate cancer.

Alternate UMich slide 68: peripheral nerve, monkey, c.s., H&E
[DigitalScope]

Alternate UMich slide 67: peripheral nerve, monkey, l.s., H&E
[DigitalScope]

The alternate UMich slides 68 and 67 are similar to WebSlide 0014 above and also show how nerves appear in H&E-stained paraffin sections. These sections are thicker, so the fine details (e.g. distinguishing between Schwann cells and endoneurial fibroblasts) are not so easy to see. However, the distinction between the perineurium and epineurium in slide 68 is quite obvious. Slide 67 is a nerve in longitudinal section, so you should try and find some examples of nodes of Ranvier. You should also start appreciating how nerves differ from the other "linear" structures that you'll encounter on microscope slides such as blood vessels, connective tissue, and muscle tissue as you will certainly need to be able to tell these tissues apart as you progress in your medical training.

Alternate Webslide 0034_O (human): lumbar spinal cord, c.s., H&E
[DigitalScope]

The alternate Webslide 0034 shows how the features described for Webslide 0302 above appear in an H&E-stained section. The neurons, glia, neuropil, and white matter fiber tracts are particularly well demonstrated. The "central" canal, however, is a bit different from that which is in Webslide 0302 due to the fact that this particular section is lower in the cord where the "central" canal is no longer a single lumen, but is instead broken up into several smaller fluid-filled channels, each lined by columnar ependymal cells.

Alternate UMich slide 65-1N: spinal cord and dorsal root ganglion, human, Trichrome
[DigitalScope]

Alternate UMich slide 65-1N shows a section of spinal cord and a dorsal root ganglion similar to that shown in WebSlide 0019 above, except this slide is stained with Masson's trichrome. Observe that the dorsal root ganglion is also comprised of pseudounipolar neurons that function in a similar manner to those in the trigeminal ganglion; i.e. they convey sensory information from the periphery (from sensory receptors in the skin, joints and muscles that respond to touch, temperature, pain, stretch) into the central nervous system. In this particular case, the fibers of the dorsal root ganglion project into the dorsal horn of the spinal cord and synapse with neurons of the dorsal horn. Like the neurons of the trigeminal ganglion, the dorsal root ganglion cells are also very large and spherical with a prominent, centrally-located nucleus and are completely encircled by a ring of satellite cells.

Alternate UMich slide 250: vagina, human, H&E
[DigitalScope]

Alternate UMich slide 250 features autonomic ganglia that can be found associated with the wall of the reproductive tract, in this particular case the vaginal wall. Look in the upper 1/3 of the section on the right side and you should see rather prominent peripheral nerve fibers and abundant autonomic ganglion cells. Like other neurons, these cells are large with euchromatic nuclei and prominent nucleoli. There are some instances where you might see a complete ring of satellite cells around a neuron, but the overall organization is more chaotic and irregular compared to that which you'll see in dorsal root ganglia.