Lab protocol. In this lab we will first examine nerve tissue before examining the blood vessels and heart.

Transcription

1 Lab protocol In this lab we will first examine nerve tissue before examining the blood vessels and heart. NERVE TISSUE 1. Slide #92 [transverse section of human or rodent spinal cord; Boudian (silver) impregnation]. This technique stains the myelinated nerve processes (axons and dendrites) and the cytoplasmic neurofibril network of larger neurons a dark brown to black color. The remainder of the tissue stains faint yellow to pinkish-orange. Observe the structure of the spinal cord and identify the gray and white matter as well as any dura or arachnoid that might be present (it is present on the section of rodent spinal cord in the virtual histology module). Is some of the dorsal root sensory ganglion present? Try to locate the central canal (surrounded by columnar-shaped ependymal cells). Find some of the larger neurons in the ventral (anterior) horn. Observe that there are nerve processes extending in all directions in the gray matter. Now observe the white matter. Note the partition of the spinal cord white matter into posterior (dorsal), lateral and anterior (ventral) columns. As in other regions of the CNS, the white matter consists of nerve fibers (myelinated and unmyelinated), neuroglial cells, and blood vessels. There are NO neuronal cell bodies in the white matter. Since the spinal cord is cut in cross section, and since the nerve fibers (axons) in the white matter are ascending or descending the spinal cord, you are looking at the circular pattern of the transected axons. Therefore, they appear as black dots. Transverse section of spinal cord at mid-lumbar level (Weigert stain for myelin) 1

2 2. Slide #90 [rodent spinal cord in situ, H&E]. Using the figure (below) as a guide, find ventral (motor) horn cells (neurons). Observe the form of the cell and characteristic structure of the nucleus and cytoplasm. This cell has many dendrites but only one axon and is called a multipolar neuron. These processes are very long and are usually cut off close to the cell in the preparation of this section. However, the proximal stumps lend an angular contour to the periphery of the perikaryon (cell body or soma). Now direct attention to the dorsal root ganglion. These contain pseudounipolar cells. They are globular in shape and vary tremendously in size. The small cells have unmyelinated processes, and the large cells have myelinated processes. A thin cellular capsule, comprised of satellite or capsule cells, surrounds the cell bodies. These cells are continuous with the neurolemmal sheaths of the cell processes. The nucleus of the sensory neurons is relatively large and vesicular and may contain more than one prominent nucleolus. The cytoplasm appears granular due to the abundant Nissl substance. If a stem process (primary neurite) can be seen note that it bifurcates in a T or Y fashion. The thick stem process divides into two secondary processes: a peripheral process ( dendrite ) which extends to some sensory receptor and a central process ( axon ) which enters the spinal cord to synapse with neurons in the gray matter. Note that large bundles of nerve fibers traverse the ganglion and isolate nerve cells into groups. These are the central and peripheral processes of the ganglion cells. No fibers of other neurons traverse this sensory ganglion. 3. Slide #91 [smear preparation of ox spinal cord; Nissl stain]. Look for the large, multipolar neurons in this preparation, which was prepared by smearing fresh spinal cord gray matter on 2

3 a slide, drying the smear, and then staining with toluidine blue and eosin. This preparation is intended primarily to show the proximal portions of the dendrites and axons of these multipolar nerve cells as well as the Nissl substance. In most instances, the axon has been broken off. The motor neurons are the largest cells in the smear. The small darkly staining nuclei with no visible nucleolus are glial cells (mostly astrocytes). Some smaller neurons can be seen as well (interneurons). The coarse Nissl material in the cell bodies of the motor neurons is readily recognizable and may be arranged linearly because of pressure exerted on it by the neurofibrils, which can t be seen with this stain. The intense basophilia of the Nissl substance is due to its high content of RNA (in ribosomes). Study the morphology of the dendrites and attempt to find an axon hillock (most likely you will be unsuccessful). 4. Slide #99 [cerebral cortex, cat] is a thin (1.5 µm) section of well-fixed, plastic resinembedded tissue stained by H&E. As indicated in the diagrams below, the section is a small vertical (perpendicular) slice of one of the ridges (gyri) of the cerebral cortex of the cat brain. First, survey the section using your 4x objective. From the periphery of the section inward identify: arachnoid matter, blood vessels (in the subarachnoid space), pia mater, gray matter, and white matter. The latter occupies the central most part of the section and displays faint linear striations (due to numerous nerve fibers coursing in this area to and from the gray matter). Now switch to the 10x objective and study the three identified regions of the section in more detail. The pia mater is the innermost and most delicate of the three fibrous meningeal coverings of the CNS. It is closely adherent to the brain tissue (impossible to strip off without destroying brain tissue) and is composed of one to several layers of flattened, modified fibroblasts and the extracellular matrix (collagen and elastic fibers, etc.) they produce. (What is the embryonic origin of these cells)? Mast cells, macrophages, lymphocytes, etc. are also found amongst the pial fibroblasts. 3

4 Notice that the larger blood vessels reside on the surface in the subarachnoid space between the pia and arachnoid. Try to find where one is penetrating the brain substance. Note that the pia continues to surround these small penetrating blood vessels as they enter the brain substance. Look at the density and organization of small blood vessels in the cortex. What is their diameter (look for red cells in the blood vessels for a gauge). Now, shift your attention to the cells in the cortex. Note, that with this stain you are only able to see nuclei. The smaller dark nuclei are glial cells, while the more open nuclei with prominent nucleoli are mostly neurons. Note that you can t really see the outline of the neurons except for those with more basophilic cytoplasm (Nissl substance). You also can t see the processes (except for occasional large proximal dendrites). Note that, when you increase the magnification, the cells seem to be sitting in a meshwork (the neuropil) that is a mix of processes of neurons and astrocytes. As you progress to the core of the specimen, note that the neuropil changes. It is not as blue because the processes that comprise it do not contain many ribosomes (axons, for example, are essentially devoid of ribosomes). There are no neuronal cell bodies or nuclei in this area. The short, eosinophilic streaks that you see in the white matter at high magnification are portions of large axons. Finally, note the density of blood vessels in the gray and white matter. 5. Slide #98 [cerebellum, monkey, PTS]. In this slide, you are looking at small portion of the cerebellar cortex. At low power identify from outside in pia mater (much of it torn away from the nerve tissue), gray matter and white matter. The gray matter can divided into 4

5 three identifiable layers (see the diagram below). The superficial, outermost layer is the molecular layer. It contains mainly dendrites and axons from cells in the deeper layers but only a few nerve cell bodies. The middle layer is dominated by the cell bodies of peculiar giant nerve cells, Purkinje cells, arranged in a single layer and is called, logically enough, the Purkinje cell layer. The deepest layer next to the white matter is called the granular layer because it is packed with the cell bodies of tiny neurons (granule cells), which are the most numerous cells in the nervous system. The white matter is deep to these three layers. 6. Slide #93 [sympathetic ganglia]. Study the sympathetic ganglia with respect to the distribution of cells and fibers, position of nucleus, shape of the nerve cell body and continuity of the cellular capsule enclosing the nerve cell bodies. Review the gross anatomical positions of these ganglia and their connections to spinal nerves, the gray and white rami communicantes. 7. Slide#28 [small intestine; H&E]. Locate parasympathetic ganglion cells (small, multipolar neurons) in Auerbach s myenteric plexus (between the longitudinal and circular smooth muscle layers). NB: Capsule (satellite) cells may be absent in parasympathetic ganglia. 8. Slide #95 [transverse section of a small nerve trunk; osmic acid]. Osmic acid blackens myelin sheaths. Note that nerve fibers tend to be bundled together by connective tissue sheaths. These can be recognized although they are not stained. Identify large, medium, and small myelinated fibers. Many non-myelinated fibers are also present but are difficult or impossible to discern in this preparation. 5

6 9. Slide #94 [transverse and longitudinal section of peripheral nerve; H&E]. Study the funicular (bundled) arrangement of nerve fibers, identifying the connective tissue sheaths: epineurium, perineurium and endoneurium (from outside to inside). Only a portion of the epineurium may be present. What are the structural differences in these sheaths? Note the distribution of blood vessels in these connective tissue sheaths. Within a funiculus, identify myelinated nerve fibers and note the size of axons, thickness of myelin and presence of the neurilemmal (Schwann cell) sheath. Correlate these structures with the development and structure of myelin as observed with the electron microscope. Keep in mind that the shape and position of the axon are usually distorted by fixation. In the longitudinal section identify nodes of Ranvier and internodal segments. Nonmyelinated nerve fibers are also present in the funiculus, but these are much smaller and difficult to distinguish with the light microscope. 10. Peripheral nerves. Try to locate peripheral nerve trunks in some of the following slides, Slide #77 (tongue look between the bundles of muscle fibers near blood vessels), Slide #33 (skin), Slide #65 (larynx look near the thyroid gland) and Slide #83 (heart look in the fat surrounding the heart). You will see nerves in many tissues and organs that you will study over the remainder of the semester. THE CIRCULATORY SYSTEM 11. Slide #106, [aorta, small mammal; elastic stain]. This is an example of a large conducting ( elastic ) artery. Analyze in detail the structure of the three tunicas (intima, media, and adventitia) of the adult aortic wall as they appear when stained for elastic tissue. Note the relatively thin intima made up of endothelium and thin layer of connective tissue (CT), the interrelationship of smooth muscle cells to the prominent lamellae of elastic fibers in the thicker media, and the type and orientation of CT fibers in the adventitia. What is the functional significance of predominantly elastic tissue in the wall of the large arteries close to the heart? Look for vasa vasorum and nervi vascularis in the adventitia. How does the tissue of the aortic wall receive its nourishment? 12. Slide #108 [Weigert elastic stain] and Slide #107 [H&E, PTS]. These are examples of medium-size distributing ( muscular ) arteries. In general the muscular arteries correspond to the smaller named arteries which you dissect in gross anatomy lab. Contrast in detail the structure of the media of these arteries to that of the aorta. Note the predominance of smooth muscle, the sparseness of elastic fibers and the prominent internal elastic lamina. What is the significance of the difference in relative amounts and organization of elastic fibers, collagen fibers and smooth muscle cells in terms of the functional role played by these two types of arteries in controlling circulation? 6

7 Diagram of a muscular artery, H & E (left) and an elastic artery, Weigert s method (right). The tunica media of a muscular artery contains predominately smooth muscle, whereas the tunica media of an elastic artery is formed by layers of smooth muscle with elastic lamina. The adventitia contains small blood vessels (vasa vasorum) and elastic and collagen fibers. NB: The relative diameters of the two arteries are NOT drawn to scale. 13. Slide #120 [lymph node, monkey; H&E]. Examine the small muscular arteries near the hilum of the organ. Then try to find some within the parenchyma of the organ that have only 1 to 4 layers of smooth muscle in the media. These vessels are termed arterioles ( µm in diameter). Identify some of the smallest muscular arteries and arterioles in the tissues and compare the caliber and histology of their walls to those of the larger muscular arteries. Next, identify lymphatic vessels and neighboring blood vessels in the capsule and the hilum of this lymph node. You may well be able to find one or more lymphatics with valves in the section. 14. Slide #109 [whole-mount preparation of mesentery]. Examine the network of arterioles, venules and capillaries as well as some lymphatic vessels (the largest vessel, with a valve). Look for individual endothelial cells, particularly in the capillaries. Identify an arteriole in this preparation, and appreciate the three dimensional architecture of this vessel, particularly the circularly-oriented layer of smooth muscle cells supporting its wall. Examine the large lymphatic vessel in this slide and see if you can find a valve. Which would be the direction of flow? 15. Slide #126 [ß]. Identify capillaries among the transversely and longitudinally sectioned skeletal muscle fibers. Capillaries in both longitudinal and transverse section also may be easily seen in the ventricular myocardium of Slide #83. This slide also provides an excellent look at the arrangement of smooth muscle cells in the wall of small arteriolar channels, and the lack of that muscular coat in their accompanying venules. 7

8 16. Slide #110 [vena cava]. Large veins are easily distinguished by their size, thinness of the media and the striking accumulation of longitudinally disposed smooth muscle in the adventitia. 17. Slides #108, #120, #65 or #83 [medium-sized veins]. Study the relative thickness of the tunicas in the walls of these veins and compare their characteristics with those of the comparable muscular artery as observed above. Next compare the distribution of elastic fibers. As compared with the accompanying artery, how do you relate the larger caliber and thinner wall of the vein to its function? Veins vary greatly in their histology according to the amount of hydrostatic pressure exerted on the wall. Thus, those located above the heart have a thinner wall than those located below the heart. 18. Small veins and venules Slides #107, #108 and #109. Find these vessels among the skeletal muscle and connective tissue. Note: You should be able to identify arteries and veins when cut in longitudinal or oblique planes as well as in the transverse. 19. Slide #114 [mouse heart; sagittal section; Gomori trichrome]. The Gomori trichrome technique renders the nuclei red, the muscle varying shades of purple, and collagen green. The plane of the section is such that portions of atrium and ventricle are present. (See diagram below). Distinguish atrium from ventricle and compare the walls of the two chambers with regard to kind, amount, and arrangement of tissues, e.g., endocardium, myocardium, epicardium. Is there any physical connection between the cardiac muscle fibers of the atria and those of the ventricles? Find the atrioventricular sulcus and note the predominance of connective tissue in this region. Review the location, arrangement and structure of the cardiac skeleton. Note the thin leaflets of the atrioventricular valve intervening between the two heart chambers. Is the endocardium of the two heart chambers continuous over the surface of the valves? Review the structure of cardiac muscle, which comprises the bulk of the section. Can you find intercalated discs? branched fibers? central nuclei? 8

9 20. Slide #113 (odd boxes) [heart valve, human; Masson s stain]. This section passes through the epicardium (visceral pericardium) and subepicardial fat with its contained extramural coronary blood vessels, the wall of the left ventricle, the left atrium and part of the left AV (atrioventricular) valve (see the above diagram). Collagen is stained green and muscle reddish-brown. Find the section of coronary artery in the pericardial fat and notice that it has a well-defined internal elastic lamina. In one area there is a subendothelial thickening (an atheromatous plaque). This is indicative of a moderate degree of atherosclerosis. Notice that the veins do not contain elastic laminae. The large vein containing a post-mortem clot is the coronary sinus. Study the structure of the valve leaflet. Of what tissues is it composed? Does it contain blood vessels? What name is given to the dense aggregation of collagenous connective tissue at the base of the valve? 21. Slide #82. Try to find the large, pale-staining modified muscle fibers called Purkinje fibers in the region beneath the ventricular endocardium. What is the function of these fibers? Purkinje fibers can also be found in the subendocardial connective tissue. 22. Please note the dense connective tissue that forms the core of the valve leaflets in Slide #82 and Slide #113, along with the density of small blood vessels supplying the myocardium. 9