Objectives:

There are 5 cardiovascular organs that you should be able to identify if presented in a practical: heart, elastic artery, muscular artery, medium vein and large vein. The level of detail required in the unknown identification is summarized just above the slide description. Students should also be able to identify the 3 organs of the lymphatic system as well as diffuse lymphatic tissue and nodules.
 

 
There are several slides of each type of cardiovascular tissue with the same number but within those numbered slides there are often several variations. Try to look at one slide of each type of vascular organ before examining the others.

A. Elastic Artery.
The slides of elastic artery are mostly of aorta. The diagnostic features that you should be looking for include the following:

1. Tunica intima relatively thick, bounded by a simple squamous epithelium (or endothelium)
2. Tunica media, the thickest layer, containing many alternating layers of smooth muscle and elastic lamina.
3. Tunica adventitia, relatively thin in comparison to t. media, containing loose connective tissue and vasculature (the vasa vasorum).

47A Aorta, rabbit, H&E
47B Aorta, rabbit, Verhoeff

Some of you have the H&E stained slide and some the Verhoeff stained slide. Both sections are from the same tissue block. The endothelial cell nuclei appear as rounded up spheres because the organ has contracted. In the Verhoeff stained slide, look for thin strands of tissue separating the smooth muscle in the t. media,. These are the elastic lamina. They do not show up well in H&E stained tissue because they do not take up stain. They look instead like pale stained curvy lines. But the Verhoeff stain brings them out very dramatically which shows them as curvy lines between the layers of smooth muscle. Note that the inner most and outermost lamina are not prominent; they look just like all the others. A mesothelium surrounds the tissue normally and has been preserved in some regions of the surrounding t. adventitia.
 

48 Aorta, elastic tissue, human, c.s.

There are two groups of slides here. Both have been stained for elastic tissue, but only one group has been stained with eosin to show cellular density. In both slides you should be able to see the division between intima, media and adventitia. In the lightly stained slide, you will notice that some elastic tissue is found in the intima, but it lacks the organization of the elastic lamellae that are seen in the media. The adventitia is unremarkable and reveals only collagen fibers in the more heavily stained slide.
 

48B Artery (Aorta), trans. sec. Cat
This slide shows the entire aorta with the attached adipose tissue. The tissue is stained for elastic fibers which show up very dark in color. There are several smaller vessels in the slide. Within this aorta, the intima is very thin and the proximity of the endothelium to the inner elastic layer makes it difficult to find. The boundary between media and adventitia is easy to find. Numerous fibroblast nuclei can be seen. The adventitia seems to be bounded by a mesothelium in places. Does this make sense?
 

48C Artery, x.s.
This is another slide of aorta that has a small muscular artery in the lower part of the slide. This allows you to compare a large elastic artery, where each layer of smooth muscle is sandwiched between an elastic lamina, with a smaller artery with only an internal elastic lamina and the vestiges of an external elastic lamina. The endothelial cells are rounded up in the large lumen which makes the intima-media boundary easy to discern.
 

B. Muscular Artery.
Muscular artery is distinguishable from elastic artery by virtue of the pronounced internal and external elastic laminae. The Verhoeff stained slide, #49a shows this elaboration of the i.e.l. and e.e.l. very clearly. The H&E stained slide, # 49b, does not show the e.e.l at all. In smaller arteries the e.e.l. is lost but the i.e.l. remains. The diagnostic features of muscular arteries that you need to keep in mind are:

1. Tunica intima lined with a simple squamous epithelium (or endothelium) with prominant internal elastic lamina.
2. Thick tunica media with smooth muscle. Elastic lamina are not prominant.
3. Tunica adventitia of loose connective tissue and prominant external elastic lamina.

49B Artery, H&E, human

This is a conventional H&E stained preparation which does not reveal the elastic tissue. However, elastic tissue, even if not specifically stained, is visible as a layering between the smooth muscle. Thus, you should be able to distinguish elastic from muscular arteries regardless of the staining. Note that the H&E brings out details of the i.e.l. that have not been visible in the other artery slides.
 

50A Blood vessel, monkey, Masson
50B Blood vessel, monkey, Verhoeff

Slide 50a and its companion, 50b contain small muscular arteries and veins of different sizes embedded in a matrix of adipose tissue. In the Masson stained slide, the collagen fibers of the adventitia appear green, smooth muscle reddish brown. Arteries can be distinguished from veins by the relatively large amount of smooth muscle in the t. media compared with the lumen diameter. There is one large artery surrounded by a number of veins of different diameter in the center and off to the side a smaller artery adjacent to a large vein cut almost longitudinally. In the Verhoeff stained slide, shown above, the elastic lamina appear black. You can see that the larger artery has a prominent internal and external elastic lamina whereas the smaller artery has only an internal elastic lamina. As arteries grade to smaller diameter, the elastic lamina of the t.media gradually disappear. There is no sharp boundary between elastic and muscular arteries. In the smallest arteries, only an internal elastic lamina remains. Note also how little Verhoeff staining there is in the t. intima of the veins.
 

C. Medium Vein:
Distinguishable by the lack of longitudinal smooth muscle in the adventitia. The medium vein diagnosis includes the following:

1. Large lumen in relation to thickness of t. media
2. Indistinct boundary between t. intima and t. media.
3. Tunica media consisting of 2-3 layers of smooth muscle
4. Tunica adventitia is thickest layer of vessel

51A Vein, Verhoeff, Human 
This slide is stained for elastic tissue, but it is so over stained that there is very little discrimination; everything is stained. Do not confuse elastic tissue for elastic lamina. The one consists of fibers, the other is organized as sheets. However, you can note that there is some layering in the media of the vein and there is a different structure to the intima so that the boundary is visible.

D. Large Vein:
Distinguishable by the pronounced t. adventitia with longitudinally organized smooth muscle. It is also worth keeping in mind that the large pulmonary veins can also have cardiac muscle in their media, the only place outside the heart proper where this occurs.

1. Large lumen in relation to thickness of t. media
2. No distinct elastic lamina. Indistinct boundary between t. intima and t. media.
3. Tunica media contains incomplete layer of circularly organized smooth muscle
4. Tunica adventitia is the thickest layer and contains longitudinally oriented smooth muscle.

51B Vein, H&E, Human

This is an excellent example of a large vein. The intima is rather thickened but there is transversely sectioned smooth muscle in the adventitia indicating that this is a "propulsive" vein. The endothelial cell lining is also clear. Note that the longitudinal smooth muscle shows very little staining of the cytoplasm in the neighborhood of the nucleus but reasonably good staining in regions that do not include the nucleus.

52B Vena cava, human, c.s.

Exactly the same orientation as the previous slide, but stained for reticular fibers and eosinophilic material.
 

52A Vena Cava, Masson, Human
This tissue is a transverse section that shows clearly the relative differences in the thickness of the different layers of a large vein. The intima is thin and not clearly separated from the media by an elastic lamina. The media is thin and has an incomplete layer of smooth muscle cells. The adventitia is very thick and has numerous bundles of smooth muscle oriented longitudinally aswell as small blood vessels, the vasa vasorum. Collagen fibers stain an intense blue-green.

E. Heart:
Distinguishable by the presence of cardiac muscle. The main histological features include:

1. Encapsulated organ.
2. Three layers: epicardium (outer), myocardium, endocardium (inner)
3. Epicardium contains irregular connective tissue, adipose tissue, vasculature and nerve fibers. Surfaced by a mesothelium (of the visceral layer of the pericardium
4. Myocardium contains cardiac muscle identifiable by cross striations, intercalated discs and central nucleus.
5. Endocardium bounded by an endothelium. Subendocardial layer of connective tissue may contain Purkinje fibers, which are modigied large cardiocytes without intercalated discs.
6. May contain cardiac valves, which are elastic connective tissue surfaced with endothelium. Valves may be connected to thread-like cords of elastic connective tissue, the chordae tendineae.

54A Ventricle wall, Masson, Human

This is a nice slide. It has a thin layer of endocardium and a glancing section through epicardium making the endocardium appear rather thick. The cardiac muscle is very well preserved and you should perhaps give it a good look as it is better than any of the slides that were looked at in the muscle lab. Where cardiocytes are sectioned transversely, the individual fibrils can be resolved as can the central nucleus. In longitudinal section, the striations and intercalated discs are easily visualized.
 

54B Ventricle, H&E, Human anatomy
This slide shows only myocardium and epicardium. There is a considerable amount of adipose tissue in the epicardium and some rather large vessels.
 

54B Papillary muscle, H&E, human.
This slide shows myocardium with a very thin endocardium. The endocardium consists of barely more than an endothelium and subendothelial layer. The cardiocytes are well preserved and show nice striations and intercalated discs. There is no epicardium. The papillary muscles lies within the ventricle chamber where they attach to the valves via the chordae tendineae which coordinate the valve opening with the ventricle contraction.
 

F. Cardiac Valves & Purkinje fibers:
You probably could not determine that the valves come from the heart unless there some cardiac muscle is present. Other wise they will look like elastic connective tissue, covered by an endothelium. Purkinje fibers are quite easy to find if the division between endocardium and myocardium is visible.
 

55A Mitral valve, Verhoeff
55B Mitral valve, Masson

Division between endocardium and underlying connective tissue is clear in places. The wavy lines are elastic fibers. There is no cardiac muscle in this tissue preparation. Chordae tendineae can also be seen in the slide as small elliptical islands.

56A Heart Purkinje fibers
56B Heart Purkinje fibers

These are two excellent slides that clearly reveal Purkinje fibers. The sections are stained with iron-hematoxylin which brings out the striations and intercalated discs clearly. The Purkinje fibers are mostly transversely sectioned through many are cut longitudinally. Note that the Purkinje fibers are generally larger than the cardiac muscle cells. They often contain myofibrils but these are not always seen in the sections. Generally the fibers are clearly located in the subendocardium.
 
 

The three lymphatic organs: lymph node, thymus and spleen, are considered "look alikes" in histology because the high concentration of lymphocytes rather dominates the first impression. However, there are clear distinguishing features that can be used to unmistakably identify each. Examine the slides with the idea of focussing on the differences.
A. Lymph Node:

Encapsulated organ with dense irregular connective tissue capsule. May have afferent lymphatic vessels immediately outside the capsule.

Cortex/Medulla organization. Cortex contains primary and secondary lymphoid follicles. Medulla contains medullary cords of lymphocytes

Subcapsular sinus, trabeculae and trabecular sinus.

Reticular fibers and reticular cells form the frame of the lymph node.

May contain a hilum with an artery, vein and efferent lymphatic

Post capillary venules with a cuboidal endothelium.

58B Lymph Nodes, H&AZ II, Human

This slide shows the characteristic features of lymph node. Encapsulation, subcapsular sinus, 1° & 2° nodules (follicles), cortex/medulla division. There is no hilus, however. High endothelial postcapillary venules are present and not too difficult to find. In the subcapsular (marginal) and trabecular sinuses look for stellate cells (red) between the reticular fibers (blue). These are the reticular cells.

B. Thymus: Distinguishable by Hassal's corpuscles and the lack of lymph follicles.

1. Multilobular organ with dense connective tissue septae surrounding lobules
2. The lobules have Cortex/Medulla organization. Cortex is densely staining. Medulla is pale staining and may contain adipose tissue (if it is an adult's thymus).
3. Diffuse lymphatic tissue only (no nodules)
5. No afferent lymphatics
6. Hassall's corpuscles found in medulla (diagnostic)

60A Thymus, primate, H&E

Most of you have this slide. It is a plastic section of thymus, which generally means better preservation. All of the features in here are typical.
 
 
 

60B Thymus, c.s.

Typical thymus. Cortex/medulla division, no nodules, Hassal’s corpuscles, thin capsule divided into lobules.
 

60C Thymus, human, adult

This thymus shows some nice Hassal’s corpuscles, cortex/medulla division and some large blood vessels. You should also be able to find adipose tissue in the medulla, a sign that this thymus comes from an older human.
 

C. Spleen:
Distinguishable by the central arteries in the lymphatic tissue.

1. No cortex/medulla organization. Instead red pulp/white pulp division.
2. No subcapsular sinus
3. Thick connective tissue capsule with trabeculae. Capsule surfaced with a simple squamous epithelium (mesothel of the peritoneum).
4. White pulp contains lymphoid tissue aggregations (diffuse and nodular). Nodules (follicles of Malpighi) are associated with a central artery (usually eccentrically located). Around the  arteries a periarteral lymphocytic sheath can be obserbed (PALS).
5. Red pulp contains a high concentration of erythrocytes, cords of Billroth and venous sinuses lined with a "cuboidal" epithelium (littoral cells).

57C Spleen, human sec.

57B Spleen, H&E, Human

The nodules in this tissue are nearly all 2°. The red pulp is easier to see. However, the cellular preservation is not as good as 57c.
 

57A Spleen, sec.
There are two groups of slides with this number. The older ones are better because the staining is richer. The older slides show a clear red pulp/white pulp division with a large splenic artery and vein entering one side through the CT capsule. The newer slide group shows the red pulp/white pulp division clearly except that the red pulp is lacking in erythrocytes (the spleen was probably "washed"[perfused] before fixation). The connective tissue capsule is clear and it is covered with mesothelium. Nearly all of the "central" arteries are eccentric. 1° and 2° nodules are present in the white pulp. Lots of trabeculae.
 

D. Aggregated lymphatic follicles and tonsils
Most of the diffuse lymphatic tissue lacks a capsular organization, which marks each of the lymphatic organs. Tonsils are lymphatic follicle aggregates covered by mucous membrane of the oral or pharyngeal cavities.

The palatine tonsil is the only tonsil, which has a connective tissue capsule. In case of the other tonsils (pharyngeal a.k.a adenoid, lingual and tubarian) tonsils, the apparent capsule is actually the subepithelial connective tissue. Note that the oropharyngeal mucous membrane ‘s epithelium sends invaginations into the tonsils. Lymphocytes usually infiltrate that epithel.

93 Ileum, Peyer’s patch, monkey, c.s.

Not the best orientation for looking at Peyer’s patches because this is a transverse section. The row’s of nodules that are diagnostic of Peyer's patches look best in longitudinal section. Note where the mucosa is completely invaded by lymphocytes. Sometimes, though not in this slide, the mucosa/submucosa division is obliterated. There are some nicely preserved regions of simple columnar epithelium displaying microvillus brush borders and goblet cells.
 
 

94B Duodenum, jejunum, ileum, c.s.
This slide shows all three regions of the small intestine in a single slide. One section is of the distal ileum and shows a nice region of Peyer’s patches, much better than 93. Note that Peyer’s patches appear on only one side of the organ (antimesenterial margin).
 

60 Adenoid, c.s. human (pharyngeal tonsil)

This tissue is almost devoid of identifying landmarks other than the lymph nodules. There are in places some bits of squamous epithelium, but mostly it is covered by pseudostratified, ciliated columnar epithel, which is characteristic to the nasopharynx as well.  It may be hard to recohgnize since it can be heavity infiltrated by lymphocyes.. This specimen is identifiable by what is missing, no clear encapsulation, no subcapsular sinus and the epithelium.
 

61C Palatine tonsil

This slide has a better set of landmarks than slide 61b.  There is a well preserved stratified squamous epithelium overlying the tonsil in several places. The presence of stratified squamous eipthelium, nonkeratinized, would be the clue that this is probably not a lymph node.
 
 

61A Palatine tonsil, human

Pretty similar to previous slide. There are two groups of slides here. Both seem about the same as far as preservation goes.
 

61D Palatine tonsil, human
A rather badly preserved specimen, but identifiable as tonsil.
 

59 Lymphatic vessel,c.s.
This slide is rather unremarkable. The vessel is lined with an endothelium. What distinguishes it from other blood vessels is the lack of anything resemblying smooth muscle in the vessel wall.
 
 

 

Spleen	Lymph Node	Thymus
Red pulp/White pulp division	Cortex/medulla division	Lobuli have cortex/medulla division
Nodules & diffuse lymphatic tissue	Nodules (follicles)& diffuse lymphatic tissue	Only diffuse lymphatic tissue
Nodules are associated with an artery	Nodules (follicles) are not associated with arteries	No nodules
No subcapsular sinus	Subcapsular sinus	No subcapsular sinus
No afferent lymphatics	Afferent lymphatics	No afferent lymphatics
Hilus	Hilus	"Pseudo"lobules
Serosa	Adventitia	Adventitia
Venous sinuses and cords of Billroth	Cuboidal endothelium in venules	Hassal's corpuscles