6th Webboard Feb 5, 2000

First. In case I left it unclear, the first exam will NOT have Neural material on written or lab part. [This means that the neural stuff will be on the next assessment and the second exam] The lab exam will be mostly spots, where you can use ONLY the fine focus control (and should use it). Put as complete answers as you can, and do not leave blanks. Second, a few comments on the Neuro lab for next week. C-8 of cerebral cortex has darkly stained blood vessels in addition to the neural tissue. C-2 use naked eye to distinguiosh the cord from the roots from the DR ganglion before you look through the 'scope Some of the large round neurons have yellow/brown aging pigment/lipofuscin in the soma. The sympathetic ganglion slide is very variable hunt for yellow neurons in the brown areas, and see if any have dendrites to show that they are multipolar, i.e. have dendrites with synapses. Fifth Histo WebBoard 20E The single lecture on Wedn , Jan 26, will start at 8.45 am prompt in the Addition Audi CARTILAGE SLIDES A-6 tendon has dense regular CT and a little loose irregular CT as endotendineum between the main collagen fiber bundles. However, the cells with the collagen fibers are thin dark fibroblasts, not round chondrocytes, so fibrocartilage is absent from all the specimens I saw in Monday’s lab A-13 these slides vary. Some are a sector out of a disc so that the nucleus pulposus, if present, is at one edge. Opposite (tracking all across the section) the pale very disorganized nucleus pulposus is sme dense regular-looking connective tissue with dark elongated fibroblasts. In between, there should be evidence of fibrous collagen type I) and some round/ovoid cells with a dark line where the edge of the lacuna has stained. This is as exciting as fibrocartilage gets - a slightly modified dense CT. The lamellae/layers of collagen in the annulus are tearing apart on many sections G-7 trachea (some have no cartilage) In ones that have cartilage, note that the perichondrium on the outer (adventitial) side of the cartilage is very wide. The adventitia is narrow, very irregular, and has some red elastic fibers stained A-20 shows a wedge of synovium, but the two cell types in the lining - like macrophages & like fibroblasts - can only be made out (and then with some debate) in EM views A-14 pubic symphysis is the right site for fibrocartilage, but at the wrong time - too soon. There is skeletal muscle attaching to periosteum on the bone. Bone is growing into mostly hyaline cartilage between the bones. Later residual hyaline cartilage in the gap will convert to fibrocartilage. At present, the only fibrocartilage consists of areas with dense, but often pale thick collagen in the background to some large ovoid cells suggestive of chondrocytes. There are many areas of transition - from hyaline to undeveloped fibrocartilage; from undeveloped fibrocartilage to the CT of the ligament joining the bones, etc. WebBoard Fourth item 20D At the weekend, your swipe cards may or may not work. (The SBLC still does not have swipe access). Try my door, if it is unlocked and the light is on, I’m in the library - down the left-hand side where the recent- journal rack is. I’m usually in Saturday am, and Sunday. Otherwise, hunt throughout the Anatomy Dept to find another faculty member who has a key. The Connective tissue lab is about the hardest, and is good training for the messiness of real tissues and real life (and the resulting analysis), which is why you are doing the work while we try to help. In the syllabus, read again (p. 56?) how the lab questions are set - name the cell, name the tissue (dense regular connective tissue), name the structure, etc. The plan for next weeks’s assessment is to have about 10 Histo & 10 Gross theory questions, and about 15-20 Histo Projection IDs, some with attached theory questions, likewise for Gross. A fairly common problem encountered in the connective tissue lab was that smooth muscle has elongated cells with elongated nuclei. Some of you had to be steered away from it to the more untidy collagen fibers of connective tissue. In E-10, the submucosa (about half-way across the thickness) had been very pulled apart, with widely separated collagen fibers and bundles. E-6 in some slides has a dense appearing connective tissue in the submucosa. However, this is tissue compaction from processing and the tissue is really loose irregular CT. Mast cells (with red granules here) are not present in all E-6s) E-15 is often unsatisfactory for plasma cells. The section has been cut too thick so that cells overlie each other, making it hard to get a clear view of individual cells. Also, the plasma cells (most cells) are shrunken and not well stained in many E-15s. I’ll try to find a good example for the demo bench. A-9 spread may have very thick as well as thin areas. Be prepared to hunt widely for mast cells, with granules stained blue-purple, but no nuclear staining. (If your section has none, borrow this slide.) Also, elastic fibers have reacted to appear as fine precise lines. Collagen was stained with “light green” instead of eosin and is the indistinct background. In places in a subcutaneous spread, there may be some fat cells (adipose tissue) overlying the primary tissue - loose irregular connective tissue. Use the special appearance of this staining combination to identify these elements on the quiz/exam. F-3 scalp (hairy skin) has variation in how well the collagen fibers are staining, ranging from pinky rad to feeble orange. Beware of mistaking ragged keratin on the epidermal surface for collagen fibers. M-9 the developing tooth is a small cup on a stalk. Close by is the pale poorly defined mesenchyme. Elsewhere in the section, one sees mesenchyme as a pale filler in between better defined structures. According to its stage of development, mesenchyme can be early and late with a matching slight increase in density. B-2 aorta is stained with a trichrome method that makes collagen blue and elastic red., so that one sees that the middle part of the arterial wall is made of alternating layers of collagen fibers and red elastic membranes/laminae. The cells in the media are smooth muscle cells that combine an ability to contract, with synthesis of both collagen and elastin, so that it is NOT only fibroblasts that make ECM materials. In the H&E-stained aorta, the elastic sheets are met edge-on as red lines, slightly better defined than the fuzzy collagen between them. Hence the use of trichrome or special elastic stained to help distinguish materials. The only human spinal ligaments comprising elastic are the scanty cervical ligamenta flava. (The vocal chord/fold also has an elastic ligament as its core, which is a rare example of regular CT in a visceral organ - the larynx) Third WebBoard C20
General comment - For light microscopy(LM), cell structures need very good fixation and staining, and observation with the X100 oil immersion lens. In the lab, we get hints of what the old-timers saw and put into the old Histo books. Some things, such as terminal bars, are in the books & lab guide mostly out of deference to tradition and earlier anatomists. For cytology, one makes much more use of the electron microscope, especially in transmission mode, i.e., beam through the specimen; and this approach will be the basis of almost all our testing on cytology. The exceptions are the things that one can see with LM - nucleolus, nuclear shape, nuclear density, cilia (in a good prep), etc In A-3 frog liver, the mitochondria need at least high-dry X40 magnification. [Slowly build up an expectation of what you can see at different magnifications: Cell membranes hardly at all with LM. The trilaminar structure of a plasma membrane only with high em mags; cells, as cells, at high dry LM mag. etc.] In A-3, mitochondria appear a very small dark blue specks in the hepatocytes. The brown cells have melanin granules in them and are a form of macrophage, i.e., they are not hepatocytes. Some F-2 slide of skin epithelium (epidermis) have grown melanin granules in the deep layers of the epithelium, but some slides do not. H-3 for PAS staining of the basal laminae (basement membranes) because of their content of glycoproteins. The basal laminae of the capillaries making up the glomerulus are a red jumble. However, the thick BM supporting the simple squamous epithelium of Bowman’s capsule shows well. The BM of the many profiles of proximal tubules are stained, but less intensely. G-7 of airway epithelium shows a wide pale Basement membrane, even without PAS staining, but there a few cilia left, and the nuclei are not really elongated for what is a pseudostrat COLUMNAR ep. E-13, E-6 for terminal bars - look at the surface epithelium. Find a clean perpendicular cut (classic view) near the top of the epithelium is either a dark line parallel to the surface, or a series of dark dots (terminal bars), which is all that one sees of a junctional complex with light microscopy E-13 ileum has a good brush border (indistinct fuzz) along the surface, which is all that one sees of microvilli with LM. However, cilia and stereocilia should, with use of the fine focusing, be resolvable into discrete hairy things. In testis slides, note that there may be two repetitive patterns seen - the seminiferous tubules where the sperm are made, and the epididymis where they are stored to mature. It is only the epididymis that has the stereocilia (sometimes visible) and the pseudostrat columnar epithelium. K-4 for sperm is another smear prep - so that there are some grey globs of secretion along with the sperm. The staining is feeble, so that the heads can sometimes barely be seen, and the tails (flagella - with another special microtubular array) are very hard to make out. Cut down the glare to help on finding them.

20A First WebBoard item

First, welcome to the Histo lab. Some issues arising out of the first lab. Most of the time, one works out where cells are, and their spacing from seeing the nuclei. The cell membranes rarely are visible. In the ovary L-2, the large oocytes have a visible zona pellucida around them: the oocyte membrane is too thin to be seen. The fine focusing needs to be used while you look, for example with the not very red granules of glycogen in hepatocytes E-32. The hepatocytes are attached to form irregular plates. This kind of pattern cannot be readily detected in the almost 2-D section. Also, not easily to make out is that the liver, like other large glands, is divided into lobules. The connective tissue strands and sheets that separate the lobules show poorly in E-29 because the collagen stain (green) in this specimen is too feeble.

In the liver and lymph node D-5 the working cells do not just hang in mid-air: They are supported on a network of reticular fibers - unseen unless a special silver stain is used to show them as a black network D- 6.

A-23 for Skeletal muscle . The cylindrical shape of the cells/muscle fibers is only apparent in cross-section. One needs various section planes to build up a three-dimensional view of Histo structures.

L-2 The ovary slide for oocytes is unsatisfactory for two main reasons. The oocytes are few. The animal was one having litters: hence, multiple synchronous ovulations, and ensuing numerous corpora lutea. So most of the ovary is glandular tissue. Next, the few oocytes are badly shrunken. Also, a thin section through a large cell (the oocyte) can miss the nucleus. In the large antral follicles, the cut may miss the oocyte. If it includes the oocyte, it may miss the nucleus. The pink rim around the oocyte is the zona pellucida, not the oocyte membrane. The small cells around the oocyte, particularly in the larger follicles, are granulosa cells. "Granulosa" because they give a granular "bitty" visual texture to the follicular lining.

The Fig & Plate #s are for useful illustrations (or close to them) in Ross' Histology Text-Atlas. You will need some sort of Histo atlas from the recent 1990s.

As time goes on, we'll put out some demo materials on the bench at the end of the entry aisle, near the printer. For example, next week there will be some Cytology atlases. A key to where the Figs are is in the Syllabus on p. 7.

Cytology will come after Epithelium - the topic for this Thursday's class. The light microscope is not too good on Cytology, so we'll come back to the topic when we've reviewed some electron-microscope ideas and views.

The lab exercise for epithelium is in two parts. The light microscope one p. 20 we do in the lab. There are some small print items on p. 20 & 21. Read these before starting the lab. . Also, as you do each item, check on p. 22 for comments related to any problems

B20 2nd WebBoard item

Sorry about the delays in the Epithelium lecture. The remotes are a kind I haven't used before, and may have low batteries and/or distance problems to the computer in the booth. In the lab, some of you may be feeling lost in sea of pink. The epithelium lab is a continuation of the 'scope & slide familiarization process. We'll have time for the hard-core identification stuff later. You will need an Atlas, preferably a Text-Atlas to help you get oriented. Get used to scanning over the slide at low power, then checking at high or medium power. If there is no match, go back to low and scan some more.

There are three main problems for now: the tissue to be identified is one component of an organs that has several tissues; the structure that the tissue helps make up is often multiple .e.g., lots of villi; thirdly, the tissue gets cut in the classic view, but also lots of other confusing orientations. Students in places 59-94 have the older (better?) Leitz microscopes. These have an extra flip-up lens at the top of the condenser under the stage. This lens should be switched OUT for the X4 low-power objective, but very definitely switched INTO use for all other objectives. After a while you'll get the hang of this. Whatever 'scope you have, try out one of the other kind between now and the first exam. The exam will be set up using both kinds, and for SOME stations you can use all the scope controls. Some Histo courses require that you draw from the 'scope views. We leave this up to you. Some find that it helps to concentrate on the features.

You can use your access cards to get into the lab at any times, except Tuesday 10-12 am, & Tuesday and Thursday 2-4 when the Dental students have class, or at their exam times. When our exam comes around , we'll work to avoid any conflicting review times.

SPECIFIC SLIDE PROBLEMS Stratified squamous epithelium can have a very pretty row of cuboidal cells as its bottom layer by the basal lamina. However, epithelia are named for the shape of the surface cells - here flat squamous. A-5 mesentery is a spread so that you are looking DOWN on a double layer of mesothelium (simple squamous ep). The method used a silver nitrate solution to deposit black material between the irregular cell margins. Sometimes the counterstain shows nuclei in the center of the cells , sometimes it hasn't taken. Mesothelial cells are quite wide across. It is only in the side view that they look flattened and squamous.

F-1 skin for strat cuboidal in sweat-gland ducts. In cross-section the dark duct can be seen to have a roughly double row of nuclei. However, the nuclei are often not a nice round shape, suggesting cuboidal cells, but are a bit squashed as the cells try to fit into the narrow space of the duct.

K-9 ductus deferens has a very small lumen. The wall is mostly muscle. It is your most muscular tube. The epithelium looks stratified (it is pseudo), but this specimen seems to have not stereocilia. E-15 colon and some of the other GI slides (E-7?) have suffered post-mortem digestion of the lining epithelium. Skip such slides. In the stomach concentrate on the actual surface epithelium, with pale mucuos columnar cells. The glands just below confuse the picture. For example, simple tubular glands in cross- section are round, and could be taken for cells, until one realizes that at X4 low-poer one is not going to see cells, but larger structures made up of many cells.

G-9 Most slides have bronchioles, but no bronchus; and bronchioles lack the pseudostratified upper airway epithelium. The staining is feeble, anyway

L-10 Cervix is the part of the uterus protruding into the vagina. The cervical canal is lined by simple columnar ep, as are the tubular glands branching down from the epithelium. The vagina and the outside of the uterus bear a stratified squamous epithelium. Somewhere, as one goes around the curving surface of the cervix the epithelium will change. Some slides have an abrupt switch from thick SSE to thin pale simple columnar ep. Other slides have a "transformation zone" where strat squam is extending into, and at the expense of, simple columnar. The instability is why the cervix is at high risk for cancer. Also, the metaplasia results in converting forms that are not typical of either classic epithelial kind.

E-21 & E-22 salivary glands. Scanning over the slide, try to find some connective tissue separating the main mass of gland into lobules. In the connective tissue there may be a large vessel and/or a large duct. Seeing the duct's lumen may be hard at first, if the lumen is not empty, but has some pale pink protein remaining. The epithelium may or may not have a double layer of nuclei. It the nuclei closer to the lumen are elongated then the epithelium is stratified columnar; if round, it is strat cuboidal. However, you may see both kinds in the one cross-section. The process of changing over can be slow and hesitant.