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Mathematics for BiologistsPart Biology

Morphometric - Stereologic Analysesof Lung Tissues

Protocol

Jan. 31st 1998

Headed by: Dr. Sänger

Handed in by:

Maricela Yip (Mat-#: 9424495)

Salzburg, January 31st 1998

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Morphologic-stereologic investigation of epithelial cilia cell, macrophage cell and alveoli in human lungs:

Introduction:The lung’s main function is to allow for efficientexchange of gases (O2 and CO2) between thesurrounding air and the blood. For this purposethe air is led through the bronchi into a largevolume made up of many small chambers, thealveoli. Similarly blood flows through arteriesinto a dense capillary network which iscontained in the alveolar walls and is thencollected and conducted back to the heart byveins.

Respiratory passages:When a mammal breaths in, air enters therespiratory system through the mouth or nose.The air is warmed and humidified by the moistmouth or the nasal cavity which are locatedposteriorly to the pharynx. The pharynxbranches into a pair of tubes; one theesophagus, leads to the stomach, while theother, the wind pipe, or trachea, is the air-wayleading to the lungs. At the anterior end of thetrachea, lies the larynx, housing the vocal cords.Just above the opening to the larynx, is a flap oftissues called the epiglottis, which normallycloses off the larynx during swallowing and thusprevents food from accidentally entering thelungs. Ventilation of lungs is brought aboutpassively by the intercostal muscle of the rib-cage, which is connected via the pleura andpleural cavity and the diaphragm.

Lungs:The trachea branches into two hollow passageways called bronchi, each of which enters alung. Finer and finer branchings of these tubescreate an inverted tree, with thousands of narrowairways, or bronchioles, that eventually leads tomillions of tiny, bubble-shaped, sacs, calledalveoli.It is in the alveoli, that gas-exchange takes place.The terminal bronchioles, the respiratorybronchioles, the alveolar ducts, and the alveolarsacs, constitute the respiratory portion of thelungs. Many of the cells that line the largerairways produce a sticky mucus ideally suitedto the capturing inhaled dirt-particles ormicroorganisms. This mucus is continuouslycleared from the bronchi by the beating of cilia,which sweep the mucus and any trapped debrisup toward the pharynx, where they can beswallowed or expelled.

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Alveolus: (L. small cavity)Gases are transferred across the thin-walled alveoli found in the region distal to the terminal bronchioles, termedacini. The airways leading to the terminal bronchiole constitute the non-respiratory portion of the lungs. Alveoli in ajoining acini are interconnected by a series of holes, the pores of kohn, allowing the collateral movement of air,which may be a significant factor in gas distribution during lung ventilation.Each alveolus is surrounded by blood capillaries, and the inside of each tiny pouch is lined with a moist layer orepithelial cells. At those places where the wall of a capillary lies near the outer wall of an alveolus, O2 easily diffusesout of the alveolus and into red blood cells squeezing down the center of the narrow capillary. Meanwhile, CO2

leaves the blood, diffusing out of the capillary and entering the alveolus. From the alveolus, it is expelled to theoutside with the next exhalation.The lung-wall tension depends on the properties of the alveolar wall and the surface tension at the liquid-airinterface. The explanation for the relatively low surface tension of the liquid lining the lungs is the presence ofsurfactant, lipo-protein complexes that bestow a very low surface tension in the liquid-air interface. Lung surfactantnot only reduce the effort associated with breathing but also help prevent alveoli from collapsing.

Finally a few words about the tabacco plant itself:Tobacco (Nicotiana tabacum) is a hardy C3 plant that grows up to 2m tall and produces very large leaves and spikesof pretty, usually pink, flowers. As a natural defense, tobacco leaves and stems produce various compounds thatdiscourage insects and other predators. Among them is a bitter-tasting nitrogen-containing compound, an alkaloidcalled nicotine.The cigarettes, cigars, pipe and chewing tobacco made from the tobacco plant seem to be addictive. This is becausethe nicotine in tobacco leaves causes a strong psychologically dependence on the taste and feel of tobacco.

Besides that, analysis of smoke from burning tobacco has shown to contain over 4000 separate compounds, includingDDT, arsenic, nitrosamines, and formaldehyde, all known carcinogens. Similar tests of chewing tobacco revealstraces of threeadditional carcinogens: cadmium, uranium, polonium. One of the most damaging compounds intobacco smoke remains as the poisonous gas carbon monoxide (CO).

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Effects of tabacco smoke in human cells:

Paralyzed Cilia: Tobacco smoke can paralyze the cilia, the microscopic hairlike projectionsfrom cells lining the airways of the human respiratory tract. Without these continuously beating cilia, germs andparticles of foreign matter can enter the lungs and cause irritation and infection. The lungs of a smoker and hisrespiratory passageways compensate by producing more mucus, which is expelled in a cough. Perennial coughing canweaken the lungs and lead to chronic bronchitis.

Lung Cell Changes: Cadmium, nitrogen dioxide, and other substances in cigarette smokecan rupture cells in the lungs' tiny, ballonlike air sacs, or alveoli. They can also prevent a cell's smooth endoplasmicreticulum from producing normal amounts of surfactant. Both of these changes can contribute to permanentshortness of breath and to lung diseases such as emphysema.

Disturbed Mitochondria: Smoke destroys the mitochondria's normal internal structure, and with it, their ability tocarry out the reactions of the Krebs cycle (an elementary process in cell respiration) and the electron transport chain.Thus the cell is starved for ATP (energy carrier within the cell) energy and eventually dies.

DNA Damage: Many of the toxic compounds in tobacco can attack and damage DNA. Repair enzymes in thenucleus attempt to fix these broken strands, and correct mismatched base pairs. However, continual exposure to thetoxins can lead to an accumulation of errors, which are implicated in the formation of cancerous tumors.

Nicotine and the Cell: Certain cells in the nervous and muscular system have receptor proteins in their membranesthat bind with nicotine, causing a different cell response to normal nerve signals, explaining how nicotine acts as astimulant.

Increased Carbon Monoxide: Smokers have elevated CO levels in their bloodstream. When CO combines withhemoglobin, the pigment delivers less oxygen to the body's tissues, including the brain, and the person's ability tothink clearly is reduced.

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Immune Cell Changes: Researchers studying the white blood cells (macrophages) that patrol and protect theairways and lungs have found an increase in the size and numbers of lysosomes within the cells and a decrease inprotein synthesis. The cells are apparently so busy ingesting foreign particles and the debris of damaged cells thatthey can't grow and function properly. Immune cell disruption helps explain why smokers catch colds, flu, andpneumonia more easily than nonsmokers, as well as experience increased cancer rates.

Further Consequences of tobacco use: Tobacco users have lowered resistance to colds and flu, and slower healing ofbroken bones and other wounds. Female smokers tend to have more miscarriages and babies of lower birth weight.Even in their teens and twenties, tobacco users tend to develop periodontal (gum) disease four times as often asnonsmokers.

A smoker’s lungs A non-smoker’s lungs

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1. Working Hypothesis: Statistical evidence show that smokers are more likely to suffer from respiratory diseases and premature death thannonsmokers. Morphologic-stereological approach should help to determine the following effects:

Hair-like cilia: In a Nonsmokers lungs, the cilia protrude from the cells that line the trachea and bronchioles sweep mucus and debrisfrom the respiratory passageways. In a smokers lungs, the cilia are paralyzed or broken by cigarette smoke. Debris can reach the lungs and accumulate,blackening and clogging the delicate tissues lining the alveoli. Stereological methods should show how the all over cilial-surface area is lower in smokers. Lung Cell Changes: Cadmium, nitrogen dioxide, and other substances in cigarette smoke can rupture cells in thelungs' tiny, ballonlike air sacs, or alveoli. They can also prevent a cell's smooth endoplasmic reticulum fromproducing normal amounts of surfactants which further deteriorates the alveolar walls. These changes can contributeto permanent shortness of breath and to lung diseases such as emphysema.

Stereology will help us to show the loss of alveolar surface area of a smoker lungs versus a nonsmoker. Macrophage Cells: in white blood cells, lysosomes enlarge after smoking. Macrophages exposed to cigarette smoke do show significant changes, lysosomes of these immunodefencee cells canbe used to show the modifications under stressed conditions; therefore, morphologic-stereological techniques can beused to prove this correlation? 2. Components of the Structures, needed for stereologic investigationThe different components must be clearly separated and identifiable. Each parameter is obtained as the ratio of twomeasurements, one estimating the size of the objects of interest (phase), the other the size of the space in which theyare contained (reference).

Structure ObjectsCiliated epithelial cell

vsCilia

Reference: A ciliated epithelial cell represents a structural unit.Phase: Cilia, part of this structure, are the site of interest on which debris and

smoked gas affects them, here as the phase.Macrophage

vsLysosome

Reference: Macrophage is the direct immune defense organelle that ingestand digest undesired material and debris that enters along with air intothe lungs, therefore considered to be the reference unit.

Phase: Lysosome are contained in the macrophage, are small membranebounded structures that contain a set of powerful hydrolytic enzymes,which are capable of breaking down most organic materials, so that theforeign body is quickly disassembled; i.e.: the phase.

Alveolar ductsvs

alveolus

Reference: Alveolar ducts as the pulmonary gas exchanger consist of at leasttwo alveoli of the same air duct system, hence, the reference.

Phase: Alveolus is a thin-walled, sacklike chamber in the vertebrate lungwhere gas exchange takes place, the single structure within the unit.

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2.1. Determination of ParametersThe quantitative description of the structure is the density of the various components within the structure (the quantityper unit volume).

Structure: surface density volume densityHair cilia: in a 2-D, SEM picture, comparison of the size ofthe surface between a nonsmoker hair cilia with a smoker haircilia reveals that a smoker’s epithelial cell is severely damagedcompared to a nonsmoker’s cilial epithelium; i.e.: ”surface” isthe criteria to look for.

X

Lysosome: the size of the lysosome of a smoker issignificantly larger than the nonsmoker’s; i.e.: the”volumetric” content determines the effect.

X

Alveolus: in a smoker’s, the alveolar walls are almost lost,resulting in shortness of breath and death; i.e.: all over surfacearea determines efficiency in gas exchange.

X

2.2. Measuring MethodsSince 2-D cuts obtained by a series of microtomial cuts do not reveal the actual 3-D structure, indirect censustechniques like point, intersection, or number counting are used to rebuild the original structure(the parameter of interest).

Structure: surface density volume densityHair cilia: irregular patches of surface area of the epithelialcell are better determined by counting the number ofintersecting lines that falls into the irregular patches.

intersectioncounting

Lysosome: the sizes of the lysosomes of the nonsmoker’s andthe smoker’s can be easily determined by this method.

point counting

Alveolus: the surface of alveolar walls can be easilydetermined by counting the intersections that fall into the airsacs of an alveolus.

intersectioncounting

2.4. TestgridThe spatial distances between testlines should not exceed dimensions of objects of interest; i.e.:• Cilium: spatial distance between testlines should be the size of average cilial length;• Lysosome: spatial distance between testlines should be the average lysosomal size;• Alveolus: spatial distance between testlines should be the distance of alveolar walls.

Structure Testgrid

Hair cilia: Damaged cilia are better determined with a finertestgrid, whereas intact cilia, with the coarse one; so that, wecan see the difference between the two SEM pictures.

double square lattice

Lysosome: Enlarged lysosomes in macrophages of smokersare determined by a coarse testgrid, whereas the finer is moresuitable for the census of a non-smoker’s tissue.

double square lattice

Alveolus: A short-lined testgrid is probably best to usebecause nonsmoker’s alveoli are far smaller (more walls inbetween them) than of a smoker’s (increase surface area peralveolus) due to the loss of alveolar walls.

short-lined multipurpose testsystem(M168)

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Healthy epithelial cilia Damaged epithelial cilia

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Loss of alveolar walls Normal alveolar walls References: • Stereological Methods Vol. 1, Ewald. R. Weibel, Academic Press London 1979, UK• Animal Physiology 4th ed., Eckert, W.H. Freeman and Company New York 1997, USA• The Nature of Life. 3rd ed., Postlethwait J.H., Hopson J.L, McGraw Hill, New York 1995, USA• Zoology, Robert L. Dorit, Warren F. Walker, Robert D. Barnes, Saunders College Publishing, Orlando 1991, USA

• Medical Physiology, Ninth Edition, Arthur C. Guyton, John E. Hall, W.B.Saunders Company 1996, USA• Cell and Tissue Ultrastructure, Patricia C. Cross, K. Lynne Mercer, W.H.Freeman and Company 1993, USA• The pathway for oxygen, E. R. Weibel, Harvard University Press, 1984, USA