APUD lung: methods, quantitation, response to injury · Grimelius or Pascual silver staining...

Thorax, 1980, 35, 363-370

APUD cells and neuroepithelial bodies in hamsterlung: methods, quantitation, and response to injuryJOHN R PALISANO, AND JEROME KLEINERMAN

From the Departments of Pathology and Pathology Research, St Luke's Hospital, Case Western ReserveUniversity, Cleveland, Ohio, and Mount Sinai School ofMedicine ofthe City ofNew York, USA

ABSTRACT A comparative study of the Falck-Hillarp Technique, a modification of Eaton-Feddeprocedure and silver staining of aldehyde-fixed tissue was carried out to determine the most efficientprocedure to demonstrate neuroendocrine cells of the hamster and rat lung. The modified Eaton-Fedde procedure is the most efficient method of observing these cells, and is also the easiest to perform.With this method, the normal hamster lung contains a total of 2 00x 10-1 to 30Ox 10-1 neuro-endocrine cells/mm in the small and large bronchioles. In the larger airways approximately 3 51 x 10-1neuroepithelial bodies (NEB)/mm are observed. Immediately after 24-hour exposure to NO2 thenumber of APUD cells dropped to approximately 25% of the control levels. These cells weredecreased to 50% of the control levels throughout the 28 days of exposure. The number of NEBdecreased transiently after 24 hours of NO2 but returned to normal numbers by day 14. We recom-mend the application of fluorescence techniques coupled with standardised sections and quantitativemethods of study for analysis ofAPUD cells and NEB.

In 1949, Frohlich' described a unique cell typein the lung epithelium which had clear cytoplasmand was given the descriptive name Helle-Zellen.Frohlich's oebservation was subsequently con-firmed by Feyrter.2 Both investigators used silverimpregnation to demonstrate these cells. Feyrterrecognised that these cells were part of a diffuseendocrine system which is scattered throughoutthe epithelium of many organs. In recent yearsPearse3 has defined these cells on the basis ofdiscrete biochemical oharacteristics. He hascalled them amine precursor uptake and de-car-boxylation (APUD) cells because of their abilityfor uptake and decarboxylation of amines. LikeAPUD cells in other organs these lung epithelialcells are presumed to origiinate from the neuralcrest and are believed to secrete polypeptidehormones.3 4 Very recently, Lauweryns andGoddeeris5 have described an organoid cluster ofcells which contain neurosecretory granules andhave neural attachments. These have been calledneuroepithelial bodies (NEB). They differ fromAPUD cells in that the latter are isolated, singlecells to which no neural attachment has beenregularly observed. Advances in this field have

Address for reprint requests: Dr Jerome Kleinerman, Depart-ment of Pathology, Mount Sinai School of Medicine, OneGustave L Levy Place, New York, New York 10029, USA.

been slow6-10 because of the paucity of APUDcells and NEB and difficulty of identifying them.The development and application of the Falck-Hililanp technique'1 for the identification APUDcells has initiated renewed interest in thesestructures which, because of their presumedorigin from neural anlage and their specialisedsecretory function, have also been termedneuroendocrine structures or cells. Severaldifferent techniques exist for identifying thesecells and it is not clear as to which of thesetechniques is most specific or sensitive. An idealtechnique should meet the following criteria: (1)simplicity, (2) speed, (3) reproducibility, (4) re-liability so as to permit quantitation, (5) main-tenance of the biochemical moieties within thecells to permit additional chemical characterisa-tion, and (6) the ability to use at least twomethods on the same tissue sample for confirma-tion. With these criteria in mind, we evaluatedseveral methods which demonstrate APUD cellsand NEB at the light microscopic level in thetracheobronchial tree and lung. The classicalFalck-Hillarp fluorescence procedure usingfreeze-dried tissue was compared to the Eaton-Fedde technique12 which uses frozen sections.These fluorescent techniques were compared toargyrophilic stain procedures using freeze-dried

363

on August 29, 2020 by guest. P

rotected by copyright.http://thorax.bm

j.com/

Thorax: first published as 10.1136/thx.35.5.363 on 1 M

ay 1980. Dow

nloaded from

http://thorax.bmj.com/

364

tissues and tissues fixed in formalin, glutaralde-hyde, or Bouin's fixative. Quantitative methodsto evaluate the number of NEB and APUD cellswere developed, and the effects of short-termexposures to 30 ppm of NO2 on the number ofthese neuroendocrine structures were evaluated.

Methods

Golden Syrian male hamsters, 4-5 months old,weighing 80-100 gm, were used. The animalswere fed and watered ad libitum. All animals wereprimed with 100 mg/kg body weight of 5-hydro-xytryptophan (5-HTP) and L-13-3-4-dihydroxy-phenylalanine (L-DOPA) 30-60 minutes beforekilling.'3 Animals were anaesthetised with Nem-butal (8 mg/100 gm body weight) intraperitone-ally (IP) and then exsanguinated under anaes-thesia by cutting the descending aorta.

FALCK-HILLARP TECHNIQUEThe lungs were removed immediately after deathand inflated with air by means of an intra-tracheal cannula attached to a syringe. After in-flation with approximately 5cc of air, the tracheawas tied and the lungs submerged in liquidnitrogen. The lungs were then transferred to afreeze-drier (Virtis, model number 10-800) andprocessed over the next two weeks. Upon com-pletion of the drying of the lungs they wereremoved from the freeze-drier, sliced with a cleanrazor blade into 2 mm thick slices, and placedin an oven at 70°C for two hours over approxi-mately 5 gm paraformaldehyde. The paraformal-dehyde was equilibrated over H2SO,4 in anatmosphere of 50% relative humidity.'4 15 Thelungs were then placed in paraffin in vacuo for10 minutes and embedded, the sections cut at5jum and mounted in Entellan. Fluorescencemicroscopy was performed on an A/O series 10Microstar microscope with an epifluorescent unitusing a 100 watt halogen lamp and exciter filtersBG-12 and KV-418 and barrier filter OG-515.Successive sections of the block were stained byroutine haematoxylin and eosin (H and E)15 andsilver stain.'7

MODIFIED EATON-FEDDE TECHNIQUEImmediately after death, the animal's lungs andtrachea were removed in toto and the lungsdistended via a tracheal cannula with a 2: 1 solu-tion of phosphate-buffered paraformaldehyde12and OCT embedding medium (Lab-Tek Pro-ducts). The lung was immediately cut coronally,both ventrally and dorsally to the trachea witha clean razor blade. A 5-8 mm slice with trachea

John R Palisano and Jerome Kleinerman

removed was taken from the midportion of thelung. The sections were submerged directly intoliquid nitrogen where they remained until placedin the freeze-drier (Virtis, model number 10-800).Subsequent processing of the tissue was identicalto that described for the Falck-Hillarp method-ology.

SILVER METHODIdentification of APUD cells and NEB by silverstaining methods was attempted on Bouin's-fixed,formaldehyde-fixed, glutaraldehyde-fixed, andfreeze-dried tissue in hamster and Bouin's-fixedand freeze-dried tissue in rabbit. The intestineand lung from the same animals were stainedsimultaneously by Pascual's,"7 Grimelius,18 andSiever-Munger's'9 silver stains. Pascual's silverstain was adopted because it proved to be thefastest, easiest, and most reproducible method.Pascual's silver stain was carried out in the fol-lowing way. Slides were deparaffinised in xyleneand then run down through a graded alcoholseries to a distilled water rinse. They were thenplaced in a 05% aqueous silver nitrate solutionfor two hours at 60°C. After two hours the slideswere rinsed in two distilled water rinses andplaced in a reducing solution (5 gm of sodiumsulphite and 1 gm hydroquinone/100 ml H20) for10 minutes at 60°C. The slides were then rinsedin running tap water and distilled water for threeminutes. If necessary the slides can be reimpreg-nanted in a fresh silver solution at 60°C for 10minutes and reduced again as described above.

QUANTITATIONQuantitation of APUD cells and NEB was car-ried out on silver-stained sections or on unstainedsections prepared for fluorescent microscopicstudy as described above. Either standard mid-coronal sections of both lungs or randomlyselected sections of peripheral lungs were used.The smallest transverse diameter of all airwayswas measured with a calibrated eyepiece micro-meter on every slide studied and airwaysseparated into three categories on the basis oftheir diameter: (1) 0-01 to 0 19 mm diameter,(2) 0-20 mm and greater but without cartilage inthe wall, and (3) bronchi. These categories ofairway size were selected arbitrarily after pre-liminary studies indicated that the separation intoa greater number of size classes did not changethe analysis or interpretation significantly. Tra-cheas were not studied because of technicaldifficulties. All airways and alveolar ducts in thestandard midcoronal sections or random peri-pheral sections were studied and the number of



j.com/


ay 1980. Dow

nloaded from


APUD cells and neuroepithelial bodies in hamster lung

APUD cells and NEB in each airway categorywere noted separately. The circumference of eachairway was measured using a calibrated eyepiecemicrometer. The total number of APUD cellsand NEB were each noted separately and de-scribed per total mm of airway circumference.These ratios of NEB/mm and APUD cells/mmwere summated for all airways in each size cate-gory. For example, if there were 10 APUD cellsin a total of 52-8 mm of airways with diametersof 0-01-0-19 mm, then the ratio was 10/52-8 or19-5 x 10-2 APUD cells per mm of airway. Manyairways contained no APUD cells or NEB; forthis reason the values are described for the totalnumber of APUD cells or NEB in the totallength of airways (mm) for each tissue sectionand each slide can be described by only one ratiovalue of

total APUD total NEBor

total mm total mm

This value is then reduced to APUD/mm orNEB/mm for each order of airway for each slidein the group studied. Because of this limitationeach order of airway can be expressed as onlyone value, without standard deviation or standarderror even though the results expressed are meanvalues.

NO2 EXPOSURESHamsters were exposed 24 hours per day to 30-40ppm of NO2 for one day, 14 days, and 28 days aspreviously described by Kleinerman and Ryn-brandt.20 While in the exposure chambers theanimals were fed four food pellets a day andwatered ad libitum. Half an hour before the ani-mals were to be killed they were primed (asdescribed earlier) and returned to the NO2chamber. At the scheduled time the animals werequickly removed from the NO2 chamber, killedand the lungs processed and quantitated asdescribed for the control animals.

Results

FALCK-HILLARP TECHNIQUEOn close observa-tion, freeze-dried tissue revealsa good state o,f preservation in most areas. Aprominent characteristic of freeze-dried tissue isthe vacuolated appearance of cytoplasm causedby ice crystal formation (fig 1). Although theepit'helium of the airways is rarely compressed,the cilia almost always are. Secretory activity isnever observed in the Clara or goblet cells (figs1 and 2). Desquamated cells on the surface are

Fig 1 Hamster airway epithelium in a smallbronchiole using the Falck-Hillarp technique.(H and E, original magnification x 2800).

preserved in situ by contrast with other methodsof fixation. With this procedure one obtainsbrightly fluorescent APUD cells and NEB as wellas mast cells. Positive argyrophilia can bedemonstrated in these cells using either theGrimelius or Pascual silver staining methods inboth rabbit and hamster intestine and lung. Themajor drawbacks of this procedure are theamount of time it takes to process the tissue(approximately two weeks in the freeze-drier) andthe fact that it is difficult to achieve standardsections of lung from animal to animal to carryout the quantitative studies.

MODIFIED EATON-FEDDE TECHNIQUEThe bronchiolar epithelium and lung tissueappear to be better preserved by using this tech-nique rather than the Falck-Hillarp procedure.Although few of the epithelial cells arecompressed using the Falck-Hilarp technique,care must be taken not to compress the epithelialcells by overdistension of the lung with thefixative (fig 1). The secretory activity which is apro,minent fea,ture of the epithelium in routint

365



j.com/


ay 1980. Dow

nloaded from



,.;:.:.Ae- .%.Fig 2 Hamster bronchiole epithelium and lungusing the nodified Eaton-Fedde technique. Noticethat the secretory activity of the epithelium ispreserved and that the cilia are not matted.(H and E, original magnification x 2800).

histology is preserved with the Eaton-Fedde tech-nique (fig 2). If the lung is carefully distended,tufts or cilia are prominent features in the airwaylumens. Positive fluorescence of the APUD cellsand NEB is easily demonstrated by this techniqueand the cells appear to fluoresce more brightlythan with the Falck-Hillarp procedure (fig 3).Although we have been unable to demonstratepositive argyrophilia of APUD cells in the lungby either the Grimelius or Pascual sil*er stains,we have demonstrated positive argyrophilia inother aldehyde tissues (such as stomach andintestine) in the same animals from which thelungs were obtained. It is, however, much easier toobtain standardised sections with this procedure.

SILVER STAINSParaffin-emtbedded freeze-dried tissue stainedreliably with all argyrophil stains. Bouin's andaldehyde-fixed lung tissue did not accept thesestains even though intestinal tissue fixed andprocessed in the same batch as lung accepted the

I)

Fig 3 Fluorescence microscopy of a neuroepithelialbody in hamster airway (a) and lung (b)demonstrated by the modified Eaton-Feddetechnique. Original magnification x 3000 (a)and 3700 (b).

366



j.com/


ay 1980. Dow

nloaded from



argyrophil stain with relialbility. The reason forthe inability to stain lung tissue treated withBouin's or aldehyde fixation is not clear. Rabbitlung, however, fixed with aldehyde fixat-ivesstained positively with the indicated silver stains.

QUANTITATIONThe number of APUD cells and NEB per mm ofairways detected by fluorescence was comparedwith those detected by silver stains. In this studyfluoTrescent sections and silver-stained sectionswere from the same freeze-dried, randomlyselected peripheral blocks of lungs. The valuesare listed in table 1. These data suggest thatgreater numbers of APUD cells and NEB areconsistently detected by fluorescence emissionthan by silver staining. These were found in spiteof the greater length of small airway surface forstudy in the silver stain group.To minimise further variability associated with

random selections of blocks of tissue, thesestructures were quantitated in a standardised mid-coronal section by fluorescence emission fromfreeze-dried paraffin-embedded sections. Sectionswere cut ifrom the same block at 100 ,mintervals, and the number of APUD cells andNEB in the airways of various sizes counted inrelation to the total airway surface for each orderof bronchus or bronchiole. The number of NEBin the bronchioles was relatively constantthrough a distance of 500jpm (six sections); theAPUD cells, however, were constant for only100 gm depth (two sections) and their numberdecreased prominently in deeper sections(table 2). There were constant numbers of0'2-0-24x10-' APUD cells/mm in the bron-chioles and 0-31 X 10-' in bronchi. Neuroepi-thlial bodies were present in similar numbers; inthe small bronchioles they numbered 0N19 x 10-';in larger bronchioles 0-28x10-1, and in thebronchi 0- 18 x 10-' per mm. The values forAPUD cells were considerably greater than thosein randomly selected peripheral sections in air-

Table 1 APUD cells and NEB in random sectionof peripheral hamster lung identified byfluorescence (n = 2) and by silver stain (n = 3)

Airway diameter Total mm NEB/mm APUD/mmairway

Fluorescence0-01 > 0-19 207-72 1-39 x 10-1 0-28 x 10-10-20 > 171-05 1-46 x 10-1 1 11 x 10-1Bronchi 34-32 0-87 x 10-1 0Silver Stain0-01 > 0-19 213-2 0-75 x 10-1 0-08 x 10-10-20 > 359 6 1-29 x 10-1 0-42 x 10-Bronchi 30-1 0-33 x 10-1 0

ways of all sizes (compare tables 1 (top section)and 2). More NEB were also detected in standardmidcoronal sections as compared with the valuesin the peripheral sections.

Table 3 contains data for NO2 exposed animals.Coronal seotions were cut from two animals foreach exposure period and epithelial surfacesmeasured. Although there was considerable vari-ation in the amount of epithelial surface availablefor study, the numiber of NEB/mm of small andlarge bronchioles was similar in the controls andthe animals exposed to NO2 for one, 14, and 28days. This was not true for the bronchi, whichreflected a marked decrease in NEB after 24hours of NO2 exposure with increase of NEB onday 28 to approximately half the values presentin control hamsters. On the other hand, thenumber or density of APUD cells decreasedmarkedly within the first day of exposure toNO2. In the small and large bronchioles thenumiber of APUD cells/mm increased by day 14of exposure and remained at this level for theentire 28 days of exposure.

In order to compare better our values withthose previously reported by Moosavi et al,8 a

Table 2 APUD cells and NEB at different depthsin airwaysDepth Airway Totalmm NEB/mm APUD/mmsection cxt diameter airway(Cm)o <0-2 115-7 1 46 x 10-1 1-72 x 10-1

>2 117-4 1-80 x 10-1 2-70 x 10-1Bronchi 74-1 2-83 x 10-1 3-77 x 10-1

100 <0-2 92-9 2-26 x 10-1 2-36 x 10-1>02 1604 3-86 x 10-1 2-05 x 10-1Bronchi 37-0 0-81 x 10-1 2-43 x 10-1

200 <0-2 39 5 2-52 x 10-1 0 75 x 10-1>0 2 74-2 1-88 x 10-1 0 94 x 10-1Bronchi - - -

300 <0-2 24-3 2-47 x 10-1 ->02 26-7 1-87 x 10-1 -

400 <0-2 41-4 1-44 x 10-1 0-48 x 10-1>02 67-5 1-18 x 10-1 0-14 x 10-1

500 <0-2 21-4 1-38 x 10-1 0-46 x 10-1>02 20-7 2-40 x 10-1 -

Table 3 Effect of NO2 on APUD cells and NEBby fluorescence

Airway Totalmm NEB/mm APUD/mmdiameter airway

Control <0-2 532-7 2-94 x 10-1 2-20 x 10-1(n = 3) >0 2 693-3 2-37 x 10-1 2-19 x 10-1

Bronchus 111-1 2-33 x 10-1 3-51 x 10-11 day <0-2 73-8 2-20 x 10-1 0 73 x 10-1(n = 2) >0-2 160-6 2-07 x 10-1 1-05 x 10-1

Bronchus 33 9 0-45 x 101 -14 day <0-2 49-0 3-76 x 10-1 1-50 x 10-1

>02 187-1 2-68 x 10-1 1-33 x 10-1Bronchus - - -

28 day <0-2 203-0 2-33 x 10-1 1-12 x 10-1>0 2 233-2 2-23 x 10-1 1-50 x 10-1Bronchus 58-6 1-35 x 10-1 2-38 x 10-1

367



j.com/


ay 1980. Dow

nloaded from


368

quantitative study of neuroendocrine cells wasperformed in neonatal rats. The data in table 4indicate that the frequency of APUD cells andNEB was similar in small and large bronchioles.However, the total number of neuroendocrinecells in the neonatal rat was substantially lessthan the number observed in adult hamster, andconsiderably less than the number reported in thisspecies by other investigators.

Discussion

FALCK-HILLARP TECHNIQUEAlthough the preservation by this freeze-dryingmethod is not comparable to standard aldehydefixation, the tissue is acceptable for light andfluorescent microscopic study. The cytoplasm ofcells is generally vacuolated, many ciliated cellshave lost their cilia, and some cells are disrupted.APUD cells and NEB are easily distinguishedfrom all other epithelial cell types in fluorescentsections and silver-stained sections. There areseveral disadvantages in the use of this technique.These include the need for specialised equipmentsuch as the freeze-dry apparatus and fluorescentmicroscope, and the difficulties associated withobtaining a standard section for study, which isboth properly fixed in inflation and processedwith adequate rapidity to preserve the intracellu-lar materials. Furthermore, tissue preserved bythe freeze-dry method is not suitable for electronmicroscopy because of its poor state of preserva-tion.

EATON-FEDDE TECHNIQUEAs with the Falck-Hillarp technique, it is easy todistinguish the brightly fluorescent APUD cellsand NEB from other fluorescent cells such asmast cells and macrophages. The enhanced fluor-escence that one obtains after this modificationof the Eaton-Fedde technique is probably a resultof the concentration of the secretory productsduring the sublimation process. The advantagesof the modified Eaton-Fedde technique is theability to prepare standard sections of lung beforethey are frozen in liquid nitrogen. The viscousOCT solution with which the lung is distendedgives structural support so that uniform coronalsections can be cut from the lung. This proce-Table 4 APUD cells and NEB in four neonatal rats

Airway diameter Totalmm NEB/mm APUD/mmairway

<0-2 102-9 0-48 x 10-1 0-87 x 10->0 2 32-9 0 30 x 10-1 0-91 x 10-1Bronchi 3-2 - -


dure like the classical Falck-Hillarp technique hasseveral disadvantages. There is a delay of up totwo weeks during the tissue processing but theconsistency and reliability of the fluorescentstructures is excellent. The Eaton-Fedde tech-nique as modified in this study is simple, repro-ducible, and consistent. A major problem usingthis method is our inability to obtain silver stain-ing of APUD cells or NEB in the hamstertracheobronchial tree and lung, whereas theFalck-Hillarp procedure stains APUD cells andNEB reliably with silver stains.

QUANTITATIONThe comparison of fluorescence and silver stainmethods for detecting neuroendocrine structuresfrom randomly selected blocks of peripheral lungtissue (table 1) indicates the greater sensitivityand reliability of the fluorescence methods. Thisanalysis was performed on successive sectionsfrom the same tissue block. The method ofexpressing numerical values for NEB and APUDcells permits only a single mean value for eachanimal in spite of the large amount of airwaysurface studied per animal. These circumstancesdo not permit valid application of the commonstatistical methods of analyses of data. However,the consistently larger values for APUD cells andNEB in the fluorescent preparations strongly sug-gest it as the method of choice for detection ofneuroendocrine structures.

Quantitative studies indicate that normal malehamsters contain 1-72-3-77 x 10-1 APUD cells/mm and between 1A46-2 83 x 10-1 NEB/mm inthe small airways and bronchioles of the lung.The number of APUD cells and NRB/mm of thebronchus in control animals is greater than in thesmall airways. The reason for this virtual doub-ling of APUD cells and NEB in the large airwaysis not known but may reflect the need forspecialised responses in epithelium of the largeairways for control of ciliary motility, goblet cellsecretion, or epithelial permeability. APUD cellsare not present in constant numbers in succes-sive sections taken at 109ium intervals from thestandard tissue block; however, NEB are con-sistently present to a depth of 600,um. These dif-ferences are believed to result from inadequateexposure to and reaction with formaldehydevapours of the APUD cells in deeper portions ofthe tissue block. The numbers of APUD cellsand NEB in the random sections of peripherallung are also less than those observed in standardmidcoronal blocks. This may reflect a true differ-ence in distribution of the neuroendocrine struc-tures favouring a greater number in the central



j.com/


ay 1980. Dow

nloaded from



airways. Additional studies are needed to confirmthis observation.

In studies of the effect of exposure to NO2the number of APUD cells is profoundly de-pressed after 24 hours of exposure, but thenumber and distribution of NEB is not altered.The decrease in APUD cells persists for the entire28-day exposure period as compared with controlvalues, but is greater at seven and 14 days thanat day 1 of NO2. Neuroepithelial bodies are notsignificantly altered in the small airways, but adecrease in number in the large airways persistsat 28 days of exposure as compared with controls.NO2 is a powerful oxidising agent producingcellular changes which vary with the concentra-tion and duration of exposure. Our data suggestthat the APUD cells are responsive to NO2 ex-posure, possibly by degranulation or desquama-tion resulting from acute APUD cell stimulation.This response is not observed in the NEB,perhaps because of the greater mass of thesestructures. APUD cells occur singly while NEBrepresent a cluster of neuroendocrine cells. It ispossible that some cells of the NEB retain theirgranules after NO2 exposure which may haveresponded by fluorescence and are more easilydetected than individual APUD cells. Additionalelectron microscopic study is required to evaluatethis hypothesis that degranulation of APUD cellsdoes inded occur after exposure to NO2.Three studies have been reported in which

quantitative values of the neurosecretory cellshave been performed. Tateishi21 found between19-41% of the human lungs examined containedargyrophilic cells in various airways from seg-mental bronchi to bronchioles, while only 1-9%of the lungs had argyrophilic cells in largerbronchi and terminal bronchioles. These data aredifficult to compare with ours because Tateishimade no attempt to quantitate the APUD cellsor NEB, nor to differentiate these two types ofneuroendocrine structures.Moosavi et all quantitated the number of

Feyrter cells/cm in bronchial epithelium of 4 to31-day-old rats and observed that the cells in-crease from 32-59/cm to 70/cm between day 4and day 7 of life and decrease to 16/cm by day31. These data indicate that neonatal and youngrats may have 10-60 times the number of similarcells which we observed in adult hamsters. Thisdiscrepancy may be in part the result of speciesand age differences. However, the studies per-formed by us on young rats indicate that speciesdifference is not significant. In our study, 3-day-old rats had 0-48 x 10-1 to 0-77 x 10-1 neuroendo-crine cells/mm in airways up to 0-2 mm and

0 30 to 2-91 x 10-1 cells/mm in larger airways.These values are only slightly less than ourvalues for adult hamsters, but one-tenth thenumber reported in rats by Moosavi et al, inneonatal rats. In that report8 only occasionalFeyrter cells were observed in adult rats andclumps of Feyrter cells were "described" in theprocess of exfoliation. These latter structuresmay represent what is now characterised as NEB.It is possible that the difference in numbers ofAPUD cells found in our studies as comparedwith that of Moosavi et al may be the result ofvariations in the number of these cells inneonatal, growing and adult animals. Moosaviet a/8 and others22-24 have demonstrated thatdegranulation of the APUD cells and NEB occurin rabbits subjected to hypoxic conditions. Ex-posure to 30-40 ppm of NO2 may lead to hypoxiaas a result of pulmonary oedema after 12-48hours. However NO2 may injure the cell mem-brane because of its oxidative potential andproduce an exocytosis of the neurosecretorygranules. Taylor24 reported an increase in thenumber of APUD cells in rabbits born and raisedin the Andes as compared to rabbits reared inEngland. Hernandez Vasquez et al,25 however,reported that the number of APUD cells in rab-bits decreases when they are exposed acutely tohypoxia. These observations suggest that adapta-tion to hypoxia during growth and developmentmay influence the APUD cells in a manner com-pletely different from that in animals not adaptedto the hypoxic environment. Degranulation of theAPUD cells is reported to occur only as a resultof acute hypoxia.The differences in the quantitative values of

APUD cells in our work as compared with thatof Moosavi et a18 are difficult to explain. Severaldifferences in technique may have contributed tothe discrepancy. Moosavi et al,8 Taylor,24 andHernandez-Vasquez et a125 used silver staining ofaldehyde-fixed tissue to detect these cells. It isnot likely that this difference is the basis of thediscrepancies since our studies indicate thatfluorescence techniques are more sensitive andreliable than are silver staining methods. Approxi-mately half as many APUD cells and NEB weredetected in our silver-stained studies of freeze-dried lung tissue as by the fluorescence method.Additional studies using acceptable quantitativetechniques may provide further information onthe role of the APUD cells and NEB in injuryand disease states of the lung.

This work was supported in part by a grant fromThe Council for Tobacco Research, USA, Grant

369



j.com/


ay 1980. Dow

nloaded from


370

number 1190, and Grant number lR01HL22202-CVA from the National Heart, Lung, and BloodInstitute of the Nationail Institutes of Health.

References

1 Frohlich F. Die "Helle Zelle" der bronchial-scheimhaut and ihre beziehungen zum problemder chemoreceptoren. Frankfurter Z Pathol1949; 60:517-59.

2 Feyrter F. Zur pathologie des argyrophilenhelle-zellen-organes in bronchialbaum des men-schen. Virchows Arch 1954; 325:723-32.

3 Pearse AGE. Common cytochemical propertiesof cells producing polypeptide-hormones, withparticular reference to calcitonin and thethyroid C cells. Vet Res 1966; 79:587-90.

4 Pearse AGE, Polak JM. Cytochemical evidencefor the neural crest origin of mammalian ulti-mobranchial omission. Histochemie 1971; 27:96-102.

5 Lauweryns JM, Goddeeris P. Neuroepithelialbodies in human child and adult lung. Am RevRespir Dis 1975; 111:469-76.

6 Lauweryns JM, Peuskins JC. Argyrophil (kininand amine producing?) cells in human infantairway epithelium. Life Sci 1969: 8:577-85.

7 Terzakis JA, Sommors SC, Anderson B. Neuro-secretory appearing cells of human segmentalbronchi. Lab Invest 1972; 26:127-32.

8 Moosavi H, Smith P, Heath D. The Feyrter cellin hypoxia. Thorax 1973; 28:729-41.

9 Hage E. Histochemistry and fine structure ofendocrine cells in foetal lungs of the rabbit,mouse and guinea pig. Cell Tissue Res 1974;149:513-24.

10 Cutz EW, Chan W, Wong V, Conen PE. Ultra-structure and fluorescence histochemistry ofendocrine (APUD-type) cells in tracheal mucosaof human and various animal species. CellTissue Res 1975; 158:425-37.

11 Falck B, Hillarp NA, Thieme G, Thorp A.Fluorescence of catecholamines and relatedcompounds condensed with formaldehyde. JHistochem Cytochem 1962; 10:348-54.

12 Eaton JA, Fedde MR. Identification of twopopulations of biogenic amine-containing cellsin the mouse lung. Cell Tissue Res 1977;176:243-49.


13 Hakanson R, Lilja B, Owman C. Cellular localis-ation of histamine and monoamines in thegastric mucosa of man. Histochemie 1969;18:74-86.

14 Hamberger B, Malmfors T, Sachs C. Standardis-ation of paraformaldehyde and of certain pro-cedures for the histochemical demonstration ofcatecholamines. J Histochem Cytochem 1965;13:147.

15 Corrodi H, Hillarp NA, Jonsson G. Fluorescencemethods for the histochemical demonstration ofmonoamines. 3. Sodium borohydride reductionof the fluorescent compounds as a specificitytest. J Histochem Cytochem 1962; 12:582-6.

16 Gray P. The microtomist's formulary and guide.New York: McGraw-Hill, 1954.

17 Pascual JSF. A new method for easy demon-stration of argyrophil cells. Stain Technol 1976;51:231-5.

18 Grimelius L. The argyrophil reaction in isletcells of adult human pancreas studied with a newsilver nitrate procedure. Acta Soc Med Upsala1968; 73:271-94.

19 Sevier AC, Munger BL. A silver method forparaffin sections of neural tissue. J NeuropatholExp Neurol 1965; 24:130-5.

20 Kleinerman J, Rynbrandt DJ. Lung proteolyticactivity and serum protease inhibition after NO2exposure. Arch Environ Health 1976; 31:37-41.

21 Tateishi R. Distribution of argyrophil cells inadult human lungs. Arch Pathol 1973; 96:198-202.

22 Lauweryns JM, Peuskins JC, Cokelaere M.Argyrophil, fluorescent and granulated (peptideand amine producing?) AFG cells in humaninfant bronchial epithelium. Light and electronmicroscopic studies. Life Sci 1970; 9:1417-29.

23 Lauweryns JM, Cokelaere M, Deleersynder D,Liebens M. Intrapulmonary neuroepithelialbodies in newborn rabbits. Cell Tissue Res 1977;182:425-40.

24 Taylor W. Pulmonary argyrophil cells at highaltitude. J Pathol 1977; 122:137-44.

25 Hernandez-Vasquez A, Will JA, Quay WB.Quantitative characteristics of the Feyrter(APUD) cells of the neonatal rabbit lung innormoxia and chronic hypoxia. Thorax 1977;32:449-56.



j.com/


ay 1980. Dow

nloaded from


APUD lung: methods, quantitation, response to injury · Grimelius or Pascual silver staining...

Documents

Transcript of APUD lung: methods, quantitation, response to injury · Grimelius or Pascual silver staining...