fit ‘5 3.; . :‘"£“v£* "' ‘H ‘53 ‘II'It'. A}... d . [‘ .......... ., 3 w. a ,m. - .s fir. kt. . :1. 00.00 a}. A.” Q." . C. an . It: ”4) 35..- ”“s «Junk Jim a a8 Whil‘ A“ I“ t a“ .x 3n )3! 3.; .. (\ .‘ur. ¢w ... . MN» .V‘ r .1... “a u... .01“ “Finn“ ,_ .3 ~ F. . 5,. .... m. E , 5. f 5 MPH” .m H on“ U‘ bl ”OW“ W. W5 £514 .3.“ “‘6 I “M “0.. AW MU mi. in” y u...“ 14.. s. . 42¢ DUE i... 4.! A» L O r“. .17. .! ’fl‘ o.. i.- e~ a: I 1- ‘3 71"' HI" I ' ‘ ‘- (IE-K... dth‘ p‘ F. F! "i g THE cwomazc RES '2' _:_:,_r::___,_2::2523::_, mmmfl Infint: LIBRARY Michigan State University THE CYTOLOGIC RESPONSES OF NORMAL BEAGLE DOGS UTILIZING THE SKIN WINDOW TECHNIC BY David T. Drees A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Anatomy I966 To JANET ACKNOWLEDGMENTS The author wishes to express his sincere appreciation and thanks to his major professor, Dr. Al N. Stinson, for his guidance and encouragement during the course of this investigation. The author also wishes to thank Dr. H. Lois Calhoun, Chairman of the Department of Anatomy, and Drs. Esther Smith and Robert F. Langham for serving on the guidance committee and giving constructive criticisms of this manuscript. Appreciation is expressed to Dr. Gabel Conner, project leader of U.S.P.H. Research Contract Number PH h3-65-l00 Leukemia Trans- mission Studies, for making dogs available for this study. Also, appreciation is extended to Dr. William S. Adam, a member of this project, for his special assistance. Dr. Jeanne M. Riddle of the Henry Ford Hospital, Detroit, Michigan, was very helpful in inter- pretation and evaluation of the slides in the skin window preparations and her encouragement was greatly appreciated. Special thanks are extended to Drs. Roger Brown, Thomas Jenkins, Heront Harcarian, Richard Notzold, and Charles Titkemeyer for their valuable advice and suggestions. The author is also indebted to Mr. Carl Oehling and Miss Sallie Dutmer for their technical assistance. TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . l REVIEW OF LITERATURE. . . . . . . . . . . . 3 NATERIALSANDNETIIODS........... 9 RESULTS AND DISCUSSION. . . . . . . . . . . 13 SUMMARY AND CONCLUSIONS . . . . . . . . . . l8 LITERATURE CITED. . . . . . . . . . . . . . 36 iv LIST OF FIGURES FIGURE IO. 12. 13. lh. IS. wverSI‘p kit. 0 O O O O O O O O O 00000 O O O Dorsal thoracic area of dog with four skin window preparations................... Two neutrophilic leukocytes. Two hour stage . . . Low power view. Four hour stage . . . . . . . . . One small mononuclear cell. Four hour stage. . . . Two neutrophilic leukocytes and five enlarging mononuclear cells. Six hour stage . . . . . . . . One eosinophilic leukocyte and two degenerating neutrophilic leukocytes. Six hour stage . . . . . Two eosinophilic leukocytes with one containing fused eosinophilic granules and two degenerating neutrophils. Six hour stage . . . . . . . . . . . Low power view. Eight hour stage. . . . . . . . . Five degenerating neutrophils and two mononuclear cells. Eight hour stage.. . . . . . . . . . . . . Six mononuclear cells in varying stages of enlargement. Eight hour stage . . . . . . . . . . One phagocytic mononuclear cell containing nuclear lobe of neutrophilic leukocyte. Ten hour stage . . . . . . . . . . . . . . . . . . . . Low power view. Fourteen hour stage . . . . . . . Eight enlarged mononuclear cells. Sixteen hwr Stage 0 O O O O O O O O O O O O O O O O 0 O 0 One neutrophilic leukocyte and five large mono- nuclear cells, four having phagocytized Pelikan ink. Twenty hour stage. . . . . . . . . . . . . . Page 2] 2l 23 23 25 25 27 27 29 29 3| 3| 33 33 35 INTRODUCTION For over a century numerous investigators have studied the structure and functions of the leukocytes and the other inflam- matory cells with a variety of technics. Today the study continues with many questions still remaining unanswered. The major problem in the study of leukocytes and inflanmatory cells in inflammation has been the deficiencies in the available technics. Laboratory animals were used in the early studies and often it was necessary to sacrifice them making continuous studies impossible.‘ In addition, it was impossible.to serially study the . cells from a single lesion. In l9h0 Rebuck introduced a new method, the skin window technic, which allowed the serial study of the leukocytes in acute inflammation in a single lesion. Since this simple technic pro- duced only superficial abrasion of the skin, the experimental animals can be used repeatedly. Rebuck first used the technic in the rabbit and later applied it to the human. Since that time it has been used many times to study the various functions and structures of the leukocytes and the other inflammatory cells in normal and diseased humans. Riddle and Barnhart (l965) used the skin window technic on dogs to Study the eosinophil as a source for profibrinolysin in acute inflammation. According to their findings they reported that the cellular exudate in the canine species resembled that reported in the human skin window. It is the purpose of this investigation to describe the cellular exudate obtained using the skin window technic in the nonmal beagle dog. REVIEH OF LITERATURE In l829, Durtrochet first reported the migration of the white cells from the blood vessels into an inflamed area (Rebuck and Crowley, l955). Since then many investigators have studied leukocytes and their functions in inflammatory reactions. In summary, their reviews stressed the importance of lymphocytes and monocytes as sources of macrophages in acute inflammation. Rebuck (l940) introduced a new‘Lguxlgg technic using the rabbit to study the role of the monocyte in acute inflammation, which later became known as the skin window technic (Sieracki and Rebuck, l960). The skin window technic allows the serial study of the cellular exudate in a single lesion over any given period of time (Rebuck and Crowley, l955; Rebuck, l958; Edwards, l959). Heretofore, this was not possible because the methods used resulted in considerable trauma or injury and the experimental animals had to be sacrificed. Using the skin window technic, Rebuck (I949) described the functional and structural changes which occurred in the neutrophils and lymphocytes in acute inflammation in man when the patient was not systemically immunized to the stimulating antigen. The neutro- phils were the first cells to migrate into a lesion. Initially they became swollen, and before they shrank and died, the neutro- phils completed their enzymatic and phagocytic activities. Fragments of neutrophilic cytoplasm were shed into the fluid exudate and these small particles were then phagocytized by the lymphocytes. 3 I, The lymphocytes probably used these pieces of neutrophilic cyto- plasm for nutrients. Lymphocytes appeared in the exudate at two hours and gradually increased in number until they became the dominant cell at lh-l8 hours. At the 9-lh hour stage the majority of the lymphocytes began to hypertropy and during the lh-l8 hour period they were indistinguishable from tissue macrophages or histiocytes. As the lymhocytes transformed into macrophages, the cytoplasm increased and became more phagocytic (Rebuck, _e_t 21,, I958; I960; l96lb). The nuclei also increased in size, the chromatin separated into finer pieces, the nuclear membrane lost the uniform smooth outline, and the nuclear indentation became larger. The cellular exudate varied with use of different antigens and with the immunological status of the patient to a specific antigen. Hhen using tuberculin as the antigen, Rebuck and Yates (i954) reported a significant decrease or absence of lymphocytes at the important l2 hour stage in patients who were tuberculin- posltlve. In contrast, the controls or tuberculin-negative patients responded with a high level of lymphocytes at the l2 hour stage of acute inflammation. Poliomyelitis vaccines produced the highest level of lympho- cytic migration at the l2 hour stage in individuals vaccinated with Salk polio vaccine, while the lymphocyte response in non- vaccinated subjects at the l2 hour stage was very poor (Rebuck and LoGrippo, l960; l96l). In individuals imune \to the typhus rickettsiae, the neutrophils remained the predominant cells for 5 2h hours. In nonimmune patients, the neutrophils were much less numerous.(Hisseman and Tabor, I965). Hu, g§,§fl, (l96l) studied Rus positive individuals and described an eosinophilia along with an increase in lymphocytes and giant cells at 33 hours of inflammation. Eitzman and Smith (I957) observed eosinophilias of IO-93%.in infants of 36 hours to 2i days of age. Riddle and Barnhart (I965), in the only report using dogs, induced eosinophilic levels up to “0% on the cover- slips by using stimulants of fibrin and fibrinogen. Investigation in diseased humans has resulted in a variety of cellular responses. The normal leukocytic response is greatly diminished in patients with a peripheral blood neutropenia, and the subsequent hypertrophied fonms of lymphocytes are also decreased in number. It is believed the time and the sequence of the cellu- lar components of the exudate is governed, at least in part, by the neutrOphil and that each step in the inflammatory cycle is subject to the preceding event (Page and Good, l958). In patients, with acute leukemia in which the circulating mature neutrophilic leukocytes were greatly decreased in number, Perillie and Finch (l960) observed markedly decreased and even acellular exudates on the coverslips. They concluded that this poor response was probably responsible for the susceptibility of these individuals to infection. Boggs (l960) also reported that immature cells were never located in the exudate. However, in patients with chronic leukemia in which the circulating mature neutrophils were near normal in number, a normal cellular exudate 6 was observed. Immature nucleated red blood cells in the peripheral blood also do not appear in the skin window exudate (Torre and Leikin, 1959). j A patient with polyarteritis nodosa exhibited increased eosinophils at seven hours (Rebuck,‘g£‘§l., l95l). Riis (I959) reported an increase in eosinophils in Felty's syndrome. Eosino- phils comprised over half of the cellular exudate in patients allergic to fish extract, grasses or ragweed (Eidinger,‘g; l., I962). The topical application of a Steroid failedito decrease the eosinophilia while oral administration greatly reduced the eosinophilic response (Eidinger, g;_§fl,, I964). The systemically acting steroid may possibly have reduced the total number of circulating eosinophils and thereby cause a decrease in eosino- phils on the skin window coverslips. The basophilic leukocyte is a rare participant in exudate of normal individuals. However, in patients with ulcerative colitis it was present at three hours of inflammation and increased steadily to peak between lh-27 hours. The peripheral blood baso- philic level in these patients was normal or only slightly increased (Priest, g$_§fl,, I960). In contrast, a patient with terminal basophilic granulocytic leukemia, whose blood contained 52% baso- phils, had very few basophils in the skin window exudate (Rebuck, 55.31., l96lb). Patients with interstitial cystitis also were characterized by an increased migration of baSOphils which peaked at 26 hours (Rebuck, stab, l96la). The cellular exudate is altered by the actions of cortisone 7 and ACTH. Rebuck and Hellinger (1953) reported a diminished cellular response after topical application of cortisone at the site of the skin window lesion. The phagocytic capabilities of the exudate were correspondingly decreased due to the diminished number of cells. The same cellular response resulted following the systemic administration of ACTH (Rebuck, g§_§fl,, l95l). Perillie,lg§.§u, (I960) reported using the skin window technic in the study of the lupus erythematosus phenomenon and evaluated its possible use as a diagnostic aid. They concluded that the value of the technic as a diagnostic aid was as good as other methods available. Several authors have questioned the ability of lymphocytes to transform into macrophages. Florey (l96l) and Hedawar (l96l) stated that lymphocytes do not stick to glass surfaces. This evidence seriously challenges Rebuck and Crowley's (I955) inter- pretation of their skin window preparations in which they reported the transformation of lymphocytes into macrophages. Volkman and Gowans (l96h) also presented evidence that small lymphocytes are not capable of transforming intoImacrophages. When tritiated thymidine labeled lymphocytes were transfused into rats at the time inflammation was created, no labeled lymphocytes appeared at the site of the lesion and no labeled macrophages were found on the coverslips. However, when labeled bone marrow cells were injected, many labeled macrophages appeared on the coverslips. This suggests precusors of macrophages other than lymphocytes. In order to avoid this controversy, these cells will be referred 8 to as small monuclear cells in this paper. HATER IALS AND METHODS A concurrent project in the College of Veterinary Medicine under contract with the National Institute of Health was the study of the transmissability of leukemic materials to dogs. Seven purebred beagle dogs from the normal colony of the leukemia project were used in this study. The dogs, three males and four females, ranged in age from h-6 months and weighed from l5-20 pounds. The animals were well cared for from birth and were in excellent health as determined by physical examination. Total red and white blood cell counts as well as a differential white cell count were made before each animal was used. The blood counts were all within the normal range (Schalm, l965). The skin window technic of Rebuck and Crowley (I955) and modified by Riddle and Barnhart (l965) for dogs was used in this investigation. The author adopted a few further modifications to facilitate the work. Number two, l5 mm square glass coverslips were alcohol cleaned. Two centimeter cardboard squares were cut from unlined file cards. A small, approximately 2 mm by ID mm, strip of masking tape was folded on itself and stuck onto the cardboard squares. The cover- slips werethen attached to the cardboard by the use of the_ remaining adhesive ends of the tape. Attaching the coverslips to the cardboard facilitated their handling and reduced the chance of contamination of the lesion.. This also allowed an easy method of identifying the cardboard squares with their accompanying coverslips. 9 l0 The coverslips and the cardboard squares were wrapped in alumi- num foil, placed in petri dishes and sterilized. Each individual kit could be removed from the petri dishes without contaminating the unused kits (Fig. I). To minimize chemical and mechanical trauma, the dog's skin was prepared for experimentation at least l5 hours prior to the creation of the lesion. The hair was clipped from the dog's back, shoulders and thorax. A commercial cream depliatory* was spread over this area and allowed to react for 15-25 minutes. The hair and cream were scraped away and washed with warm tap water. The area was then washed with Germicidal Soapflcand allowed to dry. A strip of two inch adhesive tape, twelve inches in length, was applied over the thoracic vertebral column. Another twelve inch strip of tape was applied about l5-20 mm below and parallel to the first piece of tape. Five short strips of tape about IS- 20 mm apart were arranged perpendicular to the first two strips. This resulted in four small l5-20 mm square areas of exposed skin suitable for the experimental lesions. The application of the tape could be repeated on the opposite side of the dog. This would allow the study of a total of eight lesions simultaneously. A small pledget of cotton was soaked in 70% alcohol and placed over the exposed skin. The cotton was held loosely in place with another piece of adhesive tape until the experiment A Nair, Carter Products, Inc., New York, New York. ** Park-Davis 8 Company, Detroit, Michigan. II was to begin. At this time a Elizabethian collar was cut from a cardboard box and placed around the dog's neck. The hind legs were hobbled by tying them together with a piece of gauze. Thisprevented the dog from either biting or scratching at the taped areas. A small lesion, 3-5 mm in diameter, was created by repeatedly scraping a sterile No. 20 Bard-Parker blade over the exposed skin. Uhen fine bleeding points were observed and blood began to collect on the blade, the correct depth of the lesion was reached. The experiments were timed from this point. A sterile platinum loop was used to apply one to two l00ps of an inflammatory stimulant, Diptheria Toxoid* or Old Tuberculin**. To study the phagocytic properties of the cellular exudate, trypan blue or Pelikan***ink was also applied to eight of the experimental lesions. The lesions were immediately covered with the sterile coverslip and cardboard square which was held in place with a piece of adhesive tape (Fig. 2). The cells migrated and adhered to the underside of the cover- slips. The first coverslip was removed at two hours and replaced immediately with a second coverslip. The subsequent coverslips were removed every two hours until the experiment was terminated at the 2k hour stage. *Parke, Davis 5 Company, Detroit, Michigan. **Parke, Davis 5 Company, Detroit, Michigan. John Henschel and Co., Inc., 425 Park Avenue, South New York l6, New York. I2 The coverslips were rapidly air dried and mounted on micro- scope slides with Permount*. The cells were then stained like blood smears with either Nright's-Leishman or May-GranwaId-Giemsa stains. The technic followed for the Hright's-Leishman stain was essentially that described by Schalm (l96l), however, it was necessary to increase the staining time to l2 minutes. The pro- cedure for the May-Grfinwald-Giemsa stain was described by Rebuck and Crowley (I955). A total of 384 coverslip preparations from 30 lesions were studied. * . Fisher Scientific Co., Fair Lawn, New Jersey. RESULTS AND DISCUSSIONS The diptheria and tuberculin antigens chosen for this investi- gation were agents to which the dogs were not systemically immunized. The cellular responses to the two antigens was essentially the same probably because the animals were nonimmune to them. (To seven lesions no antigen was employed and the cellular response was quali- tatively the same but quantitively decreased as compared to lesions stimulated with the nonimmune antigens. This was especially true in the latter stages of each experiment. Because the responses were very similar, the results of all these experimental lesions will be described together. _T_I_I_9_ 119043 _S_‘I_'_A.(_3_E_ The cellular exudate was sparse and the primary cell present was the neutrophilic leukocyte. The majority of the neutrophils were l2-Ih microns in diameter with the cyto- plasmic portion increased. The profiles of the cells were nearly round and the cell membranes appeared stretched. The neutrophil ic granules were more widely dispersed throughout the cytoplasm than in normal cells. A few fragments of neutrophilic cytoplasm were scattered throughout the preparation (Fig. 3). A paucity of small mononuclear cells, measuring l0-l2 microns in diameter, was present. A few'macrophages and eosinophils were also scattered throughout the exudate. FOUR HOUR STAgE The neutrophils were present in great numbers (Fig. A). They appeared much as described in the two hour 13 l4 stage, but an increase in cytoplasmic fragments was observed. The small mononuclear cells also increased in number but still measured from lO-lz microns in diameter with a few as large as 20 microns. The small mononuclear cell appeared exactly like a peripheral blood lymphocyte with a large round, dark staining nucleus that contained a coarse, clumped chromatin pattern. The nuclear membrane was smooth and thick. IThe eccentric, slightly indented nucleus was surrounded by a thin ring of baSOphiIic staining cytoplasm (Fig. 5). Some cells also contained a few azurophilic granules. I Eosinophils likewise had increased in number. These cells appeared swollen and were most numerous in areas associated with fibrin. The eosinophilic leukocyte comprised approximately 2% of the cellular exudate. §_l_l§_ HOUR STAGE The ratio of neutrophilic leukocytes to mononuclear cells was about two to one. Many of the neutrophils were less distinct and the exudate contained an increased amount of cytoplasmic fragments. A considerable number of the neutrophils had lost all their cytoplasm to the fluid exudate and their nuclei remained naked on the coverslips. The intact neutrophils often contained vacuoles within their cytoplasm. Many of the mononuclear cells increased in Size up to 20 microns and were fixed in a variety of elongated shapes suggesting they were actively motile at time of fixation. The nuclear mem- brane, which in the earlier stages was thick and round, became thin and irregular conforming to the over-all shape of the cells. IS The chromatin pattern was finer and the distribution between chromatin and parachromatin was more obvious (Fig. 6). The eosinophilic leukocyte was present at about the same percentage as in the previous stage, however, many were fragmented and free eosinophilic granules were present in the fluid exudate. They were most numerous in areas associated with fibrin (Fig. 7). This supports Riddle and Barnhart's (l965) conclusion that eosino- phils are selectively attracted to fibrin in areas of inflammation. A considerable number of the eosinophilic granules, either intra- cellular or extracellular, were fused resulting in an occasional eosinophilic mess up to 5 microns in diameter (Fig. 8). ‘gjggluuggg.§155§ The percent of neutrophils were decreased to 50% of the cellular exudate (Fig. 9). More of the neutrophils were degenerated with clumped chromatin and pyknotic nuclei their salient features. Many of the neutrophils were also dehydrated and shrunken (Fig. ID). The mononuclear cells were increased to 50% of the cellular response and like the previous stage many were fixed in irregular shapes. The chromatin was broken into finer pieces and the nuclear membrane remained irregular in outline. The cytoplasm, at this Istage, contained a few'small vacuoles and stained basophilic. The amount of cytoplasm in relation to nuclear size had increased but still remained relatively small when compared to the other cells I in the exudate (Fig. ii). The eosinophilic leukocyte decreased in number and could only l6 occasionally be located in the exudate. .1§!.fl2!§.§l駧 The neutrophils for the first time in the experiment comprised less than 50%.of the cellular exudate. A few neutrophils appeared normal but the great majority of them were shrunken with degenerate and pykndtic nuclei. Most of the cytoplasmic fragmentS'were absent, possibly because they were phagocytized by the mononuclear cells. The mononuclear cells made up over 50% of the cells on the coverslip. The majority of these cells were l2-lh microns in diameter because of an increase in cytoplasm. The basophilic: staining cytoplasm contained vacuoles and an increased number of large azurophilic granules. Some of the mononuclears also had phagocytized nuclear lobes of dead neutrophils (Fig. l2). The ten hour stage was also characterized as the point in which the majority of the lesions reached their peak cellular response. The peak response in dogs occurs approximately two hours earlier than the important peak l2 hour stage in humans (Rebuck and LoGrippo, I960). M 19 TWENTY-FOUR m m The previously described changes continued and became more pronounced. The intact or relatively normal neutrophils markedly decreased in number, while dying and shrunken forms persisted throughout the experiment. The mononuclear cells increased until they reached approxi- mately 75% of the cellular exudate (Fig. l3). The average size of the mononuclear cells continued to increase. The majority of l7 these cells reached a diameter of l8-22 microns, although small mononuclear cells such as seen at the two and four hour stages were still present on the coverslips until the experiment was terminated at 2h hours. The nucleus increased in size and the chromatin masses continued to break into smaller pieces. The cytoplasm also increased in volume and often contained many large vacuoles. Azuro- philic granules were prominent throughout the later hours. After the IA hour stage, the mononuclear cells were very similar to tissue macrophages and morphologically were indistinguishable from macrOphages (Fig. IA). The total mononuclear cell response began to decrease in number at about the l6 hour stage. wfimm Vital dyes, trypan blue or Pelikan ink, in addition to the diptheria toxoid or tuberculin were applied to eight lesions at the beginning of the experiment to study the phago- cytic ability of the exudate. The cytological response was essentially the same as previously described. The first evidence of phagocytosis occurred at four hours. A few neutrophils contained small accumu- lations of either trypan blue or Pelikan ink In their cytoplasm. At six hours more neutrophils had ingested the vital dyes and a few of the larger mononuclear cells also had phagocytized the dyes. After six hours, the neutrophils became less important in phagocytosis of the dyes, but the enlarging mononuclear cells became more phago- cytic. They retained this ability until the experiment ended at the 2A hour stage (Fig. I5). SUMMARY AND CONCLUSIONS Using Rebuck's skin window technic, a cytological study was undertaken to determine the migration of leukocytes and inflame matory cells in acute inflammation in normal beagle dogs. A few modifications of Rebuck's technic to facilitate its use on dogs were described by the author. Diptheria toxold and tuberculin, antigens to which the dogs were not systemically immunized, were used in this study. Vital dyes were also employed to study phagocytosis. The quantity of cells adhering to the coverslips was very numerous, and immature or mitotic cells were never observed. The great majority of these cells were probably carried to the area of inflammation by the blood vascular system and are of hematogenous origin. The first cells toImigrate in any appreciable number were the neutrophilic leukocytes. They increased In number and size for the first six hours. They persisted throughout the 2k hour experiment, but in relatively decreasing nunbers after six hours. The neutrophils first appeared as normal peripheral blood cells, then became swollen and later lost small pieces of their cytoplasm to the fluid exudate along with further clumping of their nuclear chromatin. They were capable of phagocytizing vital dyes.. An occasional small mononuclear cell, morphologically the same as a peripheral blood lymphocyte, was present at two hours. These cells increased in size and in number and reached their peak I8 l9 migratory point at approximately ten hours. The comparable stage in humans was not reached until l2 hours of inflammation. The small mononuclear cells changed in structure until at l6-l8 hours the majority could not be distinguished from macrophages. This transformation was accomplished first by an increase in cytoplasm accompanied by an increase in azurophilic granules and intracyto- plasmic vacuoles. As the cytoplasm increased, phagocytosis correspondingly increased. The nuclear size likewise increased and the nuclear membrane became thinner and irregular. The coarse clumped chromatin, characteristic of the early migrating small mononuclear cell, separated into finer pieces. The eosinophilic leukocyte composed about 2% of the cellular' exudate from four to six hours of inflammation. After six hours their numbers decreased rapidly. The eosinophils, like the neutrophils, first appeared as peripheral blood eosinophils, then became swollen and later lost pieces of cytoplasm and eosinophilic granules to the fluid exudate. The eosinophils were often associ- ated with fibrin suggesting an attraction for the fibrin strands present in the exudate. The cellular response in the dog was very similar to cellular response in humans (Rebuck and Crowley, I955). The most important difference was the time needed to reach the stage of peak cellular Imigration. This stage occurred at ten hours in dogs as opposed to I2 hours in humans. This indicated that the dog was capable of mobilizing its greatest cellular defense mechanism to an irritant more rapidly than man. 20 FIGURE 1 Coverslip kit. Note coverslip attached to cardboard square. FIGURE 2 Dorsal thoracic area of dog with four skin window preparations. window I and II in place. Uindow III coverslip and cardboard square in place. window IV is exposed ready for creation of lesion. 2| 22 FIGURE 3 Two neutrophilic leukocytes. Two hour stage. One neutrophil is swollen. Note cytoplasmic fragnents. May-Grfinwald-Glemsa; x I320. FIGURE 1+ Low power view. Four hour stage. Note the predominance _ of neutrophils. May-Grfinwald-Giemsa; x 330. 23 2h FIGURE 5 One small mononuclear cell. Four hour stage. May-Granwald-Giemsa; x l320. FIGURE 6 Two neutrophil ic leukocytes and five enlarging mono- nuclear cells with irregular outlines and finer chromatin pattern. Six hour stage. Wright's-Leishman; x l320. 25 26 FIGURE 7 Eosinophilic leukocyte and two degenerating neutrophilic leukocytes. Six hour stage. Note fibrin strands and free cytoplasm from eosinophil. May-Gru'nwald-Giemsa; x I320. FIGURE 8 Two eosinophilic leukocytes with one containing fused eosinophilic granules and two degenerating neutrophils. Six hour stage. May-Grfinwald-Giemsa; x I320. 27 28 FIGURE 9 Low power view. Eight hour stage. Note approximate equal numbers of neutrophils and mononuclear cells. Wright's-Leishman; x 330. FIGURE IO Five degenerating neutrophils and two mononuclear cells. Eight hour stage. Nright's-Leishman; x I320. 29 30 FIGURE ll Six mononuclear cells in varying stages of enlargement with irregular elongated outlines. Eight hour stage. Hright's-Leishman; x I320. ' FIGURE l2 Phagocytic mononuclear cell containing nuclear lobe of neutrophilic leukocyte. Ten hour stage. Uright's-Leishman; x I320. 3i 32 FIGURE I3 Low power view. Fourteen hour stage. Note predominance of mononuclear cells. Hright's-Lelshman; x 330. FIGURE IA Eight enlarged mononuclear cells. Sixteen hour stage. Note azurophil ic granules, fine chromatin pattern and int racytopl asm i c vacuoles . Wright's-Leishman; x I320. 33 34 FIGURE I5 / One neutrophilic leukocyte and five large mononuclear cells, four having phagocytized Pelikan ink. Twenty hour stage. Hright's-Leishman; x l320. 35 LITERATURE CITED Boggs, D.R. I960. The cellular composition of inflammatory exudate in human leukemias. Blood l5:h66-h75. Edwards, J.D. I959. Cytology of inflammatory exudate. Lab. Invest. 8:l09l. Eidinger, 0., M. Raff and 8. Rose. I962. Tissue eosinophilia in hypersensitivity reactions as revealed by the human skin window. Nature l96:683-684. Eidinger, 0., R. Wilkinson and 8. Rose. I96h. A study of cellular responses in immune reactions utilizing the skin window tech- nique. J. Allergy. 35:77-85. Eitzman, D.V. and R.T. Smith. I957. The non-specific inflammatory response in the newborn period. J. Dis. Child. 9hzh8h-h85. Florey, H.H. l96l. Human leucocytic functions in the tissues. in: Biological Activity of the Leucocyte. CIBA Foundation Study Group No. l0 edited by G.E.H. Holstenholme and Maeve O'Connor, pp. 27-28. Little, Brown and Company, Boston. Hu, F., R.P. Fosnaugh, H.G. Bryan and D. Jacks. l96l. A cyto- logic method of the study of allergic inflaumation. J. Invest. Dermat. 37:1IO9-lll9. Hedawar, J. l96l. Human leucocytic functions in the tissues. in: Biological Activity of the Leucocyte. CIBA Foundation Study Group No. l0 edited by G.E.H. Hblstenholme and Maeve O'Connor, p. 27. 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