THE CYTOLOGY OF THE EQUINE ENCEPHALOMYELITIS VACCINE RESPONSE IN EQUINE SKIN WINDOWS BY . P‘ 9' I ‘ Lawrence W.""‘Zarri11i A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Anatomy 1969 ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude and appreciation to Dr. M. Lois Calhoun, Professor of Anatomy and former Chairman of the Department of Anatomy, for her valued guidance and consideration during the cOurse of this study. Gratitude is also expressed to Drs. Esther M. Smith and Charles W. Titkemeyer for their suggestions and constructive-criticism of this manuscript. Special thanks and appreciation is extended to Dr. David T. Drees for his assistance in evaluation and interpretation of the skin window preparations. Thanks are also extended to Mr. Duane E. Haines and Dr. A1 W. Stinson for suggestions and assistance in the preparation of the photomicrographs. Appreciation is expressed to Dr. ‘ Fayne H. Oberst, Director of Veterinary Clinics, for his acquisition of the animals and facili- ties used in this study. Thanks are also due Miss Irene Brett, medical technologist of the Veterinary Clinics, for technical assistance. Finally, I wish to express sincere thanks to my fellow colleagues and friends whose interest has been a continuous source oi encouragement throughout this entire investigation. ii TAB LE OF CONTENTS Page INTRODUCTION --------------------- 1 REVIEW OF THE LITERATURE ------------- 2 MATERIAL AND METHODS ----------- t- - - - - 9 RESULTS'AND DISCUSSION ------------- - - 13 Two-Hour Stage ------------ - - - - l4 Four-Hour Stage ------------ - — - - 15 Six-Hour Stage --------- . ------ - - 15 Eight-Hour Stage ----------- .- - - - - 16 Ten-Hour Stage -------------- - - l7 Twelve-Hour Stage ------------- - - l7 Fourteen-Hour Stage ------------ - - l8 Sixteen-Hour Stage ------- ~ ------ - - 19 SUMMARY AND CONCLUSIONS ------------ - - 21 LITERATURE CITED ------------------- 23 ADDENDUM BIBLIOGRAPHY ------ - - - ------ 26 FIGURES ................. '. - 2 - - - - 27 iii Figure l. 10. ll. 12. l3. 14. 15. LIST OF FIGURES Two-hour stage. Neutr0philic leukocytes — - - - - - - Two-hour stage. Five neutrophils and one small mononuclear cell- - - ----- - - — .. .. - - - .. - Four-hour stage. Neutr0philic leukocytes - Four-hour stage. Swollen neutr0phil - - - - - - - - - Four-hour stage. Cyt0plasmic fragment and degenerating neutr0phil - - - - - - =- - - - - - - - - Six-hour stage. Neutr0philic leukocytes - - - - - - - Six-hour stage. Neutr0phil containing phagocytized Pelikan ink- — - - — - ------------- .. .. Six-hour stage. Two small mononuclear cells - - - - - Six-hour stage. Eosin0phil - - - - - ..... - - - Eight-hour stage. Neutrophils and mononuclear cells Eight-hour stage. Two degenerating neutr0phils - - - - Eight-hour stage. Mononuclear cell with indented nucleus-------------- ..... --- Ten-hour stage. Neutrophils containing phagocytized Pelikanink --------—- .......... Ten-hour stage. Mononuclear cell with large vacuole - Ten—hour stage. Mononuclear cell containing Pelikanink ------..---..----'--..-- iv Page 28 28 30 30 32 32 34 34 36 36 38 38 4O 4O 42 Figure 16. l7. l8. 19. 20. 21. 22. 23. 24. 25. 26. 27-28. Page Ten-hour stage. One eosinophil and two mononuclear cells surrounded by neutrophils .. .. _ - _ - - .. .. - - 42 Twelve-hour stage. Neutr0philic leukocytes and mononuclearcells------------------44 44 Twelve-hour stage. Three degenerating neutrophils Twelve—hour stage. Neutr0phil with thin nuclear filaments ----------------—--- 46 Twelve-hour stage. Two mononuclear cells fixed in ameboid motion - - - - - - - - - - - - - - - - - 46 Fourteen-hour stage. Mononuclear cells - - - - -: - 48 Fourteen-hour stage. Structurally intact neutrOphil - - 48 Sixteen-hour stage. Mononuclear cells - - - - - - - 50 Sixteen-hour stage. Mononuclear cell containing cytOplasmic vacuoles and azur0philic granules and oneneutrOphil------------------ 50 Sixteen-hour stage. Mononuclear cell containing Pelikanink---——-_--_---....-_--- 52 Peroxidase reaction. Mononuclear cell with peroxidasegranules-------..-..--..--- 52 Sudan black B reaction. Neutrophils and mononuclear cells containing sudanophilic granules .. - - .. .. .. .. .. 54 INTRODUCTION In 1955 Rebuck and Crowley described a technique which allowed the 2m serial study of the cytologic changes occurring in a single lesion over a specific time interval. This simple technique is known as the "skin window technique". The skin window technique has enhanced the study of inflamma- tion and has demonstrated itself as a possible diagnostic aid in disease, for there can occur a variation of cellular exudate in normal indivi- duals, diseased individuals, and when using different antigenic stimu- lants in experimental skin lesions. This technique has primarily been applied to human experimentation with little work done on the domestic animals. An advantage of this technique is that it requires only slight, superficial skin abrasions to the experimental animals. Since negli- gible harm to the animal occurs by this technique, studies of the cellular response in normal and diseased animals can be investigated in Species previously considered uneconomical and impractical for experimentation. A careful review of the literature revealed no reports available on skin window investigations in the equine species. The purpose of this study is to observe and record the cellular exudate on skin windows in the equine using inactivated encephalomyelitis virus as the antigenic stimulus. REVIEW OF THE LITERATURE In 1955 Rebuck and Crowley described a method of studying leukocytic functions 1_n__\:1_\_/'2 which allowed the serial study of changes in the cellular exudate in a Specific lesion for an extended period of time. This technique is commonly called the skin window technique. Rebuck and Crowley (1955), using the skin window technique, studied the transformation of lymphocytes into macrOphages in man. Functional and structural changes in the lymphocytes of the cellular exudate were observed. The transformation changes included in- creases in the amount of cytoplasm, in the phagocytic ability for cellular debris and vital dyes, in the division of coarse chromatin masses into fine angular pieces and in the irregularity of the nuclear membrane. Rebuck and Crowley concluded that macrophages of cellular exudate were derived from lymphocytes present in the exudate. Conversely, Volkman and Gowans (1965a), in skin window studies in cellular exudate in rats, suggested a monocytogenous origin of exudate macrophages. They observed exudate macrophages labeled with tritiated thymidine one day after an intravenous injection of tritiated thymidine, suggesting that macrOphages originate from rapidly dividing precursors. Small lymphocytes could not be 2 macrophage antecedents, since the proportion of labeled small- lymphocytes in the blood never rose above 2%. A high labeling of monocytes was observed. In further experiments, Volkman and Gowans (1965b) applied the skin window technique to rats to identify the tissues in which the precursors of macrophages proliferate. Lyrnphocyte-depletion by either chronic drainage from the thoracic duct or x-irradiation with 400 rads failed to suppress the emigration of macrophages or to reduce the prOportion of them which became labeled after an in- jection of tritiated thymidine. The emigration and the labeling of the exudate macrophages were suppressed following x-irradiation with 750 rads, but were restored to normal when the tibial marrow was shielded during irradiation. In recipients transfused with radioactive- label‘ed cell suspensions obtained from thoracic duct lymph, lymph nodes, thymus, spleen and bone marrow, only recipients of labeled bone marrow and spleen cells demonstrated emigration of labeled macrOphages onto coverslips. Labeled monocytes were also found in the blood of rats-which had received injections of labeled bone marrow. Trepel and Begemann (1966) studied the origin of skin window‘ macrOphages in rats by labeling the leukocytes with H3-thymidine or India ink. By Comparing the characteristic labeling pattern of the peripheral leukocytes with the labeling of the cells of the inflammatory exudate on the skin windows, a transformation of monocytes of the 4 blood into macrOphages was observed. Histochemical studies by Wulff and Sparrevohn (1966) on the origin of mononuclear cells in human skin windows revealed a strong resemblance between skin window mono- nuclear cells and blood monocytes with regard to sudanophilia, per- oxidase activity and esterase activity. A monocytogenous origin of emigrating mononuclear cells was suggested. By means of inflammatory lesions in skin windows in man, Rebuck SEQ. (1960) demonstrated the phagocytic prOperties of lymphocytes, macrOphages and monocytes in storing vital dyes (pyrrol blue, lithium carrnine, trypan blue and trypan red). Rebuck ital. (1951) and Rebuck and Mellinger (1953) using adrenalcorticotrophic hormone and cortisone, respectively, under skin windows, observed a modification of the cellular exudate. A depletion of the leukocytic and phagocytic response was reported. A marked suppression of cellular migration at the inflamma- tory site in patients with a neutrOpenia was observed in skin windows by Page and Good (1958). They concluded that the inflammatory exudate is dependent upon, and in part, a function of, the circulating neutrOphils. Rebuck and LoGrippo (1960), in a study of the leukocytic response to poliomyelitis vaccines in human skin windows, demon- strated a poor lymphocyte response in the cellular exudate of non- vaccinated individuals, and a high lymphocyte response in individuals vaccinated with Salk polio vaccine. Perillie eta}. (1960) suggested using the skin window tech- nique as a simple diagnostic aid in systemic lupus erythematosus. Lupus erythematosus cell bodies and rosettes were found on cover- slip preparations in patients afflicted with systemic lupus erythema- tosus. In studies of the inflammatory exudate produced by diph- theria toxoid under the skin window, Hu 9311: (1961) noted an early migration'of phagocytic granulocytes which were later supplemented by migrating lymphocytes and blood mononuclears. Application of Rhus oleoresin under the skin windows resulted in no difference in inflammatory response in positive and negatiVe reactors to Rhus prior to the 33-hour stage. After this time, the Rhus-positive in- dividuals demonstrated an eosin0philia, a larger number of lympho- cytes, and large multinucleated giant cells. Wolf— Jurgensen (1962) demOnstrated an increased number of eosin0phils and especially baSOphils in the cellular exudate in experi- mental contact allergy, using 2-4 dinitrochlorobenzene as the sensitizing agent. Priest and Rebuck (1962) observed a high migration of polymorphonuclear baSOphilic leukocytes in patients with acute ulcerative colitis (3 weeks) and in patients with chronic disease (over 20 years). Hu _e_t__a;1_. (1963), utilizing the skin window technique in patients with furunculosis and "Open" skin lesions, reported a high incidence of baSOphilic and eosin0philic response. The response was elicited only when suspensions of the whole staphylococci, live or killed, were uSed as the inoculum under skin windows. When a purified polysaccharide antigen prepared from the same staphylococci strain was applied to the skin lesions, a basophilic and eosin0philic response did not occur. The basophilic and eosin0philic response possibly represents a hypersensitivity reaction to the staphylococci organisms. A skin window technique was employed by Wis seman and Tabor (1964) to study the early cellular reSponse of man to typhus rickettsiae. In the first 24 hours the reactions observed were essentially those of an acute inflammatory process. In non-immune subjects, polymorphonuclear leukocytes appeared in 1. 5 to 3 hours, increased rapidly in numbers and persisted as the dominant cell type during the 24 hour period. Mononuclear‘cells appeared in increasing numbers by 6 hours and underwent development into large mononuclear cells and macrophages. The inflammatory process was intensified in immune subjects. Harman (1965), using skin windows in normal control patients, also showed an initial, almost exclusively neutrophil migration which slowly gave way to an influx of mononuclear cells. In normal skin of psoriatics, the same qualitative pattern of migration occurred but at a faster rate. Mononuclear cells predominated 9 hours after trauma instead of 24 hours as in the normal control (patients. According to Drees (1966), Eidinger in 1960 observed that the cellular exudate in patients allergic to fish extract, grasses or ragweed was composed of over 50% eosin0phils. Eidinger ital. (1964), in a study of cellular reSponses utilizing the skin window 7 technique, demonstrated a tissue eosinophilia following application of an allergen in the skin window. The eosinophilia was greatest when an allergen was used that was clinically responsible for the patient's allergic symptoms. In a similar study by Weiss (1967) the number of eosin0phils in the cellular exudate increased in a degree prOportional to the individual's sensitivity to the allergen. The cytOplasm and nuclei of the eosin0phils contained many vacuoles, and numerous degranulated eosin0phils were present. Jurgensen and Zacharias (1965) observed an emigration of eosin0phils follow- ing injections of polymyxin and histamine. Riddle and Barnhart (1965) and Drees (1966), using the skin window technique, studied the cellular response in the dog and con- cluded that it was very similar to the cellular response in man. The stage of peak cellular migration occurred at 10 hours as Opposed to 12 hours in the human species. Lymphocytes, hypertr0phied lymphocytes and macr0phages appeared in increasing numbers from about 6 to 14 hours. Small numbers of eosinophils were observed between 3 and 7 hours and seldom comprised more than 5% of the cellular exudate. The dog was capable of mobilizing its greatest cellular defense mechanism to an irritant more rapidly than man. In the same study Riddle and Barnhart suggested that specific granules in the eosinophil may function as a source for profibrinolysin in acute inflammation. Eosinophils were attracted into the skin window area of inflammation by fibrinogen or fibrin. 8 Wilkinson eta. (1966), using the skin window technique, made a comparison of the cellular exudate between 10 rheumatoid arthritic patients and 6 normal patients. A simple irritant type of reSponse was observed in each case. No significant differences between arthritic and control subjects were observed. In a skin window study in patients with chronic uremia ex- hibiting delayed homograft rejection, Lang e_t_a_l_. (1966) observed a lower percent of lymphocytes and macrophages at 8 and 24 hours in uremic patients. The lymphocytes and macr0phages showed morphologic evidence of necrobiosis. Lang and coworkers feel that the reduced number may reflect an increased rate of destruction, and/or a reduced production; and, that this phenomena may be a factor in the delayed homograft rejection in uremic patients. MATERIA LS AND METHODS The skin window technique of Rebuck and Crowley (1955) and Drees (1966) was used in this investigation. Modifications were made by the author to adapt the technique to the horse. Five horses from the Michigan State University Veterinary Clinic were used in this study. The animals consisted of 1 Quarter Horse, 2 Standardbreds, and 2 Thoroughbreds, all geldings. Age ranged from 5 to 10 years, and weight from 800 to 1200 pounds. A total white blood cell count, differential count, and'hemoglobin and hema- tocrit determinations were made on each animal to insure use of hematologically normal subjects. The site of this test was an 8 by 12 inch area at the region of the transverse processes of the lumbar vertebra on the dorsum of each horse. Only one side of each horse was used. Hair was removed from thehorse's back with an electric animal clipper. In several cases it was necessary to use a rope twitch to prevent the horse from becoming too excited. The clipped area and surrounding area were cleaned with warm water and germicidal soap. Excess water was removed with sterile gauze. The experimental 3 by 5 mm lesions were created by scraping the epidermis with a sterile No. 22 Bard-Parker blade. The depth 9 10 of each lesion was considered adequate when the papillary layer of the corium was reached. Evidence of the correct depth was indicated when fine bleeding points were observed in the lesion, and when a small amount of blood accumulated on the cutting edge of the scalpel blade. Six experimental lesions were created on the back of each horse in two rows of three each. A total of 30 lesions were prepared. The trauma of the scalpel blade creating the lesion served as an inflammatory stimulant, so the experiment was timed from this point. One drOp of an antigen, Encephalomyelitis Vaccine-Eastern and Western Avian Tissue Culture Originl, from a sterile syringe was placed on the surface of the lesion to act as an' additional inflam- matory stimulant and enhance leukocytic participation. This antigen was applied to 21 lesions. No antigen was applied to 9 of the lesions, so that a comparison could be made between the antigenic stimulated and the nonantigenic stimulated cellular exudates. The phagocytic pr0perties of the cellular exudate were studied by applying one drOp of Pelikanz ink from a sterile syringe to the surface of 6 of the experimental lesions, previously inoculated seconds earlier with the inflammatory stimulant. 1Fort Dodge Laboratories, Inc. , Fort Dodge, Iowa. 2John Henschel and Co. , Inc. , 425 Park Avenue, South New York 16, New York. 11 After each lesion was prepared, it was immediately covered with a sterile No. 2 - 15 mm square glass coverslip. The coverslips had previously been attached to a 20 mm cardboard square by means of a 2 by 8 mm strip of masking tape folded upon itself. Half of the masking tape was attached to the coverslip and half to the cardboard square. The cardboard facilitated handling of the coverslip, and reduced chance of contamination of the coverslip and the lesion (Drees, I966). The coverslip-cardboard unit was wrapped in alumi- num foil and placed in a metal container. Four hundred units were prepared in this manner and sterilized with dry heat. Each coverslip- cardboard unit could then be unwrapped from the aluminum foil and used as needed, continually maintaining sterile technique. After application of the coverslip-cardboard unit to the lesion, it was covered by a l by 3 inch strip of adhesive tape to maintain correct position and contact with‘the lesion. To reduce chance of contamination of the lesions, the tail of each horse was wrapped with gauze, and the horse was tied with a rope halter to the front of the stall to maintain a standing position. The cells of the inflammatory exudate in the lesion migrate to the undersurface of the coverslips. Every 2 hours for 16 hours the coverslips were removed from the lesions, air dried, and mounted, cell-side—up, on microsc0pe slides with a drOp of Permountl. Upon removal of the coverslip from the lesion, it was immediately replaced 1FisherScientific Co., Fair Lawn, New Jersey. 12 with another sterile coverslip. A serial study could then be made of the cytologic content of each lesion at short intervals throughout the first 16 hours of acute inflammation. Exposure of the lesion to the air for any length of time will allow the cellular exudate to dry, and will prevent continuation of that lesion. An individual serial study consisted of 8 slides covering 16 hours. Thirty such serial studies were performed. The cells on the coverslips mounted on microsc0pe slides were stained like blood sfnears. Ten series were stained using the May-Grunwald- Giemsa method (Pappenheim, 1912); 10 series were stained using Wright's-Leishman stain (Coles, 1967), modified by increasing the fixation time to 3 minutes and the staining time to 8 minutes; 6 series were stained for peroxidase according to Sato and Sekiya (Darmady and Davenport, 1958); and, 4 series were stained using Sudan black B according to Bayliff and Kimbrough (Darmady and Davenport, 1958). The phot'omicrographs (Figures 1-28) illustrate the cells which have migrated to the undersurfaces of the coverslips. RESU LT S AND DISC USSION Since encephalomyelitis vaccine antigen was applied to some skin lesions and not to others, a comparison could be made between the antigenic stimulated and the nonantigenic stimulated cellular exu- dates. This comparison revealed an essentially identical qualitative cellular response between those lesions containing the vaccine antigen and those lesions containing no vaccine antigen. However, a more in- tense quantitative response was observed in the vaccine stimulated lesion. Due to the similarity of the response between the two types of lesions, and since the majority of the skin lesions contained the vaccine antigen, the following description will be concerned with the cytology of the encephalomyelitis vaccine reaction in the equine skin windows. Since there still exists a controversy in the literature con- cerning a lymphocytogenous or monocytogenous origin of the mono- nuclear cells in skin window preparations (Rebuck and Crowley, 1955, Volkman and Gowans, 1965b, Drees, 1966, Trepel and Begemann, 1966, Wulff and Sparrevohn, 1966), all such cells in this discussion will be referred to as mononuclear cells. Most of their work would seem to indicate a monocytogenous origin of mononuclear cells. Wulff and Sparrevohn (1966) indicated that lymphocytes were devoid of peroxidase and sudanoPhila material, whereas, most blood 13 l4 monocytes contain these substances. In view of the work of Wulff And Sparrevohn (1966), several serial studies were performed on the horse utilizing a peroxidase stain and a Sudan black B stain. The peroxidase stain revealed peroxidase activity and the Sudan black B stained numerous sudanophilic granules in all mononuclear cells from the Z—hour stage to the 16-hour stage of the experiment (Figures 26-28). Since Wulff and Sparrevohn (1966) demonstrated that blood monocytes and not blood lymphocytes showed these staining characteristics, this histochemical study would seem to indicate that the mononuclear cells in skin windows are emigrated monocytes. The first coverslip from each series ‘of preparations was somewhat difficult to interpret. As a result of the initial trauma- tization of the skin and application of additives, serum, dye and antigen accumulated on the coverslip. Subsequent coverslips improved in quality and detail. Two- Hour Stage Neutr0philic leukocytes were the predominant cell type in the exudate, accounting for approximately 95% of the total cells (Figure l). The nuclear lobes appeared swollen and rounded, giving a generalized edematous appearance to the entire neutrOphil. The neutrophils measured 12-14 microns in diameter. The cytOplasm was usually colorless, but occasionally stained a pale blue. Several mononuclear cells were observed at this 2-hour 15 stage, but were an infrequent finding. Cytoplasm was sparse in these mononuclear cells, with the dense chromatin nucleus almost filling the entire cell (Figure 2). These cells measured 10-11 mic rons in diameter. Four-Hour Stage At four hours, the primary cell present was still the neutro- philic leukocyte (Figure 3). The neutrophils measured 12-14 microns, and closely resembled the 2-hour neutrOphil. The nuclear lobes still appeared swollen, often causing the cell membrane to be stretched (Figure 4). The mononuclear cells made up only a very small percentage of the cellular exudate and resembled the 2-hour mononuclear cell. Fragments of leukocytic cytoplasm were frequently observed dispersed throughout the cellular exudate, a probable result of degenerating and injured cells (Figure 5). These fragments varied in size from as small as 2 microns in diameter to as large as 10 mic rons in diameter. Six- Hour Stage At 6 hours neutrOphils still dominated the cellular exudate (Figure 6), but many of the neutrophils had lost their cytoplasm, leaving only their nuclei floating in the exudate. The first indication of phagocytic activity was observed in this 6-hour stage as small accumulations of Pelikan ink were occasionally present in the cyto- plasm of intact neutrophils (Figure 7). l6 Mononuclear cells (12-14 microns) had increased in number in the total cellular exudate. The nucleus was frequently irregular, round or oval in shape. The chromatin was deeply stained and coarse. Cytoplasm was very abundant and stained a pale blue with few vacuoles being present. The cellular outline was elongated and oval, often irregular in shape (Figure 8). An occasional eosin0philic leukocyte was observed in areas associated with fibrin. This agrees with the findings of Riddle and Barnhart ("1965) and Drees (1966). The cytOplasmic granules were few in number, and smaller than the peripheral blood eosinophil (Figure 9). Eight-Hour Stage At the 8-hour stage the percentage of neutrophils in the cellular exudate had decreased (Figure 10). The majority of these neutro- phils had also decreased in size, now measuring 11-12 microns in diameter. Many of the nuclear lobes were clumped and pyknotic. The chromatin masses stained darker than normal. Cytoplasm was Sparse (Figure 11). The mononuclear cells in this stage were larger measuring 14-20 microns. The nucleus was usually indented and contained large clumps of chromatin (Figure 12). Cytoplasm was distinct and stained baSOphilic. In a few cells vacuoles were present in the cytoplasm. A few consolidated eosin0phil granules were observed in areas 17 associated with fibrin. These granules measured 1-3 microns in diameter, and appeared as free entities in the cellular exudate. T en- Hour Stage The morphology of the neutrophils of the 10-hour stage was similar to the neutrOphils of the 8-hour stage, except that the per- centage of neutrOphils in the. cellular exudate had decreased. The small size of the degenerating neutr0phils persisted, measuring 7-9 microns in their greatest diameter. The size of the intact neutrophils was somewhat larger, averaging 12 microns. A few neutrophils contained cytoplasmic vacuoles. Phagocytic activity of the neutrOphil continued in this stage as evidenced by the presence of Pelikan ink within the neutrophilic cytoplasm (Figure 13). An increasing number of the exudative cells were mononuclear cells (16 by 22 microns), and were somewhat larger than those of the earlier stages. The number of mononuclear cells possessing cytoplasmic vacuoles appeared to increase in this stage (Figure 14). The cyt0p1asm of these mononuclear cells also contained phagocytized Pelikan ink (Figure 15). Several eosin0phils with intact cellular membranes and small granules were observed in this stage. These cells average 12 microns. Twelve-Hour Stage At 12 hours, the majority of the neutrophilic leukocytes (Figure 17) were shrunken with clumped chromatin and pyknotic nuclei. Cytoplasm was very sparse, and frequently nonexistent (Figure 18). 18 These degenerating neutrOphils averaged 7 microns in diameter. The remaining neutrophils were large (12 microns) and edematous. The nucleus was often quite well deliniated. Filaments connecting nuclear lobes could occasionally be observed (Figure 19). The cytoplasm of these cells was abundant, and sometimes vacuolated. The mononuclear cells were readily apparent at this 12-hour stage (Figure 17). The majority of these mononuclear cells were fixed in ameboid motion, as characterized by their bizarre shapes (Figure 20). Consequently, the length of these cells was much greater than the width, the average dimensions being 12 by 26 microns. The cellular outline was very irregular. The nucleus was large, accounting for half to two-thirds of the total cell. The nuclear outline was irregular, but smooth. Cytoplasm was abundant, and usually well vacuolated. The eosin0philic leukocyte reSponse diminished to a point where only a few granules could be located in the cellular exudate. The peak cellular response for the total 16-hour experiment occurred in both the lesions of the 12-hour stage and the l4-hour stage. This timed response in the equine species is similar to the reports of Rebuck and LoGrippo (1960) for the human species (12-hour stage), but 2 to 4 hours later than Drees (1966) reported for the dog (10-hour stage). Fourteen-Hour Stage At 14 hours, the percentage of neutrophilic leukocytes in the l9 cellular exudate had decreased (Figure 21). The majority of these neutrOphils were shrunken with clumped and pyknotic nuclei. Most of the cytoplasm was missing. These cells measured 7-9 microns in diameter. Structurally intact neutrOphilic leukocytes, 12-14 microns in diameter (Figure 22), were also present in moderate numbers. The nuclear chromatin was heavily plaqued which caused the nuclear membrane to assume a jagged appearance, giving a multilobulation impression. Filaments were occasionally observed. CytOplasm was quite abundant in these cells and contained evenly dispersed, fine granules. Occasionally, small, clear intracyto— plasmic vacuoles were observed. The mononuclear cells had increased .in number, and they were now a predominant cell type in the exudate. Cell size measured 14 by 26 microns. Cell characteristics closely resembled the 12- hour mononuclear cells. Sixteen- Hour Stage The cell morphology and characteristics of the neutrOphilic leukocytes in the 16-hour stage (Figure 23) were consistent with the degenerating and structurally intact neutr0phils of the l4-hour neutrophil. A slight degree of phagocytosis of Pelikan ink occurred in this stage, but its importance appeared negligible. The mononuclear cells in the skin windows had increased in number (Figure 23). Cell size averaged 22 by 27 microns. As in the preceding several stages, the cellular outline assumed an irregular 20 shape. The shape of the nucleus was also very irregular. Generally, one side of the nucleus was indented to various degrees. Any simi- larity or conformity between the nuclei of different mononuclear cells was a rare occurrence. The baSOphilic cytoplasm contained numerous vacuoles and azurOphilic granules (Figure 24). A marked degree of phagocytosis of cellular debris and Pelikan ink had occurred (Figure 25). This experiment was concluded at the 16-hour stage. SUMMARY AND C ONC LUSIONS The cytology and emigration sequence of inflammation pro- duced by a combined Eastern and Western Encephalomyelitis Vaccine was studied in the equine species using a skin window procedure over a period of 16 hours. A vital dye was used to study phagocytosis; and, a histochemical study was employed to examine the origin of emigrating mononuclear cells. Neutr0philic leukocytes were the first cells to migrate into . the skin lesions and onto the coverslips. These cells were the primary constituent of the cellular exudate for the entire 16 hours after inoculation of the vaccine stimulant. The neutrophils were able to phagocytize the vital dye. After functioning in phagocytosis, the neutrophils underwent degeneration with nuclear clumping and a loss of cyt0plasmic fragments to the fluid exudate with subsequent decrease in cell size. Emigration of new neutroPhils persisted, in decreasing amounts, throughout the 16 hours of the experiment. A slow influx of mononuclear cells occurred early in the experiment. After 6 hours, the number of mononuclear cells in- creased in rate of emigration becoming a predominant cell type in the exudate at 12 to 14 hours. It appeared that the mononuclear cells were still increasing in number, size and phagocytic activity at the 21 22 termination of the experiment at the 16-hour stage. The results of the histochemical study revealed peroxidase activity and sudanOphilia in the mononuclear cells. According to Wulff and Sparrevohn (1966), only blood monocytes show these staining characteristics. Therefore, a monocytogenous origin of emigrating mononuclear cells is probable. The cellular exudate contained only about 1% eosin0philic leukocytes. Very few of these cells were observed with intact cellular membranes. Most of the eosin0philic granules were free in the fluid exudate. The control lesions with no vaccine stimulation revealed a similar qualitative cellular response, but a diminished quantitative cellular response at each stage in the experiment. The peak cellular response in the equine species occurred at 12 to 14 hours when the greatest number of cells appeared in the exudate. This response coincides closely with that reported for man (Rebuck and LoGrippo, 1960), but is 2 to 4 hours later than the peak cellular response in the dog (Drees, 1966). LITERAT URE CITED LITERATURE CITED (totes, EC. W. i’loT. Clinical Pathology. W. B. Saunders C0,, Phi ldticlphia. Darr'narly, E. .\l. and S. G. T. Davenport. 1958. Haematological Ivehnique, 2nd Edition. Grune and Stratton. New York. Drees, D. T. l9t36. The Cytologic ReSponscs of Normal Beagle Dogs Utilizing the Skin Window Technique. M. S. Thesis. Klirhlgan State University, East Lansing. Eidinger, D. , R. Wilkinson and B. Rose. l96—l. A study of cellular responses in immune reactions utilizing the skin window technique. J. Allergy. 35:77—85. Harman, R. M. 19m"). Skin window study of inflammation in psoriasis and lichen planus. Brit. J. Dermatol. 77: "350—831. Hu, 1%}, R. P. Fosnaugh, H. G. Bryan and D. .lacks. 1961. A cytologic method of the study of allergic inflammation. .1. Invest. Dermat. 37:409-419. flu, 17., R. I). I-‘osnaugh and C. S. Livingmud. 1963. Human skin window studies. 11. CompariSon of cellular response to staphylococcus in controls and in patients with cutaneous bacterial infections. .1. Invest. Dermatol. 41:325—334. .lurgensen, P. W, and H. Zachariae. 1965. Influence of polymyxin B and histamine. on the cellular exudate m skin windows. Acta Dermato—Venereol. 45:207-211.. Lang, P. A., 8.113. Ritzmann, F. L. Merian, M. C. Lawrence, W. C. Leven and R. Gregory. l9tm. Cellular evolution in urenlic patients. Tex. Rep. iiol. Med. 24:107-lll. Page, A. R. and R. A. Good. 1958. A clinical and experimental study of the function of neutrophils in the inflammatory FGSPOHSP. Am. .1. Path. 34:!»45-oh‘l, 23 Z4 Pappenheim, A. 1912. Hematological techniques. Fol. [Haematol. 13:337—344. Perillie, P. 13., P. Calabresi and S. C. Finch. 1960. Demon- stration of the L. E. cells at local inflammatory sites in patients with systemic lupus erythematosus. New England J. Med. 263:1052-1055. Priest, R. J. and J. W. Rebuck. 1962. The role of basophilic granulocytes in ulcerative colitis. Med. et Hyg. 20:365-366. Rebuck, J. W., C. B. Boyd and J. M. Riddle. 1960. Skin windows and the action of the reticuloendothelial syStem in man. Ann. New York Acad. Sci. 88:30-42. Rebuck, J. W. and J. H. Crowley. 1955. A method of studying leukocytic functions _i_r£ vivo. Ann. New York Acad. Sci. 59:757-805. Rebuck, J. W. and G. A. LoGrippo. 1960. Leukocyte response to poliomyelitis vaccines in human skin windows. p. 23. E: Reticuloendothelial Structure and Function. Edited by J. H. Heller, Ronald Press, New York. Rebuck, J. W. and R. C. Mellinger. 1953. Interruption by t0pica1 cortisone of leukocytic cycles in acute inflammation in man. Ann. New York Acad. Sci. 56:715-732. Rebuck, J. W., R. W. Smith and R. R. Margolis. 1951. The modification of leukocytic function in human windows by ACTH. Gastroenterology 19:644-657. Riddle, J. M. and M. I. Barnhart. 1965. The eosinophil as a source for profibrinolysin in acute inflammation. Blood 25:776-794. Trepel, F. and H. Begemann. 1966. On the origin of the skin window macrophages. Acta Haematol. 36:386-398. Volkman, A. and J. L. Gowans. 1965a. The production of macrophages in the rat. Brit. J. Exper'. Path. 46:50-61. 1965b. The origin of macrOphages from bone marrow in the rat. Brit. J. Exper. Path. 46:62-70. Weiss, A. 1967. Eosinophilia as an indicator of allergic reactions. Acta Med. Pol. 8:121-128. 25 Wilkinson, R. 1)., S. Kim and A. Sargent. 1966. Skin window studies in rheumatoid arthritis. pp. 143—146. 13: Pro- ceedings of the Third Canadian Conference on Research in Rheumatic Disease, University of Toronto Press, Canada. Wisseman, C. L. and H. Tabor. 1964. Early cellular response of man to typhus rickettsiae as revealed by the skin window technic with observations of in vivo phagocytosis. J. immunol. ' 93:816-825. —__ Wolf-.lurgensen, P. 1962. Cytological examination of experimental contact allergy using the skin window technique. Acta Wulff, H. R. and S. Sparrevohn. 1966. The origin of mononuclear cells in human skin windows. Acta Path. et Microbiol. Scandinav. 68:401-407. ADDENDUM BIBLIOGRAPHY The following references making up this addendum bibliography were not directly applicable to the present study. They are included, however, because of their peripheral interest to future researchers using the skin window technique. Edwards, J. L. 1959. Cytology of inflammatory exudate. Lab. Invest. 821091-1092. Hayloe, F. G. J. 1953. The cytochemical demonstration of lipid in blood and bone marrow cells. J. Path. Bact. 65:413-421. Rebuck, J. W., H. I. Coffman, G. B. Bluhm and C. L. Barth. 1964. A structural study of reticulum cell and monocyte production with quantitation of lymphocytic modulation of nonmultiplicative type to histiocytes. Ann. New York Acad. Sci. 113:595-611. Rebuck, J. W. and G. A. LoGrippo. 1961. Characteristics and interrelationship of the various cells in the reticuloendo- thelial cell, macr0phage, lymphocyte, and plasma cell series in man. Lab. Invest. 10:1068-1093. Riis, P. and H. R. Wulff. 1960. Dynamic studies of the relation between intravascular and inflammatory neutrophils in normal subjects. Acta Haematol. 23:276—281. Schalm, O. W. 1965. Veterinary Hematology. Lea and Febiger, Philadelphia. 26 FIGURES 27 FIGURE 1 Two-hour stage. Note predominance of neutrophilic leukocytes. X 1126. FIGURE 2 Two-hour stage. Five neutr0phils and one small mononuclear cell. X 2816. 29 FIGURE 3 Four-hour stage. Cell population primarily neutrOphils. X1126. ‘ FIGURE 4 Four-hour stage. A swollen neutrOphil. X 2816. 31 FIGURE 5 Four-hour stage. Note cytOplasmic fragment adjacent to degenerating neutrOphil. This cell was identified as a neutrOphil because of the small amount of cytOplasm and the nuclear lobulation which was obvious on the coverslip, but difficult to photograph because of limited depth of focus. X 1760. FIGURE 6 Six-hour stage. Predorninance of neutrophils. X 704. 33 FIGURE 7 Six-hour stage. Three neutrOphils surrounding one neutrOphil containing phagocytized Pelikan ink in cytOplasm. X 2816. FIGURE 8 Six-hour stage. Small mononuclear cells. X 2816.' 34 FIGURE 7 FIGURE 8 35 FIGURE 9 Six-hour stage. Eosinophil with small granules. Note fusion of several granules into larger granules. X 2816. FIGURE 10 Eight-hour stage. Cell pOpulation showing two-thirds neutrOphils and one-third mononuclear cells. X 704. FIGURE 9 FIGURE 10 Eight-hour stage. 37 FIGURE 11 Two degenerating neutrOphils. of cytoplasm. X 2816. Eight-hour stage. FIGURE 12 Note sparsity Mononuclear cell. Observe indented nucleus and chromatin clumps. X 2816. 38 FIGURE 11 FIGURE 12 39 FIGURE 13 Ten-hour stage. Large neutrOphils, several containing phagocytized Pelikan ink in cytOplasm. X 2816. FIGURE 14 Ten-hour stage. Mononuclear cell with large vacuole. X 2816. 41 FIGURE 15 Ten-hour stage. Mononuclear cell containing phagocytized Pelikan ink in cytoplasm. X 2816. FIGURE 16 Ten-hour stage. One eosin0phil and two mononuclear cells surrounded by neutrOphils. X 1760. 42 FIGURE 15 FIGURE 16 43 FIGURE 17 Twelve-hour stage. Note approximate equal numbers of neutrOphilic leukocytes and mononuclear cells. X 640. FIGURE 18 Twelve-hour stage. Three degenerating neutrophils. X 2816. 44 FIGURE 18 45 FIGURE 19 Twelve-hour stage. Neutr0phil containing thin nuclear filament. X 2816. FIGURE 20 Twelve-hour stage. Two mononuclear cells fixed in ameboid motion. X 2816. 46 ' A. FIGURE 19 It“ FIGURE 20 47 FIGURE 21 Fourteen-hour stage. Mononuclear cells predominating cellular exudate. X 704. FIGURE 22 Fourteen-hour stage. Structurally intact neutrophil. X 2816. 48 FIGURE 21 FIGURE 22 49 FIGURE 23 Sixteen-hour stage. Note predominance of mononuclear cells. X1126. FIGURE 24 Sixteen-hour stage. One mononuclear cell containing numerous cytOplasmic vacuoles and azurOphilic granules, and one neutrOphil. X 2816. 50 FIGURE 24 51 FIGURE 25 Sixteen-hour stage. Ingested Pelikan ink within the cytOplasm of a mononuclear cell. X 2816. FIGURE 26 Peroxidase reaction. Mononuclear cell with positive peroxidase granules in cytoplasm. X 2816. 52 FIGURE 2 5 FIGURE 26 Sudan black B reaction. 53 FIGURES 27-28 Polymorphonuclear cells and mononuclear cells containing numerous sudanophilic granules. X 2816. M .-.— '54 F IGURE 2 7 FIGURE 28 ”'ilfllllilliilililititilllll“