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Histological changes induced in the harderian gland of the mouse by transplantation in the anterior chamber of the guinea pig eye

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Histological changes induced in the harderian gland of the mouse by transplantation in the anterior chamber of the guinea pig eye

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Content mSTOLOaiCAL CHANGES INDUCED IN THE HAHDERIAN GDAND OF THE MOUSE BY TRANSPLANTATION IN THE ANTERIOR CHAMBER OF THE GUINEA PIG EYE A THeals ^raaantod to the -iî'ftoulty of the department of ^oology The University of Southern California An Partial ^fulfillment of the Hequirementa for the degree of Master of &rts by John Rheinhardt stevena August. 1940 UMI Number: EP67176 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertatton Publishing UMI EP67176 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 This thesis, w ritten by .....JO#.™ ........... under the guidance of A.-t8- Facu lty Com m ittee, and approved by a ll its members, has been presented to and accepted by the Council on G raduate Study and Research in p a rtia l fu lfill ment of the requirements fo r the degree of .............. E. s . B o ^ D n s . .............. Dean n.,. 9 1948______ Faculty Committee ^ Chairman The writer la indebted to Ry. Rruoe m* Harrison, -t'rofesaor of ^oology at the university of south ern ualifornia. for nia painstaking guidance in the preparation of this paper. To Dr. Hruoe m. Harrison, ohalrman of the thesis oommlttee, dr. Franoia m, Baldwin, Professor of ^oology and Miss Tema ciare, Assistant Professor of bot any, the writer wisnes to express his gratitude for their valuable aid in the reading of the finished paper. To Mr.iiouis Plores, Medioal photo grapher ox the ^0 8 Angeles county General Hospital, Mr. -uioyd Matlovski, Technical photographer of the do8 Angelas County general Hospital and A, Jan sen, the writer wishes to thank for their patience and time in taking photographs of the animals and microscopic slides. ‘ £à3u£ UJj' 0HAPTE8 PASS 1 lH'£i40ji)U0'fIÜM.............. 1, il aavim uï I iItehatuhb............... s. III MÀTmiAia abb mbxbdbs ........... ii, IV UBSKHVAÏIOHS AMD i’IHDlHGS........... 23. S'IAIMIMU KBAÜ'iiüMS...... 23. COHïaui aiiûiiS ............... 24. OBSBEVATlOÏÏS VF JSMBKYOMIÜ HAHDiSHIAH TISSUE ............... 87. OBSERVATiOMS OF TRAMSPLAMTBD HARDERIAH TISSUE......... 39. V DIS003SIOH.............................. 50. VI SUMMARY................................. 04. BIBilOGHAPHY ......... 57. LIST OF FIGURES PAGE figure 1. A photomicrograph showing a section of the Harderian gland from a new born mouse, Magnification 376%. 26. figure 2. A photomicrograph showing a section of the Harderian gland from a one week old mouse. Magnification 45QA. 29. Figure 3. A photomicrograph showing a section of the Harderian gland from a two week old mouse. Magnification 188A, 31. Figure 4. A photomicrograph showing a section of the Harderian gland from a five week old mouse. Magnification 376%. 33. Figure 5. A photomicrograph showing a section of the Harderian gland from a six week old mouse. Magnification 376%. 35. Figure 6. A photomicrograph showing a section of the Harderian gland from an eight week old mouse. Magnification 376%. 36. Figure 7. A photograph showing a Harderian trans plant from a newborn mouse which has grown in the anterior chamber for seven days. Magnification !&%. 40. Figure 6. A photograph showing a Harderian gland transplant from a newoorn mouse which has grown in the anterior chamber for fourteen days. Magnification l4%. 41. II LIST OF FIGURES - continued PAGE Figure 9. A photograph showing a Harderian gland transplant from a newborn mouse after twenty-one days in the anterior chamber. Magnification l4%. 42# Figure 10.A photograph showing a Harderian gland transplant from a seven day old mouse after fourteen days in the anterior onamber. Magnification lt% 44# Figure 11#A photograph showing a Harderian gland transplant from a seven day old mouse after twenty-one days In the anterior chamber. Magnif cation li%# 45. Figure 12.A photomicrograph showing the Harderian gland in a 15.5 mm mouse embryo# Magni fication 400%. 46# Figure 13.A photomicrograph showing the anatomical relationships of the Harderian gland to the surrounding structures.Magnification 150%. 47. Figure 14.A photomicrograph of Harderian Tissue from a newborn mouse after twenty-one days in the anterior chamber.Magnification 250%. 48# Figure 15.A photomicrograph of Harderian tissue from a seven day old mouse after twenty- one days in the anterior chamber.Magni fication 300%. 49. Ill LIbT OF OHAtiTB Chart I Miorometry of Harderian PAGE Controls* 27* Chart II Miorometry of Hmhryonlo Harderian Tissue* 38. Chart III Miorometrio Comparisons. 38. UHAPTER I INTRODUCTION Considerable literature has appeared in reoent years concerning the importance of the Harderian gland in mice and rats relative to the appearance and possible relationships to malignancy# it is believed by the author that by developing a method r trans planting and growing the Harderian gland in the anterior chamber of the eye, a contribution may be made toward the solution of this problem# The writora interest was stimulated by the fact that a satisfactory histological description of the gland was lacking and by the belief that additional data might be obtained by a thorough investigation of the gland as it appears from the embryological stages which might be significant# ^he & ct tnat other tissie s have been cultivated in the anterior chamber of the eje stimulated the writer to investigate the feasibility of transplanting young Harderian tissue to the anterior Chamber of the eye. In order to pursue this investigation it was necessary to develop a ramification of the surgical procedure for operating on the anterior chamber of the eye. several changes in technic were effected before a routine was established that adapted itself well to the need, -^he type of anesthetic and the method of administration had to be investigated in conjunction with the type of surgical instruments wniob could be handled effectively, aseptic procedure was adopted to eliminate the possibility of contamination of the instruments or the animals involved, no sepsis was observed in any of the transplantations. Little information may be found in the literature pertaining to the effective staining of Harderian tissue. ^he purpose of this paper is to introduce various procedures which the writer feels will be of assistance to other investigators in this field. A wide range of staining methods have been investi gated and the four best adaptable to the objective in view have been utilized in this problem. it is hoped that the information contained in this paper will contribute to tne microtechnic of the harderian gland. From a histological viewpoint, the problem was built around establishing controls which would enable the writer to observe normal changes which occur in the Harderian gland in its process of maturation* j-he amount of changes in the material which were observed in various ages of tne mouse Harderian tissue was most gratifying, ^xtensim micrometry was performed to establish quantitatively the changes occurring in various portions of the gland, incorporating dif ferential stains it was possible to bring out structures not possible with the routine Hematoxylin and ^osin Method. 3é CHAPTER il REVIEW OF LITERATURE ^ould 4 1941) mentions that the Harderian gland was named after the Bwias Anatomist, ^ohann «^aoob Harder 4 1666- 1711) and describes the structure as a racemose gland located at the inner canthus of the eye of most vertebrates and especially those having a well-developed nictitating membrane. Bmelser 4 1943) mentioned that the guinea pig possesses a Harderian gland and at the same time does not possess a nictitating membrane. It is generally agreed 4Greene,1935) that the Harderian gland in the mouse is visible between the intraorbital lacri mal gland and the eyeball. Upon dissection this is found to be extensive, occupying a large part of the orbit and opening upon the nictitating membrane* Ag the gland is traced further it proves to be horse-shoe shaped, extending medially and internally until it encircles the optic nerve and finally ends in the dorao-lateral region of the orbit close to the bony margin. Lgydig 41850) described the secreting cells of the Harderian gland as having a small fat globule in the center. ^luter 41947) stressed the point that the oil-like secretion of the Harderian gland in the horse served to disprove the earlier investigators* theory of a common anlage for the lacrimal and Harderian gland. The lacrimal gland is known to produce an aqueous secretion While the Harderian produces an oily, protoporphyrin secretion, ^^riters who have investigated the lacrimal 4. glanda as 4 1933), Tomioka 41936), Lgplet 4 193?), and Seki 41941) have materially aided the investigators of the Harderian gland. H. G. Greene 41936) gives an excellent description of the Harderian gland in the rat which is applicable to the mouse. ü’ ekete 41941) gives a fine account of the normal histology of the Harderian gland in the mouse, ^he describes the gland as being tubulo-alveolar, divided into lobes and lobules by a thin connective tissue membrane. The tubules and alveoli are lined by tall columnar epithelial cells in wnica the pale staining nuclei are located at the base of the cells. Histological aspects of the mammalian eye were discussed in meticulous detail by anell 41941), stiles (1943), Gopenhaver 41944), and Maximow and Hioom 4 1946). HiHarp 41946) described the fiber-like strands present in epithelial cells of secreting glands as trabeculae of either polypeptides or nucleic acid chains, -^here is but one other record in the literature that the author observed which mentioned the fiber-like strands, the investigation of vgaki 4 1936), in which he described lattice-like fibers in the Harderian gland. The excretory ducts are lined with cuboidal epithelial cells and open at the base of the nictitating membrane. interest in the Harderian gland relative to the incidence of cancer in the mouse and rat has been stimulated by work 5. with Torphyrin metabolism, Harderian gland fluoresoenoe and susceptibility to carcinogenic agents, strong 41942), Hundley 41944), Jones and » ‘olfe 41944) and Hueper and figge 41945) have contributed much data on these problems* oiuter 41947) stressed the medical implications and embryological aspects of tne Harderian with special reference to development in the horse embryo. The first record in scientific literature of trans plantation into the anterior chamber was that of van Dooremal 4l877>, at which time he described the intro duction of the mucous membrane of the lip and its subsequent growth. Uohnheim 41877) transplanted tubercles into the anterior cnaraoer of the guinea pig eye to facilitate the study of Tuberculosis. The transplantation of rib fragments from human embryos was performed by T'isher 4 1882) which constituted one of the first hetero geneous transplant procedures, neiogolowy and celling 41903) stated that true tumors followed heterogeneous transfers of amphibian spawn to the eyes of fish; in which case, tne transplanted cells underwent a sarcomatous change and the sarcomas eventually killed the animals, oaltykow and retrow 4 1921) described the growtri and development of cartilage in numerous transfers. The anterior chamber was utilized oy -^enjamin, -^elt, and HriChesky 41940) for the growth of prostatio tissue from the rabbit; an experiment based on the previous work of Heokel and %retsohmer (1935). Markse made extended 6 . studies on the intra-ocular endometrial transplants into the Hhesua monkey 11940)* transplantation of a human trophoblaat was found to grow rapidly in the anterior chamber by Hrebs and Gurchot 41946). Runner 41947) succeeded in cultivating fertilized mice ova into the anterior chamber which will open new possibilities for investigations into the embryological development of mammalia. binoe the eye may be considered a heterogeneous tissue, problems relative to the most efficient micro- teohnio are numerous. The retina, whose delicacy and perishability are unsurpassed by any other animal tissue 4 Lathrop, 1944), must be fixed by the same fluid which should not superimpose hardening on the toughness and friability of the cornea, solera and durai sheath of the optic nerve. Since the implants establish their vascular supply from the iris, the structure of this portion of the eye must be considered in treating the Harderian tissue and iris from the guinea pig at the termination of the experiment. The fact that the Harderian gland is lipoid in nature presents the problem of using a stain which would have an affinity for these acini and the same time bring out the relevent cytological aspects of the gland as a whole. McManus 41946) described a cobalt-caloium-forraol fixation and Hudan Black stain to bring out the fatty substances. 7. budan III produced results on the same theoretical basis but failed to prove its value for other cytological structures in the gland as a whole. btrong 41925) described a Golgi Ghrome-silver method for staining secretory tubules which has dropped out of the literature in the past twenty years and yet it contains information which is directly applicable to such glandular structures as the lacrimal and Harderian tissues# ^reitas (1936) described a new application of iron Hematein which is valuable when applied to the Heidenhain iron Hematoxylin staining technic. In selecting the Harderian gland for this investigation, the author was cognizant of the fact that the microtechnic of the Harderian gland as well as the delicate structures in the eye presented the most difficult procedures in order to achieve admirable results. The aim was that of preparing control slides with the eye and Harderian gland mounted in their normal relationship to one another, in this manner, much more could be observed concerning the Harderian gland. Hathrop 4 1946) described in elaborate detail the microtechnic of the pathologic eye. H@ presented many new technics which were of assistance in preparing the guinea pig and mouse eye successfully, neltz (1946) described the celloidin method of processing the eyeball which preserved the internal structure of its normal relationship to the Harderian gland. Lopez (1946) gave new staining methods for histological tissue that proved 8 . helpful in the treatment of the iris. »^ilson ( 1946) introduced a celloidin mastic combination which was used to advantage when control tissues were sectioned by the frozen method utilizing carbon dioxide* This method is not desirable for paraffin technic as the permeability of the stain is decreased to a small degree and in the case of Mallory*s triple Connective stain, a portion of tne stain is taken oy the celloidin. Aalls 4 1938) contributed much to the microtechnic of the eye. staining procedures of Harrison and Aiken (1942) were used as a basis from which new staining technics and times were established. Clang 41937) gave information relative to the staining of the nervous tissue which was applied to the optic nerve in the eye as it left the retina. Gonn and Harrow 41946) was consulted for the use of budan iH and nervous tissue technics, ^onn (1946) supplied much data on the individual stains that were employed throughout the investigation. The General index of Htain Technology, a new reference covering twenty years of ^tain Technology publications, was used in many instances to find isolated articles on stains and staining methods. Hue to the close parallel between lacrimal glands and the Harderian glands, a large amount of useful data was gained by investigation into books, periodicals and papers dealing with tne lacrimal tissue. Arey 41944) y. described, the embryological development of tne lacrimal glands in tne human aa appearing during the ninth week in the form of six knobbed outgrowths of the con junctiva. They lie dorsally near the external angle of the eye. Li (1933) made a moat elaborate study of these knobbed outgrowths in tne norse embryo and presented evidence as to the rate of growth in these structures. Arey described the primordia of the lacrimal glands as first consisting of solid epithelial cords wnich soon branch and acquire lumina. ^tibbe % 1928) described the nictitating membrane in the mouse as a membrane located at the inner angle of the eye. Th@ ectoderm on the outside of the lid differentiated into epidermis. Gontraated to this is the continuation of ectoderm on the internal surface of the lid and its reflection over the front half of the solera and all of the cornea. This becomes the mucous membrane named the conjunctiva Which plays a most important part in the anterior chamber transplant in that it has to be anesthetized prior to the incision of the cornea. Leal and Hand 41939) discussed the structure of the eye and optic orbits which was applicable to the 13.0 and Ib.h millimeter mouse embryos. Tatten 41931) gave detailed information relative to the develop ment of the eye in the pig and man 4Patten,1946) both of which serve as sources of information in reading tht 1C. mouse emoryological slides. The author found no literature describing the trans plantation of the Harderian gland into the anterior chamber. Greene s work has been conoerned with such problems as the heterologous transplantation of mam malian tumors and human f ibrosaroomas % 1942). the bio logical differentiation of benign and malignant H944), the heterologous transplantation of embryonic mammalian tissues 41944), production of carcinomas and sarcomas in transplanted embryonic tissues 4 1945), variations of the Houa •'^arcoma virus in the anterior chamber 41945), the utilization of the guinea pig eye for pathological diagno sis 41946) and the growth of avian tumors in the anterior chamber. il. Til M ATEH IaùjL AND Mi^.TMüDS Two types of laboratory animals were required for this particular investigation2 white mice and guinea pigs# The animal colony was started in September of 1947, six months prior to tne actual beginning of the experi mental work as it was necessary to have a rather large number available for the efficient operation of the pro ject scheduled. The original guinea pig colony was started with a boar and seven sows. Hue to the long gestation period of sixty-three days it was questionable if the required fifteen animals of six weeks or older would be available by February, ^fortunately ten animals other than the original colony had reached the desired age by that date and others were available as the experiment progressed. The guinea pigs were housed in wooden cages with one quarter inch hardware cloth floors to rapidly facilitate the removal of feces and to prevent any mycotic diseases should they be introduced into the colony, ^resh water was kept before the animals at all times. The food of cnoice proved to oe alfalfa rabbit pellets supplemented with greens such as lettuce or cabbage. Granges were given once per week to supply the required ascorbic acid. The addition of vitamin G to the diet is instrumental in maintaining a healthy colony. The rapid growth of the young guinea pigs is aided by having containers of pellets available directly after birth as these are eaten the first even though they are nursing. At the age of one month the guinea pigs were separated from their parents. 12. when the male guinea pigs reached six weeks of age their anterior chambers were approaching the desired size for transplantation work, unless it was absolutely necessary, animals were not used until they were ten weeks of age, Tn this particular experiment only male animals were used and the females were kept for breeding stock. starting with an original mouse colony of twelve fe males and two males, toe colony was increased to one hundred and fifteen by the dead-line, February, 1948. The cages used for the mice were constructed of quarter inch hardware cloth which was cut in such a manner that two cages measuring six oy twelve by six inches could be made, using a piece of material measuring twenty-four inches by thirty-six inches. The corners of the cages were secured by means of fine wire at three inch intervals. The hinges were constructed of the same wire and a heavier guage used to con struct a aasp. ^traw was used exclusively for litter. ■The diet whica was found most satisfactory for the mice consisted of Gaines Hog pellets which have a high protein content; a necessary requirement to eliminate still-births in a mouse colony. Mioe that have a low protein intake ex hibit cannibalism. The diet was supplemented with lettuce once a week to supply the required vitamin A, it has been noted tnat a mouse colony is quickly affected by the lack of this supplement. Lear-term mice were selected as a source of embryos for controls and transplantation material. Harderian tissue was also obtained from tnose animals directly after birth 13. to maturity. The tissue was fixed in -oouin s solution for twenty-four hours after wniob it was removed to a fresh solution, an index card was prepared for eacn tissue removed and this data was available throughout the en tire procedure, «hen the tissue was started through the denydration and clearing processes up to and including infiltration, the exact intervals of time were recorded# ataining procedures were standardized with test material prior to the running of the controls. The following stains were tried on tne test material: 1. Harris* Hematoxylin and ?ü/« Hoainol. 2. Harris* Hematoxylin and 96?4> Aosinol. 3. Harris' Hematoxylin and Methylene Hiue. 4. Harris' Hematoxylin and ^etayl ned. 6. Harris' Hematoxylin and malchite Green. 6. Budan lid in 99)® isopropyl alcohol. 7..Helafield'8 Hematoxylin and 70/» Aosinol. 8. Wright 3 stain and Hematoxylin. 9. Mallory s Triple connective Btain. 10. Heidenhain s Tron Hematoxylin and 7Q7<> Hosinol. The stains which were found to have tne greatest affinity for the Harderian tissue were the following: 1. Harris Hematoxylin and ^ethylene niue. £. Harris Hematoxylin and 70/-» Hosinol. 3. Mallory's Triple Connective Btain. 4. Heidenoain s Tron Hematoxylin and 7ÜP Aosinol. The following is tne schedule of time both the control and transplanted Harderian tissues were left in the respective 14. solutions: 1. Bouln's and 70;^ isopropyl alcohol ( 1:1) 7 hours 2# 70/6 isopropyl aloohol 1 hour 3. 80/6 iaopropyl aloohol 2 hours 4. 90/6 iaopropyl aloohol 2 hours 5. Absolute iaopropyl aloohol 2 hours 6. Absolute isopropyl aloohol 4 hours 7. Absolute iaopropyl:%ylene (l:l) 3 hours 8. Absolute isopropyi:%ylene (1:1) 3 hours 9. Aylene 3 hours 10. Ayiene 3 hours 11. Ayiene 3 hours 12. Faraffin with 10/6 Bayberry 2 hours 13. Faraffin with 10/» Bayberry 2 hours 14. Faraffin with 10^ Bayberry 2 hours 16. Imbedded in paraffin with 10/^ Bayberry. The sohodule given below was used in a new staining teohnio for the Harderian gland, that of Harris* Hematoxylin and Methylene Blue: 1. Ayiene 6 min 2. Ayiene 6 min 3. Absolute iaopropyl aloohol 1 min 4. Absolute isopropyl alcohol 1 min 6. 90/6 iaopropyl aloohol 1 min 6. 80/6 iaopropyl aloohol 1 min 7. 70)^ iaopropyl aloohol 1 min 8. Listilled water 3 min 15. 9. Harris' Hematoxylin 3 min 10. Distilled water 3 min 11. Aqueous Methylene Blue ( 10/6 stock) 3 min IE. Distilled water 5 min 13. iaopropyl aloohol 10 secs 14. 80/6 iaopropyl aloohol 5 secs 15# 90/6 iaopropyl aloohol 5 secs 16. Absolute iaopropyl alcohol 6 secs 17. Absolute isopropyl alcohol 5 secs 18. Ayiene 10 min 19. Ayiene 5 min EO. Ayiene 5 min El. Gum damar. Dote: It was found that a prolonged period of time in the aloohols tended to decolorize the Methylene Blue. The purpose of tne Methylene Bitie was that of serving aa a counter-stain for the Harris* Hematoxylin. The degree of affinity exhibited by the Methylene Blue for the lipoid constituents of the Harderian tissue is much greater than the affinity exhibited by Bosinol. 'The staining schedule which was found to oe most effective for Harris' Hematoxylin and Boa in consisted of the following: 1. Ayiene 5 min E. Ayiene 5 min 3. Absolute isopropyl alcohol 3 min 4. Absolute isopropyl alcohol 3 min 6. 90/6 isopropyl alcohol 3 min 16. 6. 80/6 iaopropyl aloohol 3 min 7. 70/® isopropyl alcohol 3 min 8. Distilled water 5 min 9. Harris' Hematoxylin 3 min 10. Distilled water 5 min 11. Aoid aloohol ( 3 drops HNO3/6O00 70/6 aloohol) 15 secs 12. Distilled water 5 min 13. Alkaline wash ( 3 drops lOidOH/gOoc 70^ alcohol) 1 min 14. 70/6 iaopropyl alcohol 5 min 16. 70/6 Eosinol 1 min 16. 70/6 iaopropyl alcohol 5 min 17. 80:^ iaopropyl alcohol 5 min 18. 90/® iaopropyl alcohol 5 min 19. Absolute isopropyl alcohol 5 min 20. Absolute isopropyl alcohol 5 min 21. Ayiene 10 min 22. Ayiene 5 min 23. Ayiene 5 min 24. Gum Damar. A stain which proved effective with embryological material as well as mature Harderian tissue was that of Mallory's Triple Gonnective Tissue method. The time schedule which was used to bring out the acini consisted of the following : 1. Ayiene 6 min 2. Ayiene 6 min 3. Absolute iaopropyl alcohol 3 min 17. 4. Absolute isopropyl alcohol 3 min 5. 9076 iaopropyl alcohol 3 min 6. 80/® iaopropyl alcohol 3 min 7. 707» iaopropyl alcohol 3 min 8. Distilled water 10 min 9. Solution 1 20 secs 10. Solution 11 3 min 11. 80/6 iaopropyl aloohol 10 secs 12. 95)6 isopropyl aloohol 6 secs 13. Absolute iaopropyl aloohol 3 min 14. Absolute isopropyl alcohol 3 min 16. Ayiene 10 min 16. Ayiene 6 min 17. Ayiene 5 min 18. Gum Damar. Heidenhain*a Iron Hematoxylin stain was found to he the most effective method in bringing out the basement membrane. Although special technics were required to ob tain the desired results in this procedure,the finished slides justified the extra time spent in their preparation. The staining times were aa follows : 1. Ayiene 6 min 2. Ayiene 5 min 3. Absolute isopropyl aloohol 3 min 4. Absolute isopropyl aloohol 3 min 5. 95)6 iaopropyl aloohol 3 min 6. 80)6 iaopropyl alcohol 3 min 18. ?. 70/» isopropyl alcohol 6 min 8* Bistilled water 3 min 9. •Perrio alum (2.6/» aqueous) 30 min 10. Bistilled water 5 min 11. Heidenhai n* s Hematoxylin 60 min IE. Bistilled water 5 min 18. #errio alum:Bifferentiate under microscope. 14. Bistilled water 5 min 15. 70^ isopropyl alcohol 5 min 16. 70^ Hosinol 6 min 17. 80/0 isopropyl alcohol 6 min 18. 90;^ isopropyl alcohol 5 min 19. Absolute isopropyl alcohol 5 min 20. Absolute isopropyl alcohol 5 min 21. -Xylene 10 min 22. Aylene 5 min 23. Ayiene 6 min 24. Gum Bamar. I'he methods of transplanting tissue into the anterior ohamher as suggested by ^okel 11985^, Belt 11940^ and Greene (1988) had to he modified in many respects in order that the transplantation of the mouse ^larderian tissue might he transferred to the guinea pig suooessfully. i^he folloiiving equipment was used in the sargical pro cedure of transferring the tissue* 1. -Oard-Parker soapel handle with #11 hlade. 2. -Lrredeotomy foroep. 8. l*wo hlunt-nosed prohes. 19. 4. fwo glass depression slides. 5. ‘ fen oubio centimeter glass syringe. 6. fhree #23 guage hypodermic needles. 7. 'fwo glass eye droppers. 8. Bterile solution of & Hovaoain. 9. aterile solution of normal saline. 10. I'inoture of ^erthiolate; 1:1000. 11. isopropyl alcohol. 707». 12. Bther. u.b.i?. 13. Bouin*s solution. 14. ùQtton. 15. i'wo glass vials, two dram capacity. 16. Autoclave. sterilization was carried out at 15 pounds pressure for 20 minutes. Preparatory to beginning the excision of the Harderian tissue from the donor, normal saline was placed in the sterile depression chambers and covered to prevent contamination. ' i ' h e mouse to be used was cleansed with 70/0 isopropyl alcohol followed by i'inature of Merthiolate ten minutes prior to administration of ether. Upon removal of the Harderian glands, one group was immediately placed in a vial containing Houin's fixative while the other was trans ferred to the saline in the covered depression slide, fhe fragment of tissue was measured by means of a calibrated microscope.After the micrometry was completed transplantation to the anterior chamber of the guinea pig was oarrie d out. ‘ fhe eye of tae guinea pig had been prepared in the 20. following manner preparatory to the introduction of the implant: the entire had been cleaned with sterile saline followed by tincture of j&erthiolate. fen minutes prior to the aotual inoision of the corneoscleral juncture two cubic centimeters of 2;o Bovacain were injected at various sites of the conjunctiva and applied to the surface of the entire anterior cornea. J-n administering ether to the guinea pigs extreme care was neoessary due to their low threshold.fhe third level of anesthesia was reached in less than two minutes at which time fincture of Merthiolate was again applied to the surface of the eye. -^fter the eye had been rotated downward by gentle pressure against the tension of the extrinsic eye muscles, the initial incision was made at the oorneoseleral juncture, fhe bard-farker blade was held parallel to the surfaoe of the iris thus avoiding any trauma upon entry. Aq incision of 2 millimeters re sulted for introduction of the transplant. fhe implant was removed from the depression chamber immediately after micrometry by means of a sterile glass eye dropper to the incision in the cornea of the guinea pig.At this time a blunt probe was introduced to guide the implant into the anterior chamber and in direct contact with the iris.Upon removal of the probe an additional application of fincture of Merthiolate was made and the animal was returned to its respective cage, fhe absence of any in fections following this procedure is attributed to the use of sterile technic and the generous applications of 21 . 1‘inoture of Merthiolate. When it was desired to remove the implant at a later date the animal was sacrificed by means of ether and the entire eye was removed and placed in Bouin*s fixative for twenty-four hours, fhe stain which was employed on the controls as well as the transplanted tissues was Harris* Hematoxylin and f*osin. fhotographs were taken of the implants at various stages of development to show the gross appearance of the Harderian tissue within the anterior chamber of the guinea pig eye. A Speed Graphic camera with a f 4.5 lens was used for this work, uther pertinent details regarding the series of photographs are as follows: 1. fime -hxposure; l/50th to l/lOOth of a second. 2. Opening: #.22 3. Illuminaisiai%: Baylight. 4. jg'ilm: Ansco Huper-pan fress.St A 4^. 6. 4mul8iOD Sueed* lOC westen. 6. Developer : 5 minutes at 65 degrees Fahrenheit. 7. Fixative: 15 minutes at 65 degrees Fahrenheit. 8. wp.s&^: 30 minutes at 65 degrees Fahrenheit. I'he two embryos which were found to be most beneficial in demonstrating the development of the Harderian gland were 13.0 millimeters and 15.5 millimeters,respectively, fhe de hydration, clearing, infiltration and imbedding routines paralleled those used for the controls and transplants,All the serial sections on the embryonic tissue were cut at eight miora, 22. Harris* Hematoxylin and no a in proved to be the stain of ohoioe. Mallory*s Connective tissue stain.was used on some of the individual slides. A Spenoer #818 notary microtome and a Spencer 120 millimeter microtome knife were used for all of the sectioning done on this particular problem. Depending upon the particular histological structure desired# the thickness of the sections for the controls and the traisr planted tissue varied from 4 micra to 10 miora. 2 8 . üHâPTEH IV OBSERVATIONS AND FINDINGS STAINING REACTIONS ON HARDERIAN TISSUE The four staining technics employed in this in vestigation were : Harris* Hematoxylin and nosin, Mallory's friple connective fissue stain,Heidenhain*s Iron nematoxylin, and a combination of Harris* Hema toxylin and Methylene Hiue. from the list of staining technics described in the chapter on Materials and Methods, these four methods were selected. Hematoxylin and F»oain was the stain of choice for control and transplanted tissues, contrary to Fekete's statement % 1941) that the nuclei of the mouse Harderian tissue stain lightly, fine degrees of differentiation were obtained by this method, fhe fact that Hematoxylin and f*oain did not stain the basement membrane in some stages negated the adoption of this stain for all pro cedures. Mallory s friple connective fissue stain was found to be an excellent means of portraying the vascular structures in embryonic ^rderian tissue.-^ha disadvantage in using this method was the poor staining of the cell membrane of the columnar epithelial cells. Heidenhain a fron Hematoxylin stain proved to oe an excellent method to demonstrate the basement membrane in Harderian tissue of the more advanced stages, h problem encountered with the other stains was the inaoility to delineate the actual position of the basement membrane. 24 . A staining comoination taax haa not oeen used on Harderian tissue previously was that of Harris' Hema toxylin and Methylene Blue.Hematoxylin was chosen for its nuolear affinity, fhe Methylene Blue was used both as a nuolear and oytoplasmio stain. The selection of this combination grew out of tne niehl-Heelsen staining method employed for the detection of acid-fast micro organisms.in this particular method with Harris'Hema toxylin, the Methylene Blue serves as a counter-stain, much in the same manner as Ho sin in the Hematoxylin and Hosin method, ^he nuclear elements of the acini were clear and the lipoidcharaoter of the secreting epi thelial cells were easily discernible. CONTROL SLIDES A series of Harderian tissues from mice ranging in age from birth to maturity were fixed in -uouin's fluid. Harris* Hematoxylin and ^osin, Mallory's friple Connective Tissue method and Heidenhain*s Iron Hematoxylin were em ployed routinely on all of the control slides. Hach control was sectioned at four different thicknesses : four, six, eight, and ten micra. A. HARDERIAN TI8&UE FROM NEWBORN MOUSE The Harderian tissue removed from the newborn mouse resembled controls made from embryonic tissue in many respects, fhe interstitial mesenohyma which would event ually form the connective tissue of the gland constituted fifty percent of the entire gland at this stage.Seventeen 2 5 . percent of the lumina were open and functloni&g.fhe size of the lumina in all stages of the control was not constant due to the particular phase of secretory activity, dn this set of controls, the size of the lumina varied from thirteen to twenty-nine miora. fhe size of the acinic nuclei was one mlOra or less in diameter than the mature forms, their range toeing from four and one-tenth micra to five and four-tenths miora. The stromal nuclei remained unchanged, fhe columnar epithelial cells of the acini (Fig.l) have not developed their characteristic morphology. At this stage the oapsule has not formed around the gland. The number of stromal nuclei that will become established adjacent to each acinus cannot be determined at this stage. No basement membrane is evident, fhe stromal nuclei appear ovoid. B. HARDERIAN TISSUE FROM ONE WEEK OLD MOUSE In the interval of one week, the Harderian gland assumed many characteristics of mature tissue.fhe amount of interstitial mesenchyme had reduced materially,being replaced by mature connective tissue, fhe distance between acini was reduced by thirty micra. fifty-three percent of the acini iUhart I) were open and functioning, an increase of thirty-six percent in a period of seven days.fhe lumina varied from three and four-tenths miora to thirteen and six-tenths miora. fhe acinic nuclei ranged from four and four-tenths miora to six and one-tenth micra in diameter. fhe stromal nuclei snowed a slight decrease in size, ranging from three and seven-tenths to four and four- tenths micra. f'his appeared as a transitional stage prior 2b, üIGüRE 1. àewborn mouse A pûotomicrograpa snowing a section oi tne Harderian gland from a newborn mouse* *khe interstitial stroma con stitutes fifty percent or tne gland at tnis stage* ^he tissue was sectioned at d miora, magnifioation: 376A, GA, ....... ............. closed Moinus uTb. .... . * 'connective tissue ^troma OA, .................. upen Aoinus E6. PSSJ/>, # % ' W ^ ... 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T O T O # 0 1 © 0 0 rH ■ 4 Pi 1( T O T O a a 0 0 fti Is P i xS © 4 a P Û T O © 0 a a a 4 2; Pi Pi T O T O •n c y 0 0 •0 © Pi P i 4 2S T O c 3 c 3 T O 0 © © 0 Pi 2 i Pi f0 © 0 rH a « a a +» © C Ü t M c y e y c î > © © © © © © 2 Î 2 5 2 5 c 0 nd © m T O f-2 4 jO f0 0 a a a T O G d C d E a a © a a a 4 4 1 •H a ? H a a a 0 s a © E a 0 a a 0 a 4 4 rH © a *H c y ' a © a a 0 © a a 0 *H • * • • 0 a 0 © a 4 4 T O a a C +) a © c TO © © E a a © a a 0 © E 0 0 0 a © © a C 0 0 © D fH •H a • H © a © © *H © -H iJ a H © © E c a . Q T O0 C d a f - a © P4 T O © T O •ri 0 a © i-> fH a tP A ja P , © Ki 0 © *H '4 ■ z i i-» "4 p; © q © «H a P i s © a a # JQ C M to JQ rH rH rH 2 b . to the assumption of an elliptical shape wnich is found in the mature Harderian gland# The columnar epithelial oells had not reached their mature form, out an increase in the activity was evidenced by the appearance of mitotic figures i#ig,2) which were not found in other stages of maturation. The capsule had not formed as a definite structure, but the connective tissue had made its appearance.The basement membrane was forming, but had not acquired the adult characteristics as shown in later slides. U. HARDERIAN TISSUE FROM TWO WEEK OLD MOUSE At two weeks of age, the Harderian tissue of the mouse had a mature histological appearance. The amount of con nective tissue between the acini had decreased to such an extent that the distance between each acinus measured less than three micra (Ghart I). There was a marked difference from the one week old harderian tissue.Highty percent of the acini were open and functioning,an increase of twenty-seven percent over the seven day period. The range in size of the lumina had increased, measuring between eight and five-tenths miora and twenty-three and eight-tenths miora. i-'he acinio nuclei were approaching the mature size, the average being five micra to six and eight-tenths micra. The stromal nuclei had become stabil ized in respect to morphology and size. They had assumed an elliptical shape, three micra in width and ten and one-tenth micra in length. The columnar epithelial cells of the acini were mature 29. FiGuiüü 2, Une »<eek uid Mouse A photomicrograph snowing a section of the tarderlan gland from a one week old mouse. ^he appearance of mitotic figures at this stage indicates the rapid rate of prolifera tion. ^he tissue was sectioned at b miora, magnification 4oOA, aN . .....^cinio i*uclei GA . Gxosed Acinus 0T8, ........ ....... ..........<*onnectlve Tissue atroma L...... ....................Numen m# . . . ........... .... .Mitotic figure 29. FIGURE 2 8 0 . in structure and had a range of six and eight-tenths miora to ten miora from the basement membrane. A oapsule had formed around the Harderian tissue i#ig.3) and coonnective tissue aided in the formation of lobe and lobules. The collecting tubules were lined with cuboidal epithelial cells. The number of stromal nuclei per acinus are rare# averaging from one to two. The basement membrane appeared mature at this stage. Many of the acini were completely filled with lipoid substance. The acinic nuclei assumed a position adjacent to the basement membrane. D. HARDERIAN TISBUS FROM THREE WEEK ODD MOUSE At three weeks of age the Harderian gland of the mouse approached its maximum rate of activity, ninety percent of the acini (Ghart 1) were observed to be open and functioning, an increase of ten percent in a period of seven days. The size of the lumen ranged from thirty- four micra to ninety micra. The acinio nuclei reach their maximum size at this stage, measuring from five micra to six and eight-tenths. The height of the columnar epithelial cells varied with the phase and rate of activity. At this stage of activity, they measured ten and two-tenths micra. The average number of stromal nuclei was between one and three. The acini were separated by a narrow layer of connective tissue. The acinio nuclei were adjacent to the basement membrane. ÜI. HARDERIAN TISSUE FROM FOUR WEEK ODD MOUSE In the four week old Harderian gland, ninety-three 81. FIGURE 3. Two Meek old Mouse A photomicrograph snowing a section of the xiarderian gland from a two week old mouse. Tne relative amount of connective tissue between the acini nas decreased. The tissue was sectioned at 4 micra. Magnification 188A. A. ........................ Aoinua u .................. v^apsule uE ..... . . ..........uuboidal -epithelial ^exig uO .................... connective Tissue uf.................. .......... 'collecting Tuhule 31. n FIGURE 3 82. percent of the acini i^hart D were open and functioning. The diameter of the lumina ranged from twenty to ninety- five miora. The columnar epithelial cells measured from thirteen to twenty micra in neight. The number of stromal nuclei per acinus averaged from one to five, being deeply imbedded in tae oonneotive tissue. The acinio nuclei pre sented an elevated position in relation to the basement membrane, a distance of from one and seven-tenths miora to four miora. F. UARDERl^N T1E8ÜE FROM FIVE ODD MÜU8E. Ninety-six percent of the aoini iGhart l) were open and functioning at this stage. The range in size of the lumina was slightly diminished from that of the four week old ^rderian tissue. The lumina measured from ten to sixty-four miora. The columnw epithelial cells ranged from ten to twenty-three micra in height. There was an average of from one to five stromal nuclei per acinus. A large collecting tubule t#ig.4^ was observed filled with the lipoid seofetion from the acini. The wall of the collecting tubule was lined with cuboidal epithelial cells, tq this particular stage, the epi thelial acinic nuclei are adjacent to the basement mem brane. G. HARDERIAN TISSUE FROM SIX WEEK OLD MOUSE The Harderian gland was observed to have reached its maximum rate of activity, a h of the acini were open and functioning. The height of the columnar epithelial oells ranged from ten micra to seventeen miora. 8 2 . percent of the acini ^Ghart D were open and functioning. The diameter of the lumina ranged from twenty to ninety- five miora. The columnar epithelial cells measured from thirteen to twenty micra in neight. The number of stromal nuclei per acinus averaged from one to five, being deeply imbedded in tne oonneotive tissue. The acinio nuclei pre sented an elevated position in relation to the basement membrane, a distance of from one and seven-tenths miora to four micra. F. uAHDERli^N TlbHUE FROM FIVE wEÆ OLD MOUEE. Ninety-six percent of the aoini (Ohart l) ware open and functioning at this stage. The range in size of the lumina was slightly diminished from that of the four week old uarderian tissue. The lumina measured from ten to sixty-four micra. The columnar epithelial cells ranged from ten to twenty-three micra in height. There was an average of from one to five stromal nuclei per acinus. A large collecting tubule (#ig.4> was observed filled with the lipoid secfetion from the acini. The wall of the collecting tubule was lined with cuboidal epithelial cells, tq this particular stage, the epi thelial acinio nuclei are adjacent to the basement mem brane. G. HARDERIAN TISSUE FROM SIX V/EEK OLD MOUSE The Harderian gland was observed to have reached its maximum rate of activity, a h of the aoini were open and functioning. The height of the columnar epithelial cells ranged from ten micra to seventeen miora. 33. ¥ r - ^ % a ^ . c FIGURE 4 34. The stromal nuclei attained an average of from one to six per acinus, which became the stabilized number in following stages of development of the Harderian gland. The position of the acinio nuclei %Fig.5) in relation to the basement menbrane varied from three and four- tenths micra to six and seven-tenths micra. H. HARDERIAN TISSUE FROM SEVEN WEEK OLD MOUSE The lumina at seven weeks reached its maximum size IGhart I) of ten to one-hundred and two micra in dia meter. The heignt of the columnar epithelial cells ranged from thirteen to twenty micra. The acinic nuclei assumed a position adjacent to the basement membrane. I. HARDERIAN TISSUE FROM EIGHT WEEK OLD MOUSE The lumina showed a decrease in size from the seven week old gland, the average of ten to eighty-five micra was recorded % Uhart Ü. The columnar epithelial cells varied from ten to seventeen miora in height. The mature basement membrane i#ig.6> appears clearly. The use of Heidenhain'8 iron Hematoxylin may be credited with the staining effect obtained with this specimen. The acinic nuclei maintained a position of from one miora to ten micra from the casement membrane. U. HARDERIAN TISSUE FROM MATURE MOUSE The lumina ranged from ten to eighty-eight micra in diameter % Ghart I). Th@ columnar epithelial cells measured from thirteen micra to seventeen micra in height. The acinic nuclei were adjacent to the casement membrane. 85. Fikxüxus» 5, ôix »<eek old Mouse A pnotomiorograph snowing a section of the tarderian gland from a six week old mouse. The variation in the size of secreting columnar epithelial cells may be noted. The tissue was sectioned at b micra. magnification 876^. AN. . . ....................... .Acitiic nuclei uO. ... 0nnective Tiasue ............ .......'columnar epithelial oells OA... . . . ... . ... .... .....Open ^oinus 36. # 1 FIGURE 5 86. FiGURE 6. Tiight *<eek old mouse m photoraiorograpn showing a section of tne aarderlan glend iron an eignt week old mouse. The basement membrane may be seen clearly. The stain used on tnis tissue was Meidenbain*a iron hematoxylin. The tissue was sectioned at 4 micra. magnification 876A. AN. ..... . . ..moinic nuclei AM. .... ..**8sement membrane ...........................Oolumnar mpitnellal oeiis A.............................Lumen i^.. .... . . . .. . . . .. .. . ...JWipoid material 36. FIGURE 6 ;3?. üBSÜRVATIOHb üi ïMB'dYOmO HAHBlüRIAE 0?ISSÜE Mouse embryos that were approaohing term were selected for detailed study* Serial sections were made from a thirteen millimeter emoryo and a fifteen and five-tenths millimeter emoryo* A* HARBEHIAK TISSUE IK 13*0 mm MOUSE EMBRYO The amount of interstitial material exceeds the glandular tissue present at this stage* The acinic nuclei measured from six and eigbt-tentaa micra to ten and two-tenths micra in diameter CUhart dij.ihe stromal nuclei had an ovoid appearance,ranging from eight and five-tenths micra to nine and five-tenths micra. evidence of a capsule was noted, -i'he structure of the acini did not clearly resemble the columnar epi thelial tissue that it was destined to Become* -^he dis tance between acini varied from three micra to one- hundred and eigat micra* B* HARBERIAK TISSUE EHOM 15.6 mm MOUSE EMBRYO i-n the Marderian tissue of tnis embryo, three and three-tenths percent of tne acini were open and function ing t Ohart -Lii, The lumina measured three and four-tenths micra in diameter. Th@ aoinlo and stromal nuclei did not vary from the thirteen millimeter embryo in size, i^o capsule was noted around tne gland. :^he stromal nuclei maintained a mesencnymous appearance, ^‘ he distance between the aoini ranged from three micra to sixty-eigat micra. ^he cuboidal epitnelial cells lining the collecting tubules (Fig.IB) appeared mature. A ‘ ôb. CHART II MloxiOiiiiiiTRY ÜE ü»kj3RYüi\(iC iltui*>XAnÜH Tii^D ü E 01 ig.O mm iAWiiYOS 16.5 mm 1.Number of open 0. z . dumber of closed irtrhO. 58. 3. Percentage of 0. _ . 3.3 ......... 4. Size of lumen in . . _ 3.4 ë* ^ize of acinic jmjolei in micra. 6.8 — 10. 2 6.8- 10.2 6. Size of stromal nuclei in micra. . 8.5— 0-5..... 8 . 5— 7. distance between miara.A. 3.- 3.- -..SS^____ . . . CHART III MICROMETRIC COMPARISONS i^ewborn : Control : Kewborn trans plant : 3 heek : Contre] 1. Number of open 6.0 , .. ,55.0 2. Number of closed 28. C 15.0 . 5.0 3. Percentage of 17^0 42.3 90.1 4. &ize of lumen 13.- 3.4- 34.- 29. 29. 2 90. 5. blze of acinic 4.1- 4.4- 5.- nuclei in_ micra. 5.4 5.1 6.8 6. Size of stromal 4. 2- 3.4- , 3i A , . 5.1________ 10. L. 39. large amount of interstitial tissue was evident, mitotio figures were found in the mesencnymous nuclei, the eye and surrounding structures <Pig.13) demonstrate clearly the realtionsnip of the Marderlan gland to tne adjacent tissues. UBbEHVATIOKS UF THAKSPAANTEB nAKBEttlAJM TlbB U E Marderian tissue was removed from mice ranging from birth to fourteen days of age. tt was found that only Marderian tissue from newoorn mice could be successfully grown in the anterior chamber of the guinea pig eye. tissues from older animals failed to establish a vascular supply with tne recipient. A.dARBEHiAN TRANüIjuaNT FKùM KEwBOHN MüüSE The tissue after seven days cultivation in the anterior cnamber of the guinea pig eye (T'ig.y ) may be observed grossly. The transplant was in direct contact with the iris and became vascularized. The transplant in another guinea pig, fourteen days after implantation (ï'ig.8) revealed an increase in mass. At the end of twenty-one days (Fig.9) the implant had completely filled the anterior chamber. in a period of twenty-one days the initial implant, which had measured one-hundred and two micra, had proliferated to such an extent that it had filled the inoculation site, in a slide made from this transplant (Fig.14), it will be noted that connective tissue constitutes the larger portion of the area. The 40. Figure y. «niinea Pig No. 102 à pnotograpn snowing a aarderian gland transplant from a newborn mouse wnich nas grown in tne anterior cnamber for seven days. Magnification 1#A. f. ...... transplant 40. figure 7 41. FiGUiùü b. v»uinea Pig wo. 106 ^ pnotograpn snowing a aarderian gland transplant from a newoorn mouse after fourteen days of cultivation in tne anterior cnamber. magnification l^A. T................. transplant 41. FIGURE 8 42. FiGüKiJi 9. *ulQea Pig no. 108 A photograph snowing a aarderian gland transplant from a newborn mouse after twenty-one days cultivation in tne anterior cnamber. Rote now tne anterior chamber has become filled with tne transplanted tissue* magni fication 1^* T*................. * transplant 42. PIGUBB 9 43. lumina of tha aoini measured from three and four-tenths miora to twenty-nine and two-tenths miora in diameter (Ghart lllJ. ^he aoinio nuclei measured from four and four-tenths micra to five and one-tenth micra which may he compared with the same range of newborn control tissue. The stromal nuclei measured from three and four- tenths micra to five and one-tenth micra which is com parable to the stromal nuclei found in newborn mice. B, HÀRDEHIAK TKANSPLAKT PROM SEVER DAY OLD MOUSE The gross appearance of the transplant iT'ig.lO^ after fourteen days in the anterior chamber is similiar in many respects to the tissue from a newborn mouse. After a period of twenty-one days t^’ ig.ll). the gross appearance reveals that the size of the implant has diminalhed. A slide made from the tissue removed after twenty-one days in the anterior chamber t^'ig.lSi reveals that the majority of tne tissue is composed of connective tissue, only isolated aoini are observed. The implant originally measured two-nundred micra in length and, after the twenty-one day period, had retrogressed to seventy-four and eigot-tenths micra. 44. ificrURA 10. '^inea Tig No. 109 photograpn showing a aarderlan glund transplant from a seven day old mouse after fourteen days of culti vation in tne anterior onimber. is^agnifioation I^a , T...................................-transplant 44. FIGURE 10 46. FIGURE 11, guinea -fig ivo. 110 ^ pnotograpri snowing a narderian gland transplant from a seven day old mouse after twenty-one days culti vation in tne anterior cnamber. Magnification 1^-^. T................................transplant 45. PIOUHB 11 46# ilGuiiü 12, -e^mbryonic tiarderian «land -a pnotomicrograpn snowing tne aarderian gland in a 16.b millimeter mouse emoryo, Note the prédominance of closed aoini at tnis age. the tissue was sectioned at 8 miora. magnification 400- ^ . , A, ................. .......moinus wr................. .......» Collecting tuoule era.................. ........... connective tissue stroma UÀ.........................•••.••Open acinus 46, FIGURE 12 47. i?x^üRiSi 13. -embryo nio tarder Ian ^land A pnotomiorograpn showing tne anatomical relationship of the Harderian gland to the surrounding structures. The embryo was sectioned at 8 miora. Magnification 150A. Q . . C o r n e a ..... ............ . Conjunctival sac HG,....................... .-harderian gland nL. ...... .................. .Bower lid ...............Retina 47 FIGURE 13 48. FIGURE 14, Transplanted harderian Tissue A photomicrograph of uarderian gland tissue from a new born mouse after twenty-one days cultivation in the anterior chamber of Guinea Pig Ko* 1C8* Note the number of open acini. The tissue was sectioned at 8 micra. Magnification 250A, O L E , C o l u m n a r Epithelial «ells GO,,...... ............. 0nnective Tissue L* ............ ......Bumen 48. FIGURE 14 49. FIGURE 15, Transplanted harderian Tissue A photomicrograph of carderIan gland tissue from a seven day old mouse after twenty-one days cultivâtion in the anterior ohamber of Guinea Pig Ko. 110. Note the atrophied appearance of the acinus. Magnification 300A. A. .... ............... • Aoinus GO, ..... Connective Tissue 49. '-.-•I- FIGURE 15 50. OHAPTEH V DISCUSSION The Harderian gland was suooesafally transplanted into the anterior chamber of the guinea pig eye for the first time recorded in literature. Several different ages of tissue were attempted. The tissue which produced the desired results was taken from a newborn mouse, it was found from critical evaluation of control slides that Harderian tissue less than a week old resembled embryonic tissue histologically. Greene (1943^ found that embryonic tissue could be cultivated in the anterior chamber,how ever, Harderian tissue was not recorded. The interstitial tissue of the newborn mouse harderian tissue tPig.ii re sembles the embryo (Fig.12) in many respects. The tissues from older animals introduced into the anterior chamber developed a vascular supply and main tained their histological identity for a short period of time. At the end of twenty-one days atrophy of the active tissue was marked (Pig.15). It appeared that the rate of proliferation in the embryonic tissues was greater than in the more mature tissues. Control slides were prepared in order that a quanti tative evaluation as to the degree and rate of maturation in the Harderian gland might be obtained. A wide variety of stains were used with the preliminary control slides to determine the most effective methods. A problem which caused considerable difficulty was the staining of sec reting acini as the lipoid nature of the gland was re fractive. Heidenhain’s Iron Hematoxylin proved to be the 51. moat suooeaafal for Harderian tissue. The basement mem brane was observed to be an aid in measuring the relative position of the aoinio nuclei in various stages of cellular activity. Variations in the position of the acinic nuclei were observed at different ages («hart l). A more detailed study on the significance in the positions of the nuclei to the basement membrane is warranted. Harris* Hematoxylin and Methylene Blue Method was used for the first time recorded in literature in the staining of the Harderian gland. The successful use of this stain is dependent upon the minimum use of alcohols due to the deataining action. A number of tendencies and trends were observed in the chronological development of the Harderian gland in embryological and control material. The first acinic lumen was observed in the fifteen and five-tenths milli meter embryo (Chart iD. A period of rapid cellular activity of tne stromal nuclei was noted at this stage, evidenced by the appearance of mitotic figures. At no other time in the development of tne Harderian gland were mitotic figures observed in the stromal nuclei. The stromal nuclei maintained an ovoid shape until the second postnatal week iChart i) at which time they assuse d an elliptical morphology. The elliptical conformation became stabilized. A definite rate of maturat ion was observed in the acini of the controls. J-n the thirteen millimeter embryo all of the acini were closed and by the sixth postnatal week the entire gland was functioning as evidenced by 52. Open lumina and the presence of lipoid material within the lumina. The acinic nuclei exhibited the maximum pro liferative rate at the second week when mitotic figures (Pig.2) were observed. At no other time in the maturation of the acini were mitotic figures observed, ^t was evi dent from the variation in the appearance of mitotic figures in acinic nuclei and stromal nuclei how the morphology of the Harderian gland was being established. From the second week, the size and number of acinic nuclei remained constant. The capsule of the Harderian gland did not become established until the second postnatal week, ether in dications that the gland was approaching its mature form was indicated by ll) the aoini becoming adjacent to one another, (2) the diminished amount of inter stitial tissue, (3) the appearance of the first base ment membrane and (4) the first instance of the acinic nuclei being adjacent to the basement membrane. The Harderian tissue of a newborn mouse was trans planted into the anterior chamber of a guinea pig eye which revealed a number of interesting facts. Th@ number of mature acini had increased twenty-five and three- tenths percent in a matter of three weeks although it did not reach the same degree of function exhibited by a three week old control. The lumen of the transplant remained the same as that of the newborn control. The acinic nuclei unchanged from the appearance in newborn Harderian tissue. Fekete (1941) mentioned that the acinic nuclei were 53. Staining and rested upon the basement membrane, ^he writer has found that Doth of these facts are not always in evidence, the use of several different staining methods it was possible to stain the aoinio nuclei to the correct degree of differentiation, was found as stated earlier in this paper that the position of the aoinio nuclei vary with the phase of activity within the secreting columnar epithelial oell. i‘ he aoinio nuclei in the eight week old mouse were ten and one-tenth micra from the basement mem brane. it is believed that additional data might be obtained by the transplantation of embryonic &ar- derian tissue into the anterior chamber which would aid in establishing the complete histogenesis of this tis sue. 54. CHAPTER VI SUMMARY 1. ®he -üarderian gland from the mouse has been grown successfully in the anterior chamber of the guinea pig eye, not previously re corded in the literature. 2m A. method has been established for the effective staining of secreting ^rderian acini, Harris* Hematoxylin and ^ethylene #lue have been incor porated to bring out the lipoid character of the columnar epitnelial cells as well as the acinic nuclei. 3. It has been found that transitional stages occur in the stromal and acinic nuclei, transitional stages are evidenced by the appearance of mitotic figures in normal tLarderian tissue which indicate an increase in the proliferative rate, transitional stages in the stromal nuclei were first noted in the thirteen millimeter mouse embryo, the aoinio exhibited mitotic figures at the age of one week. 4. An extensive survey nas been made of the maturation process in the iiarderian gland of the normal mouse, the maturation process is accompanied by several well defined changes- (A) an increase in the number of functioning acini, \Hj the appearance of a basement membrane from the surrounding mesenchymous stroma, (Uj a decrease in the amount of interstitial connect ive tissue between the acini and (D) the variation in distance between the acinic nuclei and the base- 56. ment membrane. '-Observations were made possible by employing miorometric methods with the aid of a calibrated eyepiece, ^arderian tissues from mouse embryos and mice ranging in age from birth to maturity were examined. Charts I-III were made in order that an over-all view of the ^rderian gland's developmental processes might be Observed. 5. A special procedure was devised for toe trans plantation of the mouse harderian tissue into the anterior chamber of tne guinea pig eye. the com bination of a local and a general anesthetic in conjunction with a different type of surgical in strument materially aided the successful end re sults. Àhe surgical procedure required twelve minutes to perform. 6. • ‘ •he anterior chamber was selected as a site for toe transplantation of mouse uarderian tissue because of the cornea's transparent quality.By using the anterior chamber tne transplant could be Observed constantly,noting tne interval of time prior to tne establishment of a vascular supply and the rate of growth of tne transplant. 7. The applicability of this problem to future work with the -cLarderian gland is stressed from the viewpoint that the writer has established data on the normal tissue, ihis information, it is felt, may be of value to other investigators work ing with abnormal or pathological hoarderian tissue. 56. ibe writer is aware of the fact tnat normal histological standards must be established before extensions of the potentialities of the mouse ^arderian gland, in the field of cancer research, may be realized. 57. BlBIiIOGHAPHY Arey. -5, B, Armstrong, H, Heiogolowy, Helling, Gohnheim, Gonn, H. J. Barrow, m . A, Gonn, H, J, . 6 eke te, a. iJisher, A, Hreitaa, 'f. B@ Gould, G, M, ' j ’ reene, G. 1940 -k^evelopmental -«riatomy, 4th -i^dition, -deviged. W, B, launders '-Company* Philadelphia and iondon. pp 482-494. 1944 A btudy of tne Harder Ian Gland of ^elected Heptiles with Special Heferenoe to ^an** tusia vigills. Master of ^oienoe thesis. University of Southern California -uibrary. 1903 ^ur Aatiologie dor hds- artigen '^esohwulsten. Med. *<ohn- sohr. 53:1432-1444. 1885 Ueber k&nstliohe %ber- culosis. Ggg, Abhand A, Hirsoh- wald. ^erlin. 591. 1946 staining Procédures. 1st •C'dition. Bioteoh Publications, ^eneva, isew York, ppl-22. 1946 Biological Btains. 5th Edition. Hevised. -oiotech Pub lications, Geneva, Hew York, pp 11-13,215-273. 1941 -biology.of the laboratory Mouse. Bdited by the ^taff of the -ciosooe Jackson Memorial laboratory. A'he Biakiston com pany, Philadelphia, pp 109-110. 1882 Ueber transplantetionen von organ isohem material. Beutsohe "taohr.f.chir. 17:61-92 1936 A Method of Cytologioal Btaining by J-ron He mate in. Mem. Inst, cgw, Orum. 31:713-117. 1941 Gould*s Medical -Giotionarr* 5th devised -ü'dltion. i‘ he Biakis- ton Company, Philadelphia. 1935 Anatomy of the -aat. ^he American Philosophical Society, Philadelphia, pp 86-95. 56. Greene, b. S, lî, 1941 Heterologous Transplantation of Mammalian Ainora. i. The Trans fer of - ‘ ‘ ‘ abhit i'umors to ÂHen Bpecies. Axp. Med. 73:4: 461-466* 1942 -khe Participation of the Anterior chamber of the Aye in Hesietanoe Phenomena ^elated to Tumor Growth* Cancer Research, 2:10; 669-674. ^ m . - „ - - 1942 heterologous -transplantation 01 a human ^ibrosarcoma. ^ancer Research, 2:9: 649-654. ------- 1943 -«- ' h e Heterologous -transplanta tion of Ambryonio Mammalian Tissues. Gancer Hesearoh. 3:12: 809-822. ------- 1944 -the Heterologous -transplanta tion of Human Cancers, t^ancer •ctesearch. 4:6: 352-363 • 1945 The Production of Carcinoma and «carcoma in Transplanted -Em bryonic Tissue. Coience. 101:2634: 644-645. • 1945 "Ctudieg on the variation of the -ttous Aarcoma ^irus following crowth of the Tumor in the Anterior Chamber of tne Guinea Pig Aye, Cancer Research 6:6: 356-364. ------- 1946 The Microscope or the <fulnea Pig? Yale *J. -Biology and Medicine. 18:4: 239-24 2. • ^ ^ - 1947 Growth of Avian Tumors other Than the -aous Sarcoma in the Anter ior chamber of the cuinea Pig Ay@, Cancer Aesearoh. 7:1: 15-20. Barr iso n, -B, M, 1942 laboratory Outline for cqo- Aiken, J. logical Microtechnique, university of Southern California Press, pp 19- 27. Barrison, -o. M. 1936 The Prostate Gland of tne -Barnes, a , W, -c*ight CM Human with Evidence of Predisposition to Malignancy. The Urologie and cutaneous Aevicw. àL: 7:3-7. 59. Beokel, tJ. Aretsohmer, B, Biliarp, fi. A. Hundley, M, Jo ne 3, A. G, Pigge, u, H. £', Arebs, A, T, Gurohot, c. Hr io he sky, Belt, B, Benjamin, J. Bathrop, G, A, Beplat, c. Beydlg ■bopez, J. B, MoClung, c, A. 1935 Physiological A©aponses of Transplanted Prostatic Tissue in the Anterior chamber of the Ay@g of Aahbits. ^urg. Gy ne s. ard, Obst., 61-1. 1946 Structural Proteins and Orientated Blpoids in the cyto plasm of <06 ore ting and Aesorbing Apitheliai c@iig, Aota. Anat. (Basel) 2(2): 119-141. 1944Pluoresoenoe *=>tudies on Cancer, i. Porphyrin Metabolism, harderian Gland Pluoreaoence, and Susceptibility to Carcinogenic Agents. XI. The A©d fluorescence of the Gentalia of *»omen. Cancer Research. 4:8: 465-482. 1946 crowth of Trophoblaat in the Anterior chamber of the -eye of the -txabbit. ^oienoe. 103:2662: 26. 1940 Total Prostatestomy in the •«■abbit and intra-ocular Trana- plantation of Prostatic Tiasue: Anatomic-Surgical Procedure. J. of Urology. 44:1: 109-115. 1944 Miorotechnio of the Patho logic Aye. Technical -^lletin of the Aegistry of Medical Techno logists. 5:6: 131-135. 1946 Microteohnio of tne Path- logic Aye. American J. Of Clinical Pathology. 16:7: 448-456. 1937 development of Aacrimal T^uots and their Valvulae. hull. et. mem. hoc. Tranq.d‘opht. 50:9-14. 1850 Bur anatomie der mdnnlichen '^eschlechts und Analdrtisen der dfiugetiere. ^tschr. f.wiss. "ool. Ad. 2:1-57. 1946 hew Btaining ^‘ ^ethod far histologic Tissue Beotions. Amer. c. of Clinical Pathology. 16:3:53. 1939 Handbook of Microscopical Technique. Paul B, Hoeber, inc., hew Yor and -uondon. pp279-521. 60, McManus, c, £\ 1946 The demonstration of certain Patty substances in Paraffin Beo- tions. J. Pathology and Aacteriology. 58 11): 93-96. Marlcee, A, Maximow, a , A, Bloom, deal, h, V, Aa nd, n, W, Hi, T. Patten, b, M, Aunner, m . H, Saltykow, B. Petrow, d, M, Beki, M, Bluter, B, J, Bmitn, P. A, Copennaver, «». M, 1940 studies on intra-ooular endo^ metrial transplants in tne rhesus monkey, contributions to ^43bryology, ■Baltimore. 1942 A Textbook of xiiatology. »<. B, Baunders company, Phila delphia and -uondon. 27:180-193. 1939 comparative Anatomj^. ^he Blakiston company. Philadelphia, pp 688-693. 1933 The -Enbryological develop ment of the Aaorimal ''land in the dorse. master of Bcience thesis, university of Bouthern california Aibrary. 19.31 The -Embryology of the Pig. -f-he -ciakiston company. Philadelphia, pp 114, 164. 1946 o-uman -Embryology, - ‘ -he Blakiston company, -e'hi lade Ip hi a. 1947 -development of mouse -^ggs in the Anterior chamber of the Aye, Anatomical Aeoord. 98:1-13. 1921 ueber - ‘ -ransplantationen zu- sammengeaetzer theile. Arch.f.ent- wioklingsmechanik. 68:1140. 1941 determination of i-soelectric Point of Tiggue by means of stain ing Apithelial cglls of’ ^Aaorimal duota. Aeliforsch.w.Anat.(Abt.A). 31:201-2. 1947 A Btudy of the uarderian ciand in the Morse, the carter Bnake, and the human with reference to its Medical xmportanoe in malignancy. Master of science thesis. University of southern california Aibrary. 1944 Bailey s -textbook of histology, - ‘ •he ill lams and "llkins company, pp 677-718. 61. Bmelser, c. K. Bnell, c, i>. Btibbe,rA, P. Btiles, A, A, Btrong, d, C, 1943 Aeaotion of Orbital Tissues in experimental ex-op ht iialmos following removal of Mar der ' s cjancj, Anat. Mecord. 85:245-259. 1941 diology of tne Aaboratory mouse. The -oiakiston company. Philadelphia, pp 1-55,475-479. 1928 The Punotioning Kiotitating membrane in Ao^^er vertebrates. «#. of Anatomy. 62:159-176. 1942 handbook of Microscopic Characteristics of Tissues and Organs. The Blakiston company. Philadelphia, pp 172-182. 1942 The Origin of Bome inbred Mice, cancer research. 2:8:631-539. Tomioka, t. van Aooremaal, «>. G, «alls, c, Ti, 1942 Bex differences in Pigment content of harder Ian uplands in Mice. Proo. boc. Aoper. Biol, and Med. 50:123-25. 1935 dxcretion of crganio dyes Through the Tiacrimal elands, wap. castroenterol, %%ippl;. 7:656-662< 1873 dntwiokelung der in fremden grund versetzen lebedeh gewebe. Arch.ffophthl. 19:359. 1938 The Microtechnic of the My@, Btain Technology. 13:2:69. ' - r y of Ssuthmrn CAMfarnI; y

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Creator Stevens, John Rheinhardt (author)

Core Title Histological changes induced in the harderian gland of the mouse by transplantation in the anterior chamber of the guinea pig eye

Degree Master of Arts

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c39-275310

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