A QTUDY OF EOVINE SYNOVEAL FLUID FROM THE CLfNiCALLY NORMAL T'lBlO-TARSAL ARTICULATEON Thesis for {he Degree of M. 5. MICHEGAN STATE UNIVERSITY Rollo Winslow Van Pelt, Jr. 1961 masses LIBRARY Michigan State University ABSTRACT A STUDY OF BOVINE SYNOVIAL FLUID FROM THE CLINICALLY NORMAL TIBIO—TARSAL ARTICULATION by Rollo Winslow Van Pelt, Jr° An investigation has been made of certain aspects and properties of synovial fluid from the clinically normal bovine tibio—tarsal articulationo Studies of blood and plasma sugars in relation to synovial fluid sugars in fast- ing slaughter cattle and non—fasting cattle of mixed breeds, ages, and sexes have been conducted° A laboratory analysis and observation of the following synovial fluid properties has been made: (1) synovial fluid sugar levels; (2) total, absolute, and differential counts of normal synovial fluid leukocytes; (3) relative viscosity? (4) quality of mucin clot as determined by precipitation of synovial fluid with 7N glacial acetic acid; (5) nature of the synovia at the time of collection, zigo, color, absence and/or presence of flocculant material in the synovia, and degree of opacityo A review of the literature revealed few references on the properties of bovine synovial fluid, References in veterinary literature were limited primarily to gross Rollo Winslow Van Pelt, Jr“ observations. The diagnosis, prognosis, and evaluation of synovial fluid from diseased joints in man, as well as the evaluation of anti-arthritic and anti-bacterial agents, has been reported to be of practical importanceo Blood and synovial fluid samples were obtained from two sources: (1) cattle that had been fasted 12 to 18 hours prior to the time of slaughter; (2) cattle that were on full feed at the time of sample collectiono All blood, plasma, and synovial fluid sugars were determined by a modification of the Folin—Wu method, Total synovial fluid leukocyte counts were made with the use of a standard hemacytometer° Differential cell counts for synovia were made from smears stained with Wright's stain° The relative viscosity of synovial fluid was determined at 10105 degrees fahrenheit in a Krebs Constant Temperature Bath employing either the Cannon-Fenske routine or Cannon-Ubbelohde semi- micro dilution viscometerso Quality of synovial fluid, ioeo, degree of polymerization, was determined by a method of precipitation with 7N glacial acetic acido Blood, plasma, and synovial fluid sugar levels were determined for both the fasting slaughter cattle and non-fasting cattle in this study° From a diagnostic stand- point, it is the relationship between blood and/or plasma sugar levels to synovial fluid sugar levels that form a useful criteria in the diagnosis and prognosis of joint Rollo Winslow Van Pelt, Jro diseases, The results revealed that whole blood sugars in both groups of cattle most consistently paralleled syn- ovial fluid sugars and that plasma sugars greatly exceeded those of blood and synovia, as well as exhibiting a greater degree of fluctuation in relation to the lattero Cellular studies brought to light the low total number of leukocytes in the synovia of the tibio—tarsal articulationo Differential studies made on stained smears of synovia in this investigation reveal that lymphocytes and monocytes constitute the major cellular elements, fol- lowed by clasmatocytes and neutr0phils, with eosinophils rarely observed° Age and sex of the individual influenced the re— sults of blood, plasma, and synovial fluid sugars, as well as the relative viscosity and quality of mucin clot° A STUDY OF BOVINE SYNOVIAL FLUID FROM THE CLINICALLY NORMAL TIBIO-TARSAL ARTICULATION By Rollo Winslow Van Pelt, Jr, A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Surgery and Medicine 1961 ACKNOWLEDGMENTS I would like to express my sincere appreciation and gratitude to the following persons whose contributions made this thesis possible. To Doctor Gabel H. Conner, Professor, Department of Surgery and Medicine, who served as my major professor and whose advice and guidance served as a constant source of encouragement and inspiration, so necessary in the at- tainment of a successful investigation and its culmination in this thesis. To Doctor Albert R° Drury, Department of Surgery and Medicine, for the use of laboratory equipment and facil- itieso To Miss Susan W. Richards, MOTO, and Mrs° Maryann Mills for their assistance in the laboratory analysis of specimenso To the William Van Alstine Company for permitting the collection of specimens from slaughter cattle and Mro Ralph Ried, Department of Dairy Science, Nutrition Experi- mental Barn, for assistance in the collection of specimens from experimental animals° Finally to the numerous persons whose assistance in various aspects of this investigation made possible this thesiso ii Dedicated to my wife, Karen iii CHAPTER I. 110 III. TABLE OF CONTENTS INTRODUCTION 0 O O I O 0 O 0 O 0 0 O 0 REVIEW OF THE LITERATURE . Introduction. . . . . . . . . . . . . . Gross and Microscopic Anatomy . . . Physiology of Synovial Structures . . . Cytology. Sugars. . 0 0 O O O 0 O 0 O 0 0 O 0 O O O O O Viscosity and Mucin . . . . . MATERIALS AND METHODS. . . . . . . . . A. Collection Procedures . . . . . . . l. 2. BlOOdO 0 O O O O O 0 0 O O O Synovial fluid . . . . . . . . B. Laboratory Procedures . . . . . . l. 20 3. Blood sugar determinations . . Plasma sugar determinations. . Synovial sugar determinations. 0 Blood, plasma and synovial sugar procedures 0 o o" o o o o o o 0 Cellular studies . . . . . . . O a. Differential leukocyte counts. b. Total leukocyte counts . c. Absolute leukocyte counts. Relative viscosity . . . . . Mucin clot . . . . . . . . iv PAGE \0 e- as -p 16 2o 25 34 35 35 36 42 42 42 42 43 4a 4a 49 5o 51 63 CHAPTER PAGE IV. RESULTS. . . . . . . . . . . . . . . . . . . . 67 V. DISCUSSION . . . . . . . . . . . . . . . . . . 106 General Considerations and Observations . . . 106 Blood, Plasma, and Synovial Fluid Sugars. . . 108 Cellular Studies. . . . . . . . . . . . . . . 119 Viscosity and Mucin Clot. . . . . . . . . . . 127 VI. SUMMARY AND CONCLUSIONS. . . . . . . . . . . . 138 VII. BIBLIOGRAPHY . . . . . . . . . . . . . . . . . 141 TABLE II. III. IV. VI. VII. VIII. IX. LIST OF TABLES IDENTIFICATION OF ANIMALS AND COLLECTION DATA ON SYNOVIAL FLUID FROM THE CLINICALLY NORMAL TIBIO-TARSAL ARTICULATION . . . . . . . . . . BLOOD, PLASMA, AND SYNOVIAL FLUID SUGARS IN THE FASTING STATE: Sugar Values in Milligrams Per loo Milliliters O 0 O O O O 0 0 O O O O 0 BLOOD, PLASMA, AND SYNOVIAL FLUID SUGARS IN THE NON-FASTING STATE: Sugar Values in Milli— grams Per lOO Milliliters . . . . . . . . . . RELATIONSHIP OF BLOOD, PLASMA, AND SYNOVIAL FLUID SUGARS TO AGE IN THE FASTING STATE: Sugar Values in Milligrams Per 100 Milliliters RELATIONSHIP OF BLOOD, PLASMA, AND SYNOVIAL FLUID SUGARS TO AGE IN THE NON-FASTING STATE: Sugar Values in Milligrams Per 100 Milliliters COMPARISON OF THE AVERAGE BLOOD, PLASMA, AND SYNOVIAL FLUID SUGARS IN FASTING AND NON- FASTING ANIMALS: Sugar Values in Milligrams Per 100 Milliliters . . . . . . . . . . . . . RELATIONSHIP AND COMPARISON OF BLOOD, PLASMA, AND SYNOVIAL FLUID SUGARS T0 SEX IN THE FAST- ING AND NON-FASTING STATE: Sugar Values in Milligrams Per lOO Milliliters. . . . . . . CELLULAR STUDIES MADE ON THE SYNOVIAL FLUID OF THE CLINICALLY NORMAL TIBIO- TARSAL ARTICU- LATION: White Blood Cell and Differential Cell Counts . . . . . . . . . . . . . CELLULAR STUDIES MADE ON THE SYNOVIAL FLUID OF THE CLINICALLY NORMAL TIBIO- TARSAL ARTICU- LATION: Total Leukocyte and Absolute Leuko— cyte Values . . . . . . . . . . . . . . . RELATIVE VISCOSITY OF SYNOVIAL FLUID AT lOl.5° F. AND QUALITY OF MUCIN CLOT FROM THE CLINICALLY NORMAL TIBIO-TARSAL ARTICULATION . vi PAGE 79 84 87 89 9O 91 92 93 97 100 TABLE XI. XII. PAGE RELATIONSHIP OF AGE TO THE RELATIVE VIS- COSITY OF SYNOVIAL FLUID AT 101.5° F. AND QUALITY OF MUCIN CLOT FROM THE CLINICALLY NORMAL TIBIO-TARSAL ARTICULATION. . . . . . 104 COMPARISON OF THE RELATIVE VISCOSITY 0F SYNOVIAL FLUID AT 101.5° F. AND QUALITY OF MUCIN CLOT FROM THE CLINICALLY NORMAL TIBIO- TARSAL ARTICULATION IN RELATION TO SEX. . . 105 vii FIGURE 1. LIST OF FIGURES PAGE The anteromedial aspect of the hook joint has been clipped, anesthetized, and prepared for arthrocentesis . . . . . . . . . . . . . . . . 38 A 16 gauge, two—inch needle has been directed into the tibio-tarsal joint cavity from the anteromedial position. . . . . . . . . . . . . 38 A 20 milliliter metal-tip glass syringe has been attached to the needle for withdrawal of synovial fluid . . . . . . . . . . . . . . . . 40 Aspiration of synovial fluid has been completed and the external site of needle puncture into the joint cavity closed with a Michel's suture clip . . . . . . . . . . . . . . . . . . . . . 40 The Krebs Constant Temperature Bath (A.S.T.M. D445-53T) designed for use with capillary viscometers. . . . . . . . . . . . . . . . . . 53 The Cannon-Fenske Routine Viscometer (A.S.T.M. 150) with adapter for the Krebs Constant Tem- perature Bath. . . . . . . . . . . . . . . . . 57 The Cannon-Ubbelohde Semi—Micro Dilution Viscometer (A.S.T.M. 150) with adapter for the Krebs Constant Temperature Bath. . . . . . 60 Four grades of mucin clot obtained by precip- itation with 7N glacial acetic acid. . . . . . 66 The cells of normal bovine synovial fluid from the tibio-tarsal articulation. . . . . . . . . 122 viii LIST OF CHARTS CHART PAGE I. COMPARISON OF THE AVERAGE SUGAR VALUES IN TWO GROUPS OF CATTLE O O 0 0 0 0 O 0 0 O O 0 0 O O 111 II. RELATIONSHIP OF AVERAGE SUGAR VALUES TO AGE IN NON-FASTING CATTLEo o o o o o o o o o o o o 115 III. RELATIONSHIP OF AVERAGE SUGAR VALUES TO AGE IN FASTING CATTLE. . . . . . . . . . . . . . . 115 IV. COMPARISON OF THE AVERAGE SUGAR VALUES IN TWO GROUPS OF CATTLE IN RELATION TO SEX. . . . . . 116 V. RELATIVE VISCOSITY AND QUALITY OF MUCIN CLOT IN RELATION To AGE 0 O O O O O 0 0 O O 0 O O 0 134 VI. RELATIVE VISCOSITY AND QUALITY OF MUCIN CLOT IN RELATION To SEX O O 0 O O O O 0 O O O O 0 134 ix CHAPTER I INTRODUCTION To date there has been little or no research in veterinary medicine designed to arrive at either a partial or compIete analysis of bovine synovial fluid. Therefore, this study was designed to determine certain aspects of normal bovine synovial fluid. Once a true picture of the findings in normal synovial fluid is obtained, it could then be used to evaluate the variOus pathological processes encountered in diarthrodial articulations. The synovia of diarthrodial articulations serves not only as a source of nourishment to the articular sur— faces, but also plays a mechanical role by the very nature of its mucopolysaccharide content. It is conceivable then that normal synovial fluid reflects a joint that is func- tioning in a normal mechanical and physiological manner. On the other hand, joint fluid would serve to reflect changes produced in the synovial membrane and articular cartilages by disease processes. An abnormality in the exchange of substances between blood and/or lymph and the joint fluid would be reflected in the fluid as an intra-articular dis- turbance of normal metabolism. The information gained from analysis of synovial fluid would be of value in diagnosis and prognosis of joint disease as well as a means of evaluating anti—arthritic (particularly the adrenocortical steroids and their syn- thetic analogs designed for intra-articular use) and anti- bacterial agents. The combination of clinical and laboratory find- ings would be of unequivocal value in making a differential diagnosis of the various arthridites encountered in bovine practice. With these thoughts in mind, this study was con- ducted in an effort to find a series of laboratory tests that could eventually be used by the bovine practitioner as an aid to making a diagnosis and evaluation of the path— ological state of the joint or joints in question. These tests should be designed so that they could be easily and inexpensively carried out without the use of extensive lab— oratory facilities. This study was then designed to determine the following values for bovine synovial fluid in animals of all sexes, ages, and breeds, from the clinically normal tibio-tarsal articulation: (1) blood, plasma, and synovial fluid sugar levels in both fasting slaughter cattle and non-fasting cattle; (2) total, absolute, and differential count of normal synovial fluid leukocytes; (3) relative viscosity; (4) quality of mucin clot as determined by precipitation of synovial fluid with 7N glacial acetic acid; (5) nature of the synovia, i.e., color, absence or presence of flocculant material, and degree of opacity. CHAPTER II REVIEW OF THE LITERATURE Introduction A review of veterinary literature on the subject of synovial fluid serves only to reveal the paucity of in- formation on this subject. To date the work done in this field has been scant and inconclusive. The review to follow will serve to acquaint the reader with the basic fundamentals essential for a better understanding of the role synovial fluid plays in maintenance of a normal physiological balance in diarthrodial articulations. Gross and Microscopic Anatomy Diarthrodial articulations have been defined by Sisson and Grossman (1950) as those joints characterized by the presence of a joint cavity with a synovial membrane in the joint cavity and by their mobility. They are often called movable or true joints. The articular surfaces of the bones are covered by a layer of hyaline cartilage which provides a smooth gliding surface when movement occurs. The bones of the joint are maintained in direct apposition by ligaments which pass from bone to bone around the joint. This relationship- thus forms a cavity bounded on the ends by the articular cartilages of the bones and surrounded by the synovial mem- bfiane. A negative intra-articular pressure aids in main— taining the articular surfaces in apposition and drawing the villous projections of the synovial membrane in around them. Ram (1957) described the synovial membrane as smooth, glistening, and well supplied with blood vessels, lymphatics and nerves. The lining cells are called synovial cells and are of mesenchymal origin. Due to the smooth continuity of the synovial membrane, it has the appearance ‘ of being a continuous membrane; however, there are numerous collagenous fibers which lie among the synovial cells and help make up the membrane, especially in the deeper structures. Not all authors classify or term the cells lining the membrane as synovial cells. Arey (1957) described this tissue as a loose vascular layer, surfaced discontinuously with fibroblasts, sometimes called a false epithelium be— cause it is a layer made of white fibers and scattered, flattened fibroblasts, hence he terms it a connective tissue rather than epithelium. Trautmann and Fiebiger (1957) likewise described the synovial membrane as a layer of flattened fibroblasts resting on a collagenous connective tissue. Synovial villi and Haversian fringes are descrip- tive terms applied by Copenhaver and Johnson (1958) to the synovial processes that project into the joint cavity. Some of these are non-vascular and consist mainly of stel- late cells similar to those of the synovial membrane. Others have a distinct core of fibrous tissue containing blood vessels and covered with stellate connective tissue cells. From the primary villi, small secondary non-vascular villi are frequently given off. The larger blood vessels of the joint capsule lie in the outer layer of the stratum synoviale where smaller vessels and capillaries pass to the inner layer and some to the villi. The inner layer is richly supplied, and the outer layer poorly, with lymph capillaries. Non-medulated nerve fibers are found in the connective tis- sue, some of them ending in Pacinian corpuscles. Samuel (1950) made a study of twenty normal human knee joints and revealed that the nerves accompanying the blood vessels in the articular capsule migrate from one to another, forming a coarse network of nerve bundles in the capsular ligament and a more delicate network of individual fibers in the synovial membrane. His observations showed that the synovial membrane is only slightly sensitive to pain and does not give rise to pressure sensations. He concluded that it would then appear that almost all sensory impressions from the articular capsule arise in the fibrous capsular ligament. Bunn and Burch (1955) observed intra-articular pain in horses, lasting from eight to twenty-four hours following arthrocentesis. They attributed pain to increased intra-articular pressure as a direct result of air intake due to a greater negative pressure following withdrawal of synovial fluid. The hyaline cartilage that covers the ends of the bones is free of vascular supply and receives its nour- ishment by osmosis. Maximow and Bloom (1960) have shown that articular cartilages are intimately adhered to a layer of compact bone, lacking haversian systems, possessing large lacunae, and free of canaliculi. At the base of the hyaline cartilage, a small area of perichondrium is reflected back- ward into the membrane of the joint capsule. At this point there are many cartilagenous cells extending into the syn- ovial membrane. They noted that there is an increase in size and number of villi with age, with new islets of carti- lage being formed in them mainly by metaplasia of the syn- ovial fibroblasts. Johnson (1959) noted that tiny granules and threads from the superficial cartilage zone were com- monly found in normal synovial fluid. Reformation of synovial membranes following syn— ovectomy in the rabbit was demonstrated by Key (1925). It was shown that following removal of a portion of the syn— ovial lining that there was a rapid deposition of fibrin which was quickly organized by young connective tissue cells from the fibrous capsule. These were soon differentiated into synovial cells so that in sixty days, the newly formed synovial lining could not be differentiated from the undam- aged area. Synovial tissues apparently play an active role in joint metabolism. Wilson,‘gt_§l. (1956) in a study of the metabolites of intra-articular cortisone and hydrocorti- sone found this tissue to be quite active. Cortisone in the joint must be converted to hydrocortisone in order to develop anti-inflammatory activity and this reaction cannot be affected to a sufficient degree by the synovial tissues. An alternate to this theory would be that both cortisone and hydrocortisone must be transformed into available pro— ducts in order to exert their effects and that synovial tissue can make this substance from hydrocortisone, but not cortisone. The metabolites of cortisone are different than those of hydrocortisone. Harris, gt_él. (1958) injected radioactive sodium into the knee joint of patients with rheumatoid arthritis and showed that reduced clearance rates occurred in the presence of hydrocortisone. These falls in clearance rates usually coincided with good clinical response when compared with subjects who showed no significant fall in clearance rates of radioactive sodium. The author (1960) has stated previously that intra— articular adrenocortical steroids exert their greatest effect upon the synovial membrane and as a consequence change the character of the synovial fluid. Changes in the state of the synovial fluid act as an index to determine the effi- cacy of the particular steroid employed. Physiology of Synovial Structures Gardner (1959) observed that the term synovia was introduced in the sixteenth century by Paracelsus (1493- 1541) who used it to refer to various body fluids. Its use is now confined to the fluid present in certain joints (pre- sumably in bursae and synovial tendon sheaths also), which are consequently termed synovial. In 1887, McFadyean, (Hughes and Dransfield, 1953) an English veterinary anatomist, described synovial fluid as the glairy, straw-colored fluid which plays the part of a lubricant to the articular surfaces. Although somewhat oily in appearance, due to traces of fat, its glairy char- iactsr was probably due to mucin and albumin. Synovial fluid, as might be expected from the hypothesis of Ropes and Bauer (1953), is a tissue fluid, showing changes with disease which reflect alterations in the synovial tissues and intra-articular metabolism. The normal exchange of substances between the vascular and lymph— atic system may be disturbed or there may be altered util- ization, formation or destruction of various constituents 10 within the joint. Examination of synovial fluid should therefore yield information concerning the type and degree of change in the joint. Davies (1944) in studies of synovial fluid from the tibio-tarsal articulation of clinically normal cattle, observed that approximately 80 per cent were colorless and that the remainder showed a slight tinge of yellow pigment, though a deep yellow coloration was most unusual. No at- tempt was made to determine the nature of the pigment or pigments present. The volume of the synovial fluid of cattle taken immediately after slaughter is quite large and Meyer (1947) reported volumes sometimes over 50 cc. per joint. He sug- gested the possibility of the fluid actually being a mixture of synovial and edematous fluids because of long periods of standing and trauma (rough handling) of animals before slaughter. Normal human synovial fluid does not clot as Ropes, giggl. (1940) discovered. They postulated that this was presumably due to the absence of fibrinogen, since no fi- brinogen was found by precipitation with 1.1 M phosphate solution at pH 6.5. Synovial fluid has been defined by Bauer, gt_§l. (1940) as a protein-containing dialysate of blood plasma to which mucin, secreted by the synovial cells, is added as the plasma water diffuses through the synovial tissue 11 spaces into the larger tissue space, the joint cavity. Ropes, gt_§l. (1939) in making a comparison of joint fluid with that of plasma indicated that non-electrolytes diffused readily in either direction between blood and synovial fluid and that electrolytes were distributed in accord with the Gibbs-Donnan theory of membrane equilibrium. Synovial membrane permeability was defined by Moffett (1954) as the capacity of synovial tissues to allow the passage of fluids and colloids in either direction be- tween the joint cavity and the synovial blood vessels, lymph— atics and surrounding tissues. Sunderman and Boerner (1950) maintained that synovial fluid keeps in close equilibrium with the blood stream, owing to the fact that the synovial membrane is very permeable as compared with the choroid plexus or the glomerular membrane. The properties which distinguish synovial fluid from dialysates of blood plasma are attributable to mucin. This characteristic component of synovial fluid has been found to be a protein-polysaccharide complex or hyaluronic acid, that is produced by the synovial cells lining the membranous portion of the joint cavity. Kling, §t_gl. (1955) observed the presence of hyaluronic acid in about two-thirds of the supernates of tissue cultures from synovium. This was demonstrated by precipitation reactions with acetic acid, enzymatic reactions, viscosity determinations, tests for metachromasia, and electrophoretic patterns. Control 12 cultures of periarticular connective tissue were negative for hyaluronic acid and sulfonated mucopolysaccharides. Castor (1959) has demonstrated the in vitro syn- thesis of hyaluronic acid by cultures of human synovial cells. Paul (1959) stated that synthesis of a few struc- tural elements in vitro can occur, particularly that cer- tain cultured cells can synthesize large amounts of muco- polysaccharides. Asboe-Hansen (1954) in previous work does not agree with this concept. His work has shown that whenever mast cells are found in the body, there is hyaluronic acid production; thus with an increase in number and size of these cells, there is a corresponding increase in the se- cretion of the mesenchymal muco-polysaccharide-hyaluronic acid complex. In conclusion he postulated that secretion of this complex was influenced by a heparin-like precursor. The protein content of bovine hyaluronic acid (protein-poly- saccharide) complex has been identified by Curtain (1955) as being derived from serum and composed largely of alpha globulin, with smaller amounts of beta and gamma globulin as determined by immunological and electrophoretic studies. Neuhaus and Letzring (1958) in a later study ob— served that the pr0portion of albumin is greater in human and bovine synovial fluids than in their respective serums, suggesting that a slight generalized permeability (or per- haps selective permeability) exists for molecules of a l3 molecular weight of 65,000 or less. In cases of arthritis, Platt, gt_al. (1957) have shown that the proteins of synovial fluid may resemble those of the blood serum and that the electrophoretic behavior during the active stages of the disease more closely approach that of blood. The polysaccharide component has been identified by Meyer, gt_§l. (1939) as hyaluronic acid, a non—ramified polymer of D-glucuronic acid and N-acety-D-glucosamine in equimolar amounts, connected through beta-glucosidic 1-3 linkages. These workers determined that this polymer, i.e., the polysaccharide, was composed of equimolar parts of hexo- somine, a hexuronic acid and acetyl. The hemosamine has been demonstrated as D-glucosamine, and the uronic acid as glucuronic acid. Meyer (1947) in later work stated that the gel- like consistency that hyaluronic acid produced in normal joint fluid, also acts to protect surfaces and forms a vis- cous barrier which also serves to regulate the exchange of metabolites and water. Spector (1956) reports an average pH of 7.31 with a range of 7.27 to 7.43 for synovial fluid from the tibio- tarsal articulation of normal cattle. In an extensive study involving the canine species, Joseph, §t_§l. (1946) showed that the pH of joints or venous blood is more acid during exercise and that blood pH falls more rapidly than that 14 of the joint during exercise. Their studies involving intra- venous administration of acidic and basic solutions to dogs brought to light several facets of ion exchange between blood and the joint fluid. Following intravenous injections of weak solutions of Na2003 the blood pH rose rapidly but soon returned to normal, while joint pH rose more slowly and reached a maximum later than blood pH. Studies on the ef- fect of acids revealed the fact that an intravenous injec— tion of 65 cc. of a five per cent solution of NH4Cl caused blood pH to drop from 7.4 to 7.08 in four minutes, with re- covery occurring in twenty minutes. Meanwhile the joint pH fell from 7.5 to 7.25 in ten minutes and did not recover in twenty minutes. In conclusion, they observed that exer— cise played a major role in the pH of synovial fluid and that exercise apparently facilitated the exchange of ions across the synovial membrane since the pH dropped more rapidly within the joint than it did following intravenous injection of the electrolyte solutions. The fact that the composition of protein varies with the pH and ionic strength of the solution was demon- strated by Curtain (1955) who also demonstrated that the binding power of synovial proteins was greater with a lower; ing of pH. At pH 5.8, alpha globulins predominate; at pH 7 to 8, alpha and beta globulins predominate and at pH 10 the protein content is considerably reduced. At neutrality, increasing ionic strength leads to a marked increase in 15 the amount of the protein complex. In a study on intra-articular temperature, Horvath and Hollander (1949) reported that the amount of synovial fluid present appeared to make little difference in the joint temperature, except to alter the rate of metabolic exchange. A close correlation was noted between the internal joint temperature and the clinical activity of the disease. This indicated that the temperature of the synovium was directly dependent on the relative hyperemia of the synovium. These workers observed that joint temperature in degenera- tive joint disease was higher than anticipated from relative clinical activity. External heat over a joint was usually,’ but not necessarily, related to joint temperature. They concluded that this was a factor that might be related to greater joint friction effects and to retarded cooling. Hollander, gt_§;. (1951) later evaluated the ef- fects of anti-arthritic agents to reduce joint temperature. Following administration of cortisone and adrenocorticotropic hormone, serial synovial fluid counts showed a pronounced decrease in leukocytes which paralleled the fall in jdint temperature. A distinct parallelism was noted by a prompt clinical improvement followed by the fall in joint tempera- ture. This fall in joint temperature was found to be‘a more constant determination of clinical effect than a drop in cell counts. Employing the knee joint of the cat, Hunter and l6 Whillams (1951) reported that exposure to zero and subzero ambient temperatures, resulted in a significant fall in ‘joint temperature. A fall in temperature was associated with an increase in friction of the joint which was freed from muscular attachments, with the exception of cruciate ligaments. They concluded that a physiologically important component of joint stiffness resulting from exposure to cold must therefore result from a local physical change in the joint tissues. Cytology Cytological studies of synovial fluid from the tibio-tarsal and carpo-metacarpal joints of healthy young beef cattle by Warren, gt_§l. (1935) showed that a varia- tion oscurred in the total number of nucleated cells and individual cell types. The widest variation in cell types was observed in the tibio-tarsal synovial fluid. At the time, these workers stated that they knew of no real value in subdividing the large mononuclear phagocytes of synovial fluid. Observations made on 29 synovial samples in man by Bauer, gt_§1. (1940) failed to reveal either eosinophils or basophils, indicating that they are rarely present in normal synovial fluid. These workers further noted that synovial fluid cytology was never a reflection of blood cytology. 17 Robinson, gt_gl. (1955) made a study of synovial fluid changes in patients suffering from rheumatoid arth- ritis and could find no correlation between total cell count and viscosity of the synovia. Likewise there was no apparent correlation between the level of the cell count and a less satisfactory mucin clot formation. Erythrocyte and leuko— cyte cell counts were made, using the same technique as for blood counts, except that normal saline was used as the diluting fluid. Here again, no attempt was made in count- ing the mononuclear cells to differentiate between lympho- cytes, monocytes, clasmatocytes and synovial cells. Their reasons for not differentiating the various mononuclear cells may be based on work by Coggeshall (1953) who concluded that from a diagnostic standpoint, the most significant information comes from the total leukocyte count and the percentage of polymorphonuclear leukocytes. Poly- morphonuclear leukocytes should not exceed 25 per cent of the synovial fluid leukocytes in normal fluids. Davies (1945) observed in cattle that high nucle- ated cell counts generally occur in highly viscous fluids, in joints characterized by freedom from disease and degen- erative changes. Erythrocytes did not normally appear in the synovial fluid and he attributed their presence in small numbers to contamination during aspiration. This worker reported clasmatocytes and monocytes as the predominate cell type in synovial fluid. Phagocytic type cells 18 predominate in the more highly viscous fluids, with less viscous fluids such as found in the tibio-tarsal articula— tion showing a predominance of lymphocytes. -This coincided with the finding of less cellular debris noted in joints that are more acellular and exhibit a predominance of lympho— cytes. I .The work of Coggeshall, gt_§;. (1940) was substan- tiated by these findings, since they had previously deter- mined that the function of the synovial fluid leukocyte in man is the removal of the wear and tear debris incident to daily use. They postulated that variations in total and individual cell types are probably dependent upon the inconsistent tissue trauma resulting from daily use of the joint. Gardner (1950) stated that the cellular constitu- ents of synovial fluid were predominantly mononuclear and included phagocytic and non-phagocytic types. These cells were undoubtedly derived mainly from synovial tissue and reflected its cellular content and that the findings for- one joint were not necessarily similar to those of others. Anderson (1952) noted that in septic arthritis of man, the nature of the synovial fluid was at first serous, but later purulent. The synovial membrane being greatly congested, swollen, and infiltrated with inflammatory cells. Fowler and Kingrey (1956) reported that aspiration of synovial fluid reveals its purulent character in cases of pyemic tarsitis 19 of cattle. However, they made no mention of further exam- ination of the synovia. One could conclude from these ob- servations that the synovial membrane is the source of the inflammatory cells observed in septic synovial fluid. working with rabbits, Cunningham, §_t__a_l. (1924) differentiated two different types of phagocytic cells in the spleen. These two cell types were designated as clas— matocytes, macrophages or histiocytes, and the large mono- nuclear elements of blood, the so-called monocytes or transi- tional cells, which they felt were closely related if not identical. Sabin, gt_§l. (1924) also separated the phagocytic cells of the peritoneal exudate into two distinct types. They were able to demonstrate that the first reaction to take place after injection of any substance into the peri- toneal cavity was the emigration of the polymorphonuclear leukocytes, which appeared in the exudate at the end of four to six hours. In later work, Sabin, gt_§l. (1925) noted that clasmatocytes were outnumbered by monocytes in irritated connective tissue areas because they were slower to become phagocytic, as these cells must maturate before becoming functional. In advanced cases of degenerative arthritis in the bovine, Shupe (1959) noted an increased nucleated cell count, but apparently neglected to ascertain both the total number and type of nucleated cells in this disease. Studies by Mikkelsen, gt_§l. (1958) suggest that 20 the morphologic features of rheumatoid arthritis may per— sist even though the joint fluid has become essentially normal. They concluded that there was often a poor corre- lation between synovial fluid characteristics and histologic changes in the synovial membrane. DeGara (1943) reported on the bactericidal prop- erties of synovial fluid in man and observed that it con- tained phagocytic cells, but that only a few effusions were streptoccidal. Thus he concluded that most of the synovial fluids were practically free of streptoccidal antibodies. Sugars Cajori, gt_§l. (1926) reported synovial fluid sugar values in man as high as those of blood and as an argument in favor of the view that its function was partly nutritional. These workers conducted experiments in which the blood and synovial fluid were analyzed before and after administration of 100 grams of glucose orally. Results revealed a communication of the synovial fluid and alimen- tary tract that was novel and heretofore unsuspected. The rise in blood sugar following glucose ingestionpromptly caused a rise in sugar concentration within the joint, with no evidence of a lag. In two experiments, the concentration of sugar in the synovial fluid, half an hour and one hour after administration of the sugar, was higher than the con— centration of sugar in blood at these times. This may mean 21 that there is a tendency for sugar to accumulate in the joint cavity, though it seemed more likely that it repre- sents an equilibrium with respect to sugar between arterial blood and synovial fluid. The analysis was made on venous blood, which usually has a much lower sugar content, follow- ing sugar ingestion, than has arterial blood. Lower concentrations of synovial fluid glucose in relation to blood glucose were reported by Davies (1946). He felt that this fact was partly explained by a glucose consumption in the joint. 1 Working with calves, Zeller, gt_§l. (1941) noted that the initial rise of synovial fluid sugar lagged behind that of blood at least 20 minutes following intravenous administration of glucose solutions. Blood sugar in two of their experiments returned to almost pre-injection levels in less than two hours. When serum glucose fell, synovial fluid glucose again showed a lag. They concluded the rate of utilization of glucose by articular tissues and the rate of diffusion from the joint space into the synovial capil- laries is not sufficiently rapid to keep synovial sugar at or below that of serum. Hepler (1957) stated that all that is reported as blood sugar is not glucose, but the sum total of reducing substances encountered during the determination. This author listed the non-fermentable portion of the reducing substances as being composed of glutathione, ergothioneine, creatinine 22 and unknown substances. She concluded that the concentra- tion of the non-fermentable portion varied from 10 to 30 milligrams per cent. Hawk, gt_§l. (1954) stated that sugars owe their reducing ability to their free or potentially free aldehyde or ketone groups and were thus able to reduce in the pres- ence of alkaline solutions, the ions of certain heavy metals. They contended that certain organic compounds, particularly those containing one or more alcoholic hydroxy groups in the molecule, e.g., tartaric acid, citric acid, glyerol, and even sugars themselves, react in alkaline solution metalic hydroxides to form a soluble complex ion, which though rela— tively little ionized, nevertheless dissociates to yield sufficient ions of the metal for reduction to occur. To designate this group of non-fermentable copper reducing materials present in blood, Benedict (1931) proposed the term "saccharoids". The word saccharoid has been used as an adjective, and use of the word as a noun did not seem objectionable to him. He stated that the saccharoid content of plasma was almost as high as for whole blood and con- cluded that saccharoids are found in both plasma and cor- puscles in readily detectable quantities, but that in many samples of blood the analysis of unlaked blood by the usual methods may fail to reveal their presence. His observations on blood indicated that a considerable portion of the sac- charoid fraction is represented by glutathione. 23 In their work on the determination of blood sugar, Folin and Wu (1920) concluded that non-glucose reducing substances may enhance glucose values, but this saccharoid fraction appeared to be relatively constant and therefore its presence does not influence unduly the interpretation of variations in the blood sugar. Fashena (1933) determined that glutathione accounts for 37 per cent of the total saccharoid content in human blood by the Folin-Wu method and that oxidized and reduced forms of glutathione exert the same degree of reducing power towards the Folin-Wu reagent. She believed the results of her work establish that 63 per cent of the saccharoid fraction of human blood, as determined by Folin—Wu method is composed of reducing compounds other than glutathione. It is probable that different results would be obtained through the use of reagents other than Folin-Wu for measure- ments of the reduction, when the reducing value of gluta- thione in terms of glucose assumes different properties. Her work seemed to establish positively the presence of a large saccharoid fraction in blood, the composition of which was undetermined. Later Fashena and Stiff (1941) presented data that appeared to indicate that glucuronic acid or its com- pounds were responsible for practically all of the residual reducing power of tungstic acid blood filtrates not accounted for by glucose or glutathione. A very small part of the 24 reduction was due to creatinine, uric acid, and ascorbic acid; however, the total reducing value of these compounds, based on experimental determinations of glucose probably does not exceed two or three milligrams per cent in normal blood as determined by Folin-Wu methods. Cajori and Pemberton (1928) demonstrated the gly- colytic activity of cell free synovial fluid. This was much less marked than was that of the uncentrifuged material. Rapid glycolysis occurred in a portion of synovial fluid containing leukocytes, whereas the glucose level did not change for several hours in cell free fluids. Coincident with the loss of glucose, the lactic acid levels rose mark- edly. This work demonstrated that in synovial fluid con- taining leukocytes, conditions are highly favorable for the rapid disappearance of glucose and the formation of lactic acid. The presence or absence of leukocytes was of first importance in determining the level of synovial fluid sugar. The presence of glycolytic enzymes in synovial fluid appeared to be present in the polymorphonuclear leuko- cyte and confined almost entirely to these cells. This was demonstrated by Hubbard and Porter (1943). In no ex— periment in which glucose or fructose was added to cell- free fluid was there a significant change in concentration after incubation. 0n readdition of cells to a cell-free fluid, glycolytic activity was noted. Interestingly enough, 25 glucose destruction was almost twice that of fructose. In these experiments, both glucose and fructose were added in measured amounts to the synovial fluids under trial. Viscosity and Mucin Newlander (1949) defined viscosity of a substance as its resistance to flow, due to internal friction between molecules as they shear each other. Hausman and Slack (1939) stated that when a liquid flows over a surface, the layer of liquid particles in contact with the surface remains stationary because of adhesion, the next layer of particles moves slowly over the first, the third layer moves with respect to the second, and so on, the speed of each layer increasing with its distance from the solid surface. Shear- ing then occurs in the uppermost layers or layers furthest from the surface. There being a resultant decrease in vis— cosity with a rise in temperature. Eckles, gt_gl. (1936) concluded that viscosity is a property of all fluids, and that it can be expressed only in relative terms. For conven- ience, the viscosity of any fluid is therefore expressed in comparison with water. Repeated shearing or stirring of synovial fluid was reported by Blair, gt_§l. (1954) to cause a small but definite increase in viscosity, the cause of which is un- known. 26 Gortner, gt_§1. (1953) determined the rate of flow through a capillary as the basis for an important method for the measurement of viscosity. In this method, the time of flow of a known volume of liquid through a capillary under the influence of gravity is determined. The appar- atus used in measurement was the Ostwald viscometer. For determination of relative viscosity, Bingham (1922) recom- mended the Ostwald viscometer. It consists essentially of a U—tube with a capillary in the middle of one limb above which is placed a bulb. A given volume of liquid is placed in the instrument and the time required for the liquid, which is only under the influence of hydrostatic pressure, to pass between two marks, one above and one below the bulb, is determined (or measured). To reduce error in the mean head caused by devia- tion of the viscometer from the vertical, Cannon and Fenske (1935) devised a modified Ostwald viscometer in which the upper and lower bulbs lie on the same vertical axis. Such a viscometer is in common use today. Cannon and Fenske (1938) later reported that ef— flux volume is used to measure efflux time. They further stated that efflux volume and capillary diameter should be made small in order to reduce kinetic energy corrections. This factor was also reduced by increasing the driving fluid head, i.e., the distance between the efflux bulb and the lower reservoir. Drainage errors arise from the fact that 27 all liquids do not drain from a surface with equal ease; hence, the measured efflux volume for one liquid may be different than another. They attributed sources of error to too much fluid in the lower reservoir and reduction of the driving head by that amount. In conclusion they stated that relative viscosities are very accurate and if the ref- erence basis is clearly defined, little confusion will exist and reported values of viscosities may be readily compared. In later work, Cannon and Manning (1959) concluded that kinetic energy correction is insignificant in the great majority of cases. In order to remedy errors encountered in other types of viscometers, e.g., the Ostwald, Ubbelohde (1937) introduced the phenomenon of suspended level. He theorized that because of suspended level, smaller amounts can be used and capillary viscosity is therefore independent of the charge in the reservoir. In other type viscometers, the effective pressure driving the liquid through the capillary is influenced by the surface tension of the film lying on the surface of a liquid at a higher level, and by the sur- face tension effect either on the stream emerging from the capillary or on the surface of the liquid in the lower reser- voir. Application of the principle of suspended level thus allows one to balance corrections for these factors against each other and to design an instrument whose dimensions are so prOportioned as to avoid others. Suspended level 28 is the term used to designate the hanging surface of a liquid and to distinguish it from recumbent level forms by the film lying on the surface of a liquid. In the case of Ubbelohde's viscometer, this film is formed by a hemispherical surface, concave downward. It is formed at the point where a vertical capillary expands into a relatively large tube by the layer of a liquid spreading upon the interior surface at this junction. This principle therefore reduces the need for surface tension correction. Since all liquids wet the walls of this upper bulb, the meniscus formed exerts upward pull against the vessel walls because of surface tension, thus tending to diminish the effective height of the liquid col- umn. Suspended level, by a properly curved surface acts to exert traction in the opposite direction forming an in- verse meniscus to the surface tension on the surface of the liquid in the upper bulb and balancing it. Ubbelohde felt conclusively that this principle eliminated loading and surface tension errors. Barnett (1958) ascertained that synovial fluid behaves as an extremely thick oil when joint surfaces are being sheared very slowly, one upon the other, but become progressively less viscous as the rate of shear increases. In a study on the physiological function of hy- aluronic acid in synovial fluid, the results of Ogston and Stanier (1953) suggested that a major function of hyaluronic acid in synovial fluid may be its contribution to the 29 lubricant properties of the synovial fluid. These workers reasoned that a solution of hyaluronic acid is peculiarly suitable for lubricating a bearing surface of this type, required to carry a load at varying rates of movement. The viscosity is high at the lowest rates of shear, so that the joint is enabled to support a heavy load even at a low rate of movement. At higher rates of movement, the viscosity falls so that the drag of the joint is relatively lessened, the load which it will bear is not reduced, because the greater rate of movement more than offsets the fall in vis- cosity. A normal (Newtonian) fluid would not give these advantages. Furthermore hyaluronic acid produces high vis- cosity at low concentration, without any large osmotic ef- fect such as would be produced, for example, by protein at concentrations needed to give a similar viscosity. These properties of hyaluronic acid are gained from its molecular weight and its random-chain structure. The rheological and pathological behavior of syn- ovial fluids provided Levine and Kling (1956) with valuable information about the physiological and pathological nature of joints. They concluded that the concentration of hy- aluronic acid determines the intrinsic viscosity of a fluid and that this in turn reflected the mean polymerization as an index to this factor. Polymerization is of prime importance since even in the presence of increased produc- tion of hyaluronic acid, if this is low, so will be the 30 relative viscosity of the fluid. In reviewing their work, they felt that relative viscosity appeared to be as valu- able as any other single viscosity determination. Depolymerization of hyaluronic acid occurs in the presence of hyaluronidase a process that Harrow and Mazur (1954) observed, led to a decrease in viscosity of the acid and eventual hydrolysis. The effect of hyaluron- idase on the hyaluronic acid of bovine synovial fluid did not alter the hyaluronic acid glucosamine but Ogston and Stanier (1953) did show that it affected the character of the mucin precipitate. They suggested a method of judging degradation of mucin based on measurements of viscosity alone. Since glucosamine precipitated as mucin is not af- fected, even when the viscosity is strongly reduced and when the character of the mucin precipitate has been pro- foundly changed. Therefore viscosity may be compared with a normal sample having the same content of mucin, neither the weight of mucin nor its nitrogen content should be used in place of glucosamine content. The sum total of the ef- fects of hyaluronidase on hyaluronic acid is its degradation or depolymerization and resultant variation of viscosity with velocity gradient and the elastoviscosity of bovine synovial fluid. Moffett (1954) made a study on the permeability of synovial membranes in the rat and found that the addition of hyaluronidase caused a greater than two-fold increase 31 in the iodopyracet (3.5-diiodo-4-pyridone-N-acetic acid)* diffusion rate in both live and dead rats. Removal time measurements were unaffected, however, by addition of hy- aluronidase. He concluded that synovial permeability in the rat could be altered by two means: (1) a change in the synovial ground substances; (2) a change in the synovial blood supply. A quantitative study of metachromasy in synovial fluid and mucin by Hamerman and Schubert (1953) revealed that the fluid is not metachromatic even when the protein content has been reduced to levels found in mucin solutions. Hyaluronate that has been depolymerized by hyaluronidase is not metachromatic, since it is chiefly high polymers that are metachromatic and the higher the state of polymeri- zation, the greater the degree of metachromasia. They went on to state that hyaluronate is not free in synovial fluid and that native synovial fluid with a viscosity higher than the mucin, or the hyaluronate derived from it, is not meta- chromatic. Addition of alkali has been noted to free the hyaluronate and to produce a fall in viscosity. Alkali also causes synovial fluid to become metachromatic, thus the altered state of the hyaluronate is correlated with a fall in viscosity and appearance of metachromasy. The evi— dence presented indicates that in its native state in synovial *Diodrast, Winthrop Laboratories, New York, N.Y. 32 fluid, hyaluronates are either bound or their anionic groups are not entirely free. It was Gunter (1947) who described a method for assay of hyaluronidase based on the fact that the action of the enzyme on synovial fluid prevented stringiness or the formation of fibers around a rotating rod. He termed this fiber forming capacity "spinnbarkeit". Whether the spinnbarkeit of synovial fluid depended solely on the pres- ence of polysaccharide, or a protein in combination with polysaccharide, was not in his opinion altogether clear. The slight fall in spinnbarkeit after tryptic digestion suggested that this property was due mainly to the long- chain polysaccharide molecule, with protein playing only a minor role. The addition of an equal volume of undiluted normal serum had no effect on the spinnbarkeit of synovial fluid. This argument was supported by the destruction of spinnbarkeit by enzymes prepared from the testis (testicular hyaluronidase) and from Clostridium welchi, both of which are well recognized sources of hyaluronidase and therefore capable of breaking down long-chain polysaccharide molecules. Carpenter (1959) stated that hyaluronidase was formed by Clostridium perfringens as well as streptococci, pneumococci, certain micrococci, and other organisms. According to Wintrobe (1956), blood viscosity was dependent upon a number of factors, the most important ' of which was the quantity of erythrocytes. The quantity 33 of leukocytes was of significance only when they were greatly increased in number. This was particularly true in cases of myelocytic leukemia because the myeloid leukocytes were larger than lymphocytes. Furthermore, he showed that the influence of various factors which determine blood viscosity were rapidly intensified in hemoconcentration. Mann (1948) attributed variations in viscosity of human plasma to qualitative changes in protein. CHAPTER III MATERIALS AND METHODS Samples of blood and synovial fluid obtained for this study were collected from cattle, noted as to breed, sex, age, and state, i.e., either ante mortem or post mortem. Specimens taken from slaughter cattle were recorded as coming from fasted animals, since these individuals were as a rule taken off feed the afternoon prior to the morning kill. Samples taken from other than slaughter cattle were recorded as coming from non-fasting animals, in view of the fact that they were on full feed at the time of specimen collection. Determination of differences between blood and synovial sugars and plasma and synovial sugars in nonfasting cattle was deemed necessary in order to evaluate the effects of fasting in relationship to synovial fluid sugar in the slaughter animals. 'Synovial fluid was aspirated from the tibio-tarsal articulation. This joint is readily accessible and suffici- ent quantities may be obtained for a complete examination. Color and quality of the synovial fluid was noted, but no effort was made in the course of this study to determine the average quantity this articulation contained. Cattle for this study were selected after clinical examination revealed no gross enlargements of the hock joint 34 35 and no manifestations of pain or impaired gait. All indi- viduals appeared clinically to be free of disease. This procedure applied also in the case of cattle presented for slaughter. With slaughter cattle it was possible to examine the synovia, articular cartilages, and synovial membrane for pathological changes as recommended by Jones and Gleiser (1954). Age determination of animals presented for slaughter were made by examination of the teeth as correlated to the work of Sisson and Grossman (1950). Records kept on experi- mental animals not intended for slaughter verified their ages. A. Collection Procedures: 1.21mi All samples were obtained from the jug- ular vein, either by venipuncture in ante mortem cases or following incision of the jugular vein at slaughter time. Approx- imately 10 milliliters of blood was col- lected directly into screw cap vials con- taining ammonium potassium oxalate in a ratio of 0.1 milliliter of the anticoag- ulant solution to 1.0 milliliter of blood. Thus 1 cc. of the anticoagulant solution contains 1.2 per cent ammonium oxalate and 0.8 per cent potassium oxalate or in 36 round numbers a 2 per cent solution of anticoagulant. The water in the anticoag- ulant solution had previously been evap- orated to dryness in vials at 70 degrees centigrade as recommended by Hepler (1957). 2. Synovial Fluid All samples were obtained from the tibio- tarsal articulation in a manner similar to the technique used by the author (1960) for arthrocentesis of the carpus in horses. Arthrocentesis to obtain ante mortem samples was performed with the animal in the stand— ing position. The hair over the antero- medial surface of the joint was clipped closely and then the area cleansed with 70 per cent ethyl alcohol. Using a 26 gauge, % inch needle, the area was sub- cutaneously infiltrated with three to five milliliters of a two per cent solution of lidocaine hydrochloride* containing l:lO0,000 epinepherine hydrochloride. While waiting for the anesthetic to exert its maximum effect, the area was scrubbed *2% Xylocaine Hydrochloride, Astra Pharmaceutical Products, Inc., Worcester, Mass. Figure 1. Figure 2. 37 The anteromedial aspect of the hook joint has been clipped and anesthetized, then scrubbed with surgical soap, and painted with Strong Tinc- ture of Iodine, N.F., prior to arthrocentesis of the tibio-tarsal articulation. A 16 gauge, two-inch needle has been directed into the tibio-tarsal joint cavity from the anteromedial position. 38 39 Figure 3. A 20 milliliter metal-tip glass syringe has been attached to the needle for withdrawal of synovial fluid. Figure 4. Aspiration of synovial fluid has been completed and the external site of needle puncture into the joint cavity closed with a Michel's suture clip. This final step will minimize external hemorrhage as well as reduce the chance entry of microorganisms into the tissues. ho 41 with a surgical soap containing an aqueous preparation of high molecular weight alky- 1amine hydrochlorides composed of benze- thonium chloride* for five minutes, dried, and painted (Fig. 1) with Strong Iodine Tincture, N.F., which was allowed to dry. A 16 gauge, two-inch needle was then di- rected into the joint cavity (Fig. 2) from the anteromedial position at the tibio— tarsal articulation and synovial fluid then aspirated with a sterile, dry, 20 milliliter metal-tip glass syringe (Fig. 3). Following withdrawal of the needle, many cattle exhibited external hemorrhage at the site of puncture. In order to minimize this, the needle openings were closed with either a large (22 mm.) or medium (15 mm.) size Michel's suture clip (Fig. 4). The synovial fluid was then injected directly into dry, sterile, screw cap vials containing no anticoagulant. In the case of samples collected at slaughter, arthrocentesis of the tibio- tarsal articulation was performed in *Liquid Germicidal Detergent, Parke, Davis and 00., Detroit, Mich. 42 essentially the same manner, with the exception that the animal was in dorsal recumbancy and aseptic procedures were not followed. B. Laboratory Procedures 1. Blood Sugar Determinations Whole blood was used to determine the sugar levels of fasting and non-faSting animals. Protein-free filtrates of the oxalated whole blood were made within two hours after collection. Plasma Sugar Determinations Following removal of two milliliters of the oxalated whole blood to make a protein- free filtrate for blood sugar determina- tions, the remainder of the sample was centrifuged for 30 minutes at 3,000 revo- lutions per minute to obtain the plasma. Protein-free filtrates of the plasma were then made. Synovial Sugar Determinations The non-oxalated synovial fluid was centri- fuged for 30 minutes at 3,000 revolutions per minute to obtain a sample free of its cellular content as well as any organic debris accumulated in the process of col- lection. 4. Blood, 43 plasma, and synovial sugar deter— minations were ascertained by a modifi- cation of the macromethod of Folin and Wu (1919) and Folin (1929). The unknown samples were read against a standard glu- cose solution using a Bausch and Lomb Spectronic 2O Colorimeter*. a. Preparation of a Protein-Free Filtrate (l) (2) (3) (4) Place 16 milliliters of N/l2 sul- furic acid in a 50 milliliter Erlen- meyer flask. Using a two milliliter Ostwald- Folin pipette**, add slowly two milliliters of oxalated whole blood, plasma, or synovial fluid to the N/12 sulfuric acid solution. Shake gently and allow to stand for two minutes. Add, slowly and with constant shak- ing, two milliliters of 10 per cent sodium tungstate***. This will make a one to 10 dilution, i.e., in every 10 milliliters of filtrate *Bausch and Lomb Optical 00., Rochester, N.Y. **Kimble Glass 00., Toledo, Ohio ***Hartman-Leddon Co., Philadelphia, Pa. (5) (6) (7) 44 there is the equivalent of one milliliter of blood, plasma, or synovial fluid. Insert a number one rubber stopper and shake well, a metallic click and absence of foam on the mix— ture are indicative of complete precipitation of the protein. Allow the mixture to stand for five minutes. Filter, using a dry, folded number 12 Whatman Filter Paper*. The filtrate must be clear and free of any color. In the case of whole blood, a brown or brownish-yellow filtrate usually indicates that the filtrate is slightly too alka- line. b. Preparation of a Folin-Wu Filtrate Blank (l) (2) The Folin-Wu blank is prepared by placing l6 milliliters of N/12 sulfuric acid in a 50 milliliter Erlenmeyer flask. Using a two milliliter Ostwald-Folin *A.Sa Aloe 00., St. Louis, Mo. 45 pipette, add slowly two milliliters of distilled water. (3) To this mixture is then added two milliliters of 10 per cent sodium tungstate. 0. Preparation of a Glucose Standard (1) Using a one milliliter Ostwald- Folin pipette*, and a correspond- ing amount of a one per cent glu- cose stock solution** (Folin—Wu) in benzoic acid to a 100 milliliter volumetric flask and dilute to volume with distilled water (one milliliter equals 0.1 milligram). (2) Two milliliters of the glucose istandard is then equal to 0.2 mil- ligram of glucose. (3) The glucose standard may be refrig- erated for future use. d. Sugar Determinations (1) Use five Folin-Wu blood sugar tubes*** graduated at 25 milliliters. This *Kimble Glass Co., Toledo, Ohio **Hartman—Leddon Co., Philadelphia, Pa. ***Kimb1e Glass Co., Toledo, Ohio 46 number was necessary to determine the sugar levels of whole blood, plasma, and synovial fluid for each animal and at the same time run a glucose standard and filtrate blank. (2) Into three of the tubes using a two milliliter volumetric pipette, place two milliliters of the Folin- Wu filtrates of whole blood, plasma, and synovial fluid. (3) In two other tubes using a two milliliter volumetric pipette, add two milliliters of Folin-Wu blank and two milliliters of the glucose standard solution. (4) To each of the five tubes add two milliliters of alkaline copper tartrate (Folin-Wu)*. (5) Place each tube in boiling water and continue boiling for exactly eight minutes. (6) Cool in cold running tap water for two minutes, avoiding agitation. *Hartman-Leddon Co., Philadelphia, Pa. (7) (8) (9) (10) (ll) (l2) (l3) (14) 47 To each add, two milliliters of phosphomolybdic acid reagent (Folin- Wu)* and shake until dissolved. Replace in boiling water for exactly five minutes. Repeat step number six. Dilute each to the 25 milliliter mark with distilled water and mix by inversion. Transfer to absorption tubes. Wavelength of the Bausch and Lomb Spectronic 2O Colorimeter is set at 430 mu; with the Folin-Wu blank, set per cent transmittance scale at 100 per cent. The standard glucose solution should then give a reading of 60 on the rper cent transmittance scale which is then equal to 100 milligrams of glucose per 100 milliliters of the original one per cent stock solution. Replace standard with unknown and note the readings on the per cent *Hartman-Leddon Co., Philadelphia, Pa. 4e transmittance scale. (15) Concentrations in milligrams of sugar per 100 milliliters of blood, plasma, or synovial sugar, are found on the table provided by Bausch and Lomb. Cellular Studies Total leukocyte counts were made on a non—centrifuged portion of the synovial fluid. To obtain an adequate number of cells for an accurate differential leuko- cyte count, the synovial fluid sample was centrifuged for 30 minutes at 3,000 ‘ revolutions per minute. The cellular studies were made by a modification of the techniques described by Coffin (1953). All cellular studies were done on either a Dialuxt or Labolux IIIa* microscope. Cytological identifications were made according to the work of Sabin, M. (1924 and 1925) and Doan, gt_§l. (1925). a. DifferentiglLeukocyte Counts Following centrifugation the supernatant fluid was poured off and smears of the *Ernest Leitz, Inc., New York, N.Y. 49 sediment made by compressing it between two microscope slides (75 X 25 milli- meters), both smears were stained with Wright's stain. One hundred leukocytes were counted and differentiated for each sample of synovial fluid. b. Total Leukocyte Counts Total leukocyte counts were made by diluting the synovial sample in a stand- ard white cell pipette. Two types of diluting fluid were used, both employ- ing normal saline as a base. To one, two to three drops of methylene blue were added per 10 milliliters of normal saline. To the other, methyl violet crystals were added to saline, then filtered to make a final concentration of one per cent. Since normal synovial fluid is relatively acellular, the sample under test was drawn to the 1.0 mark on the pipette, making a one to ten dilution. The pipette was then shaken for five minutes on a Burton Pipette Shaker, model number 1406*. *Burton Manufacturing Co., Santa Monica, Calif. 5O Leukocytes were then counted on a stand— ard hemacytometer* in the same manner as done in the standard leukocyte count and the total leukocytes per cubic millimeter determined. In some cases where cells were more numerous, only the four large corner squares on the hemacytometer were used and in others less numerous, all nine squares were used. For excessively low counts, the technique described by Ham (1950) was employed. In all instances the total number of leukocytes was determined in cubic millimeters. c. Apsolute Leukocyte Counts These determinations were made to arrive at the actual increase or decrease in the number of cells as opposed to the' relative increase or decrease. The formula used has been described by Miale (1958) to arrive at the absolute number of leukocytes per cubic milli— meter of synovial fluid. *Sharp Line Hemacytometer, Improved Neubauer Ruling, Chicago Apparatus Co., Chicago, Ill. 51 6. Relative Viscosity Determination of the relative viscosity of synovial fluid was made on samples following centrifugation for 30 minutes at 3,000 revolutions per minute. The determinations were made using the Cannon- Fenske Routine Viscometers*, A.S.T.M. (American Society of Testing Materials) sizes 150 and 200, and the Cannon-Ubbe- lohde Semi-Micro Dilution Viscometers*, A.S.T.M. sizes 100 and 150. The visco— meters were held in a Krebs Constant Tem- perature Bath** designed for use with capillary viscometers, A.S.T.M. D445-53T (Fig. 5). Dukes (1955) gives the average body temperature of the cow as 101.5 de- grees fahrenheit; therefore, distilled water used as the bath liquid was main- tained at 101.5 degrees fahrenheit plus or minus 0.1 degree, by a vapor type thermo- regulator. Distilled water was used as the vapor source since it has an approxi- mate sensitivity range of 92 to 104 degrees *Cannon Instrument 00., State College, Pa. **Krebs Electric and Manufacturing Co., New York, N.Y. Figure 5. 52 The Krebs Constant Temperature Bath (A.S.T.M. D445-53T) designed for use with capillary vis- cometers. Both Cannon-Fenske Routine Viscometers and the Cannon-Ubbelohde Semi-Micro Dilution Viscometer are suspended in this bath. 53 _ muNH-m 54 fahrenheit. Distilled water was used as the reference liquid as recommended by Cannon (1944), since its complete stabil- ity and general availability make it an excellent reference liquid for all rela- tive viscosity measurements. The deter- minations were measurements of relative viscosity as compared to distilled water as a reference liquid, the conditions of the study being duplicated as closely as possible for water and synovial fluid. The viscometer tubes were flushed with distilled water following their use and then flushed with a solution of potassium dichromate and sulfuric acid in a concen- tration as recommended by Hepler (1957). This solution was left in the viscometer tube for a variable length of time, thus allowing digestion of any protein residue. Then the viscometer tubes were flushed with distilled water and dried in a hot air oven at 150 degrees centigrade until dry. Efflux times were measured by using a hand operated stop watch. 55 a. Determination of the Relative Viscosity Using the Cannon-Fenske Routine Vis- cometer This procedure is a modification of the technique described by Manning (1960) and Hawk, gt_§l. (1954). (1) Viscometer tubes were inserted in their adapters (Fig. 6) and placed in the constant temperature bath (Fig. 5) with the water level above bulb C and vertically aligned. (2) A five, seven or 10 milliliter sample of synovial fluid is then pipetted into the lower reservoir of the viscometer (G) through tube B. (3) The viscometer and synovial fluid sample are allowed 10 minutes to reach temperature equilibrium (101.5 degrees fahrenheit, plus or minus 0.1 degree). (4) Suction is then applied to tube A until the liquid reaches the center of bulb C. Remove suction from tube A to measure the efflux time. 56 Figure 6. The Cannon-Fenske Routine Viscometer (A.S.T.M. 150) with adapter for the Krebs Constant Tempera— ture Bath. # CANNON S7 . —ADAPTER A A —4 B t! (hf: C D E F 58 (5) To measure the efflux time, allow the liquid sample to flow freely down past etch mark D, measuring the time for the meniscus to pass from etch mark D to etch mark E, to the nearest 0.1 second. (6) Determine the efflux time for five successive measurements. (7) Relative viscosity is then deter- mined by dividing the efflux time of water measured to the nearest 0.1 second into the efflux time of the synovial fluid measured to the nearest 0.1 second. b. Determination of the Relative Viscosity Using the Cannon-Ubbelohde Semi-Micro Dilution Viscometer This procedure is a modification of the technique described by Merrington (1941). (l) The viscometer tube is inserted in its adapter (Fig. 7), placed in the constant temperature bath (Fig. 5) and vertically aligned. (2) A one milliliter sample of synovial 59 Figure 7. The Cannon-Ubbelohde Semi-Micro Dilution Visco- meter (A.S.T.M. 150) with adapter for the Krebs Constant Temperature Bath. 60 ADAPTER C ammo (3) (4) (5) (6) 61 fluid is then pipetted into the lower reservoir of the viscometer (H) through tube C. The viscometer and the synovial sample are allowed ten minutes to reach temperature equilibrium (101.5 degrees fahrenheit, plus or minus 0.1 degree). Place a finger over tube B, and apply suction to tube A until the liquid reaches the center of bulb D. Remove suction from tube A and the finger from tube B, allowing the liquid to flow freely through the capillary, while the entry of air under atmospheric pressure into tube B produces the suspended level. To measure the efflux time, allow the liquid sample to flow freely down etch mark F, measuring the time for the meniscus to pass from etch mark G to the nearest 0.1 second. Because of the tendency for a syn- ovial fluid sample to become turbu— lent and frothy on subsequent (7) 62 attempts to measure efflux time, only one reading was taken. Relative viscosity is then deter— mined by dividing the efflux time of distilled water measured to the nearest 0.1 second into the efflux time of the synovial fluid measured to the nearest 0.1 second. c. Calibration of the Cannon-Fenske Rou- tine Viscometer and the Cannon-Ubbe- lohde Semi-Micro Dilution Viscometer (l) (2) Since water is used as the reference liquid to determine the relative viscosity of synovial fluid, this then was used to calibrate the two types of viscometers used in this study. Cannon-Fenske routine viscometers were calibrated at 101.5 degrees fahrenheit, plus or minus 0.1 de- gree, with both five and ten milli- liter charges. The same procedure was followed as outlined for meas- urement of synovial fluid efflux time. 63 (3) Sterile distilled water was used because it was free of either bac- terial or mycotic growth, factors that could have a bearing on the efflux measurements. (4) Cannon-Ubbelohde semi—micro dilu- tion viscometers were calibrated at 101.5 degrees fahrenheit, plus or minus 0.1 degree with one milli— liter charges. The same procedure was followed as outlined for meas- urement of synovial fluid efflux ' time, with the exception that five successive measurements were made. Lack of turbulence and froth with sterile distilled water made this possible. Mucin Clot Determination of mucin clot quality is made on a non-oxalated one milliliter sample of synovial fluid that has been centrifuged for 30 minutes at 3,000 revolutions per minute. The test was carried out in the following manner, a modification of the method described by Furey, et a1. (1959). 64 a. 0.1 milliliter of 7/N glacial acetic acid is added to four milliliters of distilled water in a screw cap vial and thoroughly mixed. . Add one milliliter of the synovial fluid slowly, taking care that the sample does not come in contact with the glass as it is added. Gently swirl the solution so as to mix the synovial fluid thoroughly. Allow to stand for one hour and mix again by inverting the tube several times before results are determined. Four grades of clot are recognized (Fig. 8), as described by Ropes and Bauer (1953). (1) Normal - tight, ropy clump in a clear solution. (2) Fair - soft mass in a very slightly cloudy solution. (3) Poor - small, friable masses in a cloudy solution. (4) Very poor - few flecks in a cloudy solution. 65 Figure 8. Four grades of mucin clot obtained by precipita— tion with 7N glacial acetic acid: (I) normal; (II) fair; (III) poor; (IV) very poor. CHAPTER IV RESULTS General Considerations In this study, each of the 83 animals are clas- sified as clinically normal with respect to the tibio- tarsal articulation and general health. They were selected after clinical examination failed to reveal any evidence of joint disease. The writer would like to state it is conceivable that some of the test animals may have under- gone some form of joint disease prior to the collection date or were undergoing some insidious form of arthritis, inapparent at the time of sample collection. At no time did the author observe either clinical or post mortem evi— dence of arthritis in this study. Because of the availability of slaughter animals that could provide unlimited material, this source was readily sought and hence accounts for the major portion of test animals in this study. The results of laboratory determinations are pre- sented in table form in their respective classifications. Averages and standard deviations are also included with the exception of Table I. 67 68 With regard to sugar determinations, these data are classified as to findings from fasting or non-fasting animals. Plasma sugar values were sought and compared with that of blood in relation to synovial fluid to arrive at a constituent that would most closely parallel or approxi— mate synovial fluid sugar in either a fasting or non-fasting animal. In handling of animals, stress and excitement fac— tors are also taken into consideration in relation to their influence on sugarfdeterminations. All samplings were taken from the tibio-tarsal articulation, in the recumbant position in the case of slaughter cattle and standing position in the balance of the group. Table I identifies the animals in this study along with a notation as to the nature of the synovial fluid at time of collection. The animals designated by numbers from 1 to 83. Animals in the group from 1 to 52 comprise those from which samples were taken at post mortem. Because these animals were removed from feed 12 to 18 hours prior to slaughter it was necessary to make note of this fact, since interpretation and classification of data, particularly with respect to sugar values, will be made in this light. Water was available to this group of animals at all times. It was noted that the majority of the cows in this classi- fication were in some stage of lactation at the time of slaughter. The group of animals from 53 to 83 make up the 69 balance and were individuals not intended for slaughter. Many of these cattle were in active lactation and samples from all individuals in the group were collected while on full feed. The Holstein breed accounted for 56.6 per cent of all animals tested. This figure was followed by 16.9 per cent Herefords, 4.8 per cent each of Guernseys and Gal- loways, 3.6 per cent each of Brahmas and Shorthorns, 2.4 per cent Angus and 4.8 per cent animals of mixed breeding, two of which were Holstein crosses followed by one Hereford cross and one Guernsey cross. The average age of the en— tire group was 3.51 years. When subdivided the average age of fasting slaughter cattle was 2.94 years and non-fast— ing cattle 4.48 years. Females comprised 57 per cent of the entire group, followed by 23 per cent steers and 20 per cent males. These figures break down to reveal that 36.5 per cent of slaughter cattle were females, 34.6 per cent steers and 28.8 per cent males. Females again domi— nated the picture in the non-fasting series with 80.6 per cent, there being more males at 12.9 per cent than steers which only accounted for 6.5 per cent. Observations on the nature of synovial fluid were as follows: 70 per cent colorless, 9.6 per cent a clear amber, 2.4 per cent slightly Opaque, 2.4 per cent amber with flocculation and 1.2per cent an opaque—amber with flocculation. Table II gives the data for blood, plasma, and synovial fluid sugars for 52 animals in the fasting state. 70 All sugar values are expressed in milligrams per 100 milli— liters of blood, plasma, or synovial fluid. Blood sugars averaged 80.58 i 4.84 milligrams, with a low of 51 and a high of 154 milligrams of sugar per 100 milliliters of whole blood. The average plasma sugar level is considerably higher at 100.71 1 8.21 milligrams per 100 milliliters. Plasma values range from a low of 60 milligrams to 236 milligrams of sugar per 100 milliliters. Sugar values for synovial fluid are intermediate in value with an average of 81.17 I 4.37 milligrams per 100 milliliters. This average value‘ is 0.59 i 3.11 milligrams higher than the average value given for blood. Synovial fluid sugars range from a low of 42 milligrams to a high of 124 milligrams per 100 milli- liters of fluid. From a diagnostic standpoint, emphasis is placed on the difference between blood or plasma in rela- tion to the respective synovial fluid sugar for each animal. With this in mind, the blood-synovial fluid sugar difference was determined for each animal and for the entire group, with synovial fluid as previously mentioned averaging 0.59 i 3.11 milligrams of sugar higher than that of blood. Syn— ovial sugar differences range from a low of 42 milligrams of sugar less than to 22 milligrams greater than its blood counterpart. Plasma-synovial fluid differences show a re— verse, with synovial fluid sugar averaging 19.89 i 5.99 milligrams lower than the average values for plasma. This synovial fluid difference ranges from a low of 124 milligrams 71 of sugar less than to ten milligrams of sugar greater than its plasma counterpart. Table III gives the data for blood, plasma, and synovial fluid sugars, in milligrams per 100 milliliters for each animal in the non-fasting state. All 31 animals in this series were on full feed at the time of sample col- lection. Most of the animals in this series were cows in some stage of lactation. Only four males and two steers were tested, as against 25 females. This factor undoubtedly accounts for the lower averages encountered in this test series. Blood sugar values average 63.22 I 5.39 milligrams with a low of 45 milligrams and a high of 105 milligrams per 100 milliliters of whole blood. The average sugar value for plasma is 65.32 1 6.33 milligrams with a low of 40 mil- ligrams and a high of 112 milligrams per 100 milliliters. Synovial fluid sugar averaged 62.02 i 6.20 milligrams, rang- ing from a low of 36 milligrams to a high of 105 milligrams per 100 milliliters. Here again from a diagnostic stand— point, value is placed on the difference between blood and plasma in relation to their respective synovial fluid sugars. In non-fasting cattle, the blood-synovial fluid sugar dif— ference reversed the corresponding results observed in fast- ing slaughter cattle, as synovial fluid averages 1.13 i 3.79 milligrams per 100 milliliters less than that of whole blood. These values range from a low of 35 milligrams of synovial fluid sugar less than to 15 milligrams of synovial 72 fluid sugar greater than that of the corresponding values for whole blood. Plasma-synovial fluid sugars in this series average 8.08 i 3.36 milligrams of synovial fluid sugar less than that of plasma. Plasma—synovial fluid differences range from a low of 33 milligrams of synovial fluid sugar less than to eight milligrams greater than the corresponding plasma values. Table IV is designed to show the relationship of blood, plasma, and synovial fluid sugars to age in the group comprising 52 fasted cattle presented for slaughter. Blood and/or plasma-synovial fluid differences in relation to age were not determined in compilation of this data. All sugar values are expressed in milligrams per 100 milliliters of blood, plasma, or synovial fluid sugar. The number of animals tested in each age group is given in parenthesis to the left of the corresponding sugar value. The animals in this series ranged from one to seven years of age, with the greatest number falling in the 1.5 year bracket. This latter fact can be accounted for by the fact that the major— ity of animals presented for slaughter usually fall in this age range. Blood sugar values range from a low of 63.00 milligrams for the one animal in the seven year bracket to a high of 89.72 for two year olds. Plasma sugar values again show a low of 68.00 milligrams for the seven-year old animal and range to a high of 117.88 for the two-year 73 olds. The values for synovial fluid sugar follow the pat- tern set by the two previous values in the lowest range with 63.00 milligrams for the seven-year old animal on up to a high of 88.33 milligrams of sugar per 100 milliliters for three animals in the four-year age bracket. In retro— spect, one can see in this sampling, lower values for ani- mals in the one-year age group rise with age to drop progres- sively after four years to lower levels. Table V presents the dataon the relationship of blood, plasma, or synovial fluid sugars to age in the group of 31 animals on full feed at time of sample collec- tion. Blood and/or plasma—synovial fluid differences were not determined in compilation of the data presented here. All sugar values are reported in milligrams per 100 milli- liters of whole blood, plasma, or synovial fluid. The num- ber of animals tested in each age group is given in paren- thesis to the left of the corresponding sugar value. Ani— mals in this series showed a wider age range than those in the fasting group. This group ranged from 0.75 to 10 years of age; however, none were in either the 1.5 or nine—year age brackets. The majority were grouped in the four-year age bracket. This can be accounted for by the fact that the females in this series were lactating at the time samples were taken. Blood sugar values range from a low of 51.5 milligrams for two animals ten years of age to a high of 93.0 milligrams for one animal 0.75 Years of age. As usual, 74 plasma sugar values ran higher than those of blood with a low of 60 milligrams for the two oldest animals to a high of 83.5 milligrams for two animals in the two year bracket. Plasma sugar was not determined for the one animal 0.75 years of age as this individual was tested early in the' course of the study before it was decided to determine this value. Synovial fluid sugars again show an overall range intermediate between that of blood and plasma. The low value for synovial sugar at 43 milligrams is observed in one animal in the six—year age bracket and the high of 87 milligrams for the one 0.75 year old. As in the series of animals tested in the fasting group, the younger animals in this sampling show higher values that progressively drop off with advancing age. Table VI is a comparison of the average blood, plasma, and synovial fluid sugars in fasting slaughter cattle and non-fasting cattle. The material is presented in this form so that a better understanding may evolve regarding the sugar levels in the two groups. Table VII gives the relationship and comparison of blood, plasma, and synovial fluid sugars as to sex in fasting slaughter cattle and non-fasting cattle. The vari- ous sexes in the fasting group were more evenly distributed than in the non-fasting group in which females predominated. The data in this table are compiled to show the sex differ- ences in respect to sugar levels. 75 Table VIII presents findings on the cellular stud— ies of synovial fluid. It will be noted that a paradoxical situation seems to present itself in that the number of leukocytes per cubic millimeter may be given and yet no differential percentage of 100 leukocytes or vice versa. In the case of the former, an uncentrifuged sample of syn- ovial fluid was diluted one to ten with the diluent and placed in the counting chamber of the hemacytometer. In these instances no leukocytes were observed, but following centrifugation of the sample, enough cells were collected to make possible a differential leukocyte count. Conversely, instances occurred wherein no differ- ential leukocyte counts were made. Microscopic examination of the stained smear failed to reveal cells with a morphology distinct enough to arrive at any differential classification. These smears were then discarded as the writer felt it would detract from the accuracy of this study. It soon became apparent that the leukocytes ob- served in synovial fluid are of a highly fragile nature and therefore must be handled with due consideration if their identification is to be accurate. Occasionally syn- ovial cells are encountered in the course of a differential leukocyte count. These are readily identified in that they more closely resemble fibroblasts than monocytes with which they can become confused. Total leukocytes range from 0 to 725 cells, with an average number of 114 per cubic 76 millimeter. The predominant cell types observed in the differential count are: lymphocytes 48.87 per cent, mono- cytes 39.31 per cent, neutrophils 5.67 per cent, clasmato— cytes 6.13 per cent, and eosinophils 0.79 per cent. No basophils were observed in any of the stained smears. Erythrocytes were observed but not counted since they were attributed to hemorrhage at the time of aspiration in the living animal or extravasation of the cells in the course of agonal struggles at slaughter. A. Table IX gives the absolute leukocyte values or number of leukocytes per cubic millimeter. These figures represent actual numbers, not per cent of 100 leukocytes. The absolute leukocyte count is arrived at by multiplying the number of leukocytes per cubic millimeter by the per- centage values for the respective cell types as determined from a differential. Since an absolute leukocyte count is made from samples of synovial fluid from which both a total and differential leukocyte count are obtained, the average total number of leukocytes is slightly higher than the figure reported in the preceding table. This figure was 115.8 leukocytes per cubic millimeter. Total number of leukocytes ranged from 22 to 600 per cubic millimeter. The various cell types gave the following values per cubic millimeter: lymphocytes 53.7, monocytes 38.95, clasmato- cytes 12.19, neutr0phils 10.03 and eosinophils 0.91. No basophils were observed in these cellular studies. 77 Table X presents the data for the relative vis- cosity of synovial fluid at 101.5 degrees fahrenheit and quality of mucin clot for the respective sample. All rela— tive viscosity determinations were made in a constant tem— perature bath, with the majority of determinations employ- ing the Cannon-Fenske routine viscometer. Animal numbers marked with an asterisk indicate viscosity determinations arrived at with the Cannon-Ubbelohde semi-micro dilution viscometer. Relative viscosity for the tibio-tarsal articu— lation average 3.79 i 0.55, with a range of 1.53 to 13.01 for 73 determinations. Normal mucin clots were observed in 91.7 per cent of fluids and only 8.2 per cent exhibited fair quality. Mucin clot serves as an indicator of quality, i.e., the degree of polymerization of the hyaluronic acid is reflected by the firmness observed in the clot, the higher the degree of polymerization the better the quality of mucin clot or vice versa. Table XI is designed to show the relationship between the relative viscosity of synovial fluid and the age of the animal. The corresponding quality of mucin clot in per cent of normal is also given for each of the age groups. The number of animals tested in each bracket is given in parenthesis to the left of its respective value. One animal in the eight year bracket has the highest rela- tive viscosity at 9.50 and a normal mucin clot. On the other end of the scale the lowest relative viscosity is 78 observed in the one 0.75 year old at 1.99, with a normal mucin clot. Relative viscosity is quite constant from one to four years of age and at this point begins to rise only to drop after eight years to a lower than pre-peak level in the one animal tested at ten years of age. Normal mucin clot quality drops with a corresponding rise in viscosity to attain its lowest level for the entire series at 67 per cent for animals seven years of age, only to return to normal for the two remaining individuals. Table III is a comparison of the relative vis- cosity of synovial fluid in relation to sex. Quality of mucin clot is also presented to ascertain the effect sex might have on the polymerization of the hyaluronic acid. While per cent of normal mucin clot quality runs consistently high, steers rose only one per cent above the value for males and females, while relative viscosity shows more var- iation. Steers exhibit the lowest relative viscosity values at 2.99 i 0.40, almost that of males at 3.02 i 0.61.7 The 37 females are markedly higher in value at 4.55 i 0.99, a figure slightly more than half again as much for the 17 males and 19 steers. TABLE I IDENTIFICATION OF ANIMALS AND COLLECTION DATA ON SYNOVIAL FLUID FROM THE CLINICALLY NORMAL TIBIO-TARSAL ARTICULATION Animal Breed* Age Sex Fasting Ante Mortem Nature Number Yrs. or or of Non—fasting Post Mortem Fluid** 1 BRA 1.5 Steer Fasting Post Mortem 0 2 BRA 1.5 Steer Fasting Post Mortem 0 3 BRA 2 Steer Fasting Post Mortem 0 4 HOL 5 Female Fasting Post Mortem 0P 5 HER l Steer Fasting Post Mortem 0 6 HOL 3 Male Fasting Post Mortem 0 7 HOL 1.5 Male Fasting Post Mortem 0 8 G 1 Male Fasting Post Mortem 0 9 HOL 5 Female Fasting Post Mortem 0 10 HOL 5 Female Fasting Post Mortem 0 ll HER-X 1.5 Steer Fasting Post Mortem 0 l2 HER 2 Steer Fasting Post Mortem 0 13 SH 1.5 Steer Fasting Post Mortem 0 14 A l Steer Fasting Post Mortem 0 * A - Angus HOL - Holstein ** 0 - Colorless BRA - Brahma J - Jersey Synovial Fluid G - Guernsey SH - Shorthorn 0P - Slightly Opaque GAL - Galloway X - Mixed Breed Synovial Fluid HER - Hereford A - Amber Synovial Fluid F - Flocculation 79 80 TABLE I (continued...) Animal Breed Age Sex Fasting Ante Mortem Nature Number Yrs. or or of Non-fasting Post Mortem Fluid 15 HOL 3 Female Fasting Post Mortem 0 16 HOL 1.5 Steer Fasting Post Mortem 0 l7 HOL 2 Steer Fasting Post Mortem GP 18 HOL 2 Steer Fasting Post Mortem 0 19 HOL 6 Female Fasting Post Mortem 0 20 GAL 1.5 Male Fasting Post Mortem A 21 G 2 Male Fasting Post Mortem 0 22 HOL 1.5 Male Fasting Post Mortem 0 23 CAL 5 Male Fasting Post Mortem O 24 GAL 5 Male Fasting Post Mortem 0 25 GAL 1 Male Fasting Post Mortem 0 26 SH 1.5 Male Fasting Post Mortem 0 27 HER 2 Steer Fasting Post Mortem 0 28 HER 3 Male Fasting Post Mortem 0 29 HER 1.5 Male Fasting Post Mortem 0 30 G 1.5 Male Fasting Post Mortem A 31 J 2 Male Fasting Post Mortem A 32 HER 4 Female Fasting Post Mortem 0 33 HER 5 Female Fasting Post Mortem 0 34 HER 5 Female Fasting Post Mortem O 35 HER 6 Female Fasting Post Mortem 0 36 SH 6 Female Fasting, Post Mortem 0 continued... TABLE I (continued...) 81 Animal Breed Age Sex Fasting Ante Mortem Nature Number Yrs. or or of Non—fasting Post Mortem Fluid 37 HER 1.5 Female Fasting Post Mortem 0 38 HER 1.5 Steer Fasting Post Mortem 0 39 HER 1.5 Steer Fasting Post Mortem 0 40 HOL 4 Female Fasting Post Mortem O 41 HOL 5 Female Fasting Post Mortem 0 42 HOL 4 Female Fasting Post Mortem 0 43 HOL 2 Steer Fasting Post Mortem A 44 HOL 6 Female Fasting Post Mortem AF 45 HOL 7 Female Fasting Post Mortem AF 46 HOL-X 6 Female Fasting Post Mortem A 47 HOL 6 Female Fasting Post Mortem AOP 48 HOL-X 2 Steer Fasting Post Mortem A 49 HOL 1.5 Female Fasting Post Mortem O 50 A 1.5 Steer Fasting Post Mortem 0 51 HER 1.5 Female Fasting Post Mortem 0 52 HER 1.5 Steer Fasting Post Mortem 0 53 HOL 5 Female Non-fasting Ante Mortem A 54 HOL 0.75 Male Non-fasting Ante Mortem 0 55 G-X 1 Male Non-fasting Ante Mortem 0 56 HOL 3 Female Non-fasting Ante Mortem 0 57 HOL 4 Female Non-fasting Ante Mortem 0 continued... TABLE I (continued...) 82 Animal Breed Age Sex Fasting Ante Mortem Nature Number Yrs. or or of Non-fasting Post Mortem Fluid 58 G 4 Female Non-fasting Ante Mortem 0 59 HOL 4 Female Non-fasting Ante Mortem 0 60 J 3 Male Non-fasting 'Ante Mortem o 61 HOL 3 Female Non-fasting Ante Mortem 0 62 HOL 3 Male Non-fasting Ante Mortem 0 63 'HOL 2 Female Non-fasting Ante Mortem 0 64 HOL 7 Female Non-fasting. Ante Mortem o 65 HOL 10 Female Non-fasting Ante Mortem 0 66 HOL 7 Female Non-fasting Ante Mortem 0 67 HOL 7 Female Non-fasting Ante Mortem O 68 HOL 4 Female Non-fasting Ante Mortem 0 69 HOL 8 Female Non-fasting Ante Mortem 0 70 HOL l Steer Non-fasting Ante Mortem 0 71 HOL l Steer Non-fasting Ante Mortem 0 72 HOL 8 Female Non-fasting Ante Mortem 0 73 HOL 10 Female Non-fasting Ante Mortem 0 74 HOL 2 Female Non-fasting Ante Mortem 0 75 HOL 4 Female Non-fasting Ante Mortem 0 76 HOL 4 Female Non-fasting Ante Mortem 0 77 HOL 3 Female Non-fasting Ante Mortem 0 78 HOL 5 Female Non—fasting Ante Mortem 0 continued... 83 TABLE I (continued...) Animal Breed Age Sex Fasting Ante Mortem Nature Number Yrs. or or of Non-fasting Post Mortem Fluid 79 HOL 6 Female Non-fasting Ante Mortem 0 80 HOL 7 Female Non—fasting Ante Mortem 0 81. HOL 4 Female Non—fasting Ante Mortem 0 82 * HOL 4 Female Non-fasting Ante Mortem O 83 HOL 4 Female Non-fasting Ante Mortem A Ave. 3.51 ‘ TABLE II BLOODL PLASMA, AND SYNOVIAL FLUID SUGARS IN THE FASTING STATE* Sugar Values in Milligrams Per 100 Milliliters Animal Blood Plasma Synovia Blood-Synovia Plasma-Synovia Difference** Difference** 1 105 134 8g -23 -52 2 112 171 93 -19 -78 3 93 120 196 + 3 —24 4' 19 82 79 O — 3 _5 10 e5 78 + e - 7 6 90 105 87 - 3 -18 7 96 102 9g, 0 - 6 8 82 105 102 +20 - 3 _9, 73 79 7O - 3 - 9 10 73 85 65 - 8 -20 ll 96 128 90 - 6 -38 12 68 83 85 +17 + 2 13 82‘ 93 79 I -13 -14 14 - 82 63 - -19 15, 63 70 68 + 5 - 2 16 70 85 7O 0 -l5 1*Animals in this series had been taken off feed at least 12 hours prior to time of sample collection. Only water was allowed during the interim. **Milligrams of synovial fluid sugar greater (+) or less (-) than blood or plasma sugar. continued... 84 85 TABLE 11 (continued...) Animal Blood Plasma Synovia Blood-Synovia Plasma—Synovia Difference Difference 17 65 79 63 - 2 -16 18 7O 84 76 + 6 — 8 i9, 68 79 70 + 2 - 9 20 93 128 87 - 6 -41 21 79 96 87 + 8 - 9 22 68 79 68 0 -ll 23 76 9O 68 - 8 —22 24 70 73 63 - 7 -10 25 82 93 79 -.3, -14 26~ 73 85 95 +22 +10 27 63 87 76 +13 ~11 28 68 90 70' + 2 -20 29, 68 87 87 +19 0 30 109 162 112 + 3 -50 31 7o 93 82 +12 -11- 32 87 112 90 + 3 -22 33 87 112 96 + 9 -16 34 82 100 87 + 5 -13 .35 87 109 93 + 6 —16 36 87 109 93 + 6 +16 37 154 236 112 -42 -124 continued... 86 TABLE II (continued...) Animal Blood Plasma Synovia Blood—Synovia Plasma—Synovia Difference Difference A 38 85 85 90 + 5 + 5 59' 82 109. 96 +14 —13 4o 85 102 85 o -17 41‘ 79 102 79 o -23 42 85 102 90 + 5 -12 43 _57 60 48 - 9 -12 44 68 82 73 ' + 5 - 9 45 63 68 63 0 - 5 46 115 145 124 + 9 -21 47 6O 79 42 -18 -37 48 51 73 55 + 4 -18 49‘ 79 115 7O - 9 -45 so 82 102 75 - 9 -29 51 79 109 76 - 3 -33 5g 82 112 82 0 -30 Ave. 80.58 100.71 81.17 + 0.59 -19.89 SD :4.84 18.21 14.37 1 3.11 i 5.99 TABLE III BLOOD, PLASMA, AND SYNOVIAL FLUID SUGARS IN THE NON-FASTING STATE* Sugar Values in Milligrams Per 100 Milliliters Animal Blood Plasma Synovia Blood-Synovia Plasma-Synovia Difference** Difference** 53 79 - 76 - 3 - 54 93 - 87 - 6 ‘ j — 55 9Q - 105 +15 — 56 75 - 85 +12 - 57 105 - 7O -35 - 58 65 82 68 +55 -14 59. 63 76 70 + 7 - 6 60 63 77 ~10 + 7 — 7 61' 62 76 70 + 8 - 6 62 63 65 65 + 2 O 63 82 112 79 -.3. -33 64 70 - 70 0 - 65. 48 55 57 + 9 + 2 66 .57 65. 60 + 3 - 5 67 55 68 65 +10 - 3 68 60 65 52 — 8 -l3 *Animals in this series were on full feed at the time of sample collection. **Milligrams of synovial fluid sugar greater (+) or less (-) than blood or plasma sugar. continued... 87 88 TABLE III (continued...) Animal Blood Plasma Synovia Blood-Synovia Plasma-Synovia Difference Difference 69 60 63 ._50 ~10 ~13 7O 60 65 63 + 3 — 2 71 76 79. 87 +11 + 8 72 60 73 65 + 5 - 8 73 55 65 52 - 3 ~13 74 50 55 43 - 7 -12 15 45 55 45. ~ 2 ~12 76 '48 4o 43 — 5 + 3 Z7 50 50 44 ~ 6 - 6 78 48 40 #36 ~12 _ 4 79 63 57 43 -2o -14 80 952 55 48 - 4 - 7 81 45 48 43 — 2 - 5 82 52 65 40 ~12 ~15 83 68 82 76 + 8 - 6 Ave. 63.22 65.32 62.09 ~ 1.13 ~ 8.08 SD :5.39 16.33 16.20 i 3.79 i 3.36 TABLE IV RELATIONSHIP OF BLOOD, PLASMAL AND SYNOVIAL FLUID SUGARS TO AGE IN THE FASTING STATE* ‘ Sugar Values in Milligrams Per 100 Milliliters** Age: Yrs. Blood: Plasma: Synovia: 1.0 1&5 2.0 3.0 4.0 5.0 6.0 7.0 (3)*** (18) (9) (3) (3) (8) (6) (l) 78.00 89.72 68.11 73.66 85.66 77.00 80.83 63.00 (4) (18) (9) (3) " (3) <8) (6) (1) 91.25 117.88 86.11 88.33 105.33 90.37 100.50 68.00 (4) (18) (9) (3) (3) (8) (6) (1) 80.50 86.55 74.22 75.00 88.33 75.87 82.50 63.00 *Animals in this series had been taken off feed at least 12 hours prior to time of sample collection. Only water was allowed during the interim. **Sugar values measured to the nearest one-hundredth milli- gram. ***Number of animals tested in each age group. 89 9O .m50sm own some ca copmop massage Mo ampedz*** .smsmHHHHa np0os0ssnioso pmohdow one 0p ooHSmmoa modam> Hamsm** .QOHpooHHoo mamsmm mo msflp 039 vs 000% HHSH so once mofisom was» aw mamsfis 0.0 o.m 0.8 o.m o.m o.H m>.o .mmw no a saaaossaaaaas 00s was assamaaaas as mosses semen *mewem wZHHm¢MIzoz mme 2w mam OB mmmwbm QHDHm Hozwm Qz< J<2m4gm .QOOHm mo mHmmZOHewqmm > mqmHm mam muohm mmw Homo a“ mpoao :Hoda Hashes Ho pqmo Hmm* fiooa ROOH Rho Kan ROOH Raw KOCH ficoa Ram ROOM ROOH ”poao 118 118 1n8 1.8 1018 1.8 1.8 1018 1.18 1.8 ..118 mm.m om.m meow w>.m #m.v oo.m No.m ob.m mo.m mH.m mm.H “hpwwoomfl> 118 118 1.8 1.8 1018 1.8 1.8 1.18 1.18 1.8 ..118 0.0H o.w 0.5 o.w o.m o.¢ o.m O.N m.H O.H m>.o .mmw "m ¢ *ZOHHozwm mo MBHmoomH> m>Hs r 2 * I c g z I f3 0 .q FEMALE ’"' srzen RELATIVE VISCOSITY @ IUCIN‘ CLOT O III-I. lit-1!... l 135 normal synovial fluid of cattle and reported lower quantity of fluid from milking cows compared with young beef cattle. They obtained five to ten cubic centimeters from the tibio- tarsal and two to three cubic centimeters from the carpo— metacarpal articulation of milk cows. Perhaps in these instances there is greater concentration of the hyaluronic acid complex, associated with a lesser degree of molecular polymerization as noted by poorer grades of mucin clot in relation to correspondingly high relative viscosity values of the synovia.) This may explain why younger animals have greater total fluid volumes and more of them have a normal mucin clot but still have lower relative viscosity values. One might conclude from these findings that volume of syn- ovia and concentration of mucopolysaccharide are inversely proportional as reflected by a lower viscosity, in animals with greater total fluid volume. Since intrinsic viscosity was not ascertained in these studies, speculation and cor- relation of results obtained leave the only alternatives. Therefore, in instances wherein viscosity is high, it seems probable that lesser quantities of synovia would be needed for normal physiological function of the joint. Gardner (1959) has shown that the viscosity of hyaluronic acid increases exponentially relative to its concentration and that solutions (nonsulfated mucopolysac- charide or hyaluronic acid) of over one per cent form gels. Conversely he stated that viscosity decreased exponentially 136 as synovial fluid is diluted. The viscosity of synovial fluid is decreased to that of water if the hyaluronic acid is removed by precipitation with acid or hydrolized by en- zymes, or if its polymerization is destroyed by enzymes. Sex seemed to have some influence on viscosity (Table XII and Chart VI) with females exhibiting an average relative viscosity of 4.55 i 0.99. Males were next with an average relative viscosity of 3.02 i 0.61 and steers were lowest at 2.99 t 0.40. Ninety-four per cent of the males and females had a normal mucin clot, with steers only one per cent higher. It is difficult to explain why the females in this either males or study were higher in viscosity values than steers, except as previously discussed, females were generally comprised of an older group of ani- mals exhibiting lesser quantities of fluid, undoubtedly higher in concentration and polymerization of the hyaluronic acid complex. In retrospect it would be difficult to assign age as an influencing factor since the majority of females were older animals. Whether or not there is a reduction in synthesis or or an enzymatic quality was not (1959) observed about one-third concentration of the mucopolysaccharide, factor concerned with reduced synovial fluid, determined in this study. Neher and Tietz in aged bulls from a bull stud, that in of the pathologic joints, the synovial fluid was excessive in volume and of a less viscous nature than normal. These workers failed to mention how they arrived 137 at their viscosity findings as did Moulton, et a1. (1953) who aspirated 100 milliliter quantities of synovial fluid from the hook joints of calves infected with Erysipelothrix rhusiopathiae and observed that the fluid was tan to pink in color, more viscid than normal synovial fluid, and cloudy. It is conceivable that the enzyme hyaluronidase may play a role in depolymerization of the hyaluronic acid complex. Colowick and Kaplan (1955) stated that the action of hyaluronidase on hyaluronic acid when present in synovia resulted in a decrease in viscosity, the rate of which is a function of enzyme concentration. Furthermore they noted that hyaluronidase is widely distributed in nature, although it has conclusively been demonstrated only in testicular tissue of mammals. In human patients with rheumatoid arth— ritis, Bollet (1956) observed there was no correlation be— tween lowering of the intrinsic viscosity of synovia and the clinical aspects of the disease or the severity of in— flammation or the degree of cartilage and bone destruction as estimated on x-ray findings. He further observed that intrinsic viscosity and relative viscosity both increased following treatment with either oral or intra-articular adrenocortical steroids. CHAPTER VI SUMMARY AND CONCLUSIONS The blood, plasma, and synovial fluid from 83 animals of mixed ages, sexes, and breeds has been analyzed. 0f the total number, 52 of these animals comprised indi- viduals that had been fasted 12 to 18 hours prior to the time of slaughter. The remaining 31 animals in the series was made up of individuals not intended for slaughter, many of whom were in some stage of lactation. In the overall picture, breed was not considered an important factor; however, sex and age did exert some influence on blood, plasma, and synovial fluid sugar levels. Age and sex also influenced the relative viscosity of syn- ovial fluid. From the standpoint of blood-synovial fluid sugar difference, as opposed to plasma—synovial fluid sugar dif— ference, the former relationship proved to be the most con- sistent, in that both groups, i.e., fasting slaughter cattle and non-fasting cattle, showed minimal changes in a compari~ son of values. From a diagnostic standpoint, the results of this study suggest that the blood—synovial fluid sugar differences have shown the most consistent relationship, and that plasma—synovial fluid sugar differences are too great, and too fluctuant to be of value. The total blood 138 139 and synovial fluid sugars in fasting slaughter cattle were considerably higher than in non-fasting cattle but this does not seem to alter the respective relationship between these constituents in the same individual. These factors are likewise applicable when sex and age of an individual are taken into consideration. Cellular studies tend to point to lymphocytes and monocytes as the predominant leukocytes of synovial fluid from the tibio—tarsal articulation. These findings can undoubtedly be attributed to the relatively young age of the animals in this study, and accounts for the fact that few clasmatocytes were observed. The erythrocytes encountered were attributed to hemorrhage at the time of aspiration in the living animals or extravasation of the cells into the joint cavity at slaughter due to agonal strug- gles. 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