Histological aspects of the function of the malpighian body in the living frog's kidney based on studies with the fluorescence microscope.код для вставкиСкачать
HISTOLOGICAL ASPECTS OF THE FUNCTION OF THE MALPIGHIAN BODY I N T H E LIVING FROG’S KIDNEY, BASED ON STUDIES WITH THE FLUORESCENCE MICROSCOPE EDWARD SINGER WITH THE TECHNICAL ASSISTANCE O F ELIZABETH B. CUZZORT Biological Laboratory, Cold Spring Harbor and Research Laboratory, St. Clare’s Hospitat, New Pork ELEVEN FIGURES The results of the experiments of many investigators of recent years show that there are so many divergent factors that influence the different tissues crowded into the microscopical malpighian body, that one cannot suspect its function from its morphological aspect in fixed sections ; or vice versa, one cannot attempt to deduce its histological appearance from its function. Fluorescence microscopy makes it possible t o observe the cytological details and study simultaneously the function of the malpighian body, as has been reported in previous papers. This method also permits at the same time an analysis of the chemical and physical changes that perpetually occur in the living cells but does not interfere with the function of the cells or disturb their structure. Thus by showing differences in cells that appear homogeneous in white light, the use of fluorescent light permits a closer correlation of structural changes and function than could be made by previous direct examination of living glomeruli in white light. ‘This investigation has been made with the assistance of a grant from the Diamond Jubilee Fund, New York. The author wishes to acknowledge the courtesy of Dr. Hugo Fricke, director of the biophysics laboratory at Cold Spring Harbor, f o r permitting the use of the facilities of his laboratory. We are also indebted t o Prof. Max Haitinger of Vienna who supplied us with many of the dyes used in the experiments, Acknowledgment is due also to Zeiss Company, New York, who constructed parts of the instrument according to my plans. 343 T H E ANATOMICAL RECORD, VOL. 66, NO. 3 344 EDWARD SINGER AND ELIZABETH B. CTJZZORT MATERIAL USED The frogs used in our experiments were of three species : Rana catesbiana, R. pipiens, and R. clamitans. These frogs, weighing from 25 to 250 gm., were caught on the grounds of the biological laboratory in the months of July, August, and September, and were kept and fed in captivity for a few days, as we noticed that the excitement due to capture caused an interference with the glomerular circulation. The malpighian bodies of these three species did not show structural differences but varied in size, those of Rana catesbiana being largest and best suited for examination; therefore our experiments were chiefly made on this species of frog, and all the illustrations of the malpighian bodies were made from their kidneys. It was necessary to discard a number of these frogs, as they were found to have a general parasitic infection, a fact that was called attention t o by Oliver and Smith ('30). We found, however, upon the examination of three frogs that the parasitic infection, if not very extensive, did not interfere with the normal function of the malpighian body. In forty-six of the experiments we were able to conduct observations from 6 to 8 hours without any interference that would have disturbed the working plan of the experiment. METHOD In preparing the animals for observation, the same method was employed that we used in our previous experiments in which the kidney of the animal was exposed by a ventral incision of the abdominal wall after carefully ligating all blood vessels in order to prohibit bleeding. To expose the ventral side of the kidney the intestines were pushed aside with cotton moistened with Ringer's solution. Both sexes were used for the experiments, and though the ovaries of the females and the enlarged testicles of the male during the spawning period made it difficult to focus on the kidney, these organs were not excised as we noticed that their incision had a reflex effect upon the glomerular capillaries thereby interfering with the circulation. HISTOLOGY OF THE LIVING MALPIGHIAN BODY 345 Most of the observations were conducted on the malpighian bodies situated between the lateral margin of the kidney and the suprarenal gland, as in this part of the kidney the cytological details were best observed, since here the malpighian bodies lie closest to the surface. Observations on the malpighian bodies were made in reflected white light and in their fluorescent light, which was excited by ultraviolet illumination. OBSERVATIONS 1. The rnalpigh/im bodies ifi white light The malpighian bodies of healthy frogs uninfluenced by any substance will be described first as they appeared in a reflected white light, as their appearance in this light will form a basis for comparison for further observations. I n this light the capillary loops and their continuous layer of endothelial cells were visible with their nuclei slightly protruding into the lumen of the capillaries. The outside covering of the capillaries, the visceral layer of the glomerular epithelium, appeared as a homogeneous layer of uniform thickness over the capillaries. No cell boundaries or structures of any kind could be seen. It could be observed that the capillary tufts form lobes that hang into the capsular space from a common body. This common body contains the afferent and efferent vessels. The afferent vessel lies closer to the ventral side of the kidney and therefore is more superficial in this position than the efferent ;hence it may be traced for a greater distance. Bauman’s capsule appeared as a thin membrane of squamous cells with their nuclei bulging into the capsular space in bead-like protuberances. The capsular space seen between the lobes of the glomerulus and the capsule was darker and duller than its boundaries. The only function that could be observed with any great accuracy in white light was the circulation that is continuous under normal conditions. The speed of the circulation may vary, but all glomeruli have the same speed at the same time. 346 EDWARD SINGER AND ELIZABETH B. CUZZORT The circulation, however, may be stopped easily by interferences that do not have any effect on the other blood vessels of the kidney. 2. The rnalpighian bodies in autofluorescent light The autofluorescence of the malpighian bodies is very slight ; therefore all the experiments were made by inducing fluorescence by means of different substances. Our choice of fluorsecent substances was guided by our past experiences with them, by Dh6r6's work ('33) and by Haitinger's studies on fluorescent dyes ('34). These substances have to be classified, as to their ability to show fluorescence in neutral, in acid, and in alkaline solutions, as to their chemical properties, and as to their behavior in the electrical field of the blood plasma. All of these points must be taken into consideration in any attempt to correlate the appearance of fluorescence staining with function. 3. T h e rnalpighiam bodies in induced fllzcorescerct light Descriptions of the malpighian body as it appeared in induced fluorescent light will be given best by grouping our findings according to the substances employed to induce fluorescence. The optimum amount of each substance that could be injected was determined and the strength of the solution was made accordingly. Thus the relation between the weight of the animal and the water incorporated with the injection of various substances remained constant. The majority of the fluorescent colors of the various substances show only in a very definite part of the malpighian body in ultraviolet light, and this appearance of selective fluorescence staining depends upon the functional state of the glomerulus and also upon the property of the substance. HISTOLOGY OF THE LIVING MUPIGHIAN BODY 347 TABLE 1 ACID suBSTbWms ELEC'IILICAL OIiARQI IN BUXD PLASMA Aesculine ~Uranine FLIJOlLESCENCIC COWIE IN ULl'BAVIOIaET LIQHT COLOB IN WHITl LIQHT .... Positive Positive Greenish Acriflavine Negative Yellow ~Thioflavine Negative Neutral Alcaline solution solution Acid solution Adsorbate Solid Blue Yellow Blue DulI yellow Greenish yellow Blueish yellow Purplish blue Blueish yellow Strong orange Strong orange Strong yellow Strong yellow green Red ~ Blue Green Blue Yellow Blue Green AWALINE SUBSTANCES __ 8. Thiazolyellow Primulin yellow Negative - Phosphine- Negative Negative ... Yellow Strong green Yellowish Faint green Faint 0. Berberine Negative _sulphate _ Geranine 0. Negative .... Green Strong green __ Yellow Strong Strong blue blue ___ Yellow Purple Faint Faint red red ___ Yellowish Strong Strong Blueish blue white purple Reddish .... Faint Strong yellow green ... Yellow Strong yellow Faint Faint red Faint red blue Yellow Red 1. Aescuhe. One cubic centimeter saturated solution of Aesculine for each 100 gm. of the weight of the animal was injected into a lymph sac. The mode of appearance of this substance was described previously in another paper (Singer, '33) where we showed that the glomerular capillaries are clearly visible until the filtration of Aesculine reached the same concentration in the capsular space as it had in the capillaries. This time using lenses with higher numerical aperture we could also distinguish that the entire capillary wall became luminous, showing the glomerular epithelium as a homogeneous covering but without any cell differentiations. The cells of the capsule, however, were visible. One minute after this took place all details of the glomerulus disappeared and the 348 EDWARD SINGER AND ELIZABETH B. CUZZORT glomerulus looked like a luminous blue bulb. The uniformity of the color was disturbed only by the circulating blood corpuscles, erythrocytes looking dark, the leucocytes and lymphocytes appearing brilliantly sparkling. As the extra-glomerular vessels stay clearly visible all the time, the disappearance of the glomerular capillaries indicates that the capsular space has the same concentration of Aesculine as the blood. This picture of the glomerulus showed only slight variations, as the exposure to light affected it in two ways. After illumination for 15 to 20 minutes the cell boundaries and other cytological details became clearer and after that slightly hazy. The blood circulation or urine Htration, however, was never observed to suffer any changes. The same phenomenon is also characteristic of the tissues that are exposed to illumination without any fluorescent substances. This phenomenon was noted by Wels and Jokisch ('29, '31) in their studies on sperm and ova, and they also mention that the autofluorescence of the cells increases during irradiation by ultraviolet light without any damaging influence on the vitality of the cell. Aesculine is, therefore, especially adapted for the study of elimination in the glomerulus, but its ability to bring out cytological differences, is not as good as that of other substances. 2. Urawine. One cubic centimteer of 1:1000 solution for each 100 gm.of the weight of the animal was injected into the lymph sac. The behavior of Uranine is similar to that of Aesculine but causes a better cell definition. However, this substance i8 more toxic than Aesculine. 3. ThioflaviHe 8. One cubic centimeter of a 4:1000 solution for each 100 gm. of the weight of the animal was injected into the lymph sac. The glomerular capillaries appeared almost instantaneously in a light greenish-yellow color that was much brighter than the surrounding blue-violet tissues. The capillary tufts were clearly defined, as the entire capillary wall and the glomerular epithelium was stained. The blood which circulates in the capillaries had a more yellowish color than the HISTOLOGY O F THE LIVING MALPIGHIAN BODY 349 wall itself. The fluid in the capsular space was dark purple (fig. 2). In this case, the fluorescent color of both, tissue and dye, being blue we do not have color changes resulting from the mixing of different fluorescent colors. The color changes in the fluorescence of Thioflavine S. must be explained by a phenomenon that we would call according to Haitinger and Hamper1 fluorescence metachromasy, that is, a color change in the fluorescene of the dye according to the pH and electric properties of the cell, and also to whether the dye is adsorbed or absorbed into the tissue elements. Thioflavine S. is a dye that is especially adapted to show the blood vessels and blood serum. The pictures obtained by it are clearer than any histological slide could offer. This clear-cut picture, however, lasted only 3 to 5 minutes, as the dye is photosensitive, and exposure to light changed the greenish color to yellow in the capillary wall, and also made it considerably duller. The fluorescence fast became diffuse and the cytological details *disappeared. At the same time the circulation in the glomerulus stopped in a characteristic way, the efferent vessel contracting and the glomerulus and afferent vessel filling with blood. The glomerulus that was filling the capsule, especially when curare was injected previously, shrank and its wall appeared considerably thinner as the staining of the epithelium disappeared. When this occurred, the internal margin of Bauman’s capsule became very distinctly yellow (fig. 5). If the exposure to light continues, the glomerulus may lose its fluorescence in ultraviolet light entirely, and the malpighian body may appear as a dark disc, in which the shrunken coils of capillaries were visible only in white light. This phenomenon showed that this dye besides being photosensitive has a photodynamic action and makes the glomerular capillaries photosensitive also. It had a similar effect on the liver, but it did not seem to affect the color or appearance of the tubuli or their contents. Thus it seems that blood serum in vivo stimulates photosensitization; while according to Tappeiner (’23) serum in vitro retards the photodynamic effects. Non-illuminated parts re- 350 EDWARD SINGER AND ELIZABETH B. CUZZORT mained intact and were used for further study 2 to 4 hours after the injection. 4. Thiuxol yellow. One cubic centimeter of 1:1000 solution for each 100 gm.of the weight of the animal was injected into the lymph sac. It stained the glomeruli with a blue-violet color, but its fluorescence was not strong enough and soon became diffuse ; thus it was discarded after a few trials f o r use in the glomerular experiments. 5. Primzclin yellow. One cubic centimeter of 1:1000 solution f o r eaeh 100 p.of the weight of the animal was injected into the lymph sac. This like the previous dye did not stain the malpighian bodies well, although in other structures the cells contained well-stained nuclei. 6. Phosphime 0. One cubic centimeter of 1:1000 solution for each 100 gm.of the weight of the animal was injected into the lymph sac. The endothelial cells in the glomerular capillaries and the capsule were clearly visible, as they became a greenish yellow when stained by Phosphine 0. Leucocytes were brilliantly white, but the fluorescent color of this dye did not show in the blood serum or in the tubular fluid. 7. Acriflaviae. One cubic centimeter of 1:1000 solution for each 100 gm.of the weight of the animal was injected into the lymph sac. Acriflavine stained the nuclei in the glomerulus if it was injected alone, but the picture appeared very dark. If, however, Acriflavine was injected following an injection of Aesculine, a very good picture of the glomerular epithelial Fig. 1 Malpighian body of a liviiig frog which was injected with Aesculine and Acriflavine showing the cell boundaries of the individual glomerular epithelial cells. The dark space between the tubules is the distended capsular space. Fig.2 Capsular space and glomerular tufts of a living frog as they appear in the fluorescent light of Thioflavine S. in the first 3 minutes of the ultraviolet irradiation. The walls of the capillaries are thick as the glomerular epithelium and the glomerular endothelium are stained. The numerous capillary tufts are crowded. HISTOLOGY OF THE L I V I S G ;ZIATAPIGHIAW BODY T H E ANATOMICAI, RECORD, !'OL 6 6 , NO. 3 351 352 RIIWARD SINGER A N D ELIZABETH B. CTTZZORT cells could 1)c seen (fig. 1). This indicates that Acriflaviiic was contained in the cytoplasm, but it needed the illuniinatiori of another substance to make it visible, a s it did not show fluorcscencc by itself in the cytoplasm. 8. H'erherin,e sulplaate. Onc cubic centimeter of 1: 100 solution for each 100 gin. of the weight of the animal was injected into thc lymph sac. rl'liis is a photodynamic dye which before being subjected to long irradiation stained thc nuclei. After irradiation of I0 to 35 minutes the nuclei became dark and the cytoplasm was stained. When the cytoplasm begin to show staining, the glomcrular cndothelial cells became visible. 9. Gwan,ine G. One cubic centimeter of 1: 1000 solutioii for each 100 gm. of the weight of the animal was injected into tlic lyiripli sac. The fluorescence of this dye mas hardly distinguishable. Only R slight staiiiing of the nuclei was visible. H,>h,avior o f the malpighialz bodies ulzder the iiiflueiace of swake ~ t n o mcaffeine , awd cziraril, and thP ef'ect of these ngrrrts on t h e flzcorcscewe stal'niitg Of the subsiances we have already mentioned, only Tliioflavinc S. i s deleterious to the vitality of the tissue. I t s toxic efiects were definitely connected with the illumination, for whcre there was no illumination or only a short exposure to irradiation, 110 damaging effects were detected. The above suhstances, which were also used by other investigators, prodiicc injuries to the malpighiun bodies or interfcre with thcir function independcnt of the illumination. 1 . I'enom of ('rotalirs adawaanteus (Texas rattlesnake). To obswve the effects of this venom two methods of injection were used : a. For each 100 gm. of the weight of the animal 0.20 to 0.30 mg. of the d r i d vciiioin was dissolvecl in water which was half the amourit of the required volume of solution. The other half of the required volume was obtained by adding glycerine. The dose was divided into thrce parts and given in 4. HISTOLOGY OF THE LIVING MALPIGHIAN BODY 353 Fig. 3 Malpighinn body of a liring frog, whieli was injcetcd with the vciiom of Crotalus atlamanteus tlircc time8 within 3 hours, during which time i t was under continuous microscopic observation in thc fluoresecnce staiuiug of Thioflavine S. It shows the slirunkeii glonicrular r:ipillaries with brightly fluorescent endothelial cells. A t t h e lower p a r t of t h e capsular space a luminous M B ~ S is visible. A t the right upper part of thc capsular spare is the affrrcnt and a t the left tho efferent vrssel. tlii*cc scparatc injcctioiis within 3 liours’ tinie, during whicli thc frog was uiider continuous iriicroscopical c~xaniiiiatioil. By using this method we wcre able to observe the immediate iw~ctioiiof the malpigliian body to the d e c k of the vciioi~~. Td’ollowing the injection of tlw vciiorn the walls of the capillary tufts aiid other hlood vcssels became very distinct aiid w r c ~ r c sliarp ~ aiitf hrillimtly lumiiious in white light. Two or 3 houi*s after tlie iitjcctioii tlic glomcrular tufts becanie ver:s1vollc11. Their w~allswcrc thickened but their lumiiia did iiot sccwi to be iir any way influcnced and permitted a very fast c*ircnlation of blood. I n some of the malpighian bodies tlie glomcrulus filled the ciitire capsule, lcaviirg hardly any capsular sriacc; whilc in others a larger spacc ii-as seen (fig. 3). Tn the lattcr the swollen loops of the gloincruli hung loosely into tlic capsular space like fingers of‘ a heavy glove, liming large spaces between thc loops. This swelling, as shown in figure 4, may mist in sonic glomeruli for scvcral days. Iii other glomeruli, it has been observed that these swolleii loops slowly tlraw together until finally all space disappeared bctweeii t h i n . At this stage the glomerulus looked like ail irregularly shaped mass. IIowever, the loops occasioiially parted agaiii with a sutldeii cxpaiisioii, wlieii the capsular wall bii rst. Burstiiig was obsei-vcd 4 to 5 hours after the first ~ciioiriinjection. microscopic section of a ruptured capsule is shown iii fiyure 8. A t the siirne time it conld IN? observed also that the tufts of other glomeruli with intact capsule expanded b? jerk)- movements. Each pulsc wave separated tlieiii further. aiicl further as though there would havc becii some rcsistaiice tit tlw iicck of thc g1omc:rulus which liad to he o v ~ ~ i ~ o by rnc the 1iigi.horpressure of the pulse wave. This is a pheiiomcnon, Fig. 4 Malpigliiaii hody of a liviiig frog, which was iiijcctrd aitli tlw vrnoiii of (”I 0t:llus il(l:llll:llltPIlN Oil tl1roc su< sit (lays :iii(l o1)servcd i i i tlici fluoi light trf Arsculiiir, oti the iiiittli (lay :iftrr the last injrctioii. It slioms swollen l o o p (if tlie rapillary wall 1i:inging looscl\ into the capsular s p c e :ind a very bright capsular nirni1)r:iw. Fig. 5 ,Malpighiaii body of the same ftwg as in figure 4 after iii,jertion of Thiofi:iviii(> K. ant1 exposure l o ultraviolet irratliation, sliowiiig tlw fatlctl fluorcsceiiw of l’liioflarinr 8. a i d stasis in tlie gloinrrular capillaries. (3 355 356 E:I)WABD SINGER ANJ) ELIZABETH B. CUZZORT however, which is sornetimes observed in glomeruli that were iiot exposed to any poison (fig.6). The glomerular circulation was affected i n the following way: After the injection of the venom the circulation in the glomeruli of some kidneys became jerky and intermittent. The blood corpuscles crowded and pushed each other in an cff‘ort to enter the capillaries, and only a few passed at a time. An hour after the injection the blood that circulatcs in the glonwrular capillaries contained an increased number of white bloocl corpuscles. The white blood corpuscles circulated on the periplierp of the blood stmam in contrast to the normal c*irculation. (lradually a number of the white blood corpuscles stuck t o the walls of the capillaries arid in 3 or 4 hours they 10:15 70:00 Fig. 6 72:15 m50 Cliaiigc!s ill the frcv capsu1:ir SJ):IW. lined the walls. 111 othcr kidneys after the injection, the circulation became more and more sluggish with the glomerular loops d w a y s in a loonc~lyhanging pod tion. Tiijectetl substances filtered through tlie capillaries into the ~lomeriilarcapsulc in thc first few hours, but it W R S difficult l o decide wlicther there was any filtration after the fifth hour, >is most of the animals arc moribund at ihis time. h. F o r each 100 gm. of aniinal 0.06 mg. to 0.12 mg. of venom WCIT prepared in the same way. This amount w a s injected o n w H day for 3 consec*utivedays making a total arriouiit of Fig. 7 .Microscopic. scction (if :I rnalpigliian body of the sniue frog as in figi1rc.s 4 and 5 stained with H. and 15. slio\~inga granular exudate and ruptured rapsnlo. The c,xtrcrnely dil:itcd neck is also notewort1i.v. Fig. 8 Microscopic scction stained with H. and E. showing tlic shrunken glouierular capsule of a frog which rcct4ved three injections of the wnorn of (‘rotalus :rdamantcus in 3 succcssivc days :inti W R B killed t h e fourth day :ifttJr the kist injwtion. HISTOLOGY O F T H E LIVING MALPIGHIAR BODY 357 0.18 to 0.36 for each 100 gm. of tlie frog. Thc frogs were used for cxpcriments or killed for dissection 3 or 4 days after thc last injection, dcpeiidiiig upon how many days it took for t hc? frogs to rcwh a moribund stage. I n malpigliian bodies of kidneys that were in,jcctecl BCcording to this method, wc saw hypcraemia in the glomerular capi1li-lric.s and a sweIIii~gof thc gloincrular loops. Injected fluorescent substances appeared as iii tlic normal malpigliian hoclies. Most of the glomeruli showctl good circulation, but soriie were ~)lugg.cclwith 1)lood and staycd that way, unaffoctctl by adrciialinc or saline injections. The cntirc capillary \w11 was illnminatecl h;v fluorescence if Thioflaviiw 8. was injcctcd arid tlic. capsiilc~w i ~ ssurrounded by ;1 luminous border. Ilctwwn this luminous liiic and tlic sui~oundiiigv(wc1s was i i tlctiiiite chrk violet spacc (fig. 5 ) . 111 tlic glornc~i.ul;i~~ <~ii1)sUlc WC! occasioii;illy o b ~ e r ~ t~i l~ i i tlia t ap1)c~nrctlw i * ylumiiious in the flnoresccnt I igh t of ‘I’liioflaviiic S. and opaque in white light. ‘l’his could have bcen ti gloiiiernlar (?xiidate,which shows on Ihc slides made of tlicsc kidneys or it might h a r e bccii tlint tlic swollen cilii of the ncclr of t he nialpigliiaii body appeared in this manner for this substance al~--ayslies opposite to tlie lower pole of the glomerulus, whei-e cilii a r e found. Thcsc swollen rilii also tiplictir on the slides (fig. 7 ) . 2. Oafeime. (’affeinc is an alcoloicl having a slight bluish ])urplc fluoresceiwc a i d was uscd in a solution of 4:1000 of which 1 cc. was injected for cadi 100 gm. of the wcight of thc u i m a l into the lymph sac. After tlic injectioii tlic circulation in thc glomerular ciq)illtirics hecnrnc much faster, even rapid. Thc corpus(~lcs,howcvcr, did not proccccl snioothly at an even specd but riiovctl with a jerky motion. This would iiidicatc that tlicrc TWS ti high pressure iii the ;ifferelit arterial system for which the efierciit vessels c~ouldnot take care; or that the cffcrciit r c w c l wzs soiiicwliat coiitractcct a t some place i i i i d this coiit radio11 liad t o I ) c b overcome'. Glomcr*ulurtufts that did not show a n y c>irculatioiib ~ f o i ~ 110\v h , begail to havo 21 good eir*cul* <It‘1011. HISTOLOGY O F THE LIVING MALl’IGRIAX BODY 359 white light we could see very clearly the glomerular tufts with a very lumiiious iiiside margin. The inner border of the capsulc was also very luminous. This luminous line was surrounded by a darkcr, thicker line which, again, had a luminous outer lining. The luminosity of these lines which also contailled sparkling dots tirid streaks, became more and I11 Fig. Y Malpigliian body of a liviiig frog in reflected white light after injection of Caffeine showing thin dark outside margin and a broader luminous inside margin on the capillary walls of the glomerulus. The capsular epithelium is lunrinous. Its nuclei protrude into the capsular space. Around the rpithelium is :I dark linc outside of which is aiiothcr luminous limb. THE AXATOYICAL BECOBD, VOL. 6 6 , NO. 3 360 EDWARD SINGER AND ELIZABETH B. CUZZOItT more sparkling during observation (fig. 9). These streaks were also visible on the rriicroscopic section made from these kidncys (fig. 11). I n ultraviolet light the luminous lines in the capillaries had a slight greenish-yellow fluoresceuce ; those on the capsule did not show any fluorescence. If Aesculine was in,jcctecl its fluorescence appeared almost immediately in the cvipsular spti(~e. Tlie outline of the capsule was violet and distiiict. The gloniernlar capillaries, liowever, were not clistinguishable at all, evtw at the begiiiiiing, in contrast to the glomeruli iii B kidney that did not r.cwivc any caffeine. The capsule appeared, a s a homogeneous, lnrninoiis bulb, in which seemiugly free blood corpuscles circulated. The liiniinosity was higlicr than in capsulcs without caffeine, antl it reached nearly the luminosity of the distal convoluted tubuli (fig. 10). Whcn Thioflavine S. was injected and its fluorescence appearcd, the picture was that of a glomerulus without Caffeine. .?. ("umriZ. One cubic centimeter of 1: 10 of the standard Curaril solution was irijcctcd for each 100 gm. of the weight of the animal into thc lymph sac. This substance did not seem to have any other effect on the appcaraiicc or function of the glomerulus, but it should he mentioned a s it was used to paralyze the frogs in some of the experiments and affected the size of thc capsular space. Capsular spacca wcre narrower in frogs that were injected with curaril. Fig. 10 Malpighian body of R living frog, wliicli received Caffeinr in the fluorescent light of Aesculiw. The liaziness of the glomerular structures anti the slmrpness of the dark capsule are noteworthy. Tlic luminosity of tlir glomrrulus antl that of an adjoining distal eonvolutrd tubule is about the sainr. Fig. 11 Microscwpic sertioii of the ni:~lpighian body of the sanie frog as in figurr 10 stailid with H. :iud E. showing numerous luminous strcwks in tbc capillar,V wall. 361 1>1S(’1788ION 7 ’ 1 1 ~/I’M capsdar. spacc awd its variatioirs ( )i11* oliscrwitioiis of the experimciit s with fluorescent s u b poisoiis give evidence that the size of tlic capsular space that is unoccupicd by glomerular tufts tlepeiids on the functional state of the entire oi-gaii of the kidney tint1 not only oil the function of the gloiiicrulus. ‘I’hc size of t l i c free capsular spaces i n normal malpigliian bodies is .\-PI->similar, and slight increase o r decrease in ilic size of tlic (*apsulztrspaces owurs siinnltaneously in the glomcruli a s 1 0 1 1 ~ ;is tlic ltidiicy is kept uiidcr circumstances that closclp coiiforiii to its physiological state. Iiicreasc or decrease in thc free capular spztcc is due to compressioii or expansion of the 10112s of the glomerulus. This variation of the free space does iiot a i d y iiiflneiice thc circulation. LLKU and TVhite ( ’22) callcd attention to tlie ahove fact and we have also reported it in a previous paper. Variations in the size of the free catipsulttr space in the same kidney occur under pathological o r exl)aiixioii of the xlomerulus. cwiiclitions by the sli~*i~~Iiillg [’athologicol factors a r e also rcsponsihlc for the tlistciition a i d shrinkage of tlic capsule. A diminished free capsular space occurs with the 1)eginiiing of t l i ~tlarnagiiig cffecbt 011 thc g1oiriorulw~-tufts, or glomciw loiiephritis caused by Crotalus adamanteus venom, for the swolleii lobes of the glomerulus occupy more space. The free capsular space becomes larger a few hours after the begiiining of t h e iiiflmmiation. Y’liis iiicrease ill f r e c ciipsnlai- S ] ) H C C ~i i t l ~ wto the compression of the glomei*ular tufts, by iiicreasccl 1)wssure of the glomerular fluid, for it c ~ i he i seen that the cii*cunifcwxce of the capsule does not change 1)ut tlie size of tlw g1omcAriilus decreases. rncrcasetl l)ressm*c! ot’ the glouic~rularfluid indicatcs that its drainage into tlie tubuli is impaiiwl by sl g r m u l a r exudate foimed in the capsule ~ n t l pliiggiiig thc 11cck Tuercascd iiifiltmf ion may be only sccoiic1;irily rt1s1mi sihl o for the 11i g911ci. gl omci.ulai* pi’essiire as when 1)i’opei.(Irwiiiage is restoiwl the glomerulus expands. st tiiiees a i d HISTOLOGY O F THE LIVING MALPIGHIAN BODY 363 A variety of different sizes of capsular spaces can be observed when the capsule ruptures. I n our previous experiments we have observed, as have Lucas and White ( '32), the distention of the capsule itself. Never, however, did we observe the rupture of the capsule, even in capsules that were more distended than they become when the frogs are poisoned with snake venom. Rupturing of the capsule, therefore, would indicate that snake venom has a selective deleterious effect, not only on the glomerular capillaries as already has been described by Pearce ('09) but also on the capsule, which causes it to become brittle. The size of the free capsular space also diminishes by the use of curare. The diminishing is uniform and appears to be due to the shrinkage of the capsule, as the lobes of the glomerulus are close together without any interspaces. This shrinkage is so slight, however, that we were unable to make exact measurements. Enlarging of the free space occurs when the glomerular capillaries are shrunken through photosensitization. This phenomenon we shall describe more fully in the discussion of the glomerular tufts. Circurnstarzces g o v e r k g the appearalzce of fluorescelzce irc the capsular space Whether fluorescence appears or not in the fluid in the glomerular space after the administration of certain fluorescent substances depends upon the electric charge of the injected substance. The hue of the color of the fluorescence is in accordance with the di-electric constant2 of the capsular fluid. Thus fluorescence being an optical phenomenon, is a truer reflection of the electrical properties of the cell than vital stains that do not have fluorescence properties. According to the measurements made by Peterfi of the electrical charge of the glomerular fluid and the experiments Di-electric constant is a magnitude that regulates the electrostatic attraction or repulsion of electrically charged particles. The brightness of the shade of fluorescence is in reverse proportion to the di-electric constant. TEE ANATOMICAL RECORD, VOL. 66, NO. 3 364 EDWARD SINQER AND ELIZABETH B. CUZZORT of Keller ('30) made with vital stains, the glomerular fluid is positively charged. The results of our experiments agree with the findings of these two investigators, for Aesculine and Uranine, two dyes that are positively charged could be detected immediately in the capsular space after injection, but other dyes which were negatively charged took a long time to appear and never showed with a great degree of luminosity. This fact is also in agreement with the findings of Peterfi and Keller, who pointed out that the selectiveness of dyes is diminished with their raising concentration. The variation in the pH which plays an important role in the conditions controlling fluorescence will be of secondary importance, however, to the electric properties of the living organs, as it has an effect only on the intensity or color of the fluorescence and will not cause the fluorescence to appear or disappear, as the range of variation in pH, of the living tissue is very slight. The shade of the fluorescence is governed by the di-electric constant. The color of the fluorescence by having a darker shade in the capsular space than in the surrounding tissues indicates a high di-electric constant. This finding is also in harmony with the findings of Keller and Peterfi. The glomerular capillaries The various tissues of the malpighian body exercise an influence on the fluoresence of the different substances. This influence varies according to the chemical properties of the dyes and according to the functional state of the tissues; therefore the capillary wall will appear in different shapes and thicknesses at different times. The width of the loops of the glomerular capillaries can be exactly determined in white light. The different components, however, are not distinguishable, as the wall looks homogeneous. This wall also appears homogeneous in ultraviolet light for a few minutes after the injection of aesculine until the fluorescence becomes so diffuse that the margins of the loops are indefinite. HISTOLOGY O F THE L I V I N G M A L P I G H I A N BODY 365 This same homogeneous appearance of the entire capillary wall is present after the injection of Thioflavine S. but later when the tissue becomes photosensitized, only the endothelial cells are stained and the epithelial covering is unstained. Diminishing and disappearing of fluorescence in the epithelial covering indicates a damage that occurs in the epithelial cells of the capillaries. This disappearance is a complex phenomenon, and at present we are not prepared to give an explanation for it. Effects of damage on the glomerular capillaries can be manifested in the sharpening of their contour or swelling of their loops. A peculiar effect of damage is that of photosensitization, which causes a vascular spasm and is accompanied by a change of color. The nuclei of the endothelial cells are stained by all dyes grouped in our table as having a negative electric charge with the exception of Thioflavine S. The cytoplasm of glomerular epithelium appears fluorescent in ultraviolet light if Aesculine is injected previously to the injection of Acriflavine. The glomerular circulatiort An even and continuous circulation is found in glomerular capillaries of kidneys that are kept under conditions closely approximating the physiological state of the organism as was shown by the experiments of Tamura, Miyamura, Nishina and Nagasawa ( '27), and as we reported in our previous paper and for which our present experiments supply additional evidence. I n accordance with these investigators, we also observed the particular susceptibility of the glomerular circulation t o influences that have hardly any effect on the circulation in the blood vessels, a fact to which Richards and Schmidt ( '24) first called attention. This susceptibility causes a variety of interferences that can either diminish or accelerate the speed of the circulation. Diminished and jerky circulation is the first reaction following the injection of poisons. This jerky circulation gives way to a rapid circulation in the later stages, regardless of 366 EDWARD SINGER AND ELIZABETH B. CUZZORT whether vascular poisons were administered, as we observed in these experiments or tubular poisons given, as we reported in a previous paper. Intermittent circulation is caused by desiccation that may result in a stasis in the capillaries as recognized by Okkels ( '33). Stasis occurs always when hy-pertonic solutions are applied to the surface of the kidney as Ebbecke and Jaeger ( '33) showed. The strength of the solution which causes stasis is parallel with the water content of the animal. Our observation showed that stasis can also be the result of the photodynamic action of dyes. Stasis caused in this manner is the most constant, and the manner in which it occurs shows that it is the result of a spasm in the efferent vessel. This would indicate that the efferent vessel alone is capable of influencing the entire glomerular circulation, an occurrence that Bensley ( '29) postulated after demonstrating special cell elements around the efferent vessel which are capable of contraction. The extra capillary pressure seems to play a secondary and subordinate role on the circulation, as when the capsule is extremely distended, we as well as Ebbecke and Jaeger ( '33), observed very rapid circiilation. SUMMARY 1. The different cellular and humoral elements of the malpighian body were delineated by selective fluorescence staining. 2. As the appearance of the fluorescence staining and its color in the various elements of the malpghian body does not depend upon the presence of the dye only but also upon the physical and chemical properties of the structures, from the variations of the fluorescence conclusions could be drawn regarding the qualities of the different structures. 3. The pH, the electric charge, and the di-electric constant of the components of the malpighian body could be demonstrated. 4. Positively charged substances have fluorescence in the capsular fluid and in the cytoplasm, negatively charged have fluorescence in the nucleus. HISTOLOGY O F THE L I V I N G M A L P I G H I A N BODY 367 5. The variations in the electric and chemical properties manifested themselves in changes of the fluorescence staining. This caused different cell structures to be visible at different times and caused color changes in the cells and fluids. 6. In malpighian bodies of frogs poisoned with the venom of Crotalus adamanteus or in photosensitized malpighian bodies the glornerular and capsular epithelium showed more extensive changes in fluorescence than other constituents of the malpighian body. 7. The rate of the glomerular circulation is even and its flow continuous as long as the animal is kept under circumstances closely approximating the physiological state of the organism. 8. The glomerular circulation, as shown by Richards and Schmidt ( '24)is affected by influences that do not change the circulation in other blood vessels. 9. In accordance with the results of the experiments of Oliver and Smith ( '30), Mosonyi and Voith ( '35) and Reemelin and Isaacs ( '16) disturbances in the circulation and changes in the constitution of the blood plasma cause greater variations in the filtration of the fluorescent substances than changes in the histological structure of the malpighian body which are demonstrable by microscopic sections. LITERATURE CITED BENSLEY,R. D. 1929 The efferent vessels of the renal glomeruli of mammals as a mechanism for the control of glomerular activity and pressure. Am. J. Anat., vol. 44, pp. 141-169. BENSLEY,R. R. AND R. D. EENSLEY1930 The structure of the renal corpuscle. Anat. Rec., vol. 47, pp. 147-175. DE~W, CH. 1933 Naehweis der biologisch wichtigen Korper durch Fluoreseenz and Fluorescenz Spektren. Handb. biol. Arb. -4bt. 11. Phys. Meth., Teil 3, Heft 4. ' EBBECKE, U. AND A. J m m 1933 Lokale Einfliisse auf den Blutfluss im Glomerulus der Froschniere. Pfluger 's Archiv ges. Phys., Ed. 232, 8. 36-41. GIKLHORN,J. AND R. KELLER 1924 Uber elektive Vitalfarbungen. Vorldufige Mitteilung. Biochem, Zeitschr., Bd. 150, S. 2-13. HAITINQER, MAX 1930 Methoden der Fluorewnz Analyse. Mikrochemie, Bd. XI, Nf. Bd. V, S. 429-464. 1934 Die Methoden der Fluorescenz Mikroskopie. Handb. biol. Arbeitsmeth. Abt. 11. Teil 3, S. 3307-3337. 368 EDWARD SINGER AND ELIZABETH B. CUZZORT ISAACS, R. 1917 The reaction of the kidney colloids and its bearing on renal function. Am. J. Physiol., vol. 45, pp. 71-80. KELLER,R. 1930 Der electrische Faktor des Wassertransports im Lichte der Vitalfirbung. Erg. Physiol., Bd. 30, S. 294-407. LUCAS,A. AND H. L. WHI’PE 1932 Factors responsible for the abnormal distention of the glomerular capsule in the Nccturus kidney. Anat. Rec., VOI.53, pp. 371-385. MOSONYI, J. AND L. VoITri 1935 Zur Frage der Ultrafiltration i n den Glomerulis. Arch. exp. Path. Pharm., Bd. 177, S. 177-182. OKKELS,H. 1933 Lokale Einfliisse auf den Blutfluss im Glomerulus der Froschniere. Pfliiger’s Arch. ges. Physiol., Ed. 232, S. 741-742. OLIVER,J. AND P. SMITH 1930 Experimental nephritis in the frog. J. Exp. Med., vol. 52, pp. 181-193. PEARCE, R. 1909 An experimental glomerular lesion caused by venom. J. Exp. Med., vol. 11, p. 532. REEMELIN,E. B. AND R. ISAACS 1916 A study of the conditions involved in the accumulation of dissolved substances in the blood. Am. J. Physiol., VOI.42, pp. 163-174. RICHARDS, A. N. AND C. F. S C H X ~1924 A description of the glomerular circulation in the frog’s kidney and observations concerning the action of adrenalin and various other substances upon it. Am. J. Physiol., vol. 71, pp. 17%208. SINGER, E. 1932 A microscope for observation of fluorescence in living tissues. Science, vol. 75, pp. 289-291. 1933a A study of the morphological physiology ,of the frog’s kidney with the fluorescence microscope. Anat. Rec., vol. 53, p. 37. 1933 b Observations on the frog’s kidney with the fluorescence microscope. Am. J. Anat., vol. 53, pp. 469-495. TAYURA,K.,. I(. MIYAM~UIW, T. NISHINA AND H. NAGASAWA1927 The glomerular circulation i n the living frog’s kidney. Jap. J. Med. Science. IT.Pharm., vol. 1, pp. 211-228. TAPPEINER, H. 1923 Methoden beim h b e i t e n mit sensibilisierenden fluoreseierenden Farbstoffen. Handb. biol. Arbeitsmeth., Abt. IV, Teil 7, S. 1070-1082. WELS,P. AND M. JOKISCH 1939 Der Einfluss der Quartlampenbestrahlung auf die Fluorescenz von Geweben und Zellen. Pfliiger’s Arch. ges. Physiol., Bd. 223, S. 369-377. 1931 Weitere Untersuchungen fiber die Fluorescene bestrahlter Zellen. Ibid., Bd. 228, S. 671-682.