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Microcirculation of the rat placenta
Microcirculation
of the rat placenta
Scanning and transmission on fetal blood vesseh
electron
microscopic
observations
rut plmentu was studied by par&et exa,rnination with tke ~scannitzg &rtrw mirro.w$w (SEM) and the transmission electron microscope (TEM). B! e,xamining ca.stXsof vessels inr’ected with latex and prepared by corrosion in hypuchlorite ,solntions, thx f&l a,rteries, ueins, and capillaries of the placentu could be trm:ed in the .seanning electron microscope and, in addition, the ultrastructurul detail of micvoeircuiation arr,d hwtinut surjaces could be studied. Cast specimens shuzsed~fetal artrries e~~tflr~~~gthe renter of the p~centa and ranching. into tong, thaw t~esseh whick tra~~f‘~~~t,~~ almost tke e&-e th~ck~~i~.~~~ of the ~~~en~ be~~re d~~idi~~g into a ~narked~~ tortuous l,~~~lilla~ network. Tke ,smootk-surfaced ca@lary XX&~ /orated tit GUJartoio/ar end flvwed i,nto a muck less tortuous fn-euenous capillaq- netrtjork whick eondwted the blood into collecting zlenu1e.sand veins. Placental blood vessel.\ examined by tke TEM jnwided e~Gdene~etkot details observed by the SEM were urcurate fnr tke ultrastrueturaf afrj~eoranre of feta,! arterie.~, capillaries, and ueins.
IN'~RAV.~S~:~~,~R~~S~S havebeenstudied by scanning electron microscopy in such organs as kidney,t, * and lungs.’ To date. however, we are not aware of any such studies on the microcirculation of the placenta. We are, therefore, presenting some observations on the normal placental circulation in rats and suggesting that this microc~rculatiorl may be visualized in u~trastructura~ detail when corrosion preparations of latex casts are examined in scanning electron microscopy. Moreover, the great depth of field together with the high resolving power of the scanning electron microscope allow perception of three-dimensional relationships
of the microcirculatorv vesseis and of. their furnina surfaces. Corrosion-cast specimens have previously been made by Bbe4 on rat placenta+ by Art? on human placenta, and more recently by Arts and Lohman’r on rhesus monkey placenta. While these vascular studies have contributed to our knowledge of placental circulation, none of- the cast specimens was examined fat ultrastructural details. Visuahzation of the microcirculation, especially at the capillary level, would require microscopic examination. Scanning electron microscopic examination of placental tissue has been concentrated mainly on topography7 and has only recently been empIoyed for examination of the blood vessels of the human placenta.’ The present ~llvestigation correlates t.he results of the two types of microscopic examinations. We have described the ultrastructural appearances of fetal blood vessels from normal rat placenta as observed in thin sections in the transmission electron microscope and in latex casts examined m the scanning electron microscope. These observations are preiimmary to description of changes in the microcircula~ tion of the placenta when affected by pathologic lesions resulting from aging, drugs, or maternal disease. 495
496
Lee and Dempsey
Fig. 1. Diagram of the fetal circulation (F) in rat placenta. Thin, straight arteries (A] at the fetal side traverse almost the entire thickness of the placenta before branching at the maternal side into a dense, tortuous mass of capillaries (CL,,).This capillary network then forms anastomoses with a less tortuous, prevenous capillary network (L’! J. The blood is then conducted into a series of wide-lumen venules (Fr) which flow into large-size veins (&‘t) to be returned to the hilus and umbilical cord, The maternal arterial blood of the rat placenta enters through a large central artery which then divides, forming numerous branches (&t) which spread out laterally to supply the organ. The maternal blood flow is in the opposite direction to that of fetal arterial blood. This forms a counter-current flow which provides for efficient exchange of materials between mother and fetus.
MaWWs
and methods
Ten female rats (18 to 20 days pregnant) of the Sprague-Dawley strain weighing approximately ZOO grams each were used for this experiment. All animals were fed a diet of regular chow (Purma) and tap water. After the animal was anesthetized with sodium pentobarbital administered intraperitoneally (30 mg. per kiIogram), a V-shaped incision was made in the lower abdomen to expose the uterine horns. The uterine muscle layers, amnion, and yolk-sac of one side were cut with an extra-fine dissecting scalpel to expose the fetuses and placentas. The uterine horn of one side with three to four fetuses was used for TEM; the other uterine horn was used for the injection corrosion-cast technique. To obtain placenta1 tissue for TEM, the umbihcal cord was ligated and cut distally; one placenta was removed at a time from the uterine wall. Initially,
the placenta was divided at the mid-zone, with further subdivisions of the tissues made in a drop of Dalton’s fixative. The tissue was fixed in the cold at 4’ C. in Dahon’s chrome-osmium solution for 2 hours. Then the tissue was dehydrated through alcohoi series and embedded in Swiss Araldite (Durcupan). The tissue blocks were sectioned on a Porter-Blum MT-l microtome and stamed with lead citrate. The sections were examined in a RCA 3-G electron microscope. To prepare casts fcrr the scanning electron microscope, the remaining uterine horn wascut to expose the fetuses for in situ placental injections. First a small cotton swab was placed beneath the gelatinous umbihcal cord, then the umbilical artery or vein was cut to facilitate injection of a latex mixture (Cementex*) into *Cementex Corporation,
Inc., New York, New York.
Microcirculation
of rat placenta
497
Fig. 2. Scanning electron micrograph of a latex cast from placenta of rat. The stem vessel (fetal artery) has a small subdivision branch located on the right. The surface of the arterial vessel shows imprmts of nuclear impressions made by bulging nuclei of endothelial cells lining the IuminA surface. The small side-arm branch subdivides to form a capillarv network (~240.)
the umbilical attached to specimen,
arteq a 2 about
or
vein
c.c.
syringe.
0.5
c.c.
of
by a 25 gauge For 60
a per
needle
single-in-jection cent
Cementex
(diluted with distilled water) was injected into the blood vessels at a slow rate; about 3 minutes were required for the 0.5 c.c. injection. For double-injection studies, yellow Cementex was introduced into the artery and blue Cementex into the vein. In these cases the umbilical vessels were not severed and injections were made first into the artery and then into the vein. When aI1 of the placentas had been injected, the animal was left at room temperature for 3 hours to permit the Cementex to solidify: then each umbilical cord was cut and the placenta was removed from the uterus. The specimens l\‘ere fixed in IO per cent formaiin for a month to allo\\ for further hardening of’ the latex. The doubleinjection specimens were sliced in sagittal sections to be studied &jr the pattern of distribution of arterial and venous vessels in the rat placenta. The single-injection placentas were macerated by treatment in a solution of sodium hypochlorite (Clorox) for 2 to 3 days with daily changes. Following this treatment, the resulting latex casts of the blood L essels were washed gently in several rinses of distilled water, air dried, and then carefull) teased apart with a,jew~ler’s forceps into small groups
of vessels li>r detailed viewing, l’hta remaining steps were similar to those used in preparing renal gtomerular cast specimens.’ Vpon completion f:jt’ the preparations, the resulting placental vasculal casts were viewed and photographed on a C;ambridge Stereoscan Mark II SEM.
Observations By microscopic examination of the double-injection specimens, we were able to establish the general pattern of fetal arterial and venous circulation in the rat placenta. A simplified drawing representing the fetal circulation in the rat placenta is shown in Fig. 1. The arteries branch after entering the placenta and send thin? straight vessels (‘4) which traverse ahmost the entire thickness of the placenta before branching at the maternal side into a thick mass of capillaries [C,,) which is markedly tortuous in appearance. This capillary network then anastomoses with a less tortuous capillary network
((:J.
The
vessels
here
are
slightly
wavy
but
have an essentially straight course. From this capillary network, the blood is conducted through a series of wide-lumen venules CL’,) and then into large veins (V.J to
be
provided
returned
the
to
the
appearance
umbilical
of
cord.
the
The
\‘as( lriaturc.
SEJM
and
Fig- 3. Placental cast showing two arteries (center) traveling toward the maternal side of’ the organ and terminating in a rich capillary network there. The small caliber capillaries exhibit a twisting, winding appearance. (X 135.)
the course of’ blood flow was reconstructed from SEM observations as well as from double-injection specimens. The maternal arterial blood enters the placenta through a large centrally located artery which divides into numerous branches (M) to supply the organ. The maternal blood flows in opposite direction to that of the fetal arterial blood. To obtain a detailed picture of the microcirculation, it was necessary to examine the single injection cast specimens in the SEM. *axming electron microscopy. Casts prepared from the rat placenta were examined with the SEM. A difference was observed between the arterioles and the capillaries which was not noted in previous piacental injection studies for the arterial vessels in our latex casts have a rough surface contour with indentations presumably representing the bulging nuclei of endotheliaI ceils lining the vessels (Fig. 2). Similar observations on arterioles have been made in renal glomerular latex cast studies.‘, s The capillaries, in contrast, have a relatively smooth surface contour and a densely packed, tortous appearance (Figs. 3 and 5). The arterioles supplying this profuse capillary network originate from arteries which enter near the center of the placenta; these branch, sending thin, straight vessels directly across the thickness of the placenta (Fig. 4)
where, on the maternal side of the organ, the arterioles branch laterally to form a profuse network of capillaries. It is interesting to note that the long, straight conducting vessels may have side-arm branches ot- a very small caliber which can suddenly ramify f’rom the main vessel. Such a side-arm branch, coming off at right angles, is shown in Fig. 4. .Another delicate branch which has just furcated from the stem vessel is shown in Fig. 2. The abrupt change in blood vessel size may be of significance in the henlc)dynamics of the fetal arteriolar circulation (see below for additional comments). While the capillary network on the arteriolar side may be characterized as dense and tortuous, the cap& lary network on the venous side is characterized by straighter vessels, all radiating toward the larger diameter venules (Figs. 6 and i’). It is OF interest that the capillaries, whether at venous or arteriotar areas, are of fairly uniform diameter with no marked dilatations or constrictions to alter the btood flow. Transmission electron microscopy. The fetal capitlaries provide an excellent study of endothelial cells and pericytes The capillary lumen varies skghtly in diameter and may have two or three endothelial cells forming the lining. These cells contain a l’ew
Microcirculation
of rat placenta
Fig. 4. Placental cast of fetal arteries shows two long, straight vessels which branch into a thick. tortuous capillary network at the maternal side (top). The arrow (right) points to a small side-arm branch of the fetal artery which demonstrates an abrupt change in blood vessel size from the wide-diameter artery to the small-caliber off-shoot. This abrupt transition may be of significance in the hemodynanics of fetal arterial circulation. (X 180.)
499
Fig. 5. Placental capillary network and the diameter
cast of fetal capillaries located at the arteriolar side of the blood supply. The dense has a markedly tortuous appearance. The luminal surfaces appear to be smooth, of these small vessels are uniform in width. (~550.)
Fig. 6. Placental cast of venous side of fetal circulation. The capillary network (right) flows into venules and then into veins (left). The capillaries are no longer tortuous; instead, they are fairly straight in appearance. The blood is conducted directly from capillaries into largeI- venules and veins. (X 120.)
Microcirculation
of rat placenta
501
Fig. 7. A higher power view of venous side capillary network showing the essentially straight courw of the small caliber vessels. The direction of Row is downward (left) toward venules. The minor pit\ and elevations seen on the surface contour indicate that the endothehal lining of the venous side vessels is irregularly smooth. (X 2 IO.)
mitochondria, some rough endoplasmic reticulum, and a few vesicles. One endothelial cell may be attached to an acljacent one by tight junctions at the apposed surfaces. The cytoplasm is usually thinly stretched around the capillary lumen, forming a relatively smooth surface contour. A well-defined basement membrane surrounds the endothelial cells (Fig. 8). This figure also illustrates the close attachment of two pericytes to the outer surface of the endothelial cells. This close apposition of pericytes to the capillary wall, leaving no space in beth-een, was observed frequently. On the lower left of Fig. 8, the inner trophoblast cell layer can be seen lying adjacent to the basement membrane of the fetal capillary. The cytoplasm of this inner layer of trophoblast contains lipid droplets which are fairly numerous here, The venules observed in placental tissue have a fairl! wide lumen with several endothelial cells forming the lining. The cytoplasm, while attenuated, is not as smooth as the lining of capillaries. The venules have a delicately irregular endothelial lining (Fig. 9) with small bleb-like structures projecting into the lumen, and surrounding the endothelial cells in a thin basement membrane. During the course of transmission electron microscopic examination, we discovered a capillary with fetal
erythrocyte (RBC) apparently being extruded through a minute opening in the endothelial Ivail (Fig. 10). The fetal KBC, containing several mitochondria, was wedged between two adjacent endothelial cells and was evidently about to enter the maternal blood space and mix with maternal red blood cells.
Comment The placental circulation in examined by various procedures
rodents has been through our corrosion-cast preparations supportecl by REM observations. A higher degree of ultrastructual accuracy in the examination of the fetal microcirculation of rat placenta has been attained than has heen possible with previotls techniques. To study placental morphology and functions, several significant injection studies have heen done on rat placenta. In 1950, Bbe4 did a series of experiments on placental circulation in rats, using first lndia ink injections and then corrosion preparations made with vinyl acetate. By means of these two types of injection techniques, Bde demonstrated the vascular pattern of hoth fetal and maternal circulations. Our latex cast specimens of rat fetal blood vessels are in accord with the macroscopic vascular pattern demonstrated by his work.
502
Lee and Dempsey
Fi& 8. Transmission electron micrograph of a fetal capillary from nuclei are evident; the attenuated endothehal cytoplasm forms a located at the lateral surfaces which help to maintain close cell membrane surrounds the endothelial ceHs. On the lower right, two the capillary wall. On the lower left, the inner layer of trophoblast in the cytoplasm. (~5,250.)
Fig. 9. Transmission electron micrograph of a fetal containing red blood corpuscles, several of which contain a slightly irregular surface; the endothehal cell cytoplasm the lumen. (~5.000.)
rat placenta. Two endotheliaf cell smooth bning. ‘Tight junctions arc to cell contact, A thin basement pericytes lie m close apposition to is seen with lipid droplets present
venufe (near a capillary-venule junction) mitochondria. The lining of the venule has sends many small bleb-like projections into
Microcirculation
of rai placenta
503
Fig. 10. Transmission electron micrograph showing a small capillary with a fetal ervthrtlcvtte into the maternal circuiatiot~ wz IIOI observed frequently. (X7,%0.) The investigations of B& and of Mossman provided evidence that the rodent placenta has a countercurrent How: i.e., the direction of blood flow in maternal and fetal blood vessels is in opposite directions. our present anatomical findings are consistent with this concept: such a counter-current Row would provide a more efficient exchange of materials between mother and fetus, with gaseous and nutritive exchanges occurring in the dense capilIary regions. Mossman’ in his 1965 paper has discussed the various blood flow systems in mammalian placentas and concluded that the c(~unter-current type may predominate. This would appear to contradict physiologic evidence presented by Meschia and associatesLo and by Wilkin” that the blood J%ow in sheep placenta is of the concurrent type. Their physiologic data indicated that a concurrent flow system exists in mammalian placentas and also that this type of bIood flow provides for a less efficient diffusion rate between blood vesseJs. The counter-current JJow theory of Mossman* however, does not entirely contradict the work of Meschia and associates since, in monkey and human pJacentas, the counter-current flow is restricted to only a limited area of the organ: i.e., significant counter-current Row occurs only in the short villi of the terminal tufts of the placenta where
the delicate
capillaries are of short length (0.2 to 0.7 where the interviJlous maternal blood interstices are about I mm. or less. The larger blood vessels of the placenta mav operate on .I concurrent system. So mammalian placentas ma! exhibit both types of blood flow in the same organ; or. they may be exclusively of one type throughout. N’hile current physiologic data support a concurrent UC)\%sytem in most matl~rnali~in placentas. some’ pk~sct~ntas {e.g., iSodent) remain of the counter-current 1: pc. Further anatomical studies in conjunction Gth physiologic 6ndings would be helpful in ebbdating the ‘intricate patterns of placental blood flow. SEM observations demonstrated that the capilJaries in rat placenta are of fairly uniform diameter. with relati>-ely smooth surface cont,ours. This differs from the appearance of capillaries in rhesus monkey and in human placenta where Arts and J,ohm~~n” and J%$c”’ found the capillaries to be of ~ar+~g Aiber with sinusoidal dilatations. The difference, hog\ t*vt*r* mav be procedural for our ir~jections were pdi)~ tnwi 011 amw thetired animafs (with fetuses stiJJ afive) so that the resulting casts should show the fetal placental circuittion with minimum alterations. if’ any. in the sasculature. mm.)
and
504
Lee and Dempsey
An unexpected observation was seen during SEM examination of the casts of fetal arteries. These large vessels are capable of sending off very small branches directly from the main stem. It would seem that such delicate branching from a large vessel is hemodynamitally unfeasible, for the intravascular pressure within the artery must be relatively high, and at the division point of the small branch, such a pressure should be great enough to burst the delicate branch. A compensatory mechanism apparently exists to prevent vascular damage; however, no attempt was made to elucidate the intravascular pressure-regulating mechanism. The SEM has also shown that the capillaries at the arteriolar side differ in appearance from those at the venous side. The first capillary network has a markedly tortuous appearance with numerous anastomoses present. This dense, tortuous capillary network located at the arteriolar end may have functional significance. Their winding and twisting course could reduce the velocity of the blood flow and hence facilitate the exchange processes which occur at the capillary level. Then, as this network forms anastomoses with the venous-side capillary network, the need for slow circulation of blood to facilitate absorption is decreased, while the need for faster drainage of blood is increased. Thus, the second capillary network has straighter vessels, all radiating towards the venules, carrying the blood directly into the collecting venules and veins. Morphology and function would appear to correlate quite well. While we have not yet examined casts of primate placenta in the SEM, we may speculate that such a similar ultrastructural vascular pattern would exist there also. Electron microscopy of the human placenta has been done by many investigators.” Electron microscopy of rodent placenta also has been done.” These investigators have examined trophoblast cells, syncytium. and capillary tissues. Our present findings confirm and extend previous electron microscopic observations. The capillary wall is composed of endothelial cells with attenuated cytoplasm attached at one cell surface to another by tightjunctions which serve to maintain close cell-to-cell contact. A well-defined basement membrane surrounds the capillaries and, quite often, pericvtes are found closely applied to the outside of endothelial cells. In addition to these observations, we have described the appearance of venules and also the appearance of a
REFEREMCES
I.
Lee, M. L., Purkerson, E. W.: Ultrastructural
M. L., Agate, F. J., and Dempsey, changes in renal glomeruli of rats
fetal erythrocyte apparently leaving a capillary through a minute rupture in the endothelial wall. Thy occ:~sional leakage of fetal erythrocytes into the maternal circulation has been postulated as an explanation i’~r erythroblastosis fetalis and has been examined by CC perimental work in physiology,15 but not previously visualized by electron microscopy. Our observaCons provide physical evidence of the possibility that fetal erythrocytes can leave capillaries to enter the maternal circulation. A final word remains to be said about die significance of utilizing the SEM to recognize and to interpret the vascular pattern of the placenta. To date, SEM ohservations of the placenta have been limited mainly to surface examination, expecially of the villi.7 Sheppard and Bonnar” initiated SEM studies on placental blood vessels, including spiral arteries, Our present repor examines the blood vessels in the SEM by a dif’feren~ technique. Both methods could be applied to the study of placental vasculature to expand our knowledge of’ fetal and maternal circulation within the placenta. We have seen that the two microscopic techniques could be correlated. The ultrastructural findings of TEM support SEM observations as demonstrated, for example, by the smooth luminal surfaces of capillary casts accLtrately representing the smooth endothelial lining sew in the TEM. Similarly, the irregular endothehal lining seen in the TEM of fetal venules caused minor pits and elevations to be present in the venule cast specimens, In the same manner infarcts, thrombi and fibrin depositions observable in the TEM would, depending on degree of severity, be visualized as partial to complete constrictions in cast specimens seen in the SEM. An examination of the pattern of distribution of vascular lesions such as infarcts and thrombi may reveal the cause of some cases of’ fetal morbidity and death. The corrosion-cast technique coupled with TEM observations can illuminate the ultrastructural pathologic changes which may occur in the placental circulation during maternal disease states or during drug administration. Our findings suggest that this procedure w-ill be applicable to such future studies on the placenta as they have done previously in studies on the experimentally altered kidney. The advice and help of Drs. Myron Tannebaum Richard Hoar is acknowledged with appreciation.
and
during experimentzaNy induced hypertension acid uremia, Am. J. Anat. 135: 191, 1972. 2. Lee, M. L.: Morphological effects of procaine amide on
Microcirculation
~OUW kidney as observed by scanning and transmission electron microscopy, kdb. Invest. 31: 324, 1974. Nowelf, 1. A.> and Lohse, C. L.: Injection replication of the mic&vasculature for scanning -electron hicroscopy, irz fohari. 0.. amf Corvin. I., editors: Scanning Electron M&roscop~/ 1974, Chicago, 1974, ITT Res. ?nst., pp. 267-27 I. B&, F.: Studies on placental circulation in rats. II. Vascular pattern illustrated by corrosion preparations, Acta. E:ndocrinof. 5: 369, 1950. Arts, N. F. Th.: Investigation on the vascular system of the placenta, AM. J. OBSTET. GYNECOL. 82: 147, 1961. ,\rts, X. F. Th., and Lohman. A, H. M.: An injectioncorrosion stucfy ot.the f’etal and maternal vascular systems in the placenta of the rhesus monkey, Eur. .J. Obstet. Gvnecol. Reprod. Binl. 4: 133. 1974. Ludwig, H.: Surf’ace structure of the human term fllacenta ancf of’the uterine wall post partum in the screen scdrl ekrtrOn Ink ~-0scOp~. AM..J. ~hSwr. GyNEcoL. 111: 328, f!lif. Sheppard, B. L., and Bonnar, J.: Scanning electron microsc op? of tfle human f>lacenta and decidual spiral artericq in normal pregnancy, J. Obstet. Gynaecol. Br. Commons. 81: 20. 1974. Mobsman. f I. W.: The princif>af interchange vessels of the
of rai rjlacenta
505
chorioallantoic placentas of mammals. ,U f)ctl.~~n, R. I~., and Lrsprung, H., editors: Organogensis, YEW YOI-k, 1965, Hoft, Rinehart & Winston, fjp. 771.i8.3. 10. Meschia, G., Battagfia, F. C., and Bruns, I’. I).: ‘l’hcore~icat and experimental study of ~ra~~\f>la<~~~t~~l ditf’usiott, ,I. Appt. Physiol. 22: 1171, 1967. Il. Wilkin. P,: Les theories explicative\ ~II tn~xxt~ismc cfes &hanges transpfacentaires, ,I[ Sn~eck. ,J.. hfitor: IA, Placenta Humain, Paris, f97,3, \faw~t~ C: C !C pf~. 238279. M. I~,. and E;w~, G. F.: .%n 12. Martinek, J. J.. Gallagher, electron microscopic studv of fetal capillarb hasal famines , of ‘normal’ human term placenta\, ,A*“! ,I. 0asrfi.1. GYNXOL. 121: 17% 197.5. 13. Bde, F.: Studies on human placenta. f I I, ~~;is~~ll~lri7atiotl of the young fetal placenta, B. Vascufari/a~ion of the translucent villus and the syncvtiaf hnd, \\itfl special reference to the cell islands and the has;11 platr, Ac.t‘~ Obstet. Gynewl. Smnd. 48: 167. 1969. E, W’,: Efec tron micros14. F$‘islocki, G. B., and Dempsey, copy of the placenta oi the rat, Anal. Rec. 123: 33, 19.55. 15. Dan&, J., and Schneider, H.: Physiology: f’ransfer aml barrier function, it! Grutmwafcf. P., Editor l’ht. Placenta and I& Shternal Supple I,inv. ~‘niv. B~ili~~not e. l97S, L’ni\c*rsit\ f’ark f+ss.
of the rat placenta
Scanning and transmission on fetal blood vesseh
electron
microscopic
observations
rut plmentu was studied by par&et exa,rnination with tke ~scannitzg &rtrw mirro.w$w (SEM) and the transmission electron microscope (TEM). B! e,xamining ca.stXsof vessels inr’ected with latex and prepared by corrosion in hypuchlorite ,solntions, thx f&l a,rteries, ueins, and capillaries of the placentu could be trm:ed in the .seanning electron microscope and, in addition, the ultrastructurul detail of micvoeircuiation arr,d hwtinut surjaces could be studied. Cast specimens shuzsed~fetal artrries e~~tflr~~~gthe renter of the p~centa and ranching. into tong, thaw t~esseh whick tra~~f‘~~~t,~~ almost tke e&-e th~ck~~i~.~~~ of the ~~~en~ be~~re d~~idi~~g into a ~narked~~ tortuous l,~~~lilla~ network. Tke ,smootk-surfaced ca@lary XX&~ /orated tit GUJartoio/ar end flvwed i,nto a muck less tortuous fn-euenous capillaq- netrtjork whick eondwted the blood into collecting zlenu1e.sand veins. Placental blood vessel.\ examined by tke TEM jnwided e~Gdene~etkot details observed by the SEM were urcurate fnr tke ultrastrueturaf afrj~eoranre of feta,! arterie.~, capillaries, and ueins.
IN'~RAV.~S~:~~,~R~~S~S havebeenstudied by scanning electron microscopy in such organs as kidney,t, * and lungs.’ To date. however, we are not aware of any such studies on the microcirculation of the placenta. We are, therefore, presenting some observations on the normal placental circulation in rats and suggesting that this microc~rculatiorl may be visualized in u~trastructura~ detail when corrosion preparations of latex casts are examined in scanning electron microscopy. Moreover, the great depth of field together with the high resolving power of the scanning electron microscope allow perception of three-dimensional relationships
of the microcirculatorv vesseis and of. their furnina surfaces. Corrosion-cast specimens have previously been made by Bbe4 on rat placenta+ by Art? on human placenta, and more recently by Arts and Lohman’r on rhesus monkey placenta. While these vascular studies have contributed to our knowledge of placental circulation, none of- the cast specimens was examined fat ultrastructural details. Visuahzation of the microcirculation, especially at the capillary level, would require microscopic examination. Scanning electron microscopic examination of placental tissue has been concentrated mainly on topography7 and has only recently been empIoyed for examination of the blood vessels of the human placenta.’ The present ~llvestigation correlates t.he results of the two types of microscopic examinations. We have described the ultrastructural appearances of fetal blood vessels from normal rat placenta as observed in thin sections in the transmission electron microscope and in latex casts examined m the scanning electron microscope. These observations are preiimmary to description of changes in the microcircula~ tion of the placenta when affected by pathologic lesions resulting from aging, drugs, or maternal disease. 495
496
Lee and Dempsey
Fig. 1. Diagram of the fetal circulation (F) in rat placenta. Thin, straight arteries (A] at the fetal side traverse almost the entire thickness of the placenta before branching at the maternal side into a dense, tortuous mass of capillaries (CL,,).This capillary network then forms anastomoses with a less tortuous, prevenous capillary network (L’! J. The blood is then conducted into a series of wide-lumen venules (Fr) which flow into large-size veins (&‘t) to be returned to the hilus and umbilical cord, The maternal arterial blood of the rat placenta enters through a large central artery which then divides, forming numerous branches (&t) which spread out laterally to supply the organ. The maternal blood flow is in the opposite direction to that of fetal arterial blood. This forms a counter-current flow which provides for efficient exchange of materials between mother and fetus.
MaWWs
and methods
Ten female rats (18 to 20 days pregnant) of the Sprague-Dawley strain weighing approximately ZOO grams each were used for this experiment. All animals were fed a diet of regular chow (Purma) and tap water. After the animal was anesthetized with sodium pentobarbital administered intraperitoneally (30 mg. per kiIogram), a V-shaped incision was made in the lower abdomen to expose the uterine horns. The uterine muscle layers, amnion, and yolk-sac of one side were cut with an extra-fine dissecting scalpel to expose the fetuses and placentas. The uterine horn of one side with three to four fetuses was used for TEM; the other uterine horn was used for the injection corrosion-cast technique. To obtain placenta1 tissue for TEM, the umbihcal cord was ligated and cut distally; one placenta was removed at a time from the uterine wall. Initially,
the placenta was divided at the mid-zone, with further subdivisions of the tissues made in a drop of Dalton’s fixative. The tissue was fixed in the cold at 4’ C. in Dahon’s chrome-osmium solution for 2 hours. Then the tissue was dehydrated through alcohoi series and embedded in Swiss Araldite (Durcupan). The tissue blocks were sectioned on a Porter-Blum MT-l microtome and stamed with lead citrate. The sections were examined in a RCA 3-G electron microscope. To prepare casts fcrr the scanning electron microscope, the remaining uterine horn wascut to expose the fetuses for in situ placental injections. First a small cotton swab was placed beneath the gelatinous umbihcal cord, then the umbilical artery or vein was cut to facilitate injection of a latex mixture (Cementex*) into *Cementex Corporation,
Inc., New York, New York.
Microcirculation
of rat placenta
497
Fig. 2. Scanning electron micrograph of a latex cast from placenta of rat. The stem vessel (fetal artery) has a small subdivision branch located on the right. The surface of the arterial vessel shows imprmts of nuclear impressions made by bulging nuclei of endothelial cells lining the IuminA surface. The small side-arm branch subdivides to form a capillarv network (~240.)
the umbilical attached to specimen,
arteq a 2 about
or
vein
c.c.
syringe.
0.5
c.c.
of
by a 25 gauge For 60
a per
needle
single-in-jection cent
Cementex
(diluted with distilled water) was injected into the blood vessels at a slow rate; about 3 minutes were required for the 0.5 c.c. injection. For double-injection studies, yellow Cementex was introduced into the artery and blue Cementex into the vein. In these cases the umbilical vessels were not severed and injections were made first into the artery and then into the vein. When aI1 of the placentas had been injected, the animal was left at room temperature for 3 hours to permit the Cementex to solidify: then each umbilical cord was cut and the placenta was removed from the uterus. The specimens l\‘ere fixed in IO per cent formaiin for a month to allo\\ for further hardening of’ the latex. The doubleinjection specimens were sliced in sagittal sections to be studied &jr the pattern of distribution of arterial and venous vessels in the rat placenta. The single-injection placentas were macerated by treatment in a solution of sodium hypochlorite (Clorox) for 2 to 3 days with daily changes. Following this treatment, the resulting latex casts of the blood L essels were washed gently in several rinses of distilled water, air dried, and then carefull) teased apart with a,jew~ler’s forceps into small groups
of vessels li>r detailed viewing, l’hta remaining steps were similar to those used in preparing renal gtomerular cast specimens.’ Vpon completion f:jt’ the preparations, the resulting placental vasculal casts were viewed and photographed on a C;ambridge Stereoscan Mark II SEM.
Observations By microscopic examination of the double-injection specimens, we were able to establish the general pattern of fetal arterial and venous circulation in the rat placenta. A simplified drawing representing the fetal circulation in the rat placenta is shown in Fig. 1. The arteries branch after entering the placenta and send thin? straight vessels (‘4) which traverse ahmost the entire thickness of the placenta before branching at the maternal side into a thick mass of capillaries [C,,) which is markedly tortuous in appearance. This capillary network then anastomoses with a less tortuous capillary network
((:J.
The
vessels
here
are
slightly
wavy
but
have an essentially straight course. From this capillary network, the blood is conducted through a series of wide-lumen venules CL’,) and then into large veins (V.J to
be
provided
returned
the
to
the
appearance
umbilical
of
cord.
the
The
\‘as( lriaturc.
SEJM
and
Fig- 3. Placental cast showing two arteries (center) traveling toward the maternal side of’ the organ and terminating in a rich capillary network there. The small caliber capillaries exhibit a twisting, winding appearance. (X 135.)
the course of’ blood flow was reconstructed from SEM observations as well as from double-injection specimens. The maternal arterial blood enters the placenta through a large centrally located artery which divides into numerous branches (M) to supply the organ. The maternal blood flows in opposite direction to that of the fetal arterial blood. To obtain a detailed picture of the microcirculation, it was necessary to examine the single injection cast specimens in the SEM. *axming electron microscopy. Casts prepared from the rat placenta were examined with the SEM. A difference was observed between the arterioles and the capillaries which was not noted in previous piacental injection studies for the arterial vessels in our latex casts have a rough surface contour with indentations presumably representing the bulging nuclei of endotheliaI ceils lining the vessels (Fig. 2). Similar observations on arterioles have been made in renal glomerular latex cast studies.‘, s The capillaries, in contrast, have a relatively smooth surface contour and a densely packed, tortous appearance (Figs. 3 and 5). The arterioles supplying this profuse capillary network originate from arteries which enter near the center of the placenta; these branch, sending thin, straight vessels directly across the thickness of the placenta (Fig. 4)
where, on the maternal side of the organ, the arterioles branch laterally to form a profuse network of capillaries. It is interesting to note that the long, straight conducting vessels may have side-arm branches ot- a very small caliber which can suddenly ramify f’rom the main vessel. Such a side-arm branch, coming off at right angles, is shown in Fig. 4. .Another delicate branch which has just furcated from the stem vessel is shown in Fig. 2. The abrupt change in blood vessel size may be of significance in the henlc)dynamics of the fetal arteriolar circulation (see below for additional comments). While the capillary network on the arteriolar side may be characterized as dense and tortuous, the cap& lary network on the venous side is characterized by straighter vessels, all radiating toward the larger diameter venules (Figs. 6 and i’). It is OF interest that the capillaries, whether at venous or arteriotar areas, are of fairly uniform diameter with no marked dilatations or constrictions to alter the btood flow. Transmission electron microscopy. The fetal capitlaries provide an excellent study of endothelial cells and pericytes The capillary lumen varies skghtly in diameter and may have two or three endothelial cells forming the lining. These cells contain a l’ew
Microcirculation
of rat placenta
Fig. 4. Placental cast of fetal arteries shows two long, straight vessels which branch into a thick. tortuous capillary network at the maternal side (top). The arrow (right) points to a small side-arm branch of the fetal artery which demonstrates an abrupt change in blood vessel size from the wide-diameter artery to the small-caliber off-shoot. This abrupt transition may be of significance in the hemodynanics of fetal arterial circulation. (X 180.)
499
Fig. 5. Placental capillary network and the diameter
cast of fetal capillaries located at the arteriolar side of the blood supply. The dense has a markedly tortuous appearance. The luminal surfaces appear to be smooth, of these small vessels are uniform in width. (~550.)
Fig. 6. Placental cast of venous side of fetal circulation. The capillary network (right) flows into venules and then into veins (left). The capillaries are no longer tortuous; instead, they are fairly straight in appearance. The blood is conducted directly from capillaries into largeI- venules and veins. (X 120.)
Microcirculation
of rat placenta
501
Fig. 7. A higher power view of venous side capillary network showing the essentially straight courw of the small caliber vessels. The direction of Row is downward (left) toward venules. The minor pit\ and elevations seen on the surface contour indicate that the endothehal lining of the venous side vessels is irregularly smooth. (X 2 IO.)
mitochondria, some rough endoplasmic reticulum, and a few vesicles. One endothelial cell may be attached to an acljacent one by tight junctions at the apposed surfaces. The cytoplasm is usually thinly stretched around the capillary lumen, forming a relatively smooth surface contour. A well-defined basement membrane surrounds the endothelial cells (Fig. 8). This figure also illustrates the close attachment of two pericytes to the outer surface of the endothelial cells. This close apposition of pericytes to the capillary wall, leaving no space in beth-een, was observed frequently. On the lower left of Fig. 8, the inner trophoblast cell layer can be seen lying adjacent to the basement membrane of the fetal capillary. The cytoplasm of this inner layer of trophoblast contains lipid droplets which are fairly numerous here, The venules observed in placental tissue have a fairl! wide lumen with several endothelial cells forming the lining. The cytoplasm, while attenuated, is not as smooth as the lining of capillaries. The venules have a delicately irregular endothelial lining (Fig. 9) with small bleb-like structures projecting into the lumen, and surrounding the endothelial cells in a thin basement membrane. During the course of transmission electron microscopic examination, we discovered a capillary with fetal
erythrocyte (RBC) apparently being extruded through a minute opening in the endothelial Ivail (Fig. 10). The fetal KBC, containing several mitochondria, was wedged between two adjacent endothelial cells and was evidently about to enter the maternal blood space and mix with maternal red blood cells.
Comment The placental circulation in examined by various procedures
rodents has been through our corrosion-cast preparations supportecl by REM observations. A higher degree of ultrastructual accuracy in the examination of the fetal microcirculation of rat placenta has been attained than has heen possible with previotls techniques. To study placental morphology and functions, several significant injection studies have heen done on rat placenta. In 1950, Bbe4 did a series of experiments on placental circulation in rats, using first lndia ink injections and then corrosion preparations made with vinyl acetate. By means of these two types of injection techniques, Bde demonstrated the vascular pattern of hoth fetal and maternal circulations. Our latex cast specimens of rat fetal blood vessels are in accord with the macroscopic vascular pattern demonstrated by his work.
502
Lee and Dempsey
Fi& 8. Transmission electron micrograph of a fetal capillary from nuclei are evident; the attenuated endothehal cytoplasm forms a located at the lateral surfaces which help to maintain close cell membrane surrounds the endothelial ceHs. On the lower right, two the capillary wall. On the lower left, the inner layer of trophoblast in the cytoplasm. (~5,250.)
Fig. 9. Transmission electron micrograph of a fetal containing red blood corpuscles, several of which contain a slightly irregular surface; the endothehal cell cytoplasm the lumen. (~5.000.)
rat placenta. Two endotheliaf cell smooth bning. ‘Tight junctions arc to cell contact, A thin basement pericytes lie m close apposition to is seen with lipid droplets present
venufe (near a capillary-venule junction) mitochondria. The lining of the venule has sends many small bleb-like projections into
Microcirculation
of rai placenta
503
Fig. 10. Transmission electron micrograph showing a small capillary with a fetal ervthrtlcvtte into the maternal circuiatiot~ wz IIOI observed frequently. (X7,%0.) The investigations of B& and of Mossman provided evidence that the rodent placenta has a countercurrent How: i.e., the direction of blood flow in maternal and fetal blood vessels is in opposite directions. our present anatomical findings are consistent with this concept: such a counter-current Row would provide a more efficient exchange of materials between mother and fetus, with gaseous and nutritive exchanges occurring in the dense capilIary regions. Mossman’ in his 1965 paper has discussed the various blood flow systems in mammalian placentas and concluded that the c(~unter-current type may predominate. This would appear to contradict physiologic evidence presented by Meschia and associatesLo and by Wilkin” that the blood J%ow in sheep placenta is of the concurrent type. Their physiologic data indicated that a concurrent flow system exists in mammalian placentas and also that this type of bIood flow provides for a less efficient diffusion rate between blood vesseJs. The counter-current JJow theory of Mossman* however, does not entirely contradict the work of Meschia and associates since, in monkey and human pJacentas, the counter-current flow is restricted to only a limited area of the organ: i.e., significant counter-current Row occurs only in the short villi of the terminal tufts of the placenta where
the delicate
capillaries are of short length (0.2 to 0.7 where the interviJlous maternal blood interstices are about I mm. or less. The larger blood vessels of the placenta mav operate on .I concurrent system. So mammalian placentas ma! exhibit both types of blood flow in the same organ; or. they may be exclusively of one type throughout. N’hile current physiologic data support a concurrent UC)\%sytem in most matl~rnali~in placentas. some’ pk~sct~ntas {e.g., iSodent) remain of the counter-current 1: pc. Further anatomical studies in conjunction Gth physiologic 6ndings would be helpful in ebbdating the ‘intricate patterns of placental blood flow. SEM observations demonstrated that the capilJaries in rat placenta are of fairly uniform diameter. with relati>-ely smooth surface cont,ours. This differs from the appearance of capillaries in rhesus monkey and in human placenta where Arts and J,ohm~~n” and J%$c”’ found the capillaries to be of ~ar+~g Aiber with sinusoidal dilatations. The difference, hog\ t*vt*r* mav be procedural for our ir~jections were pdi)~ tnwi 011 amw thetired animafs (with fetuses stiJJ afive) so that the resulting casts should show the fetal placental circuittion with minimum alterations. if’ any. in the sasculature. mm.)
and
504
Lee and Dempsey
An unexpected observation was seen during SEM examination of the casts of fetal arteries. These large vessels are capable of sending off very small branches directly from the main stem. It would seem that such delicate branching from a large vessel is hemodynamitally unfeasible, for the intravascular pressure within the artery must be relatively high, and at the division point of the small branch, such a pressure should be great enough to burst the delicate branch. A compensatory mechanism apparently exists to prevent vascular damage; however, no attempt was made to elucidate the intravascular pressure-regulating mechanism. The SEM has also shown that the capillaries at the arteriolar side differ in appearance from those at the venous side. The first capillary network has a markedly tortuous appearance with numerous anastomoses present. This dense, tortuous capillary network located at the arteriolar end may have functional significance. Their winding and twisting course could reduce the velocity of the blood flow and hence facilitate the exchange processes which occur at the capillary level. Then, as this network forms anastomoses with the venous-side capillary network, the need for slow circulation of blood to facilitate absorption is decreased, while the need for faster drainage of blood is increased. Thus, the second capillary network has straighter vessels, all radiating towards the venules, carrying the blood directly into the collecting venules and veins. Morphology and function would appear to correlate quite well. While we have not yet examined casts of primate placenta in the SEM, we may speculate that such a similar ultrastructural vascular pattern would exist there also. Electron microscopy of the human placenta has been done by many investigators.” Electron microscopy of rodent placenta also has been done.” These investigators have examined trophoblast cells, syncytium. and capillary tissues. Our present findings confirm and extend previous electron microscopic observations. The capillary wall is composed of endothelial cells with attenuated cytoplasm attached at one cell surface to another by tightjunctions which serve to maintain close cell-to-cell contact. A well-defined basement membrane surrounds the capillaries and, quite often, pericvtes are found closely applied to the outside of endothelial cells. In addition to these observations, we have described the appearance of venules and also the appearance of a
REFEREMCES
I.
Lee, M. L., Purkerson, E. W.: Ultrastructural
M. L., Agate, F. J., and Dempsey, changes in renal glomeruli of rats
fetal erythrocyte apparently leaving a capillary through a minute rupture in the endothelial wall. Thy occ:~sional leakage of fetal erythrocytes into the maternal circulation has been postulated as an explanation i’~r erythroblastosis fetalis and has been examined by CC perimental work in physiology,15 but not previously visualized by electron microscopy. Our observaCons provide physical evidence of the possibility that fetal erythrocytes can leave capillaries to enter the maternal circulation. A final word remains to be said about die significance of utilizing the SEM to recognize and to interpret the vascular pattern of the placenta. To date, SEM ohservations of the placenta have been limited mainly to surface examination, expecially of the villi.7 Sheppard and Bonnar” initiated SEM studies on placental blood vessels, including spiral arteries, Our present repor examines the blood vessels in the SEM by a dif’feren~ technique. Both methods could be applied to the study of placental vasculature to expand our knowledge of’ fetal and maternal circulation within the placenta. We have seen that the two microscopic techniques could be correlated. The ultrastructural findings of TEM support SEM observations as demonstrated, for example, by the smooth luminal surfaces of capillary casts accLtrately representing the smooth endothelial lining sew in the TEM. Similarly, the irregular endothehal lining seen in the TEM of fetal venules caused minor pits and elevations to be present in the venule cast specimens, In the same manner infarcts, thrombi and fibrin depositions observable in the TEM would, depending on degree of severity, be visualized as partial to complete constrictions in cast specimens seen in the SEM. An examination of the pattern of distribution of vascular lesions such as infarcts and thrombi may reveal the cause of some cases of’ fetal morbidity and death. The corrosion-cast technique coupled with TEM observations can illuminate the ultrastructural pathologic changes which may occur in the placental circulation during maternal disease states or during drug administration. Our findings suggest that this procedure w-ill be applicable to such future studies on the placenta as they have done previously in studies on the experimentally altered kidney. The advice and help of Drs. Myron Tannebaum Richard Hoar is acknowledged with appreciation.
and
during experimentzaNy induced hypertension acid uremia, Am. J. Anat. 135: 191, 1972. 2. Lee, M. L.: Morphological effects of procaine amide on
Microcirculation
~OUW kidney as observed by scanning and transmission electron microscopy, kdb. Invest. 31: 324, 1974. Nowelf, 1. A.> and Lohse, C. L.: Injection replication of the mic&vasculature for scanning -electron hicroscopy, irz fohari. 0.. amf Corvin. I., editors: Scanning Electron M&roscop~/ 1974, Chicago, 1974, ITT Res. ?nst., pp. 267-27 I. B&, F.: Studies on placental circulation in rats. II. Vascular pattern illustrated by corrosion preparations, Acta. E:ndocrinof. 5: 369, 1950. Arts, N. F. Th.: Investigation on the vascular system of the placenta, AM. J. OBSTET. GYNECOL. 82: 147, 1961. ,\rts, X. F. Th., and Lohman. A, H. M.: An injectioncorrosion stucfy ot.the f’etal and maternal vascular systems in the placenta of the rhesus monkey, Eur. .J. Obstet. Gvnecol. Reprod. Binl. 4: 133. 1974. Ludwig, H.: Surf’ace structure of the human term fllacenta ancf of’the uterine wall post partum in the screen scdrl ekrtrOn Ink ~-0scOp~. AM..J. ~hSwr. GyNEcoL. 111: 328, f!lif. Sheppard, B. L., and Bonnar, J.: Scanning electron microsc op? of tfle human f>lacenta and decidual spiral artericq in normal pregnancy, J. Obstet. Gynaecol. Br. Commons. 81: 20. 1974. Mobsman. f I. W.: The princif>af interchange vessels of the
of rai rjlacenta
505
chorioallantoic placentas of mammals. ,U f)ctl.~~n, R. I~., and Lrsprung, H., editors: Organogensis, YEW YOI-k, 1965, Hoft, Rinehart & Winston, fjp. 771.i8.3. 10. Meschia, G., Battagfia, F. C., and Bruns, I’. I).: ‘l’hcore~icat and experimental study of ~ra~~\f>la<~~~t~~l ditf’usiott, ,I. Appt. Physiol. 22: 1171, 1967. Il. Wilkin. P,: Les theories explicative\ ~II tn~xxt~ismc cfes &hanges transpfacentaires, ,I[ Sn~eck. ,J.. hfitor: IA, Placenta Humain, Paris, f97,3, \faw~t~ C: C !C pf~. 238279. M. I~,. and E;w~, G. F.: .%n 12. Martinek, J. J.. Gallagher, electron microscopic studv of fetal capillarb hasal famines , of ‘normal’ human term placenta\, ,A*“! ,I. 0asrfi.1. GYNXOL. 121: 17% 197.5. 13. Bde, F.: Studies on human placenta. f I I, ~~;is~~ll~lri7atiotl of the young fetal placenta, B. Vascufari/a~ion of the translucent villus and the syncvtiaf hnd, \\itfl special reference to the cell islands and the has;11 platr, Ac.t‘~ Obstet. Gynewl. Smnd. 48: 167. 1969. E, W’,: Efec tron micros14. F$‘islocki, G. B., and Dempsey, copy of the placenta oi the rat, Anal. Rec. 123: 33, 19.55. 15. Dan&, J., and Schneider, H.: Physiology: f’ransfer aml barrier function, it! Grutmwafcf. P., Editor l’ht. Placenta and I& Shternal Supple I,inv. ~‘niv. B~ili~~not e. l97S, L’ni\c*rsit\ f’ark f+ss.