Toxemia of pregnancy is a common complication of gestation, occurring usually in late pregnancy. Whether toxemia represent an exaggeration of changes incident to pregnancy or depends upon some wholly new factor is a moot point.
Indeed, the cause of the toxemia of pregnancy, despite decades of intensive research, remains the great enigma of obstetrics and constitutes one of the most important unsolved problems in the whole field of human reproduction.
Glomerulonephritis can be induced in various animal species by numerous serums and tissue extracts. Its production by duck immune serum was first described in the rabbit by Masugi (1934).By using a potent standardized nephrotoxic duck serum or its gamma globulin, nephritis can be reproduced in a regular manner by Seegal, et. al., (1936).
Antibody Produced in rabbits by the injection of the rat placenta also is high nephrotoxic to the rat by Seegal and Loeb (1943).
The experiments recorded here show the results of injecting rabbit antidog-placenta serum into both pregnant and non-pregnant dogs as described by Seegal et al. (1955). The course o( the resulting nephritis is compared with that following the injection of rabbit antidog-kidney serum.
The large size of the animal permitted frequent bleeding and the gestation period allowed for observation of nephritis during pregnancy. The findings support the conclusion that rabbit antidog-placenta serum injected in the dog produced an acute
nephritis which usually Progressed to a chronic state compact:;bale to that which follows the injection of anti·kidney serum. Pregnancy has not been terminated by this antiserum.
Beveans et al. (1955) describe the lesions produced in these pregnant and non-pregnant dogs following injection of either rabbit anti-placenta or rabbit anti-kidney serum.
Acute and chronic phases of the nephritis have been studied over a period of 10 months. The intravenous injection of rabbit antidog-placenta or antidog-kidney serum produced immediate evidence of glomerulonephritis in dogs and rabbits.
The glomerulonephritis so induced may terminate in death within 8 days, may progress to a chronic form or may heal.
Recently, Irino et at. (1967) induced renal lesions in rats by placental extracts. These changes were observed with the electron microscope and the renal glomerular alterations in rats with a clinical syndrome resembling toxemia of pregnancy showed the characteristic changes consisting of swelling with decreased
density of the basement membrane, a dense granular deposition within and along capillary basement membranes, and marked swelling and slight proliferation of gomerular epithelium.
The glomerular lesions, designated endothelial glomerulitis are apparently a result of an antigen-antibody reaction and present further evidence that human toxemia of pregnancy has an immune mechanism as a basis for its production.
Kim (1969) had an attempt to establish the pathologic changes induced by sensitizing the rat against homologous placental tissues and to compare them with the lesions of the kidney in human toxemia. He found that renal lesions were closely related to that of human toxemia of pregnancy.
The present investigation is aimed to study the lesions in the glomerulus of the pregnant rat kidneys induced by repeated injection of homologous placental tissue as observed with the light, the fluorescent and the electron microscope and adds
further evidence for the view that the syndrome, as induced experimentally, constitutes an analog of toxemia of pregnancy as it affects the human.
Materials and Methods
Male and female albino rats weighing around 200 ems. were divided into Group Ⅰ and Ⅱ. Group Ⅰ is normal control group and was subdivided into non·pregnant, pregnant, and delivery groups, Group Ⅱ was subdivided into non-pregnant, pregnant,
delivery, and abortion groups which were then injected with homologous rat's placental tissue with complete Freund's adjuvant mixture.
The placental tissue was injected intraperitoneally once every week in a dose of 0.5 ml and a number of injections was 8 times.
Estrus cycle was determined by the vaginal smear methods of Long and Evans (1922), and matching was achieved by housing a male and female rat together in the pre-estrus cycle and rechecking the vaginal smear for the presence of spermatozoa.
In this way the matching was begun from the last injection of placental tissue.
The placental emulsion was prepared as follows. Placentae were removed on the 18 th and 20 th duty of pregnancy. After washing them with sterile isotonic saline solution several times, a 30%saline suspension of placenta was made by the tissue homogenizer and added to the same amount of Freund's complete adjuvant (Difico laboratory).
Rabbit anti-rat Placental serum of IgG fraction of high specificity and potency was conjugated with fluorescin isothiocyanate (FITC) (Microbiochemical associates I.N.C), and was used in the immune- fluorescent studies.
The animals were sacrificed and necropsied, at one or two week intervals after the fast injection of antigen.
After gross examination and the removed kidneys were divided into terce parts.
One part of the kidney was fixed in 1% osmium tetraoxide in veronal buffer at pH 7.4 for 2 hours each, and was dehydrated at 4℃ in a graded series of ethanol solution and embedded in Epon 812, and ultrathin sections were made with glass knife in 400 to 500 Å and were stained with saturated uranyl acetate. Sections
were examined in Hitachi HU-11E electron microscope.
The other parts of kidney tissue was fixed in 10% formalin and cut in 6 micron thickness after paraffin embedding, it was stained with routine hematoxylin-eosin and periodic acid Schiff (PAS) stain. And were examined with light microscope. A part of the remainder of kidney tissue was frozen quickly and was sectioned at 5 to 6 microns with the cryostat at-l5℃. The sections were thawed and are dried at room termperature for 10 minutes. The sections were overlaid with FITC conjugated rabbit
anti-rat placental serum and placed in petridishes containing moistened filter paper for one hoar at room termperature. The slides were rinsed in cold 0.01M phosphate buffered saline, pH 7.4 and placed in a 50% solution of glycerol and phosphate buffered saline and covered with cover glass. It was examined for
specific fluorescent light with a A.O spencer microscope using an Osram HBO W mercury are lamp with appropriate appropriate ultraviolet filters.
The blocking test was carried out in each set of sections by unconjugated anti-sera followed by fluorescent staining.
Result and Discussion
Body weight, blood pressure, and urine albumin were within normal range and no other pathological alterations were observed in the kidney by histologically, histochemically, immunologically, or electron microscopically in groupⅠ animals. The GroupⅡ, that had received numerous placental emulsion injections, showed a marked raise of blood pressure and an increase in urine album in net only in the pregnant and delivery groups but also the non-pregnant group.
At necropsy, the kidneys of all groupⅡ animals showed increased perirenal fat tissue, pale appearance, and no adherent renal capsule and cortex.
On cut sections, they revealed a pale and widened cortex, the renal pelvis had no gross alteations. The light microscopic findings are been mainly in the glomeruli, with variable and non-specific changes in the other structures. The glomeruli were all affected and enlarged. They are characteristically bloodless glemeruli, with the capillary lumen reduced by cytoplasmic swelling of endothelial cells. The number of cells in the glomerular tuft is either normal or slightly increased, and
inflammatory infiltrates are sparse or absent. The lobular pattern of the glomerular tuft is often accentuated in the late stage of pregnancy of GroupⅡ animals, and visceral epithelial cells are urge in size because of cytoplasmic swelling
The PAS positive material is present in the visceral epithelial cells and basement membrane of the glomerular capillary.
Endothelial cells showed swelling of their cytoplasm with the result that they may fill the capillary lumen partially or almost completely. The mesangium showed focal thickening present in some lobules. Those findings are more prominent from two weeks gestation and early delivery animals in GroupⅡ.
The capillary basement membrane of the glomerulus is thickened in all of the animals but the third week gestation group revealed characteristic thickening of the capillary walls.
The tubules showed no characteristic alteration. The PAS positive materials are contained often in the proximal convoluted tubules but the tubule usually appeared relatively normal. And renal blood vessels and the interstitial tissue showed no remarkable finding.
Intense apple- green fluorescence was observed in the basement membrane of the glomerular capillary and in the cytoplasm of the endothelial cells. The endothelial cytoplasmic fluorescence revealed more prominence in the basement membrane of capillary as linear or spot like appearance. The fluorescence was proved to be
specific anti-placental serum by the blocking test. This specific fluorescence was observed only in the GroupⅡ animals with multi-injected placental emulsion and the most intense findings showed in the late period of pregnancy and in the two weeks from last injection of placental emultion of non-pregnant animal. No specific fluorescence is evident, in the GroupⅠ.
The endothelial cells appeared increased in size. In some instances the increase in size was accompanied by a decrease in the relative density of the cytoplasmic ground substance and a reduced concentration of organelles. This findings were believed to represent cellular swelling. Some areas showed numerous caveolae intracellulares and multiple adjacent intracytoplasmic vesicles.
The endothelium showed the development of multiple small, cytoplasmic folds termed cytofotds. These cytofolds related vacuoles contained aggregates of dense, floculent material. Occasional single membrane-limited cytoplasmic inclusion bodies
containing homogenous dense material were seen.
These findings are more prominent in pregnant and delivered animals of GroupⅡ.
The basement membrane appeared relatively unaltered in GroupⅠ. The GroupⅡ showed uneveness in shape and density. Other in focal areas and on occasion diffusely, the lamina densa exhibited a patchy increase in density and marked widening. In some cases the lamina rara interna was frequently obliterated or occupied by deposit material or abnormally widened. Occasionally the complete
disappearance of the lamina rara interna in the late pregnant animal of GroupⅡ was noted. The lamina rara externa was relatively unaltered without the finding of mild increased density.
The epithelial cells were also enlarged but microvilli were less apparent. The foot process was frequently preserved, with only, focal areas that showed the portion of thickened basement membrane. Some of the epithelial cells were many and large with single membrane-limited inclusions containing various density of dense material and occasionally showing the non-membrane-limited dense bodies, and vesicles.
The data presented here indicated that there are general similarities in the structure of glomeruli of human preeclampsia or eclampsia, previously described by many authors. The dense material deposits are largely intracapillary and subendothelial portion as lamina densa and lamina rara interna of capillary
In systemic lupus erythematosus they may be intracapillary, subendothelial, or subepithelial and in acute glomerulonephritis they are almost entirely subepithelial (Faith and Trump 1966). This findings suggest that certain differences do exist between eclampsia and other renal disease in histological basis.
The appearance of the deposited material was different in other renal disease. With such a mechanism, immunologic specificity may be essential to glomerular localization as in the immuno fluorescent microscopic findings. Summary:
Homologous placental emulsion injected into pregnant rats induced perirenal fatty changes, bloodless glomeruli, and massive PAS positive material deposited in the basementmembrane and epithelial cells. Specific anti-placental antibody was observed in the basementmembrane and endotkelial cell cytoplasm. Uotrastructurally, the dense material deposits are largely intracalpillary and lamina densa and lamina rara interna of capillary basementmembranes, and swelling and slightly proliferation of glomerular endothelium and epithelium.
The glomerular lesions, designated endothelial glomerulitis and basementmembrane alterations are apparently a result of an immunological reaction and present evidence that human toxemia or eclampsia has an immunemechanism as a role for its production by auto-antiplacental antibody.