Rat Amniotic Fluid Research Articles

Simultaneous Determination of Zalcitabine and Stavudine in Maternal Plasma, Amniotic Fluid, Placental, and Fetal Tissues Using Reversed Phase on Silica Liquid Chromatography/Tandem Mass Spectrometry

In order to study the placental transfer of nucleoside reverse transcriptase inhibitors, a quick and simple reversed phase high performance liquid chromatography with tandem mass spectrometry method has been developed and validated using an underivatized silica column for the separation and analysis of DDC and D4T from rat plasma, amniotic fluid, placental, and fetal homogenate. Extraction of DDC, D4T, and their internal standard lamivudine (3TC) from the matrices was processed by protein precipitation using ice cold acetonitrile. Chromatographic separation was achieved on a Waters Spherisorb S3W silica column (4.6 mm × 100 mm) equipped with a Phenomenex guard column. The mobile phase consisted of 20% methanol in 22 mM formic acid. The flow rate was 0.4 mL/min, and MRM was used for detection. The calibration curves for each day of validation showed good linear response over the range from 2 ng/mL to 2000 ng/mL. The absolute recoveries for all the drugs are all higher than 70%, and the matrix effects are all lower than 20%. All the intra- and inter- day assay precision and accuracy were better than 10% for all the matrices.

Higher Levels of Ethyl Paraben and Butyl Paraben in Rat Amniotic Fluid than in Maternal Plasma after Subcutaneous Administration

Parabens are a group of antimicrobial preservatives widely used in cosmetics, pharmaceuticals, and in foods. Previous in vitro and in vivo studies have shown weak estrogenic effects of some parabens. Thus, especially, exposure of fetus and infants via the mother is a matter of concern. In order to obtain more knowledge about the distribution of ethyl paraben and butyl paraben in pregnant rats and pups after perinatal exposure, the presented study was designed. The data show response and distribution of ethyl paraben and butyl paraben in maternal rat plasma, pools of amniotic fluids, placenta, whole-body fetuses, and in fetal liver after dosing of dams with 100, 200, and 400 mg/kg body weight (bw)/day from gestational day 7 to 21. After cesarean section of dams, the fluids and tissues were collected, deconjugated, and purified by solid-phase extraction, and ethyl paraben and butyl paraben were analyzed by liquid chromatography-tandem mass spectrometry. Markedly higher levels of ethyl paraben compared to butyl paraben were found in all fluids and tissues. Both ethyl paraben and butyl paraben in maternal plasma, livers, and whole-body tissues from fetus seemed to be saturated after dosing with >or= 100 mg/kg bw/day, while both compounds were excreted into amniotic fluid in a dose-dependent manner. Significant difference was found between the level of ethyl paraben in maternal plasma and amniotic fluid after dosing with 200 mg/kg bw/day as well as between the levels of butyl paraben in maternal plasma and amniotic fluid after dosing with 100, 200, and 400 mg/kg bw/day.

Metabonomic and Metallomic Profiling in the Amniotic Fluid of Malnourished Pregnant Rats

Epidemiology and studies in animal models have revealed that prenatal malnutrition is highly correlated with abnormal fetal neurodevelopment. We present here a combined metabonomic and metallomic profiling technique to associate the metabolic and trace-elemental composition variations of rat amniotic fluid (AF) in malnourished pregnant rats with the retardation of fetal rat neurodevelopment. The AF samples from three groups of pregnant Sprague-Dawley rats, which were fed either a normal diet, a low-protein diet, or "a famine diet", were subjected to GC/MS and ICP/MS combined with multivariate data analysis (MVDA). PCA scores plot of both GC/MS and ICP/MS data showed similar and unique metabolic signatures of AF in response to the different diets. Rats in the famine group released increased amounts of glycine, inositol, putrescine, and rubidium and decreased amounts of methionine, dopa, tryptophan, glutamine, zinc, cobalt, and selenium in the AF. These discriminable variations in the AF may indicate the abnormality of a number of metabolic pathways in fetal rats including the folate cycle and methionine pathway, the monoamine pathway, and tri-iodothyronine (T3) metabolism. The abnormalities may be the result of metabolites or elemental differences or a combination of both. This study demonstrates the potential of combining profiling of small-molecule metabolites and trace elements to broaden the understanding of biological variations associated with fetal neurodevelopment induced by environmental perturbation.

Simultaneous Determination of Zalcitabine and Stavudine in Maternal Plasma, Amniotic Fluid, Placental, and Fetal Tissues using Reversed Phase on Silica Liquid Chromatography

In order to study the placental transfer of nucleoside reverse transcriptase inhibitors, a quick and simple reversed phase high performance liquid chromatography (HPLC) method has been developed and validated using an underivatized silica column for the separation and analysis of DDC and D4T from rat plasma, amniotic fluid, placental, and fetal homogenate. Extraction of DDC, D4T, and their internal standard lamivudine (3TC) from the matrices was processed by liquid‐liquid extraction enhanced by salting out the sample using a saturated solution of ammonium sulfate. Chromatographic separation was achieved on a Waters Spherisorb S3W silica column (4.6 mm×100 mm) equipped with a Phenomenex guard column. The mobile phase consisted of 3% methanol in 22 mM formic acid. The flow rate was 0.5 mL/min, and the detection wavelength was optimized at 265 nm. The calibration curves for each day of validation showed good linear response over the range from 0.1 µg/mL to 50 µg/mL. The absolute recoveries for all the drugs are all higher than 75%. All the intra‐ and inter‐day assay precision and accuracy were better than 5% for all the matrices.

Post-injury regeneration in rat sciatic nerve facilitated by neurotrophic factors secreted by amniotic fluid mesenchymal stem cells

Amniotic fluid mesenchymal stem cells have the ability to secrete neurotrophic factors that are able to promote neuron survival in vitro. The purpose of this study was to evaluate the effects of neurotrophic factors secreted by rat amniotic fluid mesenchymal stem cells on regeneration of sciatic nerve after crush injury. Fifty Sprague–Dawley rats weighing 250–300 g were used. The left sciatic nerve was crushed with a vessel clamp. Rat amniotic fluid mesenchymal stem cells embedded in fibrin glue were delivered to the injured nerve. Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry were used to detect neurotrophic factors secreted by the amniotic fluid mesenchymal stem cells. Nerve regeneration was assessed by motor function, electrophysiology, histology, and immunocytochemistry studies. Positive CD29/44, and negative CD11b/45, as well as high levels of expression of brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor, ciliary neurotrophic factor (CNTF), nerve growth factor, and neurotrophin-3 (NT-3) were demonstrated in amniotic fluid mesenchymal stem cells. Motor function recovery, the compound muscle action potential, and nerve conduction latency showed significant improvement in rats treated with amniotic fluid mesenchymal stem cells. ELISA measurement in retrieved nerves displayed statistically significant elevation of CNTF and NT-3. The immunocytochemical studies demonstrated positive staining for NT-3 and CNTF in transplanted cells. The histology and immunocytochemistry studies revealed less fibrosis and a high level of expression of S-100 and glial fibrillary acid protein at the crush site. Rat amniotic fluid mesenchymal stem cells may facilitate regeneration in the sciatic nerve after crush injury. The increased nerve regeneration found in this study may be due to the neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.

Amniotic Fluid and Bone Marrow Derived Mesenchymal Stem Cells Can be Converted to Smooth Muscle Cells in the Cryo-Injured Rat Bladder and Prevent Compensatory Hypertrophy of Surviving Smooth Muscle Cells

Wound healing of the cryo-injured bladder can bring about organ remodeling because of incomplete reconstitution of depleted smooth muscle cells. Stem cell transplantation could be beneficial to improve smooth muscle cell regeneration and/or modulate the remodeling process. The repair of bladder injury using adult-type stem cells would be useful for adult urological patients but unsuited for neonatal patients, in whom major benefits are likely to derive from fetal-type stem cells. The smooth muscle cell differentiation potential of fetal-type vs adult-type stem cells was evaluated by injecting green fluorescent protein labeled mesenchymal stem cells from rat amniotic fluid or bone marrow, respectively, in cryo-injured rat bladder walls. At 30 days after transplantation only a few fetal-type or adult-type mesenchymal stem cells gave rise to enteric or vascular smooth muscle cells, whereas most mesenchymal stem cells appeared incapable of specific differentiation. In vitro co-culture experiments of smooth muscle cells with fetal-type or adult-type mesenchymal stem cells selectively labeled with distinct fluorochromes showed the presence of hybrid cells, suggesting that some mesenchymal stem cells can undergo cell fusion. Surprisingly the major effect of rat bone marrow or amniotic fluid mesenchymal stem cell transplantation seemed to be preventing cryo-injury induced hypertrophy of surviving smooth muscle cells. In this model stem cell transplantation has a limited effect on smooth muscle cell regeneration. Instead it can regulate post-injury bladder remodeling, possibly via a paracrine mechanism.

Determination of fluconazole in serum and amniotic fluid of rats by gas-chromatography/mass spectometry (GC/MS)

Rats treated with oral dose of 100 mg/kg of fluconazole during pregnancy had their serum and amniotic fluid quantified for this drug using a GC/MS method. Fluconazole was extracted with ethyl acetate from samples and analysed by a GC-MS Shimadzu QP5050A system using a CBP-5 fused silica capillary column. Tioconazole was used as internal standard. Calibration curve was linear within the range 10.0 - 300.0 µg/mL. The limit of quantification was 0.1 µg/mL and no interference was observed in the blank serum and amniotic liquid. The mean concentrations of the drug in the serum and amniotic fluid were 206.01 ± 105.25 µg/mL and 125.34 ± 65.24 mug/mL, respectively. This procedure showed to be sensitive and efficient enough for the use in teratogenic studies of fluconazole and other azole drugs.

Brain tissue fragments in the amniotic fluid of rats with neural tube defect

Brain tissue nodules are occasionally seen in the lungs of neural tube defect (NTD) cases. We looked for brain tissue fragments in amniotic fluid of rats with NTD as it is the basis for the aspiration hypothesis. Eighty-seven pregnant rats were randomly divided into experimental (n=58) and control (n=29) groups. Experimental rats received 100,000 U of vitamin A in 1 mL of corn oil on gestational days 8, 9 and 10, while control rats received corn oil. On gestational days 15, 18, 19, 20 and 21, amniotic fluid was drawn from three control animals and five experimental animals and analysed. NTD was found in 22.75% of experimental fetuses and in no control fetuses. Brain tissue fragment number and volume fraction increased between gestational days 18 and 20, falling on day 21. Excessive doses of vitamin A induce a high rate of early fetal death and development of NTD. Brain tissue fragments in the amniotic fluid reflect the evolution from exencephaly to anencephaly and could support the aspiration hypothesis. However, as it is a late event in the rat, this model may not reproduce the brain tissue nodules in the lung.

Fetal Rat Amniotic Fluid: Transforming Growth Factor β and Fibroblast Collagen Lattice Contraction

Background. In several mammalian animal models, early-gestational-age fetal wounds heal without scar, but wounds of late gestational age heal with scar. This change in wound healing phenotype can be a result of both intrinsic (i.e., cellular characteristics) and extrinsic (i.e., environmental) factors. Our question was: Does amniotic fluid (AF) influence the change from scarless to scar-forming repair in the rat?Methods. Rat AF was investigated for its modulation of fibroblast-populated collagen lattice (FPCL) contraction and morphological changes of adult fibroblasts. AF was also assayed for transforming growth factor β (TGF-β) levels. Adult rat dermal fibroblasts in monolayer and incorporated into FPCLs were incubated with AF additions from gestational age 14, 16, 18, and 21 days at 10% (v/v).Results. Day 14 AF significantly stimulated FPCL contraction, but AF of 16, 18, and 21 days inhibited FPCL contraction. Fluorescence histology identified microtubules and microfilaments in AF treated adult rat dermal fibroblasts. The staining pattern of microtubules in Day 14 AF-treated fibroblasts showed denser structures at the cell center. Cells incubated with Day 16 or 18 AF showed fine peripheral microtubules. A mink lung epithelial cell bioassay was used to analyze concentrations of TGF-β in AF. TGF-β levels were greatly elevated in Day 14 AF, but were relatively low in Day 16, 18 and 21 AF. The inhibitor of FPCL contraction from AF of Days 16, 18, and 21 was not identified.Conclusion. It is proposed that the robust expression of TGF-β or cytoskeletal changes induced by Day 14 AF contributes to enhanced FPCL contraction.

First report of active renin in rat amniotic fluid.

1. Epidemiological studies indicate that a reduced birthweight increases the likelihood of human cardiovascular disease later in life. The role of hormonal factors in this finding is not known. Given that angiotensin II is believed to be a fetal regulator of growth, we have examined in the hypertensive Ren-2 transgenic rat whether it has active renin in its amniotic fluid and whether this is associated with fetal underdevelopment. 2. We found that while the Sprague-Dawley rat contained no active renin in its amniotic fluid near term (20 days), Ren-2 amniotic fluid contains high levels of active renin and is associated with a reduced fetal weight. 3. This is the first report of active renin in the rat and allows the possibility that renin overproduction plays a role in reduced fetal growth and the prenatal 'programming' of essential hypertension that has been proposed to occur in humans.

Purification, characterization, and biological compartmentalization of rat fetal antigen 1.

This study has established the rat as an animal model for the analysis of the biological role of fetal antigen 1 (FA1), a protein previously described in humans and mice. FA1 was purified from rat amniotic fluid by immunospecific affinity chromatography. Immunochemical identity between mouse and rat FA1 was established by crossed tandem immunoelectrophoresis. Molecular size was analyzed by mass spectrometry (33 kDa). The amino acid composition was determined, and the amino acid sequence was analyzed. The overall amino acid composition and sequence of the 28 first N-terminal amino acids were identical to the corresponding parts of rat preadipocyte factor 1 and rat adrenal zona glomerulosa protein. Extensive sequence similarity was found between rat and mouse FA1 (86%) and between rat and human FA1 (82%). The concentration of FA1 in fetal serum, maternal serum, urine, and amniotic fluid in rats was determined using an ELISA. The highest concentrations were found in fetal serum and amniotic fluid around Day 18 of pregnancy. This is the first report on the physicochemical characteristics and compartmentalization of rat FA1.

Detection of Novel Molecules Recognized by Anti-Placental Lactogen Antibody in Rat Amniotic Fluid.

Amniotic fluid contains various bioactive substances including the placental PRL family. In the present study, it was elucidated that rat amniotic fluid contained immunoreactive proteins which had different molecular sizes and pI values from the authentic placental lactogens (PLs) in the rat, recognized by antipeptide antibody to the N-terminal peptide of rat PL-I. Immunoreactive PLs residing in the amniotic fluid were characterized further by two-dimensional sodium dodecyl sulfate gel electrophoresis (2DE), immunoblotting and anion-exchange chromatography. Amniotic fluid collected from rats on day 12 of pregnancy contained two PL-like molecules, tentatively called A1 (MW 75 kDa, pI 4.6) and A2 (MW 99-102 kDa, pI 5.3-5.4). A1 and A2 are specific to the amniotic fluid, because no such molecules were found in the serum or placental extracts. Immunoblot analysis of amniotic fluid revealed that A1 levels increased, whereas those of A2 decreased to an undetectable level up to day 16 of pregnancy. When the A1 concentrations from days 12 to 20 were monitored intensively, they increased from day 12 to 14, were maintained until day 18, and then decreased dramatically by day 20. The expression pattern for A1 was therefore completely different from those of authentic placental PRL family members found in serum and placental tissue, indicating that the A1 is distinct from them. Partial purification by anion-exchange chromatography and 2DE revealed that A1 consisted of 5 isoforms.

Volume and glucose concentration of rat amniotic fluid: Effects on embryo nutrition and axis rotation

In order to pursue our previous studies of the changes in neural tube microvilli produced by glucose, we developed a micro method of measuring glucose concentration in the very small volumes of amniotic fluid during neurulation. The volume of amniotic fluid was found to increase nearly 10-fold during major neurulation (day 10 to day 11 in the rat). This increase in volume and our repeated observations that physical removal of the restraining amnion initiates embryonic rotation leads us to propose that the growth of the amniotic cavity is essential for conversion from the ventral- to dorsi-flexion of the embryonic axis. Amniotic fluid volume continues to increase until day 18 but dropped by day 20. A method for glucose determination was developed using the color reaction on glucose oxidase indicator paper. The intensity of the color was analyzed with a color scanner. Amniotic glucose was 27.1 +/- 1.6 mg/dl on day 10 and continued in this range with some fluctuation until day 20 when it decreased. We isolated days 10, 11, and 16 embryonic sites from their decidua and incubated them at 0 degrees C and 38 degrees C while measuring glucose concentration. The glucose concentration did not show significant decrease at 0 degrees C on day 10 or 11 or on day 16 at 38 degrees C. At 38 degrees C the day 10 embryo amniotic fluid glucose disappeared after 22 minutes and the day 11 amniotic fluid glucose was gone in 34 minutes. These depletion times were statistically different. The magnitude of glucose depletion on day 10 was shown by calculation to be approximately 323 mumoles/gm protein per hour which is a substantial portion of the glucose utilized by the embryo as determined in previous experiments (731 mumoles/gm protein per hour). This model may serve as a way to study glucose utilization by embryos after their exposure to various teratogens.

Glucose causes lengthening of the microvilli of the neural plate of the rat embryo and produces a helical pattern on their surface.

Prominent microvilli have been observed on the surface of neural plates in the embryos of many species. Since glucose is the main source of energy for embryos before neural tube closure and the onset of vascular circulation, it was of interest to study the relationship between these microvilli and glucose utilization in the neural plate. By applying microdrops of amniotic fluid to chemstrips, which colorimetrically measure glucose by glucose oxidase reaction, we determined that day 10 rat amniotic fluid glucose level was 31.6 +/- 1.6 mg/dl. On day 10 and within about 20 min from removal of the decidual sites, no glucose was found in the amniotic fluid. By use of a scanning electron microscope, the microvilli of the day 10 neural plate were found to have a 10-fold increase in length during a 40-min exposure to Hanks' solution at 21-23 degrees C. Similarly exposed embryos in Hanks' without glucose did not have microvillus elongation. However, under whole embryo culture conditions at 38 degrees C no extension of the microvilli was found. In the closed neural tube of the day 10 embryo, the microvilli were stubby and did not elongate with glucose exposure. Similarly, day 11 and 14 embryos had short microvilli which did not elongate with direct exposure to glucose at 21-23 degrees C. The short microvilli on the surface of the closed neural tube on day 11, 14, and 16 were associated with low glucose concentrations in the neural tube fluids. By use of a field emission scanning electron microscope, the surfaces of the microvilli in the extended position were seen to be covered by a right-handed helical array of globular objects the size of large molecules. The findings support the hypothesis that microvillar length may modulate glucose uptake. Shortening is associated with low concentrations of glucose in closed neural tubes, and lengthening occurs at glucose exposures of 100 mg/dl.

Trophic influences of human and rat amniotic fluid on neural tube-derived rat fetal cells

Normal human (week 17–20) and rat (E16–17) amniotic fluids were used as culture media for primary cultures of rat fetal (E 16) cortical, mesencephalic and striatal cell dissociates, or astroglial subcultures from the same brain regions. Phase-bright and dark cells were identified under phase contrast microscopy and their cell processes were measured utilizing semi-automated procedures. Subcultured astroglia were immuno-reacted against glial fibrillary acidic protein and fibronectin. Rat and human amniotic fluid allowed survival and growth of neuronal and non-neuronal cells. Human amniotic fluid samples were trophic in variable degrees. Cerebral cortex subcultured astroglia usually expressed a radial-like morphotype. Although charcoal-adsorbed human amniotic fluid was trophic for primary cultures, its ability to sustain neuritic growth depended on its degree of trophism before treatment. Growth of cell processes in neuronal- and glial-like cells in primary cultures was inhibited to different degrees by the addition of antisera towards nerve or epidermal growth factors. It is concluded that amniotic fluid constitutes a trophic medium for astroglia and neurons. Both, nerve and epidermal growth factors appear to be necessary for growth of cell processes in neuronal and glial primary cultures in amniotic fluid. Trophic effect of amniotic fluid on subcultured astroglia did not seem to be diminished by nerve growth factor antiserum. The role of amniotic fluid during the early phases of brain organogenesis is discussed.

Separate and Conjugated in vitro Effects of Clofibrate and of an Organic Extract of Rat Amniotic Fluid on Microperoxisomes of Fetal Mouse Small Intestine

Separate and conjugated effects of clofibrate and of an organic extract of rat amniotic fluid (RAF) on microperoxisomal response were studied in 15-day-old fetal mouse small intestine in organ culture. At the light-microscopic level, DAB-positive microperoxisomes were not observed in intestinal explants cultured for 48 h with Trowell T8-medium either with (group 1) or without clofibrate (control group) or with the organic extract of RAF alone (group 2). In presence of both clofibrate and the organic extract (group 3), microperoxisomes were readily identified. At the fine structural level, microperoxisomes measured 150-175 nm in diameter in controls and in group 1 and 2. In group 3, numerous microperoxisomes measured up to 0.4 micron in diameter. Total and specific catalase activities exhibited significant decreases when intestinal segments were cultured solely in Trowell T8-medium for 48 h. Addition of clofibrate (1), or of the organic extract of RAF (2), or of both (3) to Trowell T8-medium induced significant increases in total and specific catalase activities: 1.43- and 1.40-fold (1), 2.12- and 2.66-fold (2) and 2.92- and 3.17-fold (3). These results indicate that an organic extract of RAF contains a factor that (a) induces the formation of intestinal microperoxisomes and (b) potentiates the action of clofibrate.

Inhibition of placental thyroxine 5-deiodinase activity decreases amniotic fluid concentration of 3,3',5'-triiodothyronine in rat.

We investigated whether the inhibition of placental T4 5-deiodinase (5-D) activity would decrease the amniotic fluid (AF) concentration of rT3. Iopanoic acid (IOP) was used to inhibit placental T4 5-D activity. From gestation days 14 to 17, a group of rats received IOP (10 mg/100 g bw/day, sc) once daily in experiment (exp) 1, and received it (40 mg/100 g bw/day) four times daily in exp 2. In exp 2, control dams received propylthiouracil (PTU; 2 mg/100 g bw/day) instead of IOP. Methimazole and T4 were also given to all dams in exp 1 and 2. On day 17 of gestation, we collected the liver, placenta, blood, and AF of each animal. In the IOP-treated group in both experiments, the serum T4 concentration was significantly increased. Hepatic T4 5'-deiodinase activity was significantly decreased by either PTU or IOP administration. In both experiments placental T4 5-D activity was significantly decreased in the IOP-treated group. The concentration of rT3 in AF was significantly decreased in the IOP-treated group in exp 2 (1.71 vs. 0.75 nmol/l, P < 0.01) despite a higher serum T4 concentration. There was a significant positive correlation between placental T4 5-D activity and the concentration of rT3 in AF in exp 2 (r = 0.62, P < 0.05). These observations indicate that the inhibition of placental T4 5-D activity decreases the concentration of rT3 in rat AF, and that placental T4 5-D and the T4 concentration in maternal serum plays important roles in maintaining the concentration of rT3 in rat AF.

Regulation of rat insulin-like growth factor-binding protein-1: the effect of insulin-induced hypoglycemia.

Rat insulin-like growth factor-binding protein-1 (rIGFBP-1) was purified from H4IIE rat hepatoma cells by IGF-I affinity chromatography and reverse-phase HPLC. A rabbit antiserum (B2) was raised to rIGFBP-1 and a RIA established. Immunoreactive IGFBP-1 was present in rat amniotic fluid and in the medium conditioned by isolated rat hepatocytes and HTC rat hepatoma cells. To study the effect of hypoglycemia, fasting female Wistar rats were anesthetized and cannulated for multiple venous sampling after the administration of insulin or saline. Serum IGFBP-1 rose in adrenal intact rats from < 0.1 micrograms/ml to a maximum of 1.41 +/- 0.23 micrograms/ml approximately 120 min after insulin administration. Compared to adrenal-intact rats, adrenalectomized animals demonstrated a delayed rIGFBP-1 response to hypoglycemia and did not appear to have reached a maximum at 180 min. A slow rise in rIGFBP-1 levels throughout the sampling period was seen after saline injection in both adrenal-intact and adrenalectomized animals. Glucose, corticosterone, rat insulin, and human insulin levels were measured and none, alone, appeared responsible for the observed rIGFBP-1 responses. We conclude that 1) rIGFBP-1 is stimulated in response to hypoglycemia in a similar manner to glucose counterregulatory hormones, 2) an adrenal factor is required for an early rIGFBP-1 response to hypoglycemia, and 3) neither circulating glucose nor insulin levels, alone, are responsible for the observed patterns of response.

PROLONGATION OF RENAL ALLOGRAFT SURVIVAL IN THE RAT TREATED WITH AMNIOTIC FLUID

To assess the role of amniotic fluid (AMF) in the maintenance of pregnancy, immunosuppressive effects of AMF were studied in vivo, and the mechanisms of suppressor activity were analyzed immunologically in vitro in the rat. Female Lewis (LEW, RT-1l) rats mated with Brown-Norway (BN, RT-1n) rats for 14 days were sacrificed and cell-free AMF was obtained. AMF was diafiltered with PBS (PH 7.2) and reconstituted to 2 OD units measured at 280 nm. Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.2 days (n = 10). Five days of intravenous inoculation of AMF into LEW hosts remarkably enhanced BN graft survivals (MST = 20.3 +/- 4.4 days, n = 12) compared with controls (P less than 0.01), and slightly prolonged third-party DA (RT-1a) graft survivals (MST = 9.4 +/- 0.8 days, n = 7) compared with control LEW hosts engrafted with a DA kidney (MST = 7.6 +/- 0.2 days, n = 6). Five days of intravenous inoculation of pregnant sera into LEW hosts had no effect on BN graft survival. The AMF suppressed the proliferative response of LEW lymphocytes against not only irradiated BN stimulator cells but also irradiated third-party DA stimulators. The AMF also suppressed allokiller T cell generation of normal LEW lymphocytes against BN cells by 70.1% and 51.3%, and against DA cells by 64.9% and 38.9% at concentrations of 25% and 12.5%, respectively (P less than 0.01). To dissect the immunosuppressive activity of AMF, the effect of AMF on cytokine production and interleukin 2 (IL-2) receptor expression of concanavalin A-stimulated lymphocytes were investigated. AMF suppressed interferon and IL-2 production. Interestingly, however, AMF did not suppress interleukin 3 (IL-3) and interleukin 6 (IL-6) production, as well as IL-2 receptor expression. These results demonstrated that rat AMF displayed a strong immunosuppression in vivo as well as in vitro, and that AMF might play an important role in the maintenance of pregnancy.

Immunoreactive corticotropin-releasing hormone in amniotic fluid

Immunoreactive corticotropin-releasing hormone in the amniotic fluid of both human beings and rats was measured by a specific radioimmunoassay. In human subjects the hormone was detectable in all amniotic fluid samples (obtained during the sixteenth and eighteenth weeks of gestation) (2.5 +/- 1.7 fmol/ml, mean +/- SD, n = 17) and the thirty-eighth to fortieth weeks (9.3 +/- 5.4 fmol/ml, n = 24). The levels of concentration of this hormone in this amniotic fluid correlated significantly with the levels in both maternal plasma and placenta for each patient. Gel filtration of amniotic fluid extracts revealed two major peaks of immunoreactive corticotropin-releasing hormone, one at the elution position of the rat hormone and the other at a small-molecular-weight region. Immunoreactive corticotropin-releasing hormone was not detectable in rat amniotic fluid or placenta. We concluded that immunoreactive corticotropin-releasing hormone, which may be derived from the placenta, is present in human amniotic fluid and that its detection in the human placenta but not in rat placentas suggests that the mechanism of corticotropin-releasing hormone gene expression in the placenta is species specific.