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Introduction: Previous studies have shown that morphine consumption during pregnancy may delay embryo development or cause abnormal nervous system function. The present study focused on the effect of maternal morphine consumption on development of maternal and fetal portion of placent and blood corticosteron concentration in addictive pregnant mother .
Material and methods: In this research 24 female Wister rats, 170-200g weight, were used. The experimental groups after pregnancy received an oral dose of 0/05mg/ml of morphine by tap water while the control group received tap water. On 10th and 14th day of pregnancy, Wister rats were anesthetized by chloroform, and placenta with uterus removed surgically, 1ml blood was collected from each pregnant mother from Retro-orbital sinus, the concentration of blood corticosteron was determined by corticosteron Elisa kit after centrifugation. The fixed embryos underwent tissue processing, sectioning and staining with hematoxylin and eosin (H&E). Placenta was studied microscopically according to the thickness of layers, area of blood cisterns, and the number of cells in maternal and fetal portion of placenta.
Results: Comparing the plasma corticosteron concentration of the treatment and the control groups, not only a severe increase in the treatment group was detected, but also the thickness of maternal and embryonic portions of the placenta at day 10th and 14th of gestation (pregnant) was different significantly(pâ‰¤0.05). Furthermore, an increase in number of cells in maternal and embryonic portion of placenta and a decrease in blood cistern area were demonstrated in both the experimental and the control groups.
Conclusions: The effects of morphine, including an increase in blood concentration of corticosteron, in dependent pregnant mothers was seen. development of placenta in the experimental group with was delayed
Key words: Placenta fetal portion, Placenta maternal portion, Blood cisterns, Morphine, Rat, corticosteron
Dependence on addictive drugs spread all over the world and the side effects of addiction not only involve the one who is on drug but also others are indirectly affected especially placenta and fetus(1) .
It is necessary to study the function of drugs in animal trials especially in placenta.
Many behavioral problems in addicted mothers' infants indicated the effects of opioides in embryo.(1,2).The majority of studies focused on the embryo, whereas they neglected to study the placenta as an important organ.
Disruptive effects of consumption of opioides in human samples and laboratory animals were well conducted. The research showed that consumption of opiate materials by pregnant mothers cause delay in embryonic development and malfunctions, such as spinabifida (1,3). In accordance to previous studies,high blood coicosteron concentration of pregnant mother attenuate placenta and embryo. The capacity of placenta for displacement and releasing food materials depends on the placentas, shape, size and transferring factors. As morphine is small and imploring molecule can easily pass through blood barrier and placenta and then become effective on embryonic cells.(4,5,6,).
As placenta in mammals is the most important part to exchange materials between embryonic and maternal blood, the size of the placenta is directly related to food material transporting(simple an active transport ) (2 ,6,4).The morphine effects were presented with Mio, Kappa and Delta opioides receptors and activating of these receptors caused several changes, including decrease in the CAMP ,an increase in output of -K+ ion and a decrease in input of ca-ion.(8,7) On the other hand, the ca-ion has important role in secretion of estrogen and progesterone hormone from placenta ,stableness and embryonic development.(9,10) By progress of pregnancy, placenta can act as a gland that secrets progesterone, estrogen and the like that are needed for embryonic development and considered as an alternative for ovary secretion hormones.(11,6)Therefore, morphine can act as an interfering that causes dysfunction in placental secretion operating and delay in placental development.(10,5)Several experiments have shown that morphine administration cause the decrease of placenta weight in rabbits,(12,13) The presence of opioides receptors on the placental villi cells can affect placental function .On the other hand, because the placenta is a protective barrier, it can prevent the input or output of some materials. Placental barrier as a protective mechanism is often considered against pathogenic factors.(14,2) Disorders in the development of placenta cause placental disability in exchanging , endocrine and protective acts in embryo(6,). Corticosteron hormone stimulate morphine function(6,9)The importance of blood mother`s corticosteron concentration in placenta development and the effects of morphine on delay of placental development are the major reasons for the present research that consider the oral morphine administrations effect on the placenta of addictive mothers on 10th and 14th days of pregnancy.
Materials and methods:
Animals, female Wister rats were used 170-200gr weight. The animals were housed 2/cage to a temperature of (24 Â±10â„ƒ) and lighted controlled environment with a 12-h light/dark cycle and provided with food and water.
In this study, prepared morphine sulphate from Iran tamad Co, was used orally. Twenty-four female Wistar rats were divided into four equal groups consisting of six animals each
and the 24 female rats were mated into 2 groups with one adult male rat. After making sure about pregnancy (with the observation of vaginal plug and existence of sperm in vagina), they were separated from male rats the next morning and kept in the same coed-groups. Thereafter (0 day of pregnancy), experimental group received a daily dose of 0.05 mg/ml(15) (5 mg morphine per 1000 ml potable water from city pipeline for 12 rats)The amount of oral morphine were 14ml /100gr rat body weight.also, 1 ml blood were collected from retro-orbital sinus at 13th days of pregnancy. Blood samples were centrifuged. And plasma was extracted and keep in -20`C. finally plasma cortcosteron was assessed by Rat Corticosterone ELISA kit; EIA-4164; DRG Instruments GmbH, Germany) in both experimental groups.
In 10th and 14th days of pregnancy, they were anesthetized with chloroform and placenta were removed from female mice and transferred to 10% formalin solution for 10 days, then, placenta were put on in tissue processing system and were ready for molding, then blocks were sectioned with microtome and sagital sections hundred of 5Î¼m thickness serially(15,16). These slices were put on slides and stained with hematoxylin and eosin methods (H&D). After staining, slides were studied microscopically.
Data were reported as mean Â± S.E.M. Differences between all groups were calculated by a one-way analysis of variance(ANOVA) and post-hoc Duncan test by using the SPSS/PC computer program(version 9.1). Statistical significance between the two measurements was determined by the two-tailed unpaired sample T-Test Result were considered Statistically significant when P< 0.05.
The thickness of portion placenta, blood cisterns surface, and number of cells in the experimental and control groups was measured with motic software. The system used included a microscope connected to a computer and a monitor by software could take photos from slides. Subsequently, the number of cells on each layer was counted randomly and compared with that of the experimental groups.
Our results showed the oral morphine consumption at pregnant mice increased the placenta concentration of corticosteron at day 13 of pregnancy in comparison with control group. In addition morphine administration in pregnant rats placenta showed, thickness of maternal part has increased in 10th and 14th days placentae (Tabel 1) in contrast , significant decrement in thickness of placenta embryonic part was shown (Tabel 1). also, increment in cells number of placenta maternal and embryonic part in experimental groups was assessed(Tabel 1) thereby decrement in blood pools surface of placenta maternal and embryonic part in 10th day experimental groupswas shown in this study (Tabel 1).
Day Thickness ( Âµm) Number (count/ unit) Area ( Âµ Â²) ml
Pregnancy Maternal Fetal Maternal cell Fetal cell Maternal lacuna Fetal lacuna Concentration
Control placenta 10th 873.85 Â±114 677.6 Â±36 13 Â±0.1 5 Â±0.1 45307 Â± 0.1 27046. Â±0.1
Experiment placenta 10t 1064.55 Â±197* 205.2 Â±33* 13 Â±0.1** 7 Â± 0.1* 20300 Â± 1210** 19525 Â± 491**
Control placenta 14th 574.92 Â±26 1394.67 Â±0 17 Â±1 17 Â± 0.5 21979.3 Â±317 7970.54 Â±164
Experiment placenta 14th 2351 Â±173*** 533.1 Â±0*** 24 Â±5** 24 Â±2* 21935.5 Â±242 8383.1 Â±118
Control Plasma 13th 650 Â± 45
Experiment Plasma13th 1230 Â±52**
Result were considered Statistically significant when P< 0.05. (Mean Â± SEM)
( Placenta Portions Thickness in Tabel 1) : Effect of administration of oral morphine on the10, 14th days of placenta development and comparison of control and experimental group, in the thickness of maternal and fetal portion of pregnant Wistar rat placenta. The significant increase in the thickness of maternal portion (10,14th days) and significant decrease in 10,14th days fetal portion of placenta in experimental (E) group to control(C) group.( *P< 0.05, **P< 0.01, **P< 0.01, ***P< 0.001)
( Cell Number in Tabel 1) : the effect of oral morphine consumption on development of 10 to 14-day old placenta and comparison of them in the number of cells in maternal and fetal portion placenta of pregnancy Wistar rat. The significant increase in the number of 10,14th days placental maternal portion and number of 10th day fetal portion cells in experimental(E) group to control(C) group.( **P< 0.01, *P< 0.05, **P< 0.01)
( lacuna Area in Tabel 1) : Effect of administration of oral morphine on the10,14th days of placental development and comparison of them in control with experimental group, in the blood cisterns(lacuna) surface of maternal and fetal portion of Wistar rat placenta. The significant decrease in the blood cisterns surface of 10th day fetal portion of placenta and the blood cisterns surface in 10th day maternal portion of placenta in experimental(E) group to control(C) group.( **P< 0.01, **P< 0.01)
( Plasma concentration in Tabel 1 ) : Plasma corticosterone concentration in rats which received oral morphine on the 13th day of pregnancy; The experimental (E) and Control (C) groups. As it is clear from the figure, a significant increase in plasma corticosterone concentration was observed in the experimental group. (**P< 0.01) different from control group.
Morphological observations on 10-day old placenta:
Microscopic observation indicated that on the 10-day old placenta, experimental group had little thickness on embryonic part, more thickness on maternal part , more cell number at maternal and fetal part, and less area blood cisterns (lacuna) in comparison to control group (A1,A2,A3,A4, B1,B2,B3,B4,).
Fig B1: Morphologic changes of placenta part thickness decrement and maternal part thickness incrementin experimental(A1) groups in the 10-day old placenta by Ã-40(two arrowheads).
Fig B2: Morphologic changes on 10-day old placenta in experimental group, indicating increment in cells number of placenta maternal and embryonic part in experimental groups and decrement in blood lacuna of placenta by Ã-100 ( a one arrowhead)(fig2).
FigA2: Microscopic image of control group in 10 days placenta with zoom Ã-100, indicates placenta cells and blood lacuna (a one head arrow).
Morphology observations on 14-day old placenta
Microscopic observations indicated that placenta of 14 days pregnant maternal in experimental group have a less thickness of fetal portion layer, and maternal portion is more and the increase of cells in placenta maternal and decrease of blood cisterns area in experimental group and control group were seen.
Fig. B3: the changes of thickness in placenta layer in 14-day old placenta in experimental(B3) and control(A3) groups zoomed Ã-40 ( two heads arrow)
Fig. B4: The changes of the blood cisterns and the number of placenta portion cells in 14-day old placenta in experimental(B4) and control(A4) groups, zoomed Ã-100 (one head arrow)
Results of the present study showed that morphine consumption during pregnancy can cause delay in the development of embryonic and maternal portion of placenta .On pregnant mother concentration of corticosteron at blood plasma will increase at second period of pregnancy so we measured corticosteron concentration at thirteen days after pregnancy (17,18). In agreement with this subject our data also indicated the oral morphine administration increased blood plasma corticosteron concentration on addictive pregnant mothers (Tabel 1). Increase in corticosteron density of the blood plasma of the pregnant female mice can justify the side effects in embryonic of these mothers, In addition the results of these studies were consistent with previous ones, which indicated that cortisol administration could delay differentiation of placental cells.(1.5)Regarding the fact that embryonic development is the result of placental natural function and due to the essential function of that is the hormone secretion and material exchange, any disorder in normal function of fetal and maternal portion of placenta can cause abnormality on embryo growth (3,16,19).
Blood flow is a vital factor on placental function and embryo growth. Morphologic studies have shown that if physiologic changes of spiral veins alter during pregnancy, trophoblast cells attack to placenta and blood flow increase at this site and finally placental villi disrupted will appear other (6,20,21 ). Morphologic and morph metric results have shown the affect of oral morphine in both embryonic and maternal parts (Tabel 1). Nowadays, it is stabilized that corticosteron concentration increases in blood of pregnant mother. also our study indicated that oral morphine administration in pregnant mouse caused corticosteron secretion in experimental group.(5,17,10) Fowden & Forhead declared that exposure of glococorticoides will loss embryo and placenta and this lessen will directly change the cell cycle from mitotic to differentiated state (5,6,10).Also corticosteron induces proliferation of cytotrophoblast cells of maternal and fetal portion of placenta (6,22,17) by stimulation of procytotrophoblastic cells to shortening of interphase(17,5,23), so cells do not have enough time for growth, protein synthesize, replication, and enough growth, and finally cause disorder in normal function of placental fetal cells.(6,7,23) .In contrast lead to late differentiation of placental cell and embryonic development (5,19,7,10,) Results of present research together with above data showed high level of corticosteron by morphine administration raised maternal portion thickness and number of these cells in placenta of 10th and 14th pregnancy. In addition morphine consumption has decreased more placenta fetal portion in contrast to maternal portion and this increment cause severs abnormality at embryo (Tabel 1). The previous studies indicated, oral morphine administration in the 9th day of pregnancy attenuate neural tube and neural plate evolution and development of frontal cortex in embryo were reduced in the 17th day of pregnancy.(15,16,24,25)The major role of placenta is material exchange between maternal blood and placenta and secretory substances such as steroids, peptides, cytokine, glycoprotein's release from mother blood and enter to embryo by placenta .In otherwise because consumption and production of nutrition material were determined by placenta any disorder in functionality of fetal placental portion (11,5,6) cause delay in development of embryo and placenta. During pregnancy in parallel with vessel angiogenesis, thickness of fetal placenta portion will decrease, so material exchanges become more and finally morphine uptake increases and decrease in thickness of fetal placenta portion is the effect of this increment (20.14,23).So, speed of morphine transition and disruptive effects have direct ratio with decrease of fetal layer thickness. In present research , effect of morphine resulted decrease in abnormal thickness in embryonic unit of experimental to control group in 10 and 14 day old placenta, and decrease of blood cisterns surface, also number of cell in each both 10 and 14 day old placenta in experimental to control group are consistent with increase of corticosteron concentration. (Tabel 1). The other present researches of morphine effect on placenta fetal portions of both groups indicated, morphine causes decrease in blood cisterns area and they are consistent with koulin's and Doppler's researches , which the same is true in human .In other researches it was showed ,the morphine consumption orally and injection ally indicated the same effect (,20,26,27). The effective factors on blood vessel contraction are corticosteron and opioides receptors on the membrane of placental cells(3,2,7), that located on placental villi and be contracted by morphine stimulation and resulted decrease in blooding , embryonic hypoxia and delay in embryonic development(20,28,) Embryonic portion of placenta basically give rise to cyncytiotrophoblast cells and these cells play important role in embryonic development through secretory function, like estrogen and progesterone hormone (10,17) and disorder in secretory of these cells cause delay placental and embryonic development (6,19) and can aggravate embryonic abortion According to studies, morphine administration caused embryonic abortion and decrease in babies weight in pregnant rabbits (12). In total, these results indicated oral morphine consumption causes the unusual increment in plasma corticosteron density and delay in placenta fetal and maternal portion development in Wistar rat but it was not identified if present study results are due to morphine or corticosteron effect or both of them .Although behavioral disorders in infants or embryonic abortion from addictive pregnant mothers need to be studied more.
This study has been financially sponsored by Research Center of Applied Neuroscience Research Center, Medical University of Baqyiatallah (a.s.).