The effect of human menstrual blood-derived stem cells on ovarian folliculogenesis, angiogenesis and collagen volume in female rats affected by the polycystic ovary syndrome.

BACKGROUND: Infertility is one of the common problems among couples, affecting millions of people worldwide. Polycystic ovary syndrome (PCOS) is one of the main causes of infertility in women and is associated with abnormal folliculogenesis, angiogenesis and fibrosis. Common treatments may lead to numerous adverse effects on the patient's quality of life. The present study aimed to investigate the effects of human menstrual blood-derived stem cells on the ovarian histology of a PCOS model of Wistar rats. RESULTS: Based on the Papanicolaou test and H&E staining results, the number of primary, secondary and antral follicles in the PCOS and PCOS-Sham groups significantly increased compared to the control group, while they significantly decreased in the PCOS + Stem cells group compared to the PCOS and PCOS-Sham groups. Further, the number of atretic follicles in both PCOS and PCOS-Sham groups significantly increased in comparison with the control group and decreased in the PCOS + Stem cells group, compared to the two mentioned groups. Moreover, the Graafian follicles number was decreased in the PCOS and PCOS-Sham groups to significantly increase in the PCOS + Stem cells group. Based on Masson's trichrome staining, the number of blood vessels in PCOS and PCOS-Sham groups significantly increased compared to the control group, while a decrease was observed in the PCOS + Stem cells group, compared to PCOS and PCOS-Sham groups. CONCLUSION: The administration of MenSCs improved folliculogenesis in rats with polycystic ovaries. Also, MenSCs could ameliorate PCOS symptoms by improving fibrosis as well as angiogenesis and weight gain.

In utero and postnatal exposure to Foeniculum vulgare and Linum usitatissimum seed extracts: modifications of key enzymes involved in epigenetic regulation and estrogen receptors expression in the offspring's ovaries of NMRI mice.

BACKGROUND: Early-life exposure to exogenous estrogens such as phytoestrogens (plant-derived estrogens) could affect later health through epigenetic modifications. Foeniculum vulgare (fennel) and Linum usitatissimum (flax) are two common medicinal plants with high phytoestrogen content. Considering the developmental epigenetic programming effect of phytoestrogens, the main goal of the present study was to evaluate the perinatal exposure with life-long exposure to hydroalcoholic extracts of both plants on offspring's ovarian epigenetic changes and estrogen receptors (ESRs) expression level as signaling cascades triggers of phytoestrogens. METHODS: Pregnant mice were randomly divided into control (CTL) that received no treatment and extract-treated groups that received 500 mg/kg/day of fennel (FV) and flaxseed (FX) alone or in combination (FV + FX) during gestation and lactation. At weaning, female offspring exposed to extracts prenatally remained on the maternal-doses diets until puberty. Then, the ovaries were collected for morphometric studies and quantitative real-time PCR analysis. RESULTS: A reduction in mRNA transcripts of the epigenetic modifying enzymes DNMTs and HDACs as well as estrogen receptors was observed in the FV and FX groups compared to the CTL group. Interestingly, an increase in ESRα/ESRß ratio along with HDAC2 overexpression was observed in the FV + FX group. CONCLUSION: Our findings clearly show a positive relationship between pre and postnatal exposure to fennel and flaxseed extracts, ovarian epigenetic changes, and estrogen receptors expression, which may affect the estrogen signaling pathway. However, due to the high phytoestrogen contents of these extracts, the use of these plants in humans requires more detailed investigations.

Serum Scavenging Capacity and Folliculogenesis Impact following Flaxseed Consumption in the First-Generation Mice Pups.

Flaxseed is a source of antioxidants utilized for female infertility treatment in traditional medicine. This study investigated the effects of flax hydroalcoholic extract and flaxseeds during prenatal and postnatal (PND) periods on folliculogenesis and serum total antioxidant capacity (TAC). Pregnant NMRI mice received 500 and 1000 mg/kg of flax extract (LE) and the same doses of flaxseed (LS). Female pups received the same regimen for 56 days. The body, ovarian morphometry, follicle development, and TAC levels were evaluated. The ovarian weight significantly increased in the LE1000 group compared to the LS500 group. The LE500 group had a considerably lower number of primary and antral follicles compared to the CTL and LS1000 groups. The number of antral follicles significantly increased in the LE1000 group compared to the LS500 and LE500 groups. The number of preovulatory follicles was higher in the LE1000 group. A significant increase in the TAC levels was detected in the LS500, LS1000, and LE1000 groups. LE showed a dose-dependent protective effect on the folliculogenesis in F1, which is more evident with the dosage of 1000 mg/kg. This could be related to the strongest antioxidant property of LE1000, as shown by the highest levels of TAC.

Nicotine-conditioned place preference, reversal learning and social interaction in MK-801-induced schizophrenia model: Effects of post-weaning enriched environment.

Based on the clinical observations of severe cognitive deficits in schizophrenia patients and the relationship between environmental parameters and the severity of schizophrenia symptoms, the present study investigated these parameters in an dizocilpine (MK-801)-induced schizophrenia model in rats. In addition to, it evaluated whether a post-weaning enriched environment (EE) would affect the nicotine-induced conditioned place preference (CPP) and the motor and cognitive deficits caused by MK-801 treatment. Male Wistar rat pups were injected peritoneally with MK-801 (1 mg/kg) on a daily basis between the 6th and the 10th postnatal days (P) and were exposed to either an enriched or a standard cage from P21 until the end of the experiments. The rats were evaluated in open-field and three-chamber social interaction tests. Moreover, spatial and reversal learning was assessed by the Morris water maze (MWM). The animals were conditioned with 0.6 mg/kg nicotine and tested for CPP. Increased self-grooming, exploratory behaviour, potentiated nicotine-CPP and decreased social behaviours, delayed spatial learning and memory and impaired reversal learning in the water maze were observed in the MK-801 treatment group. Housing in an EE improved cognitive and behavioural deficits associated with postnatal MK-801 treatment. The results suggested that neonatal N-methyl-d-aspartate (NMDA) receptor hypofunction may cause susceptibility to these behaviours and indicated the importance of environmental conditions in the development of schizophrenia and probably other neuropsychiatric disorders.

Genetic and epigenetic modifications of F1 offspring's sperm cells following in utero and lactational combined exposure to nicotine and ethanol.

It is well established that maternal lifestyle during pregnancy and lactation affects the intrauterine programming of F1 offspring. However, despite the co-use of alcohol and nicotine is a common habit, the effects of exposure to both substances on the reproductive system of F1 male offspring and the underlying mechanisms of developmental programming have not been investigated. The present study aimed to examine pre- and postnatal concurrent exposure to these substances on genetic and epigenetic alterations of sperm cells as well as testis properties of F1 offspring compared with exposure to each substance alone. Pregnant dams in the F0 generation randomly received normal saline, nicotine, ethanol, and combinations throughout full gestation and lactation periods. Sperm cells and testes of F1 male offspring were collected at postnatal day 90 for further experiments. High levels of sperm DNA fragmentation were observed in all exposed offspring. Regarding epigenetic alterations, there was a significant increase in the relative transcript abundance of histone deacetylase 1 and 2 in all exposed sperm cells. Moreover, despite a decrease in the expression level of DNA methyltransferase (DNMT) 3A, no marked differences were found in the expression levels of DNMT1 and 3B in any of the exposed sperm cells compared to non-exposed ones. Interestingly, combined exposure had less prominent effects relative to exposure to each substance alone. The changes in the testicular and sperm parameters were compatible with genetic and epigenetic alterations. However, MDA level as an oxidative stress indicator increased in all exposed pups, which may be responsible for such outputs. In conclusion, maternal co-exposure to these substances exhibited epigenotoxicity effects on germline cells of F1 male offspring, although these effects were less marked relative to exposure to each substance alone. These counteracting effects may be explained by cross-tolerance and probably less impairment of the antioxidant defense system.

Effects of maternal consumption of morphine on rat skeletal system development.

BACKGROUND: Opioid abuse is among the most ubiquitous issues world-wide, and when it happens in mothers, it puts them at risk of diseases that can be transferred to the next generation. Previous studies have indicated that morphine addiction during pregnancy could inhibit development in rat embryos and infants. The present study focused on the effects of maternal consumption of morphine on rat skeletal system development and also investigate the molecular pathway of chondrogenesis and osteogenesis of infants from control and addicted rat groups. METHODS: Thirty-two female rats were randomly assigned to four groups. The groups consisted of one- and seven-day-old female infants which were born of morphine-dependent mothers and a control group for each of them. Experimental groups received oral morphine at the final dose of 0.4 mg/ml/day. Withdrawal signs were confirmation of morphine dependency. Female rats were crossed with male rats and coupling time was recorded. Fixed bones of all groups were processed and then stained by hematoxyline-eosin method. Thickness and cell number of proximal and distal growth plate of bones were measured. The cartilage and bone cells were stained by alcian blue/alizarin red method. Additionally, the gene expression of alkaline phosphatase, osteocalcin, and COLL2 and SOX9 gene expression were studied immuno-histochemically. RESULTS: Unfavorable effects of morphine on histological measurements were observed in one-day and seven-day infants, with more effects on seven-day infants. The thickness and cell number of the proximal and distal growth plate of morphine-dependent rat offsprings were reduced significantly. Furthermore, morphine reduced growth of primary and secondary ossification centers, and thus, longitudinal bone growth was reduced. Moreover, a decrease in the alkaline phosphatase, osteocalcin, COLL2 and SOX9 gene expression, and the number of stained cells was observed. More adverse effects of morphine in seven-day infants compared to one-day infants which showed the time dependent of morphine to the time length of administration. CONCLUSION: Histochemistry and immunohistochemistry findings on cartilage and bone matrix formation, as well as protein expression of chondrogenic and osteogenic markers suggest that morphine dependence in pregnant mothers may impair intra-cartilaginous osteogenesis in post-natal rats.

Effects of ethanol and nicotine co-administration on follicular atresia and placental histo-morphology in the first-generation mice pups during intrauterine development and lactation periods.

This study is evaluating the effects of ethanol and nicotine exposure during pregnancy and lactation on placenta histology and follicular atresia in the first-generation (f1) mice pups. The experimental groups were 5 groups of NMRI pregnant mice, including: control, vehicle (received normal saline) ethanol (3 g/kg/day, 20 % v/v intraperitoneally), nicotine (1 mg/kg/day, subcutaneously), and ethanol plus nicotine which received both. Pregnant animals in each group were then divided into two groups, one group for examining the placenta that was treated for 18 days and the other group for the ovary of one-day-old (PND1) and fifty-six-day-old (PND56) female offspring who were treated for 42 days (during intrauterine development and lactation). After the autopsy procedure, histopathological and morphometrical observations were done. Data revealed that the exposed mice had a significant change in the placenta morphometry and histology as well as a marked increase in the number of ovarian TUNEL positive cells on postnatal days 1 and 56. Therefore, maternal exposure to alcohol and nicotine during developmental and lactation periods could lead to changes in the placenta properties as well as an increase in the apoptotic ovarian follicles in f1 mice pups.

Evaluation of expression and serum concentration of anti-Mullerian hormone as a follicle growth marker following consumption of fennel and flaxseed extract in first-generation mice pups.

BACKGROUND: The aim of the present study was to assess the expression and serum level of AMH in first-generation female mice pups following fennel and flaxseed consumption. METHODS: Twenty pregnant NMRI mice were allocated into four groups including control (CTL), fennel (FV), flaxseed (LU) and FV+ LU. Sixty-four female offsprings after lactation period, received the same regimen as their mothers for 56 and 240 days. The ovarian follicles development, serum concentration of AMH, as well as gene and protein expression of AMH were evaluated in the female offsprings at post-natal day 56 (PND56) and 240 (PND240). RESULTS: The number of total growing follicles were raised in the FV group in compression to the all experimental groups. In contrast, LU group showed a marked decrease in their numbers. The highest level of serum AMH was seen in the FV-diet mice, whereas LU negatively affected it. The expression level of AMH also increased in the FV and FV + LU groups, while a reduction was observed in the LU group. As well, IHC data showed that the number of AMH-positive cells in almost ovarian follicles of FV and FV + LU-treated mice was in compared to those of the LU group. CONCLUSIONS: The overall effect of fennel treatment (alone and in combination with flaxseed) on ovary might be maintain primordial follicle storage through increased expression and serum level of AMH.

Hydroalcoholic extract and seed of Foeniculum vulgare improve folliculogenesis and total antioxidant capacity level in F1 female mice offspring.

BACKGROUND: Foeniculum vulgare (fennel) is traditionally suggested for the fertility improvement in Iranian lore due to its antioxidant and phytoestrogen compounds. The present study aimed to investigate the effects of fennel seed and its hydroalcoholic extract on the serum total antioxidant capacity (TAC) and folliculogenesis in offspring exposed to either of the treatments in utero and 56 days after birth (PND 56). METHODS: Pregnant NMRI mice were randomly divided into 5 groups of 7: extract-treated groups received 500 and 1000 mg/kg/day fennel extract (FE), seed-treated groups received 500 and 1000 mg/kg/day fennel seed (FS), and the control group (CTL) received no treatment. The treatments started from pregnancy day 1 and continued until PND 56. Body and right ovary weights and ovary dimensions were recorded. Hematoxylin and eosin stained ovary sections were prepared to calculate the proportion of different follicles. The level of TAC in the serums was also measured by fluorescence recovery after photo bleaching. RESULTS: A marked rise in the body and ovary weights of treated mice was observed compared to the CTL group. The mean number of primordial, primary, pre-antral, and pre-ovulatory follicles as well as corpus luteum size in the treated offspring was significantly higher compared to those of CTL offspring. The atretic follicle number was nonsignificantly lower in either of the treatment groups compared with that in the CTL. However, treatment of animals with 500 mg/kg FE showed a more pronounced effect. Animals in FS500, FE500 and FE1000 groups had a significantly higher level of serum TAC compared to the CTL group. CONCLUSIONS: Fennel extract and seed administration in pregnancy and lactation period improve offspring's folliculogenesis. Higher level of TAC in the serum of offspring might have positively altered the folliculogenesis milieu.

Introducing a supernumerary muscle in the anterior compartment of the hand: A cadaveric study

Many muscular variations of the upper extremity have been reported. The majority of these variations are associated with forearm muscles. However, the muscular variations of the hand are uncommon. In the current study we found a unique supernumerary muscle in the right hand of a middle-aged male cadaver. This muscle originated from the one of the tendons of flexor digitorum superficialis (FDS) muscle that goes to the index finger and then traversed 5 cm distally to attach the same tendon. The knowledge of muscular variations is very essential for surgeons and clinicians to plan the standard surgical approaches

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Alcohol and nicotine co-Administration during pregnancy and lactation periods alters sensory discrimination of adult NMRI mice offspring.

The present study investigated the impacts of alcohol, nicotine, and their co-administration during pregnancy and lactation on sensory information processing including visual, tactile, and auditory discrimination in adult NMRI mice offspring. Pregnant mice were injected with saline or 20% alcohol (3 g/kg), or nicotine (1 mg/kg) or their co-administration alcohol+nicotine, intraperitoneally until the end of lactation. The offspring were separated from their mothers after lactation period on postnatal day (PND) 28. The locomotor activity, novel object recognition-dependent on visual system (NOR-VS), novel texture discrimination- dependent on somatosensory system (NTR-SS), and acoustic startle reflex were evaluated in PND90. The results revealed no statistical significance for locomotor activity of alcohol, nicotine, and co-administration alcohol+nicotine groups compared to the saline group in the open field task. The results, however, showed a significant decline in the ability of novel object discrimination in the nicotine and co-administration alcohol + nicotine groups compared to the saline group (P < 0.05) in the NOR-VS task. In the NTR-SS and acoustic startle reflex tasks, texture discrimination and the prepulse inhibition abilities in the offspring administered with nicotine and alcohol alone were reduced when compared to the saline group. Also, co-administration of alcohol+nicotine groups showed a decline in the aforementioned tests compared to the saline group (P <0.05). Administration of alcohol and nicotine during fetal and postpartum development disrupts sensory processing of inputs of visual, tactile, and auditory systems in adult mice.

Protective effects of hydro-alcoholic extract of foeniculum vulgare and linum usitatissimum on ovarian follicle reserve in the first-generation mouse pups.

OBJECTIVE: The present study was conducted to investigate the protective effects of fennel and flaxseed during pre- and post-natal period until puberty and menopause on ovarian follicular reserve (OFR). METHODS: Pregnant NMRI mice received fennel (FV, 500 mg/kg/day), flaxseed (LU, 500 mg/kg/day), LU + FV (500 mg/kg/day) and no treatment was given to the controls. Female pups were studied on post-natal-days 1, 56 and 240 (PND1, 56, 240). Ovary weight and diameters, the number of primordial (PF), atretic (AF) and apoptotic (APF) follicles were determined. The expression of Bcl2 and STAT3 (apoptosis-related-genes), micoRNA-125a-5p, and also serum levels of sex hormones were measured. Data were analyzed using one-way ANOVA test. RESULTS: FV and FV + LU groups showed a marked rise in body and ovary weights and diameters as compared to the control group. The number of PF at PND1, PND56, and PND240 increased significantly in the FV and FV + LU groups but decreased in the LU group compared to the control mice. There was a significant reduction in the mean of AF in the FV and FV + LU group and a marked increase in the LU group compared to the controls. Also, more APF were observed in the LU group, whereas less apoptotic follicles were present in the FV group. FSH and estradiol serum levels increased significantly while LH decreased in the FV group. The anti-apoptotic-genes expression and pro-apoptotic microRNA, respectively, increased and decreased in the FV group versus control group. CONCLUSIONS: It can be concluded that fennel alone and in combination with flaxseed could improve OFR during pregnancy, lactation, and afterwards until puberty and menopause.

Preventative Effects of Vitamin E on Testicular Damage and Sperm Parameters in the First-Generation Mice Pups due to Pre- and Postnatal Mancozeb Exposure.

The present study aimed to evaluate the effects of vitamin E on mancozeb-induced testis damage of the first-generation pups during intrauterine and lactating periods. Two groups of pregnant NMRI mice received 500 mg/kg mancozeb (MNZ) as MNZ group and 200 mg/kg vitamin E as MNZ+vit.E group before receiving MNZ. In addition, a vehicle and a control group were designed every other day in gestation and lactation periods. The male pups from each group were maintained until adulthood (8-10 W). The left testes and epididymides were removed following the sacrifice of the pups. Then, they were weighed, and sperm parameters including number, viability, motility, and morphology and testis structure were evaluated. A significant decrease occurred in sperm parameters of the mancozeb-treated pups compared to the control and vehicle groups. Treatment with vitamin E reversed the deleterious effects of MNZ to a nearly normal condition. Testis parameters including the weight, gonadosomatic index, seminiferous tubule diameters, and Johnsen's score, as well as the number of germ cells such as spermatogonia, spermatocyte, spermatid, and Sertoli, decreased significantly in the MNZ group, compared to the amount in the control and vehicle groups. Interestingly, the treatment with vitamin E was reversed in most of these parameters. Based on the results, the exposure of pups to mancozeb during pregnancy and lactating periods negatively affects the reproductive system of male pups. However, the coadministration of vitamin E could prevent the deleterious effects of mancozeb on sperm and testis parameters.

Photobiomodulation and gametogenic potential of human Wharton's jelly-derived mesenchymal cells.

Recently, light emitting diode (LED) irradiation has been introduced as a new strategy to enhance proliferation and affect differentiation of stem cells. Human Wharton's jelly-derived mesenchymal (hWJM) cells have unique characteristics that make them an appropriate source of stem cells for use in basic and clinical applications. In this study, we aimed to evaluate the effect of polarized (PL) and non-polarized (NPL) red light irradiation on gametogenic differentiation of hWJM cells in the presence or absence of bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). Exposure of hWJM cells to PL and NPL red LED (625 nm, 1.9 J/cm2) with or without BMP4+RA pre-treatment effectively differentiated them into germ lineage when the gene expression pattern (Fragilis, DAZL, VASA, SCP3 and Acrosin) and protein synthesis (anti-DAZL, anti-VASA, anti-SCP3 and anti-Acrosin antibodies) of the induced cells was evaluated. These data demonstrated that photobiomodulation may be applied for gametogenic differentiation in-vitro.

Effects of polarized and non-polarized red-light irradiation on proliferation of human Wharton's jelly-derived mesenchymal cells.

Light emitting diode (LED) irradiation has recently been introduced as an encouraging strategy for promotion of cell proliferation. Human umbilical cord Wharton's jelly-derived mesenchymal (hUCM) cells are among the most available mesenchymal cells with a promising application in regenerative medicine. The aim of the present study was to examine the effect of polarized (PL) and non-polarized (NPL) red-light emitted by LED on various proliferation properties of hUCM cells. Cell proliferation was assessed 48 h after irradiation of hUCM cells by different energy densities. Cell density increased to a significant level both in PL and NPL irradiation at 0.954 J/cm2 following WST-1 assay. Staining of irradiated and non-irradiated cells with Hoechst after 3 and 6 days revealed an increased proliferation rate in irradiated cells, but the non-irradiated cells proliferated more than irradiated cells at day 9 of cultivation. Similar results were obtained in trypan blue assay. Scratch repair test for 18 h with an interval of 6 h did not reveal a significant difference between irradiated and non-irradiated cells. In addition, CFU-F assay in PL irradiated cells was higher than control when 500 cells/plate was cultivated. Totally, this study revealed that hUCM cells could be induced to achieve higher number of cells by PL and NPL red-light irradiation after 48 h.

Ovarian protection in cyclophosphamide-treated mice by fennel.

Evaluation of protective effect of fennel on mouse ovary against the destructive effects of cyclophosphamide (CP) was the aim of this study. Adult female NMARI mice were randomly divided into six groups (n = 8): (A) negative control, (B) CP200 mg/kg, (C) fennel 400 mg/kg/day, (E, F, and D) that received fennel 200, 400 and 100 mg/kg/day respectively + CP200 mg/kg. Their ovary weight, volume, and diameter (WVD) were measured. Five micron sections were stained using the H&E method. The serum levels of oestrogen and progesterone were measured using ELISA kit. The results showed that WVD significantly reduced in the CP-treated groups in comparison with the A and C, but WVD increased after treatment of the mice with fennel extract, in comparison with B group. A significant decrease of serum in terms of oestrogen and progesterone levels among CP-treated groups in comparison with the A group was observed. In the CP-treated groups a reduction in the number of different ovarian follicles in comparison with the A and C groups was observed. However, in the treated animals with fennel extract, these parameters significantly increased in comparison with the B group. Finally, it is concluded that fennel can protect ovary from cyclophosphamide side effects.

Evaluation of gametogenic potential of vitrified human umbilical cord Wharton's jelly-derived mesenchymal cells.

BACKGROUND AIMS: Vitrification as an advanced cryopreservation method is recommended for cell storage toward future applications. The purpose of this report was to appraise whether gametogenic potential of these cells is altered by vitrification. METHODS: A two steps method was applied for hUCM cells vitrification. An n-hUCM group of hUCM cells served as control. In order to differentiation of hUCM cells into male germ cells, the cells were induced by retinoic acid, testosterone and testicular-cell-conditioned medium. To evaluate induced hUCM cells toward germ cells, we used immunocytochemistry and karyotyping methods. RESULTS: v-hUCM cells similar to n-hUCM cells formed flat cells after gametogenic induction, and showed protein expression of germ-cell-specific markers DAZL, VASA (DDX4) and SCP3. Karyotyping pattern remained unchanged in the either groups. CONCLUSIONS: The analysis of these results demonstrates that vitrification does not alter differentiation potential of hUCMs to male germ like cells. These results may set an in vitro pattern to study germ-cell formation from hUCM cells and also as a potential source of sperms for male infertility.

The effects of morphine on tissue structure of the growth plate in male rats.

OBJECTIVES: Studies have shown that morphine, in addition to its analgesic properties, has several effects on cell proliferation and apoptosis. There is also evidence that opioid receptors are present on chondrocytes. Our main objective in the present study was to investigate the effects of morphine on rat femur growth cartilage. MATERIALS AND METHODS: This research was carried out on 18 4-week-old male rats. Animals were divided into four groups: groups 1 (n= 3) and 2 (n= 4) were non morphine-dependent and groups 3 (n= 6) and 4 (n= 5) were morphine-dependent. Groups 1 and 3 were followed up for 4 weeks and the others for 7 weeks. We prepared femur bone biopsies, fixed the samples in 10% formalin and 10% nitric acid and stained the samples with haematoxylin and eosin. The thickness of the growth cartilage and its proliferative zone (PZ) cell number were studied. In addition, the existence of necrosis, inflammation, fibrosis, and hyalinisation were evaluated. RESULTS: There were no signs of inflammation, fibrosis, necrosis or hyalinisation in the growth cartilages of all rats. The morphine-dependent groups had a statistically significant difference (P< 0.001) in the number of cells in the proliferative zone and thickness of the growth cartilage compared to other groups using ANOVA analysis. CONCLUSION: It seems that morphine reduces the number of cell in the proliferative zone and decreases the thickness of the growth cartilage which may alter longitudinal growth of long bones.