simple technique for three-dimensional imaging and segmentation of brain vasculature u sing fast free-of-acrylamide clearing tissue in murine

Objective: Fast Free-of-Acrylamide Clearing Tissue [FACT] is a recently developed protocol for the whole tissue three-dimensional [3D] imaging. The FACT protocol clears lipids using sodium dodecyl sulfate [SDS] to increase the penetration of light and reflection of fluorescent signals from the depth of cleared tissue. The aim of the present study was using FACT protocol in combination with imaging of auto-fluorescency of red blood cells in vessels to image the vasculature of a translucent mouse tissues

Materials and Methods: In this experimental study, brain and other tissues of adult female mice or rats were dissected out without the perfusion. Mice brains were sliced for vasculature imaging before the clearing. Brain slices and other whole tissues of rodent were cleared by the FACT protocol and their clearing times were measured. After 1 mm of the brain slice clearing, the blood vessels containing auto-fluorescent red blood cells were imaged by a z-stack motorized epifluorescent microscope. The 3D structures of the brain vessels were reconstructed by Imaris software

Results: Auto-fluorescent blood vessels were 3D imaged by the FACT in mouse brain cortex. Clearing tissues of mice and rats were carried out by the FACT on the brain slices, spinal cord, heart, lung, adrenal gland, pancreas, liver, esophagus, duodenum, jejunum, ileum, skeletal muscle, bladder, ovary, and uterus

Conclusion: The FACT protocol can be used for the murine whole tissue clearing. We highlighted that the 3D imaging of cortex vasculature can be done without antibody staining of non-perfused brain tissue, rather by a simple auto- fluorescence

Decreased expression of arginine-phenylalanine-amide-related peptide-3 gene in dorsomedial hypothalamic nucleus of constant light exposure model of polycystic ovarian syndrome

Background: An abnormality in pulse amplitude and frequency of gonadotropin releasing hormone [GnRH] secretion is the most characteristics of polycystic ovarian syndrome [PCOS]. On the other hand, arginine-phenylalanine-amide [RFamide]-related peptide-3 [RFRP3] inhibits the secretion of GnRH in mammalian hypothalamus. The current study performed in order to investigate the expression of RFRP3 mRNA in the dorsomedial hypothalamic nucleus [DMH] after the induction of PCOS in a rat model of constant light exposure, and the possible role of parity on occurrence of PCOS

Materials and Methods: In the experimental study, female nulliparous [n=12] and primiparous [n=12] rats were randomly subdivided into control and PCOS subgroups [n=6]. PCOS were induced by 90 days exposure to constant light. After 90 days, blood, brain, and ovaries were sampled. Serum levels of follicle stimulating hormone [FSH], luteinizing hormone [LH], and testosterone were evaluated. In addition, six adult female ovariectomized rats as a control of real-time polymerase chain reaction [PCR] tests were prepared and in the DMH of all rats, the relative mRNA expression of RFRP3 was assessed

Results: Histological evaluation of ovaries represented the polycystic features. In addition, serum concentrations of testosterone in the PCOS subgroups were more than the controls [P<0.05]. Furthermore, the relative expression of RFRP3 mRNA in PCOS subgroups was lower than the controls [P<0.05]

Conclusion: Constant light model of the PCOS-induced rats decreased the gene expression of RFRP3 in the DMH that suggests the decrease of RFRP3 may reduce its inhibitory effect on GnRH during the PCOS pathogenesis. This effect was stronger in the nulliparous rats than the primiparous

Endometrial mesenchymal stem stromal cells in mature and immature sheep: an in vitro study

Background: Endometrial mesenchymal stem stromal cells [EnMSCs] are critical for uterine function, repair, and regeneration

Objective: This study introduced isolation technique of EnMSCs and compared the characteristics of EnMSCs in mature and immature ewes

Materials and Methods: Endometrial tissue samples from the uterus of 10 ewes were collected from the slaughterhouse. Endometrial cells were isolated from tissue using cold incubation and then chopping and treating was performed with collagenase type I. Isolated cells were cultured in cell culture medium and then attached cells to flasks were harvested as EnMSCs and subcultured. To enumerate the cells, the population doubling time [PDT] was determined and 2.2×104 cells in passage 4 were seeded into 24-well culture plates to compare the growth curves of isolated cells. Reverse transcription polymerase chain reaction [RT-PCR] was performed for detection of CD34 and CD73 markers. The osteogenic and adipogenic potential of isolated cells were determined using differentiation tests

Results: EnMSCs adhered to the flasks and displayed spindle-shape. Based on findings of the cell count and the growth curves, the EnMSCs growth was significantly more prominent in immature ewes in comparison to mature sheep. The PDT of EnMSCs in immature ewes was about 21 hr whereas this time period was two times higher [45 hr] in mature sheep. RT-PCR analyses of EnMSCs were positive for CD73 and negative for CD34. EnMSCs were differentiated into osteoblasts and adipocytes

Conclusion: Based on mesenchymal stem cells characters confirmed in EnMSCs, they can be a candidate for cell therapy and regenerative medicine

Skeletal muscle CLARITY: a preliminary study of imaging the three-dimensional architecture of blood vessels and neurons

Objective: Passive CLARITY is a whole-tissue clearing protocol, based on sodium dodecyl sulfate [SDS] clearing, for imaging intact tissue containing transgenic or immunolabeled fluorescent proteins. In this study, we present an improved passive CLARITY protocol with efficient immunolabeling without the need for electrophoresis or complex instrumentation

Materials and Methods: In this experimental study, after perfusion of C57BL/6N mice with phosphate-buffered saline [PBS] and then with acrylamide-paraformaldehyde [PFA], the quadriceps femoris muscle was removed. The muscle samples were post-fixed and degassed to initiate polymerization. After removing the excess hydrogel around the muscle, lipids were washed out with the passive CLARITY technique. The transparent whole intact muscles were labeled for vessel and neuron markers, and then imaged by confocal microscopy. Three-dimensional images were reconstructed to present the muscle tissue architecture

Results: We established a simple clearing protocol using wild type mouse muscle and labeling of vasculatures and neurons. Imaging the fluorescent signal was achieved by protein fixation, adjusting the pH of the SDS solution and using an optimum temperature [37degree C] for tissue clearing, all of which contributed to the superiority of our protocol

Conclusion: We conclude that this passive CLARITY protocol can be successfully applied to three-dimensional cellular and whole muscle imaging in mice, and will facilitate structural analyses and connectomics of large assemblies of muscle cells, vessels and neurons in the context of three-dimensional systems

The role of sex steroid hormones in the pathophysiology and treatment of sarcopenia

Sex steroids influence the maintenance and growth of muscles. Decline in androgens, estrogens and progesterone by aging leads to the loss of muscular function and mass, sarcopenia. These steroid hormones can interact with different signaling pathways through their receptors. To date, sex steroid hormone receptors and their exact roles are not completely defined in skeletal and smooth muscles. Although numerous studies focused on the effects of sex steroid hormones on different types of cells, still many unexplained molecular mechanisms in both skeletal and smooth muscle cells remain to be investigated. In this paper, many different molecular mechanisms that are activated or inhibited by sex steroids and those that influence the growth, proliferation, and differentiation of skeletal and smooth muscle cells are reviewed. Also, the similarities of cellular and molecular pathways of androgens, estrogens and progesterone in both skeletal and smooth muscle cells are highlighted. The reviewed signaling pathways and participating molecules can be targeted in the future development of novel therapeutics.

Efficient Biomaterials for Tissue Engineering of Female Reproductive Organs

Current investigations on the bioengineering of female reproductive tissues have created new hopes for the women suffering from reproductive organ failure including congenital anomaly of the female reproductive tract or serious injuries. There are many surgically restore forms that constitute congenital anomaly, however, to date, there is no treatment except surgical treatment of transplantation for patients who are suffering from anomaly or dysfunction organs like vagina and uterus. Restoring and maintaining the normal function of ovary and uterus require the establishment of biological substitutes that can cover the roles of structural support for cells and passage of secreting molecules. As in the case of constructing other functional organs, reproductive organ manufacturing also needs biological matrices which can provide an appropriate condition for attachment, growth, proliferation and signaling of various kinds of grafted cells. Among the organs, uterus needs special features such as plasticity due to their amazing changes in volume when they are in the state of pregnancy. Although numerous natural and synthetic biomaterials are still at the experimental stage, some biomaterials have already been evaluated their efficacy for the reconstruction of female reproductive tissues. In this review, all the biomaterials cited in recent literature that have ever been used and that have a potential for the tissue engineering of female reproductive organs were reviewed, especially focused on bioengineered ovary and uterus.

Isolation, Characterization and Growth Kinetic Comparison of Bone Marrow and Adipose Tissue Mesenchymal Stem Cells of Guinea Pig

BACKGROUND: Mesenchymal stem cells (MSCs) from different sources have different characteristics. Moreover, MSCs are not isolated and characterized in Guinea pig for animal model of cell therapy. AIM OF THE WORK: was the isolating of bone marrow MSCs (BM-MSCs) and adipose tissue MSCs (AT-MSCs) from Guinea pig and assessing their characteristics. MATERIAL AND METHODS: In this study, bone marrow and adipose tissue were collected from three Guinea pigs and cultured and expanded through eight passages. BM-MSCs and AT-MSCs at passages 2, 5 and 8 were seeded in 24-well plates in triplicate. Cells were counted from each well 1~7 days after seeding to determine population doubling time (PDT) and cell growth curves. Cells of passage 3 were cultured in osteogenic and adipogenic differentiation media. RESULTS: BM-MSCs and AT-MSCs attached to the culture flask and displayed spindle-shaped morphology. Proliferation rate of AT-MSCs in the analyzed passages was more than BM-MSCs. The increase in the PDT of MSCs occurs with the increase in the number of passages. Moreover, after culture of BM-MSCs and AT-MSCs in differentiation media, the cells differentiated toward osteoblasts and adipocytes as verified by Alizarin Red staining and Oil Red O staining, respectively. CONCLUSION: BM-MSCs and AT-MSCs of Guinea pig could be valuable source of multipotent stem cells for use in experimental and preclinical studies in animal models.

Growth Kinetics, characterization, and plasticity of human menstrual blood stem cells

One of the readily available sources of mesenchymal stem cells [MSCs] is menstrual blood-derived stem cells [Men-SCs], which exhibit characteristics similar to other types of MSCs. This study was performed to determine the growth kinetics, plasticity, and characterization of Men-SCs in women. During spring 2014 in the southern Iranian city of Shiraz, menstrual blood [5 mL] was obtained from 10 women on their third day of menstruation in 2 age groups of 30 to 40 and 40 to 50 years old. Ficoll was used to separate the mononuclear cell fraction. After the Men-SCs were cultured, they were subcultured up to passage 4. Growth behavior and population doubling time were evaluated by seeding 5×10[4] cells into 12- and 24-well culture plates, and the colonies were enumerated. The expression of CD44, CD90, and CD34 was evaluated. The osteogenic potential was assessed by alizarin red staining. The Men-SCs were shown to be plastic adherent and spindle-shaped. Regarding the growth curves in the 12-24-well culture plates, it was demonstrated that in the women aged between 30 and 40 years, population doubling time was 55.5 and 62 hours, respectively, while these values in the women aged between 40 and 50 years were 70.4 and 72.4 hours, correspondingly. Positive expression of CD44 and CD90 and negative expression of CD34 were noted. In the osteogenic differentiation medium, the cells differentiated toward osteoblasts. As human Men-SCs are easily collectable without any invasive procedure and are a safe and rapid source of MSCs, they can be a good candidate for stem cell banking and cell transplantation in women

Induction of Asherman's syndrome in rabbit

Background: Uterine synechiae or Asherman's syndrome is a condition that can cause infertility. The present experimental study was designed to establish the rabbit as an animal model for human Asherman's syndrome using the endometrial curettage

Methods: In an experimental study, female adult rabbits [n=18] were randomly divided into intact and ovariectomized groups. One third of caudal part of both uteri was submitted to traumatic endometrial curettage. One group was simultaneously ovariectomized. The intact rabbits were artificially induced ovulation during 10 days after surgery. One third of cranial part of both uteri was selected as the control. Synechiae occurring, luminal area/total area [LA/TA], endometrial area/total area [EA/TA], myometrial and perimetrial area/total area [MPA/TA], endometrial area/uterine wall area [EA/UWA], and myometrial and perimetrial area/uterine wall area [MPA/UWA] ratios of both uteri in six subdivided groups [n=6] were analysed in curetted and intact control parts. On days 15, 30 and 45 following surgery by two-way ANOVA and LSD test [p<0.05]

Results: Histopathologic findings showed significant epithelial damage together with significant inflammatory reaction in the intact curettage group. The LA/TA ratios of the intact curettage group on days 15 and 45 were more than the intact control group on day 15. The EA/TA ratio of the intact curettage group on day 30 was less than the intact control group on day 30

Conclusion: Uterine fibrosis was observed in intact curettage group, and this modified animal model showed a pathogenesis condition similar to intrauterine adhesions observed in human

Induction of Spermatogenesis by Bone Marrow-derived Mesenchymal Stem Cells in Busulfan-induced Azoospermia in Hamster

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) have potential of differentiation and they secrete anti-inflammatory cytokines and growth factors which make them appropriate for cell therapy. AIM OF THE WORK: Were to evaluate the healing effect of BM-MSCs transplantation on germinal cells of busulfan-induced azoospermic hamsters. MATERIAL AND METHODS: In the present experimental case control study, BM-MSCs were isolated from bone marrow of donor albino hamsters. Five mature male recipient hamsters received two doses of 10 mg/kg of busulfan with 21 days interval to stop endogenous spermatogenesis. After induction of azoospermia, right testis of hamsters was injected with 106 BM-MSCs via efferent duct and the left one remained as azoospermia control testis. Five normal mature hamsters were selected as normal intact control. After 35 days, testes and epididymis of three groups were removed for histological evaluation. RESULTS: Histomorphological analyses of BM-MSCs treated testes and epididymis showed the epithelial tissue of seminiferous tubules had normal morphology and spermatozoa were present in epididymis tubes. Spermatogenesis was observed in most cell-treated seminiferous tubules. The untreated seminiferous tubules were empty. CONCLUSION: Transplanted BM-MSCs could successfully induce spermatogenesis in seminiferous tubules of azoospermic hamster. Therefore, BM-MSCs can be an attractive candidate in cell transplantation of azoospermia.

role of arginine-phenylalanine-amide-related peptides in mammalian reproduction

Until 2000 it was believed that gonadotropin-releasing hormone [GnRH] was the sole regulator of hypophyseal gonadotropes. In 2000, the discovery of a gonado-tropin inhibitory hormone [GnlH] initiated a revolution in the field of reproductive physiology. Identification of GnlH homologues in mammals, the arginine-pheny-lalanine-amide [RFamide]-related peptides [RFRPs], indicated a similar function. Subsequently, further works conducted in various laboratories worldwide have shown that these neuropeptides inhibit the hypothalamic-hypophyseal axis. This review discusses the role of RFRPs in mammalian reproductive processes

Increased litter size and suckling intensity stimulate mrna of rfamide-related peptide in rats

RFamide-related peptide-3 [RFRP-3] inhibits gonadotropin releasing hormone [GnRH] and luteinizing hormone [LH] secretion in rats. This study evaluates the effects of litter size and suckling intensity on RFRP mRNA expression in the dorsomedial hypothalamic nucleus [DMH] of rats. A total of 32 pregnant and 4 non-lactating ovariectomized [control group] Sprague-Dawley rats were used in this experimental study. Lactating rats were allotted to 8 equal groups. In 3 groups, the Utter size was adjusted to 5, 10, or 15 pups upon parturition. Dams were allowed to suckle their pups continuously until 8 days postpartum. In the other 3 groups, the litter size was adjusted to 5 pups following birth. These pups were separated from the dams for 6 hours on day 8 postpartum, after which the pups were allowed to suckle for 2.5, 5, or 7.5 minutes prior to killing the dams. In 2 groups, lactating rats with 10 and 15 pups were separated from their pups for 6 hours on day 8 postpartum. In these groups, the pups were allowed to suckle their dams for 5 minutes before the dams were killed. All rats were killed on day 8 postpartum and the DMH was removed from each rat. We evaluated RFRP mRNA expression using real-time polymerase chain reaction [PCR. The expression of RFRP mRNA in the DMH increased with increased litter size and suckling intensity compared to the controls. The effect of suckling intensity on the expression of RFRP mRNA was more pronounced compared to the litter size. Increased litter size and suckling intensity stimulated RFRP mRNA expression in the DMH which might contribute to lactation anestrus in rats

Expression of RFamide-related peptide-3 [RFRP-3] mRNA in dorsomedial hypothalamic nucleus and KiSS-1 mRNA in arcuate nucleus of rat during pregnancy

RFamide-related peptide-3 [RFRP-3] and kisspeptin [KiSS-1] are known to respectively inhibit and stimulate gonadotropin releasing hormone [GnRH] and luteinizing hormone [LH] secretion in rat. The aim of the present study was to evaluate the relative mRNA expression of RFRP-3 and KiSS-1 in the hypothalamus of pregnant rats. In a randomized controlled experimental study, the exact pregnancy day of 18 Sprague-Dawley rats were confirmed using the vaginal smear method and were equally assigned to three groups of days 7, 14 and 21 of pregnancy. Four non-pregnant female rats were ovariectomized and assigned as the control group. All rats were decapitated, and the dorsomedial hypothalamic nucleus [DMH] and the arcuate nucleus [ARC] for detection of KiSS-1 mRNA were separated from their hypothalamus to detect RFRP-3 and KiSS-1 mRNA respectively. Then, their relative expressions were compared between control and pregnant groups using real-time polymerase chain reaction [PCR]. The relative expression of RFRP-3 mRNA in DMH did not change significantly during pregnancy [p>0.01]. However, the relative expression of KiSS-1 mRNA in ARC was at its highest in day 7 of pregnancy and decreased until day 21 of pregnancy [p<0.01]. Decrease in GnRH and LH secretion during the pregnancy of rat may be controlled by constant expression of RFRP-3 mRNA and reduced expression of KiSS-1 mRNA in hypothalamus

Hypothalamic expression of KiSS1 and RFamide-related peptide-3 mRNAs during the estrous cycle of rats

Kisspeptin and RFamide-related peptide-3 [RFRP-3] are known to affect GnRH/luteinizing hormone [LH] in several species, including the rat. It has been hypothesized that GnRH/LH changes during the rat estrous cycle may result from changes in the expression of KiSS1 and RFRP-3 genes. Therefore, the present study investigates KiSS1 and RFRP-3 gene expression at the transcriptional level in the rat hypothalamus during the estrous cycle. In the present experimental study, 36 adult female Sprague-Dawley rats [3-4 months old] were used to study the expression of KiSS1 and RFRP-3 mRNA in the hypothalamus during the estrous cycle. Four rats were ovariectomized, whereas the remainder were allotted to four different phases of the estrous cycle [n=8 per estrus phase]. Rats were decapitated, and the hypothalami were immediately dissected and frozen in liquid nitrogen. Expressions of KiSS1 and RFRP-3 mRNAs were analyzed by real-time PCR. The expression of KiSS1 mRNA during estrus was lower than other phases of the cycle [p<0.01]. Expression of KiSS1 mRNA during the metestrus phase was lower than the proestrus phase [p<0.01]. The expression of RFRP-3 mRNA during proestrus was lower than the diestrus phase [p<0.01]. Results of the present study showed the role of coordinated expression of KiSS1 and RFRP-3 mRNA in the hypothalamus in the control of the rat estrous cycle

Characterization of Iranian isolates of canine parvovirus in fecal samples using polymerase chain reaction assay

Despite the widespread prevalence of canine parvovirus disease [CPV] in Iranian dog population, molecular diagnosis of CPV variants, and investigation of the trends of its genetic changes is a new effort. In this study 50 samples from dogs suspicious of infection with clinical signs of diarrhea and vomiting, and 25 samples from dogs suspected of infection with general symptoms such as depression and anorexia were collected from dogs presented to the veterinary clinic of Shiraz University, Shiraz, Iran. Viral DNA was extracted from feces. Three specific pairs of primers, P2, Pab, and Pb, were used in a PCR assay for differential diagnosis of the virus type. Pab primer pairs detect the new type-strains, CPV-2a and 2b. The primer pairs P2 and Pb detect CPV types 2 and 2b, respectively. Our results showed that 44 individuals with clinical signs of diarrhea and vomiting were positive for CPV-2. 39 individuals [89%] were positive for CPV-2b and 5 individuals [11%] for CPV-2a. Therefore, the CPV-2b was identified as the predominant virus type. All dogs without symptoms of diarrhea and vomiting were CPV-negative. The relationship of breed, age and sex with PCR results was not significant [P>0.05]. For the first time in the country, the causative agent of CPV-2 was identified, and presence of new antigenic variants, CPV-2a and CPV-2b was confirmed