Rat Animal Model Research Articles

Effects of 2.45 GHz Non-Ionizing Radiation on Anxiety-Like Behavior, Gene Expression, and Corticosterone Level in Male Rats.

Introduction: The effects of short-term and long-term exposures to 2.45 GHz radiofrequency electromagnetic radiation (RF-EMR) on anxiety-like behavior, corticosterone level, and gene expression were investigated. The goal of this study was to explore the effect of electromagnetic fields of 2.45 GHz on clinical signs such as body weight and anxiety-like behavior, including the elevated plus maze test and open-field test, and also on messenger RNA (mRNA) expression of Bax (Bcl2-associated x) and Bcl-2 (B-cell lymphoma 2) genes on the cognitive memory functions in an animal model of rats. Methods: The animals were classified into eight groups, sham groups and exposed groups for short-term and long-term exposures to the same dose of RF-EMR for one hour daily. The Wi-Fi equipment in the sham control group was not turned on during the experiment. Both genes were further confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). The semi-quantitative PCR method of electromagnetic fields in the 2.45 GHz range impacted the expression of Bax and Bcl-2 genes in the rat's memory. Results: The present study exhibited that short-term radiation could decrease the percentage of entry into the open arm and the percentage of time spent, while there were no substantial impacts on the long-term radiation effect. Our data support the hypothesis that short-term exposure worked as a systemic stressor, raising plasma corticosterone and changing glucocorticoid receptor expression in the hippocampus. Conclusion: Additional research on this specific frequency and amount of radiation is required to discover strategies for protecting the nervous system from the detrimental effects of RF-EMR radiation.

Transcriptomics reveal stretched human pluripotent stem cell-derived cardiomyocytes as an advantageous hypertrophy model

Left ventricular hypertrophy, characterized by hypertrophy of individual cardiomyocytes, is an adaptive response to an increased cardiac workload that eventually leads to heart failure. Previous studies using neonatal rat ventricular myocytes (NRVMs) and animal models have revealed several genes and signaling pathways associated with hypertrophy and mechanical load. However, these models are not directly applicable to humans. Here, we studied the effect of cyclic mechanical stretch on gene expression of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using RNA sequencing. hiPSC-CMs showed distinct hypertrophic changes in gene expression at the level of individual genes and in biological processes. We also identified several differentially expressed genes that have not been previously associated with cardiomyocyte hypertrophy and thus serve as attractive targets for future studies. When compared to previously published data attained from stretched NRVMs and human embryonic stem cell-derived cardiomyocytes, hiPSC-CMs displayed a smaller number of changes in gene expression, but the differentially expressed genes revealed more pronounced enrichment of hypertrophy-related biological processes and pathways. Overall, these results establish hiPSC-CMs as a valuable in vitro model for studying human cardiomyocyte hypertrophy. • Mechanical load-associated genes in human cardiomyocytes are not fully understood. • Changes in response to mechanical stretch in hiPSC-CMs were investigated by RNAseq. • hiPSC-CMs showed distinct gene expression changes compared to NRVMs or hESC-CMs. • Differentially expressed genes were restricted to hypertrophy-related processes. • GAL, LINC00648 and SLC16A9 identified as new cardiac stretch-responsive genes.

Evaluation of a Rapid and Simple Method for Assessing Retinal Vessel Structures in Adult Zebrafish

The evaluation of retinal vascular structures is important for analyzing various ophthalmic diseases. Conventional trypsin digestion was used for separating retinal vasculatures in mouse, rat, and other animal models; however, the trypsin method alone is technically difficult to perform and has not been reported in zebrafish to date. In this study, we introduced a rapid and convenient method that allows the investigation of fine vessel structures at a cellular level in the relatively intact retinal vasculature of adult zebrafish. Using an anti-ZO-1 antibody, tight junction structures in retinal vessels were examined in detail and several different cell types constituting blood vessels in arterial and capillary areas were identified. In addition, using cell type-specific antibodies, we identified smooth muscle cells, blood cells, and endothelial cells in the retinal vasculature. Finally, using the hyperglycemic model, we observed the dilation of retinal vessels, the downregulation of tight junction proteins, and the reduction in smooth muscle cells. Based on these results, we provide a rapid and convenient method for the study of retinal vasculature disease in the zebrafish animal model.

The effects of Ibuprofen and 1, 8- cineol on anxiety and spatial memory in hyperammonemic rats.

In hepatic encephalopathy, hyperammonemia (HA) causes cognitive impairment and anxiety by causing neuroinflammation. Ibuprofen and 1,8- cineol have anti-inflammatory and antioxidant properties, respectively. The aim of this study was to evaluate the effects of ibuprofen alone and in combination with 1,8- cineol on anxiety and oxidative stress in a HA rat animal model. For this purpose, 36 rats were divided into six groups (n = 6) including the HA (received intraperitoneally (IP) ammonium acetate 2.5mg/kg for four week), ibuprofen (induced HA rats that received 15mg/kg, IP), cineol (induced HA rats that received 5 and 10mg/kg, IP), Ib + cineol (induced HA rats that received 15 and 10mg/kg, respectively, IP), and the control groups (received normal saline, IP). Except the HA group, all other groups received the aforementioned treatment for two weeks.. The Morris water maze and elevated plus maze were used to assess cognitive function and anxiety in the animals, respectively. Superoxide dismutase (SOD) activity was measured to evaluate oxidative stress. The mRNA expression levels of interleukin (IL)-6 and IL-1β was assessed by real-time PCR in the animal's brain. The results showed a significant improvement in spatial memory and anxiety of the Ib group compared to the HA group (P < 0.01), but no significant change was observed in SOD activity (P > 0.05). There was a significant improvement in spatial memory and anxiety as well as a significant increase in SOD activity in the Ib + cineol group (P < 0.01) compared to the HA group. These results indicate that the Ib + cineol, not only improve cognitive function and reduce anxiety, also reduce oxidative stress, therefore, the simultaneous use of these two compounds may be useful in improving HA-induced cognitive disorders and anxiety.

Incorporation of curcumin into collagen-multiwalled carbon nanotubes nanocomposite scaffold: an in vitro and in vivo study

AimFabrication of biological substitutes to regenerate damaged tissues or organs is one of the main aims of tissue engineering (TE). In this context, the goal of this study was to fabricate a scaffold that mimicked the structure and functions of the extracellular matrix (ECM) of the native tissues. Towards this aim, a novel biomimetic three-dimensional (3D) scaffold containing collagen (COL), multi-walled carbon nanotubes (MWCNTs), and curcumin (CUR) was created using the freeze-drying technique. Materials and methodsScaffolds were prepared by adding 0.5–1.5% MWCNTs and 5–15% CUR to pure COL solutions, followed by freeze-drying. Physical and chemical characterizations of the scaffolds were evaluated by a universal testing machine, scanning electron microscope (SEM), fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). Biological studies were mainly focused on in-vitro bioactivity, biodegradability, CUR release, and in-vitro and in vivo biocompatibility using mesenchymal stem cells (MSCs) and rat animal models. ResultsFTIR and XRD confirmed the presence of MWCNTs and CUR in the COL scaffolds, while SEM revealed highly interconnected porous morphology. Moreover, the addition of up to 1% MWCNTs and 10% CUR enhanced tensile strength from 5 MPa to 19 MPa. The developed COL-MWCNTs 1%-CUR 10% composite scaffolds revealed excellent surface wettability, in vitro bioactivity, and in vitro biocompatibility using rat synovial-derived MSCs (SM-MSCs). Importantly, the in vivo study revealed reduced inflammatory response in the rat animal model after six weeks of implantation, which could be attributed to the promising in vitro biodegradability and release of CUR. ConclusionThe newly developed COL-MWCNTs 1%-CUR 10% freeze-dried scaffolds have demonstrated their high potential for TE applications.

Inflammatory mediator responses of Vaccinium corymbosum extracts on the streptokinase induced acute glomerulonephritis in rats

Blueberry (Vaccinium corymbosum) has many health benefits including anti-inflammatory and antioxidant activities. Glomerulonephritis is a commonly found kidney disease in companion animals that is characterized by glomerular proliferation and inflammation likes characteristics. The present study was carried out to evaluate the potential of blueberry against inflammatory response in the kidney of acute glomerulonephritis (AGN) in animal models. For this, twenty male Wistar rats were randomized into five groups i.e. A - E (n=4). Among these Group A has four healthy individuals administrated with aqua dest (negative control), group B individuals have streptokinase (6000IU/rat) induced acute glomerulonephritis rats treated with aqua dest (positive control) while group C-E has streptokinase (6000IU/rat) induced acute glomerulonephritis rats treated with different concentrations of blueberry extract (500, 1000, and 1500 mg/kg body weight) for 14 days, respectively. After 14 days, kidney samples were harvested for histology and immunohistochemistry examinations. One-way ANOVA followed by the Tukey test was used for statistical analysis (P&lt; 0.05). The blueberry extract treated AGN rats showed a significantly decreased in IL-1beta expression and inflammatory cell numbers compared to negative and positive control rats and 1500 mg/kg of the blueberry extract was found as the optimal dose. Results of the study can be concluded that blueberry extract has a strong anti-inflammatory effect that could depress the inflammatory responses in acute glomerulonephritis rat animal models.

Analysis of Bone Histomorphometry in Rat and Guinea Pig Animal Models Subject to Hypoxia.

Hypoxia may be associated with alterations in bone remodeling, but the published results are contradictory. The aim of this study was to characterize the bone morphometry changes subject to hypoxia for a better understanding of the bone response to hypoxia and its possible clinical consequences on the bone metabolism. This study analyzed the bone morphometry parameters by micro-computed tomography (μCT) in rat and guinea pig normobaric hypoxia models. Adult male and female Wistar rats were exposed to chronic hypoxia for 7 and 15 days. Additionally, adult male guinea pigs were exposed to chronic hypoxia for 15 days. The results showed that rats exposed to chronic constant and intermittent hypoxic conditions had a worse trabecular and cortical bone health than control rats (under a normoxic condition). Rats under chronic constant hypoxia were associated with a more deteriorated cortical tibia thickness, trabecular femur and tibia bone volume over the total volume (BV/TV), tibia trabecular number (Tb.N), and trabecular femur and tibia bone mineral density (BMD). In the case of chronic intermittent hypoxia, rats subjected to intermittent hypoxia had a lower cortical femur tissue mineral density (TMD), lower trabecular tibia BV/TV, and lower trabecular thickness (Tb.Th) of the tibia and lower tibia Tb.N. The results also showed that obese rats under a hypoxic condition had worse values for the femur and tibia BV/TV, tibia trabecular separation (Tb.Sp), femur and tibia Tb.N, and BMD for the femur and tibia than normoweight rats under a hypoxic condition. In conclusion, hypoxia and obesity may modify bone remodeling, and thus bone microarchitecture, and they might lead to reductions in the bone strength and therefore increase the risk of fragility fracture.

Downregulation of natriuretic peptide receptor-C in vascular smooth muscle cells from spontaneously hypertensive rats contributes to vascular remodeling

Hypertension is associated with vascular remodeling due to hyperproliferation and hypertrophy of vascular smooth muscle cells (VSMC). VSMC from several animal models of hypertensive rats including spontaneously hypertensive rats (SHR) exhibit hyperproliferation, hypertrophy and decreased expression of natriuretic peptide receptor-C (NPR-C). In addition, angiotensin II (Ang II) and growth factors that promotes vascular remodeling have also been shown to attenuate the expression of NPR-C in VSMC. The present study investigates the relationship between the decreased expression of NPR-C and vascular remodeling in SHR and the underlying molecular mechanisms. Aortic VSMC from SHR and their control Wistar Kyoto (WKY) rats were transfected with cDNA of NPR-C and used for the vascular remodeling studies. Transfection of VSMC with cDNA of NPR-C augmented the expression of NPR-C in both VSMC from SHR and WKY rats and resulted in the attenuation of hyperproliferation and hypertrophy of VSMC from SHR. The overexpression of NPR-C also resulted in the attenuation of increased expression of epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), cell cycle proteins, cyclin D1, cyclin-dependent kinase 4 (Cdk4), phospho-retinoblastoma (pRb) and Giα-2 proteins, all these signaling molecules implicated in the hyperproliferation/hypertrophy of VSMC from SHR. In summary, these results indicate that augmenting the decreased expression of NPR-C in VSMC from SHR improves vascular remodeling by attenuating hyperproliferation and hypertrophy through decreasing the overexpression of several signaling molecules. It may be suggested that NPR-C plays a vasculoprotective role and that the downregulation of NPR-C contributes to the vascular remodeling in SHR.

Analysis of Anti-Arrhythmic Impacts of Crocin through Estimation of Expression of Cx43 in Myocardial Infarction Using a Rat Animal Model.

Arrhythmia is an important cause of death after myocardial infarction (MI). Different substances have been evaluated for their anti-arrhythmic effect in MI. This study was performed to evaluate the anti-arrhythmic impacts of crocin in an MI animal model (rat) by estimation of the expression of connexin 43 (Cx43). Fifty male Sprague–Dawley rats were grouped into 5 groups, each composed of 10 rats. The first group was regarded as the normal control group and the second one was considered as the MI group, which was caused by ligation of the left anterior descending artery. The other three groups received crocin 50 or 10 mg/kg/day or metoprolol 100 mg/kg/day for 1 week, following ligation of the left anterior descending artery. Evaluated outcomes were cardiac Cx43 expression, arrhythmia incidence, histological findings, and myocyte resting potential. Crocin-treated MI groups showed a significantly lower arrhythmia score than the non-treated MI group, 10 mg/kg/day (1.85 ± 0.55, p < 0.01) and 50 mg/kg/day (1.70 ± 0.33, p < 0.01). Groups that received crocin 10 mg/kg/day (66.30 ± 2.59, p < 0.01), crocin 50 mg/kg/day (68.10 ± 2.43, p < 0.01), and metoprolol 100 mg/kg/day (−63.54 ± 0.63 mV, p < 0.01) significantly prevented depolarization in comparison with the non-treated MI group. Expression of Cx43 mRNA in crocin 10 mg/kg/day (1.54 ± 0.24, p < 0.01), crocin 50 mg/kg/day (1.73 ± 0.09, p < 0.01), and metoprolol 100 mg/kg/day (1.75 ± 0.14, p < 0.01) treatment groups was significantly higher in comparison with the non-treated MI group. Crocin showed a preventive effect on the arrhythmogenic impact of MI in an experimental model of ischemic injury through an increase in expression of Cx43.

Insights into the protective capacity of human dental pulp stem cells and its secretome in cisplatin-induced nephrotoxicity: effects on oxidative stress and histological changes.

Acute renal injury (AKI) is a major limiting factor for cisplatin administration. Recent evidence suggests the potential contribution of mesenchymal stem cells (MSCs) to rehabilitation from several disorders via both direct and indirect routes. Thus, the present study aimed, for the first time, to explore and compare the reno-protective potential of human dental pulp-derived stem cells (hDPSCs) vs. hDPSC-conditioned medium (hDPSC-CM) in recovery of impaired kidney tissues in a rat animal model of cisplatin-induced AKI. AKI was induced via cisplatin injection (n=36). One day after, 24 rats were treated with either hDPSCs or hDPSC-CM (n=12). An extra set of rats (n=12) served as sham group. On days 2 or 7 (n=6), rats were humanly sacrificed for further analysis. Renal injury was explored via measuring serum creatinine and BUN. Renal level of oxidative stress was assessed by determining malondialdehyde, and enzymatic activities of superoxide dismutase and catalase. Renal histopathological changes were scored for comparison among different experimental groups. A single dose of cisplatin resulted in considerable renal dysfunction and oxidative stress. Treatment with hDPSCs or hDPSC-CM resulted in significantly restored renal function, reduced level of oxidative stress, and improved histopathological manifestations. Furthermore, as compared to hDPSC-CM, administration of hDPSCs led to superior results in AKI-induced animals. The current study described the first comparative evidence of reno-protective potential of hDPSCs and their CM against cisplatin-induced nephrotoxicity in an AKI rat model, proposing them as useful adjunctive therapy in AKI. Yet, future explorations are still needed.

Expression of Transgenes in Native Bladder Urothelium using Adenovirus-Mediated Transduction

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense and transmit information about its environment to the underlying tissues, promoting voiding function and behavior. Disruption of the urothelial barrier, or its sensory/transducer function, can lead to disease. Studying these complex events is hampered by lack of simple strategies to alter gene and protein expression in the urothelium. Methods are described here that allow investigators to generate large amounts of high-titer adenovirus, which can then be used to transduce rodent urothelium with high efficiency, and in a relatively straightforward manner. Both cDNAs and small interfering RNAs can be expressed using adenoviral transduction, and the impact of transgene expression on urothelial function can be assessed 12 h to several days later. These methods have broad applicability to studies of normal and abnormal urothelial biology using mouse or rat animal models.

Expression of Transgenes in Native Bladder Urothelium using Adenovirus-Mediated Transduction.

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense and transmit information about its environment to the underlying tissues, promoting voiding function and behavior. Disruption of the urothelial barrier, or its sensory/transducer function, can lead to disease. Studying these complex events is hampered by lack of simple strategies to alter gene and protein expression in the urothelium. Methods are described here that allow investigators to generate large amounts of high-titer adenovirus, which can then be used to transduce rodent urothelium with high efficiency, and in a relatively straightforward manner. Both cDNAs and small interfering RNAs can be expressed using adenoviral transduction, and the impact of transgene expression on urothelial function can be assessed 12 h to several days later. These methods have broad applicability to studies of normal and abnormal urothelial biology using mouse or rat animal models.

Exosome loaded hydroxyapatite (HA) scaffold promotes bone regeneration in calvarial defect: an in vivo study.

In this study, hydroxyapatite (HA) scaffolds were synthesized and characterized, following the osteogenic and angiogenic effects of HA scaffolds with or without endometrial mesenchymal stem stromal cells (hEnSCs) derived Exosomes were investigated in rat animal model with calvaria defect. The X-ray diffraction (XRD) analysis of HA powder formation was confirmed with Joint Corporation of Powder Diffraction Standards (JCPDS) files numbers of 34-0010 and 24-0033A and Ball mill, and sintering manufactured Nano-size particles. Obtained results containing FE-SEM images presented that the surface of scaffolds has a rough and porous structure, which makes them ideal and appropriate for tissue engineering. Additionally, the XRD showed that these scaffolds exhibited a crystallized structure without undergoing phase transformation; meanwhile, manufactured scaffolds consistently release exosomes; moreover, in vivo findings containing hematoxylin-eosin staining, immunohistochemistry, Masson's trichrome staining, and histomorphometric analysis confirmed that our implant has an osteogenic and angiogenic characteristic. So prepared scaffolds containing exosomes can be proposed as a promising substitute in tissue engineering.

Histologic Evaluation of the Effects of Folinic Acid Chitosan Hydrogel and Botulinum Toxin A on Wound Repair of Cleft Lip Surgery in Rats.

The aim of the present study was to compare the effects of folinic acid chitosan hydrogel and botulinum toxin A on the wound repair of cleft lip surgery in rat animal models. Cleft lip defects were simulated by triangular incisions in the upper lip of 40 Wistar rats. Then, the rats were randomly assigned to four groups: control (CTRL), chitosan hydrogel (CHIT), and folinic acid chitosan hydrogel (FOLCHIT), in which the wounds were covered by a gauze pad soaked in normal saline, chitosan hydrogel, and folinic acid chitosan hydrogel, respectively for 5 min immediately after closure; and botulinum toxin A (BOT) with the injection of 3 units of botulinum toxin A in the wound region. Fibroblast proliferation, collagen deposition, inflammatory cell infiltration, neovascularization, and epithelial proliferation and each parameter were rated on days 14 and 28. Statistical analysis was performed by Kolmogorov-Smirnov test, Shapiro-Wilk test, Kruskal-Wallis, and post-hoc tests (α = 0.05). The mean score for fibroblast proliferation was significantly higher in the FOLCHIT group compared with the BOT group at days 14 and 28 (p < 0.001, p = 0.012, respectively). At day 28, collagen deposition was significantly higher in the FOLCHIT group compared with the BOT group (p = 0.012). No significant difference was observed between the inflammatory infiltration of the study groups at the two time points (p = 0.096 and p = 1.000, respectively). At day 14, vascular proliferation of group FOLCHIT was significantly higher than groups CTRL and CHIT (p = 0.001 and p = 0.006, respectively). The epithelial proliferation in the FOLCHIT group was significantly higher than groups CHIT and CTRL at day 14 (p = 0.006 and p = 0.001, respectively) and day 28 (p = 0.012). In simulated lip cleft defects, topical application of folinic acid induces faster initial regeneration by higher inflammation and cellular proliferation, at the expense of a higher tendency for scar formation by slightly higher fibroblast proliferation and collagen deposition. While injection of botulinum toxin A provides less fibroblast proliferation and collagen deposition, and thus lower potential for scar formation compared with the folinic acid group. Therefore, in wounds of the esthetic zone, such as cleft lip defects, the application of botulinum toxin A shows promising results.

Interactive Effects of Copper-Doped Urological Implants with Tissue in the Urinary Tract for the Inhibition of Cell Adhesion and Encrustation in the Animal Model Rat.

The insertion of a ureteral stent provides acute care by restoring urine flow and alleviating urinary retention or dysfunction. The problems of encrustation, bacterial colonization and biofilm formation become increasingly important when ureteral stents are left in place for a longer period of time. One way to reduce encrustation and bacterial adherence is to modify the stent surface with a diamond-like carbon coating, in combination with copper doping. The biocompatibilities of the Elastollan® base material and the a-C:H/Cu-mulitilayer coating were tested in synthetic urine. The copper content in bladder tissue was determined by atomic absorption spectroscopy and in blood and in urine by inductively coupled plasma mass spectrometry. Encrustations on the materials were analyzed by scanning electron microscopy, energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy. A therapeutic window for copper ions of 0.5–1.0 mM was determined to kill bacteria without affecting human urothelial cells. In the rat animal model, it was found that copper release did not reach toxic concentrations in the affecting tissue of the urinary tract or in the blood. The encrustation behavior of the surfaces showed that the roughness of the amorphous carbon layer with the copper doping is probably the causal factor for the higher encrustation.

Preclinical Therapeutic Evaluation of Lenvatinib-Eluting Microspheres for Transcatheter Arterial Chemoembolization of Hepatocellular Carcinoma.

To evaluate the preclinical in vivo therapeutic response of Lenvatinib-eluting microspheres (LEN-EM) transcatheter arterial chemoembolization (LEN-TACE) in an hepatocellular carcinoma (HCC) rat model. Magnetic resonance imaging (MRI) visible LEN-EM was fabricated with poly(lactide-co-glycolide) and iron oxide nanoparticles by a double-emulsion method. The morphology, LEN loading/release kinetics, and MRI contrast effect of LEN-EM were evaluated. For in vivo study, N1S1 HCC rats were treated with LEN-TACE (LEN: 2.4mg/kg, n = 5) using LEN-EM, systemic LEN (LEN: 0.4mg/kg, oral gavage daily for 7days, n = 5), control (intra-arterial (IA) saline infusion, n = 5), and non-tumor control (n = 3). Tumor size changes were measured for 2weeks. Histology, comparative LEN plasma concentration, hematologic markers, liver profile, and serum chemistry among the groups were measured. LEN-EM with 33µm in average size was prepared in an optimized emulsion process. LEN loading efficiency was 58.7%. LEN was continuously released for 500h. LEN-TACE showed the delivered LEN-EM surrounding tumor tissue in MRI-T2* images. The LEN-TACE group demonstrated a statistically significant larger tumor volume reduction compared to the other groups at 2weeks post-procedure. Quantification data of Terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells confirmed increased cancer cell death in the LEN-TACE group compared to control groups. Additional histology, hematologic markers, and liver profiles showed minimal side effects of LEN-TACE. LEN-TACE using IA delivery of LEN-EM demonstrated an effective therapeutic efficacy in an HCC rat animal model.

Distal Vagina Smooth Muscle Contractility Mechanisms: Effects of in Vivo Sex Steroids Treatment in an Animal Experimental Model of Ovariectomy

ABSTRACT Introduction Female sexual response is a complex phenomenon that involves multiple psychophysiological factors. Sex steroids play a fundamental role in the mechanisms of regulation of sexual arousal, modulating the sexual response and preserving the integrity and functionality of the vaginal tissue. For this reason, imbalances in the sexual environment of steroids in women favor the onset of important pathophysiological conditions such as hypoactive sexual desire disorder (HSDD) and genitourinary syndrome of menopause (GSM). Previous research has focused primarily on the effects of estrogen supplementation on vaginal physiology, but only few studies evaluated the effects of androgen administration, particularly on the vaginal smooth muscle compartment. The calcium sensitizing pathway of RhoA/ROCK may play a role in regulating vaginal contractility but, at present, the regulatory mechanisms of RhoA/ROCK signaling in the female genitourinary tract have yet to be fully elucidated. In particular, no specific research has been conducted on the effects of testosterone (T) on the contractility mechanism of smooth muscle in the vagina. Objective The aim of this study was to evaluate the sex steroid regulation on the distal vagina contractility mechanism, mediated by the RhoA/ROCK pathway, using a validated animal model of female ovariectomized rats. Methods Ovariectomized Sprague-Dawley rats (OVX) were treated with 17β-estradiol (OVX+E), testosterone (OVX+T), or testosterone and letrozole (OVX+T+L) and compared with an intact animals’ group (CTRL). In vitro contractility studies were performed on noradrenaline (NA) pre-contracted vaginal strips to investigate the effect of OVX and in vivo treatments on vaginal smooth muscle activity. The mRNA from vagina tissues isolated from each experimental group was analyzed by semi-quantitative RT-PCR, and membrane translocation of RhoA was evaluated by Western blot analysis. Results Y-27632 induced a dose-dependent relaxation on NA pre-contracted vaginal strips that was significantly decreased by OVX; T, with or without L, further decreased OVX-induced hyporesponsiveness to Y-27632, whilst it was fully restored up to control level by 17β-estradiol (E) supplementation. Accordingly, gene expression analysis indicated that OVX decreased the expression of RhoA/ROCK pathway markers, which was normalized by E. However, at variance with contractility results, androgen receptor (AR) activation upregulated, rather than downregulating, RhoA/ROCK expression. In Western blot analysis, OVX induced activation and membrane translocation of the active form of RhoA as compared to controls. Interestingly, T reverted this effect by reducing RhoA translocation to membrane to a level even significantly below the control one. This effect was not exerted by E administration. Considering that AR activation increases NO signaling (as measured by Prkg1 mRNA expression), we tested the effect of abolishing NO formation through the NO synthase inhibitor L-NAME. Pre-incubation with L-NAME substantially increased Y-27632 responsiveness in the OVX +T group and partially in the CTRL one and, but not in the OVX group. Conclusions This study confirms that in vagina estrogens increase the expression of the calcium-sensitizing pathway RhoA/ROCK, while androgen administration functionally decreases RhoA/ROCK activity, by hampering RhoA translocation to the membrane, a mechanism which is most probably driven by NO. Disclosure No

LB1027 An adipose-derived stem cell-engineered patch represents a promising treatment for chronic wounds

Mesenchymal stem cell (MSC)-based therapies are emerging as promising approach to treat chronic wounds, although uncontrolled/sub-optimal delivery represents a major threat to their wide-spread clinical adoption. Here we report a comprehensive pre-clinical evaluation program on the mechanism of action, efficacy and safety of an easy-to-use patch that concentrates MSC extracted from the human fat (ASCs) in a clinical-grade sponge made of porcine crosslinked-gelatin. ASC were derived from the abdominal/skin fat of four ischemic patients. Microarray data revealed that ASC grown within the patch upregulate the transcription of genes important for wound healing, including VEGF, CXCL12 and FGF7. The patch acted both as a concentrator and a reservoir of ASC-derived factors and sponge-derived fragments having healing capacity. The ASC-patch promoted dermal fibroblast survival, epidermal epithelialization and neovascularization, as shown in a chick chorioallantoic membrane model. No tumor formation was observed in immunodeficient Nude mice subcutaneously transplanted with the ASC-patch. Finally, ASC-patches prepared from syngeneic rats promoted faster healing and re-vascularization of full-thickness skin defects in a pe-clinical rat animal model of ischemic wounds. Together, we provide here compelling pre-clinical evidence that patches concentrating ASCs within crosslinked gelatin may represent a convenient and effective tool for the management of chronic wounds.

Effectiveness of exosome mediated miR-126 and miR-146a delivery on cardiac tissue regeneration.

Despite advances in the treatment of acute myocardial infarction, due to the non-proliferative nature of adult cardiomyocytes, the injured myocardium is mainly replaced by fibrotic tissue, which ultimately causes heart failure. To prevent heart failure, particularly after myocardial infarction, exosome-based therapy has emerged as one of the most promising strategies to regenerate cardiac function. Exosomes can carry microRNAs in support of neovascularization, anti-inflammatory, and endogenous cardiac regeneration. This study demonstrated that animal rat models' combination treatment with microRNA-126 and microRNA-146a mimics in exosomes is desirable for cardiac regeneration after myocardial infarction. The exosomes isolated from stem cells and loaded with microRNAs were characterized their impacts in cell migration, tube formation, and vascular endothelial growth factor degree. In the following, the usefulness of loaded microRNAs in exosomes and their encapsulation within alginate derivative hydrogel was analyzed in myocardial infarction for an animal model. Exosomes isolated and loaded with microRNAs showed the synergetic impact on cell migration, tube formation, and promoted vascular endothelial growth factor folding. Moreover, microRNAs loaded exosomes and encapsulated them in alginate hydrogel could help in reducing infarct size and improving angiogenesis in myocardial infarction. The angiogenesis markers including CD31 and connexion 43 upregulated for myocardial infarction models treated with alginate-based hydrogels loaded with exosomes and microRNAs-exosomes. Histological analysis indicated that myocardial infarction model rats treated with alginate hydrogel loaded with microRNAs-exosomes possessed lower and higher degrees of fibrosis and collagen fiber, respectively. These findings have important therapeutic implications for a myocardial infarction model through angiogenesis and vascular integrity regulation.

A standard protocol for the analysis of postmortem muscle protein degradation: process optimization and considerations for the application in forensic PMI estimation

The analysis of postmortem protein degradation has become of large interest for the estimation of the postmortem interval (PMI). Although several techniques have been published in recent years, protein degradation-based techniques still largely did not exceed basic research stages. Reasons include impractical and complex sampling procedures, as well as highly variable protocols in the literature, making it difficult to compare results. Following a three-step procedure, this study aimed to establish an easily replicable standardized procedure for sampling and processing, and further investigated the reliability and limitations for routine application. Initially, sampling and processing were optimized using a rat animal model. In a second step, the possible influences of sample handling and storage on postmortem protein degradation dynamics were assessed on a specifically developed human extracorporeal degradation model. Finally, the practical application was simulated by the collection of tissue in three European forensic institutes and an international transfer to our forensic laboratory, where the samples were processed and analyzed according to the established protocol.