Esophageal regeneration following surgical implantation of a tissue engineered esophageal implant in a pediatric model

Diseases of the esophagus, damage of the esophagus due to injury or congenital defects during fetal esophageal development, i.e., esophageal atresia (EA), typically require surgical intervention to restore esophageal continuity. The development of tissue engineered tubular structures would improve the treatment options for these conditions by providing an alternative that is organ sparing and can be manufactured to fit the exact dimensions of the defect. An autologous tissue engineered Cellspan Esophageal ImplantTM (CEI) was surgically implanted into piglets that underwent surgical resection of the esophagus. Multiple survival time points, post-implantation, were analyzed histologically to understand the tissue architecture and time course of the regeneration process. In addition, we investigated CT imaging as an “in-life” monitoring protocol to assess tissue regeneration. We also utilized a clinically relevant animal management paradigm that was essential for long term survival. Following implantation, CT imaging revealed early tissue deposition and the formation of a contiguous tissue conduit. Endoscopic evaluation at multiple time points revealed complete epithelialization of the lumenal surface by day 90. Histologic evaluation at several necropsy time points, post-implantation, determined the time course of tissue regeneration and demonstrated that the tissue continues to remodel over the course of a 1-year survival time period, resulting in the development of esophageal structural features, including the mucosal epithelium, muscularis mucosae, lamina propria, as well as smooth muscle proliferation/migration initiating the formation of a laminated adventitia. Long term survival (1 year) demonstrated restoration of oral nutrition, normal animal growth and the overall safety of this treatment regimen.

Thermal skin burn in a late gestation period in experimental animals: state of the system “mother-fetus”

Objective. To study the effect of thermal skin burns in experimental animals (rats) on the state of the system “mother-fetus” in a late gestation period.Materials and methods. An experimental study was carried out on 18 female outbred white rats weighing 300–350 g (per 9 rats in the control and experimental groups), which were exposed to third-degree thermal burns with an area of 12 cм2 in a late gestation period. The oxygen transport function and the main blood biochemical markers were studied. The effect of thermal injury on the course of pregnancy was studied.Results. The thermal skin burn in the rats in the late gestation period leads to the development of hypoxia, a decrease in the affinity of hemoglobin for oxygen and metabolic acidosis in the mother’s body. Changes in the blood biochemical markers indicate the presence of endogenous intoxication. In the experimental animal group, a decrease in fetal weight was observed, an increase in the post-implantation fetal death rate was noted.Conclusion. Impaired oxygen homeostasis and endogenous intoxication in skin burns in rats in late gestation periods lead to fetal malnutrition and a qualitative increase in the post-implantation fetal death rate.

Change of Embryotoxic Effects of Metal Citrates Depending on the Duration of Their Introduction

Given the increase in cadmium in the environment (air, soil, water) through modern industrial processes, the absorption of significant amounts from cigarette smoke is relevant to studying the effect of cadmium compounds on embryogenesis. The purpose of the study: experimental study and comparison of embryolethality and embryotoxicity of metal citrates depending on the duration of their intragastric administration (during 13 and 20 days of gestation). Materials and methods. The study was performed on 120 white adult female Wistar rats that weigh 170-200 g. They were divided into 6 groups due to the intragastric administration of solutions of the studied metals – rats treated with citrates: cadmium at a dose of 1.0 mg/kg – 1st group (nfemale = 20, nemb = 166); cadmium at a dose of 1.0 mg/kg and cerium (1.3 mg/kg) – 2nd group (nfemale = 20, nemb = 185); cadmium in a dose of 1.0 mg/kg and germanium (0.1 mg/kg) – 3rd group (nfemale = 20, nemb = 184); cadmium at a dose of 1.0 mg/kg and zinc (1.5 mg/kg) – 4th group (nfemale = 20, nemb = 179); cadmium at a dose of 1.0 mg/kg and nanocomposite (iodine + sulfur + selenium) at a dose of 2.0 mg/kg – 5th group (nfemale = 20, nemb = 180), 6th group – control (nfemale = 20, nemb = 212) – proportional volume of sterile saline in the same way. Females were divided into 2 subgroups of 10 animals each, depending on the duration of administration of test substances. The embryotoxic and embryolethal effects of the test substances were evaluated according to generally accepted criteria, which were calculated according to well-known formulas. Results and discussion. Cadmium compounds harm the embryogenesis of rats in the experimental groups and increase the rates of embryolethality. The most pronounced differences in these parameters concerning the control were found in animals with isolated exposure to cadmium citrate: the rates of total embryonic mortality increased by 4.0 times in both study periods of embryogenesis and 20% from the 13th to the 20th day; increased pre-implantation mortality by 6.0 times with an increase in post-implantation mortality by 3 times on the 13th day and by 15.0 times and 2.8 times on the 20th day of embryogenesis, respectively. Depending on the duration of introduction, the indicators of pre-implantation mortality increased by 25.0% with an increase in post-implantation mortality by 22.2% in the same period. The experimental group of exposure to cadmium citrate at a dose of 1.0 mg/kg recorded the lowest indicators of the number of live fetuses and intrauterine survival with the highest resorption rate studied of embryonic development. At the same time, in the groups of combined exposure to cadmium citrate with metal citrates, a decrease in the indicators of total embryonic mortality was 50.0% - 30.0%, pre-implantation mortality – 50.0% - 25.0%, post-implantation mortality – 60.0% - 44.4 % and increase in the number of fetuses per female – 12.7% - 25.3%. Conclusion. Analysis of the results shows a pronounced embryotoxic effect of cadmium citrate at a dose of 1.0 mg/kg on the processes of embryogenesis, which is a significant increase in overall embryonic mortality, preimplantation, and postimplantation mortality compared with the control group in all studied terms. In the groups of combined action of cadmium citrate with metal citrates, the data obtained indicate a decrease in the accumulation of cadmium under the influence of the studied citrates, which allows them to be considered as potential bioantagonists of cadmium citrate

Glycolytic flux-signaling controls mouse embryo mesoderm development

How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic Pfkfb3 (cytoPfkfb3). Overexpression of cytoPfkfb3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPfkfb3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.

Measurement of Lead Localization Accuracy Based on Magnetic Resonance Imaging

Post-implantation localization of deep brain stimulation (DBS) lead based on a magnetic resonance (MR) image is widely used. Existing localization methods use artifact center method or template registration method, which may lead to a considerable deviation of > 2 mm, and result in severe side effects or even surgical failure. Accurate measurement of lead position can instantly inform surgeons of the imprecise implantation. This study aimed to identify the influencing factors in DBS lead post-implantation localization approach, analyze their influence, and describe a localization approach that uses the individual template method to reduce the deviation. We verified that reconstructing direction should be parallel or perpendicular to lead direction, instead of the magnetic field. Besides, we used simplified relationship between magnetic field angle and deviation error to correct the localization results. The mean localization error can be reduced after correction and favors the feasibility of direct localization of DBS lead using MR images. We also discussed influence of in vivo noise on localization frequency and the possibility of using only MR images to localize the contacts.

Utero-Placental Immune Milieu during Normal and Aglepristone-Induced Parturition in the Dog

Maternal immunotolerance is required for the maintenance of pregnancy, in sharp contrast with the uterine pro-inflammatory activity observed during parturition in several species. Correspondingly, in the dog, increased immune signaling at term has been suggested, but a deeper understanding of the uterine immune milieu is still missing. Thus, the availability of 30 immune-related factors was assessed in utero-placental samples collected during post-implantation (days 18–25 of pregnancy) and mid-gestation (days 35–40) stages, and at the time of prepartum luteolysis. Gene expression and/or protein localization studies were employed. Samples collected from antigestagen (aglepristone)-treated dogs were further analyzed. Progression of pregnancy was associated with the downregulation of IL1β and upregulation of IL10 (p < 0.05) at mid-gestation. When compared with mid-gestation, a higher availability of several factors was observed at term (e.g., CD206, CD4, TLR4). However, in contrast with natural parturition, MHCII, CD25, CCR7, TNFα, IDO1 and AIF1 were upregulated after aglepristone treatment (p < 0.05), but not TNFR1 or CCL13 (p > 0.05). Altogether, these results show an increased immune activity during canine parturition, involving, i.a., M2 macrophages, Treg and Th cells, with strong support for progesterone-mediated immunomodulation. Furthermore, differences between term and induced parturition/abortion could relate to differences in placental maturation towards parturition and/or functional traits of antigestagens.

Disruption of Folate Metabolism Causes Poor Alignment and Spacing of Mouse Conceptuses for Multiple Generations

Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with the hypomorphic Mtrrgt mutation. MTRR is necessary for methyl group utilisation from folate metabolism, and the Mtrrgt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrrgt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes. Notably, we report misalignment of some Mtrrgt conceptuses within their implantation sites from E6.5. The degree of misorientation occurs across a continuum, with the most severe form visible upon gross dissection. Additionally, some Mtrrgt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Skewed growth likely influences embryo development since developmental delay and heart malformations (but not defects in neural tube closure or trophoblast differentiation) associate with severe misalignment of Mtrrgt/gt conceptuses. Typically, the uterus is thought to guide conceptus orientation. To investigate a uterine effect of the Mtrrgt allele, we manipulate the maternal Mtrr genotype. Misaligned conceptuses were observed in litters of Mtrr+/+, Mtrr+/gt, and Mtrrgt/gt mothers. While progesterone and/or BMP2 signalling might be disrupted, normal decidual morphology, patterning, and blood perfusion are evident at E6.5 regardless of conceptus orientation. These observations argue against a post-implantation uterine defect as a cause of conceptus misalignment. Since litters of Mtrr+/+ mothers display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrrgt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe conceptus skewing associates with the Mtrr genotype of either maternal grandparent. Moreover, the presence of conceptus skewing after embryo transfer into a control uterus indicates that misalignment is independent of the peri- and/or post-implantation uterus and instead is likely attributed to an embryonic mechanism that is epigenetically inherited. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. This study casts light on the complex role of folate metabolism during development beyond a direct maternal effect.

DISORDER OF SPERMATOGENESIS UNDER CONDITIONS OF EXPERIMENTAL CHRONIC KIDNEY DISEASE

Purpose - to estimate the disorder of spermatogenesis under conditions of experimentalchronic kidney disease (EChD).Material and methods. The study was carried out in two series of experiments on malemice with EChD, the model of which was created by immunizing animals with a kidneyhomogenate. The first series of experiments was devoted to the study of: sperm count(sperm concentration (mln / ml)) and the number of abnormal sperm forms; the ratio ofcells of different generations of spermatogenic epithelium (%) in the testes; pathways ofcell death of cells of the testes and epididymis (spermatocytes (primary) and spermatozoa).The fertile qualities of males were assessed in the second series of experiments, afterreplanting them to intact females. Pre- and post-implantation embryonic mortality andthe number of living fetuses per female mouse have been investigated. The research resultswere compared with the performance of animals in the control groups for each series.Results. No significant changes in the number of spermatozoa were found under EChDconditions (p> 0.05). An increase in the number of abnormal spermatozoa (22%) andthose with primary abnormalities (p <0.05) was found. Among the generations of testescells, a decrease in the number of spermatids and living spermatocytes (primary) (15%)was established, with an increase in the number of cells with apoptosis and necrosisamong them (p <0.05). The number of living cells of the epididymis (spermatozoa) alsodecreased (17.8%), with the growth of cells with apoptosis and necrosis among them (p<0.05). There was an increase in the pre- and post-implantation mortality of embryos (p<0.05); decrease in the number of living fetuses (p <0.05).Conclusions. Under conditions of four-time treatment with renal homogenate (EChD)there is a disorder of spermatogenesis in male mice. Experimental model of kidneydamage, proposed by us, can be useful for studying other aspects and consequences ofkidney pathology, and both for establishment of the features and detection of possiblepathogenetic links in the development of spermatogenesis disorder under conditions ofchronic kidney disease and search of the effective ways to correct it in future.

Repression of germline genes by PRC1.6 and SETDB1 in the early embryo precedes DNA methylation-mediated silencing

Silencing of a subset of germline genes is dependent upon DNA methylation (DNAme) post-implantation. However, these genes are generally hypomethylated in the blastocyst, implicating alternative repressive pathways before implantation. Indeed, in embryonic stem cells (ESCs), an overlapping set of genes, including germline “genome-defence” (GGD) genes, are upregulated following deletion of the H3K9 methyltransferase SETDB1 or subunits of the non-canonical PRC1 complex PRC1.6. Here, we show that in pre-implantation embryos and naïve ESCs (nESCs), hypomethylated promoters of germline genes bound by the PRC1.6 DNA-binding subunits MGA/MAX/E2F6 are enriched for RING1B-dependent H2AK119ub1 and H3K9me3. Accordingly, repression of these genes in nESCs shows a greater dependence on PRC1.6 than DNAme. In contrast, GGD genes are hypermethylated in epiblast-like cells (EpiLCs) and their silencing is dependent upon SETDB1, PRC1.6/RING1B and DNAme, with H3K9me3 and DNAme establishment dependent upon MGA binding. Thus, GGD genes are initially repressed by PRC1.6, with DNAme subsequently engaged in post-implantation embryos.