CNS macrophages differentially rely on an intronic Csf1r enhancer for their development.

The central nervous system hosts parenchymal macrophages, known as microglia, and non-parenchymal macrophages, collectively termed border-associated macrophages (BAMs). Microglia, but not BAMs, were reported to be absent in mice lacking a conserved Csf1r enhancer: the fms-intronic regulatory element (FIRE). However, it is unknown whether FIRE deficiency also impacts BAM arrival and/or maintenance. Here, we show that macrophages in the ventricular system of the brain, including Kolmer's epiplexus macrophages, are absent in Csf1rΔFIRE/ΔFIRE mice. Stromal choroid plexus BAMs are also considerably reduced. During normal development, we demonstrate that intracerebroventricular macrophages arrive from embryonic day 10.5, and can traverse ventricular walls in embryonic slice cultures. In Csf1rΔFIRE/ΔFIRE embryos, the arrival of both primitive microglia and intracerebroventricular macrophages was eliminated, whereas the arrival of cephalic mesenchyme and stromal choroid plexus BAMs was only partially restricted. Our results provide new insights into the development and regulation of different CNS macrophage populations.

Kaempferol Treatment after Traumatic Brain Injury during Early Development Mitigates Brain Parenchymal Microstructure and Neural Functional Connectivity Deterioration at Adolescence.

Targeting mitochondrial ion homeostasis using Kaempferol, a mitochondrial Ca2+ uniporter channel activator, improves energy metabolism and behavior soon after a traumatic brain injury (TBI) in developing rats. Because of broad TBI pathophysiology and brain mitochondrial heterogeneity, Kaempferol-mediated early-stage behavioral and brain metabolic benefits may accrue from diverse sources within the brain. We hypothesized that Kaempferol influences TBI outcome by differentially impacting the neural, vascular, and synaptic/axonal compartments. After TBI at early development (P31), functional magnetic resonance imaging and diffusion tensor imaging (DTI) were applied to determine imaging outcomes at adolescence (2 months post-injury). Vehicle and Kaempferol treatments were made at 1, 24, and 48 h post-TBI, and their effects were assessed at adolescence. A significant increase in neural connectivity was observed after Kaempferol treatment as assessed by the spatial extent and strength of the somatosensory cortical and hippocampal resting-state functional connectivity (RSFC) networks. However, no significant RSFC changes were observed in the thalamus. DTI measures of fractional anisotropy (FA) and apparent diffusion coefficient, representing synaptic/axonal and microstructural integrity, showed significant improvements after Kaempferol treatment, with highest changes in the frontal and parietal cortices and hippocampus. Kaempferol treatment also increased corpus callosal FA, indicating measurable improvement in the interhemispheric structural connectivity. TBI prognosis was significantly altered at adolescence by early Kaempferol treatment, with improved neural connectivity, neurovascular coupling, and parenchymal microstructure in select brain regions. However, Kaempferol failed to improve vasomotive function across the whole brain, as measured by cerebrovascular reactivity. The differential effects of Kaempferol treatment on various brain functional compartments support diverse cellular-level mitochondrial functional outcomes in vivo.

Adult and iPS-derived non-parenchymal cells regulate liver organoid development through differential modulation of Wnt and TGF-ß.

BACKGROUND: Liver organoid technology holds great promises to be used in large-scale population-based drug screening and in future regenerative medicine strategies. Recently, some studies reported robust protocols for generating isogenic liver organoids using liver parenchymal and non-parenchymal cells derived from induced pluripotent stem cells (iPS) or using isogenic adult primary non-parenchymal cells. However, the use of whole iPS-derived cells could represent great challenges for a translational perspective. METHODS: Here, we evaluated the influence of isogenic versus heterogenic non-parenchymal cells, using iPS-derived or adult primary cell lines, in the liver organoid development. We tested four groups comprised of all different combinations of non-parenchymal cells for the liver functionality in vitro. Gene expression and protein secretion of important hepatic function markers were evaluated. Additionally, liver development-associated signaling pathways were tested. Finally, organoid label-free proteomic analysis and non-parenchymal cell secretome were performed in all groups at day 12. RESULTS: We show that liver organoids generated using primary mesenchymal stromal cells and iPS-derived endothelial cells expressed and produced significantly more albumin and showed increased expression of CYP1A1, CYP1A2, and TDO2 while presented reduced TGF-ß and Wnt signaling activity. Proteomics analysis revealed that major shifts in protein expression induced by this specific combination of non-parenchymal cells are related to integrin profile and TGF-ß/Wnt signaling activity. CONCLUSION: Aiming the translation of this technology bench-to-bedside, this work highlights the role of important developmental pathways that are modulated by non-parenchymal cells enhancing the liver organoid maturation.

The effects of increasing amounts of milk replacer powder added to whole milk on mammary gland measurements using ultrasound in dairy heifers.

The aim of this study was to determine the effect of increasing total solids (TS) in the liquid diet during the preweaning phase on mammary gland development in dairy heifers. The increase in TS was obtained with the addition of milk replacer powder to whole milk. Crossbreed Holstein-Gyr heifers (n = 60) were distributed in 4 treatments with different TS concentrations: 13.5% (n = 15), 16.1% (n = 15), 18.2% (n = 15), and 20.4% (n = 15). The liquid diets were provided from 5 to 55 d of age. From 56 to 59 d of age, the total amount of liquid feed was reduced by half. Heifers were weaned at 60 d and monitored until 90 d of age. Ultrasound mammary gland evaluations were performed weekly between 5 to 11 wk of age, using a B-mode ultrasound equipped with microconvex transducer at a frequency of 6 MHz. In those same weeks, the manual palpation of mammary parenchyma (PAR) was performed. Increased TS concentration of liquid diet during the preweaning period increased body weight of heifers, but did not alter PAR growth and the deposition of adipose tissue in the mammary gland evaluated by ultrasonography. The oval-shaped structure of PAR was altered after 2 mo of age. In the evaluated period, PAR growth was isometric with respect to the body growth rate. Palpation scoring of PAR had a strong correlation with the ultrasound evaluation of the PAR.

Effects of preweaning nutrient intake in the developing mammary parenchymal tissue.

Historically, mammary gland growth has been considered isometric the first 2 mo of life and then allometric until peripuberty. However, recent work indicated that the mammary gland might be responsive to nutrient intake preweaning. The objectives of this study were to describe the effects of nutrient intake preweaning on mammary gland development and to investigate cell specific proliferation during this phase of development. Twelve dairy heifer calves were fed either a fixed amount of milk replacer (MR; control, n = 6) or an amount of MR adjusted for BW (enhanced, n = 6). Control calves received a constant amount of a 28% crude protein, 15% fat milk MR per day that was equivalent to 2.8 Mcal of metabolizable energy intake per day; enhanced calves received 0.3 Mcal of metabolizable energy intake per kilogram of metabolic body weight (from 4.2 to 8.4 Mcal of metabolizable energy intake per day). All calves had constant access to water and a 22% crude protein commercial calf starter. Calves were killed at 54 ± 2 d. Control calves consumed 32.6 ± 2.4 kg of MR and 6.7 ± 0.5 kg of calf starter per calf, whereas the enhanced calves consumed 69.5 ± 2.4 kg of MR and 1.9 ± 0.5 kg of calf starter per calf over the 54-d period. Further, to evaluate putative stem cell proliferation, BrdU (5-bromo-2'-deoxyuridine; 5 mg/kg) was injected intramuscularly once per day between 12 to 15 d and again once per day between 24 to 27 d of life. Initial and final body weight for the control and enhanced treatments were 39.2, 61.0, 39.7, and 83.2 kg, respectively. At euthanasia, weights of liver, kidneys, pancreas, whole skinned mammary gland, and mammary parenchyma were measured. The growth rate of each organ was calculated using the concept of allometry as the difference in the change in organ weight as a percentage of body weight. The mammary glands of calves fed the enhanced diet were significantly heavier at euthanasia; when mammary parenchymal weight was analyzed, enhanced calves had 5.9 times greater mammary parenchymal mass, indicating the mammary gland was responsive to nutrient intake before weaning. Allometric growth of the mammary gland was initiated preweaning in the calves fed the enhanced treatment. Further characterization of mammary cells that retained BrdU label revealed no significant differences among the tissue slices analyzed between treatments; however, as calves fed the enhanced diet had more mammary parenchymal mass, if the number of label-retaining cells per counted slide were similar between treatments then the enhanced calves had a larger total population of putative mammary stem cells present in the mammary gland.

Revisiting the human seminiferous epithelium cycle.

STUDY QUESTION: Can all types of testicular germ cells be accurately identified by microscopy techniques and unambiguously distributed in stages of the human seminiferous epithelium cycle (SEC)? SUMMARY ANSWER: By using a high-resolution light microscopy (HRLM) method, which enables an improved visualization of germ cell morphological features, we identified all testicular germ cells in the seminiferous epithelium and precisely grouped them in six well-delimitated SEC stages, thus providing a reliable reference source for staging in man. WHAT IS ALREADY KNOWN: Morphological characterization of germ cells in human has been done decades ago with the use of conventional histological methods (formaldehyde-based fixative -Zenker-formal- and paraffin embedding). These early studies proposed a classification of the SEC in six stages. However, the use of stages as baseline for morphofunctional evaluations of testicular parenchyma has been difficult because of incomplete morphological identification of germ cells and their random distribution in the human SEC. STUDY DESIGN, SIZE, DURATION: Testicular tissue from adult and elderly donors with normal spermatogenesis according to Levin's, Johnsen's and Bergmann's scores were used to evaluate germ cell morphology and validate their distribution and frequency in stages throughout human spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular tissue from patients diagnosed with congenital bilateral agenesis of vas deferens (n = 3 adults) or prostate cancer (n = 3 elderly) were fixed in glutaraldehyde and embedded in araldite epoxy resin. Morphological analyses were performed by both light and transmission electron microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: HRLM method enabled a reliable morphological identification of all germ cells (spermatogonia, spermatocytes and spermatids) based on high-resolution aspects of euchromatin, heterochromatin and nucleolus. Moreover, acrosomal development of spermatids was clearly revealed. Altogether, our data redefined the limits of each stage leading to a more reliable determination of the SEC in man. LIMITATIONS, REASONS FOR CAUTION: Occasionally, germ cells can be absent in some tubular sections. In this situation, it has to be taken into account the germ cell association proposed in the present study to classify the stages. WIDER IMPLICATIONS OF THE FINDINGS: Our findings bring a new focus on the morphology and development of germ cells during the SEC in human. Application of HRLM may be a valuable tool for research studies and clinical andrology helping to understand some testicular diseases and infertility conditions which remain unsolved. STUDY FUNDING/COMPETING INTEREST: Experiments were partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors declare that there are no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Effects of nutrient intake level on mammary parenchyma growth and gene expression in crossbred (Holstein × Gyr) prepubertal heifers.

This study investigated the effects of increased nutrient intake levels on prepubertal mammary parenchyma development in crossbreed (Holstein × Gyr) dairy heifers. Eighteen heifers age 3 to 4 mo were fed 1 of 3 nutrient intake levels (n=6 per treatment) designed to sustain an average daily gain of 0.0kg/d (maintenance, MA), 0.5kg/d (low gain, LG), or 1.0kg/d (high gain, HG). Serum blood samples collected on d 42 and 84 after a 12-h fast were analyzed for triglycerides, leptin, insulin, and insulin-like growth factor 1 (IGF-1). Liver and mammary parenchyma were biopsied on d 42 and harvested on d 84 for gene expression analysis. Parenchyma samples were also used for biochemical and histological analysis. Mammary parenchyma weight was lower in HG than in MA or LG heifers, but mammary extraparenchymal fat was greater in HG heifers than in other groups. Heifers fed the HG diet had a greater fraction of ether extract in their parenchyma than the others and a smaller fraction of crude protein in their parenchyma than MA heifers. Moreover, the HG and LG heifers had greater body fat mass than MA heifers. Nutrient intake level had no effect on the number of intraparenchymal adipocytes. Heifers fed the HG diet had greater serum IGF-1 than the others, and serum insulin was lower in the MA than the HG or LG heifers. Liver GHR, IGF1, and IGFBP3 mRNA expression was higher, but IGFBP2 mRNA was lower in HG heifers than in others. The parenchyma mRNA expression of lipogenic markers, such as CD36, ACCA, FASN, and ADIPOR1, was upregulated by nutrient intake level. Significant nutrient intake × time interactions for lipogenic genes during the experimental period indicated variable gene expression depending on the time point of prepubertal mammary gland development. Overall, our data suggest that enhancing nutrient intake increased body fat accumulation and lipogenesis in the mammary gland to the detriment of parenchyma growth. Moreover, increased lipogenesis in the parenchyma of HG heifers may indicate that fat accumulation occurred because of adipocyte hypertrophy and not differences in adipogenesis. The implications of these results for milk yield needs to be elucidated.

Parenchymal Congestion Is Important for Rapid Regeneration of the Future Liver Remnant Following the ALPPS Procedure.

AIM: To evaluate whether the congested area that develops in associating liver partition with portal vein ligation for staged hepatectomy (ALPPS) contributes to rapid future liver remnant (FLR) hypertrophy. PATIENTS AND METHODS: Eight patients undergoing liver partition and right portal vein (RPV) ligation within the FLR in the first operation of ALPPS were compared with eight patients undergoing RPV embolization in the FLR as the first operation of classical two-stage (CTS) hepatectomy. RESULTS: Extrapolated kinetic growth of the FLR in ALPPS was 32.7±18.7 ml/day, 7.8-times that in CTS (4.2±2.0 ml/day, p=0.001). Extrapolated kinetic reduction in volume of the ventral aspect of the right paramedian sector, which became congested after the first procedure in ALPPS, was 19.8±11.6 ml/day, 11-times that in CTS (1.8±1.3 ml/day, p=0.001). CONCLUSION: Production of a congested area within the deportalized liver may contribute importantly to rapid FLR hypertrophy during ALPPS.

Hypoxia-driven Hif2a coordinates mouse liver regeneration by coupling parenchymal growth to vascular expansion.

Interaction between sinusoidal endothelial cells and hepatocytes is a prerequisite for liver function. Upon tissue loss, both liver cell populations need to be regenerated. Repopulation occurs in a coordinated pattern, first through the regeneration of parenchyme (hepatocytes), which then produces vascular endothelial growth factor (VEGF) to enable the subsequent angiogenic phase. The signals that instruct hepatocytes to induce timely VEGF remain unidentified. Given that liver is highly vascularized, we reasoned that fluctuations in oxygenation after tissue loss may contribute to the coordination between hepatocyte and sinusoidal endothelial cell proliferation. To prevent drops in oxygen after hepatectomy, mice were pretreated with inositol trispyrophosphate (ITPP), an allosteric effector of hemoglobin causing increased O2 release from heme under hypoxic conditions. ITPP treatment delayed liver weight gain after hepatectomy. Comparison with controls revealed the presence of a hypoxic period around the peak of hepatocyte mitosis. Inhibition of hypoxia led to deficient hepatocyte mitosis, suppressed the regenerative Vegf wave, and abrogated the subsequent reconstruction of the sinusoidal network. These ITPP effects were ongoing with the reduction in hepatocellular hypoxia inducible factor 2a (Hif2a). In contrast, Hif1a was unaffected by ITPP. Hif2a knockdown phenocopied all effects of ITPP, including the mitotic deficiencies, Vegf suppression, and angiogenic failure. CONCLUSIONS: Oxygen is a key regulator of liver regeneration. Hypoxia-inherent to the expansion of parenchyme-activates Hif2a to couple hepatocyte mitosis with the angiogenic phase. Hif2a acts as a safeguard to initiate sinusoidal reconstruction only upon successful hepatocyte mitosis, thereby enforcing a timely order onto cell type-specific regeneration patterns. These findings portray the hypoxia-driven Hif2a-Vegf axis as a prime node in coordinating sinusoidal endothelial cell-hepatocyte crosstalk during liver regeneration. (Hepatology 2016;64:2198-2209).

Feeding a higher plane of nutrition and providing exogenous estrogen increases mammary gland development in Holstein heifer calves.

Feeding heifers a higher plane of nutrition postweaning but before puberty can negatively affect mammary gland development and future milk yield. However, enhanced nutrition preweaning may promote development and future production. Our objectives were to determine the effects of enhanced feeding preweaning and exogenous estrogen immediately postweaning on mammary gland development and the composition of the mammary parenchyma (PAR) and mammary fat pad (MFP). Thirty-six Holstein heifer calves (<1 wk old) were reared on 1 of 2 dietary treatments for 8 wk: (1) a restricted milk replacer fed at 0.45 kg/d (R; 20% crude protein, 20% fat), or (2) an enhanced milk replacer fed at 1.13 kg/d (EH; 28% crude protein, 25% fat). Upon weaning, calves from each diet (n=6) were given either a placebo or estrogen implant for 2 wk, creating 4 treatments: R, R + estrogen (R-E2), EH, and EH + estrogen (EH-E2). Calves were housed individually with ad libitum access to water. Starter feeding began at wk 5 and was balanced between treatments. Udders were evaluated by palpation and physical measurements weekly. Subsets of calves were killed at weaning (n=6 per diet) and at the conclusion of the trial (n=6 per treatment). Udders were removed, dissected, and weighed. At wk 8, EH calves had longer front and rear teats. Providing estrogen to EH calves increased the length of rear teats during wk 9 and 10. Enhanced-fed calves had 5.2-fold more trimmed mammary gland mass than R calves. Providing estrogen to EH calves further increased mammary gland weight. Masses of PAR and MFP were markedly greater for EH calves than for R calves (e.g., 7.3-fold greater PAR tissue). Estrogen increased the mass of both PAR and MFP in EH calves. Feeding a higher plane of nutrition increased total protein, DNA, and fat in the MFP and total protein and DNA in the PAR. Dual-energy x-ray absorptiometry estimates of mammary fat mass were highly correlated with biochemical analyses of fat content. From histological study, we observed that the degree of expansion of epithelium into the adjacent stromal tissue and the complexity of ductal development were minimal in R, increased in EH, and increased by estrogen in both dietary treatments. Results provide compelling evidence that preweaning nutrition and estrogen administration immediately postweaning markedly increase mammary gland development in dairy calves. Cellular and molecular mechanisms responsible for these differences are currently under study.