Modeling the development of the post-natal mouse thymus in the absence of bone marrow progenitors.

Many mathematical models have been published with the purpose of explaining aspects of T-cell development in the thymus. In this manuscript we adapted a four-compartment model of the thymus and used a range of mathematical approaches with the aim of explaining the dynamics of the four main thymocyte populations in the mouse thymus, from the emergence of the first fetal thymocyte until the death of the animal. At various pre-natal and post-natal stages we investigated experimentally the number and composition of thymocytes populations, their apoptosis and proliferation, along with data from literature, to create and validate the model. In our model the proliferation processes are characterized by decreasing proliferation rates, which allows us to model the natural involution of the thymus. The best results were obtained when different sets of parameters were used for the fetal and post-natal periods, suggesting that birth may induce a discontinuity in the modeled processes. Our model is able to model the development of both pre-natal and post-natal thymocyte populations. Also, our findings showed that the post-natal thymus is able to develop in the absence of the daily input of bone marrow progenitors, providing more evidence to support the autonomous development of the post-natal thymus.

Mesenchymal Stromal Cells Differentiating to Adipocytes Accumulate Autophagic Vesicles Instead of Functional Lipid Droplets.

Adult bone marrow mesenchymal stromal cells (BMSCs) can easily be differentiated into a variety of cells. In vivo transplantation of BMSCs-differentiated cells has had limited success, suggesting that these cells may not be fully compatible with the cells they are intended to replace in vivo. We investigated the structural and functional features of BMSCs-derived adipocytes as compared with adipocytes from adipose tissue, and the structure and functionality of lipid vesicles formed during BMSCs differentiation to adipocytes. Gas chromatography-mass spectrometry showed fatty acid composition of BMSCs-derived adipocytes and adipocytes from the adipose tissue to be very different, as is the lipid rafts composition, caveolin-1 expression, caveolae distribution in their membranes, and the pattern of expression of fatty acid elongases. Confocal microscopy confirmed the absence from BMSCs-derived adipocytes of markers of lipid droplets. BMSCs-derived adipocytes cannot convert deuterated glucose into deuterated species of fatty acids and cannot uptake the deuterated fatty acid-bovine serum albumin complexes from the culture medium, suggesting that intra-cellular accumulation of lipids does not occur by lipogenesis. We noted that BMSCs differentiation to adipocytes is accompanied by an increase in autophagy. Autophagic vesicles accumulate in the cytoplasm of BMSCs-derived adipocytes and their size and distribution resembles that of Nile Red-stained lipid vesicles. Stimulation of autophagy in BMSCs triggers the intra-cellular accumulation of lipids, while inhibition of autophagy prevents this accumulation. In conclusion, differentiation of BMSCs-derived adipocytes leads to intra-cellular accumulation of autophagic vesicles rather than functional lipid droplets, suggesting that these cells are not authentic adipocytes. J. Cell. Physiol. 231: 863-875, 2016. © 2015 Wiley Periodicals, Inc.

Accumulation of tissue macrophages and depletion of resident macrophages in the diabetic thymus in response to hyperglycemia-induced thymocyte apoptosis.

AIMS: We investigated the dynamics and morphology of thymus macrophages in response to thymus involution caused by hyperglycemia. Thymus is an organ affected early and dramatically after the onset of diabetes, losing most of the thymocyte populations but diabetes's impact on the components of the thymus stroma is largely unknown. METHODS: Rats were injected with streptozotocin and thymus weight, body weight, and glycemia were measured at various time points. The dynamics and morphology of macrophages in the diabetic thymus were investigated by histology, immunohistochemistry, qPCR, electron microscopy and flow cytometry. RESULTS: In hyperglycemic animals the involuting thymus is gradually infiltrated by tissue macrophages (ED1-positive) and depleted of resident macrophages (ED2-positive). While ED1 positive macrophages are scattered in both cortex and medulla the ED2 positive ones are limited to the cortex and cortico-medullary junction. CD4+CD11b+macrophages also accumulate. The TUNEL reaction that detects the degradation of the DNA from apoptotic thymocytes in the macrophages is enhanced. The thymic macrophages enlarge and accumulate lipid vacuoles and apoptotic bodies. qPCR measurements of the expression of macrophage markers showed a persistent increase in the diabetic thymus after the injection of streptozotocin. CONCLUSIONS: Thymus involutes rapidly and persistently after the onset of hyperglycemia because of the elevated apoptosis in the thymocytes. Tissue macrophages accumulate in the thymus and the resident macrophages decrease. This results in an overall increase in macrophage activity in the diabetic thymus in response to the elevated apoptosis of thymocytes produced by hyperglycemia.

Arachidonic acid accumulates in the stromal macrophages during thymus involution in diabetes.

Diabetes is a debilitating disease with chronic evolution that affects many tissues and organs over its course. Thymus is an organ that is affected early after the onset of diabetes, gradually involuting until it loses most of its thymocyte populations. We show evidence of accumulating free fatty acids with generation of eicosanoids in the diabetic thymus and we present a possible mechanism for the involution of the organ during the disease. Young rats were injected with streptozotocin and their thymuses examined for cell death by flow cytometry and TUNEL reaction. Accumulation of lipids in the diabetic thymus was investigated by histology and electron microscopy. The identity and quantitation of accumulating lipids was done with gas chromatography-mass spectrometry and liquid chromatography. The expression and dynamics of the enzymes were monitored via immunohistochemistry. Diabetes causes thymus involution by elevating the thymocyte apoptosis. Exposure of thymocytes to elevated concentration of glucose causes apoptosis. After the onset of diabetes, there is a gradual accumulation of free fatty acids in the stromal macrophages including arachidonic acid, the substrate for eicosanoids. The eicosanoids do not cause thymocyte apoptosis but administration of a cyclooxygenase inhibitor reduces the staining for ED1, a macrophage marker whose intensity correlates with phagocytic activity. Diabetes causes thymus involution that is accompanied by accumulation of free fatty acids in the thymic macrophages. Excess glucose is able to induce thymocyte apoptosis but eicosanoids are involved in the chemoattraction of macrophage to remove the dead thymocytes.

A pressure-controlled rat ventilator with electronically preset respirations.

Major experimental surgery on laboratory animals requires adequate anesthesia and ventilation to keep the animal alive throughout the procedure. A ventilator is a machine that helps the anesthesized animal breathe through an endotracheal tube by pumping a volume of gas (oxygen, air, or other gaseous mixtures), comparable with the normal tidal volume, into the animal's lungs. There are two main categories of ventilators for small laboratory rodents: volume-controlled and pressure-controlled ones. The volume-controlled ventilator injects a preset volume into the animal's lungs, no matter the airways' resistance (with the peak inspiratory pressure allowed to vary), while the pressure ventilator controls the inspiratory pressure and allows the inspiratory volume to vary. Here we show a rat pressure ventilator with a simple expiratory valve that allows gas delivery through electronic expiration control and offers easy pressure monitoring and frequency change during ventilation.

Retinoic acid synthesis controlled by Raldh2 is required early for limb bud initiation and then later as a proximodistal signal during apical ectodermal ridge formation.

We present evidence for the existence of two phases of retinoic acid (RA) signaling required for vertebrate limb development. Limb RA synthesis is under the control of retinaldehyde dehydrogenase-2 (Raldh2) expressed in the lateral plate mesoderm, which generates a proximodistal RA signal during limb outgrowth. We report that Raldh2(-/-) embryos lack trunk mesodermal RA activity and fail to initiate forelimb development. This is associated with deficient expression of important limb determinants Tbx5, Meis2, and dHand needed to establish forelimb bud initiation, proximal identity, and the zone of polarizing activity (ZPA), respectively. Limb expression of these genes can be rescued by maternal RA treatment limited to embryonic day 8 (E8) during limb field establishment, but the mutant forelimbs obtained at E10 display a significant growth defect associated with a smaller apical ectodermal ridge (AER), referred to here as an apical ectodermal mound (AEM). In these RA-deficient forelimbs, a ZPA expressing Shh forms, but it is located distally adjacent to the Fgf8 expression domain in the AEM rather than posteriorly as is normal. AER formation in Raldh2(-/-) forelimbs is rescued by continuous RA treatment through E10, which restores RA to distal ectoderm fated to become the AER. Our findings indicate the existence of an early phase of RA signaling acting upstream of Tbx5, Meis2, and dHand, followed by a late phase of RA signaling needed to expand AER structure fully along the distal ectoderm. During ZPA formation, RA acts early to activate expression of dHand, but it is not required later for Shh activation.

Patterning of forelimb bud myogenic precursor cells requires retinoic acid signaling initiated by Raldh2.

Limb skeletal muscle is derived from cells of the dermomyotome that detach and migrate into the limb buds to form separate dorsal and ventral myogenic precursor domains. Myogenic precursor cell migration is dependent on limb bud mesenchymal expression of hepatocyte growth factor/scatter factor (Hgf), which encodes a secreted ligand that signals to dermomyotome through the membrane receptor tyrosine kinase Met. Here, we find that correct patterning of Hgf expression in forelimb buds is dependent on retinoic acid (RA) synthesized by retinaldehyde dehydrogenase 2 (Raldh2) expressed proximally. Raldh2(-/-) forelimb buds lack RA and display an anteroproximal shift in expression of Hgf such that its normally separate dorsal and ventral expression domains are joined into a single anterior-proximal domain. Met and MyoD are expressed in this abnormal domain, indicating that myogenic cell migration and differentiation are occurring in the absence of RA, but in an abnormal location. An RA-reporter transgene revealed that RA signaling in the forelimb bud normally exists in a gradient across the proximodistal axis, but uniformly across the anteroposterior axis, with all proximal limb bud cells exhibiting activity. Expression of Bmp4, an inhibitor of Hgf expression, is increased and shifted anteroproximally in Raldh2(-/-) limb buds, thus encroaching into the normal expression domain of Hgf. Our studies suggest that RA signaling provides proximodistal information for limb buds that counterbalances Bmp signaling, which in turn helps mediate proximodistal and anteroposterior patterning of Hgf expression to correctly direct migration of Met-expressing myogenic precursor cells.