Hiromi Misawa

In Vitro Formation of Enteric Neural Network Structure in a Gut‐Like Organ Differentiated from Mouse Embryonic Stem Cells

Using an embryoid body (EB) culture system, we developed a functional organ‐like cluster—a “gut”—from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited spontaneous contractions but did not exhibit distinct peristalsis‐like movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (c‐kit [a transmembrane receptor that has tyrosine kinase activity]‐positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified; however, enteric neural ganglia were absent in the spontaneously differentiated ES gut. By adding brain‐derived neurotrophic factor (BDNF) only during EB formation, we for the first time succeeded in in vitro formation of enteric neural ganglia with connecting nerve fiber tracts (enteric nervous system [ENS]) in the ES gut. The ES gut with ENS exhibited strong peristalsis‐like movements. During EB culture in BDNF+ medium, we detected each immunoreactivity associated with the trk proto‐oncogenes (trkB; BDNF receptors) and neural crest marker, proto‐oncogene tyrosine‐protein kinase receptor ret precursor (c‐ret), p75, or sox9. These results indicated that the present ENS is differentiated from enteric neural crest‐derived cells. Moreover, focal stimulation of ES guts with ENS elicited propagated increases in intracellular Ca2+ concentration ([Ca2+]i) at single or multiple sites that were attenuated by atropine or abolished by tetrodotoxin. These results suggest in vitro formation of physiologically functioning enteric cholinergic excitatory neurons. We for the first time succeeded in the differentiation of functional neurons in ENS by exogenously adding BDNF in the ES gut, resulting in generation of distinct peristalsis‐like movements.

A 5-HT(4)-receptor activation-induced neural plasticity enhances in vivo reconstructs of enteric nerve circuit insult.

Background  It was recently reported that some 5‐HT4‐receptor agonists increased neuronal numbers and length of neurites in enteric neurons developing in vitro from immunoselected neural crest‐derived precursors. We aimed to explore a novel approach in vivo to reconstruct the enteric neural circuitry that mediates a fundamental distal gut reflex.

In vivo imaging of enteric neurogenesis in the deep tissue of mouse small intestine.

One of the challenges of using imaging techniques as a tool to study cellular physiology has been the inability to resolve structures that are not located near the surface of the preparation. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy (2PM), has overcome this limitation, providing deeper optical penetration (several hundred µm) in ex vivo and in vivo preparations. We have used this approach in the gut to achieve the first in vivo imaging of enteric neurons and nerve fibers in the mucosa, submucosa, submucosal and myenteric plexuses, and circular and longitudinal muscles of the small intestine in H-line: Thy1 promoter GFP mice. Moreover, we obtained clear three-dimensional imaging of enteric neurons that were newly generated after gut transection and reanastomosis. Neurogenesis was promoted by oral application of the 5-HT4-receptor agonist, mosapride citrate (MOS). The number of newly generated neurons observed in mice treated with MOS for one week was 421±89 per 864,900 µm2 (n = 5), which was significantly greater than that observed in preparations treated with MOS plus an antagonist (113±76 per 864,900 µm2) or in 4 week vehicle controls (100±34 per 864,900 µm2) (n = 4 both). Most neurons were located within 100 µm of the surface. These results confirm that activation of enteric neural 5-HT4-receptor by MOS promotes formation of new enteric neurons. We conclude that in vivo 2PM imaging made it possible to perform high-resolution deep imaging of the living mouse whole gut and reveal formation of new enteric neurons promoted by 5-HT4-receptor activation.

Energy expenditure by Ba2+contracture in rat ventricular slices derives from cross-bridge cycling

To clarify the energy-expenditure mechanism during Ba(2+) contracture of mechanically unloaded rat left ventricular (LV) slices, we measured myocardial O(2) consumption (VO(2)) of quiescent slices in Ca(2+)-free Tyrode solution and VO(2) during Ba(2+) contracture by substituting Ca(2+) with Ba(2+). We then investigated the effects of cyclopiazonic acid (CPA) and 2,3-butanedione monoxime (BDM) on the Ba(2+) contracture VO(2). The Ca(2+)-free VO(2) corresponds to that of basal metabolism (2.32 +/- 0.53 ml O(2). min(-1). 100 g LV(-1)). Ba(2+) increased the VO(2) in a dose-dependent manner (from 0.3 to 3.0 mmol/l) from 110 to 150% of basal metabolic VO(2). Blockade of the sarcoplasmic reticulum (SR) Ca(2+) pump by CPA (10 micromol/l) did not at all decrease the Ba(2+)-activated VO(2). BDM (5 mmol/l), which specifically inhibits cross-bridge cycling, reduced the Ba(2+)activated VO(2) almost to basal metabolic VO(2). These energetic results revealed that the Ba(2+)-activated VO(2) was used for the cross-bridge cycling but not for the Ca(2+) handling by the SR Ca(2+) pump.

Determinants of the epithelial-muscular axis on embryonic stem cell-derived gut-like structures.

Dome-like structures with epithelial-muscular layers resembling the gut have been derived from mouse embryonic stem (ES) cells. These domes have been reported to show spontaneous contractions and are called ES gut. In the present study, we examined the epithelial-muscular axis of these domes by detecting differentiation markers. A normal epithelial-muscular axis was exhibited in the domes with spontaneous motility, whereas the domes without spontaneous motility showed either an inverted or obscure axis. To investigate the factors affecting the epithelial-muscular axis, we examined the expression of hedgehog signaling factors in the domes. Expression of hedgehog family factors was detected in the epithelial components of the domes with motility, whereas this expression was inverted or obscure in the domes without motility. Out of the 25 domes, 10 of the 10 motility (+) domes showed a normal epithelial-muscular axis, whereas 14 of the 15 motility (–) domes lacked a normal epithelial-muscular axis. This implies that activin A upregulated the expression of sonic hedgehog and intestinal alkaline phosphatase in the embryoid bodies. These findings suggest that the motility of the ES gut depends on the domes’ epithelial-muscular axis.

O2 Delivery and the Venous PO2‐O2 Uptake Relationship in Pump‐Perfused Canine Muscle

Under the conditions of both an increased red cell affinity for O2 at a constant rate of O2 delivery (arterial O2 content × flow) and a decrease in the rate of O2 delivery induced by hypoxic hypoxia at constant blood flow, we have obtained a linear relationship between the partial pressure of O2 in the muscle venous effluent (Pv,O2) and O2 uptake (V̇O2). The relationship is described by the equation V̇O2= Da× Pv,O2+V̇O2,conv where Da is the apparent O2 diffusion capacity and V̇O2,conv is O2 delivery‐limited V̇O2, and Da× Pv,O2 represents the O2 diffusion‐limited V̇O2 (V̇O2,diff). From these observations, we propose the hypothesis that V̇O2 consists of two additive values, V̇O2,conv and V̇O2,diff. The mechanism underlying the reduction in V̇O2 that is induced by reducing O2 delivery to markedly below the V̇O2,conv value has only been investigated using a model based on the single compartment of diffusion‐limited V̇O2, and has not been investigated in terms of this additive V̇O2 model. The single compartment analysis appears to overestimate the role of O2 diffusion in limiting the reduction of V̇O2 that occurs in response to a decrease in O2 diffusion capacity, as reflected by the V̇O2/Pv,O2 ratio. To gain better insight into the mechanism involved, we altered the rate of O2 delivery by changing arterial PO2 from normoxia (with inhalation of air) to hypoxia (by inhalation of 10‐11% O2) and blood flow (with high and low flow rates (n = 7 for both groups), and very low and ischaemic flow rates (n = 4 for both groups)) in pump‐perfused dog gastrocnemius preparations during tetanic isometric contractions at 1 Hz. As rates of O2 delivery were reduced from 23.2 to 10.9 ml min−1 (100 g)−1, significant decreases in Pv,O2 and V̇O2 were observed (P < 0.05). From the data of Pv,O2 and V̇O2 values within this range of O2 delivery rates, we obtained the regression equation V̇O2= 0.22 × Pv,O2+ 8.14 (r = 0.58). From the equation, the intercept of the V̇O2‐axis was significantly different from zero (P < 0.05), in accordance with the observation that the V̇O2/Pv,O2 ratio (ml min−1 (100 g)−1 Torr−1) increased from 0.54 to 1.35 (P < 0.05). However, at extremely low rates of O2 delivery (5.6 and 7.3 ml min−1 (100 g)−1 the V̇O2/Pv,O2 ratio was 1.51 and 2.80 (P < 0.05), respectively. This indicates a break in the linear V̇O2‐Pv,O2 relationship as the rate of O2 delivery was reduced to below the V̇O2,conv value of the V̇O2‐axis intercept. These results suggest that the reduction in V̇O2 caused by extreme reductions in the rate of O2 delivery is not attributable to a reduction in O2 diffusion capacity, as expected from the V̇O2/Pv,O2 ratio, but to a reduction in the O2 delivery‐limited V̇O2 component, as evaluated by the V̇O2‐axis intercept of the linear V̇O2‐Pv,O2 relationship.

In vitro morphological bud formation in organ-like three-dimensional structure from mouse ES cells induced by FGF10 signaling

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. Using an embryoid body (EB) culture system, we developed a gut-like three-dimensional structure from mouse ES cells (the ES 3-D structure). Genetic studies implicate fibroblast growth factor 10 (FGF10)-FGF receptor 2b (FGFR2b) signaling as a critical regulator of lung bud morphogenesis in the embryonic foregut. The aim of the present study was to form a putative respiratory tract in the ES 3-D structure. By local application of FGF10 protein, we successfully demonstrated in vitro morphological formation of putative primitive respiratory tract-like processes, or buds, in the ES 3-D structure. Such organs that are differentiated from ES cells may provide new insights into tissue engineering and regenerative medicine.

MUSCLE VENOUS PO2 AND V.O2 ARE LINEARLY RELATED IN REPETITIVE TETANIC CONTRACTIONS OF CANINE MUSCLE DURING HYPOXIC HYPOXIA

1. It has previously been shown that perfusion with high O2‐affinity‐erythrocytes decreases venous PO2 (PVO2) and decreases O2 uptake (V.O2) in contracting muscle at the same O2 delivery (arterial O2 concentration × flow). A linear V.O2–PVO2 relationship has been obtained with a V.O2‐axis intercept, suggesting that, during this type of hypoxia, V.O2 is composed of a PVO2‐dependent and ‐independent V.O2. However, the V.O2– PVO2 relation during hypoxic hypoxia has not been examined.

Measurement of Mouse Plasma Erythropoietin by an Improved ELISA

Abstract: We have examined whether mouse plasma erythropoietin (EPO) can be measured by an improved enzyme-linked immunosorbent assay (ELISA) using milk proteins (Block Ace) both as a blocking reagent and as a diluent for standard recombinant human EPO (rHuEPO) and for plasma samples. Block Ace brought about high slope sensitivity of the standard curve, with a low background. The dose–response curves of normal or anaemic mouse plasma and of rHuEPO were linear and parallel to each other. The anaemic plasma had an additive effect with rHuEPO by increasing the absorbance at 405 nm. The coefficients of variation in the intra- and interassays ranged from 4.2% to 15.3%. The plasma EPO levels in 22 normal mice were 18.3 ± 10.3 mU/ml. An inverse relationship between the logarithm of plasma EPO concentrations and blood haemoglobin concentrations, red blood cell counts or packed cell volumes was found in normal mice and in mice with iron deficiency anaemia (IDA). These results show the validity for the use of the new improved ELISA method for measuring circulating murine EPO.

Effect of Blood Flow on PvO2-VO2 Relation in Contracting In Situ Skeletal Muscle

It has been reported that high 02 affinity of blood reduced 02 uptake (VO2) of maximally and submaximally stimulated muscle, beating heart and brain with a decrease in venous effluent PO2 (Pv02). These studies suggest that VO2 in organs of high metabolic activity is determined by the interaction of the convective 02 delivery and the subsequent diffusion of 02 from the erythrocytes to the mitochondria, rather than by 02 delivery alone (blood flow multiplied by arterial 02 content), and Pv02 reflects the diffusive 02 supply.