胚胎时期小鼠小肠的组织发生

目的　观察小鼠胚胎各个时期小肠组织的形态结构及杯状细胞在小肠内的分布规律,为小鼠小肠的组织发生提供形态学依据。 方法　采用HE染色和PAS染色,对小鼠胚胎第13.5天（E13.5d）至出生后第1天（P1d）胚胎的石蜡切片染色并行光学显微镜观察。 结果　(1)小鼠肠壁于E13.5 d已分化出现黏膜层、黏膜下层、肌层及浆膜。(2)肠绒毛于E15.5 d分化形成,杯状细胞于E16.5 d逐渐发育分化出现,肠腺于E18.5~P1 d发育分化形成,此时小肠基本结构形成。(3)杯状细胞主要分布于小肠绒毛上皮,其中以回肠末端最多,回肠、空肠、十二指肠顺次递减（P<0.05）。杯状细胞数量随胎龄逐渐增加而逐渐增多,于P1 d最多（P<0.05）。 结论　小肠上皮分化于E15.5 d至E17.5 d最为迅速,胚胎时期小肠基本结构形成,其吸收消化功能基本建立完成。     

Lymphatic development in mouse small intestine.

Lymphatic vessels in the small intestine serve as essential conduits for the absorption and transport of lipids from the intestine to the thoracic duct. Although the morphology and function of the intestinal lymphatic vasculature are well known, little is known about the embryonic development of these vessels. In this study, we examined development of lymphatic and blood vasculatures in the intestinal tube during mouse embryonic development by immunostaining with recently discovered molecular markers for lymphatic endothelial cells: LYVE-1, VEGFR3, Prox-1, and podoplanin. Immature lymphatics became detectable in mesentery, but not in intestinal tube, around E13.5-E14.5, while organized lymphatic vessel plexuses and capillaries were observed in intestinal tube and villi around E17.5. These lymphatic plexuses and capillaries in the intestinal tube appeared to be formed through an active branching process associated with activation of VEGFR3 and involvement of LYVE-1+ macrophages. Our data also reveal that the lymphatic vessels in the intestinal tube, unlike the blood vessels, have not originated from the mesoderm of intestine. All lymphatic vessels in the intestinal tube originated by extension of mesenteric lymphatic vessels through an active branching process. Although the formation of lymphatic vessels follows the formation of blood vessels in the intestine, a mature lymphatic vasculature is formed before birth. Together, our study reveals the temporal and spatial windows of intestinal lymphatic development during embryonic development in mouse.     

Selective migration of lymphocytes within the mouse small intestine

The factors which determine the migration of lymphoid cells to lamina propria or Peyer's patches of mouse small intestine have been investigated by autoradiographic tracing of intravenously injected spleen, thymus and lymph node cells. The numbers of labelled cells found in antigen-free grafts of foetal small intestine were compared with the numbers in normally sited gut. Thymus, normal spleen and B spleen lymphocytes, labelled with [³H]adenosine or [5-³H]uridine, were confined to Peyer's patches in normal and grafted gut. [³H]Thymidine-labelled lymphoblasts from the mesenteric nodes of young (19–22 days) mice and mice infected with Nippostrongylus brasiliensis were found in the lamina propria of both graft and normal small intestine, but [³H]thymidine-labelled lymphoblasts from oxazolone-primed lymph nodes did not migrate to the villi. The possible roles of intraluminal antigens, source of cells and changes in cell surface receptors during differentiation, in determining the selective migration of cells to the lamina propria and Peyer's patches, are discussed.     

In vitro potassium transport in the mouse small intestine

Ingested K+ is believed to be absorbed mainly in the small intestine by passive diffusion through the paracellular pathway. To further clarify K+ absorption in the small intestine, we determined the unidirectional flux values of Rb+ in vitro by atomic absorption spectroscopy in the mouse ileum mounted in Ussing chambers under short-circuit conditions. The mucosal-to-serosal Rb+ flux (J(ms)) was larger than the serosal-to-mucosal Rb+ flux (J(sm)), resulting in positive net Rb+ absorption (J(net)). The effect of changing the transmucosal potential (V(t)) showed that J(ms) was composed of both a V(t)-dependent diffusion component and a V(t)-independent non-diffusion component, while J(sm) was composed mainly of a V(t)-dependent component. A forskolin treatment eliminated J(net) mainly due to the increase in J(sm). When animals were fed a low-Na diet, J(net) was mainly eliminated as a result of the increase in J(sm). These findings suggest that K+ is absorbed not only by passive diffusion through the paracellular pathway, but also by an active transport mechanism operating through the cellular pathway. In addition, cAMP and aldosterone may be involved in regulating intestinal K+ transport.     

Effect of moderate exercise on IgA levels and lymphocyte count in mouse intestine

The aim of the present study was to determine the effect of moderate exercise on the production and secretion of IgA in mouse duodenum, on lymphocyte levels in the lamina propria, and on gene expression encoding for cytokines that regulate the synthesis of α-chain of IgA and the expression of pIgR in the lamina propria. Two groups of young Balb/c mice were fed ad libitum, one sedentary and the other with an exercise program (swimming) for 16 weeks. IgA levels in the duodenum were quantified by ELISA; the number of IgA containing cells as well as B cells, CD4(+) and CD8(+) T cells in the duodenal mucosa was determined by immunohistochemistry; gene expression was analyzed by real-time PCR, and the expression of proteins by Western blotting. Because of physical training, in the duodenum there was a decrease in the number of IgA producing cells, but an increase in the levels of IgA. Additionally, exercise increased the expression of the genes encoding for IL-4, IL-6, IL-10, TNF-α and TGF β, cytokines that regulate the synthesis of IgA and pIgR, the inflammatory response, and the immune response in the intestine. Thus, the increased IgA found in the duodenal lumen is probably due to the increased production of IgA in the LP and the increased transport of the pIgA-pIgR complex across epithelial cells. Possibly the increased S-IgA levels in the bile also contribute to the change in IgA levels.     

Circadian oscillation of innate immunity components in mouse small intestine

The digestive system is a major port of entry for pathogens. To detect and combat pathogens, the innate immunity in the gut utilizes pattern recognition receptors, such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) proteins, and broad-spectrum anti-bacterial polypeptides, such as defensins. We have previously shown that mouse enteric defensins (cryptdins) oscillate around the circadian cycle and peak at the end of the dark phase suggesting control by the biological clock. As the core mechanism of the biological clock has never been studied in the small intestine, our objective was to determine whether the biological clock is functional in mouse jejunum and examine whether mTlr and mNod2 mRNAs, similarly to cryptdins, oscillate throughout the circadian cycle. Mouse jejunum and Paneth-enriched crypt base cells were isolated around the circadian day and the levels of clock (mClock, mBmal1, mPer1, mPer2, mCry1) and innate immunity component (mTlr2, mTlr3, mTlr4, mTlr5, mTlr9, mNod2) genes were measured by real-time PCR. Analysis of mouse jejunum and Paneth-enriched crypt base cells revealed that all clock genes exhibited circadian oscillation. Similarly to cryptdins, mTlr2, mTlr3, mTlr4, mTlr5 displayed circadian rhythmicity in mouse jejunum. Although no circadian oscillation could be detected for mTlr9 and mNod2 in the whole jejunum, these genes oscillated in Paneth-enriched crypt base cells. In addition, mTlr3 exhibited the highest expression level. As the clock regulates intestinal motility and function, resetting of the clock in the small intestine may help not only to restore activity but also to gain better protection against pathogens.     

Macrophage-like cells in the muscularis externa of mouse small intestine

In muscularis externa of mouse small intestine, cells with ultrastructural features of macrophages were invariably observed in three layers: in the subserosal layer, between the circular and longitudinal muscle layers, and in association with the deep circular plexus. These macrophage-like cells (MLC) had a single indented nucleus, perinuclear Golgi complex, smooth and rough endoplasmic reticulum, many pits (coated and uncoated) in the plasma membrane, coated vesicles, light vesicles, and primary lysosomes, but rather few heterogeneous lysosomal vacuoles. MLC were partially enveloped by processes of interstitial cells of Cajal. FITC-dextran used in combined fluorescence stereo microscopy, fluorescence microscopy, and electron microscopy was employed as a tracer to study the endocytic qualities of the MLC. The mice were killed 5, 15, 30, and 60 min, 1 day, and 4 days after dextran administration. By fluorescence microscopy after 1 or 4 days MLC were observed as a constant cellular population with a strikingly regular distribution. By electron microscopy dextran-containing vacuoles were conspicuous after 1 h or more. MLC of the subserosal layer and between the circular and longitudinal muscle layers could be distinguished with respect to general appearance, pattern formation, and apparent dextran contents.     

Characterization of myenteric sensory neurons in the mouse small intestine

We recorded from myenteric AH/Dogiel type II cells, demonstrated mechanosensitive responses, and characterized their basic properties. Recordings were obtained using the mouse longitudinal muscle myenteric plexus preparation with patch-clamp and sharp intracellular electrodes. The neurons had an action potential hump and a slow afterhyperpolarization (AHP) current. The slow AHP was carried by intermediate conductance Ca2+ -dependent K+ -channel currents sensitive to charybdotoxin and clotrimazole. All possessed a hyperpolarization-activated current that was blocked by extracellular cesium. They also expressed a TTX-resistant Na+ current with an onset near the resting potential. Pressing on the ganglion containing the patched neuron evoked depolarizing potentials in 17/18 cells. The potentials persisted after synaptic transmission was blocked. Volleys of presynaptic electrical stimuli evoked slow excitatory postsynaptic potentials (EPSPs) in 9/11 sensory neurons, but 0/29 cells received fast EPSP input. The slow EPSP was generated by removal of a voltage-insensitive K+ current. Patch-clamp recording with a KMeSO4-containing, but not a conventional KCl-rich, intracellular solution reproduced the single-spike slow AHPs and low input resistances seen with sharp intracellular recording. Cell-attached recording of intermediate conductance potassium channels supported the conclusion that the single-spike slow AHP is an intrinsic property of intestinal AH/sensory neurons. Unitary current recordings also suggested that the slow AHP current probably does not contribute significantly to the high resting background conductance seen in these cells. The characterization of mouse myenteric sensory neurons opens the way for the study of their roles in normal and pathological physiology.     

Renewal of Paneth cells in the small intestine of the mouse

The origin and fate of Paneth cells were examined in duodenum, jejunum and ileum of adult female mice, using radioautography after administration of ³H-thymidine either in a single injection or in drinking water for four days or as a continuous infusion for up to ten days. The tissues were fixed by perfusion with 4% paraformaldehyde. One-micron thick, Eponembedded single or serial sections were stained with Regaud's hematoxylin, radioautographed, and counterstained with safranin O. Mitosis of Paneth cells is never observed, nor are these cells ever labeled one hour after ³H-thymidine. Hence, Paneth cells do not divide. However, a few days after single injection or prolonged administration of ³H-thymidine, labeled Paneth cells appear. The first labeled cells have tiny granules but, as the cells age, larger and larger granules are observed. Adjacent to Paneth cells are slender undifferentiated cells which show frequent mitoses and early labeling. The evidence points to some of these cells transforming into Paneth cells. Since occasionally Paneth cells degenerate, the newly-formed ones would provide replacement for those which die, thus insuring the steady state of the Paneth cell population. The renewal of this population is characterized by a turnover time of about three weeks.     

Effects of temperature on pacemaker potentials in the mouse small intestine

The effects of temperature on the generation of pacemaker potentials recorded from myenteric interstitial cells of Cajal (ICC-MY) distributed in the mouse small intestine were investigated using intracellular recording techniques. In response to increasing temperatures in the range of 26–40°C, the frequency and maximum rate of rise (dV/dt _max) of pacemaker potentials were increased while their duration was decreased. The resting membrane potential and amplitude of the pacemaker potentials were not affected by change in temperature. Elevation of temperature decreased the amplitude, duration, and rise time of unitary potentials generated spontaneously during intervals between the pacemaker potentials. Metabolic inhibition (KCN and iodoacetic acid) decreased the frequency of pacemaker potentials with no alteration to the amplitude and dV/dt _max. Cyclopiazonic acid (3 μM), an inhibitor of the internal Ca²⁺ pump, abolished pacemaker potentials in low-temperature conditions (<29°C) but not at high-temperature conditions (>38°C). These results suggest that the primary and plateau components of pacemaker potentials have different temperature sensitivities: the primary component is highly temperature-sensitive and is activated at higher temperatures, while the plateau component is formed by activation of temperature-insensitive mechanisms. The results also suggest that the mitochondria-induced intracellular Ca²⁺ handling system seems to be involved in the initiation of the generation of pacemaker potentials but not in their configuration.     

Phenotypic heterogeneity of intraepithelial T lymphocytes from mouse small intestine.

We have used two-colour flow cytometry to examine the heterogeneity of intraepithelial lymphocytes (IEL) from mouse small intestine. We have confirmed the predominance of CD3+ Thy 1- CD8+ IEL and show that a substantial but variable proportion of CD8+ IEL does not express the alpha beta T-cell receptor (TcR) for antigen. Simultaneous analysis of the co-expression of the alpha and beta chains of the CD8 heterodimer and of the alpha beta TcR revealed three populations of CD8+IEL. The first of these expressed both CD8 alpha and beta chains and had normal expression of V beta families and so represented conventional CD8+ alpha beta TcR+ T cells. The second population comprised alpha beta TcR- T cells (presumed gamma delta TcR+) which expressed only the alpha chain of the CD8 molecule. Finally, we identified a second, unique population of alpha beta TcR+ CD8+ IEL which were also CD8 beta-. Gamma delta + IEL predominated in mice aged less than 8 weeks, but there was a rapid increase in both populations of alpha beta TcR+ CD8+ IEL in older mice. CD8+ IEL were similar to peripheral CD8+ T cells in having high expression of the CD45RB molecule, but CD4+ IEL had generally lower expression of CD45RB than their peripheral counterparts, despite having normal expression of TcR. These findings emphasize the heterogeneity of IEL and underline the need to study phenotypically defined populations.     

Proliferative kinetics of large and small intraepithelial lymphocytes in the small intestine of the mouse

The proliferative kinetics of the intraepithelial lymphocytes (IL) of the mouse intestine have been evaluated. By inducing mitotic arrest it was found that large IL — constituting about 50% of the IL — showed a mitotic rate of 2.3. Autoradiographic results obtained after two different schedules of ³H-thymidine injections showed that 30% of the large IL were in DNA synthesis, and that the large IL were renewed at a rate comparable to that of blast cells from Peyer's patches, mesenteric lymph nodes and thoracic duct lymph. The small IL were renewed very rapidly compared to small lymphocytes of peripheral lymphoid tissues, although small lymphocytes with lifespans of several weeks were also present in the epithelial sheet. By the use of intestinal perfusion, in vivo, it was estimated that the loss of lymphocytes from intestinal villi into the lumen of the gut was negligible, and it is concluded that the most probable kinetic model for the majority of IL is: B and T lymphoblasts invade the epithelium and undergo mitosis. B lymphoblasts give rise predominantly to plasma cells, and T lymphoblasts give rise to small lymphocytes — probably long-lived — which reenter the circulation.     

Spontaneous rhythmicity in cultured cell clusters isolated from mouse small intestine

To investigate spontaneous rhythmicity in smooth muscle tissue, we have developed a cell cluster preparation. Cell clusters were enzymatically isolated from the muscle layer of mouse small intestine and cultured for several days. They included smooth muscle, neurones, and c-Kit-immunopositive interstitial cells. c-Kit-immunopositive cells in myenteric plexus, showing a networklike structure, are putative pacemaker cells. The cultured cell clusters routinely show spontaneous contraction and preserve characteristic features in this tissue: (1) high temperature dependency of contractile frequency; (2) spontaneous electrical activities measured with patch clamp techniques are insensitive to tetradotoxin (TTX) and dihydropyridine Ca(2+) antagonists. This preparation could therefore be used as a good model system to investigate the underlying mechanisms of intestinal motility and pacemaker function. The relationship between the frequency of electrical activity and cluster size suggests that the minimum unit of small intestine tissue to yield normal pacemaker activity is approximately 100 microm in diameter, or less. The applications of 100-120 microM Cd(2+) and Ni(2+) significantly suppressed the spontaneous activity. Ca(2+) influx pathways other than L-type and "classical" T-type voltage-sensitive Ca(2+) channels seem very likely to play an important role, such as nonselective cation channels and capacitative Ca(2+) entry. Furthermore, applications of heptanol reduced the amplitude and the frequency of the oscillating inward currents and eventually terminated them, suggesting that electrical cell-to-cell coupling may also make some contribution to the generation of spontaneous activity.     

Effects of Dai-kenchu-to on spontaneous activity in the mouse small intestine.

The effects of Dai-kenchu-to (DKT), a Chinese medicine, on spontaneous activity of mouse small intestine were investigated. Experiments were carried out with tension recording and intracellular recording. DKT contracted mouse longitudinal smooth muscles in a dose dependent manner (0.1-10 mg/ml). Low concentration of DKT (0.1 mg/ml) did not contract the longitudinal muscles of mouse small intestine. DKT (0.1 mg/ml) inhibited contraction elicited by transmural nerve stimulation (TNS). DKT (1 mg/ml) evoked relaxation before contraction. The initial relaxation was abolished by Nomega-nitro-L-arginine (L-NNA). DKT (10 mg/ml)-induced contraction had two components: a transient rapid contraction and a following slow contraction. Atropine inhibited DKT (1 mg/ml)-induced contraction to about 50% of control. In the presence of atropine, tetrodotoxin (TTX) inhibited the contraction elicited by DKT (1 mg/ml) to about 80%. DKT depolarized the membrane and decreased the amplitude of pacemaker potentials recorded from in situ myenteric interstitial cells of Cajal (ICC-MY) with no alteration to the frequency, duration and maximum rates of rise in the presence of nifedipine and TTX. The same results were obtained in slow waves recorded from circular smooth muscle cells. These results indicate that DKT evoked both contraction and relaxation by releasing acetylcholine, nitric oxide and other excitatory neurotransmitters in mouse small intestine. DKT had no effects on pacemaker mechanisms and electrical coupling between ICC-MY and smooth muscle cells in mouse small intestine. The results also suggest that DKT may contract smooth muscles by depolarizing the membrane directly.     

Effect of dexamethasone on the fetal mouse small intestine in organ culture

The formation of intestinal villi (organogenesis phase) may be studied in organ culture with a completely synthetic medium in 15-day fetal mouse duodenal explants. However, in these explants absorptive cells remained poorly differentiated with all the hormones studied except with epidermal growth factor. In order to elucidate the role of hormones and other factors on the maturation of absorptive cells (maturation phase) in the fetal rodent in organ culture, we have taken the explants after the organogenesis phase. We have studied different culture conditions and have found that 17-day mouse duodenal explants can be cultured during 48 hours with Leibovitz L-15 medium in a 95% O2-5% CO2 atmosphere provided that the explants are relatively large (5 X 2 mm). With this method, dexamethasone (Dx) has been shown to have a direct effect on the maturation of the fetal duodenal mucosa. The addition of Dx (300 ng/ml) to the completely synthetic medium 1) improves the morphology of the explants, 2) induces a significant increase in maltase activity in the tissues, and 3) reduces significantly the labeling index of the duodenal explants after 48 hours of culture. Direct action of Dx on the duodenal mucosa is shown for the first time in organ culture using a completely synthetic medium. This method will permit us to study the effects of other intrinsic and extrinsic factors on the regulation of enzymatic maturation in fetal small intestine.