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The fecal microbiota and short-chain fatty acids (SCFAs) play important roles in the human body. This study examined how hyperbaric conditions affect the fecal microbiota and fecal SCFAs. Fecal samples were obtained from 12 divers at three points during deep-diving training (before the diving training, at 2.1 MPa, and after decompression). At 2.1 MPa, the changes in the frequency of Clostridium cluster IV and fecal iso-valerate levels were positively correlated, and the changes in the frequencies of Bacteroides and Clostridium subcluster XIVa were inversely correlated. After decompression, positive correlations were detected between the changes in the frequency of Bifidobacterium and fecal n-valerate levels and between the changes in the fecal levels of iso-butyrate and iso-valerate. On the other hand, inverse correlations were detected between the changes in the frequency of Clostridium cluster IX and fecal iso-butyrate levels, between the changes in the frequency of Clostridium cluster IX and fecal iso-valerate levels, and between the changes in the frequencies of Bacteroides and Clostridium cluster IV plus subcluster XIVa. During the study period, the changes in fecal iso-butyrate and iso-valerate levels were positively correlated, and inverse correlations were seen between the changes in the frequency of Clostridium cluster IV and fecal propionate levels and between the changes in the frequencies of Prevotella and Clostridium subcluster XIVa. These findings suggest that hyperbaric conditions affect the fecal microbiota and fecal SCFA levels and that intestinal conditions reversibly deteriorate under hyperbaric conditions.

We aimed to determine whether the composition of the fecal microbiota changes under hyperbaric conditions. In this study, we collected fecal samples from 6 healthy divers at three points during deep diving training (before, 2.1 MPa, end). The frequency of Clostridium cluster XVIII tended to be increased after compression. The frequencies of Clostridium cluster IV and subcluster XIVa were inversely correlated with that of Bacteroides. The compositional changes in the fecal microbiota exhibited interindividual variability. These findings suggest that hyperbaric conditions affect the fecal microbiota.

Introduction Acute acoustic trauma, which is a kind of sensorineural hearing loss, is caused by acoustic overstimulation. Hyperbaric oxygen therapy (HBOT) is reported to be effective against acute acoustic trauma. Objective We aimed to evaluate the efficacy of HBOT against acoustic hearing loss based on our 20 years of experience with such cases. Methods Patients who were treated with HBOT for acute acoustic trauma between April 1997 and August 2017 were evaluated in this study. Thirty-five patients with a mean age of 25.7 ± 9.2 (range: 16–48) years were included. Thirty-nine out of 70 ears (35 patients) were damaged. We investigated the initial level of hearing loss; the extent to which hearing recovered; subjective symptoms, such as tinnitus and aural fullness; and the treatment administered. Results The planned HBOT was completed in 37 of 39 ears. Twenty-six of the 37 ears (70.2%) displayed improved hearing, and 31 of the 37 ears (83.9%) exhibited symptom improvement. Twenty-three (76.7%) and 26 (86.7%) of the 30 ears treated with steroids demonstrated improvements in hearing and subjective symptoms, respectively. Conclusion A combination of HBOT and steroids should be considered as a treatment for acute acoustic trauma in cases involving symptoms such as tinnitus and aural fullness.

Divers, pneumatic construction workers, aviators, and astronauts sometimes suffer decompression illness (DCI). DCI includes decompression sickness (DCS) and arterial gas embolism (AGE) and is treated with recompression therapy. Etiology of DCS is highly related with micro bubbles formed in the body fluid. AGE is caused by pulmonary over inflation-induced rupture of the alveoli. Recompression therapy is a kind of hyperbaric oxygen therapy, for which U.S. Navy treatment tables are widely used. The purposes of recompression therapy are (1) to reduce volume of the bubbles in the body, (2) to increase absorption of the bubbles in the body fluid and exhalation of inert gases from the lung, and (3) to increase oxygen supply via plasma to the cells in peripheral tissues. Recompression therapy has other beneficial effects, such as anti-inflammatory, wound healing, and anti-infectious effects. During the evacuation of the DCI patient to an appropriate facility, to continue administration of 100% oxygen and not to decrease environmental pressure are both critical for stabilizing the patients’ condition. It is important for all physicians who have a chance to treat DCI patients to be versed in mechanisms of recompression therapy and handling the treatment tables.

OBJECTIVE The present study aimed to combine the physiological significance of irregularly shaped initial lymphatics and mechanisms of mechanical stimulation-induced lymph formation. METHODS To confirm stretch-induced expansion of initial lymphatics, a finite element model that simulated morphological changes on a computer and fluorescent image and immunohistochemical analyses in mouse skin were adopted. Next, to quantitatively analyze the stretch-induced expansion, a simulation study was performed using a viscoelastic model of the tissue including initial lymphatics. RESULTS On the finite element model, when the tissue was stretched, irregularly shaped lymphatics were confirmed to increase luminal volume compared with round-shaped lymphatics. Stretch-induced expansion of the real initial lymphatics was demonstrated by fluorescent images and histological studies. Thereafter, with the application of a viscoelastic model of the tissue, the relationship between the lymph formation rate (Q) and massage frequency (f) could be obtained using the following equation: Q=2Af(1-e-1/2τf) , where A and τ are constants. Excellent agreement was found between the previous data and the results of the present equation. CONCLUSIONS We conclude that irregularly shaped initial lymphatics may lead to efficient lymph formation induced by mechanical stimulation of the tissue.

The effects of short-term simulated microgravity on the lymph dynamics of rat lymph nodes were investigated using a combination of Bollman's cage and head-down tilt (HDT). Efferent lymphatics of the iliac and mesenteric lymph nodes were cannulated for the collection of lymph. There was no significant difference in lymph flow rate from the iliac lymph nodes between non-HDT (control) and HDT rats. Lymph flow rate from the mesenteric lymph nodes in HDT rats was slightly higher than that obtained with the control. The cell count obtained from the iliac lymph nodes in HDT rats was significantly larger than those of the controls, while no significant difference in the number of cells from the mesenteric lymph nodes was observed between the control and HDT groups. The cells from the iliac lymph nodes in the control and HDT rats were mostly lymphocytes. The distribution of subsets of lymphocytes (CD3+, CD4+, CD8a+, and CD45R+) from the iliac lymph nodes in HDT rats was not significantly different from the subsets of lymphocytes in the control. Immunization did not affect the distribution of lymphocyte subsets from the iliac lymph nodes in the control and HDT groups. There was no significant difference in the concentrations of lymph albumin in iliac afferent or efferent lymphatics between the control and HDT groups. These findings suggest that HDT posture in Bollman's cage induces transient output of lymphocytes from the iliac lymph nodes of rats in vivo without changing the flow rate, lymphocyte subsets, or concentration of albumin.

We successfully isolated human lymphatic endothelial cells from afferent lymph vessels (HALEC) of sentinel lymph nodes in patients with breast cancer by using trypsin digestion. The cells were cultured in EGM-2 medium with 10% FBS under the condition of 5% O2, 5% CO2, and 90% N2 at 37 degrees C. The cultured cells exhibited a monolayer with cobblestone appearance and a marked phagocytosis of Dil-Ac-LDL. Immunohistochemical lymphatic vessel markers were also found, such as podoplanin, LYVE-1, VEGF receptor 3, and Prox-1. Quantitative RT-PCR analysis also showed that podoplanin, VEGF R3, and Prox-1 mRNA were expressed more selectively in the cultured cells. The cells had marked immunoreactivity to antisera of ecNOS, iNOS, COX1, and weak reactivity of COX2. Constitutively expressed cell-type specific genes of the cultured cells were also analyzed by oligonucleotide microarray methods. Compared with human umbilical vein endothelial cells (HUVEC), the cells selectively expressed 88 known genes such as angiopoietin-like 4, oxygen radicals-related enzymes, and adhesion molecules and the related proteoglycans. The findings suggest that the cultured cells seem to be human lymphatic endothelial cells. In conclusion, the isolated, cannulated and enzymatic digested method we adopted for culture of human lymphatic endothelial cells may be easy and useful for investigating cellular, molecular biological, and genomic properties of the cells.

This study was performed to investigate the heterogeneity of physiological and pharmacological properties in segments of the facial veins with special reference to selective brain cooling. Canine facial veins were isolated and the isometric tension of each segment was measured using the organ bath technique. Vessels in the segments of the facial veins that run opposite to the buccal cavity automatically produced myogenic tone and tended to show spontaneous contractions, but vessels in other segments did not. When no contractile agent was used for precontraction, noradrenaline and adrenaline produced dose-dependent relaxations in the former venous segments, but contractions in the latter ones. A Schild plot analysis for metoprolol against denopamine and for ICI118,551 against salbutamol showed that the venous segments running opposite the buccal cavity contained both beta(1)- and beta(2)-adrenoceptors, but the other venous segments contained only beta(2)-adrenoceptors. Electrical field stimulation-induced tetrodotoxin-sensitive relaxations in the former venous segments were diminished by pretreatment with metoprolol, but not with ICI118,551, indicating that the electrical stimulation-induced relaxation may be related to the activation of beta(1)-adrenoceptors in the venous smooth muscles. In conclusion, the heterogeneity of the functional properties, especially in the distribution of beta-adrenoceptors, in different segments of canine facial veins was observed in the present study, and autoregulatory mechanisms, humoral mechanisms, and neural mechanisms were suggested to affect cranial venous drainage.

We investigated the effects of cilostazol, a potent inhibitor of cGMP-inhibited cAMP phosphodiesterase, on mechanical activity of isolated pressurized rabbit cerebral penetrating arterioles with special reference to the function of the endothelium. Both cilostazol and milrinone, another inhibitor of cAMP phosphodiesterase, produced vasodilation of the cerebral penetrating arterioles in a dose-dependent manner. Pretreatment with selective inhibitors of cyclooxygenase or nitric oxide synthase, or chemical denudation of the endothelial cells caused no significant effect on the cilostazol-mediated vasodilation of the cerebral arterioles. A selective large-conductance calcium-activated potassium channel inhibitor, iberiotoxin, and a selective protein kinase A inhibitor, H-89, caused no significant effect on the cilostazol-mediated vasodilation. In the cerebral arterioles, low concentration (10–6M) of cilostazol or milrinone caused a significant shift of the dose-vasodilatory response curve for adenosine to the left. These findings suggest that cilostazol produces vasodilation independent of the presence of the endothelium or activation of endogenous vasodilative prostaglandins, nitric oxide, calcium-activated potassium channel and protein kinase A. In conclusion, the vasodilator action of cilostazol may, in part, contribute to the beneficial effect of preventing lacunar cerebral infarction in patients with functional damage of the endothelium in cerebral penetrating arterioles.

There is no reported study in which the lymphatic flow pathway in lymph nodes has been visualized and investigated in in vitro studies. The purpose of the present study is first to develop an isolated rat lymph node preparation circulated through the afferent and efferent lymph vessels in order to visualize lymphatic flow pathway within the node by using a video microscope system, and then to evaluate lymphatic flow dynamics by using fluorescence-labeled substances.In the present study, rat iliac lymph node was isolated. The afferent and efferent lymph vessels of the lymph node were cannulated with glass micropipettes in an organ chamber, while the small-sized arteries and veins connected to the lymph node were secured with sutures. Krebs-bicarbonate solution with or without fluorescent probes [FITCdextran, mol. weight; 77,000 and/or fluoresbrite carboxylate-microspheres (FC-microspheres) mean diameter of 1 microm] was circulated through the lymph vessels of the node. The time-dependent lymphatic pathway of fluorescent probes was investigated with a video microscope system. FTIC-dextran first spread through the cortex and subsequently reached the medulla of the lymph node. A follicle-like structure became evident in the cortex. FITC-dextran appeared in the efferent lymph vessel at 109 +/- 21 sec after its circulation began, while FC-microspheres distributed in the lymph node did not emerge from the node within 20 min after their introduction.In vitro preparations constructed in the present study will enable us to visualize the lymphatic flow pathway of fluorescent substances within the isolated iliac lymph nodes of rats in the absence of blood circulation.

We examined chronic effects of 17beta-estradiol (E(2)beta) on the responses of isolated rat anterior cerebral small arteries to vasoactive substances with special reference to endothelial function. Female Sprague-Dawley rats were separated into four groups: (1) sham-operated group (Sham), (2) sham-operated plus E(2)beta treated group (Sham+E), (3) ovariectomized group (OVX), (4) ovariectomized plus E(2)beta treated group (OVX+E). 5-Hydroxytryptamine (5-HT) (10(-10)-10(-3) M) and U46619 (10(-15)-10(-8) M) induced concentration-dependent contractions in the cerebral small arteries. The 5-HT- and U46619-induced contractions were not affected by pretreatment with 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME). No significant difference in high potassium (80 mM)- and the agonists-mediated contractions was observed among the four groups. Administration of acetylcholine (ACh) (10(-9)-10(-3) M) and sodium nitroprusside (SNP) (10(-8)-10(-3) M) caused dose-related relaxations in the cerebral small arteries precontracted by 10(-8) M U46619. Chronic treatment with E(2)beta caused a significant potentiation of the ACh-induced relaxations in the Sham+E and OVX+E groups. The dose-response curve for ACh in the OVX group was quite similar to that obtained with the Sham group. The ACh-induced relaxation was reduced significantly by pretreatment with 3 x 10(-5) M L-NAME, and an additional treatment with 10(-3) M L-arginine reversed significantly the L-NAME-induced inhibition. The removal of endothelial cells produced a significant reduction of the ACh-induced relaxation. Indomethacin (10(-5) M) did not alter the ACh-induced relaxation. The findings suggest that E(2)beta potentiates ACh-induced endothelium-dependent relaxation in rat anterior cerebral arteries and that the potentiation may be, in part, mediated by increasing production and release of endogenous NO from the endothelial cells.

We examined the responses of pressurized bovine retinal functional arterioles (97-185 microm in diameter and approximately 3 mm long) to vasoactive substances and the mode of action of acetylcholine (ACh) on the pressurized arterioles. The retinal arterioles were cannulated at both ends with glass micropipettes and perfused at a constant pressure of 60 mmHg. Vasoconstrictions of the retinal arterioles were induced by prostaglandin F(2 alpha) (PG F(2 alpha)), U46,619, noradrenaline (NA), and 5-hydroxytryptamine (5-HT) in a dose-dependent manner. The decreasing order of potency (pD(2) value) in the constrictive responses was as follows: 5-HT = U46,619NAPG F(2 alpha). On the other hand, sodium nitroprusside (SNP), isocarbacyclin (a stable prostaglandin I(2) analog), ACh, and isoproterenol (ISP) caused dose-dependent vasodilatation in the pressurized retinal arterioles preconstricted with high-potassium solution (40 mM K+). The decreasing order of potency in the vasodilative responses was as follows: isocarbacyclinSNPACh. The ACh-induced vasodilatation was suppressed significantly by pretreatment with N omega-nitro-L-arginine methyl ester (L-NAME) (3 x 10(-5) M). A treatment with L-arginine (10(-3) M) in the presence of 3 x 10(-5) M L-NAME reversed completely the L-NAME-induced reduction of the vasodilatation. These results suggest that ACh causes the production and release of endogenous nitric oxide or its related compounds, which results in vasodilatation of the pressurized bovine retinal functional arterioles.

We examined the effects of plasma on contractile responses of isolated dog external jugular veins to a thromboxane A(2) analog, U46619, and noradrenaline. Pretreatment with 1.0% plasma in Krebs-bicarbonate solution, but not 0.1%, caused a significant left and upward shift in the concentration-contractile response curve for U46619. The plasma-mediated potentiation of the response to U46619 was found in the venous segments without intact endothelium. The administration of 2x10(-5) M lysophosphatidylcholine in Krebs-bicarbonate solution with no plasma also produced a significant left and upward shift of the concentration-contractile response curve for U46619, the shift being quite similar to that obtained with 1.0% plasma. In contrast, pretreatment with 1.0% plasma or 2x10(-5) M lysophosphatidylcholine produced no significant effect on the noradrenaline-mediated contractions in the venous segments. Pretreatment with 10(-4) M L-ascorbate or 0.1 mg/ml alpha-tocopherol in the presence of 1.0% plasma caused a significant reduction in the plasma-mediated potentiation of the contractile responses to U46619. These findings suggest that lysophosphatidylcholine, a major phospholipid component of oxidized low-density lipoproteins, may contribute, in part, to the plasma-mediated potentiation of contractile responses of the isolated veins to U46619, and that the antioxidant vitamin, L-ascorbate, or alpha-tocopherol significantly reduces the plasma-mediated potentiation of the contractile responses to U46619, which may be related to inhibiting the production of lysophosphatidylcholine in plasma.

A mode of action of endothelin (ET) on spontaneous contractions was investigated in ring preparations of isolated bovine mesenteric lymphatics. ET-1 at concentrations between 10−10and 10−9M caused a dose-dependent increase in the frequency of spontaneous contractions. The specific ETA-receptor antagonist BQ-123 (5 × 10−7M) caused a significant inhibition of the ET-1-induced positive chronotropic effect in the ring preparations with and without the endothelium. Mechanical denudation of the lymphatic endothelial cells produced a significant potentiation of the ET-induced positive chronotropic effect. BQ-3020 (10−8–10−7M), a selective ETB-receptor agonist, induced dose dependently negative chronotropic and inotropic effects on the spontaneous contractions in the ring preparations with intact endothelium. Mechanical removal of the endothelium caused a significant reduction of the BQ-3020-induced negative chronotropic and inotropic effects. The ET-1-induced positive chronotropic effect was potentiated by pretreatment with Nω-nitro-l-arginine methyl ester (l-NAME) (10−5M) but unaffected by aspirin (10−5M). Additional treatment with l-arginine (10−4M) completely reversed the l-NAME-mediated potentiation of the ET-induced chronotropic effect. These results suggest that stimulation of ETAreceptors on the lymphatic smooth muscles causes a positive chronotropic effect on the spontaneous contractions, and stimulation of ETBreceptors on the lymphatic endothelial cells induces a release of nitric oxide, which results in the chronotropic and inotropic effects on spontaneous contractions in isolated bovine mesenteric lymphatics.

On a examine le mode d'action de la 5-hydroxytryptamine (5HT) dans les petites arteres spinales anterieures isolees de chiens. De faibles concentrations (l0 -9 -10 -7 M) de 5HT ont provoque une contraction dose-dependante, alors que des concentrations plus elevees (10 -6 -10 -3 M) ont provoque une relaxation dose-dependante des arteres precontractees par 10 -7 M de U 46619. La relaxation induite par 5HT a ete significativement antagonisee par la methiothepine (10 -9 -10 (NR> M). La ketanserine (10 -6 M) et l'ICS 205-930 (3 x 10 -6 M) n'ont pas influe sur la relaxation des arteres par 5HT. La reponse relaxante a 5HT a ete significativement reduite par le frottement mecanique des cellules endotheliales. La relaxation independante de l'endothelium induite par 5HT a aussi ete antagonisee par la methiothepine (10 -6 M). L'aspirine (5 x 10 -5 M) ou le N ω -nitro-L-arginine methyl ester (L-NAME) (10 -6 M) ont supprime significativement la relaxation dependante de l'endothelium induite par 5HT. La L-arginine (10 -3 M) a aussi renverse significativement la reduction induite par L-NAME de la relaxation dependante de l'endothelium induite par 5HT. De plus le traitement par L-NAME en presence d'aspirine a provoque une reduction beaucoup plus forte de la relaxation dependante de l'endothelium induite par 5HT. L'isocarbacycline (10 -9 -10 -5 M) a induit une relaxation dependante de la concentration des petites arteres spinales isolees, precontractees par 10 -7 M de U 46619. Ces resultats suggerent que la 5HT induit des relaxations independantes et dependantes de l'endothelium des petites arteres spinales anterieures isolees, principalement par le biais de l'activation du recepteur de type 5HT 1 , et que l'oxyde nitrique endogene et les prostaglandines vasodilatatrices pourraient participer a leur relaxation dependante de l'endothelium induite par 5HT.

Saturation diving (SD) is one of the safest techniques for tolerating hyperbaric conditions for long durations. However, the changes in the human plasma protein profile that occur during SD are unknown. To identify differential protein expression during or after SD, 65 blood samples from 15 healthy Japanese men trained in SD were analyzed by two-dimensional fluorescence difference gel electrophoresis. The expression of two proteins, one 32.4 kDa with an isoelectric point (pI) of 5.8 and the other 44.8 kDa with pI 4.0, were elevated during SD to 60, 100, and 200 meters sea water (msw). The expression of these proteins returned to pre-diving level when the SD training was completed. The two proteins were identified using in-gel digestion and mass spectrometric analysis; the 32.4 kDa protein was transthyretin and the 44.8 kDa protein was alpha-1-acid glycoprotein 1. Oxidation was detected at methionine 13 of transthyretin and at methionine 129 of alpha-1-acid glycoprotein 1 by tandem mass spectrometry. Moreover, haptoglobin was up-regulated during the decompression phase of 200 msw. These plasma proteins up-regulated during SD have a common function as anti-oxidants. This suggests that by coordinating their biological effects, these proteins activate a defense mechanism to counteract the effects of hyperbaric-hyperoxic conditions during SD.

Lymphatics are a unidirectional transport system that carries fluid from the interstitial space and back into the blood stream. Initial lymphatics take up not only fluid but also high-molecular-weight substances, such as plasma proteins and hyaluronan; immune cells, such as lymphocytes, macrophages, and dendritic cells; and colloidal particles, such as carbon particles, bacteria, and tattoo dye. Interstitially injected colloidal particles are known to accumulate in the regional lymph nodes. This phenomenon is applied to find sentinel lymph nodes in cancer patients. Lymph flow rate and composition are influenced by interstitial fluid, lymphatic pump activity, and intra-lymphatic pressure. Lymph composition is changed during its flow downstream. In this review, the main focus is on the mechanisms of lymph formation at the initial lymphatics and lymph transport through the collecting lymphatics and lymph nodes. (*English Translation of J Jpn Coll Angiol, 2008, 48: 113-123.).

Molecular mechanisms of reconnection of collecting lymph vessels were analyzed by using murine popliteal prenodal lymph vessels. At 1 and 2 weeks after being divided by cutting the lymph vessel, lymphatic reconnection was frequently observed accompanied by mesh-like lymphatic channels. Electron microscopic study also showed a monolayer of endothelial cells in the newly developed lymph vessels. Smooth muscle markers were immunofluorescently demonstrated in the wall of the new vessels. At 1 week after the procedure of cutting, augmented expressions of VEGF receptors 1, 2 and 3 were found immunohistochemically at the site of the reconnected lymph vessels. The expression of mRNA for VEGF receptor 3 was enhanced at 5 days and 1 week in small pieces of the tissues containing the reconnected lymph vessels, compared with that in the corresponding tissues obtained with sham operated ones. The administration of VEGF-C at the cutting site of the collecting lymph vessel significantly increased the rate of the reconnected lymph vessels, whereas additional treatment with Flt4/Fc chimera protein significantly reduced the rate of the reconnected ones. These results suggest that activation of VEGF-C-VEGF receptor 3 has critical roles in reconnection of the collecting lymph vessels in adult mice.

In some lymph nodes, water and water-soluble substances of smaller molecular weight are known to be absorbed into blood vessels, and consequently the protein concentration of lymph within the nodes increases. In this study, we examined pressure-flow relationships of lymph nodes in situ and exchange properties of water and water-soluble substances through the nodes with special reference to inflamed lymph nodes. A lymph perfusion model through the lymph node in situ was constructed by cannulating one of the afferent lymphatics and an efferent lymphatic. Increasing infusion pressure (0 to 150 cmH(2)O) or decreasing outflow pressure (10 to -5 cmH(2)O) in the model caused a significant increase of the lymph outflow rate through the node. This rate was also increased significantly with increases in both intranodal venous pressure (range: control, 20, 30, and 40 mmHg) and prenodal lymph albumin concentration (range: 0%, 2.6%, and 10%). When formyl-Met-Leu-Phe-OH (fMLP)-mediated acute inflammation was produced in the lymph nodes, the lymph outflow rate through the node was significantly decreased. These results indicate that colloid osmotic pressure and hydrostatic pressure within the lymph node may play important roles in the transport of water and water-soluble substances through the node. Acute fMLP-mediated inflammation of lymph nodes also produced a significant decrease of the lymph flow rate through lymph nodes.

This study was designed to examine whether mature collecting lymphatics can regenerate in the adult tissue or not.The X-ray lymphograms were used to detect network of the collecting lymphatics in rabbit hind leg. Regeneration of the lymphatics was observed after surgical removal of the popliteal lymph node or a part of the popliteal afferent lymphatic. Structure and mechanical properties of the lymphatics were also examined by light and electron microscopes and in vitro functional experiments.One week after removal of the lymph node, only an afferent lymphatic and a deposit of the contrast medium at the popliteal region were observed. Four weeks after the removal, the connection of the afferent and efferent lymphatics at the popliteal region, and collateral lymphatics were present in the leg. Further, 4 weeks after 1-mm excisions of a part of the lymphatic, recanalization was observed between the central and peripheral cut ends of the lymphatic but not after 3- and 10-mm excisions. Endothelial cells and smooth muscle cells could be observed by electron microscope, and contractile proteins, and alpha-smooth muscle actin SM1 and SM2 were immunofluorescently detected in both intact and the regenerated lymphatic walls. In both lymphatics, norepinephrine and acetylcholine induced dose-dependent constriction and dilation of the vessels, respectively.The present study demonstrated that mature collecting lymphatics are able to regenerate in the adult tissues.

We examined the effects of ATP on intrinsic pump activity in lymph vessels isolated from the rat. ATP caused significant dilation with a cessation of lymphatic pump activity. Removal of the endothelium or pretreatment with Nω-nitro-l-arginine methyl ester (l-NAME) significantly reduced ATP-induced inhibitory responses of lymphatic pump activity, whereas reduction was not suppressed completely by 10−6 M ATP. l-Arginine significantly restored ATP-induced inhibitory responses in the presence of l-NAME. ATP-induced inhibitory responses in lymph vessels with endothelium were also significantly, but not completely, suppressed by pretreatment with glibenclamide. 8-Cyclopentyl-1,3-dipropylxanthine (a selective adenosine A1 receptor antagonist), but not suramine (a P2X and P2Y receptor antagonist) or 3,7-dimethyl-1-proparglyxanthine (a selective adenosine A2 receptor antagonist), significantly decreased ATP-induced inhibitory responses. α,β-Methylene ATP (a selective P2X and P2Y receptor agonist) had no significant effect on lymphatic pump activity. In some lymph vessels with endothelium (24 of 30 preparations), adenosine also caused dose-dependent dilation with a cessation of lymphatic pump activity. l-NAME significantly reduced the inhibitory responses induced by the lower (3 × 10−8–3 × 10−7 M) concentrations of adenosine. Glibenclamide or 8-cyclopentyl-1,3-dipropylxanthine also significantly suppressed adenosine-induced inhibitory responses. These findings suggest that ATP-induced dilation and inhibition of pump activity of isolated rat lymph vessels are endothelium-dependent and -independent responses. ATP-mediated inhibitory responses may be, in part, related to production of endogenous nitric oxide, involvement of ATP-sensitive K+ channels, or activation of adenosine A1 receptors in lymphatic smooth muscle and endothelium.

The objective of the present study was to establish a rat lymphatic endothelial cell line and then to investigate the morphological and immunohistochemical properties of the cells.The lymphatic endothelial cells of rat thoracic ducts were isolated enzymatically by trypsin digestion and were cultured in endothelium growth medium (EGM)-2 in an atmosphere of low oxygen (5% O(2), 5% CO(2), and 90% N(2)) or high oxygen (21% O(2), 5% CO(2), and 74% N(2)).The number of the cells cultured in the low-oxygen atmosphere was significantly larger than that obtained in the high-oxygen atmosphere. The cultured cells in the low-oxygen atmosphere showed a monolayer with uniform cobblestone appearance, suggesting the morphological properties of endothelial cells. Factor VIII-related antigen and cell surface carbohydrates (i.e., D-galactose alpha and D-N-acetylgalactosamine alpha) were found on the lymphatic cultured cells. The phagocytosis of 1,1-diocadecyl1-3,3,3',3'-tetramethylindo-carbocyanine perchlorate-labeled acetylated low-density lipoprotein also was observed in the cultured cells. The cytoskeleton protein F-actin was located on the plasma membrane of the cultured cells as circumferential thin bundles and in the cytoplasma as filamentous bundles.The present study indicates that the choice of EGM-2 as a culture medium and the hypoxic atmosphere ( approximately 5%) enabled us to establish rat lymphatic endothelial cell line.

The contractile effects of alpha-adrenoceptor agonists and field electrical stimulation were investigated pharmacologically in canine isolated tracheobronchial lymph nodes. Addition of noradrenaline (NA), phenylephrine (Phe) and UK14,304 caused dose-related contractions of the isolated lymph nodes. Pretreatment with prazosin (3 x 10(-9), 3 x 10(-8) and 3 x 10(-7) M) shifted the concentration-response curve for phenylephrine to the right, whereas yohimbine (3 x 10(-7) M) did not affect the contractile responses to phenylephrine. On the other hand, the concentration-response curve for UK 14,304 shifted to the right by treatment with yohimbine (3 x 10(-9), 3 x 10(-8) and 3 x 10(-7) M) but not by prazosin (3 x 10(-7) M). Field electrical stimulation (25 V, 0.7 ms, 16 Hz) produced contractile responses of the lymph nodes. The electrical stimulation-induced contractions were completely reduced by pretreatment with tetrodotoxin (TTX) (3 x 10(-7) M) or bretylium (10(-6) M). Furthermore, phentolamine (10(-8), 10(-7) and 10(-6) M), prazosin (3 x 10(-9) and 3 x 10(-8) M) and yohimbine (3 x 10(-8) and 3 x 10(-7) M) significantly reduced the electrical stimulation-induced contractions in the preparations. The present findings suggest that there are both alpha1- and alpha2-adrenoceptors located on the smooth muscle cells in canine tracheobronchial lymph nodes, and that the adrenoceptors-mediated excitatory mechanisms of adrenergic innervation exist in the lymph nodes.

Physiological roles of endogenous nitric oxide (NO) in the lymphatic pump activity of rat mesenteries in vivo were evaluated using an intravital video microscope system. Changes in the pumping frequency ( F), the end diastolic diameter (EDD), and the end systolic diameter (ESD) of the mesenteric lymph microvessels were measured with the microscope system and then the pump flow index (PFI) was calculated. A 15-min superfusion of 30 μM N ω-nitro-l-arginine methyl ester (l-NAME) in the mesenteries caused significant increases of F and PFI and a significant decrease of the EDD and ESD. Simultaneous superfusion of 1 mM l-arginine with 30 μMl-NAME produced a significant reversal of thel-NAME-mediated increase of F and decrease of ESD. A 15-min superfusion of 100 μM aminoguanidine caused no significant effects on F, EDD, and ESD of the mesenteric lymph vessels in vivo. These findings suggest that endogenous NO has physiologically modulated the lymphatic pump activity in rat mesentery in vivo and that the production and release of NO may be mediated by constitutive NO synthase but not by inducible NO synthase.

The electrical stimulation-induced responses of isolated dog thoracic ducts were investigated using an organ bath technique. Electrical stimulation (0.7 ms in pulse width, 25 V in nominal voltage, 10 s in duration time, 1–32 Hz at frequency) produced frequency-related contractions in the lymphatic preparations. The contractions were abolished by pretreatment with tetrodotoxin (10 −7 M), guanethidine (10 −7 , 10 −6 M), and bretylium (10 −7 , 10 −6 M). Cocaine (10 −6 M) significantly potentiated the electrical stimulation−induced contractions. Phentolamine (10 −8 –10 −5 M), prazosin (10 −8 –10 −5 M), bunazosin (10 −6 , 10 −5 M), yohimbine (10 −8 –10 −6 M) and rauwolscine (10 −8 –10 −6 M) also dose−dependently reduced the contractions. On the other hand, propranolol (10 −8 –10 −6 M), atropine (10 −6 M), hexamethonium (10 −6 M), aspirin (3×10 −5 M), Nω -nitro- l -arginine methyl ester (L-NAME) (3×10 −5 M) and L-NAME (3×10 −5 M)+ l -arginine (10 −4 M) caused no significant effect on electrical stimulation-induced contractions. No significant difference in the electrical stimulation-induced responses was observed between the lymphatic preparations with and without an intact endothelium. The electrical stimulation caused only a small contraction with no relaxation in the thoracic duct preparation precontracted with 10 −8 M U46619. The small contraction was abolished by 10 −5 M phentolamine. These findings suggest that there exists α 1 - and α 2 -adrenoceptors-mediated excitatory innervation, but no NO-ergic inhibitory nerve fiber in dog thoracic ducts.

Cancer metastases are commonly found in the lymphatic system. Like tumor blood angiogenesis, stimulation of tumor lymphangiogenesis may require the interplay of several tumor-derived growth factors. Here we report that members of the PDGF family act as lymphangiogenic factors. In vitro, PDGF-BB stimulated MAP kinase activity and cell motility of isolated lymphatic endothelial cells. In vivo, PDGF-BB potently induced growth of lymphatic vessels. Expression of PDGF-BB in murine fibrosarcoma cells induced tumor lymphangiogenesis, leading to enhanced metastasis in lymph nodes. These data demonstrate that PDGF-BB is an important growth factor contributing to lymphatic metastasis. Thus, blockage of PDGF-induced lymphangiogenesis may provide a novel approach for prevention and treatment of lymphatic metastasis.