Distribution of diaphragmatic lymphatic lacunae.

The morphology of the submesothelial lymphatic lacunae on the pleural and peritoneal surface over the tendinous and muscular portion of the diaphragm was studied in 10 anesthetized rabbits. The lymphatic network was evidenced by injecting 1 ml of colloidal carbon solution in the pleural (n = 5) or the peritoneal (n = 5) space. After 1 h of spontaneous breathing, the animal was killed and the diaphragm was fixed in situ by injection of approximately 5 ml of fixative in pleural and peritoneal spaces. Then both cavities were opened and the diaphragm was excised and pinned to a support. According to which cavity had received the injection, the peritoneal or the pleural side of the diaphragm was scanned by sequential imaging of the whole surface by use of a video camera connected to a stereomicroscope and to a video monitor. The anatomic design appeared as a network of lacunae running either parallel or perpendicular to the major axis of the tendinous or muscular fibers. The lacunae were more densely distributed on the tendinous peritoneal area than on the pleural one. Scanty lacunae were seen on the muscular regions of both diaphragmatic sides, characterized by large areas without lacunae. The average density of lacunae on tendinous and muscular regions was 6 and 1.7/cm2 for the pleural side and 25 and 3.4/cm2 for the peritoneal side, respectively. The average width of lacunae was 137.9 +/- 1.6 and 108.8 +/- 1.7 microns on the tendinous pleural and the peritoneal side, respectively, and 163 +/- 1.8 microns on the muscular portion of the pleural and peritoneal surfaces.     

一氧化氮对大鼠胸膜淋巴孔调控及淋巴吸收的影响

实验研究了一氧化氮（nitric oxide,NO）对大鼠胸膜淋巴孔的调控和胸膜腔淋巴吸收的影响。NO供体和NOS（nitric oxide synthase）抑制剂分别经腹腔给药,示踪剂（台盼蓝）胸膜腔内注射后,处死大鼠,测定血清NO和台盼蓝浓度;在扫描电镜下观察各组胸膜淋巴孔,用计算机图像处理,统计学分析。结果显示,NO供体组血清NO浓度为49.34±18.47μmol/L,淋巴孔的面积和密度分别为6.80±1.13 μm2和170.24±66.60/0.1mm2;NOS抑制剂组血清NO浓度为17.72±6.58μmol/L,淋巴孔的面积和密度分别为5.72±1.54μm2和61.71±12.73/0.1mm2。血清NO浓度与淋巴孔开放的面积和密度成正相关（P<0.05）。在胸膜腔给示踪剂后,NO供体组血清台盼蓝的浓度为74.68±33.67mg/L,与对照组比较有显著差异（P<0.05）。提示,NO可以调控胸膜淋巴孔,促进胸膜腔淋巴吸收。     

Functional arrangement of rat diaphragmatic initial lymphatic network

Fluid and solute flux between the pleural and peritoneal cavities, although never documented under physiological conditions, might play a relevant role in pathological conditions associated with the development of ascitis and pleural effusion and/or in the processes of tumor dissemination. To verify whether a pleuroperitoneal flux might take place through the diaphragmatic lymphatic network, the transdiaphragmatic pressure gradient (Delta P(TD)) was measured in five spontaneously breathing anesthetized rats. Delta P(TD) was -1.93 cmH2O (SD 0.59) and -3.1 cmH2O (SD 0.82) at end expiration and at end inspiration, respectively, indicating the existence of a pressure gradient directed from the abdominal to the pleural cavity. Morphometrical analysis of the diaphragmatic lymphatic network was performed in the excised diaphragm of three additional rats euthanized with an anesthesia overdose. Optical and electron microscopy revealed that lymphatic submesothelial lacunae and lymphatic capillaries among the skeletal muscles fibers show the ultrastructural features of the so-called initial lymphatic vessels, namely, a discontinuous basal lamina and anchoring filaments linking the outer surface of the endothelial cells to connective tissue or to muscle fibers. Primary unidirectional valves in the wall of the initial lymphatics allow entrance of serosal fluid into the lymphatic network preventing fluid backflow, while unidirectional intraluminar valves in the transverse vessels convey lymph centripetally toward central collecting ducts. The complexity and anatomical arrangement of the two valves system suggests that, despite the existence of a favorable Delta P(TD), in the physiological condition no fluid bulk flow takes place between the pleural and peritoneal cavity through the diaphragmatic lymphatic network.     

小鼠卵巢囊淋巴孔的超微结构及其吸收功能

为研究小鼠卵巢囊及卵巢囊淋巴孔的超微结构 ,探讨其吸收功能 ,本实验用常规扫描电镜和透射电镜观察小鼠卵巢囊组织 ,应用Elscope软件对卵巢囊淋巴孔进行定量处理。并通过曲利本蓝吸收实验 ,测定其吸收功能。本实验首次发现小鼠卵巢囊上皮的立方细胞和扁平细胞之间存在淋巴孔 ,淋巴孔直径为 3 6 12± 1 132μm ,周长为 10 86 2± 3 4 15 μm。同时也发现小鼠卵巢囊上存在闭合淋巴孔。小鼠卵巢囊淋巴孔能吸收曲利本蓝 ,并随着时间的延长 ,曲利本蓝吸收增多。本实验的结果提示 :小鼠卵巢囊淋巴孔的大小、分布都与金仓鼠、豚鼠的卵巢囊淋巴孔有一定的差异 ,其淋巴孔的物质吸收功能具有时间依赖性。同时 ,闭合淋巴孔的发现表明淋巴孔的可调控性。卵巢囊淋巴孔使卵巢囊腔、卵巢囊淋巴系和腹膜腔沟通 ,在维持卵巢正常发育所需的体液环境方面发挥了重要作用     

Kinetics of fluid flux in the rat diaphragmatic submesothelial lymphatic lacunae

The specific role of loops and/or linear segments in pleural diaphragmatic submesothelial lymphatics was investigated in seven anesthetized, paralyzed, and mechanically ventilated rats. Lymphatic loops lay peripherally above the diaphragmatic muscular plane, whereas linear vessels run over both the muscular and central tendineous regions. Lymph vessel diameter, measured by automatic software analysis, was significantly greater (P < 0.01) in linear vessels [103.4 +/- 8.5 microm (mean +/- SE), n = 18] than in loops (54.6 +/- 3.3 microm, n = 21). Conversely, the geometric mean of intraluminal flow velocity, obtained from the speed of distribution of a bolus of fluorescent dextrans injected into the vessel, was lower (P < 0.01) in linear vessels (26.3 +/- 1.4 microm/s) compared with loops (51.3 +/- 3.2 microm/s). Lymph flow, calculated as the product of flow velocity by vessel cross-sectional area, was similar in linear vessels and in individual vessels of a loop, averaging 8.6 +/- 1.6 nl/min. Flow was always directed from the diaphragm periphery toward the medial tendineous region in linear vessels, whereas it was more complex and evidently controlled by intraluminal unidirectional valves in loops. The results suggest that loops might be the preferential site of lymph formation, whereas linear vessels would be mainly involved in the progression of newly formed lymph toward deeper collecting diaphragmatic ducts. Within the same hierarchic order of diaphragmatic lymphatic vessels, the spatial organization and geometrical arrangement of the submesothelial lacunae seem to be finalized at exploiting the alternate contraction/relaxation phases of diaphragmatic muscle fibers to optimize fluid removal from serosal cavities.     

腹膜淋巴孔与淋巴转归NO-cGMP-Ca2+信号转导途径研究

为研究一氧化氮(nitric oxide,NO)调节大鼠腹膜淋巴孔和淋巴吸收的细胞内信号转导机制,在体外培养的间皮细胞上,利用cGMP检测试剂盒和激光共聚焦扫描显微镜,研究NO对间皮细胞内cGMP和游离钙离子浓度(Ca^2 )的影响;并利用动物实验研究NO-cGMP-Ca^2 通路对大鼠腹膜淋巴孔和淋巴吸收的影响。结果发现：(1)与对照组相比,NO供体Sper NO （spermine／nitric oxide complex)可以剂量依赖性地升高间皮细胞内cGMP的浓度(P＜0．01)。此作用可被可溶性鸟苷酸环化酶(soluble guanylyl cyclase,sGC)特异性抑制剂1H-[1,2,4]噁二唑[4．3-a]喹喔啉-1-one酮(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one,ODQ)阻断(P＜0．05,P＜0．01)。Sper／NO可使间皮细胞内[Ca^2 ],相对水平降低(P＜0．05),但此效应可被ODQ阻断;L-型电压依赖性钙通道阻滞剂nifedipine,可使细胞内的[Ca^2 ];在短时间内迅速明显下降(P＜0．05),达平衡后再加入Sper／NO并不能引起[Ca^2 ];的进一步改变(P＞0．05);(2)NO可以剂量依赖性地提高大鼠膈组织淋巴孔最大分布面积(P＜0．01)和对示踪剂的吸收量(P＜0．05),ODQ可明显抑制NO引起的淋巴孔和淋巴吸收的改变(P＜0．01)。该结果首次发现nifedipine能显著增加淋巴孔最大分布面积(P＜0．01)及膈组织对台盼蓝的吸收量(P＜O．05),而且nifedipine预处理后Sper／NO并不能使淋巴孔的淋巴吸收进一步升高(P＞0．05)。结果提示,NO可以通过降低cGMP水平降低大鼠腹膜间皮[Ca^2 ],且此过程和L-型电压依赖性钙通道有关;NO可通过NO-cGMP-[Ca^2 ],通路,促进淋巴孔的开放和吸收。     

妊娠和激素干预对豚鼠卵巢囊淋巴孔的影响

In order to study the influence of pregnancy and hormone on the lymphatic stomata of guinea pig's ovary bursa, the lymphatic stomata of ovary bursa were observed during pregnancy and by using exogenous hormone, including serum gonadotrophin (PMSG) combined with human chorionic gonadotropin (HCG) to promote ovulation and androgen to inhibit the ovary. Then the lymphatic stomata in the inner layer of ovary bursa and their absorption functions were observed with scanning electron microscope (SEM) and trypan blue as a tracer respectively. The lymphatic stomata were also counted by the Elescope computer image processing system. We found that the quantity of tracer absorbed by the lymphatic stomata of ovary bursa in pregnant group was more than that in ovulation-promoted group, and least in the androgen-treated group, which had statistical significance between each couples of them (P < 0.05). The opening area of the lymphatic stomata in pregnant, ovulation-promoted and androgen-treated groups were 189.9 +/- 48.7 micron 2/1000 micron 2, 104.4 +/- 31.2 micron 2/1000 micron 2 and 40.5 +/- 18.7 micron 2/1000 micron 2 respectively, which had remarkable statistical difference between each couples (P < 0.01). Under SEM observation, the secretory granules in ciliated columnar epithelium were less in pregnant group than the other two, while both the secretory granules and the cilium were most active in ovulation-promoted group. The results indicated that both the openings and absorption functions of the lymphatic stomata in guinea pig's ovary bursa could be affected by pregnancy and exogenous homone. There existed relationship between ovary function and the regulation of the lymphatic stomata in ovary bursa.     

Distribution of stomata on the second leaf ofZea mays following root hypoxia

The changes in leaf dimensions, transverse and longitudinal gradients in stomatal density and the total number of stomata under the influence of root hypoxia were followed. In spite of considerably reduced leaf area following hypoxia the total number of stomata per leaf was not changed significantly. The resulting increase in stomatal density was not uniform being the most prominent in the basal part of the leaf where the distances between stomata and between rows of stomata became shorter.     

Distribution of prosaposin in rat lymphatic tissues

Prosaposin (PSAP) is as a trophic factor and an activator protein for sphingolipid hydrolase in lysosomes. We generated a specific antibody to PSAP and examined the spatiotemporal distribution of PSAP-immunoreactive (PSAP-IR) cells in the lymphatic tissues of Wistar rats. Immunoblots of tissue homogenates separated electrophoretically showed a single band for PSAP in brain but two bands in spleen. PSAP-IR cells were distributed in both the red and white pulp of the spleen, in both the cortex and medulla of the thymus and in mesenteric lymph nodes. Many PSAP-IR cells were found in the dome portion of Peyer’s patches and the number of PSAP-IR cells increased with the age of the rat. To identify the PSAP-IR cells, double- and triple-immunostainings were performed with antibodies against PSAP, CD68 and CD1d. The large number of double- and triple-positive cells suggested that antigen-presenting cells contained much PSAP in these lymphatic tissues. Intense expression of PSAP mRNA, examined by in situ hybridisation, was observed in the red pulp and corona of the spleen. In rats, the PSAP gene generates two alternative splicing forms of mRNA: Pro+9 containing a 9-base insertion and Pro+0 without the insertion. We examined the expression patterns of the alternative splicing forms of PSAP mRNA in the spleen. The presence of both types of mRNA (Pro+9 and Pro+0) indicated that the spleen contains various types of prosaposin-producing and/or secreting cells. These findings suggest diverse functions for PSAP in the immune system.     

Immunohistochemical localisation of ET-1 and eNOS in lymphatic stomata of the porcine broad ligament of the uterus

Immunohistochemical localization and distribution of endothelin (ET-1) and nitric oxide synthase (eNOS) were investigated in lymphatic stomata in areas of their special accumulation in the porcine broad ligament during the estrous cycle. The study was performed using polyclonal antibody for ET-1 and monoclonal antibody for eNOS. ET-1 and eNOS immunoreactivities were demonstrated in some thin endothelial lymphatic lacuna walls throughout the estrous cycle. In the mesothelial cell layer, ET-1 and eNOS were detected only in stomata-related cuboidal mesothelial cells, however, the intensity of the immunostaining and distribution of the positive cells varied during the cycle. These results suggest that ET-1 and eNOS can play a role in mechanisms regulating the tone of lymphatic stomata during the absorption and passage of fluids, particles and cells from the peritoneal cavity to lymphatic vessels in the porcine broad ligament.     

豚鼠卵巢囊淋巴孔的发现及卵巢囊超微结构研究

In order to explore its structure and speciality of species, the ovary bursa of guinea pig was studied by using dissecting microscopy and scanning electron microscopy. In this paper, the lymphatic stomata on both internal and external layer of the ovary bursa was frist reported in guinea pig. The results suggested that the stomata in bursa not only provided a pathway to connect the bursal cavity with the lymphatic vessels of bursa and the peritoneal cavity, but also may be involved in the reproduction and local immunity of the ovary. The stomata may play an important role in physiological function of the ovary. At the same time, the structural differences were identified in ovary bursa between guinea pig and golden hamster.     

Distribution of lymphatic vessels in mouse thymus: immunofluorescence analysis

Thymic blood and lymphatic vessels in humans and laboratory animals have been investigated in morphological studies. However, occasionally a clear distinction between blood vessels and lymphatic vessels cannot be made from morphological characteristics of the vasculature. To visualize thymic lymphatics in normal adult BALB/c mice, we used antibodies against specific markers of lymphatic endothelial cells. Expression of vascular endothelial growth factor receptor–3 (VEGFR–3) was detected throughout the thymus, i.e., the capsule, cortex, and medulla. Most thymic lymphatics were present in capillaries of ~20 μm in caliber. The plexuses of lymphatic capillaries were occasionally detectable. Lymphatic vessels were frequently adjacent to CD31–positive blood vessels, and some lymphatic vessels were seen in the immediate vicinity of or within the perivascular spaces around postcapillary venules. The identity of VEGFR–3–positive vessels as lymphatics was further confirmed by staining with additional markers: LYVE–1, Prox–1, neuropilin–2, and secondary lymphoid tissue chemokine (SLC). The distributions of LYVE–1 were similar to those of VEGFR–3. Most lymphatic vessels were also identified by Prox–1. Neuropilin–2 was restricted to lymphatic vessels in the thymus. The most abundant expression of SLC in the thymus was in medullar epithelial cells; SLC was also expressed in lymphatic vessels and blood vessels. Thus, lymphatic endothelium in mouse thymus was characterized by positive staining with antibodies to VEGFR–3, LYVE–1, Prox–1, neuropilin–2, or SLC, but not with an antibody to CD31. Our results suggest the presence of lymphatic capillary networks throughout the thymus.     

The transverse orientation of stomata

The orientation of cell walls co-determines development. The orientation of the slits of the stomata can be used for analyzing the factors involved. A comprehensive and annotated list is given of those plant species most of whose stomata are known to be oriented transversely to the long axis of an organ or a main rib. Included are also species showing only a trend toward transverse orientation. Transverse orientation is known from a few mosses, from Bennettitatae, fromAzolla and some other ferns, and from species of about 45 families of spermatophytes. It could be confirmed that succulent species show the trait more often than do other plants. Two thirds of the species listed belong to the Caryophyllales and Santalales, a few only to Asteraceae, but none to Rubiaceae, Cyperaceae, Poaceae, or Orchidaceae. Hence, the incidence of succulent species or of species with some succulent traits within the two orders and the lack of such species among other taxa may account in part for the distribution. On the other hand, many succulent species do not show transverse orientation whereas in, e.g.,Casuarina and Tamaricaceae transverse orientation goes together with non-succulent xeromorphy;Azolla shows no xeromorphy at all. Various factors, separately or together, may be involved. Proposed mechanisms determining the orientation of cell walls have been compiled from literature and are discussed.     

Postnatal development of the ovarian bursa of the golden hamster (Mesocricetus auratus): its complete closure and morphogenesis of lymphatic stomata

The golden hamster ovarian bursa was studied by light and electron microscopy to clarify the process of its complete closure and the development of lymphatics that leads to morphogenesis of stomata. The results were as follows. 1) The bursa completely closed at 9 days of age primarily due to development of the mesotubarium superius. 2) With the closure, the ovary and bursa became closely apposed, and most of the original bursal cavity disappeared. 3) Between 9 and 12 days of age U-shaped folds of the bursal mesothelium began to invade the connective tissue of the bursa. 4) Widening of the internal angle of the U-shaped folds contributed to reappearance of the bursal cavity, and thus separation of the bursa from the ovary. It also contributed to future geometrical proximity of lymphatics to the cavity of the bursa. 5) The separation of the bursa from the ovary began as early as 12 days of age in the cephalic half of the bursa. It occurred remarkably late in the caudal half. Juxtaposition of the window portion of the bursa to the ovary remained in some adult animals. 6) Development of lymphatics in the cephalic half of the bursa was divided into two stages, before and after days 21-24 of life. In the first stage, lymphatics grew in the submesothelial connective tissue, and the framework of lymphatics was formed. In the second stage, lymphatics extended small branches to form the submesothelial plexus or lymphatic lacuna. 7) Intercellular junctions between contiguous lymphatic endothelial cells were mostly tight and desmosomelike. Open junctions were, if they occurred at all, rare. (8) A smooth-surfaced area lined with the lymphatic endothelium was found in the bursa on day 27 of life, before the initiation of ovulation. Valvelike stomal orifices were absent before the initiation of ovulation and extremely rare even after the first ovulation. They were commonly present in the bursae after the fourth ovulation, however. The process of complete closure of the ovarian bursa is very complex and may be related to the later development of the bursal mesothelium and lymphatics. Some liplike stomal orifices are of purely developmental origin. However, all valvelike stomal orifices are assumed to be formed as a result of damage to the bursal mesothelium, as well as to the submesothelial connective tissue and lymphatics, by repetition of ovulation. It is possible that liplike stomal orifices may be formed in the process of repairing the damage.