Mast-cell secretion and angiogenesis, a quantitative study in rats and mice

The activation of the autogenous mast cells (MCs) in situ in intact mesenterial windows was elicited by the intraperitoneal injection of the MC secretagogue Compound 48/80 over a period of 1, 3 and 5 days in Sprague-Dawley rats and in C57 BL/6 and CBA/Ca mice. As a probe of MC secretion, the release of histamine was quantified fluorometrically at predetermined intervals during the treatment. Fourteen days after the start of the treatment, the angiogenic response was quantified histologically as the number of vessel profiles per unit length of mesenteric window. Both the MC-activating and the angiogenic effect of the 48/80-treatment was greater in the rats than in the mice. The occurrence of MC-mediated angiogenesis in the mouse is demonstrated here for the first time. In the rat, 48/80-induced MC mediated angiogenesis increased in a distinctly dose-dependent manner. Two daily doses of 48/80 was the most efficient angiogenic protocol tested; a single day's treatment increased the number of vessels almost fivefold. The remarkable potency of the angiogenic reaction following MC secretion supports our previous notion that MC-mediated angiogenesis may have therapeutic implications in poorly vascularized tissues.     

Myometrial cells induce angiogenesis and salvage damaged myocardium

Characteristically, uterine myometrial cells (MCs) are proliferative, inducing angiogenesis within the female reproductive organ. We evaluated whether MCs implanted into myocardium could also induce angiogenesis and restore heart function after injury. MCs were isolated from the adult rat uterus and cultured for three studies: 1) Intracellular VEGF levels were measured in MCs cultured with progesterone (10(-11), 10(-9), and 10(-7) M) (n = 6 tests per group). 2) Blood vessel density was evaluated 8 days after MCs (3 x 10(6) or 6 x 10(6)), smooth muscle cells (SMCs), or endothelial cells (n = 6 rats per group) were injected with matrigel into the subcutaneous tissue of adult rats. 3) MCs, SMCs (5 x 10(6)/rat), or media were injected into a transmural scar 3 wk after cryoinjury in rat hearts (n = 12 rats per group), and heart function, blood vessel density, and myocardial scar size and thickness were evaluated 5 wk later. In study 1, cultured MCs expressed VEGF, with levels significantly (P < 0.05) upregulated by progesterone at an optimal dose of 10(-11) M. In study 2, MCs injected into the subcutaneous tissue with matrigel induced significantly more blood vessels, especially large-diameter vessels, than did SMCs or endothelial cells (P < 0.01 for all groups). This angiogenic effect was greatest (P < 0.01) at higher doses of MCs and was enhanced by progesterone (10(-11) M). In study 3, MCs implanted into the injured myocardium increased blood vessel density at the implant area, reduced scar size, and improved cardiac function relative to SMCs and media. Overall, MCs induced angiogenesis in vitro and in vivo, prevented cardiac remodeling, and improved heart functional recovery after cardiac injury.     

Changes in nurse cell nuclei during synchronous infection with Trichinella spiralis

The rate of enlargement of nuclei was determined on 4-microns-thick sections of synchronously infected mouse thigh muscle. Normal muscle nuclei had a geometric mean volume of 84 microns and a range of 42-170 microns 3. At days 5, 6, 7, 8, and 10 and 6 mo after infection, mean nuclear volume was 177 (100-315) microns 3, 254 (140-462) microns 3, 278 (172-447) microns 3, 681 (407-1,138) microns 3, 512 (326-804) microns 3, and 509 (298-870) microns 3, respectively. Size of nuclei for any given day followed a log normal distribution. On days 7 and 8 after infection, 31% of enlarged nuclei had 2 nucleoli, whereas only 15% had 2 nucleoli on day 10. One percent of enlarged nuclei in 6-mo-old nurse cells had double nucleoli. The number of enlarged nuclei in 6-mo-old nurse cells was determined from serial sections of infected tongue muscle. Each nurse cell contained an average of 40 enlarged nuclei. Sixty-four percent of nurse cells examined (n = 55) had between 30 and 60 enlarged nuclei. However, there was great variation in the range (7-142). These results are discussed in relation to the development of the nurse cell.     

Biology and pathogenicity of Eimeria spinosa Henry, 1931 in experimentally infected pigs.

A single-species isolate of E. spinosa from a diarrheic weaned pig was used to determine the endogenous development and pathogenicity of this swine coccidium. Seven out of 14 inoculated pigs developed endogenous stages or passed oocysts of E. spinosa in their feces. Immunosuppressive treatment with cyclophosphamide had no effect on the susceptibility to infection with E. spinosa in young pigs. The endogenous stages developed within the apical cytoplasm of the enterocytes lining the distal part of the villi in the posterior jejunum. The asexual development comprised three generations of meronts, which were seen at 5, 7 and 9 days post-infection (DPI). Meronts of the first generation measured 6-8 microns and produced 10-14 merozoites 4-6 microns in length. The second generation of meronts measured 6-8 microns and contained 10-20 merozoites 4-6 microns in length. Third generation mature meronts (8-10 microns) on DPI 9 contained 12-20 merozoites measuring 5-7 microns, which were more crescent-shaped and less blunt than the merozoites at 5 and 7 DPI. Merogony continued after formation of the gametes and the first fully developed macrogametes (10-14 microns), microgametes (9-12 microns), and oocysts were also seen at 9 DPI. The prepatent period was 8 or 9 days, but the patent period was not determined. In the present study E. spinosa infection did not produce overt clinical signs. Pathological changes consisted of an inflammatory infiltration in the lamina propria of the posterior jejunum, Peyer's patches activation and sporadic erosions scattered at the villous tips. No villous atrophy in association with a large number of endogenous stages was observed.     

In vitro growth of oocyte-granulosa cell complexes isolated from cryopreserved ovine tissue.

A culture system has been designed in which enzymatically isolated oocyte-granulosa cell complexes from fresh and frozen-thawed ovine ovarian tissue can be grown to antral size in vitro. Oocyte-granulosa complexes ranging from 100 to 240 microns in diameter were dissected from stromal tissue and grown individually in serum-free medium for 30 days. Complexes < 190 microns generally excluded their oocytes or lost three-dimensional structure early in the culture period. In contrast, complexes isolated from fresh or frozen-thawed tissue and measuring 190-240 microns on the day of isolation formed antral cavities in 25 +/- 9% and 18 +/- 6% (mean +/- SEM) of cases, respectively. The effect of gonadotrophin supplementation to the culture medium was tested on frozen-thawed oocyte-granulosa cell complexes only. In cultures supplemented with both FSH and LH or FSH alone, there was no significant difference in the number of oocyte-granulosa cell complexes that formed antral cavities (18 +/- 7%). However, antrum formation was significantly less frequent in cultures lacking gonadotrophin stimulation (7 +/- 4%). All oocyte-granulosa cell complexes maintained a three-dimensional structure throughout culture and developed a functional P450 aromatase enzyme complex, as revealed by the induction of oestradiol production during 8 days of culture after antrum formation in serum-free medium containing testosterone. Oocytes recovered after 30 days of culture were viable and had increased in diameter from 78 +/- 2 microns on the day of isolation, to 131 +/- 3 microns at the end of culture. These results show that oocyte-granulosa cell complexes isolated from cryopreserved ovarian tissue can be grown to antral size in vitro with similar efficiency to those isolated from fresh tissue.     

The early revascularization of membranous bone

The experimental finding that membranous onlay bone grafts maintain volume and viability to a greater extent than do endochondral grafts may be related to the more rapid vascularization of membranous bone. Microangiographic techniques were used to study the rates of vascularization of membranous and endochondral bone grafts in adult white New Zealand rabbits at 1, 3, 7, 14, and 21 days after bone grafting. Vascularization patterns were quantified microscopically using a modified point-counting technique. At 3 days, membranous bone grafts demonstrated vessel ingrowth from both soft tissue and host bone. Little ingrowth was seen in endochondral grafts. By day 7, 2.5 vessels per square were identified entering membranous grafts, while an average of 0.6 vessels per square were counted for endochondral bone grafts. At day 14, there was an average of greater than 20 vessels per square for membranous grafts versus 1.8 for their endochondral counterparts. At 21 days, the endochondral grafts demonstrated persistent avascular central areas not seen in membranous grafts. Membranous onlay bone grafts in the rabbit are more rapidly vascularized than endochondral grafts. This factor may affect the greater volume maintenance seen in experimental membranous grafts.     

Quantitative studies of the vasculature of the carotid body in fetal and newborn sheep

Resetting of the hypoxic sensitivity of the carotid chemoreceptors from the fetal to the adult arterial PO2 range follows the rise in PO2 which occurs after birth. The mechanism of this resetting is unknown. To study whether it is accompanied by a change in the carotid body microvasculature, 2 pairs of carotid bodies from fetal sheep (145 days gestation) and 2 pairs from 7-8 days-old lambs were examined. The ratio of the area of small vessels (6-16 microns diameter) or of larger vessels (greater than 16 microns diameter) to the total area of individual lobules of the carotid body was measured, using a semi-automatic image analysis system. This quantified the number and total cross-sectional area of small vessels and of larger vessels in 20 sections of 5 microns thickness taken at random from 200-350 sections cut from each carotid body. When the carotid bodies of the fetus and neonate were compared, the neonates showed increases in the percentage of the lobule area occupied by both small and large vessels, but the difference was only significant in the case of the larger vessels. There was no difference in the ratio of the area occupied by smaller vessels to the extravascular area of the lobule. Our results do not support the idea that the post-natal resetting of chemoreceptor sensitivity from the fetal to the post-natal range is accompanied by a change in the perfusion of the carotid body chemoreceptor cells.     

用显微穿刺技术直接测量微血管压力的伺服零方法研究

本实验制作了尖端的直径为０５－５０μｍ和斜面为２５°的玻璃微插管,成功地建立了用显微穿刺（ｍｉｃｒｏｐｕｎｃｔｕｒｅ）技术直接测量微血管压力（Ｐｍ）的伺服零方法（ｓｅｒｖｏｎｕｌｍｅｔｈｏｄ）,对自发高血压大鼠（ＳＨＲ）和正常血压大鼠（ＷＫＹ）肠系膜Ｐｍ进行了测量研究。结果表明,ＳＨＲ的平均动脉压（ＰＡ）和微动脉的Ｐｍ均明显大于ＷＫＹ的ＰＡ和Ｐｍ;ＰＡ在微动脉中明显降低,最大压降在直径小于５０μｍ的微动脉以及毛细血管中。     

High density VERO cell culture on microcarriers in a cell bioreactor

VERO cells were cultivated in microcarriers (MCs) using a bioreactor with a working capacity of 3.7 l. We studied the effect of MCs concentration in the proportion of MC-bearing cells and on the kinetics of cell growth, as well as the cell growth with a batch, fed-batch and perfusion mode operation. The data show that, in a batch system, with an initial VERO cell inoculum of 3×10⁴ cells/mg of MCs, 65%, 55% and 50% of the MCs were shown to be totally covered by cells after 7 days in cultures with respectively 2, 5 and 10 mg/ml of MCs. It was observed, that higher concentrations of MCs produced higher final yields of VERO cells. With 10 mg/ml of MCs, after 7 days of culture, a final yield of 10¹⁰ cells in the bioreactor, was obtained. The study of cell growth with a batch, fed-batch or perfusion system showed that the medium renewal allowed the continuous cell growth with the obtention of a final yield of 4×10⁹ cells in a culture with 2 mg/ml of MCs. The number of cells that can be easily reached in these culture systems, which can be even further improved, indicates its usefulness for cell propagation and the preparation of cell products such as viral antigens.This work was supported in part by grants from the Fundação de Amparo à Pesquisa do Estado de S. Paulo (FAPESP), Fundação Instituto Butantan, and European Economic Community (EEC). C.A. Pereira is recipient of a Conselho Nacional de Pesquisa (CNPq) fellowship. We thank A.C. Barbosa for technical assistance.     

Effects of interleukin 3, interleukin 4, and fibroblasts on cultures of human lung mast cells in bronchoalveolar lavage fluids

The effects of T cell factors, including interleukin (IL)-3 and IL-4, and fibroblasts on the growth and differentiation of human lung mast cells (MCs) obtained by bronchoalveolar lavage (BAL) were examined. The number of MCs identified by alcian blue-safranin staining was twice that of the control culture without conditioned medium (CM) when BAL cells were cultured for 2 weeks in RPMI 1640 containing 10% fetal calf serum and partially purified CM derived from PHA-stimulated lymphocytes. In the presence of both recombinant (r) IL-3 and rIL-4, the number of MCs was twice as high as the control without increase in the per-cell histamine content after 2 weeks' culture. In umbilical cord blood cultures, IL-3 plus IL-4 augmented basophilic cells about 20-fold more than the control when cultured for 2 weeks. In some cases, the percentage of safranin-positive MCs was about 2-5 fold greater, with 2-7 fold higher histamine content, when cultured for 10 days with CM and fibroblasts derived from human embryonic lung. However, in all BAL experiments, there was no increase in the total number of MCs after culture compared with the initial number of MCs, unlike the umbilical cord blood cultures. These results suggest that T cell factors, including IL-3 and IL-4, and fibroblasts may influence the phenotype and the survival of lung mast cells in BAL, whereas there was no evidence for the presence of MC precursors in BAL fluids.     

Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37v°C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37v°C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 2 10⁵, 8.8 2 10⁵ and 1.8 2 10⁶ cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm² T-flasks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.     

Rat mesentery exteriorization: a model for investigating the cellular dynamics involved in angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth. The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions.     

Dexamethasone blocks the systemic inflammation of alveolar hypoxia at several sites in the inflammatory cascade

Alveolar hypoxia produces a rapid and widespread systemic inflammation in rats. The inflammation is initiated by the release into the circulation of monocyte chemoattractant protein-1 (MCP-1) from alveolar macrophages (AMO) activated by the low alveolar Po(2). Circulating MCP-1 induces mast cell (MC) degranulation with renin release and activation of the local renin-angiotensin system, leading to microvascular leukocyte recruitment and increased vascular permeability. We investigated the effect of dexamethasone, a synthetic anti-inflammatory glucocorticoid, on the development of the systemic inflammation of alveolar hypoxia and its site(s) of action in the inflammatory cascade. The inflammatory steps investigated were the activation of primary cultures of AMO by hypoxia, the degranulation of MCs by MCP-1 in the mesentery microcirculation of rats, and the effect of angiotensin II (ANG II) on the leukocyte/endothelial interface of the mesentery microcirculation. Dexamethasone prevented the mesentery inflammation in conscious rats breathing 10% O(2) for 4 h by acting in all key steps of the inflammatory cascade. Dexamethasone: 1) blocked the hypoxia-induced AMO activation and the release of MCP-1 and abolished the increase in plasma MCP-1 of conscious, hypoxic rats; 2) prevented the MCP-1-induced degranulation of mesentery perivascular MCs and reduced the number of peritoneal MCs, and 3) blocked the leukocyte-endothelial adherence and the extravasation of albumin induced by topical ANG II in the mesentery. The effect at each site was sufficient to prevent the AMO-initiated inflammation of hypoxia. These results may explain the effectiveness of dexamethasone in the treatment of the systemic effects of alveolar hypoxia.     

Induction of angiogenesis by intraperitoneal injection of asbestos fibers

Tumors and activated macrophages release angiogenic factors that stimulate migration and proliferation of capillaries. We studied the development of angiogenesis before the appearance of mesotheliomas in C57B1/6 mice. Weekly i.p. injections of crocidolite asbestos fibers produced mesotheliomas after 30-50 wk. The initial histologic response to asbestos fibers was a nodular lesion on the peritoneal lining composed of clusters of fibers, activated macrophages, and proliferating mesenchymal cells. The earliest visible evidence of angiogenesis was seen surrounding 7% of these lesions 14 days after a single injection of 200 micrograms of crocidolite asbestos fibers. After six weekly injections, 30% of the lesions containing asbestos fibers were surrounded by a capillary network radiating toward the center of the lesion. Other mineral fibers, including chrysotile asbestos and fiberglass, also induced angiogenesis after six weekly injections. In contrast, only 8% of the lesions containing short asbestos fibers (90.6% less than or equal to 2.0 microns) and 9% of the lesions containing silica particles showed evidence of angiogenesis. We conclude that tumorigenic mineral fibers induce angiogenesis in the peritoneal lining, whereas nontumorigenic mineral particles or short asbestos fibers are less effective. Ingrowth of new blood vessels around clusters of asbestos fibers may facilitate the later emergence of mesotheliomas at these sites.     

Appearance of adipose cells in connective tissue at the implantation site of bone matrix gelatin and bupivacaine injection.

Two types of adipose cells were found in the connective tissue on day 7 after bone matrix gelatin (BMG) implantation and an injection of bupivacaine: mature adipose cells with a large lipid droplet (2-140 microns) and immature adipose cells with many small lipid droplets (0.1-2 microns). On day 10 after BMG implantation, typical adipose tissue was observed near the implant. The immature adipose cells had small, spherical mitochondria, glycogen granules and cytoplasmic microvesicles, and they might differentiate from undifferentiated mesenchymal cells in the connective tissue or the peripheral cells around the vessels as a white adipose tissue. These findings suggest that the differentiation of adipose cells in the connective tissue near heterotopic bone formation might be induced not only by mechanical and/or bupivacaine injury, but also by some factor or factors of the BMG.     

Morphological characteristics of the microvasculature in healing myocardial infarcts.

Myocardial infarction (MI) is associated with an angiogenic response, critical for healing and cardiac repair. Using a canine model of myocardial ischemia and reperfusion, we examined the structural characteristics of the evolving microvasculature in healing MI. After 7 days of reperfusion, the infarcted territory was rich in capillaries and contained enlarged, pericyte-poor "mother vessels" and endothelial bridges. During scar maturation arteriolar density in the infarct increased, and a higher percentage of microvessels acquired a pericyte coat (60.4 +/- 6.94% after 28 days of reperfusion vs 30.17 +/- 3.65% after 7 days of reperfusion; p<0.05). The microvascular endothelium in the early stages of healing showed intense CD31/PECAM-1 and CD146/Mel-CAM immunoreactivity but weak staining with the Griffonia simplicifolia lectin I (GS-I). In contrast, after 28 days of reperfusion, most infarct microvessels demonstrated significant lectin binding. Our findings suggest that the infarct microvasculature undergoes a transition from an early phase of intense angiogenic activity to a maturation stage associated with pericyte recruitment and formation of a muscular coat. In addition, in the endothelium of infarct microvessels CD31 and CD146 expression appears to precede that of the specific sugar groups that bind the GS-I lectin. Understanding of the mechanisms underlying the formation and remodeling of the microvasculature after MI may be important in designing therapeutic interventions to optimize cardiac repair.     

The density of myocardial and pericardial rat mast cells in isoproterenol-induced heart failure

Myocardial mast cells (MC) respond to cardiovascular pathology. The behavior of MC population in myocardium and pericardium of rats has been studied 24 h, 14, 28 and 60 days after two isoproterenol injections (at 24 h intervals). The extent of heart failure has been estimated by supersonic inspection 28 and 60 days after isoproterenol injections. The density of MCs of different degrees of maturity was estimated on paraffin sections stained with Alcian blue--Safranin. The MC density in myocardium of intact and experimental rats was relatively low: from 4 to 6 cells/mm2. The MC density in pericardium of intact rats was several times higher than in myocardium: 48.6 +/- 13.0 cells/mm2. In 24 h and 14 days after isoproterenol injections the pericardial MC density was 1.5 times higher than in control rats (P < 0.05) at the expense of increase in the number of mature MCs with Safranine-positive granules without the increase in the number of immature cells with Alcian blue-positive granules. In 28 days the pericardial MC density was 2 times higher than in intact rats (P < 0.05) at the expense of increase in number of immature and mature cells. In 60 days after isoproterenol injections the pericardial MC density and the ratio of immature and mature cells compared with control did not reach statistical significance. The changes in pericardial MC population corresponded to severity of heart failure according to functional indices. The findings show active reaction of pericardial MCs on myocardium dysfunction that stimulates the maturation of resident immature MCs in pericardium and migration of immature cells to pericardium of damage heart.     

Direct and prolonged effect of thyroliberin in ultra small doses on contractility of the white rat mesentery lymphatic vessels

The prolonged effect of thyroliberin in ULD after single intramuscular injection on contractility of lymphatic vessels directly was investigated. The controlled group of animals received injection of 0.2 ml of physiological solution. The experimental group was injected by 0.2 ml of thyroliberin in concentrations of 10(-10) or 10(-16) mol/l (1 x 10(-4) and 1 x 10(-10) micrograms/kg of the body weight respectively). During the experiment the animals were grouped in the following way: 1) directly after the injection; 2) 3 hours later; 3) on the 1st day and then every day during 2 weeks. Lymphatic vessels reactivity of the experimental animals as well as controlled was studied by application of thyroliberin and noradrenalin (in concentrations of 1 x 10(-16) and 1 x 10(-6) mol/l respectively) directly on mesentery lymphatic vessels. The lymphatic vessels reaction in control group of animals on the noradrenalin and thyroliberin was the same during the period of observation. Thyroliberin stimulated contractility at concentration of 1 x 10(-16) mol/l. The reaction of experimental group was dramatically decreased to 10(-4) mol/l on the 1st and the 3rd day (in the case i.m. injected concentration 1 x 10(-10) mol/l) and to 10(-10) mol/l (in the case of i.m. injected concentration 10(-16) mol/l). The lymphatic vessels reactivity to exogenous thyroliberin gradually established at the 6-7th days till 12th day from the moment of thyroliberin injection. The mechanisms of the action of thyroliberin in ULD are discussed.     

Kinetics of follicle growth in the prepubertal gilt.

Follicular growth rates were determined by histological examination of ovaries of five prepubertal gilts following treatment with the stathmokinetic agent colchicine. One ovary from each of five gilts was removed surgically and then colchicine (n = 3) or saline (n = 2) was infused i.v. Precisely 2 h after treatment with colchicine, the remaining ovary was removed. Ovaries were processed for histological analyses and sectioned at 10 microns; every twentieth section was stained with hematoxylin and periodic acid-Schiffe's. Sections were viewed with a projection microscope and individual follicles were measured. Eight classes of follicles were established such that the number of granulosa cells per cross section doubled in each class. Diameters of follicles for each class were as follows: 1) less than 106 microns, 2) 106-148 microns, 3) 148-206 microns, 4) 206-287 microns, 5) 287-400 microns, 6) 400-657 microns, 7) 657-1480 microns, and 8) 1480-3130 microns. A layer of thecal cells was first seen in class 2 follicles, and 76% of class 3 follicles had a thecal layer. Oocyte diameter increased through the first four classes and reached a maximum diameter of approximately 110 microns. Almost all follicles greater than 400 microns had an antrum. Preantral follicles had a lower mitotic index and a higher mitotic time and class time than antral follicles. Growth rate increased with increasing size of follicles. Preantral follicles grew at a rate of 5.2 microns/day whereas antral follicles grew at 313 microns/day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in lung permeability after chronic pulmonary artery obstruction.

We have shown that left pulmonary artery ligation (LPAL) in mice causes a prompt angiogenic response, with new systemic vessels from intercostal arteries penetrating the pleura within 6 days. Because angiogenic vessels in other organs have been shown to exhibit increased permeability, we studied vascular permeability (Evans blue dye extravasation, lung wet weight-to-dry weight ratio, and lavaged protein) in naive C57BL/6 mice and 4 h, and 14 and 21 days after LPAL (4-6 mice/time point). We also measured radiolabel clearance as an index of functional perfusion after LPAL. Tracer clearance from the left lung was maximal by 6 days after LPAL and not different from right lungs. Thus a functional vasculature is established before 6 days of LPAL that results in normal tracer clearance. By 21 days after LPAL, Evans blue-albumin was significantly increased in the left lung relative to both 4 h (no vasculature) and 14 days after LPAL. Only after 21 days of LPAL was left lung wet weight-to-dry weight ratio significantly different from naive lungs. Additionally, lavaged protein was significantly increased both 4 h and 21 days after LPAL relative to control mice. Thus, using three different methods, results consistently demonstrated increased permeability to protein and water 21 days after LPAL. Although changes in surface area of perfusion might affect the interpretation of these results, blood flow measured with labeled microspheres indicated no change in left lung perfusion between 14 and 21 days of LPAL. Thus the lung vasculature, remodeled as a consequence of chronic pulmonary artery obstruction, demonstrates increased water and protein permeability.