The measurement of endometrial perfusion in norplant users: a pilot study.

The use of progestogens without oestrogen is commonly associated with irregular menstrual bleeding. Oestrogens and progestogens are both thought to influence endometrial perfusion; changes in endometrial perfusion may contribute to vascular fragility and breakdown. In this study, endometrial perfusion was measured using laser-Doppler fluxmetry in women in the secretory phase of the menstrual cycle before and 4-6 weeks after insertion of the low-dose long-acting levonorgestrel contraceptive implant system, Norplant. Endometrial perfusion was also measured in women exposed to Norplant for up to 19 months. There was no significant difference between endometrial perfusion in control cycles (27.2 flux units +/- 5.5, SEM) and at 4-6 weeks after Norplant insertion (16.3 flux units +/- 5.0), a time when irregular bleeding and spotting are common. Endometrial perfusion was no different from controls after longer periods of Norplant exposure (35.7 flux units +/- 7.2). No direct relationships between endometrial perfusion and plasma concentrations of ovarian steroid hormones were demonstrated. Short-term endometrial vasomotion was largely abolished during Norplant exposure.

Aquaporin-1 expression in visceral smooth muscle cells of female rat reproductive tract.

The aquaporins (AQ-s) are a group of intrinsic membrane proteins which facilitate movement of water across cell membranes; their recent identification in the kidney has led to the reappraisal of the mechanisms and pathways of water movement across epithelia. Aquaporin-1, (CHIP-28) is reported distributed in cardiac myocytes and vascular smooth muscle cells of large arteries. A related protein, AQ-4, has been identified in the sarcolemma of skeletal muscle fibres. We report aquaporin expression in the cell membrane of smooth muscle cells of the rat genital tract; fluorescence immunohistochemistry of rat uterine (fallopian) tube and vagina demonstrated AQ-1 in visceral smooth muscle of these tissues. In the uterine tube, AQ-1 labelling is most pronounced in the innermost longitudinal and the inner cells of the circular muscle layer and is absent from the outer longitudinal muscle layer of the myosalpinx. The possibility of a specific role for AQ-1 in tubal transport by altering the tubal luminal diameter during the estrus cycle is suggested.

Morphometric analysis of postnatal lung development in a marsupial: transmission electron microscopy.

Postnatal lung development in the tammar wallaby was investigated using transmission electron microscopy and stereological morphometry. Volume densities of interstitial, epithelial and endothelial tissue and capillaries in the parenchymal septa were measured as were surface densities of the airspaces and gas exchange capillaries. Absolute changes in these parameters were related to body mass. Three phases of development were identified. During the ectothermic period, in the first 70 days after birth when the lung was in the terminal air sac phase, the most marked change was an increase in volume density of septal interstitium. The transitional period between ectothermy and endothermy, between 70 and 180 days after birth, corresponded to the alveolar phase and was characterised by accelerated increase in air space surface area. Maturation of the parenchymal septa and establishment of the mature capillary system occurred largely after 180 days when endothermy was established. The anatomical diffusion factor in the tammar wallaby adult is similar to that for eutherians.

Oxygen transport and acid-base balance during exercise in the tammar wallaby.

Rates of oxygen consumption (V(O)2), body temperatures and pulmonary blood temperatures, blood gases and blood pH were measured for seven 4.9+/-0.8 (SE) kg tammar wallabies (Macropus eugenii) during rest and during treadmill hopping. For animals resting on the treadmill V(O)2 averaged 0.030+/-0.003 L min(-1). During hopping V(O)2 increased linearly with speed up to 2.5 m sec(-1). Above 2.5 m sec(-1) V(O)2 was independent of hopping speed and averaged 0.340+/-0.004 L min(-1). At rest, rectal temperatures and pulmonary blood temperatures averaged 36 degrees C. During treadmill hopping, rectal temperatures and pulmonary blood temperatures increased similarly, to 39 degrees C. The Pv(CO)2 increased and pHv decreased in proportion to the increased V(O)2. The Pa(CO)2 and pHa were not significantly changed from values for animals resting on the treadmill. Cardiac output (Vb) averaged 0.97+/-0.04 L min(-1) when the wallabies were at rest on the treadmill and increased linearly with treadmill speeds up to 2.5 m sec(-1). Above 2.5 m sec(-1) Vb was independent of hopping speed and averaged 2.9+/-0.04 L min(-1). When data for all speeds were combined, Vb increased linearly with V(O)2. Thus, in spite of their unique mode of locomotion wallabies have maintained relationships between pulmonary ventilation and V(O)2 and between Vb and V(O)2 that are similar to those reported for eutherian mammals.

Morphometric analysis of postnatal lung development in the tammar wallaby: light microscopy.

Postnatal growth of the lung in the tammar wallaby, Macropus eugenii, was investigated using morphometric techniques with light microscopy. Lung volume, parenchymal and non-parenchymal volume densities were measured. Volume densities of parenchymal airspace and tissue and non-parenchymal conducting airways and large blood vessels were determined. Lung volume and all the other parameters that were measured showed a biphasic increase in relation to increase in body mass. All parameters, with the exception of airway volume, increased relatively slowly in relation to increase in mass in the first 70 days after birth, when the pouch young are ectothermic. Between 70 and 180 days, during the period of transition from ectothermy to endothermy, the parameters increased more rapidly, suggesting accelerated lung growth in preparation for the extra metabolic demands associated with the establishment of thermoregulatory control in the pouch young. Specific lung volume in the adult tammar is lower than that of eutherians of equivalent mass, however, the parenchymal volume is relatively high.

Morphometric estimate of gas-exchange tissue in the new-born tammar wallaby, Macropus eugenii.

The lung of the new-born marsupial is at the terminal air sac stage of development. The maturational status of the lung of new-born tammar wallaby was assessed using established morphometric techniques and the results were compared with data from a morphometric study of the lung of the rat. Volume densities of the parenchyma and non-parenchyma, conducting airways and blood vessels, the relative volumes of airspace and tissue, the thickness and the composition of the septa differed between the two species. In addition the volume of capillaries and the surface area of the effective gas-exchange tissue was greater in the new-born rat than in the new-born tammar pouch young. The lung of the new-born tammar appears to be at an earlier phase of the terminal air sac stage than that of the new-born rat. Lung development up to birth appears to be commensurate to the metabolic needs of the organism at birth.

Human endometrial perfusion after tubal occlusion.

We examined variations in human endometrial microvascular perfusion across one menstrual cycle in women who had undergone tubal ligation and did not report unusual menstruation. Endometrial red blood cell flux was monitored by laser Doppler fluxmetry via a fibreoptic probe atraumatically inserted transvaginally into the uterus of each of 13 conscious volunteers. The observations obtained have been compared with those previously reported from a matched control group of women [B.J. Gannon et al., Hum. Reprod., 12, 132-139 (1997)]. Women who had undergone tubal occlusion for sterilization exhibited greater endometrial perfusion during menstruation (cycle days 0-5), at the time of ovulation (cycle days 13-16) and in the late secretory phase (cycle days 23-28) than occurred in controls. In addition, vasomotion in the study group was lower than that in controls in the early and late secretory phase (cycle days 17-22 and 23-28). Tubal occlusion appeared to alter endometrial perfusion. It is possible that the reported menstrual changes in women following tubal ligation are a consequence of altered endometrial perfusion; a possible causative relationship is discussed.

PIP: Tubal ligation has been associated with an increase in postsurgical dysmenorrhea or menorrhagia. The present study used laser Doppler fluximetry to measure endometrial perfusion in 13 South Australian women who had undergone tubal ligation an average of 5.7 years earlier and did not report subsequent menstrual dysfunction. This technique provides a measure of the flux of red blood cells through a small sphere of tissue immediately adjacent to a probe placed over the endometrium. Measurements were taken once a week over 4 weeks (usually 1 menstrual cycle) and were compared with those obtained earlier by the authors from a matched control group of 19 women. Women who had undergone tubal occlusion exhibited greater endometrial perfusion than controls during menstruation (cycle days 0-5), at the time of ovulation (cycle days 13-16), and in the late secretory phase (cycle days 23-28). In addition, vasomotion was lower in the study group than among controls in the early (cycle days 17-22) and late (cycle days 23-28) secretory phase. Further exploration of the potential pathophysiology associated with tubal sterilization is warranted.

Endometrial perfusion across the normal human menstrual cycle assessed by laser Doppler fluxmetry.

This study investigated variations in microvascular perfusion of human endometrium across the menstrual cycle, using a laser Doppler technique to assess red blood cell (RBC) flux. Endometrial RBC flux was monitored by laser Doppler fluxmetry via a fibre optic probe inserted transvaginally into the uteri of 19 conscious normal volunteer women, on four occasions at weekly intervals over one menstrual cycle. Regional variation in RBC flux was investigated in 16 surgical patients under general anaesthesia and in five excised uteri. Endometrial perfusion exhibited short-term temporal variations consistent with the cardiac cycle and often also showed vasomotion (5-12 cycles/min). Mean endometrial perfusion differed between phases of the menstrual cycle in conscious women, being highest during early proliferative and early follicular phases. There were no significant regional differences in local mean endometrial perfusion in anaesthetized patients. No evidence of endometrial ischaemia/reperfusion episodes was found in any subject using this technique. This study provides benchmark data of variations in RBC flux per unit volume of tissue in the luminal approximately 1 mm of endometrium, across the normal human menstrual cycle. Flux values were highest at times associated with endometrial growth and preparation for implantation, indicating that RBC flux may be a useful parameter for assessment of endometrial physiology.

Postnatal development of the lung parenchyma in a marsupial: the tammar wallaby.

BACKGROUND: Marsupials are born at an early stage of development, and lung development from an air-sac stage to maturity occurs in the air-breathing environment, the pouch. METHODS: The morphology of the lung parenchyma in pouch young of the tammar wallaby, Macropus eugenii, at four stages of development (0, 30, 70, and 180 days) after birth and in the adult animal was investigated using light, scanning, and transmission electron microscopy, and developmental changes were documented. RESULTS: The lung at birth is at the terminal-sac stage of development. In the first 70 days, development involves tissue proliferation and septal development leading to subdivision of the terminal sacs, terminal-sac expansion, and septal thinning. Between 70 and 180 days, when the pouch young make the transition from ectothermy to endothermy, remodelling of the septa and microvascular maturation occurs. There is an overlap with further tissue proliferation and terminal-sac subdivision and expansion. Alveoli are found at 180 days, but they are small in comparison to those in the adult lung. CONCLUSIONS: Lung growth occurs slowly, and the lung remains at the terminal-sac stage of development during the ectothermic period. True alveoli appear only during the transition from ectothermy to endothermy.

Central cardiovascular shunts in the perinatal marsupial.

BACKGROUND: Marsupials are born at an early stage of development after a short period of gestation. In this study the nature and timing of closure of the central cardiovascular shunts was investigated. METHODS: Light and scanning electron microscopy were used to determine changes in central cardiovascular shunts in eight marsupial species with gestation periods of between 12.5 and 36.5 days and birth weights ranging from 12.5 mg to 740 mg. Laboratory mice with a birth weight of about 1,000 mg and a gestation period of 21 days were included for comparison. RESULTS: Marsupials have a ductus arteriosus and an interatrial communication. The former closes rapidly after birth in the marsupial; however the interatrial communication is in the form of a fenestrated septum, which closes as a result of tissue proliferation over a period of days after birth. An additional central shunt, an interventricular foramen, was found to persist in three species for a short time after birth. In one species, the eastern native cat, Dasyurus viverrinus, which has a gestation period of about 19 days and low birth weight of about 12.5 mg, in addition to the two common shunts there was a large interventricular communication and septation of the outflow tract was incomplete. CONCLUSION: In adapting from intra-uterine life, it seems that marsupials have adopted different, but equally effective strategies, with regard to the circulatory system.

The effects of radiation on the contractile activity of guinea pig mesenteric lymphatics.

In order to assess the effects of irradiation on lymphatic function, the contraction frequency and maximum and minimum diameters of guinea pig mesenteric collecting lymphatic vessels were measured in vivo 4 hours after 1000 rads of abdominal irradiation. The mean contraction frequency for lymphatics from irradiated guinea pigs (7.6 +/- 0.7) was significantly higher than for normals (non-irradiated) (4.7 +/- 0.7) during an initial control observation period, but there was no difference in maximum or minimum diameter between the two groups during this period. Topical application of 10(-4) M noradrenaline (NA) significantly increased contraction frequency in both groups; lymph vessel diameter significantly decreased after NA in irradiated, but not in normal guinea pigs. Intravenous infusion of calcium dobesilate (200 mg/kg) caused a significant increase in the contraction frequency of lymphatic vessels in both normal (to 9.4 +/- 1.5) and irradiated (to 9.8 +/- 1.2) animals, but diameter was not significantly altered. Thus, lymphatic vessels from irradiated guinea pigs were still responsive to exogenous stimuli 4 hours post-irradiation and were initially pumping more actively than those from normal guinea pigs, presumably in response to radiation-induced edema. They also exhibited a supersensitivity to the vasoconstrictive effects of NA, perhaps due to an alteration of the pacemaker or smooth muscle cells by irradiation.

Effect of carbon monoxide intoxication on skin perfusion in anaesthetized rats.

Carbon monoxide intoxication decreases systemic blood pressure and peripheral resistance. In order to assess the role of the skin in this process, we measured the perfusion of hind limb shaven skin in anaesthetized rats during acute moderate carbon monoxide intoxication. At a steady blood level of 25% carboxyhaemoglobin, the red blood cell flux was measured as an index of tissue perfusion, using laser Doppler fluxmetry. The mean blood pressure decreased by 30% during carbon monoxide exposure, but there was no change in mean red blood cell flux of the hind limb skin microvessel bed. Thus, rat hind limb skin perfusion was not affected by acute moderate steady state carbon monoxide intoxication.

Noradrenergic and peptidergic innervation of the extrinsic vessels and microcirculation of the rat cremaster muscle.

The noradrenergic and peptidergic innervation of the extrinsic vessels and microcirculation of the rat cremaster muscle was examined. Catecholamine-containing nerves were identified histochemically by glyoxylic acid-induced fluorescence and tyrosine hydroxylase immunoreactivity (TH-IR). The extrinsic pudic-epigastric artery and vein as well as the entire intramuscular arteriolar network was innervated by noradrenergic axons. The capillaries and intramuscular venules of the cremaster muscle were devoid of a noradrenergic innervation. Immunohistochemical double-labeling demonstrated that most, if not all, of the TH-IR axons also possessed neuropeptide Y immunoreactivity (NPY-IR), implying colocalization of the norepinephrine and NPY in the perivascular nerves. No vasoactive intestinal peptide immunoreactivity (VIP-IR) was found, except for occasional VIP-IR axons associated with the pudic-epigastric artery. Substance P immunoreactive (SP-IR) axons formed a sparse plexus around the arteries and larger arterioles. Calcitonin gene-related peptide immunoreactivity (CGRP-IR) had a similar distribution to the SP-IR axons. CGRP-IR was also observed in axons alongside some smaller arterioles and capillaries. The extrinsic vessels and intramuscular arteriolar network of the rat cremaster muscle are innervated by noradrenergic axons which contain NPY and by presumed sensory nerves containing SP and/or CGRP. Both types of nerves may contribute to regulation of microvascular function.

Changes in metabolic rates and blood respiratory characteristics during pouch development of a marsupial, Macropus eugenii.

Oxygen levels in the pouch of the tammar wallaby, Macropus eugenii, have been related to the metabolic rate of the young at pouch temperatures and the oxygen transport characteristics of its haemoglobin. Until 100 days of age, pouch young show mass-specific rates of oxygen consumption lower than predicted on the basis of body mass. This contrasts with data presented for laboratory mice. Oxygen levels in the pouch approximate atmospheric levels for the first 20 days and at times approaching pouch evacuation at 200 days. However, levels of around 17-18% occur at about the mid-point of pouch life. The oxygen-haemoglobin equilibrium curve shows a high P50 (up to 50 Torr at 36 degrees C) in the neonate, but after 2-3 weeks of age levels drop to those representative of the adult. Concentrations of 2,3-diphosphoglycerate in early pouch young are higher than in the adult animal and peak at around 30 mumol/g Hb. There is a significant decrease in the concentration of this compound with the age of the young.

Changes in macromolecular permeability of microvessels in rat small intestine after total occlusion ischemia/reperfusion.

We investigated the role of free radicals in total occlusion ischemia/reperfusion injury (IRI) of rat small intestine (SI), as measured by changes in macromolecular permeability following pretreatment with free radical blockers or scavengers. We also compared susceptibilities to IRI both along the length of the small intestine, and between tissue layers of its wall. In anaesthetised rats, loops of SI were made ischemic for 1-15 min, followed by reperfusion for 0-10 min. Five min prior to killing, 250 mg/kg fluorescein isothiocyanate bovine serum albumin (FITC-BSA) was injected i.v. Tissue was formalin-fixed, sectioned and examined by fluorescence microscopy. FITC-BSA fluorescence was restricted to discrete focal spots (i.e. blood vessel lumina) in control intestine. In tissue made ischemic, but not reperfused, there was little extravasation of FITC-BSA. In reperfused tissue, there was extensive tracer leakage from vessels, predominantly in the mucosa and submucosa. There was also histological evidence of villus tissue damage. The jejunum was more susceptible to IRI than was the mid small intestine which was more susceptible than the ileum. Superoxide or hydroxyl generation blockers or scavengers (allopurinol, dimethyl sulphoxide or superoxide dismutase) failed to prevent extravasation of FITC-BSA in the mucosa and submucosa of reperfused tissue, indicating that these radicals appear to have no crucial role in total occlusion IRI of rat SI.

Do cardiorespiratory frequencies show entrainment with hopping in the tammar wallaby?

Breathing, heart and gait frequencies, tidal volume, cardiac output, and rates of oxygen consumption were measured in tammar wallabies (Macropus eugenii Desmarest) hopping on a treadmill. At speeds greater than 1.6 ms-1 the rate of metabolic power consumption was independent of hopping speeds. Blood lactate levels within the speed range where VO2 was independent of speed showed a mean increase of 4.8 mmol l-1. During bipedal hopping, the frequencies of breathing and limb movement are phase-locked in the ratio of 1:1. Inspiration begins as the animal leaves the ground and may be a passive process driven by a visceral piston. A relatively large central tendon in the diaphragm may correlate this function. Unlike breathing frequencies, cardiac frequencies show no entrainment with hopping. The site of dissipation of the presumed large arterial pressure excursion is unknown.

Preparation of cytoplasmic bodies (cytospheres) from isolated hepatocytes and their biochemical properties.

The controlled centrifugation of isolated rat hepatocytes at 260 000 g results in the formation of membrane-bounded cell fragments that we have termed 'cytospheres'. A method is described for the isolation of these cytospheres. Cytospheres are spherical, have a mean diameter of 9.2 +/- 3.2 microns (SD) and a protein content of 225 +/- 12 mg/g wet wt. About 3% of the protein from the original isolated hepatocyte suspension is recoverable. Transmission electron microscopy (TEM) shows cytospheres to possess a trilaminar membrane, and a finely granular hyaloplasm generally devoid of organelles, filaments and microtubules. Freeze-fracture studies reveal a membrane structure typical of a plasma membrane. Ouabain and wheat germ agglutinin (WGA)-binding studies indicate that the original orientation of the plasma membrane is maintained throughout the formation of the cytospheres. The cytospheres have also been characterized biochemically. Cytospheres are enriched in the enzymes normally associated with the hyaloplasm, whereas the activities of enzymes localized in organelles are greatly diminished. Lipid analysis of the cytosphere membrane indicates that it is derived from the plasma membrane of the hepatocyte. Cytospheres are sensitive to changes in the osmolarity and ionic composition of their environment. Cytospheres should therefore prove a useful preparation for the study of hyaloplasm metabolism and of plasma membrane receptor and permeability properties.

A technique for the insertion of an implantable portal system for the long-term epidural administration of opioids in the treatment of cancer pain.

The feasibility of using a subcutaneously implanted portal system attached to a conventional 16-gauge epidural catheter has been evaluated in 50 patients with sever pain associated with cancer. This technique allowed for the percutaneous epidural administration of morphine at 8-12-hourly intervals for pain control. The mean duration of implantation was 12 weeks and the longest period a portal remained in situ was 36 weeks. Five portals had to be removed for various reasons. The injection system has blocked on eight occasions due to catheter blockage (six times) and portal blockage (two occasions). These patients have continued to obtain excellent analgesia when either catheter or portal were replaced. In a cadaver, 300 injections were simulated using either 22-gauge Huber point needles or disposable needles (25 gauge) and the injectate examined by both light and scanning electron microscopy. Both needle types resulted in particulate contamination which was greater with the recommended Huber point needles.

Vascular perfusability and capillary macromolecular permeability in the mechanically induced rabbit hydrosalpinx.

The mechanically induced rabbit hydrosalpinx, a frequently studied animal model of human hydrosalpinges, was examined to determine the variations, in vascular perfusion and capillary albumin permeability, which occur in hydrosalpinges. At laparotomy, 4 adult female virgin rabbits underwent isthmic and ampullary occlusion with small tantalum clips. 4 weeks later, fluorescein isothiocyanate-labelled bovine serum albumin (FITC BSA: molecular weight 67,000) was injected intravenously 5 min before oviduct excision. Examination of tubal sections by incident light fluorescent microscopy demonstrated poor interplical vascular perfusability and markedly reduced interplical capillary permeability to FITC BSA in both isthmic and ampullary segments of hydrosalpinx. These observations imply that, in the experimental rabbit hydrosalpinx, interplical deciliation is probably vascular in origin; furthermore the marked decrease in capillary macromolecule permeability may explain the serous fluid collection within the hydrosalpinx. Poor fecundity following microsurgical restoration of tubal patency in hydrosalpinges is possibly due to the failure of this decrease in submucosal capillary perfusability and macromolecular permeability to resolve.

Variations in rabbit oviduct microvascular architecture after ovulation induced by hCG.

Rabbits were induced to ovulate by injection with hCG and vascular corrosion casts of the oviducts were examined by scanning electron microscopy after 24 and 48 h, when the ova would be expected to be at the ampullary-isthmic junction, and traversing the isthmus respectively. At 24 h there was dilatation of the isthmic subserosal venous plexus. It is suggested that venous distension in the isthmic subserosal venous plexus, due to raised venous pressure or to reduced venous wall tone, may occlude the isthmic lumen to ova, and thus explain the known pre-isthmic delay in ovum transport. By 48 h after hCG, distension was no longer evident, consistent with the possibility of ovum transport.