A new method of monitoring ventilatory activity in mussels and its use in a study of the ventilatory patterns of Mytilus edulis L.

An apparatus for measuring pumping rates in mussels, based on the delivery of exhaled sea water into a constant flow of fresh water, is described. When food is available, pumping is continuous for several days although there are signs of satiation after ≈ 1 wk. When food is withdrawn pumping does not cease for several hours especially in animals which have been food-deprived for some time before feeding. Food-deprived mussels often fail to pump for periods of > 24 h and ventilatory bursts are carried out at flow rates well below maximum. Shell valve movements and diffusion of oxygen through the gape between open valves can supplement ciliary ventilation.     

Effects of food availability on the valve movements and feeding behavior of juvenile Crassostrea Virginica (Gmelin). I. Valve movements and periodic activity

The effects of changes in the availability of algal food on the valve movements of juvenile Crassostrea virginica (Gmelin) were investigated. Individual oysters were subjected to different conditions: (1) unfed in filtered sea water; (2) continuously fed, in which oysters were given an algal suspension maintained at a constant level (50 000 cells/ml) by an automatic turbidostat; (3) discontinuously fed, where oysters were alternately fed with the turbidostat for 12 h and unfed for 12 h; and (4) batch fed, in which oysters were given specific daily rations at two ration levels (maximum and 40%). Valve movements were concurrently recorded and periods of activity (valves open) and quiescence (valves closed) measured.Oysters fed continuously remained open an average of 94.3% of the time, whereas unfed oysters were open 35.1% of the time. Discontinuously fed animals were open 95.7% of the time during the times of feeding, but 60.6% of the time during the unfed periods. Activity in the batch-fed oysters was related to the ambient algal concentration in that both the percentage time open/hour and the duration of valve openness decreased with decreasing concentration. Time series analysis of valve movements showed that oysters exhibited a 24-h periodicity of activity when subjected to a 24-h feeding schedule (discontinuous and batch groups) but exhibited no consistent periodicity under the continuous feeding or unfed conditions.     

Neuromuscular coordination and proprioceptive control of rhythmical abdominal ventilation in intactLocusta migratoria migratorioides

Summary Within the fifth abdominal segment of intact locusts a group of dorso-ventral expiratory muscles and one inspiratory antagonist display alternating ventilatory patterns of three basic types. Accelerated movements in the dorso-ventral plane are supported by isometric activity of the intersegmental muscles which prevent extensions in the longitudinal axis.The intersegmental coupling of ventilatory motor patterns is strict during strong ventilation and loose and more metachronal with weaker pumping movements.In resting animals ventilatory rhythms are discontinuous and the long intervening pauses are interrupted by miniature inspirations only. Pumping series have a tendency to prolong the later ventilatory cycles, and interfering rhythms of different pumping types occur. Low concentrations of atmospheric CO₂ up to 3 % do not accelerate ventilatory rhythms.Afferent activity from proprioceptors could be related to ventilatory motor bursts and stimulation of the sensory nerve produces inspiratory bursts via the segmental ganglion.The neuronal mechanisms of synergistic and antagonistic muscle control as well as the segmental and intersegmental coordination and the effect of autonomous ganglionic oscillators in ventilation are discussed.     

Temporal variation in the heart activity of Scrobicularia plana (Da Costa) in constant and tidal conditions

A marked pattern of short-term variation in heart activity is described in Scrobicularia plana (Da Costa). This is unique to Scrobicularia among the bivalves so far investigated. Heart activity, shell movement, and pumping have been recorded during suspension- and deposit-feeding activity and during inactivity. Heart activity in Scrobicularia responds markedly to gross changes in pumping level found during feeding, both heart rate and amplitude, as measured by impedance techniques, increasing rapidly when pumping commences after short periods of cessation. Heart amplitude falls rapidly and heart rate more gradually once pumping ceases. Scrobicularia shows considerable flexibility in behaviour under any given set of conditions. Tidal bradycardia occurs consistently in Scrobicularia only when the water level in the mud falls below the level of the animal in its burrow, pumping activity being maintained if surface water is present.     

Protocols for thawing and cryoprotectant dilution of heart valves

PURPOSE: To reduce the time taken for thawing and removal of cryoprotectant from heart valves. METHODS: Three sets of experiments were carried out using porcine heart valves. The valves in all three experiments were first exposed to 10% (v/v) dimethyl sulphoxide (DMSO) by a 2-step protocol. Outcome was determined after the various experimental treatments by monitoring the outgrowth of cells from valve leaflet explants. Experiment 1-Dilution protocol. Valves exposed to 10% DMSO were subjected to 4-, 2- or 1-step dilution to remove the DMSO. Experiment 2-Warming rate. The rate of warming was increased by reducing the volume of cryoprotectant medium in which the valves were frozen. Valves were exposed to 10% DMSO, frozen in different volumes (100, 50, 25 or 0 ml) of cryoprotectant medium, and warmed in a 37 degrees C water bath. The DMSO was removed by 4-step dilution. Experiment 3-Standard vs. Modified protocol. Valves were either frozen in 100 ml 10% DMSO, thawed, and subjected to 4-step dilution (Standard) or frozen in 50 ml 10% DMSO, thawed, and the DMSO removed by single-step dilution (Modified). RESULTS: Neither the rate of warming nor the rate of dilution of DMSO had any influence on the subsequent outgrowth of valve leaflet fibroblasts. There were no differences in the outgrowth of cells from valve leaflets cryopreserved by the Standard or Modified protocols. CONCLUSION: The time taken for thawing and dilution of heart valves could be reduced from >20 min to <10 min without detriment to the viability of the leaflet fibroblasts. This should have a positive impact on valve replacement surgery as the thawing and dilution of valves are typically carried out while the patients are on cardiopulmonary bypass.     

Activity and respiration in the anemone, Metridium senile (L.) exposed to salinity fluctuations

Activity and respiration in the anemone, Metridium senile (L.), were monitored under both constant and fluctuating salinity conditions. During constant exposure to 50% sea water it was found that the animals retracted the tentacles and that the rate of oxygen consumption decreased by ≈50%. The same response was elicited from animals in 100% sea water in a contracted state. Animals exposed to continually fluctuating salinities were found to retract the tentacles, contract the body wall, and produce amounts of mucus during periods of decreasing salinities. These reactions were reversed during exposure to increasing salinity. Oxygen consumption never ceased entirely in animals exposed to dilute sea water and it was found that during declining oxygen tension M. senile regulated its oxygen consumption until the environmental oxygen tension fell to ≈30% saturation.     

Ice-free cryopreservation of heart valve allografts: better extracellular matrix preservation in vivo and preclinical results

The purpose of this study was evaluation of an ice-free cryopreservation method for heart valves in an allogeneic juvenile pulmonary sheep implant model and comparison with traditionally frozen cryopreserved valves. Hearts of 15 crossbred Whiteface sheep were procured in Minnesota. The valves were processed in South Carolina and the pulmonary valves implanted orthotopically in 12 black faced Heidschnucke sheep in Germany. The ice-free cryopreserved valves were cryopreserved in 12.6?mol/l cryoprotectant (4.65, 4.65, and 3.31?mol/l of dimethylsulfoxide, formamide and 1,2-propanediol) and stored at ?80°C. Frozen valves were cryopreserved by controlled slow rate freezing in 1.4?mol/l dimethylsulfoxide and stored in vapor-phase nitrogen. Aortic valve tissues were used to evaluate the impact of preservation without implantation. Multiphoton microscopy revealed reduced but not significantly damaged extracellular matrix before implantation in frozen valves compared with ice-free tissues. Viability assessment revealed significantly less metabolic activity in the ice-free valve leaflets and artery samples compared with frozen tissues (P?<?0.05). After 3 and 6?months in vivo valve function was determined by two-dimensional echo-Doppler and at 7?months the valves were explanted. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (P?=?0.0403, P?=?0.0591). Histo-pathology showed significantly thickened leaflets in the frozen valves (P?<?0.05) and infiltrating CD3+ T-cells (P?<?0.05) compared with ice-free valve leaflets. Multiphoton microscopy at explant revealed reduced inducible autofluorescence and extracellular matrix damage in the frozen explants and well preserved structures in the ice-free explant leaflets. In conclusion, ice-free cryopreservation of heart valve transplants at ?80°C avoids ice formation, tissue-glass cracking and preserves extracellular matrix integrity resulting in minimal inflammation and improved hemodynamics in allogeneic juvenile sheep.     

Are heart valves from donors over 65 years of age morphologically suitable for transplantation?

Since there is no upper age limit for general organ donation, unlike heart valve donation, and since a quarter of all organ donors are 65 years and older, we examined whether the heart valves from these donors are suitable as allografts. In the period 1999–2004 the aortic valve and pulmonary valve of 100 organ donors above 65 years of age were examined to establish whether they would have been suitable as valve grafts. To compare the valve grafts above and below the age limit of 65 years, we used data on the aortic and pulmonary valves of 380 organ donors below the age limit in the same time period. Examination of the 200 heart valves showed that – just like valves from donors below the age limit – 100 of them would have met the medical quality standards for transplantation, which discriminate among optimal, suitable and unsuitable tissue morphology. The morphological suitability of the aortic valves decreases rapidly during the 4th decade of life and near to the age limit only 6% of them are accepted as grafts. The rate of potentially acceptable aortic valve grafts from organ donors aged over 65 years of 15% is also small. By contrast, the pulmonary valves are not affected by age-related tissue changes that might reduce their transplantability. The predominant majority (85%) of potential pulmonary valve grafts from organ donors over 65 years of age fulfilled the acceptance criteria, half of them (48%) even showing good tissue quality. In light of these results the age limit was raised to 70 years in 2005.     

Risk factors for rejection for morphological reasons of heart valves for transplantation

The study aim was to identify risk factors for morphological rejection of aortic and pulmonary valves for transplantation that could be used to optimize donor selection. The files of all Dutch heart valve donors, donating in a 2.5 years period, whose hearts were processed at Heart Valve Bank Rotterdam, were reviewed for all factors that could be relevant for valve rejection and related to outcome of morphological assessment of the valves. Valves were retrieved from 813 deceased Dutch donors, 24.1% also donating organs. For 797 aortic and 767 pulmonary valves, who met retrieval criteria, morphological assessment was done. 69.5% of aortic and 37.5% of pulmonary valves were considered unsuitable for transplantation at morphological assessment. Backward stepwise multivariate logistic regression analysis, showed age, cardiac cause of death, cerebrovascular accident as cause of death or in medical history, and number of cardiovascular risk factors in a donor to be independent risk factors for morphological rejection of aortic valves. Age, sex, weight >100 kg and ruptured aortic aneurysm as cause of death were independent risk factors for morphological rejection of pulmonary valves. Being an organ donor was an independent predictor of morphological approval of aortic and pulmonary valves, while hypertension was an independent predictor for morphological approval of aortic valves. Thus, independent factors were identified that are associated with morphological rejection of aortic and pulmonary valves for transplantation, and that could be used to optimize donor selection by preventing unnecessary retrievals, limiting costs, while improving yield per donor with minimal compromise for availability.     

Interaction of mineral elements in sea water and shell of oysters (Crassostrea virginica (Gmelin)) cultured in controlled and natural systems

The interaction is reported of selected chemical elements (cadmium, calcium, copper, iron, magnesium, manganese, strontium, and zinc) in cultured sea water, with soft tissues, prismatic calcite of the right valve, and foliated calcite of right and left valves of genetically similar American oysters, Crassostrea virginica (Gmelin) grown in a natural habitat and in two environmentally controlled experimental systems (flow-through and recycle). The addition of trace elements as algal nutrients in ambient sea water was reflected in higher concentrations of trace metals in shells and soft tissues of oysters grown in experimental systems. Calcium was relatively uniformly distributed in major regions of valves from the three habitats, even though its concentration fluctuated widely in sea water in experimental systems. Magnesium and strontium were most concentrated in valves of oysters grown in the recycle system (magnesium in the prismatic layer of the shell and strontium in the foliated calcite). Iron was uniformly distributed. Cadmium, copper, manganese, and zinc were most concentrated in the prismatic calcite of valves from the flow-through system. In soft tissues, calcium was more concentrated in oysters from experimental systems than in those from the natural habitat. Manganese was about equally distributed in soft tissues from the three habitats, whereas copper and iron were more concentrated in soft tissues in experimental systems than in the natural habitat, and were many times more concentrated in soft tissues than in valves from all three habitats. As concentrations of magnesium, strontium, mangenese, zinc, and cadmium increased in valves in experimental systems, pigmentation of valves decreased. The study confirmed the capacity of oysters to concentrate several elements in their valves as concentration of these elements increased in ambient sea water and disclosed the heterogeneous distribution of these elements in major regions of the valves.     

Heart valve cryopreservation: protocol for addition of dimethyl sulphoxide and amelioration of putative amphotericin B toxicity

Aim

To investigate the need for stepwise addition of dimethyl sulphoxide to heart valves and amelioration of putative amphotericin B toxicity.

Methods

There were four groups: an untreated control (Group 1) and three experimental groups. For the latter, porcine heart valves were exposed to the antibiotic/antimycotic mixture used for disinfecting heart valves in the Bristol Heart Valve Bank, for 24 h at 22 °C. Dimethyl sulphoxide (Me₂SO, 10% v/v) was added either in two steps (5% then 10%) (Group 2) or in a single step. For single-step addition, valves were either first placed in Hanks’ balanced salt solution for 10 min before transfer to the cryoprotectant solution (Group 3) or immersed directly in the 10% cryoprotectant solution (Group 4). The valve leaflets were dissected from the valves and frozen in 10% Me₂SO in multi-well tissue culture plates at 1 °C/min to −80 °C. After storage overnight, the valve leaflets were warmed at approximately 11 °C/min and the cryoprotectant was removed by single-step dilution in excess Hartmann’s solution. Each leaflet was then divided into four pieces, which were placed in separate wells of a culture plate. Outgrowth of cells from the explants was monitored daily and graded according to the extent of cell growth.

Results

After freezing and thawing, only 77% of the explants from valves placed directly into 10% Me₂SO (Group 4) showed outgrowth of cells after freezing compared with 89% with two-step addition of Me₂SO (Group 2) and 95% with one-step addition after the extra rinse in Hanks’ solution (Group 3) (χ², p = 0.001). 92% of unfrozen control explants showed outgrowth of cells (Group 1). Only 37% of Group 4 explants reached confluence compared with 63 and 56%, respectively, of Groups 2 and 3 explants (χ², p = 0.007). The rates of cell growth in Group 2 (two-step addition of Me₂SO) and Group 3 (one-step addition of Me₂SO with additional Hanks’ solution rinse) were similar and faster than the Group 4 (one-step addition of Me₂SO without the additional Hanks’ rinse).

Conclusion

Single-step addition of Me₂SO before freezing gave similar results to two-step addition provided an additional rinse in Hanks’ solution was introduced after exposure to the antibiotic/antimycotic mixture. This suggests that antibiotic/antimycotic carryover may have been harmful during freezing and that the additional rinse in Hanks before one-step addition of Me₂SO, and the 5% Me₂SO step in the two-step protocol, merely served to reduce this carryover.     

Adaptation of trout to seawater. Effects on sodium plasma concentration, gill exchange and intestinal transport]

10. Trout Salmo irideus are adapted to sea water (S W) within four weeks by submitting them to a stepwise increase in external salinity (successively: 1/3 SW, 1/2 SW, 3/4 SW, full SW). 20. In completely adapted individuals mean plasma electrolyte concentrations vary only slightly (by a few %) from one medium to another. Thus, the trout may be regarded as a euryhaline, eventually homeosmotic species. 30. With increasing outside salinity there is a progressive diminution in the overall gill permeability to ions which is suggested by saturation curves obtained for sodium fluxes (maximum at about 500 muEq/h. 100 g at 15 degrees C, for unshocked fish). 40. Disturbance of the animals provokes a striking elevation and desequilibrium in these exchanges and this in turn induces an abnormal rise in plasma concentrations and a subsequent failure to adapt to hypertonic media. 50. In vitro absorption of water and sodium by intestinal everted-sacs increases only after transfer to full sea water. Mucosal entry of ions into intestinal epithelial cells measured by the technique of Schultz et al. (1967), is diminished in sea water-adapted animal (by 42% in the case of sodium). 60. These results demonstrate that Salmo irideus possesses efficient osmoregulatory mechanisms which operate with minimal energy expenditure in hypertonic media.     

Laboratory feeding and egg production in the chaetognath Sagitta crassa Tokioka

Egg production, daily ration, and growth efficiency in Sagitta crassa Tokioka are described along with the morphological characteristics of the ovary and body during laboratory observations. Feeding by S. crassa in small containers containing 50 or 100 ml sea water and feeding rates bore a linear relationship to food concentration from 5 to 20 copepods per 100 ml. Among 100 animals individually isolated in containers, 3 animals were the longest lived and the best egg producers. One of these produced eggs on more than 30 consecutive days, producing almost 1000 eggs. Egg production showed periodic change at intervals from 7 to 10 days. Seminal vesicles also became periodically filled or empty. Fluctuations in ovary length and morphological changes of ovary and body were observed through life. Daily ration of S. crassa ranged from 8.7 to 10.4 prey per day or ≈ 60 μg in dry wt. The specific daily ration ranged from 0.347 to 0.568. S. crassa had a growth efficiency (dry-wt basis) which increased during the early life of the animal (≈28%), then decreased with age (22 → nearly 0%). Reproductive efficiency ranged from 7.0 to 16.4%, with a mean of 12.5%.     

Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01

An unsterile and continuous fermentation process was developed based on a halophilic bacterium termed Halomonas TD01 isolated from a salt lake in Xinjiang, China. The strain reached 80 g/L cell dry weight containing 80% poly(3-hydroxybutyrate) (PHB) on glucose salt medium during a 56 h fed-batch process. In a 14-day open unsterile and continuous process, the cells grew to an average of 40 g/L cell dry weight containing 60% PHB in the first fermentor with glucose salt medium. Continuous pumping of cultures from the first fermentor to the second fermentor containing the nitrogen-deficient glucose salt medium diluted the cells but allowed them to maintain a PHB level of between 65% and 70% of cell dry weight. Glucose to PHB conversions were between 20% and 30% in the first fermentor and above 50% in the second one. This unsterile and continuous fermentation process opens a new area for reducing the cost in polyhydroxyalkanoates production.     

Enkephalin-induced changes in ventilation and ventilatory pattern in adult dogs

We studied the changes in ventilation induced by intracisternal administration of enkephalins in four unanesthetized adult dogs. Instantaneous minute ventilation (VT/TT) decreased markedly after D-Ala-Met-enkephalinamide (DAME). Mean VT/TT decreased maximally by 20-50 min after DAME and lasted an additional 15-60 min; by 2 h, VT/TT had returned to base line. Four doses (5, 25, 60, and 125 micrograms/kg) of DAME were used, and the ventilatory response depended on the dose. Mean inspiratory time decreased but mean expiratory time and mean TT showed a marked prolongation. Periodic breathing (2-3 breaths separated by long apneic pauses) occurred in every study and the frequency of sighs increased considerably. All these ventilatory changes were reversed by low doses of naloxone or naltrexone; in addition, VT/TT increased well above base line after the administration of these antagonists. However, naloxone did not increase VT/TT when injected without prior administration of DAME. We conclude that 1) the decrease in VT/TT is due to a decrease in respiratory duty cycle; 2) periodic breathing and increased frequency of sighs constitute part of the changes in the ventilatory pattern induced by DAME; 3) a ventilatory withdrawal reaction may occur after a receptor-agonist interaction of short duration; and 4) although enkephalins can modulate ventilation and the breathing pattern in a major way, these data provide no evidence suggesting that this modulation is tonic.     

Using KCl to determine size at competence for larvae of the marine gastropod Crepidula fornicata (L.)

Competent larvae of the marine gastropod Crepidula fornicata (L.) were induced to metamorphose (i.e., lose the velum) by elevating sea-water [KCl] by 5–50 mM. The response was optimal at 15–20-mM elevations, at which 50% metamorphosis was obtained in <4 h. Larvae that did not metamorphose during brief exposures (1–5 h) to elevated [KCl] generally maintained the larval form following transfer to control sea water, suggesting that competent larvae must be continuously immersed in the test solutions for metamorphosis to occur. The smallest larvae to respond to elevated [KCl] had shell lengths of ≈700–800 μm, the range of shell lengths within which larvae of this species become responsive to natural inducers. All larvae >≈1125 μm shell length metamorphosed in response to increased [KCl]. Rearing temperature may affect the size at which larvae of this species become responsive to K⁺. CaCl₂ (20-mM concentration elevations), GABA (4×10⁻⁷, 4×10⁻⁶ M), and NaCl (10–20-mM concentration elevations) generally failed to trigger metamorphosis. Twenty-mM elevations of [RbCl] and [CsCl] induced 100% metamorphosis but the juveniles were immobile and died after several days. Elevating [KCl] appears to be a reliable way to assess competence and trigger metamorphosis in larvae of C. fornicata.     

The cardiac responses of the scallop Pecten maximus (L.) to respiratory stress

Heart activity of Pecten maximus (L.) has been recorded during various forms of experimentally induced respiratory stress. There was considerable variation in the responses of individual scallops but bradycardia generally occurred in response to all forms of respiratory stress, with the rate of fall in heart rate dependent upon the severity of hypoxia.When oxygen tension declined slowly in a closed respirometer there was regulation of both heart rate and oxygen consumption. The critical tension, P_c, for oxygen consumption lay between 70 and 80 mm Hg, and corresponded with a slight regulatory upswing of the heart rate, whereas the P_c for heart rate was much lower at 20–30 mm Hg. Sudden transfer to deoxygenated water for 3 h resulted in very rapid bradycardia and there was a rapid recovery and initial overshoot of the normal rate on return to well-oxygenated sea water. Aerial exposure for 3 h produced more gradual bradycardia followed by gradual recovery on return to sea water.The results of this work are compared in some detail with previous work on other species of bivalve from different geographical areas and habitats, and the mechanisms controlling cardiac and respiratory regulation are discussed. It is concluded that there are few clear-cut general differences between littoral and sublittoral species in their behavioural and physiological adaptations to hypoxia; the main distinguishing feature of littoral-adapted species is their ability to control air-gaping. Changes in heart activity generally indicate variations in metabolic rate, the speed at which the metabolic rate may be altered reflecting the degree of adaptation to the littoral environment.     

Analysis of regurgitant jets in natural and bio-prosthetic heart valves.

Artificial bio-prosthetic heart valves are prone to fatigue tearing, having a 50% failure rate in ten years. Tears in valves give rise to pulsing reverse flow back through the valve. This is termed regurgitant flow and the resultant jet of blood a regurgitant jet. The regurgitant volume of the jet during the pulsing cycle gives a measure of the severity of the valve defect and clinical significance. Hence, it is important for the cardiologists to be able to quantify this volume. Although the velocity of the regurgitant jet can be determined using Doppler ultrasound, the dimensions of the heart valve lesion cannot be measured directly; hence, the volumetric flow rate cannot be quantified accurately. At present the severity of the regurgitant jet is assessed qualitatively from the intrusion of the jet into the cardiac chamber. In the present study, classical mathematical theories of turbulent jets have been used to describe the velocity distributions for the types of jets expected in defective heart valves and these distributions have been verified experimentally. One of these models has been developed to enable the regurgitant volumetric flow through an axisymmetric orifice of unknown radius to be calculated from the velocity distribution of the jet. This relationship may be used in conjunction with ultrasound techniques to quantify the regurgitant volume within defective artificial heart valve implants. The present study shows that there is a significant difference in the velocity distributions in jets emanating from axisymmetric and high aspect ratio slots.(ABSTRACT TRUNCATED AT 250 WORDS)