An alternative model to study the association of rainbow trout (Oncorhynchus mykiss L.) pathogens with the gill tissue

This study describes the development of a trout gill perfusion model, consisting of an excised branchial arch from rainbow trout (Oncorhynchus mykiss L.), perfused via the afferent branchial artery and suspended in a circular organ chamber filled with Ringer solution. Different perfusion fluids were tested: Ringer, Cortland, Ringer + procaine, Ringer + adrenalin, Cortland + procaine, Cortland + adrenalin and Cortland + dextran 1%. The latter perfusion fluid proved to be satisfactory, maintaining the gill tissue in a healthy condition outside the body of the fish for at least 180 min. Using this model, the interaction of damaging agents with the trout gill tissue may be studied under carefully controlled conditions. The trout gill perfusion model leads effectively to a reduction in the number of experimental animals to be used and also involves an elimination of pain and/or suffering, which is as good as complete.     

Arterial perfusion of frog tongue for intracellular recording of taste cell receptor potential

The frog tongue was perfused through its artery with a Ringer solution using a peristaltic pump, and a method was developed to record stable intracellular receptor potentials of taste cells. Perfusing at 0.05 ml/min with a Ringer solution containing 5% dextran did not cause tongue edema, but perfusing at the same rate with Ringer without dextran caused edema. After perfusion at 0.05 ml/min with 100 mM K Ringer, the membrane potential of taste cells gradually decreased and reached a constant level in about 30 min, indicating that the intercellular fluid of the tongue could be replaced within this time period. While the artery of the frog tongue was perfused at 0.05 ml/min with Ringer containing 5% dextran, intracellular receptor potentials of taste cells elicited by four basic taste stimuli (1 M NaCl, 10 mM quinine-HCl (Q-HCl), 1 mM acetic acid and 1 M galactose) were similar to those obtained from the control taste cells under normal blood flow.     

Synthesis of stress-induced protein in isolated and perfused rat hearts

Isolated and perfused rat hearts were examined by two-dimensional gel electrophoresis and liquid scintillation counting for alterations in protein synthesis following incubation with L-[3H]leucine at 0.5-2.5, 2.5-4.5, or 4.5-6.5 h of perfusion. When 35-mL volumes of three different buffers were recycled for a 2-h period from 0.5 to 2.5 h, by fluorography little effect was seen on the normal patterns of protein synthesis and there was a moderate synthesis of a stress-induced protein (heat-shock protein) with a molecular mass of 71 X 10(3) daltons (SP71). However, hearts perfused with Krebs-improved Ringer 1 bicarbonate had the highest incorporation of L-[3H]leucine. When buffers were recycled for 30-min periods from 0.5 to 2.5 h, SP71 was synthesized in hearts perfused with Krebs-Henseleit original Ringer bicarbonate. Hearts perfused in a similar fashion with Krebs-improved Ringer 1 bicarbonate had the lowest incorporation of label into SP71 and in fact SP71 was undetectable on fluorograms. Overall protein synthesis was decreased and the ratio of SP71 to the total synthesis was increased at 4.5-6.5 h of perfusion when 35-mL volumes of Krebs-improved Ringer 1 bicarbonate was recycled for 2-h periods. A similar result was observed at 2.5-4.5 h of perfusion when this buffer was recycled for either the duration of the experiment or 30-min periods.     

Enhanced Human Tissue Microdialysis Using Hydroxypropyl-?-Cyclodextrin as Molecular Carrier

Microdialysis sampling of lipophilic molecules in human tissues is challenging because protein binding and adhesion to the membrane limit recovery. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) forms complexes with hydrophobic molecules thereby improving microdialysis recovery of lipophilic molecules in vitro and in rodents. We tested the approach in human subjects. First, we determined HP-ß-CD influences on metabolite stability, delivery, and recovery in vitro. Then, we evaluated HP-ß-CD as microdialysis perfusion fluid supplement in 20 healthy volunteers. We placed 20 kDa microdialysis catheters in subcutaneous abdominal adipose tissue and in the vastus lateralis muscle. We perfused catheters with lactate free Ringer solution with or without 10% HP-ß-CD at flow rates of 0.3–2.0 µl/min. We assessed tissue metabolites, ultrafiltration effects, and blood flow. In both tissues, metabolite concentrations with Ringer+HP-ß-CD perfusate were equal or higher compared to Ringer alone. Addition of HP-ß-CD increased dialysate volume by 10%. Adverse local or systemic reactions to HP-ß-CD did not occur and analytical methods were not disturbed. HP-ß-CD addition allowed to measure interstitial anandamide concentrations, a highly lipophilic endogenous molecule. Our findings suggest that HP-ß-CD is a suitable supplement in clinical microdialysis to enhance recovery of lipophilic molecules from human interstitial fluid.     

Oxygen transfer, gill resistance and structural changes in rainbow trout (Salmo gairdneri, Richardson) gills perfused with vasoactive agents

1. Gill resistance (Rg) and oxygen transfer (To2) were measured in perfused rainbow trout gills. After perfusion the gills were analysed morphometrically. 2. Rg increased with 40% and To2 decreased with 91% during 60 min perfusion without vasoactive substances in the perfusion Ringer. 3. In presence of 10 micro M adrenaline both Rg and To2 remained stable at their starting levels throughout the experimental period (60 min). 4. With 0.2 micro M acetylcholine and 10 micro M adrenaline in Ringer Rg increased nearby to the same extent as in perfusions without vasoactive agents, while To2 was not significantly affected. 1 micro M acetylcholine increased Rg with 76% and decreased To2 with 51%. 5. The changes in Rg and To2 could be explained by structural changes in the secondary lamellae.     

On the interaction of NH+4 and Na+ fluxes in the isolated trout gill.

1. Sodium influx was measured in isolated, previously perfused gill arches of rainbow trout, Salmo gairdneri, by measuring incorporation of 22Na into gill tissue following timed exposure to a 1 mM 22NaCl medium. Transport rates approximated those estimated for intact fish and were linear for at least one min. 2. NH4Cl-containing perfusates at pH 7 and 8 stimulated Na+ influx equally, indicating that only ionized ammonia is important in the transport process. A Na+/NH4+ exchange at basal and/or lateral membranes of the transporting cells is suggested. 3. Low-sodium Ringer perfusate augmented Na+ influx; in one group of gills the transport rate was more than double that of NaCl Ringer controls. The increase in transport induced by internal NH4+ was not additive with the low sodium augmentation. A reduction in intracellular (Na+) is postulated as the mechanism operating in both cases. 4. Ouabain had no appreciable effect on Na+ influx, either with or without NH4+ in the perfusate. Diamox partially blocked the augmented Na+ influx induced by NH4+. Amiloride completely inhibited Na+ influx, both with and without NH4+ in the perfusate.     

Transfer of magnesium across the perfused choroid plexus of sheep

Isolated perfused choroid plexus preparations from sheep were used to study the effects of low concentrations of magnesium in the perfusion fluid on the transfer of magnesium into choroid plexus fluid (CPF). A perfusion fluid of similar electrolyte composition to sheep blood resulted in CPF similar to ventricular cerebrospinal fluid at a rate of 2.2 microliter min-1 mg-1 dry choroidal tissue. Decreasing the concentration of magnesium in the perfusion fluid caused a fall in the concentration of magnesium in the CPF, although it remained higher than in the perfusion fluid. The rate of transfer of magnesium from the perfusion fluid to the CPF decreased in the presence of high levels of potassium in the perfusion fluid. But decreasing the concentration of calcium in the perfusion fluid had no effect on magnesium transfer rates. These results suggest that the ability of the choroid plexus to transport magnesium against a concentration gradient is an important control of the concentration of the cerebrospinal fluid. However, this ability is insufficient to maintain cerebrospinal fluid concentrations of magnesium at normal levels when the blood magnesium concentration is below about 0.5 mmol l-1.     

Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models

Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient-rich oxygenated blood through the vasculature to support cell metabolism within most cell-dense tissues. Since scaffold-free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue-like structures, we generated a generalizable biofabrication method resulting in self-supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO-A5 osteoblast-based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico.     

Effect of a hypertonic mannitol solution on the peri-infarct area of an isolated frog heart

The influence of the hypertonic (3%) solution of mannitol on the process of the excitability recovery (in the near necrotic zone) appeared as a result of the necroses tissue effect on the ventricle has been investigated in the experiments on the isolated frog's heart. The analogous perfusion was conducted by Ringer solution in the control experiments. The process of isopotential phase S-T recovery was practically the same at heart perfusion with Ringer solution or with mannitol. Simultaneous registration of ventricular mechanogram showed the pronounced negative inotropic effect of mannitol. It is suggested that the positive effect of mannitol which has been noted under clinic conditions during the treatment of ischemic disease, doesn't connect with the effect of hypertonic solution on the cells excitability of periinfarcted (near necrotic) zone.     

Temporal Profile of Levels of Monoamines and Their Metabolites in Striata of Rats Implanted with Dialysis Tubes

We have measured the levels of monoamines and their metabolites in rat striata implanted with a dialysis tube, in contralateral nonimplanted striata, and in dialysates obtained from the dialysis tube. The perfusion was done with Ringer solution. The animals were perfused either for a continuous period of 7 h at 1 day after implantation or for periods of 2 h on days 1, 4, and 7 after implantation. In animals perfused for 7 h, levels of monoamine metabolites in dialysates remained stable for the first 4 h of perfusion, but a reduction was observed during the last 3 h. In animals perfused for 2 h on days 1, 4, and 7 after implantation, we observed a progressive reduction in levels of metabolites in dialysates with respect to the first day of perfusion. The levels of dopamine and its metabolites in the striata in which a dialysis cannula had been implanted showed a progressive reduction during the period postimplantation comparable to that observed in dialysates. The levels of 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid were elevated 24 h after implantation in the implanted striata with respect to the contralateral nonimplanted striata, but 7 days after implantation, the levels of dopamine were decreased in the implanted striata, and the levels of metabolites were unchanged.     

THE SELECTIVE ABSORPTION OF POTASSIUM BY ANIMAL CELLS : II. THE CAUSE OF POTASSIUM SELECTION AS INDICATED BY THE ABSORPTION OF RUBIDIUM AND CESIUM.

1. Frog muscles perfused with Ringer solution in which potassium chloride has been replaced by an equivalent amount of rubidium or cesium chloride take up rubidium or cesium and incorporate them into the tissue substance in such form as to be retained during a subsequent perfusion with potassium-free Ringer solution, provided the muscles contract during the first perfusion. Retention of rubidium or cesium by a resting muscle does not occur. 2. Rats on synthetic diets, adequate in all respects except that potassium was replaced by an equivalent amount of rubidium or cesium, died after a period varying from 10 to 17 days with characteristic symptoms including tetanic spasms. Muscle, heart, liver, kidney, spleen, and lung tissues were then found to contain significant amounts of rubidium or cesium. The concentration of these metals in the muscle amounted, in some cases, as shown by a spectroscopic estimation, to about half the concentration of potassium normally found in mammallian muscle. 3. The results are regarded as tending to confirm the theory that the peculiarities in the physiological effects of potassium, including the facility with which it is "selected" by living cells in preference to sodium, are related to the electronic structure of the potassium ion as compared with that of similar ions. The possible relationship of the comparative migration velocity, a function of the electronic structure, to physiological effects is suggested.     

Effects of hypothermic perfusion of kidneys on tissue and mitochondrial phospholipids

The changes in the level of phospholipids in kidney tissue and isolated mitochondria from dog kidneys perfused hypothermically (6-8 degrees C) for 1, 3, and 5 days were compared. Following 1 day of perfusion there was no change in total tissue phosphatidylserine (PS), a 25% decrease in the level of phosphatidylethanolamine (PE), and a 16% decrease in phosphatidylcholine (PC). No further decrease was observed with longer perfusion times. In fact, an increase in the level of PE occurred between the third and fifth days. Mitochondria isolated from perfused kidneys also showed a slight decrease in PE and PC following 1 day, no further change at 3 days, and an increase at Day 5. The loss of tissue phospholipids does not appear related to the viability of perfused kidneys. The major loss occurs within 1 day of perfusion and kidneys perfused up to 3 days are fully viable. Five-day perfused kidneys are nonviable, but show no greater loss of phospholipids than the viable 1- or 3-day perfused kidneys.     

用于核磁共振检测的离体心脏灌流模型

本报告是关于核磁共振研究生理学问题的灌流模型。我们解决了Ｌａｎｇｅｎｄｏｒｆｆ灌流装置用于核磁共振研究时存在的问题,如远距离灌流、保温、保氧和灌流液回收等,建立了稳定的可供核磁共振测量用的灌流模型。在这个装置中离体大鼠心脏的节律性活动可维持９０ｍｉｎ以上。     

Oxygen delivery to skeletal muscle fibers: effects of microvascular unit structure and control mechanisms

The number of perfused capillaries in skeletal muscle varies with muscle activation. With increasing activation, muscle fibers are recruited as motor units consisting of widely dispersed fibers, whereas capillaries are recruited as groups called microvascular units (MVUs) that supply several adjacent fibers. In this study, a theoretical model was used to examine the consequences of this spatial mismatch between the functional units of muscle activation and capillary perfusion. Diffusive oxygen transport was simulated in cross sections of skeletal muscle, including several MVUs and fibers from several motor units. Four alternative hypothetical mechanisms controlling capillary perfusion were considered. First, all capillaries adjacent to active fibers are perfused. Second, all MVUs containing capillaries adjacent to active fibers are perfused. Third, each MVU is perfused whenever oxygen levels at its feed arteriole fall below a threshold value. Fourth, each MVU is perfused whenever the average oxygen level at its capillaries falls below a threshold value. For each mechanism, the dependence of the fraction of perfused capillaries on the level of muscle activation was predicted. Comparison of the results led to the following conclusions. Control of perfusion by MVUs increases the fraction of perfused capillaries relative to control by individual capillaries. Control by arteriolar oxygen sensing leads to poor control of tissue oxygenation at high levels of muscle activation. Control of MVU perfusion by capillary oxygen sensing permits adequate tissue oxygenation over the full range of activation without resulting in perfusion of all MVUs containing capillaries adjacent to active fibers.     

An isolated rat liver model for the evaluation of thermal techniques to quantify perfusion

An isolated, thermally regulated, perfused rat liver model system is presented. The model was developed to evaluate thermal methods to quantify perfusion in small volumes of tissue. The surgically isolated rat liver is perfused with an isothermal oxygenated Krebs-Ringer bicarbonate buffer solution via the cannulated portal vein. A constant-pressure head variable-resistance scheme is utilized to control the total flow to the liver. Total flow is quantified by hepatic vein collection. The spatial distribution of perfusion within the liver is determined using two independent methods. In the first method, radio-labelled microspheres are injected into the portal vein, and the regional flow distribution is determined from the relative radioactivity of each section of tissue. In the second method, the tissue is thermally perturbed, and the time constant of the tissue temperature recovery is measured. The regional distribution is determined from the relative time constants of each section of tissue. Both methods require the measurement of total liver flow to determine the absolute perfusion at each point. Results obtained by the two methods were well correlated (0.973). The rat liver system offers a stable, controllable, and measurable perfusion model for the evaluation of new perfusion measurement techniques.     

Evidence of extensive lymphocyte death in sheep Peyer's patches. II. The number and fate of newly-formed lymphocytes that emigrate from Peyer's patches

The emigration of newly produced lymphocytes from Peyer's patches (PP) of lambs was studied. Mesenteric lymph nodes (MLN) were excised from most animals a few weeks after birth, and then at 8 to 10 wk of age, the dividing cells in 3 to 4 m of the small intestine were labeled in situ with [3H]thymidine. An extracorporeal perfusion system was used to restrict the 15-min period of labeling to the perfused lengths of intestine, which included either the large continuous ileal PP or a number of smaller jejunal PP. One or 3 days later, the number of labeled cells in the perfused tissue and in other lymphoid organs was studied by autoradiography. In the perfused tissues, labeled lymphocytes accounted for 63.7% of ileal PP cells by 1 day and for 86.7% by 3 days compared with only 9.6% of lymphocytes in the perfused MLN. Labeled lymphoid cells in the perfused PP were nearly all in the follicles. Labeled lymphocytes that must have been produced in the segments of ileum or jejunum at the time of the perfusion, subsequently emigrated via the lymphatics, and were identified in the spleen, MLN, other lymph nodes, blood, jejunal PP, and at a lower frequency in the thymus, nonperfused ileal PP, and bone marrow. In lymph nodes, spleen, and nonperfused PP, more than 80% of the immigrant newly formed PP-derived cells were small- and medium-sized lymphocytes, and about 15% were large lymphocytes. The nature of the labeled cells in the lamina propria of the nonperfused small intestine was quite different in that approximately 50% were plasma cells as early as 24 hr after the cells were born in the perfused gut. It is proposed that terminal B cell differentiation was most likely initiated within the PP in response to the entry of antigen. It was estimated that at both 1 and 3 days after perfusion there were about 100 times more labeled cells in the perfused ileal PP than could be accounted for by emigration to other organs. It was concluded that these results provide additional support for the view that PP in lambs produce a tremendous number of lymphocytes, but relatively few leave their site of production; most apparently die in situ.     

Ultrastructural study of the permeability of the guinea-pig placenta to horseradish peroxidase

Summary Horseradish peroxidase (HRP) was used to study macromolecule permeation into the guinea-pig placenta perfused in situ. When tissue culture medium 199 (TC 199) was used as fetal-side perfusate, the tracer reaction product was found only lining the fetal endothelium. When a longer period of perfusion with HRP in TC 199 was used, a small amount of reaction product was found in the subendothelial space and syncytiotrophoblastic vesicles, but not in maternal lacunae. In similar experiments using a Krebs bicarbonate Ringer (KRBG) as perfusate the tracer was found (i) lining the fetal endothelium, (ii) in the lateral intercellular spaces of the endothelium, (iii) in the subendothelial space, and (iv) in the maternal lacunae.It is therefore evident that the vehicle influenced the permeability of the guinea-pig placenta to horseradish peroxidase. As other studies have shown that perfusion of the fetal side with salt solution increases pore size, the results with TC 199 are regarded as more representative of the situation in the intact animal. It is therefore suggested that the fetal endothelium of the guinea-pig placenta may be largely impermeable to molecules of the size of horseradish peroxidase (4 nm) or larger.     

A transient heating technique for the measurement of thermal properties of perfused biological tissue

Knowledge of tissue thermal transport properties is imperative for any therapeutic medical tool which employs the localized application of heat to perfused biological tissue. In this study, several techniques are proposed to measure local tissue thermal diffusion by heating with a focused ultrasound field. Transient as well as near steady-state heat inputs are discussed and examined for their suitability as a measurement technique for either tissue thermal diffusivity or perfusion rate. It is shown that steady-state methods are better suited for the measurement of perfusion; however the uncertainty in the perfusion measurement is directly related to knowledge of the tissue's intrinsic thermal diffusivity. Results are presented for a transient thermal pulse technique for the measurement of the thermal diffusivity of perfused and nonperfused tissues, in vitro and in vivo. Measurements conducted in plexiglas, animal muscle, kidney and brain concur with tabulated values and show a scatter from 5-15 percent from the mean; measurements made in perfused muscle and brain compare well with the nonperfused values. An estimate of the error introduced by the effect of perfusion shows that except for highly perfused kidney tissue the effect of perfusion is less than the experimental scatter. This validation of the tissue heat transfer model will allow its eventual extension to the simultaneous measurement of local tissue thermal diffusivity and perfusion.     

Gender dimorphic tissue perfusion response after acute hemorrhage and resuscitation: role of vascular endothelial cell function

Proestrous female rodents are protected from the deleterious effects of trauma-hemorrhage that are observed in males. We hypothesized that the gender dimorphic outcome after trauma-hemorrhage might be related to gender differences in endothelial function and organ perfusion under such conditions. Male and cycle-matched proestrous female Sprague-Dawley rats underwent a midline laparotomy, hemorrhagic shock (40 mmHg for approximately 90 min), and resuscitation (Ringer lactate, 4x shed blood volume over 60 min). Various parameters were measured 2 h after completion of resuscitation. In the first set of animals, the left ventricle was cannulated and heart performance (maximal rate of left ventricular pressure increase) as well as cardiac output and organ perfusion rates were determined with (85)Sr microspheres. In the second set of animals, aortic vessel rings were harvested and relaxation in response to acetylcholine and nitroglycerin was measured. In the third set of animals, in situ isolated small intestine was perfused to measure the response of the splanchnic vessel bed to acetylcholine and nitroglycerin. After trauma-hemorrhage and resuscitation, females maintained cardiac output and demonstrated increased splanchnic and cardiac perfusion compared with males. Moreover, female intestines did not manifest the endothelial dysfunction that was observed in male intestines after hemorrhagic shock. We conclude that proestrous females show improved endothelial function and tissue perfusion patterns after hemorrhagic shock and that this gender-specific response might be a potential mechanism contributing to the beneficial effects of the proestrus stage under such conditions.     

Regulation of perfused capillary density in canine intestinal mucosa during endotoxemia.

When O2 delivery (blood flow X arterial O2 content) is reduced, many tissues respond by increasing perfused capillary density. This facilitates the increase in O2 extraction required to maintain tissue O2 consumption in the face of limited O2 supply. In a previous study of isolated canine small intestine (J. Appl. Physiol. 64: 2410-2419, 1988), endotoxin administration was associated with an impaired ability to increase O2 extraction in response to progressive reductions in O2 delivery. The aim of the present study was to determine whether reductions in perfused capillary density occur after endotoxin administration. Fourteen male dogs were anesthetized with chloralose (150 mg/kg iv) and urethan (750 mg/kg iv), and a segment of small intestine was exteriorized through a midline laparotomy. The segment was isolated vascularly, autoperfused, and maintained at body temperature. Escherichia coli endotoxin (5 mg/kg) or sham challenge was administered, and the animals were allowed to stabilize. Blood flow and arterial and gut venous blood O2 contents were measured after 3 h. Perfused vessels were then labeled by injecting colloidal carbon (less than 0.8 microns) through the arterial cannula and clamping the artery and vein as the bolus passed through the tissue. In some of the experiments a second gut segment was successfully obtained within 1 h of the first, yielding a total of 14 gut segments in nine endotoxin animals and nine segments in five control animals. Morphological analysis of capillary surface density in mucosal villi and crypts showed a significantly higher perfused capillary density in control tissue blocks (77.8 +/- 9.2%) than in blocks from endotoxin-treated animals (68.8 +/- 8.0%, P less than 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)