A comparison of the effects of different perfusion regimes on the structure of the isolated human placental lobule

Summary Isolated lobules of normal term human placentas were perfused using two different procedures. In the first more conventional system, open-circuit perfusion of both the maternal and the fetal circulations with Earle's solution containing dextran was established and maintained for either 30 min or 1 h. In the second series of experiments both circulations were perfused in separate closed circuits with a mixture of fresh autologous fetal blood and Earle's solution for 0, 1, 2 or 3 h. In both series the lobule was then fixed by perfusion through the fetal circulation.Light and electron-microscopic examination of a set of tissue samples from each perfused lobule showed substantial differences between the effects of these two types of perfusion procedure. Tissue from lobules perfused by the open-circuit blood-free procedure showed patchy but severe cell swelling and vacuolation of the trophoblast after only one hour's perfusion. Particularly striking was swelling and disruption of a large proportion of the mitochondria in all placental cell types. By contrast, placental tissue from the closed-circuit perfusion with blood-containing medium showed little change over a period of two hours, while after three hours it showed oedema and microvillous damage, but no sign of cell swelling and little mitochondrial damage.It is concluded that the viability of the perfused human placental lobule depends on the type of perfusate used, and that the use of a fetal blood-enriched perfusate is of considerable value in maintenance of the preparation as assessed by structural criteria.     

Increase in portal flow induces c-myc expression in isolated perfused rat liver

We examined expression of the c-myc oncogene in isolated perfused livers to elucidate the mechanisms involved in triggering the proliferation of hepatocytes after partial hepatectomy (PH). During perfusion with a 1:1 mixture of Dulbecco's modified Eagle's medium and the oxygen transport fluid FC-43, rat livers were two-thirds resected (PH), and further perfused for 1 1/2 hours at the physiological portal flow throughout the perfusion. Expression of c-myc in the perfused livers with PH(+) was ten times higher than in those with PH(?). Furthermore, expression of c-myc in the PH(?) livers perfused with a threefold volume of the physiological portal flow was 5–10 times higher than that in the livers perfused with the physiological portal flow. The perfusates that passed through the livers did not induce DNA synthesis of primary cultured hepatocytes. These results suggest that an increase in the portal flow volume may act as a trigger for hepatocyte proliferation after PH. © 1993 Wiley-Liss, Inc.     

Fluorometric analysis of an attempt to reclaim ischemic flaps in rats with Fluosol

An experiment was designed to answer two questions as they apply to random skin-flap survival: Is there a therapy that can improve random skin-flap survival when given postoperatively? And if so, when does one start such a therapy? Fluosol-DA 20% (Fluosol) has increased random skin-flap survival when given preoperatively in our laboratory. An experiment was devised to see if it could rescue failing flaps. One-hundred Sprague-Dawley rats were divided into a control (N = 25) and five experimental groups (N = 15). All had 10 X 13 cm reverse McFarlane random flaps raised and reinset. The experimental groups underwent hemodilution with either Ringer's lactate or Fluosol at 4, 8, and 12 hours after flap elevation. All were kept in 50% oxygen for 72 hours postoperatively. The flaps and their corresponding necrotic areas were measured on day 7. As to when to institute a therapy, we simultaneously evaluated the use of a microfluorometer as a monitor of flap survival. Analysis of flap survival showed little difference between control and experimental Ringer's lactate or Fluosol groups. Analysis of the microfluorometric data led to the following points. First, as a monitor of flap viability, it is limited by a lack of specificity and sensitivity. Second, comparison of the data from portions of the flap destined to live with those destined to die suggests that it may not be failure of circulatory inflow that leads to flap death.     

Effects of shear stress on eicosanoid gene expression and metabolite production in vascular endothelium as studied in a novel biomechanical perfusion model

This study investigated the effects of shear stress on gene expression of prostacyclin synthesis-related enzymes cyclooxygenases (COX-1 and COX-2), prostacyclin synthase (PGS), and thromboxane synthase (TXS) and their metabolites prostaglandin (PGI(2)) and thromboxane A(2) (TXA(2)) in endothelium of intact conduit vessels. Paired human umbilical veins were perfused at high/low shear stress (25/<4 dyn/cm(2)) at identical intraluminal pressure (20 mmHg) for 1.5, 3, or 6 hours in a computerized vascular model. High shear perfusion induced a significant, monophasic upregulation of PGS and TXS gene expressions after 6 hours. COX-1 and COX-2 mRNA showed a biphasic response with peaks at 1.5 and 6 hours, with a nadir level at 3 hours. Shear-induced gene expression was associated with a significantly greater accumulation of 6-keto prostaglandin F(1alpha) and TXA(2) in the perfusion medium. Thus, shear stress independently of perfusion pressure alters the expression of prostacyclin synthesis-related enzymes and the biosynthesis of their vasoactive metabolites.     

Retrograde coronary venous perfusion at low pressure

The effectiveness of localized retrograde coronary venous perfusion (RCVP) in preventing or reversing myocardial ischemia after acute ligation of a coronary artery is described. Ten domestic pigs (Group I) underwent aorto left anterior coronary vein grafting with RCVP at systemic pressure. In another set of ten pigs (Group II), the coronary vein was similarly grafted, but the proximal end of the graft was perfused with oxygenated blood by means of an external pump at reduced pressure and flow. In both groups, RCVP successfully reversed the mechanical or electrical effects produced by 2 to 5 minutes of acute arterial ligation. After several hours, Group I animals showed evidence of acute ischemia and developing infarction. Group II animals, however, were maintained for 7 hours or more with regional RCVP and no evidence of ischemia. Retrograde coronary venous perfusion at reduced pressure may be more effective than perfusion at systemic pressure in providing myocardial blood flow.     

Microcirculatory disturbances following the passage of emboli in an experimental free-flap model.

Following completion of arterial repair in an experimental free-flap model, platelet emboli have been observed passing through the microcirculation downstream. The purpose of this experimental study was to observe and quantitate changes in capillary perfusion occurring subsequent to these events. The isolated rat cremaster model was used. For 6 hours subsequent to surgical injury of the main artery in this model, the number of emboli and the number of perfused capillaries downstream were counted. In eight rats having an intentional arterial wall injury, emboli were consistently seen during the first hour of reflow. In the nine control animals having no arterial injury, no emboli were seen. The presence of emboli in the cremaster muscle, resulting from the arterial injury, was associated with a significant reduction in the number of perfused capillaries. We suggest that the observed decrease in capillary perfusion was associated with microemboli that produced an adverse effect for several hours after their initial presence in the circulation.     

Effect of chronic ethanol treatment and the abstinence period on the ethanol elimination by isolated rat liver lesioned by carbon tetrachloride

The study was carried out on perfused livers isolated from rats receiving ethanol (EtOH) as their only drinking fluid for the period of 4 weeks. Twelve, 24, 72 and 120 hours after EtOH withdrawal the livers were isolated and perfused with 100 ml of perfusion mixture with addition of EtOH (0.2% final concentration). After 12 hours of EtOH withdrawal acceleration of the EtOH elimination from perfusate was observed. It returned to the control level after 24 hours, but after 72 and 120 hours of abstinence the rate of EtOH elimination from perfusate was found to diminish. CCl4 injected to the rats in doses of 2 and 5 mmoles/kg once a week for the period of 4 or 8 weeks, resulted in decreased EtOH elimination from the perfusate. In the EtOH-drinking group previously treated with CCl4 we found that irrespective of the time of EtOH withdrawal, EtOH elimination did not differ from that in the respective CCl4 treated group, only 12 hours after its withdrawal EtOH elimination was decreased in livers injured with CCl4 in dose of 5 mmoles/kg.     

Degradation of [3H]chondroitin 4-sulphate and re-utilization of the [3H]hexosamine component by the isolated perfused rat liver.

Radiolabelled chondroitin 4-sulphate was isolated after incubation of rat rib cartilage with N-acetyl-D-[6-3H]galactosamine. After proteolytic digestion of the tissue with either papain or trypsin the released [3H]chondroitin 4-sulphate was added to an isolated perfused rat liver system. Analysis of perfusate after several hours perfusion showed that radiolabelled amino sugars were secreted by the liver in a low-molecular-weight form and as components of glycoproteins.     

Renal preservation by hypothermic perfusion. III. The lack of influence of pulsatile flow

Rabbit kidneys were perfused at 5 °C with a plasma-like solution containing dextran 70 and bovine serum albumin, and were autografted 24 hr later. One experimental group was perfused at a constant pressure of 40 mm Hg, while the second group was perfused with a pulsatile pressure having a root-mean-square (rms) equivalent of 40 mm Hg; the pulse pressure was 15 mm Hg and the pulse rate 60 min^?1 The behaviour of the two groups during perfusion and after transplantation was similar. It is concluded that pulsatile flow is without benefit during renal preservation by hypothermic perfusion.     

Ultrastructural and hormonal metabolic studies of rat liver maintained in vitro by perfusion at 30 degrees C and 37 degrees C: a time course study by TEM, SEM and RIA

Isolated rat liver perfusion system has been extensively used for metabolic and functional studies. Results derived from the application of this system may reflect true biochemical changes but they may also be associated with some structural changes. This study was undertaken to correlate the cytological changes and functional integrity of isolated rat liver perfused in vitro at normal physiological temperature (37 degrees C) and 30 degrees C, using a non-recirculating system. The livers were perfused for 3 hours with modified Ham's F10 culture medium supplemented with thyroxine hormone (T4). The hepatocyte structural integrity was studied by light microscopy, transmission and scanning electron microscopy. The triiodothyronine (T3) and T4 hormones in the perfusion medium and the effluent fractions were assessed by radioimmunoassay. The livers perfused at 30 degrees C remained morphologically intact at the ultrastructural level for 3 hours whilst at 37 degrees C, hepatocytes in the centrilobular zone exhibited marked structural alterations. The percentage of T4 uptake was significantly higher (P less than 0.01) in livers perfused at 30 degrees C (50.8 +/- 7.7% vs 38 +/- 7.7%, 37 degrees C), but the net T3 output (3.16 +/- 1.04 micrograms) and the conversion of T4 to T3 (4 +/- 0.62%) were significantly higher (P less than 0.001) in livers perfused at 37 degrees C in comparison to livers perfused at 30 degrees C (1.61 +/- 0.84 micrograms and 1.68 +/- 0.76%, respectively). In conclusion, at 30 degrees C the hepatic T4 uptake is not inhibited, but the rate of T4 to T3 conversion has decreased, additionally the livers remain morphologically well preserved throughout the experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Silica-free silicone rubber in isolated heart perfusion with blood at 13 °C

Isolated lamb hearts were perfused at 13 °C for 24 hr with whole fresh blood using a silicone rubber circuit and a membrane lung (N = 7); there was formation of thrombi, deposition of fibrin, and an increase in resistance to blood flow in the membrane lung. The perfused hearts fibrillated at hypothermia and showed unequal recovery of function upon final rewarming.There was less rise in membrane lung resistance when the perfusion circuit was primed with blood at 38 instead of 13 °C and then cooled progressively to 13 °C. Some hearts perfused in these circuits were well preserved but others became edematous with loss of ventricular contractility (N = 6).Coating the perfusion circuit with a hypothrombogenic material, silica-free silicone rubber and priming the circuit at 38 °C prevented any rise in membrane lung resistance during blood perfusion. All the hearts perfused in these circuits (N = 6) had the same left ventricular function before and after cold perfusion.Thus isolated hearts can be perfused in vitro with whole blood at hypothermic temperature without loss in function when attention is paid to thrombogenicity of materials used to construct the perfusion circuit.     

The extracorporeal perfusion of swine uterus as an experimental model: the effect of oxytocic drugs.

The objective of this study was to establish an experimental model for extracorporeal perfusion of swine uterus. In order to validate this model, we examined some biochemical parameters and determined the effect of oxytocic drugs (Oxytoxin, Prostaglandin E (2)) on extracorporeal perfused swine uteri. Thirty swine uteri were perfused with Krebs-Ringer bicarbonate-glucose buffer for a period up to eleven hours with the aim to preserve a viable organ, which should be responsive to hormones. The intrauterine pressure was recorded after administration of various concentrations of oxytocin and prostaglandin E (2). Perfusate pH, perfusate lactate, partial oxygen and carbon dioxide tensions, oxygen saturation, and hydrogencarbonate levels in the perfusate, all indicators of tissue ischemia or cell necrosis, showed good preservation of the organ for up to seven hours. We examined the relation of intrauterine pressure to oxytocin and prostaglandin E (2). Both were able to induce contractions of the uterus, whereas prostaglandin E (2) produced rhythmical contractions of smaller amplitude and a higher frequency. We could demonstrate that our perfusion system was able to preserve the swine uterus in a functional condition appropriate for the study of physiological questions.     

Combined perfusion and percolation of embalmed animal bodies for removing formaldehyde

Excess formaldehyde was extracted from embalmed animal cadavers by vascular perfusion or combined perfusion and percolation with 20% ethanol. The perfusion was undertaken through a carotid artery and the percolation through the serous body cavities for 1-2 h. Nineteen goats were perfused once and one goat and three cows were perfused and percolated twice a day for 3 days. The extracted cadavers were stored in 20% ethanol or in a freezer at -20 degrees C. The mean value of the atmospheric concentration of formaldehyde after extraction was 0.27 +/- 0.05 mg/m3 for goats and 0.39 +/- 0.12 mg/m3 for cows.     

Influence of chronic ethanol treatment and the abstinence period on the ethanol elimination by the isolated perfused rat liver

Rats received ethanol as their only drinking fluid for 4 weeks in a concentration ranging from 6-20%. After the ethanol-free intervals of 24, 48, 72, 96, 120 and 144 hours, the livers were isolated and perfused with 100 ml of perfusion fluid with an addition of ethanol (0.2% f.c.) and the elimination of ethanol from the perfusate was studied. The livers of animals chronically exposed to ethanol showed significant diminution of ethanol elimination from perfusate after 72, 96 and 120 hours of withdrawal. After 144 hours the rate of the ethanol elimination returned to the control level.     

Revascularization of peripheral nerve autografts and allografts

The timing and mechanisms of peripheral nerve revascularization were investigated using a 2-cm sciatic nerve graft model in 58 rats. Epineurial perfusion was consistently established by 48 hours and endoneurial perfusion by 72 hours. The pattern of endoneurial perfusion was "all-or-none"--either all or none of the vessels in a fascicle exhibited blood flow. Conventional allografts exhibited similar revascularization dynamics and patterns. Capping the ends of the autograft with Silastic significantly delayed revascularization; no flow was observed at 4 days, and only a peripheral rim of perfused fascicular vessels was observed at 7 days. These patterns suggested that the primary method of revascularization in the conventional graft was longitudinal inosculation; no evidence of peripheral neovascularization or dependence on the graft bed as a source of revascularization was observed. The introduction of a major histocompatibility complex barrier between the grafted tissue and the recipient animal did not alter the timing or the mechanics of blood flow reestablishment.     

Salient effects of l-carnitine on adenine-nucleotide loss and coenzyme A acylation in the diabetic heart perfused with excess palmitic acid: A phosphorus-31 NMR and chemical extract study

The beneficial effects of l-carnitine perfusion on energy metabolism and coenzyme A acylation were studied in isolated hearts from control and diabetic rats. All hearts were perfused at a constant flow rate with a glucose/albumin buffer which contained 2.0 mM palmitate. ³¹P-NMR was utilized to assess sequential phosphocreatine and ATP metabolism during 1 h of recirculation perfusion. l-Carnitine (5.0 mM final concentration) was added after 12 min of baseline recirculation perfusion. Frozen samples were taken after 1 h of recirculation perfusion for spectrophotometric analysis of high-energy phosphates and the free and acylated fractions of coenzyme A. l-Carnitine perfusion of diabetic hearts attenuated or prevented the reduction of ATP observed in untreated diabetic hearts. It also attenuated the accumulation of long-chain fatty-acyl coenzyme A. Although l-carnitine improved myocardial function in diabetic hearts, this was independent of any direct effect on physiological indices. Thus, the salutory effect of acute perfusion with l-carnitine on energy metabolism in the isolated perfused diabetic rat heart appears to be a direct effect on lipid metabolism.     

Permeability characteristics of isolated perfused rat lungs

We examined the factors that influence the permeability characteristics of isolated perfused rat lungs and compared the ex vivo permeability-surface area product (PS) with that obtained in vivo. In lungs perfused for 20 min with homologous blood or a physiological salt solution (PSS) containing 4 g/100 ml albumin, mean PS values, obtained by the single-sample method of Kern et al. [Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H229-H236, 1983], were 9.9 +/- 0.6 (SE) and 6.8 +/- 0.3 cm3.min-1.g wet lung-1.10(-2), respectively. These values were similar to lung PS obtained in intact rats (7.7 +/- 0.4 cm3.min-1.g wet lung-1.10(-2). In perfused lungs, PS values were influenced by the perfusate albumin concentration, the length of perfusion time, and the degree of vascular recruitment. Twenty minutes after lung isolation, PS was 126% higher in lungs perfused with albumin-free PSS containing Ficoll than in lungs perfused with albumin-PSS. Moreover, PS in Ficoll-PSS-perfused lungs increased even higher after 2 h of perfusion, and this time-dependent increase in PS was attenuated by addition of 0.1 g/100 ml albumin to the perfusate. Two hours of ex vivo ventilation with hypoxic (0 or 3% 0(2)) or hyperoxic (95% 0(2)) gas mixture did not affect PS values in perfused lungs. However, PS was elevated in lungs perfused ex vivo with protamine, which causes endothelial cell injury, or in lungs from rats exposed in vivo to human recombinant tumor necrosis factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The application of fluidics technology for organ preservation

A prototype design of a portable, pulsatile, perfusion preservation device based on a novel application of fluidics technology was tested to evaluate its ability to oxygenate preservation solution and to examine the relationship between organ resistance, perfusion pressure, and perfusion flow characteristics. The effects of organ resistance on pulse rate, perfusion pressure, and perfusion flow were modeled. Interstitial PO2 in canine hearts stored at 4 degrees C for 12 hours in the fluidics device (n = 5) and in static hypothermic storage (n = 5) was also compared. Increasing outflow resistance did not have an effect on operating frequency of the fluidics actuator. Perfusion pressure rose as outflow resistance was increased, and the flow of preservation solution decreased proportionately. The PO2 of the preservation solution increased to 300 mm Hg in two hours and reached a plateau that exceeded 400 mm Hg within six hours. The aortic flow profile during pulsatile perfusion resembled a square wave function with a mean pulse duration of 0.30 +/- 0.05 seconds. Oxygen delivery by the fluidics perfusion device exceeded the oxygen requirements of the hypothermically preserved organs at all resistance levels. Initial interstitial PO2 in the hearts of both groups was greater than 150 mm Hg. In perfused hearts, PO2 declined 30% by the 12th hour, whereas complete depletion of oxygen was noted in the static storage group within six hours. The fluidics organ perfusion/transport apparatus weighs less than 18 kg, uses no electrical power, and can operate continuously for 10 to 12 hours expending 780 L of oxygen.     

Improvement in the preservation of ischemically impaired renal transplants of pigs by iloprost (ZK 36 374)

Kidneys of five pigs were preoperatively perfused with iloprost in situ (infusion of 0.5 μg.kg⁻¹ for 15 minutes) and exposed to a warm ischemic period of 45 minutes. The behaviour of these kidneys during initial perfusion by gravity and hypothermic mechanical perfusion for 24 hours was comparable to that of kidneys having had a warm ischemic. period of less than 5 minutes. After retransplantation all kidneys showed a sufficient blood flow and immediate production of urine during the test period of 3 hours.     

The effects of combined cooling and perfusion on experimental free-flap survival in rabbits

The viability of the rabbit epigastric flap was investigated after a period of cold ischemia of 3, 4, or 5 days. Groups of flaps were either perfused with Ross's solution before ischemia (early perfusion), after ischemia (late perfusion), or not at all (control). Significantly more flap necrosis was formed at exploration 7 days after revascularization in the early perfusion group than in the control group (p less than 0.01). Late perfusion was not significantly detrimental to flap survival as compared with cold storage alone (0.1 less than p less than 0.5). It is concluded that early perfusion is detrimental to the ultimate fate of the cold ischemic flap, whereas late perfusion offers no advantage over cold storage alone.