Fixation of Vervet Monkey Oral Mucosa for Ultrastructural Investigation-Tem

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecui pygerythrta) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolalities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

Fixation of vervet monkey oral mucosa for ultrastructural investigation--TEM

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecus pygerythrus) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolarities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

Effect of increased concentrations of ca++ and mg++ in the fluid perfusing the cerebral ventricles, and hypoxia on evoked tongue jerks

Effect of increased concentrations of Ca++ and Mg++ in the fluid perfusing the cerebral ventricles, and hypoxia on evoked tongue jerks. Acta Physiol. Pol., 1978, 29 (1): 27-36. The infraorbital nerve, the sensory part of the trigeminal nerve, was stimulated in rats under chloralose anaesthesia. Electric stimuli of 0.2 Hz caused retractive movements of the stretched tongue. These evoked tongue jerks (ETJ) were recorded directly on a kymograph or on a linear recorder. Using a stereotaxic apparatus cannulas were inserted into both lateral ventricles of the brain for infusion of McIlwain-Rodnight's fluid at a rate of about 50 microliter/minute. The cannula for outflow of the perfusing fluid was inserted into the cerebellomedullary cistern. The ETJ was enhanced by 43%, on the average, during perfusion of the cerebral ventricles with solutions with fivefold increased concentration of calcium ions, and decreased by a mean value 24% when the perfusing solution contained a higher concentration of magnesium ions. After 10 min of breathing with increased respiratory dead space, which caused hypoxia and hypercapnia, the amplitude of ETJ diminished by 65%, on the average.     

Fixation of Vervet Monkey Oral Mucosa for Ultrastructural Investigation–Tem

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecui pygerythrta) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolalities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

Release of Catecholamines from Superfused Bovine Adrenal Chromaffin Cells Cultured on Microcarrier Beads

A procedure is described for the establishment of stable primary cultures of bovine chromaffin cells on microcarrier beads. The cells flatten and send out processes with varicosities over a few days and maintain their catecholamine content for 2 weeks. The beads may be incorporated into a superfusion apparatus with a chamber volume of about 150 microliters, enabling the efficient perfusion of a high density of cells. The response to the introduction of nicotine and high potassium into the perfusing medium is shown to be more rapid and more transient than hitherto described, with each secretagogue producing a different degree of preferential stimulation of noradrenaline-secreting cells.     

Modification of intramuscular pH oscillations in the isolated perfused rat heart by different interventions.

Changes in the intramuscular pH oscillations were examined by the use of an antimony electrode upon perfusing the isolated rat heart under different experimental conditions. The pH oscillations were decreased upon perfusing the hearts with Na+- or Ca2+-free medium and increased upon perfusing with K+-free medium. Increasing the temperature of perfusion medium from 25 to 40 degrees C or omitting glucose from the perfusing medium decreased the magnitude of oscillations. On the other hand, complete interruption of the perfusion flow resulted in an increase in the amplitude of pH oscillation. An initial increase followed by a decrease in the pH oscillation was seen when hearts were perfused with medium containing lactic acid at pH 6.6. These results suggest that pH oscillations reflect fluctuations in myocardial metabolism.     

清醒山羊颈动脉体灌流模型及其评估

本文对清醒山羊颈动脉体灌流模型作了详细介绍。手术步骤包括:(1)在颈部任一侧结扎枕动脉、切断窦神经及切除颈动脉体;(2)完成对供应另一侧(灌流侧)脑和颈部血管的改道手术,以便从血液供应上游离颈动脉体;(3)在灌流侧安置颈静脉插管和颈动脉插管,建立灌流通路。实验中应用体外循环方法以动物自身血液灌流颈动脉体。该模型具有两个显著优点:(1)实验中可保持动物处于正常生理状态;(2)对脑组织和颈动脉体的血液血气及pH能分别加以人为控制。实验前动物均经过氰化钠试验检查颈动脉体和主动脉体的化学敏感性,用以评价灌流模型的有效性。文中还对这一模型的应用进行了讨论。     

Liver glycogen metabolism during intestinal perfusion in the rat.

The effects of metabolic constituents on glycogen metabolism in the liver can be studied by perfusing the small intestine with those constituents capable of being absorbed across the gut mucosa. Perfusion of the gut with glucose to give concentrations of approx. 20 mM in the portal vein produced a 5-fold increase in liver glycogen after 60 min. The rate of synthesis over the 15–30 min period of perfusion of 1.24 μmol/min/g liver fell to 0.34 in the 30–60 min period. This stimulation was accompanied by a transient activation of glycogen synthase which occurred in the first 30 min period of perfusion but which had disappeared by 60 min. Glucose perfusion had no effect on total and active phosphorylase at the time intervals studied. While the pattern of insulin secretion during perfusion could not be ascertained, the activation of glycogen synthase and increase in glycogen synthesis correlated with the production of gastric inhibitory polypeptide (GIP), a potent stimulator of insulin secretion. Perfusion of the gut with fructose did not stimulate glycogen synthesis or activate glycogen synthase or stimulate GIP secretion, but instead it produced a marked activation of phosphorylase after 30 min of perfusion.     

Method for determining solutes in the cell walls of leaves

A perfusion method is described whereby large discs of amphistomatous leaves are vacuum-perfused with water so that either successive fractions of perfusate may be analyzed for solutes or the infused water may be displaced and collected after equilibration with the leaf cells. With castor bean leaves, estimates of electrolyte concentration in cell wall water by the two methods were similar. Total electrolytes in leaf cell wall water of castor beans (Ricinus communis), sunflower (Helianthus annuus), and cabbage (Brassica oleracea capitata) from nonsaline cultures were about 2, 2, and 10 milliequivalents per liter, respectively, increasing to 4, 10, and 30 milliequivalents per liter under saline conditions. Electrolytes recovered in successive fractions were similar in composition, and continuous perfusion resulted in a steady release of solutes, the concentration in the perfusate varying inversely with the perfusion rate. Diffusional release of solutes from cells was less than expected at low perfusion rates, suggesting that solute reabsorption may increase as solute concentration in the perfusate increases with decreased perfusion rates. Perfusate concentration and composition were essentially unaffected by temperature (2 and 23 C) or by perfusing with 0.5 mm CaSO₄ rather than with water. Electrolytes in perfusates on an equivalent basis were Ca²⁺, 30%; Mg²⁺, 10%; and Na⁺ + K⁺, 60%, the proportions of sodium increasing from 10 to 50% in leaves (cabbage) that accumulated sodium under saline conditions. Salinity (added NaCl) of the root culture medium caused a 3- to 5-fold increase in total cell wall electrolyte concentration, but this amounted to an increase from less than 1 or a few per cent to no more than 7% (in cabbage) of the cell sap electrolyte concentrations. Solutes in the cell wall appear to be in dynamic equilibrium with intracellular solutes.     

The involvement of calmodulin in the secretion of calcitonin from the porcine thyroid gland

The effect of the calmodulin antagonist W7 has been studied in the pig in vivo by measuring directly the secretion rate of calcitonin (CT) in the thyroid venous blood after surgical isolation of the thyroid and subsequent perfusion of the gland in situ. Over the concentration range of W7 9-100 mumol/l in the perfusing plasma there was a significant increase in CT secretion rate associated with the addition of W7 to the perfusing blood. There was no significant change in the perfusing plasma calcium concentration. It is suggested that calmodulin plays an important role in calcium homeostasis within the porcine thyroid C-cell.     

Histochemical Demonstration of Endogenous Dehydrogenase Systems by Supravital Perfusion

Endogenous dehydrogenase activity is demonstrated in fresh, intact organs by supravital perfusion with a tetrazolium solution. The animal is first injected intravenously with 1.5 mg Heparin/100 gm body weight. It is then anesthetized and a fine polyethylene cannula (PE50, Intramedic) is inserted into a major artery and secured with a ligature. An initial perfusion with warm (37°C) M/20 phosphate buffer (pH 7.6) to remove the blood from the tissues is followed by a 10 min perfusion with the same kind of buffer to which has been added 0.25% neotetrazolium chloride (Dajac Laboratories). The tetrazolium solution is delivered to the tissue at the rate of 1 ml/minute. A final perfusion with 10% formalin in warm phosphate buffer (pH 7.6) flushes and fixes the tissues. Frozen sections can then be cut and mounted in glycerol jelly. Fine, colored formazan crystals are deposited at the sites of enzyme activity. The method is simple and yields excellent histochemical preparations.     

Evidence for electronogenic sodium pumping in the ductal epithelium of rabbit salivary gland and its relationship with (Na+ plus K+)-ATPase.

The temperature dependence of the transepithelial potential difference (PD) of the main duct of the submaxillary gland has been measured during in vitro perfusion studies. The magnitude of the PD depends strongly on the anion composition of the perfusing and bathing fluids. The following combinations of perfusion and bathing fluids respectively were used: (1) Na2SO4/NaCl, (2) Na2SO4, (3) NaCl/-NaCl, (4) NaCl/Na2SO4. The mean transepithelial potential differences at 35 degrees C with these four sets of conditions were respectively: 144, 148, 10 and - 15 mV, serosal side positive with respect to lumen. From the data obtained it was possible to construct Arrhenius plots of temperature dependence of the PD for the four sets of experimental conditions. They all show a breakpoint between 16 and 19 degrees C. The apparent activation energies in the four situations above the breakpoint are 4.2, 1.4, 12.0 and 10.6 kcal/mol, respectively. Below the breakpoint they are 29.9, 37.5, 29.0 and 31.3 kcal/mol, respectively. The rapid change in the PD as a function of temperature (which can also be achieved by the addition of ouabain), the effects of the removal of K+ on the serosal side on the PD, the decrease in the PD after the addition of ouabain or CN-, and the activation energies and breakpoints all lead to the conclusion that a large part of the PD is caused by an electrogenic sodium pump which is very probably the enzyme (Na+ plus K+)-ATPase. When the duct is perfused with Na2SO4 we find, above the breakpoint in the Arrhenius plots, a lower activation energy than is found when perfusing with NaCl.     

An airlift pump device for low pressure perfusion storage of the isolated heart

A portable apparatus for the continuous hypothermic perfusion of the isolated heart is described. The system has been used successfully to store pig and baboon hearts for periods of up to 48 hr, and to store human donor hearts for periods of 7 to 17 hr before being transplanted. The perfusate is both oxygenated and circulated by gas flow from a pressurized oxygen cylinder, using the air-lift pump principle. The apparatus has no moving parts and requires no electrical energy supply; malfunction is, therefore, extremely unlikely. A regulator has been incorporated which can be adjusted to increase or decrease the myocardial perfusion pressure. The system and environmental variables which can affect flow and pressure within the apparatus are discussed. The storage time allowed by this system will enable transportation of donor hearts between most of the world's major cities.     

Postinhibitory overshoot of insulin release by gastric inhibitory polypeptide

Following intravenous infusion of somatostatin $\text{in vivo}$ occasionally there is a large rebound overshoot of insulin release. An $\text{in vitro}$ model to simulate this phenomenon was made by perfusing rat pancreas with gastric inhibitory polypeptide (GIP) during simultaneous perfusion with somatostatin. Adding GIP (100 ng/ml) to the perfusate for 2 minutes beginning either 3 or 9 minutes before terminating the somatostatin perfusion produced a large overshoot in insulin release. The magnitude of overshoot was greater when medium contained 300 mg/dl glucose that when it contained 150 mg/dl glucose. Perfusion with GIP for 2 minutes beginning 9 minutes before increasing the glucose concentration of the medium from 30 to 300 mg/dl elicited a large increase in both the acute and second-phase release of insulin. These suggest that post-inhibitory overshoot of insulin release after somatostatin may be produces $\text{in vitro}$ by the suppressed action of stimulatory hormones such as GIP. Prior infusion with GIP can also potentiate glucose-stimulated insulin increase.     

Systemic perfusion: a method of enhancing relative tumor uptake of radiolabeled monoclonal antibodies

We evaluated the feasibility of systemic vascular perfusion with saline (mimicking plasmapheresis) as a method to enhance tumor-specific monoclonal antibody (MoAb) tumor/background ratios. Initially, groups of rats were injected intravenously (i.v.) with ¹³¹I-5G6.4 MoAb (murine IgG2aK reactive with ovarian carcinoma). These animal's radioactivity levels were determined by dose calibrator and they were imaged before and after perfusion which was conducted at 4 or 24 h post-antibody injection. Animals were sacrificed after perfusion, as were controls, and normal organ radioactivity levels determined. In addition, nude mice bearing HTB77 ovarian cancers subcutaneously were injected i.v. with ¹³¹I-5G6.4 MoAb and were imaged before and after systemic perfusion with saline 24 h post-5G6.4 injection. Perfusion in rats dropped whole-body 5G6.4 levels significantly at both perfusion times (P < 0.0005). The drop in whole-body radioactivity with perfusion was significantly greater for the animals perfused at 4 h post i.v. 5G6.4 antibody injection (48.3 ± 5.1%) than for those perfused at 24 h post i.v. antibody injection (32.9 ± 2.9%) (P < 0.025). In the nude mice with ovarian cancer xenografts, γ camera images of tumors were visually and quantitatively (by computer image analysis) enhanced by perfusion, with a 2.33-fold greater decline in whole body uptake than in the tumor (P < 0.05). These studies show that (1) much background antibody radioactivity can be removed using whole-body perfusion with saline, (2) that the decline in whole body activity is larger with 4 than 24 h perfusion and (3) tumor imaging can be enhanced by this approach. This and similar approaches that increase relative tumor antibody uptake such as plasmapheresis may be useful in imaging and therapy with radiolabeled antibodies.     

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient’s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.     

Perfusion flow rate substantially contributes to the performance of the HepaRG‐AMC‐bioartificial liver

Bioartificial livers (BALs) are bioreactors containing liver cells that provide extracorporeal liver support to liver‐failure patients. Theoretically, the plasma perfusion flow rate through a BAL is an important determinant of its functionality. Low flow rates can limit functionality due to limited substrate availability, and high flow rates can induce cell damage. This hypothesis was tested by perfusing the AMC‐BAL loaded with the liver cell line HepaRG at four different medium flow rates (0.3, 1.5, 5, and 10 mL/min). Hepatic functions ammonia elimination, urea production, lactate consumption, and 6β‐hydroxylation of testosterone showed 2–20‐fold higher rates at 5 mL/min compared to 0.3 mL/min, while cell damage remained stable. However, at 10 mL/min cell damage was twofold higher, and maximal hepatic functionality was not changed, except for an increase in lactate elimination. On the other hand, only a low flow rate of 0.3 mL/min allowed for an accurate measurement of the ammonia and lactate mass balance across the bioreactor, which is useful for monitoring the BAL's condition during treatment. These results show that (1) the functionality of a BAL highly depends on the perfusion rate; (2) there is a universal optimal flow rate based on various function and cell damage parameters (5 mL/min for HepaRG‐BAL); and (3) in the current set‐up the mass balance of substrate, metabolite, or cell damage markers between in‐and out‐flow of the bioreactor can only be determined at a suboptimal, low, perfusion rate (0.3 mL/min for HepaRG‐BAL). Biotechnol. Bioeng. 2012; 109: 3182–3188. © 2012 Wiley Periodicals, Inc.     

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage,Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.     

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19^th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease^1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals^1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes – a key component of any experiment utilizing this device^1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user’s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.