Canine Red Blood Cells Research Articles

Perfused rat intestine for study of norepinephrine release.

Previous preparations for studying the neuronal release of norepinephrine (NE) employed relatively large vessels, nonsanguinous perfusates, and the preloading of [3H]NE. To study the stimulated release of endogenous NE and the responses of true resistance vessels, we developed a rat intestine preparation that is pump perfused with canine red blood cells suspended in bicarbonate buffer with 6% albumin. In a pentobarbital-anesthetized rat, the duodenum, colon, and cecum are extirpated to isolate the ileum vascularly. After the superior mesenteric artery and vein are cannulated, the perivascular nerves are isolated to stimulate the postganglionic sympathetic fibers. To evaluate the preparation, we stimulated the sympathetic fibers at 1-10 Hz with supramaximal pulses. Resistance changes were assessed by monitoring perfusion pressure, and the concentration of NE was assayed in the venous effluent by the single-isotope radioenzymatic method. During nerve stimulation, the increases in both resistance and NE release rate were frequency dependent. Repetitions of electrical stimulation yielded reproducible frequency-response curves. Pretreatment with phentolamine (10 microM) abolished the resistance response and enhanced stimulated NE release, which roughly tripled at 10 Hz. Phentolamine at smaller doses (1 microM) eliminated the resistance responses to stimulation but did not enhance NE release. Cocaine alone (30 microM) increased base-line resistance and unstimulated NE release. After cocaine pretreatment, phentolamine at 1 microM enhanced the stimulated NE release rate. We conclude that the isolated rat intestine contains postsynaptic alpha-adrenoceptors that mediate vasconstriction and prejunctional alpha 2-adrenoceptors that mediate the inhibition of NE release. Thus the rat intestine is a responsive preparation for studying the release of endogenous NE and noradrenergic neurotransmission.

Evaluation of artificial plasma for maintaining the isolated canine brain.

The use of canine erythrocytes suspended in artificial plasma to maintain the isolated brain was investigated in 18 preparations. Two plasmas were studied: One (AP1) contained electrolytes, amino acids, and albumin; the other (AP2) was similar to CSF and contained a mixture of 37 organic nutrients plus electrolytes and albumin. The CMRO2, CMRglu, and cerebral vascular resistance (CVR) were measured during 2 h of perfusion, and tissue high-energy phosphates were measured at the end of perfusion. The AP1 and AP2 groups were compared with control preparations perfused with canine red blood cells suspended in buffy coat-poor canine plasma. Both CMRO2 and ATP decreased to 60% of the control value; CVR increased to 187% of the control value in both groups following 2 h of perfusion. After 2 h of perfusion, the calculated value of intracellular pH (pHi)--based on creatine kinase equilibrium--remained normal (6.96) for the control brains, but decreased to 6.49 and 6.63, respectively, for the AP1- and AP2-perfused brains. Thus, there appears to be an eventual disruption of normal oxidative metabolism resulting in energy failure, possibly caused by the absence of an essential nutrient from the artificial plasma. For studies of intermediary metabolism in isolated normothermic brain, diluted whole blood appears to be the perfusate of choice.

Erythrocyte swelling after rapid dilution of cryoprotectants and its prevention

These in vitro studies on canine red blood cells confirm that cell swelling occurs after rapid dilution of Me 2SO and glycerol. Cells loaded with a penetrating cryoprotectant in a medium with low Na +, high K + composition present significantly less swelling after rapid dilution of the cryoprotectant than cells exposed to an electrolyte medium characterized by high Na +, low K + composition. The osmotic cell stress during rapid dilution of Me 2SO can be completely counteracted by the simultaneous use of the nonpenetrating sorbitol during exposure and loading. However, the addition of sorbitol is of no important benefit when glycerol is used as the intracellular cryoprotectant. This is probably due to the slower elution of glycerol. Thus utilizing a perfusion solution containing sorbitol during loading and dilution of Me 2SO reduces the osmotic injury and may greatly improve the survival prospects of cryopreserved organs by avoiding “out-flow” block.

Effects of hematocrit on oxygenation of the isolated perfused rat liver.

The isolated perfused rat liver is used ubiquitously for metabolic and endocrine studies of hepatic function, yet few data are available regarding the inadequacy of the oxygenation of such preparations. Moreover, the isolated rat liver is usually deprived of its arterial supply and perfused via the hepatic portal vein with low-hematocrit or cell-free solutions. To investigate the efficacy of the oxygen supply, we determined the effect of hematocrit on the relation between oxygen consumption and perfusate flow. We then attempted to define a hematocrit at which hepatic oxygenation was maximal. Livers of male rats anesthesized with pentobarbital sodium were perfused via the portal vein with fresh canine red blood cells suspended in Krebs-Ringer-bicarbonate buffer. Perfusions were carried out at various flow rates, and the relation between perfusate flow and oxygen uptake was determined. At flow rates above 100 ml X min-1 X 100 g liver-1, oxygen uptake was independent of flow but below that value was flow limited, regardless of whether the hematocrit was 10, 20, or 40%. To determine the optimal hematocrit for hepatic oxygen uptake, hepatic portal venous and hepatic venous pressures were held at 10 and 0 mmHg, respectively. The hematocrit was lowered in steps from 80 to 10%. Blood flow increased exponentially as hematocrit fell while oxygen uptake increased to a maximum at approximately 20%. It is concluded that an hematocrit of approximately 20% provides the optimal combination of blood flow and oxygen-carrying capacity while maintaining physiological perfusion pressures, e.g., 10 mmHg.

Polymorphism at the glucosephosphate isomerase locus in the dog.

A polymorphism of the enzyme glucosephosphate isomerase (GPI) in canine red blood cells is reported. This polymorphism was detected by horizontal starch gel electrophoresis utilizing a tris-EDTA-borate buffer system of pH 8.0. Four out of 92 dogs examined appeared to be heterozygous at this locus.

Determination of sedimentation potentials of canine erythrocytes

A knowledge of sedimentation potentials of blood elements is of value in obtaining their interfacial electrical characteristics. An apparatus was designed to determine sedimentation potentials. It was tested by measuring sedimentation potentials of silver iodide powder and glass powder in distilled water and in some aqueous electrolytes by varying the weight of sediment in the liquid. Sedimentation potentials of canine erythrocytes in Krebs solution and in plasma were determined as a function of weight of sediment in each media. Linear relationships between the sedimentation potential ( E) and weight of particles ( W) were obtained in all the cases. Further, negative sedimentation potentials were obtained indicating that the surfaces of silver iodide particles, glass particles, and canine erythrocytes are negatively charged. The slopes of these lines ( dE dW ) for canine red blood cells in Krebs solution and in plasma were approximately the same, showing that non-ionic constituents in plasma have no influence on the interfacial electrical characteristics of erythrocytes. By a comparison of the results of erythrocytes with glass powder, it was concluded that the zeta potential of erythrocytes in Krebs solution or in plasma is about 70 mV.

Immunologic Response to Subarachnoid and Intracerebral Injection of Antigens

Summary Antibody response in rabbits was measured after canine red blood cells (RBC) and Brucella antigen were injected s.c., i.v., into the cisterna magna (i.c.m.), and into the substance of the brain (i.c.). The following average agglutinin titers were observed after injection of RBC: s.c., 1:320; i.v., 1:5,120; i.c.m., 1:30,000; and i.c., 1:160. The following average hemolysin titers were observed after injection of RBC: s.c., 1:10; i.v., 1:16; i.c.m., 1:28; i.c., 1:8. After injection of Brucella antigen, agglutinin titers were s.c., 1:200; i.v., 1:900; and i.c.m., 1:2,560. In splenectomized rabbits agglutinin and hemolysin titers after s.c. injection were similar to titers after s.c. injection in normal rabbits. After i.v. injections in splenectomized rabbits, titers were similar to those attained after s.c. injection. Titers after i.c.m. injection in splenectomized rabbits were similarly lower than titers produced by i.c.m. injection in normal rabbits. Magnitude of the antibody response was thought to be related to the distribution of antigen in the body; the extremely high titers after i.c.m. injection, to a “slow leak” of antigen from the cerebrospinal fluid into the blood.

Effect of polycythemia on gastric secretion

Vagally innervated and denervated gastric pouches were constructed in normal mongrel dogs. After control secretions were obtained, the animals were made polycythemic by the administration of packed washed canine red blood cells. During polycythemia the quantitative daily output of acid either decreased or remained near control levels. The animals responded in a quantitatively similar manner to histamine and feeding tests before, during, and after polycythemia. Factors other than a hypersecretion of gastric juice in the formation of peptic ulcers are discussed.