Sequestration of heat damaged rat erythrocytes during experimental action of methyl palmitate on RES

The effect of an i.v. injection of methyl palmitate emulsion (MP) on the clearance of heat damaged erythrocytes from the blood and their sequestration in organs of rats was followed. Twenty-four hours following application of MP in the dose of 1.5 g/kg of body weight the survival half time of erythrocytes (T1/2(51)Cr) significantly increased, whereas the amount of sequestrated red cells in the spleen decreased. The subsequent intervals of examination, however, showed no differences as compared to the controls.     

The inhibitory effect of methyl palmitate on heteroagglutinin formation in rats.

In Wistar rats the immunosuppressive effect of methyl palmitate on the formation of heteroagglutinins against human O-group erythrocytes was followed. An i.v. injection of methyl palmitate both delayed the heteroagglutinin formation and decreased its intensity. The inhibitory effect of methyl palmitate was not accompained by the subsequent hyperactive phase as could be observed in the previous experiments using ethyl palmitate.     

The formation of heteroagglutinins in rats "chemically" splenectomized by ethyl palmitate, in comparison to animals surgically splenectomized.

In Wistar rats "chemically splenectomized" by an i.v. injection of ethyl palmitate the formation of heteroagglutinins against human O-erythrocytes has been followed. Ethyl palmitate applied 2 hrs before immunization induced on the 5th day a significant decrease of the antibody level. This temporary inhibition, however, was at a later stage replaced by a significant increase of antibody level with the peak of 8--14 days. Animals surgically splenectomized 24 hrs prior to immunization displayed a weak antibody response throughout the experiment. An injection of the ethyl palmitate on the 6th day after immunization induced a pronounced and persistent decrease of the antibody level. Surgical splenectomy performed within the same interval had a comparable effect. The experiments revealed that the inhibitory effect of ethyl palmitate on antibody production was temporary only, and at a later stage could be compensated by an enhanced antibody activity.     

Erythrocyte survival and haematocrit values in rats after i.v. application of ethyl palmitate and methyl palmitate emulsions

In the experiments on rats the effect of repeated application of ethyl palmitate emulsion (EP) and methyl palmitate emulsion (MP) on the survival rate of51Cr labelled erythrocytes and haematocrit values of the peripheral blood was studied. EP was applied in a total amount of 0.8 g, while MP due to its higher toxicity in a dose of 0.4 g per 100 g of body weight. The application of EP caused mild, though significant shortening of the survival period of the erythrocytes; the haematocrit values remained unchanged. Following application of MP no differences as compared to control animals could be found.     

Endotoxaemia in rats: role of leukocyte sequestration in rapid pulmonary nitric oxide synthase-2 expression.

Nitric oxide (NO), depending on the amount, time and source of generation may exert both, protective and deleterious actions during endotoxic acute lung injury (ALI). Evaluation of the expression and localization of NOS isoforms in the lung of lipopolysaccharide (LPS)-treated rats may contribute to understanding the role of NO in pathogenesis of ALI. Tissue samples (lung, heart, liver, kidney and spleen) as well as peripheral blood polymorphonuclear cells (PMNs) were collected from control male Wistar rats and LPS - treated animals, 15, 30, 60, 120 and 180 min after LPS injection (2 mg kg(-1) min(-1) for 10 minutes, i.v.). Levels of NOS-2 and NOS-3 mRNA and protein in tissues and PMNs were estimated by RT-PCR, Northern blotting and Western blotting. Additionally, myeloperoxidase (MPO) activity in tissue samples was assayed. NOS-3 mRNA as well as protein were detected in lungs of control animals; pulmonary NOS-3 expression was not influenced by LPS. The induction of NOS-2 mRNA in rat lungs and in PMNs isolated from peripheral blood was observed 15 minutes after LPS challenge. In contrast, increase of NOS-2 mRNA in the heart, kidneys, liver and spleen was observed 2-3 hours after LPS injection. In all tissues rise in NOS-2 mRNA was followed after 1-2 hours by increase of NOS-2 protein. Importantly, progressive leukocyte sequestration in the lung parenchyma that started as early as 15 min after LPS injection was revealed only in the lungs; in other organs no significant changes in MPO activity were detected up to 180 min after LPS injection. In conclusion, infusion of LPS caused much more rapid expression of NOS-2 in lungs as compared to the heart, kidneys, liver and spleen. Early induction of NOS-2 may depend on the LPS-stimulated rapid neutrophil sequestration within lung vasculature and fast induction of NOS-2 in sequestrated neutrophils.     

Changes of lipofuscin-like pigments in erythrocytes and spleen after whole-body gamma irradiation of rats

Whole-body gamma irradiation of rats induced the formation of lipofuscin-like pigments in erythrocytes. Erythrocytes that were damaged by oxidation were scavenged in the spleen, and lipofuscin-like pigments were transferred from erythrocytes to the spleen during this process. The time course of lipofuscin-like pigments in erythrocytes and spleen indicates that the pigments were not induced by the action of free radicals produced by ionizing radiation but rather were a sequela of postirradiation metabolic changes.     

Immunological studies of the mechanism of anaemia in experimental Trypanosama evansi infection in rats

Pathological changes in the blood of rats acutely infected with Trypanosoma evansi and the probable mechanism of the accompanying anaemia, were investigated. A severe anaemia, together with rreticulocytosis and hepato-splenomegaly, were regularly observed. Histological examination of the liver, spleen and bone-marrow confirmed the increasedin erythropoietic activity that the observed anaemia was due to increasedextravascular destruction of erythrocytes rather than by inhibition of haemopoietic activity. All the infected rats showed significant immune responses to the infecting trypanosome peak agglutinin titres occurring 10–12 days after injection, coincidentally with maximun destruction of erythrocytes. Serological examination of sera and erythrocytes from all infected and control rats did not reveal the presence of either circulating or adsorbed erythrocyte auto--antibodies. Furthermore, there was no in vivo trypanosomal antigen coating of the erythrocytes from either infected or multiple antigen-injected rats. Repeated intraveoous injections into rats of more than 100 μg per g body weight of soluble T. evansi antigen resulted in moderately severe, probably antibody-mediated, haemolytic anaemia. It is considered that an immunologically-mediated mechanism may be responsible for the development of the anaemia accompanying T. evansi infection.     

The micronucleus test in rat erythrocytes from bone marrow, spleen and peripheral blood: the response to low doses of ionizing radiation, cyclophosphamide and vincristine determined by flow cytometry

The frequency of micronucleated polychromatic erythrocytes (fMPCE) was determined in samples from bone marrow, spleen and peripheral blood of rats exposed to low doses of X-rays, cyclophosphamide or vincristine. The fMPCE values were lower in the peripheral blood than in bone marrow or spleen. This is due to the elimination of MPCE from the circulating blood, which was confirmed by the results from prolonged exposure of rats to gamma-radiation. When the analysis was restricted to the youngest PCE in peripheral blood, the sensitivity of the assay was considerably improved. This can be reproducibly achieved with the flow cytometric analysis.     

Filtrability of erythrocytes in "hypersplenic" rats.

The values of haemoglobin, reticulocytes and half-times of the filtrability of non-washed and washed erythrocytes were examined in male albino rats, Wistar strain, after i.p. injections of methylcellulose (MC) and compared with controls. In individual experiments the rats received 2 to 32 injections of MC. In injected animals, the filtrability of non-washed erythrocytes was altered. The filtrability half-times of the washed erythrocytes did not differ from the controls. Thus, the filtrability is altered for extracorpuscular reasons. "Hypersplenism" being completely developed, (after 32 MC injections), the filtrability of non-washed erythrocytes repaired when the application of MC had been discontinued, the reticulocyte values remained however increased. Problems of the mechanism of anaemia in experimental "hypersplenism" after MC injections in rats and relations between the altered filtrability of the erythrocytes and the haemolysis are discussed.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 μl of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats.

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 microliters of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

Adrenaline-induced damage to the myocardium in rats with genetically determined arterial hypertension

Genetically hypertensive and normotensive rats were subjected to acute myocardial injury by a single subcutaneous injection of adrenaline (0.5 mg/100 g bw). The animals were sacrificed one day later. The lesions showed the signs of focal coagulative necrosis and intracellular myocytolysis. The damaged cardiomyocytes with high sarcolemmal permeability for blood plasma proteins were more widespread in the hypertensive versus normotensive rats. Intracellular myocytolysis, which is not associated with alterations in the cell membrane, was found in both experimental groups at an equal rate. The data agree with the concepts of alterations in biological membranes in genetically determined arterial hypertension.     

Prolonged blood glucose reduction in mrp-2 deficient rats (GY/TR(-)) by the glucose-6-phosphate translocase inhibitor S 3025

Chlorogenic acid derivatives are potent inhibitors of hepatic glucose production by inhibition of the glucose-6-phosphate translocase component of the hepatic glucose-6-phosphatase system. The pharmacological proof of concept was clearly demonstrated during i.v. infusion of potent derivatives (S 4048, S 3483) in rats. However, the blood glucose lowering effect of S 4048 after bolus i.v. injection lasted only 60-90 min. Plasma clearance of S 4048 was very high, and the parent compound was rapidly and efficiently excreted into the bile of Wistar and GY/TR(-) rats, indicating that mrp-2 was not involved in this hepatobiliary elimination process. About 72% of the total administered radioactivity appeared in the bile within 20 min after i.v. bolus injection of the radiolabeled analogue [(3)H]S 1743 in a Wistar rat. However, in GY/TR(-) rats the dicarboxylic analogue of S 4048, S 3025, was cleared from the plasma less rapidly than its parent compound and its biliary elimination was comparatively low. In contrast, S 3025 exhibited comparable pharmacokinetics and biliary elimination profile as S 4048 in Wistar rats, suggesting that biliary elimination of S 3025 is facilitated by mrp-2, functionally absent in GY/TR(-) rats. Targeting to mrp-2 resulted in a significantly prolonged reduction of blood glucose levels in GY/TR(-) rats after i.v. bolus administration of S 3025.     

Vanadium exposure enhances lipid peroxidation in the kidney of rats and mice

Vanadium (V) as sodium orthovanadate induces an increase in lipid peroxidation in the kidneys after a single subcutaneous or intraperitoneal injection to rats or mice. The rate of malondialdehyde (MDA) formation, an index of lipid peroxidation, by kidney homogenates increased by more than 100% 1 h after injection. Chronic exposure of rats to vanadium sulfate, initially through maternal milk and later in the drinking water, resulted after 10 weeks in a significant increase in MDA formation by kidney but not by other tissues. In both acute and chronic studies in rats and mice, no significant increase in lipid peroxidation by V treatment was detected in brain, heart, lung, spleen, or liver. In mice, administration of ascorbate prior to acute exposure to V diminished both toxicity, i.e., respiratory depression and limb paralysis, and the formation of MDA in kidney.     

Comparative effect and fate of non-entrapped and liposome-entrapped neuraminidase injected into rats

Non-entrapped and liposome-entrapped Clostridium perfringens neuraminidase (0.5-0.6 unit) was injected into rats and its fate as well as its effect on plasma and erythrocyte N-acetylneuraminic acid was investigated. The following observations were made. (1) Although removal of both non-entrapped and liposome-entrapped neuraminidase from the circulation was completed within 5h after injection, their recovery in tissues was distinctly different; 7-10% of the injected non-entrapped enzyme was found in the liver and none in the liver lysosomal fraction or the spleen. In contrast, 20-26% of the liposome-entrapped enzyme was found in the liver of which 60-69% was in the lysosomal fraction. Spleen contained 3.6-5.0% of the enzyme. (2) The presence of the non-entrapped neuraminidase in blood led to the extensive desialylation of plasma and to a decrease in the concentration or total removal from the circulation of some of the plasma glycoproteins. (3) Injection of non-entrapped neuraminidase also led to the partial desialylation of erythrocytes the life span of which was diminished and their uptake by the liver and spleen augmented. (4) Entrapment of neuraminidase in liposomes before its injection prevented the enzyme from acting on its substrate in plasma or on the erythrocyte surface, and values obtained for plasma glycoproteins and erythrocyte survival were similar to those observed in control rats. (5) Entrapment in liposomes of therapeutic hydrolases intended for the degradation of substances stored within the tissue lysosomes of patients with storage diseases could prevent the potentially hazardous enzymic action of hydrolases in blood and at the same time direct the enzymes to the intracellular sites where they are needed.     

Metabolism of adenosine, AMP and ATP in rats

Metabolism of [14C]adenosine in a dose of 100 mg per 1 kg of mass and [14C]ATP in the equimolar quantity was studied in rats after intraperitoneal administration. Adenosine is shown to enter tissues of the liver, spleen, thymus, heart and erythrocytes where it phosphorylates into adenine nucleotides (mainly ATP) and deaminates into inosine. The content of adenosine increases for a short period in the above tissues, except for erythrocytes and plasma. The latter accumulates a considerable amount of inosine and hypoxanthine, but only traces of uric acid, xanthine and adenine nucleotides. ATP administered to rats catabolizes through the adenosine formation. The exogenic adenosine and ATP replace in tissues and erythrocytes only a slight part (1-12%) of their total adenine nucleotide pool. The content of these metabolites and ADP in the blood plasma does not change essentially under the effect of adenosine, ATP and AMP. It is shown on rats whose adenine nucleotide pool of cells is marked by the previous administration of [14C]adenine that injections of adenosine, ATP and inosine do not accelerate catabolism of adenine nucleotides in tissues and erythrocytes as well as do not increase the level of catabolism products in the blood plasma. Adenosine enhances and ATP lowers the content of cAMP in spleen and myocardium, respectively.     

Humoral factors of the spleen in the regulation of the calcium content in blood plasma of rats]

Introduction of protein-free peptides-enriched spleen extract increases the calcium content in blood plasma of rats. After the effect of stress factor (long swimming) it falls. The value of the index under study increases in the splenectomized rats and remains unchanged after stress and introduction of the spleen factors. The most pronounced increase of the calcium concentration is observed in the case of experimental hypersplenism induced by methylcellulose introduction. The calcium-regulating effects of introduction of the enriched protein-free spleen extract and pharmacopoeial splenin preparation are compared. It is supposed that spleen contains two humoral factors of different chemical nature which are able to influence somewhat differently the calcium content in blood plasma.     

Macromolecular covalent binding of [14C]nitrobenzene in the erythrocyte and spleen of rats and mice

Nitrobenzene exposure is known to produce red blood cell damage as well as engorgement and sinusoidal congestion of the spleen in male Fischer-344 (F-344) rats but not in male B6C3F1 mice. These studies were conducted to investigate the species differences in the covalent binding of [¹⁴C]nitrobenzene in the erythrocyte and spleen and to assess the contribution of nitrobenzene-induced erythrocytic damage to the splenic effects. Total and covalently bound ¹⁴C concentrations in erythrocytes of rats were 6–13 times greater than those of mice following a single oral dose of 75, 150, 200 or 300 mg/kg [¹⁴C]nitrobenzene, suggesting that species differences in nitrobenzene-induced red blood cell toxicity may be related to differences in erythrocytic accumulation of nitrobenzene and its metabolites. Covalently bound ¹⁴C in erythrocytes of rats peaked 24 h following administration of 200 mg [¹⁴C]nitrobenzene/kg; in contrast, bound radio-label in erythrocytes from mice plateaued at 10 h. Splenic engorgement increased in a time-related manner in treated rats but not in mice. Species specificity was also observed in the accumulation of bound radiolabel in the spleen. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of lysed, dialyzed erythrocytes from treated rats revealed that hemoglobin was the primary, if not the exclusive, site of macromolecular covalent binding following nitrobenzene treatment. SDS-PAGE of dialyzed rat spleens revealed that 82% of total bound ¹⁴C migrated identically to hemoglobin. These data indicate that covalent binding of [¹⁴C]nitrobenzene and its metabolites in the spleen is primarily derived from bound ¹⁴C from scavenged erythrocytes. Therefore, the species differences in splenic engorgement and accumulation of [¹⁴C]nitrobenzene may be related to differences in susceptibility to nitrobenzene-induced red blood cell damage.     

Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)-nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin-receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (K d1), low affinity (K d2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.     

An experimental study on the pathway of iron transfer from macrophages to erythrocytes in rat liver

In order to reveal the pathway of iron release from macrophages, a 59Fe-labelled ferric hydroxide-potassium polyvinyl sulfate complex (Fe-PVS) was injected intravenously into anemic rats and the level of radioactivity in the liver, spleen, bone marrow, blood plasma and red blood cells (RBC) was estimated at various time intervals after the injection. Histochemical observation of ferric iron and ferritin in the liver was also made on anemic rats treated using unlabelled Fe-PVS. Fe-PVS injection promoted the recovery of anemia causing a rapid increase in the RBC number, with activated erythropoiesis occurring in the spleen and bone marrow. Soon after the injection, most of the radio iron was found in the liver with a small amount in the circulating erythrocytes, bone marrow and spleen. The iron level in the liver decreased gradually with a rapid increase in the iron level of the erythrocytes which reached a very high level 6 days after the 59Fe-PVS injection. Histochemical observations showed a heavy deposition of ferritin in the Kupffer cells 3 days after Fe-PVS injection. This deposition was minimized after 6 days with an increase in the level of ferritin in the parenchymal cells in the central area of acini. The level of radioferritin estimated biochemically in the nonparenchymal cell fractions of the liver revealed that the level dropped by about one third approximately 3.5 days after the Fe-PVS injection, showing the stimulated ferritin release at this stage. Results indicate that Kupffer cells in the liver play an important role in ferritin synthesis from the phagocytized iron compounds and that the iron is supplied for erythroid cell proliferation.