Hypotonic contrast media is more toxic than isotonic contrast media on endothelial cells in vivo and in vitro.

The aim of the current study was to investigate the cytotoxic effects of hypotonic (iopamidol) and isotonic (iodixanol) contract media (CMs) in vitro and in vivo. A total of 60 Wistar rats were included and were randomly divided into three groups (20 rats per group). Iodixanol (4 g iodine/kg), iopamidol (4 g iodine/kg) or equal volume of normal saline was injected via tail vein. HUVEC and H5V cell viability was determined by Cell Counting Kit­8 agents. Western blotting was performed to detect ATP­binding cassette subfamily G member 1 (ABCG1) expression. For histological analysis, hematoxylin and eosin staining was performed. Plasma endothelin, von Willebrand factor, tissue type plasminogen activator, plasminogen activator inhibitor, D­Dimer, fibrinogen, anti­thrombin III, plasminogen and nitric oxide synthase (NOS) were measured by using ELISA. Both iopamidol and iodixanol treatments deceased cell viability and increased apoptosis of HUVEC and H5V cells, along with downregulated NOS and ABCG1. The injection of iopamidol or iodixanol into rats changed the endothelium­related plasma levels of biomarkers, including endothelin, von Willebrand factor, tissue type plasminogen activator, plasminogen activator inhibitor, D­Dimer, fibrinogen and anti­thrombin III. However, endothelia isolated from rat abdominal aorta in the iodixanol group retained their normal structure, whereas endothelial structure in the iopamidol group was injured and disrupted. The findings in the present study suggested that both hypotonic and isotonic CMs may lead to endothelial dysfunction and thrombin and fibrinolytic system disorder. However, hypotonic CMs may be more toxic than isotonic CMs. Therefore, additional cautions should be taken when selecting hypotonic CMs and their dosages during cardioangiography.

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets.

Blockade of chloride ion transport enhances the cytocidal effect of hypotonic solution in gastric cancer cells.

BACKGROUND: Cancer cells that are exfoliated into the peritoneal cavity during surgery are viable and have the potential to produce peritoneal recurrence. Although peritoneal lavage with distilled water is applied in some cancer surgeries to kill tumor cells, there is no consensus regarding the optimal methodology and its effects. METHODS: Three human gastric cancer cell lines, MKN28, MKN45, and Kato-III, were exposed to distilled water, and the resultant morphologic changes were observed using a microscope. Analysis of cell volume changes was performed using a flow cytometer. To investigate the cytocidal effects of the water, re-incubation of the cells was performed after exposing them to hypotonic solution. Additionally, the effects of 5-nitro-2-3-phenylpropylamino)-benzoic acid (NPPB), a Cl(-) channel blocker, and R(+)-[(dihydroindenyl)oxy] alkanoic acid (DIOA), a blocker of the K(+)/Cl(-) co-transporter, on the cells during their exposure to hypotonic solution were analyzed. RESULTS: After the cells had been exposed to the distilled water, a rapid increase in cell volume occurred followed by cell rupture. In the MKN45 and Kato-III cells, treatment with NPPB increased cell volume by inhibiting regulatory volume decrease and enhanced the cytocidal effects of the hypotonic solution, whereas no such effects were observed in the MKN28 cells. On the other hand, treatment of the MKN28 cells with DIOA inhibited RVD and enhanced the cytocidal effects of hypotonic shock. CONCLUSION: These findings support the efficacy of peritoneal lavage with distilled water during surgery for gastric cancer and suggest that the regulation of Cl(-) transport enhances the cytocidal effects of hypotonic shock.

Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos.

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at -5°C for 30min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60min at 25°C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25°C for 30min, the maturation rate decreased in solution with 0.51Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25°C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at -5°C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.

Halorubrum kocurii sp. nov., an archaeon isolated from a saline lake.

A Gram-negative, non-motile, neutrophilic, rod-shaped, extremely halophilic archaeon, designated strain BG-1(T), was isolated from a salt lake, Lake Bagaejinnor, in Inner Mongolia, China. Strain BG-1(T) was able to grow at 25-55 degrees C, required at least 2.5 M NaCl for growth (with an optimum at 3.4 M NaCl) and grew at pH 6.0-9.0 (with an optimum at pH 7.5). Hypotonic treatment with less than 2.0 M NaCl caused cell lysis. Phylogenetic analysis of the almost-complete 16S rRNA gene sequence positioned the isolate within the genus Halorubrum in the family Halobacteriaceae. Strain BG-1(T) was most closely related to Halorubrum aidingense 31-hong(T) (98.8% sequence similarity), Halorubrum saccharovorum NCIMB 2081(T) (98.6%), Halorubrum lacusprofundi ACAM 34(T) (98.6%) and Halorubrum lipolyticum 9-3(T) (98.4%). However, values for DNA-DNA hybridization between strain BG-1(T) and the most closely related members of the genus Halorubrum were below 40%. Analysis of the polar lipids of strain BG-1(T) revealed the presence of mannosyl-2-sulfate-(1-4)-glycosyl-archaeol, the main glycolipid found in neutrophilic species of the genus Halorubrum. The G+C content of the genomic DNA was 69.4 mol% (T(m)). Comparison of the phenotypic characteristics of the strain with those of Halorubrum species supported the conclusion that BG-1(T) represents a novel species within this genus, for which the name Halorubrum kocurii sp. nov. is proposed. The type strain is BG-1(T) (=CECT 7322(T) =CGMCC 1.7018(T) =JCM 14978(T)).

Halobacterium piscisalsi sp. nov., from fermented fish (pla-ra) in Thailand.

A Gram-negative, motile, rod-shaped, extremely halophilic archaeon, designated strain HPC1-2(T), was isolated from pla-ra, a salt-fermented fish product of Thailand. Strain HPC1-2(T) was able to grow at 20-60 degrees C (optimum at 37-40 degrees C), at 2.6-5.1 M NaCl (optimum at 3.4-4.3 M NaCl) and at pH 5.0-8.0 (optimum at pH 7.0-7.5). Hypotonic treatment with less than 1.7 M NaCl caused cell lysis. The major polar lipids of the isolate were C(20)C(20) derivatives of phosphatidylglycerol, methylated phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether, sulfated triglycosyl diether and sulfated tetraglycosyl diether. The G+C content of the DNA was 65.5 mol%. 16S rRNA gene sequence analysis indicated that the isolate represented a member of the genus Halobacterium in the family Halobacteriaceae. Based on 16S rRNA gene sequence similarity, strain HPC1-2(T) was related most closely to Halobacterium salinarum DSM 3754(T) (99.2%) and Halobacterium jilantaiense JCM 13558(T) (97.8%). However, low levels of DNA-DNA relatedness suggested that strain HPC1-2(T) was genotypically different from these closely related type strains. Strain HPC1-2(T) could also be differentiated based on physiological and biochemical characteristics. Therefore, strain HPC1-2(T) is considered to represent a novel species of the genus Halobacterium, for which the name Halobacterium piscisalsi sp. nov. is proposed. The type strain is HPC1-2(T) (=BCC 24372(T)=JCM 14661(T)=PCU 302(T)).

Endothelial cell density in porcine corneas after exposure to hypotonic solutions.

PURPOSE: To evaluate exposure to sucrose solution (1.8%) and hypotonic balanced salt solution (BSS) for its effects on endothelial cell density of porcine corneas. METHODS: Two groups of central discs from pig corneas were organ-cultured for 24 h. Twelve corneas per group were exposed to sucrose solution (1.8%) or hypotonic BSS for 4 min each. The paired corneal discs were not treated and served as controls. After further organ culture with and without dextran for 48 h, corneal endothelium was stained with alizarin red and examined by light microscopy. The endothelial cell densities were determined manually on three central images. RESULTS: The endothelial cell density differed significantly between corneas exposed to sucrose and the control corneas (3982+/-382 cells/mm(2) and 4360+/-331 cells/mm(2) respectively, and 3876+/-364 cells/mm(2) versus 4374+/-168 cells/mm(2) respectively with 6% dextran). In contrast, the endothelial cell density did not differ significantly between corneas exposed to hypotonic BSS and the control corneas (4374+/-296 cells/mm(2) and 4317+/-193 cells/mm(2) respectively, and 4348+/-151 cells/mm(2) versus 4426+/-175 cells/mm(2), respectively with 6% dextran). CONCLUSIONS: Exposure to 1.8% sucrose for 4 min induces a significant endothelial cell loss of 10% on average, whereas exposure to hypotonic BSS did not significantly influence the endothelial cell density.

Hypoosmolar conditions reduce extracellular volume fraction and enhance epileptiform activity in the CA3 region of the immature rat hippocampus.

The osmolarity of the extracellular space (ECS) compartment is an important factor determining the excitability of neuronal tissue. In the adult hippocampus an important role of osmolarity and ECS diffusion parameters on the susceptibility to epileptic events is well established, but the influence of hypo- and hyperosmolar conditions on the immature hippocampus remains elusive. To investigate the influence of osmolarity on epileptiform activity, extracellular field potentials were recorded in the CA3 region of hippocampal slices of immature (postnatal days 4-7) Wistar rats. The ECS diffusion parameters were determined by the real-time tetramethylammonium (TMA+) iontophoretic method with ion-selective microelectrodes in immature hippocampal slices and showed a lack of diffusion anisotropy; a tortuosity of about 1.39; and a volume fraction, alpha, of 0.41 +/- 0.01 (n = 10 slices). A reduction in osmolarity of -90 mOsm induced a decrease in alpha to 0.17 +/- 0.02 (n = 4 slices). The frequency of epileptiform activity elicited in 10-50 microM 4-AP-containing low-Mg2+ solution was increased under -90 mOsm and -40 mOsm hypoosmolar conditions by 39.9% +/- 8.1% (n = 16) and 24.1% +/- 4.8% (n = 10), respectively, whereas hyperosmolar solutions decreased the frequency. A -90-mOsm reduction in the osmolarity of low-Mg2+ solution induced epileptiform activity in nine of 19 slices. In summary, these results demonstrate that hypoosmolar conditions increased excitability and susceptibility to epileptiform activity in immature hippocampal slices, suggesting a functional role of the larger alpha in suppression of seizures.

Peritoneal toxicity of water: a model of chronic peritonitis caused by osmotic dysequilibrium in rats.

The purpose of this work was to determine whether the osmotic dysequilibrium created by intraperitoneal (i.p.) injection of pure water has any permanent, damaging toxic sequelae. Rats were injected i.p. with pure water on five successive days. Necropsies were performed 1 week after the last injection. Necropsies revealed fibrosis of peritoneal surfaces of liver and spleen, similar to the effects of chemical irritants but milder. The severity of the lesions depended on the dose of water and the number of injections. A few minutes of contact with pure water was sufficient to ensure subsequent development of fibrosis. Soon after the initial injury, the inoculum became less hypotonic and then isotonic. Isotonic or moderately hypotonic electrolyte solutions did not produce peritoneal fibrosis but very hypotonic solutions were toxic. Injection of the synthetic compound 48/80, which is known to cause discharge of mast cell granules, did not produce peritonitis, nor was contamination by endotoxin or by blood responsible for the lesions. Injection of water may be a useful method for investigating the role, if any, of mast cells and/or mesothelial cells in the toxic effects of osmotic dysequilibrium.

Osmolality and nonsynaptic epileptiform bursts in rat CA1 and dentate gyrus.

In several clinical situations, such as hyposmolar states and hypoxia-ischemia, reductions in the size of the extracellular space are associated with increased seizure susceptibility. Nonsynaptic interactions provide a likely means of mediating the effect of extracellular space on seizure susceptibility. Synchronous bursting of CA1 hippocampal neurons occurs via nonsynaptic mechanisms in solutions containing very low [Ca2+] and excitatory amino acid antagonists. We tested the hypothesis that lowering the osmolality of the extracellular medium could induce nonsynaptic bursting in the dentate gyrus, even though it is normally resistant to this treatment. Extracellular field potentials were recorded in the dentate gyrus and CA1 area of rat hippocampal slices. In the low-[Ca2+] solution with normal osmolality, bursts of population spikes were recorded from the dentate gyrus in only 7% of the slices, but solutions with decreased osmolality induced bursting in 63%. Corresponding values for the CA1 area were 60 and 73%, respectively. Mannitol, which reversed the hyposmolar state, abolished bursting in both regions. This study demonstrates that reducing the size of the extracellular space by lowering extracellular osmolality can transform a seizure-resistant area into one that exhibits robust epileptiform activity.

The effect of heparin on endothelial stability.

Heparin in ultra low doses had a stabilizing effect on endothelial lining in rats. This effect was demonstrated as prevention of increases in counts of circulating endothelial cells after a standard intravenous citrate injection. The same effect of heparin was observed even with other provoking agents /adrenaline, hypotonic saline/. The duration of heparin effect was very prolonged, a 50% effect having been observed 5 hours after the administration. Subcutaneous heparin was effective only in relatively high doses and had a lower maximum effect compared with intravenous heparin.

[Pathology of cerebral edema. II. Experimental models and modifying agents]. / Patología del edema cerebral. II. Modelos experimentales y agentes modificadores.

Current experimental models of brain edema are described and evaluated for their contribution to the knowledge of basic processes involved in its production as well their contribution to the understanding of different clinical forms. The participation of each main pathogenic mechanism in a given experimental model is analyzed and proves to vary with each particular model and site studied. The importance of various experimental models in the evaluation of different therapeutic procedures directed to control the genesis and evolution of brain edema is stressed.