NaCl-treated Controls Research Articles

In vitro evaluation of antibacterial efficacy of vancomycin-loaded suture tapes and cerclage wires

Usage of implants containing antibiotic agents has been a common strategy to prevent implant related infections in orthopedic surgery. Unfortunately, most implants with microbial repellent properties are characterized by accessibility limitations during daily clinical practice. Aim of this in vitro study was to investigate whether suture tapes and cerclage wires, which were treated with vancomycin, show a sustainable antibacterial activity. For this purpose, we used 24 stainless steel wire cerclages and 24 ultra-high molecular weight polyethylene and polyester suture tape test bodies. The test bodies were incubated for 30 min. in 100 mg/ml vancomycin solution or equivalent volumes of 0.9% NaCl. After measuring the initial solution uptake of the test bodies, antibacterial efficacy via agar diffusion test with Staphylococcus aureus and vancomycin elution tests were performed 1, 2, 3, and 6 days after incubation. Vancomycin-loaded tapes as well as vancomycin-loaded cerclage wires demonstrated increased bacterial growth inhibition when compared to NaCl-treated controls. Vancomycin-loaded tapes showed an additional twofold and eightfold increase of bacterial growth inhibition compared to vancomycin-loaded wires at day 1 and 2, respectively. Elution tests at day 1 revealed high levels of vancomycin concentration in vancomycin loaded tapes and wires. Additionally, the concentration in vancomycin loaded tapes was 14-fold higher when compared to vancomycin loaded wires. Incubating suture tapes and cerclage wires in vancomycin solution showed a good short-term antibacterial activity compared to controls. Considering the ease of vancomycin application on suture tapes or wires, our method could represent an attractive therapeutic strategy in biofilm prevention in orthopedic surgery.

CD95-ligand contributes to abdominal aortic aneurysm progression by modulating inflammation.

Abdominal aortic aneurysm (AAA) is one of the number of diseases associated with a prominent inflammatory cell infiltration, matrix protein degradation, and smooth muscle cell apoptosis. CD95 is an inflammatory mediator and an apoptosis inducer. Previous studies have shown elevated expression of CD95 or CD95L in the aortic tissue of AAA patients. However, how the CD95L/CD95 contributes to aneurysm degeneration and whether blocking its signalling would be beneficial to disease progression remains largely unknown. In the present study, we sought to determine the role of CD95L and its downstream target, caspase 8, in AAA progression. By using the CaCl2 murine model of AAA, abdominal aortic aneurysms were induced in C57BL/6 mice. We found that both mRNA and protein levels of CD95L were increased in aneurysm tissue compared with NaCl-treated normal aortic tissue. To determine whether CD95L contributes directly to aneurysm formation, we used CD95L null (CD95L-/-) mice to examine their response to CaCl2 aneurysm induction. Six weeks after periaortic application of CaCl2, aortic diameters of CD95L-/- mice were significantly smaller compared to CaCl2-treated wild-type controls. Connective tissue staining of aortic sections from CaCl2-treated CD95L-/- mice showed minimal damage of medial elastic lamellae which was indistinguishable from the NaCl-treated sham control. Furthermore, CD95L deficiency attenuates macrophage and T cell infiltration into the aortic tissue. To study the role of CD95L in the myelogeous cells in AAA formation, we created chimaeric mice by infusing CD95L-/- bone marrow into sub-leathally irradiated wild-type mice (WT/CD95L-/-BM). As controls, wild-type bone marrow were infused into sub-leathally irradiated CD95L-/- mice (CD95L-/-/WTBM). WT/CD95L-/-BM mice were resistant to aneurysm formation compared to their controls. Inflammatory cell infiltration was blocked by the deletion of CD95L on myeloid cells. Western blot analysis showed the levels of caspase 8 in the aortas of CaCl2-treated wild-type mice were increased compared to NaCl-treated controls. CD95L deletion inhibited caspase 8 expression. Furthermore, a caspase 8-specific inhibitor was able to partially block aneurysm development in CaCl2-treated aneurysm models. These studies demonstrated that inflammatory cell infiltration during AAA formation is dependent on CD95L from myelogeous cells. Aneurysm inhibition by deletion of CD95L is mediated in part by down-regulation of caspase 8.

Acute Hypernatremia Exerts an Inhibitory Oxytocinergic Tone That Is Associated With Anxiolytic Mood in Male Rats

Anxiety disorders are the most common psychiatric illnesses and are associated with heightened stress responsiveness. The neuropeptide oxytocin (OT) has garnered significant attention for its potential as a treatment for anxiety disorders; however, the mechanism mediating its effects on stress responses and anxiety is not well understood. Here we used acute hypernatremia, a stimulus that elevates brain levels of OT, to discern the central oxytocinergic pathways mediating stress responsiveness and anxiety-like behavior. Rats were rendered hypernatremic by acute administration of 2.0 M NaCl and had increased plasma sodium concentration, plasma osmolality, and Fos induction in OT-containing neurons relative to 0.15 M NaCl-treated controls. Acute hypernatremia decreased restraint-induced elevations in corticosterone and created an inhibitory oxytocinergic tone on parvocellular neurosecretory neurons within the paraventricular nucleus of the hypothalamus. In contrast, evaluation of Fos immunohistochemistry determined that acute hypernatremia followed by restraint increased neuronal activation in brain regions receiving OT afferents that are also implicated in the expression of anxiety-like behavior. To determine whether these effects were predictive of altered anxiety-like behavior, rats were subjected to acute hypernatremia and then tested in the elevated plus maze. Relative to controls given 0.15 M NaCl, rats given 2.0 M NaCl spent more time in the open arms of the elevated plus maze, suggesting that acute hypernatremia is anxiolytic. Collectively the results suggest that acute elevations in plasma sodium concentration increase central levels of OT, which decreases anxiety by altering neuronal activity in hypothalamic and limbic nuclei.

Hippocampal synapsin isoform levels are linked to spatial memory enhancement by SGS742

Synapsins are essential proteins for synaptic plasticity and there is no information available for their role in cognitive enhancement (CE) of spatial memory formation. It was therefore the aim of the study to link individual synapsin proteins and their isoforms to spatial memory formation enhanced by SGS742 in the mouse. Extracted hippocampal proteins from a cognitive study treating OF1 mice with the cognitive enhancer SGS742 and tested in the Morris water maze, were run on two-dimensional gel electrophoresis. Subsequently, protein spots were unambiguously identified by qQ-TOF mass spectrometry. Quantification of proteins from four groups (NaCl-treated mice, SGS742-treated mice, SGS742-treated yoked controls, and NaCl-treated yoked controls) was carried out according to an in-gel stable isotope labeling method. A total of 17 protein spots representing synapsin isoforms were identified and quantified. Using quantification of individual synapsin isoforms showed that these can be clearly assigned to CE by the GABAB antagonist SGS742. Quantitative determination of individual synapsin isoform showed an increase in SGS742-treated mice (mean+/-SD) of ratios between light and heavy stable isotope labeled synapsin protein (SGS742 vs. controls: 2.19+/-0.41 for synapsin Ia, and 1.41+/-0.81 for synapsin IIa). Synapsins Ib and IIb were not linked to CE. The NaCl-treated controls and the use of yoked controls that were ruling out swimming- and stress-mediated changes of synapsins, unequivocally allow to propose a role for synapsins Ia and IIa in the mechanism of CE of spatial memory formation.

Demineralized bone alters expression of Wnt network components during chondroinduction of post-natal fibroblasts

Objective: The Wnt family of secreted proteins, their receptors (Fzd proteins) and antagonists (secreted Fzd-related proteins, or Sfrp) regulate chondrocyte differentiation and chrondrogenesis during embryonic development. Here, the hypothesis that the Wnt regulatory network contributes to chondrocyte differentiation of post-natal cells was tested in an in vitro model of chondroinduction by demineralized bone powder (DBP). Design: Human dermal fibroblasts (hDFs) were cultured in porous, three-dimensional (3D) collagen sponges with or without chondroinductive DBP. In some experiments, lithium chloride (LiCl), an agonist of the Wnt/β-catenin signaling pathway, was added to the culture media. Sponges were cultured for intervals (0.5–21 days) before processing for molecular, histologic, and biochemical analyses. Expression of wnt, fzd, and sfrp genes was characterized by semi-quantitative RT-PCR. Fibroblasts’ contacts with DBP were documented by histology. Accumulation of proteoglycan in extracellular matrix was evaluated by histology (metachromasia in toluidine blue-stained sections) and quantitative immunoassay (chondroitin 4-sulfate ELISA). Results: Expression of 15 wnt, fzd, and sfrp family members was detected in hDFs by RT-PCR. A subset of those genes ( wnt2b, wnt5b, wnt10b, fzd6, fzd7) showed altered expression in hDFs exposed to DBP for 3 days. wnt and fzd gene expression was not altered before hDFs contacted the DBP within the collagen sponge. Human DFs cultured in plain collagen sponges and treated with LiCl accumulated significantly more metachromatic matrix than NaCl-treated controls on day 10, and showed a trend towards increased matrix chondroitin-4 sulfate content. Conclusions: These data suggest that changes in Wnt signaling contribute to chondroinduction of post-natal fibroblasts by DBP. This is the first evidence that Wnt components, which are essential regulators of pre-natal chondrocyte differentiation, may also influence post-natal chondrocyte differentiation induced by DBP.

Dichloroacetic acid pretreatment of male and female rats increases chloroform metabolism in vitro

The role of metabolism in dichloroacetic acid (DCA) potentiation of CHCl 3 hepatotoxicity was investigated. Male and female Sprague-Dawley rats were given three doses (09:00, 16:00 and 09:00 the next morning) of DCA (each 2.45 mmol/kg) by gavage. The rats were euthanized 3 h after the last dose, hepatic microsomes were prepared and 14CHCl 3 metabolism was measured in vitro. The binding of 14CHCl 3-derivatives to microsomal proteins and lipids was increased 65 and 100%, respectively, in DCA-treated rats compared to their respective NaCl-treated controls. The formation of CO 2 (nmol 14CO 2/incubation) was significantly elevated in DCA-treated rats compared to controls (10.4 vs 6.3 in males; 10.8 vs 6.1 in females). DCA treatment decreased the apparent Michaelis constant (K m app ) for conversion of 14CHCl 3 to 14CO 2 in rats (0.665 vs 0.415 mM in males, P<0.05; 0.161 vs 0.081 in females). 14CO 2 production and 14C binding were observed under N 2 atmosphere, indicating that reactive metabolites of 14CHCl 3 were formed by oxidation as well as reduction. Male and female rats metabolized CHCl 3 differently. The K m app for CO 2 production was up to 5-fold higher in the males than in the females, regardless of DCA treatment. Inhibition by SKF 525-A and piperonyl butoxide was gender dependent in both control and DCA-treated groups. The results showed, that increased bioactivation of CHCl 3 by DCA treatment is one element in the DCA-CHCl 3 interaction.

Sodium nitrite and potassium nitrate in control of nonproteolytic Clostridium botulinum outgrowth and toxigenesis in vacuum-packed cold-smoked rainbow trout

The effect of sodium nitrite (NaNO 2) and potassium nitrate (KNO 3) on the outgrowth and toxigenesis of nonproteolytic Clostridium botulinum in vacuum-packed cold-smoked rainbow trout stored for six weeks was studied in two inoculation studies at slightly abusive storage temperatures of 4 °C and 8 °C. The depletion rate of nitrite and the reduction rate of nitrate to nitrite as well as the effect of nitrite and nitrate on the shelf-life of the product during eight weeks' storage period were also determined. The nitrite concentrations were reduced from 166 mg kg ± 9 (mean ± SE), to a final concentration of 34 mg kg ± 2 and 11 mg kg ± 2, and the nitrate concentrations from 686 mg kg ± 67 to 465 mg kg ± 140 and 427 mg kg ± 33 at 4 ° C and 8 ° C respectively. The nitrite depletion rate was more rapid at 8 °C; nitrate depletion was not significantly affected by temperature. A considerable amount of nitrite was detected in the nitrate-treated samples in the latter half of the storage period. At 4 °C the aerobic plate counts were significantly lower in the samples treated with NaNO 2 + NaCl and with KNO 3 + NaCl as compared to the NaCl-treated controls, while at 8 °C the differences were smaller. The sensorial shelf-life of the product was considerably extended by nitrite and nitrate curing. The nitrite and nitrate concentrations used in the present study did not completely inhibit the toxigenesis of nonproteolytic C. botulinum during the six-week storage period, although the number of toxic samples was considerably reduced by nitrite and nitrate curing.

Effect of cyclosporin A, azathioprine, and prednisolone on carbohydrate metabolism of rat hepatocytes

The effect of different immunosuppressive drugs (prednisolone, azathioprine, cyclosporin A) on liver carbohydrate metabolism in the rat was investigated. Daily administration of prednisolone (3 mg/kg body weight) and azathioprine (2 mg/kg body weight) intraperitoneally for 2 weeks caused significantly lower liver glycogen content than that in NaCl-treated controls. Liver glucose and lactate content, as well as plasma glucose, glucagon, and serum insulin concentration of these animals, remained unchanged. There were no differences in any of these parameters between cyclosporin A (15 mg/kg body weight)-treated and vehicle (olive oil/ethanol)-treated animals. Prednisolone caused significantly lower glucose production in isolated rat hepatocytes using Na-pyruvate as the substrate, whereas glucose production was unchanged in hepatocytes of azathioprine-treated rats using pyruvate or L-serine as substrates. Glucose production from pyruvate or serine was significantly inhibited by cyclosporin A compared to the vehicle, but did not differ from the effects of azathioprine and prednisolone. Lactate production was significantly lower in cyclosporin-treated animals than in those given either the vehicle or azathioprine. Cyclosporin A completely reversed the inhibition of hepatocyte glycogen consumption caused by the vehicle. However, glycogen production in the presence of cyclosporin A was comparable to the effects of prednisolone and azathioprine. Finally, hepatocyte ketone body production using pyruvate as the substrate was higher in the presence of all immunosuppressive drugs. In the presence of serine, acetoacetate production increased in rats treated with 50 mg/kg body weight cyclosporin A, and beta-hydroxybutyrate production in animals receiving 15 and 50 mg/kg body weight cyclosporin A.

Effect of ACTH suppression on adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in 4-aminopyrazolopyrimidine-treated rats

4-Aminopyrazolopyrimidine (4-APP) treatments to rats for 3 days induced 2-fold increase of circulating ACTH and 11-fold increase of adrenal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA compared to NaCl-treated controls. This in vivo model was used to study the effect of the suppression of ACTH secretion on the adrenal HMG-CoA reductase mRNA level. Dexamethasone (Dex) administration to 4-APP-treated rats caused a rapid and parallel decline of the levels of plasma ACTH and adrenal HMG-CoA reductase mRNA to 50% within 2.5 h, whereas the free and esterified cholesterol content was increased 5 and 9.4 times respectively. These changes could be counteracted by the co-administration of ACTH with Dex. Aminoglutethimide (AG) administration to 4-APP-treated rats, which increased the adrenal esterified cholesterol content (7.5 times), decreased the HMG-CoA reductase mRNA level (44%), despite plasma ACTH level remaining elevated. Moreover, the participation of newly synthesized protein(s) in the lowering of adrenal HMG-CoA reductase mRNA level induced by ACTH suppression is suggested by the fact that cycloheximide (Cyclo), when co-administered with AG, completely blocked the decrease of HMG-CoA reductase mRNA level, despite the plasma ACTH level decreasing by 68% and the free and esterified cholesterol content increasing 3.9 and 12.3 times, compared to 4-APP-treated rats. Furthermore, the specificity of these effects was established by the fact that the β-actin mRNA level was not affected by the administration of either Dex, AG, Cyclo, or AG + Cyclo to 4-APP-treated rats. In conclusion, it is hypothesized that the decrease in adrenal HMG-CoA reductase mRNA level caused by the suppression of ACTH secretion could be mediated through both the enhancement of the adrenal cholesterol (or its metabolites) content and newly synthesized protein(s).

Subacute and Chronic In Vivo Lithium Treatment Inhibits Agonist‐ and Sodium Fluoride‐Stimulated Inositol Phosphate Production in Rat Cortex

We have investigated the effects of in vivo lithium treatment on cerebral inositol phospholipid metabolism. Twice-daily treatment of rats with LiCl (3 mEq/kg) for 3 or 16 days resulted in a 25-40% reduction in agonist-stimulated inositol phosphate production, compared with NaCl-treated controls, in cortical slices prelabelled with [3H]inositol. A small effect was also seen with 5-hydroxytryptamine (5-HT) 24 h after a single dose of LiCl (10 mEq/kg). Dose-response curves to carbachol and 5-HT showed that lithium treatment reduced the maximal agonist response without altering the EC50 value. This inhibition was not affected by the concentration of LiCl in the assay buffer. Stimulation of inositol phosphate formation by 10 mM NaF in membranes prepared from cortex of 3-day lithium-treated rats was also inhibited, by 35% compared with NaCl-treated controls. Lithium treatment did not alter the kinetic profile of inositol polyphosphate formation in cortical slices stimulated with carbachol. Muscarinic cholinergic and 5-HT2 bindings were unaltered by lithium, as was cortical phospholipase C activity and isoproterenol-stimulated cyclic AMP formation. [3H]Inositol labelling of phosphatidylinositol 4,5-bisphosphate was significantly enhanced by 3-day lithium treatment. The results, therefore, indicate that subacute or chronic in vivo lithium treatment reduces agonist-stimulated inositol phospholipid metabolism in cerebral cortex; this persistent inhibition appears to be at the level of G-protein-phospholipase C coupling.

Repetitive treatment with gonadotrophin releasing factor or a long-acting analogue upon gonadotrophin secretion in the ferret.

The changes in plasma levels of LH and FSH were examined after treatment of intact and ovariectomized females ferrets with a long-acting analogue of gonadotrophin releasing factor (Gn-RF) (D-Ser(But)6-LH-releasing hormone (1-9)-nonapeptide ethylamide; Hoe 766) either as a single intravenous injection or daily for 6 days. The responses were compared with those induced by daily injections of Gn-RF or 0.9% NaCl (w/v). Treatment with Gn-RF consistently induced rises in both LH and FSH release with peak levels of both hormones being reached 20 min after injection and being of similar size from day to day in individual animals. Thereafter, the gonadotrophin levels declined rapidly to approach basal values by the end of each sampling period. Treatment with Hoe 766, however, produced very high values on day 1 of treatment, with LH being raised for 10-12 h and FSH for up to 24 h. Subsequent injections, on the other hand, produced an abbreviated LH response of similar size to that induced by Gn-RF and little, if any, FSH response. In ovariectomized ferrets, Hoe 766 induced a variable LH response and little FSH response at any time. In addition, basal FSH levels in the first three samples taken on each day from day 2 onwards tended to decline markedly in all of the Hoe 766-treated animals, an effect not seen in Gn-RF or 0.9% NaCl-treated controls.

Zellkinetische Untersuchungen zur Interferenz zwischen Proliferations- und Funktionsstoffwechsel in der regenerierenden Rattenleber nach Applikation von Phenobarbital

To investigate the correlation between function and proliferation, cell kinetic studies were carried out in regenerating liver of phenobarbital (PB) treated rats. Male adult Wistar rats received i.p. injections of 90 mg PB/kg body weight daily for five days (group A). Rats of group B received additional injections of PB at a reduced dose (30 mg/kg) every 12 hrs after partial hepatectomy, starting immediately after the operation. 60 min after a single injection of3H-TdR at different intervals after surgery, specific activity of DNA,3H-TdR labeling index, and mitotic index were determined. Intralobular distribution of DNA-synthesizing hepatocytes was analyzed in 22 h regenerating liver. Stimulation of functional activities by PB results in a transitional synchronization of the start of DNA synthesis in liver parenchymal cells after partial hepatectomy. The labeling indices of group A and B increase to 36% and 30%, respectively, as compared to 18.5% of NaCl-treated controls. Analysis of intralobular distribution of DNA-synthesizing hepatocytes revealed that higher levels of synchronization are limited to periportal zones of the liver lobule which provide optimal conditions for G1-S transition. PB treatment apparently does not influence duration of S, G2 or M under these conditions. The results stress the possibility that transitional synchronization of DNA synthesis by functional load might simulate stimulation of growth in a proliferating population.

Glutamate-induced brain damage in infant primates.

The brains of 9 infant rhesus monkeys were examined for acute cytopathology following treatment with monosodium L-glutamate (MSG) (6 infants) or NaCl (3 infants). Hypothalamie lesions identical to those described elsewhere in MSG-treated rodents were identified by light microscopy and verified with the electron microscope in each infant monkey given MSG. No cellular pathology was detected in the hypothalami of NaCl-treated controls. Infants given relatively low oral doses of MSG (1 and 2 g/kg) sustained small focal lesions confined primarily to the rostro-ventral aspect of the infundibular nucleus. Those treated with high subcutaneous doses developed lesions which spread throughout and sometimes beyond the infundibular nucleus. At all doses tested (1–4 g/kg) and by either route of administration, rapid necrosis of neurons (within 5 hours) was a striking feature of the MSG-induced reaction pattern. Blood glutamate curves from time of treatment to time of sacrifice (5 hours) suggest that the threshold for lesion formation in 1-week-old rhesus monkeys may be in the range of 20 mg%. Differences in research approach which might account for the failure of others to locate the lesions we describe are discussed as are some of the toxicological implications of our findings.

Source of the Histamine Released During Damage to the Gastric Mucosa by Acetic Acid

Summary The stomachs of pylorus-ligated rats were filled with 154 mm NaCl or 100 mm acetic acid plus 54 mm NaCl. After various intervals, the stomach contents were removed and the amount of histamine present was determined by bioassay on the guinea pig ileum. Homogenates were made from some rat stomachs. These were centrifuged, and the supernatant fluids were used as preparations of the enzyme, histidine decarboxylase. The activity of the enzyme was determined by counting the 14 CO 2 evolved when 14 C-carboxyl histidine was incubated with the enzyme. This measurement was converted to the histamineforming capacity (HFC). Acetic acid damage released significant amounts of histamine into the gastric contents. Release was complete after 30 min of irrigation. After 15 min of irrigation the HFC of the rats treated with acetic acid was increased 3-fold and was twice that of the NaCl-treated controls. After 2 hr the HFC of damaged stomachs had increased 12-fold as compared with a 6-fold increase in the control animals. Rats were treated with the mast cell histamine depletor, compound 48/80, and their stomachs were filled with either NaCl or acetic acid. At the end of 30 min there was no histamine present in NaCl irrigation fluids and less than one-tenth the amount normally found in the contents of acetic acid damaged stomachs. Since the releasable histamine pool is exhausted within 30 min, 48/80 prevents histamine release, and the HFC remains high after release is complete, it is concluded that the mucosal mast cells are the source of the histamine. It is hypothesized that the histamine release triggers an adaptive increase in the HFC. The increased enzyme activity furnishes more histamine to the damaged mucosa. Since activity is high throughout injury, the elevated HFC may serve some function during later stages of damage.