Customized vascular catheters for rodents

Custom arterial and venous catheters were made for rodents from polyurethane tubing. The low thrombogenicity and toxicity index, chemical stability and resiliency of polyurethane made this tubing an ideal catheter material. The tubing is shaped using peanut oil heated to 160 degrees C. Sealing these catheters is accomplished simply by heating the tube end with a hot object and pinching it between the fingers. Chronic catheter patency was maintained using Burr's solution (9:1 mixture of glycerine and heparin). No flushing was necessary.     

Ileal amino acids digestibility of sorghum in weaned piglets and growing pigs

The objective of the study was to determine the coefficients of ileal apparent digestibility (CIAD) of sorghum protein and amino acids (AA) in weaned piglets and growing pigs. Digestibility coefficients were estimated using the regression and difference methods for the weaned piglets; and the direct and difference methods for the growing pigs. To test the hypothesis that CP and AA digestibility of sorghum is lower in weaned piglets than in growing pigs, two experiments were conducted. In experiment one, 20 weaned piglets were fitted with a 'T' cannula at 21 days of age and were fed for 2 weeks one of five dietary treatments: a reference or control diet providing 200 g of CP/kg from casein (C) as the sole protein source, and four casein-sorghum (C-S) diets kept isoproteic to C by the appropriate adjustment of C and maize starch proportions; the amount of sorghum (S) in these diets was 135, 307, 460 and 614 g/kg. In experiment 2, fifteen castrated pigs weighing 57.8 ± 2.8 kg were used and randomly allotted to one of three dietary treatments: a reference casein-maize starch diet containing C as the sole protein source, a C-S diet, both diets containing 160 g of CP/kg, and a fortified S diet containing 68 g of CP/kg. In piglets the CIAD for CP and AA decreased linearly (P < 0.05) as the amount of S in the diet increased. The average ileal digestibility of AA from C was 0.858 ± 0.111, and decreased to 0.663 ± 0.191 at the higher S level. The CIAD estimated using the regression or difference methods were similar for leucine, cysteine, glutamic acid, serine, alanine and tyrosine, and different for the other AA. In growing pigs the CIAD of protein and AA (except alanine and cysteine) were similar (P > 0.05) for the C and the C-S diets, but higher (P < 0.05) than those for the S diet. The CIAD for S obtained by the difference method were higher (P < 0.05) than those obtained using the direct method, except for lysine, isoleucine, valine, methionine, threonine and cysteine. The results indicate that except for lysine and cysteine, growing pigs' ability to digest AA and protein is superior than weaned piglets.     

A model of growing vascular structures

Increasing attention is being paid to the configuration and development of vascular structures and their possible correlations with physiological events. The study of angiogenesis in normal and pathological states as well as in the embryo and adult has provided new insights into the mechanism of vessel growth and organization of the vasculature. Various mathematical branching models have been developed. These constructions are mainly geometrical and only involve a branching phenomenon. We propose the use of a deterministic non-linear model based on physiological laws and hydrodynamics. Growth, branching and anastomosis, the three actual main events occurring in vascular growth, are included in this model. Space growth, including cells and vessels, is defined by a decreasing transformation. Space density and the length of new sprouts are controlled by a set of parameters. The conditions on these parameters are well established, which allows the production of realistic patterns.     

Upregulation of vascular calcium channels in neonatal piglets with hypoxia-induced pulmonary hypertension

Inhibition of voltage-gated, L-type Ca(2+) (Ca(L)) channels by clinical calcium channel blockers provides symptomatic improvement to some pediatric patients with pulmonary arterial hypertension (PAH). The present study investigated whether abnormalities of vascular Ca(L) channels contribute to the pathogenesis of neonatal PAH using a newborn piglet model of hypoxia-induced PAH. Neonatal piglets exposed to chronic hypoxia (CH) developed PAH by 21 days, which was evident as a 2.1-fold increase in pulmonary vascular resistance in vivo compared with piglets raised in normoxia (N). Transpulmonary pressures (DeltaPtp) in the corresponding isolated perfused lungs were 20.5 +/- 2.1 mmHg (CH) and 11.6 +/- 0.8 mmHg (N). Nifedipine reduced the elevated DeltaPtp in isolated lungs of CH piglets by 6.4 +/- 1.3 mmHg but only reduced DeltaPtp in lungs of N piglets by 1.9 +/- 0.2 mmHg. Small pulmonary arteries from CH piglets also demonstrated accentuated Ca(2+)-dependent contraction, and Ca(2+) channel current was 3.94-fold higher in the resident vascular muscle cells. Finally, although the level of mRNA encoding the pore-forming alpha(1C)-subunit of the Ca(L) channel was similar between small pulmonary arteries from N and CH piglets, a profound and persistent upregulation of the vascular alpha(1C) protein was detected by 10 days in CH piglets at a time when pulmonary vascular resistance was only mildly elevated. Thus chronic hypoxia in the neonate is associated with the anomalous upregulation of Ca(L) channels in small pulmonary arteries in vivo and the resulting abnormal Ca(2+)-dependent resistance may contribute to the pathogenesis of PAH.     

Tolerance ofStaphylococcus epidermidis grown from indwelling vascular catheters to antimicrobial agents

During a prospective study of indwelling vascular catheter-related infections, 134 isolates ofStaphylococcus epidermidis were grown from 700 catheter tips.In vitro antimicrobial susceptibility testing of these isolates to oxacillin, vancomycin and ofloxacin was performed using the standard broth microdilution technique. These results were compared to those for the same organisms grown in biofilm before the addition of antimicrobial agents. In 96-well flat bottom microtiter plates, 10⁴–10⁵ colony forming units ofS. epidermidis in 0.1 ml broth were grown for 18 h at 37°C, at which time a biofilm was observed for all isolates. Different concentrations of antimicrobial agents (0.1 ml) were then added to the plates. The plates were incubated for 18 h at 37°C. Since MICs could not be estimated in these plates, all the wells were subcultured after mixing the biofilm with the broth. Minimum bactericidal concentrations (MBCs) were defined as 99.9% reduction in colony forming units. For organisms grown in suspension, 100% of the isolates were susceptible to vancomycin, 81% to ofloxacin and 40% to oxacillin. MBCs of susceptible isolates were within four-fold differences for vancomycin (53%), oxacillin (50%), and ofloxacin (51%). When grown as a biofilm, 78%, 93% and 71% of isolates had MBCs of 2048 g ml^–1 of oxacillin, vancomycin and ofloxacin respectively. These data demonstrate the reduced bactericidal activity of antimicrobial agents againstS. epidermidis in a biofilm and a simple method for its detection in the microbiology laboratory.     

Chronic ethanol exposure induces alterations in the nucleocytoplasmic transport in growing astrocytes

Nucleocytoplasmic transport is a crucial process for cell function. We assessed the general effect of chronic alcohol exposure on this transport in growing astrocytes for the first time. Import and export of proteins to the nucleus were examined by pulse-chase experiments using ³H-methionine, and we showed that ethanol induces a delay in both processes. Furthermore, we took an approach to evaluate the mechanisms involved in this effect. Whereas alcohol did not affect the amount and the distribution of several representative proteins that participate in nuclear import, such as RanBP1, RanGAP1 and the importins α2 and β3, it decreased the amount of Exp1/CRM1, which is a general export receptor involved in the nuclear export. In addition, the density and distribution of nuclear pore complexes, which contribute to nucleocytoplasmic transport, were also affected by ethanol. These effects can be related with changes found in the content of several proteins associated with the nuclear envelope and the nuclear pore complex structure such as lamins A/C, and nucleoporins p62 and RanBP2, respectively. These results suggest that ethanol could interfere with some of the important processes regulated by nucleocytoplasmic transport in astrocytes and support the idea that one of the main ethanol targets is intracellular transport.     

Prostanoid synthesis and vascular responses to exogenous arachidonic acid following cerebral ischemia in piglets

In newborn pigs, cerebral ischemia abolishes both increased cerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuation of prostaglandin endoperoxide synthase activity could account for the failure to increase prostanoid synthesis and loss of responses to these stimuli. To test this possibility, arachidonic acid (3, 6, or 30 micrograms/ml) was placed under cranial windows in newborn pigs that had been exposed to 20 min of cerebral ischemia. The conversion to prostanoids and pial arteriolar responses to the arachidonic acid were measured. At all three concentrations, arachidonic acid caused similar increases in pial arteriolar diameter in sham control piglets and piglets 1 hr postischemia. Topical arachidonic acid caused dose-dependent increases of PGE2 in cortical periarachnoid cerebral spinal fluid. 6-keto-PGF1 alpha and TXB2 only increased at the highest concentration of arachidonic acid (30 micrograms/ml). Cerebral ischemia did not decrease the conversion of any concentration of arachidonic acid to PGE2, 6-keto-PGF1 alpha, or TXB2. We conclude that ischemia and subsequent reperfusion do not result in inhibition of prostaglandin endoperoxide synthase in the newborn pig brain. Therefore, the mechanism for the impaired prostanoid production in response to hypercapnia and hypotension following cerebral ischemia appears to involve reduction in release of free arachidonic acid.     

Prostanoid synthesis and vascular responses to exogenous arachidonic acid following cerebral ischemia in piglets

In newborn pigs, cerebral ischemia abolishes both increased cerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuation of prostaglandin endoperoxide synthase activity could account for the failure to increase prostanoid systhesis and loss of responses to these stimuli. To test this possibility, arachidonic acid (3,6, or 30μg/ml) was placed under cranial windows in newborn pigs that been exposed to 20 min of cerebral ischemia. The conversion to prostanoids and pial arteriolar responses to the arachidonic acid were measured. At all three concentration, arachidonic acid caused similar increases in pial arteriolar diameter in sham control piglets and piglets 1 hr postischemia. Topical arachidonic acid caused dosedependent increases of PGE₂ in cortical periarachnoid cerebral spinal fluid. 6-keto-PGF_1α and TXB₂ only increased at the highest concentration of arachidonic acid (30 μg/ml). Cerebral ischemia did not decrease the conservation of any concentration of arachidonic acid to PGE₂, 6-keto-PGF_1α, or TXB₂. We conclude that ischemia and subsequent reperfusion do not result in inhibition of prostaglandin endoperoxide synthase in the newborn pig brain. Therefore, the mechanism for the impaired prostanoid production in response to hypercapnia and hypotension following cerebral ischemia appears to involve reduction in release of free arachidonic acid.     

Chronic administration of indomethacin increases role of nitric oxide in hypercapnic cerebrovasodilation in piglets.

Hypercapnia-induced cerebral vasodilation is associated with prostanoids in the piglet, but is a primarily nitric oxide (NO) associated response in many adult models. Hypercapnia-induced cerebral vasodilation is both NO and prostanoid associated in the juvenile pig. We hypothesized that with chronic administration of indomethacin the piglet would advance the role of the NO system in cerebrovascular responses. The closed cranial window technique was used in piglets to determine pial arteriolar response. Chronically indomethacin treated newborn animals dilated in response to CO2 similarly to control newborns (40.9+/-4.4% vs 48.4+/-4.1%). Topical n-nitro L-arginine (L-NA, 10(-3) M), attenuated CO2 induced dilation in the chronically indomethacin treated animals (11.7+/-3.3% vs 40.9+/-4.4%; p < 0.001), but had no effect on the response to hypercapnia of piglets not treated with indomethacin. Neither indomethacin nor L-NA altered response to topical isoproterenol (10(-6) M). We conclude that with chronic indomethacin administration there develops a significant hypercapnia-induced cerebral vasodilation in which NO has an important role. The chronic inhibition of the newborn's principal dilator system appears to increase the role of NO in newborn cerebral hemodynamics.     

Chronic ammonia exposure does not influence hepatic gene expression in growing pigs

Housed pigs are often exposed to elevated concentrations of atmospheric ammonia. This aerial pollutant is widely considered to be an environmental stressor that also predisposes to reduced growth rates and poor health, although evidence to support this view is limited. Hepatic gene expression is very responsive to stress and metabolic effects. Two batches of growing pigs were therefore exposed to a nominal concentration of atmospheric ammonia of either 5 ppm (low) or 20 ppm (high) from 4 weeks of age for 15 weeks. Growth rates were monitored. Samples of liver were taken after slaughter (at ∼19 weeks of age). Samples from the second batch were analysed for global gene expression using 23 K Affymetrix GeneChip porcine genome arrays. Samples from both batches were subsequently tested for five candidate genes using quantitative real-time PCR (qPCR). The array analysis failed to detect any significant changes in hepatic gene expression following chronic exposure to atmospheric ammonia. Animals clustered into two main groups but this was not related to the experimental treatment. There was also no difference in growth rates between groups. The qPCR analyses validated the array results by showing similar fold changes in gene expression to the arrays. They revealed a significant batch effect in expression of lipin 1 (LPIN1), Chemokine (C-X-C motif) ligand 14 (CXCL14), serine dehydratase (SDS) and hepcidin antimicrobial peptide (HAMP). Only CXCL14, a chemotactic cytokine for monocytes, was significantly down-regulated in response to ammonia. As chronic exposure to atmospheric ammonia did not have a clear influence on hepatic gene expression, this finding implies that 20 ppm of atmospheric ammonia did not pose a significant material risk to the health or metabolism of housed pigs.     

Chronic hypoxia attenuates ischemia-reperfusion-induced increase in pulmonary vascular resistance

This study explored the effect of chronic hypoxia on the elevation of pulmonary vascular resistance caused by ischemia-reperfusion (IR) in anesthetized rats. Experiments were separated into five parts. In Part 1, we examined the increase in left pulmonary vascular resistance (Rpl) after ischemia of the left lung and localized the major site for the increased resistance of the left pulmonary vasculature in both the normoxic and chronic hypoxia groups. Here, IR induced a significant increase in Rpl in the normoxic but not the chronic hypoxia group. This increased Rpl in the normoxic group was attributed to contraction of pulmonary arterial segments. Part 2 and Part 3 were focused on the changes in plasma nitrate/nitrite (NOx) and thromboxane B(2) (TxB(2)) levels. TxB(2) increased significantly in the normoxic group, whereas NOx increased significantly in the chronic hypoxia group, following ischemia. Indomethacin (Part 4) prevented IR-induced increase in Rpl in the normoxic group, whereas the IR-induced increase in Rpl appeared in the chronic hypoxia group after N(G)-nitro-L-arginine methyl ester treatment (Part 5). We conclude that IR elicited increases in the cyclooxygenase products such as TxB(2), which in turn caused an increase in Rpl. However, this increased Rpl was attenuated by elevated NOx in the chronic hypoxia group.     

Chronic mevastatin modulates receptor-dependent vascular contraction in eNOS-deficient mice

We tested the hypothesis that endothelial nitric oxide (NO) synthase (eNOS)-derived NO modulates rho-kinase-mediated vascular contraction. Because 3-hydroxy-3-methylglutaryl (HMG)-CoA-reductase inhibition can both upregulate eNOS expression and inhibit rhoA/rho-kinase function, a second hypothesis tested was that statin treatment modulates rho-kinase-mediated contraction and that this can occur independently of eNOS. Contractile responses to the receptor-dependent agonists serotonin and phenylephrine but not to the receptor-independent agent KCl were greater in aortic rings from eNOS-null (eNOS(-/-)) vs. wild-type (eNOS(+/+)) mice. Similarly enhanced responses were seen in eNOS(+/+) rings after acute NOS inhibition. The rho-kinase inhibitor Y-27632 abolished or profoundly attenuated responses to receptor agonists in both eNOS(+/+) and eNOS(-/-) rings, but responses in eNOS(+/+) were more sensitive to Y-27632. Mevastatin treatment (20 mg/kg sc per day, 14 days) reduced responses to serotonin and phenylephrine in female mice of both strains. KCl-induced contractions were slightly smaller in eNOS(+/+)-derived aortic rings only. Levels of plasma cholesterol, and aortic expression of rhoA and rho-kinase, did not differ between groups. Thus eNOS-derived NO suppresses rhoA/rho-kinase-mediated vascular contraction. Moreover, a similar suppressive effect on rho-kinase-mediated vasoconstriction by statin therapy occurs independently of effects on eNOS or plasma cholesterol.     

Chronic hypercapnia inhibits hypoxic pulmonary vascular remodeling

Chronic hypercapnia is commonly found in patients with severe hypoxic lung disease and is associated with a greater elevation of pulmonary arterial pressure than that due to hypoxia alone. We hypothesized that hypercapnia worsens hypoxic pulmonary hypertension by augmenting pulmonary vascular remodeling and hypoxic pulmonary vasoconstriction (HPV). Rats were exposed to chronic hypoxia [inspiratory O(2) fraction (FI(O(2))) = 0.10], chronic hypercapnia (inspiratory CO(2) fraction = 0.10), hypoxia-hypercapnia (FI(O(2)) = 0.10, inspiratory CO(2) fraction = 0.10), or room air. After 1 and 3 wk of exposure, muscularization of resistance blood vessels and hypoxia-induced hematocrit elevation were significantly inhibited in hypoxia-hypercapnia compared with hypoxia alone (P < 0.001, ANOVA). Right ventricular hypertrophy was reduced in hypoxia-hypercapnia compared with hypoxia at 3 wk (P < 0.001, ANOVA). In isolated, ventilated, blood-perfused lungs, basal pulmonary arterial pressure after 1 wk of exposure to hypoxia (20.1 +/- 1.8 mmHg) was significantly (P < 0.01, ANOVA) elevated compared with control conditions (12.1 +/- 0.1 mmHg) but was not altered in hypoxia-hypercapnia (13.5 +/- 0.9 mmHg) or hypercapnia (11.8 +/- 1.3 mmHg). HPV (FI(O(2)) = 0.03) was attenuated in hypoxia, hypoxia-hypercapnia, and hypercapnia compared with control (P < 0.05, ANOVA). Addition of N(omega)-nitro-L-arginine methyl ester (10(-4) M), which augmented HPV in control, hypoxia, and hypercapnia, significantly reduced HPV in hypoxia-hypercapnia. Chronic hypoxia caused impaired endothelium-dependent relaxation in isolated pulmonary arteries, but coexistent hypercapnia partially protected against this effect. These findings suggest that coexistent hypercapnia inhibits hypoxia-induced pulmonary vascular remodeling and right ventricular hypertrophy, reduces HPV, and protects against hypoxia-induced impairment of endothelial function.     

Emergent vascular network inhomogeneities and resulting blood flow patterns in a growing tumor

Tumors acquire sufficient oxygen and nutrient supply by coopting host vessels and neovasculature created via angiogenesis, thereby transforming a highly ordered network into chaotic heterogeneous tumor specific vasculature. Vessel regression inside the tumor leads to large regions of necrotic tissue interspersed with isolated surviving vessels. We extend our recently introduced model to incorporate Fahraeus-Lindqvist- and phase separation effects, refined tissue oxygen level computation and drug flow computations. We find, unexpectedly, that collapse and regression accelerates rather than diminishes the perfusion and that a tracer substance flowing through the remodeled network reaches all parts of the tumor vasculature very well. The reason for decreased drug delivery well known in tumors should therefore be different from collapse and vessel regression. Implications for drug delivery in real tumors are discussed.