Reversal of acute coagulopathy during hypotensive resuscitation using small-volume 7.5% NaCl adenocaine and Mg2+ in the rat model of severe hemorrhagic shock

OBJECTIVE: : Acute traumatic coagulopathy occurs early in hemorrhagic trauma and is a major contributor to mortality and morbidity. Our aim was to examine the effect of small-volume 7.5% NaCl adenocaine (adenosine and lidocaine, adenocaine) and Mg on hypotensive resuscitation and coagulopathy in the rat model of severe hemorrhagic shock. DESIGN: : Prospective randomized laboratory investigation. SUBJECTS: : A total of 68 male Sprague Dawley Rats. INTERVENTION: : Post-hemorrhagic shock treatment for acute traumatic coagulopathy. MEASUREMENTS AND METHODS: : Nonheparinized male Sprague-Dawley rats (300-450 g, n = 68) were randomly assigned to either: 1) untreated; 2) 7.5% NaCl; 3) 7.5% NaCl adenocaine; 4) 7.5% NaCl Mg; or 5) 7.5% NaCl adenocaine/Mg. Hemorrhagic shock was induced by phlebotomy to mean arterial pressure of 35-40 mm Hg for 20 mins (~40% blood loss), and animals were left in shock for 60 mins. Bolus (0.3 mL) was injected into the femoral vein and hemodynamics monitored. Blood was collected in Na citrate (3.2%) tubes, centrifuged, and the plasma snap frozen in liquid N2 and stored at -80°C. Coagulation was assessed using activated partial thromboplastin times and prothrombin times. RESULTS: : Small-volume 7.5% NaCl adenocaine and 7.5% NaCl adenocaine/Mg were the only two groups that gradually increased mean arterial pressure 1.6-fold from 38-39 mm Hg to 52 and 64 mm Hg, respectively, at 60 mins (p < .05). Baseline plasma activated partial thromboplastin time was 17 ± 0.5 secs and increased to 63 ± 21 secs after bleeding time, and 217 ± 32 secs after 60-min shock. At 60-min resuscitation, activated partial thromboplastin time values for untreated, 7.5% NaCl, 7.5% NaCl/Mg, and 7.5% NaCl adenocaine rats were 269 ± 31 secs, 262 ± 38 secs, 150 ± 43 secs, and 244 ± 38 secs, respectively. In contrast, activated partial thromboplastin time for 7.5% NaCl adenocaine/Mg was 24 ± 2 secs (p < .05). Baseline prothrombin time was 28 ± 0.8 secs (n = 8) and followed a similar pattern of correction. CONCLUSIONS: : Plasma activated partial thromboplastin time and prothrombin time increased over 10-fold during the bleed and shock periods prior to resuscitation, and a small-volume (~1 mL/kg) IV bolus of 7.5% NaCl AL/Mg was the only treatment group that raised mean arterial pressure into the permissive range and returned activated partial thromboplastin time and prothrombin time clotting times to baseline at 60 mins.

Ability of recombinant human bone morphogenetic protein 2 to enhance bone healing in the presence of tobramycin : evaluation in a rat segmental defect model

Objective To determine whether locally applied tobramycin influences the ability of recombinant human bone morphogenetic protein 2 (rhBMP-2) to heal a segmental defect in the rat femur. Methods The influence of tobramycin on the osteogenic differentiation of mesenchymal stem cells was first evaluated in vitro. For the subsequent, in vivo experiments, a 5-mm segmental defect was created in the right femur of each of 25 Sprague-Dawley rats and stabilized with an external fixator and four Kirschner wires. Rats were divided in four groups: empty control, tobramycin (11 mg)/absorbable collagen sponge, rhBMP-2 (11 μg)/absorbable collagen sponge, and rhBMP-2/absorbable collagen sponge with tobramycin. Bone healing was monitored by radiography at 3 and 8 weeks. Animals were euthanized at 8 weeks and the properties of the defect were compared with the intact contralateral femur. Bone formation in the defect region was assessed by dual-energy x-ray absorptiometry, microcomputed tomography, histology, and mechanical testing. Results Tobramycin exerted a dose-dependent inhibition of alkaline phosphatase induction and calcium deposition by mesenchymal stem cells cultured under osteogenic conditions. The inhibition was reversed in the presence of 500 ng/mL of rhBMP-2. Segmental defects in the rat femora failed to heal in the absence of rhBMP-2. Tobramycin exerted no inhibitory effects on the ability of rhBMP-2 to heal these defects and increased the bone area of the defects treated with rhBMP-2. Data obtained from all other parameters of healing, including dual-energy x-ray absorptiometry, microcomputed tomography, histology, and mechanical testing, were unaffected by tobramycin. Conclusions Although our in vitro results suggested that tobramycin inhibits the osteogenic differentiation of mesenchymal stem cells, this could be overcome by rhBMP-2. Tobramycin did not impair the ability of rhBMP-2 to heal critical-sized femoral defects in rats. Indeed, bone area was increased by nearly 20% in the rhBMP-2 group treated with tobramycin. This study shows that locally applied tobramycin can be used in conjunction with rhBMP-2 to enhance bone formation at fracture sites.

Exercise alters the profile of phospholipid molecular species in rat skeletal muscle

We have determined the effect of two exercise-training intensities on the phospholipid profile of both glycolytic and oxidative muscle fibers of female Sprague-Dawley rats using electrospray-ionization mass spectrometry. Animals were randomly divided into three training groups: control, which performed no exercise training; low-intensity (8 m/min) treadmill running; or high-intensity (28 m/min) treadmill running. All exercise-trained rats ran 1,000 m/session for 4 days/wk for 4 wk and were killed 48 h after the last training bout. Exercise training was found to produce no novel phospholipid species but was associated with significant alterations in the relative abundance of a number of phospholipid molecular species. These changes were more prominent in glycolytic (white vastus lateralis) than in oxidative (red vastus lateralis) muscle fibers. The largest observed change was a decrease of ∼20% in the abundance of 1-stearoyl-2-docosahexaenoyl-phosphatidylethanolamine [PE(18:0/22:6); P < 0.001] ions in both the low- and high-intensity training regimes in glycolytic fibers. Increases in the abundance of 1-oleoyl-2-linoleoyl phopshatidic acid [PA(18:1/18:2); P < 0.001] and 1-alkenylpalmitoyl-2-linoleoyl phosphatidylethanolamine [plasmenyl PE (16:0/18:2); P < 0.005] ions were also observed for both training regimes in glycolytic fibers. We conclude that exercise training results in a remodeling of phospholipids in rat skeletal muscle. Even though little is known about the physiological or pathophysiological role of specific phospholipid molecular species in skeletal muscle, it is likely that this remodeling will have an impact on a range of cellular functions.

Phospholipid composition of the rat lens is independent of diet

Dietary fatty acids are known to influence the phospholipid composition of many tissues in the body, with lipid turnover occurring rapidly. The aim of this study was to investigate whether changes in the fatty acid composition of the diet can affect the phospholipid composition of the lens. Male Sprague-Dawley rats were fed three diets with distinct profiles in both essential and non-essential fatty acids. After 8 weeks, lenses and skeletal muscle were removed, and the lenses sectioned into nuclear and cortical regions. In these experiments, the lens cortex was synthesised during the course of the variable lipid diet. Phospholipids were then identified by electrospray ionisation tandem mass spectrometry, and quantified via the use of internal standards. The phospholipid compositions of the nuclear and cortical regions of the lens differed slightly between the two regions, but comparison of the equivalent regions across the diet groups showed remarkable similarity. In contrast, the phospholipid composition of skeletal muscle (medial gastrocnemius) in these rats varied significantly. This study provides the first direct evidence to show that the phospholipid composition of the lens is tightly regulated and thus appears to be independent of diet. As phospholipids determine membrane fluidity and influence the activity and function of integral membrane proteins, regulation of their composition may be important for the function of the lens. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

Estrogen deficiency-associated bone loss in the maxilla: A methodology to quantify the changes in the maxillary intra-radicular alveolar bone in an ovariectomized rat osteoporosis model

The effects of estrogen deficiency on bone characteristics are site-dependent, with the most commonly studied sites being appendicular long bones (proximal femur and tibia) and axial bones (vertebra). The effect on the maxillary and mandibular bones is still inconsistent and requires further investigation. This study was designed to evaluate bone quality in the posterior maxilla of ovariectomized rats in order to validate this site as an appropriate model to study the effect of osteoporotic changes. Methods: Forty-eight 3-month-old female Sprague-Dawley rats were randomly divided into two groups: an ovariectomized group (OVX, n=24) and Sham-operated group (SHAM, n=24). Six rats were randomly sacrificed from both groups at time points 8, 12, 16 and 20 weeks. The samples from tibia and maxilla were collected for Micro CT and histological analysis. For the maxilla, the volume of interest (VOI) area focused on the furcation areas of the first and second molar. Trabecular bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th.), trabecular number (Tb.N.), trabecular separation (Tb.Sp.), and connectivity density (Conn.Dens) were analysed after Micro CT scanning. Results: At 8 weeks the indices BV/TV, Tb.Sp, Tb.N and Conn.Dens showed significant differences (P<0.05) between the OVX and SHAM groups in the tibia. Compared with the tibia, the maxilla developed osteoporosis at a later stage, with significant changes in maxillary bone density only occurring after 12 weeks. Compared with the SHAM group, both the first and second molars of the OVX group showed significantly decreased BV/TV values from 12 weeks, and these changes were sustained through 16 and 20 weeks. For Tb.Sp, there were significant increases in bone values for the OVX group compared with the SHAM group at 12, 16 and 20 weeks. Histological changes were highly consistent with Micro CT results. Conclusion: This study established a method to quantify the changes of intra-radicular alveolar bone in the posterior maxilla in an accepted rat osteoporosis model. The degree of the osteoporotic changes to trabecular bone architecture is site-dependent and at least 3 months are required for the osteoporotic effects to be apparent in the posterior maxilla following rat OVX.

Cloning and gastrointestinal expression of rat hephaestin: Relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

The effects of dietary fish oil on inflammation, fibrosis and oxidative stress associated with obstructive renal injury in rats.

Scope: We examined whether dietary supplementation with fish oil modulates inflammation, fibrosis and oxidative stress following obstructive renal injury. Methods and results: Three groups of Sprague-Dawley rats (n = 16 per group) were fed for 4 wk on normal rat chow (oleic acid), chow containing fish oil (33 g eicosapentaenoic acid and 26 g docosahexaenoic acid per kg diet), or chow containing safflower oil (60 g linoleic acid per kg diet). All diets contained 7% fat. After 4 wk, the rats were further subdivided into four smaller groups (n = 4 per group). Unilateral ureteral obstruction was induced in three groups (for 4, 7 and 14 days). The fourth group for each diet did not undergo surgery, and was sacrificed as controls at 14 days. When rats were sacrificed, plasma and portions of the kidneys were removed and frozen; other portions of kidney tissue were fixed and prepared for histology. Compared with normal chow and safflower oil, fish oil attenuated collagen deposition, macrophage infiltration, TGF-beta expression, apoptosis, and tissue levels of arachidonic acid, MIP-1 alpha, IL-1 beta, MCP-1 and leukotriene B(4). Compared with normal chow, fish oil increased the expression of HO-1 protein in kidney tissue. Conclusions: Fish oil intake reduced inflammation, fibrosis and oxidative stress following obstructive renal injury.

Localization of Mineralocorticoid Receptors at Mammalian Synapses

In the brain, membrane associated nongenomic steroid receptors can induce fast-acting responses to ion conductance and second messenger systems of neurons. Emerging data suggest that membrane associated glucocorticoid and mineralocorticoid receptors may directly regulate synaptic excitability during times of stress when adrenal hormones are elevated. As the key neuron signaling interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. The lateral amygdala is a key site for synaptic plasticity underlying conditioned fear, which can both trigger and be coincident with the stress response. A large body of electrophysiological data shows rapid regulation of neuronal excitability by steroid hormone receptors. Despite the importance of these receptors, to date, only the glucocorticoid receptor has been anatomically localized to the membrane. We investigated the subcellular sites of mineralocorticoid receptors in the lateral amygdala of the Sprague-Dawley rat. Immunoblot analysis revealed the presence of mineralocorticoid receptors in the amygdala. Using electron microscopy, we found mineralocorticoid receptors expressed at both nuclear including: glutamatergic and GABAergic neurons and extra nuclear sites including: presynaptic terminals, neuronal dendrites, and dendritic spines. Importantly we also observed mineralocorticoid receptors at postsynaptic membrane densities of excitatory synapses. These data provide direct anatomical evidence supporting the concept that, at some synapses, synaptic transmission is regulated by mineralocorticoid receptors. Thus part of the stress signaling response in the brain is a direct modulation of the synapse itself by adrenal steroids.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated.

Serum bone formation marker correlation with improved osseointegration in osteoporotic rats treated with simvastatin

Objective: Simvastatin has been shown to enhance osseointegration of pure titanium implants in osteoporotic rats. This study aimed to evaluate the relationship between the serum level of bone formation markers and the osseointegration of pure titanium implants in osteoporotic rats treated with simvastatin. Materials and methods: Fifty-four female Sprague Dawley rats, aged 3 months old, were randomly divided into three groups: Sham-operated group (SHAM; n=18), ovariectomized group (OVX; n=18), and ovariectomized with Simvastatin treatment group (OVX+SIM; n=18). Fifty-six days after ovariectomy, screw-shaped titanium implants were inserted into the tibiae. Simvastatin was administered orally at 5mg/kg each day after the placement of the implant in the OVX+SIM group. The animals were sacrificed at either 28 or 84 days after implantation and the undecalcified tissue sections were processed for histological analysis. Total alkaline phosphatase (ALP), bone specific alkaline phosphatase (BALP) and bone Gla protein (BGP) were measured in all animal sera collected at the time of euthanasia and correlated with the histological assessment of osseointegration. Results: The level of ALP in the OVX group was higher than the SHAM group at day 28, with no differences between the three groups at day 84. The level of BALP in the OVX+SIM group was significantly higher than both OVX and SHAM groups at days 28 and 84. Compared with day 28, the BALP level of all three groups showed a significant decrease at day 84. There were no significant differences in BGP levels between the three groups at day 28, but at day 84 the OVX+SIM group showed significantly higher levels than both the OVX and SHAM groups. There was a significant increase in BGP levels between days 28 and 84 in the OVX+SIM group. The serum bone marker levels correlated with the histological assessment showing reduced osseointegration in the OVX compared to the SHAM group which is subsequently reversed in the OVX+SIM group.

Metabolomic analysis characterizes tissue specific indomethacin-induced metabolic perturbations of rats

In this study, the promising metabolomic approach integrating with ingenuity pathway analysis (IPA) was applied to characterize the tissue specific metabolic perturbation of rats that was induced by indomethacin. The selective pattern recognition analyses were applied to analyze global metabolic profiling of urine of rats treated by indomethacin at an acute dosage of reference that has been proven to induce tissue disorders in rats, evaluated throughout the time-course of -24-72 h. The results preliminarily revealed that modifications of amino acid metabolism, fatty acid metabolism and energetically associated metabolic pathways accounted for metabolic perturbation of the rats that was induced by indomethacin. Furthermore, IPA was applied to deeply analyze the biomarkers and their relations with the metabolic perturbations evidenced by pattern recognition analyses. Specific biochemical functions affected by indomethacin suggested that there is an important correlation of its effects in kidney and liver metabolism, based on the determined metabolites and their pathway-based analysis. The IPA correlation of the three major biomarkers, identified as creatinine, prostaglandin E2 and guanosine, suggested that the administration of indomethacin induced certain levels of toxicity in the kidneys and liver. The changes in the levels of biomarker metabolites allowed the phenotypical determination of the metabolic perturbations induced by indomethacin in a time-dependent manner.

Ingenuity pathways analysis of urine metabonomics phenotypes toxicity of gentamicin in multiple organs

We introduce the use of Ingenuity Pathway Analysis to analyzing global metabonomics in order to characterize phenotypically biochemical perturbations and the potential mechanisms of the gentamicin-induced toxicity in multiple organs. A single dose of gentamicin was administered to Sprague Dawley rats (200 mg/kg, n = 6) and urine samples were collected at -24-0 h pre-dosage, 0-24, 24-48, 48-72 and 72-96 h post-dosage of gentamicin. The urine metabonomics analysis was performed by UPLC/MS, and the mass spectra signals of the detected metabolites were systematically deconvoluted and analyzed by pattern recognition analyses (Heatmap, PCA and PLS-DA), revealing a time-dependency of the biochemical perturbations induced by gentamicin toxicity. As result, the holistic metabolome change induced by gentamicin toxicity in the animal's organisms was characterized. Several metabolites involved in amino acid metabolism were identified in urine, and it was confirmed that gentamicin biochemical perturbations can be foreseen from these biomarkers. Notoriously, it was found that gentamicin induced toxicity in multiple organs system in the laboratory rats. The proof-of-knowledge based Ingenuity Pathway Analysis revealed gentamicin induced liver and heart toxicity, along with the previously known toxicity in kidney. The metabolites creatine, nicotinic acid, prostaglandin E2, and cholic acid were identified and validated as phenotypic biomarkers of gentamicin induced toxicity. Altogether, the significance of the use of metabonomics analyses in the assessment of drug toxicity is highlighted once more; furthermore, this work demonstrated the powerful predictive potential of the Ingenuity Pathway Analysis to study of drug toxicity and its valuable complementation for metabonomics based assessment of the drug toxicity.

Effect of high-frequency resistance exercise on adaptive responses in skeletal muscle

PURPOSE: Regulation of skeletal muscle mass is highly dependent on contractile loading. The purpose of this study was to examine changes in growth factor and inflammatory pathways following high-frequency resistance training. METHODS: Using a novel design in which male Sprague-Dawley rats undertook a "stacked" resistance training protocol designed to generate a summation of transient exercise-induced signaling responses (four bouts of three sets × 10 repetitions of squat exercise, separated by 3 h of recovery), we determined the effects of high training frequency on signaling pathways and transcriptional activity regulating muscle mass. RESULTS: The stacked training regimen resulted in acute suppression of insulin-like growth factor 1 mRNA abundance (P < 0.05) and Akt phosphorylation (P < 0.05), an effect that persisted 48 h after the final training bout. Conversely, stacked training elicited a coordinated increase in the expression of tumor necrosis factor alpha, inhibitor kappa B kinase alpha/beta activity (P < 0.05), and p38 mitogen-activated protein kinase phosphorylation (P < 0.05) at 3 h after each training bout. In addition, the stacked series of resistance exercise bouts induced an increase in p70 S6 kinase phosphorylation 3 h after bouts ×3 and ×4, independent of the phosphorylation state of Akt. CONCLUSIONS: Our results indicate that high resistance training frequency extends the transient activation of inflammatory signaling cascades, concomitant with persistent suppression of key mediators of anabolic responses. We provide novel insights into the effects of the timing of exercise-induced overload and recovery on signal transduction pathways and transcriptional activity regulating skeletal muscle mass in vivo.

Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity

Introduction The clinically known importance of patient sex as a major risk factor for compromised bone healing is poorly reflected in animal models. Consequently, the underlying cellular mechanisms remain elusive. Because mesenchymal stem cells (MSCs) are postulated to regulate tissue regeneration and give rise to essential differentiated cell types, they may contribute to sex-specific differences in bone healing outcomes. Methods We investigated sex-specific variations in bone healing and associated differences in MSC populations. A 1.5 mm osteotomy gap in the femora of 8 male and 8 female 12-month-old Sprague-Dawley rats was stabilized by an external fixator. Healing was analyzed in terms of biomechanical testing, bridging and callus size over time (radiography at 2, 4, and 6 weeks after surgery), and callus volume and geometry by μCT at final follow-up. MSCs were obtained from bone marrow samples of an age-matched group of 12 animals (6 per gender) and analyzed for numbers of colony-forming units (CFUs) and their capacity to differentiate and proliferate. The proportion of senescent cells was determined by β-galactosidase staining. Results Sex-specific differences were indicated by a compromised mechanical competence of the callus in females compared with males (maximum torque at failure, p = 0.028). Throughout the follow-up, the cross-sectional area of callus relative to bone was reduced in females (p ≤ 0.01), and the bridging of callus was delayed (p 2weeks = 0.041). μCT revealed a reduced callus size (p = 0.003), mineralization (p = 0.003) and polar moment of inertia (p = 0.003) in female animals. The female bone marrow contained significantly fewer MSCs, represented by low CFU numbers in both femora and tibiae (p femur = 0.017, p tibia = 0.010). Functional characteristics of male and female MSCs were similar. Conclusion Biomechanically compromised and radiographically delayed bone formation were distinctive in female rats. These differences were concomitant with a reduced number of MSCs, which may be causative for the suboptimal bone healing.