In vivo NMR detection of diet-induced changes in adipose tissue composition

We introduce an in vivo spectroscopic method to assess the effects of diet on fatty acid composition of the predominant chemical constituent of adipocytes in mice. To do this, we make use of a nonlinear NMR signal that, unlike a standard NMR signal, is intrinsically insensitive to local magnetic field inhomogeneities and which naturally suppresses the large water signal from nonfatty tissues. Our method yields fat composition information from fat depots distributed over large sample volumes in a single experiment, without requiring the use of tedious shimming procedures, voxel selection, or water suppression. Our results suggest that this method can reveal clear differences in adipose tissue composition of mice fed a standard chow diet compared with mice fed a diet rich in polyunsaturated fatty acids. With further developments this method could be used to obtain information on human lipid composition noninvasively and to track changes in lipid composition induced by diet intervention, pharmaceutical drugs, and exercise.     

In vivo MRI quantification of individual muscle and organ volumes for assessment of anabolic steroid growth effects

This study aimed to develop a quantitative and in vivo magnetic resonance imaging (MRI) approach to investigate the muscle growth effects of anabolic steroids. A protocol of MRI acquisition on a standard clinical 1.5 T scanner and quantitative image analysis was established and employed to measure the individual muscle and organ volumes in the intact and castrated guinea pigs undergoing a 16-week treatment protocol by two well-documented anabolic steroids, testosterone and nandrolone, via implanted silastic capsules. High correlations between the in vivo MRI and postmortem dissection measurements were observed for shoulder muscle complex (R=0.86), masseter (R=0.79), temporalis (R=0.95), neck muscle complex (R=0.58), prostate gland and seminal vesicles (R=0.98), and testis (R=0.96). Furthermore, the longitudinal MRI measurements yielded adequate sensitivity to detect the restoration of growth to or towards normal in castrated guinea pigs by replacing circulating steroid levels to physiological or slightly higher levels, as expected. These results demonstrated that quantitative MRI using a standard clinical scanner provides accurate and sensitive measurement of individual muscles and organs, and this in vivo MRI protocol in conjunction with the castrated guinea pig model constitutes an effective platform to investigate the longitudinal and cross-sectional growth effects of other potential anabolic steroids. The quantitative MRI protocol developed can also be readily adapted for human studies on most clinical MRI scanner to investigate the anabolic steroid growth effects, or monitor the changes in individual muscle and organ volume and geometry following injury, strength training, neuromuscular disorders, and pharmacological or surgical interventions.     

Monitoring changes in lung air and liquid volumes with electrical impedance tomography

Adler, A., R. Amyot, R. Guardo, J. H. T. Bates, and Y. Berthiaume. Monitoring changes in lung air and liquid volumes withelectrical impedance tomography. J. Appl.Physiol. 83(5): 1762-1767, 1997.Electricalimpedance tomography (EIT) uses electrical measurements at electrodesplaced around the thorax to image changes in the conductivitydistribution within the thorax. This technique is well suited tostudying pulmonary function because the movement of air, blood, andextravascular fluid induces significant conductivity changes within thethorax. We conducted three experimental protocols in a total of 19 dogsto assess the accuracy with which EIT can quantify changes in thevolumes of both gas and fluid in the lungs. In the first protocol, lungvolume increments from 50 to 1,000 ml were applied with a largesyringe. EIT measured these volume changes with an average error of 27 ± 6 ml. In the second protocol, EIT measurements were made at endexpiration and end inspiration during regular ventilation with tidalvolume ranging from 100 to 1,000 ml. The average error in the EITestimates of tidal volume was 90 ± 43 ml. In the third protocol,lung liquid volume was measured by instilling 5% albumin solution intoa lung lobe in increments ranging from 10 to 100 ml. EIT measured thesevolume changes with an average error of 10 ± 10 ml and was alsoable to detect into which lobe the fluid had been instilled. These results indicate that EIT can noninvasively measure changes in thevolumes of both gas and fluid in the lungs with clinically usefulaccuracy.

     

Preventing mediastinal shift after pneumonectomy impairs regenerative alveolar tissue growth

To examine the effects of mechanical lung strain on regenerative growth of alveolar septal tissue after pneumonectomy (PNX), we replaced the right lungs of adult dogs with a custom-shaped inflatable silicone prosthesis. The prosthesis was either inflated (Inf) to maintain the mediastinum at the midline or deflated to allow mediastinal shift. The animals were euthanized approximately 15 mo later, and the lungs were fixed at a constant distending pressure. With the Inf prostheses, lung expansion, alveolar septal tissue volumes, surface areas, and diffusing capacity of the tissue-plasma barrier were significantly lower than with the deflated prostheses; the expected post-PNX tissue responses were impaired by 30-60%. Capillary blood volume was significantly higher with Inf prostheses, consistent with microvascular congestion. Measurements in the Inf group remained consistently and significantly higher than those expected for a normal left lung, indicating persistence of partial compensation. In one dog, delayed deflation of the prosthesis 9-10 mo after PNX led to vigorous lung expansion and septal tissue growth, particularly of type II epithelial cells. We conclude that mechanical lung strain is a major signal for regenerative lung growth; however, other signals are also implicated, accounting for a significant fraction of the compensatory response to PNX.     

In vivo and in organello assessment of OXPHOS activities

Mitochondrial OXPHOS defects are responsible for a large group of human diseases and have been associated with degenerative disorders and aging. The accurate in vivo and in organello biochemical assessment of the OXPHOS system is necessary for the diagnosis and investigation of such conditions. Here I describe a set of accurate polarographic and spectrophotometric assays that use relatively small amounts of biological material (cells or isolated mitochondria) and discuss the biochemical parameters appropriate for discriminating partial OXPHOS defects.     

In vivo tissue engineering of musculoskeletal tissues

Tissue engineering of musculoskeletal tissues often involves the in vitro manipulation and culture of progenitor cells, growth factors and biomaterial scaffolds. Though in vitro tissue engineering has greatly increased our understanding of cellular behavior and cell-material interactions, this methodology is often unable to recreate tissue with the hierarchical organization and vascularization found within native tissues. Accordingly, investigators have focused on alternative in vivo tissue engineering strategies, whereby the traditional triad (cells, growth factors, scaffolds) or a combination thereof are directly implanted at the damaged tissue site or within ectopic sites capable of supporting neo-tissue formation. In vivo tissue engineering may offer a preferential route for regeneration of musculoskeletal and other tissues with distinct advantages over in vitro methods based on the specific location of endogenous cultivation, recruitment of autologous cells, and patient-specific regenerated tissues.     

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes

The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation-evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.     

In vitro and in vivo assessment of herb drug interactions

Herbal products contain several chemicals that are metabolized by phase 1 and phase 2 pathways and also serve as substrates for certain transporters. Due to their interaction with these enzymes and transporters there is a potential for alteration in the activity of drug metabolizing enzymes and transporters in presence of herbal components. Induction and inhibition of drug metabolizing enzymes and transporters by herbal component has been documented in several in vitro studies. While these studies offer a system to determine the potential for a herbal component to alter the pharmacokinetics of a drug, they cannot always be used to predict the magnitude of any potential effect in vivo. In vivo studies are the ultimate way to determine the clinical importance of herb drug interactions. However, lack of content uniformity and lack of documentation of the bioavailability of herbal components makes even in vivo human studies difficult to interpret as the effect may be product specific. It appears that St. John's wort extract is probably one of the most important herbal product that increases the metabolism and decreases the efficacy of several drugs. Milk thistle on the other hand appears to have minimal effect on phase 1 pathways and limited data exists for phase 2 pathways and transporter activity in vivo. Further systematic studies are necessary to assess the significance of herb drug interactions.     

In vivo measurement of protein functional changes

Conformational changes in proteins are fundamental to all biological functions. In protein science, the concept of protein flexibility is widely used to describe protein dynamics and thermodynamic properties that control protein conformational changes. In this study, we show that urea, which has strong sedative potency, can be administered to fish at high concentrations, and that protein functional changes related to anesthesia induction can be measured in vivo. Ctenopharyngodon idellus (the grass carp) has two different types of N-methyl d-aspartate (NMDA) receptors, urea-insensitive and urea-sensitive, which are responsible for the heat endurance of fish. The urea-sensitive NMDA receptor showed high protein flexibility, the gamma aminobutyric acid (GABA) receptor showed less flexibility, and the protein that is responsible for ethanol anesthesia showed the lowest flexibility. The results suggest that an increase in protein flexibility underlies the fundamental biophysical mechanisms of volatile general anesthetics.     

In vivo measurements of changes in pH triggered by oxalic acid in leaf tissue of transgenic oilseed rape

Oxalic acid (OA), a non-host-specific toxin secreted by Sclerotinia sclerotiorum during pathogenesis, has been demonstrated to be a major phytotoxic and pathogenic factor. Oxalate oxidase (OXO) is an enzyme associated with the detoxification of OA, and hence the introduction of an OXO gene into oilseed rape (Brassica napus L.) to break down OA may be an alternative way of increasing the resistance of the plant to Sclerotinia sclerotiorum. In order to investigate the activation of OXO in transgenic oilseed rape, a convenient and accessible method was used to monitor changes in pH in response to stress induced by OA. The pH sensor, a platinum microcylinder electrode modified using polyaniline film, exhibited a linear response within the pH range from 3 to 7, with a Nernst response slope of 70 mV/pH at room temperature. The linear correlation coefficient was 0.9979. Changes induced by OA in the pH values of leaf tissue of different oilseed rape species from Brassica napus L. were monitored in real time in vivo using this electrode. The results clearly showed that the transgenic oilseed rape was more resistant to OA than non-transgenic oilseed rape.     

In vivo quantitative assessment of catheter patency in rats

Formation of fibrin sleeves around catheter tips is a central factor in catheter failure during chronic implantation, and such tissue growth can occur despite administration of anticoagulants. We developed a novel method for monitoring catheter patency. This method recognizes the progressive nature of catheter occlusion, and tracks this process over time through measurement of changes in catheter resistance to a standardized 1 mL bolus infusion from a pressurized reservoir. Two indirect measures of catheter patency were used: (a) reservoir residual pressure and (b) reservoir discharge time. This method was applied to the study of catheter patency in rats comparing the effect of catheter material (silastic, polyurethane, Microrenathanetrade mark), lock solution (heparin, heparin/dexamethasone) and two different cannulation sites (superior vena cava via the external jugular vein, inferior vena cava via the femoral vein). Our findings reveal that application of flexible smaller-size silastic catheters and a dexamethasone lock solution resulted in prolonged catheter patency. Patency could be maintained over nine weeks with the femoral vein catheters, compared with five weeks with the external jugular vein catheters. The current method for measuring catheter patency provides a useful index for the assessment of tissue growth around the catheter tip. The method also provides an objective and quantitative way of comparing changes in catheter patency for different surgical methods and catheter types. Our method improves on the conventional method of assessing catheter occlusion by judging the ability to aspirate from the catheter.     

Biomechanical assessment of tissue retrieved after in vivo cartilage defect repair: tensile modulus of repair tissue and integration with host cartilage

Failure to restore the mechanical properties of tissue at the repair site and its interface with host cartilage is a common problem in tissue engineering procedures to repair cartilage defects. Quantitative in vitro studies have helped elucidate mechanisms underlying processes leading to functional biomechanical changes. However, biomechanical assessment of tissue retrieved from in vivo studies of cartilage defect repair has been limited to compressive tests. Analysis of integration following in vivo repair has relied on qualitative histological methods. The objectives of this study were to develop a quantitative biomechanical method to assess (1) the tensile modulus of repair tissue and (2) its integration in vivo, as well as determine whether supplementation of transplanted chondrocytes with IGF-I affected these mechanical properties. Osteochondral blocks were obtained from a previous 8 month study on the effects of IGF-I on chondrocyte transplantation in the equine model. Tapered test specimens were prepared from osteochondral blocks containing the repair/native tissue interface and adjacently located blocks of intact native tissue. Specimens were then tested in uniaxial tension. The tensile modulus of repair tissue averaged 0.65 MPa, compared to the average of 5.2 MPa measured in intact control samples. Integration strength averaged 1.2 MPa, nearly half the failure strength of intact cartilage samples, 2.7 MPa. IGF-I treatment had no detectable effects on these mechanical properties. This represents the first quantitative biomechanical investigation of the tensile properties of repair tissue and its integration strength in an in vivo joint defect environment.     

In vivo evaluation of bioprinted prevascularized bone tissue

Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three-dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose-derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion-based bioprinting and drop-on-demand (DoD) bioprinting, respectively. The computer-generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical-sized bone defects.     

In vivo and in vitro assessment of brain bioenergetics in aging rats

Brain energy disorders can be present in aged men and animals. To this respect, the mitochondrial and free radical theory of aging postulates that age‐associated brain energy disorders are caused by an imbalance between pro‐ and anti‐oxidants that can result in oxidative stress. Our study was designed to investigate brain energy metabolism and the activity of endogenous antioxidants during their lifespan in male Wistar rats. In vivo brain bioenergetics were measured using ³¹P nuclear magnetic resonance (NMR) spectroscopy and in vitro by polarographic analysis of mitochondrial oxidative phosphorylation. When compared to the young controls, a significant decrease of age‐dependent mitochondrial respiration and adenosine‐3‐phosphate (ATP) production measured in vitro correlated with significant reduction of forward creatine kinase reaction (kfor) and with an increase in phosphocreatine (PCr)/ATP, PCr/Pi and PME/ATP ratio measured in vivo. The levels of enzymatic antioxidants catalase, GPx and GST significantly decreased in the brain tissue as well as in the peripheral blood of aged rats. We suppose that mitochondrial dysfunction and oxidative inactivation of endogenous enzymes may participate in age‐related disorders of brain energy metabolism.     

In vivo assessment of kynurenate neuroprotective potency and quinolinate excitotoxicity

Three complementary questions related to the kynurenine pathway and excitotoxicity were addressed in this study: (i) Which extracellular levels of quinolinic acid (QUIN) may be neurotoxic? (ii) Which extracellular levels of kynurenic acid (KYNA) may control excessive NMDA-receptor function? (iii) Can "anti-excitotoxic" levels of KYNA be reached by inhibition of kynurenine-3-hydroxylase (i.e. inhibition of QUIN synthesis and shunts of kynurenine metabolism toward KYNA)? Multifunctional microdialysis probes were used in halothane anaesthetised rats to apply NMDA or QUIN directly to the brain, with or without co-perfusion of KYNA, to record the resulting local depolarisations, and to monitor changes in dialysate KYNA after kynurenine-3-hydroxylase inhibition. QUIN produced concentration-dependent depolarisations with an estimated EC50 (i.e. concentration in the perfusion medium) of 1.22mM. The estimated ED50 for KYNA inhibition of NMDA-responses was 181microM. Kynurenine-3-hydroxylase inhibition (Ro-61-8048, 100mg/kg i.p.) increased dialysate KYNA 11 times (to 33.8nM) but without any reduction of NMDA-responses. These data challenge the notion that extracellular accumulation of endogenous QUIN may contribute to excessive NMDA-receptor activation in some neurological disorders, and the suitability of kynurenine-3-hydroxylase inhibition as an effective anti-excitotoxic strategy.     

In vivo diffusion of lidocaine through tissue expanders.

It has been suggested that the addition of lidocaine to the saline used to fill tissue expanders will reduce the pain often associated with the expansion process. In vitro experiments have shown that lidocaine as it is normally supplied will diffuse through an expander only at a very slow rate, which would probably be inadequate for a clinical effect. We found that the addition of sodium bicarbonate resulted in a substantial increase in the rate of diffusion. Studies in rabbits demonstrated that at a pH of 8.0, 75 percent of the lidocaine dose crossed the silicone elastomer membrane at 24 hours and greater than 95 percent had left the expander at 1 week. We have concluded that intraluminal lidocaine can be effective only when the pH is close to the pKa of lidocaine.