In vivo assessment of muscle fascicle length by extended field-of-view ultrasonography

The present study examined the reliability and validity of in vivo vastus lateralis (VL) fascicle length (L(f)) assessment by extended field-of-view ultrasonography (EFOV US). Intraexperimenter and intersession reliability of EFOV US were tested. Further, L(f) measured from EFOV US images were compared to L(f) measured from static US images (6-cm FOV) where out-of-field fascicle portions were trigonometrically estimated (linear extrapolation). Finally, spatial accuracy of the EFOV technique was assessed by comparing L(f) measured on swine VL by EFOV US to actual measurements from digital photographs. The difference between repeated VL L(f) measurements by the same experimenter was 2.1 ± 1.7% with an intraclass correlation (ICC) of 0.99 [95% confidence interval (CI) = 0.95-1.00]. In terms of intersession reliability, no difference (P = 0.48) was observed between L(f) measured on two different occasions, with ICC = 0.95 (CI = 0.80-0.99). The average absolute difference between L(f) measured by EFOV US and using linear extrapolation was 12.6 ± 8.1% [ICC = 0.76 (CI = -0.20-0.94)]; EFOV L(f) was always longer than extrapolated L(f). The relative error of measurement between L(f) measured by EFOV US and by dissective assessment (digital photographs) in isolated swine VL was 0.84% ± 2.6% with an ICC of 0.99 (CI = 0.94-1.00). These results show that EFOV US is a reliable and valid method for the measurement of long muscle fascicle in vivo. Thus EFOV US analysis was proven more accurate for the assessment of skeletal muscle fascicle length than conventional extrapolation methods.     

In vivo regional diaphragm function in dogs

A biplane videofluorographic system was used to track the position of metallic markers affixed to the abdominal surface of the left hemidiaphragm in supine anesthetized dogs. Regional shortening was determined from intermarker distances of rows of markers placed along muscle bundles in the ventral, middle, and dorsal regions of the costal diaphragm and of one row on the crural diaphragm. Considerable variability of regional shortening was seen in a given row, which was reproducible on repeat study in individual dogs but which differed between mechanical ventilation and spontaneous breathing. There were no consistent patterns among dogs. Regional shortening obtained from the change in length of rows extending from chest wall to central tendon showed no consistent differences among dogs during spontaneous breathing. At equal tidal volumes, all regions (except the ventral costal diaphragm) shortened more during spontaneous breathing than during mechanical ventilation.     

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 Structure/Function analysis of the Drosophila fat facets deubiquitinating enzyme gene

The Drosophila Fat facets protein is a deubiquitinating enzyme required for patterning the developing compound eye. Ubiquitin, a 76-amino-acid polypeptide, serves as a tag to direct proteins to the proteasome, a protein degradation complex. Deubiquitinating enzymes are a large group of proteins that cleave ubiquitin-protein bonds. Fat facets belongs to a class of deubiquitinating enzymes called Ubps that share a conserved catalytic domain. Fat facets is unique among them in its large size and also because Fat facets is thought to deubiquitinate a specific substrate thereby preventing its proteolysis. Here we asked which portions of the Fat facets protein are essential for its function. P-element constructs that express partial Fat facets proteins were tested for function. In addition, the DNA sequences of 12 mutant fat facets alleles were determined. Finally, regions of amino acid sequence similarity in 18 Drosophila Ubps revealed by the Genome Project were identified. The results indicate functions for specific conserved amino acids in the catalytic region of Fat facets and also indicate that regions of the protein both N- and C-terminal to the catalytic region are required for Fat facets function.     

In vivo imaging of subcutaneous structures using functional photoacoustic microscopy

Functional photoacoustic microscopy (fPAM) is a hybrid technology that permits noninvasive imaging of the optical absorption contrast in subcutaneous biological tissues. fPAM uses a focused ultrasonic transducer to detect high-frequency photoacoustic (PA) signals. Volumetric images of biological tissues can be formed by two-dimensional raster scanning, and functional parameters can be further extracted from spectral measurements. fPAM is safe and applicable to animals as well as humans. This protocol provides guidelines for parameter selection, system alignment, imaging operation, laser safety and data processing for in vivo fPAM. It currently takes approximately 100 min to carry out this protocol, including approximately 50 min for data acquisition using a 10-Hz pulse-repetition-rate laser system. The data acquisition time, however, can be significantly reduced by using a laser system with a higher pulse repetition rate.     

In vivo recording of monophasic action potentials in awake dogs

Assessment of cardiac repolarization in dogs in vivo is of interest in numerous experimental canine models. Previous studies have used monophasic action potentials (MAPs) to investigate repolarization in vitro and in vivo in anesthetized animal models. Therefore, an approach for recording MAPs in awake dogs without the interference of anesthesia is desirable. We describe an experimental technique to record MAPs in conscious dogs by means of conventional rubber introducers which are implanted into the internal jugular vein. Atrial as well as ventricular MAPs can be simultaneously measured without complications. Pacing thresholds are low and stable over time. Continuous MAP recordings of stable amplitude can be achieved from the same endocardial site for periods up to 1h. Antegrade and retrograde atrioventricular nodal conduction properties can be assessed. Programmed stimulation can be performed to simultaneously determine local refractory periods and MAP duration at cycle lengths from 500 to 200ms. In awake, unsedated dogs measuring MAPs via rubber introducers permits safe, long-term recording of MAPs. Such recordings may be useful for safety pharmacologic studies in evaluating cardiovascular and noncardiovascular drugs with respect to their effects on repolarization. In various canine in vivo models including in vivo models of long QT syndrome, heart failures or sudden cardiac death, the present technique permits electrophysiologic measurements without the interference of anesthesia.     

In vivo lipogenesis in genetically fat and lean chickens of various ages

Lipogenesis was studied in vivo in genetically fat and lean male chickens at 2, 5 and 15 weeks of age using tritiated water. At 2 and 5 weeks of age, the liver was the major site of de novo fatty acid synthesis rather than the carcass. Per 100 g of tissue lipogenesis was greater in the carcass at 15 weeks of age than at 2 or 5 weeks. Lipogenesis in the liver did not vary according to age. At 15 weeks of age there was no observable exaggeration in the degree of fattening (as in genetically obese rodents) of fat line chickens. The regulation of fattening in fat line chickens, at least at the onset of sexual maturity, appears to be different to that in genetically obese rodents.     

Skin and subcutaneous fat morphology alterations under the LED or laser treatment in rats in vivo

The main objective of this work is to quantify the impact of photodynamic/photothermal treatment by using visible LED and NIR laser irradiation through the skin of subcutaneous fat in vivo followed up by tissue sampling and histology. The optical method may provide reduction of regional or site‐specific accumulations of abdominal or subcutaneous adipose tissue precisely and least‐invasively by inducing cell apoptosis and controlled necrosis of fat tissue. As photodynamic/photothermal adipose tissue sensitizers Brilliant Green (BG) or Indocyanine Green (ICG) dyes were injected subcutaneously in rats. The CW LED device (625 nm) or CW diode laser (808 nm) were used as light sources, respectively. Biopsies of skin together with subcutaneous tissues were taken for histology. The combined action BG‐staining and LED‐irradiation (BG + LED) or ICG‐staining and NIR‐laser irradiation (ICG + NIR) causes pronounced signs of damage of adipose tissue characterized by a strong stretching, thinning, folding and undulating of cell membranes and appearance of necrotic areas. As a posttreatment after 14 days only connective tissue was observed at the site of necrotic areas. The data obtained are important for safe light treatment of site‐specific fat accumulations, including cellulite. This work provides a basis for the development of fat lipolysis technologies and to move them to clinical applications. Schematics of animal experiment.      

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 human lipolytic activity in preperitoneal and subdivisions of subcutaneous abdominal adipose tissue

We studied eight normal-weight male subjects to examine whether the lipolytic rate of deep subcutaneous and preperitoneal adipose tissues differs from that of superficial abdominal subcutaneous adipose tissue. The lipolytic rates in the superficial anterior and deep posterior subcutaneous abdominal adipose tissues and in the preperitoneal adipose tissue in the round ligament were measured by microdialysis and (133)Xe washout under basal, postabsorptive conditions and during intravenous epinephrine infusion (0.15 nmol. kg(-1). min(-1)). Both in the basal state and during epinephrine stimulation, the superficial subcutaneous adipose tissue had higher interstitial glycerol concentrations than the two other depots. Similarly, the calculated glycerol outputs from the superficial depot were significantly higher than those from the deep subcutaneous and the preperitoneal depots. Thus, it is concluded that the lipolytic rate of the superficial subcutaneous adipose tissue on the anterior abdominal wall is higher than that of the deep subcutaneous adipose tissue on the posterior abdominal wall and that of the preperitoneal adipose tissue in the round ligament.     

Morphological characterization and viability assessment of Trichoderma reesei by image analysis

The production of cellulase from the filamentous fungus Trichoderma reesei is a critical step in the industrial process leading to cellulose ethanol. As a result of the lack of quantitative analysis tools, the intimate relationship that exists between the morphological and physiological states of the microorganism, the shear field in the bioreactor, and the process performance is not yet fully understood. A semiautomatic image analysis protocol was developed to characterize the mycelium morphology and to estimate its percentage viability during the fermentation process based on four morphological types (unbranched, branched, entangled, and clumped microorganisms). Pictures taken under bright field microscopy combined with images of fluorescein diacetate stained fungi were used to assess the morphological parameters and the percentage viability of microorganisms simultaneously. The method was tested during the course of fed-batch fermentation in a reciprocating plate bioreactor. The use of the image analysis protocol was found to be successful in quantifying the variations in the morphology and the viability of T. reesei throughout the fermentation.     

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.     

In vivo and in vitro correlation of trachealis muscle contraction in dogs.

Maximal trachealis muscle shortening in vivo was compared with that in vitro in seven anesthetized dogs. In addition, the effect of graded elastic loads on the muscle was evaluated in vitro. In vivo trachealis muscle shortening, as measured using sonomicrometry, revealed maximal active shortening to be 28.8 +/- 11.7% (SD) of initial length. Trachealis muscle preparations from the same animals were studied in vitro to evaluate isometric force generation, isotonic shortening, and the effect of applying linear elastic loads to the trachealis muscle during contraction from optimal length. Maximal isotonic shortening was 66.8 +/- 8.4% of optimal length in vitro. Increasing elastic loads decreased active shortening and velocity of shortening in vitro in a hyperbolic manner. The elastic load required to decrease in vitro shortening to the extent of the shortening observed in vivo was similar to the estimated load provided by the tracheal cartilage. We conclude that decreased active shortening in vivo is primarily due to the elastic afterload provided by cartilage.