Computer-assisted Videodensitometry Research Articles

Ultrasound-mediated delivery of echogenic immunoliposomes to porcine vascular smooth muscle cells in vivo

Vascular smooth muscle cells (VSMCs) are important targets in the treatment of atherosclerosis. However, the arterial media, where the majority of VSMCs reside, have proven to be a difficult target for drug/gene delivery. We have demonstrated that ultrasound enhances drug/gene delivery to VSMCs in vitro by using echogenic immunoliposomes (ELIPs) as the vector. This study aimed to evaluate whether ultrasound can similarly enhance the delivery of an agent to VSMCs, particularly within the arterial media, in vivo, using ELIP. Anti–smooth-muscle cell actin-conjugated calcein-loaded ELIP were injected into the peripheral arteries of Yucatan miniswine (n = 8 arterial pairs). The right-sided porcine arteries were treated with 1-MHz continuous-wave ultrasound at a peak-to-peak pressure amplitude of 0.23 ± 0.05 MPa for 2 minutes. The contralateral arteries served as controls. Arteries were harvested after 30 minutes and imaged with fluorescence microscopy. Image data were converted to grayscale and analyzed by using computer-assisted videodensitometry. There was significant improvement in calcein uptake in all three arterial layers in the arteries exposed to ultrasound (> 300%). This enhanced uptake was site specific and appeared limited to the ultrasound-treated arterial segment. We have demonstrated enhanced delivery of a small molecule to VSMCs in all arterial wall layers, particularly the arterial media, using ultrasound and targeted ELIP. The combined effect of ultrasound exposure and ELIP as a contrast agent and a drug/gene-bearing vector has the potential for site-specific therapy directed at VSMC function.

Echogenic Liposome Compositions for Increased Retention of Ultrasound Reflectivity at Physiologic Temperature

Targetable echogenic liposomes (ELIP) for ultrasound enhancement of atheroma have recently been developed; however, their retention of echogenicity at physiological temperature is less than desirable. The purpose of this study was to improve ELIP stability and increase clinical potential. The approach utilized the original procedures but involved manipulation of the lipid composition by reducing the level of unsaturation of the phospholipids components to minimize the rate of loss of echogenicity. Echogenicity was measured using a 20 MHz intravascular ultrasound (IVUS) catheter and quantified (as mean gray scale values) using computer-assisted videodensitometry. The optimal preparation for retention of echogenicity stability at physiologic temperature was egg phosphatidylcholine/dipalmitoylphosphatidylcholine/dipalmitoylphos-phatidylethanolamine/dipalmitoylphosphatidylglycerol/cholesterol (27:42:8:8:15, molar percent). This preparation retained 51 +/- 3.5% of its echogenicity after 1 h at 37 degrees C, more than 5x that retained by the previously descried preparation. In this composition nearly 2/3 of the phosphosphatidylcholine is fully saturated. Such an increase in saturation is anticipated to stiffen the lipid acyl chains. The air pockets that are responsible for reflection of ultrasound waves can be assumed to be stabilized by a lipid monolayer at the interface between the air and bulk water. The increased rigidity of that monolayer is presumed to be responsible for reducing the loss of air and extending the duration of echogenic activity. The stability of this improved formulation now appears to be more than adequate for clinical applications.

Effects of trigeminal ganglion stimulation on the central auditory system

A projection from the trigeminal ganglion to the ventral cochlear nucleus (VCN) of the guinea pig was recently described. The synaptic terminals of this projection terminate in the granule and magnocellular regions of the VCN. Stimulation of this projection has been shown to result in activation of neurons of the ventral cochlear nucleus. We investigated the effect of electrically stimulating the trigeminal ganglion on the central auditory system activity using 2-deoxyglucose (2-DG) autoradiographic techniques. Electrical stimuli were applied to the left trigeminal ganglion as bipolar pulses, 100 μs per phase, at intervals of 200 ms and an amplitude of 100 μA. Negative control animals were not stimulated. A positive control animal was stimulated in the left ear using a 1 kHz tone burst with 200 ms duration and an amplitude of 80 dB SPL. 2-DG was administered by intramuscular injection. Following a 1 h incorporation period, animals were sacrificed, the brains rapidly harvested, and prepared for autoradiography using standard techniques. Autoradiographs were analyzed using computer-assisted video densitometry to determine film optical density in the central auditory regions of interest. The cerebellum was also sampled as a gray matter indifferent intra-brain control region. Results showed systematic and significant differences between 2-DG uptake in the cochlear nucleus and higher auditory centers between control and stimulated animals. Trigeminally stimulated animals showed significantly higher uptake than unstimulated animals in all auditory centers examined, especially ipsilateral to the stimulation site. The activation pattern differs qualitatively from that seen with sound stimulation in that mainly contralateral pathways are activated with sound stimulation. These results demonstrate that a projection from the predominantly somatosensory trigeminal ganglion can influence the activity of central auditory neurons in a manner distinct from acoustic stimulation, suggesting activation of non-classical auditory pathways.

Novel echogenic drug-immunoliposomes for drug delivery.

We have developed novel echogenic immunoliposomes (ELIPs) that can be antibody-conjugated for the specific highlighting of atheroma and atheroma components. The utility of these agents for regional drug delivery has not been evaluated previously. We chose to use an antibiotic as the prototype drug. The concept that an infectious agent may affect the development and progression of atherosclerosis has stimulated trials on the use of antibiotics for coronary syndromes. However, these agents are given systemically with concomitant problems. Development of an agent for local drug delivery may obviate adverse effects and improve treatment efficacy. The aim of this study was to evaluate the potential of our ELIPs for drug incorporation and to demonstrate efficient drug delivery to cultured cells. Azithromycin was incorporated into the ELIPs during development. Free drug was removed with a Sephadex G-50 column. Acoustic properties were evaluated using an intravascular ultrasound catheter and quantified by computer-assisted videodensitometry. Human umbilical arterial endothelial cells were infected with Chlamydia pneumoniae. Cells were treated with the drug-ELIP complexes, and infection-forming units were counted using fluorescence techniques. We were able to incorporate a drug into the ELIPs with retention of acoustic properties. The drug-ELIP complex demonstrated effective inhibition of microbial growth in endothelial cells (P < 0.001 vs. empty liposomes and control). We have developed a novel acoustic drug-liposomal agent that can deliver drugs to cultured cells. Although in vivo translation is required, this technique has potential for site-specific drug delivery.

Physical correlates of the ultrasonic reflectivity of lipid dispersions suitable as diagnostic contrast agents

The objective of this study was to determine the physical basis of ultrasound (US) reflectivity of echogenic lipid dispersions. These dispersions were made using a process previously described involving sonication of the lipid in water, addition of mannitol, freezing, lyophilization and rehydration. The component lipids were egg phosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylglycerol and cholesterol in a molar ratio of 69:8:8:15. Ultrasound reflectivity, as assessed with a 20-MHz intravascular US catheter and analyzed using computer-assisted videodensitometry, was found to be sensitive to variations in ambient pressure; echogenicity was greatly reduced by exposure to 0.5 atm vacuum for 10 s or 1.5 atm pressure for 10 s. Pressure changes of the magnitude that obtain in the arterial circulation had little effect on echogenicity. Vacuum treatment resulted in the release of approximately 100 μL air from a standard preparation of 10 mg lipid in 1 mL. Maximum ultrasonic reflectivity required the presence of 0.1–0.2 mol/L mannitol during the lyophilization step; mere addition of mannitol to the lipid lyophilized in the absence of mannitol produced nonreflective dispersions. Inclusion of sodium phosphate or other electrolytes reduced echogenicity. High echogenicity was associated with the presence of large-volume freeze-dried cakes and fusion of liposomes (which led to a 10 times increase in liposome diameters) during freezing before lyophilization. Lyophilization from water led to liposome fusion, but the cakes were small and US reflectivity was weak. Lyophilization from solutions of cryoprotectants such as trehalose produced large cakes, but little liposome fusion and also led to weak US reflectivity. Filtration through defined pores revealed that approximately 50% of the echogenicity originated from particles smaller than 1 μm and about 2/3 from particles smaller than 3 μm. These results indicate that lyophilization from 0.2 mol/L mannitol solution generates a disrupted array of lipid bilayers that, upon rehydration, fuse and trap small amounts of air distributed among liposome-size particles.

A computer-assisted videodensitometric method to visualize mineral distributions in in vitro and in vivo formed root caries lesions.

A computer-assisted videodensitometry (CAV) method is presented for the microradiographical mineral determination and mineral visualization of in vitro and in vivo formed dentin lesions. The method employs as essential steps (1) image digitizing by a CCD camera, (2) conversion of gray values to equivalent aluminium thicknesses and (3) conversion of aluminium thickness to vol% of mineral. Procedure and accuracy are described. The CAV method is illustrated for dentin lesions formed in vitro as well as for clinical root caries lesions. The CAV method is especially advantageous for clinical lesions because it can be employed for very complex mineral distributions and irregular lesion outlines. In given areas of interest, the mineral distribution (in vol%) can be plotted in 3D form in pseudo-colors. Conventional microradiographical profiles can be obtained directly from the CAV images. For in vitro lesions, the mineral distribution parameters, lesion depth, mineral loss, and mineral acquisition values can be generated from CAV images with a reproducibility of better than 7%. The CAV method is a valuable tool in quantitative mineral studies of clinical lesions in extracted teeth as well as in in situ lesion investigations.

Femoral cortical remodeling after uncemented total hip arthroplasty: A prospective radiologic study of 26 hips followed for 2 to 4 years

Twenty-six consecutive, hydroxyapatite-coated Bi-Metric femoral stem prostheses (Biomet, Warsaw, IN) were studied in 20 patients. The follow-up period averaged 37 months (range, 24–48 months). Clinical rating (Harris score) was determined and radiography were taken at regular intervals. Digital imaging and computer-assisted videodensitometry were used to quantify the cortical remodeling. The anteroposterior radiographs of the femur were scanned and digitized, and the pictures were analyzed by a digital image processing software program. The periosteal diameter, the cortices of the femur, and the total endosteal diameter in five regions were determined. The mean preoperative Harris score was 50 and improved to 97 at the latest follow-up evaluation. After surgery, there was hypertrophy of the periprosthetic cortical bone and an increase in the periosteal diameter. The medullary canal of the operated femur did not change.

Bone remodeling around cementless hip implants.

Bone remodeling results from the adaptation of bone to the stresses that act upon it. The insertion of an endoprosthesis into a femur changes the stress distribution within the femur, causing the bone to remodel. In this paper, we show how radiographic bone remodeling changes can be used to determine implant fixation and how bone remodeling changes can be quantitated by using dual-energy x-ray absorptiometry analysis as well as computer-assisted videodensitometry.

Changes in femoral cortical dimensions after uncemented revision hip arthroplasty: Radiological results with the porous-coated Bi-Metric femoral stem prosthesis in 15 asymptomatic cases

The radiographic results of 15 uncemented femoral revisions after a minimum follow-up period of 2 years are reported. Digital imaging was used to quantify the remodeling of the cortical bone. A method using computer-assisted video densitometry was developed and radiographs were scanned and digitized by a computer. The femoral cortex in five regions, as well as the endosteal diameter, was determined. After rearthroplasty, there was a significant loss in cortical bone with widening of the medullary canal. The periosteal diameter did not change. This endosteal resorption could contribute to further aseptic loosening. In addition, it could be an explanation of the primary prosthetic failure; one mode of failure could be an accelerated rate of endosteal expansion in a subgroup of the general population after hip arthroplasty.

Videodensitometry of osteons in females with femoral neck fractures

The medial femoral cortices of 10 females with femoral neck fractures and 10 age-matched female autopsy cases were studied using computer-assisted videodensitometry. Radiographic mineral density was determined using the calibration method described by Martin et al. [20]. Measurements were made of porosity, osteon and haversian canal dimensions, and of interstitial and osteon mineral density across the cortical wall of the orthopedic calcar region. There were no differences between the fracture and nonfracture groups in the overall mean mineral density of the bone averaged over the microstructure, excluding pore space, or in the interstitial bone mineral density. The porosity in the fracture group was greater than in the autopsy group, especially in the periosteal region, where the porosity was 2.4 times greater and where there were also 27% fewer osteons per unit area than in the autopsy group (P < 0.05). Mean osteon mineral density was 2.5% higher in the fracture group (P < 0.05) compared with the autopsy group in the endosteal region, and 4% higher in the periosteal region. Osteon and haversian canal areas were also larger in the fracture group, especially in the middle region of the cortical wall (17% and 23%, respectively, P < 0.05).

Reproducibility of measurements of coronary narrowings by videodensitometry and by digital calipers.

Computer-assisted videodensitometry has been shown to be a reliable and reproducible method of measuring absolute and relative coronary narrowings. Using a commercially available analyzer (Vanguard XR70) we confirmed intra- and interobserver reproducibilities in 34 narrowings in 9 patients. Analyses were performed on normal area and diameter, stenotic area and diameter, percent area stenosis and percent diameter stenosis. For all 6 analyses, excellent intra- and interobserver correlations were found (r = 0.93-0.98), with slopes close to 1 and intercepts close to zero. Caliper measurements (Mitutoyo Digimatic) of the same lesions by the same observers showed good inter- and intraobserver reproducibility for percent diameter stenosis (r = 0.90 and 0.86), with mean interobserver difference of 1.67 +/- (SD) 6.4% and intraobserver difference of 2.97 +/- (SD) 7.9%. However, less good correlations were found between caliper and videodensitometric measurements of percent diameter stenosis; r = 0.61 and 0.76 for the two observers. These data suggest that videodensitometry is a highly reproducible quantitative angiographic method, suitable for documenting changes in the severity of coronary artery lesions, both spontaneous or related to interventions. Caliper measurements do not provide the same degree of accuracy, but they have acceptable reproducibility in measuring diameter stenosis. As such, they are also suitable for assessing changes in severity of coronary artery lesions in individual patients.

Reproducibility of measurements of coronary narrowings by videodensitometry: Unreliability of single view measurements

Computer-assisted videodensitometry has been shown to be a reliable and reproducible method of measuring absolute and relative coronary narrowings. Using a commercially available analyzer (Vanguard XR70) we confirmed the intra- and interobserver reproducibilities in 34 narrowings in 9 patients. Analyses were performed on normal area and diameter, stenotic area and diameter, percent area stenosis and percent diameter stenosis. For all 6 analyses, excellent intra- and interobserver correlations were found (r = 0.93-0.98), with slopes close to 1 and intercepts close to zero. In a separate study of 16 lesions in 11 patients, each lesion was analyzed in both the RAO and LAO planes. Correlation between the measurements was reasonable with r = 0.76 for the stenotic area and r = 0.75 for the absolute diameter stenosis. However, with suboptimal slopes (0.54 and 0.63 respectively), actual differences between measurements in the two planes were often unacceptably large. These data suggest that videodensitometry is a highly reproducible quantitative angiographic method; however, single view analyses are inadequate for comparative studies.

Bone mass determination from microradiographs by computer-assisted videodensitometry

Bone mass determination from microradiographs by computer-assisted videodensitometry

Bone Mass Determination from Microradiographs by Computer-Assisted Videodensitometry

Aluminium was evaluated as a reference substance in the assessment of rabbit cortical bone by microradiography followed by videodensitometry. Ten dense, cortical-bone specimens from the same tibia diaphysis were microradiographed using prefiltered 27 kV roentgen radiation together with aluminium step wedges and bone simulating phantoms for calibration. Optimally exposed and processed plates were analysed by previously described computer-assisted videodensitometry. For comparison, the specimens were analysed by physico-chemical methods. A strict proportionality was found between the 'aluminium equivalent mass' and the ash weight of the specimens. The total random error was low with a coefficient of variation within 1.5 per cent. It was concluded that aluminium is an appropriate reference material in the determination of cortical bone, which it resembles in effective atomic number and thus x-ray attenuation characteristics. The 'aluminium equivalent mass' is suitably established as the standard of expressing the results of bone assessment by microradiography.

Bone Mass Determination from Microradiographs by Computer-Assisted Videodensitometry

The mass of hard-tissue specimens is assessed by optimised microradiography followed by computer-assisted videodensitometry. Radiographs are produced with 27 kV roentgen radiation, filtered through 0.5 to 1 mm of aluminium, on high-resolution glass plates. Aluminium step wedges are used for density calibration, the result being expressed as 'aluminium equivalent mass'. The plates are subjected to digital image analysis by an operator in interaction with a computer-based image processor, a television camera being used for image input. The program stored in the computer provides calibration of geometry and--via the step wedge images--of densitometry, background adjustment, etc. Measurement takes place by integration over the specimen-image area. No assumptions being needed concerning a specific attenuation model, the errors stemming from the common presumption of exponential attenuation are eliminated. Density profiles or maps of mass distribution are readily obtained from the computer. From measurements of aluminium phantom samples, the total random error (coefficient of variation) was determined at 1.8 per cent, the microradiographic and videodensitometric procedures contributing 1.6 per cent and 0.9 per cent, respectively, whereas the systematic error was negligible. Since specimens need not be sectioned, this method does not interfere with subsequent histologic preparation.

Bone mass determination from microradiographs by computer-assisted videodensitometry

Bone mass determination from microradiographs by computer-assisted videodensitometry

Bone mass determination from microradiographs by computer-assisted videodensitometry

Bone mass determination from microradiographs by computer-assisted videodensitometry

Effect of thyroid hormones and high carbohydrate feeding on gene expression in rat epididymal adipose tissue.

We have determined the messenger RNA activity profiles of epididymal fat in euthyroid, hypothyroid, and hyperthyroid rats as well as in animals fed a high carbohydrate fat-free (lipogenic) diet. Radioautographs of two-dimensional gels of the in vitro translational products of RNA were quantitated by computer-assisted videodensitometry and analyzed by multivariate statistics. Of the 250 spots observed, each presumably representing the translational product of a separate messenger RNA, 21 were responsive to changes in hormonal state. Eighteen increased and two decreased at some stage in the transition from the hypothyroid to the hyperthyroid state. One spot changed in a biphasic manner. Eight products responded to the lipogenic diet, six increasing and two decreasing. Six of these eight spots responded in a similar fashion to thyroid hormone administration. As previously shown for liver, there appears to be a substantial overlap between the genomic response to carbohydrate and thyroid hormone administration. Whereas the carbohydrate-generated changes are largely included in the thyroid-hormone induced alterations, the majority of thyroid hormone responsive changes are not duplicated by the diet. Multivariate analysis showed a clear separation of each state from the others and revealed that it was necessary to monitor only nine spots to achieve an effective separation of the states analyzed.