James Wasvary

Quantitative and qualitative assessment of articular cartilage in the goat knee with magnetization transfer imaging

We investigated the role of collagen in the magnetization transfer (MT) effect in contrast to other macromolecules. By means of phantoms made of collagen, chondroitin sulfate (CS) and albumin, MR parameters have been optimized in order to reduce the acquisition time and improve the sensitivity, as well as to minimize the contributions from CS and albumin to the MT induced signal attenuation. The same method was used to study cartilage ex vivo (bovine articular and nasal cartilage plugs) and in vivo (goat knee femoral chondyle). In phantom samples, the MT signal attenuation depended on the collagen concentration while contributions from the other macromolecules were found to be minimal. In average, analysis of MT images revealed a approximately 25%, approximately 35% and approximately 30% signal attenuation in 10% w/v type I collagen gels, cartilage plugs, and cartilage from the weight-bearing areas of the goat knee, respectively. Biochemical data revealed that treatment of cartilage plugs with bacterial collagenase led to collagen depletion and correspondingly to a decrease of the MT response. In contrast, trypsin-induced proteoglycan loss in cartilage plugs did not alter the MT effect. A significant correlation was observed between the collagen content in these plugs and their respective MT ratios and the rate constant k for the exchange process bound versus free water. Finally, data obtained from in vivo MT measurement of the goat knee demonstrated that intra-articular injection of papain might not only cause degradation of proteoglycans but also a change in collagen integrity in a dose-dependent manner. We conclude that in vivo measurement of MT ratios gives quantitative and qualitative information on the collagen status and may be applied for the routine evaluation of normal and abnormal articular cartilage.

Effect of essential hypertension on kidney function as measured in rat by dynamic MRI.

In this study, dynamic MRI was applied to investigate the effect of genetic hypertension on the renal clearance of Gd(DTPA). Assuming that the paramagnetic agent was essentially cleared through the kidney by glomerular filtration, a first‐order kinetic model was used to estimate a rate constant kcl describing the transport from the renal cortex to the inner medulla. Experiments were carried out on 12–15‐week‐old spontaneously hypertensive rats (young‐SHR) and age‐matched Wistar‐Kyoto rats (young‐WKY). An additional group of 22–25‐week‐old SHR (old‐SHR) was investigated to assess the long‐term effect of chronic hypertension on kidney function. The glomerular filtration rate as measured by the inulin method correlated well with kcl results as measured by MRI. A clearance index 30% lower than young‐WKY was observed in young‐SHR, reflective of a kidney dysfunction. In old‐SHR, kcl was only 50% of the young‐WKY value. As computed from Gd(DTPA) concentration profiles, this translated into delayed time‐to‐peak (TTP) values, decreased peak Gd(DTPA) concentrations ([Gd]peak), and a slow elimination of Gd(DTPA) from the blood pool, renal cortex, and inner medulla (e.g., high t1/2 values). Finally, using the same MRI approach, the data showed that the acute administration of the calcium antagonist verapamil at an antihypertensive dose was followed by a near normalization of SHR renal function. These results indicate that chronic hypertension remains a major pathogenic factor in the progression of glomerular degeneration, as opposed to a primary glomerular defect independent of systemic pressure. Magn Reson Med 47:127–134, 2002.

In vivo qualitative assessments of articular cartilage in the rabbit knee with high-resolution MRI at 3 T.

Proteoglycan (PG) loss and disruption of the collagen framework in cartilage are early events associated with osteoarthritis (OA). The feasibility of in vivo high‐resolution MRI assessments probing both macromolecules was explored in articular cartilage of the rabbit knee. One‐millimeter thick coronal images were obtained at 3 T with a 97 × 97 μm2 pixel size. A 22% decrease in the magnetization transfer (MT) exchange rate along with an ∼2‐fold greater Gd(DTPA)2‐‐induced decrease in T1 relaxation time were measured in response to papain injection 1 day prior to the MRI session, indicative of an alteration of collagen integrity and PG depletion, respectively. A two‐point method was tested as an alternative to the more time‐consuming multipoint method typically used to measure T1 changes. Kinetics of Gd(DTPA)2‐ uptake were observed with a 10‐min time resolution. The diffusive transport of Gd(DTPA)2‐ was characterized by a T1 decrease ∼2‐fold faster in papain‐treated knees. These data suggest that kinetics of tracer diffusion may be used as an informative marker of PG loss, in addition to the amplitude of T1 variations. When applied to a relevant OA model, the combination of MT and Gd(DTPA)2‐‐MRI may help in identifying new active compounds during efficacy studies on cartilage protection. Magn Reson Med 50:541–549, 2003.

In vivo assessment of macromolecular content in articular cartilage of the goat knee

Loss of proteoglycans (PGs) from the extracellular matrix of cartilage is an early event of osteoarthritis. The capability of Gd(DTPA)2–‐enhanced MRI to quantitatively assess PG content was explored in a goat model of cartilage degeneration. Partial to total PG depletion was induced by an intraarticular injection of papain 1 day prior to the MRI session. A close correlation was found between the extent of the PG loss and the Gd(DTPA)2–‐induced T1 decrease. Papain‐induced PG depletion was confirmed by post‐mortem histological and biochemical assessments. A 2‐hr delay after Gd(DTPA)2– injection was found to be optimal for an accurate quantitation of the cartilage defect. A series of knee flexions were performed post‐Gd(DTPA)2– injection to facilitate penetration of the contrast agent into cartilage. However, ΔT1s observed in cartilage of exercised goat knees were not affected by papain or IL1β pretreatment. Therefore, as long as a preinjection T1 map was obtained, the Gd(DTPA)2–‐enhanced MRI technique provided good sensitivity in detecting partial loss of PG in articular cartilage. This was true only when the animal was maintained in a resting state during diffusion of the Gd(DTPA)2–. This approach is of particular interest for long‐term evaluations of cartilage degeneration and regeneration. Magn Reson Med 49:1037–1046, 2003.

Superoxide anion production from human neutrophils measured with an improved kinetic and endpoint microassay

Superoxide dismutase (SOD)-inhibitable reduction of cytochrome c is the basis for a widely used assay to measure superoxide production. We report novel modifications leading to a dual wavelength, high throughput simultaneous kinetic and endpoint microplate assay with high reproducibility. Neutrophils were isolated using a modified elutriation procedure to minimize priming and adherence during isolation. Cytochrome c reduction was measured in a microplate reader using 96-well polystyrene plates with a modified (Plastek A*) surface to prevent the adherence and consequent activation of PMNs. Comparison of Plastek A* treated and untreated plates revealed a statistically significant difference in basal as well as stimulated levels of superoxide production. Absorption measurements were made at both 550 nm, the absorption maximum of reduced cytochrome c, and 557 nm, an isosbestic point. A significant increase in both well-to-well reproducibility and sensitivity (detection limit) was realized by using the normalized 550-557 nm difference values compared to the 550 nm absorbance values alone. These modifications represent an improved method for handling and assessing the function of superoxide production, providing greater experimental reproducibility and lessening the perturbations caused by the microplate.

Basement membrane biosynthesis. Secretion without deposition of underhydroxylated basement membrane collagen by parietal yolk sacs.

1. 1. In contrast to published results with other collagen-synthesizing systems the rate of secretion of basement membrane collagen synthesized by the rat parietal yolk sac was essentially the same in the presence or absence of either dipyridyl or GPA 1734 (8,9-dihydroxy-7-methyl-benzo(b)quinolizinium bromide), with no evidence for intracellular accumulation of underhydroxylated basement membrane collagen. This was shown by measuring total radioactivity and [14C]hydroxyproline in collagenase digests of tissue and medium proteins after incubation for 3 and 6 h with [14C]proline or [14C]proline plus [3H]glycine. The secreted underhydroxylated basement membrane collagen contained 2–10% of the hydroxyproline found in controls without hydroxylation inhibitors. The identity of the non-dialyzable radioactivity in the medium after incubation with dipyridyl or GPA 1734 with underhydroxylated basement membrane collagen was established (a) by tripeptide analyses of the collagenase digests, which showed about 20% of the radioactivity to be Gly-Pro-Pro and (b) by incubation with partially purified prolyl hydroxylase from rat skin, which converted the material to hydroxyproline-containing protein with a [14C]hydroxyproline to total [14C]protein ratio close to that of control basement membrane collagen. 2. 2. Normal 14C-labelled basement membrane collagen, whether found intracellularly, secreted into the medium or deposited on Reicherts membrane, after reduction and denaturation appears to be the same molecular size on SDS-agarose chromatography. Similarly, 14-labeled basement membrane collagen from the medium synthesized in the presence of hydroxylation inhibitors and chromatographed under conditions which prevent proteolytic digestion appears as a single peak with the same molecular size as control basement membrane collagen. 3. 3. Underhydroxylated basement membrane collagen was not deposited on Reicherts membrane as evidenced by measurements of total [14C]proline and [14C]hydroxyproline in collagenase-digests of membranes free of trophoblast and epithelial cells. 4. 4. The inhibition of hydroxylation did not affect the synthesis of the glucosamine-containing moiety of basement membrane collagen. This was shown by pulsing with [14C]glucosamine in the presence of dipyridyl and chasing with 0.5 mM glucosamine in fresh medium containing Fe2+ and cycloheximide. Glucosamine incorporated intor basement membrane collagen during the pulse stage was found to deposited on Reicherts membrane after hydroxylation of proline and lysine and secretion at the chase stage.