VALIDATION OF A SUBENDOCARDIAL ISCHAEMIC SHEEP MODEL BY INTRACORONARY FLUORESCENT MICROSPHERES

1. We evaluated the use of non-radioactive fluorescent-labelled microspheres (FM) for the measurement of regional myocardial blood flow (RMBF) in an ischaemic sheep model. 2. Injection of FM directly into the coronary artery was compared with left atrial injection. There was a good correlation in the measurement of RMBF between these two injection methods (r = 0.92; n= 107 data points). Injection into the coronary artery requires less FM (one twentieth of that required by atrial injection) and is more economical. 3. The use of a fluorescent technique without filtering myocardial tissue was investigated. Calibration curves from the fluorescence plus myocardial tissue samples were similar to those of the pure fluorescence samples and both showed a linear relationship between fluorescent intensity and the number of microspheres (r > 0.97). These results indicate that the extraction of six fluorescent dyes (blue-green, yellow-green, green, orange, red and crimson) directly from the aqueous solution using ethyl acetate is effective. 4. A subendocardial ischaemic model was produced by partially occluding the circumflex artery (CxA) with concomitant left atrium (LA) pacing. During ischaemia, the endocardium/epicardium (Endo/Epi) flow ratios in the ischaemic area changed from 1.04 ± 0.12 to 0.47 ± 0.17 (P < 0.05; CxA injection) and from 1.08 ± 0.12 to 0.51 ± 0.05 (P < 0.05; LA injection). The ratio in the non-ischaemic area remained unchanged (1.12 ± 0.26 to 1.01 ± 0.22; not significant). 5. RMBF calculation using coronary inflow as the reference flow was also compared with that using the traditional method. We found that, in this study in which a non-filtering technique was applied, using coronary inflow as the reference flow was superior to the conventional distal sampling method.     

Nonenzymatic glycation of basement membranes from human glomeruli and bovine sources. Effect of diabetes and age

The nonenzymatic glycation of glomerular basement membranes (GBMs) from 14 diabetic and 19 nondiabetic human subjects was determined after boronic acid affinity and high-performance cation-exchange chromatography of their NaB[3H]4-reduced ketoamine adducts. The glucitol-lysine (Glc-Lys) and the glucitol-hydroxylysine (Glc-Hyl) content of diabetic GBM was found to be about twofold higher than that of nondiabetic samples (P less than .001). The content of these glycated amino acids did not correlate with age over the range examined (20-91 yr) or with the length of disease in diabetic subjects (2-16 yr). However, analyses of Glc-Lys and Glc-Hyl in calf and adult bovine GBM and lens capsules indicated that the levels of these glycated amino acids were several times greater in basement membranes from older animals. We also observed that guanidine-insoluble collagen of bovine GBM is more extensively glycated (approximately 4-fold) than primarily noncollagenous proteins that are extracted by this reagent. In all of the basement membranes examined, the percentage of glycation of lysine was greater than of hydroxylysine. Characterization of the components released by alkaline hydrolysis indicated that O-glycosylated hydroxylysine residues are nonenzymatically N-glycated to the same extent as those without an enzymatically attached carbohydrate unit. Our study indicates that more than a hundred times as many hydroxylysine residues are enzymatically glycosylated in human and bovine GBM as those containing the nonenzymatically formed ketoamine adduct.     

The penetration of inulin, sucrose, mannitol and tritiated water from the interstitial space in muscle into the vascular system

1. An isolated gastrocnemius muscle from cat was perfused with a modified Ringer fluid containing the extracellular tracers mannitol, sucrose and inulin and in some cases tritiated water (HTO). When equilibration of the tracers between the perfusate and the tissue was obtained, perfusion with tracer-free fluid was started.2. The fractional rate of escape, Phi (t), of the tracers was followed. For all tracers it declined steadily with time. The rate of decline was faster for the smaller extracellular molecules when any combination of two extracellular tracers was studied.3. Arrest of flow for 1-2 min lead to an increase in Phi (t) for the extracellular tracers when flow was resumed. There was little or no effect upon Phi (t) for HTO.4. Doubling the rate of flow had no effect upon Phi (t) for the extracellular tracers, while that of HTO increased.5. We conclude that the cause of the decline in Phi (t) for the extracellular tracers is concentration gradients in the interstitial space. The findings raise problems in methods for studying capillary permeability by means of the fractional rate of escape of tracers from the interstitial space into the vascular system.     

Assessment of unilateral afferent pupillary defects by pupillography.

A pupillographic technique, an adaptation of the swinging light test, is described for the measurement of unilateral afferent pupillary defects. In normal persons it yielded accurate, reproducible estimates of the magnitude of artificial afferent defects made with neutral filters of 1 to 4 log units density. In 15 studies on 8 patients with unilateral optic neuritis and 2 with unilateral compressive lesions the measured afferent pupillary defects correlated closely with both visual acuity and visual threshold deficits. Measurement of the pupillary defect underestimated severe degrees of impairment revealed by visual threshold determination. Discrepancies between pupillary defect and visual acuity were observed in the 2 patients with compressive lesions and in 1 patient who had a central scotoma 4 months after an attack of optic neuritis.     

Nonenzymatic glycation of human lens crystallin. Effect of aging and diabetes mellitus

We have examined the nonenzymatic glycation of human lens crystallin, an extremely long-lived protein, from 16 normal human ocular lenses 0.2-99 yr of age, and from 11 diabetic lenses 52-82-yr-old. The glucitol-lysine (Glc-Lys) content of soluble and insoluble crystallin was determined after reduction with H-borohydride followed by acid hydrolysis, boronic acid affinity chromatography, and high pressure cation exchange chromatography. Normal lens crystallin, soluble and insoluble, had 0.028 +/- 0.011 nanomoles Glc-Lys per nanomole crystallin monomer. Soluble and insoluble crystallins had equivalent levels of glycation. The content of Glc-Lys in normal lens crystallin increased with age in a linear fashion. Thus, the nonenzymatic glycation of nondiabetic lens crystallin may be regarded as a biological clock. The diabetic lens crystallin samples (n = 11) had a higher content of Glc-Lys (0.070 +/- 0.034 nmol/nmol monomer). Over an age range comparable to that of the control samples, the diabetic crystallin samples contained about twice as much Glc-Lys. The Glc-Lys content of the diabetic lens crystallin samples did not increase with lens age.     

Bacterial meningitis in infants: sonographic findings

A retrospective study was performed on 78 patients (newborn to 2 years old) with clinically proved bacterial meningitis. Sonograms were obtained during the acute illness and medical records were reviewed. The spectrum of sonographic features of meningitis included normal scans (30 patients), ventriculomegaly (11 patients), echogenic sulci (31 patients), extra-axial fluid collections (26 patients), abnormal parenchymal echogenicity (9 patients), evidence of ventriculitis (5 patients), and brain abscess (1 patient). In 46 patients, correlation between the sonographic findings and neurologic outcome on clinical follow-up (6 months to 4 years) was made. Findings of abnormal parenchymal echogenicity and/or moderate-to-marked ventriculomegaly were associated with significant neurologic sequelae; however, echogenic sulci and small extra-axial fluid collections did not appear to have any prognostic significance. Twenty-nine of the 78 patients had sonography without clinical indication of complications of meningitis, and in no patient was a significant abnormality found. Our study suggests that sonography is indicated only when there is clinical suspicion of complications.