Profiles of matrix metalloproteinase activity in equine tear fluid during corneal healing in 10 horses with ulcerative keratitis.

OBJECTIVE: Levels of tear film matrix metalloproteinases (MMPs) activity are significantly elevated in horses with ulcerative keratitis and contribute to the excessive breakdown of stromal collagen. Changes in the amount of proteolytic activity in horse tear film during corneal healing and stromal remodeling have not yet been reported, but we hypothesize they should decrease. In the present study we analyzed serial tear fluid from horses with ulcerative keratitis to identify any changes in MMP activity during corneal healing and stromal remodeling. PROCEDURES: Samples of tear fluid were obtained from both eyes of 10 horses with ulcerative keratitis on the day of admission (day 1) at the hospital and then at various time points until complete healing of the cornea. Tear film MMP2 and MMP9 activity was determined by quantitative gelatin zymography. In all cases medical treatment included topical applications of equine serum, antibiotics, atropine and systemic administration of anti-inflammatory drugs. Surgical procedures were performed in several cases on day 2 in addition to the medical treatment. RESULTS: The mean total MMP activity (+/- SD) measured in relative standard units (RSU) in the tear fluid of the ulcerated eye (2.44 +/- 1.44) of the 10 horses was significantly higher than the mean in the contralateral eye (0.81 +/- 0.68) (P = 0.006), on the day of admission at the VMTH. The mean MMP activity in these ulcerated eyes significantly decreased (-82.4%) between the first day of admission and the day when the ulcer had completely healed (P = 0.0002). The activity level in the healed eye (0.43 +/- 0.17) was not significantly different to the one in the contralateral eye (0.36 +/- 0.18) on the day of complete corneal healing (P = 0.374). The level of MMP activity in the contralateral eye also decreased from 0.81 +/- 0.68-0.36 +/- 0.18 but this decrease (56%) was not significant (P = 0.069). CONCLUSIONS: Ulcerative keratitis in horses is associated with initially high levels of tear film proteolytic activity that decrease as the ulcers heal. The success of medical and surgical treatment of the corneal ulcers is reflected by the enzyme activity in tears. In horses successful treatment does lead to a rapid reduction in tear film proteolytic activity that corresponded with the improvement in the clinical signs of corneal ulceration. Measurement of MMP activity in the tear film might represent a way to monitor the progression of corneal healing in horses with ulcerative keratitis.

Time-specific intraocular pressure curves in Rhesus macaques (Macaca mulatta) with laser-induced ocular hypertension.

OBJECTIVE: To detect and categorize time-specific variations in daytime intraocular pressure (IOP) found in Rhesus monkeys with laser-induced ocular hypertension. PROCEDURES: Ten male monkeys with argon laser-induced ocular hypertension in one eye were anesthetized with ketamine hydrochloride, and the IOP measured in both eyes at 7 a.m., 7.30 a.m., and then hourly until 1 p.m. with a Tonopen trade mark XL applanation tonometer. Intraocular pressure time profiles for both eyes in each animal were developed. The means +/- SD of the IOPs for both eyes were calculated for the whole 6-h study period, and the values compared statistically. The difference between the lasered eye mean IOP standard deviation and the normal eye mean IOP standard deviation for each animal during the 6-h follow-up was also calculated and compared. RESULTS: Mean IOP (+/- SD) in the glaucoma and normal eyes for the 10 animals during the 6-h study was 32.6 +/- 2.5 and 14.9 +/- 2.5 mmHg, respectively. The IOP was significantly higher in the experimental eye than in the normal eye (P = 0.0008). The mean IOP in the lasered eye did not significantly change during the study period, whereas a slight but significant increase in IOP of the normal eye over the study period was recorded (P = 0.003). The variance in IOP in the hypertensive eyes was considerably greater than that in the untreated control eyes. From 7 a.m. to 1 p.m. the IOP declined in five eyes and increased in the other five eyes with laser-induced ocular hypertension. CONCLUSIONS: The time-specific IOP variation pattern in the daytime in the laser treated eyes is significantly greater than the variation in the normotensive eyes. This shows that in order to detect statistical differences between IOP variations induced by an IOP-reducing drug, and the exaggerated spontaneous IOP variations present in the laser-induced hypertensive eye, sufficient animals should be included in any study. Understanding the time-specific IOP variation present in a group of monkeys with laser-induced ocular hypertension is essential prior to using the model for the evaluation of IOP-reducing drugs.

Magnetic resonance imaging in patients with central nervous system pathology: a comparison of OptiMARK (Gd-DTPA-BMEA) and Magnevist (Gd-DTPA).

RATIONALE AND OBJECTIVES: The objective of the two pivotal phase 3 studies was to evaluate the safety and efficacy of OptiMARK (Gd-DTPA-bis(methoxyethylamide) [Gd-DTPA-BMEA]) compared with Magnevist (Gd-DTPA) in magnetic resonance imaging of the central nervous system. METHODS: Two multicenter, randomized, double-blind, parallel group studies were conducted in 395 patients with known or suspected central nervous system pathology. Subjects were randomized to receive a single 0.1 mmol/kg intravenous injection of either Gd-DTPA-BMEA or Gd-DTPA. The safety of Gd-DTPA-BMEA and Gd-DTPA was monitored for up to 72 hours after study drug administration. Precontrast and postcontrast administration magnetic resonance scans were acquired using identical imaging planes and techniques. RESULTS: No deaths or unexpected adverse events were reported in either group. A comparison of adverse events by intensity and relation demonstrated no statistically significant differences between the two groups. Gd-DTPA-BMEA and Gd-DTPA were equivalent with respect to confidence in diagnosis, conspicuity, and border delineation. CONCLUSIONS: Gd-DTPA-BMEA and Gd-DTPA demonstrated comparable efficacy profiles, and the safety profiles were considered similar.

Pharmacokinetics, safety, and tolerability of gadoversetamide injection (OptiMARK) in subjects with central nervous system or liver pathology and varying degrees of renal function.

The pharmacokinetic parameters, safety, and tolerability of OptiMARK (gadoversetamide injection), a gadolinium-based magnetic resonance imaging (MRI) contrast agent, were evaluated in 163 subjects with either central nervous system (CNS) or liver pathology with and without renal insufficiency, for which a contrast-enhanced MRI was indicated. A multicenter, double-blind, randomized, placebo-controlled, parallel-group design was used in which subjects received 0.1, 0.3, or 0.5 mmol/kg of OptiMARK or placebo intravenously. Samples were analyzed for total gadolinium by inductively coupled plasma/mass spectrometry. Gadolinium pharmacokinetics were affected by renal impairment: area under the curve, half-life, and steady-state distribution volume significantly increased with declining renal function, while total body clearance decreased. In subjects with normal renal function, neither age, gender, nor liver versus CNS pathology altered gadolinium pharmacokinetics. No clinically significant changes from baseline were noted in vital signs, laboratory measures, electrocardiograms, or physical examinations. OptiMARK is safe and well-tolerated following a single intravenous injection in subjects with either liver or CNS pathology despite a prolonged elimination half-life in subjects with renal impairment.

CNS neuronal focal adhesion kinase forms clusters that co-localize with vinculin.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that appears to play a central role in integrin-mediated signal transduction in non-neuronal cells, linking the extracellular matrix to the actin-based cytoskeleton at focal adhesion contacts. Biochemical analysis has revealed the presence of FAK immunoreactivity in cells of neuronal lineage (Zhang et al., 1994) and in the CNS (Burgaya et al. 1995; Grant et al., 1995). In the current work, we have examined the immunodistribution of FAK in nerve cell cultures and tissue sections from the rat CNS. Cultures of B103 CNS neuroblastoma cells and primary cultures of hippocampal neurons both showed abundant FAK immunoreactivity in nerve cell bodies. Immunoreactivity also extended into neurites and growth cones. The most striking feature of FAK distribution was the presence of short, punctate clusters of high FAK concentration. These FAK clusters were maintained in triton-extracted cell ghosts, indicating association with the cytoskeleton. Double-label confocal imaging showed that clusters of FAK coincided with clusters of vinculin, another actin-associated signal transduction molecule implicated in control of growth cone motility. Data from hippocampal sections verified the presence of FAK in adult neurons where it was enriched in somato-dendritic domains and showed a non-uniform distribution. Quantitative FAK immunoprecipitation to compare adult with embryonic brain showed a 7-fold developmental down-regulation of FAK and a 21-fold down-regulation of FAK TyrP. The data suggest that neuronal FAK may participate in signal transduction complexes relevant to neuronal morphogenesis and plasticity.

Locomotor effects in sheep of alkaloids identified in Australian Tribulus terrestris.

Fresh, mature, ungrazed Tribulus terrestris plant material was subjected to a standard alkaloid extraction procedure. The extract was fractionated by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Two major alkaloid fractions were demonstrated. These fractions were identified by means of TLC, ultraviolet spectrofluorimetry (UVS) and HPLC, as the beta-carboline indoleamines harmane and norharmane. The extractable alkaloid content was determined to be 44 mg/kg dry matter. Synthetic harmane and norharmane were administered subcutaneously to sheep at a dose rate of 54 mg/kg. Both compounds caused similar nervous effects. The main effect observed was limb paresis, which in some sheep was body side blased. The clinical signs observed in the experimental sheep were consistent with those described for naturally occurring cases of Tribulus terrestris staggers. It was proposed that harmane and norharmane accumulate in tryptamine-associated neurones of the central nervous system, during months of tribulus ingestion, and gradually interact irreversibly with a specific neuronal gene DNA sequence.

Particle size and protein content of six fractions of the Sf 20 plasma lipoproteins isolated by density gradient centrifugation.

A procedure is described for the separation of plasma S(f) > 400 and S(f) 20-400 lipoproteins each into three fractions. Serum samples are overlayered with a sodium chloride density gradient in a preparative ultracentrifuge tube and thin layers are removed at the top of the tube after successive centrifugations at different speeds in a swinging bucket rotor. The procedure was evaluated by electron microscopy of the S(f) > 400 lipoprotein fractions and schlieren analysis of the S(f) 20-400 lipoprotein fractions. Protein content of each fraction was measured by elemental N, C, H, and lipid-P analysis. Protein coverage was calculated for all fractions on the assumption that there is a surface layer 20 A thick. For the entire S(f) > 400 lipoprotein spectrum and for a part of the S(f) 20-400 lipoprotein distribution the proportion of surface covered by protein was constant (approximately 20% coverage). Therefore, for these portions of the lipoprotein spectrum, the increase in surface: volume ratio as particle size decreases is approximately compensated for by an increase in the weight percentage of protein.

Ultracentrifugal isolation of serum chylomicron-containing fractions with quantitation by infrared spectrometry and NCH elemental analysis.

An ultracentrifugal method for isolating chylomicron-containing fractions from serum by flotation, using either standard Spinco swinging-bucket rotors or a specially fabricated swinging-bucket rotor, is described. Lower limits of the S(f) rates of the chylomicron fractions are evaluated using a computer technique to define lipoprotein flotation over a nonlinear NaCl density gradient. The latter is prepared by a special overlayering technique.Quantitation within a 9-50 mug region of mass assay is accomplished by both infrared spectrometry and elemental analysis for N, C and H. Results indicate that the chylomicron concentration in serum for a small population of nonfasting male adults ranges from approximately 0-50 mg %.