Osteonecrosis in a chemically induced rat model of human hemolytic disorders associated with thrombosis--a new model for avascular necrosis of bone.

Bone injury occurs in human hemolytic disorders associated with thrombosis, such as beta-thalassemia and sickle cell disease. Exposure of rats to 2-butoxyethanol (BE) has been associated with hemolytic anemia, disseminated thrombosis, and infarction in multiple organs including bone. This rat model apparently mimics acute hemolysis and thrombosis in humans. To elucidate the extent of bone injury, male and female Fischer F344 rats were given 4 daily doses of 250 mg BE/5 ml water/kg of body weight. Tail vertebrae were studied by histopathology and magnetic resonance imaging (MRI). Thrombosis and infarction were seen in both sexes, but females were more severely affected. Lesions were characterized by extensive medullary fat necrosis, granulomatous inflammation, fibroplasia, growth plate degeneration, and new woven bone formation adjacent to necrotic bone trabeculae. MRI mean and standard deviation tissue-density data for both sexes indicated a significant (P < or = 0.05) decrease following 4-days treatment and a significant increase (P < or = 0.05) following an additional 24 days without treatment. Thus, MRI was useful in revealing BE-induced bone injury, which was predominantly necrotic initially and subsequently regenerative with proliferation of connective tissue and bone following postischemia recovery.

Experimental studies on viscofluids for intraocular surgery.

PURPOSE: To investigate the feasibility and efficacy of using diluted viscofluids during intraocular surgery. SETTING: Laboratory for Intraocular Microsurgery and Implants, Goldschleger Eye Research Institute, and the Sackler School of Medicine, Tel-Hashomer, Israel. METHODS: Diluted hyaluronic acid (0.06 to 0.12%) in balanced salt solutions were irrigated through the phacoemulsification machine or a separate irrigation line (i.e., anterior chamber maintainer). Irrigation was facilitated by increasing the atmospheric pressure in the bottle using compressed air. Experimental surgery was done in rabbit eyes. RESULTS: A viscous fluid could be irrigated at any practical rate by increasing the height of the bottle, increasing the atmospheric pressure within the bottle, or both. Experimental surgeries showed that viscofluids maintained intraocular pressure and anterior chamber volume, allowed safe intraocular manipulations, reduced turbulence in the anterior chamber, and probably provided tissue protection. CONCLUSION: Viscofluids irrigated using a high-pressure system combined the advantages of continuous irrigation of fluids and the protective qualities of viscoelastics in this rabbit eye study.

Recombinant polypeptides derived from the fibrin binding domain of fibronectin are potential agents for the imaging of blood clots.

Thrombus formation in the circulation is accompanied by covalent linkage of fibronectin (FN) through transglutamination of glutamine no. 3 in the fibrin binding amino terminal domain (FBD) of FN. We have exploited this phenomenon for thrombus detection by the employment of radioactively-labelled recombinant polypeptide molecules derived from the 5-finger FBD of human FN. Three recombinant FBD polypeptides, 12 kDa ("2 fingers"), 18.5 kDa ("3 fingers") and 31 kDa FBD ("5 fingers"), were prepared and compared to native FN-derived 31 kDa-FBD with respect to their ability to attach to fibrin clots in vitro and in vivo. The accessibility of Gln-3 in these molecules was demonstrated by the incorporation of stoichiometric amounts of 14C-putrescine in the presence of plasma transglutaminase. Competitive binding experiments to fibrin have indicated that, although the binding affinities of the FBD molecules are lower than that of FN, substantial covalent linkage was obtained in the presence of transglutaminase, and even in the presence of excess FN or heparin. The biological clearance rates of radioactively labelled FBD molecules in rats and rabbits were much higher than those of FN and fibrinogen, thus indicating their potential advantage for use as a diagnostic imaging tool. Of the three molecules, the 12 kDa FBD exhibited the highest rate of clearance. The potential of the 12 kDa and 31 kDa FBDs as imaging agents was examined in a stainless steel coil-induced thrombus model in rats and in a jugular vein thrombus model in rabbits, using either [125I] or [111In]-labelled materials. At 24 h, clot-to-blood ratios ranged between 10 and 22 for [125I]-12 kDa FBD and 40 and 60 for [111In]-12 kDa FBD. In the rat model, heparin did not inhibit the uptake of FBD. Taken together, the results indicate that recombinant 12 kDa FBD is a good candidate for the diagnosis of venous thrombosis.

Cellular and secretory mechanisms related to delayed radiation-induced microvessel dysfunction in the spinal cord of rats.

PURPOSE: This study aimed to investigate long-term, radiation-induced changes in microvessel permeability, the profile of the vasoactive mediators endothelin and nitric oxide, and the response of specific cell systems in the irradiated spinal cord of rats. METHODS AND MATERIALS: The thoracolumbar spinal cords of Fischer rats were irradiated to a dose of 15 Gy, and the rats were sacrificed at various times afterward. Endothelin levels and nitric oxide-synthase (NOS) activity were assayed in extracts of spinal cords. Microvascular permeability and the effect of treatment with recombinant human manganese superoxide dismutase (r-hMnSOD) were assessed quantitatively. Immunohistochemistry evaluated astrocytes, microglia, vascular basal membrane, and neurofilaments. RESULTS: None of the rats developed neurologic dysfunction. Endothelin levels were significantly reduced at 18 h after irradiation and markedly attenuated after 10 days (p < 0.007). Thereafter, endothelin levels returned to normal values at 56 days after radiation and escalated to markedly high levels after 120 and 180 days (p < 0.002). NOS activity remained very low throughout the period of follow-up and failed to counterbalance the shifts in endothelin levels. Treatment with r-hMnSOD had no effect on normal vascular permeability but it abolished the abnormally increased permeability measured at 18 h after radiation and again after 120 and 180 days. Standard microscopic evaluation failed to reveal abnormalities in the irradiated spinal cord, but immunohistochemical staining showed a progressive increase in the number of microglial cells per field after 120 and 180 days (p < 0003). A similar increase in the number of astrocytic cells per field was noted after more than 180 days, but an earlier short lasting peak was also noted at 14 days after radiation. No abnormalities were found in blood vessel configuration, density, diameter, and basal membrane staining, or in the neurofilaments. CONCLUSION: Marked imbalance in the regulatory function of endothelium-derived mediators of the vascular tone is present after radiation therapy probably inducing chronic vasoconstriction. This imbalance favors localized procoagulation that may enhance the consequent loss of function measured as increased permeability. Microglial proliferation may account for continuous release of superoxide that may enhance disruption of normal permeability. The latter is corrected by SOD treatment. Astrocytic proliferation may present a response to the mitogenic effect of endothelin and to microglial-derived paracrine effect of cytokines.

Sodium hyaluronate as a tool in strabismus surgery in rabbits.

BACKGROUND AND OBJECTIVE: The authors tested whether coating tissue with sodium hyaluronate (Na-HA) reduced postoperative adhesions and accelerated the healing process in strabismus surgery. MATERIALS AND METHODS: The surgical technique was tested during recession and resection operations performed on 30 rabbits and was compared with the use of NaCl 0.9%. Clinical, biomicroscopic examinations were performed on postoperative days 1, 2, 7, and 30 and histopathologic examinations were performed on postoperative days 2, 7, and 30. RESULTS: Clinically, there were no statistically significant differences between the study group and the control group. Also, there were no statistically significant differences between the two groups for most of the histopathologic criteria; however, new vessel formation was smaller with Na-HA than without it. Statistical significance was defined as P < .05. CONCLUSION: The authors found no significant positive effect of Na-HA on postoperative healing in rabbits.

Absorption, distribution, metabolism, and excretion of bacteria-derived hyaluronic acid in rats and rabbits.

The feasibility of using bacteria-derived hyaluronate solution as a viscous aid for anterior chamber surgery was examined by studying the pharmacokinetic behavior and metabolic fate of 14C-labelled material, following administration to rats and rabbits. Intravenously-administered HA disappeared rapidly from the blood of rabbits and rats with a mean t1/2 of 5.3 and 3.7 min, respectively. The labelled material has concomittantly accumulated in the liver, where it was digested to oligomeric sugar subunits; these were further utilized metabolically either for energy generation or for incorporation into new high molecular weight species. Metabolic cage studies has indicated that most of the 14C-HA label administered intravenously to rats was excreted as CO2 via the respiration within 24h, while a smaller portion was excreted in the urine. The disposition of viscous 14C-HA administered into the anterior eye chamber of rabbits was slow and followed first-order kinetics with a t1/2 of 10.5h. No degradation occurred in the aqueous humour. Low blood levels of 14C-labeled material were found during 72h after intra-ocular administration. The results indicate that the absorption, distribution, metabolism and excretion of bacteria-derived HA is similar to those of the currently used ophthalmic surgery HA aids extracted from rooster combs.