Minocycline and Diacetyl Minocycline Eye Drops Reduce Ocular Neovascularization in Mice.

Purpose: To evaluate the efficacy of minocycline and a novel, modified minocycline analogue that lacks antimicrobial action, diacetyl minocycline (DAM), on choroidal neovascularization (CNV) in mice of both sexes. Methods: CNV was induced via laser injury in female and male C57BL/6J mice. Minocycline, DAM, or saline was administered via topical eye drops twice a day for 2 weeks starting the day after laser injury. CNV volume was measured using immunohistochemistry labeling and confocal microscopy. Results: Minocycline reduced lesion volume by 79% (P ≤ 0.0004) in female and male mice. DAM reduced lesion volume by 73% (P ≤ 0.001) in female and male mice. There was no significant difference in lesion volume between minocycline and DAM treatment groups or between female and male mice. Conclusions: Both minocycline and DAM eye drops significantly reduced laser-induced CNV lesion volume in female and male mice. While oral tetracyclines have been shown to mitigate pathologic neovascularization in both preclinical studies and clinical trials, the present data are the first to suggest that tetracycline derivatives may be effective to reduce pathologic CNV when administered via topical eye drops. However, the action is unrelated to antimicrobial action. Targeted delivery of these medications via eye drops may reduce the potential for systemic side effects. Translational Relevance: Topical administration of minocycline and/or DAM via eye drops may represent a novel therapeutic strategy for disorders involving pathologic CNV.

2,3-Butanedione monoxime facilitates successful resuscitation in a dose-dependent fashion in a pig model of cardiac arrest.

PURPOSE: Ischemic contracture compromises the hemodynamic effectiveness of cardiopulmonary resuscitation (CPR) and resuscitability from cardiac arrest. In a pig model of cardiac arrest, 2,3-butanedione monoxime (BDM) attenuated ischemic contracture. We investigated the effects of different doses of BDM to determine whether increasing the dose of BDM could improve the hemodynamic effectiveness of CPR further, thus ultimately improving resuscitability. METHODS: After 16minutes of untreated ventricular fibrillation and 8minutes of basic life support, 36 pigs were divided randomly into 3 groups that received 50mg/kg (low-dose group) of BDM, 100mg/kg (high-dose group) of BDM, or an equivalent volume of saline (control group) during advanced cardiovascular life support. RESULTS: During advanced cardiovascular life support, the control group showed an increase in left ventricular (LV) wall thickness and a decrease in LV chamber area. In contrast, the BDM-treated groups showed a decrease in the LV wall thickness and an increase in the LV chamber area in a dose-dependent fashion. Mixed-model analyses of the LV wall thickness and LV chamber area revealed significant group effects and group-time interactions. Central venous oxygen saturation at 3minutes after the drug administration was 21.6% (18.4-31.9), 39.2% (28.8-53.7), and 54.0% (47.5-69.4) in the control, low-dose, and high-dose groups, respectively (P<.001). Sustained restoration of spontaneous circulation was attained in 7 (58.3%), 10 (83.3%), and 12 animals (100%) in the control, low-dose, and high-dose groups, respectively (P=.046). CONCLUSION: 2,3-Butanedione monoxime administered during CPR attenuated ischemic contracture and improved the resuscitability in a dose-dependent fashion.

Reactive skin decontamination lotion (RSDL) for the decontamination of chemical warfare agent (CWA) dermal exposure.

Rapid decontamination of the skin is the single most important action to prevent dermal absorption of chemical contaminants in persons exposed to chemical warfare agents (CWA) and toxic industrial chemicals (TICs) as a result of accidental or intentional release. Chemicals on the skin may be removed by mechanical means through the use of dry sorbents or water. Recent interest in decontamination systems which both partition contaminants away from the skin and actively neutralize the chemical has led to the development of several reactive decontamination solutions. This article will review the recently FDA-approved Reactive Skin Decontamination Lotion (RSDL) and will summarize the toxicity and efficacy studies conducted to date. Evidence of RSDL's superior performance against vesicant and organophosphorus chemical warfare agents compared to water, bleach, and dry sorbents, suggests that RSDL may have a role in mass human exposure chemical decontamination in both the military and civilian arenas.

Industry-wide medical surveillance of California flavor manufacturing workers: Cross-sectional results.

BACKGROUND: Two cases of bronchiolitis obliterans in flavor manufacturing workers prompted California health and labor agencies to initiate industry-wide surveillance. METHODS: Companies' physicians submitted cross-sectional questionnaire and spirometry data for 467 workers in 16 workplaces. We compared prevalence ratios of respiratory symptoms, diagnoses, and abnormal spirometry to a general population sample. We calculated odds ratios for risk factors for spirometric obstructive abnormality. RESULTS: Flavoring workers were 2.7 times more likely than the general population to have severe airways obstruction. Risk factors identified for 18 cases with obstruction from six companies included younger age, Hispanic ethnicity, liquid and powder production work, greater company diacetyl usage, and having a coworker with obstruction. Severity of obstruction was related to tenure. At least 12 workers had probable occupational fixed airways obstruction. CONCLUSIONS: The flavoring industry risk of severe lung disease justifies lowering flavoring exposures and medical screening for secondary prevention until worker safety is demonstrated.

Treatment with tertiary oximes prevents seizures and improves survival following sarin intoxication.

The capability of the tertiary oximes, monoisonitrosoacetone (MINA) and diacetylmonoxime (DAM), to reactivate acetylcholinesterase (AChE) inhibited by sarin (GB) in the blood, brain, and peripheral tissues of guinea pigs was compared with that of the quaternary oximes 2-PAM, HLö7, and MMB-4. Animals were injected subcutaneously (s.c.) with 1.0 x LD(50) of GB and treated intramuscularly (i.m.) 5 min later with one of these oximes. Sixty minutes after GB exposure, tissues were collected for AChE analysis. At low doses, MINA and DAM produced significant increases in AChE activity in all brain areas examined, but no significant AChE reactivation in peripheral tissues or blood. At higher doses, MINA and DAM increased AChE activity in the brain, peripheral tissues, and blood. In contrast, the quaternary oximes produced significant reactivation in peripheral tissues and blood AChE, but no significant reactivation of brain AChE. In another study, animals were pretreated i.m. with pyridostigmine 30 min prior to s.c. challenge with 2.0 x LD(50) of GB and treated i.m. 1 min later with atropine sulfate (2.0 mg/kg), plus a varied dose of oximes. MINA and DAM prevented or terminated GB-induced seizure activity and protected against GB lethality in a dose-dependent fashion. In contrast, none of the quaternary oximes prevented or stopped GB-induced seizures. Thus, tertiary oximes reactivated AChE in the brain, improved survival, and terminated seizures following GB intoxication.

Protection of ischemic myocardium in dogs using intracoronary 2,3-butanedione monoxime (BDM).

BACKGROUND: Actomyosin ATPase is one of the major ATP consuming enzymes in the myocardium. We tested whether 2,3-butanedione monoxime (BDM), a reversible inhibitor of actomyosin ATPase, given before coronary occlusion, limits infarct size in anesthetized open-chest dogs. METHODS AND RESULTS: After circumflex artery catheterization using fluoroscopic guidance, BDM (125 mM) or buffer vehicle was infused (12.0 ml/min) for 20 min (BDM-20, n = 5 and Buffer-20, n = 6) or for 5 min (BDM-5, n = 6 and Buffer-5, n = 6) prior to 60 min of ischemia and 3 h of reperfusion. BDM administration increased subendocardial blood flow 271% above baseline flow (radioactive microspheres), and systolic wall thickening was converted to wall bulging (wall thickening by sonomicrometry was -27 +/- 29% and -22 +/- 13% of baseline in BDM-20 and BDM-5, respectively). Adjusted mean infarct size (% area-at-risk) was 11.0 +/- 2.8% and 11.9 +/- 2.6% in BDM-20 and BDM-5 vs. 20.2 +/- 2.5% and 20.5 +/- 2.5% in Buffer-20 and Buffer-5 (ancova, P < 0.05 for each BDM vs. Buffer group). Measurement of glycolytic metabolites and the adenine nucleotide pool of myocardium paced electronically at 150 beats per minute during total ischemia at 37 degrees C following BDM showed a metabolic response similar to that seen in ischemic preconditioning. ATP depletion, nucleoside production, and lactate accumulation were slowed in ischemic tissue treated with BDM. CONCLUSION: BDM given before the onset of ischemia markedly limited infarct size and reduced energy demand after the onset of ischemia. The explanation for the reduced infarct size induced by BDM treatment is hypothesized to be the persistent reduction in energy demand found in ischemic BDM treated myocardium.

Time-dependent efficacy of initial reperfusion with 2,3 butanedione monoxime (BDM) on release of cytosolic enzymes and ultrastructural damage in isolated hearts.

BACKGROUND: Reperfusion injury after cardioplegia may not be sufficiently addressed by conventional cardioplegic techniques in open heart surgery. 2,3-butanedione monoxime (BDM) has the potential to reduce myocardial reperfusion injury by uncoupling myocyte contraction from the intracellular calcium concentration, thus reducing reperfusion contracture. The aim of this study was to investigate the effects of different application periods of BDM during initial reperfusion on myocardial tissue injury after cardioplegia. METHODS: Isolated guinea-pig hearts underwent 50 min of cardioplegic arrest in St. Thomas' Hospital II solution at 37 C. Control hearts (n = 8) were immediately reperfused with normal Krebs-Henseleit solution for 30 min. In the therapy groups BDM-5, BDM-20, and BDM-40 (n = 8, each), hearts were initially reperfused with BDM (20mmol/L) for either 5, 20, or 40 min, respectively, followed by 30 min of reperfusion with normal Krebs-Henseleit solution. Coronary venous effluent was collected to estimate myocardial tissue damage through release of cytosolic enzymes (LDH and CK) and cardiac troponin 1. Ultrastructural alterations were qualitatively assessed by electron microscopy. RESULTS: Initial reperfusion with BDM markedly reduced LDH and CK release, as long as BDM was present. After washout of the protective agent a rebound of enzyme release occurred in BDM-5 hearts which was effectively reduced in BDM-20 and BDM-40 hearts. Troponin I release was similarly increased in all groups at the onset of reperfusion and rapidly decreased thereafter. Myocardial ultrastructural damage was most pronounced in control hearts, intermediate in BDM-5 and BDM-40 hearts, but markedly attenuated in BDM-20 hearts. CONCLUSIONS: Both 20 and 40 min of initial reperfusion effectively protected the hearts from reperfusion damage as indicated by cytosolic enzyme release, while 5 min of treatment were clearly insufficient. Toxic effects of BDM during the longer treatment period of 40 min or induction of edema by the long-term perfusion of non-beating hearts in this group may account for the worse preservation of myocardial ultrastructure in BDM-40 hearts. Thus, contraction uncoupling during initial reperfusion by BDM or similarly acting drugs may prove a viable principle for reduction of myocardial reperfusion injury. However, the ideal duration of treatment for the best therapeutic effect must be carefully evaluated.

Efficacy of myocardial initial reperfusion with 2,3 butanedione monoxime after cardioplegic arrest is time-dependent.

OBJECTIVE: In a previous study, initial reperfusion of isolated hearts after cardioplegic arrest with 2,3 butanedione monoxime (BDM) for 5 min was markedly superior to warm hyperkalemic reperfusion in improving the initial oxygen balance and reducing reperfusion arrhythmias. However, left ventricular contractility was only marginally enhanced. The goal of the present study was to test, wether the efficacy of BDM reperfusion can be enhanced by prolonging the application period. METHODS: 32 Langendorff perfused guinea pig hearts were subjected to 50 min of cardioplegic arrest in St. Thomas Hospital II solution at 37 degrees C for 50 min. Control hearts (n = 8) were immediately reperfused with normal Krebs solution for 30 min. In BDM-5, BDM-20, and BDM-40 hearts (n = 8, each), a 5, 20, or 40 min period of initial BDM reperfusion preceded perfusion with normal Krebs. RESULTS: BDM markedly improved the O2 balance during initial reperfusion by reducing O2 demand by over 50% (p < 0.01) in all treatment groups while coronary flow was maintained. Reperfusion contracture, estimated by the end-diastolic balloon pressure was inhibited by more than 50% in BDM-20 and BDM-40 hearts. Recovery of left ventricular developed pressure, dP/dtmax, and -dP/dtmax was significantly enhanced throughout the reperfusion period only in the BDM-20 group (p < 0.05). Myocardial ultrastructure was best preserved in BDM-20 hearts. CONCLUSIONS: 20 min of initial BDM reperfusion were clearly superior to immediate Krebs reperfusion or a shorter (5 min) or longer (40 min) BDM treatment period in attenuating reperfusion damage. Thus, contraction uncoupling during initial reperfusion by BDM or similarly acting drugs may prove a viable technique to reduce myocardial reperfusion damage in patients undergoing open heart surgery.

Effects of butanedione monoxime and temperature on prolonged cardiac storage.

BACKGROUND: The optimal temperature for cardiac allograft storage remains controversial. We conjectured that supplementation of the potent cardioprotective agent 2,3-butanedione monoxime with calcium may improve allograft storage and make the precise storage temperature less critical. METHODS: Hearts were harvested from Sprague-Dawley rats (250 to 350 g), mounted on a Langendorff apparatus, and instrumented with an intraventricular balloon. Hearts were flushed and stored with either unmodified University of Wisconsin solution (UWS) or UWS supplemented with 10 mmol/L of 2,3-butanedione monoxime and calcium 0.1 mmol/L (BDM). Hearts were then subjected to 12 hours of storage at one of five temperatures (0 degree, 4 degrees, 8 degrees, 12 degrees, or 16 degrees C) in a complete 2 x 5 factorial design (n = 6/group). Data are reported either as a percentage of the prestorage results or as an absolute value (mean +/- standard deviation). RESULTS: Recovery of developed pressure (p < 0.0001), coronary flow (p < 0.0001), and diastolic volume (p < 0.001) were significantly enhanced, whereas creatine kinase (p < 0.0001) and lactate dehydrogenase release (p < 0.0001) were reduced in the BDM versus the UWS groups. In both the BDM and UWS storage groups, recovery was better at temperatures of 8 degrees C or less than at 12 degrees C or more. The single preferred temperature was 4 degrees C, significantly better than 0 degree C with unmodified UWS, while similar to 0 degree and 8 degrees C with BDM. Adenine nucleotide values were decreased equally in the BDM and UWS hearts, but preservation was enhanced at 0 degree C compared with all warmer temperatures. CONCLUSIONS: We conclude that 4 degrees C is the preferred temperature for prolonged cardiac storage with UWS and that the inclusion of 2,3-butanedione monoxime with calcium 0.1 mmol/L markedly enhances recovery for storage temperatures of 8 degrees C or less.

Poststorage diastolic abnormalities of heart transplants: is vascular dysfunction or myocardial contracture the culprit?

BACKGROUND: Vascular dysfunction and myocardial contracture can both contribute to posttransplantation diastolic abnormalities commonly exhibited by heart transplants, but their respective importance remains incompletely elucidated. To address this issue, we assessed the effects of supplementing a new heart preservation solution, Celsior, with a nitric oxide precursor (L-arginine) and a compound known to uncouple excitation from contraction (2, 3-butanedione monoxime). METHODS: Fifty isolated buffer-perfused rat hearts were divided into four groups. In group 1, hearts were arrested with St. Thomas' Hospital solution No. 2 (Plegisol) and stored in normal saline solution. In group 2, Celsior solution was used for cardiac arrest and storage. Group 3 hearts were arrested with and stored in Celsior solution supplemented with 2 mmol/L of L-arginine. In group 4, Celsior solution used for arrest and storage was supplemented with both 2 mmol/L of L-arginine and 30 mmol/L of 2, 3-butanedione monoxime. All hearts were stored for 10 hours, subsequently reperfused for 1 hour on a Langendorff column, and left ventricular pressure-volume curves were constructed. 5-Hydroxytryptamine (10(-7) mol/L) and papaverine (5 x 10(-6) mol/L) were used to test changes in endothelium-dependent and endothelium-independent coronary vascular responses, respectively, and compared with those obtained during the preischemic period. RESULTS: After 10 hours of cold storage, a major postischemic contracture was found in group 1. Left ventricular diastolic function was best preserved in group 4 at the end of storage and over the entire period of reperfusion. Coronary vasodilatory response to 5-hydroxytryptamine was completely lost in all groups after cold storage and reperfusion. Endothelium-independent vasodilatory response to papaverine was preserved in 2, 3-butanedione monoxime-treated hearts, whereas it was reduced in other groups. CONCLUSIONS: Our results suggest that myocardial contracture plays a major role in posttransplantation diastolic abnormalities shown by cardiac allografts. Alleviation of contracture significantly improves the responsiveness of coronary smooth muscles but does not affect that of the vascular endothelium which needs to be handled by separate interventions.

Regional contractile blockade at the onset of reperfusion reduces infarct size in the dog heart.

An important mechanism of lethal myocardial reperfusion injury is the development of cellular hypercontracture at the onset of reperfusion. Hypercontracture can lead to cytolysis by mutual mechanical disruption of myocardial cells. 2,3-Butanedione monoxime (BDM) inhibits myofibrillar cross-bridge cycling and may therefore reduce infarct size in ischaemic reperfused myocardium. This study investigated whether a temporary presence of BDM protects against myocardial reperfusion injury in an intact-animal preparation. Anaesthetized open-chest dogs (n = 10) underwent 1 h of left anterior descendent artery (LAD) occlusion and received intracoronary BDM (25 mM, n = 5) or vehicle (n = 5) for 65 min starting with an anoxic local infusion 5 min before reperfusion. Infarct size was assessed by triphenyltetrazolium staining after 6 h reperfusion. The infusion of BDM was accompanied by a transient reduction of left ventricular systolic pressure from 84.3 +/- 11.2 mm Hg during occlusion to 66.4 +/- 9.9 mm Hg at 30 min reperfusion (mean +/- SD, P < 0.01 vs. control). LAD-flow and regional wall motion in the area at risk showed no difference between groups. Infarct size (% of area at risk) was reduced from 24.4 +/- 8.7 (control) to 6.6 +/- 2.0% (BDM) (P < 0.01). The results demonstrate that development of necrosis in reperfused myocardium can be greatly reduced by temporary presence of the contractile inhibitor BDM at the onset of reperfusion.

Effects of 2,3-butanedione monoxime in isolated hearts: protection during reperfusion after global ischemia.

The cardiac effects of 2,3-butanedione monoxime on electrical and mechanical function, rhythm, oxygen utilization, and coronary flow responsiveness, particularly during severe ischemia and reperfusion, have not been studied. After perfusing hearts at 55 mm Hg, coronary perfusion was interrupted for 30 minutes and was then reestablished at the control perfusion pressure for 40 minutes. Hearts were divided into four groups (n = 10 each) treated with 0, 3, 5, or 10 mmol/L of 2,3-butanedione monoxime added to the perfusate for 10 minutes before and during ischemia and for the first 10 minutes of reperfusion. An additional nonischemic group served as a time control. Variables monitored were heart rate, atrioventricular conduction time, cardiac rhythm, isovolumetric systolic and diastolic left ventricular pressure, maximum rate of left ventricular pressure change, coronary flow, myocardial oxygen consumption, and the ratio of oxygen delivery to myocardial oxygen consumption. Before ischemia, 2,3-butanedione monoxime significantly decreased isovolumetric left ventricular systolic pressure and increased the ratio of oxygen delivery to myocardial oxygen consumption in a dose-dependent manner, with only slight changes in heart rate and atrioventricular time with 10 mmol/L of 2,3-butanedione, monoxime. After 40 minutes of reperfusion, isovolumetric left ventricular systolic pressure recovered to 81 +/- 5% and 83 +/- 2% of the initial control values for the 5 and 10 mmol/L 2,3-butanedione monoxime groups. This was significantly greater than the recovery for the 0 and 3 mmol/L 2,3-butanedione monoxime groups, 59 +/- 3% and 63 +/- 4%, respectively. Similarly, the duration of ventricular fibrillation and of tachycardia was significantly lower, coronary flow reserve was better preserved, and myocardial oxygen consumption was greater with reperfusion in the 5 and 10 mmol/L 2,3-butanedione monoxime groups than in the 0 mmol/L 2,3-butanedione monoxime group. This study shows that relatively low concentrations of 2,3-butanedione monoxime, given before global ischemia and early during reperfusion of isolated hearts, can protect against dysrhythmias and improve return of myocardial and vascular function.

Selective inhibition of the contractile apparatus. A new approach to modification of infarct size, infarct composition, and infarct geometry during coronary artery occlusion and reperfusion.

BACKGROUND: Myocardial reperfusion is associated with calcium overload and cell contracture, mechanisms that may precipitate cell death. In this study, we tested the hypothesis that in vivo inhibition of this contracture could lead to cell preservation in an open-chest large animal model. METHODS AND RESULTS: Regional myocardium function was measured during a selective intracoronary infusion of 2,3-butanedione monoxime (BDM), a specific inhibitor of actin-myosin coupling, in the control state (10 pigs) and in a protocol of a 51-minute coronary occlusion followed by reperfusion (40 pigs). The effects on coronary artery blood flow in the basal state were also studied (seven pigs). Intramyocardial distribution of the infusate during coronary occlusion, myocardial water content after 30 minutes of reperfusion and area at risk, infarct size, type of histological necrosis, and infarct geometry after 24 hours of reperfusion were assessed. Methods used included electromagnetic flowmeter, radiolabeled microspheres, subendocardial sonomicrometers, fluorescein, triphenyl tetrazolium chloride and Masson's trichrome staining, and computer quantification of infarct edges. In the absence of ischemia, BDM infusion inhibited regional shortening in a dose-dependent manner up to full systolic bulging while producing marked regional increase in coronary blood flow. During early reperfusion, BDM reduced end-diastolic length 76% more than the control infusion (p less than 0.05) and increased systolic bulging by 420% compared with no change in control animals. The ratio of infarct size/area at risk was reduced by 31% with BDM (p less than 0.05), with striking modifications of infarct histology and infarct geometry; specifically, the extent of contraction band necrosis was reduced by 63% from 105.5 +/- 18.2 to 39.2 +/- 13.6 mm2 (p less than 0.02), and more patches of necrosis (6.5 +/- 2.1 versus 1.6 +/- 0.4, p less than 0.05) and higher contour (7.7 +/- 1.2 versus 5.03 +/- 0.2, p less than 0.05) and fractal (12.1 +/- 1.3 versus 7.8 +/- 0.2, p less than 0.05) indexes were found. CONCLUSIONS: Selective intracoronary infusion of BDM at doses inhibiting regional wall motion decreased infarct size after reperfusion. The effects of BDM on regional function, the reduction in contraction band necrosis at histology, and the peculiar configuration of these infarcts all suggest that inhibition of contracture can interfere with cell-to-cell progression of myocardial necrosis, supporting a role for contracture in reperfusion-induced cell death.

The therapeutic effects of 2,3-butanedione monoxime and atropine in severe dichlorvos intoxication in buffalo calves.

2,3-Butanedione monoxime and atropine alone or in combination were evaluated for their ability to alleviate the toxicity and to reverse the biochemical changes induced by dichlorvos in the blood of buffalo calves. Treatment with 2,3-butanedione monoxime plus atropine 30 min after oral administration of dichlorvos (160 mg/kg) eliminated the apparent toxic signs within 10-15 min, completely prevented lethality, and reversed the dichlorvos-induced alterations in the concentrations of serum carboxylesterase, total plasma proteins, blood glucose and plasma cholinesterase within 2, 4, 12 and 168 h, respectively. Treatment with either 2,3-butanedione monoxime or atropine alone was less effective but the former was the more potent of the two in counteracting the biochemical effects of dichlorvos. These antidotal studies suggest that 2,3-butanedione monoxime in conjunction with atropine would provide effective therapy against severe dichlorvos intoxication in buffalo.

Effectiveness of certain drugs in acute malathion intoxication in rats.

The protective effects of atropine, diacetylmonoxime (DAM), and diazepam separately and in combination were investigated in rats exposed to malathion. Malathion (500 mg/kg, ip) inhibited acetylcholinesterase (AchE) activity in RBC and brain and produced hyperglycemia and hyperlactacidemia with depletion of glycogen in liver, triceps, and brain of animals 2 hr after its administration. Atropine (20 mg/kg, ip) given immediately after malathion abolished hyperglycemia and glycogenolytic effect but exhibited no effect on the recovery of inhibited AchE activity. DAM (100 mg/kg ip) given immediately after malathion significantly reactivated the inhibited AchE activity both in RBC and brain. It also partially modified hyperglycemia and glycogenolytic effect. Diazepam (50 mg/kg, ip) slightly modified AchE and abolished hyperglycemia, hyperlactacidemia, and glycogenolytic effects. A combination of these drugs protected the animals from the acute toxic effects of malathion.