Measuring mitochondrial respiration in vivo: From mouse to human

Introduction: The mitochondrial energy ecosystem can be non-invasively interrogated in photoreceptors by combing a clinical tool, optical coherence tomography (OCT), with a mitochondrial protonophore (2,4 dinitrophenol, DNP). It remains unclear if only supra-clinical doses of DNP will be useful for mouse studies or if lower but clinically relevant doses of DNP would facilitate translation from mice to humans. Methods: The experiment was a paired longitudinal design that took place over 2 days. On day 1, C57BL/6J mice were overnight dark adapted, then light-adapted for 5 h before OCT examination before regaining consciousness; a similar procedure was followed on day 2 but mice were injected IP with either saline or an acceptable human dose of DNP (0.5 mg/kg) 1 hour before OCT examination. Pre- and post-injection retinal laminae thickness were compared for evidence of toxicity. In particular, we measured the external limiting membrane – retinal pigment epithelium to look for thinning between day 1 and 2, which has a confirmed basis in DNP modulation of rod photoreceptor mitochondrial respiration upstream based on pH-triggering of RPE co-transporter-based water efflux. Results: Compared to uninjected controls, saline evoked no changes in retina layer thickness between days 1 and 2. On the other hand, a clinical dose of DNP caused a reduction in ELM-RPE thickness in the absence of any other changes in retinal layer thickness, a finding consistent with increased mitochondrial respiration. Conclusions: The promising results herein raise the possibility that combining a low dose of DNP with a standard clinical imaging tool will facilitate first-in-kind measurements of neuronal mitochondria in patients suffering from a range of diseases and morbidities

Effect of Methylimidazole-Induced Hypothyroidism in a Model of Low Retinal Neovascular Incidence

PURPOSE. To determine the effect of methylimidazole (MMI)-induced hypothyroidism in a newborn rat model of low retinal neovascular (NV) incidence. METHODS. Control and MMI-exposed newborn rats were raised either in room air or variable oxygen (40/15) until P14. All groups were then exposed to room air between postnatal day (P)14 and P20. Dams drank either tap water or water containing MMI. Eyes of animals in all groups were enucleated, and retinas were removed and stained with adenosine diphosphatase and analyzed for peripheral avascularity, vascular density, and NV incidence and severity. RESULTS. In the control group, MMI treatment did not promote the development of retinal NV although a linear relationship (r ϭ 0.99, P Ͻ 0.01) was found between increased MMI dose and lower peripheral retinal vascular densities. In all the 40/15 groups, peripheral retinal vascular densities were lower (P Ͻ 0.05) than normal and were not a function of MMI dose. Increased MMI dose produced increased retinal incidence of NV (r ϭ 0.99, P Ͻ 0.05). CONCLUSIONS. These data are consistent with the notions that thyroid function contributes to normal retinal vascular density and that hypothyroidism can play a permissive role in the development of retinal NV. (Invest Ophthalmol Vis Sci. 2004; 45:919 -921) DOI:10.1167/iovs.03-0914 V ery-low-birth-weight infants are at substantially higher risk for blinding complications, such as the development of retinal neovascularization (NV) associated with retinopathy of prematurity (ROP). To minimize the impact of retinal NV on vision, photocoagulation is currently used, but it is a destructive approach that is not always effective. A better understanding is needed of the pathogenic factors involved in the formation of retinal NV in ROP so that new methods of prevention and treatment can be developed. To date, studies have demonstrated an important link between insulin-like growth factor (IGF)-1 and normal and abnormal retinal vascular development. 1-3 IGF-1 is, among other functions, a downstream modulator of thyroid activity. Infants born very prematurely (Ͻ27 weeks) are more likely to have low thyroxine (T 4 ) levels, indicating an abnormal hypothalamus-pituitary-thyroid axis function. In this study, we used a clinically relevant model of ROP involving newborn rats exposed to a variable oxygen environment from postnatal day (P)0 to P14 and then to room air between P15 and P20. 6 -8 In animals exposed to an oxygen environment that alternates between 40% and 15% every other day (the 40/15 model), only a small percentage (Ͻ10%) of the rat pups exhibit 1 clock hour of NV in the peripheral retina. -14 METHODS The animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. MMI Between P0 and P20, rat pups were raised in room air and received MMI (0.05%, 0.1%, 0.13%, or 0.15% wt/vol; Sigma-Aldrich, St. Louis, MO), added to the drinking water of the dam. Control dams and their pups drank untreated water. In the control and 0.13% and 0.15% MMI-treated 40/15 groups, measurements of T 4 , thyroid stimulating hormone (TSH), and IGF-1 were performed (Anilytics Inc., Gaithersburg, MD) on pooled blood samples. Pooled blood samples were used because of the limited amount of blood available from individual newborn rats (Ͻ30 g). However, retinal histopathology was not investigated, because 0.13% and 0.15% MMI doses usually resulted in substantial (Ͼ90%) pup attrition (data not shown). Animal Model 7 Briefly, Sprague-Dawley dams and litters (12-15 pups per litter) were housed in modified pediatric incubators where the oxygen levels were varied between 40% and 15% (40/15) every 24 hours for the first 14 days after birth. Rats were then allowed to recover in room air (21%) during the next 6 days until P20. The drinking water in one of two cages per incubator was supplemented with MMI between P0 and P20. Holes in the sides of the pediatric incubators were purposely not sealed so that the incubators would be somewhat leaky and minimize the unwanted buildup of carbon dioxide. Although we did not directly assess whether airflow at the holes reversed during the variable oxygen exposure, it is unlikely that this happened, because the computercontrolled (Oxycycler; Biospherix, Ltd., Redfield, NY) variable oxygen procedure constantly maintain positive pressure inside the incubator by injecting the appropriate mixture of 100% oxygen or nitrogen to maintain either a 40% or 15% oxygen environment. In addition, each incubator housed one untreated and one MMI-treated 40/15 cage and so these groups experienced similar variable oxygen exposures. From th

Refractive Development in the “ROP Rat”

Although retinopathy of prematurity (ROP) is clinically characterized by abnormal retinal vessels at the posterior pole of the eye, it is also commonly characterized by vascular abnormalities in the anterior segment, visual dysfunction which is based in retinal dysfunction, and, most commonly of all, arrested eye growth and high refractive error, particularly (and paradoxically) myopia. The oxygen-induced retinopathy rat model of ROP presents neurovascular outcomes similar to the human disease, although it is not yet known if the “ROP rat” also models the small-eyed myopia characteristic of ROP. In this study, magnetic resonance images (MRIs) of albino (Sprague-Dawley) and pigmented (Long-Evans) ROP rat eyes, and age- and strain-matched room-air-reared (RAR) controls, were examined. The positions and curvatures of the various optical media were measured and the refractive state (℞) of each eye estimated based on a previously published model. Even in adulthood (postnatal day 50), Sprague-Dawley and Long-Evans ROP rats were significantly myopic compared to strain-matched controls. The myopia in the Long-Evans ROP rats was more severe than in the Sprague-Dawley ROP rats, which also had significantly shorter axial lengths. These data reveal the ROP rat to be a novel and potentially informative approach to investigating physiological mechanisms in myopia in general and the myopia peculiar to ROP in particular

Oral rivaroxaban versus standard therapy for the treatment of symptomatic venous thromboembolism : a pooled analysis of the EINSTEIN-DVT and PE randomized studies

Background: Standard treatment for venous thromboembolism (VTE) consists of a heparin combined with vitamin K antagonists. Direct oral anticoagulants have been investigated for acute and extended treatment of symptomatic VTE; their use could avoid parenteral treatment and/or laboratory monitoring of anticoagulant effects. Methods: A prespecified pooled analysis of the EINSTEIN-DVT and EINSTEIN-PE studies compared the efficacy and safety of rivaroxaban (15 mg twice-daily for 21 days, followed by 20 mg once-daily) with standard-therapy (enoxaparin 1.0 mg/kg twice-daily and warfarin or acenocoumarol). Patients were treated for 3, 6, or 12 months and followed for suspected recurrent VTE and bleeding. The prespecified noninferiority margin was 1.75. Results: 8282 patients were enrolled. 4151 received rivaroxaban and 4131 received standard-therapy. The primary efficacy outcome occurred in 86 rivaroxaban-treated patients (2.1%) compared with 95 (2.3%) standard-therapy-treated patients (hazard ratio, 0.89; 95% confidence interval [CI], 0.66-1.19; pnoninferiority<0.001). Major bleeding was observed in 40 (1.0%) and 72 (1.7%) patients in the rivaroxaban and standard-therapy groups, respectively (hazard ratio, 0.54; 95% CI, 0.37-0.79; p=0.002). In key subgroups, including fragile patients, cancer patients, patients presenting with large clots and those with a history of recurrent VTE, the efficacy and safety of rivaroxaban was similar compared with standard-therapy. Conclusion: The single-drug approach with rivaroxaban resulted in similar efficacy to standard-therapy and was associated with a significantly lower rate of major bleeding. Efficacy and safety results were consistent among key patient subgroups

Synthesis and Cytotoxicity of Cyanoborane Adducts of N 6 -Benzoyladenine and 6-Triphenylphosphonylpurine

N 6 -Benzoyladenine-cyanoborane (2), and 6-triphenylphosphonylpurine-cyanoborane (3) were selected for investigation of cytotoxicity in murine and human tumor cell lines, effects on human HL-60 leukemic metabolism and DNA strand scission to determine the feasibility of these compounds as clinical antineoplastic agents. Compounds 2 and 3 both showed effective cytotoxicity based on ED50 values less than 4 μg/ml for L1210, P388, HL-60, Tmolt3, HUT-78, HeLa-S3 uterine, ileum HCT-8, and liver Hepe-2. Compound 2 had activity against ovary 1-A9, while compound 3 was only active against prostate PL and glioma UM. Neither compound was active against the growth of lung 549, breast MCF-7, osteosarcoma HSO, melanoma SK2, KB nasopharynx, and THP-1 acute monocytic leukemia. In mode of action studies in human leukemia HL-60 cells, both compounds demonstrated inhibition of DNA and protein syntheses after 60 min at 100 μM. These compounds inhibited RNA synthesis to a lesser extent. The utilization of the DNA template was suppressed by the compounds as determined by inhibition of the activities of DNA polymerase α, m-RNA polymerase, r-RNA polymerase and t-RNA polymerase, which would cause adequate inhibition of the synthesis of both DNA and RNA. Both compounds markedly inhibited dihydrofolate reductase activity, especially in compound 2. The compounds appeared to have caused cross-linking of the DNA strands after 24 hr at 100 μM in HL-60 cells, which was consistent with the observed increased in ct-DNA viscosity after 24 hr at 100 μM. The compounds had no inhibitory effects on DNA topoisomerase I and II activities or DNA-protein linked breaks. Neither compound interacted with the DNA molecule itself through alkylation of the nucleotide bases nor caused DNA interculation between base pairs. Overall, these antineoplastic agents caused reduction of DNA and protein replication, which would lead to killing of cancer cells