Ronald W. Wood

Inhibition of Lymphangiogenesis and Lymphatic Drainage via Vascular Endothelial Growth Factor Receptor 3 Blockade Increases the Severity of Inflammation in a Mouse Model of Chronic Inflammatory Arthritis

OBJECTIVE This study was undertaken to investigate the effect of lymphatic inhibition on joint and draining lymph node (LN) pathology during the course of arthritis progression in mice. METHODS Tumor necrosis factor (TNF)-transgenic mice were used as a model of chronic inflammatory arthritis. Mice were subjected to contrast-enhanced magnetic resonance imaging to obtain ankle and knee joint synovial volumes and draining popliteal LN volumes before and after 8 weeks of treatment with vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody, VEGFR-2 neutralizing antibody, or isotype IgG. Animals were subjected to near-infrared lymphatic imaging to determine the effect of VEGFR-3 neutralization on lymph transport from paws to draining popliteal LNs. Histologic, immunohistochemical, and reverse transcriptase-polymerase chain reaction analyses were used to examine lymphatic vessel formation and the morphology of joints and popliteal LNs. RESULTS Compared with IgG treatment, VEGFR-3 neutralizing antibody treatment significantly decreased the size of popliteal LNs, the number of lymphatic vessels in joints and popliteal LNs, lymphatic drainage from paws to popliteal LNs, and the number of VEGF-C-expressing CD11b+ myeloid cells in popliteal LNs. However, it increased the synovial volume and area of inflammation in ankle and knee joints. VEGFR-2 neutralizing antibody, in contrast, inhibited both lymphangiogenesis and joint inflammation. CONCLUSION These findings indicate that lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis. Lymphatic drainage plays a beneficial role in controlling the progression of chronic inflammation.

Vascular endothelial growth factor C attenuates joint damage in chronic inflammatory arthritis by accelerating local lymphatic drainage in mice

OBJECTIVE To investigate whether the enhancement of joint lymphangiogenesis by injection of vascular endothelial growth factor C (VEGF-C) adeno-associated virus (AAV) into the affected joints has therapeutic efficacy in chronic inflammatory arthritis in mice. METHODS Tumor necrosis factor-transgenic (TNF-Tg) mice were used as a model of chronic inflammatory arthritis. Human VEGF-C was cloned into an AAV expression vector to generate AAV-VEGF-C. The joints of TNF-Tg mice were injected with AAV-VEGF-C or AAV-luciferase (AAV-Luc) as a control. During the 4 months following injection, magnetic resonance imaging of the joints and lymphatic imaging were performed to assess changes in synovial volume and lymph flow from the joint tissues to local draining lymph nodes. Joint inflammation, bone erosion, and cartilage loss were examined by histologic analyses. Lymphatic vessel formation was assessed using immunohistochemistry. RESULTS Intraarticular administration of AAV-VEGF-C virus significantly attenuated the increase in synovial volume and increased lymphatic vessel number in the joint sections, as compared with that in control AAV-Luc-injected joints, during the 4-month period. This was accompanied by a reduction in the area of inflammation, bone erosion, cartilage loss, and osteoclast numbers. Lymph flow from the joints to local draining lymph nodes was slower in TNF-Tg mice than in wild-type littermates, and was significantly improved with AAV-VEGF-C treatment. CONCLUSION Intraarticular injection of AAV-VEGF-C increased lymphangiogenesis and improved lymphatic drainage from the inflamed joints of mice, resulting in attenuation of joint tissue damage. Thus, improvement of joint lymphatic function by local administration of lymphatic growth factors represents a new therapeutic approach for chronic inflammatory arthritis.

Methylecgonidine coats the crack particle

Crack is a form of cocaine base self-administered by smoking. When heated, it volatilizes and may partially pyrolyze to methylecgonidine (MEG). Upon cooling, a condensation aerosol forms. Heating cocaine base in model crack pipes produced particles of about 1 micron in diameter, regardless of the amount heated; however, MEG concentration increased from < or = 2% at 10 mg per heating to as much as 5% at 30 mg per heating. Methylecgonidine was < or = 1% of the recovered material when cocaine was vaporized off a heated wire coil, but the particles were larger (2-5 microns), and the distribution disperse. The vapor pressure of MEG was higher [log P(mm Hg) = 9.994 - 3530/T] than cocaine base, consistent with MEG coating the droplet during condensation, and with evaporation during aging or dilution. Disappearance of MEG from a chamber filled with crack smoke was a two-component process, one proceeding at the rate of cocaine particle removal, and the other at the desorption rate from other surfaces. Particle diameter influences the deposition site in the respiratory tract; thus, the likely different patterns of deposition in the respiratory tract of humans and animals of crack aerosols produced by different techniques warrant consideration, as they may influence our understanding of immediate and delayed sequelae of the inhalation of cocaine and its pyrolysis product, MEG.

Effects of intradermal foot and forearm capsaicin injections in normal and vulvodynia-afflicted women

&NA; Cutaneous response to capsaicin has been used to assess central sensitization in pain research. This study compared the response to intradermal capsaicin in the forearm and foot of vulvar vestibulitis (vestibulodynia)‐afflicted cases and controls. We hypothesized that cases will experience greater spontaneous pain, larger cutaneous areas of punctate hyperalgesia and dynamic allodynia, and greater vascular flow than controls. We also hypothesized enhanced post‐injection pain in the foot compared to the forearm based on dermatome proximity of the foot and vulva. Methods. Ten vulvar vestibulitis syndrome (VVS) cases and 10 age and ethnically matched controls underwent two randomized, cross‐over trials with intra‐dermal injections of capsaicin or a saline placebo in the forearm and foot. Outcome measures included spontaneous pain level, surface area of punctate hyperalgesia, surface area of dynamic allodynia, cutaneous blood flow, regional skin temperature and vital signs. Results. VVS cases experienced greater spontaneous pain, punctate hyperalgesia and dynamic allodynia than pain‐free controls. Within the VVS group, post‐capsaicin spontaneous pain, punctate hyperalgesia and dynamic allodynia were similar in the forearm and foot. Post‐capsaicin blood flow did not differ between cases and controls by anatomic site. Measures of depression and anxiety correlated with spontaneous pain intensity but did not correlate with measures of hyperalgesia, allodynia, or blood flow. VVS cases had higher resting pulse rates and lower resting systolic blood pressures than in controls. Conclusion. VVS patients show enhancement of post‐capsaicin pain response extending far beyond the anatomic location of the primary complaint.

Hand Tremor Induced by Industrial Exposure to Inorganic Mercury

Hand tremor induced by industrial exposure to inorganic mercury vapor was studied during recovery in two women patients, Subjects were instructed to maintain a force with the forefinger between two limits (10 to 40 gm). Performance progressively improved after the cessation of exposure, and were asymptomatic in about 3.5 months. The dispersion of the amplitude distribution decreased, and percent time within the specified limits increased. Power spectral analyses revealed a marked reduction in power associated with a change in the shape of the spectra. These changes were associated with a reduction in plasma levels of mercury.

Near-infrared lymphatic imaging demonstrates the dynamics of lymph flow and lymphangiogenesis during the acute versus chronic phases of arthritis in mice.

OBJECTIVE To develop an in vivo imaging method to assess lymphatic draining function in the K/BxN mouse model of inflammatory arthritis. METHODS Indocyanine green, a near-infrared fluorescent dye, was injected intradermally into the footpads of wild-type mice, mouse limbs were illuminated with an 806-nm near-infrared laser, and the movement of indocyanine green from the injection site to the draining popliteal lymph node (LN) was recorded with a CCD camera. Indocyanine green near-infrared images were analyzed to obtain 5 measures of lymphatic function across time. Images of K/BxN arthritic mice and control nonarthritic littermates were obtained at 1 month of age, when acute joint inflammation commenced, and again at 3 months of age, when joint inflammation became chronic. Lymphangiogenesis in popliteal LNs was assessed by immunochemistry. RESULTS Indocyanine green and its transport within lymphatic vessels were readily visualized, and quantitative measures were derived. During the acute phase of arthritis, the lymphatic vessels were dilated, with increased indocyanine green signal intensity and lymphatic pulses, and popliteal LNs became fluorescent quickly. During the chronic phase, new lymphatic vessels were present near the foot. However, the appearance of indocyanine green in lymphatic vessels was delayed. The size and area of popliteal LN lymphatic sinuses progressively increased in the K/BxN mice. CONCLUSION Our findings indicate that indocyanine green near-infrared lymphatic imaging is a valuable method for assessing the lymphatic draining function in mice with inflammatory arthritis. Indocyanine green-near-infrared imaging of K/BxN mice identified 2 distinct lymphatic phenotypes during the acute and chronic phase of inflammation. This technique can be used to assess new therapies for lymphatic disorders.

Near Infrared Fluorescence Imaging After Intravenous Indocyanine Green: Initial Clinical Experience With Open Partial Nephrectomy for Renal Cortical Tumors

OBJECTIVE To evaluate the safety of near infrared fluorescence (NIRF) of intravenously injected indocyanine green (ICG) during open partial nephrectomy, and to demonstrate the feasibility of this technology to identify the renal vasculature and distinguish renal cortical tumors from normal parenchyma. METHODS Patients undergoing open partial nephrectomy provided written informed consent for inclusion in this institutional review board-approved study. Perirenal fat was removed to allow visualization of the renal parenchyma and lesions to be excised. The patients received intravenous injections of ICG, and NIRF imaging was performed using the SPY system. Intraoperative NIRF video images were evaluated for differentiation of tumor from normal parenchyma and for renal vasculature identification. RESULTS A total of 15 patients underwent 16 open partial nephrectomies. The mean cold ischemia time was 26.6 minutes (range 20-33). All 14 malignant lesions were afluorescent or hypofluorescent compared with the surrounding normal renal parenchyma. NIRF imaging of intravenously injected ICG clearly identified the renal hilar vessels and guided selective arterial clamping in 3 patients. No adverse reactions to ICG were noted, and all surgical margins were negative on final pathologic examination. CONCLUSION The intravenous use of ICG combined with NIRF is safe during open renal surgery. This technology allows the surgeon to distinguish renal cortical tumors from normal tissue and highlights the renal vasculature, with the potential to maximize oncologic control and nephron sparing during open partial nephrectomy. Additional study is needed to determine whether this imaging technique will help improve the outcomes during open partial nephrectomy.

Generation of stable test atmospheres of cocaine base and its pyrolyzate, methylecgonidine, and demonstration of their biological activity

Generating controlled test atmospheres of known chemical identity and airborne concentration upon demand is a significant technical obstacle that limits the scope and repeatability of studies of inhaled substances. We addressed this problem as applied to the generation of atmospheres that result from heating crack cocaine, which include both cocaine and its pyrolyzate methylecgonidine (MEG). A condensation aerosol generator was used to generate atmospheres comprised of monodisperse particles of cocaine, MEG, or mixtures of both that are of submicron size suitable for deposition in the alveolar region of primates. Compressed air seeded with nanometer-size sodium chloride particles was passed through a constant depth of molten cocaine or MEG in a bead bed, reheated, and condensed to an aerosol within an annulus of cold air. To achieve control of a mixture of both compounds, MEG was condensed onto cocaine particles in a separate coating step. On-line analytical instruments provided verification of airborne concentration, estimates of particle size, and dispersion as well as chemical identity. Specific airway conductance (SGaw), heart rate, and rectal and skin temperatures were measured in squirrel monkeys breathing atmospheres containing condensation aerosols of cocaine or MEG free base. SGaw was reduced after inhalation of either base, and both induced temperature and cardiovascular changes, demonstrating that the aerosols so generated had biological activity.

Biphasic Changes in Mouse Motor Activity during Exposure to Toluene

There are few careful studies of the effects of solvents on unlearned animal behavior during acute exposure, despite the importance of the prevention of acute behavioral or neurological effects in the workplace. To examine the effects of toluene on the locomotor activity of mice, we divided a plastic vacuum desiccator into six wedge-shaped compartments with diffusing plena above and below. A phototransistor in each wedge measured the movement of individual mice, thus each mouse could serve as its own control. Six groups of six mice were exposed to each of five concentrations of toluene (300-3000 ppm) or air in a Latin-square design for 1 hr on Tuesdays and Fridays. Individual animals differed in their sensitivity to toluene, and the use of each subject as its own control permitted the detection of effects at lower concentrations. The magnitude of the effect was related to concentration, the duration of exposure, and the control rate of activity. Activity increases were obvious at 560 ppm, and decreases at 3000 ppm. The concentrations at which these reversible activity increases occurred are the lowest reported to date and are only slightly greater than those that have been reported to alter human reaction time. This preparation displays sensitivity comparable to that observed in published studies of the effects of toluene on learned behavior in the rat.

Empirical Modeling of Particle Deposition in the Alveolar Region of the Lungs: A Basis for Interspecies Extrapolation

Different species exposed to the same particle atmosphere may not receive identical initial doses in comparable respiratory tract regions, and the selection of a certain species for toxicologic evaluation of inhaled particles may, thus, influence the estimated human lung, or systemic, dose, as well as its relationship to potential adverse health effects. Estimating regional particle deposition patterns is important for establishing the comparability of animal models, for understanding interspecies differences in the expression of chemical toxicities, and, ultimately, for the human risk assessment process. A method is described which offers a strategy for summarizing published data on regional deposition of particles of different diameters and calculating a deposited fraction for a particular particle size distribution. This involved the construction of nomograms to allow estimation of alveolar deposition fractions in three species, namely the human, monkey, and rat. A regression model was then developed to permit the calculation of more exact deposition fractions. Although this paper describes the procedure for one region of the lungs, the same technique can be applied to other regions of the respiratory tract or to the total system for which deposition data are available. While this technique may facilitate the interpretation of available experimental results and their application to human health risk assessment, appropriate caution should be exercised in applying the developed nomograms given limitations of the deposition database upon which it is based.