Ann L. Baldwin

Inhibition of nitric oxide synthesis increases venular permeability and alters endothelial actin cytoskeleton

Inhibition of nitric oxide (NO) synthesis using NG-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA) increases venular permeability in the rat mesentery (I. Kurose, R. Wolf, M. B. Grisham, T. Y. Aw, R. D. Specian, and D. N. Granger. Circ. Res. 76: 30-39, 1995), but the cellular mechanisms of this response are not known. This study was performed to determine whether such venular leaks are associated with changes in the endothelial actin cytoskeleton. In anesthetized Sprague-Dawley rats, the microvasculature of a mesenteric window was perfused with buffered saline, with or without 10(-5) M L-NAME, L-NMMA, or the inactive enantiomer NG-nitro-D-arginine methyl ester for 3 or 30 min. FITC-albumin was added to the perfusate for the last 3 min. The vasculature was perfusion fixed, stained for filamentous actin and for mast cells, and viewed microscopically. In control preparations, venules showed few FITC-albumin leaks and the endothelial actin cytoskeleton consisted of a peripheral rim along the cell-cell junctions. Preparations treated with L-NAME or L-NMMA showed significantly more leakage, the actin rims in leaky venules were discontinuous, and short, randomly oriented fibers appeared within the cells. In nonleaky venules, the peripheral actin rims sometimes contained small, equally spaced discontinuities not seen in control preparations. Although a mast cell stabilizer was used, 27-70% of the mast cells were degranulated in the presence of L-NMMA. Thus inhibition of NO synthesis alters the endothelial cytoskeleton and increases albumin leakage from mesenteric venules, either directly or indirectly via the involvement of mast cells.Inhibition of nitric oxide (NO) synthesis using N G-nitro-l-arginine methyl ester (l-NAME) or N G-monomethyl-l-arginine (l-NMMA) increases venular permeability in the rat mesentery (I. Kurose, R. Wolf, M. B. Grisham, T. Y. Aw, R. D. Specian, and D. N. Granger. Circ. Res. 76: 30-39, 1995), but the cellular mechanisms of this response are not known. This study was performed to determine whether such venular leaks are associated with changes in the endothelial actin cytoskeleton. In anesthetized Sprague-Dawley rats, the microvasculature of a mesenteric window was perfused with buffered saline, with or without 10-5M l-NAME,l-NMMA, or the inactive enantiomer N G-nitro-d-arginine methyl ester for 3 or 30 min. FITC-albumin was added to the perfusate for the last 3 min. The vasculature was perfusion fixed, stained for filamentous actin and for mast cells, and viewed microscopically. In control preparations, venules showed few FITC-albumin leaks and the endothelial actin cytoskeleton consisted of a peripheral rim along the cell-cell junctions. Preparations treated withl-NAME orl-NMMA showed significantly more leakage, the actin rims in leaky venules were discontinuous, and short, randomly oriented fibers appeared within the cells. In nonleaky venules, the peripheral actin rims sometimes contained small, equally spaced discontinuities not seen in control preparations. Although a mast cell stabilizer was used, 27-70% of the mast cells were degranulated in the presence ofl-NMMA. Thus inhibition of NO synthesis alters the endothelial cytoskeleton and increases albumin leakage from mesenteric venules, either directly or indirectly via the involvement of mast cells.

Cadherin-5 redistribution at sites of TNF-α and IFN-γ-induced permeability in mesenteric venules

The response of the endothelial permeability barrier in microvascular networks of the rat mesentery to perfused immune inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was examined. TNF-α (12.5 U/ml) treatment did not change albumin permeability, but in combination with IFN-γ (20 U/ml), there was a marked increase in the number of sites of extravascular albumin in postcapillary venules. Endothelial integrity was characterized by cadherin-5 immunoreactivity, which was localized to the continuous intercellular junctions of endothelium in arterioles, capillaries, and venules. Perfusion with the combined cytokines showed that the increased albumin permeability was dose dependent and correlated with the focal disorganization of cadherin-5 at intercellular junctions of venular endothelium. No correlation was found between the increase in albumin permeability and the localization of intravascular leukocytes or extravascular mast cells. These results show that the combination of TNF-α and IFN-γ induces an endothelial phenotype with focal loss of cadherin-5 intercellular adhesion, which, in part, facilitates passage of blood macromolecules and cells to the interstitium.

Environmental Stress Causes Mast Cell Degranulation, Endothelial and Epithelial Changes, and Edema in the Rat Intestinal Mucosa

Objective: Mental stress has been shown to produce intestinal disease, but the effects of a mild environmental stress on intestinal physiology have not been elucidated. This study was performed to determine the effects of environmental stress on the ultrastructure of the intestinal mucosa, using the rat as an experimental model.

Effect of pressure on aortic hydraulic conductance.

This study was performed to determine whether the transmural hydraulic conductance (Lp) of the rabbit aortic wall depends on its distension. In 19 rabbits, the aorta was cannulated in situ and perfused at a given pressure with a physiologically buffered solution containing 4% bovine serum albumin. The output cannula was then occluded to limit fluid flow to that traversing the artery wall. External diameter and transmural fluid flow were measured at three pressures (eight rabbits, group 1) or at four pressures (12 rabbits, group 2) in each vessel. Transmural fluid flow was determined by monitoring the velocity of an air bubble within a buffer-filled tube leading to the input cannula. From group 1 measurements, Lp values (mean +/- SD) at 50, 100, and 150 mm Hg were calculated to be 3.8 +/- 2.8, 3.5 +/- 1.3, and 4.1 +/- 1.2 x 10(-8) cm/sec/mm Hg, respectively. Group 2 measurements gave values of 4.2 +/- 1.6, 3.8 +/- 1.1, 3.8 +/- 1.1, and 4.2 +/- 1.1 x 10(-8) cm/sec/mm Hg at 75, 100, 125, and 150 mm Hg, respectively. Paired Students t tests indicated no significant change in Lp with pressure. However, linear regression analysis demonstrated a weak correlation between Lp values obtained at 50 and 100 mm Hg (r2 = 0.30) and at 75 and 100 mm Hg (r2 = 0.36). Values of Lp at 100 and 150 mm Hg and at 125 and 150 mm Hg were closely correlated in each case. These results suggest that between 50 and 100 mm Hg the structural properties of the aortic wall change so as to alter Lp but not in the same way in each vessel. Lp may increase or decrease depending on which structural change predominates in a particular vessel.

Effect of Atherosclerosis on Transmural Convection and Arterial Ultrastructure Implications for Local Intravascular Drug Delivery

Local infusion of agents through perforated catheters may reduce neointimal formation following vascular angioplasty. Such treatment will succeed only if the drug is retained within the arterial intima long enough to promote repair. Drugs will be dispersed throughout the wall predominantly by transmural convection instead of diffusion if the Peclet number, Pe = J (1-delta f)/P, is greater than unity, where J is the transmural fluid flow per unit surface area and delta(f) and P are the reflection and permeability coefficients to the drug, respectively. Although the targets of local drug delivery will be atherosclerotic vessels, little is known about the transport properties of these vessels. Accordingly, we evaluated the effects of hypercholesterolemia and atherosclerosis on J per unit pressure (hydraulic conductance, Lp) and on ultrastructure in femoral arteries. Measurements were made at 30, 60, and 90 mm Hg in anesthetized New Zealand white rabbits fed a normal diet (n = 6) and after 3 weeks of lipid feeding (n = 19). Atherosclerosis was induced in six lipid-fed animals by air desiccation of a femoral artery. Hydraulic conductance was significantly greater in vessels from hypercholesterolemic than from normal animals and decreased with pressure only in hypercholesterolemic arteries. Atherosclerosis did not augment hydraulic conductance compared with hypercholesterolemia alone. Electron microscopic examination demonstrated damaged endothelium in hypercholesterolemic arteries and both altered endothelium and less tightly packed medial tissue, compared with controls, in atherosclerotic vessels, at least at lower pressures. Peclet numbers for macromolecules exceeded unity for all three groups of arteries and reached 0.3 to 0.4 for molecules as small as heparin. Thus, convection plays a dominant role in the distribution of macromolecular agents following local delivery and may result in their rapid transport to the adventitia in the femoral artery.

Biofield Research: A Roundtable Discussion of Scientific and Methodological Issues

Richard Hammerschlag: This discussion will be among the participants of a symposium on clinical and basic science research on the biofield, held as part of the International Research Congress on Integrative Medicine and Health in Portland, Oregon in May 2012. The 7 of us are conventional biomedical researchers in physiology, clinical psychology, cell biology, biophysics, and neurobiology, who through various paths have expanded our research interests to include performing preclinical studies, clinical trials, and systematic reviews of biofield therapies, as well as basic research and reviews on what we call biofield physiology. We should begin with a few definitions. Biofield therapies, which most commonly include external Qigong, Healing Touch, Johrei, Reconnective Healing, Reiki, and Therapeutic Touch, are a family of health care practices that involve either, or both, hands-on and hands-off treatment. We infer that such healing can occur since living systems coexist within and co-contribute to a biofield, which we define in terms of electric, magnetic, and electromagnetic fields as well as subtle energies (energies that appear to exist but have not yet been measured). I’d like to ask Jim to provide us with a brief overview of how the concept of the biofield has evolved.

Endothelial surface charge of intestinal mucosal capillaries and its modulation by dextran

Capillary endothelial surface charge was investigated by perfusing the intestinal circulation of anesthetized rats in situ with 0.1 or 10 mg/ml native ferritin (NF, pI = 3.8-4.2) or with 0.1 mg/ml cationized ferritin (CF, pI greater than 10.0) for 5 min, with or without 5% Dextran 40. Ferritin binding was quantified by electron microscopy. All electron micrographs of capillaries perfused with NF showed some NF binding. Mean NF particle densities (particles/microns) were significantly greater at vesicle necks (PDv) than elsewhere on the endothelial surface (PD). Capillaries perfused with CF showed binding in only 60% of the transverse sections examined. The binding was very marked in a large proportion of these vessels. Mean CF particle densities were significantly greater at fenestrae (PDf) than elsewhere. These results demonstrate that mucosal capillaries have a variable negative electrostatic charge on the endothelial surface and support the hypothesis that some vesicle diaphragms act as preferential attractors for anionic macromolecules. Such structures could promote transendothelial vesicular transport of albumin. Dextran significantly decreased PD for NF from 25.4 +/- 2.4 (SEM) to 13.1 +/- 0.9 and PD, PDv, and PDf for CF from 45.9 +/- 4.6 to 31.0 +/- 2.7, from 62.1 +/- 6.2 to 43.6 +/- 5.4, and from 152.9 +/- 15.5 to 114.5 +/- 8.6, respectively. The above responses result from dextrans weak interaction with the endothelial surface. Dextran reduces access of ferritin molecules to the cell surface by steric hindrance and/or electrostatic shielding of the glycocalyx.

Reiki Improves Heart Rate Homeostasis in Laboratory Rats

OBJECTIVES To determine whether application of Reiki to noise-stressed rats can reduce their heart rates (HRs) and blood pressures. RATIONALE In a previous study, we showed that exposure of rats to 90 dB white noise for 15 minutes caused their HRs and blood pressures to significantly increase. Reiki has been shown to significantly decrease HR and blood pressure in a small group of healthy human subjects. However, use of humans in such studies has the disadvantage that experimental interpretations are encumbered by the variable of belief or skepticism regarding Reiki. For that reason, noise-stressed rats were used as an animal model to test the efficacy of Reiki in reducing elevated HR and blood pressure. DESIGN Three unrestrained, male Sprague-Dawley rats implanted with radiotelemetric transducers were exposed daily for 8 days to a 15-minute white noise regimen (90 dB). For the last 5 days, the rats received 15 minutes of Reiki immediately before the noise and during the noise period. The experiment was repeated on the same animals but using sham Reiki. SETTING/LOCATION The animals were housed in a quiet room in University of Arizona Animal Facility. OUTCOME MEASURES Mean HRs and blood pressure were determined before Reiki/sham Reiki, during Reiki/sham Reiki, and during the noise in each case. RESULTS Reiki, but not sham Reiki, significantly reduced HR compared to initial values. With Reiki, there was a high correlation between change in HR and initial HR, suggesting a homeostatic effect. Reiki, but not sham Reiki, significantly reduced the rise in HR produced by exposure of the rats to loud noise. Neither Reiki nor sham Reiki significantly affected blood pressure. CONCLUSION Reiki is effective in modulating HR in stressed and unstressed rats, supporting its use as a stress-reducer in humans.

Simultaneous measurement of capillary distensibility and hydraulic conductance

Capillaries are often assumed to be indistensible. Only recently has the error caused by capillary distensibility in the measurement of hydraulic conductance been considered. An oil-drop method was used to measure simultaneously distensibility and hydraulic conductance (Lp) of mesenteric capillaries of 40 cranially pithed Rana pipiens. All vessels studied were distensible in the range 5-80 mm Hg, with a mean equilibrium time, teq, of 12.4 +/- 0.5 sec (SEM, n = 7). Capillary distensibility decreased with increasing pressure. Mean capillary distensibilities at 10, 30, and 60 mm Hg were 18.9 +/- 4.9 X 10(-4) (mm Hg)-1, 13.5 +/- 1.2 X 10(-4) (mm Hg)-1 (mean +/- SEM, n = 9), and 7.6 +/- 2.7 X 10(-4) (mm Hg)-1 (mean +/- SEM, n = 7), respectively. Mean values of capillary Lp, uncorrected and corrected for distensibility, were 0.0113 +/- 0.0017 microns(sec.mm Hg)-1 and 0.0083 +/- 0.0016 microns(sec.mm Hg)-1 (SEM, n = 8 capillaries), respectively. A paired t test demonstrated that uncorrected and corrected values of Lp were statistically different from one another (P less than 0.005). Both distensibility and Lp varied widely between capillaries indicating the necessity of measuring and correcting for distensibility in each individual capillary when estimating Lp. The finding of a slow component of vessel distensibility demonstrates that previous filtration data taken not only from single vessels, but also from whole organs, should be reinterpreted.

The Touchstone Process: an ongoing critical evaluation of reiki in the scientific literature.

Background:Reiki is used by a growing number of people but little is known about the scientific basis for its use. Purpose:The Touchstone Process was developed as an ongoing process to systematically analyze published, peer-reviewed studies of Reiki, the results being made accessible to the public online. Method:Thirteen scientifically qualified experts in the field of Reiki were assembled into 3 teams to retrieve, evaluate, and summarize articles using standardized, piloted evaluation forms. Results:Summaries of 26 Reiki articles, including strengths and weaknesses, were posted on a newly developed Web site (www.centerforreikiresearch.org), together with an overall summary of the status of Reiki research and guidelines for future research: The Touchstone Process determined that only 12 articles were based on a robust experimental design and utilized well-established outcome parameters. Of these articles, 2 provided no support, 5 provided some support, and 5 demonstrated strong evidence for the use of Reiki as a healing modality. Conclusion:There is a need for further high-quality studies in this area.