Three-dimensional distributions of elements in biological samples by energy-filtered electron tomography.

By combining electron tomography with energy-filtered electron microscopy, we have shown the feasibility of determining the three-dimensional distributions of phosphorus in biological specimens. Thin sections of the nematode, Caenorhabditis elegans were prepared by high-pressure freezing, freeze-substitution and plastic embedding. Images were recorded at energy losses above and below the phosphorus L2,3 edge using a post-column imaging filter operating at a beam energy of 120 keV. The unstained specimens exhibited minimal contrast in bright-field images. After it was determined that the specimen was sufficiently thin to allow two-window ratio imaging of phosphorus, pairs of pre-edge and post-edge images were acquired in series over a tilt range of +/-55 degrees at 5 degrees increments for two orthogonal tilt axes. The projected phosphorus distributions were aligned using the pre-edge images that contained inelastic contrast from colloidal gold particles deposited on the specimen surface. A reconstruction and surface rendering of the phosphorus distribution clearly revealed features 15-20 nm in diameter, which were identified as ribosomes distributed along the stacked membranes of endoplasmic reticulum and in the cytoplasm. The sensitivity of the technique was estimated at < 35 phosphorus atoms per voxel based on the known total ribosomal phosphorus content of approximately 7000 atoms. Although a high electron dose of approximately 10(7)e/nm2 was required to record two-axis tilt series, specimens were sufficiently stable to allow image alignment and tomographic reconstruction.

Oral nalmefene therapy reduces scratching activity due to the pruritus of cholestasis: a controlled study.

BACKGROUND: Intravenous naloxone frequently ameliorates the pruritus of cholestasis, but its low oral bioavailability precludes its use as a long-term therapy. Nalmefene is an orally bioavailable opiate antagonist. OBJECTIVE: We assessed the efficacy of oral nalmefene in ameliorating the pruritus of cholestasis. METHODS: In a prospective controlled study conducted in a tertiary referral hospital, 11 patients with generalized pruritus complicating chronic liver disease were randomized to receive either nalmefene or placebo in a double-blinded fashion for 2-month periods. Scratching activity was measured continuously for 24-hour periods at baseline and at the end of each treatment period. RESULTS: Data on 8 patients who received at least 1 course of nalmefene were available for comparison with corresponding control data, which consisted of observations obtained during a course of placebo and/or at baseline. Nalmefene therapy was associated with a 75% reduction in the geometric mean hourly scratching activity (P <.01) and a decrease in the mean of a visual analogue score of the perception of pruritus in all 8 patients (mean decrease 77%, P <.01). CONCLUSION: Oral administration of nalmefene can ameliorate pruritus complicating chronic liver disease.

Open-label trial of oral nalmefene therapy for the pruritus of cholestasis.

The aims of this study were to determine whether long-term oral administration of the opiate antagonist nalmefene is associated with any beneficial effects in patients with pruritus secondary to cholestatic liver disease and to assess the safety of long-term administration of this drug to these patients. Fourteen patients with unrelieved chronic pruritus of cholestasis were studied. Scratching activity, independent of limb movements, was recorded continuously for 24-hour periods before and during treatment with an initial ameliorating dose of nalmefene. Simultaneously, during these periods, visual analogue scores (VASs) of pruritus were recorded every 4 hours while patients were awake. The dose of nalmefene, which initially was 2 mg orally twice daily, was increased during the study, usually until a satisfactory clinical response was achieved. Five patients experienced a transient opioid withdrawal-like reaction that did not preclude continuing with nalmefene therapy. Serum biochemical indices of cholestasis did not change appreciably during treatment. Thirteen patients reported amelioration of the perception of pruritus on nalmefene. In 5 patients, exacerbations of pruritus occurred approximately 4 weeks after an initial ameliorating dose had been reached; these exacerbations were managed by increasing the dose. Baseline mean values for VAS and scratching activity were higher than corresponding means during nalmefene therapy in 13 (P = .002) and 12 (P = .013) patients, respectively. Possible tolerance to nalmefene occurred in 3 patients. Three patients experienced marked exacerbation of pruritus after nalmefene therapy was suddenly discontinued. Blood levels of nalmefene were consistent with normal pharmacokinetics of the drug. These results suggest that nalmefene may have a favorable risk-to-benefit ratio when it is administered orally long-term to patients with the pruritus of cholestasis.

Effects of naloxone infusions in patients with the pruritus of cholestasis. A double-blind, randomized, controlled trial.

OBJECTIVE: To determine whether endogenous opioids contribute to the pruritus of cholestasis by studying the effect of the opiate antagonist naloxone on the perception of pruritus and on scratching activity in patients with this form of pruritus. DESIGN: Double-blind, placebo-controlled, crossover trial with four periods. SETTING: Clinical research referral center. PATIENTS: 29 pruritic patients with liver diseases of various causes. INTERVENTION: Each patient received as many as two naloxone and two placebo solution infusions consecutively in random order. Each infusion lasted 24 hours. MEASUREMENTS: During the infusions, visual analog scores of pruritus were recorded every 4 hours while patients were awake; scratching activity independent of limb movements was recorded continuously. RESULTS: One patient had a mild reaction consistent with a naloxone-precipitated syndrome similar to opiate withdrawal. A significant 24-hour rhythm of scratching activity was seen in 7 of 11 patients for whom complete 96-hour data were collected. The mean of a visual analog score of the perception of pruritus (maximum, 10.0) recorded during naloxone infusions was 0.582 lower than that recorded during placebo infusions (95% CI, 0.176 to 0.988; P < 0.01). Furthermore, the ratio of the geometric mean hourly scratching activity during naloxone infusions to that during placebo infusions was 0.727 (CI, 0.612 to 0.842; P < 0.001) and was greater than 1.0 in only five patients. CONCLUSIONS: Naloxone administration is associated with amelioration of the perception of pruritus and reduction of scratching activity in cholestatic patients. Because of the opioid receptor specificity of the action of naloxone, these findings support the hypothesis that a mechanism underlying the pruritus of cholestasis is modulated by endogenous opioids and suggest that opiate antagonists may have a role in the management of this complication of cholestasis.

Collaborative development of a patient simulator for educating nurses in hemofiltration therapies.

As the complexity and variety of the treatment modalities in critical care continue to expand, the clinician is faced with the task of providing a safe environment for the education of staff and the evaluation of these new modalities. The use of simulators, similar to the one described in this article, is one economical solution that can provide this education and evaluation. For instance, the major components of the hemofiltration simulator can be fabricated and purchased for under $1,000. This estimate excludes the medical cart, which retails for $437. The success of such a solution, however, necessitates that the simulator meet the defined clinical needs while meeting or exceeding the biomedical equipment standards set by AAMI or other regulatory agencies that might govern the use of such devices.

A controlled trial of naloxone infusions for the pruritus of chronic cholestasis.

To test the hypothesis that opioid agonist activity contributes to the pruritus of cholestasis, a placebo-controlled single-blinded trial of naloxone, an opioid antagonist, was conducted in eight patients with primary biliary cirrhosis. After discontinuation of all conventional antipruritic medications, one or two continuous (24-hour) IV infusions of naloxone (0.2 micrograms.kg-1.min-1) and placebo solution were administered consecutively in an order that was not predetermined. Pruritus was assessed subjectively by means of four hourly recordings of a visual analogue score. In addition, objective measurements of scratching activity that were independent of gross body movements were continuously recorded using an apparatus specifically designed to measure the frequencies associated with this activity. No side effects associated with naloxone infusions were observed. Only scratching activity data obtained for the same periods of day and night during both naloxone and placebo infusions were compared. Naloxone infusions were consistently associated with a decrease in values of the scratching activity index. In addition, in 50% of the patients the infusions were associated with a decrease in visual analogue score. The mean decrease in scratching activity ranged from 29% to 96% (mean, 50%; P less than 0.001). These findings imply that increased opioid agonist activity contributes to scratching activity in cholestatic patients.

Application of piezo film technology for the quantitative assessment of pruritus.

A method to quantitate scratching has been developed for assessing the efficacies of interventions purported to ameliorate pruritus. The technique employs a piezo film sensor attached to the finger and the supporting electronics to amplify, transmit, and process the signals generated by the piezo film. The piezo film is essentially a contact microphone on the fingernail of the scratching subject. The method is currently incorporated into clinical trials, and preliminary results are promising.

Errors in pressure gradient measurement by continuous wave Doppler ultrasound: type, size and age effects in bioprosthetic aortic valves.

The accuracy of continuous wave Doppler ultrasound in deriving pressure gradients across bioprosthetic heart valves was evaluated in an in vitro pulse duplicator. Simultaneous pressure transducer and Doppler measurements were made in new and explanted aortic bioprosthetic valves of several sizes and four types: Carpentier-Edwards, Ionescu-Shiley, Hancock standard and Hancock modified. The mean and peak gradients calculated by the modified Bernoulli equation from Doppler velocity measurements were always greater than those measured manometrically, despite corrections for location dependence of the manometric gradient (or pressure recovery). The relation between manometric and ultrasonically determined gradient was found to be statistically dependent on the valve type (mean gradient p less than 0.0001; peak gradient p = 0.0003) and size (mean gradient p = 0.0089; peak gradient p = 0.0107). Effects of implantation were observed, but were not shown to be significant. It is concluded that the continuous wave Doppler velocity data overestimated prosthetic valve pressure gradient in all cases, even when pressure recovery was taken into account. Clinicians should be wary of Doppler data when making major diagnostic or therapeutic decisions.

The local effects of cachectin/tumor necrosis factor on wound healing.

Previous experimental studies have suggested that tumor necrosis factor (TNF) may have either a beneficial or a detrimental role in wound healing. Control and doxorubicin-treated (6 mg/kg, intravenously) rats underwent paired dorsal 5-cm linear wounds and had either vehicle or recombinant (r)TNF (0.5, 5, or 50 micrograms) applied locally to the wound. Paired wounds were harvested at 7 and 14 days after wounding and analyzed for wound-bursting strength (WBS) and activity of the gene for type 1 collagen and TNF. Doxorubicin treatment decreased WBS at 14 days but not at 7 days after wounding. Local application of 50 micrograms of rTNF decreased WBS in saline-treated rats and concentrations of 5 and 50 micrograms decreased WBS in doxorubicin-treated rats when measured 7 days after wounding. These effects dissipated when WBS was measured 14 days after wounding. Doxorubicin decreased wound collagen gene expression and local TNF treatment decreased wound collagen gene expression in saline-treated rats and further decreased it in doxorubicin-treated rats. The decrement in collagen gene expression induced by rTNF increased as the local dose of rTNF increased. The gene for TNF was not detectable in wounds from normal or doxorubicin-treated rats at 3, 7, 10, or 14 days after wounding. These data suggest that the gene for TNF is not expressed in wounds and that the local application of TNF is detrimental to wound healing as it decreases WBS and activity of the gene for collagen.

A computerized physiologic pulse duplicator for in-vitro hydrodynamic and ultrasonic studies of prosthetic heart valves.

A physiologic pulse duplicator for the simultaneous in-vitro hydrodynamic and ultrasonic evaluation of aortic prosthetic heart valves is described. The system is interfaced to a personal computer, which provides greater efficiency over manual techniques in system calibration, data acquisition, and analysis. The data analysis program aids selection of start and end systole and calculates pressure difference across the valve, the closing and regurgitant volumes, flow rates, and the Gabbay and Swanson performance indices. The pulse duplicator is designed to accommodate the ultrasonic measurement of fluid velocities, including pressure difference via the Bernoulli equation, and color-flow imaging. In tests of 19-mm, 23-mm, and 27-mm Bjork-Shiley spherical occluder valves, continuous-wave Doppler ultrasound was found to overestimate by about 50% the pressure difference measured directly by pressure transducers, a finding that is clinically important.

Cross-sectional tongue shape during the production of vowels.

This study used ultrasound imaging to examine the cross-sectional shape of the tongue during the production of the ten English vowels ( see text ) in two consonant contexts--/p/ and /s/--and at two scan angles--anterior and posterior. Results were compared with models of sagittal tongue shape. A newly built transducer holder and head restraint maintained the ultrasound transducer in a fixed position inferior to the mandible at a chosen location and angle. The transducer was free to move only in a superior/inferior direction, and demonstrated reliable tracking of the jaw. Since the tongue is anisotrophic along its length, anterior and posterior scan angles were examined to identify differences in tongue shape. Similarly, the coarticulatory effects of the sibilant /s/ versus the bilabial /p/ were examined, to assess variability of intrinsic tongue shape for the vowels. Results showed that the subject's midsagittal tongue grooving was almost universal for the vowels. Posterior grooves were deeper than anterior grooves. In /s/ context, posterior tongue grooves were shallower than in /p/ context. Anteriorly, /s/ context caused deeper grooves for low vowels. Cross-sectional tongue shape varied with tongue position similarly to sagittal tongue shape.

Wound healing in sarcoma-bearing rats: tumor effects on cutaneous and deep wounds.

Many surgeons think that cancer causes a higher incidence of wound complications. Wound healing was examined in a cutaneous and deep model in control and sarcoma-bearing rats to evaluate this concept. In a dorsal incisional wound, a significant decrease in wound breaking strength was observed from 19 days after tumor implantation onward. The amount of the breaking strength deficit increased with the size of tumor and the day post-tumor implant. In a deeper wound chamber, hydroxyproline levels, 3H-thymidine incorporation into DNA, histology, and collagen types were examined, and tumor produced no significant change in any parameter. The presence of tumor appeared to inhibit wound healing in cutaneous wounds but had no apparent effect on deeper wounds. This difference in healing in the two wound models is important to surgical oncologists. Because there is no demonstrable tumor-induced healing deficit in deep wounds, cancer-bearing organisms probably still heal these wounds normally.

Preoperative or postoperative doxorubicin hydrochloride (adriamycin): which is better for wound healing?

The purpose of this study was to determine if any interval between preoperative or postoperative administration of doxorubicin hydrochloride (Adriamycin) and wounding would limit the impairment in healing induced by Adriamycin. The question was evaluated in 7 cm incisional wounds on the dorsal midline of rats. Preoperative Adriamycin was administered 42, 28, 21, 14, and 7 days before wounding, and wound breaking strengths (WBS) of the incisional wounds were measured 7, 18, and 28 days after wounding. Postoperative Adriamycin was administered 0, 7, 14, 21, and 28 days after wounding, and WBS were measured 7, 14, 28, 35, and 42 days after wounding. A similar and significant degree of impairment was induced in healing by all preoperative Adriamycin regimens evaluated. Postoperative Adriamycin administered up to 21 days after wounding induced a healing deficit that manifested 1 to 2 weeks after Adriamycin administration. Adriamycin administered 28 days after surgery induced no demonstrable impairment in WBS. In addition, Adriamycin never decreased the WBS of a wound after a certain degree of strength had been obtained. These results lead us to warn against the use of preoperative Adriamycin and support the use of postoperative Adriamycin administered 28 days after a surgical procedure.

Doxorubicin-induced impairment of wound healing in rats.

The mortality rate induced by 3 doses of iv doxorubicin was evaluated in F344 rats, and a dose of 8 mg doxorubicin/kg body weight was the maximum dose tolerated with an acceptable mortality rate. Rats treated with 8 mg doxorubicin/kg prior to or on the day of wounding demonstrated decreased wound breaking strength in incisional wounds at all intervals after wounding. Decreased amounts of collagen and DNA and less cellularity were noted in wound chambers from rats treated in the same manner. In both the incisional wound and wound chamber models, rats treated with doxorubicin 7 days after wounding showed a less dramatic healing impairment. No difference in collagen types was noted between chambers from the doxorubicin-treated and untreated rats. Doxorubicin also produced a significant reduction in platelet and white blood cell counts 1 week after it was administered. The data indicate that doxorubicin impedes healing by decreasing wound cellularity and collagen synthesis.

Hemodynamic principles in the control of coronary blood flow.

The effects of atherosclerotic epicardial stenoses on coronary vascular resistance can be understood in terms of basic principles of fluid mechanics. Resistance is directly related to the pressure drop across the stenosis and inversely related to flow. Even with a fixed anatomic stenosis, however, resistance is not fixed; it increases as flow across the stenosis increases. This exacerbates the pressure drop across the stenosis that develops as a result of flow; at high flows, large pressure drops can occur. This characteristic of flow through stenotic lesions can contribute to a "steal" phenomenon between either epicardial or intramural coronary arteries. Studies have also shown the clinical importance and influence of dynamic alterations in coronary resistance, occurring either at the large or small vessel level. In addition, compressive forces exerted by the myocardium or by elevated intraventricular pressures can increase coronary vascular resistance, and thus interfere with myocardial perfusion. All of these factors must be considered in order to obtain a comprehensive understanding of the mechanisms leading to myocardial ischemia and, therefore, to the clinical syndrome of angina pectoris.

A practical graphical method for estimating the fraction of cells in S in DNA histograms from clinical tumor samples containing aneuploid cell populations.

A graphical method for the analysis of unperturbed DNA histograms is presented in which the area of the normalized histogram subtended by the fraction of cells in S is represented by a trapezoid whose dimensions are dependent on features common to all such histograms. The technique takes measurement variability into account. This method was applied to a variety of synthetic DNA histograms. Overall, calculated values for the fraction of cells in S correlated well with actual values. This method was applied to 36 diploid cases of non-Hodgkin lymphoma; results correlated well with those obtained by a computer-based method. The results of the graphical-method were also highly reproducible between different observers. The graphical method can be used in the presence of aneuploid cell populations. Techniques for calculating S fractions in the presence of aneuploidy in clinical samples are described. These techniques were applied to synthetic histograms of mixed diploid and aneuploid populations. Calculated values correlated well with actual values.

The effect of ultrasonics and thermal treatment on wounds.

A method of local treatment of wounds to accelerate healing would be a major benefit in those patients in whom abnormal healing is expected. Earlier studies has suggested that local ultrasonic treatment of wounds would stimulate the healing process. We have evaluated the effect of local ultrasound (5 MHz) and thermal treatments on healing in a dermal wound model. Various intensities of ultrasound and heat were employed for 5 min/day (0.05 W/cm2) and 10 min/day (0.05W/cm2), with healing assessed by wound breaking strength measurements obtained 14 days after injury. Subcutaneous temperature measurements demonstrated that equal intensities of ultrasound and heat produced equivalent temperature changes in the tissues. None of the treatments employed resulted in greater wound breaking strengths than the controls, and the higher intensities of ultrasound (0.1 and 0.15 W/cm2) and heat (0.15 W/cm2) resulted in decreased wound breaking strength. Our results failed to support brief daily treatments of local ultrasound or heat as stimulants of wound healing.