Fast axonal transport in amyotrophic lateral sclerosis: an intra-axonal organelle traffic analysis.

Fast transport of intra-axonal organelles was studied in motor nerve from amyotrophic lateral sclerosis (ALS) patients. Organelle traffic in ALS nerves demonstrated a significant increase in anterograde mean speed, while retrograde mean speed was decreased compared with that of controls. Retrograde traffic density (organelles per unit time) was also significantly decreased in the ALS specimens. Anterograde transport machinery is therefore intact and may be responding to the increased physiologic demand of larger motor units. Diminished retrograde speed and organelle traffic density are consistent with a defect in retrograde transport and could impair communication between axon terminals and perikarya.

Developing microcomputer-based quality assurance systems for health care.

Points out some important issues that must be dealt with when developing microcomputer-based QA systems for health care. While no single solution to the problems associated with quality assurance (QA) system development exists, the first step towards an efficient and effective approach is problem identification and commitment to developing a strategy which addresses the issues discussed in this paper. Examines potential solution alternatives and associated pitfalls, and gives an example of designing a QA system, including a database management system with suggestions for spreadsheet templates.

A non-invasive off-line method of measuring cardiac output.

The measurement of cardiac output has many clinical applications and the development of a reliable, non-invasive measurement technique would be of considerable value to clinicians, cardio-respiratory physiologists and cardiovascular pharmacologists. Currently-used methods of measuring cardiac output are either invasive, and therefore potentially dangerous, or require the use of expensive, sophisticated, complex equipment which often has to be kept exclusively for the purpose of measuring cardiac output. We describe a method based on the recently modified and validated acetylene rebreathing technique which avoids the necessity for on-line computer acquisition of data by employing semi-manual digitization of hard copy recordings. The method is non-invasive, accurate, sensitive and relatively inexpensive. In addition, the whole technique can be rapidly performed by minimally trained personnel.

Rapid resetting of aortic nerves in conscious rabbits.

The effect of increased arterial pressure on aortic depressor nerve activity was studied in the conscious rabbit. Aortic baroreceptor resetting was observed following 15 min of sustained pressure elevation. At 15 min, there was a significant increase in the threshold arterial pressure for aortic nerve activity, but peak nerve activity did not change. This resulted in an increase in the slope of the pressure-nerve activity relationship. Therefore, except for peak nerve activity, aortic nerve activity was reduced at all pressures following 15 min of sustained pressure elevation. At 30 min, peak nerve activity also increased, resulting in a parallel shift in the pressure-activity curve. The early increase in slope of the pressure-activity relationship may be due to an early resetting of low threshold fibers with a high pressure differential between their threshold pressure and the sustained pressure elevation used to induce resetting. A late resetting of high threshold fibers with a low pressure differential between their threshold pressures and the pressure used to cause resetting of baroreceptors results in a parallel shift in the curve observed at 30 min. These data obtained from multiunit recordings provide unexpected evidence about baroreceptor resetting, which is not apparent from single-unit recordings.

Influence of translocation track on the motion of intra-axonally transported organelles in human nerve.

The mechanism by which organelles are transported bidirectionally in axoplasm is still unknown; however, evidence of a key role for microtubules in many nonmammalian models has been established. We have observed common or shared tracks within the axoplasm of human nerves along which multiple organelles of varying size and shape are bidirectionally transported. Organelles traveling anterogradely and retrogradely were visualized by video-enhanced differential interference contrast optics and analyzed with the aid of computer-image-processing techniques. Speeds of translocating organelles were determined at eight to 16 translocation points along a path or "track." Each translocation speed was plotted against its corresponding position on the track to develop a "speed/position diagram." Regardless of mean organelle speed or direction of motion, organelles sharing a common track exhibited similar patterns of "speeding up" and "slowing down" relative to position along the track. Speed position data for organelles translocating the local axonal region of a common track showed no unique patterns (not different from a uniform distribution, p less than 0.05). The unique speed/position patterns exhibited by common tracks were not necessarily related to the patterns of other tracks in the immediate vicinity (distance between tracks of less than 0.50 micron). These findings suggest that there are "common tracks" shared by organelles moving retrogradely and anterogradely; both the organelles and the "track" associated with its translocation play a role in the resultant motion of that organelle; the influence exerted by a common track on the motion of an organelle results in a pattern of speed changes related to position along the track.

Spatial distribution and accumulation of low density lipoproteins in the abdominal aorta of swine: determination by a novel electrotransfer procedure.

An immunotransfer procedure has been developed which can determine both the spatial distribution of low density lipoproteins (LDL) along the intima-media of large blood vessels such as the aorta, and can quantify LDL accumulation along its length. Aortas which were opened longitudinally along their ventral aspect were positioned so that their intimal side abutted against a gel containing glyoxyl agarose to which anti-LDL had been covalently coupled. LDL was electrophoresed out of the agarose gel where it was immunofixed. This distribution was then visualized first by incubating the gel with 125I-anti-LDL which bound to free epitopes on the immunofixed LDL, and second by subjecting the washed and dried gel to autoradiography. Plasma LDL was applied to wells of different shapes and sizes in an agarose gel substituting for aortic tissue, and the transfer procedure was performed as described. The resultant patterns matched those of the original wells, suggesting that the spatial distribution of LDL in the autoradiogram probably mimicked that in the aortic tissue. The transfer procedure appeared to be specific for the antigen under study since minimal silver grains were observed in autoradiograms when an IgG fraction of nonimmune serum was used in place of anti-LDL. Application of increasing concentrations of LDL to wells in a gel substituting for tissue, resulted in a dose-dependent increase in autoradiographic grain density. If such standards were applied to gels adjacent to tissue samples, the amounts of LDL in the tissue could be quantified from the standard curve of grain density versus LDL concentration. The distribution of LDL along the abdominal aortas of 10- and 31-week-old swine was determined by converting autoradiographic grain densities to isopleths of LDL concentrations by computer assisted image analysis. These distributions were focal and were found to range between 10 and 225 ng of apoB/mm2 of intimal surface area. This procedure lends itself not only to studies relating lipoprotein accumulation to atherogenesis, but also to any studies dealing with tissue accumulation of macromolecules.

The relationship of the extrinsic blood supply to regeneration in graft reconstructed peripheral nerves.

Median nerves of rats were reconstructed with conventional or vascular nerve grafts. After 2, 3 and 4 weeks, Allen Video-Enhanced Contrast, Differential Interference Contrast (AVEC-DIC) microscopy revealed axonal transport in most preparations, with varying degrees of myelination. Radio-isotope tracer was measured in the nerve. Two and 4 week measurements revealed no difference between the graft types. At 3 weeks the vascular graft group exhibited transport along the entire length of the nerve in contrast to a relatively abbreviated path length in the conventional graft group. Nerve conduction velocities (NCV) were measured proximal to, within and distal to the grafts. Three week NCV showed no difference between the graft types. The 4 week NCV revealed normal values in the vascular graft group at points distal to and within the graft. Significant slowing was seen in the conventional grafts at both points.

Baroreflex control of sympathetic nerve activity after elevations of pressure in conscious rabbits.

The purpose of this study was to assess the effect of rapid baroreceptor resetting on the baroreflex control of renal sympathetic nerve activity in conscious rabbits. Renal sympathetic nerve activity was recorded and used as an index of the efferent limb of the baroreflex. Heart rate and arterial pressure were also recorded. Arterial pressure was raised with either phenylephrine or angiotensin II to a level that eliminated renal sympathetic nerve activity and was maintained at this level for periods of time ranging from 1 to 60 min. On returning pressure to control levels, renal sympathetic nerve activity remained suppressed for up to 90 min, with the duration of the suppression dependent on the magnitude and duration of the pressure stimulus. During this period of suppressed nerve activity, baroreflex curves were generated. The curves produced at this time were also suppressed as compared with control baroreflex curves. With time, the suppressed baroreflex curves returned to control. Further studies were performed to show that the suppression of renal sympathetic nerve activity was mediated via the prolonged increase in baroreceptor afferent activity during the pressure stimulus and was not due to a central effect of phenylephrine. This study indicates that although baroreceptor afferent activity may reset rapidly, there does not appear to be an augmentation of renal sympathetic nerve activity as would be expected.

Alterations in cardiac function and cardiopulmonary blood volume in chronic sodium depletion in dogs.

1. Chronic sodium depletion in dogs is associated with a blunting of the pressor response to carotid occlusion. AFter section of the vagus nerves this pressor response reverts to normal although atropine is without effect, suggesting a possible role for increased activity of cardiopulmonary vagal afferents in suppressing sympathetic outflow. Since increases in central blood volume stimulate vagal afferents, cardiopulmonary haemodynamics were studied by radioisotope dilution before and after 3 and 4 weeks of dietary sodium restriction together with frusemide. 2. Sodium depletion was associated with significant decreases in cardiac output, ejection fraction and total blood volume; the cardiopulmonary blood volume increased but the change was not statistically significant (P < 0.2). These changes were accompanied by a significant increase in the ratio cardiopulmonary blood volume/total blood volume and a decrease in the ratio cardiac output/cardiopulmonary blood volume. 3. Results indicate that sodium depletion may be accompanied by a paradoxical translocation of blood to the cardiopulmonary region in part due to depressed ventricular performance.

Factors affecting cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile.

We explored the influence of several compositional factors considered capable of influencing the nucleation time of model biles supersaturated in cholesterol. In addition to the classical techniques, e.g., electron microscopy and quasielastic light scattering, employed for size measurement and structural assessment, we employed a novel technique, i.e., video-enhanced microscopy, for particle evaluation in these polydisperse systems which often may simultaneously contain isolated small vesicles, their complex aggregates, and small cholesterol monohydrate crystals. The factors we studied included dilution, degree of cholesterol supersaturation, bile salt/lecithin molar ratio, and Ca2+ concentration. Dilution markedly raised the degree of cholesterol saturation, prolonged nucleation time for cholesterol monohydrate crystals, and favored formation of metastable small unilamellar vesicles. Increasing the degree of cholesterol supersaturation as an independent variable in more concentrated systems both shortened the nucleation time and favored spontaneous formation of a relatively small number of isolated vesicles. A decrease in bile salt/lecithin molar ratio within the physiologically relevant range was accompanied by a prolonged nucleation time and favored spontaneous vesicle formation. Large numbers of small unilamellar vesicles were observed even in concentrated model bile solutions (total lipids: 20 g/dl) when the bile salt/lecithin molar ratio was 1.9 or less. At physiological concentrations, Ca2+ promoted nucleation of cholesterol monohydrate crystals only in vesicle-containing solutions. Taken together, the following conclusions can be drawn. First, spontaneous vesicle formation in dilute systems prolongs solid cholesterol crystal nucleation. It can thus provide a supplementary non-micellar mode of cholesterol transport in micellar systems of supersaturated human bile. Second, dilution, degree of cholesterol supersaturation, and a decrease in bile salt/lecithin ratio prolong cholesterol crystal nucleation time and favor spontaneous vesicle formation. With increasing calcium concentrations, opposite effects are observed. Third, the presence of vesicles may help to account for the frequently observed and otherwise unexplained remarkable degree of metastable supersaturation and prolonged metastability (delayed nucleation time) for cholesterol in human bile.

Vesicle aggregation in model systems of supersaturated bile: relation to crystal nucleation and lipid composition of the vesicular phase.

The presence of small vesicles composed of phospholipid and cholesterol has recently been demonstrated in super-saturated model and in dilute native human biles by several groups using differing methods. Among compositional factors shown to favor spontaneous vesicle formation and prolong the cholesterol monohydrate nucleation time in model bile systems are dilution, a raised cholesterol saturation index (CSI), and a low bile salt/phospholipid ratio. Time-lapse video-enhanced microscopy of a series of model bile systems representing systematically designed variations in the above factors revealed strong evidence for an essential linkage between antecedent vesicle aggregation and subsequent crystal nucleation. Stability of vesicles was inversely related to their degree of cholesterol saturation, i.e., the greater the degree of vesicular cholesterol saturation, the less their stability (metastability). Instability of vesicles was reflected by their early aggregation followed by rapid cholesterol crystal nucleation. The lowest degree of vesicular cholesterol saturation was found in dilute systems which also exhibited the greatest metastability despite a high degree of cholesterol solubility (raised CSI). Conversely, the more concentrated and least metastable systems exhibited both rapid vesicle aggregation and rapid onset of crystal nucleation. These systems, while influenced by the other compositional factors, were found to have a high degree of vesicular cholesterol saturation, i.e., cholesterol/phospholipid molar ratio = 2.0. An additional finding was the extreme variability in the proportionate distribution of total solution cholesterol distributed to the vesicular phase, i.e., from zero to as high as 37%. Higher solute concentration, raised bile salt/lecithin ratio, and raised CSI were interactive and almost equally capable of increasing the proportionate amount of cholesterol in the vesicular phase. In conclusion, lipid compositional differences in model bile systems drastically affect the cholesterol saturation of spontaneously formed phospholipid-cholesterol vesicles. This effect, in turn, exerts a potent influence upon the metastability of vesicles, subsequently affecting the cholesterol crystal nucleation time.

Rapid vesicle formation and aggregation in abnormal human biles. A time-lapse video-enhanced contrast microscopy study.

Rapid nucleation of cholesterol crystals has previously been shown to provide a sharp discrimination between abnormal (cholesterol gallstone-associated) and normal human gallbladder bile. In the present study, we sought to further clarify the crystal nucleation process by time-lapse microscopy using a novel high-resolution video-enhanced microscopy technique. Using a previously described method for removal of particles from abnormal biles, we found a strikingly rapid rate of de novo formation of unilamellar vesicles, soon followed by massive vesicular aggregation, culminating in crystal formation. In normal biles, by contrast, this rapid aggregation process was not observed and the isolated unilamellar vesicles showed prolonged stability. Morphometric analysis of interval particle counts showed statistically significant differences. The process of cholesterol monohydrate crystal nucleation in supersaturated human bile is characterized by a sequential combination of vesicle formation, vesicle aggregation, and subsequent crystal formation. The primary distinction between abnormal and normal biles resides only in the consistent rapidity of onset and completion of these events in the abnormal biles.

Long-term analysis of organelle translocation in isolated axoplasm of Myxicola infundibulum.

Moving intra-axonal organelles demonstrate frequent variations in speed when viewed over several seconds. To evaluate these and other motion variations, a long-term analysis of organelle motion in isolated axoplasm of Myxicola infundibulum was carried out using differential interference contrast optics and analog and digital image enhancement techniques. Motion characteristics of individual organelles were analyzed for periods of up to 58 minutes. Three principle observations on organelle motion were made: 1) Classes of organelles of the same size demonstrated a 5- to 25-fold variation of speed, with the slowest speeds occurring most frequently; 2) organelle speeds over individual translocations (motion without stopping) are inversely proportional to their size, but the speeds calculated for the long-term analysis of organelle motion (total distance travelled/total observation time, including pauses) did not reflect this observation; and 3) organelles displayed variable trip lengths, durations, mean speeds, and pause durations, and the relationships between these variations showed no repetitive patterns. In contrast to reported observations of uniform velocities of organelles moving on isolated microtubule preparations, these observations suggest that a variety of factors must play a role in organelle translocation in Myxicola axoplasm.