Detection of unique transmural architecture of human idiopathic cardiomyopathy by ultrasonic tissue characterization.

Noninvasive approaches to the evaluation of idiopathic cardiomyopathy are limited. Recent work from our laboratory has used quantitative ultrasound to define the three-dimensional structure of normal human myocardium and the myocardial remodeling associated with infarction. Our goal was to define the role of ultrasonic tissue characterization for detection of specific alterations in the three-dimensional transmural architecture of idiopathic dilated cardiomyopathy. We measured frequency-dependent backscatter from 22 cylindrical biopsy specimens from nine explanted fixed hearts of patients who underwent heart transplantation for idiopathic cardiomyopathy, seven specimens from normal portions, and 12 specimens of infarcted tissue from six explanted fixed human hearts. Consecutive transmural levels from each specimen were insonified with a 5-MHz broadband transducer. The dependence of apparent (uncompensated for attenuation) backscatter, B(f), on frequency (f) was computed from radiofrequency (rf) data as: magnitude of B(f)2 = afn, where n is an index that reflects in part the size of the dominant scatterers in myocardial tissue. Myofiber diameter and percentage fibrosis were determined at each transmural level for each specimen. For cardiomyopathic tissue, the frequency dependence of backscatter (n) increased progressively from epicardial to endocardial (0.02 +/- 0.37 to 1.01 +/- 0.12, p less than 0.05) levels in conjunction with a progressive decrease in myofiber diameter (29.5 +/- 0.9 to 21.4 +/- 0.6 microns, p less than 0.0001). In contrast, in tissue from areas of infarction, the frequency dependence decreased progressively from epicardium to endocardium (0.91 +/- 0.20 to 0.23 +/- 0.21, p less than 0.05) in conjunction with a progressive increase in the percentage of fibrosis (23.5 +/- 9.4% to 54.5 +/- 4.9%, p less than 0.005). Normal tissue exhibited no significant transmural trend for frequency dependence, myofiber diameter, or percentage fibrosis. These data indicate the presence of a heterogenous transmural distribution of scattering structures associated with human idiopathic cardiomyopathy and myocardial infarction that may be detected by ultrasonic tissue characterization. The divergence of these transmural trends for frequency dependence of backscatter reflects distinct mechanisms of structural heterogeneity for different pathological processes that comprise a transmural gradation of cell size and fibrosis for idiopathic cardiomyopathy and infarction, respectively.


Regional left ventricular wall thickening. Relation to regional uptake of 18fluorodeoxyglucose and 201Tl in patients with chronic coronary artery disease and left ventricular dysfunction.

In previous studies comparing regional 201Tl (201Tl) and 18fluorodeoxyglucose (FDG) activity in patients with chronic coronary artery disease and left ventricular dysfunction, we hypothesized that regions with mild-to-moderate reduction in FDG activity and regions with mild-to-moderate irreversible 201Tl defects after 3- to 4-hour redistribution represent viable myocardium. In the present study, regional FDG and 201Tl activities were compared with regional systolic wall thickening by gated magnetic resonance imaging (MRI) to confirm the presence of viable myocardium in these territories. Twenty-five patients with chronic stable coronary artery disease and left ventricular dysfunction (ejection fraction, 28 +/- 10) underwent exercise 201Tl tomographic imaging (SPECT), using a reinjection protocol, positron emission tomography (PET) with FDG and H2(15)O, and gated MRI. Matched SPECT, PET, and MRI tomograms were analyzed. From the PET data, 105 regions had matched reduction in FDG and blood flow, of which 69 regions had moderately reduced FDG uptake (50-79% uptake relative to a normal reference region) and 36 had severely reduced FDG uptake (less than 50% of normal activity). Regions with moderately reduced as compared with severely reduced FDG activity had greater end-diastolic wall thickness (9.4 +/- 2.6 versus 8.0 +/- 3.7 mm; p less than 0.05) and regional systolic wall thickening (1.7 +/- 2.7 versus -0.7 +/- 2.1 mm; p less than 0.01). From the SPECT data, 169 irreversible 201Tl defects after 3-4 hour redistribution were identified, of which 70 were mild (greater than 65 to less than 85% of maximal 201Tl activity), 52 were moderate (50-65% of maximal activity), and 47 were severe (less than 50% of maximal activity). Regional systolic wall thickening was greater in regions with normal 201Tl uptake (3.3 +/- 2.3 mm) as compared with all other regions. Regions showing only mild or moderate irreversible defects at redistribution, however, showed wall thickening (2.4 +/- 2.4 and 2.2 +/- 2.5 mm, respectively), which was similar to that observed in regions with reversible 201Tl defects (2.1 +/- 2.2 mm). Only regions with severe irreversible defects at redistribution showed absence of thickening (-0.1 +/- 2.9 mm, p less than 0.01 versus all other groups). After 201Tl reinjection, 12 of 47 (26%) regions with severe irreversible defects showed enhanced 201Tl uptake. The impairment in regional systolic wall thickening was not significantly different between 201Tl defects with and without enhanced 201Tl uptake after reinjection. FDG activity, however, was present in all 12 regions (100%) with enhanced 201Tl uptake after reinjection as compared with only five of 35 (14%) that were unchanged after reinjection (p less than 0.01). Therefore, preserved wall thickness and systolic wall thickening in regions with moderate reduction in blood flow and FDG activity, and in irreversible 201Tl defects that are only mild-to-moderate, provide additional evidence that such regions represent viable myocardium. Moreover, the finding of metabolic activity and 201Tl uptake in regions with reduced blood flow and absent wall thickening provides clinical evidence of hibernating myocardium in humans.


Transcatheter ablation of ectopic atrial tachycardia in young patients using radiofrequency current.

Ectopic atrial tachycardia (EAT) is a reversible cause of cardiomyopathy but may be quite difficult to control with conventional therapy. Transcatheter ablation with radiofrequency current was tested as an alternative to medical or surgical treatment of this condition. Twelve young patients (aged 10 months to 19 years) with drug-resistant EAT were treated with direct transcatheter ablation of the ectopic focus using radiofrequency (RF) energy. All had depressed left ventricular contractility by echocardiographic criteria, involving shortening fractions of 10-26% (median, 20%; normal, 28-35%). The EAT was mapped to the left atrium in seven cases and to the right atrium in five. Local atrial activation at the ectopic site preceded the onset of the surface P wave by 20-60 msec (median, 42 msec). Tachycardia terminated 0.5-13.0 seconds (median, 2.0 seconds) into a successful RF application. The ablation effectively eliminated EAT in 11 of 12 patients (92%), all of whom were discharged in sinus rhythm without medications after a median hospital stay of 48 hours. Ablation was unsuccessful in one patient with diffuse dysplasia of the anterior right atrium, who eventually did well after surgical resection of abnormal atrial tissue. Transient depression of sinus node function was noted in one patient who had successful ablation of an EAT focus in close proximity to the sinus node, although normal sinus node function returned within 72 hours. No other complications were encountered. During follow-up (3-21 months; median, 13 months), one patient had recurrence of a slower and less-sustained EAT that was successfully eliminated at a second ablation session. All others remained in sinus rhythm, and all 12 subjects recovered normal ventricular function. RF ablation appears to be a safe and effective therapeutic option for drug-resistant ectopic atrial tachycardia and may be the preferred first-line therapy for those patients with depressed ventricular function.


Effect of Ebstein's anomaly on short- and long-term outcome of surgically treated patients with Wolff-Parkinson-White syndrome.

Ebstein's anomaly is the most commonly occurring congenital abnormality associated with the Wolff-Parkinson-White (WPW) syndrome. However, the effects of Ebstein's anomaly on the risks and benefits of surgical ablation of accessory pathways in patients with WPW syndrome are unknown. This study compared the long-term outcome of 38 WPW patients with Ebstein's anomaly undergoing accessory pathway ablation to a reference population of 384 similarly treated patients without the anomaly. Ebstein's anomaly was mild in 21 patients (55%) and moderate-to-severe in 17 patients (45%). Sixteen patients (42%) required tricuspid valve surgery, and 23 (61%) had an atrial septal defect or patent foramen ovale repaired. Baseline clinical characteristics and preoperative clinical arrhythmias were similar in both groups. Ten-year survival was 92.4% and 91.2% for patients with and without Ebstein's anomaly, respectively (p = NS). During a mean follow-up of 6.2 +/- 3.8 and 5.3 +/- 3.6 years, 82% of patients with and 90% without Ebstein's anomaly had either clinically insignificant or no arrhythmias, and 18% versus 10% reported symptoms suggesting arrhythmias lasting longer than 1 minute, respectively. Atrial fibrillation was reduced postoperatively to 9% (p less than 0.001) in patients with and to 4% (p less than 0.001) in those without the anomaly. Fewer hospitalizations were reported postoperatively by 90% versus 96% of patients with and without Ebstein's anomaly; 9.4% versus 6.0% of patients were disabled at follow-up, respectively (p = NS). Patients with Ebstein's anomaly are improved significantly after accessory pathway ablation. The presence of this anomaly should not preclude accessory pathway ablation in these patients.


Loss of the coronary microvascular response to acetylcholine in cardiac transplant patients.

The coronary arteries of transplanted hearts frequently develop accelerated diffuse arteriosclerosis. The effects of this disease on resistance vessel function are unknown. To investigate the integrity of endothelium-dependent small-vessel vasodilation in transplanted hearts, coronary blood flow (CBF) responses to the endothelium-dependent dilator acetylcholine (10(-8) to 10(-6) M) and the essentially endothelium-independent dilator adenosine (10(-6) to 10(-4) M) were assessed in 40 studies of 29 transplant patients 1-3 years after transplantation and in seven nontransplanted controls. CBF was measured at constant arterial pressure with a Doppler catheter in the left anterior descending coronary artery. Controls, year 1 transplant patients, and year 2 transplant patients had similar increases in CBF in response to acetylcholine (232 +/- 40%, 200 +/- 41%, and 201 +/- 54%, respectively; p = NS), whereas year 3 transplant patients had increased CBF of only 100 +/- 39% (p less than 0.05 versus controls). An index of the proportion of CBF reserve attributable to endothelium-dependent dilation was obtained by normalizing each patient's peak acetylcholine flow response by the peak adenosine flow response. In patients receiving both acetylcholine and adenosine, endothelium-dependent flow responses declined over time [57 +/- 9% in controls, 56 +/- 10% for year 1, 47 +/- 12% for year 2, and 29 +/- 9% for year 3 (p less than 0.05 versus controls)]. An increased mean cyclosporine level (range, 99-261 ng/ml) (r = 0.67, p = 0.004) and increased transplant recipient age (range, 20-63 years) (r = 0.51, p = 0.004) predicted a preserved endothelium-dependent microvascular response. Thus, microvascular endothelium-dependent dilation deteriorates over time in the transplanted heart, which may reflect underlying graft arteriosclerosis and contribute to ischemic damage of the myocardium.


Long-term predictors of subsequent cardiovascular events with coronary artery disease and 'desirable' levels of plasma total cholesterol.

Patients with coronary artery disease (CAD) are at considerable risk for subsequent cardiovascular events. Although hyperlipidemia accentuates the risk, predictors of subsequent events with CAD and desirable total cholesterol (TC) (less than 5.2 mmol/l) have not been assessed. A survival analysis was performed in a subset of 740 consecutive patients who underwent diagnostic coronary arteriography between 1977 and 1978. Eight-three men and 24 women with angiographically documented CAD and desirable TC were followed for subsequent cardiovascular events, including myocardial infarction and cardiovascular death. Over a 13-year period, 75% of CAD subjects with reduced high density lipoprotein cholesterol (HDL-C) (less than 0.9 mmol/l) developed a subsequent cardiovascular event compared with 45% of those with HDL-C greater than or equal to 0.9 mmol/l (p = 0.002). A Kaplan-Meier analysis revealed significantly greater survival from cardiovascular end points in patients with baseline levels of HDL-C greater than or equal to 0.9 mmol/l (p = 0.005). After 11 variables were tested, an age-adjusted Cox proportional-hazards model identified two pairs of independent predictors of subsequent cardiovascular events: they were a left ventricular ejection fraction (LVEF) less than 35% (relative risk [RR], 6.5; 95% confidence interval [CI], 2.8, 15.3; p less than 0.001) and reduced HDL-C (RR, 2.0; 95% CI, 1.2, 3.3; p = 0.01) in the first model and LVEF less than 35% (RR, 6.5; 95% CI, 2.7, 15.6; p less than 0.001) and TC:HDL ratio greater than or equal to 5.5 (RR, 1.9; 95% CI, 1.1, 3.1; p = 0.02) in the second model. Low HDL-C (or high TC:HDL-C) is strongly predictive of subsequent cardiovascular events in subjects with CAD, despite desirable TC. As such, identification of this potentially modifiable risk factor should be actively pursued in this high-risk subgroup.


Endothelial dysfunction early after heart transplantation. Assessment with intravascular ultrasound and Doppler.

Allograft vasculopathy after heart transplantation is thought to represent a response to endothelial injury in the graft vessels. To assess endothelial function before the onset of anatomic disease, coronary vasomotor responses to adenosine, acetylcholine, and nitroglycerin were evaluated in transplant recipients by intravascular ultrasound imaging and Doppler flow studies. Nine patients were studied 1 year after heart transplantation. Acetylcholine provoked significant vasoconstriction to 82% of maximal coronary diameter but was associated with an increase in mean coronary blood flow from 63.1 to 204 ml/min. Coronary blood flow increased fivefold in response to adenosine, a normal response. The vasomotor response to acetylcholine at 1 year after heart transplantation is consistent with endothelial dysfunction in the epicardial conduit vessels. Microvascular function as judged by coronary flow reserve appears to be normal.


Platelet hyperaggregability across the coronary bed in response to rapid atrial pacing in patients with stable coronary artery disease.

Platelet aggregation is believed to contribute to the precipitation of acute ischemic syndromes. Because physical activity has been proposed as one possible trigger in converting a patient with chronic coronary artery disease to one with an acute ischemic syndrome, we examined the hypothesis that platelets become activated when coronary blood flow velocities (and thereby shear stress) increase across an atherosclerotic bed. During catheterization, 82 patients (36 with left coronary artery disease, 12 with only right coronary artery disease, and 34 with normal coronary arteries) had measurement of whole blood platelet aggregation performed on blood samples obtained simultaneously from the coronary sinus and aorta at rest, 2 minutes after onset of rapid atrial pacing, and 10 minutes after pacing was terminated. There was no arteriovenous difference in platelet aggregation under resting conditions in patients with versus those without coronary artery disease. Atrial pacing in patients with left coronary artery disease (greater than or equal to 50% stenosis in a major epicardial vessel) caused an increase in platelet aggregation in the coronary sinus blood (+64 +/- 9%, p less than 0.01) but not in arterial blood (2 +/- 8% decrease, p = NS). This increase was transient and returned nearly to baseline 10 minutes after termination of pacing. Patients with nonsignificant left coronary artery disease, those with normal coronary arteries, and patients with significant disease only in the right coronary artery (venous drainage not into the coronary sinus) did not show any changes in either the coronary sinus or arterial blood with atrial pacing. There is no evidence of platelet activation across a normal or an atherosclerotic coronary bed at rest. When coronary blood flow increases in the presence of significant (greater than or equal to 50%) narrowing of epicardial coronary arteries, however, platelets are activated and aggregate more easily. This mechanism may play a role in the precipitation of acute ischemic syndromes in patients with coronary artery disease.


Clinical characteristics and outcome of patients with high defibrillation thresholds. A multicenter study.

Successful defibrillation by an implantable cardioverter-defibrillator (ICD) depends on its ability to deliver shocks that exceed the defibrillation threshold. This study was designed to identify clinical characteristics that may predict the finding of an elevated defibrillation threshold and to describe the outcome of patients with high defibrillation thresholds. The records of 1,946 patients from 12 centers were screened to identify 90 patients (4.6%) with a defibrillation threshold greater than or equal to 25 J. Excluding three patients who received ICDs that delivered greater than 30 J, there were 81 men and six women with a mean age of 59.5 +/- 10.1 years, a mean left ventricular ejection fraction of 0.32 +/- 0.14, and a 76% prevalence of coronary artery disease. Sixty-one patients (70%) were receiving antiarrhythmic drugs, and 45 (52%) were receiving amiodarone. Seventy-one patients (82%) received an ICD. Death occurred in 27 patients--19 of the 71 (27%) with an ICD (eight arrhythmic), and eight of the 16 (50%) without an ICD (four arrhythmic). Actuarial survival for all patients at 5 years was 67%. Actuarial survival rates at 2 years for patients with and without an ICD were 81% and 36%, respectively (p = 0.0024). Actuarial survival at 5 years for the ICD patients was 73%; no patient without an ICD has lived longer than 32 months. Actuarial survival free of arrhythmic death in the ICD patients at 5 years was 84%. Although the only variable to predict survival was ICD implantation (p = 0.003), it is entirely possible that in this retrospective analysis, clinical selection decisions to implant or to not implant an ICD differentiated patients destined to have better or worse outcomes, respectively. Antiarrhythmic drug use may be causally related to the finding of an elevated defibrillation threshold. When patients with high defibrillation thresholds receive an ICD, arrhythmic death remains an important risk (42% of deaths in these patients were arrhythmia related, with 16% actuarial incidence at 5 years). Vigorous testing to optimize patch location can potentially benefit patients by enhancing the margin of safety for effective defibrillation.


Failure of an implantable cardioverter-defibrillator to redetect ventricular fibrillation in patients with a nonthoracotomy lead system.

Shock delivery of an implantable defibrillator may cause a change in the amplitude of endocardial electrograms and impair the detection of ventricular fibrillation. Thus, the effects of shock discharges on the amplitude of endocardial electrograms were evaluated in five patients undergoing implantation of a cardioverter-defibrillator in combination with a new nonthoracotomy lead system. At implant, bipolar endocardial electrograms were recorded before each shock application, during ventricular fibrillation, during redetection of ventricular fibrillation in case the applied shock was ineffective, and at intervals of 5, 10, 20, 30, 60, and 120 seconds after each shock delivery. The amplitude of the endocardial electrograms decreased from 10.5 +/- 3.8 mV during sinus rhythm to 6.3 +/- 1.9 mV during initial ventricular fibrillation and declined to 2.2 +/- 1.3 mV during redetection of ventricular fibrillation. After successful termination, the following bipolar electrograms could be obtained at the predetermined intervals: 1.9 +/- 1.2 mV, 3.1 +/- 1.8 mV, 4.5 +/- 1.9 mV, 6.5 +/- 2.9 mV, 9.5 +/- 3.3 mV, and 10.4 +/- 3.8 mV. At predischarge testing, failure of redetection of ventricular fibrillation could be documented in two patients, requiring rescue external defibrillation in both cases to restore sinus rhythm. These findings demonstrate that the implantable cardioverter-defibrillator did not ensure reliable redetection of ventricular fibrillation in patients using the implanted nonthoracotomy lead system. Thus, the potential risk of sudden cardiac death may persist in these patients despite defibrillator therapy.


Radiofrequency catheter ablation for the treatment of human type 1 atrial flutter. Identification of a critical zone in the reentrant circuit by endocardial mapping techniques.

Recent studies of human type 1 atrial flutter demonstrated reentry in the right atrium and an area of slow conduction in the low posteroseptal right atrium. Direct-current catheter ablation of this area has been only moderately successful in preventing recurrence. Therefore, we performed endocardial activation mapping and entrainment pace mapping during atrial flutter to determine the critical site for radiofrequency ablation of this arrhythmia. Twelve consecutive patients (seven men and five women; age, 21-73 years) with type 1 atrial flutter (mean cycle length, 253 +/- 39 msec) underwent right atrial endocardial activation and entrainment pace mapping using standard transvenous catheter techniques to localize the atrial flutter reentrant circuit, the area of slow conduction, and the exit site from the area of slow conduction. Upon identifying appropriate sites, radiofrequency energy (16-29 W) was applied via a 4-mm tipped catheter. Activation mapping of atrial flutter revealed a counterclockwise reentrant wave front originating just inferior or posterior to the coronary sinus ostium, proceeding superiorly in the atrial septum to the right atrial free wall, then inferiorly toward the tricuspid annulus and finally medially between the inferior vena cava and the tricuspid annulus, where low-amplitude fragmented electrical activity was noted. Entrainment pace mapping from this area produced an exact P wave match to atrial flutter on 12-lead ECG with a long (greater than 40 msec) stimulus-to-P interval indicating slow conduction, whereas pacing just inferior or posterior to the coronary sinus ostium produced an exact P wave match with a short stimulus-to-P interval (less than 40 msec), presumably identifying the exit site from the area of slow conduction. Radiofrequency energy (one to 14 applications) was effective in terminating and preventing reinduction of atrial flutter in 10 patients. In two patients, atrial flutter was not terminated during radiofrequency energy application but during subsequent pacing attempts. Sites where ablation was successful, located just inferior or posterior to the coronary sinus ostium, were characterized by discrete electrograms with activation times of -20 to -50 msec before P wave onset and exact entrainment pace maps with a stimulus-to-P interval of 20 to 40 msec, consistent with the exit site from the area of slow conduction. Follow-up (mean, 16 +/- 9 weeks; range, 2-31 weeks) revealed recurrence of the original atrial flutter in two patients, one of whom underwent repeat ablation without further recurrence, self-limited infrequent recurrence of a new atrial flutter or atrial fibrillation in three suppressed by beta-blocker or digoxin, and no recurrence in seven. 1) Radiofrequency energy applied to a critical area in the atrial flutter reentrant circuit, inferior or posterior to the coronary sinus ostium, will terminate and prevent arrhythmia reinduction. 2) Long-term follow-up in a larger series of patients will be required to confirm efficacy of this technique, although short-term results look promising.


Endogenous fibrinolytic system in chronic large-vessel thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a disorder characterized by pulmonary arterial hypertension as a consequence of organized thrombotic material in the central pulmonary arteries. Incomplete resolution of acute pulmonary emboli is believed to be pathogenically important; however, the mechanism for poor thrombus dissolution remains to be explained. We undertook this study to assess the major determinants of plasma fibrinolysis in patients with CTEPH (n = 32). Immunological and functional levels of tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) were quantified in platelet-poor plasma (PPP) from patients with CTEPH as well as age-matched controls. Although basal PPP t-PA antigen levels (CTEPH mean, 29.5 ng/ml; control mean, 2.7 ng/ml) and PAI-1 antigen levels (CTEPH mean, 55.8 ng/ml; control mean, 21.0 ng/ml) were higher in the CTEPH group, no between-group differences were detected in the enzymatic activities of these two molecules. The CTEPH group demonstrated a greater rise in t-PA antigen (CTEPH mean rise, 53.0 ng/ml; control mean rise, 5.6 ng/ml) and PA activity (CTEPH mean rise, 10.5 IU/ml; control mean rise, 1.2 IU/ml) than controls in response to an experimentally induced venous occlusion. Immunoprecipitation and fibrin autography of PPP from two patients with markedly elevated basal t-PA antigen levels demonstrate that the t-PA antigen was present in PPP primarily in complex with PAI-1. Although abnormalities of the fibrinolytic system were detected, neither a high resting plasma PAI-1 activity nor a blunted response of t-PA to venous occlusion can be invoked as an etiology for CTEPH:


Dose-dependent smooth muscle cell proliferation induced by thermal injury with pulsed infrared lasers.

Recently, laser-heated and radio frequency-heated balloon angioplasty techniques have been proposed as a means to treat or minimize dissection and elastic recoil but have been associated with a high rate of clinical restenosis. Similarly, pulsed laser angioplasty techniques proposed to minimize thermal injury while ablating obstructing atheroma have failed to reduce clinical restenosis. Because "hot balloon" and pulsed laser angioplasty create both mechanical and thermal injury, it has been difficult to discern the cause of the smooth muscle cell (SMC) proliferation resulting in restenosis and whether such magnitude of proliferation is predictable and dose related. This study was undertaken to explore these issues. Localized thermal lesions accompanying efficient ablation were created with a pulsed Tm:YAG laser in nine rabbit aortas, which consistently led to a focal proliferation of SMC that filled the ablated region by 4 weeks. Transcutaneous Ho:YAG pulsed laser irradiation at multiple independent sites of 24 central rabbit ear arteries without ablation led to brief approximately 30 degrees C thermal transients and thermal damage to the artery wall resulting in significant neointimal proliferation by 3 weeks and a mean cross-sectional narrowing of 59 +/- 17% at a dose of 390 mJ/mm2. Acute and chronic responses to varying total energy deposition were studied by histology after the rabbits were killed at 2 hours to 4 weeks. Arterial segments midway between laser injuries were unaffected and served as internal controls. Neointimal proliferation at 3 weeks after laser injury exhibited a clear dose dependence. Mean cross-sectional narrowing increased from 34 +/- 10% to 85 +/- 15% as laser fluence increased from 240 mJ/cm2 to 640 mJ/cm2 (r = 0.84). Similarly, cross-sectional narrowing caused by SMC neointimal proliferation increased from 20 +/- 10% to 77 +/- 17% for a fixed surface irradiation as the depth of the most superficial arterial media decreased from 600 microns to 330 microns (r = 0.94). Thermal injury to the arterial wall is a potent stimulus for SMC proliferation and may necessitate reduction in laser or thermal energy used for angioplasty. Moreover, a dose-response relation exists between the degree of thermal injury and SMC proliferative response. Hence, this technique could be used as a practical model of restenosis suitable for screening therapies for inhibition of SMC proliferation.


Effect of ultrasound on tissue-type plasminogen activator-induced thrombolysis.

The efficacy of fibrinolytic therapy is limited by the small surface area of the clot that is available for the binding of the thrombolytic agent, such as tissue-type plasminogen activator (t-PA). We hypothesized that exposure of the clot to ultrasound during thrombolytic treatment could enhance lysis through perturbation of the thrombus, which would expose additional fibrin binding sites for t-PA. Whole human blood clots containing radiolabeled fibrinogen were incubated in vitro for 200 minutes with Tris-albumin buffer containing t-PA at concentrations ranging from 3 to 3,000 IU/ml. In paired experiments, one of the clots also was exposed to intermittent ultrasound (1 MHz, 1.75 W/cm2) throughout the experiment. The ultrasound was delivered as a 2-second exposure followed by a 2-second rest interval. The overall difference in mean clot lysis between thrombi receiving ultrasound and those receiving no ultrasound was significant (p less than 0.001) at all concentrations of t-PA. For clots incubated with t-PA at a concentration of 300 IU/ml, ultrasound increased the percent lysis at 200 minutes from 42 +/- 5% (mean +/- SEM) to 64 +/- 10%. In six paired experiments in a rabbit jugular vein thrombosis model, rabbits received 1 mg t-PA alone or t-PA and intermittent ultrasound (1 MHz, 1.75 W/cm2) for 200 minutes. For rabbits receiving ultrasound and t-PA, lysis was 55 +/- 11% at 100 minutes compared with 30 +/- 12% for rabbits receiving only t-PA. Lysis was 6 +/- 10% for rabbits (n = 4) receiving ultrasound alone. No evidence for tissue damage was noted in rabbits exposed to intermittent ultrasound. Exposure of whole blood clots in vitro to intermittent ultrasound combined with t-PA caused a significant enhancement of thrombolysis compared with t-PA alone. Intermittent ultrasound also showed a trend toward enhancement of t-PA-induced clot lysis in an animal thrombosis model. These data suggest that noninvasive intermittent ultrasound may be a useful adjunct to thrombolytic therapy.


Myocardial function and transmural blood flow during coronary venous retroperfusion in pigs.

The degree of recovery of regional myocardial contraction during coronary venous retroperfusion has not been well established, particularly in the absence of coronary collateral channels. Therefore, the maximal functional benefit attainable with coronary venous retroperfusion was assessed in pigs by means of using selective pump retroperfusion of the left anterior descending vein, with venting of the left anterior descending artery to zero pressure. In eight anesthetized open-chest pigs during selective left anterior descending venous retroperfusion over a range of retroperfusion flows, regional myocardial function (percent systolic wall thickening by sonomicrometry) increased progressively to an average of 62% of control values at a retroperfusion flow rate 200% of control arterial flow. Progressive thickening of the end-diastolic dimension of the anterior wall was observed with increasing retroperfusion flow (from 8.7 +/- 0.9 to 10.7 +/- 2.3 mm, p less than 0.001). Perfusion pressures within the left anterior descending vein increased linearly with increased retroperfusion flow rates (up to 132 +/- 57 mm Hg with retroperfusion flow 200% of control). A gradual increase of retrograde left anterior descending arterial outflow was observed with increasing retroperfusion flows; however, the absolute amount (maximum, 8.3 +/- 4.1 ml/min) was much too low to explain the extent of functional recovery. Transmural myocardial capillary blood flows in the anterior wall with retroperfusion flows of 100% and 200% of control arterial flow were 0.22 and 0.42 ml/min/g with corresponding subendocardial blood flows of 0.14 and 0.29 ml/min/g; ratios of endocardium to epicardium were 0.51 and 0.61, respectively. Thus, capillary blood flows during selective retroperfusion were relatively low despite considerable restoration of regional systolic wall thickening, and a significant difference was noted in the slopes of the relations between regional systolic wall thickening and myocardial blood flow during retroperfusion and anterograde arterial perfusion (p less than 0.05). With retrograde injection of silicone elastomer at different retroperfusion pressures (50, 75, and 100 mm Hg) in three pigs, capillaries were well visualized, and profuse intramyocardial venous anastomotic connections were seen at the highest retroperfusion pressure (100 mm Hg), whereas there was filling of small venules but little capillary filling at the lowest retroperfusion pressure (50 mm Hg). Considerable recovery of regional myocardial function with low regional capillary blood flows were observed during acute venous retroperfusion with high retroperfusion flows with arterial blood. These findings together with low levels of retrograde arterial outflow and visualization of retrograde capillary filling with a rich venous network provide evidence for possible oxygen delivery via the intramyocardial venous plexus.


Changes in the radius of curvature of the ventricular septum at end diastole during pulmonary arterial and aortic constrictions in the dog.

At end diastole, the position and shape of the ventricular septum depend on the transseptal pressure gradient. It is not clear, however, how the septal radius of curvature changes in response to the gradual change in transseptal pressure gradient during progressive pulmonary arterial constriction (PAC) and aortic constriction (AC). In 11 anesthetized open-chest dogs, the septal radius of curvature was measured from the short-axis two-dimensional echocardiogram, and the transseptal pressure gradient (left ventricular [LV] pressure minus right ventricular [RV] pressure) was calculated from ventricular pressures measured with micromanometers. Seven dogs were studied with both PAC and AC (group 1) and four dogs only with PAC, which was initiated before and after volume loading (group 2). The transseptal pressure gradient decreased during PAC. As the transseptal pressure gradient decreased, the septum shifted continuously leftward with decreases in the LV septum-free wall diameter and in LV cross-sectional area. The septal radius of curvature (Rs) increased until the septum became flat. The flat septum (i.e., Rs = infinity) occurred at a relatively constant value of transseptal pressure gradient (-4.6 +/- 1.4 mm Hg) independently of the absolute values of LV pressures when between 2 and 9 mm Hg, although necessarily a greater RV pressure was needed to make the septum flat when LV pressure was higher. After inversion, the septum again became curved, with a decrease in the absolute value of septal radius of curvature as the transseptal pressure gradient became increasingly negative. The septum was still concave to the LV cavity at zero transseptal pressure gradient, and its curvature decreased (i.e., its radius of curvature increased) with increases in ventricular pressures. During AC, the septal radius of curvature also increased, but with an increase in transseptal pressure gradient accompanied by increases in LV septum-free wall diameter and in LV area. In group 2 animals, at zero transseptal pressure gradient, the normalized septal radius of curvature was greater (p less than 0.005) at high LV pressure than at low LV pressure. The transseptal pressure gradient required to make the septum flat was not significantly different between low and high LV pressure, which confirmed the results of group 1. The results of the present study show that the shape and position of the ventricular septum are determined by the transseptal pressure gradient but that the shape of the septum is also affected by the ventricular pressures. The septum was not flat but rather still concave to the LV cavity at zero transseptal pressure gradient. Approximately 5 mm Hg of negative transseptal pressure gradient was required to displace the septum farther leftward and make it flat. The septal radius of curvature increased during both PAC (which decreased transseptal pressure gradient) and AC (which increased transseptal pressure gradient), indicating that the mechanisms involved in changing septal radius of curvature are different during PAC and AC.


Mechanoenergetic effects of pimobendan in canine left ventricles. Comparison with dobutamine.

We hypothesized that the effect of pimobendan (UD-CG 115 BS) to increase calcium sensitivity of contractile protein might result in less myocardial oxygen consumption (VO2) in comparison with dobutamine when they enhance ventricular contractility to the same extent. To examine this hypothesis, we compared the effects of pimobendan and dobutamine on left ventricular contractility and energetics using the frameworks of Emax (contractility index) and the relation between VO2 and PVA (systolic pressure-volume area, a measure of left ventricular total mechanical energy). We measured VO2, Emax, PVA, and force-time integral (FTI) in excised, cross-circulated, nonfailing dog hearts. The slope of the VO2-PVA relation reciprocally indicates the efficiency from PVA-dependent VO2 to the total mechanical energy (contractile efficiency). The VO2 intercept of the VO2-PVA relation, i.e., PVA-independent VO2, reflects energy utilization for excitation-contraction coupling. The ratio of FTI to PVA-dependent VO2 can be called contractile economy. Both drugs comparably enhanced Emax. Although the contractile economy was greater by 14 +/- 19% (p less than 0.05) for pimobendan than for dobutamine, the contractile efficiency was similar between the two drugs. Oxygen cost of contractility, defined as the slope of the relation between the PVA-independent VO2 and Emax, was the same between the two drugs. Other mechanoenergetic effects of both drugs were similar except for a greater coronary vasodilating effect of pimobendan. Pimobendan has almost the same mechanoenergetic effects as dobutamine but slightly greater contractile economy and coronary vasodilation. The calcium-sensitizing effect of pimobendan did not save the oxygen cost of contractility.


Endogenous nitric oxide protects against platelet aggregation and cyclic flow variations in stenosed and endothelium-injured arteries.

This study was designed to test the hypothesis that endogenously produced nitric oxide protects against platelet aggregation and cyclic flow variations in stenosed and endothelium-injured arteries of mongrel dogs. NG-Monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide formation, was administered at 5 mg/kg to 15 dogs after the left anterior descending coronary artery was mechanically injured and narrowed by external constrictors and to nine dogs before endothelial injury of the femoral artery and after injury and moderate arterial constriction. Treatment with L-NMMA resulted in cyclic flow variations (as detected by external Doppler flow probes) in the left anterior descending artery of seven of 15 dogs and in the femoral artery of four of nine dogs after endothelial injury. L-Arginine, the precursor for nitric oxide synthesis, was administered at 60 mg/kg and abolished cyclic flow variations in each of the 11 dogs. D-Arginine did not change the L-NMMA-induced cyclic flow variations. Saline infusion did not induce or change cyclic flow variations in any of the animals. Acetylcholine (1, 10, and 100 micrograms/min; n = 9) was administered in the femoral artery of nine additional dogs before and after endothelial injury in moderately stenosed femoral arteries. Acetylcholine did not induce cyclic flow variations in any animal; however, it did increase the severity of cyclic flow variations that developed in severely stenosed arteries. The diameter of the femoral artery was measured by intravascular ultrasound imaging. L-NMMA caused vasoconstriction of normal arteries, but no change was detected in endothelium-injured arteries. In contrast, L-arginine caused vasodilation of normal arteries, but, again, no change was noted in endothelium-injured arteries. Acetylcholine dilated normal femoral arteries but constricted arteries with endothelial injury. In both in vitro and ex vivo platelet studies, L-NMMA enhanced platelet aggregation, whereas L-arginine significantly reduced platelet aggregation. D-Arginine and acetylcholine showed no effect on platelet aggregation. Promotion of nitric oxide production decreases platelet aggregation and may eliminate cyclic flow variations, whereas a reduction in nitric oxide formation enhances platelet aggregation and may induce cyclic flow variations. Acetylcholine causes vasoconstriction at the femoral arterial site of endothelial injury and may increase the severity of cyclic flow variations.


Role of myocardial ATP-sensitive potassium channels in mediating preconditioning in the dog heart and their possible interaction with adenosine A1-receptors.

A brief period of myocardial ischemia can result in an increased resistance to subsequent, more severe episodes of ischemia. Recent studies have indicated that activation of adenosine A1-receptors may mediate this preconditioning effect. It is also known that A1-activation can lead to ATP-sensitive potassium channel (KATP) opening via a G(i) protein-mediated effect. Thus, we determined whether the KATP blocker glyburide could abolish preconditioning or the protective effects of A1-receptor activation. Anesthetized dogs were subjected to 5 minutes of left circumflex coronary artery (LCx) occlusion (or sham) followed by 10 minutes of reperfusion. The hearts were then subjected to 60 minutes of LCx occlusion and 5 hours of reperfusion. Glyburide (5 micrograms/kg/min) or vehicle was given directly into the LCx 20 minutes before preconditioning or sham preconditioning. Preconditioning resulted in a significantly reduced infarct size compared with nonpreconditioned animals. Glyburide abolished the protective effect of preconditioning. To establish a link between KATP and A1-receptor activation, the effect of the A1-agonist R-PIA with or without glyburide on infarct size was determined. R-PIA (0.4 microgram/kg/min, directly into the LCx) significantly reduced infarct size, and this protective effect was abolished by glyburide. None of the treatments described above had a significant effect on peripheral hemodynamic status or myocardial blood flow. Preconditioning may be mediated by KATP activation, and this may be linked to A1-receptor stimulation.


Left atrial contribution to ventricular filling during the course of evolving heart failure.

Abnormal left ventricular (LV) filling has been observed in patients with heart failure and is characterized by marked heterogeneity of mitral inflow velocity. In the present study, the contribution of the left atrium to LV filling was examined in eight dogs during the course of evolving heart failure. Heart failure was produced by multiple sequential intracoronary embolizations with microspheres. Pulsed Doppler echocardiography was used to measure mitral inflow velocity at baseline, before embolization, and at 3, 8, 15, 23, and 33 weeks after initiation of microembolization. The early rapid LV filling (Ei) and late left atrial filling (Ai) components were quantitated based on the time-velocity integral of the early and late mitral inflow velocity waveforms, respectively. Ei decreased progressively from 7.6 +/- 1.5 cm at baseline to 4.0 +/- 0.4 cm at 33 weeks (p less than 0.01). In contrast, Ai initially increased from 1.8 +/- 0.9 cm at baseline to 2.7 +/- 0.4 cm at 3 weeks (p less than 0.01) and subsequently decreased gradually to below baseline values reaching 0.8 +/- 0.4 cm at 33 weeks (p less than 0.01). These temporal changes of Ei and Ai were accompanied by a gradual reduction of LV ejection fraction (56 +/- 5% versus 22 +/- 2%) (p less than 0.01) (baseline versus 33 weeks) and by a gradual increase of LV end-diastolic wall stress (24 +/- 7 versus 92 +/- 8 g/cm2) (p less than 0.01), left atrial dimension (2.4 +/- 0.2 cm to 3.3 +/- 0.3 cm) (p less than 0.01), and left atrial fractional shortening (22 +/- 3% versus 15 +/- 2%) (p less than 0.01). The initial rise in left atrium contribution to LV filling may represent a compensatory response to the diminution of the rapid early component of LV filling. With further progression of LV dysfunction, the left atrium contribution to LV filling gradually decreased. This reduction may be mediated by increased workload imposed on the left atrial myocardium due to increased LV diastolic wall stress, which, over time, may have lead to intrinsic left atrium dysfunction.


[Normalization of acupuncture anesthesia used in neurosurgery].

From March 1975 to February 1982 and from April 1987 to October 1990, the national cooperative neurosurgical acupuncture research group had already accumulated the clinic data of 5,244 cases totally, consisting of 2,107 cases in frontal fossa, 1951 cases in the temporo-parieto-occipital region and 1,186 in posterior fossa. By the same manipulative procedures and scaling criteria, the indications, choices of acupoints, stimulus parameters, adjuvants, preoperative measurements, and physiological and biochemical changes during operations were studied. Practically, the results was not only reliable, but repetitive highly. 95% of the cases in frontal fossa belonged to grade I (success), 91.5% of the cases in temporo-parieto-occipital region was grade I and 89.38% of the cases in posterior fossa was grade I. We suggest that acupuncture anesthesia should be widely used as one of the usual methods of anesthesia. In this paper, the relative specificity of acupoints, the mechanism of adjuvants, personal differences and preoperative measurements were discussed. In the meantime, the advantages and the remaining problems of acupuncture anesthesia in craniocerebral operations were also mentioned.


[Effect of prefrontal cortex on the neuronal activity of the nucleus raphe magnus in rats].

The unit discharge of neurons in nucleus raphe magnus (NRM) were recorded with glass microelectrodes in 33 waking and tubocurarine-immobilized rats. It is observed that effects of the noxious stimulation and prefrontal cortex (PFC) stimulation on spontaneous discharges of NRM neurons and effects of PFC stimulation on noxious-evoked discharges of NRM neurons by stimulating sural nerve. The results indicated that noxious stimulation and PFC stimulation could increase spontaneous discharge of most (68.18% and 60.82% respectively) NRM neurons. It was also noted that most (49/80) NRM neurons altered their spontaneous discharge in response to noxious stimulation and PFC stimulation in a similar manner. The remainder (31/80) opposite manners. PFC stimulation could alter the response of NRM neurons to noxious stimulation. PFC stimulation could enhance or reduce noxious-evoked discharge in NRM neurons which alter their spontaneous discharge in response to noxious stimulation and PFC stimulation in a similar manner. PFC stimulation could reduce noxious-evoked discharge in NRM neurons which alter their spontaneous discharge in opposite manners. The above results suggest that PFC and NRM have a functional relation. PFC could modulate nociceptive response and it might be partly responsible for influence on activity of neurons in NRM.


[Comparison of effects of EA at "hegu" on nociceptive responses of NRM neurons to noxious stimulation of the face and tail].

The experiments were performed on the rats. Unit discharges in NRM and their nociceptive responses to dental pulp and tail noxious stimulations were recorded extracellularly with glass microelectrodes. We observed that EA at the "Hegu" acupoint could increase spontaneous discharges of NRM neurons and inhibit their nociceptive responses to noxious stimulation acting on dental pulp and tail. It is interestingly noted that the inhibitory effect of EA on noxious stimulation applying on the pulp is stronger than the tail.


Influence of acupuncture on the discharge of PHA neurons in the rabbit.

This paper deals with whether the posterior hypothalamus area (PHA) can receive signals from electroacupuncture (EA) at "Neiguan", and whether a relative specificity does exist between acupoints or acupoint and non-acupoint. The neuron activity was recorded extracellularly for analyzing the influence of EA at different acupoints and non-acupoint on the discharge of PHA neurons.


[Effects of head point needling on cardiac function and hemodynamics].

The present investigation was undertaken to observe the effects of head point-cardiovascular point needling on the cardiac function and hemodynamics in 8 anesthetized dogs, and to elucidate the underlying mechanisms and its clinical implications. Cardiovascular point needling might induce significant changes in cardiac function and hemodynamic parameters: arterial systolic pressure decreased by 22.4 +/- 8.88 from 104.6 +/- 20.55 mmHg, diastolic pressure by 16.7 +/- 8.04 from 66.5 +/- 18.03 mmHg, mean arterial pressure by 18.6 +/- 8.16 from 79.2 +/- 18.36 mmHg, left ventricular end-diastolic pressure by 0.3 +/- 0.47 from 3.6 +/- 1.94 mmHg, Lv dp/dt max by 300.1 +/- 200.1 from 2000.1 +/- 700.8 mmHg/s, coronary resistance by 399 +/- 310 from 1959 +/- 1150 dyn.s.cm-5, femoral arterial resistance by 242 +/- 634 from 2438 +/- 1595 dyn.s.cm-5, total peripheral resistance by 1570.7 +/- 691.0 from 9000.2 +/- 2537.4 dyn.s.cm-5, and left ventricular work index by 2.7 +/- 1.9 from 11.4 +/- 5.17 kg.m/min/m2, whereas the coronary sinus blood flow increased by 0.7 +/- 1.6 from 33.6 +/- 14.7 ml/min. There were significant differences between the values of all parameters before and after cardiovascular point needling (p greater than 0.005). The results indicated that cardiovascular point needling was capable of lowering the cardiac preload and afterload with resultant fall in blood pressure and cardiac oxygen consumption, as well as relaxing the coronary vessels and consequent increase in coronary blood flow with enhanced oxygen supply. These beneficial effects induced by cardiovascular point needling provided the basis for the treatment of hypertension, coronary artery diseases and cardiac failure in clinic.


[Effect of electroacupuncture on myocardial oxygen metabolism and pH of coronary sinus blood during experimental angina pectoris].

The experiments were performed on 30 healthy mongrel dogs, intubation was taken in the left anterior descending coronary (LAD), the blood in carotid was transported to LAD through a peristalic pump, the blood flow was reduced to 3-5 ml/min, thus acute myocardial ischemia was produced. Basing on this condition, 0.1-0.16 ml bradykinin (2 ug/ml) was given into LAD coronary before recording to produce angina pectoris. The effects of electroacupuncture (EA) at "Neiguan" area on myocardial oxygen metabolism, pH of coronary sinus blood and myocardial contractile force were observed (EA intensity 5 volts, frequency 1-20 Hz). The results are as follows: 1. EA could reduce obviously A-V difference of blood oxygen capacity (Ca-vO2) and the rate of myocardium extracting oxygen (O2E), thus reduced obviously oxygen consumption of ischemic myocardium. 2. EA could reduce V-A difference of carbon dioxide partial pressure (Pv-aCO2), prevent the decrease of pH of coronary sinus blood (PHv), this indicated that EA could prevent accumulation of acidic metabolic products. 3. EA could increase myocardial developed tension (DT) of ischemic area, strengthen myocardial contractile force of ischemic area. Above results indicated that EA could reduce oxygen consumption of ischemic myocardium, prevent the decrease of pH of coronary sinus blood, thus myocardial cell acidosis was prevented, myocardial contractile force was strengthened. It might be the mechanism of acupuncture treating coronary heart disease.


[Effect of electroacupuncture point on the value of ventricular fibrillation threshold in rats].

The Neiguan and Lingdao were stimulated by electroacupuncture in anesthetized Wistar rats. We observed that the value of ventricular fibrillation threshold (VFT) was increased significantly in 30 min of electro-acupuncturing (P less than 0.01) and in 15 min of stopping the acupuncturing (P less than 0.05). Although the value of VFT was increased until 30 min of stopping acupuncture, it was not significantly different compared with its basic value. The control group were dealt with the same way as the experimental group except electroacupuncture. The value of VFT measured in control group were not significantly different compared with its basic value.


[The role of ear electroacupuncture on arterial pressure and respiration during asphyxia in rabbits].

This paper reports the study of the effect of ear electroacupuncture (EA) inserted in the "Er Jian" point on arterial pressure and integrated phrenic nerve discharge (IPND). The result showed that the arterial pressure was decreased and frequency of IPND was increased. The differences as follows: During spontaneous breathing, the blood pressure and IPND were -36.56 +/- 26.63 mmHg and, 2.42 +/- 1.42 spikes/10S (P less than 0.001); in excitatory period, -24.31 +/- 6.01 mmHg and, 2.16 +/- 1.12 spikes/10S (P less than 0.01) and in inhibitory period -18.26 +/- 7.04 mmHg (P less than 0.01) and, 2.07 +/- 1.07 spikes/10S (P less than 0.05). The lasting time was 26 min, 330S and 280S. We observed 20 cases with hypertension by ear electro-acupuncture. The systolic pressure and diastolic pressure of hypertension patients were decreased (P less than 0.001) and respiratory rate increased (P less than 0.05).


[A study on the effect of stimulating auricular points on the biliary tract].

We have developed an animal model to study the effect of stimulating auricular points on the function of hepato-biliary system. The preliminary result shows that 14 of 18 rabbits acquired a marked increase (P less than 0.05) in the amount of heptic bile secretion 3 min and 7 min after point No. 3 stimulated with 58V.


[The effect of electroacupuncture "zusanli" and "neiguan" points on membrane fluidity of red cells in rabbits].

The effect of electroacupuncture "ZUSANLI" and "NEIGUAN" points on membrane fluidity of red cells in rabbits, has been determined by fluorescence polarization method. Results showed that the membrane fluidity of red cells was developed evidently after electro-acupunctured "NEIGUAN", but "ZUSANLI" had no effect on it, and it was significantly developed by electroacupuncture "ZUSANLI" and "NEIGUAN" points at the same time.


[The role of peripheral C afferent fiber in electroacupuncture analgesia].

Experiments were carried out in rats anaesthetized with urethane (1 g/kg). Late discharges of spinal dorsal horn neurons were recorded as noxious responses to strong stimulation of right tibial nerve (a train of 3 pulses, 25V, 1ms). Left St.36 and Sp.6 were stimulated electrically (100Hz, 3V or 6V, 1ms). Left sciatic nerve was exposed and soaked for 15 min in 1.5% capsaicin or vehicle 24 hours before experiment. Left femoral nerve was sectioned. Spinal cord was transected at T4 level in spinal rat. Intact rats: 1) EA group (N = 12). Sciatic nerve was not treated. Late discharges of neuron were reduced to 44.6 +/- 18.3% of control (P less than 0.02) by EA(3v). 2) Capsaicin group (N = 15). Late discharges were reduced to 89.8 +/- 6.0% of control (P greater than 0.05) by EA(3V). 3) Vehicle group (N = 10). Late discharges were reduced to 51.2 +/- 15.6% of control (P less than 0.05) by EA(3V). 4) There was a statistical difference (P less than 0.05) between capsaicin and vehicle group. Spinal rats: 1) EA group. Late discharges of neuron were reduced to 67.6 +/- 10.3% of control (P less than 0.02) in 11 neurons by EA(3V), 72.7 +/- 8.8% (P less than 0.01) in 9 neurons by EA(6V). 2) Capsaicin group. Late discharges were 110 +/- 21.0% of control (P greater than 0.05) in 13 neurons after termination of EA (3V), 90.7 +/- 12.9% (P greater than 0.05) in 10 neurons after EA (6V). 3) Vehicle group. Late discharges were reduced to 67.7 +/- 8.1% of control (P less than 0.02) in 12 neurons by EA (3V), 68.1 +/- 5.0% (P less than 0.01) in 10 neurons by EA (6V). 4) There was a statistical difference (P less than 0.05) between capsaicin and vehicle group for 3V, no statistical difference (P greater than 0.05) for 6V. C fiber of peripheral nerve is main component involved in effect of EA analgesia in intact rats. Both A and C fiber of peripheral nerve involved in effect of EA analgesia in spinal rats.


[Effect of He-Ne laser acupuncture on lymph-nodes in rats].

The lymphocytes and antigen presenting cells in lymph node of rats stimulated by He-Ne laser acupuncture were observed by using TEM and SEM to investigate the ultrastructural changes of them. There were numerous activated T-cells which showed deeply indented nucleus, abundant small void mitochondria and free ribosomes in the paracortex area. The B-cells were gradually differentiated into large lymphocytes, immature and mature plasmatic cells which with a lot of rough endoplasmic reticulum. They were prominently increased in the pulp area. The macrophages had short processes with numerous folds and microvilli and tended to neighboring lymphocytes. The nucleus pores were increased. There were a lot of pinocytosomes, phagosomes, lysosomes in various size of macrophages. The bundles (5-6 nm in diameter) of microfilaments of the macrophages were extended from the cytoplasm to the processes. The interdigitating cells which contained the characterized single layer of rER, numerous polysomes, mitochondria and well-developed Golgi-complex were closed to macrophages and lymphocytes. In conclusion the activities of the cellular immunity and humoral immunity were enhanced by laser.


[The influence of different acupuncture manipulations on the plethysmogram of the patient with kidney deficiency].

In order to prove the specific property between the tonifying manipulation (TM) and reducing manipulation (RM), and search further for the key in improving the effect of manipulation, the influence of to on two different manipulations. The plethysmogram in the same body-condition (kidney-deficiency) One was TM with slow-trusting and quick lefting; the other was RM with the converse movement. Randomized block design, cross-over design and single-blind design were used. The result showed that the TM increased the amplitude of the plethysmogram obviously, whereas the RM had no significant influence on it. There was significant difference not only between the TM group and the central group, but also between the TM group and RM group (P less than 0.01 or P less than 0.05). The difference between the RM group and the control group was not significant (P greater than 0.05). Meanwhile, it was recommended that "mind concentration" on acupuncture manipulation is important. In conclusion, the experiment demonstrated the experimental evidence for explaining the mechanism of TM and RM. It was suggested that acupuncture manipulations are worthy to pay attention so as to improve clinical effect.


[Electron microscopic observation on the effect of electroacupuncture (EA) on the ultrastructure of nucleus raphe dorsalis (NRD) in rats].

Nucleus raphe dorsalis (NRD) plays an important role in acupuncture analgesia. The aim of this investigation is to determine the effects of electroacupuncture (EA) analgesia on the ultrastructure of NRD. 12 Wistar rats (220-250g) were divided into control and experiment groups. Pain threshold was determined by potassium iontophoretic colorimetry. EA was applied at bilateral "Zusanli" points. The effective analgesia animals and control animals were sacrificed and the NRD were taken out for electron microscopic observation. In EA analgesia groups, NRD was observed the number of the clear round vesicle-containing terminals and clear round vesicles with the granular vesicle-containing terminals showed a significant decrease and sometimes vesicles were emptied. The area of presynaptic terminals were expanded. Some synaptic gaps appeared narrow profiles. Part of the mitochondria and endoplasmic reticulum in neuron and neurogliocytes appeared the expanded profiles. These ultrastructural change of the NRD in EA analgesia might indicate the neurons and neurogliocytes were in active functional state.


[Preliminary observation of imaging of facial temperature along meridians].

Visualization of meridians has been carried out by means of infrared thermography and radionuclide scintigraphy, but real success was hardly achieved on some special regions including facial channels. In this paper, we would represent some imagings of thermal line along channels by means of infrared thermography. Distribution of the facial temperature in 139 cases of healthy volunteers and 305 cases of patients with facial paralysis was observed with model AGA-782 infrared thermovision and TC-800 computer. In all cases of patients and 15 cases of healthy persons, acupuncture test was conducted. Special program, DISCO3.1 was used to process the thermographic data and the results were as follows: 1. Facial thermal line along channel was presented in 95 out of 444 cases. High temperature line along bladder channel appeared in 34 cases of them; 2. In 26 out of 95 cases, the thermal line appeared after acupuncture, which could improve the imaging of the line; 3. The range of the temperature was within 1.3 degrees C, in some cases only 0.2-0.3 degrees C, with +/- 0.5-1.0 degrees C difference to surrounding temperature; the width of the line was 1-1.5 cm in most cases; 4. The line could appear stably, in some cases, last for several days, weeks or months; 5. Certain relationship between the appearing of the line and facial paralysis was found. As mentioned above, the thermal characteristics of meridians in face could be visualized by means of infrared thermography, the special imaging of facial temperature along bladder meridian was easily found, related to facial troubles and promoted by acupuncture.


[Preliminary observation on the relation among needling sensation, propagated sensation along meridian (PSM), and acupuncture effect when acupuncture neiguan].

This paper reports 300 coronary heart disease patients with different needling sensations, mainly with distension, when acupuncturing Neiguan by identical acupuncture doctor with same manipulation. The higher rate of PSM appearance and better acupuncture effect were observed, in patients with compound sensation, such as sourness-distension and distension-numbness. All of the patients who felt pain did not appear PSM and the acupuncture effect was poor. The appearance rate of PSM and acupuncture effect of other kind needling sensations were between the two. The results showed the kind of needling sensation while acupuncture had close relation with the appearance of PSM and the acupuncture effect.


Expression of inherent neuronal shape characteristics after transient sensitivity to epigenetic factors.

We investigated effects of different substrates and culture media on the early morphological differentiation of rat neocortical neurons in culture. In particular, we examined the effects of homotypic astrocytes, the adhesive glycoprotein laminin and the polycationic substrate poly-L-lysine, as well as diffusible astrocyte-derived conditioned medium factors and serum on (1) soma area, (2) total neuron area and (3) primary neurite number. To assess variations in morphological reactions of neurons with a defined neurotransmitter phenotype, we analyzed the differentiation of GABAergic neurons. The morphology of young neocortical neurons was dramatically affected by both substrate and culture medium. Replacement of the astrocytic monolayer or the astrocyte-conditioned medium by other substrates or non-conditioned medium, respectively, was accompanied by (1) spreading and flattening of neuronal somata, (2) a marked decrease in total neuron area and (3) an increase in the number of primary neurites. The various morphological parameters studied exhibited different sensitivities to changes of these external factors. Moreover, the influences of epigenetic factors on the generation of primary neurites depended on the transmitter phenotype of the neuron. The induced morphological alterations were transient. At the end of the first week in culture, the surviving neurons underwent substantial remodeling of their morphology leading to an expression of in vivo shape characteristics. These observations suggest that despite an early, transient sensitivity to environmental influences, the neuronal differentiation with respect to the morphological parameters studied in culture is to a large degree determined by intrinsic factors.


Pre- and postnatal development of high-affinity [3H]nicotine binding sites in rat brain regions: an autoradiographic study.

The ontogeny of high affinity nicotinic cholinergic binding sites was studied in Long-Evans rat brain by in vitro autoradiography, using [3H]nicotine (10 nM) and cold (-)nicotine bitartrate to assess specificity. The first binding sites become detectable in spinal cord and caudal medulla oblongata at gestational day (GD) 12. Until GD 14, labelling spreads throughout lower brainstem, mesencephalon and parts of diencephalon, with higher densities in ventral areas (including the area of developing mesencephalic dopamine neurons). Matrix zones remain unlabelled. Receptor sites appear in the cerebellar anlage by GD 15, and in caudal caudate-putamen by GD 16. During development from late gestational to early postnatal stages, labelling is reduced in many lower brainstem areas and increases in forebrain, in particular in neocortex. Receptor density remains high in thalamus. In neocortex, nicotinic receptor sites are first seen in the subplate layer by GD 20. Labelling of this zone remains prominent until PN 14, when an additional band of increased receptor density is seen in cortical layers III/IV which contain high receptor levels in adulthood. At PN 27, the pattern has become similar to the adult one. The development of [3H]nicotine-binding sites in individual brain regions, with a general caudo-rostral gradient, accompanies cell differentiation and early synapse formation, e.g., in neocortex. The ontogenetic pattern differs in detail from that of muscarinic-cholinergic binding sites. The early presence of binding sites provides a basis for specific actions of nicotine on the fetal brain. As a consequence of the ontogenetic changes, different brain structures become targets for the action of this drug at different stages of development.


Administration of tryptophan-enriched diets to pregnant rats retards the development of the serotonergic system in their offspring.

It is well established that an increased availability of tryptophan stimulates serotonin synthesis not only in the adult but also in the developing brain. In order to study the influence of a permanently increased supply of tryptophan on the developing central 5-HT-system, female rats were fed a tryptophan-enriched diet from mating throughout pregnancy and lactation. The effect of this dietary regime was assessed by measurements of neurochemical markers of 5-HT innervation in the developing brain of their offspring. A diminished content of 5-HT, a decreased activity of tryptophan hydroxylase and a reduction of crude synaptosomal high-affinity 5-HT uptake was found in the cortex and in the brain stem of 5 day old rat pups of mothers fed the tryptophan-enriched diet. The postnatal increase of all three markers of serotonergic innervation in the offspring of these mothers was retarded. Both the initial depletion and the delayed maturation of 5-HT content, of tryptophan hydroxylase activity and of synaptosomal serotonin uptake were more pronounced in the cortex than in the brain stem. Apparently, the increased dietary intake of tryptophan throughout pregnancy and lactation caused a delayed outgrowth of 5-HT axons and/or reduced collateral sprouting and synapse formation in the brain of the developing offspring.


Newly generated neurons of the adult songbird brain become functionally active in long-term culture.

The vocal control nucleus, HVc, of the songbird forebrain undergoes neurogenesis in adulthood, as ventricular zone precursor cells divide and their daughter cells migrate into the subjacent forebrain, where they differentiate into neurons. We have previously demonstrated that the migration and development of these new neurons can proceed in vitro, in HVc ventricular zone explant cultures derived from the adult canary HVc. By a combination of electron microscopy and electrophysiology, we now report that these newly produced neurons become functionally mature and synaptically competent in culture. These cells developed synaptic contacts which became morphologically evident during the second week in culture, and which preceded the development of both stimulus-evoked and spontaneous action potentials during the second and third weeks in vitro. Thus, the newly generated neurons of the adult avian forebrain can form structurally complex, electrically interactive networks in long-term culture.


Expression of the F84.1 glycoprotein in the spinal cord and cranial nerves of the developing rat.

The monoclonal antibody designated as F84.1 was used for an immunohistochemical study of the developing rat nervous system. The most prominent neural components recognized by F84.1 are motor and sensory components of the spinal cord and cranial nerves. F84.1 is first detected in the dorsal root ganglia of embryonic day 11 spinal cord. The expression in the dorsal roots persists in the adult. In contrast, a more transient expression of F84.1 is found in the spinal motor system. F84.1 labels primary neurons of cranial nerves V, VIII, IX and X. F84.1 is also expressed by the non-neuronal cells of the notochord and the floor plate. Immunoprecipitation experiments from several types of cells in culture show that the F84.1 antigen is a cell-surface glycoprotein with a molecular weight of 90-105 kDa. An analysis of the amino terminal sequence demonstrates that the F84.1 antigen is similar to the chick cell adhesion molecule SC1/DM-GRASP, a member of the immunoglobulin superfamily. The pattern of expression of F84.1 in the rat differs in several aspects from that of the chick molecules, leaving a possibility that F84.1 may be a variant of SC1/DM-GRASP.


Altered development of basal and forskolin-stimulated adenylate cyclase activity in brain regions of rats exposed to nicotine prenatally.

Exposure of the fetus to nicotine is known to affect cellular development, synaptogenesis and synaptic activity of a wide variety of neurotransmitter pathways in the central nervous system. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipumps. After birth, offspring of the nicotine infused dams displayed marked alterations in membrane-associated adenylate cyclase activity; the regional selectivity correlated both with nicotinic cholinergic receptor concentration and the maturational timetable of each region. In the midbrain and brainstem, which display relatively high receptor concentrations and earliest cell development, basal adenylate cyclase activity in the nicotine group was elevated in the immediate period postpartum, returned to normal by the end of the first month, but then became subnormal in young adulthood. The initial promotion of basal activity was mirrored by forskolin-stimulated activity, suggesting that in this phase, the alterations were occurring at the level of the adenylate cyclase catalytic unit itself. The lack of effect on forskolin stimulation in the later phase, where basal activity was subnormal in the nicotine group, suggests that some alterations in regulatory subunits are responsible for the maturational switch in nicotine's effects on adenylate cyclase. In the cerebellum, where cell replication occurs primarily after birth and receptor concentrations are low, basal adenylate cyclase showed only a deficit in the nicotine group; again, although forskolin stimulation was significantly affected, the actions on basal activity were much more prominent, suggesting defects at the level of G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)


The immunolocalisation of ubiquitin carboxyl-terminal hydrolase (PGP9.5) in developing paraneurons in the rat.

Protein gene product (PGP9.5) has been detected by means of immunocytochemistry in the rat fetus in intra- and extra-adrenal chromaffin cells (E12.5), pancreatic islet cells (E12.5), anterior pituitary cells (E14.5), carotid body chief cells (E15.5), thyroid C cells (E17.5) and respiratory neuroendocrine bodies (E15.5). It was expressed early in ontogenesis coincidental with other known morphological and biochemical signs of differentiation.


Embryonic brain-derived heparan sulfate inhibits cellular membrane binding and biological activity of basic fibroblast growth factor.

We have investigated the ability of glycosaminoglycans from embryonic chick brain (15 days old) to interact with basic fibroblast growth factor (bFGF). 35SO4 metabolically labeled glycosaminoglycans were purified and separated on DEAE-cellulose chromatography. Material which eluted between 0.20 and 0.35 M NaCl displaced the binding of [125I]bFGF to brain membrane. This activity was dose-dependent and on the basis to its heparinase sensitivity and chondroitinase insensitivity, has been attributed to heparan sulfate. CL-6B-Sepharose chromatography of this material revealed two glycosaminoglycans of molecular masses of about 15,000 and 65,000. Incubation with [125I]bFGF followed or not by heparinase and chondroitinase treatment of electrotransfert from SDS-PAGE revealed that both of these forms correspond to heparan sulfate chains and bind bFGF. In vitro, embryonic brain-derived heparan sulfate inhibited both bFGF induced [3H]thymidine incorporation in CCL39 cells and neurite outgrowth in PC12 cells. These results suggest that heparan sulfate play an important function in the control of the biological activity of bFGF during brain development.


Expression of necdin, an embryonal carcinoma-derived nuclear protein, in developing mouse brain.

Necdin is a polypeptide sequence encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells. We have examined the expression of necdin and its mRNA in cultured cells and mouse brain by Northern blot analysis and immunohistochemistry. Among various established cell lines including neuroblastoma and glioma cells, only differentiated embryonal carcinoma cells (P19 and F9) expressed necdin mRNA. Necdin immunoreactivity was localized in the nuclei of differentiated neurons derived from P19 cells. Necdin mRNA was detected throughout brain regions of adult mouse; the relative abundances in the hypothalamus and midbrain were the highest, whereas those in the olfactory bulb and cerebellum were the lowest. In developing mouse brain, necdin mRNA was expressed during early periods of neuronal generation and differentiation, and the peak levels were attained during postnatal days 1-4. Necdin immunoreactivity was not detected in the neural stem cells on embryonic day 10, but was concentrated in the nuclei of brain cells, mostly neurons, at advanced stages of differentiation. The majority of differentiated neurons in the brain had necdin-immunoreactive nuclei on postnatal day 33. Thus, necdin may represent a valuable molecular marker for differentiated neurons both in vitro and in vivo.


Widespread lateral processes of glial cells in the immature striate cortex of the cat.

The topographies of intrinsic tangential connections of cells in the striate cortex of cats were determined in animals ranging in age between 1 day and 30 days. Implants of the carbocyanin dye, DiI, were found to label cellular elements which are classified by morphological criteria as astrocytes, in addition to neurons. Labeled astrocytes are seen in all cortical layers and in the superficial and deep white matter. Most labeled astrocytes occur underneath the DiI implant, but a number of them are also located horizontally at various distances from the implant. The horizontal span of the glial processes is the same as that for neuronal processes; in contrast to neurons however, the laterally distributed astrocytes assume a flat and unclustered distribution in the horizontal plane. Our observations suggest that the program for determining the span of laterally directed projections in the cat striate cortex is likely to be the same for neurons and astrocyte-like neuroglia.


Effect of milk on dopamine release in the newborn rat: an in vivo microdialysis study.

Newborn rats exhibit a rich behavioral repertoire to access the nipple and obtain milk. In older pups, catecholamines including dopamine (DA) mediate the behavioral effects of milk. In the present study, pups were delivered at term by caesarean section and instrumented with the microdialysis probe. Microdialysis samples were collected at 15 min intervals and K(+)-evoked levels of DA were measured with HPLC-ED. Pups received either no infusion, single or multiple intraoral infusions of saline or milk during subsequent samples. A decrease in K(+)-evoked DA release was evident after the first infusion in all subjects. Repeated milk infusions continued to reduce levels of extracellular DA, which remained evident 30 min after the last milk infusion. The rat neonate's first exposure to milk exerts lasting effects on neostriatal DA activity in the absence of prior suckling experience.


Serum creatinine as an index of renal function: new insights into old concepts.

The serum creatinine concentration is widely interpreted as a measure of the glomerular filtration rate (GFR) and is used as an index of renal function in clinical practice. Glomerular filtration of creatinine, however, is only one of the variables that determines its concentration in serum. Alterations in renal handling and metabolism of creatinine and methodological interferences in its measurement may have a profound impact on the serum concentration of creatinine. We review the fundamental principles of physiology, metabolism, and analytical chemistry that are necessary to correctly interpret the serum creatinine concentration. These principles are then applied to important clinical circumstances, including aging, pregnancy, diabetes mellitus, drug administration, and acute and chronic renal failure. Despite numerous limitations, serum creatinine remains a useful clinical tool, but more accurate measures of renal function are frequently necessary.


Highly sensitive, specific enzyme-linked immunosorbent assay of neopterin and biopterin in biological samples.

An enzyme immunosorbent assay of neopterin and biopterin on a polystyrene microtiter plate has been developed. A conjugate of neopterin or biopterin to bovine serum albumin was used to raise a specific antiserum against neopterin or biopterin in rabbits. An incubation mixture of the antiserum and samples prepared from human serum underwent another antigen-antibody reaction with the hapten fixed on the microtiter plate. The amount of antibody bound to the fixed hapten, which is inverse to the amount of hapten in the sample, was determined by using anti-rabbit IgG-horseradish peroxidase conjugate in a usual manner by measuring absorbance at 490 nm after reaction with o-phenylenediamine and hydrogen peroxide. The minimal detectable amounts of neopterin and biopterin were approximately 0.1 pmol. The specificity of the assay was so high that the assay system for neopterin completely distinguished it from biopterin, as judged from the cross-reaction of 0.002%, and vice versa. The amounts of neopterin and biopterin in human serum determined by the present method agreed well with those determined by high-performance liquid chromatography. We used the present method to determine the concentrations of neopterin in serum from healthy control subjects and patients with cancers and systemic lupus erythematosus; the results were consistent with literature data.


Two-site direct immunoassay specific for active renin.

A sensitive immunoradiometric assay, without an enzymatic step and specific for active human renin, was developed with use of two monoclonal antibodies (MAbs). In this assay system, the first MAb was coupled to magnetic beads (Magnogel); the second one, directed against the active form of the enzyme, was radiolabeled with 125I. The specificity of this assay was demonstrated in experiments measuring the active plasma renin concentration in the presence or absence of inactive renin. The assay, performed in two steps, was sensitive enough to detect 0.9 pg of renin per tube (3.5 ng/L). Intra- or interassay CVs were < 10%. Concentrations of active plasma renin measured in normotensive subjects were between 7 and 40 ng/L.


Direct, simplified, and sensitive assay of angiotensin II in plasma extracts performed with a high-affinity monoclonal antibody.

A very simple, fast, and sensitive RIA of angiotensin (Ang) II has been developed, based on a monoclonal antibody with high affinity and specificity, making possible the direct measurement of circulating Ang II in human plasma after solid-phase extraction. The purified monoclonal antibody 4D8 has an association constant of 1.3 x 10(11) L/mol with Ang II and a cross-reactivity of < 1% for Ang I. The assay can detect as little as 0.8 fmol of Ang II in 2 mL of plasma and is not influenced by the presence of Ang I. Analytical recoveries between 112% and 116% were obtained for Ang II added to human plasma at physiological concentrations. Comparison of the RIA with a reversed-phase, high-performance liquid chromatographic method followed by RIA to measure Ang II in human plasma samples from normal and hypertensive subjects--and from normotensive subjects before and after an acute inhibition of angiotensin-converting enzyme with captopril (50 mg)--showed a high degree of correlation (r2 = 0.93) between the two methods.


Avidin-biotin enzyme immunoassay of osteocalcin in serum or plasma.

We describe a competitive enzyme immunoassay, the ExtrAvidin-biotin system, for determining osteocalcin in human serum or plasma. Antibodies were raised against bovine osteocalcin. Binding of the antibodies to osteocalcin was calcium-dependent. Limit of detection is 0.07 nmol/L (0.4 microgram/L). The standard curve for method is linear between 0.3 and 17.6 nmol/L (1.9 and 100 micrograms/L). Interassay CV over the range 0.9 to 14.8 nmol/L (5.3 to 84 micrograms/L) is 7.5% to 11.7%. Analytical recovery is 105% +/- 5% (mean +/- SD). The measurement, which is adapted to microtiter plates, requires only 20 microL of serum and 5 h. The coefficient of correlation between the concentrations measured by this method and by a commercially available radioimmunoassay kit (CIS Biointernational) is 0.91. Osteocalcin can be measured in serum or heparinized plasma. Hemolysis (174 mumol/L hemoglobin) reduces osteocalcin concentration by 54%. High concentrations of triglycerides (7 mmol/L) give an overestimation of 63%. Serum concentrations of osteocalcin measured in 130 healthy subjects (ages 15-64 years) and 86 children (ages 4-14 years) were 1.4 +/- 0.8 and 4.0 +/- 1.5 nmol/L (8.1 +/- 4.6 and 22.5 +/- 8.6 micrograms/L), respectively (mean +/- SD).


Sensitive, specific radioimmunoassay for quantifying pergolide in plasma.

Pergolide, a synthetic ergoline with potent dopaminergic activity, is used to treat Parkinson disease. The low plasma concentrations of pergolide achieved during therapy complicate the development of a method for its analysis. Because radioimmunoassay successfully measures other structurally related ergolines in physiological fluids, we undertook the development of a radioimmunoassay of pergolide. The detection limit of the radioimmunoassay is 21 ng/L with an optimal working range from 100 to 1000 ng/L. We maximized assay specificity by using a monoclonal antibody that displayed low cross-reactivity with pergolide sulfoxide, a major metabolite found in animals. The radioimmunoassay has performed acceptably for > 2 years during toxicology studies with rats and rhesus monkeys and in clinical studies involving patients with Parkinson disease. We consider the radioimmunoassay a valid method for quantifying therapeutic concentrations of pergolide in plasma.


Spectrotype distributions of circulating IgG from patients with systemic lupus erythematosus.

Pathogenic autoantibodies from patients with systemic lupus erythematosus (SLE) may represent a relatively cationic fraction of IgG. We compared the spectrotype distributions of affinity-purified IgG from the sera of 10 SLE patients and 10 age- and sex-matched control subjects. Purified IgG was subjected to isoelectric focusing between pI 3 and 9. No significant difference was observed for pI 6.0-6.5 and 7.5-8.0. However, control subjects had a higher percent of total IgG at 6.5-7.0 (15.4 +/- 4.0 vs 11.1 +/- 2.0, P = 0.008) and at 7.0-7.5 (22.4 +/- 4.8 vs 18.2 +/- 3.8, P = 0.04) whereas SLE patients had a higher percent of total IgG at 8.0-8.5 (24.3 +/- 3.0 vs 20.5 +/- 4.0, P = 0.03) and at 8.5-9.0 (21.9 +/- 5.9 vs 15.1 +/- 3.7, P = 0.006). Spectrotype distributions of circulating IgG from SLE patients are skewed toward higher pI, providing further evidence of proliferation of B-cell clones that express more cationic IgG in patients with SLE. Longitudinal studies of serum IgG from several patients for > 1 year reveal distinct changes in both cationic and anionic clonotypes, suggesting clonal expansion of antiidiotypes to autoantibodies.


Direct determination of cadmium and lead in whole blood by potentiometric stripping analysis.

The commercially available equipment for potentiometric stripping analysis (PSA) was tested for routine lead and cadmium determination in whole-blood samples. In contrast to anodic stripping voltammetry, PSA is not subject to background interferences from organic electroactive constituents in the sample or to the presence of dissolved oxygen (i.e., oxygen removal is not necessary). To determine lead and cadmium by PSA, it is sufficient to dilute the blood sample with an appropriate supporting electrolyte (0.5 mol/L HCl). The detection limit changes with deposition time and volume of blood sample used. For 1 mL of blood and a 1-min deposition time, the detection limit is 1 microgram/L for both elements. If the deposition time increases to 10 min, cadmium can be determined at its normal concentration in blood (the detection limit is improved to < 0.1 microgram/L). Procedures for routine determination of lead and cadmium in whole blood are presented.


Magnesium, total calcium, phosphorus, copper, and zinc in plasma and erythrocytes of venous cord blood from infants of diabetic mothers: comparison with a reference group by logistic discriminant analysis.

Concentrations of magnesium (Mg), total calcium (Ca), phosphorus (P), copper (Cu), and zinc (Zn) were investigated in plasma (Pl) and erythrocytes (Erc) of venous cord blood of 44 infants of diabetic mothers (IDMs). These same concentrations plus total glycohemoglobin and fructosamine were determined at delivery in a subset of 15 mothers of these infants. Mineral results for IDMs were compared with those for 66 apparently healthy newborns. The duration of gestation in the two groups was significantly different (P < 0.001). After adjustment for gestational age, the mean (+/- SD) differences between groups were significant for birth weight, head circumference, Erc-Mg (1.71 +/- 0.17 for IDMs vs 1.76 +/- 0.15 mmol/L for control subjects), Pl-Ca (1.96 +/- 0.32 vs 2.48 +/- 0.22 mmol/L), Pl-P (1.99 +/- 0.40 vs 1.57 +/- 0.25 mmol/L), and Erc-Cu (10.9 +/- 2.41 vs 12.9 +/- 3.00 mumol/L), but not for Erc-Zn (33.0 +/- 18.3 vs 40.4 +/- 13.6 mumol/L). The variable that best discriminated between the two infant groups after adjustment for gestational age was Pl-Ca. In the 15 mothers, Pl-Mg (0.67 +/- 0.07 mmol/L) and Pl-Ca (1.66 +/- 0.21 mmol/L) concentrations were low, Pl-Zn (9.81 +/- 3.40 mumol/L) was normal, and Pl-Cu (33.5 +/- 10.7 mumol/L) was above normal. Correlations between total glycohemoglobin and mineral values of the mothers or paired IDM mineral values were not significant. The concentration of Pl-Ca was positively correlated with Erc-Cu (P < 0.001) and Pl-Cu (P < 0.05) in the comparison group newborns but not in the IDMs.


Analysis for cerebrospinal fluid proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Cerebrospinal fluid (CSF) proteins with molecular masses of < 150,000 Da were identified by immunoblotting after two kinds of nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). With PAGE 1 (17-27% gradient gel), CSF proteins were clearly separated into seven to nine bands with molecular masses of 3000-67,000 Da; seven bands were identified as beta 2-microglobulin, lysozyme, prealbumin, free kappa and lambda chain, apolipoprotein A-I, glycoproteins, and albumin by immunoblotting. With PAGE 2 (10-20% gradient gel), proteins were clearly separated into 11-16 bands with molecular masses of 15,000-150,000 Da; 11 were identified as prealbumin, free kappa and lambda chain, apolipoprotein A-I, glycoproteins, albumin, alpha 1-antitrypsin, transferrin (separated into two bands), immunoglobulin fragments, haptoglobin, and IgG. We analyzed CSF samples collected from 81 patients with cerebrospinal signs by these SDS-PAGE methods and observed prominent bands in some cases.


Precipitation and characterization of an aluminosilicate from AlCl3-Na2SiO3-HCl in serum, of interest for Alzheimer disease.

A precipitation experiment was performed with human serum to model aluminosilicate formation in brains of patients with Alzheimer disease. Aluminum and (or) silicate ions were added to serum in a 1:2 molar ratio at pH 7.4. Precipitates formed immediately and were left for 24 h at 37 degrees C before filtration. Silicate and aluminosilicate formed precipitates with human serum proteins albumin, transferrin, and IgG. In untreated samples, the IgG/albumin ratio increased slightly compared with the ratio in dried serum. Diethylbarbiturate-washed precipitates had a significantly lower protein content than did untreated ones. The IgG/albumin ratio increased considerably in the sample containing aluminosilicate. We conclude that IgG is the sodium dodecyl sulfate-soluble human protein most firmly bound to the aluminosilicate matrix. From 27Al magic-angle-spinning nuclear magnetic resonance (MAS NMR), a pronounced peak was found at 52.79 ppm and a minor peak at 0.53 ppm, suggesting that 4-coordinated aluminum predominates and that 6-coordinated aluminum is present in a smaller proportion. The 29Si MAS NMR spectrum shows a poorly ordered structure. The aluminosilicate formed also contains the cations Na+ > K+ > Ca2+ > Mg2+ and anions Cl- > PO4(3-). Rather than looking for aluminum toxicity to explain the effects of Alzheimer disease, one should consider that by precipitating such a composite phase, the balance of cations, anions, and proteins in human serum is changing.


Using changes in attributable risk to predict long-term efficacy of simvastatin treatment.

It is a common perception that coronary heart disease (CHD) deaths will decline by 2% for every 1% decrease in serum total cholesterol (TC). We investigated the changes in serum TC concentrations obtained with simvastatin, an inhibitor of hydroxymethylglutaryl-CoA reductase, in 70 hypercholesterolemic subjects. Although simvastatin reduced serum TC concentrations by 30% after 6 months, calculations from age- and risk-related TC quintiles show that the absolute difference in attributable CHD deaths would be just > 1%. These results are reminiscent of the experience from several large prospective primary drug trials that demonstrated impressive relative benefit ratios but much smaller absolute differences (< 2%) in CHD endpoints between the treated and untreated groups. This epidemiologically based approach of assessing efficacy and potential long-term benefit of a lipid-lowering drug may be more appropriate and potentially less misleading than just the percentage change of TC (or other lipoproteins) so frequently reported in short-term drug trials.


Simultaneous quadruple-label fluorometric immunoassay of thyroid-stimulating hormone, 17 alpha-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM isoenzyme in dried blood spots.

We describe a quadruple-label fluorometric immunoassay for simultaneously measuring four analytes: thyroid-stimulating hormone (TSH), 17 alpha-hydroxyprogesterone (17 alpha-OHP), immunoreactive trypsin (IRT), and creatine kinase MM (CK-MM). The assay is based on immunoreagents labeled with four different lanthanide ions (Eu3+, Tb3+, Sm3+, and Dy3+), on dissociative fluorescence enhancement applying the principle of co-fluorescence, and on time-resolved fluorometry. The monoclonal anti-alpha-TSH and anti-IRT antibodies and the polyclonal anti-CK-MM antibody were labeled with Eu3+, Sm3+, and Dy3+, respectively; 17 alpha-OHP was labeled with Tb3+. The assay was performed in microtitration strip wells coated with a mixture of monoclonal antibodies against beta-TSH, IRT, and CK-MM and a polyclonal goat anti-rabbit IgG for capture of the rabbit anti-17 alpha-OHP antibodies. After completion of the immunoreactions, the bound fractions of the lanthanides were dissociated into the co-fluorescence enhancement solution, creating highly fluorescent chelates. The four lanthanide-specific signals were subsequently measured in a time-resolved fluorometer. The detection limits of the assay were 0.1 mIU/L for TSH, 2 nmol/L for 17 alpha-OHP, 2 micrograms/L for IRT, and 4 U/L for CK-MM.


Homologous radioimmunoassay of human osteocalcin.

Osteocalcin or bone gamma-glutamic acid-containing protein (GLA protein) was isolated from human bone and used to develop a homologous radioimmunoassay of human osteocalcin. The effect of age on serum osteocalcin was studied in 380 normal children and adolescents and 330 normal adults. The mean (+/- SD) values in adults were higher in men [25 +/- 5 micrograms/L (4.3 +/- 0.8 nmol/L)] than in premenopausal women [20 +/- 6 micrograms/L (3.4 +/- 1.0 nmol/L); P < 0.01], but both were lower than in postmenopausal women [29 +/- 2 micrograms/L (5.0 nmol/L)]. The highest concentrations were seen in girls [ages 10-12 years: 99 +/- 38 micrograms/L (17.0 nmol/L)] and boys [ages 14-16 years: 107 +/- 57 micrograms/L (18.4 nmol/L)]. These mean values were substantially higher than those previously reported for results of heterologous osteocalcin radioimmunoassays but the correlation (r = 0.87, n = 77, P < 0.001) between both sets of results was excellent. In patients with metabolic bone diseases characterized by high or low bone turnover, the increase or decrease in serum osteocalcin observed was as expected. This homologous radioimmunoassay of human osteocalcin thus reflects bone turnover but reports serum concentrations higher than previously suspected.


Flow-injection analysis for malondialdehyde in plasma with the thiobarbituric acid reaction.

A simple, precise, and rapid method to measure plasma malondialdehyde (MDA) was developed by use of solvent extraction--flow-injection analysis. The reagent solution, containing thiobarbituric acid (TBA), 5 g/L in 100 mL/L phosphoric acid, and extraction solvent (methylisobutyl ketone, MIBK) were propelled with a double-plunger micropump at a flow rate of 0.3 mL/min, and 20 microL of sample was introduced into the reagent stream. After TBA-MDA reactant was extracted into MIBK, the organic phase was continuously separated by a successive phase-separation system equipped with two phase separators, and the absorbance of the TBA-MDA reactant was measured at 532 nm. This approach resulted in excellent sensitivity, a CV of < 1.5%, a good correlation with the conventional manual method, and a sampling frequency of 7 samples/h, suggesting that this semiautomated method is suitable for measuring plasma MDA.


Improved measurement of low-density-lipoprotein susceptibility to copper-induced oxidation: application of a short procedure for isolating low-density lipoprotein.

Low-density-lipoprotein (LDL) oxidation may provide the crucial link between plasma LDL and atherosclerotic-lesion formation. Oxidation can be induced in vitro by incubating LDL with cells or metal ions and can be measured by continuously monitoring conjugated-diene absorbance at 234 nm. Measurement of LDL oxidizability was improved by performing the assay with 0.05 g of LDL-protein per liter of phosphate buffer containing 1 mumol of EDTA, by initiating oxidation by adding CuCl2 (5 mumol/L) at 30 degrees C, and by using a short-run ultracentrifugation method for isolating LDL, which reduced the time needed for obtaining purified LDL and thus reduced in vitro oxidation. LDL apolipoprotein analysis and oxidizability determination showed that this method is better than the longer sequential-isolation procedure. Adding butylated hydroxytoluene (BHT) to plasma as an antioxidant unpredictably increased the LDL oxidation lag time, making BHT unsuitable as an antioxidant. Adding EDTA appeared to be sufficient to prevent in vitro oxidation. Additionally, the diene production correlated highly with the concentration of thiobarbituric acid-reactive substances (r = 0.97). No relation between the vitamin E content of LDL and the oxidation lag time was found.