Combined mepacrine-hydroxychloroquine treatment in patients with systemic lupus erythematosus and refractory cutaneous and articular activity.

Aim The aim of this study was to evaluate the clinical response to combined therapy with hydroxychloroquine and mepacrine in patients with systemic lupus erythematosus and refractory joint and/or skin disease. Methods Mepacrine was added to 46 systemic lupus erythematosus patients unresponsive to treatment with the following drug combinations: hydroxychloroquine + prednisone + immunosuppressive drugs ( n = 24), hydroxychloroquine + prednisone ( n = 16), hydroxychloroquine + prednisone + retinoids ( n = 2), hydroxychloroquine alone ( n = 1), hydroxychloroquine + one immunosuppressive drug ( n = 1), hydroxychloroquine + prednisone + one immunosuppressive drug + belimumab ( n = 1) or hydroxychloroquine + prednisone + belimumab ( n = 1). The outcome variable was the clinical response, either complete or partial, based on clinical judgement. The Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score were additionally used. Results A total of 91% patients showed complete/partial response, with similar rates among those with joint or skin disease. In patients with cutaneous activity, a statistically significant decrease in the CLASI was seen. There also was a statistically significant decrease in the SLEDAI. The mean daily dose of prednisone decreased from 5.8 to 3.4 mg/d ( p = 0.001). Prednisone could be discontinued in 20% of patients. No serious adverse events were seen. Smoking was the only predictor of complete response. Conclusion In the setting of refractory skin and/or joint disease, the addition of mepacrine to previous therapy including hydroxychloroquine was safe and effective in reducing disease activity and decreasing prednisone doses. The fact that smokers responded better opens the door to further studying the combination of mepacrine-hydroxychloroquine as a first-line therapy in such patients.

How common is ballooning? Hiatal area on 3D transperineal ultrasound in urogynecological patients and its association with lower urinary tract symptoms.

INTRODUCTION: Pelvic floor ultrasound plays a major role in urogynecologic diagnostics. Using 3D ultrasound we can identify integrity of levator ani and measure hiatal area in the axial plane. The main goal of our study was to measure hiatal area on Valsalva in a cohort of urogynecological patients. Furthermore, we aimed to correlate hiatal area with urogynecological symptoms, levator integrity and evaluate cut-off values for pelvic organ prolapse. MATERIALS AND METHODS: In a retrospective analysis, we included 246 patients seen for urogynecological problems in our tertiary urogynecological unit. After a standardized interview and physical examination, a 3D pelvic floor ultrasound was performed. According to the cardinal urogynecological symptoms and signs, patients were categorized into three groups: pelvic organ prolapse, stress urinary incontinence and overactive bladder symptoms. RESULTS: Median age of our study population was 66 (range 29-94) years, median parity was 2.1 (range 0-9) with 17 (6.9 %) nulliparous women. Symptoms of overactive bladder in 71.1 % were most common, followed by 54.5 % symptoms of stress incontinence and 32.1 % symptoms of prolapse. On examination 49.2 % showed signs of prolapse. Levator avulsions on 3D ultrasound were detected in 20.7 %. Hiatal area was normally distributed with a median of 28.7 cm2 (range 10.4-50.0 cm2). Patients with levator avulsion had a significantly larger hiatal area (p < 0.001). Also patients with signs of prolapse had a significantly larger hiatal area (p < 0.001). There was no correlation between hiatal area and symptoms of overactive bladder (p = 0.374). Although not reaching statistical significance there was evidence of a smaller hiatal area for patients with stress incontinence (p = 0.016). In our cohort there were 33.7 % (83) women without ballooning, 27.2 % (67) showed mild, 18.3 % (45) moderate, 12.3 % (30) marked and 8.5 % (21) severe ballooning. The ROC curve analysis for hiatal area on patients with prolapse yielded an AUC of 0.755 [95 % CI (0.696-0.814)]. Using the Youden-Index we obtained 27.53 cm2 as a cut-off with a sensitivity of 0.70 and a specificity of 0.69. DISCUSSION: Hiatal area is a new repeatable diagnostic parameter. Its clinical application could improve our understanding of the pathophysiology of pelvic organ prolapse as a form of hiatal hernia.

Improved recovery of Listeria monocytogenes from stainless steel and polytetrafluoroethylene surfaces using air/water ablation.

AIMS: To develop a gentle ablation technique to recover Listeria monocytogenes biofilms from stainless steel (SS) and polytetrafluoroethylene (PTFE) surfaces by using compressed air and water injection. METHODS AND RESULTS: Biofilms were grown for 4, 24 and 48 h or 7 days and a compressed air and water flow at 2, 3 and 4 bars was applied for cell removal. Collected cells were quantified for total/dead by staining with SYTO 9/PI double staining and cultivable populations were determined by plating onto brain heart infusion (BHI) agar, while coupon surfaces also were stained with DAPI to quantify in situ the remaining cells. The recovery efficiency was compared to that of conventional swabbing. Results showed that the air/water ablation is able to collect up to 98·6% of cells from SS surfaces while swabbing only recovered 11·2% of biofilm. Moreover, air/water ablation recovered 99·9% of cells from PTFE surfaces. CONCLUSIONS: The high recovery rate achieved by this technique, along with the fact that cells were able to retain membrane integrity and cultivability, indicate that this device is suitable for the gentle recovery of viable L. monocytogenes biofilm cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This work presents a highly efficient technique to remove, collect and quantify L. monocytogenes from surfaces commonly used in the food industry, which can thus serve as an important aid in verifying cleaning and sanitation as well as in reducing the likelihood of cross-contamination events.

Effect of red pepper on symptoms of irritable bowel syndrome: preliminary study.

BACKGROUND: Abdominal pain, that characterizes irritable bowel syndrome (IBS) together with bloating and disordered defecation, is mainly related to a visceral hypersensitivity due to an increase of TRPV(1) nociceptive nerve fiber activity. AIM: As capsaicin contained in red pepper is able to desensitize the TRPV(1) fibres, we evaluated whether the red pepper oral administration can decrease the symptoms of visceral hypersensitivity in IBS patients. METHODS: The study was performed on 50 patients with IBS diagnosed following Rome II criteria. After a 2-week washout period, 23 patients were planned to receive 4 pills/day, for 6 weeks randomly and in a double blind manner, each containing 150 mg of red pepper powder with a coat that dissolves in the colon, and 27 patients placebo. The patients scored each day in a diary the abdominal pain and bloating intensities following the 5-point Likert scale. The weekly symptom mean scores and the final patient subjective evaluation on treatment effectiveness were statistically compared among groups and intra-groups with appropriate tests. RESULTS: Eight patients dropped from the study: 6 in the red pepper group for abdominal pain and 2 in the placebo group. In 8 patients, the pills were reduced to 2/day, because of the abdominal pain at the onset of treatment. The intra-group comparisons showed that in patients taking red pepper the abdominal pain and bloating mean score values of the last weeks of treatment were significantly improved with respect to pre-treatment values, unlike patients taking placebo. The final patient subjective evaluation on the treatment effectiveness showed that red pepper group scored significantly better than placebo. CONCLUSIONS: The results of this preliminary study indicate that the chronic administration of red pepper powder in IBS patients with enteric-coated pills was significantly more effective than placebo in decreasing the intensity of abdominal pain and bloating and was considered by the patients more effective than placebo.

Relevance of sex, age and gait kinematics when predicting fall-risk and mortality in older adults.

Approximately one-third of elderly people fall each year with severe consequences, including death. The aim of this study was to identify the most relevant features to be considered to maximize the accuracy of a logistic regression model designed for prediction of fall/mortality risk among older people. This study included 261 adults, aged over 65 years. Men and women were analyzed separately because sex stratification was revealed as being essential for our purposes of feature ranking and selection. Participants completed a 3-m walk test at their own gait velocity. An inertial sensor attached to their lumbar spine was used to record acceleration data in the three spatial directions. Signal processing techniques allowed the extraction of 21 features representative of gait kinematics, to be used as predictors to train and test the model. Age and gait speed data were also considered as predictors. A set of 23 features was considered. These features demonstrate to be more or less relevant depending on the sex of the cohort under analysis and the classification label (risk of falls and mortality). In each case, the minimum size subset of relevant features is provided to show the maximum accuracy prediction capability. Gait speed has been largely used as the single feature for the prediction fall risk among older adults. Nevertheless, prediction accuracy can be substantially improved, reaching 70% in some cases, if the task of training and testing the model takes into account some other features, namely, sex, age and gait kinematic parameters. Therefore we recommend considering sex, age and step regularity to predict fall-risk.

Amniotic fluid stem cell-derived vesicles protect from VEGF-induced endothelial damage.

Injection of amniotic fluid stem cells (AFSC) delays the course of progression of renal fibrosis in animals with Alport Syndrome, enhancing kidney function and improving survival. The mechanisms responsible for these protective outcomes are still largely unknown. Here, we showed that vascular endothelial growth factor (VEGF) signaling within the glomeruli of Alport mice is strongly elevated early on in the disease, causing glomerular endothelial cell damage. Intraventricular injected AFSC that homed within the glomeruli showed strong modulation of the VEGF activity, particularly in glomerular endothelial cells. To investigate this phenomenon we hypothesized that extracellular vesicles (EVs) produced by the AFSC could be responsible for the observed renoprotection. AFSC derived EVs presented exosomal and stem cell markers on their surface membrane, including VEGFR1 and VEGFR2. EVs were able to modulate VEGF in glomerular endothelial cells by effectively trapping the excess VEGF through VEGFR1-binding preventing cellular damage. In contrast, VEGFR1/sVEGFR1 knockout EVs failed to show similar protection, thus indicating that VEGF trapping is a potentially viable mechanism for AFSC-EV mediated renoprotection. Taken together, our findings establish that EVs secreted by AFSC could target a specific signaling pathway within the glomerulus, thus representing a new potential glomerulus-specific targeted intervention.

Comparative study on shelf life of whole milk processed by high-intensity pulsed electric field or heat treatment.

The effect of high-intensity pulsed electric fields (HI-PEF) processing (35.5 kV/cm for 1,000 or 300 micros with bipolar 7-micros pulses at 111 Hz; the temperature outside the chamber was always < 40 degrees C) on microbial shelf life and quality-related parameters of whole milk were investigated and compared with traditional heat pasteurization (75 degrees C for 15 s), and to raw milk during storage at 4 degrees C. A HIPEF treatment of 1,000 micros ensured the microbiological stability of whole milk stored for 5 d under refrigeration. Initial acidity values, pH, and free fatty acid content were not affected by the treatments; and no proteolysis and lipolysis were observed during 1 wk of storage in milk treated by HIPEF for 1,000 micros. The whey proteins (serum albumin, beta-lactoglobulin, and alpha-lactalbumin) in HIPEF-treated milk were retained at 75.5, 79.9, and 60%, respectively, similar to values for milk treated by traditional heat pasteurization.

Modeling high-intensity pulsed electric field inactivation of a lipase from Pseudomonas fluorescens.

The inactivation kinetics of a lipase from Pseudomonas fluorescens (EC 3.1.1.3.) were studied in a simulated skim milk ultrafiltrate treated with high-intensity pulsed electric fields. Samples were subjected to electric field intensities ranging from 16.4 to 27.4 kV/cm for up to 314.5 micros, thus achieving a maximum inactivation of 62.1%. The suitability of describing experimental data using mechanistic first-order kinetics and an empirical model based on the Weibull distribution function is discussed. In addition, different mathematical expressions relating the residual activity values to field strength and treatment time are supplied. A first-order fractional conversion model predicted residual activity with good accuracy (A(f) = 1.018). A mechanistic insight of the model kinetics was that experimental values were the consequence of different structural organizations of the enzyme, with uneven resistance to the pulsed electric field treatments. The Weibull model was also useful in predicting the energy density necessary to achieve lipase inactivation.

Hemodynamic and neuroendocrine predictors of lower body negative pressure (LBNP) intolerance in healthy young men.

Exposure to LBNP results in body fluid shift to lower extremities similarly as under influence of orthostatic stress. In susceptible persons it leads to syncope. For better understanding why certain individuals are more susceptible to orthostatic challenges it seemed necessary to collect more data on hemodynamic and neuroendocrine adjustments occurring before onset of presyncopal symptoms Accordingly, in this study heart rate (HR), blood pressure (BP), stroke volume (SV), cardiac output (CO), hematocrit, plasma catecholamines, adrenomedullin, ACTH and plasma renin activity (PRA) were measured in 24 healthy men during graded LBNP (-15, -30 and -50 mmHg). Thirteen subjects completed the test (HT group) whereas 11 had presyncope signs or symptoms at -30 mmHg or at the beginning of -50 mmHg (LT group). Comparison of these groups showed that LT subjects had lower baseline total peripheral resistance and higher plasma adrenomedullin. During LBNP plasma catecholamine and PRA increases were even greater in LT than in HT group while plasma adrenomedullin elevations were similar in both groups. Plasma ACTH increased only in LT group following presyncope symptoms. Low tolerant group showed more rapid decline of SV and CO than HT subjects from the beginning of LBNP. It is suggested that measurements of SV at the level of LBNP which did not evoke any adverse symptoms may be of predictive value for lower orthostatic tolerance.

Detection of Subpubic Tumor Causing Bladder Outlet Obstruction by 3D Perineal Ultrasound.

This case report shows that 3D perineal ultrasound can be superior to clinical examination and routine 2D perineal ultrasound in the detection of an unusual subpubic tumor. A 73-year-old female patient was referred to our urogynecological outpatient unit complaining of over-active bladder symptoms and voiding dysfunction for 3 years. Gynecological examination found no signs of pelvic organ prolapse or abnormality in the vaginal cavity. Routine 2D perineal ultrasound showed substantial residual urine (ca. 300 ml on catheter) and limited bladder neck mobility, but no signs of pelvic organ prolapse. Use of standardized 3D perineal ultrasound revealed a 24 × 26 × 32 mm spherical, hypoechoic tumor below the pubic symphysis between the distal urethra and the pubic bones. This structure was mistaken for the pubic symphysis in the midline on 2D ultrasound performed earlier. At surgery, the tumor was completely excised through a vaginal incision between the urethra and the pubic symphysis. After an uneventful postoperative recovery the patient developed de-novo stress urinary incontinence, which was corrected successfully by the insertion of a retropubic tension-free suburethral sling after an interval of 8 weeks. After a further follow-up of 8 weeks the patient reported well-being, urinary continence and no voiding dysfunction; no abnormalities were found on examination. In conclusion, 3D perineal ultrasound is a useful additional tool for the diagnostic workup of bladder outlet obstruction.

Daily oral magnesium supplementation suppresses bone turnover in young adult males.

This study examined the effects of daily oral magnesium (Mg) supplementation on bone turnover in 12 young (27-36 yr old) healthy men. Twelve healthy men of matching age, height, and weight were recruited as the control group. The study group received orally 15 mmol Mg (Magnosolv powder, Asta Medica) daily in the early afternoon with 2-h fasting before and after Mg intake. Fasting blood and second void urine samples were collected in the early morning on days 0, 1, 5, 10, 20, and 30, respectively. Total and ionized Mg2+ and calcium (Ca2+), and intact PTH (iPTH) levels were determined in blood samples. Serum biochemical markers of bone formation (i.e. C-terminus of type I procollagen peptide and osteocalcin) and resorption (i.e. type I collagen telopeptide) and urinary Mg level adjusted for creatinine were measured. In these young males, 30 consecutive days of oral Mg supplementation had no significant effect on total circulating Mg level, but caused a significant reduction in the serum ionized Mg+ level after 5 days of intake. The Mg supplementation also significantly reduced the serum iPTH level, which did not appear to be related to changes in serum Ca2+ because the Mg intake had no significant effect on serum levels of either total or ionized Ca2+. There was a strong positive correlation between serum iPTH and ionized Mg2+ (r = 0.699; P < 0.001), supporting the contention that decreased serum iPTH may be associated with the reduction in serum ionized Mg2+. Mg supplementation also reduced levels of both serum bone formation and resorption biochemical markers after 1-5 days, consistent with the premise that Mg supplementation may have a suppressive effect on bone turnover rate. Covariance analyses revealed that serum bone formation markers correlated negatively with ionized Mg2+ (r = -0.274 for type I procollagen peptide and -0.315 for osteocalcin), but not with iPTH or ionized Ca2+. Thus, the suppressive effect on bone formation may be mediated by the reduction in serum ionized Mg2+ level (and not iPTH or ionized Ca2+). In summary, this study has demonstrated for the first time that oral Mg supplementation in normal young adults caused reductions in serum levels of iPTH, ionized Mg2+, and biochemical markers of bone turnover. In conclusion, oral Mg supplementation may suppress bone turnover in young adults. Because increased bone turnover has been implicated as a significant etiological factor for bone loss, these findings raise the interesting possibility that oral Mg supplementation may have beneficial effects in reducing bone loss associated with high bone turnover, such as age-related osteoporosis.

Adrenergic suppression of peripheral blood T cell reactivity in the rat is due to activation of peripheral alpha 2-receptors.

A 20-h treatment of rats with catecholamines using s.c.implantable retard tablets markedly suppresses the in vitro reactivity of peripheral blood (PBL) T lymphocytes, provided that beta-receptors are blocked with propranolol (Felsner et al., 1992). The results can be summarized as follows: (i) the suppressive effect of noradrenaline+propranolol to the concanavalin A (ConA) response of PBL was abolished by the simultaneous application of the alpha-blocker phentolamine. Using selective agonists, the relevant receptor was identified to belong to the alpha 2-subtype. (ii) The alpha-adrenergic suppression of the PBL T cell response was likewise observed in adrenalectomized animals, which rules out the participation of secondarily induced glucocorticoids. Furthermore, the combination of noradrenaline with the watersoluble beta-blocker nadolol was equally effective to suppress the ConA response of PBL. (iii) An analogous alpha-mediated suppression of T cell function of PBL, but not spleen cells, was observed 1 h after i.p. treatment with tyramine, which leads to the release of endogenous noradrenaline. From these results it is concluded that the adrenergic suppression of PBL T cell functions is primarily due to the activation of peripheral alpha 2-receptors and that it is likewise observed under acute indirect sympathomimetic treatment.

The in vivo effects of beta-3-receptor agonist CGP-12177 on thyroxine deiodination in cold-exposed, sympathectomized rat brown fat.

OBJECTIVE: The effects of the beta-3-receptor agonist CGP-12177 on thyroxine (T4) deiodination in sympathectomized (SX) interscapular brown adipose tissue (BAT) were assessed in 300 g body weight (BW) Wistar rats. DESIGN: Seven days after SX, groups of rats were implanted s.c. with pellets containing 5mg CGP-12177 or 5mg norepinephrine (NE) and were immediately placed at 4 degrees C for 24h. Other SX groups were injected with CGP-12177 or NE 1mg/kg BW i. p. and placed in the cold for 4h. The latter group was injected, in addition, with prazosin 0.4 mg/100g BW i.p. or propranolol 0.5mg/100g BW i.p. 15 min before and 2h after the administration of CGP-12177 or NE. METHODS: Two hours after the last injection of prazosin or propranolol, animals were killed and BAT was removed, homogenized and centrifuged at 500 g for 10 min at 4 degrees C. The infranatants were incubated during 60 min in the presence of dithiothreitol and 1 microCi [(125)I]T4. Aliquots were chromatographed on paper for the measurement of [(125)I]T4 and its deiodinated subproducts. RESULTS: CGP-12177 restored normal T4 deiodination in SX BAT from both groups, but NE was slightly more effective. Propranolol, although not prazosin, blocked the CGP-12177 effects. Contrariwise, the NE-induced rise in deiodination was blocked by prazosin and to a lesser extent by propranolol. CONCLUSIONS: The results indicate that CGP-12177 stimulated the in vivo activation of 5'-deiodinase type II activity predominantly via beta-3-receptor, without participation of alpha-1-receptors.

Sulfoconjugated and free plasma catecholamine levels at rest and during exercise in patients with idiopathic dilated cardiomyopathy.

Plasma levels of sulfoconjugated (sc) catecholamines (CA) have been shown to be increased with activation of the sympathoadrenal system in a number of clinical settings. We evaluated the relation between scCA and clinical or hemodynamic parameters of patients with idiopathic dilated cardiomyopathy (IDC) at rest and during incremental exercise testing. Eleven healthy subjects, nine patients in New York Heart Association (NYHA) functional class I (IDC-A group) and 11 in NYHA functional class II and III (IDC-B group) performed a symptom-limited, graded bicycle exercise test. Resting, peak and various postexercise levels of plasma free and scCA were determined by high-pressure liquid chromatography. Resting CA levels obtained in the supine position were remarkable for elevations of free norepinephrine (NE) in IDC-B patients (355 +/- 157 ng/l) as compared to IDC-A patients (177 +/- 54, p = 0.006) or healthy controls (193 +/- 74, p = 0.007). Similarly, scNE was highest in IDC-B patients with 1856 +/- 1089 ng/l, followed by IDC-A (1028 +/- 187, p = 0.025) and control subjects (1109 +/- 440, p = 0.025). There was a highly significant correlation between free and scNE (r = 0.76, p < 0.0005). Whereas resting free dopamine (DA) levels were comparable in all three groups, scDA was found to be elevated clearly in IDC-B patients (8772 +/- 2097 ng/l) and significantly different to IDC-A (5786 +/- 2481, p = 0.01) or control subjects (4892 +/- 1575, p = 0.0005). The NYHA functional class and maximum exercise performance correlated best with resting scDA (r = 0.68, p = 0.001 and r = 0.56, p = 0.005, respectively). At peak exercise, IDC-B patients exhibited a significant decrease in scNE and sc epinephrine (E) (from 1856 +/- 1089 to 1495 +/- 932 ng/l, p < 0.005 and from 491 +/- 173 to 282 +/- 143 ng/l, p < 0.01) compared to controls (from 1109 +/- 444 to 1094 +/- 548 ng/l and from 379 +/- 200 to 329 +/- 134 ng/l). In IDC-B patients this decrease in scNE and scE at peak exercise was related inversely to the rise in free NE and E (r = -0.81, p < 0.005 and r = -0.68, p < 0.05). Resting hemodynamic indices generally were reflected better by some free CA rather than by conjugated forms or by parameters of clinical performance. These findings suggest that in addition to free or scNE levels, resting scDA is elevated in symptomatic patients with IDC.(ABSTRACT TRUNCATED AT 400 WORDS)

Pivagabine-induced increases in the abundance of CRF mRNA in the cerebral cortex and hypothalamus of rats.

The effect of treatment of rats with pivagabine (4-[(2,2-dimethyl-1-oxopropyl) amino] butanoic acid) for 4 days on the abundance of corticotropin-releasing factor (CRF) mRNA in the brain was investigated. Such treatment resulted in dose-dependent (100-300 mg/kg, i.p.) increases in the amount of CRF mRNA in both the hypothalamus and cerebral cortex. The maximal increases were thus apparent with the dose of 300 mg/kg in the hypothalamus (+108%) and cerebral cortex (+49%) 30 or 60 min, respectively, after the last drug injection. Foot-shock stress administered 30 min after the final drug injection had no effect on the pivagabine-induced increases in the abundance of CRF mRNA in the hypothalamus or cerebral cortex. Such stress also had no effect on the amounts of CRF mRNA in these brain regions of vehicle-treated rats. These results demonstrate that pivagabine increases the amount of CRF mRNA in both the hypothalamus and cerebral cortex of rats, effects that might be relevant to the action of this drug in preventing the stress-induced changes in CRF hypothalamic concentration.

Changes in the gene expression of GABAA receptor subunit mRNAs in the septum of rats subjected to pentylenetetrazol-induced kindling.

Chemical kindling was induced in rats by long-term administration of pentylenetetrazol (PTZ) (30 mg/kg three times a week for 9 weeks). The effects of such kindling on the abundance of transcripts encoding subunits of the gamma-aminobutyric acid type A (GABAA) receptor in the brain were measured by RNase protection assay. Kindled rats were examined either 3 or 30 days after discontinuation of PTZ treatment. The amounts of gamma2L and gamma2S subunit mRNAs were significantly increased in the hippocampus and cerebral cortex of kindled rats 3 and 30 days after treatment discontinuation, compared with those observed in control rats, and these effects were prevented by the concomitant administration of the anticonvulsant abecarnil. In contrast, the amounts of alpha1 and beta2 subunit mRNAs in these two brain regions did not differ significantly between kindled and control rats. The abundance of alpha1, beta2, gamma2L and gamma2S subunit mRNAs was decreased in the septum of rats 3 or 30 days after discontinuation of treatment with PTZ either alone or in combination with abecarnil. The amounts of none of the four subunit mRNAs measured differed significantly between the striatum or frontal cortex of kindled rats and control rats 3 days after drug discontinuation. Immunohistochemical analysis with antibodies to choline acetyltransferase revealed a marked decrease in the number of cholinergic neurons in the septum of kindled rats 30 days after discontinuation of PTZ treatment; this effect was not prevented by the administration of abecarnil. These results suggest that long-term treatment with PTZ induces a loss of GABAA receptors in the septum.

A tail-artery cannulation method for the study of blood parameters in freely moving rats.

The aim of this study was to improve the chronic tail-artery cannulation technique for repeated substance administration and blood sampling under stress-free conditions in freely moving rats. For that purpose, a special metal capsula and spring-tube system were developed, which not only protects the inserted cannula but also provides a less injurious insertion procedure and improves the animals' welfare as illustrated by normal cleaning and feeding behavior. Intraarterial application of test substances and repeated blood sampling can be done under minimal stress conditions as shown by unaltered plasma levels of highly stress-sensitive prolactin and the catecholamines (CA), epinephrine (E), norepinephrine (NE), and dopamine (DA), after infusion of 300 microL saline.

Computer-controlled flushing for long-term cannulation in freely moving rats.

An in vivo long-term perfusion system is presented, which is based on automated, computer-controlled high-frequency heparin (10 U/mL) flushing of the cannula inserted into the tail artery of freely moving rats. The short flushing intervals as achieved with this system guarantee stable conditions throughout an experiment of several days, and reduce blood clotting and the infiltration of fibroblasts. In addition, a specially developed software enables the periodical application of test substances into the blood stream under time-controlled conditions over long periods. The system can be used continuously to follow up blood parameters in single animals without significant elicitation of stress-sensitive plasma catecholamines for up to 12 days.

Prolonged administration in vivo of alpha and beta adrenergic agonists decreases insulin binding to rat myocardial membranes in vitro by different mechanisms.

Male Sprague Dawley rats were continuously treated in vivo for 6, 12 and 20 hours with a combination of an alpha- (beta-) adrenoreceptor agonist and a beta- (alpha-) adrenoreceptor antagonist in subcutaneously implanted depot tablets. Crude membranes prepared from myocardial cells exhibited a decreased maximum binding of [125I]-insulin after 20 hours irrespective of the treatment applied. Scatchard and non-linear regression analysis of the displacement curves assuming two non-cooperative binding sites revealed a downregulation of the high affinity receptors for about 85% and a concomitant 2.5-fold increased receptor affinity under beta-adrenergic influence. In contrast, alpha-adrenergic treatment did not affect the receptor number but decreased the high affinity by 70%. The low affinity binding sites were virtually unaffected by the different treatments. The phospholipid and cholesterol contents of the membranes were not significantly altered. The phospholipid/cholesterol ratios after 12 and 20 hours of alpha-adrenergic treatment, however, were decreased. We suggest that the decreased binding activity of insulin receptors on rat myocardial membranes after continuous in vivo treatment with alpha- and beta- adrenergic agonists is mediated by different mechanisms.

Norepinephrine triggers medullar epinephrine depletion during normoglycemia.

Our experiments did show that chronic 12 hours administration of norepinephrine (NE) to rats by means of subcutaneously implantable retard tablets, led to a highly significant epinephrine (E) depletion of the adrenal medulla during normoglycemia. The expected rise of free plasma NE at 6 and 12 hours was accompanied by increased free plasma E values at 12 hours. At this very time point the liver contents of glycogen and free intracellular glucose showed their most pronounced decrease. Since at 12 hours both liver glycogen and medullar E values were at their lowest, a second experiment was performed to examine a possible causal relationship. In order to curb the breakdown of liver glycogen, rats were force fed with 50% glucose solution 9 hours after NE tablet implantation. Glucose feeding not only caused a much less pronounced liver glycogen fall at 12 hours, but, at the same time also prevented E depletion of the adrenal medulla. These observations suggested that rapid fall of liver glycogen and/or liver intracellular free glucose might be the trigger for medullar E depletion, even before hypoglycemia.