Cholinergic and purinergic contribution to the micturition reflex in conscious rats with long-term bladder outlet obstruction

The urethra of female Wistar rats was partially obstructed for 15 weeks. The effects of atropine (1 mg/kg i.v.), suramin (100 mg/kg i.v.), and a combination of atropine and suramin on the peak micturition pressure (MP) were compared during cystometry in conscious rats controls or subjected to outlet obstruction. On the isolated bladder dome, we studied the inhibitory effect of 1 micromol/L atropine, 1 mmol/L suramin, and the combination of the two drugs on contractions induced by electrical field stimulation (EFS). We studied also the contractile response to 80 mmol/L KCl and the concentration-response curves to noradrenaline, phenylephrine, and carbachol on the bladder dome and bladder neck and alpha, beta-methylene adenosine triphosphate on the bladder dome. In conscious rats, the MP, bladder capacity, and micturition volume were significantly higher in obstructed rats than in controls. Suramin induced the same inhibition in the two groups of animals (-30.7 +/- 13.3% in controls and -29.2 +/- 8.5% in obstructed rats). Atropine decreased the MP, but this effect was twofold greater in obstructed animals (-28.1 +/- 3.1% and -65.1 +/- 6.9% in control and obstructed animals, respectively). However, the combined effect of atropine and suramin was additive in controls but not in obstructed (-56.7 +/- 5.4% and -55.9 +/- 9.4%, respectively). Similar results were obtained in vitro using 1 micromol/L atropine and 1 mmol/L suramin. In the obstructed bladder dome and bladder neck, we found a great reduction in KCl- and carbachol-induced contractility but no difference in the response to EFS. Responses to noradrenaline and phenylephrine were moderately reduced in the bladder neck only, whereas responses to alpha, beta-methylene adenosine triphosphate in the bladder dome were not reduced except at the concentration of 300 micromol/L. We conclude that long-term obstruction in rats could induce cholinergic nerve fiber proliferation as suggested by the decrease in M(3) muscarinic receptor contractility (desensitization) and by a greater sensitivity of the MP to atropine.     

Serial microPET measures of the metabolic reaction to a microdialysis probe implant

Despite the widespread use of chronic brain implants in experimental and clinical settings, the effects of these long-term procedures on brain metabolism and receptor expression remain largely unknown. Under the hypothesis that intracerebral microdialysis transiently alters tissue metabolism, we performed a series of 18FDG microPET scans prior to and following surgical implantation of microdialysis cannulae. Parallel microPET measures using the competitive dopamine (DA) D2 receptor antagonist, 11C-raclopride, provided an assay of DA stability in these same animals. 18FDG scans were performed prior to microdialysis cannulation and again at 2, 12, 24, 48, 120, 168, 360 and 500 h (0.2, 0.5, 1, 2, 5, 7, 15 and 25 days). Separate animals received a sham surgery and the control group had no surgical intervention. For the first 24 h (scans at 2, 12 and 24 h post-surgery) uptake was reduced in both hemispheres. However, by 48 h, contralateral uptake had returned to pre-surgical levels. The striking finding was that from 48 to 500 h, the microdialysis cannulation produced widespread ipsilateral reductions in 18FDG uptake that encompassed the entire hemisphere. Despite the extent and persistence of these reductions, 11C-raclopride binding and ECF DA concentrations remained stable.     

Significance of acellular mucin pools in rectal carcinoma after neoadjuvant chemoradiotherapy

The presence of mucin pools lacking neoplastic epithelium ("acellular" mucin) in resection specimens of rectal carcinoma after neoadjuvant chemoradiotherapy (CRT) is a well-recognized phenomenon. The current recommendation by the College of American Pathologists is to regard acellular mucin as a type of treatment response and not as residual tumor. However, data-based evidence for or against such an approach is incomplete. In this study, we systematically analyzed the pattern and significance of mucin pools in 108 consecutive, prospectively collected resection specimens from patients who had uT3-4 and/or uN1 rectal cancer and were treated with preoperative long-course CRT. The 108 patients, 39 female and 69 male, had a median age of 58.5 years. With every tumor entirely examined in whole-mount sections, mucin pools were identified in 33 cases (33 of 108, 31%); in 16 (15%) they were all acellular. The mucin pools were focal (10% to 50% of the entire lesion) in 25 cases and extensive (>50%) in 8 cases. Mucin pools were also noted in the lymph nodes in 6 cases (6%); 3 of these were entirely acellular. Five cases had mucin pools in both the primary site and the lymph nodes. When acellular mucin was considered as "no residual tumor," the complete pathologic response rate for the entire cohort was 22% (24 of 108). The pathologic stage of the residual tumor (ypT) was 0 or 1 for 27 cases (25%) and 2 to 4 for 81 cases. The pathologic stage of nodal disease (ypN) was 0 for 83 (77%) and 1 or 2 for 25 cases. When acellular mucin was considered as "residual tumor," the complete pathologic response rate dropped to 17%; ypT was upstaged in 10 tumors and ypN was upstaged in 2 tumors. With a median follow-up of 31 months, the 3-year recurrence-free survival (RFS) was 73% for the entire group. Advanced pathologic response and low pathologic stage of the residual tumor (determined based on the depth of only viable tumor cells) correlated significantly with better RFS. However, the correlation between pathologic response and RFS became insignificant when acellular mucin pools were considered as residual tumor. Neither the presence of mucin pools nor their extent or cellularity had an impact on RFS. Furthermore, none of the 12 patients whose ypT or ypN was upstaged by acellular mucin had recurrent disease (3-y RFS of 100%). Thus, our results suggest that mucin pools in rectal carcinoma after neoadjuvant CRT do not have a significant impact on patient outcome, supporting the College of American Pathologists recommendation that only viable tumor cells, not acellular mucin, are to be interpreted as residual disease in the tumor pathologic staging.     

Acute hyperglycemia induces a global downregulation of gene expression in adipose tissue and skeletal muscle of healthy subjects

To define the effects of acute hyperglycemia per se (i.e., without the confounding effect of hyperinsulinemia) in human tissues in vivo, we performed global gene expression analysis using microarrays in vastus lateralis muscle and subcutaneous abdominal adipose tissue of seven healthy men during a hyperglycemic-euinsulinemic clamp with infusion of somatostatin to inhibit endogenous insulin release. We found that doubling fasting blood glucose values while maintaining plasma insulin in the fasting range modifies the expression of 316 genes in skeletal muscle and 336 genes in adipose tissue. More than 80% of them were downregulated during the clamp, indicating a drastic effect of acute high glucose, in the absence of insulin, on mRNA levels in human fat and muscle tissues. Almost all the biological pathways were affected, suggesting a generalized effect of hyperglycemia. The induction of genes from the metallothionein family, related to detoxification and free radical scavenging, indicated that hyperglycemia-induced oxidative stress could be involved in the observed modifications. Because the duration and the concentration of the experimental hyperglycemia were close to what is observed during a postprandial glucose excursion in diabetic patients, these data suggest that modifications of gene expression could be an additional effect of glucose toxicity in vivo.     

Expression of galectin-3 and its regulation in the testes

Although spermatogenesis is a complex process under hormonal control, which includes mainly follicle stimulating hormone (FSH) and androgens, little is known about the intra-testicular mediators of these hormones. In the present study, galectin-3 (Gal-3) expression has been identified in human, rat and porcine testes where it is under hormonal control. Gal-3 is present in Sertoli cells and appears to be absent in human and (probably) in rat germ cells. Gal-3 expression was evidenced in the testes, in terms of both mRNA and protein (31 kDa). Gal-3 expression in cultured porcine Sertoli cells was shown to be under the positive control of FSH as well as of two cytokines epidermal growth factor (EGF) and tumour necrosis factor-alpha (TNF-alpha). Gal-3 expression in Sertoli cells is also potentially under the control of mature germ cells as an increased expression was observed in adult rat testes depleted in spermatocytes or spermatids. Although the function of testicular Gal-3 remains to be investigated, a potential role of Gal-3 in germ cell survival/regeneration is suggested based on its increased expression 1 month after a transient germ cell death process triggered by 10 days of treatment with the antiandrogen flutamide. Finally, although in the normal human testes, Gal-3 is exclusively located in the Sertoli cell cytoplasm, a nuclear localization is observed in the infertile testes. Together, the present findings have shown that (i) Gal-3 is expressed in the porcine, rat and human Sertoli cells; (ii) Gal-3 is under the positive control of FSH as well as of EGF and TNF-alpha and possibly of adult germ cells. These observations are compatible with a potential pro-survival role of Gal-3 in the testes.     

Laparoscopic gastrointestinal anastomoses using knotless barbed sutures are safe and reproducible: a single-center experience with 201 patients

Background

Intestinal anastomosis is a complex procedure during laparoscopy, mainly due to the difficulties knotting the sutures. Unidirectional barbed sutures have been proposed to simplify wall and mesentery closure, but the results for intestinal anastomosis are not clear. This study aimed to establish the feasibility and the safety of laparoscopic intestinal anastomosis using barbed suture.

Methods

Between June 2011 and May 2012, 15-cm-long unidirectional absorbable barbed sutures (V-Loc; Covidien, Mansfield, MA, USA) were used for all laparoscopic intestinal anastomoses: one suture for closure of intestinal openings after mechanical anastomoses and two sutures for hand-sewn anastomoses.

Results

Over a 1-year period, 201 consecutive patients required 220 laparoscopic anastomoses for gastrojejunostomy (n = 177; 172 during Roux-en-Y gastric bypass and 5 after gastrectomy), ileocolostomy (n = 15), colocolostomy (n = 1), esophagojejunostomy (n = 5), and jejunojejunostomy (n = 22; 4 after small bowel resection and 18 during gastric bypass or gastrectomy). Senior and training surgeons performed 209 closures of intestinal openings and 11 hand-sewn anastomoses. There was no conversion to usual sutures. One fistula occurred in an esophagojejunostomy and was managed conservatively. One self-limited anastomotic bleeding occurred, and no anastomotic stenosis occurred during 6 months of follow-up evaluation.

Conclusions

The use of knotless barbed suture for laparoscopic intestinal anastomosis is safe and reproducible.     

Risk of pulmonary embolism after a negative spiral CT angiogram in patients with pulmonary disease: 1-year clinical follow-up study

PURPOSE: To evaluate the effect of pulmonary disease on diagnostic utility of spiral computed tomographic (CT) angiography in clinical practice. MATERIALS AND METHODS: Three hundred thirty-four patients, including 215 patients with pulmonary disease (group 1) and 119 patients with no history of respiratory disorder (group 2), were referred for thin-collimation CT angiography of the pulmonary circulation as the first-line diagnostic test. Patients with negative angiograms who had not received anticoagulation therapy and who could be clinically followed up at 3 months, 6 months, and 1 year were considered in the final study groups (n = 185); 135 patients had lung disease (group 3), and 50 patients had no history of a respiratory disorder (group 4). RESULTS: Between groups 3 and 4, no significant differences were found in the referral location, age, and risk factors. Confident evaluation of pulmonary arteries down to the subsegmental level was performed in 31 (23%) patients in group 3 and in 15 (30%) in group 4 (P =.5). Three episodes of acute pulmonary embolism (PE), all fatal, were diagnosed in group 3 patients; two cases occurred 14 days and one case occurred 6 months after the negative spiral CT scan. The negative predictive value of spiral CT angiography was 98% (175 of 178) in the study group in which follow-up was performed, with no significant difference between the values in groups 3 (98% [132 of 135]) and 4 (100% [50 of 50]). CONCLUSION: Underlying respiratory disease does not affect the negative predictive value of thin-collimation CT angiography, which appears to be a reliable tool in the work-up in this subgroup of patients with acute PE.     

Évaluation de la reproductibilité et de la validité de construit d’une forme modifiée de l’indice algofonctionnel de Lequesne dans l’arthrose du genouAssessment of the test-retest reliability and construct validity of a modified Lequesne index in knee osteo-arthritis

Objectives. – To assess the test-retest reliability and the construct validity of a modified version of the Lequesne index.Methods. – Patients with symptomatic knee osteo-arthritis fulfilling the revised criteria of the american college of rheumatology completed the Lequesne index twice at a 3 h interval. Impairment outcome measures and patients’ perceived discomfort in walking and handicap were recorded. An item by item analysis was performed. Items having insufficient psychometric properties were excluded. Test-retest reliability was assessed using the intraclass correlation coefficient (ICC) and the Bland and Altman method. Construct validity was investigated using Spearman rank correlation coefficient and a factor analysis was performed.Results. – Eighty-eight patients were included. One question assessing pain (question IE) had a weak reliability (Kappa = 0.39) and was excluded. The test-retest reliability of the modified questionnaire was excellent (ICC = 0.95). Expected convergent and divergent correlations were achieved excepted for Vas pain and Vas handicap (0.46 and 0.40 respectively), and the “a priori” double stratification was confirmed by factor analysis, explaining 48.7% of the variance.Conclusion. – The modified form of the Lequesne index has sufficient psychometric properties to be used to assess pain and function in knee osteo-arthritis in a french population.     

Bladder telemetry: A new approach to evaluate micturition behavior under physiological and inflammatory conditions

Aims

To establish a new approach to cystometry using telemetry in conscious rats and to use this technique to determine the role of conscious decision making processes with respect to the initiation of voiding in physiological, inflammatory, and painful conditions.

Methods

The pressure transducer of a telemetric transmitter was implanted in the dome of the urinary bladder. After a recovery period of at least 1 month, several investigations of urodynamic parameters were performed after diuresis activation by a pulse of furosemide. The model was characterized by tolterodine and mirabegron under physiological conditions and same animals were reused to evaluate the modification of the voiding pattern under bladder inflammation induced by cyclophosphamide.

Results

The quality of traces and measurement of parameters recorded telemetrically were comparable to those with conventional cystometry. Furosemide induced a reproducible transient increase of urine production and a series of voids that persisted for 60 min. Tolterodine reduced the amplitude of micturition contractions although mirabegron was devoid of any effect. Seven hours after injection of CYP, voiding frequency increased significantly and the micturition amplitude contraction was not altered. However, the mean volume voided during individual micturitions and the total voided volume decreased. During a second exposure to furosemide 24H after CYP injection, the micturition pattern returned to control, however, the micturition volume was still lower than in control.

Conclusion

This telemetric model appears to be as accurate as previously described in conscious conventional cystometry, and allows the repeated evaluation of compounds which may modulate the voiding patterns. Neurourol. Urodynam. 36:308–315, 2017. © 2016 The Authors. Neurourology and Urodynamics published by Wiley Periodicals, Inc.     

The common P446L polymorphism in GCKR inversely modulates fasting glucose and triglyceride levels and reduces type 2 diabetes risk in the DESIR prospective general French population

OBJECTIVE— Hepatic glucokinase (GCK) is a key regulator of glucose storage and disposal in the liver, where its activity is competitively modulated, with respect to glucose, by binding to glucokinase regulatory protein (GCKR) in the presence of fructose 6-phosphate. Genome-wide association studies for type 2 diabetes identified GCKR as a potential locus for modulating triglyceride levels. We evaluated, in a general French population, the contribution of the GCKR rs1260326-P446L polymorphism to quantitative metabolic parameters and to dyslipidemia and hyperglycemia risk.RESEARCH DESIGN AND METHODS— Genotype effects of rs1260326 were studied in 4,833 participants from the prospective DESIR (Data from an Epidemiological Study on the Insulin Resistance syndrome) cohort both at inclusion and using the measurements at follow-up.RESULTS— The minor T-allele of rs1260326 was strongly associated with lower fasting glucose (−1.43% per T-allele; P = 8 × 10⁻¹³) and fasting insulin levels (−4.23%; P = 3 × 10⁻⁷), lower homeostasis model assessment of insulin resistance index (−5.69%; P = 1 × 10⁻⁸), and, conversely, higher triglyceride levels (3.41%; P = 1 × 10⁻⁴) during the 9-year study. These effects relate to a lower risk of hyperglycemia (odds ratio [OR] 0.79 [95% CI 0.70–0.88]; P = 4 × 10⁻⁵) and of incident cases during the study (hazard ratio [HR] 0.83 [0.74–0.95]; P = 0.005). Moreover, an additive effect of GCKR rs1260326(T) and GCK (−30G) alleles conferred lower fasting glycemia (P = 1 × 10⁻¹³), insulinemia (P = 5 × 10⁻⁶), and hyperglycemia risk (P = 1 × 10⁻⁶).CONCLUSIONS— GCKR-L446 carriers are protected against type 2 diabetes despite higher triglyceride levels and risk of dyslipidemia, which suggests a potential molecular mechanism by which these two components of the metabolic syndrome can be dissociated.The enzyme glucokinase (GCK) is the major glucose sensor of the pancreatic β-cells, where it adapts insulin secretion to blood glucose levels. GCK also participates in regulating glycogen synthesis and gluconeogenesis in the liver (1). GCK activity is allosterically controlled in hepatocytes by the glucokinase regulatory protein (GCKR), which reversibly binds to GCK and inhibits its activity in the presence of fructose 6-phosphate. GCKR acts as a competitive inhibitor of GCK with respect to glucose and is suppressed by the specific metabolite fructose 1-phosphate (2).In GCKR-deficient mice, the disruption of this regulation and the subsequent decrease in GCK activity leads to altered glucose metabolism and impaired postprandial glycemic control (3,4), although no change in fasting blood glucose concentration is observed. Adenoviral-mediated hepatic overexpression of GCKR in mice with high-fat diet–induced diabetes improves fasting and glucose-induced glycemia and leads to a concomitant increase in insulin sensitivity and triglyceride levels and a decrease in leptin levels (5). Heterozygous mutations in GCK resulting in a reduction of enzymatic activity are responsible for a subtype of monogenic diabetes (maturity-onset diabetes of the young-2) (1,6). Previous genetic studies at the GCKR locus have not reported intragenic mutations associated with type 2 diabetes in humans (7,8).The Diabetes Genetics Initiative (DGI) genome-wide association study for type 2 diabetes and quantitative metabolic traits reported an intronic polymorphism of GCKR (rs780094) explaining interindividual variability in plasma triglyceride (TG) levels and a trend toward association with lower fasting glycemia, less insulin resistance, and lower risk for type 2 diabetes (9). The HapMap II CEU data (www.hapmap.org) showed that rs780094 is in strong linkage disequilibrium (r² = 0.932) with a non-synonymous GCKR variant (Pro446Leu, rs1260326) that we previously identified by the DNA sequencing of French individuals (7). Marju Orho-Melander and colleagues recently communicated their fine-mapping data showing the strongest signal for TG levels at the coding single nucleotide polymorphism (SNP) (rs1260326; P = 1.5 × 10⁻⁹), with lesser associations to fasting glycemia and insulin sensitivity (M. Orho-Melander, O. Melander, V. Lyssenko, for the DGI, unpublished data). Similar findings for the intronic variant rs780094 were reported in a Danish population (10).We evaluated the association between GCKR rs1260326-P446L and TG levels in a middle-aged general French population with a follow-up of 9 years (11). Given the key role of GCKR in hepatic glucose metabolism, we also assessed the effect of rs1260326 on glucose homeostasis parameters and on the risk of impaired fasting glycemia and type 2 diabetes. Furthermore, as we previously showed a strong association of the GCK (−30A) promoter variant (rs1799884) to increased fasting glycemia and type 2 diabetes risk in the same study cohort (11), we also assessed possible additive effects of GCKR rs1260326-P446L and GCK −30G/A SNPs on fasting glucose, insulin, TG levels, and hyperglycemia risk.