Two-photon microscopy of vital murine elastic and muscular arteries. Combined structural and functional imaging with subcellular resolution

Understanding vascular pathologies requires insight in the structure and function, and, hence, an imaging technique combining subcellular resolution, large penetration depth, and optical sectioning. We evaluated the applicability of two-photon laser-scanning microscopy (TPLSM) in large elastic and small muscular arteries under physiological conditions. Elastic (carotid) and muscular (uterine, mesenteric) arteries of C57BL/6 mice were mounted in a perfusion chamber. TPLSM was used to assess the viability of arteries and to visualize the structural components elastin, collagen, nuclei, and endothelial glycocalyx (EG). Functionality was determined using diameter changes in response to noradrenaline and acetylcholine. Viability and functionality were maintained up to 4 h, enabling the assessment of structure-function relationships. Structural vessel wall components differed between elastic and muscular arteries: size (1.3 vs. 2.1 microm) and density (0.045 vs. 0.57 microm(-2)) of internal elastic lamina fenestrae, smooth muscle cell density (3.50 vs. 1.53 microm(-3)), number of elastic laminae (3 vs. 2), and adventitial collagen structure (tortuous vs. straight). EG in elastic arteries was 4.5 microm thick, covering 66% of the endothelial surface. TPLSM enables visualization and quantification of subcellular structures in vital and functional elastic and muscular murine arteries, allowing unraveling of structure-function relationships in healthy and diseased arteries.     

Endotracheal temperature and humidity measurements in laryngectomized patients: intra- and inter-patient variability

This study assesses intra- and inter-patient variability in endotracheal climate (temperature and humidity) and effects of heat and moister exchangers (HME) in 16 laryngectomized individuals, measured repeatedly (N = 47). Inhalation Breath Length (IBL) was 1.35 s without HME and 1.05 s with HME (P < 0.0001). With HME, end-inspiratory (minimum) humidity values increased 5.8 mg H₂O/L (P < 0.0001) and minimum temperature values decreased 1.6°C (P < 0.0001). For the temperature and humidity minimums, the inter-patient variability was much smaller than the short- and long-term intra-patient variability. For exhalation breath length and full breath length, the opposite was the case. Conclusions: (1) Because inter-patient variability is smaller than intra-patient variability, investigating endotracheal climate in a limited number of laryngectomized subjects is justified, provided repeated measurements per patient are accomplished; (2) main contributor to intra-patient variability is the positioning of the catheter tip in the trachea; (3) an HME leads to a shortened IBL which enhances the HME effect.     

Randomized phase III trial of chemoimmunotherapy in patients with previously untreated stages III and IV suboptimal disease ovarian cancer: a Southwest Oncology Group Study

Between 1979 and 1984, 185 fully evaluable patients with stage III or IV epithelial type ovarian cancer and suboptimal surgical resections were randomly assigned to treatment with doxorubicin + cyclophosphamide + BCG (DC + BCG) vs doxorubicin + cyclophosphamide + cisplatin (DCP) vs. doxorubicin + cyclophosphamide + cisplatin + BCG (DCP + BCG). Patients with measurable disease (119) were analyzed separately from those with nonmeasurable disease (66). In measurable disease patients the overall clinical complete plus partial response rates for DC + BCG, DCP, and DCP + BCG-treated patients were 36, 57, and 59%, respectively. Although there were no significant patient characteristic differences between the DCP and DCP + BCG treatment groups, the addition of cisplatin to the DC + BCG regimen resulted in significantly prolonged response (P less than 0.03) and survival (P less than 0.002) durations. To the contrary, the addition of BCG to the DCP regimen did not improve objective response rates or response or survival durations. For patients with nonmeasurable, suboptimal disease there were no significant differences between the three treatments with respect to response or survival parameters; however, patients in this disease category fared generally better than those with clinically measurable disease. We conclude that cisplatin adds significantly to the efficacy of DC + BCG, but BCG does not add to the efficacy of DCP in patients with measurable, stage III or IV disease.     

Adequate cytogenetic examination in myelodysplastic syndromes: analysis of 529 patients

In myelodysplastic syndromes (MDS), the karyotype is one of the most significant prognostic markers with profound impact on differential diagnosis and therapeutic decisions. In a retrospective study, we examined karyotypes of bone marrow specimens of an oligocentric cohort comprising 529 patients with MDS to address the question how many metaphases need to be analyzed to detect even small cell clones with an appropriate expenditure. We found a statistically significant difference of the frequency of normal karyotypes in the patient group with 19 or less analyzed metaphases compared to the group with 20 or more metaphases analyzed (56% versus 47%, p=0.041). Furthermore, we demonstrate that the analysis of 25 or more metaphases can further improve the sensitivity of karyotype analysis and leads to the identification of additional clinically relevant abnormal clones or subclones in a substantial proportion of patients. In summary, our data suggest the examination of at least 20 metaphases in MDS.     

Use of perfluorohexyloctane as a long-term internal tamponade agent in complicated retinal detachment surgery.

PURPOSE: To report the use of perfluorohexyloctane, a liquid semifluorinated alkane that is heavier than water, as an internal tamponade agent in surgery for complicated retinal detachments. DESIGN: A consecutive interventional case series from three study centers. METHODS: In 23 consecutive eyes (23 patients, 19 men and four women, mean +/- standard deviation (SD) age of 58.5 years +/- 16.1) perfluorohexyloctane was used for long-term internal tamponade. Included were eyes with complicated retinal detachment involving the lower two quadrants of the fundus. Excluded were patients with diseases in the fellow eye or severe systemic disease. A pars plana vitrectomy was performed, including membrane peeling and retinotomy where necessary. RESULTS: The mean duration for perfluorohexyloctane being left in situ was 76 days (SD 37.64) (range, 35-202 days). Four weeks following the removal of perfluorohexyloctane 19 of the 23 patients had total reattachment of the retina; three eyes had a recurrence of retinal detachment. One patient was lost to follow-up. The mean follow-up after perfluorohexyloctane removal was 97 days (range, 48 to 169 days). Cataract formation or progression was noted in nine of the 10 eyes. There were two cases with high intraocular pressures. Dispersion into small droplets was observed as early as 3 days postoperatively in three of the 23 patients. At least 12 of the 23 patients had an obvious dispersion by the time of perfluorohexyloctane removal. There was no sign of optic atrophy, retinal necrosis, or retinal vascular occlusion. CONCLUSION: Perfluorohexyloctane was tolerated as a long-term internal tamponade agent without obvious signs of damage to the retina or optic disk. Of all the complications noted, the most common was that of dispersion of the perfluorohexyloctane bubble into droplets.     

Hepatic derangement following N‐Acetylcysteine enemas in an infant with cystic fibrosis

We discuss an infant with MI secondary to cystic fibrosis, who was managed surgically by a double barrel ileostomy for mid – small bowel atresia and developed severe faecal impaction in the post – operative period. The faecal impaction was treated successfully with oral NAC and 0.2% NAC contrast enemas. The patient's liver function tests revealed a dramatic increase in transaminases and bilirubin contemporaneous with the administration of the enemas. The levels showed a spontaneous improvement after discontinuation. This is only the second reported case of hepatotoxicity secondary to NAC enemas in the literature. While our experience offers modest support for the use of NAC, its efficacy is not yet proven and paediatric surgeons using NAC in the enema form need to closely monitor liver function contemporaneous with this agent's administration and adjust their treatment accordingly.     

Increased PDZ-RhoGEF/RhoA/Rho kinase signaling in small mesenteric arteries of angiotensin II-induced hypertensive rats

BACKGROUND: The phosphorylation of myosin light chain (MLC) maintains the contracted state of vascular smooth muscle. Dephosphorylation results in relaxation and is determined by the activity of myosin light chain phosphatase (MLCP), which is negatively regulated by Rho kinase. METHODS: We tested whether an increased Rho kinase activity, and hence a decreased contribution of MLCP, results in an increased contractility of small fourth-order mesenteric arteries (MA) during the early onset of angiotensin II (Ang II)-induced hypertension (Ang II-14d). RESULTS: Calcium sensitivity was similar, but contractile tension in response to [Ca]ex (5 mmol/l) in endothelium-denuded and depolarized MA was greater, in Ang II-14d rats compared to sham-operated normotensive (SHAM) and Ang II-1d. The Rho kinase inhibitor Y-27,632 caused a significantly greater inhibition of the contractile response to various agents (phenylephrine, norepinephrine, U46,619 and K) in MA of Ang II-14d compared to SHAM. Protein expression levels of the GDP/GTP exchange factor PDZ-RhoGEF, which co-immunoprecipitated with RhoA, were increased in MA of Ang II-14d compared to SHAM. RhoA translocation was greater in U46,619 (1 micromol/l)-stimulated MA of Ang II-14d compared to SHAM. Expression levels of Rho kinase beta were higher in MA of Ang II-14d. The MLCP inhibitor calyculin A (100 nmol/l) caused a greater contraction in MA of SHAM compared to Ang II-14d. Phosphorylation of the target subunit of MLCP (MYPT1) was enhanced in U46,619-stimulated MA of Ang II-14d compared to SHAM. CONCLUSION: This is the first study demonstrating enhanced PDZ-RhoGEF/RhoA/Rho kinase signaling during hypertension at the level of resistance-sized arteries. This enhanced signaling leads to increased MLCP phosphorylation, resulting in vascular hyper-reactivity.     

Putative role of epithelial sodium channels (<Emphasis Type="Italic">ENaC</Emphasis>)in the afferent limb of cardio renal reflexes in rats

Abstract. Recent studies suggest a role of ion channels of the DEG/ENaC family for mechanosensation in different species and in baroreceptor reex control in rats. We tested the hypothesis that ENaC within the cardiac sensory network are mandatory for mechanosensation. Experiments were performed in male Sprague-Dawley rats, isolated nodose ganglion cells with cardiac afferents and isolated vagus nerves.Epicardial delivery of the amiloride analogue benzamil intended to specifically inhibit ENaC presumably located on cardiac sensory afferents indeed blunted the mechanosensitive (i. e., sympathoinhibition by intravenous volume loading [–32% and –42% in treated groups vs. –67% in controls; n = 7 each; p < 0.05]) as well as—though to a lesser extent—the 5-HT₃-mediated chemosensitive cardiorenal reex in vivo in a dose-dependent manner. Using patch clamp technique, however, it turned out that neither amiloride nor benzamil inuenced mechanically induced currents in ganglion nodosum cells in vitro, stimulated by hypoosmotic stress. The unspecific stretch activated ion channel blocker gadolinium completely abolished mechanically induced currents, indicating respective cells were mechanosensitive. In isolated vagus nerves benzamil impaired action potentials obtained by electrical stimulation (C-spike amplitude [–33%]; latency [+12%]; n = 8; p < 0.05).Our ndings at least cast doubt on ENaC exclusively playing a specific role as mechanotransducers within the cardiac sensory network. Other ion channels might be involved. Furthermore the observed ndings in vivo could also be due to unspecific disturbance of afferent signal conduction.