ENaC inhibition stimulates Cl- secretion in the mouse cortical collecting duct through an NKCC1-dependent mechanism.

In cortical collecting ducts (CCDs) perfused in vitro, inhibiting the epithelial Na(+) channel (ENaC) reduces Cl(-) absorption. Since ENaC does not transport Cl(-), the purpose of this study was to determine how ENaC modulates Cl(-) absorption. Thus, Cl(-) absorption was measured in CCDs perfused in vitro that were taken from mice given aldosterone for 7 days. In wild-type mice, we observed no effect of luminal hydrochlorothiazide on either Cl(-) absorption or transepithelial voltage (V(T)). However, application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid or application of a Na(+)-K(+)-ATPase inhibitor to the bath reduced Cl(-) absorption by ∼66-75% and nearly obliterated lumen-negative V(T). In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1a(loxlox)). Whereas benzamil-sensitive Cl(-) absorption did not depend on CFTR, application of a Na(+)-K(+)-2Cl(-) cotransport inhibitor (bumetanide) to the bath or ablation of the gene encoding Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) blunted benzamil-sensitive Cl(-) absorption, although the benzamil-sensitive component of V(T) was unaffected. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl(-) absorption is benzamil sensitive, whereas thiazide-sensitive Cl(-) absorption is undetectable. Second, benzamil-sensitive Cl(-) absorption occurs by inhibition of ENaC, possibly due to elimination of lumen-negative V(T). Finally, benzamil-sensitive Cl(-) flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.

The Macrocirculation and Microcirculation of Hypertension.

Changes in vascular structure that accompany hypertension may contribute to hypertensive end-organ damage. Both the macrovascular and microvascular levels should be considered, as interactions between them are believed to be critically important. Regarding the macrocirculation, the article first reviews basic concepts of vascular biomechanics, such as arterial compliance, arterial distensibility, and stress-strain relationships of arterial wall material, and then reviews how hypertension affects the properties of conduit arteries, particularly examining evidence that it accelerates the progressive stiffening that normally occurs with advancing age. High arterial stiffness may increase central systolic and pulse pressure by two different mechanisms: 1) Abnormally high pulse wave velocity may cause pressure waves reflected in the periphery to reach the central aorta in systole, thus augmenting systolic pressure; 2) In the elderly, the interaction of the forward pressure wave with high arterial stiffness is mostly responsible for abnormally high pulse pressure. At the microvascular level, hypertensive disease is characterized by inward eutrophic or hypertrophic arteriolar remodeling and capillary rarefaction. These abnormalities may depend in part on the abnormal transmission of highly pulsatile blood pressure into microvascular networks, especially in highly perfused organs with low vascular resistance, such as the kidney, heart, and brain, where it contributes to hypertensive end-organ damage.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

Glibenclamide enhances neurogenesis and improves long-term functional recovery after transient focal cerebral ischemia

Glibenclamide is neuroprotective against cerebral ischemia in rats. We studied whether glibenclamide enhances long-term brain repair and improves behavioral recovery after stroke. Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (MCAO) for 90 minutes. A low dose of glibenclamide (total 0.6mg) was administered intravenously 6, 12, and 24 hours after reperfusion. We assessed behavioral outcome during a 30-day follow-up and animals were perfused for histological evaluation. In vitro specific binding of glibenclamide to microglia increased after pro-inflammatory stimuli. In vivo glibenclamide was associated with increased migration of doublecortin-positive cells in the striatum toward the ischemic lesion 72 hours after MCAO, and reactive microglia expressed sulfonylurea receptor 1 (SUR1) and Kir6.2 in the medial striatum. One month after MCAO, glibenclamide was also associated with increased number of NeuN-positive and 5-bromo-2-deoxyuridine-positive neurons in the cortex and hippocampus, and enhanced angiogenesis in the hippocampus. Consequently, glibenclamide-treated MCAO rats showed improved performance in the limb-placing test on postoperative days 22 to 29, and in the cylinder and water-maze test on postoperative day 29. Therefore, acute blockade of SUR1 by glibenclamide enhanced long-term brain repair in MCAO rats, which was associated with improved behavioral outcome.

Safety of combined heat and moisture exchanger filters in long-term mechanical ventilation.

STUDY OBJECTIVE: To evaluate the safety of a combined heat and moisture exchanger filter (HMEF) for the conditioning of inspired gas in long-term mechanical ventilation (MV). DESIGN: Randomized controlled trial. SETTING: Medical ICU in a large teaching hospital. PATIENTS: One hundred fifteen consecutive patients who required > or = 48 h of MV. INTERVENTIONS: Patients were randomized at intubation time (day 1) to receive inspired gas conditioned either by a water-bath humidifier heated at 32 degrees C (HWBH) or by an HMEF (Hygroster; DAR; Mirandola, Italy). MEASUREMENTS AND MAIN RESULTS: The two study groups were comparable in terms of primary pathologic condition at the time of hospital admission, disease severity as measured by the Simplified Acute Physiology Score, and ICU mortality. They did not differ with respect to ventilator days per patient (mean +/- SD: HMEF, 7.6 +/- 6.5; HWBH, 7.8 +/- 5.8), incidence of endotracheal tube obstruction (HMEF, 0/59; HWBH, 1/56), and incidence of hypothermic episodes (HMEF, five; HWBH, two). In 41 patients receiving MV for > or = 5 days, the morphologic integrity of respiratory epithelium was evaluated on day 1 and day 5, using a cytologic examination of tracheal aspirate smears. The state of ciliated epithelium was scored on a scale from 0 (poorest integrity) to 1,200 (maximum integrity), according to a well-described method. In both patient groups, the scores slightly but significantly decreased from day 1 to day 5 (mean +/- SD: HWBH, from 787 +/- 104 to 745 +/- 88; HMEF, from 813 +/- 79 to 739 +/- 62; p < 0.01 for both groups); there were no statistically significant differences between groups. CONCLUSIONS: These data indicate acceptable safety of HMEFs of the type used in the present study for long-term mechanical ventilation.

Angiotensin-II mediates norepinephrine and neuropeptide-Y secretion in a human pheochromocytoma.

The potential role of angiotensin-II in mediating catecholamine and neuropeptide-Y release in a human pheochromocytoma has been investigated. Angiotensin-II type I receptors are transcribed and translated into functional proteins in a surgically removed pheochromocytoma. Primary cell culture of the tumor has been studied in a perfused system. Angiotensin-II increased the release of norepinephrine and neuropeptide-Y by the pheochromocytes. Activation of the angiotensin-II type I receptors by angiotensin-II was associated with a rise in cytosolic free calcium. The renin-angiotensin system may, therefore, contribute to the secretion of catecholamines and NPY occurring in patients with pheochromocytoma and when stimulated trigger hypertensive crisis.

Comparative assessment of intimal hyperplasia development after 14 days in two different experimental settings: tissue culture versus ex vivo continuous perfusion of human saphenous vein.

BACKGROUND: Intimal hyperplasia (IH) is a vascular remodeling process which often leads to failure of arterial bypass or hemodialysis access. Experimental and clinical work have provided insight in IH development; however, further studies under precise controlled conditions are required to improve therapeutic strategies to inhibit IH development. Ex vivo perfusion of human vessel segments under standardized hemodynamic conditions may provide an adequate experimental approach for this purpose. Therefore, chronically perfused venous segments were studied and compared to traditional static culture procedures with regard to functional and histomorphologic characteristics as well as gene expression. MATERIALS AND METHODS: Static vein culture allowing high tissue viability was performed as previously described. Ex vivo vein support system (EVVSS) was performed using a vein support system consisting of an incubator with a perfusion chamber and a pump. EVVSS allows vessel perfusion under continuous flow while maintaining controlled hemodynamic conditions. Each human saphenous vein was divided in two parts, one cultured in a Pyrex dish and the other part perfused in EVVSS for 14days. Testing of vasomotion, histomorphometry, expression of CD 31, Factor VIII, MIB 1, alpha-actin, and PAI-l were determined before and after 14days of either experimental conditions. RESULTS: Human venous segments cultured under traditional or perfused conditions exhibited similar IH after 14 days as shown by histomorphometry. Smooth-muscle cell (SMC) was preserved after chronic perfusion. Although integrity of both endothelial and smooth-muscle cells appears to be maintained in both culture conditions as confirmed by CD31, factor VIII, and alpha-actin expression, a few smooth-muscle cells in the media stained positive for factor VIII. Cell-proliferation marker MIB-1 was also detected in the two settings and PAI-1 mRNA expression and activity increased significantly after 14 days of culture and perfusion. CONCLUSION: This study demonstrates the feasibility to chronically perfuse human vessels under sterile conditions with preservation of cellular integrity and vascular contractility. To gain insights into the mechanisms leading to IH, it will now be possible to study vascular remodeling not only under static conditions but also in hemodynamic environment mimicking as closely as possible the flow conditions encountered in reconstructive vascular surgery.

Substance P and calbindin D-28k-immunoreactivity in primary sensory neurons of chick embryos: differential neuronal birthdates and transient co-localization.

During the ontogenesis of dorsal root ganglia (DRG), the immunoreactivity to substance P (SP) and calbindin D-28k (CaBP) appears in chickens at embryonic day 5 (E5) and E10 respectively. To establish the birthdates of primary sensory neurons expressing SP or CaBP, chick embryos were given repetitive intra-amniotic injections of [3H]-thymidine. The neuroblasts giving rise to SP-expressing neurons were labeled up to E6 while those generating CaBP-immunoreactive neurons stopped to incorporate [3H]-thymidine before E5.5. This finding indicates that neurons exhibiting distinct phenotypes may originate from neuroblasts which arrest to proliferate at close but distinct stages of development. To determine whether SP and CaBP are co-expressed or not in DRG neurons, chick embryos at E12, E18, and chickens two weeks after hatching were perfused and fixed to detect simultaneously SP- and CaBP-immunoreactivity in DRG sections. The results showed that SP and CaBP were transiently co-expressed by a subset of neurons at E12. Later, however, the SP-immunoreactivity was gradually lost by these ganglion cells, so that the SP- and CaBP-immunoreaction defined two distinct neuronal subpopulations after hatching. In conclusion, most CaBP-immunoreactive DRG cells derive from a subset of neurons in which SP and CaBP are transiently co-localized.

The Macklin effect: a frequent etiology for pneumomediastinum in severe blunt chest trauma.

STUDY OBJECTIVES: To review the etiology and pathophysiology of pneumomediastinum in severe blunt trauma, with a special interest in one of its possible origins, the Macklin effect. The Macklin effect relates to a three-step pathophysiologic process: blunt traumatic alveolar ruptures, air dissection along bronchovascular sheaths, and spreading of this blunt pulmonary interstitial emphysema into the mediastinum. The clinical relevance of the Macklin effect was also evaluated. SETTING: A university hospital serving as a reference trauma center. PATIENTS: A selection of 51 patients with severe blunt trauma between 1995 and 2000. Inclusion criteria: Severe trauma or high-speed deceleration justifying chest CT; if chest CT demonstrated a pneumomediastinum, bronchoscopy and esophagoscopy were performed to rule out tracheobronchial or esophageal injury. DESIGN: Retrospective analysis of patients' clinical files, chest CT, and bronchoscopy and esophagoscopy reports. The Macklin effect was diagnosed when an air collection adjacent to a bronchus and a pulmonary vessel could be clearly identified on the chest CT. Clinical relevance of the Macklin effect was statistically evaluated regarding its repercussions on the pulmonary gas exchange function, the respective durations of intensive care and total hospital stay, and the associated injuries. RESULTS: Twenty (39%) Macklin effects and 5 tracheobronchial injuries (10%) were identified. One tracheobronchial injury occurred simultaneously with the Macklin effect. The presence of the Macklin effect affected neither the clinical profile nor the result of pulmonary gas analysis on hospital admission, but was associated with a significant (p < 0.001) lengthening of the intensive care stay. CONCLUSIONS: The Macklin effect is present in 39% of severe blunt traumatic pneumomediastinum detected by CT. Its identification does not rule out a tracheobronchial injury. The Macklin effect reflects severe trauma, since it is associated with significantly prolonged intensive care stay.

Role of hemodynamic forces in the ex vivo arterialization of human saphenous veins.

BACKGROUND: Human saphenous vein grafts are one of the salvage bypass conduits when endovascular procedures are not feasible or fail. Understanding the remodeling process that venous grafts undergo during exposure to arterial conditions is crucial to improve their patency, which is often compromised by intimal hyperplasia. The precise role of hemodynamic forces such as shear stress and arterial pressure in this remodeling is not fully characterized. The aim of this study was to determine the involvement of arterial shear stress and pressure on vein wall remodeling and to unravel the underlying molecular mechanisms. METHODS: An ex vivo vein support system was modified for chronic (up to 1 week), pulsatile perfusion of human saphenous veins under controlled conditions that permitted the separate control of arterial shear stress and different arterial pressure (7 mm Hg or 70 mm Hg). RESULTS: Veins perfused for 7 days under high pressure (70 mm Hg) underwent significant development of a neointima compared with veins exposed to low pressure (7 mm Hg). These structural changes were associated with altered expression of several molecular markers. Exposure to an arterial shear stress under low pressure increased the expression of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 at the transcript, protein, and activity levels. This increase was enhanced by high pressure, which also increased TIMP-2 protein expression despite decreased levels of the cognate transcript. In contrast, the expression of plasminogen activator inhibitor-1 increased with shear stress but was not modified by pressure. Levels of the venous marker Eph-B4 were decreased under arterial shear stress, and levels of the arterial marker Ephrin-B2 were downregulated under high-pressure conditions. CONCLUSIONS: This model is a valuable tool to identify the role of hemodynamic forces and to decipher the molecular mechanisms leading to failure of human saphenous vein grafts. Under ex vivo conditions, arterial perfusion is sufficient to activate the remodeling of human veins, a change that is associated with the loss of specific vein markers. Elevation of pressure generates intimal hyperplasia, even though veins do not acquire arterial markers. CLINICAL RELEVANCE: The pathological remodeling of the venous wall, which leads to stenosis and ultimately graft failure, is the main limiting factor of human saphenous vein graft bypass. This remodeling is due to the hemodynamic adaptation of the vein to the arterial environment and cannot be prevented by conventional therapy. To develop a more targeted therapy, a better understanding of the molecular mechanisms involved in intimal hyperplasia is essential, which requires the development of ex vivo models of chronic perfusion of human veins.

Effect of chronic ethanol feeding on rat hepatocytic glutathione. Compartmentation, efflux, and response to incubation with ethanol.

Hepatocytes from rats that were fed ethanol chronically for 6-8 wk were found to have a modest decrease in cytosolic GSH (24%) and a marked decrease in mitochondrial GSH (65%) as compared with pair-fed controls. Incubation of hepatocytes from ethanol-fed rats for 4 h in modified Fisher's medium revealed a greater absolute and fractional GSH efflux rate than controls with maintenance of constant cellular GSH, indicating increased net GSH synthesis. Inhibition of gamma-glutamyltransferase had no effect on these results, which indicates that no degradation of GSH had occurred during these studies. Enhanced fractional efflux was also noted in the perfused livers from ethanol-fed rats. Incubation of hepatocytes in medium containing up to 50 mM ethanol had no effect on cellular GSH, accumulation of GSH in the medium, or cell viability. Thus, chronic ethanol feeding causes a modest fall in cytosolic and a marked fall in mitochondrial GSH. Fractional GSH efflux and therefore synthesis are increased under basal conditions by chronic ethanol feeding, whereas the cellular concentration of GSH drops to a lower steady state level. Incubation of hepatocytes with ethanol indicates that it has no direct, acute effect on hepatic GSH homeostasis.

Cricotracheal resection for adult and pediatric subglottic stenoses: Similarities and differences

Since the mid-1970s thoracic surgeons have used cricotracheal resection (CTR) in the adult population. Similar positive results have from then on been reported by different investigators using this technique. In contrast, otolaryngologists have typically used an anterior midline approach to the larynx and trachea for their laryngotracheal reconstructions (LTRs), both in adults and children. The fear of injury to the recurrent laryngeal nerve (RLN), the risk of a dehiscence of the anastomosis, and the interference with normal growth of the larynx in the pediatric age group were the main reasons put forward for not using a CTR for the cure of subglottic stenosis. In this article, we describe the similarities and differences in CTRs performed for adult and pediatric cases. The high rate (>90%) of success for severe pediatric SGS using CTR compares most favorably with LTR, which is still used in most otolaryngology departments. The reasons lie in the full resection of the stenosis and the restoration of a steady cartilaginous framework of the larynx and trachea with full mucosal lining on both sides of the anastomosis. Furthermore, CTR can be associated with a Rethi procedure for the cure of combined posterior glottic and subglottic stenoses. Good results from series of two different centers suggest that CTR will become the treatment of choice for pediatric severe SGS, as is presently the case in the adult population.

Procedure for human saphenous veins ex vivo perfusion and external reinforcement.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

The αVβ3/αVβ5 integrin inhibitor cilengitide augments tumor response to melphalan isolated limb perfusion in a sarcoma model.

Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor (TNF)-α is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better-tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha-V integrins, has both anti-angiogenic and direct anti-tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra-peritoneal (i.p.) injections of cilengitide or saline 2 hr before and 3 hr after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a three to sevenfold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting.

Partial cricotracheal resection for pediatric subglottic stenosis: a single institution's experience in 60 cases.

In our study, 60 infants and children, each with a severe subglottic stenosis (SGS), underwent partial cricotracheal resection (PCTR) with primary thyrotracheal anastomosis. According to the Myer-Cotton classification, two were grade II, 41 were grade III and 17 were grade IV stenoses. Of the 60 patients, 57 (95%) are presently decannulated, and one patient sustained a complete restenosis. Two patients with better than 80% subglottic airways still are waiting for decannulation: one because of bilateral cricoarytenoid joint fixation and the second because of temporary stenting of the subglottis with a Montgomery T-tube. The rate of decannulation is 97% (36 of 37 cases) in primary PCTRs, 100% (13 of 13 cases) in salvage PCTRs for failed laryngotracheal reconstructions (LTR) and 70% (7 of 10 cases) in extended PCTRs (i.e., PCTR associated with an additional open-airway procedure).