Custom-engineered hydrogels for delivery of human iPSC-derived neurons into the injured cervical spinal cord.

Cervical damage is the most prevalent type of spinal cord injury clinically, although few preclinical research studies focus on this anatomical region of injury. Here we present a combinatorial therapy composed of a custom-engineered, injectable hydrogel and human induced pluripotent stem cell (iPSC)-derived deep cortical neurons. The biomimetic hydrogel has a modular design that includes a protein-engineered component to allow customization of the cell-adhesive peptide sequence and a synthetic polymer component to allow customization of the gel mechanical properties. In vitro studies with encapsulated iPSC-neurons were used to select a bespoke hydrogel formulation that maintains cell viability and promotes neurite extension. Following injection into the injured cervical spinal cord in a rat contusion model, the hydrogel biodegraded over six weeks without causing any adverse reaction. Compared to cell delivery using saline, the hydrogel significantly improved the reproducibility of cell transplantation and integration into the host tissue. Across three metrics of animal behavior, this combinatorial therapy significantly improved sensorimotor function by six weeks post transplantation. Taken together, these findings demonstrate that design of a combinatorial therapy that includes a gel customized for a specific fate-restricted cell type can induce regeneration in the injured cervical spinal cord.

Selective inhibition of guanylate cyclase prevents impairment of hypoxic pulmonary vasoconstriction in endotoxemic mice.

Nitric oxide (NO) may cause sepsis-induced impairment of hypoxic pulmonary vasoconstriction (HPV). Although NO exerts many of its actions by activating soluble guanylate cyclase (sGC), there are several cGC-independent mechanisms that may lead to NO-induced vasodilation during endotoxemia. We investigated the role of sGC for the regulation of HPV during lipopolysaccharide (LPS) induced endotoxemia using 1H-(1,2,4)oxadiazole(4,3-alpha)quinoxaline-1-one (ODQ), a specific inhibitor of sGC, in isolated, perfused, and ventilated mouse lungs. Without ODQ, lungs from LPS-challenged mice constricted significantly less in response to hypoxia as compared to lungs from mice not treated with LPS (26 +/- 27% vs. 134 +/- 37%, respectively, p < 0.05). 20 mg/kg ODQ, but not 2 mg/kg or 10 mg/kg, restored the blunted HPV response in LPS-challenged mice as compared to mice not challenged with LPS (80+/-14 % vs. 98+/-21 %). ODQ had no effect on baseline perfusion pressures under normoxic conditions. Analysis of pulmonary vascular P-Q relationships suggested that the restoration of pulmonary vascular response to hypoxia by ODQ is associated with a restoration of pulmonary vascular properties during normoxia. Our data show in a murine model that specific inhibition of sGC may be a new approach to restore HPV during endotoxemia.

Preconditioning, but not postconditioning, with Sevoflurane reduces pulmonary neutrophil accumulation after lower body ischaemia/reperfusion injury in rats.

BACKGROUND AND OBJECTIVES: Aortic ischaemia and reperfusion may induce pulmonary sequestration of neutrophil granulocytes. Preconditioning and postconditioning with volatile anaesthetics confer protection against reperfusion injury in various organs, such as heart, kidneys or brain. We tested the hypothesis that pre- or postconditioning with Sevoflurane attenuates pulmonary neutrophil accumulation after ischaemia/reperfusion injury of the aorta. METHODS: Anaesthetized and mechanically ventilated Wistar rats underwent laparotomy and were randomly assigned to one of the following groups: Sham (n = 10), ischaemia/reperfusion (n = 8, lower body ischaemia by clamping of the infrarenal aorta for 2 h followed by 3 h of reperfusion), preconditioning (n = 10, 2.0% Sevoflurane administered over 30 min prior to ischaemia) and postconditioning (n = 9, 2.0% Sevoflurane during reperfusion). Following reperfusion, the lungs were removed for microscopic determination of neutrophil accumulation. RESULTS: Ischaemia/reperfusion induced a significant increase in pulmonary neutrophil accumulation (mean +/- SD, 29.9 +/- 7.4 vs. 15.8 +/- 6.6 neutrophils per microscopic field in ischaemia/reperfusion vs. Sham, respectively, P < 0.001). Sevoflurane preconditioning resulted in a lower neutrophil count (20.3 +/- 7.1 neutrophils, P < 0.001 vs. ischaemia/reperfusion), while postconditioning showed no effects (25.8 +/- 9.8 neutrophils vs. ischaemia/reperfusion, not significant). CONCLUSIONS: Preconditioning, but not postconditioning, with Sevoflurane reduces pulmonary neutrophil accumulation after ischaemia/reperfusion injury of the lower body. Since neutrophil accumulation plays a major role in the pathophysiology of acute lung injury, our data suggest a protective effect of Sevoflurane preconditioning on remote pulmonary ischaemia/reperfusion injury.

Worsening of long-term myocardial function after successful pharmacological pretreatment with cyclosporine.

Pretreatment with cyclosporine (CsA) decreases infarct size 24h after myocardial ischemia/reperfusion (I/R). The goal of this study was to determine effects of CsA pretreatment on long-term cardiac function after I/R-injury. Rats were randomly assigned to group-1: vehicle-only, group-2: CsA-5mg/kg/day, and group-3: CsA-12.5mg/kg/day given orally for three days prior to I/R-injury (30 min of left anterior descending coronary artery occlusion). Post-I/R survival and cardiac function were evaluated 14 days after I/R-injury by echocardiography and invasive hemodynamic measurements. Rats with I/R-injury showed increased left ventricular pressure (LVEDP) compared to rats without I/R-injury (p<0.005). Although CsA initially decreased infarct size, no differences of LVEDP were seen 14 days after I/R-injury (vehicle: 21.2+/-8.9 mmHg, CsA-5mg/kg/day: 21.5+/-0.7 mmHg, CsA-12.5mg/kg/day: 20.5+/-9.4 mmHg). Ejection fraction and fractional shortening were decreased compared to baseline, but showed no differences between groups. On day 14, a dose-dependent increase in left ventricular end diastolic diameter was seen (p<0.001). CsA pretreatment was associated with a dose-dependent decrease in post-I/R-survival (vehicle: 56%, CsA-5mg/kg/day: 32%, CsA-12.5mg/kg/day: 16%; p=0.017). CsA pretreatment did not improve long-term cardiac function despite decreased infarct size 24h after I/R-injury, but increased post-I/R mortality significantly. Poor cardiac function after CsA pretreatment might be caused by left ventricular dilation.

[Hydrofluoric acid burns. A rare chemical emergency situation]. / Verätzung mit Flusssäure. Ein seltener chemischer Notfall.

Burns caused by hydrofluoric acid can be life-threatening. Of special significance is the often underestimated local and sometimes delayed deep action of the highly diffusible free fluoride ions and the accompanying systemic toxicity. The specific antidote calcium gluconate can be topically applied, injected into tissue or infused intra-arterially. Because of the extreme danger of systemic toxicity even after seemingly trivial injuries, monitoring in the intensive care station, especially by measuring the calcium concentration in blood and electrocardiography, and therapy is recommended.

[Can lung protective ventilation methods modify outcome?--A critical review]. / Können lungenprotektive Beatmungsstrategien das "Outcome" beeinflussen?--Eine kritische Ubersicht.

A large body of experimental and clinical work leaves no room for doubt that mechanical ventilation can contribute to the progression of a lung disease or, in the worst case, produce acute pulmonary damage. The pathophysiological processes involved have been described as barotrauma, volutrauma, atelectrauma and biotrauma. In response, a socalled lung-protective ventilation strategy has been proposed, especially for patients with acute respiratory distress syndrome (ARDS). Such an approach seeks to apply limited airway pressures, small tidal volumes and appropriate levels of positive end-expiratory pressures even if, as a consequence, non-physiological gas exchange values (i.e. elevated PaCO2-levels) need to be tolerated. A recent large prospective randomized trial demonstrated reduced mortality rates using such a strategy. To support lung-protective ventilation in ARDS patients, an array of therapeutic measures has been proposed, including meticulous attention to fluid and transfusion management, prone position, extracorporeal membrane oxygenation (ECMO), inhalation of nitric oxide, implementation of spontaneous breathing, partial liquid ventilation and tracheal gas insufflation. Of these, only prone positioning has become part of routine clinical management, while ECMO is applied in selected cases only. Unfortunately, thus far, none of these measures has passed the litmus test of a randomized controlled trial. Recent large prospective observational studies, however, suggest that only an optimized concert of therapeutic interventions, but not a single measure alone, may improve the outcome of ARDS patients.

[Imported tropical malaria after a sojourn in Kenya. Serious consequences of neglected chemoprophylaxis and delayed diagnosis]. / Importierte Malaria tropica nach Keniaaufenthalt - Schwerwiegende Folgen von unterlassener Chemoprophylaxe und verspäteter Diagnose -

HISTORY AND ADMISSION FINDINGS: A 56-year-old man was admitted to the hospital 11 days after returning from Kenya because of recurrent fever attacks. The patient had not taken malaria chemoprophylaxis and had previously received symptomatic treatment for suspected viral infection by his general practitioner. Physical findings on admission included enlargement of liver and spleen, marked dehydration and a body temperature of 40.1 degrees C. INVESTIGATIONS: Initial chest radiography showed no abnormalities. Thick and thin blood smears were positive for Plasmodium falciparum. Initial parasitemia was 0.5 per thousand. TREATMENT AND COURSE: Despite immediate quinine therapy including loading dose and intensive care treatment complicated malaria with multiorgan failure developed. The patient required mechanical ventilation, high-dose catecholamine treatment and hemodialysis for several days. The course of parasitemia peaked on treatment day 2 at a level of 31.1 per thousand. CONCLUSION: Our case shows serious consequences and important complications of Plasmodium falciparum malaria in a patient without chemoprophylaxis and with delayed diagnosis. Fever following a stay in the tropics requires immediate testing for malaria infection.

Inhibition of lung phosphodiesterase improves responsiveness to inhaled nitric oxide in isolated-perfused lungs from rats challenged with endotoxin.

OBJECTIVES: To investigate the ability of phosphodiesterase (PDE) selective inhibitors to improve responsiveness to inhaled nitric oxide (NO) in isolated-perfused lungs of rats pretreated with endotoxin/lipopolysaccharide (LPS). DESIGN AND SETTING: Prospective, controlled animal study in the animal research facility of a university hospital. INTERVENTIONS: Sixteen hours after adult Sprague-Dawley rats were injected intraperitoneally with 0.4 mg/ kg E. coli 0111:B4 LPS administration, lungs were isolated and perfused, and the thromboxane mimetic U46619 was employed to increase the mean pulmonary artery pressure by 5-7 mmHg. The lungs were then ventilated with or without 0.4 ppm NO, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; PDE type 2 inhibitor), milrinone (PDE type 3 inhibitor), or zaprinast (inhibitor of PDE types 5 and 9) were added to the perfusate. MEASUREMENTS AND RESULTS: In the presence of EHNA (12.5, 25, 50 microM) the vasodilator response to inhaled NO was not greater than in its absence (0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg vs. 0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg, respectively). In the presence of milrinone (125, 250, 500 nM), the vasodilator response to inhaled NO was also not improved. In contrast, zaprinast (3.7, 7.4, 14.8 microM) augmented the pulmonary vasodilatory effect of inhaled NO in lungs from LPS-pretreated rats from 0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg to 0.75 +/- 0.25, 1.5 +/- 0.5, 1.75 +/- 0.75 mmHg, respectively (p < 0.05). CONCLUSIONS: Our results demonstrate that inhibition of pulmonary PDE enzyme activity with zaprinast increases vasodilator responsiveness to inhaled NO in lungs obtained from rats 16 h after LPS challenge.

Effects of oxygen and nitric oxide inhalation in a porcine model of recurrent microembolism.

BACKGROUND: Inhalation of nitric oxide (iNO) has been proposed for the treatment of acute pulmonary embolism. The present study evaluates the effects of oxygen (O2) and nitric oxide inhalation in a porcine model of sustained pulmonary hypertension induced by recurrent pulmonary microembolism. METHODS: Twelve pigs were embolized under general anesthesia with 300-microm microspheres intravenously three times over a period of seven weeks. Five pigs served as untreated controls. Hemodynamic and gas exchange responses to 100% oxygen and 40 ppm NO inhalation, and their combination (O2+iNO) were measured seven days after the last embolization. RESULTS: Recurrent microembolism caused sustained pulmonary hypertension (pulmonary vascular resistance index; PVRI 408 +/- 57 dyn x s x cm(-5) x m(-2)) as compared to the control group (PVRI 143 +/- 20 dyn x s x cm(-5) m(-2); P<0.05). PVRI was significantly reduced by O2, iNO, and O2+iNO inhalation by 29 +/- 3, 28 +/- 4, and 32 +/- 3%, respectively. CONCLUSION: We conclude that both O2 and iNO are selective pulmonary vasodilators in a porcine model of sustained pulmonary hypertension following recurrent pulmonary microembolism and, therefore, may be useful in the treatment not only in the acute phase of pulmonary embolism but also later in the time course of the disease.

Exhaled nitric oxide production by nitric oxide synthase-deficient mice.

Nitric oxide (NO) is produced in the nasal cavities, airways, and lungs and is exhaled by normal animals and humans. Although increased exhaled NO concentrations in airway inflammation have been associated with increased airway expression of nitric oxide synthase 2 (NOS 2), it is uncertain which NOS isoform is responsible for baseline levels of exhaled NO. We therefore studied wild-type mice and mice with a congenital deficiency of NOS 1, NOS 2, or NOS 3. By studying a closed chamber in which the exhaled gas of a group of mice was collected, gaseous NO production rates were measured. Wild-type mice exhaled 362 +/- 35 x 10(-15) mol g(-1) min(-1) NO (mean +/- SE, n = 16 groups of five mice), NOS 1-deficient mice exhaled 592 +/- 74 x 10(-15) mol g(-1) min(-1) NO (n = 15 groups, p < 0.05 versus wild-type and NOS 2-deficient mice), NOS 2-deficient mice 330 +/- 74 x 10(-15) mol g(-1) min(-1) NO (n = 14 groups) and NOS 3-deficient mice 766 +/- 101 x 10(-15) mol g(-1) min(-1) NO (n = 16 groups, p < 0.001 versus wild-type and NOS 2-deficient mice). Pharmacological NOS inhibition with L-NAME decreased (p < 0.05) the exhaled NO production rate of wild-type and NOS 3-deficient but not of NOS 2-deficient mice. L-Arginine administration increased exhaled NO production rate in all but NOS 2-deficient mice. Absence of NOS 1 or 3 is associated with increased murine exhaled NO production rates. Since NOS 2-deficient mice were the only genotype to lack substrate- and inhibitor-regulated changes of NO exhalation, we suggest that NOS 2 is an important isoform contributing to exhaled NO exhalation in healthy mice.

Nitric oxide inhalation decreases pulmonary artery remodeling in the injured lungs of rat pups.

Vascular injury causes the muscularization of peripheral pulmonary arteries, which is more pronounced in the infant than in the adult lung. Although inhaled NO gas attenuates pulmonary artery remodeling in hypoxic rats, whether or not it protects the lung by mitigating vasoconstriction is unknown. This investigation tested whether inhaled NO decreases the muscularization of injured pulmonary arteries in rat pups by modulating vascular tone. One week after monocrotaline administration, the percentage of muscularized rat pup lung arteries was increased by >3-fold. Nevertheless, monocrotaline exposure did not cause right ventricular hypertrophy, pulmonary hypertension, or vasoconstriction. In addition, it did not increase the expression of markers of inflammation (interleukin-1beta, intercellular adhesion molecule-1, and E-selectin) or of platelet-mediated thrombosis (GPIbalpha). Continuous inhalation of 20 ppm NO gas prevented the neomuscularization of the pulmonary arteries in pups with lung injury. Moreover, a 3-fold increase in cell proliferation and 30% decrease in cell numbers in pulmonary arteries caused by monocrotaline exposure was prevented by NO inhalation. These data indicate that inhaled NO protects infants against pulmonary remodeling induced by lung injury by mechanisms that are independent of pulmonary tone, inflammation, or thrombosis.

Sildenafil is a pulmonary vasodilator in awake lambs with acute pulmonary hypertension.

BACKGROUND: Phosphodiesterase type 5 (PDE5) hydrolyzes cyclic guanosine monophosphate in the lung, thereby modulating nitric oxide (NO)/cyclic guanosine monophosphate-mediated pulmonary vasodilation. Inhibitors of PDE5 have been proposed for the treatment of pulmonary hypertension. In this study, we examined the pulmonary and systemic vasodilator properties of sildenafil, a novel selective PDE5 inhibitor, which has been approved for the treatment of erectile dysfunction. METHODS: In an awake lamb model of acute pulmonary hypertension induced by an intravenous infusion of the thromboxane analog U46619, we measured the effects of 12.5, 25, and 50 mg sildenafil administered via a nasogastric tube on pulmonary and systemic hemodynamics (n = 5). We also compared the effects of sildenafil (n = 7) and zaprinast (n = 5), a second PDE5 inhibitor, on the pulmonary vasodilator effects of 2.5, 10, and 40 parts per million inhaled NO. Finally, we examined the effect of infusing intravenous l-NAME (an inhibitor of endogenous NO production) on pulmonary vasodilation induced by 50 mg sildenafil (n = 6). RESULTS: Cumulative doses of sildenafil (12.5, 25, and 50 mg) decreased the pulmonary artery pressure 21%, 28%, and 42%, respectively, and the pulmonary vascular resistance 19%, 23%, and 45%, respectively. Systemic arterial pressure decreased 12% only after the maximum cumulative sildenafil dose. Neither sildenafil nor zaprinast augmented the ability of inhaled NO to dilate the pulmonary vasculature. Zaprinast, but not sildenafil, markedly prolonged the duration of pulmonary vasodilation after NO inhalation was discontinued. Infusion of l-NAME abolished sildenafil-induced pulmonary vasodilation. CONCLUSIONS: Sildenafil is a selective pulmonary vasodilator in an ovine model of acute pulmonary hypertension. Sildenafil induces pulmonary vasodilation via a NO-dependent mechanism. In contrast to zaprinast, sildenafil did not prolong the pulmonary vasodilator action of inhaled NO.

Randomized, placebo-controlled, blinded and cross-matched study on the antiplatelet effect of inhaled nitric oxide in healthy volunteers.

The platelet inhibitory effect of 0-40 ppm inhaled nitric oxide (NO) was investigated in healthy men and women. In both groups, ADP-and collagen-induced platelet aggregation was significantly inhibited 20 (T20) and 40 min (T40) after the beginning of inhalation of 5, 10, and 40 ppm. Moreover, in both men and women, the in vitro bleeding time was significantly prolonged at T20 and T40 during inhalation of 40 ppm. Inhalation of NO also inhibited P-selectin expression at 5, 10, and 40 ppm and fibrinogen binding to the GPIIb/IIIa-receptor at 40 ppm. In conclusion, in healthy volunteers, the platelet inhibitory effect of inhaled NO was not dose-related, since it was significant at 5 and 10 ppm but did not increase during the administration of higher NO concentrations. In addition, gender-related differences were only observed in ADP-induced platelet aggregation at 10 ppm and in bleeding time prolongation at 40 ppm.

Right ventricular upregulation of the Ca(2+) binding protein S100A1 in chronic pulmonary hypertension.

The Ca(2+) binding protein S100A1 increases the Ca(2+) release from the sarcoplasmatic reticulum by interacting with the ryanodine receptor. In order to understand whether this effect might be operative in the early course of hypertrophy, when myocardium is able to meet increased workload, we investigated the expression of S100A1 in a model of moderate right ventricular hypertrophy. The pulmonary arteries of nine pigs were embolised three times with Sephadex G-50. After 70 days, all pigs showed a moderate pulmonary hypertension. Right ventricular tissue of embolised animals showed a significant increase of connective tissue and enlargement of myocyte diameters. In controls, we found a differential expression of S100A1 with significantly lower S100A1 protein levels in right ventricular compared to left ventricular tissue. In pulmonary hypertension, S100A1 expression increased significantly in hypertrophied right ventricles while it was unchanged in left ventricular tissue. No change was observed in the expression of SERCA2a and phospholamban. Our data show, for the first time, that moderate pressure overload results in an upregulation of S100A1. This may reflect an adaptive response of myocardial Ca(2+) homeostasis to a higher workload.

Congenital NOS2 deficiency protects mice from LPS-induced hyporesponsiveness to inhaled nitric oxide.

BACKGROUND: In animal models, endotoxin (lipopolysaccharide) challenge impairs the pulmonary vasodilator response to inhaled nitric oxide (NO). This impairment is prevented by treatment with inhibitors of NO synthase 2 (NOS2), including glucocorticoids and L-arginine analogs. However, because these inhibitors are not specific for NOS2, the role of this enzyme in the impairment of NO responsiveness by lipopolysaccharide remains incompletely defined. METHODS: To investigate the role of NOS2 in the development of lipopolysaccharide-induced impairment of NO responsiveness, the authors measured the vasodilator response to inhalation of 0.4, 4, and 40 ppm NO in isolated, perfused, and ventilated lungs obtained from lipopolysaccharide-pretreated (50 mg/kg intraperitoneally 16 h before lung perfusion) and untreated wild-type and NOS2-deficient mice. The authors also evaluated the effects of breathing NO for 16 h on pulmonary vascular responsiveness during subsequent ventilation with NO. RESULTS: In wild-type mice, lipopolysaccharide challenge impaired the pulmonary vasodilator response to 0.4 and 4 ppm NO (reduced 79% and 45%, respectively, P < 0.001), but not to 40 ppm. In contrast, lipopolysaccharide administration did not impair the vasodilator response to inhaled NO in NOS2-deficient mice. Breathing 20 ppm NO for 16 h decreased the vasodilator response to subsequent ventilation with NO in lipopolysaccharide-pretreated NOS2-deficient mice, but not in lipopolysaccharide-pretreated wild-type, untreated NOS2-deficient or untreated wild-type mice. CONCLUSIONS: In response to endotoxin challenge, NO, either endogenously produced by NOS2 in wild-type mice or added to the air inhaled by NOS2-deficient mice, is necessary to impair vascular responsiveness to inhaled NO. Prolonged NO breathing, without endotoxin, does not impair vasodilation in response to subsequent NO inhalation. These results suggest that NO, plus other lipopolysaccharide-induced products, are necessary to impair responsiveness to inhaled NO in a murine sepsis model.

Trypsin and activation of circulating trypsinogen contribute to pancreatitis-associated lung injury.

Pancreatic proteases are secreted in acute pancreatitis, but their contribution to associated lung injury is unclear. Applying models of mild edematous (intravenous caerulein) and severe necrotizing (intraductal glycodeoxycholic acid) pancreatitis in rats, we showed that both trypsinogen and trypsin concentrations in peripheral blood, as well as lung injury, correlate with the severity of the disease. To isolate the potential contribution of proteases to lung injury, trypsin or trypsinogen was injected into healthy rats or trypsinogen secreted in caerulein pancreatitis was activated by intravenous enterokinase. Pulmonary injury induced by protease infusions was dose dependent and was ameliorated by neutrophil depletion. Trypsinogen activation worsened lung injury in mild pancreatitis. In vitro incubation of leukocytes with trypsinogen showed that stimulated leukocytes can convert trypsinogen to trypsin. In conclusion, this study demonstrates that the occurrence and severity of pancreatitis-associated lung injury (PALI) corresponds to the levels of circulating trypsinogen and its activation to trypsin. Neutrophils are involved in both protease activation and development of pulmonary injury.

Selective vasodilation by nitric oxide inhalation during sustained pulmonary hypertension following recurrent microembolism in pigs.

PURPOSE: This study establishes a new model of sustained pulmonary hypertension induced by recurrent microembolism in pigs and evaluates the effects of nitric oxide (NO) inhalation in this model. MATERIALS AND METHODS: Fourteen pigs were embolized under general anesthesia with 300-microm microspheres intravenously three times over a period of 7 weeks. Four pigs served as untreated controls. Hemodynamic and gas exchange measurements were performed on days 1 and 7 after the last embolization. RESULTS: Recurrent microembolism caused sustained pulmonary hypertension (mean pulmonary artery pressure [MPAP] 26 +/- 2 and 18 +/- 1 mm Hg on days 1 and 7, respectively) compared with the control group (MPAP 13 +/- 1 mm Hg each for days 1 and 7; P < .05, respectively). Right heart hypertrophy was present at autopsy as indicated by an increase in minimal myocyte diameter. Inhaled NO (5 and 40 parts per million [ppm]) was administered on days 1 and 7. On both days, inhaled NO significantly reduced MPAP and pulmonary vascular resistance without affecting systemic hemodynamics. There were no differences in responses to 5 and 40 ppm inhaled NO. CONCLUSION: We conclude that recurrent microembolization in pigs provides a reliable model of sustained pulmonary hypertension. In this model inhaled NO is a selective pulmonary vasodilator, indicating that active vasoconstriction significantly contributes to sustained pulmonary hypertension after recurrent microembolism.

Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.

To visualize the movements of cells and their processes in developing vertebrates, we constructed replication-incompetent retroviral vectors encoding green fluorescent protein (GFP) that can be detected as a single integrated copy per cell. To optimize GFP expression, the CMV enhancer and avian beta-actin promoter were incorporated within a retrovirus construct to drive transcription of redshifted (F64L, S65T) and codon-modified GFP (EGFP), EGFP tagged with GAP-43 sequences targeting the GFP to the cell membrane, or EGFP with additional mutations that increase its ability to fold properly at 37 degrees C (S147P or V163A, S175G). We have used these viruses to efficiently mark and follow the developmental progression of a large population of cells in rat neocortex and whole avian embryos. In the chick embryo, the migration and development of GFP-marked neural crest cells were monitored using time-lapse videomicroscopy. In the neocortex, GFP clearly delineates the morphology of a variety of neuronal and glial phenotypes. Cells expressing GFP display normal dendritic morphologies, and infected cells persist into adulthood. Cortical neurons appear to form normal local axonal and long-distance projections, suggesting that the presence of cytoplasmic or GAP-43-tagged GFP does not significantly interfere with normal development.

Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets.

Information processing in the nervous system depends on the creation of specific synaptic connections between neurons and targets during development. The homeodomain transcription factor Otx1 is expressed in early-generated neurons of the developing cerebral cortex. Within layer 5, Otx1 is expressed by neurons with subcortical axonal projections to the midbrain and spinal cord. Otx1 is also expressed in the precursors of these neurons, but is localized to the cytoplasm. Nuclear translocation of Otx1 occurs when layer 5 neurons enter a period of axonal refinement and eliminate a subset of their long-distance projections. Otx1 mutant mice are defective in the refinement of these exuberant projections, suggesting that Otx1 is required for the development of normal axonal connectivity and the generation of coordinated motor behavior.