The effect of botulinum toxin on ureteral inflammation.

PURPOSE: The impact of onabotulinum toxin type A (BoNT-A) on bladder afferent nerve pathways and chemosensory functions is an active area of investigation. There may be a role for BoNT-A in disorders of the ureter; however, no histologic studies have assessed the effects of BoNT-A on ureteral tissue. Our objective was to develop an animal model of ureteral inflammation and determine the impact of ureteral BoNT-A instillation on known mechanisms of inflammation. METHODS: The safety and feasibility of a novel animal model of ureteral inflammation was assessed. Through open cystotomy, the effect of ureteral BoNT-A instillation on inflammation was determined through H&E, masson's trichrome, Ki-67 stain, and prostaglandin E (PGE) synthase expression, a known marker of pain and inflammation in ureteral tissue. Urothelial microstructure was assessed using electron microscopy and standard histologic techniques. RESULTS: All experiments were carried to completion, and no systemic signs of botulinum toxicity were seen. BoNT-A exposure was associated with a decrease in PGE synthase expression in a dose-dependent fashion. BoNT-A exposure was not found to impact collagen deposition or cell proliferation. Disruption of tight junctions between urothelial cells was observed under conditions of inflammation. CONCLUSION: We describe the feasibility of a novel in vivo model of ureteral inflammation and report the first histologic study of the effects of BoNT-A on the ureter. Preliminary findings show that BoNT-A attenuates ureteral PGE synthase expression under conditions of inflammation. The application of BoNT-A may provide anti-inflammatory and analgesic effects in the context of ureteral disorders.

Treatment of Canine Oral Melanoma with Nanotechnology-Based Immunotherapy and Radiation.

The presence and benefit of a radiation therapy-associated immune reaction is of great interest as the overall interest in cancer immunotherapy expands. The pathological assessment of irradiated tumors rarely demonstrates consistent immune or inflammatory response. More recent information, primarily associated with the "abscopal effect", suggests a subtle radiation-based systemic immune response may be more common and have more therapeutic potential than previously believed. However, to be of consistent value, the immune stimulatory potential of radiation therapy (RT) will clearly need to be supported by combination with other immunotherapy efforts. In this study, using a spontaneous canine oral melanoma model, we have assessed the efficacy and tumor immunopathology of two nanotechnology-based immune adjuvants combined with RT. The immune adjuvants were administered intratumorally, in an approach termed "in situ vaccination", that puts immunostimulatory reagents into a recognized tumor and utilizes the endogenous antigens in the tumor as the antigens in the antigen/adjuvant combination that constitutes a vaccine. The radiation treatment consisted of a local 6 × 6 Gy tumor regimen given over a 12 day period. The immune adjuvants were a plant-based virus-like nanoparticle (VLP) and a 110 nm diameter magnetic iron oxide nanoparticle (mNPH) that was activated with an alternating magnetic field (AMF) to produce moderate heat (43 °C/60 min). The RT was used alone or combined with one or both adjuvants. The VLP (4 × 200 µg) and mNPH (2 × 7.5 mg/gram tumor) were delivered intratumorally respectively during the RT regimen. All patients received a diagnostic biopsy and CT-based 3-D radiation treatment plan prior to initiating therapy. Patients were assessed clinically 14-21 days post-treatment, monthly for 3 months following treatment, and bimonthly, thereafter. Immunohistopathologic assessment of the tumors was performed before and 14-21 days following treatment. Results suggest that addition of VLPs and/or mNPH to a hypofractionated radiation regimen increases the immune cell infiltration in the tumor, extends the tumor control interval, and has important systemic therapeutic potential.

Warming barium sulfate improves esophageal leak detection in pig model.

BACKGROUND: Barium esophagograms have poor sensitivity in detecting leaks. We hypothesized that heating barium would decrease viscosity, facilitate extravasation, and enhance its sensitivity in detecting esophageal leaks. METHODS: We characterized the viscosity of barium at increasing temperatures. We measured the radiopacity of barium at 25°C and 50°C. We determined the smallest diameter defect in esophagus that barium can detect by perforating a porcine esophageal segment with angiocatheters of various diameters, injecting barium at 25°C, and observing extravasation of contrast. We repeated this with barium heated to 30°C, 40°C, 50°C, and 70°C. To determine the ability of barium to detect a staple line leak, we perforated a stapled esophageal segment by air insufflation, injected barium at different temperatures, and monitored extravasation. We used Visipaque, a water-soluble contrast agent, for comparison in all experiments. RESULTS: The viscosity of barium decreased with increasing temperature. The radiopacity of barium did not change with increasing temperature and was higher than that of Visipaque (P < 0.001). The size of the smallest detectable leak decreased from 2.1 mm with barium at 25°C to 1.3 mm at 40°C and 1.1 mm with Visipaque (P < 0.0001). The sensitivity of staple line leak detection increased from 0% for barium at 25°C to 80% (P = 0.02) with barium at 40°C. There was no significant difference in sensitivity between barium at 40°C and Visipaque. CONCLUSIONS: Barium warmed to 40°C offers the best sensitivity of esophageal leak detection without compromising radiopacity. Barium at 40°C may be the optimum choice for swallow study to detect esophageal leaks.

Porcine-human glioma xenograft model. Immunosuppression and model reproducibility.

BACKGROUND: Glioblastoma is the most common primary malignant and treatment-resistant human brain tumor. Rodent models have played an important role in understanding brain cancer biology and treatment. However, due to their small cranium and tumor volume mismatch, relative to human disease, they have been less useful for translational studies. Therefore, development of a consistent and simple large animal glioma xenograft model would have significant translational benefits. METHODS: Immunosuppression was induced in twelve standard Yucatan minipigs. 3 pigs received cyclosporine only, while 9 pigs received a combined regimen including cyclosporine (55 mg/kg q12 h), prednisone (25 mg, q24 h) and mycophenolate (500 mg q24 h). U87 cells (2 × 106) were stereotactically implanted into the left frontal cortex. The implanted brains were imaged by MRI for monitoring. In a separate study, tumors were grown in 5 additional pigs using the combined regimen, and pigs underwent tumor resection with intra-operative image updating to determine if the xenograft model could accurately capture the spatial tumor resection challenges seen in humans. RESULTS: Tumors were successfully implanted and grown in 11 pigs. One animal in cyclosporine only group failed to show clinical tumor growth. Clinical tumor growth, assessed by MRI, progressed slowly over the first 10 days, then rapidly over the next 10 days. The average tumor growth latency period was 20 days. Animals were monitored twice daily and detailed records were kept throughout the experimental period. Pigs were sacrificed humanely when the tumor reached 1 - 2 cm. Some pigs experienced decreased appetite and activity, however none required premature euthanasia. In the image updating study, all five pigs demonstrated brain shift after craniotomy, consistent with what is observed in humans. Intraoperative image updating was able to accurately capture and correct for this shift in all five pigs. CONCLUSION: This report demonstrates the development and use of a human intracranial glioma model in an immunosuppressed, but nongenetically modified pig. While the immunosuppression of the model may limit its utility in certain studies, the model does overcome several limitations of small animal or genetically modified models. For instance, we demonstrate use of this model for guiding surgical resection with intraoperative image-updating technologies. We further report use of a surrogate extracranial tumor that indicates growth of the intracranial tumor, allowing for relative growth assessment without radiological imaging.

Intracranial electrical impedance tomography: a method of continuous monitoring in an animal model of head trauma.

BACKGROUND: Electrical impedance tomography (EIT) is a method that can render continuous graphical cross-sectional images of the brain's electrical properties. Because these properties can be altered by variations in water content, shifts in sodium concentration, bleeding, and mass deformation, EIT has promise as a sensitive instrument for head injury monitoring to improve early recognition of deterioration and to observe the benefits of therapeutic intervention. This study presents a swine model of head injury used to determine the detection capabilities of an inexpensive bedside EIT monitoring system with a novel intracranial pressure (ICP)/EIT electrode combination sensor on induced intraparenchymal mass effect, intraparenchymal hemorrhage, and cessation of brain blood flow. Conductivity difference images are shown in conjunction with ICP data, confirming the effects. METHODS: Eight domestic piglets (3-4 weeks of age, mean 10 kg), under general anesthesia, were subjected to 4 injuries: induced intraparenchymal mass effect using an inflated, and later, deflated 0.15-mL Fogarty catheter; hemorrhage by intraparenchymal injection of 1-mL arterial blood; and ischemia/infarction by euthanasia. EIT and ICP data were recorded 10 minutes before inducing the injury until 10 minutes after injury. Continuous EIT and ICP monitoring were facilitated by a ring of circumferentially disposed cranial Ag/AgCl electrodes and 1 intraparenchymal ICP/EIT sensor electrode combination. Data were recorded at 100 Hz. Two-dimensional tomographic conductivity difference (Δσ) images, rendered using data before and after an injury, were displayed in real time on an axial circular mesh. Regions of interest (ROI) within the images were automatically selected as the upper or lower 5% of conductivity data depending on the nature of the injury. Mean Δσ within the ROIs and background were statistically analyzed. ROI Δσ was compared with the background Δσ after an injury event using an unpaired, unequal variance t test. Conductivity change within an ROI after injury was likewise compared with the same ROI before the injury making use of unpaired t tests with unequal variance. RESULTS: Eight animal subjects were studied, each undergoing 4 injury events including euthanasia. Changes in conductivity due to injury showed expected pathophysiologic effects in an ROI identified within the middle of the left hemisphere; this localization is reasonable given the actual site of injury (left hemisphere) and spatial warping associated with estimating a 3-dimensional conductivity distribution in 2-dimensional space. Results are shown as mean ± 1 SD. When averaged across all 8 animals, balloon inflation caused the mean Δσ within the ROI to shift by -11.4 ± 10.9 mS/m; balloon deflation by +9.4 ± 8.8 mS/m; blood injection by +19.5 ± 11.5 mS/m; death by -12.6 ± 13.2 mS/m. All induced injuries were detectable to statistical significance (P < 0.0001). CONCLUSION: This study confirms that the bedside EIT system with ICP/EIT combination sensor can detect induced trauma. Such a technique may hold promise for further research in the monitoring and management of traumatically brain-injured individuals.

Detection of subclinical hemorrhage using electrical impedance: a porcine study.

Objective.Analyze the performance of electrical impedance tomography (EIT) in an innovative porcine model of subclinical hemorrhage and investigate associations between EIT and hemodynamic trends.Approach. Twenty-five swine were bled at slow rates to create an extended period of subclinical hemorrhage during which the animal's heart rate (HR) and blood pressure (BP) remained stable from before hemodynamic deterioration, where stable was defined as <15% decrease in BP and <20% increase in HR-i.e.hemorrhages were hidden from standard vital signs of HR and BP. Continuous vital signs, photo-plethysmography, and continuous non-invasive EIT data were recorded and analyzed with the objective of developing an improved means of detecting subclinical hemorrhage-ideally as early as possible.Main results. Best area-under-the-curve (AUC) values from comparing bleed to no-bleed epochs were 0.96 at a 80 ml bleed (∼15.4 min) using an EIT-data-based metric and 0.79 at a 120 ml bleed (∼23.1 min) from invasively measured BP-i.e.the EIT-data-based metric achieved higher AUCs at earlier points compared to standard clinical metrics without requiring image reconstructions.Significance.In this clinically relevant porcine model of subclinical hemorrhage, EIT appears to be superior to standard clinical metrics in early detection of hemorrhage.

Selective regulation of arterial branching morphogenesis by synectin.

Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.

The FGF system has a key role in regulating vascular integrity.

The integrity of the endothelial monolayer is essential to blood vessel homeostasis and active regulation of endothelial permeability. The FGF system plays important roles in a wide variety of physiologic and pathologic conditions; however, its role in the adult vasculature has not been defined. To assess the role of the FGF system in the adult endothelial monolayer, we disrupted FGF signaling in bovine aortic endothelial cells and human saphenous vein endothelial cells in vitro and in adult mouse and rat endothelial cells in vivo using soluble FGF traps or a dominant inhibitor of all FGF receptors. The inhibition of FGF signaling using these approaches resulted in dissociation of the VE-cadherin/p120-catenin complex and disassembly of adherens and tight junctions, which progressed to loss of endothelial cells, severe impairment of the endothelial barrier function, and finally, disintegration of the vasculature. Thus, FGF signaling plays a key role in the maintenance of vascular integrity.

The antiangiogenic activity of rPAI-1(23) inhibits vasa vasorum and growth of atherosclerotic plaque.

Plaque vascularity has been implicated in its growth and stability. However, there is a paucity of information regarding the origin of plaque vasculature and the role of vasa vasorum in plaque growth. To inhibit growth of vasa vasorum in atherogenic mice and assess its effect on plaque growth, we used a truncated plasminogen activator inhibitor (PAI)-1 protein, rPAI-1(23), that has significant antiangiogenic activity. Female LDLR(-/-)ApoB-48-deficient mice fed Paigen's diet without cholate for 20 weeks received rPAI-1(23) treatment (n=21) for the last 6 weeks. Plaque size and vasa vasorum density were compared to 2 controls: mice fed Paigen's diet and treated with saline for the last 6 weeks (n=16) and mice fed Paigen's diet until the onset of treatment (n=14). The rPAI-1(23) treatment significantly reduced plaque area and plaque cholesterol in the descending aorta and plaque area in the innominate artery. Measurements of reconstructed confocal microscopy images of vasa vasorum demonstrate that rPAI-1(23) treatment decreased vasa vasorum area and length, which was supported by microCT images. Confocal images provide evidence for vascularized plaque in the saline-treated group but not in rPAI-1(23)-treated mice. The increased vessel density in saline-treated mice is attributable, in part, to upregulated fibroblast growth factor-2 expression, which is inhibited by rPAI-1(23). In conclusion, rPAI-1(23) inhibits growth of vasa vasorum, as well as vessels within the adjacent plaque and vessel wall, through inhibition of fibroblast growth factor-2, leading to reduced plaque growth in atherogenic female LDLR(-/-)ApoB-48-deficient mice.

Epinephrine plus chest compressions is superior to epinephrine alone in a hypoxia-induced porcine model of pseudo-pulseless electrical activity.

AIM: Pseudo-pulseless electrical activity (pseudo-PEA) is a global hypotensive ischemic state with retained coordinated myocardial contractile activity and an organized ECG with no clinically detectable pulses. The role of standard external chest compressions (CPR) and its associated intrinsic hemodynamics remains unclear in the setting of pseudo-PEA. We undertook an experimental trial to compare epinephrine alone versus epinephrine with CPR in the treatment of pseudo-PEA. METHODS: Using a porcine model of hypoxic pseudo-PEA, we randomized 12 Yorkshire male swine to resuscitation with epinephrine only (control) (0.0015 mg/kg) versus epinephrine plus standard CPR (intervention). Animals who achieved return of spontaneous circulation (ROSC) were stabilized, fully recovered to hemodynamic and respiratory baseline, and rearrested up to 6 times. Primary outcome was ROSC defined as a sustained systolic blood pressure (SBP) of 60 mmHg for 2 min. Secondary outcomes included time to ROSC, coronary perfusion pressure (CoPP), and end-tidal carbon dioxide (ETCO2). RESULTS: Among 47 events of pseudo-PEA in 12 animals, we observed significantly higher proportion of ROSC when treatment included CPR (14/21 - 67%) compared to epinephrine alone (4/26 - 15%) (p = 0.0007). CoPP, aortic pressures and ETCO2 were significantly higher, and right atrial pressures were lower in the intervention group. CONCLUSIONS: In a swine model of hypoxia-induced pseudo-PEA, epinephrine plus CPR was associated with improved intra-arrest hemodynamics and higher probability of ROSC. Thus, epinephrine plus CPR may be superior to epinephrine alone in the treatment of patients with pseudo-PEA.

Myocardial hypertrophy in the absence of external stimuli is induced by angiogenesis in mice.

Although studies have suggested a role for angiogenesis in determining heart size during conditions demanding enhanced cardiac performance, the role of EC mass in determining the normal organ size is poorly understood. To explore the relationship between cardiac vasculature and normal heart size, we generated a transgenic mouse with a regulatable expression of the secreted angiogenic growth factor PR39 in cardiomyocytes. A significant change in adult mouse EC mass was apparent by 3 weeks following PR39 induction. Heart weight; cardiomyocyte size; vascular density normalization; upregulation of hypertrophy markers including atrial natriuretic factor, beta-MHC, and GATA4; and activation of the Akt and MAP kinase pathways were observed at 6 weeks post-induction. Treatment of PR39-induced mice with the eNOS inhibitor L-NAME in the last 3 weeks of a 6-week stimulation period resulted in a significant suppression of heart growth and a reduction in hypertrophic marker expression. Injection of PR39 or another angiogenic growth factor, VEGF-B, into murine hearts during myocardial infarction led to induction of myocardial hypertrophy and restoration of myocardial function. Thus stimulation of vascular growth in normal adult mouse hearts leads to an increase in cardiac mass.

Syx, a RhoA guanine exchange factor, is essential for angiogenesis in Vivo.

Rho GTPases play an important and versatile role in several biological processes. In this study, we identified the zebrafish ortholog of the mammalian Rho A guanine exchange factor, synectin-binding guanine exchange factor (Syx), and determined its in vivo function in the zebrafish and the mouse. We found that Syx is expressed specifically in the vasculature of these organisms. Loss-of-function studies in the zebrafish and mouse point to a specific role for Syx in angiogenic sprouting in the developing vascular bed. Importantly, vasculogenesis and angioblast differentiation steps were unaffected in syx knockdown zebrafish embryos, and the vascular sprouting defects were partially rescued by the mouse ortholog. Syx knockdown in vitro impairs vascular endothelial growth factor-A-induced endothelial cell migration and angiogenesis. We have also uncovered a potential mechanism of endothelial sprout guidance in which angiomotin, a component of endothelial cell junctions, plays an additive role with Syx in directing endothelial sprouts. These results identify Syx as an essential contributor to angiogenesis in vivo.

Quantitative evaluation of the in vivo biocompatibility and performance of freeze-cast tissue scaffolds.

Quantitative methods are little used for the in vivo assessment of tissue scaffolds to evaluate biocompatibility. To complement current histological techniques, we introduce as a measure of biocompatibility a straightforward, geometric analysis for the quantitative assessment of encapsulation thickness, cross-sectional area, and biomaterial shape. Advantages of this new technique are that it enables, on the one hand, a more complete and objective comparison of scaffolds with differing compositions, architectures, and mechanical properties, and, on the other, a more objective approach to their selection for a given application. In this contribution, we focus on freeze-cast polymeric scaffolds for tissue regeneration and their subcutaneous implantation in mice for biocompatibility testing. Initially, seven different scaffold types are screened. Of these, three are selected for systematic biocompatibility studies based on histopathological criteria: EDC-NHS-crosslinked bovine collagen, EDC-NHS-crosslinked bovine collagen-nanocellulose, and chitin. Geometric models developed to quantify scaffold size, ovalization, and encapsulation thickness are tested, evaluated, and found to be a powerful and objective metric for the in vivo assessment of biocompatibility and performance of tissue scaffolds.

Intravenous calcium as a pressor in a swine model of hypoxic pseudo-pulseless electrical mechanical activity-a preliminary report.

BACKGROUND: Pseudo-pulseless electrical activity (pseudo-PEA) is a lifeless form of profound cardiac shock characterized by measurable cardiac mechanical activity without clinically detectable pulses. Pseudo-PEA may constitute up to 40% of reported cases of cardiac arrest. Resuscitation from pseudo-PEA is often associated with hypotension refractory to catecholamine pressors. We hypothesized that this post-resuscitation state may be associated with hypocalcemic hypotension responsive to intravenous calcium. METHODS: Using pre-existing data from our hypoxic swine pseudo-PEA model, we measured blood pressure, hemodynamics, and electrolytes. Physiological data were analyzed on a heartbeat by heartbeat basis. The midpoint of the calcium response was defined using change of curvature feature detection. Hemodynamic parameters were shifted such that the value at the midpoint was equal to zero. RESULTS: In 9 animals with refractory hypotension, we administered 37 boluses of intravenous calcium in the dosage range of 5-20 mg. Comparisons were made between the average values in the time period 40-37 s before the midpoint and 35-40 s after the midpoint. Of the 37 administered boluses, 34 manifested a change in the blood pressure, with mean aortic pressure, systolic and diastolic pressures all increasing post bolus administration. CONCLUSIONS: Administration of intravenous calcium may be associated with a pressor-like response in refractory hypotension after resuscitation from pseudo-PEA. Relative ionized hypocalcemia may cause hypotension after resuscitation from pseudo-PEA. Therapy with intravenous calcium should be further investigated in this setting.

Experimentally Observed Cherenkov Light Generation in the Eye During Radiation Therapy.

PURPOSE: Patients have reported sensations of seeing light flashes during radiation therapy, even with their eyes closed. These observations have been attributed to either direct excitation of retinal pigments or generation of Cherenkov light inside the eye. Both in vivo human and ex vivo animal eye imaging was used to confirm light intensity and spectra to determine its origin and overall observability. METHODS AND MATERIALS: A time-gated and intensified camera was used to capture light exiting the eye of a patient undergoing stereotactic radiosurgery in real time, thereby verifying the detectability of light through the pupil. These data were compared with follow-up mechanistic imaging of ex vivo animal eyes with thin radiation beams to evaluate emission spectra and signal intensity variation with anatomic depth. Angular dependency of light emission from the eye was also measured. RESULTS: Patient imaging showed that light generation in the eye during radiation therapy can be captured with a signal-to-noise ratio of 68. Irradiation of ex vivo eye samples confirmed that the spectrum matched that of Cherenkov emission and that signal intensity was largely homogeneous throughout the entire eye, from the cornea to the retina, with a slight maximum near 10 mm depth. Observation of the signal external to the eye was possible through the pupil from 0° to 90°, with a detected emission near 2500 photons per millisecond (during peak emission of the ON cycle of the pulsed delivery), which is over 2 orders of magnitude higher than the visible detection threshold. CONCLUSIONS: By quantifying the spectra and magnitude of the signal, we now have direct experimental observations that Cherenkov light is generated in the eye during radiation therapy and can contribute to perceived light flashes. Furthermore, this technique can be used to further study and measure phosphenes in the radiation therapy clinic.

Design, Manufacture, and In vivo Testing of a Tissue Scaffold for Permanent Female Sterilization by Tubal Occlusion.

Current FDA-approved permanent female sterilization procedures are invasive and/or require the implantation of non-biodegradable materials. These techniques pose risks and complications, such as device migration, fracture, and tubal perforation. We propose a safe, non-invasive biodegradable tissue scaffold to effectively occlude the Fallopian tubes within 30 days of implantation. Specifically, the Fallopian tubes are mechanically de-epithelialized, and a tissue scaffold is placed into each tube. It is anticipated that this procedure can be performed in less than 30 minutes by an experienced obstetrics and gynaecology practitioner. Advantages of this method include the use of a fully bio-resorbable polymer, low costs, lower risks, and the lack of general anaesthesia. The scaffold devices are freeze-cast allowing for the custom-design of structural, mechanical, and chemical cues through material composition, processing parameters, and functionalization. The performance of the biomaterial and de-epithelialization procedure was tested in an in vivo rat uterine horn model. The scaffold response and tissue-biomaterial interactions were characterized microscopically post-implantation. Overall, the study resulted in the successful fabrication of resilient, easy-to-handle devices with an anisotropic scaffold architecture that encouraged rapid bio-integration through notable angiogenesis, cell infiltration, and native collagen deposition. Successful tubal occlusion was demonstrated at 30 days, revealing the great promise of a sterilization biomaterial.

Freeze-cast Porous Chitosan Conduit for Peripheral Nerve Repair.

A novel freeze-cast porous chitosan conduit for peripheral nerve repair with highly-aligned, double layered porosity, which provides the ideal mechanical and chemical properties was designed, manufactured, and assessed in vivo. Efficacies of the conduit and the control inverted nerve autograft were evaluated in bridging 10-mm Lewis rat sciatic nerve gap at 12 weeks post-implantation. Biocompatibility and regenerative efficacy of the porous chitosan conduit were evaluated through the histomorphometric analysis of longitudinal and transverse sections. The porous chitosan conduit was found to have promising regenerative characteristics, promoting the desired neovascularization, and axonal ingrowth and alignment through a combination of structural, mechanical and chemical cues.

PKCalpha activates eNOS and increases arterial blood flow in vivo.

Endothelial nitric oxide synthase (eNOS) plays an important role in control of vascular tone and angiogenesis among other functions. Its regulation is complex and has not been fully established. Several studies have emphasized the importance of phosphorylation in the regulation of eNOS activity. Although it is commonly accepted that protein kinase C (PKC) signaling inhibits eNOS activity by phosphorylating Thr497 and dephosphorylating Ser1179, the distinct role of different PKC isoforms has not been studied so far. The PKC family comprises roughly 12 different isozymes that activate distinct downstream pathways. The present study was designed to investigate the role of PKCalpha isoform in regulation of eNOS activity. Overexpression of PKCalpha in primary endothelial cells was associated with increased eNOS-Ser1179 phosphorylation and increased NO production. Inhibition of PKCalpha activity either by siRNA transfection or by overexpression of a dominant negative mutant resulted in a marked decrease in FGF2-induced Ser1179 phosphorylation and NO production. In vivo, PKCalpha transduction in rat femoral arteries resulted in a significant increase in the resting blood flow that was suppressed by treatment with L-NAME, an eNOS inhibitor. In conclusion, these data demonstrate for the first time that PKCalpha stimulates NO production in endothelial cells and plays a role in regulation of blood flow in vivo.

Endoscopy after esophagectomy: Safety demonstrated in a porcine model.

BACKGROUND: Endoscopy is useful in assessing conduit ischemia and anastomotic leaks after esophagectomy but poses a theoretical threat of anastomotic disruption. We used a porcine model to evaluate the safety of endoscopy after esophagectomy. METHODS: We performed esophagectomies in 10 live pigs and performed endoscopy with progressive air insufflation and continuous intraluminal pressure monitoring. We stopped insufflation when the intraluminal pressure reached a plateau. We assessed the integrity of the conduit and anastomosis via endoscopy. We also performed pulse oximetry of the stomach and Doppler velocimetry of the right gastroepiploic artery on 5 live pigs to study the effects of endoscopic gastric insufflation. RESULTS: With gentle air insufflation, there was no measurable increase in intraluminal pressure, disruption of the conduit or anastomosis, or significant gastric distension. With progressive insufflation, the intraluminal pressure reached a plateau at a maximum of 8.7 ± 2.1 cm H2O (95% confidence interval, 7.2-10.2). At this plateau, air leaked retrograde via the mouth, which prevented further gastric distension. There were no significant changes in oxyhemoglobin saturation along various regions in the stomach even with maximal insufflation sustained for 10 minutes. There was a momentary reduction in gastroepiploic flow from 12.0 ± 1.0 [95% confidence interval, 10.8-13.2] mL/min/100 g to 9.6 ± 1.5 [95% confidence interval, 7.8-11.4] mL/min/100 g immediately after maximal insufflation, but flow recovered to 11 ± 1.3 [9.6, 12.8] mL/min/100 g after 10 minutes of sustained insufflation. CONCLUSIONS: Endoscopy after esophagectomy with gentle or maximal air insufflation results in safe endoluminal pressures and minimal disturbance of blood flow and oxygenation.