Cholestasis in a murine experimental model: lesions include hepatocyte ischemic necrosis

OBJECTIVE: To establish a murine experimental model of bile duct obstruction that would enable controlled observations of the acute and subacute phases of cholestasis. METHODOLOGY: Adult male isogenic BALB/c mice underwent a bile duct ligation (22 animals) or a sham operation (10 animals). Fifteen days after surgery, or immediately after the animal's death, macroscopic findings were noted and histological study of the liver, biliary tree, and pancreas was performed (hematoxylin-eosin and Masson trichromic staining). RESULTS: Beginning 24 hours after surgery, all animals from the bile duct ligation group presented progressive generalized malaise. All animals presented jaundice in the parietal and visceral peritoneum, turgid and enlarged liver, and accentuated dilatation of gallbladder and common bile duct. Microscopic findings included marked dilatation and proliferation of bile ducts with accentuated collagen deposits, frequent areas of ischemic necrosis, hepatic microabscesses, and purulent cholangitis. Animals from the sham operation group presented no alterations. CONCLUSION: We established a murine experimental model of induced cholestasis, which made it possible to study acute and subacute tissue lesions. Our data suggests that in cholestasis, hepatic functional ischemia plays an important role in inducing hepatic lesions, and it also suggests that the infectious process is an important factor in morbidity and mortality.

The effect of the N-methyl-D-aspartate receptor antagonist dextromethorphan on perioperative brain injury in children undergoing cardiac surgery with cardiopulmonary bypass: results of a pilot study.

Experimental evidence indicates a role of the N-methyl-D-aspartate receptor in the pathogenesis of brain injury occurring during cardiac surgery with cardiopulmonary bypass (CPB). Dextromethorphan is a noncompetitive antagonist of this receptor with a favorable safety profile. Thirteen children age 3-36 months undergoing cardiac surgery with expected CPB of 60 minutes or more were randomly assigned to treatment with dextromethorphan (36-38 mg/kg/day) or placebo administered by naso-gastric tube. Dextromethorphan was absorbed well and reached putative therapeutic levels in blood and cerebrospinal fluid. Adverse effects were not observed. Mild hemiparesis developed after operation in one child of each group, and severe encephalopathy in one of the placebo group. Sharp waves were recorded in postoperative continuous electroencephalography in all placebo (n = 7) but only in 2/6 dextromethorphan treated children (p = 0.02). Pre- and postoperative cranial magnetic resonance imaging (MRI) revealed less pronounced ventricular enlargement in the dextromethorphan group (not significant). An increase of periventricular white matter lesions was visible in two placebo-treated children only. No elevations of cerebrospinal fluid enzymes were observed in either group. Although children with dextromethorphan showed less abnormalities in electroencephalography and MRI, dissimilarities of the treatment groups by chance diminished conclusions to possible protective effects of dextromethorphan at this time.

Repair of non-circumferential cervical trachea defects by three different latissimus dorsi flaps. A comparative studyin an experimental setting.

BACKGROUND: Large intrathoracic airway defects may be closed using a pedicled latissimus dorsi (LD) flap, with rewarding results. This study addresses the question of whether this holds true for extrathoracic non-circumferential tracheal defects. METHODS: A cervical segment of the trachea of 4 x 1 cm was resected in 9 white male pigs. The defect was stented with a silicone stent for 3 months and closed either by an LD flap alone (group a, n = 3), an LD flap with an attached rib segment covered by pleura (group b, n = 3), or an LD flap reinforced by a perforated polylactide (MacroPore) plate (group c, n = 3). The trachea was assessed by rigid endoscopy at 3 and 4 months and histologically at 4 months postoperatively. RESULTS: The degree of stenosis at the level of the reconstruction at 4 months was 25, 50 and 75% in group a, 15, 50 and 60% in group b, and 20, 95 and 95% in group c, respectively. The percentage of the defect covered by columnar epithelium was 100% in all animals of group a, 60, 100 and 100% in group b, and 10, 0 and 0% in group c. Resorption of the rib was seen in all animals of group b and obstructive inflammatory polyps were found in 2 animals of group c. CONCLUSION: Pedicled LD flaps provided less satisfactory results for closure of large non-circumferential extrathoracic airway defects than observed after intrathoracic reconstruction. A pedicled rib segment added to the LD flap did not improve the results obtained from LD flap repair alone, and an embedded MacroPore prosthesis may result in severe airway stenosis due to plate migration and intense inflammatory reaction protruding into the tracheal lumen.

Characterization of Nedd4-2 ubiquitin ligase expression in experimental neuropathic pain

Background: Neuropathic pain is associated with altered expression of voltage-gated sodium channels (VGSCs). The ubiquitin ligase Nedd4-2 regulates sodium channels and we have previously demonstrated in expression systems that this protein decreases the Nav1.7 current. Nav1.7 is the most abundant VGSC in dorsal root ganglion (DRG) and is a major contributor to pain perception. We hypothesize that Nedd4-2 modulates Nav1.7 channel density at the neuronal cell membrane and the goal of this present experiment is to characterize Nav1.7 and Nedd4-2 expression in the context of neuropathic pain. Methods: Biotinylation, Western Blot and Immunohistochemistry experiments for Nav1.7 and Nedd4-2 were performed in HEK transfected cells or in rodent DRGs 7 days after SNI surgery. We used antibodies against Nedd4-2 and Nav1.7 and several comarkers of DRG neurons (Peripherin for nociceptors, NF-200 for large myelinated cells, ATF3 for injured neurons). Data are expressed in proportion of positive cells (%) and protein signal ratio } SEM, n = 3-4 in each condition. Results: In HEK293 cells, upon co-expression of Nedd4-2, a decrease of 50% of Nav1.7 signal at the membrane is demonstrated (p ≤0.005). Immunofluorescence on DRGs neurons reveals a decreased number of positive Nedd4-2 cells in the SNI model (27.0 } 1.2%) versus sham group (43.4 } 3.5%) (p <0.005). Nedd4-2 is mainly colocalized with markers of small neurons and almost absent in large neurons. In addition, Nedd4-2 is predominantly decreased in injured ATF3 positive cells. Conclusion: Our results indicate that Nedd4-2 decreases Nav1.7 channels and currents at the cell membrane and that it is mainly expressed in nociceptors and downregulated after nerve injury. Taken together, our data suggest that the reduction of Nedd4-2, after nerve injury, modulates Nav1.7 activity and can contribute to neuropathic pain. We will further try to restore a normal level of Nedd4.2 via a gene therapy approach with viral vectors in order to soothe symptoms of neuropathic pain.

Preretinal partial pressure of oxygen gradients before and after experimental pars plana vitrectomy.

PURPOSE: To evaluate preretinal partial pressure of oxygen (PO2) gradients before and after experimental pars plana vitrectomy. METHODS: Arteriolar, venous, and intervascular preretinal PO2 gradients were recorded in 7 minipigs during slow withdrawal of oxygen-sensitive microelectrodes (10-μm tip diameter) from the vitreoretinal interface to 2 mm into the vitreous cavity. Recordings were repeated after pars plana vitrectomy and balanced salt solution (BSS) intraocular perfusion. RESULTS: Arteriolar, venous, and intervascular preretinal PO2 at the vitreoretinal interface were 62.3 ± 13.8, 22.5 ± 3.3, and 17.0 ± 7.5 mmHg, respectively, before vitrectomy; 97.7 ± 19.9, 40.0 ± 21.9, and 56.3 ± 28.4 mmHg, respectively, immediately after vitrectomy; and 59.0 ± 27.4, 25.2 ± 3.0, and 21.5 ± 4.5 mmHg, respectively, 2½ hours after interruption of BSS perfusion. PO2 2 mm from the vitreoretinal interface was 28.4 ± 3.6 mmHg before vitrectomy; 151.8 ± 4.5 mmHg immediately after vitrectomy; and 34.8 ± 4.1 mmHg 2½ hours after interruption of BSS perfusion. PO2 gradients were still present after vitrectomy, with the same patterns as before vitrectomy. CONCLUSION: Preretinal PO2 gradients are not eliminated after pars plana vitrectomy. During BSS perfusion, vitreous cavity PO2 is very high. Interruption of BSS perfusion evokes progressive equilibration of vitreous cavity PO2 with concomitant progressive return of preretinal PO2 gradients to their previtrectomy patterns. This indicates that preretinal diffusion of oxygen is not altered after vitrectomy. The beneficial effect of vitrectomy in ischemic retinal diseases or macular edema may be related to other mechanisms, such as increased oxygen convection currents or removal of growth factors and cytokines secreted in the vitreous.

Medicine and Science in a New Medical-surgical Context: The Royal College of Surgery of Barcelona (1760–1843)

Taking the Royal College of Barcelona (1760 -1843) as a case study this paper shows the development of modern surgery in Spain initiated by Bourbon Monarchy founding new kinds of institutions through their academic activities of spreading scientific knowledge. Antoni Gimbernat was the most famousinternationally recognised Spanish surgeon. He was trained as a surgeon at the Royal College of Surgery in Cadiz and was later appointed as professor of theAnatomy in the College of Barcelona. He then became Royal Surgeon of King Carlos IV and with that esteemed position in Madrid he worked resiliently to improve the quality of the Royal colleges in Spain. Learning human bodystructure by performing hands-on dissections in the anatomical theatre has become a fundamental element of modern medical education. Gimbernat favoured the study of natural sciences, the new chemistry of Lavoisier and experimental physics in the academic programs of surgery. According to the study of a very relevant set of documents preserved in the library, the so-called “juntas literarias”, among the main subjects debated in the clinical sessions was the concept of human beings and diseases in relation to the development of the new experimental sciences. These documents showed that chemistry andexperimental physics were considered crucial tools to understand the unexplained processes that occurred in the diseased and healthy human bodyand in a medico-surgical context. It is important to stress that through these manuscripts we can examine the role and the reception of the new sciences applied to healing arts.

Experimental endocarditis following dental extractions in rats with periodontitis.

This study analyzed the development of bacterial endocarditis following dental extraction in rats with periodontal disease. Periodontal disease was produced in rats by tying silk ligatures around the two maxillary first molars, and placing the animals on a high sucrose diet. Sterile aortic valve vegetations were produced by means of a transaortic catheter, and 24 hours later the maxillary first molars were extracted. The animals were killed 72 hours after the extractions. In rats with periodontal disease induced for 10 and 14 weeks, extractions resulted in an incidence of bacterial endocarditis of 24% and 50%, respectively, most of which were due to streptococcal species (two were caused by Staphylococcus [corrected] aureus). The difference, though not statistically significant (p = 0.10, chi 2 with Yates correction), shows a trend toward increased incidence of endocarditis with increasing severity of periodontal disease. This model demonstrates that one can reliably induce bacterial endocarditis after dental extractions in rats with periodontal disease.

Sustained release of nanosized complexes of polyethylenimine and anti-TGF-beta 2 oligonucleotide improves the outcome of glaucoma surgery.

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-beta2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-beta2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

Stent-assisted coiling of bifurcation aneurysms may improve endovascular treatment: a critical evaluation in an experimental model.

BACKGROUND AND PURPOSE: Endovascular treatment of wide-neck bifurcation aneurysms often results in incomplete occlusion or aneurysm recurrence. The goals of this study were to compare results of coil embolization with or without the assistance of self-expandable stents and to examine how stents may influence neointima formation. MATERIALS AND METHODS: Wide-neck bifurcation aneurysms were constructed in 24 animals and, after 4-6 weeks, were randomly allocated to 1 of 5 groups: 1) coil embolization using the assistance of 1 braided stent (n = 5); 2) coil embolization using the assistance of 2 braided stents in a Y configuration (n = 5); 3) coil embolization without stent assistance (n = 6); 4) Y-stenting alone (n = 4); and 5) untreated controls (n = 4). Angiographic results were compared at baseline and at 12 weeks, by using an ordinal scale. Neointima formation at the neck at 12 weeks was compared among groups by using a semiquantitative grading scale. Bench studies were performed to assess stent porosities. RESULTS: Initial angiographic results were improved with single stent-assisted coiling compared with simple coiling (P = .013). Angiographic results at 12 weeks were improved with any stent assistance (P = .014). Neointimal closure of the aneurysm neck was similar with or without stent assistance (P = .908), with neointima covering coil loops but rarely stent struts. Y-stent placement alone had no therapeutic effect. Bench studies showed that porosities can be decreased with stent compaction, but a relatively stable porous transition zone was a limiting factor. CONCLUSIONS: Stent-assisted coiling may improve results of embolization by allowing more complete initial coiling, but these high-porosity stents did not provide a scaffold for more complete neointimal closure of aneurysms.

Nedd4-2 ubiquitin ligase: a contributor to experimental neuropathic pain?

Background: Neuropathic pain is associated with altered expression of voltage-gated sodium channels (VGSCs) leading to peripheral nerve hyperexcitability. Interestingly, in cell expression systems, the ubiquitin ligase Nedd4-2 regulates the cell membrane density of the most abundant peripheral and pain-related VGSC, namely Nav1.7, and decreases its sodium current. Yet nothing is known about the involvement of Nedd4-2 in nociception and chronic pain. Therefore, the goal of this study is (i) to characterize Nedd4-2 and Nav1.7 expression in an experimental model of neuropathic pain (ii) to design by viral vector-mediated gene therapy an approach to depict the implication of Nedd4-2 in chronic pain. Methods: Western Blot and immunohistochemistry experiments detecting Nav1.7 and Nedd4-2 were performed in rodent DRGs 7 days after spared nerve injury (SNI). For the viral vector-mediated gene therapy, a recombinant Adeno-Associated Virus (rAAV2/6) was generated expressing the Nedd4-2 gene. Intrathecal injection of rAAV2/6 was followed 2 weeks after by the SNI surgery. Data are expressed in mean ± SEM, n = 4 in each condition. Results: Immunofluorescence on DRGs neurons reveals a decreased number of positive Nedd4-2 cells in the SNI model (27.0 ± 1.2%) versus sham group (43.4 ± 3.5%; p <0.005), as well as an increase in positive Nav1.7 cells in SNI (50.1 ± 2.9%) versus Sham (41.6 ± 1.8%; p <0.05). The change of Nedd4-2 expression was confirmed by western-blot analysis. In addition, we show that Nedd4-2 and Nav1.7 are largely expressed in overlapping cell populations, chiefly colocalizing with markers of small nociceptive neurons. Furthermore, we report that intrathecal injection of rAAV is able to counteract the reduction of Nedd4-2 expression in SNI animals. Conclusion: Our results indicate that Nedd4-2 is mainly expressed in nociceptors and downregulated after nerve injury. Moreover, our data suggest that the reduction of Nedd4-2, after nerve injury, may modulate Nav1.7 activity and contribute to hyperexcitability in neuropathic pain. A normal level of Nedd4-2 can be restored using a viral vector and we will further assess its functional effect on pain sensitivity.

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.

How to improve flow during cardiopulmonary bypass in an acardia experimental model.

OBJECTIVES In extreme scenarios, such as hyperacute rejection of heart transplant, an urgent heart explantation might be necessary. The aim of this experimental study was to determine the feasibility and to improve the haemodynamics of a venoarterial cardiopulmonary bypass after cardiectomy. METHODS A venoarterial cardiopulmonary bypass was established in seven calves (56.4 ± 7 kg) by the transjugular insertion to the caval axis of a self-expanding cannula, with a carotid artery return. After baseline measurements (A), ventricular fibrillation was induced (B), great arteries were clamped (C), the heart was excised and the right and left atria remnants, containing the pulmonary veins, were sutured together leaving an atrial septal defect over the cannula in the caval axis (D). Measurements were taken with the pulmonary artery clamped and declamped. RESULTS Initial pump flow was 4.16 ± 0.75 l/min dropping to 2.9 ± 0.63 l/min (P(AB )< 0.001) 10 min after induction of ventricular fibrillation. After cardiectomy with the pulmonary artery clamped, the pump flow increased non-significantly to 3.20 ± 0.78 l/min. After declamping, the flow significantly increased close to baseline levels (3.61 ± 0.73 l/min, P(DB )= 0.009, P(DC )= 0.017), supporting the notion that full cardiopulmonary bypass in acardia is feasible only if adequate drainage of pulmonary circulation is assured to avoid pulmonary congestion and loss of volume from the left-to-right shunt of bronchial vessels.

Experimental photodynamic therapy for malignant pleural mesothelioma with pegylated mTHPC.

BACKGROUND AND OBJECTIVES: Experimental assessment of photodynamic therapy (PDT) for malignant pleural mesothelioma using a polyethylene glycol conjugate of meta-tetrahydroxyphenylchlorin (PEG-mTHPC). STUDY DESIGN/MATERIALS AND METHODS: (a) PDT was tested on H-meso-1 xenografts (652 nm laser light; fluence 10 J/cm(2); 0.93, 9.3, or 27.8 mg/kg of PEG-mTHPC; drug-light intervals 3-8 days). (b) Intraoperative PDT with similar treatment conditions was performed in the chest cavity of minipigs (n = 18) following extrapleural pneumonectomy (EPP) using an optical integrating balloon device combined with in situ light dosimetry. RESULTS: (a) PDT using PEG-mTHPC resulted in larger extent of tumor necrosis than in untreated tumors (P < or = 0.01) without causing damage to normal tissue. (b) Intraoperative PDT following EPP was well tolerated in 17 of 18 animals. Mean fluence and fluence rates measured at four sites of the chest cavity ranged from 10.2 +/- 0.2 to 13.2 +/- 2.3 J/cm(2) and 5.5 +/- 1.2 to 7.9 +/- 1.7 mW/cm(2) (mean +/- SD). Histology 3 months after light delivery revealed no PDT related tissue injury in all but one animal. CONCLUSIONS: PEG-mTHPC mediated PDT showed selective destruction of mesothelioma xenografts without causing damage to intrathoracic organs in pigs at similar treatment conditions. The light delivery system afforded regular light distribution to different parts of the chest cavity.

Transvaginal specimen extraction in colorectal surgery: current state of the art.

Aim  Background The expected benefit of transvaginal specimen extraction is reduced incision-related morbidity. Objectives A systematic review of transvaginal specimen extraction in colorectal surgery was carried out to assess this expectation. Method  Search strategy The following keywords, in various combinations, were searched: NOSE (natural orifices specimen extraction), colorectal, colon surgery, transvaginal, right hemicolectomy, left hemicolectomy, low anterior resection, sigmoidectomy, ileocaecal resection, proctocolectomy, colon cancer, sigmoid diverticulitis and inflammatory bowel diseases. Selection criteria Selection criteria included large bowel resection with transvaginal specimen extraction, laparoscopic approach, human studies and English language. Exclusion criteria were experimental studies and laparotomic approach or local excision. All articles published up to February 2011 were included. Results  Twenty-three articles (including a total of 130 patients) fulfilled the search criteria. The primary diagnosis was colorectal cancer in 51% (67) of patients, endometriosis in 46% (60) of patients and other conditions in the remaining patients. A concurrent gynaecological procedure was performed in 17% (22) of patients. One case of conversion to laparotomy was reported. In two patients, transvaginal extraction failed. In left- and right-sided resections, the rate of severe complications was 3.7% and 2%, respectively. Two significant complications, one of pelvic seroma and one of rectovaginal fistula, were likely to have been related to transvaginal extraction. The degree of follow up was specified in only one study. Harvested nodes and negative margins were adequate and reported in 70% of oncological cases. Conclusion  Vaginal extraction of a colorectal surgery specimen shows potential benefit, particularly when associated with a gynaecological procedure. Data from prospective randomized trials are needed to support the routine use of this technique.