Polyethylene Catheter Research Articles

Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.

P-314: Do interventions necessary to overcome technical difficulties encountered in trial embryo transfer and employed during real embryo transfer really improve outcome in IVF cycles?

To evaluate the impact of intervention techniques employed in real embryo transfer to overcome technical difficulties presented during trial transfer on the outcome of IVF cycles. Prospective observational study of 418 consecutive IVF/ET cycles. Trial embryo transfer (TET) to map the cervical canal was performed with a soft polyethylene catheter and a firm outer Teflon introducer (Wallace, SIMS Portex, UK) in all cycles immediately before the real transfer. All trial and real embryo transfers (RET) were performed under abdominal ultrasound guidance 72 hours after oocyte retrieval and only RETs considered uneventful (n=364) were compared. Interventions suggested by TET that were employed in RET to overcome difficulties included the use of: Group A (n=184)- soft catheter, no interventions; Group B (n=97)- soft catheter, insertion of the outer introducer through the endocervical canal and internal os, with or without the help of a tenaculum and; Group C (n=83)- firm catheter (Tefcat, Cook, USA), with or without the help of a tenaculum. All catheters were loaded with a maximum of four embryos in 20-30μl of culture medium after embryo morphology evaluation and grading. After RET, the following variables were recorded: presence of blood and/or mucus in or on the transfer catheter; cramping or patient discomfort; retention of embryos in the transfer catheter. There was no difference between groups with respect to patients’ main demographic and baseline characteristics, embryo development rate, mean number and quality at transfer. The overall clinical pregnancy rate/transfer for the different interventions was 48.4%, 41.2% and 25.3% in groups A, B and C, respectively, showing a significant difference (p=0.02) between groups A and C. Likewise, the implantation rate was significantly higher (p=0.005) in group A (26.2%) than in group C (15.6%) and revealed a trend (p=0.08) toward better results when compared to group B (19.7%). Clinical pregnancy/implantation rates showed no difference when groups B and C were compared. Contamination of the catheter with blood and/or cervical mucus was more frequent in group B (68.0%) when compared to groups A (37.5%) and C (41.0%) (p=0.0001, p=0.0004; respectively). The occurrence of uterine cramping was 3.6%, 10.2% and 18.6% in groups A, B and C, respectively, showing a significant difference (p=0.001) between groups A and C and no difference when groups A and B or B and C were compared. There was no difference between groups in regard to the incidence of retained embryos. The use of more invasive and potentially traumatic interventions by clinicians to efficiently overcome a difficult TET during RET may, by themselves, significantly impair the outcome of IVF cycles. This is reflected in the lower pregnancy and implantation rates observed in the study, regardless of degree of ease in execution of the procedure. Every maneuver should be tried with a softer catheter before changing it to a firmer one, since it is clearly shown by this study that the latter has a negative impact on the overall outcome. A balance between the choice of the intervention necessary to overcome the difficulties encountered during TET, and the expected decrease in pregnancy/implantation rates should always be taken into account by the clinician in charge of the embryo transfer, since different intervention techniques may have negative influence on the outcome of IVF cycles to varying degrees.

No major role of BKCa channels in the modulation of renal blood flow in rats

The aim of the present study was to investigate the role of large calcium activated potassium (BKCa) channels in the maintenance of basal renal vascular tone in vivo in anesthetized adult Sprague-Dawley rats. We also examined whether the vasoconstriction elicited by angiotensin II (Ang II) and norepinephrine (NE) was buffered by simultaneous activation of BKCa. Vasoactive agents, BKCa blockers or a stimulator were infused directly into the renal artery via a polyethylene (PE-10) catheter introduced into the left femoral artery and advanced through the abdominal aorta and approximately 1 mm into the left renal artery. Renal blood flow (RBF) was measured by using an electromagnetic flow sensor. Renal preinfusion of BKCa blockers tetraethylammonium chlorid (TEA) (3.0 μmol/min) for 1 (n = 8) or 5 min (n = 5) and iberiotoxin for 5 min (n = 6) (3.3 nmol/min) did not significantly affect the RBF baseline. Renal arterial injection of a 10 μL bolus of Ang II (n = 18) (1-4 ng/10 μL) or NE (n = 17) (10-40 ng/10 μL) produced a significant dose dependent temporary decrease in RBF. These responses were not significantly affected by 1 or 4 min preinfusion of TEA (n = 8) or a 4 min preinfusion of iberiotoxin (n = 6). Renal addition of the BKCa opener NS 1619 (n = 5) (30 and 90 μmol/min) did not significantly affect basal renal blood flow or the responses to Ang II and NE. The present results do not indicate any major role for BKCa channels in the control of basal renal tone in vivo. Furthermore, there is no support for the hypothesis that BKCa channels have a buffering effect on the responses to Ang II and NE in rat renal vasculature. (Supported by Danish Heart Foundation)

Linezolid in Prophylaxis against Experimental Aortic Valve Endocarditis Due to Streptococcus oralis or Enterococcus faecalis

There are no experimental studies regarding the prophylactic efficacy of linezolid against infective endocarditis. Nonbacterial thrombotic endocarditis of the aortic valve was induced in rabbits by the insertion of a polyethylene catheter. Twenty-four hours later, animals were randomly assigned to a control group, and groups receiving either ampicillin (two doses of 40 mg/kg of body weight each, given intravenously, 2 h apart) or linezolid (a single per os dose of 75 mg/kg). The first dose of ampicillin and the single dose of linezolid were administered 0.5 and 1 h, respectively, prior to the intravenous inoculation of approximately 10(7) CFU of Streptococcus oralis or Enterococcus faecalis. Linezolid peak levels in rabbit serum were similar to the peak serum levels in humans following a 600-mg oral dose of linezolid. Linezolid prevented endocarditis in 87% of S. oralis-challenged rabbits (P < 0.001 versus controls; P = 0.026 versus ampicillin). In rabbits challenged with E. faecalis, linezolid prevented endocarditis in 73% (P = 0.003 versus controls; P = 0.049 versus ampicillin). Ampicillin prevented endocarditis due to S. oralis or due to E. faecalis in 47% (P = 0.005 versus controls) and in 30% (P = not significant versus controls) of the challenged animals, respectively. In conclusion, linezolid was effective as prophylaxis against endocarditis caused by a strain of S. oralis and to a lesser degree against that caused by a strain of E. faecalis. Its prophylactic efficacy was superior to that of ampicillin.

Biliary Duct Ablation with N-Butyl Cyanoacrylate

To assess the efficacy of percutaneous insertion of n-butyl cyanoacrylate (NBCA) in the ablation of bile ducts in patients with persistent postsurgical bile leaks in which traditional means of treatment have failed. Ablation of bile ducts with NBCA was performed in six patients (two men and four women). The average length of follow-up was 27 months (range, 13-46 months). Four patients presented after hepatic lobectomy with a persistent bile leak, one patient presented after cholecystectomy with a chronically obstructed bile duct, and one patient presented after cholecystectomy from intraoperative bile duct injury. After access to the biliary system was obtained, a cholangiogram was obtained. After the desired duct was isolated, it was copiously irrigated with saline solution. A glue solution containing NBCA glue, Ethiodol, and tantalum powder was delivered into the duct through a polyethylene catheter that had been irrigated with dextrose solution. Four patients had problems arising from isolated segmental ductal systems that had no communication with the normal biliary ductal system and were treated successfully on the first attempt. In two patients, there was communication to the main biliary ductal system and a persistent bile leak occurred that required placement of a coil and a second final gluing procedure. The only complication observed was unintentional spillage of glue into the main biliary system in one patient, which was ultimately clinically insignificant. The use of NBCA glue in obliteration of bile ducts is a safe procedure with excellent results in patients with complications from isolated segmental ducts. Although a repeat procedure may be necessary if the duct communicates with the main biliary tree, the procedure can decrease the morbidity associated with chronic external biliary drainage.

History of Peritoneal Access Development

The first peritoneal accesses were devices that had been used in other fields (general surgery, urology, or gynecology): trocars, rubber catheters, and sump drains. In the period after World War II, numerous papers were published with various modifications of peritoneal dialysis. The majority of cases were treated with the continuous flow technique; rubber catheters for inflow and sump drains for outflow were commonly used. At the end of the 1940s, intermittent peritoneal dialysis started to be more frequently used. Severe complications of peritoneal accesses created incentive to design accesses specifically for peritoneal dialysis. The initial three, in the late 1940s, were modified sump drains; however, Ferris and Odel for the first time designed a soft, polyvinyl intraperitoneal tube with metal weights to keep the catheter tip in the pelvic gutter where the conditions for drain are the best. In the 1950s, intermittent peritoneal dialysis was established as the preferred technique; polyethylene and nylon catheters became commercially available and peritoneal dialysis was established as a valuable method for treatment of acute renal failure. The major breakthrough came in the 1960s. First of all, it was discovered that the silicone rubber was less irritating to the peritoneal membrane than other plastics. Then, it was found that polyester velour allowed an excellent tissue ingrowth creating a firm bond with the tissue. When a polyester cuff was glued to the catheter, it restricted catheter movement and created a closed tunnel between the integument and the peritoneal cavity. In 1968, Tenckhoff and Schechter combined these two features and designed a silicone rubber catheter with a polyester cuff for treatment of acute renal failure and two cuffs for treatment of chronic renal failure. This was the most important development in peritoneal access. Technological evolution never ends. Multiple attempts have been made to eliminate remaining complications of the Tenckhoff catheter such as exit/tunnel infection, external cuff extrusion, migration leading to obstruction, dialysate leaks, recurrent peritonitis, and infusion or pressure pain. New designs combined the best features of the previous ones or incorporated new elements. Not all attempts have been successful, but many have. To prevent catheter migration, Di Paolo and his colleagues applied the old idea of providing weights at the catheter tips to Tenckhoff catheters. In another modification, Twardowski and his collaborators created a permanent bend to the intra-tunnel portion of the silicone catheter to eliminate cuff extrusions. The Tenckhoff catheter continues to be widely used for chronic peritoneal dialysis, although its use is decreasing in favor of swan-neck catheters. Soft, silicone rubber instead of rigid tubing virtually eliminated such early complications as bowel perforation or massive bleeding. Other complications, such as obstruction, pericatheter leaks, and superficial cuff extrusions have been markedly reduced in recent years, particularly with the use of swan-neck catheters and insertion through the rectus muscle instead of the midline. However, these complications still occur, so new designs are being tried.

Improvement of bladder storage function by α1‐blocker depends on the suppression of C‐fiber afferent activity in rats

Alpha1-blockers improve voiding symptoms through the reduction of prostatic and urethral smooth muscle tone; however, the underlying mechanism of improvement of storage symptoms is not known. Using a rat model of detrusor overactivity caused by cerebral infarction (CI), we undertook the present study to determine whether the effect of an alpha1-blocker, naftopidil, is dependent on the suppression of C-fiber afferents. To induce desensitization of C-fiber bladder afferents, we injected resiniferatoxin (0.3 mg/kg, RTX) sub-cutaneously to female Sprague-Dawley rats 2 days prior to left middle cerebral artery occlusion (MCAO) (RTX-CI rats). As controls we used rats without RTX treatment (CI rats). MCAO and insertion of a polyethylene catheter through the bladder dome were performed under halothane anesthesia. We investigated the effects on cystometrography (CMG) of intravenous (i.v.), intracerebroventricular (i.c.v.), or intrathecal (i.t.) administration of naftopidil in conscious CI rats. Bladder capacity (BC) was markedly reduced after MCAO in both RTX-CI and CI rats. I.v. administration of naftopidil significantly increased BC in CI rats without an increase in residual volume, but it had no effects on BC in RTX-CI rats. I.t. administration of naftopidil significantly increased BC in CI but not in RTX-CI rats. These results suggest that naftopidil has an inhibitory effect on C-fiber afferents in the lumbosacral spinal cord, improving BC during the storage phase.

Simplified Rat Model of Intestinal Transplantation

Rat intestinal transplantation is an optimal model for research; nevertheless, it requires a complex and difficult microsurgical technique (1–5). We have recently developed a simplified rat model for intestinal transplantation using portal cuff anastomosis to overcome these technical difficulties (Fig. 1A).FIGURE 1.: (A) Figure illustrating the present model of intestinal transplantation. G, intestinal graft in shady color; SMA, superior mesenteric artery of the graft; VC, recipient vena cava; AO, recipient aorta. (B) Micropicture (16×) showing details of the portal cuff.Preoperative procedures, anesthesia and laparotomy are performed as previously described (1–4). In the donor operation, middle and descending colons are isolated from small bowel and ascending colon. Superior mesenteric vein is dissected and encircled. Splenic and pyloric veins are divided and portal vein is dissected up to hepatic hilum. Abdominal aorta is dissected and celiac trunk, right renal artery and lumbar branches are divided. Aorta is clamped at the diaphragm, tied below the mesenteric artery and this arterial segment is resected allowing a long aortomesenteric conduit. Entire small bowel, cecum and ascending colon are harvested on a vascular pedicle consisting of right colonic vessels, aortomesenteric conduit and portal vein. The graft is flushed and stored in cold preservation solution. Polyethylene catheter is used to create the cuff as described elsewhere (5). In this modified technique, the vein is introduced into the cuff and everted, covering the outer wall and fixed with three equidistant stitches (Fig. 1B). In the recipient, the superior mesenteric vein is exposed, and a segment between the first and second jejunal branch is dissected and prepared for cuff anastomosis. Eventually, the second jejunal branches and the right colonic vein may be divided in order to increase this segment. The graft is implanted in the recipient with an end-to-side aorto-aorta anastomosis as previously described (1–5). The dissected segment of mesenteric vein is isolated between clamps to insert the portal cuff and a 6-0 suture is used to fix the cuff into recipient’s mesenteric vein. The clamps are released and immediate graft reperfusion is observed. The recipient jejunum, ileum, cecum, and ascending colon are removed en bloc. Finally, anastomosis between the graft and the remaining intestine restores the digestive tract (Fig. 1A). The present model offers many possibilities for intestinal transplantation research with the following advantages: reconstitutes the natural and physiologic portal graft drainage; the portal cuff anastomosis is performed within few minutes without bleeding or venous thrombosis. This alternative technique avoids recipient visceral ischemia and hemodynamic instability because there is no obstruction on inferior vena cava and portal vein. Furthermore, it maintains grafts ileocecal valve and preserves the extrinsic enervation along the mesenteric-aortic segment that may improve bowel movement. This modified technique can be easily performed within two hours after a short period of training (8±3 week) and may reach excellent success rates. This rat model may surmount technical obstacles precluding microsurgical techniques and may ultimately spread this model in centers with low technical resources. Flávio H.F. Galvão Rafael M.N. Santos Marcel A.C. Machado Telesforo Bacchella Marcel C.C. Machado Transplantation and Liver Surgery Discipline University of Sao Paulo School of Medicine Sao Paulo, Brazil

EFFECTS OF TOLTERODINE ON AN OVERACTIVE BLADDER DEPEND ON SUPPRESSION OF C-FIBER BLADDER AFFERENT ACTIVITY IN RATS

We determined whether the effects of antimuscarinics depend on the suppression of C-fiber bladder afferent nerves. We administered tolterodine intravenously or intravesically. To induce C-fiber bladder afferent nerve desensitization resiniferatoxin (RTX) (0.3 mg/kg) was injected subcutaneously in female Sprague-Dawley rats 2 days prior to left middle cerebral artery occlusion (MCAO). As controls, we used rats treated with ethanol and saline vehicle (VEH). Insertion of a polyethylene catheter through the bladder dome and MCAO were performed using halothane anesthesia. The effects of intravenous (0.2 to 2000 nM/kg) or intravesical (0.2 or 2 nM) tolterodine, an antimuscarinic agent, on cystometrography were investigated in conscious rats with a cerebral infarct (CI). Tolterodine was instilled intravesically for 30 minutes and cystometry was repeated. Bladder capacity (BC) was markedly decreased after MCAO in RTX treated (RTX-CI) and VEH treated (VEH-CI) rats. Low tolterodine doses (0.2 or 2 nM/kg) significantly increased BC in VEH-CI rats without increasing residual volume but it had no effects on BC in RTX-CI rats. At the highest dose (2,000 nM/kg) the drug significantly decreased bladder contraction pressure and increased residual volume in RTX-CI and VEH-CI rats. Intravesical administration of tolterodine (0.2 or 2 nM) significantly increased BC in VEH-CI rats. However, tolterodine had no effect on BC in RTX-CI rats. These results suggest that at low doses tolterodine exerts an inhibitory effect on C-fiber bladder afferent nerves, thereby, improving BC during the storage phase.

Successful moxifloxacin prophylaxis against experimental streptococcal aortic valve endocarditis

Studies related to the prophylactic efficacy of fluoroquinolones against infective endocarditis are scarce. The aim of this study was to evaluate the efficacy of moxifloxacin, a quinolone active in vitro against Gram-positive cocci, in preventing streptococcal aortic valve endocarditis. Non-bacterial thrombotic endocarditis of the aortic valve was induced by the insertion of a polyethylene catheter. Twenty-four hours later, rabbits were randomly assigned to a control group, and groups receiving either two doses of ampicillin (40 mg/kg, intravenously), 2 h apart, or a single dose of moxifloxacin (15 mg/kg, intravenously). Ampicillin and moxifloxacin were administered 0.5 and 1 h, respectively, prior to the intravenous inoculation of 10(7) cfu of Streptococcus oralis. Eighty-nine percent of the control animals developed infected vegetations. In rabbits challenged with this very high inoculum, moxifloxacin and ampicillin prevented endocarditis in 80% (P < 0.001 versus controls) and in 50% (P = 0.022 versus controls) of animals, respectively. The difference between ampicillin and moxifloxacin was not statistically significant (P = 0.128). Moxifloxacin was at least as effective as ampicillin in preventing streptococcal endocarditis.

Short-Term Angiotensin(1-7) Receptor Mas Stimulation Improves Endothelial Function in Normotensive Rats

In this study we evaluated the effect of angiotensin(1-7) and its nonpeptide analog, AVE 0991, on the endothelial function in vivo. The experiments were performed in conscious adult male Wistar rats, with polyethylene catheters implanted into the descending aorta (through left carotid artery), for injection of acetylcholine or sodium nitroprusside, femoral artery for mean arterial pressure and heart rate measurement; and femoral vein for drug administration. Increasing doses of acetylcholine (3.1 ng to 25.0 ng) or nitroprusside (1.0 microg to 10.0 microg) were administered before and 30 minutes after the start of the infusion of: angiotensin(1-7) (0.7 and 7.0 pmol/min); A-779 (180 pmol/min); angiotensin(1-7) (7.0 pmol/min) combined with A-779 (180 pmol/min); AVE 0991 (11, 45, and 230 pmol/min); AVE 0991 (45 pmol/min) combined with A-779 (180 pmol/min), or vehicle (6 microL/min). Baseline mean arterial pressure and heart rate were not altered during angiotensin(1-7) or AVE 0991 infusion. Angiotensin(1-7) (0.7 pmol/min) infusion produced a significant potentiation of the hypotensive effect of acetylcholine (3.1 ng: -9+/-1 mm Hg before; -18+/-2 mm Hg after; P<0.05). A similar potentiation was observed with the higher dose of angiotensin(1-7). As observed for angiotensin(1-7), infusion of AVE 0991 at 230 pmol/min potentiated the acetylcholine effect (3.1 ng: -8+/-2 mm Hg before; -16+/-2 mm Hg after; P<0.05). The potentiating effect was not observed for nitroprusside. A-779 or l-NAME treatment blocked the potentiation produced by angiotensin(1-7) or AVE 0991. Our data indicate that short-term stimulation of angiotensin(1-7) receptors improve endothelial function through facilitation of nitric oxide release.

Abnormal monocyte recruitment and collateral artery formation in monocyte chemoattractant protein-1 deficient mice

Monocyte chemoattractant protein 1 (MCP-1) has been shown to be effective for the stimulation of collateral artery formation in small and large animal models. The availability of a genetic knockout mouse enables evaluation of the importance of the role of MCP-1 in the natural course of collateral artery growth. In a total of 21 MCP-1 -/- as well as 13 of the appropriate genetic background controls ([129Sv/J X C57BI/6J]F1), a femoral artery ligation was performed. Subsequently, a polyethylene catheter, connected to an osmotic minipump, was inserted retrogradely into the occluded femoral artery with the tip pointing upstream. Using this technique, PBS (MCP-1 -/-: n = 13 and C57BI/6J: n = 13) or MCP-1 (JE; MCP-1 -/-: n = 8) was delivered intra-arterially. Seven days after ligation, determination of hind limb flow was assessed by controlled tissue perfusion using differently labeled fluorescent microspheres. MCP-1 -/- mice exhibited a reduction of hind limb flow of 32.9 +/- 9.2% of the unligated hind limb, compared with 55.4 +/- 6.8% in C57BI/6J mice (p<0.01). MCP-1 -/- mice that underwent a subsequent 'rescue' treatment with MCP-1 showed a restoration of flow to a level of 47.4 +/- 9.8% (p = NS compared with PBS-treated C57BI/6J). Specific immunohistochemical staining for monocytes (MOMA-2: MCP-1 -/-, n = 5 and C57BI/6J, n = 5) showed a reduced number of monocytes around developing collateral arteries in the MCP-1 -/- mice. In conclusion, our data show that the absence of MCP-1 causes a strong reduction in flow restoration after femoral artery occlusion, coinciding with a reduced monocyte attraction, emphasizing the central role of this chemokine in the multifactorial process of collateral artery formation.

Enhanced abdominal aortic aneurysm in TIMP-1-deficient mice 1

Matrix metalloproteinases (MMPs) are known elastolytic mediators of abdominal aortic aneurysm (AAA) degeneration, and their activity is tightly regulated by the presence of tissue inhibitors of MMPs (TIMPs). Imbalances in this system may be instrumental in compromising arterial wall integrity. The aim of this study was to show that, in an elastase-induced murine model of aneurysm formation, TIMP-1 has a protective effect. Twenty-four wild-type (TIMP-1+/+) and 22 knockout (TIMP-1-/-) mice underwent laparotomy and isolation of the infrarenal aorta. A polyethylene catheter was inserted into the aorta and dilute pancreatic elastase (0.39 Units/ml) was infused over 5 min using a perfusion pump. Pre- and postinfusion maximal aortic diameters were obtained in triplicate for each animal using NIH Image. Final aortic measurements were obtained 14 days later, prior to perfusion fixation with 10% buffered Formalin. Aortic specimens were sectioned and stained. Statistical analysis was performed using the Student's t test. TIMP-1-/- mice demonstrated a significant postinfusion diameter increase compared to wild-types after elastase, which was not seen after saline infusion. At sacrifice, TIMP-1-/- mice, following both saline and elastase infusion, showed a significant increase in maximal aortic diameter relative to postinfusion measurements compared to TIMP-1+/+ mice. TIMP-1-/- mice develop larger aneurysms than TIMP-1+/+ mice. This study illustrates the protective effects of TIMP-1 in an experimental AAA model and may provide a means for pharmacologically controlling aneurysm growth.

Avaliação intra-operatória da pressão portal e resultados imediatos do tratamento cirúrgico da hipertensão portal em pacientes esquistossomóticos submetidos a desconexão ázigo-portal e esplenectomia

The main cause of portal hypertension in Brazil is the hepato-splenic form of mansonic schistosomiasis and the most employed technique for the surgical approach of this disease is the esophagogastric devascularization with splenectomy, generally associated to postoperative endoscopical esophageal varices sclerotherapy. The hemodynamic alterations after surgical treatment and its possible influence on the outcome are not well established. To evaluate the immediate impact of esophagogastric devascularization with splenectomy upon portal pressure as well as the results of the surgical treatment on digestive hemorrhage recurrence and on esophageal varices. Nineteen patients with mean age of 37.9 years and portal hypertension and previous episodes of digestive hemorrhage caused by esophageal varices rupture due to hepato-splenic schistosomiasis were studied. None of the patients had received any treatment prior to the surgery and underwent to elective esophagogastric devascularization with splenectomy. Portal pressure was assessed at the beginning and at the end of esophagogastric devascularization with splenectomy through portal vein catheterization with a polyethylene catheter introduced through a jejunal branch vein. All patients were submitted to digestive endoscopy before and after the surgery, in order to classify the size of esophageal varices after esophagogastric devascularization with splenectomy according to Palmer's classification. In all patients the portal pressure had diminished with a mean decrease of 31.3% after esophagogastric devascularization with splenectomy. In the postoperative endoscopic follow-up (1 month), the esophageal varices showed a statistically significant reduction in their size, when compared to the pre-surgical measurements. These results have demonstrated that the esophagogastric devascularization with splenectomy promotes immediate decrease in the portal pressure and a consequent reduction in the esophageal varices size. We also observed that the risk of mortality and severe complications related to this technique is not insignificant.

Evaluation of protein C and other biomarkers as predictors of mortality in a rat cecal ligation and puncture model of sepsis.

To evaluate protein C and other factors associated with the septic response as predictors of mortality in a clinically relevant animal model of sepsis. Laboratory investigation. Eli Lilly and Company discovery research laboratory. Forty female Sprague Dawley rats weighing 245-265 g. Polyethylene catheters were surgically implanted into the femoral vein and sepsis was induced by cecal ligation and puncture (CLP). A solution of 5% dextrose in 0.9 % saline was continuously infused via femoral catheters immediately following surgery. Blood sampling was done before surgery and at 6 and 20 hrs after surgery. Rats were then monitored for survival out to 4 days. Blood collections were used to measure blood glucose, bacteremia, plasma protein C, D-dimer, hormones, chemokines, cytokines, and myoglobin (as a marker of organ damage). Mortality was categorized into three groups: early death (before 30 hrs post-CLP), late death (after 30 hrs post-CLP), and survivors (96 hrs post-CLP). Compared with survivors, early death rats had statistically significant differences in 30 variables indicative of severe inflammation, coagulopathy, and muscle damage including less bacterial clearance, hypoglycemia, lower plasma protein C, higher plasma D dimer, higher plasma cytokine/ chemokines, and higher plasma myoglobin concentrations. Twenty variables had a moderate to strong correlation with time of death. Receiver operator characteristic curves generated from a simple logistic regression model indicated that KC and macrophage inflammatory protein-2, rodent homologues of the human growth related oncogene CXC chemokine family, and protein C were the best predictors of mortality in this model. The data from this study indicate that an early decrease in protein C concentration predicts poor outcome in a rat sepsis model. The data further indicate that increases in the CXC chemokines macrophage inflammatory protein-2 and KC precede poor outcome.

495. Efficient In Vivo Transduction of Mouse Airway Epithelial Cells by the Simian Immunodeficiency Virus Vector Pseudotyped with Sendai Virus F and HN Proteins

We have described gene delivery in sheep as a large animal model for airway gene transfer. This model allows comparison of different GTAs in terms of efficacy and safety using doses, delivery methods and assays, relevant to human studies. Proof-of-principle for non-viral gene transfer has been established however, levels of transgene expression are too low on a per cell basis to characterise which cells are targeted. Our preferred method for delivery is by aerosol however, not all GTAs are stable following aerosolisation in conventional jet or ultrasonic nebulisers and the cost of producing GTAs in sufficient quantities for whole lung aerosol studies can be prohibitive. These issues were behind our initial studies with delivery by instillation to individual segments. Unfortunately this approach is associated with increased toxicity as a result of pooling of material in the terminal bronchiole/alveolar duct regions. The Trudell AeroProbe* catheter develops an aerosol at the tip and can be passed through a bronchoscope. This allows localised delivery of GTAs, reducing the amount of material required. A further advantage of the AeroProbe* is that it is a single-pass aerosol device and subjects the GTA to less shear stress. An important question to be addressed when comparing delivery with different devices, is how their respective distribution patterns compare. Here we used a recombinant Sendai virus (DF/SeV-LacZ) vector, shown to be highly efficient at transducing airway epithelial cells in a variety of species, to investigate distribution patterns of delivery methods. SeV was delivered either by instillation to single lung segments via a polyethylene catheter (PEC), by AeroProbe* directed at segments or by AeroProbe* directed at the whole lung. In terms of stability, 100% and 49 +/- 3% of infectious virus particles survived the PEC and AeroProbe* respectively (n=3/group). In vivo studies show the variation in distribution of the different delivery methods. Instillation of 5ml SeV (3e9 CIU) to the same segment of different animals (n=3) results in varied distribution. Data from these animals confirm the absence of spillover as determined by histological assessment of adjacent segments. Segmentally-directed delivery of 5ml SeV (1e10 CIU) via AeroProbe* gives a different distribution. We observed areas of expression at airway bifurcations from impaction of aerosol particles at these sites and expression around sub-mucosal gland openings and within the glands themselves. Spillover to adjacent segments was observed in this case. Delivery of 24ml SeV (4e10 CIU) to the whole lung via the AeroProbe* gave a different type of expression pattern again. SeV was distributed throughout the lung. An even pattern of distribution all the way to the peripheral alveolar regions can be observed in several segments although the extent varies. These distribution patterns will have implications for sampling. It is clear from these studies that the properties of SeV make it an ideal vector to study the distribution pattern of different devices in our ovine model.

Hemoglobin-vesicles suspended in recombinant human serum albumin for resuscitation from hemorrhagic shock in anesthetized rats.

Hemoglobin-vesicle (HbV) has been developed to provide oxygen-carrying ability to plasma expanders. Its ability to restore the systemic condition after hemorrhagic shock was evaluated in anesthetized Wistar rats for 6 hrs after resuscitation. The HbV was suspended in 5 g/dL recombinant human serum albumin (HbV/rHSA) at an Hb concentration of 8.6 g/dL. Prospective, randomized, controlled trial. Department of Surgery, School of Medicine, Keio University. Forty male Wistar rats. The rats were anesthetized with 1.5% sevoflurane inhalation throughout the experiment. Polyethylene catheters were introduced through the right jugular vein into the right atrium for infusion and into the right common carotid artery for blood withdrawal and mean arterial pressure monitoring. Shock was induced by 50% blood withdrawal. The rats showed hypotension (mean arterial pressure = 32 +/- 10 mm Hg) and significant metabolic acidosis and hyperventilation. After 15 mins, they received HbV/rHSA, shed autologous blood (SAB), washed homologous red blood cells (wRBC) suspended in rHSA (wRBC/rHSA, [Hb] = 8.6 g/dL), or rHSA alone. The HbV/rHSA group restored mean arterial pressure to 93 +/- 8 mm Hg at 1 hr, similar to the SAB group (92 +/- 9 mm Hg), which was significantly higher compared with the rHSA (74 +/- 9 mm Hg) and wRBC/rHSA (79 +/- 8 mm Hg) groups. There was no remarkable difference in the blood gas variables between the resuscitated groups; however, two of eight rats in the rHSA group died before 6 hrs. After 6 hrs, the rHSA group showed significant ischemic changes in the right cerebral hemisphere relating to the ligation of the right carotid artery followed by cannulation, whereas the HbV/rHSA, SAB, and wRBC/rHSA groups showed less changes. HbV suspended in recombinant human serum albumin provides restoration from hemorrhagic shock that is comparable with that using shed autologous blood.

Longitudinal MRI tracking of the angiogenic response to hind limb ischemic injury in the mouse.

Ischemic injury and revascularization are frequently associated with hyperpermeability. Although extravasation of plasma proteins may promote tissue recovery through the generation of the provisional matrix that supports angiogenesis, edema may also result in progressive damage to the muscle. The aim of this research was to determine the time course of hyperpermeability associated with the angiogenic response induced by ligation of the femoral artery at the right posterior limb in mice. Hyperpermeability was followed noninvasively by MRI using an in-house-built permanent polyethylene catheter that enabled daily intravenous administration of biotin-BSA-Gd-DTPA. The mice were scanned once prior to ligation and five times during the week post-ligation. The MRI data, along with histopathology, indicated that the early hemodynamic compensation over loss of arterial blood supply occurred by angiogenesis and dilation of vessels in the skin and subcutaneous fat, and was accompanied by vascular hyperpermeability around the site of ligation. Functional recovery of the ischemic limb (i.e., regaining the ability to step on the limb), and the color and shape of the toes correlated with regeneration as shown by histopathology and MRI analysis. Thus, MRI provided valuable information on the transient hyperpermeability induced during the early stages of angiogenesis, and its subsequent resolution along with functional recovery from acute hind limb ischemia in mice.

Microscopic Changes of the Spinal Cord after the Intrathecal Administration of Ondansetron in the Rat

Background: Serotonin type 3 receptors are abundant in the dorsal horn of the spinal cord and in the spinal tract of the trigeminal nerve in the medulla. Moreover, the intrathecal (IT) or epidural administration of ondansetron, a selective 5-hydroxytryptamine 3 (5-) receptor antagonist, might prevent postoperative nausea, vomiting and intrathecal opioid-induced pruritus more effectively than the drug administered systemically. Before new drugs for spinal administration are accepted for clinical practice, experimental neurotoxicity studies must be undertaken. This study was performed in rats in order to reveal the behavioral and morphological signs of spinal cord damage after chronic IT ondansetron administration. Methods: Rats were anesthetized using enflurane in oxygen and an 8 cm polyethylene catheter was advanced caudally through the atlanto-occipital membrane under aseptic surgical conditions. Twelve rats were randomly divided into two groups, an ondansetron (O) group (n = 6) and a normal saline (N) group (n = 6). After postoperative 7 days, we started the IT injection of ondansetron 40 (20) or normal saline 20 once a day over two weeks. Potential changes of spinal cords were evaluated morphologically by light microscopy (LM) and electron microscopy (EM) in addition to behavioral changes. Results: No rat in either groups showed any motor or behavioral changes during the administration of ondansetron. By LM and EM, all six rats in the O group showed massive neovascularization over the white and gray matters of spinal cords. Conclusions: The results suggest that chronic IT administration of ondansetron causes pathologic changes in the rat spinal cord.

Enhanced abdominal aortic aneurysm (AAA) formation in timp-1-deficient (timp-1-/-) mice after local elastase perfusion

Background: Matrix metalloproteinases (MMPs) are known elastolytic mediators of abdominal aortic aneurysm (AAA) degeneration, and their activity is tightly regulated by the presence of tissue inhibitors of MMPs (TIMPs). TIMPs act as a negative regulator of MMPs. Imbalances in this system may be instrumental in compromising arterial wall integrity. The aim of this study is to show that, in an elastase-induced murine model of aneurysm formation, TIMP-1 has a protective effect. Methods: 24 wild-type (TIMP-1+/+) and 22 knockout (TIMP-1−/−) mice underwent laparotomy and isolation of the infrarenal aorta. A polyethylene catheter was inserted into the aorta and dilute pancreatic elastase (0.39 Units/ml) was infused over 5 minutes using a perfusion pump. Pre- and postperfusion maximal aortic diameters were obtained in triplicate for each animal using NIH Image. Final aortic measurements were obtained 14 days later, prior to perfusion fixation with 10% buffered-formalin. Aortic specimens were sectioned and stained with Verhoeff-van Giessen and H&E. Statistical analysis was performed using the Student’s t-test; data are presented as mean ± SEM. Results: Mean aortic diameter change was not significantly different between groups immediately following perfusion: 84 ± 4% (TIMP-1+/+) vs. 92 ± 5% (TIMP-1−/−). However, 14 days later, TIMP-1−/− mice showed a two-fold increase in maximal aortic diameter relative to postperfusion measurements, as compared to TIMP-1+/+ mice (108 ± 13% vs. 54 ± 9%, p = 0.001). A significant difference in mean aortic diameter between TIMP-1+/+ and TIMP-1−/− mice was also observed when comparing size change from preperfusion to time of sacrifice (p = 0.001). Histologically, TIMP-1−/− mice had a pronounced inflammatory infiltrate and extensive elastin fragmentation, whereas TIMP-1+/+ mice maintained an intact elastic lamellar structure and absence of inflammatory cells. Conclusions: TIMP-1−/− mice developed significantly larger aneurysms compared to TIMP-1+/+ mice. This study demonstrates the protective effects of TIMP-1 by inhibiting the effects of MMPs in an experimental AAA model and may provide a means for pharmacologically controlling the progressive growth in aneurysms.