Processed porcine collagen tubulization versus conventional suturing in peripheral nerve reconstruction: an experimental study in rabbits.

In peripheral nerve reconstruction, various procedures are used. One of the procedures that received the most interest in the past decade is the tubulization technique for small nerve gaps. A disadvantage in the use of non-biodegradable tubes is that the material often has to be removed owing to its mechanical properties. Some investigators, in exploring the use of collagen tubes, being a natural biodegradable material, found either allogenicity or xenogenicity and immune responses that may inhibit nerve regeneration. Processed porcine collagen (PPC) is a new inert and biodegradable material that has a favorable effect on wound healing, as demonstrated by experiments on other tissues. The aim of our study was to compare the healing of nerve sutures with PPC tubes with conventional end-to-end sutures. In our experiments, we reconstructed the saphenous nerves of 27 rabbits. In series 1 (n = 12) and 2 (n = 12), PPC tubes were slid over an end-to-end nerve suture without or with a 10-mm nerve gap, respectively. In series 3 (n = 12), conventional suturing was performed in the collateral saphenous nerves of the animals of series 1. Epineurial suturing was performed. Three other non-operated saphenous nerves served as controls. The healing was studied after 3, 6, and 12 months in sections stained by monoclonal antibodies and by conventional histologic staining. Morphometric analysis of the regenerating axons was done by using confocal scanning laser microscopy (CLSM). Data analysis was carried out using a software program especially developed for this purpose. All results were evaluated statistically. Our results showed that during the healing period in the distal nerve stump, the number of axons of the PPC procedure with a 10-mm gap was significantly higher than that in the procedure without a gap. At 12 months, the mean number of axons of all procedures was significantly lower than in the non-operated nerve, and the mean axon diameter in all distal stumps did not differ significantly from that of the non-operated nerve. In the distal nerve stump, the ratio of total axon area to total fascicle area in the PPC procedure with a gap was significantly higher than that in the conventional suturing procedure. After 12 months, there was no significant difference between the percentages of axon outgrowth of the PPC procedure without a gap, the conventional suturing procedure, and the non-operated nerve (100%). The percentage of axon outgrowth in PPC with a gap was significantly higher than in the other procedures.

Silicone rubber tubulization in peripheral sensory nerve reconstruction: an experimental study in rabbits.

Silicone rubber (polydimethyl siloxane) tubes are used clinically in peripheral nerve reconstruction. A disadvantage of this procedure is that the material often has to be removed owing to its mechanical properties. The aim of our study was to investigate the healing of reconstructed sensory nerves tubulized by silicone rubber in an animal model. In our experiments, we reconstructed the saphenous nerves in 27 rabbits. In series 1 (n = 12), silicone rubber tubes were slid over a nerve suture without a gap. In series 2 (n = 12), silicone rubber tubes were slid over a 10-mm nerve gap. In series 3 (n = 12), conventional suturing was performed in the collateral saphenous nerves of the animals of the series 1. Epineurial suturing was performed. Three other collateral nonoperated saphenous nerves served as controls. The healing was studied after 3, 6, and 12 months. Morphometric analysis of the regenerating axons was performed by using our new method for quantification of nerve fibers in cross sections stained by immunohistochemistry and using confocal laser scanning microscopy. Data analysis was carried out using a software program especially developed for this purpose. Our results showed in the silicone procedures that at 12 months significantly fewer axons per fascicle area were present compared with conventional suturing. However, mean axon diameters in the distal nerve stump of the silicone procedures did not differ significantly compared with the conventional suturing procedure. The ratio of total axon area to total fascicle area in the distal nerve stumps of the silicone procedure without gap was significantly smaller compared with the conventionally sutured nerve. The percentage outgrowing axons from the proximal nerve stump into the distal one in the silicone rubber procedure without gap was 57%. This was significantly higher than in the silicone rubber procedure with 10-mm gap (48%). However, in conventional suturing, the percentage of outgrowing axons (99%) was significantly higher than in both tubulization procedures. It appeared that tubulization by silicone rubber of sutured nerves without gap did not enhance axon regeneration. Conventional suturing gave significantly better results. If a gap was present, the use of a silicone rubber tube was preferable to non-suturing.

Method for morphometric analysis of axons in experimental peripheral nerve reconstruction.

A new method for morphometric analysis of axons in experimental peripheral nerve reconstruction is presented. Twelve adult female rabbits were used. In nine animals the saphenous nerve was transected and stitched epineurially. Three animals functioned as control. After 3, 6, and 12 months, the nerves were harvested, fixed in Kryofix and embedded in Histowax. Transverse sections of 6 microm were cut, immunohistochemically stained for NF 90, and counterstained by Sirius Red. Quantification of nerve fibers in cross sections was performed by using a confocal laser scanning microscope (CLSM), and the images were stored digitally. Data analyzing was performed by the Optimas program (5.2). Calculations were done with Microsoft Excel. The total number of axons, the mean axon diameter and the percentage axon area/fascicle area were evaluated statistically. This method for morphologic analysis provides automatically complete registration of axons and so different methods of experimental nerve reconstruction can be compared in a fast and reliable way.

Morphology and function of preserved microvascular arterial grafts: an experimental study in rats.

The aim of this study is to examine the morphology and function and small-caliber, arterial grafts after preservation in the University of Wisconsin solution (UW). Rat carotid arteries were stored in UW (n = 10) or in phosphate-buffered saline (PBS) (n = 10) for 1, 3, 7, and 14 days and were examined with light microscopy (LM) and scanning electron microscopy (SEM). Rat aortic preparations were stored in UW or PBS for 1 hour, 24 hours, 72 hours, 7 days, and 14 days and assessed for functional responses (stimulated contraction and endothelium-dependent relaxation). Segments (5 mm) of rat carotid arteries were stored in UW or PBS for 3 days, 7 days, and 14 days and orthotopically implanted as autografts and allografts. No immunosuppressive or anticoagulant agents were used. After 28 days of implantation, the grafts were assessed for patency and excised for LM and SEM. In UW, the endothelial layer remained intact up to 9 days of storage. In PBS, the endothelial layer showed deterioration after 1 day and was completely lost after 3 days. Functional responses were demonstrated to exist for as long as 7 days storage in UW. In PBS, no responses could be evoked after 24 hours storage. Autografts preserved in UW for 3 days (n = 6), 7 days (n = 6), and 14 days (n = 6) showed patency rates of 83.3%, 66.6%, and 66.6%, respectively, whereas patency rates of allografts were 66.6%, 33.3%, and 33.3%, respectively. Autografts stored in PBS for 3 days (n = 6), 7 days (n = 6), and 14 days (n = 6) showed patency rates of 33.3%, 33.3%, and 50%, respectively, whereas patency rates of allografts were 16.7%, 0%, and 33.3%, respectively. The UW preserved autografts showed normal morphology. All other groups showed vessel wall degeneration which in the allograft groups, were accompanied by lymphocellular infiltration. In conclusion, the endothelial layer and vessel wall of arteries are adequately preserved in UW. Functional responses are retained up to 14 days storage in UW, but, are lost after 24 hours storage in PBS. Autograft implantation studies accordingly show good performance of arterial segments preserved in UW, whereas allografts are subject to degradation as a result of rejection.

Homologous testis transplantation in dogs.

There is growing interest in the possible use of homologous testis transplantation for the treatment of anorchia and male infertility. In order to test the surgical and immunological feasibility of this therapy, three series of experimental studies of homologous testis transplantation were carried out in dogs. In the first pilot study, four beagles from the same litter were transplanted using microsurgical techniques for end-to-end anastomosis of the testicular vessels and the vas deferens. These dogs received cyclosporin A (CyA) for 3 months after transplantation. The longest functional graft survival in this series was 163 days, strongly suggesting that long-term survival of a homologously transplanted testis graft is possible. A second series of operations was performed on ten mongrel dogs. The same surgical technique was employed and the series was divided into three groups. Group 1 received CyA monotherapy, group 2 a combination of CyA and prednisolone, and group 3 received no immunosuppression. The average graft survival time in this series was 28 days, significantly less than the 71 days in the first series. The dogs in group 2, however, had graft survival times that were three times longer than those in the other two groups, suggesting that CyA in combination with prednisolone yields the best graft survival. In the third series, five littermates received a testis graft after castration. Immunosuppression was achieved by administration of CyA and prednisolone for 3 months. In three out of five animals, the graft survived until the immunosuppressive therapy was suspended. Histological biopsies of the graft 3 months after transplantation showed the same maturation of sperm cells as in the control testis of the same dog. The results of the last series suggest that long-term survival of homologously transplanted testis grafts in dogs is, indeed, possible with the aid of CyA and prednisolone.

Experimental arterial allografting under low and therapeutic dosages of cyclosporine for immunosuppression.

The aim of this study was to investigate performance of preserved arterial allografts under the protection of a high-dose and a low-dose immunosuppressive regimen, with cyclosporine (CsA). Dog carotid arteries were harvested and stored for 14 days at 4 degrees C in University of Wisconsin organ preservation solution. Segments (6 cm) of carotid artery were orthotopically and bilaterally implanted in mongrel dogs (n = 18). CsA was given in two dosage regimens: 25 mg/kg/day (group I, n = 7) and 10 mg/kg/day (group II, n = 7). The control group received no CsA (group III, n=4). After 3 months of implantation, patency was assessed by angiography. The grafts were excised for investigation of vessel wall and endothelial function and morphology. For assessment of function in vitro, slices of arterial segments were connected as ring preparations to an isometric force transducer and immersed in a 5 ml organ bath (37 degrees C) containing Tyrode's solution. The contractile response was examined by adding 40 mM KCl and phenylephrine (100 microM) to the organ bath; endothelium-dependent relaxation was examined by adding methacholine (100 microM). Morphology was assessed semiquantitatively. The functional responses to KCl, phenylephrine (Phe) and methacho- line (Met) after 14 days of storage in UW, were 30.2 +/- 1.2 mN, 26.9 +/- 1.0 and 45 +/- 1.2% (means +/- SEM, n=9), respectively. Patency after three months of implantation for group I was 100% (14/14), for group II 50% (7/14), and for group III 75% (6/8). In vitro functional responses of preserved arteries, after 3 months of implantation in group I were 58.5 +/- 10.6 mN (KCl), 36.5 +/- 5.8 mN (Phe), and 57.4 +/- 9.7% (Met), respectively. Functions in group II were 1.2 +/- 0.1 mN (KCl, 0.0 mN (Phe), and 0.0% (Met). Grafts in group III showed no function. Measurement of medial thickness showed significant thinning (P <0.05) in groups II and III. Patency and function of arterial allografts under a therapeutic dose of CsA were superior to grafts implanted under low-dose CsA or no immunosuppressive treatment.

High failure rate of cementless threaded acetabular cups: a radiographic and histologic study in the goat.

We studied the fixation of the Mecron cementless titanium screw cup radiographically and histologically in 20 dwarf-goats after periods of 0, 6, 26 and 52 weeks. In only 3 goats did histology show good bone-implant contact, whereas in the other 17 goats a fibrous membrane interface was seen. This high failure rate is caused by the poor primary fixation and should be a warning against the use of this implant.

Reconstruction of the flexor tendon sheath. An experimental study in rabbits.

The role of the tendon sheath in flexor tendon healing was investigated in rabbits. Tendon sheath was reconstructed with syngeneic parietal peritoneum or a non-tanned processed porcine collagen membrane. Resection of the tendon sheath led to adhesions. Reconstruction of the sheath with either graft resulted in a synovial-like lining, resembling a neo-tendon sheath. Even when combined with tendon repair a neo-tendon sheath was seen after reconstruction with both grafts, without adhesions. Subcutaneously implanted processed porcine collagen membrane was completely resorbed in less than 3 months.

Hyaluronic acid uptake in the assessment of sinusoidal endothelial cell damage after cold storage and normothermic reperfusion of rat livers.

The uptake of hyaluronic acid (HA) was used to assess preservation damage to sinusoidal endothelial cells (SEC) during cold storage and subsequent normothermic reperfusion of rat livers. After 8, 16, 24, and 48 h storage in University of Wisconsin (UW) solution, livers were gravity-flushed via the portal vein with a standard volume of cold UW solution containing 50 micrograms/l HA. The effluent was collected for analysis of HA, aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). The mean uptake of HA at 0 h was 59.1% +/- 4.6% (mean +/- SEM). After 8 h of storage, HA uptake was similar (55.5% +/- 7.3%), whereas after 16 h of storage it was reduced to 34.7% +/- 5.8%. At 24 and 48 h of storage, no uptake of HA was found. In a second series of experiments, livers were stored in UW solution and subsequently reperfused for 90 min with a Krebs-Henseleit solution (37 degrees C) in a recirculating system containing 150 micrograms/l HA. Following 8 h of storage, 34.6% +/- 8.0% of the initial HA concentration was taken up from the perfusate. After 16 and 24 h of storage, no uptake of HA was found. The results of this study indicate that damage to SEC occurs progressively during storage, leading to zero uptake of HA by the rat livers at 24 h of cold ischemia time. Additional reperfusion injury to the SEC was demonstrated by the reduced ability of the SEC to take up HA following normothermic reperfusion. The uptake of exogenous HA in preserved livers, used as a tool to assess SEC injury, enables the detection of early preservation damage.

Morphology and function of dog arterial grafts preserved in UW-solution.

OBJECTIVES: To assess the function of arterial grafts after prolonged preservation in the University of Wisconsin solution (UW), in vitro and in vivo. METHODS: Carotid arteries were harvested from dogs and stored for 1-21 days at 4 degrees C in UW (n = 10) or in PBS (0.9% NaCl, pH 7.4), (PBS) (n = 10). Slices were examined by lightmicroscopy (LM) and scanning electron microscopy (SEM). For viability testing, specimens were connected to an isometric force transducer (2 x n = 9). Contractile and relaxation responses were examined by adding phenylephrine (200 microM) and metacholine (200 microM), respectively. For in vivo studies (n = 41), 2.5cm carotid artery segments were implanted or orthotopically, as autografts and allografts, after 14 days of storage in UW or in PBS. Autologous veins were used as controls. After 28 days or 56 days, arteriography was performed and the grafts were excised for LM and SEM. RESULTS: The arterial endothelial layer remained intact after up to 14 days of storage in UW. In PBS, the endothelium was lost after 3 days. The functional response after 14 days storage in UW was approximately 50% vs. 0% after 14 days in PBS. In the autografts, total patencies (28 days + 56 days) were 100% (8/8) and 63% (5/8) for UW and PBS stored grafts, respectively. In the allografts, the UW and PBS preserved grafts showed total patencies of 86% (12/14) and 83% (5/6), respectively. Microscopically, the allografts showed fibrotic degeneration. CONCLUSIONS: Arteries are well preserved in UW up to 14 days of storage. Arterial autografts preserved in UW showed good patency and better integrity of the vessel wall after implantation, than grafts stored in PBS or allografts (without immunosuppressive therapy).

The influence of anaesthetic drugs on the lower oesophageal sphincter in propofol/nitrous oxide anaesthetized dogs. Pressure profilometry in an animal model.

The lower oesophageal sphincter (LES) is the main barrier against gastro-oesophageal reflux. Various anaesthetic drugs have been reported to affect the LES pressure. In this study, the effects of cisapride, atropine, suxamethonium, vecuronium and pancuronium on the LES pressure of six mongrel dogs anaesthetized with propofol and nitrous oxide were investigated. By means of eight-channel pressure profilometry the LES pressure was measured in consecutive sessions before and after administration of each drug. Compared to basal values, atropine and suxamethonium significantly decreased LES pressure, pressure vector volume and sphincter length. Cisapride significantly increased all sphincter parameters, vecuronium significantly increased LES pressure and pressure vector volume while pancuronium had no significant effects. A significant decrease of the LES pressure and pressure vector volume was observed when nitrous oxide was omitted from the ventilation mixture. Three-dimensional imaging showed an asymmetric shape of the LES pressure which resulted from radial differences of the LES pressure. The results from this study show that both the anaesthetic animal model and the eight-channel pressure profilometry are useful procedures in studying the effects on sphincter function of different drugs during anaesthesia.

Laparoscopy without pneumoperitoneum. Effects of abdominal wall retraction versus carbon dioxide insufflation on hemodynamics and gas exchange in pigs.

Laparoscopic surgery with CO2 insufflation is associated with adverse effects on hemodynamics and gas exchange. The abdominal wall retractor (AWR) is an alternative for pneumoperitoneum. Hemodynamics and gas exchange during the use of an AWR were compared to those of CO2 pneumoperitoneum. In eight pigs subjected to 1 h of CO2 pneumoperitoneum or abdominal wall retraction, hemodynamics, gas exchange, and oxygen transport were studied in a randomized cross-over study design. The only change observed during abdominal wall retraction was mild respiratory alkalosis. In contrast, during CO2 pneumoperitoneum mean arterial blood pressure increased 13%, central filling pressures doubled, and a small increase in cardiac output was observed. Peak airway pressures increased 50%, end-tidal CO2 increased 20%, and respiratory acidosis was induced (arterial pH from 7.46 +/- 0.07 to 7.31 +/- 0.06 and pCO2 from 33 +/- 3 mmHg to 53 +/- 4 mmHg). Arterial PO2 decreased but mixed venous oxygen saturation and oxygen consumption were unaffected. In contrast with CO2 pneumoperitoneum, laparoscopy using abdominal wall retraction was not associated with adverse effects on hemodynamics or gas exchange.

Critical tissue oxygen tension defines tissue oxygen debt in the isolated hindlimb of the pig during progressive ischemia.

OBJECTIVE: To determine a critical skeletal muscle oxygen tension (PO2) value below which a tissue oxygen debt develops. DESIGN: Descriptive study of oxygen transport values in relation to skeletal muscle PO2 throughout progressive ischemia by means of arterial blood flow reductions in an isolated hindlimb model in the pig. SETTING: Surgical Research department of the University of Amsterdam. SUBJECTS: Six female Yorkshire pigs weighing 26 to 35 kg (average 33). INTERVENTIONS: Controlled blood flow to the isolated hindlimb was achieved by means of extracorporeal circulation. The hindlimb was studied during progressive flow reduction. MEASUREMENTS AND MAIN RESULTS: Oxygen delivery (DO2) and oxygen consumption were calculated as the product of extracorporeal blood flow and, respectively, arterial oxygen content and arterial-venous oxygen content difference. In this fashion, an oxygen supply dependency could be determined in all animals. A critical DO2 value was determined below which a tissue oxygen debt developed. Skeletal muscle PO2 was measured continuously, using a Clark-type polarographic oxygen sensor. Combining the critical DO2 value with the corresponding skeletal muscle PO2 value resulted in a critical skeletal muscle PO2 value of 15.2 +/- 0.4 torr (2.0 +/- 0.1 kPa). CONCLUSION: In this pig model, a critical skeletal muscle PO2 value could be determined below which a tissue oxygen debt presumably developed.

Hemodynamics during laparoscopy in the supine or prone position. An experimental study.

During laparoscopy elevations in arterial pressure and a decrease in cardiac output have been reported. Laparoscopic surgery performed in the prone position may be advantageous for some surgical procedures, but the hemodynamic effects of pneumoperitoneum in this position have not been studied. We studied the effects of different levels of increased intraabdominal pressure on hemodynamics and oxygen transport in eight pigs in the prone and the supine position. Increases in intraabdominal pressure did not result in decreased cardiac output or in a reduction of oxygen transport and consumption in either position. These results suggest that laparoscopy in the prone position does not result in more severe hemodynamic depression than laparoscopy in the supine position.

Extraperitoneal laparoscopic paraaortic lymph node sampling in prone position: development of a technique.

Paraaortic lymph node sampling has been found to be efficient in the staging of genitourinary cancers. However, the complications associated with this procedure using the traditional transperitoneal or extraperitoneal approach are considerable. Developments in endoscopic technology and instrumentation have allowed an extraperitoneal approach. Presented is a porcine model for extraperitoneal endoscopic paraaortic lymph node dissection as a staging procedure for genitourinary cancers. The pig is placed in a prone position, and an extraperitoneal pneumoperitoneum is created. Using a three-port technique, we were able to remove almost 95% of all paraaortic lymph nodes laparoscopically without any complications. The prone position allows for a fast and safe procedure because it minimizes the need for extra entry ports and gives a clear view, unobstructed by the bowel, of the back wall of the abdomen.

The prone position. Using gravity for a clear view.

Obtaining a clear view during laparoscopic surgery in the posterior abdominal cavity, on the abdominal backwall, or during colon surgery is time consuming and therefore one of the major objections to these procedures. In an experimental setting we positioned the animals in the prone position using the abdominal flanks to introduce the trocars. Our experience with this position is that a clear and unobstructed view of the abdominal back wall and the large intestines is obtained, facilitating laparoscopic procedures in these areas.

Mechanical and metabolic responses to extracorporeal regional hypoperfusion of the porcine heart.

Controversy exists as to whether hibernating myocardium is ischemic (with evidence of lactate production and ATP breakdown) during sustained coronary hypoperfusion or whether the oxygen supply is balanced by the oxygen requirements of contractile function. To investigate the mechanical and metabolic response to a moderate reduction in regional coronary blood flow, selective coronary perfusion was performed by a carotid-coronary shunt using a small roller pump circuit in six pigs. Flow was reduced for 45 minutes to 40% of base line followed by 2 hours reperfusion at normal blood flow. No hemodynamic changes occurred during flow reduction and reperfusion. Reduction of coronary blood flow to 40% resulted in a reduction in wall motion to 40.8 +/- 6.1% of base line. Two hours of reperfusion resulted in myocardial stunning shown by persistence of wall motion abnormalities (reduction to 64.6 +/- 6.0% of base line) without histologic and electron microscopic evidence of necrosis. The metabolic response to hypoperfusion varied from nil to substantial, measured as nucleotide catabolism and lactate production. We found no correlation between the base line normoxic contractile state and the magnitude of ischemic metabolite efflux. The efflux of lactate, inosine and uridine did not correlate with wall motion at each time during coronary flow reduction. Initial contractile recovery correlated with maximal lactate and uridine efflux during hypoperfusion. The results provide evidence that, in the in-vivo porcine myocardium, moderate coronary hypoperfusion can exist without metabolic evidence of ischemia.

Effects of biliary endoprostheses on the extrahepatic bile ducts in relation to subsequent operation of the biliary tract.

Despite the widespread use of transpapillary biliary endoprostheses, little is known about their effect on the extrahepatic bile ducts. In an experimental study in dogs, we induced inflammatory changes in the bile ducts by stent insertion and studied the reversibility of these changes after stent removal. In addition, the consequences of a period of preoperative stenting for subsequent operation of the biliary tract and the eventual detrimental effects of stenting on the histologic factors of the liver were studied. Twenty-six mongrel dogs were randomly divided into four groups: group 1, stenting during four weeks; group 2, after four weeks stenting, construction of a hepaticojejunostomy; group 3, four days of common bile duct (CBD) ligation, four weeks stenting and hepaticojejunostomy, and group 4, four days of CBD ligation and hepaticojejunostomy. All dogs were sacrificed two months after the last procedure. Hepatic biopsies were obtained during each procedure and bile duct biopsies during hepaticojejunostomy and upon sacrifice. Four weeks of stenting of a normal or obstructed CBD resulted in fibrosed bile ducts, showing severe chronic inflammation with papillary hyperplasia of the epithelium. All bile cultures grew fecal bacteria. Two months after stent removal, inflammation was still present, albeit less severe. Stenting and subsequent surgical treatment resulted in a higher incidence of postoperative complications (54 percent) compared with the control group (14 percent), although this did not reach statistical significance. Hepatic histologic factors were not markedly changed after transpapillary endoprosthesis placement, but after hepaticojejunostomy cholangiolitis was observed. Whenever transpapillary biliary endoprostheses are used, the local effects on the extrahepatic bile ducts and the subsequent bacterial contamination of the bile should be considered.