Recovery of fibrinogen concentrate after intraosseous application is equivalent to the intravenous route in a porcine model of hemodilution.

BACKGROUND: Fibrinogen concentrate is increasingly considered as a hemostatic agent for trauma patients experiencing bleeding. Placing a venous access is sometimes challenging during severe hemorrhage. Intraosseous access may be considered instead. Studies of intraosseous infusion of coagulation factor concentrates are limited. We investigated in vivo recovery following intraosseous administration of fibrinogen concentrate and compared the results with intravenous administration. METHODS: This study was performed on 12 pigs (mean [SD] body weight, 34.1 [2.8] kg). Following controlled blood loss (35 mL/kg) and fluid replacement with balanced crystalloid solution, intraosseous (n = 6) administration of fibrinogen concentrate (80 mg per kilogram of bodyweight) in the proximal tibia was compared with intravenous (n = 6) administration of the same dose (fibrinogen infusion time approximately 5 minutes in both groups). The following laboratory parameters were assessed: blood cell count, prothrombin time index, activated partial thromboplastin time, and plasma fibrinogen concentration (Clauss assay). Coagulation status was also assessed by thromboelastometry. RESULTS: All tested laboratory parameters were comparable between the intraosseous and intravenous groups at baseline, hemodilution, and 30 minutes after fibrinogen concentrate administration. In vivo recovery of fibrinogen was also similar in the two groups (89% [23%] and 91% [22%], respectively). There were no significant between-group differences in any of the thromboelastometric parameters. Histologic examination indicated no adverse effects on the tissue surrounding the intraosseous administration site. CONCLUSION: This study suggests that intraosseous administration of fibrinogen concentrate results in a recovery of fibrinogen similar to that of intravenous administration. The intraosseous route of fibrinogen concentrate could be a valuable alternative in situations where intravenous access is not feasible or would be time consuming. LEVEL OF EVIDENCE: Prospective, randomized, therapeutic feasibility study in an animal model, level V.

Small intestine submucosa (SIS) implants in experimental IPOM repair.

BACKGROUND: Synthetic meshes can cause adverse effects (e.g., adhesions, mesh infection) in intraperitoneal onlay mesh repair (IPOM). Although data for its biocompatibility as well as degradation behavior is still scarce, small intestine submucosa (SIS) implants have been suggested as a favorable alternative for IPOM repair. The aim of the study was to assess safety and efficacy of SIS used as allo- or xenograft in an experimental model of IPOM repair, with the purpose of creating a critical awareness for specific aspects of the biomesh concept among researchers and surgeons alike. Main outcome parameters were adhesion formation, tissue integration, shrinkage, and dislocation. MATERIALS AND METHODS: Open IPOM repair was performed in 16 Sprague Dawley rats and two minipigs. SIS implants were 2 x 2 cm in rats (one per animal) and 6 x 8 cm in pigs (four per animal). All implants were fixed with six nonresorbable sutures. Observation period was 17 and 28 d (n =8) in rats and 28 d in pigs. Outcome parameters were assessed macroscopically, and histologic samples (H and E staining) were obtained. RESULTS: Upon autopsy, SIS appeared to be only moderately integrated. Dislocation of five SIS implants in the rats and of two SIS implants in the pigs were observed although all sutures were still in place. No seroma formation or infection was detected macroscopically, but substantial shrinkage and adhesion formation at the margins of implants and suture sites were frequently observed. Histology confirmed the macroscopic finding of limited integration and substantial shrinkage. The pathomorphology was similar in both species. CONCLUSIONS: Small intestine submucosa implants are susceptible to shrinkage, dislocation, and adhesion formation in experimental IPOM repair in rats and pigs. These findings are in accordance with literature and warrant further investigations of SIS implants in hernia repair.

Fibrin sealant (Tisseel) for hiatal mesh fixation in an experimental model in pigs.

BACKGROUND: This study was designed to assess the efficacy of the fibrin sealant fixation of titanized polypropylene mesh in experimental hiatal mesh closure in pigs. Prosthetic hiatal closure is recommended for the repair of large hiatal/paraesophageal hernias as well as for antireflux surgery. However, only limited data exist on the favorable choice of meshes and fixation devices. Migration of the implant and trauma to neighboring organs due to perforating devices, such as sutures or tacks, present potentially lethal complications. In this study, we propose the fixation of titanized polypropylene meshes (TS) specifically developed for hiatal closure (TISure; GfE Medizintechnik GmbH, Nuremberg, Germany) with human fibrin sealant (FS, Tisseel; Baxter Biosciences, Vienna, Austria). MATERIALS AND METHODS: A laparotomy was carried out in 7 mini-pigs (27-30 kg bodyweight) under general anaesthesia, and a TS was implanted after precise dissection of the right and left crura and the crural commissure. The key hole of the TS was placed around the esophagus at the gastroesophageal junction. One mL of FS was applied with the Easy Spray system (Baxter Biosciences, Vienna, Austria) for circular and three dimensional mesh fixation onto the diaphragm. Due to the lack of accepted gold standards of hiatal mesh reinforcement, no control group was used. Animals were sacrificed after 4 wk, and meshes were explanted after macroscopical assessment of the correct position and tissue integration. Histology was performed. RESULTS: All meshes showed excellent tissue integration and no signs of migration or dislocation. FS was completely degraded and replaced by well vascularized fibroblastic tissue. CONCLUSIONS: Titanized polypropylene mesh with FS fixation was found to be a safe and efficient combination for reinforcement of the hiatal closure in this preliminary experimental model.

Endotoxin causes functional endoplasmic reticulum failure, possibly mediated by mitochondria.

Inflammatory response has recently been shown to induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which either recovers proper ER function or activates apoptosis. Here we show that endotoxin (lipopolysaccharide = LPS) can lead to functional ER failure tentatively via a mitochondrion-dependent pathway in livers of rats. Histological examination did not reveal significant damage to liver in form of necroses. Electron microscopy displayed transparent rings appearing around morphologically unchanged mitochondria, which were identified as dilated ER. The spliced mRNA variant of X-box protein-1 (XBP1) and also the mRNA of 78 kDa glucose-regulated protein (GRP78) were up-regulated, both typical markers of ER stress. However, GRP78 was down-regulated at the protein level. A pro-apoptotic shift in the bax/bcl-XL mRNA ratio was not accompanied by translocation of apoptosis inducing factor (AIF) to the nucleus, suggesting that the cells entered a pre-apoptotic state, but apoptosis was not executed. Monooxygenase activity of p450, representing the detoxification system in ER, was decreased after administration of endotoxin. Biochemical analysis of proteins important for ER function revealed the impairment of protein folding, transport, and detoxification suggesting functional ER failure. We suggest that functional ER failure may be a reason for organ dysfunction upon excessive inflammatory response mediated by endotoxin.

Fibrin sealant (Tissucol) enhances tissue integration of condensed polytetrafluoroethylene meshes and reduces early adhesion formation in experimental intraabdominal peritoneal onlay mesh repair.

BACKGROUND: The laparoscopic intraabdominal peritoneal onlay mesh repair (IPOM) is a common technique for the reinforcement of multiple ventral hernias or defined defects after laparotomies. However, the placement of synthetic meshes in the intraabdominal cavity can be associated with severe complications. Adhesions frequently originate from the implant and protruding parts of fixation devices, presenting a serious clinical problem with potentially detrimental consequences. This study was designed to assess the impact of fibrin sealing with Tissucol (FS; Baxter, Vienna, Austria) on adhesion formation to condensed polytetrafluoroethylene meshes (Motif Meshes, MM; Proxy Biomedical, Galway, Ireland) as well as on tissue integration of these implants in experimental IPOM repair in rats. It was tested whether FS application allowed the reduction of sutures for mesh fixation without increasing the risk of mesh dislocation. MATERIALS AND METHODS: Sixteen rats were assigned to the implantation of MM with four nonresorbable sutures (Synthofil; Ethicon, Norderstedt, Germany) with additional fibrin coating with 0.2 mL FS or to MM fixation with six nonresorbable sutures without FS (n = 8 per group). MM with 2 cm in diameter were implanted in open IPOM by a laparatomy. The observation period of 17 days ensured assessment of adhesions after the full degradation of FS. Adhesions were rated with the score suggested by Vandendael. Histology was performed. RESULTS: All eight MMs without FS sealing elicited severe (grade III) adhesions, whereas fibrin-sealed MM were rated mild in 1, moderate in 5, and severe in 2 cases. The superior finding in the FS group was statistically significant. Impaired integration of sutured-only MM was observed in four cases, whereas all FS-sealed MM were well integrated. CONCLUSIONS: FS improves the tissue integration, reduces early adhesion formation to cPTFE implants, and allows reduction of perforating fixation devices in experimental IPOM repair.

Adverse effects associated with the use of porcine cross-linked collagen implants in an experimental model of incisional hernia repair.

BACKGROUND: Porcine cross-linked collagen (PermaCol, PCL; TSL, Aldershot, United Kingdom) has been proposed as permanent biomaterial in incisional hernia repair. We evaluated the biocompatibility of PCL in an established animal model. MATERIAL AND METHODS: In 10 Sprague Dawley rats, two hernias per animal were created in the abdominal wall left and right of the linea alba (1.5 cm in diameter), and the peritoneum was spared. The lesions were left untreated for 10 days, until incisional hernias developed. These defects were covered with non-perforated (out-of-the-box, n = 12) or perforated (modified; n = 8) PCL (2 x 2 cm). In a first step, 12 non-perforated implants were tested in a short-term observation period of 17 days. Eight of these non-perforated implants were fibrin sealed (0.3 mL, Tissucol; Baxter, Vienna, Austria), whereas four non-perforated implants were sutured with non-resorbable material. In a second step, perforations were added as modification to PCL to facilitate drainage of fluids, cell ingrowth, and transgression of fibrin sealant. All perforated implants were fibrin sealed and included in a long-term observation period of 3 months. The observation periods allowed the evaluation of the complete degradation of the fibrin sealant fixation after 2 weeks and of the implant integration in a chronic timeframe. Implant sites were analyzed macroscopically and histologically. RESULTS: All PCL samples elicited strong local inflammation with signs of foreign body reaction. Integration of perforated PCL appeared limited after 3 months. Three animals had to be euthanized prior to intended time points because of transcutaneous migration of implants. CONCLUSIONS: In an experimental model of incisional hernia repair, PCL does not integrate well in the abdominal wall and shows poor biocompatibility.

Opposite effects of endotoxin on mitochondrial and endoplasmic reticulum functions.

In this study, we determined functional integrity and reactive oxygen species generation in mitochondria and endoplasmic reticulum in liver of rats subjected to endotoxic shock to clarify whether intracellular reactive oxygen species (ROS) destabilize cellular integrity causing necrosis in rats challenged with lipopolysaccharide (LPS). LPS caused drastically increased plasma levels of alanine aminotransferase, suggesting damage to plasma membranes of liver cells. Liver necrosis was confirmed by histological examination. LPS induced a significant increase in ROS production in rat liver mitochondria (RLM), but did not impair mitochondrial function. In contrast to mitochondria, enzymatic activity and ROS production of cytochrome P450 were lower in microsomal fraction obtained from LPS-treated animals, suggesting the dysfunction of endoplasmic reticulum. Protein patterns obtained from RLM by two-dimensional electrophoresis showed significant upregulation of mitochondrial superoxide dismutase by LPS. We hypothesize that upregulation of this enzyme protects mitochondria against mitochondrial ROS, but does not protect other cellular compartments such as endoplasmic reticulum and plasma membrane causing necrosis.

Different effects of endotoxic shock on the respiratory function of liver and heart mitochondria in rats.

This study was designed to clarify whether mitochondrial function/dysfunction and reactive oxygen species (ROS) production have a temporal relationship with organ failure during endotoxic shock. Adult male Sprague-Dawley rats were divided into three groups receiving 1) isotonic saline (control group, n = 16); 2) 8 mg/kg lipopolysaccharide (LPS; n = 8); or 3) 20 mg/kg LPS (n = 8) intraperitoneally under short anesthesia with 3.5% of isoflurane. After 16 h, animals were killed to analyze plasma, rat liver mitochondria (RLM), and rat heart mitochondria (RHM). In accordance with plasma analysis, LPS-treated rats were divided into "responders" and "nonresponders" with high and low levels of alanine aminotransferase and creatine, respectively. RHM from responders had significantly lower respiratory activity in state 3, suggesting a decreased rate of ATP synthesis. In contrast, RLM from responders had significantly higher respiratory activity in state 3 than both nonresponders and the control group. This increase was accompanied by a decrease in phosphate-to-oxygen ratio values, which was not observed in RHM. ROS generation determined with a spin probe, 1-hydroxy-3-carboxypyrrolidine, neither revealed a difference in RHM between LPS and control groups nor between responders and nonresponders. In contrast, RLM isolated from responders showed a marked increase in ROS production compared with both the control group and nonresponders. Our data demonstrate that 1) RHM and RLM respond to endotoxic shock in a different manner, decreasing and increasing respiratory activity, respectively, and 2) there is a temporal relationship between ROS production in RLM (but not in RHM) and tissue damage in rats subjected to LPS shock.

Increased plasma D-lactate is associated with the severity of hemorrhagic/traumatic shock in rats.

D-lactate is produced by indigenous bacteria in the gastrointestinal tract. Mammals do not have the enzyme systems to metabolize D-lactate rapidly. The present study was designed to determine the kinetics of circulating D-lactate levels and to examine whether the severity of shock affects circulating D-lactate levels in rats subjected to hemorrhagic/traumatic shock. Anesthetized rats underwent midline laparotomy (duration 30 min) and were bled to 30-35 mmHg mean arterial pressure (MAP). After the onset of decompensation, MAP was either increased to 40-45 mmHg immediately by administration of Ringer's solution (moderate shock) or after 40% of shed blood volume had been re-infused as Ringer's solution (severe shock). MAP was then maintained at 40-45 mmHg for 40 min by further administration of Ringer's solution (inadequate resuscitation). Subsequently, adequate resuscitation was performed for 60 min with shed blood and additional Ringer's solution. Metabolic acidosis was significantly more pronounced in severe than in moderate hemorrhagic/traumatic shock. Plasma D-lactate levels were already significantly increased at the end of severe hemorrhagic/traumatic shock and remained high during inadequate resuscitation. D-lactate levels were significantly higher after severe than after moderate shock. Endotoxin levels did not correlate with shock severity. Damage to the intestinal mucosa was more profound in severe shock than in moderate shock. Our data suggest that hemorrhagic/traumatic shock is associated with mucosal damage and increased plasma D-lactate levels. The severity of shock affects D-lactate concentrations in plasma. Plasma D-lactate may be a useful marker of intestinal injury after hemorrhagic/traumatic shock.