Evaluation of the hemostatic effect of a combination of hemostatic agents and fibrin glue in a rabbit venous hemorrhage model.

BACKGROUND: In neurosurgery, it is important to use local hemostatic agents. We have explored a more powerful method of hemostasis by the combination of commercially available hemostatic agents with fibrin glue in the hopes of synergistic effects. METHOD: A bleeding model was constructed by puncturing the rabbit posterior vena cava with a needle. After applying the sample to the bleeding point, compression was performed for 10 s. If temporary hemostasis was achieved after pressure release, a 30 s wash was performed to confirm that ultimate hemostasis was achieved. Up to three hemostasis attempts were performed on the same bleeding point until hemostasis was achieved, and the number of attempts required for hemostasis was counted. If hemostasis was not achieved after three attempts, it was counted as four times. Four groups were evaluated: (1) gelatin sponge alone, (2) gelatin sponge + fibrin glue, (3) oxidized cellulose alone, and (4) oxidized cellulose + fibrin glue; each group was tested 16 times. RESULTS: The median value (range minimum value-maximum value) of the number of hemostatic attempts in Group 1 to Group 4 was 3 (1-4), 1 (1-1), 4 (4-4), and 4 (2-4). In Group 2, there were two test exclusions owing to deviations of the test procedure. CONCLUSIONS: The compatibility of gelatin sponge and fibrin glue was very good, with a very strong and rapid hemostatic effect compared to other methods, showed its usefulness. This combination method may be effective for a variety of venous hemorrhages in neurosurgery.

Potent Hemostatic Efficacy of a Novel Recombinant Fibrin Sealant Patch (KTF-374) in Rabbit Bleeding Models.

PURPOSE: Fibrin sealants are used for hemostasis during surgery. Commercially available fibrin sealants are made of materials of human or animal origin. We developed a novel recombinant fibrin sealant patch (KTF-374) that has thin and flexible properties. This study evaluated the hemostatic efficacy of KTF-374 for various patterns of bleeding in rabbits, as compared with that of the existing fibrin-coated collagen fleece (FCCF). MATERIALS AND METHODS: Test hemostats used were KTF-374 and FCCF. Laparotomy was performed under general anesthesia in rabbits. We created wounds in the liver, caudal vena cava, and ventral aorta under anticoagulating conditions with heparin. Test hemostats were then applied to the wound site and compressed manually for 3 min. Hemostatic efficacy was evaluated with the success rate of hemostasis at 3 min. RESULTS: In all bleeding models, the success rate of hemostasis was significantly higher with KTF-374 than FCCF. The hemostatic success rate of KTF-374 and FCCF was 100% vs. 25% (p = .007) in the partial hepatectomy model (n = 8); 100% vs. 12.5% (p = .001) in the caudal vena cava resection model (n = 8); and 100% vs. 25% (p = .004) in the ventral aortic puncture model (n = 8). The wound site could clearly be recognized through the patch after the application of KTF-374 but not FCCF. CONCLUSIONS: These results suggest that KTF-374 possesses more potent hemostatic properties than FCCF for various patterns of bleeding. KTF-374 is a promising hemostat due to its potent efficacy and good visibility of the wound site through the patch.

Freeze-dried equine-derived redback spider antivenom: a local irritation study by intramuscular injection in rabbits and a repeated-dose toxicity study in rats.

The redback spider (Latrodectus hasseltii) is nonindigenous to Japan but has now spread throughout the country. Bites to humans are rare but can be fatal. We prepared freeze-dried redback spider antivenom for therapeutic use against bites in Japan by immunization of horse plasma. This study included two nonclinical tests of the antivenom: a local irritation study involving a single intramuscular administration to rabbits (with injections of physiological saline and an existing freeze-dried diphtheria antitoxin as control and comparison substances, respectively) and a 2-week repeated intermittent intravenous-dose toxicity study in rats. The irritation study showed the antivenom's irritancy to be comparable with that of the saline and the existing antitoxin preparations under the test conditions. In a repeated-dose toxicity study, no toxicity change was found in male or female rats, and the no-observed-adverse-effect level (NOAEL) was judged to be a dose volume of 20 mL/kg (1082 units/kg antivenom activity) in both male and female rats. In addition, there was no toxicological difference between proteinaceous diphtheria antitoxin and redback spider antivenom prepared to have the same protein content and the same additive composition. Based on these findings, we will further advance our research towards clinical application of the redback spider antivenom. This research was supported by the Research Program on Emerging and Re-emerging Infectious Disease of the Japan Agency for Medical Research and Development.

A novel and effective delivery method for polyglycolic acid sheets to post-endoscopic submucosal dissection ulcers.

Background and aims Shielding methods for post-endoscopic submucosal dissection (ESD) ulcers have delivery-related problems. We developed an enveloped device for this purpose and evaluated its usefulness. Materials and methods Polyglycolic acid (PGA) sheets were delivered to six 3.0-cm ulcers in two resected porcine stomachs and six 5.0-cm ulcers in another three stomachs. In the regular method group, small PGA sheets were delivered via forceps. In the novel method group, a large PGA sheet was delivered via the new device. The methods were compared in terms of time, and macroscopic and histological findings of the ulcer floor. Results The median time required to cover a 3.0-cm ulcer was 0.39 min/cm2 in the novel method group and 1.03 min/cm2 in the regular method group (P = 0.03), and to cover a 5.0-cm ulcer was 0.38 min/cm2 and 0.85 min/cm2, respectively (P = 0.03). In the novel method group, the PGA sheets were in close contact, fully covering the ulcer floor. In the regular method group, the sheets were partly elevated from the ulcer floor. Conclusions This novel technique seems promising in this preliminary study.

A Total Pleural Covering for Lymphangioleiomyomatosis Prevents Pneumothorax Recurrence.

BACKGROUND: Spontaneous pneumothorax is a major and frequently recurrent complication of lymphangioleiomyomatosis (LAM). Despite the customary use of pleurodesis to manage pnenumothorax, the recurrence rate remains high, and accompanying pleural adhesions cause serious bleeding during subsequent lung transplantation. Therefore, we have developed a technique of total pleural covering (TPC) for LAM to wrap the entire visceral pleura with sheets of oxidized regenerated cellulose (ORC) mesh, thereby reinforcing the affected visceral pleura and preventing recurrence. METHODS: Since January 2003, TPC has been applied during video-assisted thoracoscopic surgery for the treatment of LAM. The medical records of LAM patients who had TPC since that time and until August 2014 are reviewed. RESULTS: TPC was performed in 43 LAM patients (54 hemithoraces), 11 of whom required TPC bilaterally. Pneumothorax recurred in 14 hemithoraces (25.9%) from 11 patients (25.6%) after TPC. Kaplan-Meier estimates of recurrence-free hemithorax were 80.8% at 2.5 years, 71.7% at 5 years, 71.7% at 7.5 years, and 61.4% at 9 years. The recurrence-free probability was significantly better when 10 or more sheets of ORC mesh were utilized for TPC (P = 0.0018). TPC significantly reduced the frequency of pneumothorax: 0.544 ± 0.606 episode/month (mean ± SD) before TPC vs. 0.008 ± 0.019 after TPC (P<0.0001). Grade IIIa postoperative complications were found in 13 TPC surgeries (24.1%). CONCLUSIONS: TPC successfully prevented the recurrence of pneumothorax in LAM, was minimally invasive and rarely caused restrictive ventilatory impairment.

Tissue shielding with polyglycolic acid sheets and fibrin glue on ulcers induced by endoscopic submucosal dissection in a porcine model.

BACKGROUND AND STUDY AIMS: The safety and efficacy of the application of polyglycolic acid (PGA) sheets with fibrin glue to ulcers induced by endoscopic submucosal dissection (ESD) have not been established in the treatment of lesions of the gastrointestinal tract, in which the influence of digestive fluid and peristalsis may affect treatment, and there may also be a risk of infection. The aims of this study were to evaluate the healing process of ESD-induced ulcers in animals treated with the application of a PGA sheet with fibrin glue and to verify experimentally the safety of this treatment procedure. MATERIALS AND METHODS: Gastric ESD was performed in nine pigs under general anesthesia. Two ulcer sites were prepared in each pig; one ulcer was treated by applying a PGA sheet with fibrin glue (treated ulcer site), while the other ulcer was left untreated (control ulcer site). Three pigs were euthanized at week 1, three at week 4, and three at week 8 after ESD, and the ulcer sites were macroscopically and histopathologically evaluated. RESULTS: Of the nine treated ulcer sites, seven ulcer sites, to which a PGA sheet had been applied without exposure to the mucosal fluid, showed no peeling of the sheet despite the influence of peristalsis and gastric acid. Histopathologic examination revealed abundant, newly formed blood vessels in the treated ulcers and good granulation. In the treated ulcers, no excessive inflammation, necrosis, or infection was observed. CONCLUSIONS: Our animal study experimentally demonstrated that this treatment technique can be safely applied to ESD-induced ulcers.

[Enhancement of regulatory T cell induction by intravenous S-sulfonated Immunoglobulin during the treatment of experimental autoimmune encephalomyelitis].

Intravenous immunoglobulin (IVIg) has been shown to be effective for a variety of autoimmune diseases. Despite its widespread use and therapeutic success, the precise mechanisms for the anti-inflammatory therapeutic effects of IVIg are not well understood. In particular, few reports have examined the mechanism of IVIg on regulatory T cells (Treg: CD4(+)CD25(+)FoxP3(+) T cells). In the present study, to clarify the effect of intravenous S-sulfonated immunoglobulin (S-IVIg) on Treg, we investigated experimental autoimmune encephalomyelitis (EAE), the representative animal model of autoimmune disease. First, when we evaluated the effect of S-IVIg in an acute EAE model, the prophylactic treatment of S-IVIg dose-dependently controlled the symptoms of EAE. Next, we measured Treg in EAE mice spleen by flow cytometry. The percentage of Treg in S-IVIg-treated mice was significantly increased compared with Saline-treated mice. Finally, in reinduced EAE, S-IVIg not only prevented EAE progression, but also increased the percentage of Treg in the spleen. The increase in percentage of Treg in S-IVIg-treated EAE might be associated with protection against EAE. These observations provide important evidence that IVIg is effective in T-cell-mediated control of autoimmunity.

Improvement of the tissue-adhesive and sealing effect of fibrin sealant using polyglycolic acid felt.

Although fibrin sealant (FS) has an advantage of high biocompatibility, its adhesive force and sealing effect have been generally considered to be inadequate. In the present study, a high adhesive force and sealing effect were obtained by first rubbing fibrinogen solution into the target tissue, attaching polyglycolic acid (PGA) felt to the treated area, and finally spraying it with FS. This method was compared with three conventional FS application methods and a method using fibrin glue-coated collagen fleece. The adhesive force resulting from the present method was 12 times higher than that for the sequential application method, 4.5 times higher than the spray method, 2.5 times higher than the rubbing and spray method, and 2.2 times higher than the use of fibrin glue-coated collagen fleece. The high adhesive force of FS with PGA felt seemed to be due the high fibrin content of the fibrin gel (FG). Light and electron microscopic observations suggested that the formation of FG in closer contact with the muscle fibers was a factor contributing to this superior adhesive force. Comparison of the sealing effect of the present method with other methods using various biomaterials in combination with FS showed that the sealing effect of FS with PGA felt was 1.4 times higher that of polyglactin 910, 1.8 times that of polytetrafluoroethylene, and 6.7 times that of oxidized regenerated cellulose.

Hemostatic efficacy of a recombinant thrombin-coated polyglycolic acid sheet coupled with liquid fibrinogen, evaluated in a canine model of pulmonary arterial hemorrhage.

BACKGROUND: In thoracic surgery, although infrequent, we encounter unexpected damage to the pulmonary artery (PA). In the present study, we evaluated the hemostatic efficacy of a newly developed fibrin-based sheet material, thrombin sheet, coupled with liquid fibrinogen (TSF), in an experimental model of PA hemorrhage. METHODS: Female beagles (n = 8) were used for the study. Left thoracotomy was performed under general anesthesia. PA injury (approximately 4 x 2 mm) was created, and repaired by TSF (TSF group) or TachoComb (TC group). The animals were allowed to survive, and the repaired site was evaluated 4 weeks after the experiment. RESULTS: The number of sheet application and compression procedures required for hemostasis was increased in the TC group compared with in the TSF group (TC vs. TSF, 4 +/- 1 vs. 1 +/- 0.5, p = 0.01, unpaired t test). The time required to achieve hemostasis was increased in the TC group compared with in the TSF group (TC vs. TSF, 7 +/- 3 vs. 1 +/- 0.5 minutes, p = 0.01, unpaired t test). The amount of bleeding during the hemostasis procedure was increased in the TC group compared with in the TSF group (TC vs. TSF, 48 +/- 22 vs. 3 +/- 3 g, p = 0.01, unpaired t test). At 4 weeks, rethoracotomy revealed no apparent indication of delayed bleeding, such as intrathoracic hematoma formation or excessive adhesion formation in the vicinity of PA, in either group. Histologically, the vessel lumen was well sustained in both groups, with no apparent stenosis or thrombus formation. CONCLUSION: The hemostatic efficacy of TSF was superior to TC in this particular experiment. Single application of TSF was sufficient to achieve hemostasis in all but one animal. Compression time of approximately 1 minute was also very short albeit that the bleeding was from the PA and not an artery. These results were presumably because the adhesion was stronger, faster, and the sheet was more pliable in TSF compared with TC.

Fibrin glue and polyglycolic Acid nonwoven fabric as a biocompatible dural substitute.

OBJECTIVE: A novel biocompatible dural substitute created using fibrin glue and polyglycolic acid nonwoven fabric was examined for closing ability and histology. METHODS: A rabbit skin model of dural defect was repaired using fibrin glue-covered polyglycolic acid fabric without suture and subjected to a water leakage test to investigate closing ability. In addition, the dural defects created on 12 hemispheres in 6 beagle dogs were repaired with the dural substitute and subjected to macroscopic and histological examination of the dural substitute and adjacent tissue 1 and 2 months later. RESULTS: The dural substitute showed a breaking pressure of 109.9 +/- 37.1 mmHg. Macroscopically, no cases of excessive granulation, infection, or liquorrhea, either on the dural substitute or surrounding tissue, were observed. Histology indicated favorable tissue replacement of the dural substitute with collagenous fiber, although slight foreign body reaction was associated with its absorption. There was no evidence for adhesion to the brain surface or influence on nerve cells. CONCLUSION: Dural substitute created using fibrin glue and polyglycolic acid fabric is advantageous in that it exerts excellent closing ability without requiring suture and can replace biological tissue without causing incompatibility.

The sealing effect of fibrin glue against alveolar air leakage evaluated up to 48 h; comparison between different methods of application.

OBJECTIVE: There is little experimental evidence to show how much positive airway pressure fibrin sealants can actually withstand, and in particular, how this effect changes over time. In the present study, we experimentally evaluated the sealing effect of fibrin glue against alveolar air leakage up to 48 h after application. METHODS: Beagles were used (n = 48). Under thoracotomy, approximately 5 x 10 mm defects (2 mm depth) were made on the lung surface. Fibrin glue sealants were applied to this defect in three ways. In rubbing and spray method, fibrinogen was rubbed, followed by spraying of both fibrinogen and thrombin solutions. In double layer method, fibrinogen was dripped, followed by thrombin. Collagen fleece, coated with fibrinogen and thrombin (TachoComb) was also tested. The minimum positive airway pressure which produced air leakage was measured for each sealed defect (seal breaking pressure, cmH2O) at 0, 3, 6, 12, 24, and 48 h after application (n = 6 at each time point). RESULTS: The seal-breaking pressure increased over time in all of the application methods. At 6 h, differences between methods were not significant but three defects in RS reached 70 cmH2O, the maximum pressure tested, compared with none in other two methods. At 12h, the seal-breaking pressure was significantly higher in RS compared with the other two methods (rubbing and spray method vs TachoComb; 66+/-3 vs 47+/-17, P = 0.047, rubbing and spray method vs double layer method; 66+/-3 vs 42+/-18, P = 0.024). Beyond 24 h, sealing pressure reached close to 70 cmH2O in all the methods. CONCLUSIONS: The results show that the sealing effect of fibrin glue is relatively unstable up to 12h after its application. Rubbing and spray method may help the fibrin seal to reach its full strength faster compared with the other two methods.

A novel coating biomaterial for intracranial aneurysms: effects and safety in extra- and intracranial carotid artery.

Methyl-2-cyanoacrylate, a widely used material for coating cerebral aneurysm, was recently withdrawn. The aim of the present study was to develop an alternative coating material for cerebral aneurysm, which is safe, effective and stable within the brain. In the first experiment, an aneurysm model of the common carotid artery was produced in a rabbit by the local application of elastase. The aneurysm produced was covered by no material (Group A), a cellulose cotton sheet and conventional methyl-2-cyanoacrylate (Group B), a newly produced polyglycolic acid felt and fibrin glue (Group C), or a cellulose cotton sheet and fibrin glue (Group D). Histological examination showed that the materials resulted in the formation of tight connective tissue around the artery, and that the material was completely replaced by the connective tissue after 12 weeks. This change was found exclusively in Group C, but not in Group A or the other materials, although a temporary thickening of the intima was also observed at the site of the elastase application in Group C. In Group D, a long-term, marked thickening of the intima was observed. In the second experiment, using an intracranial internal carotid artery from a beagle, the applied polyglycolic acid felt and fibrin glue to the intracranial artery induced the formation of connective tissue around the artery that was completely absorbed 16 weeks after surgery. There were no signs of intimal thickening or of adverse reactions in nervous tissue. The present results suggest that polyglycolic acid felt and fibrin glue is a possible candidate for a safe, effective biomaterial to wrap or coat cerebral aneurysm.

Rabbit aneurysm model mediated by the application of elastase.

The concentrations and application methods of elastase in the rabbit aneurysm model were optimized to control the initiation of aneurysms and to cause rupture in a stepwise, controlled fashion. The common carotid artery of male Japanese albino rabbits was exposed. No aneurysm was generated if the adventitia was not dissected. After gentle removal of the adventitia, a two-fold dilution series of elastase was applied to the lesion and observed over a period of 2 hours. Various stages of aneurysmal lesions, from spindle-shaped enlargement to rupture, were produced in proportion to the elastase concentration. Application of elastase stock solution (5 U/mg of type I porcine pancreatic elastase) resulted in rupture within 30 minutes in all six animals. Elastase 1:2 solutions caused oozing in all animals, but subsequent rupture in only three of six animals. Histological examination found serious destruction of the internal elastic lamina and media, with expansion of the very thin wall. Elastase 1:4 to 1:16 solutions caused spindle-like distention of the entire artery and the development of tortuosity at the lesion. Elastase 1:32 or weaker solutions caused only localized dilatations. Overall, the destruction of the tunica media became less severe with decreased elastase concentration. Furthermore, the bursting pressure of the aneurysms decreased with increasing elastase concentrations. In particular, aneurysms produced by the elastase 1:2 solution ruptured at less than 150 mmHg, whereas aneurysms induced by the elastase 1:4 or weaker solutions did not rupture within the physiological range of blood pressure. The present aneurysm model requires shorter preparation time and enables accurate control of aneurysm development and rupture.