Polylactic acid nanosheets in prevention of postoperative intestinal adhesion and their effects on bacterial propagation in an experimental model.

BACKGROUND: Ultrathin films (nanosheets) adhere tightly to organ surfaces but prevent adhesion to other organs. The antiadhesive effect of nanosheets and their effect on bacterial propagation were investigated in a murine intestinal adhesion model. METHODS: Polylactic acid nanosheets (approximately 80 nm thick) were produced. Serosal defects were created by peeling off the intestinal serosa; these were left open or covered with nanosheets or Seprafilm® and the formation of intestinal adhesions was analysed. To examine bacterial propagation, a nanosheet or Seprafilm® was placed on intact murine jejunum followed by Escherichia coli inoculation at the site. RESULTS: Treatment both with nanosheets and with Seprafilm® reduced postoperative intestinal adhesion (mean adhesion score 0·67 for nanosheets, 0·43 for Seprafilm® and 2·87 for no antiadhesive treatment; P < 0·001 for nanosheets or Seprafilm® versus no adhesive treatment). Nanosheet treatment did not affect bacterial propagation in the peritoneal cavity, whereas Seprafilm®-treated mice showed bacterial propagation, leading to increased mortality. CONCLUSION: Nanosheets may be effective novel antiadhesive agents even in the presence of bacterial contamination. Surgical relevance Intra-abdominal adhesions following surgical contamination can trigger postoperative complications and lead to deterioration in long-term quality of life. However, currently there are no effective antiadhesion materials to prevent the formation of adhesions. Treatment with ultrathin nanosheets effectively reduced postoperative intestinal adhesion in an experimental mouse model, and did not affect bacterial propagation in the peritoneal cavity. These nanosheets are potent novel antiadhesive materials that potentially can be applied even in contaminated conditions.

Photodynamic therapy mediates innate immune responses via fibroblast-macrophage interactions.

Antibacterial photodynamic therapy (PDT) has come to attract attention as an alternative therapy for drug-resistant bacteria. Recent reports revealed that antibacterial PDT induces innate immune response and stimulates abundant cytokine secretion as a part of inflammatory responses. However, the underlying mechanism how antibacterial PDT interacts with immune cells responsible for cytokine secretion has not been well outlined. In this study, we aimed to clarify the difference in gene expression and cytokine secretion between combined culture of fibroblasts and macrophages and their independent cultures. SCRC-1008, mouse fibroblast cell line and J774, mouse macrophage-like cell line were co-cultured and PDT treatments with different parameters were carried out. After various incubation periods (1-24 h), cells and culture medium were collected, and mRNA and protein levels for cytokines were measured using real-time PCR and ELISA, respectively. Our results showed that fibroblasts and macrophages interact with each other to mediate the immune response. We propose that fibroblasts initially respond to PDT by expressing Hspa1b, which regulates the NF-κB pathway via Tlr2 and Tlr4. Activation of the NF-κB pathway then results in an enhanced secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß) and neutrophil chemoattractant MIP-2 and KC from macrophages.

Fibrinogen γ-chain peptide-coated, ADP-encapsulated liposomes rescue thrombocytopenic rabbits from non-compressible liver hemorrhage.

BACKGROUND: We developed a fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV [H12])-coated, ADP-encapsulated liposome (H12-[ADP]-liposome) that accumulates at bleeding sites via interaction with activated platelets via glycoprotein IIb-IIIa and augments platelet aggregation by releasing ADP. OBJECTIVE: To evaluate the efficacy of H12-(ADP)-liposomes for treating liver hemorrhage in rabbits with acute thrombocytopenia. METHODS: Thrombocytopenia (platelets < 50 000 µL(-1)) was induced in rabbits by repeated blood withdrawal (100 mL kg(-1) in total) and isovolemic transfusion of autologous washed red blood cells. H12-(ADP)-liposomes with platelet-poor plasma (PPP), platelet-rich plasma (PRP), PPP, ADP liposomes with PPP or H12-(PBS)-liposomes/PPP, were administered to the thrombocytopenic rabbits, and liver hemorrhage was induced by penetrating liver injury. RESULTS: Administration of H12-(ADP)-liposomes and of PRP rescued all thrombocytopenic rabbits from liver hemorrhage as a result of potent hemostasis at the liver bleeding site, although rabbits receiving PPP or ADP liposomes showed 20% survival in the first 24 h. Administration of H12-(ADP)-liposomes and of PRP suppressed both bleeding volume and time from the site of liver injury. H12-(phosphate-buffered saline)-liposomes lacking ADP also improved rabbit survival after liver hemorrhage, although their hemostatic effect was weaker. In rabbits with severe thrombocytopenia (25 000 platelets µL(-1)), the hemostatic effects of H12-(ADP)-liposomes tended to be attenuated as compared with those of PRP treatment. Histologic examination revealed that H12-(ADP)-liposomes accumulated at the bleeding site in the liver. Notably, neither macrothombi nor microthrombi were detected in the lung, kidney or liver in rabbits treated with H12-(ADP)-liposomes. CONCLUSIONS: H12-(ADP)-liposomes appear to be a safe and effective therapeutic tool for acute thrombocytopenic trauma patients with massive bleeding.

Hyperthermia: is it an ominous sign after cardiac arrest?

OBJECTIVE: To clarify the clinical characteristics of hyperthermia at an early stage after resuscitation from cardiac arrest (CA). MATERIALS AND METHODS: We reviewed the medical records of 43 adult patients with non-traumatic out-of-hospital CA, who survived for longer than 24 h after admission to our intensive care unit (ICU) between January, 1995, and December, 1998. The patients were divided into two groups: a clinical brain death (CBD) group (n=23) and a non-CBD group (n=20), and various factors relating to hyperthermia were compared between the two groups. RESULTS: The mean value of peak axillary temperatures within 72 h of admission was 39.8+/-0.9 degrees C for the CBD group, which was significantly greater than 38.3+/-0.6 degrees C for the non-CBD group (P<0.0001). The time of occurrence of the peak axillary temperature was at 19+/-16 h of admission in the CBD group and 20+/-18 h in the non-CBD group (not significantly different). There were no significant differences in risk factors relating to the occurrence of hyperthermia between the two groups, except for the number of patients who received epinephrine at ICU. In 23 patients with a peak axillary temperature of > or =39 degrees C during the first 72 h of hospitalization, brain death was diagnosed in 20 patients, whereas only 3 of 20 patients having a peak axillary temperature of <39 degrees C developed brain death (odds ratio, 37.8; 95% confidence interval, 6.72-212.2). CONCLUSION: Hyperthermia at an early stage after resuscitation from CA may be associated with the outcome of brain death.

Significance of elevated serum interleukin-8 in patients resuscitated after cardiopulmonary arrest.

The objectives of this study were to analyze changes in serum interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) levels in patients that restored spontaneous circulation after cardiopulmonary arrest (CPA), and to clarify the cause and significance of elevated serum cytokines after resuscitation. Twenty-eight patients who were admitted to our hospital after out of hospital CPA were studied. Patients' IL-8 levels and TNF-alpha levels in serum increased to a peak within 12 h and within 6 h after the return of spontaneous circulation (ROSC), respectively. Serum IL-8 levels in patients who died or became brain dead within 1 week after ROSC were significantly higher than those in other patients. In stepwise multiple regression analysis, maximum IL-8 values were significantly correlated with maximum TNF-alpha values within post-ROSC 24 h, with the total dose of administered epinephrine and with peripheral neutrophil counts. It is especially noteworthy that the total dose of epinephrine administered during and after resuscitation markedly influenced the elevation of serum IL-8 after ROSC. The increases in serum IL-8 induced by excessive administration of epinephrine might be harmful in the ROSC-patients resuscitated after CPA.

Analysis of plasma nitrite/nitrate in human thermal injury.

The aim of this study is to examine the characteristics of nitrite/nitrate (NOx), the final metabolite of nitric oxides, in plasma after burn injury. A total of 83 blood samples were collected from 19 patients on arrival, day 1, day 3, and day 5 after suffering burn injuries and from 7 non-burned volunteers. We measured the NOx levels in plasma using the Griess method, and analyzed the relationships among plasma the NOx levels, the burn-magnitude, and the blood examination data using a stepwise multivariate regression analysis. The plasma NOx levels at hospital-arrival after burns significantly exceeded those of non-burned volunteers, and the NOx levels in the plasma returned to normal range after day 1. Based on the findings of a multivariate analysis, the plasma NOx levels at admission to the hospital were not found to be related to the total burn surface area, the burn index or inhalation injury, but they were significantly related to age. Furthermore, these plasma NOx levels were also related to the platelet count, neutrophil count and blood urea nitrogen. The increase in the plasma NOx level may therefore play an important role in the pathophysiology of elderly burned patients, while the nitric oxide levels in the plasma might also play a role in inhibiting the constriction of microvascular smooth muscle in extensively burned patients.

Characteristics of plasma extracellular SOD in burned patients.

The aim of this study was to examine three types of superoxide dismutase (SOD) in plasma after burns, and especially to clarify the characteristics of plasma extracellular SOD (EC-SOD) in burned patients. A total of 71 blood samples were collected from 18 patients on arrival, day 1, day 3 and day 5 after burns. We measured three types of SOD (Mn, Cu/Zn, EC) in plasma using ELISA, and the relationships among the three types of SOD concentrations were examined. We next analyzed the characteristics of plasma EC-SOD using stepwise multivariate regression analysis. Any plasma SOD isoenzyme concentration measured after burns was beyond the normal range and EC-SOD accounted for the major part of plasma SODs. EC-SOD and Cu/Zn-SOD were positively correlated, whereas Mn-SOD was not related to the other SODs. Also, plasma EC-SOD was significantly related to existence of inhalation injury, %TBSA and age, respectively. The plasma EC-SOD might therefore play some roles in the pathophysiology of burned patients.

Changes in the tissue and plasma superoxide dismutase (SOD) levels in a burned rat model.

To examine whether thermal injury alters the superoxide dismutase (SOD) concentrations in various types of tissue or plasma, we studied the plasma and tissue Mn- and Cu/Zn-SOD levels in a rodent burn model. The animals were resuscitated with saline (50 mg/kg, i.p.) immediately following thermal injury and thereafter were sacrificed at either 6 or 24 hours post-burn. The Mn- and Cu/Zn-SOD levels were measured using an enzyme-linked immunosorbent assay (ELISA). The plasma Mn- and Cu/Zn-SOD concentrations significantly increased 6 hours after the injury and positively correlated with the burn size. The kidney Mn-SOD concentrations were significantly higher 24 hours after the injury in the animals with 30% burns than in those with either sham or 50% burn injuries. The lung Cu/Zn-SOD concentrations were also significantly higher 6 hours after the injury in animals with 30% burns than in the other two types above. These findings suggest that the changes in the SOD concentrations after burn injury vary according to the type of SOD and also the type of tissue. As a result, the SOD concentrations may play some role in the early response to thermal trauma.

Is emergency open chest cardiopulmonary resuscitation accepted by patients' families?

Emergency open chest cardiopulmonary resuscitation (OCCPR) is sometimes performed on patients with cardiopulmonary arrest (CPA), especially those resulting from trauma. Since OCCPR is frequently carried out without the permission of patients' families, we surveyed the opinions of the families. A total of 1058 CPA patients were transferred to our department during the last 15 years. We sent questionnaires individually to the families of these patients to ask their opinions about OCCPR. The questionnaire provided the six questions allowing multiple answers; (1) Do you unconditionally agree with OCCPR? (2) Do you agree with OCCPR in children? (3) Do you agree with OCCPR in elderly patients? (4) Do you agree with OCCPR without permission from patient's families? (5) Do you entrust OCCPR to the doctors in charge? and (6) others. The questionnaire reached 846 families, of which 277 (32.7%) responded. The percentage response to each question was (1) 70.2, (2) 5.8, (3) 21.8, (4) 7.1, (5) 4.2 and (6) 5.0%. The younger the age of the responders the more they agreed with OCCPR. All the responders less than 30 years old agreed with the procedure. Of the 277 families, 95 had CPA patients treated with OCCPR. This group of families responded to six questions at the following rates: (1) 79.5, (2) 6.0, (3) 13.3, (4) 2. 4, (5) 4.8 and (6) 4.8%, suggesting that families with OCCPR patients are more cooperative to our treatment than those with non-OCCPR patients. The results of this study suggest that OCCPR in CPA patients is generally accepted by the patients' families, especially by young generations, although post-OCCPR careful explanation to patients' families is still indispensable.

Differential effect of short-term REM sleep deprivation on NGF and BDNF protein levels in the rat brain.

It is well known that REM sleep is associated with memory consolidation, especially, procedural skill learning. Neurotrophic factors are known to be involved in synaptic plasticity. We therefore investigated the effects of selective REM sleep deprivation (RSD) on NGF and BDNF proteins in the hippocampus, cerebellum and brainstem in the rat. NGF and BDNF were detected by an ELISA. Our findings show that 6 h RSD affected the NGF and BDNF protein levels in different manner. In the cerebellum and brainstem, BDNF was significantly decreased, while NGF was not changed. Conversely, in the hippocampus, NGF was significantly decreased while BDNF was not changed. This study indicates that REM sleep may be associated with the secretion of neurotrophic factors and thus contribute to the memory functions.

Significance of shock in head-injured patients with skull fracture.

The clinical differences between patients with skull base and convexity fractures were retrospectively investigated in 324 patients, of whom 110 had suffered head injury resulting in skull fracture. These 110 patients were divided into the skull base and convexity groups. There were no significant differences between the groups with respect to sex, age, Glasgow Coma Scales, injury severity scores, pupil abnormalities, and outcomes. Automobile collisions were the most common causes in the skull base group, and falls in the convexity group. Traumatic Coma Data Bank diffuse 1 type injuries were more frequent in the skull base group and evacuated masses were more frequent in the convexity group. Multiple injuries, shock on admission, lower hemoglobin concentrations, and increased transfusion requirements were evident in the skull base group. Controlling for shock, the outcomes in the skull base group were favorable. Convexity fractures were usually associated with isolated severe head injuries and require brain protection therapy. Skull base fractures were caused by a significant force distributed over a large area of the body with a tendency to induce shock, and require a multidisciplinary approach to treatment.

Expression of CD44 variants and its association with survival in pancreatic cancer.

Since the CD44 variant 6(v6) molecule has been noted as a marker for tumor metastasis and prognosis in several tumors, we examined whether or not v6 is a useful marker for evaluating the prognosis of pancreatic cancer patients. In addition, we attempted to assess the clinicopathological implications for pancreatic cancer of the variant 2 (v2) isoform using a recently developed monoclonal antibody against a v2 epitope. The expression of CD44 variants was evaluated immunohistochemically in paraffin-embedded pancreatic cancer tissues from 42 patients who were confirmed surgically and histologically to have received curative resection. An indirect immunoperoxidase method was used with monoclonal antibodies against epitopes of the standard (CD44s) portion, v6 and v2. Protein expression data were evaluated statistically for any correlations with the length of survival or with histological parameters. The expression of CD44v6 and v2 was observed only in tumor cells, if at all. On the other hand, expression of total CD44 (including CD44v, as well as CD44s) was observed in both tumors and adjacent normal sites. Tumor tissue from 21 (50%) and 16 (38%) patients showed positive immunoreactivity with mAb 2F10 (anti-CD44v6) and mAb M23.6.1 (anti-CD44v2), respectively. The expression of CD44v6 and v2 was correlated with decreased overall survival (P = 0.0160 and P = 0.0125, respectively). A significant correlation was obtained between CD44v2 peptide expression and vessel invasion (P = 0.026). These results suggest that CD44v2 and CD44v6 may be useful markers for poor prognosis in curatively resected primary pancreatic cancer.

Plasma activated clotting time as an indicator of dangerous hypocoagulability in warfarin-treated trauma patients: a preliminary study.

This study was conducted to determine the possibility of detecting dangerous hypocoagulability in trauma victims given warfarin by measuring plasma activated clotting time (ACT). Sensitivity of the plasma ACT to warfarin was tested using lyophilized plasmas and plasma samples from nontraumatized but anticoagulated patients. Lyophilized plasmas were also used to evaluate the effect of defects in the intrinsic coagulation system on the plasma ACT. The plasma ACT, measured using a 4.4-mM calcium solution, showed satisfactory prolongation when the thrombotest of matched samples was within the therapeutic range for warfarin therapy. Conversely, the plasma ACT was not prolonged when the only abnormality of matched samples was mild to moderate prolongation of the activated partial thromboplastin time (APTT). These findings suggest that the plasma ACT could be a reliable indicator of dangerous hypocoagulability in trauma patients receiving warfarin therapy during the immediate postinjury period.

Effects of burns on inhalation injury.

BACKGROUND: There are few studies of smoke injury combined with thermal burn. METHODS: Seven sheep (G1) received smoke injury alone; eight (G2) received a 40% full-thickness scald burn immediately after smoke injury. All animals were resuscitated with lactated Ringer's solution and killed 48 hours after injury. Cardiopulmonary variables and blood gases were measured serially. Ventilation perfusion distribution was analyzed using the multiple inert gas elimination technique. Lung wet to dry weight ratio and malondialdehyde levels were determined. RESULTS: G2 resulted in early significant hemodynamic changes. Serum total protein concentration was significantly lower and malondialdehyde significantly higher in G2. However, PaO2, lung wet to dry weight ratio, and ventilation perfusion mismatching in G2 did not differ from those in G1. CONCLUSIONS: Although the addition of burn injury exaggerated the lung lipid peroxidation and hypoproteinemia in the presence of more pronounced hemodynamic changes, the pulmonary dysfunction was not accentuated.

Norepinephrine stimulates the expression of fibroblast growth factor 2 in rat brown adipocyte primary culture.

To elucidate the role of norepinephrine (NE) in the hyperplasia of brown adipose tissue (BAT), we investigated the effects of NE on the expression of fibroblast growth factor-2 (FGF-2) in rat brown adipocyte primary culture and on capillary growth in an in vitro angiogenesis model in which microvascular fragments and brown adipocyte precursor cells isolated from rat BAT were grown in coculture. NE significantly increased the number of brown adipocyte precursor cells. The NE effect on cell proliferation was greatly inhibited by anti-FGF-2-specific antibody. Likewise,NE considerably increased the levels of FGF-2 mRNA and the antigen in brown adipocyte primary culture. The ability of NE to stimulate the expression of FGF-2 mRNA was blocked by actinomycin D or was inhibited partly by propranolol. Moreover, NE considerably increased the in vitro capillary growth and the level of FGF-2 antigen in the coculture. These results suggest that NE is a crucial factor to mediate FGF-2 production, in part via the beta-adrenergic receptor, in rat brown adipocytes and to stimulate the cell proliferation and capillary growth in BAT by an autocrine/paracrine mechanism.

Insulin stimulates the expression of basic fibroblast growth factor in rat brown adipocyte primary culture.

The effect of insulin on the hyperplasia of brown adipose tissue (BAT) was investigated using the primary culture of rat brown adipocyte precursor cells (RBAC). Results showed insulin to significantly increase the number of RBAC, but not bovine capillary endothelial cells, in the presence of fetal bovine serum. Insulin also increased the expression of basic fibroblast growth factor (bFGF) mRNA and the related protein in the primary culture of RBAC. In addition, insulin enhanced the capillary growth in an in vitro angiogenesis model in which microvascular fragments and RBAC isolated from rat BAT were grown in coculture. The level of bFGF-related protein in the coculture was higher in the presence of insulin than in the absence of insulin. These findings suggest that insulin may play an important role in the proliferation as well as in the differentiation of brown adipocytes, with resulting hyperplasia of BAT (including the formation of new capillaries) through increased production of bFGF in brown adipocytes.

Direct evidence for the occurrence of superoxide radicals in the small intestine of the burned rat.

To determine if superoxide radicals (O2-) and related metabolites are generated in extradermal tissues of burned animals, 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazol [1,2-å]pyrazin-3-one (MCLA) was infused intravenously into rats, and change in the chemiluminescence (CL) intensity of the small intestine was determined by using a sensitive photodetector. When animals were challenged with burn stress of 40% total body surface area (TBSA), the CL intensity of the intestine gradually increased, reaching a maximum within 1 hour and remaining elevated for up to 3 hours. Pretreatment of animals with a long-acting superoxide dismutase (SOD) derivative (SM-SOD) significantly inhibited the increase in CL intensity. Administration of SM-SOD immediately after inducing burn injury also significantly inhibited the increase in CL. These results suggest that superoxide radicals are generated in extradermal tissues, such as the small intestine, in the early stage after burn injury.

The effect of inhaled nitric oxide on pulmonary ventilation-perfusion matching following smoke inhalation injury.

BACKGROUND: We previously reported that inhaled nitric oxide (NO) improved pulmonary function following smoke inhalation. This study evaluates the physiologic mechanism by which inhaled NO improves pulmonary function in an ovine model. METHODS: Forty-eight hours following wood smoke exposure to produce a moderate inhalation injury, 12 animals were anesthetized and mechanically ventilated (FIO2, 0.40; tidal volume, 15 mL/kg; PEEP, 5 cm H2O) for 3 hours. For the first and third hours, each animal was ventilated without NO: for the second hour, all animals were ventilated with 40 ppm NO. Cardiopulmonary variables and blood gases were measured every 30 minutes. The multiple inert gas elimination technique (MIGET) was performed during the latter 30 minutes of each hour. The data were analyzed by ANOVA. RESULTS: Pulmonary arterial hypertension and hypoxemia following smoke inhalation were significantly attenuated by inhaled NO compared with the values without NO (p < 0.05, ANOVA). Smoke inhalation resulted in a significant increase in blood flow distribution to low VA/Q areas (VA/Q < 0.10) with increased VA/Q dispersion. These changes were only partially attenuated by the use of inhaled NO. The SF6 (sulfur hexafluoride) retention ratio was also decreased by inhaled NO. Peak inspiratory pressures and pulmonary resistance values were not affected by inhaled NO. CONCLUSIONS: Inhaled NO moderately improved VA/Q mismatching following smoke inhalation by causing selective pulmonary vasodilation of ventilated areas in the absence of bronchodilation. This modest effect appears to be limited by the severe inflammatory changes that occur as a consequence of smoke exposure.

The pulmonary effect of nitric oxide synthase inhibition following endotoxemia in a swine model.

OBJECTIVE: To evaluate the pulmonary effect of treatment with N-nitro-L-arginine methyl ester (NAME) with and without inhaled nitric oxide (NO) in a swine model of endotoxemia. DESIGN: Randomized controlled trial. SETTING: Laboratory. INTERVENTIONS: Following a 20-minute intravenous infusion of Escherichia coli lipopolysaccharide (LPS) (200 micrograms/kg), animals were resuscitated with saline solution (1 mL/kg per minute) and observed for 3 hours while mechanically ventilated (fraction of inspired oxygen [FIO2], 0.6; tidal volume, 12 mL/kg; positive end-expiratory pressure, 5 cm H2O). Group 1 (LPS, n = 6) received no additional treatment; group 2 (NAME, n = 5) received NAME (3 mg/kg per hour) for the last 2 hours; group 3 (NO, n = 6) received NAME (3 mg/kg per hour) and inhaled NO (40 ppm) for the last 2 hours; and group 4 (control, n = 5) received only saline solution without LPS. MAIN OUTCOME MEASURES: Cardiopulmonary variables and blood gases were measured serially. The multiple inert gas elimination technique was performed at 3 hours. The wet-to-dry lung weight ratio was measured following necropsy. RESULTS: Administration of LPS resulted in pulmonary arterial hypertension, pulmonary edema, and hypoxemia with increased ventilation perfusion ratio mismatching. None of these changes were attenuated by NAME treatment alone but all were significantly improved by the simultaneous administration of inhaled NO. CONCLUSIONS: Systemic NO synthase inhibition failed to restore hypoxic pulmonary vasoconstriction following LPS administration. The deleterious effects of endotoxemia on pulmonary function can be improved by inhaled NO but not by systemic inhibition of NO synthase.