Safety of the injectable expanding biopolymer foam for non-compressible truncal bleeding in swine.

BACKGROUND: A novel hydrophobically modified chitosan (hm-chitosan) polymer has been previously shown to improve survival in a non-compressible intra-abdominal bleeding model in swine. We performed a 28-day survival study to evaluate the safety of the hm-chitosan polymer in swine. METHODS: Female Yorkshire swine (40-50 kg) were used. A mild, non-compressible, closed-cavity bleeding model was created with splenic transection. The hm-chitosan polymer was applied intra-abdominally through an umbilical nozzle in the same composition and dose previously shown to improve survival. Animals were monitored intraoperatively and followed 28 days postoperatively for survival, signs of pain, and end-organ function. Gross pathological and microscopic evaluations were performed at the conclusion of the experiment. RESULTS: A total of 10 animals were included (hm-chitosan = 8; control = 2). The 2 control animals survived through 28 days, and 7 of the 8 animals from the hm-chitosan group survived without any adverse events. One animal from the hm-chitosan group required early termination of the study for signs of pain, and superficial colonic ulcers were found on autopsy. Laboratory tests showed no signs of end-organ dysfunction after exposure to hm-chitosan after 28 days. On gross pathological examination, small (<0.5 cm) peritoneal nodules were noticed in the hm-chitosan group, which were consistent with giant-cell foreign body reaction in microscopy, presumably related to polymer remnants. Microscopically, no signs of systemic polymer embolization or thrombosis were noticed. CONCLUSION: Prolonged intraperitoneal exposure to the hm-chitosan polymer was tolerated without any adverse event in the majority of animals. In the single animal that required early termination, the material did not appear to be associated with end-organ dysfunction in swine. Superficial colonic ulcers that would require surgical repair were identified in 1 out of 8 animals exposed to hm-chitosan.

Chitosan-based lifefoam improves survival in lethal noncompressible abdominal bleeding in swine.

BACKGROUND: In military combat settings, noncompressible closed cavity exsanguination is the leading cause of potentially survivable deaths, with no effective treatment available at point of injury. The aim of this study was to assess whether an expanding foam based on hydrophobically modified chitosan (hm-chitosan) may be used as a locally injectable hemostatic agent for the treatment of noncompressible bleeding in a swine model. METHODS: A closed-cavity, grade V hepato-portal injury was created in all animals resulting in massive noncoagulopathic, noncompressible bleeding. Animals received either fluid resuscitation alone (control, n = 8) or fluid resuscitation plus intraperitoneal hm-chitosan agent through an umbilical port (experimental, n = 18). The experiment was terminated at 180 minutes or death (defined as end-tidal CO2 <8mmHg or mean arterial pressure [MAP] <15mmHg), whichever came first. RESULTS: All animals had profound hypotension and experienced a near-arrest from hypovolemic shock (mean MAP = 24 mmHg at 10 minutes). Mean survival time was higher than 150 minutes in the experimental arm versus 27 minutes in the control arm (P < .001). Three-hour survival was 72% in the experimental group and 0% in the control group (P = .002). Hm-chitosan stabilized rising lactate, preventing acute lethal acidosis. MAP improved drastically after deployment of the hm-chitosan and was preserved at 60 mmHg throughout the 3 hours. Postmortem examination was performed in all animals and the hepatoportal injuries were anatomically similar. CONCLUSION: Intraperitoneal administration of hm-chitosan-based foam for massive, noncompressible abdominal bleeding improves survival in a lethal, closed-cavity swine model. Chronic safety and toxicity studies are required.

Heparin reduces overcirculation-induced pulmonary artery remodeling through p38 MAPK in piglet.

BACKGROUND: Artery remodeling is the principal change of pulmonary artery hypertension. Heparin has been shown to inhibit vascular smooth muscle cell proliferation. We hypothesized that heparin may modulate vascular remodeling in pulmonary artery hypertension, and explored the mechanism. METHODS: A localized overcirculation-induced artery remodeling was created in piglets by anastomosing the left lower lobe pulmonary artery (LLLPA) to the thoracic aortic artery. Piglets were treated with heparin or saline for 4 weeks. Hemodynamic data were collected, and histology of the lung was assessed. We investigated the expressions of several candidate genes in lung and further observed the involvement of P38 mitogen-activated protein kinases (MAPK). The effects of heparin on the growth of cultured pulmonary arterial vascular smooth muscle cell and P38 MAPK expression were further determined under various conditions. RESULTS: Four weeks after the shunt setup, overcirculation caused significant LLLPA remodeling, pressure increase, and pulmonary vascular resistance increase, and LLLPA flow reduction compared with that immediately after the shunt setup. Heparin reduced the LLLPA remodeling, pressure, and pulmonary vascular resistance, and increased the LLLPA flow compared with that not heparin treated. Shunt and heparin treatment did not change the piglet's systemic hemodynamics. Shunt increased the expression of P38 MAPK and heparin decreased its expression in the shunted LLLPA. Both heparin and P38 MAPK inhibitor suppressed VSMC growth and P38 MAPK expression in the cultured VSMC, but they did not present additive effects when the two treatments were combined. CONCLUSIONS: Heparin reduces overcirculation-induced pulmonary artery remodeling through a P38 MAPK-dependent pathway.

Efficacy of a prehospital self-expanding polyurethane foam for noncompressible hemorrhage under extreme operational conditions.

BACKGROUND: Noncompressible abdominal hemorrhage is a significant cause of battlefield and civilian mortality. We developed a self-expanding polyurethane foam intended to provide temporary hemorrhage control and enable evacuation to a definitive surgical capability, for casualties who would otherwise die. We hypothesized that foam treatment would be efficacious over a wide range of out-of-hospital operational conditions. METHODS: The foam was tested in an established lethal, closed-cavity hepatoportal injury model in four groups as follows. Group 1 involved baseline conditions, wherein foam was deployed from a pneumatically driven, first-generation delivery device at room temperature (n = 6). Group 2 involved foam deployment from a field-relevant, handheld delivery prototype (n = 12). Group 3 involved foam components that were conditioned to simulate 1-year shelf-life (n = 6). Group 4 involved foam that was conditioned to a range of temperatures (10 °C and 50 °C; n = 6 per group). In all studies, survival was monitored for up to 180 minutes and compared with an ongoing and accumulating control group with no intervention (n = 14). RESULTS: In Group 1 with a first-generation delivery system, foam treatment resulted in a significant survival advantage relative to the control group (p < 0.001), confirming previous results. In Group 2 with a handheld delivery system, survival was also improved, 83% at 3 hours, compared with 7% in the control group (p < 0.001). In Group 3, survival was 83% at 3 hours (p = 0.002). In Group 4 at temperature extremes, 3-hour survival was 83% (p = 0.002) and 67% (p = 0.014) in the low- and high-temperature groups, respectively. Temperature extremes did not result in hypothermia, hyperthermia, or thermal injury. In all studies, the bleeding rate in foam groups was significantly lower than in the control group (p < 0.05). CONCLUSION: Under a range of military operational conditions, foam treatment resulted in a survival advantage relative to the control group. This supports the feasibility of foam treatment as a prehospital hemostatic bridge to surgery for severely bleeding causalities.

Diagnosis and deployment of a self-expanding foam for abdominal exsanguination: Translational questions for human use.

BACKGROUND: We have previously described the hemostatic efficacy of a self-expanding polyurethane foam in lethal venous and arterial hemorrhage models. A number of critical translational questions remain, including prehospital diagnosis of hemorrhage, use with diaphragmatic injury, effects on spontaneous respiration, the role of omentum, and presence of a laparotomy on foam properties. METHODS: In Experiment 1, diagnostic blood aspiration was attempted through a Veress needle before foam deployment during exsanguination (n = 53). In Experiment 2: a lethal hepatoportal injury/diaphragmatic laceration was created followed by foam (n = 6) or resuscitation (n = 10). In Experiment 3, the foam was deployed in naïve, spontaneously breathing animals (n = 7), and respiration was monitored. In Experiments 4 and 5, the foam was deployed above (n = 6) and below the omentum (n = 6) and in naïve animals (n = 6). Intra-abdominal pressure and organ contact were assessed. RESULTS: In Experiment 1, blood was successfully aspirated from a Veress needle in 70% of lethal iliac artery injuries and 100% of lethal hepatoportal injuries. In Experiment 2, in the presence of a diaphragm injury, between 0 cc and 110 cc of foam was found within the pleural space. Foam treatment resulted in a survival benefit relative to the control group at 1 hour (p = 0.03). In Experiment 3, hypercarbia was observed: mean (SD) Pco2 was 48 (9.4) mm Hg at baseline and 65 (14) mm Hg at 60 minutes. In Experiment 4, abdominal omentum seemed to influence organ contact and transport in two foam deployments. In Experiment 5, there was no difference in intra-abdominal pressure following foam deployment in the absence of a midline laparotomy. CONCLUSION: In a series of large animal studies, we addressed key translational issues surrounding safe use of foam treatment. These additional data, from diagnosis to deployment, will guide human experiences with foam treatment for massive abdominal exsanguination where no other treatments are available.

Self-expanding foam for prehospital treatment of intra-abdominal hemorrhage: 28-day survival and safety.

BACKGROUND: Intracavitary noncompressible hemorrhage remains a significant cause of preventable death on the battlefield and in the homeland. We previously demonstrated the hemostatic efficacy of an in situ self-expanding poly(urea)urethane foam in a severe, closed-cavity, hepatoportal exsanguination model in swine. We hypothesized that treatment with, and subsequent explantation of, foam would not adversely impact 28-day survival in swine. METHODS: Following a closed-cavity splenic transection, animals received either fluid resuscitation alone (control group, n = 6) or resuscitation plus foam treatment at doses of 100 mL (n = 6), 120 mL (n = 6), and 150 mL (n = 2). Foam was allowed to polymerize in situ and was explanted after 3 hours. The animals were recovered and monitored for 28 days. RESULTS: All 18 animals in the 100-mL, 120-mL, and control groups survived to the 28-day endpoint without complications. The 150-mL group was terminated after the acute phase (n = 2). En bloc explantation of the foam took less than 2 minutes and was associated with millimeter-sized remnant particles. All foam animals required some level of enteric repair (imbrication or resection). Excluding the aborted 150-mL group, all animals survived, with no differences in renal or hepatic function, serum chemistries, or semiquantitative abdominal adhesion scores. Histologic analysis demonstrated that remnant particles were associated with a fibrotic capsule and mild inflammation, similar to that of standard suture reaction. In addition, safety testing (including genotoxicity, pyrogenicity, and cytotoxicity) was performed consistent with the ISO-10993 standard, and the materials passed all tests. CONCLUSION: For a distinct dose range, 28-day recovery after foam treatment and explantation for noncompressible, intra-abdominal hemorrhage is not associated with significant physiologic or biochemical evidence of end-organ dysfunction. A foam volume exceeding the maximum tolerable dose was identified. Bowel repair is required to ensure survival.

Self-expanding foam improves survival following a lethal, exsanguinating iliac artery injury.

BACKGROUND: Noncompressible abdominal bleeding is a significant cause of preventable death on the battlefield and in the civilian setting, with no effective therapies available at point of injury. We previously reported that a self-expanding polyurethane foam significantly improved survival in a lethal hepatoportal injury model of massive venous hemorrhage. In this study, we hypothesized that foam treatment could improve survival in a lethal iliac artery injury model in noncoagulopathic swine. METHODS: In swine with a closed abdomen, an iliac artery transection was created, resulting in massive noncompressible exsanguination. After injury, animals were treated with damage-control fluid resuscitation alone (n = 14) or foam treatment in addition to fluids. Two doses of foam treatment were studied: 100 mL (n = 12) and 120 mL (n = 13); all animals were monitored for 3 hours or until death. RESULTS: Foam treatment at both doses resulted in a significant survival benefit and reduction in hemorrhage rate relative to the control group. Median survival time was 135 minutes and 175 minutes for the 120-mL and 100-mL doses, compared with 32 minutes in the control group (p < 0.001 for both groups). Foam resulted in an immediate, persistent improvement in mean arterial pressure and a transient increase in intra-abdominal pressure. The median hemorrhage rate was 0.27 g/kg per minute in the 120-mL group and 0.23 g/kg per minute in the 100-mL group, compared with 1.4 g/kg per minute in the control group (p = 0.003 and 0.006, respectively, as compared with the control). CONCLUSION: Self-expanding foam treatment significantly improves survival in an otherwise lethal, noncompressible, massive, arterial injury. This treatment may provide a prehospital intervention for control of noncompressible abdominal hemorrhage.

Self-expanding foam for prehospital treatment of severe intra-abdominal hemorrhage: dose finding study.

BACKGROUND: Noncompressible abdominal bleeding is a significant cause of preventable death on the battlefield and in the civilian trauma environment, with no effective therapies available at point of injury. We previously described the development of a percutaneously administered, self-expanding, poly(urea)urethane foam that improved survival in a lethal Grade V hepatic and portal vein injury model in swine. In this study, we hypothesized that survival with foam treatment is dose dependent. METHODS: A high-grade hepatoportal injury was created in a closed abdominal cavity, resulting in massive noncompressible hemorrhage. After injury, the animals were divided into five groups. The control group (n = 12) was treated only with fluid resuscitation, and four polymer groups received different dose volumes (Group 1, n = 6, 64 mL; Group 2, n = 6, 85 mL; Group 3, n = 18, 100 mL; and Group 4, n = 10, 120 mL) in addition to fluids. Ten minutes after injury, the foam was percutaneously administered, and animals were monitored for 3 hours. RESULTS: Survival with hepatoportal injury was highest in Group 4 (90%) and decreased in a dose-dependent fashion (Group 3, 72%; Group 2, 33%; Group 1, 17%). All polymer groups survived significantly longer than the controls (8.3%). Hemorrhage rate was reduced in all groups but lowest in Group 4 versus the control group (0.34 [0.052] vs. 3.0 [1.3] mL/kg/min, p < 0.001). Increasing foam dose volume was associated with increased peak intra-abdominal pressure (88.2 [38.9] in Group 4 vs. 9.5 [3.2] in the controls, p < 0.0001) and increased incidence of focal bowel injuries. CONCLUSION: The self-expanding foam significantly improves survival in a dose-dependent fashion in an otherwise lethal injury. Higher doses are associated with better survival but resulted in the need for bowel resection.

Self-expanding polyurethane polymer improves survival in a model of noncompressible massive abdominal hemorrhage.

BACKGROUND: Intracavitary noncompressible hemorrhage remains a significant cause of preventable death on the battlefield. Two dynamically mixed and percutaneously injected liquids were engineered to create an in situ self-expanding polymer foam to facilitate hemostasis in massive bleeding. We hypothesized that intraperitoneal injection of the polymer could achieve conformal contact with sites of injury and improve survival in swine with lethal hepatoportal injury. METHODS: High grade hepatoportal injury was created in a closed abdominal cavity, resulting in massive noncoagulopathic, noncompressible hemorrhage. Animals received either standard battlefield fluid resuscitation (control, n = 12) or fluid resuscitation plus intraperitoneal injection of hemostatic foam (polymer, n = 15) and were monitored for 3 hours. Blood loss was quantified, and all hepatoportal injuries were inspected for consistency. RESULTS: Before intervention, all animals initially experienced severe, profound hypotension and near-arrest (mean arterial pressure at 10 minutes, 21 [5.3] mm Hg). Overall survival at 3 hours was 73% in the polymer group and 8% in the control group (p = 0.001). Median survival time was more than 150 minutes in the polymer group versus 23 minutes (19-41.5 minutes) in the control group (p < 0.001), and normalized blood loss in the polymer group was 0.47 (0.30) g/kg per minute versus 3.0 (1.3) g/kg per minute in the controls (p = < 0.001). All hepatoportal injuries were anatomically similar, and the polymer had conformal contact with injured tissues. CONCLUSION: Intraperitoneal polymer injection during massive noncompressible hemorrhage reduces blood loss and improves survival in a lethal, closed-cavity, hepatoportal injury model. Chronic safety and additional efficacy studies in other models are needed.

Low molecular weight (LMW) heparin inhibits injury-induced femoral artery remodeling in mouse via upregulating CD44 expression.

OBJECTIVE: The mechanism of postangioplasty restenosis remains poorly understood. Low molecular weight (LMW) heparin has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), which is the principal characteristic of restenosis. Studies have shown that LMW heparin could bind to CD44. We hypothesized that LMW heparin might modulate CD44 expression thereby decreasing vascular remodeling. METHODS: Vascular remodeling was induced in CD44(+/+) and CD44(-/-) mice and treated with LMW heparin. The arteries were harvested for histologic assessment and determination of CD44 expression. Bone marrow transplantation was introduced to further explore the role and functional sites of CD44. Effects of LMW heparin on growth capacity, CD44 expression were further studied using the cultured mouse VSMCs. RESULTS: Transluminal injury induced remarkable remodeling in mouse femoral artery (sham wall thickness percentage [WT%]: 3.4 ± 1.2% vs injury WT%: 31.8 ± 4.7%; P < .001). LMW heparin reduced the remodeling significantly (WT%: 17.8 ± 3.5%, P < .005). CD44(-/-) mice demonstrated considerably thicker arterial wall remodeling (WT%: 46.2 ± 7.6%, P = .0035), and CD44-chimeric mice exhibited equal contributions of the local and circulating CD44 signal to the neointima formation. LMW heparin markedly upregulated CD44 expression in the injured femoral arteries. In vitro, LMW heparin decreased mouse VSMC growth capacity and upregulated its CD44 expression simultaneously in a dose-dependent and time-dependent manner, which could be partially blocked by CD44 inhibitor. CONCLUSIONS: LMW heparin inhibits injury-induced femoral artery remodeling, at least partially, by upregulating CD44 expression.

Inhibition of HA synthase 3 mRNA expression, with a phosphodiesterase 3 inhibitor, blocks lung injury in a septic ventilated rat model.

Low-molecular-weight hyaluronan produced by hyaluronan synthase 3 (HAS3) has been shown to play a role in acute lung injury secondary to high-tidal-volume ventilation. Phosphodiesterase 3 inhibitors have been shown to decrease HAS3 expression. We hypothesized that low-molecular-weight hyaluronan (LMW HA) produced by HAS3 mediates LPS-induced lung injury in the mechanically ventilated rat and that milrinone (MIL), by blocking HAS3 mRNA expression, would prevent the injury. Rats were randomized to four groups: controls with mechanical ventilation at 7 cc/kg MV, MV+LPS, MV+MIL, and MV+LPS+MIL. Rats were intubated and ventilated without PEEP for 4 h. Lipopolysaccharide (LPS) (1 mg/kg) was infused into the arterial line 1 h prior to MV. MIL 10 microg/kg/min (or an equivalent volume of saline) was infused through the venous line at the beginning of MV. Bronchoalveolar lavage fluid (BAL) was collected after 4 h of ventilation and lungs were saved for histopathology. LPS significantly increased neutrophil infiltration and protein concentration in the BAL and augmented lung injury score on histology. MIL significantly lowered alveolar protein and neutrophil infiltration as well as lung injury in response to LPS. Furthermore, MIL decreased the mRNA expression for HAS3 and MIP2 in lung tissue and decreased the protein content in BAL. MIL, a commonly used inotropic agent, inhibited LPS-induced lung inflammation and lung injury in mechanically ventilated rats. The anti-inflammatory properties of MIL may be mediated by inhibition of HAS3 and/or MIP2 and could be beneficial in the treatment of sepsis.

The role of phosphodiesterase 3 in endotoxin-induced acute kidney injury.

BACKGROUND: Acute kidney injury frequently accompanies sepsis. Endotoxin is known to reduce tissue levels of cAMP and low levels of cAMP have been associated with renal injury. We, therefore, hypothesized that endotoxin induced renal injury by activating phosphodiesterase 3 (PDE3) which metabolizes cAMP and that amrinone an inhibitor of PDE3 would prevent the renal injury. METHODS: Animals were divided into three groups (n = 7/group): 1) Control (0.9% NaCl infusion without LPS); 2) LPS (0.9% NaCl infusion with LPS); 3) Amrinone+LPS (Amrinone infusion with LPS). Either lipopolysaccharide (LPS) or vehicle was injected via the jugular vein and the rats followed for 3 hours. We explored the expression of PDE3 isoenzymes and the concentrations of cAMP in the tissue. RESULTS: The PDE3B gene but not PDE3A was upregulated in the kidney of LPS group. Immunohistochemistry also showed that PDE3B was expressed in the distal tubule in the controls and LPS caused PDE3B expression in the proximal as well. However, PDE3A was not expressed in the kidney either in the control or LPS treated groups. Tissue level of cAMP was decreased after LPS and was associated with an increase in blood urea nitrogen, creatinine, ultrastructural proximal tubular changes, and expression of inducible nitric oxide synthase (iNOS) in the endotoxemic kidney. In septic animals the phosphodiesterase 3 inhibitor, amrinone, preserved the tissue cAMP level, renal structural changes, and attenuated the increased blood urea nitrogen, creatinine, and iNOS expression in the kidney. CONCLUSION: These findings suggest a significant role for PDE3B as an important mediator of LPS-induced acute kidney injury.