John E. Kalns

Thermal injury resulting from application of a granular mineral hemostatic agent.

BACKGROUND Uncontrolled hemorrhage accounts for the majority of deaths in combat. Effective topical hemostatic agents suitable for use on the battlefield may be valuable in controlling hemorrhage until definitive surgical intervention is possible. In an effort to identify a hemostatic agent suitable for battlefield use, we evaluated several potential hemostatic agents in a swine injury model and noted thermal injury to tissues with a granular mineral hemostatic agent (QuikClot). METHODS Anesthetized swine were maintained with a mean arterial pressure in excess of 60 mm Hg. Cutaneous, muscular, hepatic, splenic, venous, and arterial wounds were created in a standardized fashion. Topical hemostatic agents were immediately applied to the wounds and the amount of bleeding and time to hemostasis were noted. RESULTS The results reported here are part of a larger study in which a variety of hemostatic agents were evaluated. Only the findings related to the granular mineral hemostatic agent are discussed here. Application of the agent resulted in elevated tissue surface temperatures in excess of 95 degrees C and internal tissue temperatures exceeding 50 degrees C, 3 mm deep to the bleeding surface. Necrosis of fat and muscle were noted as well as full and partial thickness cutaneous burns. CONCLUSIONS Topical administration of a granular mineral hemostatic agent to a variety of wounds in an experimental swine model resulted in thermal tissue injury and necrosis. Suggestions for reducing the extent of injury with this product are offered.

Hyperbaric oxygen exposure temporarily reduces Mac-1 mediated functions of human neutrophils.

Highly elevated partial pressures of oxygen achievable during hyperbaric oxygenation (HBO) have been shown to reduce leukocyte sequestration following ischemia/reperfusion injury suggesting a clinical role for HBO in treatment of various disease states characterized by transient ischemia. Previous studies have suggested that this effect may be due to inhibition of beta2-integrin function. In this study the effect of HBO on various CD11b/CD18 (Mac-1) mediated neutrophil functions was investigated in healthy human subjects. HBO 3.0 ATA, 23 m reduced adhesion 50% at 2 h with return to pre-HBO levels by 6 h. Homotypic aggregation, a Mac-1 dependent function, under fluid shear following stimulation with f-MLP was reduced from 20+/-2.6 to 3.4+/-1.0% 2 h after HBO. However, HBO did not inhibit adhesion to IL-1beta stimulated HUVEC. Mac-1 mediated oxidative burst induced by opsonized zymosan was reduced 38.2+/-10.6% (P<0.05) by HBO. However, oxidative burst induced by PMA or f-MLP was not affected. HBO did not alter the distribution of neutrophils displaying morphologies associated with stimulation (ruffled, bipolar, uropod) over a 24 h period after HBO nor did HBO change the percentages of mature versus immature cells. Taken together these findings demonstrate that HBO specifically inhibits Mac-1 mediated functions.

Oxidative stress precedes circulatory failure induced by 35-GHz microwave heating.

Sustained whole-body exposure of anesthetized rats to 35-GHz radio frequency radiation produces localized hyperthermia and hypotension, leading to circulatory failure and death. The physiological mechanism underlying the induction of circulatory failure by 35-GHz microwave (MW) heating is currently unknown. We hypothesized that oxidative stress may play a role in the pathophysiology of MW-induced circulatory failure and examined this question by probing organs for 3-nitrotyrosine (3-NT), a marker of oxidative stress. Animals exposed to low durations of MW that increased colonic temperature but were insufficient to produce hypotension showed a 5- to 12-fold increase in 3-NT accumulation in lung, liver, and plasma proteins relative to the levels observed in control rats that were not exposed to MW. 3-NT accumulation in rats exposed to MW of sufficient duration to induce circulatory shock returned to low, baseline levels. Leukocytes obtained from peripheral blood showed significant accumulation of 3-NT only at exposure levels associated with circulatory shock. 3-NT was also found in the villus tips and vasculature of intestine and within the distal tubule of the kidney but not in the irradiated skin of rats with MW-induced circulatory failure. The relationship between accumulation in liver, lung, and plasma proteins and exposure duration suggests either that nitro adducts are formed in the first 20 min of exposure and are then cleared or that synthesis of nitro adducts decreases after the first 20 min of exposure. Taken together, these findings suggest that oxidative stress occurs in many organs during MW heating. Because nitration occurs after microwave exposures that are not associated with circulatory collapse, systemic oxidative stress, as evidenced by tissue accumulation of 3-NT, is not correlated with circulatory failure in this model of shock.

EXPOSURE TO HYPERBARIC OXYGEN INDUCES CELL CYCLE PERTURBATION IN PROSTATE CANCER CELLS

SummaryCell cycle synchronization of tumor cells by exposure to hyperbaric oxygenation (HBO) may increase the efficacy of chemotherapy or radiation by placing cells into a chemosensitive portion of the cycle. The purpose of the current study was to examine oxygen pressure-dependent relationships with respect to the cell cycle in prostate tumor cells in vitro. LNCaP cells were grown in an incubator at 21% O2 and then exposed to 100% oxygen at pressures up to 6 atmospheres (atm) for 1.5 h. Cells were then returned to the incubator and evaluated for DNA content by propidium iodide and new DNA synthesis with a pulse-chase experiment. Cell cycle effects were evaluated by flow cytometry. Exposure to HBO increased the percentage of cells synthesizing new DNA in a dose-dependent fashion: 0 atm, 44%; 6 atm, 65%. Cells that synthesize new DNA accumulate in G2/M as a function of partial pressure of oxygen. These results suggest that HBO induces cells to enter the cell cycle and accumulate in G2/M. Cell cycle synchronization and entry of senescent cells into the cell cycle suggest that HBO may be a useful adjuvant to chemotherapy or radiation in the treatment of prostate cancer. There are two potential mechanisms of action that may make HBO efficacious in the treatment of prostate cancer. HBO may potentiate cancer chemotherapeutic agents that cause damage to DNA during DNA synthesis or HBO may inhibit cell division causing accumulation in G2/M.

Delayed treatment with doxycycline has limited effect on anthrax infection in BLK57/B6 mice

Blk57/B6 mice were infected with LD90 dose of Sterne strain anthrax spores subcutaneously and then treated with doxycycline. Doxycycline at a dose of 1.5mg/kg, by intra-peritoneal injection, protected mice from death when given at the same time as spores. When doxycycline administration was delayed 4h survival is 90%. Delay of 24h increased survival time but had no impact on eventual mortality. When doxycycline was delayed 48h, mortality and time to death were comparable to sham injection. Peritoneal macrophages harvested from Blk57/B6 mice were examined for response to anthrax lethal toxin and are shown to be deficient in their ability to produce TNF-alpha and have increased expression of IL-6 compared to RAW 264.7 murine macrophage cell line. These findings suggest that antibiotic therapy has limited effects following lethal anthrax spore challenge, even when the host is of a phenotype that does not produce TNF-alpha in response to anthrax lethal toxin exposure.

Comparison Of Blood Pressure And Thermal Responses In Rats Exposed To Millimeter Wave Energy Or Environmental Heat

ABSTRACT Electromagnetic fields at millimeter wave lengths are being developed for commercial and military use at power levels that can cause temperature increases in the skin. Previous work suggests that sustained exposure to millimeter waves causes greater heating of skin, leading to faster induction of circulatory failure than exposure to environmental heat (EH). We tested this hypothesis in three separate experiments by comparing temperature changes in skin, subcutis, and colon, and the time to reach circulatory collapse (mean arterial blood pressure, 20 mmHg) in male Sprague-Dawley rats exposed to the following conditions that produced similar rates of body core heating within each experiment: (1) EH at 42°C, 35 GHz at 75 mW/cm2, or 94 GHz at 75 mW/cm2 under ketamine and xylazine anesthesia; (2) EH at 43°C, 35 GHz at 90 mW/cm2, or 94 GHz at 90 mW/cm2 under ketamine and xylazine anesthesia; and (3) EH at 42°C, 35 GHz at 90 mW/cm2, or 94 GHz at 75 mW/cm2 under isoflurane anesthesia. In all three experiments, the rate and amount of temperature increase at the subcutis and skin surface differed significantly in the rank order of 94 GHz more than 35 GHz more than EH. The time to reach circulatory collapse was significantly less only for rats exposed to 94 GHz at 90 mW/cm2, the group with the greatest rate of skin and subcutis heating of all groups in this study, compared with both the 35 GHz at 90 mW/cm2 and the EH at 43°C groups. These data indicate that body core heating is the major determinant of induction of hemodynamic collapse, and the influence of heating of the skin and subcutis becomes significant only when a certain threshold rate of heating of these tissues is exceeded.ABBREVIATIONS-MMW - millimeter wave, EH - environmental heat, MAP - mean arterial pressure, HR - heart rate, TC - colonic temperature, TSQ - subcutaneous temperature, Tsurf - skin surface temperature

Hyperbaric oxygen therapy in a mouse model of implant-associated osteomyelitis

Implant associated osteomyelitis (OM) is difficult to treat with antibiotics, and outcomes remain poor. Some reports suggest that hyperbaric oxygen treatment is a safe and effective means of treating OM. We tested this hypothesis in a murine model. Clinical isolates of methicillin‐resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Klebsiella pneumoniae were used. The mice were infected with each of the three pathogens, treated with 100% oxygen at high pressure, hyperbaric oxygen (HBO), and monitored for the ability of HBO to prevent and/or clear the OM infection. Assessments included bacterial burden of the tibias and lesion scores, as well as receptor activator of NF‐κB ligand (RANKL) and myeloperoxidase (MPO) concentrations. HBO resulted in more severe lesion scores and higher RANKL and MPO concentrations for MRSA. A significant positive correlation was found between RANKL concentration and lesion score. No significant difference was found with HBO in P. aeruginosa infections and K. pneumoniae seems to either not infect bone well or get cleared before establishing an infection. The model is useful for studying OM infections caused by MRSA and P. aeruginosa, but HBO does not appear to be an efficacious treatment of an implant‐associated OM infection.

Threshold Model for Extremity Compartment Syndrome in Swine

BACKGROUND Extremity compartment syndrome occurs when swelling develops within a muscle compartment to such an extent that the microvasculature is compressed and tissue perfusion is compromised. Untreated, this condition can result in widespread tissue destruction and loss of the affected limb. METHODS Swine were subjected to diffuse muscle compression injury using a balloon catheter inserted between the anterior muscle compartment of the hind limb and the anterior face of the tibia. Balloons were inflated with saline to produce a sustained intramuscular pressure (IMP) of approximately 30 mmHg greater than mean arterial pressure. Following injury the IMP was monitored for up to 8 h. At the end of the monitoring period, the tibialis anterior muscle was collected and examined for injury. RESULTS One animal receiving 6 h injury dislodged the implanted pressure transducers and was dropped from the data analysis. In all other limbs (n = 8) receiving 6 h injury, significant spontaneous increases in IMP were observed following injury. The tibialis anterior in all of the 6 h injury limbs also showed extensive tissue damage. In the limbs injured for 5 h (n = 10), only three showed a significant increase in IMP. The magnitude and duration of this increase closely resembled that seen following 6 h injury. Tissue damage was reduced in comparison with 6 h injury. CONCLUSIONS The injury technique described here provides a potential useful threshold model for studying extremity compartment syndrome and the influence of related factors on the progression of this condition.

Hyperbaric oxygen treatment prevents up-regulation of angiogenesis following partial-thickness skin grafts in the pig

We hypothesized that hyperbaric oxygen treatment (HBO) would reduce neovascularization following partial‐thickness skin grafting in the Yucatan pig. Results show that capillary density 4 days postgraft is increased twofold in grafts not treated with HBO, compared to normal, ungrafted skin or skin grafts from pigs treated with HBO, p < 0.05. Similarly, the expression of vascular endothelial growth factor, a growth factor associated with neovascularization, was also reduced by HBO. Cell density in the graft boundary increased gradually after grafting, reaching a maximum 2.7‐fold increase, relative to normal skin, at day 4, p < 0.05. Cell nuclei positive for proliferating cell nuclear antigen, a marker of proliferation, increased threefold by day 2, p < 0.05, and then declined to normal levels by day 7. HBO had no effect on cell density or proliferation in the boundary region or on shear strength of the graft. In the epidermis, proliferation declined 80% 2 days after grafting and then returned to levels observed in normal skin by day 4, p < 0.05; however, in pigs treated with HBO, we observed no decline in proliferation. These findings confirm the hypothesis that HBO reduces neovascularization in the partial‐thickness skin graft while preserving regenerative capacity in the graft boundary and normal proliferative capacity of the epidermis. (WOUND REP REG 2003;11:139–144)

A Comparison of Cognitive Performance Decreases During Acute, Progressive Fatigue Arising From Different Concurrent Stressors

Fatigue is known to impair cognitive performance, but it remains unclear whether concurrent common stressors affect cognitive performance similarly. We used the Stroop Color-Word Conflict Test to assess cognitive performance over 24 hours for four groups: control, sleep-deprived (SD), SD + energy deficit, and SD + energy deficit + fluid restricted. Fatigue levels were quantified using the Profile of Mood States (POMS) survey. Linear mixed-effects (LME) models allowed for testing of group-specific differences in cognitive performance while accounting for subject-level variation. Starting fatigue levels were similar among all groups, while 24-hour fatigue levels differed significantly. For each cognitive performance test, results were modeled separately. The simplest LME model contained a significant fixed-effects term for slope and intercept. Moreover, the simplest LME model used a single slope coefficient to fit data from all four groups, suggesting that loss in cognitive performance over a 24-hour duty cycle with respect to fatigue level is similar regardless of the cause.