Molecular Imaging of Smoke-Induced Changes in Nuclear Factor-Kappa B Expression in Murine Tissues Including the Lung.

Many inflammatory responses are mediated by activation of the transcription factor, nuclear factor-kappa B (NF-κB), and a wide variety of human diseases involve abnormal regulation of its expression. In this investigation, we evaluated the effect of smoke inhalation injury on NF-κB expression in lung using two strains of NF-κB reporter mice. Groups of reporter mice with viral thymidine kinase (TK) or "fire fly" luciferase (Luc) genes under control by the NF-κB promoter (TK/NF-κB mice and Luc/NF-κB mice) were subjected to nonlethal smoke inhalation injury. Sham-treated animals served as controls. Twenty-four hours (each animal was injected intravenously with either 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (FHBG) (~ 1.0 mCi) or luciferin (1.0 mg). One hour later, the TK/NF-κB mice were studied by micro-positron emission tomography (µ-PET) imaging using a Concord P4 µ-PET camera, and the Luc/NF-κB mice were studied by bioluminescence imaging with a charge-coupled device camera. The µ-PET data demonstrated that smoke injury produced massive increases in NF-κB expression (FHBG-standardized uptake value: 3.1 vs 0.0) 24 hours after smoke inhalation, which was reduced 48 hours after smoke inhalation, but still significantly different than the control. Qualitative analysis of the bioluminescence data revealed a remarkably similar effect of burn NF-κB luciferase expression in vivo. Biodistribution studies of FHBG uptake and luciferase activity in lung tissue demonstrated a similar increase 24 hours after injury, which was reduced 48 hours later, but still significantly higher than the sham. The present data with these models providing longitudinal imaging data on the same mouse may prove useful in the examination of the factors producing lung injury by smoke inhalation, as well as the treatment(s) for the damage produced with and without burn injury.

Single hind limb burn injury to mice alters nuclear factor-κB expression and [¹8F] 2-fluoro-2-deoxy-D-glucose uptake.

Burn trauma to the extremities can produce marked systemic effects in mice. Burn injury to the dorsal surface of mice is also associated with changes in glucose metabolism ([18F] 2-fluoro-2-deoxy-D-glucose [18FDG] uptake) by brown adipose tissue (BAT) and nuclear factor (NF)-κB activity in several tissues including skeletal muscle. This study examined the effect of a single hind limb burn in mice on 18FDG uptake by NF-κB activity in vivo, and blood flow was determined by laser Doppler techniques. Male NF-κB luciferase reporter mice (28-30 g) were anesthetized, both legs were shaven, and the right leg was subjected to scald injury by immersion in 90°C water for 5 seconds. Sham-treated animals were used as controls. Each burned and sham mouse was resuscitated with saline (2 mL, i.p.). The individual animals were placed in wire bottom cages with no food and free access to water. After 24 hours, the animals were imaged with laser Doppler for measuring blood flow in the hind limb. The animals were then unanesthetized with 50 µCi of FDG or luciferin (1.0 mg, i.v.) via tail vein. Five minutes after luciferin injection, NF-κB mice were studied by bioluminescence imaging with a charge-coupled device camera. One hour after 18FDG injection, the animals were killed with carbon dioxide overdose, and 18FDG biodistribution was measured. Tissues were also analyzed for NF-κB luciferase activity. The scalding procedure used here produced a full-thickness burn injury to the leg with sharp margins. 18FDG uptake by the burned leg was lower than that in the contralateral limb. Similarly, luciferase activity and blood flow in the burned leg were lower than those in the contralateral leg. 18FDG uptake by BAT and heart increased, whereas that by brain decreased. In conclusion, the present study suggests that burn injury to a single leg decreased FDG uptake by skeletal muscle but increased 18FDG uptake by BAT. The injury to the leg reduced NF-κB expression compared with the contralateral leg and the uninjured skeletal muscle of the sham but activated NF-κB expression in a number of other organs. These findings are consistent with the hypothesis that burn trauma to the extremities can produce marked systemic effects, including activation of NF-κB expression and activation of 18FDG uptake by BAT.

Effect of exercise on burn-induced changes in tissue-specific glucose metabolism.

Exercise is a component of the clinical management for burn patients, to help reduce muscle wasting associated with prolonged hospitalization. In the present study the authors examined 2-deoxy-2-[18F] fluoro-D-glucose (18FDG) uptake in mice subjected to burn injury with and without exercise. Mice had their the dorsums shaven, were placed in molds, and the exposed area was immersed in 90°C water for 9 seconds followed by resuscitation with saline (2 ml) to produce a 30% full-thickness burn injury. Twenty-four hours later, the mice were subjected to treadmill exercise for 1 hour. Before exercise, mice were injected with ~50 µCi 18FDG. Mice were killed after running and a complete biodistribution was performed. Exercise produced a stimulation of 18FDG update by skeletal muscle and heart, while reducing 18FDG accumulation in brain. Burn injury had no significant effect on 18FDG update by skeletal muscle, but did increase 18FDG accumulation in heart, while reducing 18FDG accumulation in brain. However, exercise combined with a burn injury produced a significant increase in 18FDG uptake in the skeletal muscle compared with the burned mice, as great as that produced in the sham animals subjected to exercise. The combination of burn plus exercise appeared to prevent the stimulation of 18FDG uptake by the heart produced by burn injury alone. Exercise treatment did not correct the changes in 18FDG uptake in the brain produced by burn injury. Separately, exercise and burn injury significantly increased serum interleukin-6 levels, increases that were higher when exercise was combined with the burn injury. These findings suggest that exercise may exert some therapeutic effects in burn patients by tissue-specific modulation of glucose metabolism, and these changes may be related to interleukin-6.

Combination of radiation and burn injury alters [¹8F] 2-fluoro-2-deoxy-D-glucose uptake in mice.

Radiation exposure and burn injury have both been shown to alter glucose utilization in vivo. The present study was designed to study the effect of burn injury combined with radiation exposure on glucose metabolism in mice using [¹8F] 2-fluoro-2-deoxy-D-glucose (¹8FDG). Groups of male mice weighing approximately 30 g were studied. Group 1 was irradiated with a ¹³7Cs source (9 Gy). Group 2 received full thickness burn injury on 25% TBSA followed by resuscitation with saline (2 ml, IP). Group 3 received radiation followed 10 minutes later by burn injury. Group 4 were sham-treated controls. After treatment, the mice were fasted for 23 hours and then injected (IV) with 50 µCi of ¹8FDG. One hour postinjection, the mice were sacrificed, and biodistribution was measured. Positive blood cultures were observed in all groups of animals compared to the shams. Increased mortality was observed after 6 days in the burn plus radiated group as compared to the other groups. Radiation and burn treatments separately or in combination produced major changes in ¹8FDG uptake by many tissues. In the heart, brown adipose tissue, and spleen, radiation plus burn produced a much greater increase (P < .0001) in ¹8FDG accumulation than either treatment separately. All three treatments produced moderate decreases in ¹8FDG accumulation (P < .01) in the brain and gonads. Burn injury, but not irradiation, increased ¹8FDG accumulation in skeletal muscle; however, the combination of burn plus radiation decreased ¹8FDG accumulation in skeletal muscle. This model may be useful for understanding the effects of burns plus irradiation injury on glucose metabolism and in developing treatments for victims of injuries produced by the combination of burn plus irradiation.

Previous burn injury predisposes mice to lipopolysaccharide-induced changes in glucose metabolism.

In mice, it has been demonstrated that at 7 days after burn injury, injection of lipopolysaccharide (LPS) is more lethal than the same dose at 1 day after injury. In the present study, we examined the effect of LPS injection to mice burned 7 days previously on glucose metabolism ([(18)F] 2-fluoro-2-deoxy-D-glucose [(18)FDG] uptake) in vivo. CD-1 male mice (25-28 g, Charles River Breeding Laboratories, Wilmington, MA) were anesthetized, backs shaven, and subjected to dorsal full thickness burn on 25% TBSA. Sham-treated animals were used as controls. Six days after burn injury, all mice were fasted overnight. One half of the burned and sham controls were subsequently injected IP with LPS (10 mg/kg; Escherichia coli). The remaining animals were injected with saline IP. Two hours later, all mice were injected IV with 50 µCi of (18)F FDG. One hour later, the animals were euthanized, and biodistribution was measured. Tissues were weighed, and radioactivity was measured with a well-type γ counter. Results were expressed as %dose/g tissue, mean ± SEM. The combination of burn 7 days previously and LPS significantly increased mortality compared to animals with burn alone, LPS alone, or sham controls. Burn injury 7 days previously caused a significant decrease in (18)FDG uptake by the brain compared to sham controls. The combination of LPS and burn injury 7 days previously produced a significant increase in (18)FDG uptake by brown adipose tissue and heart compared with either treatment separately. LPS produced a significant increase in (18)FDG uptake by lung, spleen, and gastrointestinal tract of the sham animals, changes that were different in mice burned 7 days previously and injected with LPS. The present results suggest that burn injury 7 days previously predisposes mice to alterations in (18)FDG uptake produced by LPS. These changes may relate, in part, to the increased lethality of LPS injection in previously burned mice.

Gram-negative bacterial infection in thigh abscess can migrate to distant burn depending on burn depth.

Sepsis remains the major cause of death in patients with major burn injuries. In the present investigation we evaluated the interaction between burn injuries of varying severity and preexisting distant infection. We used Gram-negative bacteria (Pseudomonas aeruginosa and Proteus mirabilis) that were genetically engineered to be bioluminescent, which allowed for noninvasive, sequential optical imaging of the extent and severity of the infection. The bioluminescent bacteria migrated from subcutaneous abscesses in the leg to distant burn wounds on the back depending on the severity of the burn injury, and this migration led to increased mortality of the mice. Treatment with ciprofloxacin, injected either in the leg with the bacterial infection or into the burn eschar, prevented this colonization of the wound and decreased mortality. The present data suggest that burn wounds can readily become colonized by infections distant from the wound itself.

Association of heat production with 18F-FDG accumulation in murine brown adipose tissue after stress.

Previous studies have demonstrated that cold stress results in increased accumulation of (18)F-FDG in brown adipose tissue (BAT). Although it has been assumed that this effect is associated with increased thermogenesis by BAT, direct measurements of this phenomenon have not been reported. In the current investigation, we evaluated the relationship between stimulation of (18)F-FDG accumulation in BAT by 3 stressors and heat production measured in vivo by thermal imaging. Male SKH-1 hairless mice were subjected to full-thickness thermal injury (30% of total body surface area), cold stress (4°C for 24 h), or cutaneous wounds. Groups of 6 animals with each treatment were kept fasting overnight and injected with (18)F-FDG. Sixty minutes after injection, the mice were sacrificed, and biodistribution was measured. Other groups of 6 animals subjected to the 3 stressors were studied by thermal imaging, and the difference in temperature between BAT and adjacent tissue was recorded (ΔT). Additional groups of 6 animals were studied by both thermal imaging and (18)F-FDG biodistribution in the same animals. Accumulation of (18)F-FDG in BAT was significantly (P < 0.0001) increased by all 3 treatments (burn, ∼5-fold; cold, ∼15-fold; and cutaneous wound, ∼15-fold), whereas accumulation by adjacent white adipose tissue was unchanged. Compared with sham control mice, in animals exposed to all 3 stressors, ΔTs showed significant (P < 0.001) increases. The ΔT between stressor groups was not significant; however, there was a highly significant linear correlation (r(2) = 0.835, P < 0.0001) between the ΔT measured in BAT versus adjacent tissue and (18)F-FDG accumulation. These results establish, for the first time to our knowledge, that changes in BAT temperature determined in vivo by thermal imaging parallel increases in (18)F-FDG accumulation.

Evaluation of the antioxidant peptide SS31 for treatment of burn-induced insulin resistance.

After severe burn injury and other major traumas, glucose tolerance tests demonstrate delayed glucose disposal. This 'diabetes of injury' could be explained by insulin deficiency, and several studies have shown that soon after trauma (ebb phase) insulin concentrations are reduced in the face of hyperglycemia. After resuscitation of trauma patients (flow phase), ß-cell responsiveness normalizes and plasma insulin levels are appropriate or even higher than expected, however, glucose intolerance and hyperglycemia persist. In the acute care setting, several approaches have been used for treating insulin resistance, including insulin infusion, propranolol and glucagon-like-peptide-1 (GLP-1). Recently, it was demonstrated that a tetrapeptide with antioxidant properties D-Arg-Dmt-Lys-Phe-NH2 (SS31), but not its inactive analogue Phe-D-Arg-Phe-Lys-NH2 (SS20) attenuates insulin resistance in mice maintained on a high fat diet. In this report the effects of SS31 and SS20 on burn-induced insulin resistance was studied in mice. Oral glucose tolerance tests (OGTT) were performed in 4 groups of 6 mice with thermal injury with or without pre-treatment with SS31 or SS20 and sham controls. In addition, biodistribution of 18FDG was measured in burned mice with and without SS31 treatment and shams (subsets of these animals were also studied by µPET). For comparison purposes, groups of 6 cold-stressed mice with and without SS31 treatment were also studied. The results of these studies demonstrate that SS31 but not SS20 ameliorated burn-induced insulin resistance. In addition, SS31 treatment resulted in marked reduction in the increased 18FDG uptake by brown adipose tissue (BAT) in burned but not cold-stressed animals; suggesting that the stressors act by different mechanisms. Overall, these studies confirmed that SS31 can be used to reverse burn-induced insulin resistance and provide a firm pre-clinical basis for future clinical trials of SS31 for the treatment of insulin resistance in patients with burn injury.

Effects of burn injury, cold stress and cutaneous wound injury on the morphology and energy metabolism of murine brown adipose tissue (BAT) in vivo.

AIMS: Cold stress has been shown to produce dramatic increases in 2-fluoro-2-deoxy-D-Glucose ((18)FDG) accumulation by brown adipose tissue (BAT) in rodents. However, neither the effects of other types of stress on (18)FDG accumulation nor the effects of stressors on the accumulation of tracers of other aspects of energy metabolism have been evaluated. In this report we studied the effects of cold stress, burn injury and cutaneous wounds on murine BAT at the macroscopic, microscopic and metabolic level. MAIN METHODS: Glucose metabolism was studied with (18)FDG, fatty acid accumulation was evaluated with trans-9(RS)-(18)F-fluoro-3,4(RS,RS)-methyleneheptadecanoic acid (FCPHA) and tricarboxcylic acid cycle (TCA) activity was evaluated with (3)H acetate. KEY FINDINGS: All three stressors produced dramatic changes in BAT at the macroscopic and microscopic level. Macroscopically, BAT from the stressed animals appeared to be a much darker brown in color. Microscopically BAT of stressed animals demonstrated significantly fewer lipid droplets and an overall decrease in lipid content. Accumulation of (18)FDG by BAT was significantly (p<0.01) increased by all 3 treatments (Cold: ~16 fold, burn ~7 Fold and cutaneous wound ~14 fold) whereas uptake of FDG by white fat was unchanged. This effect was also demonstrated non invasively by µPET imaging. Although less prominent than with (18)FDG, BAT uptake of FCPHA and acetate were also significantly increased by all three treatments. These findings suggest that in addition to cold stress, burn injury and cutaneous wounds produce BAT activation in mice. SIGNIFICANCE: This study demonstrates brown fat activated by several stressors leads to increased uptake of various substrates.

Simvastatin treatment improves survival in a murine model of burn sepsis: Role of interleukin 6.

Infection is the most common and most serious complication of a major burn related to burn size. Recent studies have demonstrated that statin treatment can decrease mortality in murine or human sepsis. In the current study mice were anesthetized and subjected to a dorsal 30% TBSA scald burn. Simvastatin or placebo were administered by intraperitoneal injection once daily or every 12h. On post burn day 7 cecal ligation and puncture with a 21-gauge needle (CLP) was performed under ketamine/xylazine anesthesia, the two different dosing schedules were continued and survival was monitored. In other groups of mice, interleukin-6 (IL-6) levels in blood were measured in mice at 7 days after injury. A simvastatin dependent improvement in survival was observed in the burn sepsis model. This protection was found to be dose and time dependent. In addition, statin treatment reduced the elevation in IL-6 levels of mice burned 7 days previously. However, IL-6 levels in burned mice with or without statin treatment were elevated by CLP to the same degree. The results of these studies suggest that statin treatment reduces mortality in mice with burns and CLP and that this effect may not be mediated via IL-6 levels.

Effect of simvastatin on burn-induced alterations in tissue specific glucose metabolism: implications for burn associated insulin resistance.

In addition to their primary role in lowering plasma cholesterol, statins have a variety of other actions. We studied the effect of simvastatin treatment on burn injury-induced changes in regional glucose metabolism. Groups of six CD-1 mice (male, approximately 25 g) were subjected to full thickness 30% total body surface area (TBSA) burn injury. The animals were treated with simvastatin at various doses (0.02, 0.2 and 2.0 microg/kg, i.p.) for seven days. The following morning, mice were injected with 18F labeled 2-fluoro-2-deoxy-D-glucose (18FDG) (50 microCi) via the tail vein. Approximately 60 min after tracer injection, the animals were sacrificed and biodistribution was measured. A sub-set of burned mice with and without statin treatment and sham controls was injected with approximately 1.0 mCi of FDG and tracer distribution was evaluated by microPET. In addition, oral glucose tolerance tests (OGTT) were performed in other groups of burned mice with and without statin treatment and sham controls. In the heart and brown adipose tissue (BAT), burn injury produced a highly significant increase in 18FDG accumulation (p<0.01), whereas tracer accumulation in brain was markedly reduced (p<0.01). In the heart and BAT, simvastatin treatment produced dose-dependent reductions in 18FDG accumulation. In contrast, simvastatin did not affect 18FDG accumulation in the brain. There was no effect of simvastatin treatment on 18FDG accumulation in the heart, BAT or brain of sham-treated mice. Less pronounced effects were detected in other tissues that were studied. All animals had normal plasma glucose levels (approximately 90 mg/dl). The OGTTs demonstrated insulin resistance in burn injured mice which was reversed by statin treatment. Our results indicate that simvastatin reverses burn-induced increases in 18FDG accumulation by the heart and BAT in a dose-dependent manner but does not affect burn-induced reductions of 18FDG accumulation by the brain. These findings suggest that statins exert some of their effects by tissue specific modulation of glucose metabolism.