Sex differences in the hypothalamic-pituitary-adrenal axis response following a single or multiple days of sleep restriction.

One in three adults reports experiencing inadequate or disrupted sleep throughout the night, with the incidence being higher in women than in men. Disturbances in nightly sleep result in physiological alterations that contribute to a number of disorders. Poor sleep quality is believed to contribute to the pathogenesis of these disorders through interactions with the hypothalamic-pituitary-adrenal (HPA) axis. The present study investigated the effect of one and three days of restricted sleep on HPA axis reactivity. Male and female C57BL/6J (n = 8/group) mice were sleep-deprived for a 20 h period for one day or three consecutive days using the modified multiple platform method, and then subjected to acute restraint stress. In response to sleep restriction, males showed blunted restraint-induced rises in CORT relative to controls. After three days of restricted sleep, females showed a similar attenuation in restraint-induced CORT. However, this effect was ablated after a single day of sleep restriction. Analyses of gene expression revealed significant elevations in the expression of pituitary HPA axis regulatory genes proopiomelanocortin and corticotropin releasing factor receptor 1 in both sexes following sleep restriction. In males, but not females, adrenal mRNA expression of 11ß-hydroxylase and melanocortin receptor 2 were also increased. Altogether, these data suggest several possible mechanisms are involved in the HPA axis dysregulation following sleep restriction, and that there are sex differences in how the HPA axis responds to sleep loss.Lay summarySleep restriction alters the stress response differently in males and females following varying nights of sleep restriction. These alterations are accompanied by changes in gene expression in the pituitary and adrenal glands.

Multimodal noninvasive monitoring of soft tissue wound healing.

Here we report results of non-invasive measurements of indirect markers of soft tissue healing of traumatic wounds in an observational swine study and describe the quantification of analog physiological signals. The primary purpose of the study was to measure bone healing of fractures with four different wound treatments. A second purpose was to quantify soft tissue wound healing by measuring the following indirect markers: (1) tissue oxygenation, (2) fluid content, and (3) blood flow, which were all measured by non-invasive modalities, measured with available devices. Tissue oxygenation was measured by near infrared spectroscopy; fluid content was measured by bipolar bio-impedance; and blood flow was measured by Doppler ultrasound. Immediately after comminuted femur fractures were produced in the right hind legs of thirty anesthetized female Yorkshire swine, one of four wound treatments was instilled into each wound. The four wound treatments were as follows: salmon fibrinogen/thrombin-n = 8; commercial bone filler matrix-n = 7; bovine collagen-n = 8; porcine fibrinogen/thrombin-n = 7. Fractures were stabilized with an external fixation device. Immediately following wound treatments, measurements were made of tissue oxygenation, fluid content and blood flow; these measurements were repeated weekly for 3 weeks after surgery. Analog signals of each modality were recorded on both the wounded (right) hind leg and the healthy (left) hind leg, for comparison purposes. Data were processed off-line. The mean values of 10-s periods were calculated for right-left leg comparison. ANOVA was applied for statistical analysis. Results of the bone healing studies are published separately (Rothwell et al. in J Spec Oper Med 13:7-18, 2013). For soft tissue wounds, healing did not differ significantly among the four wound treatments; however, regional oxygenation of wounds treated with salmon fibrinogen/thrombin showed slightly different time trends. Further studies are needed to establish standards for healthy wound healing and for detection of pathological alterations such as infection. Non-invasive measurement and quantification of indirect markers of soft tissue wound healing support the goals and principles of evidence-based medicine and show potential as easy to administer tools for clinicians and battlefield medical personnel to apply when procedures such as the PET scan are not available or affordable. The method we developed for storing analog physiological signals could be used for maintaining electronic health records, by incorporating vital signs such as ECG and EEG, etc.

Alterations in the activation of corticotropin-releasing factor neurons in the paraventricular nucleus following a single or multiple days of sleep restriction.

Sleep disturbances are common among disorders associated with hypothalamic pituitary-adrenal (HPA) axis dysfunction, such as depression and anxiety. This comorbidity may partly be the result of the intersection between the role of the HPA axis in mediating the stress response and its involvement in sleep-wake cyclicity. Our previous work has shown that following 20 h of sleep restriction, mice show a blunting of the HPA axis in response to an acute stressor. Furthermore, these responses differ in a sex-dependent manner. This study sought to examine the effect of sleep restriction on corticotropin-releasing factor (CRF)-containing neurons in the paraventricular nucleus (PVN) of the hypothalamus. Male and female Crf-IRES-Cre: Ai14 (Tdtomato) reporter mice were sleep restricted for 20 h daily for either a single or three consecutive days using the modified multiple platform method. These mice allowed the visualization of CRF+ neurons throughout the brain. Animals were subjected to acute restraint stress, and their brains were collected to assess PVN neuronal activation via c-Fos immunohistochemistry. Analyses of cell counts revealed an ablation of the restraint-induced increase in both CRF/c-Fos colocalization and overall c-Fos expression in female mice following both a single day and three days of sleep restriction. Males showed an overall decrease in restraint-induced c-Fos levels following a single day of sleep restriction. However, male mice examined after three days of sleep restriction showed a recovery in PVN-CRF and overall PVN neuronal activation. These data suggest the sex dependent dysregulation in CRF function following sleep restriction.

The long term immunological response of swine after two exposures to a salmon thrombin and fibrinogen hemostatic bandage.

Experimental salmon thrombin/fibrinogen dressings have been shown to provide effective hemostasis in severe hemorrhage situations. The hypothesis for this study was that swine would still remain healthy without coagulopathy six months after exposure to salmon thrombin/fibrinogen dressings. Initial exposure was by insertion of the salmon dressing into the peritoneal cavity. Three months after the initial exposure, the same animals were subjected to two full thickness dermal wounds on the dorsal surface. One wound was bandaged with the salmon thrombin/fibrinogen bandage and the other wound was dressed with a standard bandage. The animals were monitored for an additional three months. Blood was drawn every 14 days over the six months for immunological and coagulation function analysis. All of the animals (8 pigs) remained healthy during the six month period and the dermal wounds healed without incidence. Lymph nodes and spleen showed signs of normal immune response and Western blots showed development of antibodies against salmon fibrinogen, but none of the animals made antibodies that recognized any species of thrombin. Coagulation parameters (fibrinogen concentration, thrombin time, PT and aPTT) and hematological parameters remained normal over the course of the study when compared to initial values of the subject swine.

Wound healing and the immune response in swine treated with a hemostatic bandage composed of salmon thrombin and fibrinogen.

We investigated the inflammatory response in pigs exposed to salmon fibrinogen/thrombin dressings. Animals were exposed to the material in 3 ways: (a) thrombin and fibrinogen were injected intravenously, (b) dual full-thickness skin lesions were surgically created on the dorsal aspect of the swine and treated with the fibrinogen/thrombin bandage and a commercial bandage or (c) a fibrinogen/thrombin bandage was inserted through an abdominal incision into the peritoneal cavity. Blood was collected twice weekly and animals were sacrificed at 7, 10 or 28 days. Animals in the 28-day dermal lesion group were given an injection of salmon fibrinogen/thrombin at the 10 day point to simulate a second bandage application. The immune response manifested itself as induction of germinal centers in mesenteric lymph nodes and in the white pulp of the spleen. Examination of the histology of the skin and organs showed a cellular inflammatory response with granulation tissue and signs of edema that resolved by the 28-day stage. Antibodies reactive to salmon and human thrombin and fibrinogen were detected, but fibrinogen levels and coagulation processes were not affected. In conclusion, animals treated with salmon fibrinogen/thrombin bandages demonstrated a smooth recovery course in terms of both tissue healing and the immune response without adverse effects from the exposure to the fish proteins.

Human dendritic cells and macrophages exhibit different intracellular processing pathways for soluble and liposome-encapsulated antigens.

The intracellular fates of soluble and liposomal antigens in human macrophages and dendritic cells are not well defined. Previous studies using murine macrophages have demonstrated that liposomal antigens can enter the MHC class I pathway. The Golgi complex is a major organelle in this pathway. Phagocytosis of the antigens is followed by translocation of antigen-derived peptides to the trans-Golgi where they can complex with MHC class I molecules. In contrast, soluble antigens are normally processed through the MHC class II pathway. Therefore, in the present study, ovalbumin and a synthetic Ebola peptide were used either in a soluble form or encapsulated in liposomes to investigate the intracellular trafficking and localization of these antigens to the Golgi complex in human macrophages and dendritic cells. While liposome-encapsulated antigens were transported to the trans-Golgi region in 59-78% of macrophages, soluble antigens remained diffuse throughout the cytoplasm with only 3-11% of the macrophages exhibiting trans-Golgi localization. The majority of dendritic cells localized both soluble (Ebola, 75%; ovalbumin, 84%) and liposomal antigens (58% and 65%), and irradiated Ebola virus to the trans-Golgi. These studies demonstrate that the intracellular fate of soluble and liposomal antigens can differ depending upon the antigen-presenting cell.

Functional microtubules are required for antigen processing by macrophages and dendritic cells.

Antigen-presenting cells readily phagocytose antigens and channel them through various membrane-bound organelles within the cell. In previous studies, we demonstrated that macrophages concentrated and localized particulate antigens to the trans-Golgi prior to displaying the MHC-class I-antigenic peptides on the cell surface. In this study, we evaluated the importance of cytoskeletal elements in the intracellular trafficking of soluble and liposome-encapsulated ovalbumin in murine bone marrow-derived macrophages and human dendritic cells. F-actin, as identified by staining with fluorescein phalloidin, was observed at the point of contact between soluble or liposomal antigen and the cell membrane, suggesting that a rearrangement of the cytoskeleton occurs to facilitate the uptake of the antigens. Cells were incubated with colchicine, a microtubule depolymerizing agent, or paclitaxel, a microtubule polymerizing agent, before the addition of Texas Red-labeled ovalbumin or liposome-encapsulated Texas Red-labeled ovalbumin. Colchicine disrupted the trans-Golgi, whereas the trans-Golgi complexes were intact in paclitaxel treated cells. In either paclitaxel or colchicine-treated macrophages, internalized liposomal ovalbumin was not concentrated in the area of the trans-Golgi as determined by staining with fluorescent ceramide. In contrast, soluble ovalbumin was concentrated in the region of the trans-Golgi in 15% of the dendritic cells treated with paclitaxel, whereas 6% of the dendritic cells were able to concentrate liposomal antigen. In colchicine-treated dendritic cells, both soluble and liposomal antigens were internalized but did not localize to the area of the trans-Golgi. These data suggest that trafficking of soluble and liposome-encapsulated ovalbumin requires a functional microtubule-dependent translocation system.

Platelet adhesion to dengue-2 virus-infected endothelial cells.

Platelets in circulation normally do not adhere to resting endothelial cells. However, in response to vascular injury they adhere to stimulated endothelium and thereby play an essential role in hemostasis and thrombosis. Infection with dengue-2 virus can cause illness accompanied by thrombocytopenia and hemorrhage. Increased adherence of platelets to stimulated endothelial cells could contribute to the thrombocytopenia. In this study, adherence of radioisotopically labeled platelets to 1) unstimulated, 2) lipopolysaccharide (LPS)-stimulated, and 3) dengue-2 virus-infected human umbilical vein endothelial cells (HUVEC) was measured in an in vitro assay. Primary HUVEC were cultured in 96-well tissue culture plates in the presence or absence of LPS or dengue-2 virus. These cells were co-incubated with 3H-adenine-labeled fresh platelets for 30 min after which the cells were assayed for adherent platelets. Within 30 min there was maximum adherence of platelets to confluent LPS-stimulated HUVEC (36 +/- 4% over controls; P = 0.005). In comparison, there was a significant increase in adherence to dengue-2 infected HUVEC (78 +/- 7%; P < or = 0.001). Additionally, platelet adherence was visualized using fluorescent microscopy. Dengue-2 infection stimulated the HUVEC as monitored by expression of E-selectin. Platelets that adhered to dengue-2 or LPS-stimulated HUVEC were activated as visualized by dual fluorescent probes. These data demonstrate that human platelets adhere to dengue-2 virus-stimulated HUVEC and this interaction could contribute to the thrombocytopenia observed during infection.

Epsilon-amino caproic acid additive decreases fibrin bandage performance in a swine arterial bleeding model.

epsilon -Amino caproic acid (EACA), a lysine analog that inhibits the activity of plasmin, was added to Nycomed (TC-S) fibrin bandages, and the bandages were tested for hemostatic efficacy in a swine femoral artery bleeding model. The blood loss using the bandage with EACA (TC-S+EACA; 728.8+/-132 g, n=5) was much greater as compared to the TC-S bandage alone (TC-S; 237.8+/-47.9 g, n=6, p=.02). The time to "cessation of bleeding" (T(b)) was also increased for animals treated with TC-S+EACA bandages compared to the TC-S controls (33.6+/-10.8 min vs. 9.2+/-2.2 min; p=.05). Although plasma fibrinogen concentration decreased in animals treated with the TC-S+EACA bandage, activated partial thromboplastin times (aPTT) and thrombin times (TT) were decreased. Animals treated with the TC-S control bandage exhibited no changes in fibrinogen, aPTT or TT. Prothrombin times (PT) were unchanged in either group. In conclusion, addition of EACA to the Nycomed product decreased fibrin bandage efficiency.

Assessment of omega-fatty-acid-supplemented human platelets for potential improvement in long-term storage.

Uptake of omega (omega)-3 fatty acids can influence membrane stability and cell mobility. We investigated the effects of omega-3 and -6 fatty acids on the hemostatic efficacy of human platelets using an in vivo rabbit bleeding model. In vitro assays such as platelet aggregation, vWF bead-mediated ATP release and platelet adhesion to beads (measured by the residual platelet count [RPC] [free platelet count after reacting with the beads]/[baseline platelet count]x 100=%RPC; a high %RPC indicates reduced platelet function) were conducted on platelets treated with 1% fish oil (omega-3); 2% fish oil emulsion or 1% soy oil (omega-6). Oil treatment of platelets reduced the vWF bead-induced ATP release insignificantly. Addition of omega-3 agents reduced physical reactivity (%RPC) with the vWF beads by a factor of 1.2 (oil) and 1.9 (emulsion). The omega-6 oil enhanced reactivity by a factor of 1.7. After washing to remove excess reagent, platelet resuspension was most efficient with the omega-3 emulsion. Platelet function was higher with the omega-3-treated platelets (%RPC=52.3%, omega-3 oil; 63.3%, omega-3 emulsion vs. 85%, omega-6 oil; 82% untreated platelets). Ethyl-palmitate-treated thrombocytopenic rabbits were infused with human platelets. Survival times of the treated platelets, as monitored by flow cytometry (6.2-8.2 h) were comparable to untreated platelets (8.6 h). In the rabbit kidney injury model, blood loss after infusion of the treated platelets was similar to that of saline-infused rabbits (75.3+/-3.4 g). However, platelets washed prior to infusion reduced blood loss to a value comparable to that of fresh platelets (48.3+/-5 g). Furthermore, the presence of the infused platelets at the injury site was clearly visualized using FITC-tagged anti CD42a antibody. Thus, the omega-3-based agents protect the platelets from damage during the washing procedure as demonstrated in vitro by improved platelet resuspension, low %RPC, high stimulus-responsive ATP secretion and a reduction in blood loss in vivo.

Addition of a propyl gallate-based procoagulant to a fibrin bandage improves hemostatic performance in a swine arterial bleeding model.

Fibrin bandages manufactured by Nycomed Austria (TC-S) were modified by the addition of Hemostyptin (HS), a proprietary platelet-activating reagent containing propyl gallate. HS was added as an additional layer to TC-S fibrin bandages and the bandages were tested for hemostatic efficacy in a swine femoral artery bleeding model. Injuries were treated with a TC-S+HS bandage preparation using HS lyophilized onto a bandage surface that was then attached to the fibrin dressing. This preparation qualitatively and quantitatively exhibited more robust blood clotting at the surgical site than the control bandages. TC-S+HS bandages were more effective than control bandages with a difference in blood loss of 251.8+/-66.5 g for TC-S bandage alone, n=12 vs. 121+/-40.7 g, n=13 for the TC-S+HS bandage, P=0.05. Bleeding times were shortened for animals treated with the HS fortified bandages and residual platelets counts in these animals were higher.

Femoral strength after induced lesions in rats (Rattus norvegicus).

Rats are a common model for the study of bone healing, with the cranium, femur, and tibia being the bones studied most frequently. This study examines noncritical-sized lesions that would allow rats to continue to bear weight without the need for fixation but that are sufficiently large to enable characterization of the healing process. We compared the femoral bone strength associated with 3 lesion sizes selected for use in future studies. Sprague-Dawley rats (age, 10 to 16 wk) were used to assess the ultimate breaking strength, stress, and break force of normal, unmanipulated femurs. We then created lesions of 3 different sizes in the mid- to distal diaphysis of the left and right femurs and characterized the associated decreases in bone strength. Femurs (n = 85) for this study were collected through tissue sharing from rats used in other acute surgical procedures and were tested by using a 3-point bending flexural materials-testing machine. Our hypothesis was that, as a model for bone healing, 3 induced lesions of different sizes would show incremental and proportional decreases in femoral strength, with the intermediate-sized (1.5-mm) lesion demonstrating a decrease of 20% to 40%. A lesion of 1.5 mm yielded a decrease in strength of 17% for both the left and right femurs. The strength of left femurs carrying intermediate lesions was significantly less than that of control, uninjured femur bones. In addition to providing validation for our own future bone-healing project, these data are a useful baseline for other investigators studying bone healing in a rat femur model.

Comparison of fibrinogen- and collagen-based treatments for penetrating wounds with comminuted femur fractures in a Swine model.

INTRODUCTION: Military servicemembers in combat operations often sustain injuries to the extremities from highspeed projectiles, resulting in bleeding and comminuted open fractures. Severe injury with bone fragmentation can result in limb amputation. Surgical treatment options include materials that promote osteogenesis and bone proliferation, such as growth hormones, stem cells, or mineralized matrix adjuncts. However, none of these are amenable to use by the first responder, nor do they address the question of hemorrhage control, which is a common problem in traumatic injuries. HYPOTHESIS: Our hypothesis was that treatment with a fibrinogen-based protein mixture at the time of the bone injury will provide both hemostasis and a supportive environment for preservation of injured bone. METHODS: A comminuted femur fracture was produced in 28 female Yorkshire swine, and one of four treatments was instilled into the wound immediately after injury. Each animal was evaluated for the following parameters: inflammation, new bone growth, osteoclast proliferation, callus formation, and femur wound cavity fill, using post-mortem computed tomography and analysis of histological sections. RESULTS: Overall, salmon fibrinogen?thrombin and porcine fibrinogen?thrombin showed a trend for improved healing based on bone filling and calcification. However, statistically significant differences could not be established between treatment groups. CONCLUSIONS: These findings indicate that a fibrinogen?thrombin matrix may be a useful as an immediate response product to enhance fracture healing. Salmon fibrinogen?thrombin has the advantages of cost and a pathogen profile compared to mammalian fibrinogens.

Assessment of postoperative analgesia after application of ultrasound-guided regional anesthesia for surgery in a swine femoral fracture model.

Management of pain in research swine used for studies involving painful procedures is a considerable challenge. Here we assessed whether a regional anesthesia method is effective for pain control of hindlimb injuries in pigs used for research in bone fracture healing. For this randomized controlled study, we administered regional anesthesia before an experimental femoral injury was produced. Using ultrasound guidance, we placed sterile infusion catheters near the sciatic and femoral nerves and administered local anesthetic (bupivacaine) for the first 24 h after surgery. We evaluated various behavioral and physiologic parameters to test the hypothesis that this regional anesthesia would provide superior analgesia compared with systemic analgesia alone. We also collected blood samples to evaluate serum levels of cortisol and fentanyl postoperatively. At the end of the study period, we collected sciatic and femoral nerves and surrounding soft tissues for histopathologic evaluation. Treatment pigs had lower subjective pain scores than did control animals. Control pigs had a longer time to first feed consumption and required additional analgesia earlier in the postoperative period than did treatment pigs. Ultrasound-guided regional anesthesia is a viable and effective adjunct to systemic analgesics for providing pain control in swine with experimental femoral fractures.

A salmon thrombin-fibrinogen dressing controls hemorrhage in a swine model compared to standard kaolin-coated gauze.

BACKGROUND: Battlefield hemorrhage remains the primary cause of death in potentially survivable combat injuries with noncompressible hemorrhage. Fibrin dressings have great potential for reducing mortality, however are limited by cost, availability, and disease transmission. METHODS: Dressings comprising a soluble dextran dressing with lyophilized salmon thrombin and fibrinogen (STF) were tested against Combat Gauze (CG) as a control in a standard swine femoral artery hemorrhage model. Ten female swine were used in each arm of the study. RESULTS: Survival, blood loss, and time to hemostasis were similar between the two dressings. Two of the CGtreated animals that survived exsanguinated during the simulated walking maneuver. Three CG-treated animals formed a clot within the wound, but the clot did not adhere to the femoral artery injury. All ten of the STF-treated animals formed a clot in the wound that adhered and sealed the arterial injury site, even in three animals that did not survive. None of the STF-treated animals bled following the simulated walking maneuver. Three of five STF-treated animals reestablished blood flow distal to the injury as demonstrated by angiography. CONCLUSIONS: The STF dressing is as efficacious as CG in treating hemorrhage in this model of a lethal injury. Further, the STF dressing formed a fibrin sealant over the injury, whereas CG achieved hemostasis by occlusive compression of the artery. The sealant property of the STF dressing allowed reestablishment of antegrade blood flow into the distal limb, demonstrating that this dressing has the potential of limb salvage in addition to control of life-threatening hemorrhage.

Salmon thrombin-fibrinogen dressing allows greater survival and preserves distal blood flow compared to standard kaolin gauze in coagulopathic Swine with a standardized lethal femoral artery injury.

We have previously shown that lyophilized salmon thrombin and fibrinogen (STF) embedded in a dissolvable dextran dressing is as efficacious as Combat Gauze (CG) with regard to controlling hemorrhage and survival in non-coagulopathic swine with femoral artery lacerations. A major limitation of currently available advanced field dressings is the inability to control hemorrhage in coagulopathic casualties because of the exhaustion of host coagulation proteins. We tested the hypothesis that the STF dressing would be better able to control hemorrhage and prolong survival in coagulopathic swine compared to CG. Survival rate was 50% in CG-treated animals versus 90% in STF-treated animals. Survival time was significantly greater in STF-treated animals. Clots formed over the arterial injury in 100% of STF-treated animals compared to 0% in CG-treated animals (p < 0.001). STF-treated animals consumed less host coagulation factors, including platelets (p = 0.03). Survival after limb manipulation that simulated casualty evacuation was significantly higher with the STF dressing (p < 0.005). Angiographic observation of distal blood flow was seen twice as often with the STF dressing as with CG. The STF dressing allows a high survival rate, significantly greater survival time, and a significantly more stable dressing than CG in coagulopathic swine. The clot formed by the STF dressing also enables restoration of distal blood flow to the limb potentially resulting in higher limb salvage.

Plasma Proteomic Signature in Overweight Girls Closely Correlates with Homeostasis Model Assessment (HOMA), an Objective Measure of Insulin Resistance.

Obesity is known to be associated with a large number of long-term morbidities, and while in some cases the relationship of obesity and the consequences is clear (for example, excess weight and lower extremity orthopedic problems) in others the mechanism is not as clear. One common system of categorizing overweight in terms of the likelihood of negative consequences involves using the concept of "metabolic syndrome". We hypothesized that the development of a plasma protein profile of overweight adolescents with and without the metabolic syndrome might give a more precise and informative picture of the disease process than the current clinical categorization and permit early targeted intervention. For this paper, we used antibody microarrays to analyze the plasma proteome of a group of 15 overweight female adolescent patients. Upon analysis of the proteome, the overweight patients diverged from the nonoverweight female controls. Furthermore, the overweight patients were divided by the analysis into two population clusters, each with distinctive protein expression patterns. Interestingly, the clusters were characterized by differences in insulin resistance, as measured by HOMA. Categorization according to the presence or absence of the metabolic syndrome did not yield such clusters.