Flexible auditory training, psychophysics, and enrichment of common marmosets with an automated, touchscreen-based system.

Devising new and more efficient protocols to analyze the phenotypes of non-human primates, as well as their complex nervous systems, is rapidly becoming of paramount importance. This is because with genome-editing techniques, recently adopted to non-human primates, new animal models for fundamental and translational research have been established. One aspect in particular, namely cognitive hearing, has been difficult to assess compared to visual cognition. To address this, we devised autonomous, standardized, and unsupervised training and testing of auditory capabilities of common marmosets with a cage-based standalone, wireless system. All marmosets tested voluntarily operated the device on a daily basis and went from naïve to experienced at their own pace and with ease. Through a series of experiments, here we show, that animals autonomously learn to associate sounds with images; to flexibly discriminate sounds, and to detect sounds of varying loudness. The developed platform and training principles combine in-cage training of common marmosets for cognitive and psychoacoustic assessment with an enriched environment that does not rely on dietary restriction or social separation, in compliance with the 3Rs principle.

Properties of an ideal burn dressing: A survey of burn survivors and front-line burn healthcare providers.

OBJECTIVES: The objective of this study is to identify what burn survivors and front-line staff indicate would improve satisfaction with burn dressings, and the ranking of importance of different burn dressing characteristics. These findings will guide the development of future dressings to meet these needs. METHODS: Burn survivors (including the person injured and their family) and front-line burn healthcare providers completed a questionnaire on the importance given to different burn dressing characteristics (non-stick, absorbent, able to wear for a long time, flexible, easy to put on, easy to take off, antimicrobial, and non-bulky), and about the adequacy of pain management during dressing changes. RESULTS: A total of 99 individuals filled out the questionnaire (31 caregivers/survivors and 68 front-line burn healthcare providers). The most important dressing characteristics by both groups were "non-stick" and "fights infection". There was a significant difference between burn survivors and front-line burn healthcare providers pertaining to adequacy of pain management during dressing change. Adequate pain management was reported by 59% of burn survivors, which was significantly higher than that reported by the 25% front-line burn healthcare providers (p=0.002). CONCLUSIONS: Our study suggests that burn survivors and front-line burn providers have similar views on what constitutes an ideal dressing. A significantly proportion of caregiver/survivors felt that pain associated with dressing changes is being adequately managed despite healthcare providers' perception.

The impact of operative time and hypothermia in acute burn surgery.

BACKGROUND: Prolonged operative time and intraoperative hypothermia are known to have deleterious effects on surgical outcomes. Although millions of burn injuries undergo operative treatment globally every year, there remains a paucity of evidence to guide perioperative practice in burn surgery. This study evaluated associations between hypothermia and operative time on post-operative complications in acute burn surgery. METHOD: A historical cohort study from January 1, 2006 to October 31, 2015 was completed at an American Burn Association verified burn centre. 1111 consecutive patients undergoing acute burn surgery were included, and 2171 surgeries were analyzed. Primary outcomes included post-operative complications, defined a priori as either infectious or noninfectious. Statistical analysis was undertaken using a modified Poisson model for relative risk, adjusted for total body surface area, inhalation injury, co-morbidities, substance abuse, and age. RESULTS: The mean operative time was 4.4h (SD 3.7-4.7h; range 0.58-11h), and 18.6% of patients became hypothermic intra-operatively. Operative time was independently associated with the incidence of hypothermia (p<0.05), and both infectious (RR1.5; 1.2-1.9, p<0.0004) and non-infectious complications (RR2.3; 1.3-4.1, p<0.0066). In patients with major burns (TBSA≥20%), hypothermia predisposed to infectious (RR1.3; 1.1-1.5, p<0.0017) and non-infectious complications (RR1.7; 1.2-2.5; p<0.0049). Risk stratification revealed that hypothermic patients with major burns undergoing prolonged surgery had an increased risk of both infectious (RR1.4; 1.1-1.7, p<0.0068) and non-infectious complications (RR1.8; 1.1-3.0, p<0.0132) when compared with those without these risk factors. CONCLUSIONS: Patients who undergo prolonged surgeries and become hypothermic are more likely to develop complications. We therefore advocate for diligent adherence to strategies to prevent hypothermia and recommend limiting operative time in clinical circumstances where intraoperative measures are unlikely to adequately prevent hypothermia.

Animal models in burn research.

Burn injury is a severe form of trauma affecting more than 2 million people in North America each year. Burn trauma is not a single pathophysiological event but a devastating injury that causes structural and functional deficits in numerous organ systems. Due to its complexity and the involvement of multiple organs, in vitro experiments cannot capture this complexity nor address the pathophysiology. In the past two decades, a number of burn animal models have been developed to replicate the various aspects of burn injury, to elucidate the pathophysiology, and to explore potential treatment interventions. Understanding the advantages and limitations of these animal models is essential for the design and development of treatments that are clinically relevant to humans. This review aims to highlight the common animal models of burn injury in order to provide investigators with a better understanding of the benefits and limitations of these models for translational applications. While many animal models of burn exist, we limit our discussion to the skin healing of mouse, rat, and pig. Additionally, we briefly explain hypermetabolic characteristics of burn injury and the animal model utilized to study this phenomena. Finally, we discuss the economic costs associated with each of these models in order to guide decisions of choosing the appropriate animal model for burn research.

The effect of obesity on adverse outcomes and metabolism in pediatric burn patients.

HYPOTHESIS: Obesity influences metabolism and increases the incidence of clinical complications and worsens outcomes in pediatric burn patients. DESIGN: Retrospective, single-center study. SUBJECTS: In all, 592 severely burned pediatric patients who had burns covering more than 30% of the total body surface area and who were treated between 2001 and 2008 were enrolled in this study. Patients were divided into ≥85th percentile (n=277) and normal (n=315) weight groups based on body mass index (BMI) percentiles. RESULTS: Patients stratified below (normal) and ≥85th percentile had similar age, gender distribution and total burn size. No significant differences were detected in the incidence of sepsis (11% for obese vs 10% for normal), the incidence of multiple organ failure (MOF) (21% for obese and 16% for normal) or mortality (11% for obese vs 8% for normal). Compared with the normal group, the ≥85th percentile group had low levels of constitutive proteins (α2macroglobulin and Apolipoprotein A1) (P<0.05 for both) as well as high levels of triglycerides and the acute-phase protein, C-reactive protein (P<0.05 for both) up to 60 days after injury. Patients ≥85th percentile showed a significant higher loss of bone mineral density and lipolysis compared with normal individuals. Stepwise logistic regression analysis revealed that BMI had a positive predictive value towards the maximum DENVER2 score, an index of organ failure (P<0.001). CONCLUSIONS: BMI≥85th percentile altered the post-burn acute phase and catabolic response but did not increase the incidence of sepsis, MOF or mortality in pediatric burn patients. Our results suggest that impaired metabolism and an altered inflammatory response already exists in patients starting at the 85th percentile BMI.

Pre-clinical evaluation of liposomal gene transfer to improve dermal and epidermal regeneration.

Liposomal gene transfer effectively enhances dermal and epidermal regeneration in burned rodents. To advance this treatment to clinical studies, we investigated the efficacy of liposomal gene transfer in a clinically relevant porcine wound model. Mimicking the clinical scenario, six female Yorkshire pigs (40-50 kg) received up to 12 burns of 50 cm(2) area that were fully excised and covered with skin autograft meshed at 4:1 ratio 24 h post-burn. Animals received control injections (empty liposomes), liposomes (DMRIE-C) containing 1 mg LacZ-cDNA, or liposomes (DMRIE-C) with 1 mg of platelet-derived growth factor (PDGF)-cDNA, or the naked PDGF gene. Serial biopsies were taken from different wound sites at multiple time points up to 12 days post-wounding. Transfection efficacy and transfection rate of LacZ and localization of beta-gal were determined by immunohistochemical and immunofluorescent techniques. RT-PCR and multiplex protein analysis (ELISA) were used to measure levels of growth factor mRNA transcribed and growth factor protein translated. Wound re-epithelialization and graft adhesion was evaluated using planimetric analysis and clinical scores. We found that peak transfection of liposomal beta-galactosidase occurred on day 2, with a fluorescence increase of 154% to baseline (P<0.001). Transfection intensity dropped to 115% above baseline on day 4 (P<0.001) and 109% on day 7. Immunohistochemistry showed a maximum transfection rate of 34% of cells in wound tissue. Gene transfer of liposomal PDGF-cDNA resulted in increased PDGF-mRNA and protein expression on days 2 and 4, and accelerated wound re-epithlialization as well as graft adhesion on day 9 (P<0.05). In this study, we showed that liposomal cDNA gene transfer is possible in a porcine wound model, and by using PDGF-cDNA we further showed that dermal and epidermal regeneration can be improved. These data indicate that liposomal gene transfer can be a new therapeutic approach to improve wound healing in humans.

The combination of IGF-I and KGF cDNA improves dermal and epidermal regeneration by increased VEGF expression and neovascularization.

Insulin-like growth factor-I (IGF-I) and keratinocyte growth factor (KGF) cDNA gene transfer individually improves dermal and epidermal regeneration. The aim of the present study was to determine whether the combination of IGF-I plus KGF cDNA further improves wound healing and by which mechanisms these changes occur. Rats received an acute wound and were divided into four groups to receive weekly subcutaneous injections of liposomes plus Lac Z cDNA, liposomes plus IGF-I cDNA, liposomes plus KGF cDNA, or liposomes plus IGF-I/KGF cDNA. Planimetry, immunological assays, histological and immunohistochemical techniques were used to determine IGF-I, KGF, platelet-derived growth factor, fibroblast growth factor (FGF), transforming growth factor-beta and vascular endothelial growth factor (VEGF) expression and different types of collagen (I, III and IV). IGF-I, KGF and their combination cDNA treatment significantly (P<0.05) accelerated re-epithelization, increased IGF-I, KGF, FGF, VEGF and collagen type IV expression, while it had no effect on collagen type I and III expression. The combination of IGF-I plus KGF cDNA increased (P<0.05) neovascularization and VEGF expression when compared to IGF-I cDNA, KGF cDNA groups and controls. In conclusion, exogenous administration of liposomal IGF-I plus KGF cDNA enhanced dermal and epidermal regeneration which is due to increased neovascularization.

Beta-blockade in burns.

A significant proportion of the mortality and morbidity of severe burns is attributable to the ensuing hypermetabolic response that typically lasts for at least 9-12 months post-injury. This is associated with impaired wound healing, increased infection risks, erosion of lean body mass, hampered rehabilitation and delayed reintegration of burn survivors into society. The endocrine status is markedly altered during this period with an initial and then sustained increase in proinflammatory 'stress' hormones such as cortisol and other glucocorticoids, and catecholamines including epinephrine and norepinephrine by the adrenal medulla and cortex. These hormones exert catabolic effects leading to muscle wasting, the intensity of which depends upon the percentage of total body surface area (TBSA) involved, as well as the time elapsed since initial injury. Pharmacological and non-pharmacological strategies may be used to reverse the catabolic effect of thermal injury. Of these, beta-adrenergic blockade with propranolol has been the most efficacious anti-catabolic therapy in the treatment of burns. The underlying mechanism of action of propranolol is still unclear, however its effect appears to occur due to an increased protein synthesis in the face of a persistent protein breakdown and reduced peripheral lipolysis. This article aims to review the current understanding of catecholamines in postburn muscle wasting and focuses on the clinical and metabolic effects of beta-blockade in severe burns.

Gene therapy in wound healing: present status and future directions.

Gene therapy was traditionally considered a treatment modality for patients with congenital defects of key metabolic functions or late-stage malignancies. The realization that gene therapy applications were much vaster has opened up endless opportunities for therapeutic genetic manipulations, especially in the skin and external wounds. Cutaneous wound healing is a complicated, multistep process with numerous mediators that act in a network of activation and inhibition processes. Gene delivery in this environment poses a particular challenge. Numerous models of gene delivery have been developed, including naked DNA application, viral transfection, high-pressure injection, liposomal delivery, and more. Of the various methods for gene transfer, cationic cholesterol-containing liposomal constructs are emerging as a method with great potential for non-viral gene transfer in the wound. This article aims to review the research on gene therapy in wound healing and possible future directions in this exciting field.

The structure and composition of liposomes can affect skin regeneration, morphology and growth factor expression in acute wounds.

Liposomal gene transfer is an effective therapeutic approach to improve dermal and epidermal regeneration. The purpose of the present study was to define whether the biological or chemical structure of a liposome influences cellular and biological regeneration in the skin, and to determine by which mechanisms possible changes occur. Rats were inflicted a full-excision acute wound and divided into three groups to receive weekly subcutaneous injections of DMRIE liposomes plus the Lac Z gene, or DOTAP/Chol liposomes plus the Lac Z gene, or saline. Planimetry, immunological assays, histological and immunohistochemical techniques were used to determine cellular responses after gene transfer, protein expression, dermal and epidermal regeneration. DOTAP/Chol increased IGF-I and KGF protein concentration and caused concomitant cellular responses, for example, by increasing IGFBP-3, P<0.05. DOTAP/Chol liposomes improved epidermal regeneration by exhibiting the most rapid area and linear wound re-epithelization compared to DMRIE or control, P<0.001. DOTAP/Chol and DMRIE exerted promitogenic and antiapoptotic effects on basal keratinocytes, P<0.05. Dermal regeneration was improved in DOTAP/Chol-treated animals by an increased collagen deposition and morphology, P<0.001. DOTAP/Chol liposomes increased vascular endothelial growth factor concentrations and thus neovascularization when compared with DMRIE and saline, P<0.001. In the present study, we showed that different liposomes have different effects on intracellular and biological responses based on its chemical and molecular structure. For gene transfer in acute wounds, the administration of DOTAP/Chol liposomes appears to be beneficial.

Amaranthus hybridus can be pollinated frequently by A. tuberculatus under field conditions.

Recent studies have confirmed that weedy Amaranthus species are capable of interspecific hybridization, and such hybridization may foster the evolution of herbicide resistance. However, the extent to which hybridization among these species occurs in nature is unknown. The purpose of this study was to determine the frequency under field conditions at which A. hybridus, a monoecious and predominantly self-pollinated species, would be pollinated by A. tuberculatus, a dioecious species. To do this, parents carrying different alleles at the ALS locus, which encodes a herbicide target site, were used. Male A. tuberculatus parents were homozygous for a dominant herbicide-insensitive allele, while A. hybridus parents were homozygous for a sensitive form. Hybrid progeny therefore could be detected via herbicide selection. Mean hybridization frequencies between 0.4 and 2.3% were obtained, depending on the proximity between parents (P=0.02). The robustness of the hybrid selection assay was verified using a molecular marker and DNA content analyses. Using these techniques, more than 99% of the progeny that survived the herbicide were confirmed to be hybrids. Frequencies obtained in this study were many times higher than the generally expected rate of mutation. Therefore, even minimal fertility in hybrid progeny would support the view that hybridization could play a role in adaptive evolution of weedy Amaranthus species.

[Indication and clinical results of buried skin grafting to treat problematic wounds]. / Indikation und klinische Ergebnisse der Hautpfropfung zur Behandlung problematischer Wunden.

In 1920 Braun described a technique of skin grafting particularly designed for areas where shearing forces, high pressure and extensive secretion cause repetitive loss of conventionally transplanted skin. During the last 10 years we successfully used this technique when impaired wound healing was encountered due to various reasons. Clinical examples of application and results are presented. By combining this technique with vacuum therapy, formation of granulation tissue can be accelerated, thereby resulting in successful transplantation of problematic and therapy resistant wounds.

Composition and characteristics of an autologous thrombocyte gel.

BACKGROUND: The purpose of the current study was to characterize and compare an autologous thrombocyte gel containing several blood components with a commercially available glue. MATERIALS AND METHODS: Twenty-five volunteers had blood drawn, and lab values, characteristics of the platelet-enriched plasma (PRP), thrombocyte aggregation, electron microscopic examinations, and the breaking strength were determined and compared to a commercial glue. RESULTS: Overall 65% of the total thrombocytes could be isolated from the volunteers and an enrichment of 300% with an autotransfusion device could be achieved. Thrombocyte aggregation as a marker for thrombocyte function decreased from 92% in patients to 71% in the PRP. The autologous glue demonstrated a significant reduced breaking strength (0,76 N/cm(3)) compared to the commercial glue (7.42 N/cm(2)), P < 0.05. The decrease in breaking strength could be correlated with the thrombocyte concentration, P < 0.05. CONCLUSIONS: In the present study we have shown that an autologous platelet-enriched plasma cannot be used as a glue in the common sense to seal stitches or prosthesis. Platelet gels, however, have a high concentration of platelets that release the bioactive proteins and growth factors are necessary to initiate and accelerate tissue repair and enhance dermal and epidermal regeneration. To evaluate the possible clinical implication prospective, randomized studies should be performed to examine the effect of autologous plasma platelet-enriched plasma on wound healing.

Liposomal gene transfer of multiple genes is more effective than gene transfer of a single gene.

Liposomal gene transfer is an effective therapeutic approach for the treatment of several pathophysiologic states. The purpose of the present study was to define whether gene transfer of multiple genes is a feasible approach and whether this approach would be more effective than the single transfer of cDNA. Rats were inflicted an acute wound and divided into four groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.22 microg, vehicle), or liposomes plus the insulin like-growth factor-I (IGF-I)cDNA (2.2 microg) and Lac Z gene (0.22 microg), or liposomes plus the keratinocyte growth factor (KGF) cDNA (2.2 microg) and Lac Z gene (0.22 microg), or liposomes plus the IGF-I/KGF cDNA (2.2 microg) and Lac Z gene (0.22 microg). Planimetry, immunological assays, histological and immunohistochemical techniques were used to determine molecular mechanisms after gene transfer, protein expression, dermal and epidermal regeneration. IGF-I/KGF cDNA transfer increased IGF-I and KGF protein concentration and caused concomitant cellular responses, for example,by increasing IGFBP-3, P<0.05. IGF-I/KGF cDNA gene transfer improved epidermal regeneration by exhibiting the most rapid area and linear wound re-epithelization by almost 250% compared to control and each growth factor given individually, P<0.001, which was probably due to promitogenic and antiapoptotic effects on basal keratinocytes when compared to controls, P<0.001. Dermal regeneration was improved in IGF-I/KGF cDNA-treated animals by an increased collagen deposition and morphology when compared with vehicle, IGF-I and KGF, P<0.001. IGF-I/KGF cDNA increased VEGF concentrations and thus neovascularization when compared with vehicle, IGF-I and KGF, P<0.001. In the present study, we showed that exogenous gene transfer of multiple cDNA sequences have an additive effect on intracellular and biological responses when compared to the same gene administered as a single cDNA sequence. Our findings demonstrate that gene therapy with multiple genes is feasible, and that the gene transfer of multiple genes can enhance and accelerate physiologic and biological effects.

Non-viral liposomal keratinocyte growth factor (KGF) cDNA gene transfer improves dermal and epidermal regeneration through stimulation of epithelial and mesenchymal factors.

Keratinocyte growth factor (KGF) stimulates epithelial cell differentiation and proliferation, which are of major importance for wound healing. Local protein administration, however, has been shown to be ineffective due to enzymes and proteases in the wound fluid. We hypothesized that delivering KGF as a non-viral liposomal cDNA gene complex is a new approach that would effectively enhance dermal and epidermal regeneration. Twenty-two rats were given an acute wound and divided into two groups to receive weekly subcutaneous injections of liposomes plus the LacZ gene (0.2 microg, vehicle), or liposomes plus the KGF cDNA (2.2 microg) and LacZ cDNA (0.2 microg). Transfection was confirmed by histochemical assays for beta-galactosidase. Planimetry, histological and immunohistochemical techniques were used to determine protein expression, dermal and epidermal regeneration. Transfection and subsequent KGF expression was found in diving cells in the granulation tissue. Epidermal regeneration was improved by 170% in rats receiving the KGF cDNA constructs by exhibiting the most rapid area and linear wound re-epithelialization, P < 0.0001. KGF improved epidermal cell net balance by increasing skin cell proliferation and decreasing skin cell apoptosis, P < 0.0001. Dermal regeneration was further improved in KGF cDNA treated animals by an increased collagen deposition and morphology, P < 0.0001. KGF cDNA increased neo-vascularization and concomitant VEGF concentrations when compared with vehicle, P < 0.01. KGF cDNA did not only stimulate epithelial cells, but also mesenchymal cells through increases in IGF-I concentration, P < 0.005. Liposomes containing the KGF cDNA gene constructs were effective in improving epidermal and dermal regeneration. KGF gene transfer to acute wounds may represent a new therapeutic strategy to enhance wound healing.

Fluoroaluminate stimulates phosphorylation of p130 Cas and Fak and increases attachment and spreading of preosteoblastic MC3T3-E1 cells.

Fluoroaluminate is a G-protein activator, it stimulates osteoblastic cells in culture, and is a bone-forming agent in vivo. To elucidate the mechanisms of G-protein-mediated action of fluoroaluminate in osteoblasts, we studied protein tyrosine phosphorylation in the preosteoblastic cell line MC3T3-E1. Fluoroaluminate, lysophosphatidic acid (LPA; an agonist for G-protein-coupled receptor), or adhesion to type I collagen all stimulated phosphorylation of a similar set of proteins, including p130, p120, p110 (previously identified as proline-rich tyrosine kinase 2, Pyk2), and p70. The phosphorylation of these proteins was sensitive to an Src inhibitor, but not to a Gi-protein inactivator, pertussis toxin. By purification/mass spectrometry and by immunodepletion, p130 protein was identified as p130 Cas (Crk-associated protein), a Src substrate and a protein involved in signaling by cell-adhesion receptors, integrins. Phosphorylation of immunoprecipitated p130 Cas increased upon stimulation with fluoroaluminate and with agonists of G-protein-coupled receptors, but not with growth factors. By immunodepletion, the p120 protein was identified as focal adhesion kinase, Fak. The addition of fluoroaluminate during cell attachment to type I collagen further stimulated phosphorylation of p130 Cas and of Fak. Simultaneously, fluoroaluminate increased the number of attached MC3T3-E1 cells and their spreading. These novel aspects of fluoroaluminate action in cell culture may be important for the bone-forming action of fluoroaluminate in vivo.