Various effects of 11,12 EET rescue wound healing in a combined model of diabetes and ischemia.

Chronic non healing wounds in diabetic patients still impose a major problem in modern medicine. Especially additional peripheral vascular disease complicates treatment success in these patients. Thus, we analyzed the effects of 11,12 epoxyeicosatrienoic acid (EET) in a combined model of hyperglycemia and ischemia in mice. Hyperglycemia was induced by Streptozotozin 2 weeks prior to wounding. 3 days before wound creation 2 of the 3 suppling vessels of the moue ear were cautherized for ischemia. Either 11,12 EET or solvent for control was applied. Wound closure as well as TNF-α, TGF-ß, SDF-1α, VEGF, CD31, and Ki67 were measured. The wounds closed on day 14.4 ± 0.4 standard deviation (SD). 11,12 EET treatment enhanced healing to 9.8 ± 0.6 SD. TNF-α level was augmented on day 9 compared to control and receded on day 18. TGF-ß seemed to be elevated all days observed after 11,12 EET treatment. SDF-1α was enhanced on day 6 and 9 by 11,12 EET, and VEGF on day 6 and 18 as well as CD13 on day 3, 6, and 18. 11,12 EET did not alter Ki67. 11,12 EET are able to rescue deteriorated wound healing in a combined model of hyperglycamia and ischemia by resolution of inflammation, augmentation of neovascularization and increasing expression of TGF-ß as well as SDF-1α.

Local application reduces number of needed EPC for beneficial effects on wound healing compared to systemic treatment in mice.

INTRODUCTION: Stem cell transplantation is one of the most promising strategies to improve healing in chronic wounds as systemic administration of endothelial progenitor cells (EPC) enhances healing by promoting neovascularization and homing though a high amount of cells is needed. In the following study, we analysed whether local application can reduce the number of EPC needed achieving the same beneficial effect on wound healing. MATERIAL AND METHODS: Wound healing after local or systemic treatment with EPC was monitored in vivo by creating standardized wounds on the dorsum of hairless mice measuring wound closure every second day. Systemic group received 2 × 106 EPC i.v. and locally treated group 2 × 105 EPC, locally injected. As control PBS injection was performed the same way. Expression of CD31, VEGF, CD90 and, SDF-1α was analysed immunohistochemically for evaluation of neovascularisation and amelioration of homing. RESULTS: Local (7.1 ± 0.45 SD) as well as systemic (6.1 ± 0.23 SD) EPC transplantation led to a significant acceleration of wound closure compared to controls (PBS local: 9.7 ± 0.5 SD, PBS systemic 10.9 ± 0.38 SD). Systemic application enhanced CD31 expression on day 6 after wounding and local EPC on 6 and 9 in comparison to control. VEGF expression was not significantly affected. Systemic and local EPC treatment resulted in a significantly enhanced SDF-1α and CD90 expression on all days investigated. CONCLUSION: Local as well as systemic EPC treatment enhances wound healing. Moreover, beneficial effects are obtained with a tenfold decrease number of EPC when applied locally. Thus, local EPC treatment might be more convenient way to enhance wound healing as number of progenitor cells is limited.

11,12 Epoxyeicosatrienoic Acid Rescues Deteriorated Wound Healing in Diabetes.

Epoxyeicosatrienoic acids (EET) facilitate regeneration in different tissues, and their benefit in dermal wound healing has been proven under normal conditions. In this study, we investigated the effect of 11,12 EET on dermal wound healing in diabetes. We induced diabetes by i.p. injection of streptozotocin 2 weeks prior to wound creation on the dorsal side of the mouse ear. 11,12 EET was applied every second day on the wound, whereas the control groups received only solvent. Epithelialization was monitored every second day intravitally up to wound closure. Wounds were stained for VEGF, CD31, TGF-ß, TNF-α, SDF-1α, NF-κB, and Ki-67, and fibroblasts were counted after hematoxylin-eosin stain on days 3, 6, 9, and 16 after wounding. After induction of diabetes, wounds closed on day 13.00 ± 2.20 standard deviation (SD). Local 11,12 ETT application improved wound closure significantly to day 8.40 ± 1.39 SD. EET treatment enhanced VEGF and CD31 expression in wounds on day 3. It also seemed to raise TNF-α level on all days investigated as well as TGF-ß level on days 3 and 6. A decrease in NF-κB could be observed on days 9 and 16 after EET application. The latter findings were not significant. SDF-1α expression was not influenced by EET application, and Ki-67 was significantly less in the EET group on day 9 after EET application. The number of fibroblasts was significantly increased on day 9 after the 11,12 EET application. 11,12 EET improve deteriorated wound healing in diabetes by enhancing neoangiogenesis, especially in the early phase of wound healing. Furthermore, they contribute to the dissolution of the initial inflammatory reaction, allowing the crucial transition from the inflammatory to proliferative phase in wound healing.

11,12 and 14,15 epoxyeicosatrienoic acid rescue deteriorated wound healing in ischemia.

INTRODUCTION: Epoxyeicosatrienoic acids (EETs) are able to enhance angiogenesis and regulate inflammation that is especially important in wound healing under ischemic conditions. Thus, we evaluated the effect of local EET application on ischemic wounds in mice. METHODS: Ischemia was induced by cautherization of two of the three supplying vessels to the mouse ear. Wounding was performed on the ear three days later. Wounds were treated either with 11,12 or 14,15 EET and compared to untreated control and normal wounds. Epithelialization was measured every second day. VEGF, TNF-α, TGF-ß, matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP), Ki67, and SDF-1α were evaluated immunohistochemically in wounds on day 3, 6, and 9. RESULTS: Ischemia delayed wound closure (12.8 days ± 1.9 standard deviation (SD) for ischemia and 8.0 days ± 0.94 SD for control). 11,12 and14,15 EET application ameliorated deteriorated wound healing on ischemic ears (7.6 ± 1.3 SD for 11,12 EET and 9.2 ± 1.4 SD for 14,15 EET). Ischemia did not change VEGF, TNF-α, TGF-ß, SDF-1α, TIMP, MMP7 or MMP9 level significantly compared to control. Local application of 11,12 as well as 14,15 EET induced a significant elevation of VEGF, TGF-ß, and SDF-1α expression as well as proliferation during the whole phase of wound healing compared to control and ischemia alone. CONCLUSION: In summary, EET improve impaired wound healing caused by ischemia as they enhance neovascularization and alter inflammatory response in wounds. Thus elevating lipid mediator level as 11,12 and 14,15 EET in wounds might be a successful strategy for amelioration of deranged wound healing under ischemia.

[Thoracic injuries in severely injured children : Association with increased injury severity and a higher number of complications]. / Thorakale Verletzungen beim schwer verletzten Kind : Assoziation mit erhöhter Verletzungsschwere und vermehrten Komplikationen.

BACKGROUND: Severely injured children and adolescents in clinical practice are rare. For adequate treatment of these patients, detailed knowledge of anatomical and physiological peculiarities, as well as abundant injury patterns, are indispensable. Traumatic brain injuries are known to lead to an unfavorable outcome. In addition, thoracic trauma is regarded as prognostically unfavorable. OBJECTIVES: This study depicts epidemiology and injury patterns of severely injured children and adolescents focusing on peculiarities in the severely injured with associated thoracic injuries. MATERIALS AND METHODS: A retrospective analysis of underaged patients with suspicion of severe injuries who obtained emergency-room treatment in our level-one trauma center during a four-year time period was performed. The data was collected prospectively using the TraumaRegister® of the German Trauma Society as well as an extended house-internal dataset including data of daily clinical routine. The patients were divided into subgroups with (TT) and without (KT) thoracic trauma based on whether a thoracic injury was present or not. For further analysis, four age groups were established. RESULTS: In all, 256 patients younger than 18 years were eligible. Of these, 46 patients revealed thoracic injuries. The mean age of patients with thoracic trauma (12.4 ± 4.9 years) was significantly higher than for patients without thoracic trauma (8.0 ± 5.2 years). In both subgroups, most patients were male (TT: 69.9%, KT: 64.8%). Patients with concomitant thoracic trauma showed a significantly higher injury severity score (ISS) than patients without thoracic trauma (ISS: TT: 26.7 ± 15.8 vs. KT: 8.1 ± 6.8 points). Mortality was higher for TT as well (TT: 6.9% vs. KT: 1.9%). For both groups, traffic accidents were the most common cause of injury. Of patients with thoracic injuries, 52.2% developed at least one complication during their hospital stay (KT: 12.9%). CONCLUSIONS: Thoracic trauma is a relevant factor in children with regard to the severity of total injury and complications. Particular attention should therefore be paid to early diagnosis and treatment.

Delayed wound repair in sepsis is associated with reduced local pro-inflammatory cytokine expression.

Sepsis is one of the main causes for morbidity and mortality in hospitalized patients. Moreover, sepsis associated complications involving impaired wound healing are common. Septic patients often require surgical interventions that in-turn may lead to further complications caused by impaired wound healing. We established a mouse model to the study delayed wound healing during sepsis distant to the septic focus point. For this reason cecal ligation and puncture (CLP) was combined with the creation of a superficial wound on the mouse ear. Control animals received the same procedure without CPL. Epithelialization was measured every second day by direct microscopic visualization up to complete closure of the wound. As interplay of TNF-α, TGF-ß, matrix metalloproteinases (MMP), and tissue inhibitors of metalloproteinases (TIMP) is important in wound healing in general, TNF-α, TGF-ß, MMP7, and TIMP1 were assessed immunohistochemical in samples of wounded ears harvested on days 2, 6, 10 and 16 after wounding. After induction of sepsis, animals showed a significant delay in wound epithelialization from day 2 to 12 compared to control animals. Complete wound healing was attained after mean 12.2± standard deviation (SD) 3.0 days in septic animals compared to 8.7± SD 1.7 days in the control group. Septic animals showed a significant reduction in local pro-inflammatory cytokine level of TNF-α on day 2 and day 6 as well as a reduced expression of TGF-ß on day 2 in wounds. A significant lower expression of MMP7 as well as TIMP1 was also observed on day 2 after wounding. The induction of sepsis impairs wound healing distant to the septic focus point. We could demonstrate that expression of important cytokines for wound repair is deregulated after induction of sepsis. Thus restoring normal cytokine response locally in wounds could be a good strategy to enhance wound repair in sepsis.

Effects of acute ethanol gavage on intestinal integrity after hemorrhage/resuscitation.

BACKGROUND: In hemorrhagic shock with subsequent resuscitation (H/R), increased pro-inflammatory changes contribute to tissue injury and mortality in rodent models. Ethanol (EtOH) is assumed to modulate the inflammatory response and the subsequent organ injury after H/R. Therefore, we determined the contribution of acute ethanol gavage on intestinal inflammation and injury as well as survival after H/R in rats. METHODS: Fourteen hours before H/R, female LEWIS rats were gavaged with single dose of EtOH or saline (5 g/kg, 30% EtOH, H/R_EtOH group or H/R_ctrl group). Then, rats were hemorrhaged to a mean arterial blood pressure of 30 ± 2 mmHg for 60 min and resuscitated. Control groups underwent surgical procedures and gavage without H/R (sham_ctrl group and sham_EtOH group). Tissue was harvested 2 h after resuscitation. Mortality was assessed 72 h after H/R. RESULTS: Ethanol gavage increased survival after H/R from 20% to 80%, but amplified plasma alanineaminotransferase (ALT) release compared to saline gavage (2847 ± 406 vs. 1159 ± 200 IU/L, p < 0.05). Intestinal mucosal damage index, intestinal permeability, ileal myeloperoxidase levels as indicators of polymorphonuclear leukocyte (PMNL) infiltration and systemic IL-6 levels as well as ileal IL-6 and TNF gene expressions after H/R were reduced and partly restored after ethanol gavage when compared to the saline gavaged group after H/R. CONCLUSIONS: Taken together, we propose that acute ethanol gavage prior to H/R 1) did not enhance intestinal mucosa injury after H/R and 2) suppressed the H/R-induced inflammatory response. Both findings seem to contribute to the ethanol-induced survival benefit after H/R in our model.

The role of TNXB single-nucleotide polymorphisms in recurrent shoulder dislocation.

Tenascin-X (TNX) is an extra-cellular matrix glycoprotein associated with collagen fibril deposition. Recent reports have linked truncated TNX mutations (TNXB) to generalized joint hypermobility and most importantly recurrent joint dislocation. In the present study, we investigated whether there is an association between joint dislocation recurrence rate and the frequency of TNXB single-nucleotide polymorphisms (SNPs). Seventy-eight patients treated for post-traumatic shoulder instability and 82 healthy controls were genotyped for selected TNXB SNP using TaqMan® Genotyping Assays. At a mean follow-up of 24 months recurrence rate and clinical outcomes were evaluated using the Constant and Murley, Rowe, and DASH scores. The association between genotypes and joint dislocation was tested using the dominant, recessive and additive models, and the model-free approach. Genotype distribution of the examined SNPs did not significantly deviate from the Hardy-Weinberg equilibrium (HWE) neither in patients nor in the controls. Moreover, there was no significant difference in genotype and allele distribution between patients and controls. Finally, no difference in genotype frequency was detected between patients who experienced a re-dislocation after the initial surgery and patients who did not sustain a re-dislocation. The SNPs investigated in this study have no clinically relevant influence on TNXB gene expression and/or TNX function. Therefore, these SNPs could not be used for predicting individual risk of recurrent shoulder dislocation.

Soluble epoxide hydrolase disruption as therapeutic target for wound healing.

BACKGROUND: Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) possess angiogenic effects. However, the effect of CYP-derived EETs and soluble epoxide hydrolase (sEH) deletion on wound healing in vivo has not been rigorously investigated. In this study, we measured the effect of exogenous CYP-derived EETs and targeted disruption of sEH in an in vivo wound model. MATERIALS AND METHODS: Standardized full-thickness dermal wounds were created on the dorsum of mouse ears. Wound epithelialization was directly viewed and measured using intravital microscopy and computerized planimetry every second day until healing was complete. Wound sections were analyzed by immunostaining for metalloproteinase (MMP) 2, MMP7, MMP9, tissue inhibitor of metalloproteinases (TIMP) 1, and tumor necrosis factor (TNF) α on days 2, 4, and 12. RESULTS: Treatment with 11,12-EETs, 14,15-EETs, and sEH deletion significantly accelerated wound closure. This effect was attenuated by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) in sEH(-/-) mice. Neither 11,12- nor 14,15-EETs caused significant alterations in MMP9 expression in wounds. In contrast, MMP2 and MMP7 were significantly upregulated in the EET-treated groups, whereas TIMP1 and TNF-α were downregulated. CONCLUSIONS: Collectively, these data demonstrated that potentiation of the CYP epoxy-genase pathway by either exogenous CYP-derived EETs or sEH deletion significantly accelerated wound epithelialization in vivo. This beneficial effect might be due to downregulation of TNF-α production and, to a lesser degree, to the release of MMPs and could be used as a viable angiogenic therapeutic strategy.

Time dependency and topography of hepatic nuclear factor κB activation after hemorrhagic shock and resuscitation in mice.

The leading causes of death in people aged 1 to 44 years are unintentional injuries with associated hemorrhagic shock. Hemorrhagic shock followed by resuscitation (H/R) activates the nuclear factor κB (NF-κB) pathway. To further address the association between liver damage and NF-κB activation, we analyzed the H/R-induced activation of NF-κB using cis-NF-κB reporter gene mice. In these mice, the expression of green fluorescent protein (GFP) is linked to the activation of NF-κB, and therefore tracing of GFP colocalizes NF-κB activation. Mice were hemorrhaged to a mean arterial blood pressure of 30mmHg for 90 min, followed by resuscitation. Six, 14, or 24 h after resuscitation, mice were killed. Compared with sham-operated mice, H/R led to a profound hepatic and cellular damage as measured by aspartate aminotransferase, creatine kinase, and lactate dehydrogenase levels, which was accompanied by an elevation in interleukin 6 levels and hepatic leukocyte infiltration. Interleukin 10 levels in plasma were elevated 6 h after H/R. Using serial liver sections, we found an association between necrotic areas, oxidative stress, and enhanced GFP-positive cells. Furthermore, enhanced GFP-positive cells surrounded areas of necrotic liver tissue, predominantly in a penumbra-like-shape pericentrally. These results elucidate spatial relationship between oxidative stress, liver necrosis, and NF-κB activation, using an in vivo approach and therefore might help to further analyze mechanisms of NF-κB activation after resuscitated blood loss.

Cytochrome P450-derived epoxyeicosatrienoic acids accelerate wound epithelialization and neovascularization in the hairless mouse ear wound model.

PURPOSE: Epoxyeicosatrienoic acids (EETs) are known to modulate proliferation and angiogenesis in vitro. Tissue levels of EETs are regulated by the cytochrome P450 (CYP) epoxygenases that generate them as well as by the soluble epoxide hydrolase metabolizes them to their less active diols. The aim of this study was to determine the effect of locally administered EETs (11,12- and 14,15-EETs) and the selective sEH inhibitor (sEHI) trans-4-[4-(3-adamantan-1-ylureido)-cyclohexyloxy]-benzoic acid (t-AUCB) on wound healing in vivo. METHODS: Standardized full thickness dermal wounds were created on the dorsum of hairless mouse ears. Wound epithelialization was directly viewed and measured using intravitalmicroscopy and computerized planimetry every second day until healing was complete. Wound sections were analyzed by immunostaining for endothelial lineage marker CD31, vascular endothelial growth factor (VEGF), and angiogenic cytokine stromal cell-derived factor (SDF) 1α on days 2, 4, and 13. RESULTS: Treatment with EETs and t-AUCB, respectively, significantly accelerated wound epithelialization and neovascularization by synergistic upregulation of SDF1α and VEGF in vivo. CONCLUSIONS: These findings demonstrated that exogenous CYP-derived EETs and globally decreased EET hydrolysis by sEH inhibition significantly accelerated wound epithelialization and neovascularization in unimpaired healing wounds. Given that hypoxia induces CYP expression and subsequently EET-dependent angiogenesis, EETs and sEHIs provide a promising new class of therapeutics for ischemic non-healing wounds.

Systemic transplantation of progenitor cells accelerates wound epithelialization and neovascularization in the hairless mouse ear wound model.

BACKGROUND: Impaired wound healing due to local injury, infection, or systemic diseases, such as diabetes, is a major clinical problem. Recent studies have shown that endothelial progenitor cells (EPC) isolated from peripheral blood, bone marrow, as well as the spleen accumulate in granulation tissue at the site of neovascularization, causing secretion of growth factors and cytokines and thus accelerating wound healing. MATERIALS AND METHODS: In the present study, we transplanted systemic EPC and then measured epithelialization and neovascularization in the hairless mouse ear wound model. RESULTS: Systemic EPC transplantation significantly accelerated epithelialization and neovascularization compared with control wounds receiving phosphate-buffered saline without calcium and magnesium (PBS). The EPC group had significantly higher vascular density than did the PBS-treated group as determined by immunohistochemistry for CD31 and CD90. Fluorescence microscopy revealed accumulation "homing" of the transplanted EPC at the sites of neovascularization in the granulation tissue throughout healing. Furthermore, transplantation of EPC also increased the expression of the angiogenic cytokine stromal cell-derived factor 1α (SDF1α). CONCLUSIONS: This appears to be the first demonstration of EPC recruitment to the site of wound neovascularization throughout the healing process. These findings demonstrate that transplanting systemic EPC into "normal" healing wounds promotes epithelialization and neovascularization and thus could be an useful method for accelerating wound healing.

Prehospital and Early Clinical Care of Infants, Children, and Teenagers Compared to an Adult Cohort : Analysis of 2,961 Children in Comparison to 21,435 Adult Patients from the Trauma Registry of DGU in a 15-Year Period.

BACKGROUND: Although the incidence of pediatric patients in emergency services is as low as 5-10%, trauma remains one of the leading causes of death during childhood. Only a few reports exist about the quality of the initial treatment of pediatric trauma patients. Therefore, we tested the hypothesis of whether prehospital treatment and emergency management in pediatric trauma patients is similar to the treatment that is provided for adult patients. MATERIALS AND METHODS: We performed a retrospective data analysis of the German Trauma Registry of the DGU from January 1993 to December 2007. Exclusion criteria were missing information about injury severity and/or age and patients older than 50 years. All pediatric patients were subdivided into five groups (infants 0-1 year, toddlers 2-5 years, children 6-9 years, pupils 10-13 years, teenagers 14-17 years) with regard to their age and were compared with the adult cohort (18-50 years). From 24,396 patients, 2,961 were below 18 years of age, thus, about 12% of the whole population of injured patients below the age of 50 years. RESULTS: 66.4% of infants sustained relevant head injuries (Abbreviated Injury Scale [AIS] ≥3), and this rate declined with increasing age. The mean Injury Severity Score (ISS) increased from 21.0 (±11.6) in the group of infants to 26.7 (±13.9) in the adult cohort. In all groups, the majority of patients were male. The injury pattern differed according to age, with predominant traumatic brain injury (TBI) in infants. During the preclinical treatment, infants were less often intubated and this was contrasted by a higher rate of cardiopulmonary resuscitation in this group (infants 16.2%, toddlers 6.8%, adults 3.1%). Diagnostic multislice computed tomography (CT) examination was less often performed in infants as compared to the other groups (infants 57.1%, toddlers 77.2%, adults 77.8%). Mortality and quality indicators such as timelines show no significant differences between children and adults. CONCLUSION: We observed typical age-dependent differences regarding the injury pattern and severity and differences referring to the preclinical and initial treatment. With respect to the high rate of serious TBI in the infants and toddlers age groups, a more focused education and training of emergency physicians and paramedics should be considered.

Pediatric Polytrauma Management.

Caring for pediatric trauma patients requires an understanding of the distinct anatomy and pathophysiology of the pediatric population compared to adult trauma patients. Initial evaluation, management, and resuscitation are performed as a multidisciplinary approach including pediatric physicians, trauma surgeons, and pediatric intensive care physicians. Head injury severity is the principle determinant of outcome and mortality in polytraumatized children. Abdominal injuries rarely require surgery in contrast to adults, but need to be detected. Spine and pelvic injuries as well as injuries of the extremities require age-adapted surgical procedures. However, the degree of recovery in polytraumatized children is often remarkable, even after apparently devastating injuries. Maximal care should, therefore, be rendered under the assumption that a complete recovery will be made.

Epidemiology and Management of Injuries to the Spinal Cord and Column in Pediatric Multiple-Trauma Patients.

Injuries to the spinal column and cord in children are a rare condition. Epidemiological data could help to establish an evidence-based assessment and therapy of these patients. We present a retrospective chart analysis of children with spinal injuries who were admitted to the emergency room. The patients were analyzed regarding age, mechanism, and distribution of their injuries to all spinal regions and treatment strategies. Thirty-five children met the inclusion criteria with severe spinal injuries (Abbreviated Injury Scale [AIS] for Region 6 [spine]; AIS region 6) in a period from January 2003 to December 2009. The incidence was extremely low in younger children, with increasing numbers during adolescence. Neurological deficit without fracture accounted for almost 25% of all patients. The majority of patients were treated conservatively; operative treatment was performed in 25% of patients with unstable fractures, particularly in adolescents. Treatment strategies differ according to the type and degree of injury, age, and level of spine maturation.

Fracture and retrosternal dislocation of the medial clavicle in a 12-year-old child--case report, options for diagnosis, and treatment in children.

We report a retrosternally displaced epiphysiolysis in a 12-year-old child and discuss options for the most appropriate diagnostic and therapeutic approach. If a standard anteroposterior view of the shoulder girdle shows abnormalities or if appropriate clinical suspicion is present, we strongly favor an magnetic resonance imaging study for further workup instead of a computed tomographic scan, which is currently the diagnostic method of choice in clinical algorithms. Radiation exposure is thereby limited and maximum information about possible mediastinal soft tissue complications is obtained using a single diagnostic tool. If open reduction is indicated, retention of the joint or approximation of physeal fracture or treatment of ligamentous injury should be performed without metal devices. These suggestions for future management of these patients should further reduce the use of x-rays in children. In addition, applying a suture instead of using metal for stabilization can avoid the extensively described complications in literature that can potentially result from metal devices. In addition, sutures circumvent the need for a second operation for metal removal.