Targeted metabolic profiling of wounds in diabetic and nondiabetic mice

While cellular metabolism is known to regulate a number of key biological processes such as cell growth and proliferation, its role in wound healing is unknown. We hypothesized that cutaneous injury would induce significant metabolic changes and that the impaired wound healing seen in diabetes would be associated with a dysfunctional metabolic response to injury. We used a targeted metabolomics approach to characterize the metabolic profile of uninjured skin and full‐thickness wounds at day 7 postinjury in nondiabetic (db/‐) and diabetic (db/db) mice. By liquid chromatography mass spectrometry, we identified 129 metabolites among all tissue samples. Principal component analysis demonstrated that uninjured skin and wounds have distinct metabolic profiles and that diabetes alters the metabolic profile of both uninjured skin and wounds. Examining individual metabolites, we identified 62 with a significantly altered response to injury in the diabetic mice, with many of these, including glycine, kynurenate, and OH‐phenylpyruvate, implicated in wound healing for the first time. Thus, we report the first comprehensive analysis of wound metabolic profiles, and our results highlight the potential for metabolomics to identify novel biomarkers and therapeutic targets for improved wound healing outcomes.     

Effect of a diode laser on wound healing by using diabetic and nondiabetic mice

The purpose of this study was to evaluate a 980-nm gallium-aluminum-arsenide diode laser for wound healing. Using genetically diabetic and nondiabetic mice, two 6-mm wounds were created on the back of each mouse by using a punch biopsy. The mice were assigned to 1 of 4 subgroups for laser treatment at different fluence and frequency of treatment: 5 W (18 J/cm2) every 2 days, 5 W (18 J/cm2) every 4 days, 10 W (36 J/cm2) every 2 days, and 10 W (36 J/cm2) every 4 days. In addition, control mice were used and the wounds were allowed to heal naturally. Wound healing was evaluated on days 5, 12, and 19 by percentage of wounds healed and percent wound closure. A maximum of 5 mice per subgroup were killed at days 7, 14, and 21, and histology was conducted on the wound sites. For diabetic mice receiving 5 W every 2 days, the percentage of wounds healed after 19 days was 100% versus 40% in the control group. Only 20% of wounds in the 10-W diabetic subgroups achieved healing during the same period. For the subgroups whose wounds did not completely heal, all but the 10 W every 2 days subgroup had average closure of >90%. The 100% closure for the 5 W every 2 days subgroup was significantly greater than the other subgroups. For nondiabetic mice, 100% of the wounds in the 5 W every 4 days and control subgroups were completely healed, whereas 90% of the wounds from the 5 W every 2 days and the 10 W every 4 days subgroups were completely healed. In the latter 2 subgroups, wound closure was 99.4% and 98.8%, respectively. These differences were not significant. The histologic results confirmed these findings. In conclusion, treatment at 18 J/cm2 shows a beneficial effect on wound healing in diabetic mice and does not have a detrimental effect in nondiabetic mice.     

胸腺瘤细胞中PCNA、HSP70的表达水平的研究及意义

目的 研究PCNA、HSP70在胸腺瘤细胞及正常胸腺组织中的表达水平及其临床意义。方法 用免疫组织化学链霉菌抗生物素蛋白－过氧化物酶染色法（S—P法）检测20例恶性胸腺瘤、18例良性胸腺瘤及12例正常胸腺组织细胞中PCNA、HSP70蛋白的表达水平。结果 20例恶性胸腺瘤中PCNA、HSP70蛋白阳性表达分别为80．0％（16／20）、75．0％（15／20）；18例良性胸腺瘤中PCNA、HSP70蛋白阳性表达分别为38．9％（7／18）、27．8％（5／18）；12例正常胸腺组织细胞中分别为8．3％（1／12）、0（0／12），PCNA蛋白及HSP70蛋白的表达在良、恶性胸腺瘤和正常胸腺组织三者之间差异都有显著性（P〈0．001），且在良、恶性胸腺瘤之间相比差异也均有显著性（P〈0．05）。但良性胸腺瘤和正常胸腺组织相比，PCNA及HSP70蛋白的表达差异均无显著性（P〉0.05）。结论 通过检测HSP70与PCNA在胸腺瘤的表达水平，可以判断胸腺瘤的良、恶性，为术后进一步治疗提供了有价值的参考指标。     

热休克蛋白70和27在非小细胞肺癌中的表达和临床意义

目的研究热休克蛋白(heatshockprotein,HSP)70和27在非小细胞肺癌(nonsmallcelllungcancer,NSCLC)组织中的表达,探讨HSP70和HSP27与NSCLC细胞的组织学类型、分化和转移之间的关系。方法采用免疫组化SP法检测60例NSCLC标本中HSP70和HSP27的表达情况。结果NSCLC中HSP70和HSP27的表达与肿瘤细胞组织学类型无关。HSP70和HSP27的表达正相关。HSP70的表达与肿瘤细胞分化程度和淋巴结转移明显相关,HSP27的表达则无关。结论HSP70在NSCLC中的高表达与肿瘤细胞的分化和转移明显正相关,提示HSP70可以作为NSCLC辅助诊断和估计预后的指标。而HSP27尽管在多种恶性肿瘤中发挥促进细胞恶变的作用,但在NSCLC中的作用需要进一步研究。     

In vivo pH of induced soft-tissue abscesses in diabetic and nondiabetic mice

Infections in the diabetic host have been shown to persist longer than those in the nondiabetic host. To investigate whether intra-abscess milieu might be a contributing factor to this persistence, the in vivo intra-abscess pH was measured in induced soft-tissue abscesses in diabetic and nondiabetic mice. Two models (female genetically obese insulin-resistant and male streptozocin-induced diabetic mice) were used with appropriate controls. The bacteria injected to produce the soft-tissue abscesses were Bacteroides fragilis and Enterococcus (B + E), Staphylococcus epidermidis and Enterococcus (S + E), and S. aureus (SA). Intra-abscess pH measured on day 3 was consistently and significantly lower in all diabetic mice compared with their controls. In the diabetic mice, the pH of an abscess induced with B + E, S + E, and SA was 6.28 (n = 17), 6.79 (n = 10), and 6.52 (n = 10), respectively; the pH in the controls was 7.21 (n = 20), 7.30 (n = 10), and 7.17 (n = 10), respectively. Differences in all groups between diabetic and nondiabetic mice were significant. The blood glucose values of the diabetic mice averaged 722 mg/dl, and in the nondiabetic mice were 210 mg/dl. No animals were ketotic. There were no significant differences in total colony counts between any groups. In conclusion, there is a significantly lower pH in the abscess of the diabetic host compared with the nondiabetic host that is not related to the numbers or types of causative bacteria.     

Nerve growth factor accelerates wound healing in diabetic mice.

Patients with diabetic neuropathy have reduced numbers of cutaneous nerves, which may contribute to an increased incidence of nonhealing wounds. Nerve growth factor (NGF) has been reported to augment wound closure. We hypothesized that topical 2.5S NGF, a biologically active subunit of the NGF polymer, would accelerate wound repair, augment nerve regeneration, and increase inflammation in excisional wounds in diabetic mice. A full-thickness 6-mm punch biopsy wound was created on the dorsum of C57BL/6J-m+ Leprdb mice (db/db) and heterozygous (db/-) littermates and treated daily with normal saline or 2.5S NGF (1 microg/day or 10 microg/day) on post-injury days 0-6. Time to closure, wound epithelialization, and degree of inflammation were compared using a Student's t-test. Color subtractive-computer-assisted image analysis was used to quantify immunolocalized nerves in wounds. Non-overlapping (20x) digital images of the wound were analyzed for nerve profile counts, area density (number of protein gene product 9.5 positive profiles per unit dermal area) and area fraction (protein gene product 9.5 positive area per unit dermal area). Healing times in db/db mice decreased from 30 days in normal saline-treated mice to 26 days in mice treated with 1 microg/day NGF (p<0.05) and 24 days in mice treated with 10 microg/day NGF (p<0.02). A similar trend in db/- mice was not significant. NGF treatment augmented epithelialization in the db/db mice (p<0.05). Histological evaluation of inflammation in healed wounds showed no statistical difference between treatment groups. Total nerve number, area density, and area fraction were increased in NGF-treated wounds at 14, 21, and 35 days (p<0.05). The 2.5 NGF subunit may improve wound closure kinetics by promoting epithelialization and nerve regeneration. Further studies to determine the role of nerves in wound repair are warranted.     

氯胺酮诱导的热休克蛋白70在大鼠脑组织中不同区域的表达

目的 研究氯胺酮诱导的热休克蛋白70(HSP70)在大鼠脑组织中不同区域的表达.方法 SD大鼠20只,随机均分为氯胺酮组(K组)和生理盐水组(N组),分别在腹腔注射氯胺酮80mg/kg和生理盐水3ml.24h后取鼠脑,应用HSP70单克隆抗体免疫组化染色检测胼胝体压部后皮质、海马、大鼠后扣带回和枕叶皮质中HSP70的表达,并用MIAS-2000图文分析系统分析上述区域HSP70阳性细胞百分率、阳性细胞密度(个/mm2)和阳性产物灰度值.结果 氯胺酮可诱导大鼠大脑特定区域表达HSP70;大脑各区域HSP70表达不同.结论 氯胺酮诱导HSP70在大脑特定区域的表达,提示可能产生神经细胞损害;氯胺酮对大脑特定区域产生损害.     

经高温处理后大鼠肝中热休克蛋白70的表达

目的　研究热休克蛋白 70 (HSP 70 )在高温处理后大鼠肝中不同时段的表达。方法　SD大鼠随机分成A组 (对照组 )和B组 (高温组 ,42℃ ,2 0min)。A组分别在麻醉后 4,8,2 0 ,3 6,72h取肝脏组织 ;B组置于自行设计的加热装置分别热处理后 4,8,2 4,3 6,72h取肝脏组织。用免疫组织化学、原位杂交方法检测A ,B两组不同时段HSP70表达 ,并对比其差异。结果　高温处理组各时段HSP70的表达差异有显著性 (P <0 .0 5 ) ;8～ 2 4h达最高峰 (P <0 .0 1)。高温处理后HSP70的表达明显高于对照组 (P <0 .0 5 )。结论　高温可诱导HSP70在大鼠肝中的高表达 ,处理后不同时段表达的量不同。     

The healing of liver wounds.

Cases of traumatic hemobilia are often characterized by a protracted history with recurrent episodes of hemorrhage over many years. It seems that while the liver regenerates quickly and profusely it heals slowly and poorly. This is in contradistinction to the kidneys where lesions often heal within a short time. In order to investigate the cause of this peculiar behavior a series of experiments was performed where local lesions were produced in the musculature, in the kidneys and in the liver of dogs. The healing process was studied at different intervals. There was essentially no difference between the rate and character of healing in the three localities. As the lesions that produce hemobilia are open to the biliary tract, another series of experiments was performed where the healing took place in presence of bile. The effect of this was striking with a very diminished production of fibrinous exudate, granulating tissue and fibrous scar.     

Measurement of optical properties to quantify healing of chronic diabetic wounds

Measurement of scattering and absorption characteristics of tissue by near infrared spectroscopy may provide a clinically applicable, quantitative method of assessing healing of diabetic wounds. Twenty SICH-1 hairless rats were divided into a control group and a streptozotocin-induced diabetic group. Full-thickness wounds were made on the dorsal surface of each animal. Wounds were examined using a near infrared device with three wavelengths of incident light. Amplitude and phase of scattered light were obtained at four different source detector distances at each wavelength. The probe was positioned at six different locations. Tissue absorption and scattering coefficients were calculated from amplitude and phase data. Wound dimensions were calculated by cross and parallel polarization. Thirty-eight wounds were evaluated during the experiment. Wound size decreased at twice the rate in the control animals, whereas the average absorption coefficient was higher by a factor of two while the average value of the reduced scattering coefficient was 30% higher in the diabetic wounds. During healing, both scattering and absorption coefficients increased faster in the diabetics. Higher absorption in diabetic rats suggests impaired blood flow. Scattering may reflect tissue disorganization observed in delayed wound healing. We conclude that near infrared spectroscopy may differentiate between healing diabetic and nondiabetic wounds.     

Impaired healing of nitrogen mustard wounds in CXCR2 null mice

To examine the significance of chemokine activation of CXCR2 in wound healing after chemical burn, cutaneous injury was created by topical application of nitrogen mustard on CXCR2 wild type (+/+), heterozygous (+/-), and knockout (-/-) mice. Wounds were analyzed histologically for neutrophil and monocyte infiltration and for reepithelialization at postwound days 4, 7, and 10. Neutrophil recruitment to the wound site was reduced through postwound day 7 in CXCR2 -/- mice as indicated by myeloperoxidase assay and by visual quantitation. Because there is always concern that mice with targeted deletion of a specific receptor may undergo developmental adaptations to offset the loss of the receptor, we also accessed chemical wound repair in the presence of a small molecule antagonist of CXCR2. Dietary supplementation with a CXCR2 antagonist (SB-265610) during the wound repair process also markedly delayed healing parameters in CXCR2 +/+ mice, even greater than treatment with glucocorticoids. These parallel studies further establish that mice deficient in CXCR2 function exhibit delayed cutaneous wound healing that may be primarily linked to impaired neutrophil recruitment after chemical burn with nitrogen mustard. Thus, there may be a potential therapeutic benefit of treating nitrogen mustard-induced skin lesions with agonists of CXCR2 to facilitate the wound repair process.     

Wound healing potential of topical bacteriophage therapy on diabetic cutaneous wounds

Chronic wounds that fail to heal are a common complication of diabetes mellitus and the most common precipitating reason for nontraumatic lower limb amputation. Unfortunately, the bacterial species that cause these infections are becoming more resistant to antibiotics, making them increasingly difficult to treat. We assessed the feasibility of combating chronic bacterial infections with a topically delivered bacteriophage cocktail in two animal models of diabetes mellitus. Microbiological, planimetric, and histological parameters were compared in debrided infected wounds with or without topical bacteriophage treatment. We determined that bacteriophage treatment effectively decreased bacterial colony counts and improved wound healing, as indicated by smaller epithelial and dermal gaps, in Staphylococcus aureus and Pseudomonas aeruginosa infections but was not as effective against Acinetobacter baumannii. Although the improvements were more significant in the rodent model than in the porcine model, our results suggest that topically administered bacteriophage treatment may be effective in resolving chronic infections, especially when applied in conjunction with wound debridement. These findings have important implications for the feasibility of using topical antimicrobial therapies to safely treat chronic infections in diabetes mellitus patients.     

Myofibroblasts in healing laser excision wounds.

BACKGROUND AND OBJECTIVE: The lack of myofibroblasts, cells responsible for wound contraction, has been suggested to be the underlying factor to the clinically observed minimal contraction in CO2 laser wounds. However, the histologic background to this phenomenon in laser excisions has not been thoroughly clarified. Therefore, we analyzed the expression of myofibroblasts in healing laser excisions and control excisions made by scalpel. STUDY DESIGN/MATERIALS AND METHODS: CO2 laser (continuous wave, 5 W) or scalpel excision wounds were created in the dorsal tongue mucosa of 144 rats. Sixteen additional rats were kept as untreated controls. Specimens from the tongues were cut at 16 different healing time points and fixed in 10% formalin. Immunohistochemical stainings with monoclonal antibodies to vimentin and to alpha-smooth muscle actin were done to determine microscopically the contractile type of myofibroblasts. RESULTS: The maximum amount of myofibroblasts was almost three times higher in scalpel than in laser excisions. The peak value was reached at 4 days in laser and at 3 days in scalpel wounds. The increase reverted to normal levels at 14 days in laser and at 6 days in scalpel wounds, respectively. CONCLUSION: Myofibroblasts appeared and disappeared slower in laser wounds. There were clearly fewer myofibroblasts in CO2 laser than in corresponding scalpel excisions known to heal by contraction. The lack of contractile myofibroblasts, therefore, is suggested as the reason for the minimal degree of contraction in CO2 laser excision wounds.