Genetic background, gender, age, body temperature, and arterial blood pH have a major impact on myocardial infarct size in the mouse and need to be carefully measured and/or taken into account: results of a comprehensive analysis of determinants of infarct size in 1,074 mice

In order to determine whether the myocardial response to ischemia/reperfusion (I/R) injury varies depending on genetic background, gender, age, body temperature, and arterial blood pH, we studied 1,074 mice from 19 strains (including 129S6/SvEvTac (129S6), B6/129P2-Ptgs2(tm1Unc), B6/129SvF(2)/J, B6/129/D2, B6/CBAF1, B6/DBA/1JNcr, BALB/c, BPH2/J, C57BL/6/J (B6/J), C3H/DBA, C3H/FB/FF, C3H/HeJ-Pde6b(rd1), FVB/N/J [FVB/N], FVB/B6, FVB/ICR and Crl:ICR/H [ICR]) and distributed them into 69 groups depending on strain and: (1) two phases of ischemic preconditioning (PC); (2) coronary artery occlusion (O) time; (3) gender; (4) age; (5) blood transfusion; (6) core body temperature; and (7) arterial blood pH. Mice underwent O either without (non-preconditioned [naive]) or with prior cyclic O/reperfusion (R) (PC stimulus) consisting of six 4-min O/4-min R cycles 10?min (early PC, EPC) or 24?h (late PC, LPC) prior to 30 or 45-min O and 24?h R. In B6/J and B6/129/D2 mice, almost the entire risk region was infarcted after a 60-min O. Of the naive mouse hearts, B6/ecSOD(WT) and FVB/N mice had infarct sizes significantly smaller than those of the other mice. All strains except FVB/N benefited from the cardioprotection afforded by the early phase of PC; in contrast, development of LPC was inconsistent amongst groups and was strain-dependent. Female gender (1) was associated with reduced infarct size in ICR mice, (2) determined whether LPC developed in ICR mice, and (3) limited the protection afforded by EPC in 129S6 mice. Importantly, mild hypothermia (1?°C decrease in core temperature) and mild acidosis (0.18 decrease in blood pH) resulted in a striking cardioprotective effect in ICR mice: 67.5 and 43.0?% decrease in infarct size, respectively. Replacing blood losses with crystalloid fluids (instead of blood) during surgery also reduced infarct size. To our knowledge, this is the largest analysis of the determinants of infarct size in mice ever published. The results demonstrate that genetic background, gender, age (but not in ICR), body temperature and arterial blood pH have a major impact on infarct size, and thus need to be carefully measured and/or taken into account when designing a study of myocardial infarction in mice; failure to do so makes results uninterpretable. For example, core temperature and blood pH need to be measured, respiratory acidosis (or alkalosis) and hypothermia (or hyperthermia) must be avoided, and comparisons cannot be made between mouse strains or genders that exhibit different susceptibility to I/R injury (e.g., FVB/N male mice and ICR female mice are inherently protected against I/R injury).     

Identification of inducible nitric oxide synthase in peripheral blood cells as a mediator of myocardial ischemia/reperfusion injury

Although the late phase of ischemic preconditioning is known to be mediated by increased inducible nitric oxide synthase (iNOS) activity, controversy persists regarding the role of iNOS in ischemia/reperfusion (I/R) injury and, specifically, whether this protein is protective or detrimental. We hypothesized that iNOS is protective in myocytes but detrimental in inflammatory cells. To test this hypothesis, we created chimeric mice with iNOS-deficient peripheral blood cells by transplanting iNOS knockout (KO) bone marrow in wild-type (WT) mice after lethal irradiation. 2 months later, the mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion. In WT naïve mice (iNOS^+/+ naïve; group I, n = 17), infarct size was 56.9 ± 2.8% of the risk region. In iNOS KO naïve mice with whole-body iNOS deletion (iNOS^?/? naïve; group II, n = 10), infarct size was comparable to group I (53.4 ± 3.5%). When irradiated WT mice received marrow from WT mice (iNOS^+/+ chimera; group III, n = 10), infarct size was slightly reduced versus group I (44.3 ± 3.2%), indicating that irradiation and/or transplantation slightly decrease I/R injury. However, when WT mice received marrow from iNOS KO mice (iNOS^?/? chimera; group IV, n = 14), infarct size was profoundly reduced (22.8 ± 2.1%, P < 0.05 vs. group III), indicating that selective deletion of iNOS from peripheral blood cells (with no change in myocardial iNOS content) induces protection against myocardial infarction. Together with our previous work showing the cardioprotective actions of NO donors, iNOS gene therapy, and cardiac-specific overexpression of iNOS, these data support a complex, dual role of iNOS in myocardial infarction (i.e., protective in myocytes but deleterious in blood cells). To our knowledge, this is the first study to identify a critical role of iNOS in peripheral blood cells as a mediator of myocardial I/R injury. The results support heretofore unknown differential actions of iNOS depending on cell source and have important translational implications.     

透明质酸钠预防兔膝关节固定后关节软骨退行性改变的作用

目的:多项研究成果已证明透明质酸钠在软骨修复及重建过程中具有重要作用。观察关节内注射透明质酸钠对兔膝关节固定后关节软骨退行性改变中的预防作用,并评估其效率。方法:实验于2004-10/2005-05在泰山医学院基础研究所完成。①实验分组:雄性新西兰大白兔24只,体质量2.5～3.0kg,随机分为生理盐水对照组和透明质酸治疗组,每组12只。②实验方法:透明质酸治疗组给予左膝关节内注射0.3mL透明质酸,1次/周,共行5次;生理盐水对照组给予左膝关节注射同等剂量生理盐水,1次/周,共行5次。各组均于初次注射后将左下肢给予石膏管型固定,膝部预留窗口以备下次注射。于末次注射1周后麻醉处死动物,采集左膝关节股骨髁关节软骨制成石蜡切片。同时采集右膝同部位软骨作为正常对照。③实验评估:普通光学显微镜下观察苏木精-伊红染色切片的形态变化;对甲苯胺蓝染色切片利用Image-ProPlus6.0图像分析系统进行平均染色灰度质分析;激光共聚焦显微镜分析免疫荧光标记的Ⅱ型胶原荧光强度。结果:纳入兔24只,均进入结果分析。透明质酸治疗组苏木精-伊红染色显示软骨层无变薄现象,软骨细胞同源细胞簇排列整齐,细胞大小均匀,细胞核染色均匀,潮线较为完整,无血管翳增生;生理盐水对照组苏木精-伊红染色显示软骨层变薄,细胞排列紊乱无规律,细胞大小不等,核染色浓淡不一,潮线不完整,血管翳有侵入软骨基底部的迹象。透明质酸治疗组软骨甲苯胺蓝染色灰度值及Ⅱ型胶原荧光强度均明显高于生理盐水对照组软骨(P<0.01),与正常软骨亦有显著差异(P<0.01)。结论:透明质酸能够有效地预防兔膝关节固定后的软骨退行性改变,但这种预防作用所产生的效果与正常软骨尚有差距。     

p16, p53, EGFR expression and KRAS mutation status in squamous cell cancers of the anus: Correlation with outcomes following chemo-radiotherapy

Background and Purpose

Squamous cell carcinomas of the anal canal are associated with infection with Human Papilloma Viruses (HPVs). Chemo-radiotherapy (CRT) gives 70% 3-year relapse-free survival. Improved predictive markers and therapeutic options are required.

Methods

Tumours from 153 patients treated with radical chemo-radiotherapy (50.4 Gy in 28# with concurrent Mitomycin and 5-Fluorouracil between 2004 and 2009) were retrieved and immunohistochemistry performed for p16^INK4A, p53 and EGFR and correlated with outcome. Primary and relapsed samples were analysed for mutations in KRAS.

Results

137/153 (89.5%) stained moderately or strongly for p16^INK4A. p16^INK4A correlated strongly with outcome. 37/137 patients demonstrating moderate/strong p16^INK4A expression relapsed (27.0%), as opposed to 10/16 (62.5%) with absent/weak staining (log rank test p < 0.001). p16 and p53 expression were inversely correlated. p16^INK4A negative tumours were more frequent in men. p16^INK4A negative patients had significantly worse overall survival (p < 0.001). No mutations in KRAS were identified in primary tumours or relapses following treatment.

Conclusions

p16^INK4A is strongly associated with relapse in SCC of the anus and identifies patients with very poor rates of relapse-free and overall survival. Primary and recurrent anal cancer expresses wild type KRAS, unaffected by treatment, supporting trials targeting EGFR in poor risk/recurrent anal cancer.     

Collection of fungi samples from nails: comparative study of curettage and drilling techniques

Background Onychomycosis is a common problem. Obtaining a positive laboratory test before treatment is important in clinical practice because the treatment of onychomycosis requires expensive oral antifungal therapy with potentially serious side‐effects. Objective The purpose of this study was to compare curettage and subungual drilling techniques of nail sampling in the diagnosis of onychomycosis. Methods We evaluated 194 patients suffering from distal and lateral subungual onychomycosis and lateral subungual onychomycosis using curettage and subungual drilling sampling techniques. Nail samples were obtained in each case from proximal, medial and distal parts of the nail. KOH examination and fungal culture were used for detection and identification of fungal infection. Results With each technique, the culture sensitivity improved as the location of the sample was more proximal (drilling proximal vs. distal, χ² = 5.15, P = 0.023; curettage proximal vs. distal, χ² = 4.2, P = 0.041). In each sample location, the drilling technique has a better culture sensitivity (drilling vs. curettage proximal, χ² = 11.9, P = 0.001; drill vs. curettage distal, χ² = 13.7, P < 0.0001). Trichophyton rubrum was by far the most common pathogen detected by both techniques from all sampling sites. Conclusion The drilling technique was found to be statistically better than curettage at each site of sampling. With each technique, we found that the culture sensitivity improved as the location of the sample was more proximal. More types of pathogens were detected in samples taken by both methods from proximal parts of the affected nails.