Histological evaluation of healing tendons is primarily focused on monitoring restoration of longitudinal collagen alignment, although the elastic property of energy-storing flexor tendons is largely attributed to interfascicular sliding facilitated by the interfascicular matrix (IFM). The objectives of this study were to explore the utility of second harmonic generation (SHG) imaging to objectively assess cross-sectional tendon fascicle architecture, to combine SHG microscopy with elastin immunofluorescence to assess the ultrastructure of collagen and elastin in longitudinal and transverse sections, and lastly, to quantify changes in IFM elastin and fascicle collagen alignment of normal and collagenase-injured flexor tendons. Paraffin-embedded transverse and longitudinal histological sections (10-μm thickness) derived from normal and collagenase-injured (6- and 16-week time-points) equine superficial digital flexor tendons were de-paraffinized, treated with Tris EDTA at 80°C for epitope retrieval, and incubated with mouse monoclonal anti-elastin antibody (1:100 dilution) overnight. Anti-mouse IgG Alexa Flour 546 secondary antibody was applied, and sections were mounted with ProLong Gold reagent with 4′,6-diamidino-2-phenylindole (DAPI). Nuclei (DAPI) and elastin (Alexa Fluor 546) signals were captured by using standard confocal imaging with 405 and 543 nm excitation wavelengths, respectively. The SHG signal was captured by using a tunable Ti:Sapphire laser tuned to 950 nm to visualize type I collagen. Quantitative measurements of fascicle cross-sectional area (CSA), IFM thickness in transverse SHG-DAPI merged z-stacks, fascicle/IFM elastin area fraction (%), and elastin–collagen alignment in longitudinal SHG-elastin merged z-stacks were conducted by using ImageJ software. Using this methodology, fascicle CSA, IFM thickness, and IFM elastin area fraction (%) at 6 weeks (∼2.25-fold; ∼2.8-fold; 60% decrease; p < 0.001) and 16 weeks (∼2-fold; ∼1.5-fold; 70% decrease; p < 0.001) after collagenase injection, respectively, were found to be significantly different from normal tendon. IFM elastin and fascicle collagen alignment characterized via fast Fourier transform (FFT) frequency plots at 16 weeks demonstrated that collagen re-alignment was more advanced than that of elastin. The integration of SHG-derived quantitative measurements in transverse and longitudinal tendon sections supports comprehensive assessment of tendon structure. Our findings demonstrate the importance of including IFM and non-collagenous proteins in tendon histological evaluations, tasks that can be effectively carried out by using SHG and immunofluorescence microscopy.

中文翻译：

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二次谐波产生和免疫荧光显微镜相结合的肌腱层级结构定量评估。

愈合肌腱的组织学评估主要集中在监测纵向胶原蛋白排列的恢复，尽管储能屈肌腱的弹性主要归因于束间基质（IFM）促进的束间滑动。这项研究的目的是探索二次谐波生成（SHG）成像在客观评估肌腱横断面束结构中的实用性，将SHG显微镜与弹性蛋白免疫荧光结合以评估纵断面和横断面胶原和弹性蛋白的超微结构以及最后，量化正常和胶原酶损伤的屈肌腱的IFM弹性蛋白和束胶原排列的变化。将源自正常和胶原酶损伤（6周和16周时间点）马浅指屈肌腱的石蜡包埋的横向和纵向组织切片（厚度为10μm）去石蜡化，在80°C下用Tris EDTA处理取抗原决定簇，并与小鼠单克隆抗弹性蛋白抗体（1：100稀释）孵育过夜。应用抗小鼠IgG Alexa Flour 546二抗，并用ProLong Gold试剂和4'，6-diamidino-2-phenylindole（DAPI）固定切片。通过使用分别具有405和543 nm激发波长的标准共聚焦成像来捕获核（DAPI）和弹性蛋白（Alexa Fluor 546）信号。SHG信号是通过使用可调至950 nm的可调式Ti：Sapphire激光器捕获的，以显示I型胶原蛋白。束横截面积（CSA）的定量测​​量–使用ImageJ软件在纵向SHG-弹性蛋白合并的Z堆栈中进行胶原蛋白排列。使用这种方法，在6周（〜2.25倍;〜2.8倍;降低60％; p  <0.001）和16周（〜2倍;〜）时，分册CSA，IFM厚度和IFM弹性蛋白面积分数（％）1.5倍;降低70％; p 分别在注射胶原酶后<0.001）与正常肌腱明显不同。通过16周快速傅里叶变换（FFT）频率图表征的IFM弹性蛋白和束状胶原排列表明，胶原蛋白重新排列比弹性蛋白更先进。SHG派生的横向和纵向肌腱截面定量测量结果的集成支持对肌腱结构的全面评估。我们的发现表明，在肌腱组织学评估中包括IFM和非胶原蛋白的重要性，这些任务可以通过使用SHG和免疫荧光显微镜有效地完成。