Bennett Rachel E, Brody David L
Department of Neurology, Washington University in St. Louis, 660 S. Euclid Avenue, Saint Louis, MO 63110, USA.
Department of Neurology, Washington University in St. Louis, 660 S. Euclid Avenue, Saint Louis, MO 63110, USA.
J Neurosci Methods. 2015 Apr 30;245:25-36. doi: 10.1016/j.jneumeth.2015.02.005. Epub 2015 Feb 14.
Axonal injury is a key feature of several types of brain trauma and neurological disease. However, in mice and humans, many axons are less than 500 nm in diameter which is at or below the resolution of most conventional light microscopic imaging methods. In moderate to severe forms of axon injury, damaged axons become dilated and therefore readily detectible by light microscopy. However, in more subtle forms of injury, the damaged axons may remain undilated and therefore difficult to detect.
Here we present a method for adapting array tomography for the identification and quantification of injured axons. In this technique, ultrathin (∼70 nm) plastic sections of tissue are prepared, labeled with axon injury-relevant antibodies and imaged using conventional epifluorescence.
To demonstrate the use of array-tomography-based methods, we determined that mice that received two closed-skull concussive traumatic brain injury impacts had significantly increased numbers of non-dilated axons that were immunoreactive for non-phosphorylated neurofilament (SMI-32; a marker of axonal injury), compared to sham mice (1682±628 versus 339±52 per mm(2), p=0.004, one-tailed Mann-Whitney U test). Tubulin loss was not evident (p=0.2063, one-tailed Mann-Whitney U test). Furthermore, mice that were subjected to more severe injury had a loss of tubulin in addition to both dilated and non-dilated SMI-32 immunoreactive axons indicating that this technique is suitable for the analysis of various injury conditions.
With array tomography we could detect similar overall numbers of axons as electron microscopy, but accurate diameter measurements were limited to those with diameters >200 nm. Importantly, array tomography had greater sensitivity for detecting small non-dilated injured axons compared with conventional immunohistochemistry.
Imaging of individual axons and quantification of subtle axonal injury is possible using this array tomography method. This method may be most useful for the assessment of concussive injuries and other pathologies in which injured axons are not typically dilated. The ability to process moderately large volumes of tissue, label multiple proteins of interest, and automate analysis support array tomography as a useful alternative to electron microscopy.
轴突损伤是多种类型脑外伤和神经疾病的关键特征。然而，在小鼠和人类中，许多轴突直径小于500纳米，这处于或低于大多数传统光学显微镜成像方法的分辨率。在中度至重度形式的轴突损伤中，受损轴突会扩张，因此通过光学显微镜很容易检测到。然而，在更轻微的损伤形式中，受损轴突可能不会扩张，因此难以检测。
在此，我们提出一种使阵列断层扫描适用于识别和量化受损轴突的方法。在该技术中，制备组织的超薄（约70纳米）塑料切片，用与轴突损伤相关的抗体进行标记，并使用传统落射荧光成像。
为了证明基于阵列断层扫描方法的用途，我们确定，与假手术小鼠相比，接受两次闭合性颅骨冲击性脑损伤的小鼠中，对非磷酸化神经丝（SMI - 32；轴突损伤标志物）呈免疫反应的未扩张轴突数量显著增加（每平方毫米1682±628个对339±52个，p = 0.004，单尾曼 - 惠特尼U检验）。微管蛋白损失不明显（p = 0.2063，单尾曼 - 惠特尼U检验）。此外，遭受更严重损伤的小鼠除了有扩张和未扩张的SMI - 32免疫反应性轴突外，还出现了微管蛋白损失，这表明该技术适用于分析各种损伤情况。
通过阵列断层扫描，我们能检测到与电子显微镜相似的轴突总数，但精确的直径测量仅限于直径>200纳米的轴突。重要的是，与传统免疫组织化学相比，阵列断层扫描在检测小的未扩张受损轴突方面具有更高的灵敏度。
使用这种阵列断层扫描方法可以对单个轴突进行成像并对细微的轴突损伤进行量化。该方法可能对评估脑震荡损伤和其他受损轴突通常不扩张的病理情况最为有用。处理中等体积组织、标记多种感兴趣蛋白质以及自动分析的能力支持将阵列断层扫描作为电子显微镜的一种有用替代方法。
