Array tomography for the detection of non-dilated, injured axons in traumatic brain injury

Background: 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. New method: 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. Results: 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 mm², 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. Comparison with existing method: 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. Conclusion: 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.