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

Abstract

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 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.

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.

Keywords: Array tomography; Axonal injury; Electron microscopy; Neurofilament; Traumatic brain injury; Tubulin.

Figure 1. Array tomography workflow

(1) Tissue is cut into 5 × 1.5 × 1 mm blocks containing corpus callosum and external capsule (Franklin and Paxinos, 2004) which are embedded in LR white media in gelatin capsules (inset, top). An ultramicrotome is used to produce 70–90 nm section ribbons using a histojumbo diamond knife. (2) Standard immunofluorescent techniques are used to label each ribbon and images from sections are captured using a 63× lens on an epifluorescent microscope. (3) Each image can then be further subdivided into smaller regions for analysis, excluding cell bodies and tissue processing artifacts.

Figure 2. Example of a short array containing uninjured mouse external capsule labeled with anti-tubulin and Alexa 488

(A–K) Images of eleven 70 nm thick ultrathin sections labeled with anti-tubulin and Alexa 488. Images have been co-registered so that each represents the same 19.5 × 19.5 µm area. (L) A projection of the 11 image stack shows a reconstruction of individual, longitudinally/transversely cut axons within this stack.

Figure 3. Side by side comparison of tubulin labeling in thick sections and ultrathin sections

(A) A confocal X–Y projection image of a section of uninjured mouse corpus callosum mouse cut at a 50 µm thickness on a freezing microtome and labeled with anti-tubulin and Alexa 488. (B) A confocal X–Y projection image of an ultrathin section from a similar region of corpus callosum cut at 90 nm on ultramicrotome and labeled with anti-tubulin and Alexa 488. (C and D) are X–Z images showing the improved spatial resolution along the Z axis.

Figure 4. Electron microscopy versus tubulin-labeled ultrathin sections in uninjured wild-type mouse corpus callosum and external capsule

(A–C) Electron micrographs or (DF) tubulin-Alexa 488 fluorescence images were obtained of axonal cross-sections and were used for axon diameter measurements. (G) Frequency distribution of axon diameter measurements from electron micrographs (EM) and ultrathin sections used in array tomography (AT). An equal tissue area was examined by each technique. A total of 5 uninjured mice were included in this analysis, with the right hemisphere being prepared for EM and the left for AT.

Figure 5. Axonal injury markers SMI-31, SMI-32 and APP 24 hours in uninjured sham or 1.0 mm CCI in the ipsilateral external capsule

(A) SMI-31-Alexa 488 labels axons in an uninjured mouse while (B) SMI-32-Alexa 488 does not. Both images are co-labeled for tubulin- Alexa 594 to visualize axons and DAPI to indicate cell nuclei. After injury, (C) APP Cy3 labeled axons (inset is from CCI injured APP knockout mouse indicating non-specific binding). (D) SMI- 31 and (E) composite image of DAPI, SMI-31, and APP. (F) APP Cy3 labeled axons. (G) SMI-32 Alexa 488 labeled axons. (H) Composite image of DAPI, SMI-32, and APP. All images are from wild-type mice except panel C inset.

Figure 6. Conventional immunohistochemistry for APP and SMI-32 in external capsule

Representative images of axonal injury from 1 day CCI (A, E), 7 day sham (B, F), 2 day rcTBI (C, G) or 7 day rcTBI (D, H) in wild-type mice. Sections were labeled for amyloid precursor protein (A, B, C, D) or with antibodies to SMI-32 (E, F, G, H). Images in CCI mice were captured from pericontusional corpus callosum. All other images were taken from external capsule directly underlying the site of impact (or sham injury) near the lateral ventricle, which was the only region where axonal varicosities were observed using these markers.

Figure 7. Projection images of arrays (~20 sections each) from external capsule labeled with the axonal injury marker SMI-32-Alexa 594 and tubulin-Alexa 488

(A, B) Axons from uninjured wild-type mice displayed little SMI-32 labeling. (C,D) Mice subjected to repetitive concussive traumatic brain injury had punctate areas of SMI-32 labeling and occasional colocalization of SMI-32 and tubulin in swollen axons at 7 days. (E,F) Larger axonal varicosities >3 µm in diameter were apparent in a model of 1.5 mm CCI moderate traumatic brain injury at 7 days. Tubulin loss was also evident. Panels in each row represent images of arrays from two separate mice.

Figure 8. Quantification of injured axons following repetitive concussive traumatic brain injury

(A) Example of a tubulin labeled section and the scheme for imaging corpus callosum and external capsule beginning at the medial edge of the cingulum and continuing laterally until 5 images are captured. (B) Example of a field included in the analysis for tubulin-Alexa 488 (top) and SMI-32-Alexa 594 (bottom) shown pre- and post-thresholding (“Moments” threshold selected for tubulin and “MaxEntropy” threshold selected for SMI-32 in Image J). (C) Quantification of SMI-32 puncta and tubulin labeled axons per mm² in sham and rcTBI mice 7 days post injury (Error bars represent standard error of the mean, **p<0.01).

Figure 9. Additional axonal markers for use with array tomography

(A–C) Myelin basic protein and tubulin labeling in the external capsule of an uninjured wild-type mouse. (A) Tubulin- Alexa 594 labeled axons. (B) Myelin basic protein (MBP)-Alexa 488 labeled axons. (C) Composite image of DAPI, myelin basic protein, and tubulin. Inset shows an enlarged view of the box in (C), where individual myelinated axonal cross-sections are clearly visible. (D–F) PHF-1 tau and tubulin labeling in the entorhinal cortex of a 12-month-old Tau P301S mouse. (D) PHF1-Cy3 labeled punctae. Inset shows the absence of PHF1 labeling in cortex from a tau knockout mouse. (E) Tubulin Alexa 488 labeled neuropil. (F) Composite image of DAPI, tubulin, and PHF1.