Optic tract injury after closed head traumatic brain injury in mice: A model of indirect traumatic optic neuropathy

Abstract

Adult male C57BL/6J mice have previously been reported to have motor and memory deficits after experimental closed head traumatic brain injury (TBI), without associated gross pathologic damage or neuroimaging changes detectable by magnetic resonance imaging or diffusion tensor imaging protocols. The presence of neurologic deficits, however, suggests neural damage or dysfunction in these animals. Accordingly, we undertook a histologic analysis of mice after TBI. Gross pathology and histologic analysis using Nissl stain and NeuN immunohistochemistry demonstrated no obvious tissue damage or neuron loss. However, Luxol Fast Blue stain revealed myelin injury in the optic tract, while Fluoro Jade B and silver degeneration staining revealed evidence of axonal neurodegeneration in the optic tract as well as the lateral geniculate nucleus of the thalamus and superior colliculus (detectable at 7 days, but not 24 hours, after injury). Fluoro Jade B staining was not detectable in other white matter tracts, brain regions or in cell somata. In addition, there was increased GFAP staining in these optic tract, lateral geniculate, and superior colliculus 7 days post-injury, and morphologic changes in optic tract microglia that were detectable 24 hours after injury but were more prominent 7 days post-injury. Interestingly, there were no findings of degeneration or gliosis in the suprachiasmatic nucleus, which is also heavily innervated by the optic tract. Using micro-computed tomography imaging, we also found that the optic canal appears to decrease in diameter with a dorsal-ventral load on the skull, which suggests that the optic canal may be the site of injury. These results suggest that there is axonal degeneration in the optic tract and a subset of directly innervated areas, with associated neuroinflammation and astrocytosis, which develop within 7 days of injury, and also suggest that this weight drop injury may be a model for studying indirect traumatic optic neuropathy.

Fig 1. Experimental timeline.

Animals were divided into two main cohorts, with study performed at 1 day post-TBI or 7 days post-TBI. Animals were sacrificed at 1 day or 7 days post-TBI (n = 8 sham and 8 TBI at each time point). TBI: traumatic brain injury.

Fig 2. Weight change.

Animals with TBI lost a slight amount of weight between pre-surgery weights and 7 days post-injury. There was no significant difference overall between TBI and sham animals, and there was no difference between day 1 and day 7 sham animals (n = 16 per group at each time point). * p < 0.05 vs pre-surgery weight.

Fig 3. NeuN and Nissl staining.

There was no discernible difference in the appearance of NeuN or Nissl staining between TBI and sham animals 7 days post-injury. A, C, E, G are from sham animals and B, D, F, H are from TBI animals. Typical NeuN patterns are shown for prefrontal cortex (A, B) and lateral geniculate nucleus of the thalamus (LGN, C, D). Typical appearance of Nissl stained sections are also illustrated, from prefrontal cortex (E, F) and LGN (G, H).

Fig 4. Luxol Fast Blue myelin stain.

Representative micrographs from sham (A) or TBI (B) animals are shown, centered over the optic tract. There is a subjective decrease in the intensity of staining in the optic tract of TBI animals, 7 days after injury. In addition, there are globular collections of positive staining seen within the optic tract, consistent with the appearance of myelin after white matter injury. For clarity, several of these myelin globules are indicated by arrows in part B of the figure. OT–optic tract. Scale bar indicates 50 μm.

Fig 5. Fluoro Jade B staining for neurodegeneration.

Representative staining from sham (A, C, E) and TBI (B, D, F) animals, from the 7 day post-injury group. Sections from optic tract (A, B), LGN (C, D), and superior colliculus (E, F) are shown. Inset images are higher magnification photographs from the same region. There is a punctate staining pattern present in all three areas in animals with TBI, 7 days after injury, which is absent in TBI mice 1 day post-injury and in sham animals at either time point. The 50 μm scale bar applies to all low-magnification micrographs and the 20 μm bar applies to all inset micrographs.

Fig 6. NeuroSilver degeneration stain.

Representative staining from sham (A, C, E) and TBI (B, D, F) animals, using a silver stain for neurodegeneration (NeuroSilver II stain). Sections from optic tract (A, B), LGN (C, D), and superior colliculus (E, F) are shown. There is positive staining in a similar pattern to that seen in Fig 5 for Fluoro Jade B staining.

Fig 7. GFAP staining.

Mean gray levels from TBI and sham animals in optic tract, LGN, SC, SCN, or visual cortex at either 1 day (A, E, I, M, Q) or 7 days (B, F, J, N, R) post-injury (n = 8 mice per group at each time point). * p < 0.05 vs sham group in the same day. Also shown are representative photomicrographs from each of these areas in sham (C, G, K, O, S) and TBI (D, H, L, P, T) animals, at 7 days post-injury.

Fig 8. Iba-1 staining and microglial morphology analysis.

Representative photomicrographs of the optic tract of sham (A) and TBI (B) animals, 7 days post-injury. (C–H) show mean area of microglia in optic tract, LGN, and SC (n = 8 per group at each time point). * p < 0.05.

Fig 9. MicroCT analysis.

Skull shape and measurements were evaluated using micro CT scanning either at rest (A, C, E, G), or under dorsal-ventral compression using a wooden clamp (B, D, F, H). Images were in the sagittal (A, B), coronal (C–F), or axial (G, H) planes. Measurements recorded in Table 3 were taken from these planes as indicated in A, C, and E. The approximate position of the eye and optic nerve are schematized in G and H.