Inflammation and white matter degeneration persist for years after a single traumatic brain injury

Abstract

A single traumatic brain injury is associated with an increased risk of dementia and, in a proportion of patients surviving a year or more from injury, the development of hallmark Alzheimer's disease-like pathologies. However, the pathological processes linking traumatic brain injury and neurodegenerative disease remain poorly understood. Growing evidence supports a role for neuroinflammation in the development of Alzheimer's disease. In contrast, little is known about the neuroinflammatory response to brain injury and, in particular, its temporal dynamics and any potential role in neurodegeneration. Cases of traumatic brain injury with survivals ranging from 10 h to 47 years post injury (n = 52) and age-matched, uninjured control subjects (n = 44) were selected from the Glasgow Traumatic Brain Injury archive. From these, sections of the corpus callosum and adjacent parasaggital cortex were examined for microglial density and morphology, and for indices of white matter pathology and integrity. With survival of ≥3 months from injury, cases with traumatic brain injury frequently displayed extensive, densely packed, reactive microglia (CR3/43- and/or CD68-immunoreactive), a pathology not seen in control subjects or acutely injured cases. Of particular note, these reactive microglia were present in 28% of cases with survival of >1 year and up to 18 years post-trauma. In cases displaying this inflammatory pathology, evidence of ongoing white matter degradation could also be observed. Moreover, there was a 25% reduction in the corpus callosum thickness with survival >1 year post-injury. These data present striking evidence of persistent inflammation and ongoing white matter degeneration for many years after just a single traumatic brain injury in humans. Future studies to determine whether inflammation occurs in response to or, conversely, promotes white matter degeneration will be important. These findings may provide parallels for studying neurodegenerative disease, with traumatic brain injury patients serving as a model for longitudinal investigations, in particular with a view to identifying potential therapeutic interventions.

Figure 1

Representative images of observed CR3/43 immunoreactivity in the corpus callosum following TBI versus control subjects. (A–C) Representative images showing increasing CR3/43 reactivity with age as has been previously reported. (A) Virtually absent CR3/43 immunoreactivity in an 18-year-old female control subject who died as a result of leukaemia. (B) Minimal, highly ramified microglia observed in a 36-year-old female who died following a sudden cardiac event, and (C) numerous microglia with shortened, thickened processes and hypertrophy of the cell body, indicative of activation in a 92-year-old female who died as a result of bronchopneumonia. (D–E) Clusters of activated microglia with decreased ramifications in a (D) 23- and (E) 31-year-old male, who each died 4 weeks after TBI. Note the occasional cell displaying amoeboid morphology. In addition, cells can be seen arranging in parallel lines, likely along the length of an injured axon. (F–H) Extensive and densely packed amoeboid CR3/43 immunoreactive cells displaying minimal or no processes in (F) a 43-year-old male, 4 years post-TBI, (G) a 67-year-old male 8 months post-TBI and (H) a 64-year-old male 16 years post-TBI. All scale bars = 100 µm.

Figure 2

Percentage of cases displaying extensive amoeboid CR3/43 and CD68 immunoreactive cells in the corpus callosum following TBI by survival time versus control subjects.

Figure 3

CR3/43 versus CD68 immunoreactivity in the corpus callosum in a case of long-term survival after TBI. (A) Dense CR3/43 reactive microglia with an amoeboid morphology in a 37-year-old male, 4 years post-injury. CR3/43 is an major histocompatibility complex Class II specific antibody and as such binds to the cell surface as can be observed. Scale bar = 100 µm. (B) The same region in this case displays a virtually identical cell population when subjected to immunohistochemistry specific for CD68. In contrast to CR3/43, CD68 immunoreactivity is predominantly intracellular, given that it is a lysosomal protein. Scale bar = 100 µm. (C–D) Low magnification photomicrographs displaying CR3/43 and CD68 immunoreactivity, respectively, in the same case as above. Cells tend to form bands along the inferior and superior border of the corpus callosum. Scale bar = 200 µm.

Figure 4

Representative images of axonal pathology in the corpus callosum as demonstrated by amyloid precursor protein immunoreactivity. (A) Normal white matter displaying no abnormal amyloid precursor protein immunoreactivity in a 67-year-old female who died following volvulus of the colon. (B) Axonal pathology with a distribution and morphology consistent with a traumatic origin in a 20-year-old male, 2 days post-injury. (C) Extensive axonal pathology in a pattern and distribution consistent with acute hypoxia-ischaemia in an 18-year-old male 10 h after TBI. (D) Small clusters of amyloid precursor protein immunonoreactivity consistent with the appearance of axonal bulbs in an 89-year-old female, 7 years post-TBI. On serial sections, the same region displayed extensive activated microglia as determined using CR3/43 and CD68 immunohistochemistry. (E) A similar pattern of multiple axonal bulbs in the corpus callosum of a 28-year-old male, 9 months post-TBI. Again, the same region displayed extensive activated microglia in serial sections. All scale bars = 100 µm.

Figure 5

Extent of axonal pathology identified using amyloid precursor protein immunohistochemistry following TBI by survival time versus control subjects.

Figure 6

CR3/43, amyloid precursor protein and Luxol fast blue staining in the corpus callosum of representative cases with TBI versus control subjects. (A) CR3/43 (B) amyloid precursor protein and (C) Luxol fast blue staining in a 24-year-old male control subject who died following cardiomyopathy. Minimal CR3/43 immunoreactivity is accompanied by an absence of axonal pathology and white matter of a normal density and uniform distribution. Scale bars = 200 µm. (D) CR3/43 (E) amyloid precursor protein and (F) Luxol fast blue staining in a 37-year-old male who died 4 years post-TBI. Extensive amoeboid CR3/43 immunoreactivity is accompanied by multiple axonal bulbs and Luxol fast blue staining showing a decreased density of fibres and a non-uniform distribution of fibres within inflamed regions. Scale bars = 200 µm. (G) CR3/43 (H) amyloid precursor protein and (I) Luxol fast blue staining in a 43-year-old male who died 4 years post-TBI. Again, extensive amoeboid CR3/43 immunoreactivity is accompanied by minimal axonal pathology and a patchy loss of integrity of the white matter in association with regions of amoeboid microglia. Scale bars = 1 mm. (J) High magnification Luxol fast blue staining in the corpus callosum in the same case as A–C. (K) High magnification Luxol fast blue staining in the corpus callosum in the same case as D–F. (L) High magnification Luxol fast blue staining in the corpus callosum in the same case as G–I. Scale bars = 100 µm.

Figure 7

Representative images showing CR3/43 and myelin basic protein double labelling in the corpus callosum following survival from TBI. For all cases, CR3/43 is displayed in green and myelin basic protein (MBP) in red. (A–C) A 67-year-old male 8 months following TBI caused by a fall. (D–F) A 44-year-old female 2 years post-TBI caused by a fall. (G–I) A 37-year-old male 4 years post-TBI caused by a fall. Note the co-localization of myelin basic protein immunoreactivity within CR3/43 reactive cells, indicating phagocytosed myelin fragments.

Figure 8

Representative low magnification images showing CR3/43 immunoreactivity in the hemi-corpus callosum following long-term survival from TBI versus representative age-matched control subjects. The thickness of the corpus callosum is markedly decreased following TBI. All images are presented at the same magnification and scale. All scale bars = 1 mm. (A) A 67-year-old male 8 months post-TBI compared with (B) a 60-year-old male control subject who died following heart failure. (C) A 44-year-old female who died 2 years post-TBI compared with (D) a 50-year-old male who died following bronchopneumonia. (E) A 37-year-old male who died 4 years post-TBI compared with (F) a 33-year-old female who died following an acute cardiac event.

Figure 9

Thickness of the corpus callosum (CC) following survival from TBI versus uninjured control subjects. (* = p < 0.05).