Evidence of traumatic brain injury in headbutting bovids

Abstract

Traumatic brain injury (TBI) is a leading cause of neurologic impairment and death that remains poorly understood. Rodent models have yet to produce clinical therapies, and the exploration of larger and more diverse models remains relatively scarce. We investigated the potential for brain injury after headbutting in two combative bovid species by assessing neuromorphology and neuropathology through immunohistochemistry and stereological quantification. Postmortem brains of muskoxen (Ovibos moschatus, n = 3) and bighorn sheep (Ovis canadensis, n = 4) were analyzed by high-resolution MRI and processed histologically for evidence of TBI. Exploratory histological protocols investigated potential abnormalities in neurons, microglia, and astrocytes in the prefrontal and parietal cortex. Phosphorylated tau protein, a TBI biomarker found in the cerebrospinal fluid and in neurodegenerative lesions, was used to detect possible cellular consequences of chronic or acute TBI. MRI revealed no abnormal neuropathological changes; however, high amounts of tau-immunoreactive neuritic thread clusters, neurites, and neurons were concentrated in the superficial layers of the neocortex, preferentially at the bottom of the sulci in the muskoxen and occasionally around blood vessels. Tau-immunoreactive lesions were rare in the bighorn sheep. Additionally, microglia and astrocytes showed no grouping around tau-immunoreactive cells in either species. Our preliminary findings indicate that muskoxen and possibly other headbutting bovids suffer from chronic or acute brain trauma and that the males' thicker skulls may protect them to a certain extent.

Keywords: CTE; Chronic traumatic encephalopathy; Concussion; MRI; TBI; Tau protein.

Fig. 1

Sampling site and serial sections in bovid brains. a The brains were extracted from three muskoxen. Here, a block from the prefrontal cortex of the old male muskox is represented. Sections were cut at 50 µm-thickness from a block in the prefrontal cortex of the right hemisphere (purple shading). In serial sections each 500 µm apart, tau pathologies were counted exhaustively after pSer202 tau immunostaining. Neuropil threads are represented by yellow points and neuritic thread clusters by green points. Note the difference in shape between the sections with progression through the block. Muskox image courtesy of the Musk Ox Farm, AK. b Brain sections from the male bighorn sheep were pSer396/Ser404 tau-immunostained and tau pathologies were counted exhaustively. Neuropil threads are represented by yellow points and neurons are represented by blue points. No neuritic thread clusters were found in the bighorn sheep. Bighorn sheep image source: rawpixel.com. Scale bar = 1 cm

Fig. 2

Magnetic resonance imaging (MRI) coronal scans of a muskox and a human brain. a Male muskox brain ex vivo, before sampling; the right prefrontal cortex is shaded in purple. Deformation and damage to the cerebellum occurred postmortem. Image obtained by coronal T2-weighted turbo spin-echo images obtained from an ex vivo old male muskox brain at 0.57 mm in-plane resolution and 1 mm slice thickness using a 7 T MRI scanner. b Human brain with a history of TBI, frame taken through the human prefrontal cortex at the level of the corpus callosum. Image obtained by ex vivo coronal 3D T2-weighted fluid attenuated inversion recovery (FLAIR) turbo spin-echo image acquired at 3 T at 0.63 mm isotropic resolution. Scale bar = 1 cm

Fig. 3

Micrographs of abnormally phosphorylated tau immunostained using different antibodies in brains of human with late-stage Alzheimer’s disease (a–c), human with CTE (d–f), old male muskox (g–i), and a male bighorn sheep (j–l). Samples were taken from the prefrontal cortex and immunostained for pSer396/Ser404 tau (a, d, g, j), pSer202/Thr205 tau (b, e, h, k), and pSer202 tau (c, f, i, l). In the human AD specimen, pSer396/Ser404 tau-immunoreactive (a) neuropil threads, (b) pSer202/Thr205 tau-immunostained neuritic thread clusters, neuropil threads, and neurons, and (c) pSer202 tau-immunostained neurons and neuropil threads. In the human CTE specimen, pSer396/Ser404 tau-immunoreactive (d) neuron, (e) pSer202/Thr205 tau-immunoreactive neurons and neuropil threads, and (f) pSer202 tau-immunoreactive neuritic threads. In the old male muskox pSer396/Ser404 tau-immunoreactive (g) neuropil threads, (h) a pSer202/Thr205 tau-immunoreactive neuron, and (i) pSer202 tau-immunoreactive neuritic thread clusters, neuropil threads, and neurons in layer II near a blood vessel. In the male bighorn sheep one of the only pSer396/Ser404 tau-immunoreactive structures (j) a neuron and associated neuropil threads; (k) pSer202/Thr205 tau-immunoreactive neuropil threads, circled; (l) pSer202 tau-immunoreactive neuropil threads, circled. Scale bar = 50 µm. Controls in Fig. S1

Fig. 4

Distribution of pSer202 tau-immunoreactive structures among neocortical layers in the muskox. Brain samples from the prefrontal (a–c) and parietal cortex (d–f) of an old male, middle-aged female, and old female muskox. Sections were pSer202 tau immunostained, and an exhaustive count of tau-immunoreactive (a, d) neuropil threads, (b, e) neuritic thread clusters, and (c, f) neurons was performed using StereoInvestigator on ten slices, each 500 µm apart. Estimated population was corrected for volume per brain layer (cm³). Note the scale difference for each graph

Fig. 5

Distribution of pSer202 tau-immunoreactive structures recorded in neocortical sections of muskox brain. Heatmaps represent the total distribution (not the estimated population) of pSer202 tau-immunoreactive neuropil threads throughout 500 µm of brain tissue in 10 sections. Tissue is from the prefrontal cortex in a the male muskox, b the middle-aged female muskox, and c the old female muskox, and from the parietal cortex of the right hemisphere in the d male muskox, e middle-aged female, and f old female muskox. Yellow lines mark the average outline of white and grey matter in each individual throughout the ten slides. Grey points indicate pSer202 tau-immunoreactive neuritic thread clusters and pink diamonds represent pSer202 tau-immunoreactive neurons. Note that the highest densities of neurites are at the base of the sulci and that higher neuropil thread counts in c does not necessarily equate higher density. The highest densities of neuropil threads are in yellow, note the different scale in the two brain regions in a–c and d–f, calculated separately because of the difference in scale, which would have hidden density patterns. Scale bar = 1 cm

Fig. 6

Tau-containing neurons in a human late-stage Alzheimer’s disease and b a human CTE case and c muskox brain. All sections were taken from the prefrontal cortex and pSer202 tau immunostained. Asterisks represent the magnified areas shown in d–f. Note the accumulation of pSer202 tau-immunoreactive structures in the AD human are well visible whereas in the CTE case, it is more sparse, similar to the muskox. Scale bar = 100 µm. No pSer202 tau-immunoreactive lesions appeared in a clinically healthy human case

Fig. 7

Photomicrographs of pathological structures in the parietal cortex of muskoxen. a–c pSer202 tau-immunoreactive pretangle-like features in pyramidal cells and neurites in the old female muskox. d pSer202/Thr205 tau-immunoreactive pretangle like features in a pyramidal cell and neurites in the old female muskox. e pSer396/Ser404 tau-immunoreactive NFT-like lesion in a layer III pyramidal cell in the old female muskox. f Iba1-immunoreactive microglial aggregation on the border of the white matter in the middle-aged muskox. Scale = 100 µm

Fig. 8

Immunofluorescence photomicrographs of pSer202 tau-immunoreactive structures and GFAP-immunoreactive astrocytes in human and muskox brain. In Brodmann area 10 of the human specimen with late-stage AD, a anti-pSer202 tau (red), b astrocytes are immunostained for GFAP (green), c nuclei are stained with DAPI (blue), and all three are combined in d. In the human specimen with CTE, tissue was immunostained with the same antibodies (e–h), as was the old male muskox prefrontal cortex (i–l) and the old female muskox parietal cortex tissue (m–p). In d note the activated astrocytes, in l and p the autofluorescence of blood vessels and in l astrocyte activation in the male muskox. Astrocytes and neuropil threads are encircling a blood vessel in the old female muskox represented by an asterisk (p). Scale bar = 50 µm

Fig. 9

Photomicrographs of microglia (a, c, e) and astrocytes (b, d, f) in brains of human with late-stage AD (a, b), old male muskox (c, d), and a male bighorn sheep (e, f). Samples were taken from the prefrontal cortex and immunostained for Iba1 to detect microglia, or GFAP to detect astrocytes. Note astrocyte clumping in b but not in f. Note the astrocytic processes with endfeet around the blood vessel in f. Scale bar = 50 µm. Controls in Fig. S2