A total of nine brain specimens were used in this study: three from muskoxen, four from bighorn sheep, one from a human with late-stage Alzheimer’s disease (AD), and one from a human with CTE, the latter two used as positive controls. The brains of three wild adult muskoxen were collected during a field expedition to Ittoqqortoormiit, Greenland by TMW and all ages were estimated from tooth wear. During the time of brain recovery, the ambient temperature remained always below 7 °C, mitigating tissue deterioration. The male muskox (older adult, collected in summer 2019) was shot in the neck directly after a goring injury to the flank sustained while headbutting another male muskox. One female muskox was shot in the top of the head (middle-aged adult, collected in summer 2018) and the second female muskox was shot in the neck (very old adult, collected in summer 2018), for subsistence hunting (Table 1). The male muskox’s skull was approximately 4 cm thick in the frontal region (including sinuses), and 2 cm thick in the parietal cortex. The four bighorn sheep brains were collected as follows. The brain of an adult male bighorn sheep (Bighorn 1, five years old, collected in winter 2020) was acquired from Colorado Parks and Wildlife from a captive research herd after it was humanely euthanized (darted with NalMedA for sedation, euthanized with 20 ml Euthasol IV) due to a leg fracture. Formalin was injected around the brainstem and into the carotid artery for preservation before shipping. The male bighorn sheep’s skull was approximately 3 cm thick in the frontal region (including sinuses) and 1.5 cm thick in the parietal region. The brain of one wild adult female bighorn sheep (Bighorn 2, four years old, collected in winter 2020) was acquired from Utah Fish and Wildlife after euthanasia due to a Mycoplasma infection. The skull of the bighorn ewe was approximately 1.3 cm thick in the frontal region (including sinuses) and 0.8 cm thick in the parietal cortex. Two additional female bighorn sheep brains (Bighorn 3, five years old, collected in fall 1987; Bighorn 4, adult, collected in winter 2008) were archived in our collection and were provided by the Buffalo Zoo in 2003. After the muskoxen and sheep brains were removed from the skull, all samples were fixed in 10% formalin, either hours after death for the muskoxen, or within 36 h after death for the bighorn sheep. As a positive control for presence of phosphorylated tau, an archived human brain specimen with AD (male, 85 years old, Clinical Dementia Rating 3, Mini-Mental State Exam 11, Thal amyloid stage 4, Braak tangle stage V, postmortem interval 11 h, clinical diagnosis: severe cognitive impairment)  and one with CTE (male, 69 years old, postmortem interval 9 h, clinical history: repetitive athletic head injuries, post-mortem diagnosis of advanced CTE, moderate cerebrovascular disease—athero-arteriolosclerosis; moderate hypoxic–ischemic encephalopathy, severe postmortem autolysis) were used in this study.
Magnetic resonance imaging (MRI)
MRI with superior soft tissue visualization was used to image the anatomical structure of the muskox and bighorn sheep brains. Coronal T2-weighed turbo spin-echo images were performed on a whole-body 7 Tesla (7 T) MRI scanner (Siemens Magnetom, Siemens Healthcare, Erlangen Germany) using a 1-channel transmit and 32-channel receive head coil (Nova Medical, Wilmington, Massachusetts) with the following parameters: repetition time (TR) 8000 ms, echo time (TE) 64 ms, number of sections 24, section thickness 1 mm, field-of-view 16 × 14 cm2, voxel size 0.5 × 0.5 × 1 mm3, scanning duration 6 min 20 s. Human tissue specimen imaging was performed in compliance with all institutional requirements.
The tau-immunoreactive antibodies selected for this study were developed against human antigens. Their specificity in bighorn sheep and muskox tissues required validation to confirm their immunoreactivity. A basic local alignment tool (BLAST) compares protein sequences to sequence databases and calculates the statistical significance. The MAPT gene or protein sequence for tau was not available in bighorn sheep or muskoxen, therefore BLAST was applied to the predicted proteome of the domestic sheep (Ovis aries), the closest related species with MAPT sequence available on NCBI. Protein isoforms with the closest sequence homology and molecular weight to the requested sequence were ranked in terms of degree of identity in percent and E value. Homology values over 95% are considered acceptable, values over 85% are considered moderate.
Immunohistochemistry and histochemical staining
Brain tissue from the human subjects was sampled from Brodmann area 10. In the bovids, tissue was taken from the anterior region of the prefrontal cortex of the right hemisphere (Fig. 1), and additionally from the parietal cortex of the right hemisphere for the three muskoxen. Each block was cut into 50 µm-thick sections on a vibratome (Leica VT1000S) and stored in phosphate-buffered saline (PBS, pH 7.0) solution with 0.1% sodium azide. Multiple phosphorylated tau antibodies were tested in this study, as no protocols existed for these bovid species, making it uncertain which epitopes were present in each. Antibodies recognizing ionized calcium-binding adaptor molecule-1 (Iba1) and glial fibrillary acidic protein (GFAP) were used to investigate microglial and astrocytic morphology, respectively. Three antibodies were tested to detect the presence of phosphorylated tau protein (p-tau). Anti-CP13 is a p-tau antibody clone that binds an epitope around Serine 202 (pSer202 tau), the AT8 clone recognizes epitopes around Serine 202 and Threonine 205 (pSer205/Thr205 tau), and the PHF-1 clone was raised against epitopes including Serine 396 and Serine 404 (pSer396/Ser404 tau). Both CP13 and PHF-1 antibody clones were generously supplied by the Davies laboratory (Feinstein Institute for Medical Research, Northwell Health, Manhasset, New York, USA). Anti-neurofilament clone SMI-312 detects medium- and heavy-chain phosphorylated neurofilament proteins (pNFP) and was used to investigate axonal damage. Anti-denatured myelin basic protein (dMBP) was applied to assess possible demyelination. The MOAB-2 antibody clone was applied against amyloid beta protein (Aβ), and anti-TDP43 clone binds an epitope around phosphorylated Serine 409 and Serine 410 (pSer409/410) to detect pathological proteins. Anti-collagen IV was applied to highlight blood vessel morphology. Additional details on antibodies are reported in Table 2. Sections were stained either as single instances or, in the case of the muskoxen, as series of ten sections, each 500 µm apart for stereological quantification. Primary antibody controls were performed by omitting the primary antibody and assessed that the secondary antibody did not generate non-specific staining (supplementary figures S1 and S2, provided as an online resource).
All washes were performed with either PBS (Iba1, GFAP, dMBP, collagen IV) or Tris-buffered saline (TBS, pH 7.0) (AT8, CP13, PHF1, TDP43, MOAB-2, SMI-312) at room temperature on a shaker at 80 rpm for five minutes each. Antigen retrieval for Iba1 was performed by submerging free-floating tissue sections in a 10 mM EDTA solution (pH 8.0) in closed 15-ml tubes. The tubes were immersed in a water bath at 80 °C for 10 min. For GFAP, AT8, MOAB-2, dMBP, and SMI-312, antigen retrieval was performed by submerging free-floating tissue sections in citrate buffer (pH 6.0), then boiled at 100 °C for 10 min, followed by 5 min of cooling down. Antigen retrieval for collagen IV was performed by submerging sections in a 0.5 M acetic acid and 10 mg/ml pepsin solution at 37 °C for 8 min.
After antigen retrieval, sections were transferred to 12-well plates using a glass hook and washed three times. Sections were then incubated with 0.3% hydrogen peroxide and 0.3% Triton X-100 in buffer for 30 min at room temperature, to inhibit endogenous peroxidase activity. The sections were then washed four times and blocked in buffer with 5% normal donkey serum (017000121, JacksonImmuno, West Grove, PA, USA) or normal goat serum (for TDP43 and collagen IV. 1002635372, Sigma, St. Louis, MD, USA) for 1 h, followed by an incubation in primary antibody (Iba1, GFAP, CP13, AT8, PHF1, MOAB-2, TDP43, SMI-312, dMBP, or collagen IV) in buffer with 5% normal donkey serum and 0.3% Triton X-100, at 4 °C overnight (Iba1, GFAP, AT8, MOAB-2, dMBP, SMI-312, collagen IV) or for 64 h (CP13, PHF1, TDP43); the control sections were incubated in buffer. After primary antibody incubation, sections were washed three times in buffer and 0.3% Triton X-100, then incubated with the appropriate secondary antibody (biotinylated donkey anti-rabbit, secondary antibody, 1:1000, 715065152, JacksonImmuno; biotinylated donkey anti-mouse, secondary antibody, 1:1000, 715065150, JacksonImmuno, biotinylated goat anti-rat secondary antibody 1:1000, BA9400, Vector laboratories, Burlingame, CA, USA) in a 5% normal donkey or goat serum and 0.3% Triton X-100 solution in buffer at room temperature for one hour (Iba1, GFAP, collagen IV, SMI-312) or two hours (AT8, CP13, PHF1, TDP43, MOAB-2, dMBP). After incubation with the secondary antibody, the sections were washed in buffer four times, then incubated with avidin–biotin solution (according to the manufacturer’s instructions, Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) for one hour (Iba1, GFAP, MOAB-2, dMBP, collagen IV, SMI-312) or two hours (AT8, CP13, PHF1, TDP43) at room temperature. The sections were then washed four times, followed by an incubation with DAB peroxidase substrate (Vector Laboratories, according to the manufacturer’s instructions, DAB kit SK-4100) to reveal immunostaining. Samples were then mounted on gelatin-coated slides and left to dry overnight. After drying, the samples were counterstained with cresyl violet, dehydrated through an ethanol gradient, and coverslipped with DPX mounting medium.
Luxol Fast-Blue was applied to observe demyelination as follows. Mounted sections were incubated in 0.1% Luxol Fast-Blue solution for one hour at 56 °C, then rinsed in distilled water. Sections were then differentiated in a 0.05% lithium carbonate solution for 30 s, followed by 70% ethanol for 30 s. Sections were rinsed in distilled water and checked microscopically for differentiation. Finally, slides were rinsed and differentiated in 95% ethanol then coverslipped as above.
The blocking solution was increased from 5 to 10% normal donkey serum and primary antibody incubation was performed as in the immunohistochemistry protocol for both the CP13 and GFAP antibodies. After primary antibody incubation, sections were washed four times, protected from light, and incubated in biotinylated donkey anti-mouse antibody (1:1000, 715065150, JacksonImmuno) and anti-rabbit-AlexaFluor 488 (1:1000, A31570, ThermoFisher Scientific), followed by streptavidin-AlexaFluor 555 (1:500, A21206, ThermoFisher Scientific) in 10% normal donkey serum and 0.1% Triton X-100 in TBS at room temperature for two hours. The sections were washed four times and then incubated in 10% normal donkey serum and 0.1% Triton X-100 in TBS at room temperature for two hours. Sections were then washed four more times, mounted on SuperFrost slides and dried for one hour at 50 °C. Wells were drawn around the sections with an ImmEdge pen and sections were washed for 10 min. Sections were then incubated with TrueBlack (diluted 20 × in 70% ethanol) for 30 s each to reduce autofluorescence, then washed four times. The sections were then incubated with 4′,6-diamino-2-phenylindole dihydrochloride (DAPI, 250 ng/ml) for ten minutes in a humid chamber to stain cell nuclei and then washed a final time, after which they were mounted under Vectashield (H1000, Vector Laboratories) and coverslipped (24 × 50 mm No.1.5 ThermoFisher Scientific).
Microscopy and stereology
Brightfield microscopy images were taken on an Axiophot brightfield microscope (Carl Zeiss Microscopy, Jena, Germany), with a 10×/0.32 Plan-Apochromat objective using StereoInvestigator (version 11.03, MBF Bioscience, Williston, VT, USA). Fluorescence images were taken on a CLSM 780 confocal microscope (Carl Zeiss Microscopy), using a 20×/0.8 DICII objective and DPSS 561-10 diode and Argon lasers at excitation wavelengths of 461, 555, and 488 nm. Confocal stacks in layers II and III of the cerebral cortex were imaged at 512 × 512 pixel resolution with a Z-step of 1 µm and a pinhole setting of 1 Airy unit for the red wavelength and optimal settings for gain and contrast. Images are presented as maximum intensity projections of the Z-stack using ZenBlue (version 3.3, Carl Zeiss Microscopy).
Stereological quantification of tau immunostaining was performed using the optical fractionator workflow probe in StereoInvestigator (magnification × 10, counting frame size 700 × 700 µm, SRS grid layout at 100% of the region of interest, optical dissector height 11 µm with 2 µm of top and bottom guard zones, manual focus), on each muskox specimen in a series of 10 sections, each separated by 500 µm, and counted exhaustively. Cortical layers were manually contoured into layers I, II, III, IV–VI, and white matter. In each layer, different markers were placed for tau-immunoreactive neuropil threads (axonal or dendritic filaments composed of abnormally phosphorylated microtubule-associated tau protein), neuritic thread clusters (circular dense cluster of neuritic threads), and neurons (neuronal cells with tau-immunoreactive inclusions in the cytoplasm). Section contours were aligned manually and the coordinates were exported to create individual and combined heatmaps of tau-immunoreactive neuropil density distribution in Rstudio  using the ggplot2() package . Annotated R code and raw data is available on GitHub (https://github.com/NLAckermans/Ackermans2022BovidTBI.git).
To quantify tau-immunoreactive pathology accumulation in the sulci as opposed to gyri in the muskox specimens, sulcal depths were delimitated as 1/3 of the sulcus as in  and pSer202 tau-immunoreactive markers in each region were quantified in StereoInvestigator using the same specifications as above. To quantify pSer202 tau-immunoreactive pathology around blood vessels in the muskoxen as compared to the CTE human control, one section from each individual was subjected to exhaustive counting of vessels larger than 30 µm in diameter at 2.5 × magnification with StereoInvestigator’s Optical Fractionator probe. The percentage of vessels immunostained with pSer202 tau located within 100 µm of the edge of the vessel and the average distance from the edge of the vessel were calculated and reported.