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Acta Neuropathologica (2024) 147:79 https://doi.org/10.1007/s00401-024-02735-9

ORIGINAL PAPER

Sex differences in the extent of acute axonal pathologies after experimental concussion

Hailong Song1 · Alexandra Tomasevich1 · Andrew Paolini1 · Kevin D. Browne1,2 · Kathryn L. Wofford1,2 · Brian Kelley1 · Eashwar Kantemneni1 · Justin Kennedy1 · Yue Qiu1 · Andrea L. C. Schneider4,5 · Jean‑Pierre Dolle1 · D. Kacy Cullen1,2,3 · Douglas H. Smith1

Received: 22 January 2024 / Revised: 16 April 2024 / Accepted: 17 April 2024 © The Author(s) 2024

Abstract Although human females appear be at a higher risk of concussion and suffer worse outcomes than males, underlying mechanisms remain unclear. With increasing recognition that damage to white matter axons is a key pathologic substrate of concussion, we used a clinically relevant swine model of concussion to explore potential sex differences in the extent of axonal pathologies. At 24 h post-injury, female swine displayed a greater number of swollen axonal profiles and more widespread loss of axonal sodium channels than males. Axon degeneration for both sexes appeared to be related to indi- vidual axon architecture, reflected by a selective loss of small caliber axons after concussion. However, female brains had a higher percentage of small caliber axons, leading to more extensive axon loss after injury compared to males. Accordingly, sexual dimorphism in axonal size is associated with more extensive axonal pathology in females after concussion, which may contribute to worse outcomes.

Keywords Concussion · Sex difference · Diffuse axonal injury · Amyloid precursor protein · Voltage-gated sodium channel isoform 1.6 · Axon ultrastructure

Introduction

Each year, approximately 50 million individuals world- wide suffer a concussion, also commonly referred to as mild traumatic brain injury (TBI) [32]. However, there is nothing ‘mild’ about this condition for the more than 15%

of individuals who suffer persisting neurocognitive dysfunc- tion [34, 36]. There is mounting consensus that concussion induces acute structural and physiologic disruption of brain- network connectivity and function. In particular, the multi- focal appearance of axonal pathology across the white mat- ter, generally referred to as diffuse axonal injury (DAI) [1, 47, 51], is thought to be an important pathologic substrate underlying the clinical manifestations of concussion [5, 6, 25, 27, 33, 37, 44, 51].

While post-mortem examination of concussion in humans is limited due to its generally non-lethal nature, one study of six individuals who died shortly after injury due to other causes has identified DAI as the only neuropathological change [6]. The principal mechanical force associated with the induction of DAI is head rotational acceleration [35]. While these forces induce dynamic mechanical deforma- tion of tissue across the brain, white matter axons appear selectively vulnerable to mechanical damage. This is due in part to their very thin, delicate, and elongated architec- ture and high anisotropic organization in white matter tracts. Indeed, dynamic deformation of axons induces immediate mechanical breaking of axonal microtubules [54], thereby

* Douglas H. Smith [email protected]

1 Department of Neurosurgery, Center for Brain Injury and Repair, University of Pennsylvania, 3320 Smith Walk, 105 Hayden Hall, Philadelphia, PA 19104, USA

2 Center for Neurotrauma, Neurodegeneration and Restoration, Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, PA 19104, USA

3 Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104, USA

4 Department of Neurology, University of Pennsylvania, Philadelphia, PA 19104, USA

5 Department of Epidemiology, Biostatistics, and Informatics, University of Pennsylvania, Philadelphia, PA 19104, USA

Acta Neuropathologica (2024) 147:79 79 Page 2 of 17

inducing axonal transport interruption and accumulation of proteins in hallmark periodic swellings along the axon, which historically have been considered the morphologic signature of DAI [20, 25]. However, DAI may represent a spectrum of axonal injury phenotypes [27, 51]. Using a clin- ically relevant swine model of concussion via head rotational acceleration biomechanically scaled to human concussion [8, 14, 18], we recently also observed disruption of axonal sodium channels (NaChs) across the white matter, similar to axonal NaCh loss we identified in higher severities of TBI in humans [51]. Collectively, these acute pathologic processes of DAI are thought to play key roles in the physical and functional loss of brain connectivity after concussion.

Although males dominate emergency department visits for concussion, this has been primarily attributed to their greater exposure to activities with a risk of head impacts compared to females [19]. In contrast, it has recently been observed that female athletes have a higher rate of concus- sion and appear to have worse outcomes than their male counterparts participating in the same sport [4, 7, 13, 53]. While many factors are likely at play here, this raises the intriguing possibility that sex-based differences in the extent of axonal pathology could contribute to differences in con- cussion outcome, potentially related to sex differences in average axon size and architecture.

It is long proposed that axons in the corpus callosum of human females might contain a greater percentage of small diameter axons compared to males [58], which has now been demonstrated by direct measurement in post-mortem micros- copy examinations [39]. In addition, magnetic resonance imaging (MRI) studies have also shown smaller white mat- ter volumes in human females than in males [21, 31]. Since female brains are approximately 8–13% smaller than male brains [41], it has been suggested that by having a reduced axon size, females can accommodate a similar number of axons forming the brain’s networks as males. Recently, we found that sexual dimorphism of axon structure also appears in both rat and human-induced pluripotent stem cell (iPSC) neurons in vitro [16]. Further, using an in vitro model that is based on concussion biomechanics [16], dynamic stretch injury of micropatterned unmyelinated axonal tracts induced

greater microtubule damage and ionic disruption in female axons compared to male axons under the same level of injury. However, it has not been known if this link between potential sex differences in axon architecture and extent of axonal pathogenesis might also occur in vivo in brain myeli- nated axons due to concussion.

Here, using a well-characterized swine model of concus- sion [9, 14, 27, 28, 51], we explored potential sex differences in the extent of axonal pathologies in relation to axon size. Scaled to closely mimic the head rotational acceleration kin- ematics of human concussion, this model induces selective axonal pathologies in the absence of neuron death or gross pathologic changes [9, 14, 27, 28, 51]. We first examined the extent of axonal pathology at 24 h post-injury using the ‘gold standard’ immunohistochemical (IHC) staining for amyloid precursor protein (APP), which identifies periodic axonal swellings that arise due to microtubule damage and impaired axonal transport [20]. In addition, we examined the extent of loss of the predominant axonal NaCh that popu- lates the nodes of Ranvier (NOR), Nav1.6 [51]. Moreover, using transmission electron microscopy (TEM), we evalu- ated potential differences in axonal diameter between female and males with no injury and changes in the average caliber of white matter axons after injury between the sexes.

Materials and methods

Experimental design and the swine head rotational acceleration model of concussion

Due to their relatively large gyrencephalic brain with extensive white matter, swine are ideally suited to model concussion to be clinically relevant to human concussion [14]. A total of 16 swine (Hanford strain, Sinclair Research Center, Inc.), aged approximately 6 to 8 months, were used and randomly assigned into four groups: sham female (F) (n = 3), sham male (M) (n = 3), injury F (n = 5), and injury- M (n = 5). All detailed animal characteristics, including age, body weight, brain mass, and injury level (maximum angular velocity) were listed in Table 1. For the injury group, swine

Table 1 Animal characteristics, injury kinematics, and recovery duration

Sham-F (n = 3) Sham-M (n = 3) Injury-F (n = 5) Injury-M (n = 5)

Age (months) Mean (range) ± SD 6.2 (6.1–6.3) ± 0.1 6.3 (6.1–6.5) ± 0.2 6.6 (5.9–7.4) ± 0.7 7.1 (6.2–7.9) ± 0.6 Body weight (kg) Mean (range) ± SD 36.9 (34.0–41.5) ± 4.0 34.1 (31.2–37.2) ± 3.0 29.2 (25.0–35.4) ± 4.0 36.1 (29.4–43.0) ± 4.9 Brain mass (g) Mean (range) ± SD 98.4 (90.1–

111.3) ± 11.3 96.7 (91.6–104.4) ± 6.8 92.2 (85.6–109.4) ± 9.7 100.9 (91.6–

107.2) ± 6.2 Injury level-max angu-

lar velocity (rad/s) Mean (range) ± SD N/A N/A 259 (257–261) ± 1.6 257 (253–262) ± 3.4

Recovery duration (minutes)

Mean (range) ± SD 8.3 (4.0–13.0) ± 4.5 13.3 (7.0–18.0) ± 5.7 36.2 (21.0–50.0) ± 10.9 14.8 (2.0–26.0) ± 10.7

Acta Neuropathologica (2024) 147:79 Page 3 of 17 79

were subjected to experimental concussion via rapid head rotational acceleration scaled to closely mimic human con- cussion biomechanics [8, 9, 14, 18, 27, 28, 51]. Extensive previous characterization demonstrated that this rotational acceleration resulted in selective axonal pathologies, which is morphologically identical to that observed in human [6, 20, 27, 51]. In addition, in this model, axonal pathology is seen in the absence of other pathologic features, e.g., hemor- rhage, raised intracranial pressure (ICP), brain swelling or neuron death [14, 27, 28].

To compare potential sex differences, all injured animals were examined at 24 h post-injury when extensive axonal pathologies are expected based on prior observation [14, 27, 28, 51]. In addition, this early time point is selected based on known sex differences in structural disruption of white mat- ter at acute phases of clinical concussion [11] and its close relevance to our previous in vitro findings of sex differences in axonal structures/ outcomes [16]. The proposed animal number was anticipated to provide > 80% power and a Type I error of 0.05, especially considering the absence of axonal pathology in sham animals as demonstrated before [27, 28, 51]. All histologic experiments, analyses and quantification were performed blind to the injury status of the animal. All animal experiments were conducted in accordance with ARRIVE guidelines and protocols approved by the Insti- tutional Animal Care and Use Committee at the University of Pennsylvania.

Animal preparation, injury and recovery procedure

The animal preparation was carried out as previously described [9, 14, 27, 28, 51]. Animals were fasted for over 12 h before any surgical procedures and anesthesia was induced by intramuscular administration of midazolam (0.5 mg/kg) with dexmedetomidine (0.05 mg/kg) for injury procedure and midazolam with ketamine (20 mg/kg) for sacrifice procedure. Anesthesia was then maintained on a surgical plane via 2–5% isoflurane with snout mask and intubation. Animals’ statuses were monitored throughout the procedure.

Experimental concussion was performed using a HYGE pneumatic actuator, which can convert linear motion to angular (rotational) motion to produce impulsive head rotation of 110 degree in the coronal plane in 20 ms. In this model, the animal’s mouth was positioned with a pad- ded bite plate and then the head was secured to the HYGE device with adjustable snout straps. This padded linkage assembly allowed precise control of swine head movement in the coronal plane. In this study, swine were subjected to precisely controlled coronal rotational injuries that ranged from 253 to 262 rad/s, with no difference between female

and male injury group (Table 1). Rotational kinematics were recorded using angular velocity transducers (Applied Technology Associates) and calculated as previously noted [60].

Following injury, swine were immediately removed from the HYGE device, then extubated as prompted by either chewing on the endotracheal tube, swallowing or cough- ing, and continuously monitored in the housing unit for the duration of the recovery process. Preemptive analgesia of sustained release buprenorphine (0.2 mg/kg) was injected subcutaneously post-injury. Following previous descrip- tion [60], recovery duration was defined as the time elapsed between extubation of animals and when animals were weight-bearing on all four limbs. Sham animals received all procedures described above except for the head rotation.

Tissue preparation

Under terminal anesthesia, all animals were sacrificed at 24 h post-injury through transcardial perfusion with heparin- ized saline (2 L) followed by 10% neutral buffered formalin (8 L), as previously described [27, 28, 51]. The brain was then removed, extracted, and post-fixed in 10% formalin for 1 week. Subsequently, the brain was blocked at 5 mm in the coronal plane and tissue block was processed for either standard paraffin embedding in an automated tissue proces- sor (Shandon Scientific Instruments) or standard TEM prep- aration (described in below section). Serial Sects. (8 µm) were cut on a Leitz rotary microtome (Leica) then mounted on Fisherbrand Superfrost/Plus microscope slides (Fisher Scientific).

The brain coronal level described in this study was des- ignated according to the stereotaxic atlas of the pig brain such that in the antero-posterior direction, the anterior bor- der of the posterior commissure was presented in the plane A 0.00 mm [17]. Then, the planes anterior to it were labeled as anterior (A) with the distance to this level. Here, continu- ous tissue blocks at plane A 5.50 mm, 10.50 mm, 15.50 mm, and 20.50 mm were used to systematically assess axonal pathologies (Supplementary Fig. 1a, online resource). These coronal levels include white matter that contains radiation of the corpus callosum (deep white matter) and subcallosal fasciculus, which were selected for analysis given their established biomechanical vulnerability to the injury [14, 27, 28, 51]. In addition, to test the hypothesis that female axons may be more selectively vulnerable than male axons related to axon size, tissue block at plane A 0.50 mm was selected to examine axon ultrastructure under TEM. Based on our extensive prior characterization, a consistently high number of swollen axonal profiles were observed at this level after injury [9, 27, 28].

Acta Neuropathologica (2024) 147:79 79 Page 4 of 17

Immunohistochemical (IHC) staining and quantification

Standard IHC techniques were performed as previously described [27, 28, 49, 51, 52]. Swine brain tissue sec- tions were deparaffinized with xylene and rehydrated using a series of graded ethanol (100% and 95%) and water. Sections were then incubated with 3% hydrogen peroxide (Carolina Biological) for 15  min to quench endogenous peroxidase activity. Antigen retrieval was followed by heating in Tris EDTA buffer (pH 8.0) using TintoRetriever Pressure Cooker (Bio SB). Sections were then blocked with normal horse serum (Vector Labs) in Optimax buffer (BioGenex) and incubated with primary antibody of amyloid precursor protein (APP) (Millipore, MAB348, 1:60,000) or myelin basic protein (MBP) (Cell Signaling Technology, 78896, 1:2000) overnight at 4 °C. Next day, biotinylated secondary antibody (Vector Labs) was applied for 30 min followed by avidin biotin complex (Vector Labs) incubation for another 30 min. Visualization was done by DAB peroxidase substrate kit (Vector Labs) and counterstaining with hematoxylin (Surgipath, Leica Biosystems). Sections were scanned at 20 × with an Aperio ScanScope and annotation of each APP-immunoreactive profile was achieved using Aperio ImageScope software (Leica Biosystems).

Quantification of APP-immunoreactive axonal profile was followed by previous studies that have extensively characterized the pattern and distribution of APP axonal pathology in this swine model of concussion [9, 14, 27, 28, 51]. Specifically, an individual APP-immunoreactive axonal profile was defined as a swollen bulb, morphologi- cally injured, or varicose profile and counted once for anal- ysis. First, we mapped individual APP-immunoreactive profile in whole brain section of both injured female swine and males. Given the known biomechanical vulnerability of this model, we further performed focused examina- tion and quantitative analysis of white matter regions that contain left radiation of the corpus callosum (deep white matter) and subcallosal fasciculus adjacent to the left lat- eral ventricle, where extensive axonal pathologies were consistently shown for both sexes (Supplementary Fig. 1b, online resource). We provided an estimation of the No. of APP-immunoreactive profiles spanning four continu- ous coronal levels of the brain. For each of the coronal levels, approximately 4 mm2 area at the center of left deep white matter and 1 mm2 area at subcallosal fasciculus were systematically surveyed for positive APP-immunoreactive axonal profiles (Supplementary Fig. 1b, online resource, inset white box). The No. of APP-immunoreactive profiles was counted for whole section and in a given field, con- verted to per unit area (mm2), and averaged for individual

animal for statistical analysis. Quantification was per- formed independently by two investigators, blind to the injury status, with results showing excellent inter-rater reliability (interclass correlation coefficient (ICC) of 0.94).

Immunofluorescent (IF) staining, confocal imaging, and quantification

IF staining was carried out similarly to the IHC procedures, except for the hydrogen peroxide quenching step was omit- ted as previously described [27, 28, 49, 51]. Sections were then incubated overnight at 4 °C with primary antibodies of Nav1.6 (Alomone Labs, ASC-009, 1:200), Caspr (from Ste- phen Waxman, Yale University, generated against the GST fusion protein composed of the entire cytoplasmic domain of rat p190/Caspr (contactin-associated protein) (GST–190CT), 1:2000) [42], and APP (Millipore, MAB348, 1:1000). The next day, secondary antibodies conjugated with Alexa Fluor 488/568/647 (ThermoFisher, 1:250) were applied on sec- tions for 60 min. VECTASHIELD Vibrance antifade mount- ing medium (Vector Labs) was used for coverslipping.

For visualization, sections were imaged using a Zeiss LSM 880 Airyscan confocal microscope with a 63 × oil immersion objective (1.40 NA) and 488, 561, and 633 laser lines for each fluorophore as previously described [51]. The Airyscan confocal used a multichannel area detector with 32 elements to collect all the light from an Airy disk simultaneously with no need to set the pinhole size. Images were acquired with z-stack (numerical zoom of 2, a total of 10 steps with each z-step of 0.22 μm) and prepared using ImageJ. Surface rendering was performed for 3D reconstruc- tion using Imaris software (GraphPad, Bitplane, v.9.7.2).

Quantitative analysis of Nav1.6 was conducted indepen- dently by two investigators, blind to the injury status, again showing excellent inter-rater reliability (ICC of 0.97)). Fol- lowing previous description [51], for individual animal, two non-overlapping regions of interests (ROIs) from left deep white matter and one ROI from subcallosal fasciculus were surveyed in each of the four coronal levels (approximately covering a total area of 0.12 mm2 for one level and 0.48 mm2 per animal). These ROIs were selected in close prox- imity to existing APP-immunoreactive axonal profiles. The loss of Nav1.6 was quantified by counting the numbers of Nav1.6 void nodes and then calculated as a percentage by dividing the numbers of void nodes by the numbers of total NOR observed. Caspr staining labels septate-like paranodal space surrounding the NOR and was used as a reference to characterize the void node morphologic phenotype (paired Caspr domain in the absence of Nav1.6). In addition, the flu- orescent intensities of Nav1.6 were measured using ImageJ and normalized against background fluorescent signals to generate relative expression [Integrated Density—(Area

Acta Neuropathologica (2024) 147:79 Page 5 of 17 79

of selected area * Mean fluorescence of background read- ings)]. Since the presence of Nav1.6 is diffuse and its loss after experimental concussion is widespread across the brain white matter [51], measurements of Nav1.6 were then aver- aged for each animal for statistical comparison.

Transmission electron microscopy (TEM) procedure and quantification

Brain tissue at the coronal level of plane A 0.50 mm was used for TEM preparation, processing, and analysis. The tissue block was post-fixed with a mixture of 2.5% glutaral- dehyde and 2% paraformaldehyde (Electron Microscopy Sci- ences) in 0.1 M sodium cacodylate buffer (pH 7.4). Samples cut from the corpus callosum were selected for cross-sec- tional examination of white matter axons. After subsequent washes with 0.1 M sodium cacodylate buffer, tissues were post-fixed in 2% osmium tetroxide for 1 h at room tempera- ture and rinsed in diH2O prior to en bloc staining with 2% uranyl acetate. After dehydration through a graded ethanol series, samples were infiltrated and embedded in EMbed- 812 (Electron Microscopy Sciences). Thin Sects. (75 nm) were then stained with uranyl acetate and lead citrate.

Ultrastructural images were acquired using a JEOL JEM 1010 electron microscope fitted with a Hamamatsu digital camera and AMT Advantage image capture software at 10,000 × magnification for quantitative analysis. A higher magnification at 25,000 × was used for figure illustration. For analysis, an approximately 6000 μm2 area selected in a sys- tematic random fashion was surveyed for individual injury-F and injury-M group animals, and an 11,000 μm2 area was surveyed for each sham-F and sham-M group. While there may be some artifact in myelin lamination possibly owing to fixation, normal myelinated axons were selected for measur- ing axon area, g-ratio, and frequency of fibers based on their intact axolemma and no presence of apparent loss of axon content, including neurofilament and normal mitochondria as previously described [16, 30, 50]. In contrast, injured axons were distinguished based on the presence of empty of axon cytoplasm/ neurofilament or apparent formation of vacuoles/ degenerated mitochondria/ lysosomes in the appearance of a swollen axon. Note that when measuring axon area in injured animals, only axons without the mor- phologic changes described above were included in analysis to capture caliber changes after injury.

Axon area was measured using ImageJ (NIH, USA). Axon diameter was then converted by 2 ×

√

area ÷ � and g-ratio was calculated as

√

inner myelin area ÷ outer myelin area . The percentage of injured axons was quantified as numbers of injured axons/ numbers of total axons surveyed. For sta- tistical analysis, the averaged measurements from individual animals were used to compare differences between groups.

All TEM quantitative analyses were performed indepen- dently by multiple investigators, blind to the injury status, with results demonstrating excellent inter-rater reliability (ICC of 0.95).

Statistical analysis

Statistical analysis was performed using GraphPad Prism statistical software (GraphPad Software Inc.). Data were presented as scatter plots or bar graphs and expressed as mean ± standard error of mean. A two-sample t test was used to determine differences between two groups [28, 51]. Linear regression was used to correlate recovery duration and the extent of axonal pathologies. The frequency of axon fibers in TEM studies was depicted by Gaussian distribution and compared using Kolmogorov–Smirnov test. A p value less than 0.05 was considered significant for all analyses, with * indicating p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001. The inter-rater reliability was assessed by ICC. The ICC values were interpreted as follows: < 0.40, poor; 0.4–0.6, fair; 0.6–0.75, good; and 0.75–1.0, excellent [12].

Results

Animal characteristics, injury kinematics, and recovery duration following experimental concussion

Both female and male swine were selected at a similar range of age (6 to 7 months) (Table 1). While the female swine averaged slightly lower body weight than the males, there was no difference in brain tissue mass (Supplementary Fig. 2a, b, online resource). Closely matched brain mass between animals was important for this study, since larger brains are predicted to undergo greater tissue deformation and axonal pathology than smaller brains under the same parameters of head rotational acceleration [22, 35]. Here, setting the same head rotation parameters for each injury, the maximum angular velocities were found to be the same between sexes (Table 1 and Supplementary Fig. 2c, online resource). In addition, there was no significant difference between sexes in additional kinematic parameters, including injury velocity magnitude, acceleration peaks, and accelera- tion duration, as reflected by the captured velocity and accel- eration traces (Supplementary Fig. 2d–i, online resource). Immediately following injury, female swine sustained a sig- nificantly longer recovery duration than males, which was also greater than sham animals (Table 1 and Supplementary Fig. 3a, online resource).

Acta Neuropathologica (2024) 147:79 79 Page 6 of 17

Experimental concussion results in greater number of swollen axonal profiles in females, identified by APP immunohistochemistry

Historically, the ‘gold standard’ histopathological evi- dence of DAI has been the identification of APP-immu- noreactive swollen axonal profiles in a multifocal pat- tern across the white matter [1, 6, 20]. These swellings

form as a result of impaired axonal transport arising from mechanical disruption of the axon cytoskeleton [20]. Here, we quantified the numbers (No.) of APP-immunoreactive axonal profile following previous description [27, 51], defined as a swollen axonal bulb or individual morphologi- cally injured or varicose profile, particularly in brain white matter where established biomechanical vulnerability was shown in this model [9, 14, 27, 28, 51].

No APP-immunoreactive profiles were identified in the sham animals in either sex (Fig. 1a, b and Supplementary

Fig. 1 Extent of APP axonal pathology following experimental con- cussion. Representative whole brain section map of annotated APP- immunoreactive profiles (at A 15.50  mm level) in sham female (a) and male (b) swine, as well as injured female (c) and male swine (d). Red-dot labels annotate individual APP-immunoreactive axonal

profiles. Scale bar = 10  mm. e Significantly greater numbers of APP-immunoreactive axonal profiles per whole brain section were observed in injured females than injured males at 24  h post-injury. Each dot represents individual animal’s mean

Acta Neuropathologica (2024) 147:79 Page 7 of 17 79

Fig. 4, online resource). In contrast, whole-section map of APP-immunoreactive profiles showed axonal pathology in both injured female swine and males, with a similar distri- bution relevant to the dynamic mechanical deformation of brain tissue but differing in extent (Fig. 1c, d). Notably, at 24 h post-injury, a greater extent of APP-immunoreactive axonal profiles were identified in female swine compared to males throughout whole section (Fig. 1e), and particularly at white matter localized to the left radiation of the corpus callosum (deep white matter) and subcallosal fasciculus adjacent to the lateral ventricle (Fig. 2). This difference was consistent throughout continuous coronal levels of the brain. The extent of APP axonal pathology was positively corre- lated with injury recovery duration (Supplementary Fig. 3b and Supplementary Table 1, online resource). As in previ- ous studies using this model, these swollen axonal profiles were morphologically similar to those found in human TBI [6, 20, 27, 51] and presented as characteristic periodic vari- cosities or beading along the axon, fusiform profiles, and terminal bulbs (Fig. 2a, c). In addition, overt loss of myelina- tion (MBP) was not detected in the tissue (Supplementary Fig. 5, online resource).

Females display more widespread loss of axonal NaChs after 

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