Chronic traumatic encephalopathy neuropathologic change in former Australian rugby players

We applied the 2021 consensus criteria for both chronic traumatic encephalopathy neuropathological change and traumatic encephalopathy syndrome in a small case series of six former elite‐level Australian rugby code players.


INTRODUCTION
Chronic traumatic encephalopathy neuropathologic change (CTE-NC) is defined as a specific regional pattern of tau pathology that has been identified in the brains of former combat, collision and contact sport athletes after death [1,2].It has been identified in virtually all brain donations from former American professional football players [3], but it is uncommon in former high school football players [4,5].It has also been identified in small samples of former boxers [6,7] and former elite or professional soccer [8], rugby [8] and ice hockey [9] players.
It is not known if, or the extent to which, CTE-NC is the underlying cause or contributing underlying cause of specific clinical problems during life, especially psychiatric problems [5,18].In a large-scale clinicopathological association study [19], there was no association between CTE-NC and depression, suicidality, anxiety, paranoia, apathy, impulsivity, explosivity, violence or motor symptoms or problems during life.The authors of that study reported that there was an association between CTE-NC and cognitive impairment during life [19].
Traumatic encephalopathy syndrome (TES) is a clinical diagnosis that has been used historically to describe the clinical features of chronic brain damage experienced by some ultrahigh exposure boxers [20,21].There are no validated diagnostic criteria for TES, although consensus criteria for TES were published in 2021 [22].These TES criteria can be used in clinical studies, neuroimaging studies and, especially, post mortem neuropathology studies.The purpose of this study is to document the post mortem neuropathology in a case series of former elite-level rugby code (i.e., rugby league and rugby union) players in Australia.We applied the 2021 consensus criteria for CTE-NC [2] and the 2021 consensus criteria for TES [22] to the sample.

Brain donor recruitment and tissue collection
As part of the Former Elite-Level Athlete Brain Health Research Programme, participants are offered an opportunity to donate their brains through the prospective brain donor programme that is affiliated with the Sydney Brain Bank.Subject consent was obtained according to the Declaration of Helsinki, and ethical approval was granted by the ethical committee of the institutions where the data were collected.
In particular, the prospective Athlete Brain Donor Programme has ethics approval through the University of Newcastle, and the Sydney Brain Bank has ethical approval from the University of New South Wales, to collect, characterise, store and distribute human brain tissue for medical research.One donor lived in the state of Victoria, Australia, and was collected by the Victorian Brain Bank.The Victorian Brain Bank has ethical approval from Melbourne University.
As there can be methodological differences between brain banks, formalin-fixed, paraffin-embedded tissue blocks from this case were sent to the Sydney Brain Bank for standardised processing.

Key Points
• In this clinicopathological case series of former Australian rugby code players, four of six had post mortem chronic traumatic encephalopathy neuropathological change.
• All cases met current criteria for traumatic encephalopathy syndrome and had multiple pathologies at post mortem.
• Future studies are needed to improve the specificity of the traumatic encephalopathy syndrome consensus diagnostic criteria for predicting the presence of chronic traumatic encephalopathy neuropathological change.
• All neuropathological comorbidities should be clearly reported in future studies investigating chronic traumatic encephalopathy neuropathological change and traumatic encephalopathy syndrome.

Microscopic analysis
We assessed pathologies based on published diagnostic criteria [2].
According to the current 2021 consensus criteria, we identified CTE-NC as the presence of one or more pathognomonic lesions in the cortex consisting of phosphorylated tau aggregates in neurons, with or without glial tau in thorn-shaped astrocytes, at the depth of a cortical sulcus around a small blood vessel [2].Pathology staging was performed based on the 2021 (low or high) consensus criteria [2].Features of CTE-NC were also assessed, meaning that some of the characteristics of CTE-NC were present but the case did not meet strict criteria for CTE-NC, as previously described [13].
The presence of thorn-shaped or granular/fuzzy astrocytes indicating age-related tau astrogliopathy (ARTAG) [32] was also assessed and the distribution noted, specifically whether it was situated in the cortex, basal ganglia and brainstem and whether the arrangement was subpial, subependymal, perivascular or located in the white matter.
The brains of six individuals were collected between 2019 and 2022 for neuropathological examination.The demographic details of these individuals can be seen in Table 1.

Clinical information
For five out of the six cases, the information pertaining to medical history, including clinical diagnoses, was collected during an in-person research visit as part of the Former Elite-Level Athlete Brain Health Research Programme.Each participant that attended the in-person research visit was accompanied by their next of kin.For the sixth case, this information was collected from the next of kin posthumously.Information was collected via clinical interview and health questionnaires completed by the participant and/or their next of kin, and neuropsychological testing was conducted with the participant.Information regarding the lifetime history of sport participation was collected from the participant (if they were capable of doing so) or the informant.

Neuropathological assessment
Of the six donors whose brains were collected, all met neuropathological criteria for more than one neuropathological entity or disease (see Could not be reliably staged due to the presence of coexisting pathologies.

CTE-NC IN AUSTRALIAN RUGBY PLAYERS
Table 1).Four of the six cases met the criteria for CTE-NC (Cases 1-3 and 6; Figure 1), but due to the presence of significant coexisting tau pathology, CTE-NC could only be staged in two of the four cases.Tau pathology levels were high in both cases (see Table 1).All six cases had TDP-43 pathology consistent with limbic age-related TDP-43 encephalopathy (LATE) [36].Three of the cases (Cases 2, 3 and 6) had TDP-43 pathology that spread beyond the medial temporal lobe and into the neocortex (Stage 3 LATE), where TDP-43 was sometimes distributed in a similar pattern to the tau pathology and colocalised with astrocytic tangles (Figure 2).The amyloid-beta pathology was varied in the cases, with two cases having no or minimal beta-amyloid pathology (Cases 2 and 3).Four cases (Cases 1-4) showed remote infarctions of either cortical, cerebellar, hippocampal or deep white matter regions.
All of the cases had at least some ARTAG in the form of granular fuzzy or thorn-shaped astrocytes in the cortex (Figure 3).ARTAG was also present in the brainstem and, to a lesser extent, in the basal ganglia of most cases (Cases 1-4 and 6).Thorn-shaped astrocytes dominated in the brainstem and were present throughout the midbrain tegmentum where they showed some perivascular localisation (Figure 3).Despite the presence of significant tau pathology throughout the hippocampus and entorhinal cortex, CA4 dendritic swellings were not identified in any of the cases (data not shown).The neuropathological findings of the six cases are presented in Table 1.

Sport exposure and clinical information
All former players had extensive exposure to collision sports.The duration of their rugby playing career ranged from 22 to 33 years.
Their duration of play at the elite level ranged from 8 to 19 years.The games they played at the elite level ranged from 28 to 383.
All six cases had a documented neurological disease or condition for which they had received health care services.The onset of their neurological problems was many years after the end of their playing careers, and all had a progressive worsening over several years.The clinical characteristics of the cases are presented in Table 2.All six cases had dementia prior to death.Depigmentation of the substantia nigra (B, F, J, N, R and V) and varying degrees of hippocampal atrophy (C, G, K, O, S and W) were also consistently seen.Superior surfaces of the cerebrum show cerebral atrophy was often minimal (D, H, L, P, T and X).Case 5 substantia nigra was not shown as image not available.
5 had evidence of neurobehavioral dysregulation reported by informants.Cases 1, 3 and 4 did not have evidence of neurobehavioral dysregulation reported by an informant.Case 6 is 'unknown' because there was no reported evidence available from an informant.It is possible that some of the men in our case series experienced aspects of neurobehavioral dysregulation during their lives that we did not document, because a lifetime history of behaviours and problems comprising this criterion was not specifically examined for during their research visits.It was only documented if it came up as a notable and important problem area for the men during their research interview or with their informant.

DISCUSSION
We applied the consensus criteria for CTE-NC [2] to a sample of former elite-level Australian rugby code players based on microscopic tissue examination after death.CTE-NC was identified in four out of the six cases, and coexisting neuropathologies were present in all cases.LATE and ARTAG were present in all cases, and intermediateto-high Alzheimer's disease neuropathologic change (AD-NC) was present in four cases.Hippocampal sclerosis was present in two cases.
We also applied the new consensus criteria for TES [22] to these cases using evaluation data obtained during life to examine the methodology for the clinical diagnosis of TES and the TES criteria for predicting the provisional levels of certainty for the presence of CTE-NC.
All individuals included in this study appeared to meet clinical diagnostic criteria for TES because they had far more than 5 years' exposure to repetitive head impacts (RHI) through participation in collision sports during youth and early adulthood and they exhibited progressive cognitive impairment later in life.We identified several methodological challenges associated with applying the TES diagnostic criteria, particularly the criterion that requires ruling out other conditions that could fully account for the core clinical features.In all cases, the T A B L E 2 Clinical characteristics relating to the 2021 consensus criteria for traumatic encephalopathy syndrome.Note: All cases had dementia and were receiving care in supported care facilities (i.e., nursing homes) at the end of their lives.Abbreviation: CTE-NC, chronic traumatic encephalopathy neuropathologic change.
subjects had clinical conditions and diagnoses during life that are associated with cognitive impairment.
Asken et al. applied the 2021 TES criteria [22] to an autopsy cohort of nine patients (age range 49-84 years) [37].They reported CTE-NC in five patients, with three classified as probable TES and two with uncertain TES (due to unknown exposure) despite having high CTE-NC.Of the remaining cases without CTE-NC, two were classified as probable TES but had a neuropathological diagnosis of AD-NC and frontotemporal lobar degeneration with tau (corticobasal degeneration).Similar to our findings, Asken et al. emphasised that their cases had multiple neuropathologies that likely contributed to the clinical signs and symptoms and cautioned against attributing clinical findings, neuroimaging findings or other biomarker changes associated with RHI or a TES diagnosis solely to CTE-NC [37].
The presence of multiple pathologies has been previously reported in neuropathological studies of former athletes, including a study of 21 Australian collision sports players where two or more pathologies were identified in 88% of aged (75 ± 19 years) and 50% of younger (48 ± 19 years) individuals [38].Outcomes from the UNITE study also identified AD-NC and Lewy body disease (LBD) in 39% of participants over the age of 50 years, with a similar pattern of LBD pathology in cases with CTE-NC compared with LBD alone [39].
Other clinicopathological studies have reported that AD-NC, TDP-43 and alpha-synuclein pathologies all commonly co-occur with CTE-NC [3], particularly those cases with higher CTE-NC stage [3].In one study, when the clinical and neuropathological information is used to generate an integrated clinicopathological diagnosis, findings suggest that CTE-NC is a common comorbidity but less frequently believed to be a primary cause of dementia in former high-exposure athletes [8].Consistent with this finding, a recent study of 571 former athletes (age 19-97 years, average 65 years) reported two or more pathologies in 62.5% of cases.Furthermore, the greatest contributors to dementia were AD-NC and LBD [40].
Additionally, some neuropathologies are observed to coexist with CTE-NC with high frequency that require further investigation.TDP-43 inclusions characterise several disorders including amyotrophic lateral sclerosis, frontotemporal lobar degeneration and LATE [33,36].
LATE-NC is seen in $25% of elderly individuals [41] and is characterised by neuronal cytoplasmic inclusions in the amygdala (Stage 1), hippocampus (Stage 2) and middle frontal gyrus (Stage 3) [36] where Stages 2 and 3 have been associated with impairments in memory and other cognitive abilities [41,42].It is commonly seen in association with AD-NC, where its frequency increases with greater AD-NC disease stages [41].TDP-43 pathology was also considered to be a supportive feature of CTE-NC according to the 2016 consensus criteria [1], but not in the revised consensus criteria published in 2021 [2].TDP-43 pathology has recently been reported to co-occur in 43% of individuals with CTE-NC, and hippocampal sclerosis was present in $one in four cases (i.e., 23.4%) [43].In addition to being a frequent feature of the normal aged brain [14,32], ARTAG is a significant feature in those with CTE-NC and it is frequently seen in neurodegenerative diseases, including Alzheimer's disease [45].One study suggests that the presence of astroglial tauopathy reportedly facilitates CTE-NC diagnosis over neuronal pathology alone, indicating it may play a significant role in the pathology of CTE-NC [46].CTE-NC and ARTAG have been shown to share molecular and histopathological features, indicating a potential common aetiology [47].However, it has been suggested that astrocyte pathology is more common in more advanced stages of CTE-NC [48] and is less common in younger individuals with CTE-NC [49].Of the two cases that did not meet the criteria for CTE-NC, both had significant ARTAG in the form of granular fuzzy astrocytes in the cortex (Case 4) and thorny astrocytes in the subpial, subependymal, perivascular (Case 4) and white matter (Cases 4 and 5) [32].The perivascular ARTAG seen in Case 4 was in a patchy distribution in the sulcal depth and consequently met the criteria for 'features of CTE-NC' but for the lack of tau-positive neurons (Figure 4).Whether ARTAG or 'features of CTE-NC' represent a precursor to the diagnostic neuropathology is still unknown, although previous animal studies indicate that astrocytes play an important role in tau seeding and may play a crucial role in the pathogenesis of tauopathies [50].Furthermore, ARTAG-like neuropathology has been reported to persist decades after surgical brain injury, indicating that brain injury of any type may predispose to ARTAG [47].Although the presence of significant ARTAG seen in Case 4 is not unusual for an 82-year-old man, the patchy distribution at the sulcal depths is not consistent with the subpial, subependymal, perivascular and white matter distribution usually observed in normal ageing [45].Future molecular and genomic studies assessing regional variations will assist with better characterising the similarities and differences between ARTAG and other astroglial tauopathies.

CTE-NC IN AUSTRALIAN RUGBY PLAYERS
A strength of this study lies in the clinical evaluation (for five out of the six cases) conducted for research purposes during life.Another strength is the extensive bilateral tissue sampling that was carried out on all of the donors.According to the authors of the 2021 consensus criteria for CTE-NC, a methodological limitation of previous studies using limited sampling is that further sampling of recommended diagnostic regions may reveal pathologies that are missed from screening limited cortical regions [2].A weakness of this study is its small size and the inability to carry out any statistical analysis to examine the strength of association between any clinical and pathological variables.Future studies should include larger samples of individuals who have undergone clinical assessments prior to neuropathological examination.
Multiple pathologies are common in former athletes with a history of RHI [40], which will likely complicate the clinical diagnosis and make it difficult for researchers to draw inferences relating to the provisional levels of certainty for the possible presence of CTE-NC.It can be assumed that all former elite and professional athletes who participated in American football, soccer, rugby and ice hockey will meet the exposure criterion for RHI, which was certainly the case for the former rugby players in this study.Therefore, former elite and professional contact and collision sport athletes who develop memory and/or executive functioning later in life may meet the criteria for TES but may also be likely to meet criteria for a neurodegenerative disease, such as AD, which should not be neglected.In fact, recent research suggests that AD-NC and LBD may be the greatest contributors to dementia in these individuals [40].
Neurobehavioural dysregulation is a core feature of TES [22], defined as poor regulation or control of emotions and/or behaviour, including (but not limited to) explosiveness, impulsivity, rage, violent outbursts, having a short fuse (exceeding what might be described as periodic episodes of minor irritability) or emotional lability (often reported as mood swings) (p.852).However, a large-scale clinicopathological study found no association between features of neurobehavioural dysregulation and the presence of CTE-NC [19].Specifically, when comparing a sample of 244 brain donors who had CTE-NC (mean age 63.6) to 92 donors who did not have CTE-NC (mean age 50.6), there was no significant difference in the percentages who experienced verbal violence, physical violence, explosivity or impulsivity during life [19].The TES criteria acknowledge that neurobehavioural dysregulation alone limits the diagnostic certainty of CTE-NC to 'suggestive of CTE' [22].However, a recent study of 152 young contact and collision sport athletes under 30 years of age also did not find an association between clinical symptoms, including neurobehavioral dysregulation, and CTE-NC status (41.4% had CTE-NC), despite a high frequency of cognitive and neurobehavioural symptoms in this convenience sample [49].Similarly, psychiatric features are also used to draw inferences relating to provisional levels of certainty that a research subject might harbour CTE-NC.This, too, will be an important area for future research-to determine if psychiatric features are clinical correlates of CTE-NC that should be used for drawing inferences about the possible presence of CTE-NC during life.The best available evidence suggests that the TES-supportive psychiatric features are not associated with CTE-NC.Specifically, when comparing a sample of 244 brain donors who had CTE-NC to 92 donors who did not have CTE-NC, there was no significant difference in the percentages who experienced anxiety, apathy, depressive symptoms, hopelessness, suicidality or paranoia during life [19].

CONCLUSION
In this clinicopathological case series of former Australian rugby code players, four of six had post mortem CTE-NC.All met current diagnostic criteria for TES and had multiple pathologies at post mortem.
Future studies are needed to improve the specificity of the TES consensus diagnostic criteria for predicting the presence of CTE-NC, and we encourage future researchers to report all neuropathological comorbidities clearly in their clinicopathological studies.

Cases 4 and 5
Figure 5C,G,K,O,S,W) was present in the majority of the cases, but cortical atrophy was more variable and often mild (Cases 1-4; Figure 5D,H,L,P,T,X).

Case 1
had a clinical diagnosis of Alzheimer's disease.He was seen for two research visits and underwent neuropsychological testing.In his early 70s, he performed in the broadly normal range on memory tests, and he had low scores on tests of executive functioning.When tested a few years later, he had a clear decline in memory performance and multiple other areas of cognitive functioning, consistent with dementia.Case 2 experienced Parkinsonism.He underwent neuropsychological testing several times from his early to mid-60s.He showed evidence of memory impairment and executive functioning across multiple evaluations.His mobility deteriorated over a number of years, to the point that he was confined to a wheelchair.He had F I G U R E 2 TDP-43 and tau pathology.TDP-43 and tau were seen in a similar distribution in TSAs at the pial surface of neocortical regions.(A), (D) and (G) show tau-positive TSAs, (B), (E) and (H) show TDP-43 positive TSAs from an adjacent section and (C), (F) and (I) show high magnification TDP-43 immunopositive TSAs.(A), (B), (D), (E), (G) and (H) = 100Â magnification and (C), (F) and (I) = 400Â magnification.Cases 2 and 6 = inferior temporal cortex and case 3 = superior frontal cortex.F I G U R E 3 Age-related tau astrogliopathy was a feature of all six cases.All cases had age-related tau astrogliopathy in the cortex as TSAs (A) or GFAs (B).TSAs were also seen frequently in the brainstem in a perivascular arrangement (C) and were present less frequently in the striatum (D).(A) = 200Â, (B) = 400Â and (C) and (D) = 100Â magnification.(A) and (B) = superior frontal cortex, (C) = midbrain tegmentum and (D) = caudate.F I G U R E 4 Two cases showed features of chronic traumatic encephalopathy neuropathologic change (CTE-NC).These two cases did not meet the criteria for CTE-NC.Case 4 showed patchy TSAs in the sulcal depth of the right middle frontal gyrus, although the distribution was not perivascular and there was no visible neuronal tau (A and B).The black arrowhead in (A) shows the sulcus.Case 5 did not show any features of CTE-NC.Neuronal tau was evenly distributed throughout the cortex, meeting criteria for Alzheimer's disease neuropathologic change (C), with astrocytic tau mostly confined to white matter (D, black arrows).(A) and (C) = 25Â and (B) and (D) = 100Â magnification.(A) and (B) = middle frontal cortex and (C) and (D) = superior frontal cortex.severe dementia during the final year of his life.Case 3 had memory impairment and a clinical diagnosis of Alzheimer's disease, and he also experienced a stroke and had seizures.He underwent neuropsychological testing in his early 80s and had some low scores on tests of executive functioning, but not below 1.5 standard deviations from the mean.Case 4 had a clinical diagnosis of Alzheimer's disease and was living in a nursing home for many years prior to his death.He was too cognitively impaired to undergo neuropsychological testing for research purposes.Case 5 had memory impairment and a clinical diagnosis of posterior cortical atrophy and a seizure disorder during life.He underwent neuropsychological testing in his early 50s.Case 6 had a clinical diagnosis of Alzheimer's disease.Cognitive problems became apparent in his early 80s, according to information provided by a family member after his death.The core clinical feature, neurobehavioral dysregulation, was present in two of our cases, one of whom had post mortem CTE-NC (Case 2) and one who did not (Case 5).Case 2 had behavioural control problems that were difficult to manage in the nursing home.CaseF I G U R E 5 Macroscopic features of cases.Macroscopic images show all six cases with a cavum septum pellucidum (A, E, I, M, Q and U).
Demographic and neuropathological details of all cases.
Abbreviations: A score, β-amyloid plaque distribution, A0-A3; AD-NC, Alzheimer's disease neuropathologic change; ARTAG, age-related tau astrogliopathy; B score, neurofibrillary tangle distribution, B0-B3; C score, CERAD score, C0-C3; CTE-NC, chronic traumatic encephalopathy neuropathologic change; LATE, limbic-predominant age-related TDP-43 encephalopathy; LBD, Lewy body disease.a [43]s et al. reportedTDP-43 pathology occurred frequently in frontal cortex sulcal depths in association with CTE-NC, even in the absence of limbic inclusions, leading the authors to speculate that TDP-43 deposition in CTE-NC might differ from prototypical LATE-NC based on the distribution and frequency of pathology and its premature deposition in the neocortex[43].Although hippocampal sclerosis was seen in cases with CTE-NC, it was always in the presence of LATE-NC, supporting the theory that hippocampal sclerosis is more commonly, but not exclusively, associated with LATE-NC rather than CTE-NC[43].Nicks et al. also noted a preferential distribution of TDP-43 in the CA2 in CTE-NC compared with LATE-NC.All the cases in our study had significant