The Neuropathology of Chronic Traumatic Encephalopathy

INTRODUCTION

Over the last decade, there has been increased awareness regarding the long-term consequences of mild traumatic brain injury (mTBI), including concussion and subconcussion, associated with sports participation and exposure to explosive blast (59). Although the concept that brain trauma from boxing might induce a chronic neurodegenerative condition was recognized nearly a century ago, it is only recently that modern sports, such as American football, soccer, ice hockey and rugby (57, 59, 74, 75), have been associated with a similar deterioration. The first report in the medical literature was made by Harrison Martland, a New Jersey pathologist and medical examiner, who described a clinical syndrome consisting of poor balance, floppiness of the extremities, mental confusion and slowing of muscular action that developed in “nearly half of the fighters who have stayed in the game long enough” (53). Moreover, Martland also noted that some affected boxers went on to develop a severe and progressive syndrome manifest as Parkin-sonism and mental deterioration that necessitated a “commitment to an asylum” (53). Although Martland referred to this condition as “punch drunk” (53), other terms were gradually introduced to describe the condition, including “traumatic progressive encephalopathy” (79) and “dementia pugilistica” (63). From the 1940s onward, the broader designation “chronic traumatic encephalopathy” or “CTE” was in common use, recognizing that the condition could arise from brain trauma from a variety of sources in addition to boxing (22, 27, 43).

The neuropathological aspects of CTE were first reported as case reports or small case series of boxers who developed Parkinsonism and dementia (9, 18, 21, 32, 55, 70). Gross neuropathological findings included cerebral atrophy, enlarged ventricles, cavum septum pellucidum and depigmentation of the substantia nigra, with microscopic evidence of neurofibrillary tangles (NFT) in the cortex and brainstem, sometimes associated with senile plaques or neuronal loss. In 1973, Corsellis et al published a large clinicopathological series of CTE in 15 boxers, ranging in age from 57 to 91 years (19). This landmark paper further described enlarged lateral and third ventricles, thinning of the corpus callosum, cavum septum pellucidum with fenestrations and scarring of the cerebellar tonsils as important gross neuropathological findings. Microscopically, Corsellis et al used cresyl violet stains and Von Braunmühl’s silver stains to characterize neuronal loss in the cerebral cortex, substantia nigra and cerebellar tonsils, neurofibrillary degeneration of the substantia nigra and cerebral cortex and senile plaques in 27% of cases (19). Subsequent re-examination of Corsellis’ original series of boxers and additional cases using beta-amyloid (Aβ) immunohistochemistry determined that 95% of CTE cases showed widespread diffuse Aβ deposits (82, 96).

In 1990, Roberts et al reported the neuropathological findings of CTE in the first female subject, a nonboxer. The case involved a woman who has been physically abused for decades who developed dementia with prominent memory loss and confusion several years before her death at age 76 years (81). Necropsy revealed the “cauliflower ears” of pugilists and old rib fractures, while brain examination revealed cerebral atrophy, ventricular dilatation, septal cavum with fenestrated leaves and multiple lacunes. NFT were present in the hippocampus and brainstem; senile plaques, primarily of the diffuse type, were found in the frontal and temporal cortex.

In 1991, Hof et al reported CTE in a young woman, 24 years old, who was autistic and displayed prominent head-banging behaviors (38). Neuropathological examination was notable for cerebral atrophy, cavum septum, NFT in the perirhinal and entorhinal cortex, amygdala, prepiriform and orbitofrontal cortex in the absence of Aβ deposits.

Five young men, ranging in age from 23 to 28 years with neuropathological alterations of early CTE were reported by Geddes et al (27). The subjects included a soccer player, a “mentally subnormal” individual with a long history of head banging, an epileptic who frequently hit his head during seizures and two boxers. There were no grossly detectable changes to the brain, however, microscopic examination demonstrated argyrophilic, phosphorylated tau (p-tau)-positive NFTs and neuropil threads (NT) “strikingly arranged in groups, sited predominantly around small intracortical blood vessels, often found at the depths of the cortical sulci” (27). NFT were not found in any of the 21 age-matched control cases that Geddes et al examined. Geddes et al also noted that the arrangement and distribution of NFT was very unlike the early p-tau pathology of Alzheimer’s disease (AD) (27). Furthermore, no hemosiderin, astrocytosis or microglial proliferation was evident around the vessels surrounded by p-tau pathology nor was there any evidence of any Aβ and the hippocampus was normal in all cases.

Hof et al reported the neuropathological findings in three boxers with severe memory disturbance, cognitive abnormalities, dysphoria and Parkinsonism and drew attention to the superficial distribution of the NFT to layer II and the upper third of layer III of the neocortex, unlike the distribution of NFT in AD (39).

In 1996, Williams and Tannenberg (99) reported the findings in a 33-year-old achondroplastic dwarf who worked for 15 years as a clown in a circus, participated in dwarf-throwing events and had been knocked unconscious “a dozen times.” Autopsy showed dilatation of the lateral and third ventricles and cerebral aqueduct, a thinned corpus callosum, fenestrated septum pellucidum, shrinkage of the medial dorsal nucleus of the thalamus and atrophy of the vermis of the cerebellum. Microscopic examination revealed occasional NFT in the hippocampus without Aβ (99).

Omalu et al (74, 75) first described the clinical and pathological findings of two retired National Football League (NFL) football players. The men had played football for 14–24 years including 8–17 years in the NFL as offensive linemen. Both had developed behavioral and mood changes, memory loss and cognitive impairment. Histologically, in one subject, there were NFT and NT in neocortex, associated with sparse diffuse Aβ plaques (74). The brain of the other subject showed NFT in the frontal, temporal, inferior parietal, insular and entorhinal cortices, hippocampus, amygdala, diencephalon, substantia nigra and locus coeruleus (75). Omalu et al also reported the finding of CTE in a 40-year-old professional wrestler characterized by NFT and NT in the cerebral cortex, deep nuclei and brainstem without Aβ deposits (76).

By 2014, the list of sports whose participants had been diagnosed with CTE expanded to include rugby, professional baseball, soccer, ice hockey and American football at the high school, college and professional level (30, 59, 60).

Moreover, early stage CTE was reported in military personnel exposed to repetitive mTBI from explosive blast injuries and repetitive concussion (30, 56, 73). The early p-tau changes associated with blast neurotrauma share similarities with the changes of early CTE reported in young American football and soccer players, boxers, head-bangers and others (27, 30, 59, 60), although it is likely that there are additional brain pathologies associated with blast exposure that are not characteristic of impact injuries. Four of the five veterans with early stage CTE were also diagnosed with posttraumatic stress disorder (PTSD) suggesting that PTSD might share biological and pathological underpinnings with CTE. Advanced CTE has also been reported after TBI sustained during military service (56).

In a large case series, McKee et al reported the clinical and immunocytochemical characteristics of 68 men with CTE, ranging in age 17–98 years, and compared them to 18 age-matched men without a history of repetitive mTBI (59). McKee et al also introduced pathological criteria for the neuropathological diagnosis of CTE (Table 1) and a staging system for grading pathological severity (Figure 1) (59).

Figure 1

Stages of chronic traumatic encephalopathy (CTE) [adapted from McKee et al (59)]. In stage I CTE, p-tau pathology is found in discrete foci in the cerebral cortex, most commonly in the superior or lateral frontal cortices, typically around small vessels at the depths of sulci. In stage II CTE, there are multiple foci of p-tau at the depths of the cerebral sulci and there is localized spread of neurofibrillary pathology from these epicenters to the superficial layers of adjacent cortex. The medial temporal lobe is spared neurofibrillary p-tau pathology. In stage III CTE, p-tau pathology is widespread; the frontal, insular, temporal and parietal cortices show widespread neurofibrillary degeneration with greatest severity in the frontal and temporal lobes, concentrated at the depths of the sulci. Also in stage III CTE, the amygdala, hippocampus and entorhinal cortex show substantial neurofibrillary pathology that is found in earlier CTE stages. In stage IV CTE, there is widespread severe p-tau pathology affecting most regions of the cerebral cortex and the medial temporal lobe, sparing calcarine cortex in all but the most severe cases. All images, CP-13 immunostained 50 μ tissue sections.

Neuropathology of CTE

Gross pathology

Grossly identifiable changes in the brain are unusual in early or mild CTE; if present, they are most often cavum septum pellucidum and mild enlargement of the frontal and temporal horns of the lateral ventricles. There may also be prominent perivascular spaces in the white matter, particularly in the temporal lobe. In intermediate and advanced CTE, macroscopic changes include a reduction in brain weight, gray and white matter atrophy (typically most severe in the frontal and anterior temporal lobes), enlargement of the lateral and third ventricles, cavum septum pellucidum, septal fenestrations, atrophy of the thalamus, hypothalamus and mammillary bodies, thinning of the isthmus of the septum corpus callosum and depigmentation of the locus coeruleus and substantia nigra (Figure 2). Although grossly identifiable cerebellar abnormalities were described in the initial reports of CTE affecting boxers, macroscopic cerebellar abnormalities are rarely present in CTE associated with other sports or activities (59).

Figure 2

Gross neuropathological features of advanced chronic traumatic encephalopathy (CTE). A. CTE stage IV in a former professional football player. Despite the relatively large size of the brain, there is generalized, symmetrical brain atrophy that is most severe in the frontal and temporal lobes. There is also flattening of the hypothalamic floor, atrophy of the mammillary bodies and pallor of the substantia nigra. B. The brain of a subject with Alzheimer’s disease, by contrast, also shows generalized, symmetrical brain atrophy but the hypothalamic floor, mammillary bodies and substantia nigra are normal. C. There is depigmentation of the substantia nigra and locus ceruleus and degeneration of the frontopontine tracts in the cerebral peduncle. D, E, F. Coronal sections show pronounced frontal and temporal lobe atrophy, marked ventricular dilatation, as well as cavum septum (A) and absence of the posterior septum (B) and (C). G. The floor of the hypothalamus is severely thinned and the mammillary bodies are markedly atrophic. H. Multiple septal perforations may be present. I. The posterior septum may be absent with only small blood vessels remaining. J. A large single septal perforation.

Microscopic pathology

In the mildest forms of disease, CTE is characterized by the deposition of p-tau protein as NFT and NT in conspicuous clusters in the neocortex, often arranged along small blood vessels at the depths of the sulci. The NFT typically follow the penetrating small cortical vessels as linear accumulations extending from the surface of the brain to the lowest layers of the cortical gray matter, or when observed in cross-section, as clusters NFT, pre-tangles and dot-shaped and thread-like NT in a penumbra around small arterioles (Figure 3). Mild forms of the disease affect primarily the neurons, although there may be focal subpial p-tau immunopositive astrocytes at the sulcal depths and occasional p-tau immunoreactive glia around the affected vessel (Figure 3). The pathological significance of isolated subpial p-tau astrocytes at the sulcal depths unaccompanied by perivascular NFT and NT in the underlying cortex is unknown, although subpial p-tau astrocytes at the depths of the cortical sulci is not a phenomenon found in normal aging and has not been found in the brains of subjects without a history of repetitive brain trauma.

Figure 3

Microscopic findings in stage II chronic traumatic encephalopathy (CTE). A. Whole mount coronal sections in stage II CTE show multiple foci of p-tau pathology primarily located at the depths of the cortical sulci of the frontal and temporal lobes (free floating 50 μ sections, AT8 immunostain). B–H. The p-tau pathology consists of neurofibrillary tangles and dot-like and thread-like dystrophic neurites and is characteristically found around small blood vessels (B–F, free floating 50 μ sections, AT8 immunostain; G, H, 10 μ paraffin embedded sections, AT8 immunostain). (I). Subpial astrocytic tangles are also found at the cortical depths (free floating 50 μ sections, AT8 immunostain). Other pathologies include pretangles (J), dystrophic neurites in the white matter (K) and occasional p-tau astrocytes (L) (free floating 50 μ sections, AT8 immunostain). There may be marked astrocytosis of the white matter (M, N) (free floating 50 μ sections, Glial fibrillary acidic protein immunostain). Hemosiderin-laden macrophages (O) (10 μ paraffin section, Luxol fast blue hematoxylin and eosin stain) and multiple perivascular foci of reactive microglia (P) are found around small vessels in the cerebral white matter (free floating 50 μ sections, LN3 immunostain).

Provisional criteria for the neuropathological diagnosis of CTE are listed in Table 1 (59). These criteria are currently being validated by a panel of expert neuropathologists as part of a UO1 funded by the National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Biomedical Imaging and Bioengineering (NIBIB) (1U01NS086659-01). The provisional criteria are founded on the hypothesis that the irregular, perivascular p-tau pathology found in clusters at the depths of the cortical sulci distinguishes CTE from other tauopathies, including AD (27, 28, 57–59); this hypothesis will be formally assessed at the NINDS/NIBIB-sponsored conference. One of the more difficult tasks will be deciphering the point at which the pathology is sufficient to make a diagnosis of CTE, as opposed to CTE-related changes. This conundrum is reminiscent of the early phases of AD neuropathology when it was difficult to distinguish between AD-related changes and a neuropathological diagnosis of low, intermediate and high likelihood AD (71). Similarly, stage I CTE is likely a preclinical pathology, sometimes asymptomatic, and sometimes associated with prolonged post-concussive symptoms, but not necessarily progressive. Stage II CTE may occasionally be asymptomatic, although in many cases, it is associated with florid behavior and mood changes. Determining the degree and pattern of microscopic features necessary to diagnose comorbid CTE when another tauopathy is present will be another challenge for the future, such as when CTE is present in addition to AD, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease or many others.

The tau isoform profile and phosphorylation state in CTE are similar to AD (84) and the neuronal p-tau pathology shows immunoreactivity to both 3 repeat and 4 repeat (4R) tau (59, 91). The 4R isoform of tau is predominantly expressed in astrocytes located in the subpial region of the sulcal depths (59, 91), although some of the neuronal abnormalities in the hippocampus also appear to be primarily 4R tau in CTE (Figure 4).

Figure 4

Patterns of 3R and 4R tau in chronic traumatic encephalopathy (CTE) [adapted from McKee et al (59)]. A, D. 3R immunostaining shows scattered RD3 immunoreactive neurons in middle frontal cortex. B, E. 4R immunostaining shows many ET3 immunoreactive neurons and astrocytic tangles in the subpial region of the middle frontal cortex and at the depth of the sulcus. C, F. AT8 immunostaining shows 3R and 4R immunopositive neurons and astrocytic tangles in middle frontal cortex. G, J, M. 3R immunostaining shows scattered RD3 immunore-active neurons in CA1 (G), CA2 (J) and CA4 hippocampus (M). H, K, N. 4R immunostaining shows many ET3 immunoreactive neurons in CA1 (H), CA2 (K) and CA4 hippocampus (N). I, L, O. I. AT8 immunostaining shows 3R and 4R immunopositive neurons in CA1 (I), CA2 (L) and CA4 hippocampus (O). All 10 μ paraffin embedded sections, magnification bars 50 μm.

Staging of pathology in CTE

Stage I

Brains with stage I CTE are grossly unremarkable but microscopically show rare, isolated perivascular focal epicenters of p-tau NFTs and neuropil neurites (Figure 1). The NFTs and neuropil neurites predominate and the neurites are characteristically dot-like as well as thread-like. Occasional p-tau immunoreactive glia and glial processes may be present. Tau pathology is most commonly localized to the depths of the cerebral sulci of the frontal, temporal, insular, septal and parietal cortices, although there may also be sparse NFT scattered throughout the adjacent cortex. There also may be subpial p-tau astrocytic tangles in clusters at the depths of sulci. NFT and neurites are also found in the locus coeruleus. Approximately one-half of cases with stage I p-tau pathology show rare TDP-43 neurites. Clusters of reactive microglia are found in the white matter with axonal swellings and distorted profiles in the subcortical U-fibers. Aβ plaques are not found in subjects with stage I CTE, unless over the age of 50 years. Hemosiderin-laden macrophages are characteristically found around small vessels in the white matter, usually not associated with p-tau abnormalities.

Stage II

Subtle macroscopic changes may be found in stage II CTE cases including mild enlargement of the frontal horns of the lateral ventricles and third ventricle, cavum septum pellucidum and pallor of the locus coeruleus and substantia nigra. Microscopically, multiple epicenters of perivascular foci of p-tau NFT and neurites are found at the depths of the frontal, temporal, parietal, insular and septal cortices. These cerebral epicenters typically consist of perivascular p-tau NFT, pre-tangles, neurites and occasional glia (Figure 3) and may be associated with p-tau astrocytic tangles in the overlying subpial region. In the cortex adjacent to the focal epicenters, NFT are scattered throughout the cerebral layers with a tendency to be most numerous in the superficial cortical layers. There are also NFT in the locus coeruleus and substantia innominata. Mild TDP-43 pathology may be found in stage II CTE cases as abnormal neurites and neuronal inclusions. Clusters of reactive microglia are often found in the subcortical white matter with axonal swellings and distorted profiles in the subcortical U-fibers. Aβ plaques are found in 19% of subjects with stage II CTE, only if older than 50 years.

Stage III

Most brains with stage III CTE show macroscopic changes although the gross structural changes are minor relative to the changes found microscopically. Typically, there is a reduction in brain weight, mild frontal and temporal atrophy and enlargement of the lateral and third ventricles. One-half of subjects have septal abnormalities including cavum septum pellucidum, septal perforations or fenestrations. Pallor of the locus coeruleus and substantia nigra, atrophy of the mammillary bodies, thalamus and hypothalamus and thinning of the corpus callosum are characteristic. Microscopically, confluent patches of NFT, NTs and astrocytic tangles are found around blood vessels at the sulcal depths, as well as in linear arrays in the superficial laminae of cortex. NFT are found in the superior, dorsolateral and inferior frontal, septal, insular, temporal pole, superior, middle and inferior temporal and inferior parietal cortices. NFT are also found in the olfactory bulbs, hippocampus, entorhinal cortex, amygdala, hypothalamus, mammillary bodies, nucleus basalis of Meynert, substantia nigra, dorsal and median raphe nuclei and locus coeruleus. Neurofibrillary degeneration in the hippocampus includes CA4 and CA2, as well as CA1.

Approximately one-third of stage III cases have occasional NFT in the dentate nucleus of the cerebellum and the spinal cord gray matter. TDP-43-positive neurites and inclusions are found in the majority of cases. Thirteen percent of stage III CTE cases show sparse diffuse and neuritic Aβ plaques. White matter abnormalities, loss of myelinated nerve fibers, axonal dystrophy and axonal loss may be conspicuous (Figure 5).

Figure 5

Microscopic changes in stage III chronic traumatic encephalopathy (CTE). (A) Whole mount coronal sections in stage III CTE show multiple cortical foci of p-tau pathology throughout the frontal and temporal cortices. The cortical epicenters and depths of the sulci often consist of confluent masses of neurofibrillary tangles (NFT) and astrocytic tangles (ATs). (B–D) The p-tau pathology consists of NFTs, ATs and dot-like and thread-like dystrophic neurites clustered around the penetrating cortical vessels. (E) Cortex adjacent to the cortical epicenters shows scattered NFT. (F) The hippocampus shows dense neurofibrillary pathology. Subpial ATs (G) and p-tau immunoreactive astrocytes may be prominent (H). There may be dystrophic neurites in the white matter (I). SMI-34 immunostaining shows reduction in axonal staining and numerous large, irregular axonal varicosities (J). (A–I. free floating 50 μ sections, AT8 immunostain; J. SMI-34 immunostain, 10-μ paraffin section).

Stage IV

Brains with stage IV CTE show a decrease in brain weight, which in some instances may be substantial. Brain weights of 1000 g or less are not uncommon. In addition to generalized cerebral atrophy, there is pronounced atrophy of the frontal and temporal lobes, medial temporal lobe and anterior thalamus. There is also diffuse atrophy of the white matter and thinning of the corpus callosum, particularly the isthmus. There is typically severe thinning of the hypothalamic floor and atrophy of the mammillary bodies. Most cases of stage IV CTE show septal abnormalities including cavum septum, perforations, fenestrations or total absence of the posterior septum. The locus coeruleus and substantia nigra are pale. Microscopically, in stage IV CTE, there is widespread myelin loss, astrocytosis of the white matter and neuronal loss in the cerebral cortex, hippocampus and substantia nigra. Neuronal loss and astrocytosis may also be prominent in the frontal and temporal cortices associated with microvacuolation of layer II.

P-tau pathology is densely distributed throughout the cerebrum, thalamus, hypothalamus, mammillary bodies, basal ganglia, brainstem, cerebellar dentate nucleus and occasionally, spinal cord. Primary visual cortex is typically spared. NFT, most of which are extracellular, are found throughout the hippocampal formation including the dentate gyrus, CA3, CA2 and CA4. CA1 typically shows severe loss of neurons with substantial numbers of extracellular tangles. P-tau pathology also involves the cerebellum, including the dentate nucleus, granular cell layer, and sometimes, Purkinje cells. Irregular tau neurites are often found in the white matter of the anterior cerebellar vermis lining the fourth ventricle. Widespread TDP-43 deposits are also found with dot-like and thread-like inclusions in neurites and intraneuronal cytoplasmic inclusions. There is marked loss of myelinated nerve fibers, axonal dystrophy and loss throughout the cerebral and cerebellar white matter (Figure 6).

Figure 6

Microscopic changes in stage IV chronic traumatic encephalopathy (CTE). Whole mount coronal sections in stage IV CTE show widespread p-tau pathology affecting most regions of the cerebral cortex and medial temporal lobe. Astrocytic tangles are prominent and there is marked neuronal loss in the cortex, amygdala and hippocampus. There are also widespread pTDP-43 abnormalities. All images: 50 μ tissue sections, CP-13 or p-TDP-43 immunostain.

CTE with comorbid degenerative disease

Similar to most other neurodegenerative diseases (31, 42, 45, 88, 98), CTE is associated with the development of other neurodegenerations, especially as a function of increased age (37, 58, 59). Of 142 neuropathologically confirmed cases of CTE, 15 (10.56%) had coexistent motor neuron disease (MND), 15 (10.56%) had AD, 10 (7.04%) had Lewy Body disease, 4 (2%) had FTLD and 7 (4.51%) had two or more of these comorbidities; 89 (63.16%) of the cases were diagnosed only with CTE (Figure 7).

Figure 7

Chronic traumatic encephalopathy (CTE) with comorbid neuropathology. Of 142 neuropathologically confirmed cases of CTE at the VA-BU-SLI CTE Brain Bank, 15 (10.56%) had coexistent motor neuron disease (MND), 15 (10.56%) had Alzheimer’s disease (AD), 10 (7.04%) had Lewy Body disease, 4 (2.%) had frontotemporal lobar degeneration (FTLD) and 7 (4.51%) had two or more of these comorbidities; 89 (63.16%) cases were diagnosed only with CTE.

Imaging and biomarkers

Although there are no validated imaging techniques or biomarkers to confirm the diagnosis of CTE in living subjects, potentially useful imaging and biomarkers include positron emission tomography (PET) ligands for p-tau and neuroinflammation (13, 64), diffusion tensor imaging (4, 50), magnetic resonance spectroscopy (51) and CSF markers (10). P-tau-specific PET ligands, such as T-807 and T-808 reported by Chien et al (13) might prove to identify primary tauopathy, such as CTE, and p-tau PET imaging combined with PET imaging for Aβ might be useful to differentiate between CTE and AD (3, 44, 67). Recently, this technique was used to evaluate a retired NFL player with clinical symptoms suggestive of AD. He was found to have a negative florbetapir scan for Aβ and a positive scan for T-807, results supportive of CTE, although the pattern of T-807 positivity most likely represented non-specific off-target labelling (64). Additionally, more conventional imaging techniques and biomarkers might be useful in identifying some of the pathological features of “probable CTE” in vivo, such as abnormalities of the septum pellucidum, cortical thinning and/or cortical atrophy (Figure 8) (67). Other potential biomarkers for “probable CTE” include normal Aβ CSF fluid levels and an elevated CSF p-tau/tau ratio (67).

Figure 8

Coronal T1-weighted Magnetic resonance imaging shows a cavum septum pellucidum, cortical and white matter atrophy in former professional football player with neuropathologically confirmed chronic traumatic encephalopathy stage IV (arrows indicate the location of the cavum septum).

Neuropathology of CTE

Gross pathology

Grossly identifiable changes in the brain are unusual in early or mild CTE; if present, they are most often cavum septum pellucidum and mild enlargement of the frontal and temporal horns of the lateral ventricles. There may also be prominent perivascular spaces in the white matter, particularly in the temporal lobe. In intermediate and advanced CTE, macroscopic changes include a reduction in brain weight, gray and white matter atrophy (typically most severe in the frontal and anterior temporal lobes), enlargement of the lateral and third ventricles, cavum septum pellucidum, septal fenestrations, atrophy of the thalamus, hypothalamus and mammillary bodies, thinning of the isthmus of the septum corpus callosum and depigmentation of the locus coeruleus and substantia nigra (Figure 2). Although grossly identifiable cerebellar abnormalities were described in the initial reports of CTE affecting boxers, macroscopic cerebellar abnormalities are rarely present in CTE associated with other sports or activities (59).

Figure 2

Gross neuropathological features of advanced chronic traumatic encephalopathy (CTE). A. CTE stage IV in a former professional football player. Despite the relatively large size of the brain, there is generalized, symmetrical brain atrophy that is most severe in the frontal and temporal lobes. There is also flattening of the hypothalamic floor, atrophy of the mammillary bodies and pallor of the substantia nigra. B. The brain of a subject with Alzheimer’s disease, by contrast, also shows generalized, symmetrical brain atrophy but the hypothalamic floor, mammillary bodies and substantia nigra are normal. C. There is depigmentation of the substantia nigra and locus ceruleus and degeneration of the frontopontine tracts in the cerebral peduncle. D, E, F. Coronal sections show pronounced frontal and temporal lobe atrophy, marked ventricular dilatation, as well as cavum septum (A) and absence of the posterior septum (B) and (C). G. The floor of the hypothalamus is severely thinned and the mammillary bodies are markedly atrophic. H. Multiple septal perforations may be present. I. The posterior septum may be absent with only small blood vessels remaining. J. A large single septal perforation.

Microscopic pathology

In the mildest forms of disease, CTE is characterized by the deposition of p-tau protein as NFT and NT in conspicuous clusters in the neocortex, often arranged along small blood vessels at the depths of the sulci. The NFT typically follow the penetrating small cortical vessels as linear accumulations extending from the surface of the brain to the lowest layers of the cortical gray matter, or when observed in cross-section, as clusters NFT, pre-tangles and dot-shaped and thread-like NT in a penumbra around small arterioles (Figure 3). Mild forms of the disease affect primarily the neurons, although there may be focal subpial p-tau immunopositive astrocytes at the sulcal depths and occasional p-tau immunoreactive glia around the affected vessel (Figure 3). The pathological significance of isolated subpial p-tau astrocytes at the sulcal depths unaccompanied by perivascular NFT and NT in the underlying cortex is unknown, although subpial p-tau astrocytes at the depths of the cortical sulci is not a phenomenon found in normal aging and has not been found in the brains of subjects without a history of repetitive brain trauma.

Figure 3

Microscopic findings in stage II chronic traumatic encephalopathy (CTE). A. Whole mount coronal sections in stage II CTE show multiple foci of p-tau pathology primarily located at the depths of the cortical sulci of the frontal and temporal lobes (free floating 50 μ sections, AT8 immunostain). B–H. The p-tau pathology consists of neurofibrillary tangles and dot-like and thread-like dystrophic neurites and is characteristically found around small blood vessels (B–F, free floating 50 μ sections, AT8 immunostain; G, H, 10 μ paraffin embedded sections, AT8 immunostain). (I). Subpial astrocytic tangles are also found at the cortical depths (free floating 50 μ sections, AT8 immunostain). Other pathologies include pretangles (J), dystrophic neurites in the white matter (K) and occasional p-tau astrocytes (L) (free floating 50 μ sections, AT8 immunostain). There may be marked astrocytosis of the white matter (M, N) (free floating 50 μ sections, Glial fibrillary acidic protein immunostain). Hemosiderin-laden macrophages (O) (10 μ paraffin section, Luxol fast blue hematoxylin and eosin stain) and multiple perivascular foci of reactive microglia (P) are found around small vessels in the cerebral white matter (free floating 50 μ sections, LN3 immunostain).

Provisional criteria for the neuropathological diagnosis of CTE are listed in Table 1 (59). These criteria are currently being validated by a panel of expert neuropathologists as part of a UO1 funded by the National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Biomedical Imaging and Bioengineering (NIBIB) (1U01NS086659-01). The provisional criteria are founded on the hypothesis that the irregular, perivascular p-tau pathology found in clusters at the depths of the cortical sulci distinguishes CTE from other tauopathies, including AD (27, 28, 57–59); this hypothesis will be formally assessed at the NINDS/NIBIB-sponsored conference. One of the more difficult tasks will be deciphering the point at which the pathology is sufficient to make a diagnosis of CTE, as opposed to CTE-related changes. This conundrum is reminiscent of the early phases of AD neuropathology when it was difficult to distinguish between AD-related changes and a neuropathological diagnosis of low, intermediate and high likelihood AD (71). Similarly, stage I CTE is likely a preclinical pathology, sometimes asymptomatic, and sometimes associated with prolonged post-concussive symptoms, but not necessarily progressive. Stage II CTE may occasionally be asymptomatic, although in many cases, it is associated with florid behavior and mood changes. Determining the degree and pattern of microscopic features necessary to diagnose comorbid CTE when another tauopathy is present will be another challenge for the future, such as when CTE is present in addition to AD, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease or many others.

The tau isoform profile and phosphorylation state in CTE are similar to AD (84) and the neuronal p-tau pathology shows immunoreactivity to both 3 repeat and 4 repeat (4R) tau (59, 91). The 4R isoform of tau is predominantly expressed in astrocytes located in the subpial region of the sulcal depths (59, 91), although some of the neuronal abnormalities in the hippocampus also appear to be primarily 4R tau in CTE (Figure 4).

Figure 4

Patterns of 3R and 4R tau in chronic traumatic encephalopathy (CTE) [adapted from McKee et al (59)]. A, D. 3R immunostaining shows scattered RD3 immunoreactive neurons in middle frontal cortex. B, E. 4R immunostaining shows many ET3 immunoreactive neurons and astrocytic tangles in the subpial region of the middle frontal cortex and at the depth of the sulcus. C, F. AT8 immunostaining shows 3R and 4R immunopositive neurons and astrocytic tangles in middle frontal cortex. G, J, M. 3R immunostaining shows scattered RD3 immunore-active neurons in CA1 (G), CA2 (J) and CA4 hippocampus (M). H, K, N. 4R immunostaining shows many ET3 immunoreactive neurons in CA1 (H), CA2 (K) and CA4 hippocampus (N). I, L, O. I. AT8 immunostaining shows 3R and 4R immunopositive neurons in CA1 (I), CA2 (L) and CA4 hippocampus (O). All 10 μ paraffin embedded sections, magnification bars 50 μm.

Gross pathology

Grossly identifiable changes in the brain are unusual in early or mild CTE; if present, they are most often cavum septum pellucidum and mild enlargement of the frontal and temporal horns of the lateral ventricles. There may also be prominent perivascular spaces in the white matter, particularly in the temporal lobe. In intermediate and advanced CTE, macroscopic changes include a reduction in brain weight, gray and white matter atrophy (typically most severe in the frontal and anterior temporal lobes), enlargement of the lateral and third ventricles, cavum septum pellucidum, septal fenestrations, atrophy of the thalamus, hypothalamus and mammillary bodies, thinning of the isthmus of the septum corpus callosum and depigmentation of the locus coeruleus and substantia nigra (Figure 2). Although grossly identifiable cerebellar abnormalities were described in the initial reports of CTE affecting boxers, macroscopic cerebellar abnormalities are rarely present in CTE associated with other sports or activities (59).

Figure 2

Gross neuropathological features of advanced chronic traumatic encephalopathy (CTE). A. CTE stage IV in a former professional football player. Despite the relatively large size of the brain, there is generalized, symmetrical brain atrophy that is most severe in the frontal and temporal lobes. There is also flattening of the hypothalamic floor, atrophy of the mammillary bodies and pallor of the substantia nigra. B. The brain of a subject with Alzheimer’s disease, by contrast, also shows generalized, symmetrical brain atrophy but the hypothalamic floor, mammillary bodies and substantia nigra are normal. C. There is depigmentation of the substantia nigra and locus ceruleus and degeneration of the frontopontine tracts in the cerebral peduncle. D, E, F. Coronal sections show pronounced frontal and temporal lobe atrophy, marked ventricular dilatation, as well as cavum septum (A) and absence of the posterior septum (B) and (C). G. The floor of the hypothalamus is severely thinned and the mammillary bodies are markedly atrophic. H. Multiple septal perforations may be present. I. The posterior septum may be absent with only small blood vessels remaining. J. A large single septal perforation.

Microscopic pathology

In the mildest forms of disease, CTE is characterized by the deposition of p-tau protein as NFT and NT in conspicuous clusters in the neocortex, often arranged along small blood vessels at the depths of the sulci. The NFT typically follow the penetrating small cortical vessels as linear accumulations extending from the surface of the brain to the lowest layers of the cortical gray matter, or when observed in cross-section, as clusters NFT, pre-tangles and dot-shaped and thread-like NT in a penumbra around small arterioles (Figure 3). Mild forms of the disease affect primarily the neurons, although there may be focal subpial p-tau immunopositive astrocytes at the sulcal depths and occasional p-tau immunoreactive glia around the affected vessel (Figure 3). The pathological significance of isolated subpial p-tau astrocytes at the sulcal depths unaccompanied by perivascular NFT and NT in the underlying cortex is unknown, although subpial p-tau astrocytes at the depths of the cortical sulci is not a phenomenon found in normal aging and has not been found in the brains of subjects without a history of repetitive brain trauma.

Figure 3

Microscopic findings in stage II chronic traumatic encephalopathy (CTE). A. Whole mount coronal sections in stage II CTE show multiple foci of p-tau pathology primarily located at the depths of the cortical sulci of the frontal and temporal lobes (free floating 50 μ sections, AT8 immunostain). B–H. The p-tau pathology consists of neurofibrillary tangles and dot-like and thread-like dystrophic neurites and is characteristically found around small blood vessels (B–F, free floating 50 μ sections, AT8 immunostain; G, H, 10 μ paraffin embedded sections, AT8 immunostain). (I). Subpial astrocytic tangles are also found at the cortical depths (free floating 50 μ sections, AT8 immunostain). Other pathologies include pretangles (J), dystrophic neurites in the white matter (K) and occasional p-tau astrocytes (L) (free floating 50 μ sections, AT8 immunostain). There may be marked astrocytosis of the white matter (M, N) (free floating 50 μ sections, Glial fibrillary acidic protein immunostain). Hemosiderin-laden macrophages (O) (10 μ paraffin section, Luxol fast blue hematoxylin and eosin stain) and multiple perivascular foci of reactive microglia (P) are found around small vessels in the cerebral white matter (free floating 50 μ sections, LN3 immunostain).

Provisional criteria for the neuropathological diagnosis of CTE are listed in Table 1 (59). These criteria are currently being validated by a panel of expert neuropathologists as part of a UO1 funded by the National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Biomedical Imaging and Bioengineering (NIBIB) (1U01NS086659-01). The provisional criteria are founded on the hypothesis that the irregular, perivascular p-tau pathology found in clusters at the depths of the cortical sulci distinguishes CTE from other tauopathies, including AD (27, 28, 57–59); this hypothesis will be formally assessed at the NINDS/NIBIB-sponsored conference. One of the more difficult tasks will be deciphering the point at which the pathology is sufficient to make a diagnosis of CTE, as opposed to CTE-related changes. This conundrum is reminiscent of the early phases of AD neuropathology when it was difficult to distinguish between AD-related changes and a neuropathological diagnosis of low, intermediate and high likelihood AD (71). Similarly, stage I CTE is likely a preclinical pathology, sometimes asymptomatic, and sometimes associated with prolonged post-concussive symptoms, but not necessarily progressive. Stage II CTE may occasionally be asymptomatic, although in many cases, it is associated with florid behavior and mood changes. Determining the degree and pattern of microscopic features necessary to diagnose comorbid CTE when another tauopathy is present will be another challenge for the future, such as when CTE is present in addition to AD, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease or many others.

The tau isoform profile and phosphorylation state in CTE are similar to AD (84) and the neuronal p-tau pathology shows immunoreactivity to both 3 repeat and 4 repeat (4R) tau (59, 91). The 4R isoform of tau is predominantly expressed in astrocytes located in the subpial region of the sulcal depths (59, 91), although some of the neuronal abnormalities in the hippocampus also appear to be primarily 4R tau in CTE (Figure 4).

Figure 4

Patterns of 3R and 4R tau in chronic traumatic encephalopathy (CTE) [adapted from McKee et al (59)]. A, D. 3R immunostaining shows scattered RD3 immunoreactive neurons in middle frontal cortex. B, E. 4R immunostaining shows many ET3 immunoreactive neurons and astrocytic tangles in the subpial region of the middle frontal cortex and at the depth of the sulcus. C, F. AT8 immunostaining shows 3R and 4R immunopositive neurons and astrocytic tangles in middle frontal cortex. G, J, M. 3R immunostaining shows scattered RD3 immunore-active neurons in CA1 (G), CA2 (J) and CA4 hippocampus (M). H, K, N. 4R immunostaining shows many ET3 immunoreactive neurons in CA1 (H), CA2 (K) and CA4 hippocampus (N). I, L, O. I. AT8 immunostaining shows 3R and 4R immunopositive neurons in CA1 (I), CA2 (L) and CA4 hippocampus (O). All 10 μ paraffin embedded sections, magnification bars 50 μm.

Staging of p-tau pathology in CTE

Using the provisional criteria for CTE, it is possible to identify a relatively stereotyped sequence of pathological progression of p-tau pathology that can be classified into four pathological stages (59). These stages of p-tau pathology are accompanied by progressive macroscopic changes and progressive increases in axonal and 43 kDa TAR DNA-binding protein (TDP-43) pathologies and neuroinflammation. The staging of p-tau pathology in CTE is adapted from the work of Braak and Braak who examined a series of 83 autopsy brains with AD for neurofibrillary changes and found that there was a characteristic distribution pattern of NFT and NT that permitted the differentiation of six stages (7). This staging system forms the basis for the neuropathological diagnosis of AD used by the National Institute on Aging (40) and similar staging schemes are now in use for Aβ plaques in AD (95) and Lewy bodies in Parkinson’s disease (8).

Using the pathological staging system for CTE, we found a significant correlation between the pathological stage of CTE severity and the duration of the football career, age at death and years since retirement from football among our series of former American football players (59).

Staging of pathology in CTE

Stage III

Most brains with stage III CTE show macroscopic changes although the gross structural changes are minor relative to the changes found microscopically. Typically, there is a reduction in brain weight, mild frontal and temporal atrophy and enlargement of the lateral and third ventricles. One-half of subjects have septal abnormalities including cavum septum pellucidum, septal perforations or fenestrations. Pallor of the locus coeruleus and substantia nigra, atrophy of the mammillary bodies, thalamus and hypothalamus and thinning of the corpus callosum are characteristic. Microscopically, confluent patches of NFT, NTs and astrocytic tangles are found around blood vessels at the sulcal depths, as well as in linear arrays in the superficial laminae of cortex. NFT are found in the superior, dorsolateral and inferior frontal, septal, insular, temporal pole, superior, middle and inferior temporal and inferior parietal cortices. NFT are also found in the olfactory bulbs, hippocampus, entorhinal cortex, amygdala, hypothalamus, mammillary bodies, nucleus basalis of Meynert, substantia nigra, dorsal and median raphe nuclei and locus coeruleus. Neurofibrillary degeneration in the hippocampus includes CA4 and CA2, as well as CA1.

Approximately one-third of stage III cases have occasional NFT in the dentate nucleus of the cerebellum and the spinal cord gray matter. TDP-43-positive neurites and inclusions are found in the majority of cases. Thirteen percent of stage III CTE cases show sparse diffuse and neuritic Aβ plaques. White matter abnormalities, loss of myelinated nerve fibers, axonal dystrophy and axonal loss may be conspicuous (Figure 5).

Figure 5

Microscopic changes in stage III chronic traumatic encephalopathy (CTE). (A) Whole mount coronal sections in stage III CTE show multiple cortical foci of p-tau pathology throughout the frontal and temporal cortices. The cortical epicenters and depths of the sulci often consist of confluent masses of neurofibrillary tangles (NFT) and astrocytic tangles (ATs). (B–D) The p-tau pathology consists of NFTs, ATs and dot-like and thread-like dystrophic neurites clustered around the penetrating cortical vessels. (E) Cortex adjacent to the cortical epicenters shows scattered NFT. (F) The hippocampus shows dense neurofibrillary pathology. Subpial ATs (G) and p-tau immunoreactive astrocytes may be prominent (H). There may be dystrophic neurites in the white matter (I). SMI-34 immunostaining shows reduction in axonal staining and numerous large, irregular axonal varicosities (J). (A–I. free floating 50 μ sections, AT8 immunostain; J. SMI-34 immunostain, 10-μ paraffin section).

Stage IV

Brains with stage IV CTE show a decrease in brain weight, which in some instances may be substantial. Brain weights of 1000 g or less are not uncommon. In addition to generalized cerebral atrophy, there is pronounced atrophy of the frontal and temporal lobes, medial temporal lobe and anterior thalamus. There is also diffuse atrophy of the white matter and thinning of the corpus callosum, particularly the isthmus. There is typically severe thinning of the hypothalamic floor and atrophy of the mammillary bodies. Most cases of stage IV CTE show septal abnormalities including cavum septum, perforations, fenestrations or total absence of the posterior septum. The locus coeruleus and substantia nigra are pale. Microscopically, in stage IV CTE, there is widespread myelin loss, astrocytosis of the white matter and neuronal loss in the cerebral cortex, hippocampus and substantia nigra. Neuronal loss and astrocytosis may also be prominent in the frontal and temporal cortices associated with microvacuolation of layer II.

P-tau pathology is densely distributed throughout the cerebrum, thalamus, hypothalamus, mammillary bodies, basal ganglia, brainstem, cerebellar dentate nucleus and occasionally, spinal cord. Primary visual cortex is typically spared. NFT, most of which are extracellular, are found throughout the hippocampal formation including the dentate gyrus, CA3, CA2 and CA4. CA1 typically shows severe loss of neurons with substantial numbers of extracellular tangles. P-tau pathology also involves the cerebellum, including the dentate nucleus, granular cell layer, and sometimes, Purkinje cells. Irregular tau neurites are often found in the white matter of the anterior cerebellar vermis lining the fourth ventricle. Widespread TDP-43 deposits are also found with dot-like and thread-like inclusions in neurites and intraneuronal cytoplasmic inclusions. There is marked loss of myelinated nerve fibers, axonal dystrophy and loss throughout the cerebral and cerebellar white matter (Figure 6).

Figure 6

Microscopic changes in stage IV chronic traumatic encephalopathy (CTE). Whole mount coronal sections in stage IV CTE show widespread p-tau pathology affecting most regions of the cerebral cortex and medial temporal lobe. Astrocytic tangles are prominent and there is marked neuronal loss in the cortex, amygdala and hippocampus. There are also widespread pTDP-43 abnormalities. All images: 50 μ tissue sections, CP-13 or p-TDP-43 immunostain.

Stage I

Brains with stage I CTE are grossly unremarkable but microscopically show rare, isolated perivascular focal epicenters of p-tau NFTs and neuropil neurites (Figure 1). The NFTs and neuropil neurites predominate and the neurites are characteristically dot-like as well as thread-like. Occasional p-tau immunoreactive glia and glial processes may be present. Tau pathology is most commonly localized to the depths of the cerebral sulci of the frontal, temporal, insular, septal and parietal cortices, although there may also be sparse NFT scattered throughout the adjacent cortex. There also may be subpial p-tau astrocytic tangles in clusters at the depths of sulci. NFT and neurites are also found in the locus coeruleus. Approximately one-half of cases with stage I p-tau pathology show rare TDP-43 neurites. Clusters of reactive microglia are found in the white matter with axonal swellings and distorted profiles in the subcortical U-fibers. Aβ plaques are not found in subjects with stage I CTE, unless over the age of 50 years. Hemosiderin-laden macrophages are characteristically found around small vessels in the white matter, usually not associated with p-tau abnormalities.

Stage II

Subtle macroscopic changes may be found in stage II CTE cases including mild enlargement of the frontal horns of the lateral ventricles and third ventricle, cavum septum pellucidum and pallor of the locus coeruleus and substantia nigra. Microscopically, multiple epicenters of perivascular foci of p-tau NFT and neurites are found at the depths of the frontal, temporal, parietal, insular and septal cortices. These cerebral epicenters typically consist of perivascular p-tau NFT, pre-tangles, neurites and occasional glia (Figure 3) and may be associated with p-tau astrocytic tangles in the overlying subpial region. In the cortex adjacent to the focal epicenters, NFT are scattered throughout the cerebral layers with a tendency to be most numerous in the superficial cortical layers. There are also NFT in the locus coeruleus and substantia innominata. Mild TDP-43 pathology may be found in stage II CTE cases as abnormal neurites and neuronal inclusions. Clusters of reactive microglia are often found in the subcortical white matter with axonal swellings and distorted profiles in the subcortical U-fibers. Aβ plaques are found in 19% of subjects with stage II CTE, only if older than 50 years.

Stage III

Most brains with stage III CTE show macroscopic changes although the gross structural changes are minor relative to the changes found microscopically. Typically, there is a reduction in brain weight, mild frontal and temporal atrophy and enlargement of the lateral and third ventricles. One-half of subjects have septal abnormalities including cavum septum pellucidum, septal perforations or fenestrations. Pallor of the locus coeruleus and substantia nigra, atrophy of the mammillary bodies, thalamus and hypothalamus and thinning of the corpus callosum are characteristic. Microscopically, confluent patches of NFT, NTs and astrocytic tangles are found around blood vessels at the sulcal depths, as well as in linear arrays in the superficial laminae of cortex. NFT are found in the superior, dorsolateral and inferior frontal, septal, insular, temporal pole, superior, middle and inferior temporal and inferior parietal cortices. NFT are also found in the olfactory bulbs, hippocampus, entorhinal cortex, amygdala, hypothalamus, mammillary bodies, nucleus basalis of Meynert, substantia nigra, dorsal and median raphe nuclei and locus coeruleus. Neurofibrillary degeneration in the hippocampus includes CA4 and CA2, as well as CA1.

Approximately one-third of stage III cases have occasional NFT in the dentate nucleus of the cerebellum and the spinal cord gray matter. TDP-43-positive neurites and inclusions are found in the majority of cases. Thirteen percent of stage III CTE cases show sparse diffuse and neuritic Aβ plaques. White matter abnormalities, loss of myelinated nerve fibers, axonal dystrophy and axonal loss may be conspicuous (Figure 5).

Figure 5

Microscopic changes in stage III chronic traumatic encephalopathy (CTE). (A) Whole mount coronal sections in stage III CTE show multiple cortical foci of p-tau pathology throughout the frontal and temporal cortices. The cortical epicenters and depths of the sulci often consist of confluent masses of neurofibrillary tangles (NFT) and astrocytic tangles (ATs). (B–D) The p-tau pathology consists of NFTs, ATs and dot-like and thread-like dystrophic neurites clustered around the penetrating cortical vessels. (E) Cortex adjacent to the cortical epicenters shows scattered NFT. (F) The hippocampus shows dense neurofibrillary pathology. Subpial ATs (G) and p-tau immunoreactive astrocytes may be prominent (H). There may be dystrophic neurites in the white matter (I). SMI-34 immunostaining shows reduction in axonal staining and numerous large, irregular axonal varicosities (J). (A–I. free floating 50 μ sections, AT8 immunostain; J. SMI-34 immunostain, 10-μ paraffin section).

Stage IV

Brains with stage IV CTE show a decrease in brain weight, which in some instances may be substantial. Brain weights of 1000 g or less are not uncommon. In addition to generalized cerebral atrophy, there is pronounced atrophy of the frontal and temporal lobes, medial temporal lobe and anterior thalamus. There is also diffuse atrophy of the white matter and thinning of the corpus callosum, particularly the isthmus. There is typically severe thinning of the hypothalamic floor and atrophy of the mammillary bodies. Most cases of stage IV CTE show septal abnormalities including cavum septum, perforations, fenestrations or total absence of the posterior septum. The locus coeruleus and substantia nigra are pale. Microscopically, in stage IV CTE, there is widespread myelin loss, astrocytosis of the white matter and neuronal loss in the cerebral cortex, hippocampus and substantia nigra. Neuronal loss and astrocytosis may also be prominent in the frontal and temporal cortices associated with microvacuolation of layer II.

P-tau pathology is densely distributed throughout the cerebrum, thalamus, hypothalamus, mammillary bodies, basal ganglia, brainstem, cerebellar dentate nucleus and occasionally, spinal cord. Primary visual cortex is typically spared. NFT, most of which are extracellular, are found throughout the hippocampal formation including the dentate gyrus, CA3, CA2 and CA4. CA1 typically shows severe loss of neurons with substantial numbers of extracellular tangles. P-tau pathology also involves the cerebellum, including the dentate nucleus, granular cell layer, and sometimes, Purkinje cells. Irregular tau neurites are often found in the white matter of the anterior cerebellar vermis lining the fourth ventricle. Widespread TDP-43 deposits are also found with dot-like and thread-like inclusions in neurites and intraneuronal cytoplasmic inclusions. There is marked loss of myelinated nerve fibers, axonal dystrophy and loss throughout the cerebral and cerebellar white matter (Figure 6).

Figure 6

Microscopic changes in stage IV chronic traumatic encephalopathy (CTE). Whole mount coronal sections in stage IV CTE show widespread p-tau pathology affecting most regions of the cerebral cortex and medial temporal lobe. Astrocytic tangles are prominent and there is marked neuronal loss in the cortex, amygdala and hippocampus. There are also widespread pTDP-43 abnormalities. All images: 50 μ tissue sections, CP-13 or p-TDP-43 immunostain.

TDP-43 pathology

Abnormal TDP-43 pathology is found in most cases of CTE. The pathology ranges from cytoplasmic neuronal inclusions that partially colocalize with p-tau inclusions to glial inclusions and abnormal neurites (58). In stage I disease, TDP-43 immunopositive neurites are found in the subcortical white matter and fornix. In stage II disease, TDP-43 immunopositivity consists of isolated neurites or inclusions in the subcortical white matter, brainstem or medial temporal lobe, often in a subpial, periventricular or perivascular distribution. By stage III CTE, most cases show TDP-43 immunoreactive neurites in the cerebral cortex, medial temporal lobe or brainstem. TDP-43 immunoreactivity is found in nearly all stage IV cases, as TDP-43-positive, rounded and thread-like neurites, intraglial and intraneuronal inclusions in cerebral cortex, medial temporal lobe, diencephalon, basal ganglia, brainstem and occasionally, spinal cord. In cases with the most severe TDP-43 deposition, dense accumulations of TDP-43 inclusions and neurites are found in all layers of the neocortex, particularly layer II, as well as occasional TDP-43-positive inclusions in the dentate fascia of the hippocampus, a distribution pattern that overlaps with the distribution of TDP-43 found in frontotemporal lobar degeneration (FTLD) with TDP-43 (11).

As TDP-43 is an RNA-binding protein that regulates RNA metabolism (85, 86) and binds to tau (83), TDP-43 dysregulation might play an influential role in tau isoform expression and the development of tau pathology (68). Diseases caused by abnormal TDP-43 metabolism have shown altered tau metabolism including hyperphosphorylation, tau phosphatase resistance and deposition of p-tau intracellular aggregates (93). Furthermore, animal models have shown TDP-43 to be upregulated and relocated from the neuronal nucleus to the neuronal cytoplasm (65, 66) after traumatic injury.

Aβ in CTE

Aβ-containing plaques, especially diffuse Aβ plaques, are present in some cases of CTE, although they are not a consistent feature of CTE, are not found in early stage disease and are significantly associated with age at death (59). Compared with a normal aging population, Aβ deposition occurs at an earlier age and at an accelerated rate in CTE and is associated with increased clinical and pathological severity (T. Stein, pers. comm.).

CTE with comorbid degenerative disease

Similar to most other neurodegenerative diseases (31, 42, 45, 88, 98), CTE is associated with the development of other neurodegenerations, especially as a function of increased age (37, 58, 59). Of 142 neuropathologically confirmed cases of CTE, 15 (10.56%) had coexistent motor neuron disease (MND), 15 (10.56%) had AD, 10 (7.04%) had Lewy Body disease, 4 (2%) had FTLD and 7 (4.51%) had two or more of these comorbidities; 89 (63.16%) of the cases were diagnosed only with CTE (Figure 7).

Figure 7

Chronic traumatic encephalopathy (CTE) with comorbid neuropathology. Of 142 neuropathologically confirmed cases of CTE at the VA-BU-SLI CTE Brain Bank, 15 (10.56%) had coexistent motor neuron disease (MND), 15 (10.56%) had Alzheimer’s disease (AD), 10 (7.04%) had Lewy Body disease, 4 (2.%) had frontotemporal lobar degeneration (FTLD) and 7 (4.51%) had two or more of these comorbidities; 89 (63.16%) cases were diagnosed only with CTE.

CTE with MND

Approximately 10% of individuals with CTE develop a progressive MND characterized by weakness, atrophy, spasticity and fasciculations that appears clinically indistinguishable from Amyotrophic lateral sclerosis (ALS) (58, 59). In addition, a recent screen of ALS cases from the Mayo Clinic Jacksonville ALS brain bank and the Boston Veterans Affairs (VA) ALS brain bank found that six of the 91 (6.6%) and five of the 113 (4.4%) cases, respectively, had pathological features of CTE (all Caucasian males) (5). Most individuals with CTE and MND present with symptoms of ALS and develop mild cognitive and behavioral symptoms several years after the onset of motor weakness and fasciculations. Individuals with CTE and MND often die from respiratory failure at younger ages and in earlier stages of CTE (stage II–III) compared with CTE subjects without MND. Approximately one-third of subjects with CTE and MND present with depression, behavioral or cognitive changes related to CTE years before developing MND symptoms. Subjects with CTE and MND usually show more severe TDP-43 pathology than subjects with CTE alone. The marked accumulation of pTDP-43 aggregates in advanced stages of CTE, the partial immunohistochemical colocalization of p-tau with pTDP-43, and the development of MND in some individuals with CTE suggests that CTE might share some pathogenic mechanisms with FTLD (20, 49).

Relationship of tau pathology to trauma

Although all cases of neuropathologically verified CTE have been associated with repetitive mild brain trauma, the pathophysiological mechanisms critical to developing a latent and progressive neurodegeneration and tauopathy after repetitive mTBI, including concussion and subconcussion, are only beginning to be identified. When the semisolid brain, suspended in cerebrospinal fluid (CSF) inside a boney skull, is subjected to the rapid acceleration, deceleration and rotational forces associated with impact injury, the brain elongates and deforms, stretching the microstructural elements, including neurons, glial cells and blood vessels. These stretch forces predominantly affect long fibers, specifically axons and blood vessels (54, 61) and are most severe where the direction of axons changes or where a change in tissue-density occurs (e.g. around blood vessels or at the gray-white matter interface) (16, 26, 80). Experimental studies of acceleration-deceleration injuries in gyrencephalic animals further demonstrate that axonal injury and vascular disruption is most severe at focal stress points of the brain—particularly the perivascular region and the depths of the cerebral sulci (17, 87). Moreover, the early and predominant involvement of the superior and dorsolateral frontal lobes in CTE affecting former football players parallels the high frequency of impacts to the top of the head compared with those to the front, back and side of the head (35, 62). Functional MRI data in football players also shows activation impairments in dorsolateral prefrontal cortex that is associated with significantly higher numbers of head collisions to the top-front of the head (94).

Pathological studies of concussion and post-concussion syndrome in human subjects have found that multifocal traumatic axonal injury is often perivascular (6, 60, 77). Other changes found in acute and subacute concussion are microvascular injury and microhemorrhages (60, 77), astrocytosis, most severe in the cerebral white matter and brainstem white matter tracts, and clusters of activated microglia, most prominent in the white matter around small vessels (59, 77). Less common changes include focal clusters of p-tau in NFTs, pre-tangles and neurites or stage I CTE (60).

It is known from the experimental literature that acceleration-deceleration injury causes tau protein, normally associated with microtubules in axons, to become abnormally phosphorylated, misfolded, aggregated and cleaved, all of which generate neurotoxic tau peptide fragments (1, 12, 46, 47, 59, 101). Nevertheless, how this combination of factors: axonal injury, breach of blood–brain barrier, neuroinflammation and aggregation of abnormally truncated and hyperphosphorylated proteins, including p-tau and TDP-43, develop into a progressive deterioration remains to be determined.

There are most likely additional factors that play a role in the initiation of p-tau pathology in CTE, including genetic susceptibility or resistance, gender, physiological stress, additional environmental exposures and age at exposure. Studies have suggested that children and adolescents experience prolonged recovery rates after TBI compared with adults (24) as well as poorer outcomes (29). Animal models have shown that stress can lead to increased tau phosphorylation (72, 90). Environmental influences, such as alcohol, opiates or performance-enhancing drugs, could also play a role in tau aggregation, deposition and propagation. P-tau immunoreactivity in opiate user brains has been shown to be significantly higher than in controls (2). Glucocorticoids have also been associated with increased tau phosphorylation and greater cognitive deficits in rodent models (33, 89).

Spread of tau pathology

Tau phosphorylation and misfolding are potentially reversible processes (92, 97, 100); correspondingly, stage I or even stage II CTE might represent a reversible or nonprogressive pathology, at least in some subjects. However, repeated traumatic injuries and progressively greater accumulations of toxic p-tau fragments have the potential to induce a self-perpetuating process. We know that athletes who develop CTE develop a progressive neurodegeneration that typically produces clinical symptoms years to decades after retiring from the sport. This progressive post-traumatic neurodegeneration may involve spreading of tau pathology intercellularly and extracellularly throughout the brain. Direct and indirect evidence for interneuronal tau transmission in animal models suggests that the transfer of toxic tau species between neurons might be caused by the interneuronal spreading of tau mediated by a prion-like templated misfolding of tau (14, 15, 23, 34, 36, 48, 52). Other possible modes of transmission involve oligomeric or toxic N-terminal tau in the receiving neuron with dysregulation of intracellular calcium (25, 78). Spreading of tau pathology may occur between neuronal synapses, but may also involve astrocytes, microglia and CSF pathways. CSF is present in the Virchow–Robin spaces surrounding penetrating arteries and brain interstitial fluid is cleared along paravenous drainage pathways, allowing the possibility that p-tau transmission might involve this clearance pathway as has been shown for Aβ (41).

Axonal pathology

Axonal injury, axonal degeneration, myelinated fiber loss and white matter atrophy are constant features of CTE. Moreover, axonal injury most likely plays a critical role in initiating the p-tau pathology (59, 69). The degree of axonal dysintegrity parallels the severity of the neurodegeneration. In early stage CTE, stages I and II, scattered axonal varicosities are found in the deep layers of the frontal and temporal cortices, subcortical white matter and deep white matter tracts of the diencephalon and axons in the white matter are occasionally immunoreactive for p-tau (Figure 3). In more advanced disease, stages III and IV, axonal loss is severe with frequent distorted axonal profiles in the cortex and subcortical white matter, macrophages and widespread p-tau abnormalities in white matter.

Imaging and biomarkers

Although there are no validated imaging techniques or biomarkers to confirm the diagnosis of CTE in living subjects, potentially useful imaging and biomarkers include positron emission tomography (PET) ligands for p-tau and neuroinflammation (13, 64), diffusion tensor imaging (4, 50), magnetic resonance spectroscopy (51) and CSF markers (10). P-tau-specific PET ligands, such as T-807 and T-808 reported by Chien et al (13) might prove to identify primary tauopathy, such as CTE, and p-tau PET imaging combined with PET imaging for Aβ might be useful to differentiate between CTE and AD (3, 44, 67). Recently, this technique was used to evaluate a retired NFL player with clinical symptoms suggestive of AD. He was found to have a negative florbetapir scan for Aβ and a positive scan for T-807, results supportive of CTE, although the pattern of T-807 positivity most likely represented non-specific off-target labelling (64). Additionally, more conventional imaging techniques and biomarkers might be useful in identifying some of the pathological features of “probable CTE” in vivo, such as abnormalities of the septum pellucidum, cortical thinning and/or cortical atrophy (Figure 8) (67). Other potential biomarkers for “probable CTE” include normal Aβ CSF fluid levels and an elevated CSF p-tau/tau ratio (67).

Figure 8

Coronal T1-weighted Magnetic resonance imaging shows a cavum septum pellucidum, cortical and white matter atrophy in former professional football player with neuropathologically confirmed chronic traumatic encephalopathy stage IV (arrows indicate the location of the cavum septum).

CTE IN THE MILITARY SETTING

Most individuals with neuropathologically verified CTE were exposed to repetitive brain trauma through athletic participation. Although CTE has also been reported in association with military exposures (56), typically CTE affects veteran athletes with considerable traumatic exposure from sports. Sports-related injuries are relatively stereotyped and predictable, whereas military-related mTBI is acquired in widely heterogeneous ways, including recreational activities, physical training practices, falls, motor vehicle accidents, athletics and exposure to explosive blasts. Injury from explosive blasts varies depending on the strength of the explosive, whether the injury occurs in an open field, in or near buildings or in a motor vehicle. Military mTBI is also random; an individual may experience a single injury or many thousands of injuries over a similar time period depending on chance exposure.

There have been case reports of CTE occurring in young veterans of the recent conflict in Iraq and Afghanistan, five of the veterans were exposed to blast and one veteran experienced concussive injury (30, 56, 73). In the cases of CTE reported in young military veterans (n = 5 males; ages 22 to 45 years; mean, 31.0 years), the changes of CTE were mild and ranged from stage I to II CTE (30, 56). In all five, there was also evidence of myelinated fiber loss, axonal degeneration, microvascular degeneration and neuroinflammation. Symptoms included headaches, verbal and physical violence, irritability, attentional deficits, difficulty in sleeping, short-term memory loss and depression. Four of the five were also diagnosed with PTSD.

The outcome of military-related mTBI tends to be less predictable and less homogeneous than sports-related mTBI and the neurodegenerative pathologies that occur after pure military mTBI remain largely unknown. It is likely that military-related mTBI will give rise to neurodegenerations other than CTE and that post-traumatic neurodegenerations are variable. Axonal injury after military mTBI is likely more severe than apparent pathologically, as mild axonal damage and loss are not well visualized histologically and are difficult to quantitate. Neuroimaging of white matter tracts, such as with diffusion tensor imaging and related modalities, may prove to be the best strategy for assessing the contribution of axonal injury to military-related post-traumatic neurodegeneration.

CONCLUSION

CTE is a distinct neurodegenerative disease that currently can only be diagnosed with certainty by post-mortem neuropathological examination. Given the importance of sports participation and physical exercise to physical and psychological health as well as disease resilience, it is critical to identify the genetic risk factors for CTE as well as the contribution of exposure variables, stress, alcohol, substance abuse and other factors to the development of CTE. The acute consequences of mTBI, including concussion, subconcussion and blast injuries, are known to produce axonal injury, microvascular disruption, breach of the blood–brain barrier, neuroinflammation and, in some cases, deposition of small amounts of p-tau (60). Further research is needed to identify the pathogenetic mechanisms responsible for developing a long-term, often progressive, neurodegeneration after acute, repetitive mTBI. The neuropathology of CTE is characterized by a focal perivascular deposits of p-tau in the neocortex that appear to spread to affect the superficial layers of adjacent cortex, and eventually, the medial temporal lobe, diencephalon and brainstem in susceptible individuals. The mechanisms underlying the spread of tau and the accumulation of TDP-43 in the brain remain to be defined. Clinical and pathological diagnostic criteria have been proposed for CTE but remain to be validated. In vivo biomarkers and neuroimaging techniques hold the promise for developing an in vivo diagnostic technique for CTE. Additional prospective, longitudinal, epidemiological and animal studies will be essential to establishing the incidence and prevalence of CTE and to identifying underlying pathogenic mechanisms that will help target future preventative and therapeutic strategies (102–105).

Acknowledgments