Animal model of repetitive mild traumatic brain injury for human traumatic axonal injury and chronic traumatic encephalopathy

Chronic traumatic encephalopathy (CTE) is a chronic neurodegenerative disease featured with tauopathy. CTE is tightly related with repetitive mild traumatic brain injury (mTBI), which is interchangeably known as concussion (McKee et al., 2009, 2013). This disease is diff erentiated by neuropathological features from other neurological diseases that involve tau protein aggregation and tangle formation abnormalities like Alzheimer’s disease (AD), frontotemporal dementia, and Parkinsonism linked to chromosome 17 (FTDP-17). The diff erentiation of CTE from other neurodegenerative diseases provides helps on exploring the mechanism of etiology and seeking potential treatment for this disease. Repetitive mTBI is usually evident in civilians participating in contact sports and in military personnel engaging in combat. Due to the number of individuals partaking in these high-risk environments, the general public concern has heightened in concordance to the reveal of many new CTE cases. Despite increased awareness, the mechanisms of CTE are still unknown and there are no eff ective therapies to help slow the progression of the devastating disease. The heterogeneity of human CTE cases and the complexity of patients’backgrounds make it diffi cult for scientists to specify the links between mTBI and CTE using epidemiological or clinical approaches; appropriate animal models are eff ective in exploring cause-and-eff ect relationships.

We developed this repetitive mTBI mouse model (IA model) (Xu et al., 2014) with the hope of clarifying relationships between multiple concussions and tauopathy, to clarify CTE mechanisms, and to eventually serve as vehicles for experimental therapeutics or for biomarker discovery and validation. The IA model incurs traumatic axonal injuries (TAI) approximate to the ones induced in contact-related repetitive concussions, which supports the notion that this model can be used as a tool to investigate repeated concussive events in human subjects. We did not fi nd any signs of tauopathy in the mouse IA model after repetitive mTBI (Xu et al., 2014), even though tau hyperphosphorylation was evident in a single blast injury in another study experimenting on the same mouse strain (Goldstein et al., 2012).

The incidence and prevalence of CTE is currently still unknown. Even though there is a large population of repetitive mTBI victims (1.4–3.8 million mTBI annually in USA), only a small fraction of these cases are diagnosed as CTE (only a couple of hundred cases are identifi ed from post-mortem analyses and reported) (Baugh et al., 2012; McKee et al., 2013). Therefore there must be a number of factors that have decisive roles in CTE formation; one suggested factor is the genetic risk of mutations leading to abnormal tau aggregation. A variety of transgenic mice, which carry mutant human tau genes that induce tauopathy, can be used with this IA model to investigate if repetitive mTBI triggers or expedites tau aggregation. These mutations may include, but not limited to P301S, V337M, and the deletion mutation ΔK280. Our studies with the P301S mouse resulted in signifi cantly increased numbers of hyperphosphorylated tau in retina ganglion cells (RGCs) after repetitive mTBI. However, there is no signifi cant increased tau hyperphosphorylation in the cortex even though the corticospinal tract and corpus callosum were the major white matter TAI targets as optic tract in this IA model (Xu et al., 2014). One possible explanation for this fi nding is that cortical TAI is more diff usive than visual system TAI, and therefore does not generate experimental signifi cance. In addition to P301S mouse, we observed similar results in tauopathy-prone transgenic mice, which harbored all six isoforms of the wild-type human tau gene but were knocked out for the mouse tau gene. Due to the absence of mouse tau, any possible interaction between mouse tau and human tau could be excluded in this transgenic mouse. These fi ndings indicate that repetitive mTBI accelerates tauopathy under diverse number of genetic conditions that predispose a subject to tau aggregation; more transgenic mice with various mutations and with or without mouse tau need to be tested to confi rm this theory. Other tau-related proteins should also be investigated to see if they contribute to abnormal tau aggregation. For example, evidence supports that aggregated Aβ protein, one feature of AD, increases tau phosphorylation and elevates the number of tangles in Tau p301L transgenic mice (Gotz et al., 2001). TBI is also one factor in inducing AD, but its causative role is still not confi rmed. Therefore, transgenic mice that carry mutant amyloid precursor protein (APP), which then produces aggregate Aβ protein, can also help verify if repetitive mTBI expedites plaque formation in genetically vulnerable environments.

Other mechanisms may also be involved in tauopathy induction and progression after repetitive mTBI. Evidence supports that tau proteolysis plays an important role in tau aggregation; after TBI, excessive amounts of activated caspase and calpain were found (Knoblach et al., 2002; Saatman et al., 2010), and these activated enzymes are capable of cleaving tau at various sites. The resulting truncated tau fragments form oligomers and trigger tau aggregation (Wang et al., 2007). Another tau protease, thrombin, mobilizes into the brain parenchyma through the TBI-disrupted blood brain barrier (BBB). Thrombin induces the proteolysis of tau proteins that leaked into the extracellular space after injury; these tau fragments following a pathological event may initiate tau aggregation as caspase or calpain mentioned above. In our repetitive mTBI model, signifi cantly increased BBB permeability is evident in both single and repetitive injury paradigms. Therefore, questions regarding truncated tau and tau aggregation post-repetitive mTBI and also which proteases are most signifi cant in tauopathy are still under investigation.

Neuroinflammation, which includes microglia activation, concomitantly occurs at the lesioned area after TBI. In our repetitive mTBI model, there is a signifi cantly elevated number of activated microglia in the TAI location, although their specifi c role in repetitive concussive injury remains unclear. The activation of microglia from their resting, surveillance state can be detrimental and may contribute to a secondary injury. Activated microglia M1-type release a variety of pro-infl ammatory and neurotoxic molecules such as reactive oxygen species (ROS) and tumor necrosis factor-α (TNFα), which induces further damage to the nervous system. Microglia also mediate retrograde axonal degeneration post-TBI (Busch et al., 2009), and chronic microglia activation has been linked to various neurodegenerative diseases like AD, amyotrophic lateral sclerosis (ALS), and Parkinson’s disease (PD). Studies involving subjects with neurodegenerative diseases or TBI have shown that inhibiting microglial activation both clinically and experimentally with minocycline or other anti-neuroinflammatory therapies improve disease state (Sanchez Mejia et al., 2001; Homsi et al., 2010). In TBI, activated microglia can remain activated for a long period time,sometimes from months to years (Gentleman et al., 2004; Johnson et al., 2013); this pattern indicates chronic neuroinfl ammation. CTE subjects have activated microglia in the tau tangle area, which suggests microglia may contribute to tauopathy severity. This relationship may exist since there is evidence to support that activated microglia is involved in tau phosphorylation and, perhaps, tau aggregation through interleukin1 (IL1)/p38 mitogen-activated protein kinases (MAPK) signaling (Bhaskar et al., 2010). Despite these hypotheses, the role of microglia in tauopathy induction and chronicity still needs to be confi rmed; the mechanistic clarification of how microglia take part in tauopathy may lead to an eff ective therapy for CTE.

The development of eff ective therapies for TAI after concussion is still ongoing. The Wallerian degeneration slow (WLDS) protein has experimental support for its protective effects on axons in experimental animal studies (Avery et al., 2012). Another therapeutic target may be dual leucine zipper kinase (DLK), which is a key mediator of axonal degeneration that is robustly upregulated after axonal injury; DLK inhibition can theoretically reduce retina ganglion cell (RGC) death caused by optic nerve transection (Welsbie et al., 2013). DLK is a kinase of c-Jun N-terminal kinase (JNK) and is the key activator of JNK signaling following axotomy. JNK is activated after TBI and activated JNK can lead to cell apoptosis (Greer et al., 2011). Vision disorder is also frequently experienced in TBI patients. In our repetitive mTBI model, the optic tract is one of the main white matter targets that show signifi cant axonal injury; this localization thus led to distinct RGC death (Xu et al., 2014). Our pilot study showed that multiple mTBI can dramatically increase JNK in the RGCs. Therefore, blocking or disrupting DLK/JNK signaling may protect axons from degeneration and also improve the chances for RGCs survival. Floxed DLK or JNK transgenic mouse and corresponding pharmacologic reagents are now being used with our repetitive mTBI model to verify this therapeutic potential. If the pharmaceutical aff ectability is validated in the visual system, the next study will focus on cortical TAI, which is another major target for concussion therapy.

In summary, this repetitive mTBI model can be served as a tool for a wide range of both basic and translational repetitive concussion studies, due to its ability to replicate the traumatic nature of human concussion.

Leyan Xu*

Division of Neuropathology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

*Correspondence to: Leyan Xu, Ph.D., lxu9@jhmi.edu.

Accepted: 2015-07-17

orcid: 0000-0001-5613-5420 (Leyan Xu)

Avery MA, Rooney TM, Pandya JD, Wishart TM, Gillingwater TH, Geddes JW, Sullivan PG, Freeman MR (2012) WldS prevents axon degeneration through increased mitochondrial fl ux and enhanced mitochondrial Ca2+buff ering. Curr Biol 22:596-600.

Baugh CM, Stamm JM, Riley DO, Gavett BE, Shenton ME, Lin A, Nowinski CJ, Cantu RC, McKee AC, Stern RA (2012) Chronic traumatic encephalopathy: neurodegeneration following repetitive concussive and subconcussive brain trauma. Brain Imaging Behav 6:244-254.

Bhaskar K, Konerth M, Kokiko-Cochran ON, Cardona A, Ransohoff RM, Lamb BT (2010) Regulation of tau pathology by the microglial fractalkine receptor. Neuron 68:19-31.

Busch SA, Horn KP, Silver DJ, Silver J (2009) Overcoming macrophage-mediated axonal dieback following CNS injury. J Neurosci 29:9967-9976.

Gentleman SM, Leclercq PD, Moyes L, Graham DI, Smith C, Griffin WS, Nicoll JA (2004) Long-term intracerebral inflammatory response after traumatic brain injury. Forensic Sci Int 146:97-104.

Goldstein LE, Fisher AM, Tagge CA, Zhang XL, Velisek L, Sullivan JA, Upreti C, Kracht JM, Ericsson M, Wojnarowicz MW, Goletiani CJ, Maglakelidze GM, Casey N, Moncaster JA, Minaeva O, Moir RD, Nowinski CJ, Stern RA, Cantu RC, Geiling J, et al. (2012) Chronic traumatic encephalopathy in blast-exposed military veterans and a blast neurotrauma mouse model. Sci Transl Med 4:134ra60.

Gotz J, Chen F, Van Dorpe J, Nitsch RM (2001) Formation of neurofi brillary tangles in P3011 tau transgenic mice induced by Abeta fi brils. Science 293:1491-1495.

Greer JE, McGinn MJ, Povlishock JT (2011) Diff use traumatic axonal injury in the mouse induces atrophy, c-Jun activation, and axonal outgrowth in the axotomized neuronal population. J Neurosci 31:5089-5105.

Homsi S, Piaggio T, Croci N, Noble F, Plotkine M, Marchand-Leroux C, Jafarian-Tehrani M (2010) Blockade of acute microglial activation by minocycline promotes neuroprotection and reduces locomotor hyperactivity after closed head injury in mice: a twelve-week follow-up study. J Neurotrauma 27:911-921.

Johnson VE, Stewart JE, Begbie FD, Trojanowski JQ, Smith DH, Stewart W (2013) Infl ammation and white matter degeneration persist for years after a single traumatic brain injury. Brain 136:28-42.

Knoblach SM, Nikolaeva M, Huang X, Fan L, Krajewski S, Reed JC, Faden AI (2002) Multiple caspases are activated after traumatic brain injury: evidence for involvement in functional outcome. J Neurotrauma 19:1155-1170.

McKee AC, Cantu RC, Nowinski CJ, Hedley-Whyte ET, Gavett BE, Budson AE, Santini VE, Lee HS, Kubilus CA, Stern RA (2009) Chronic traumatic encephalopathy in athletes: progressive tauopathy after repetitive head injury. J Neuropathol Exp Neurol 68:709-735.

McKee AC, Stern RA, Nowinski CJ, Stein TD, Alvarez VE, Daneshvar DH, Lee HS, Wojtowicz SM, Hall G, Baugh CM, Riley DO, Kubilus CA, Cormier KA, Jacobs MA, Martin BR, Abraham CR, Ikezu T, Reichard RR, Wolozin BL, Budson AE, et al. (2013) The spectrum of disease in chronic traumatic encephalopathy. Brain 136:43-64.

Saatman KE, Creed J, Raghupathi R (2010) Calpain as a therapeutic target in traumatic brain injury. Neurotherapeutics 7:31-42.

Sanchez Mejia RO, Ona VO, Li M, Friedlander RM (2001) Minocycline reduces traumatic brain injury-mediated caspase-1 activation, tissue damage, and neurological dysfunction. Neurosurgery 48:1393-1399.

Wang YP, Biernat J, Pickhardt M, Mandelkow E, Mandelkow EM (2007) Stepwise proteolysis liberates tau fragments that nucleate the Alzheimer-like aggregation of full-length tau in a neuronal cell model. Proc Natl Acad Sci U S A 104:10252-10257.

Welsbie DS, Yang Z, Ge Y, Mitchell KL, Zhou X, Martin SE, Berlinicke CA, Hackler L Jr., Fuller J, Fu J, Cao LH, Han B, Auld D, Xue T, Hirai S, Germain L, Simard-Bisson C, Blouin R, Nguyen JV, Davis CH, et al. (2013) Functional genomic screening identifi es dual leucine zipper kinase as a key mediator of retinal ganglion cell death. Proc Natl AcadSci U S A 110:4045-4050.

Xu L, Nguyen JV, Lehar M, Menon A, Rha E, Arena J, Ryu J, Marsh-Armstrong N, Marmarou CR, Koliatsos VE (2014) Repetitive mild traumatic brain injury with impact acceleration in the mouse: Multifocal axonopathy, neuroinfl ammation, and neurodegeneration in the visual system. Exp Neurol doi: 10.1016/j.expneurol.2014.11.004.

10.4103/1673-5374.165319 http://www.nrronline.org/

Xu L (2015) Animal model of repetitive mild traumatic brain injury for human traumatic axonal injury and chronic traumatic encephalopathy. Neural Regen Res 10(11):1731-1732.