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Memantine Mitigates Oligodendrocyte Damage after Repetitive Mild Traumatic Brain Injury
Journal Pre-proofs Research Article Memantine mitigates oligodendrocyte damage after repetitive mild traumatic brain injury Guixian Ma, Changyun Liu, Jumana Hashim, Grace Conley, Nicholas Morriss, William P. Meehan, Jianhua Qiu, Rebekah Mannix PII: DOI: Reference:
S0306-4522(19)30703-1 https://doi.org/10.1016/j.neuroscience.2019.10.016 NSC 19319
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Neuroscience
Received Date: Revised Date: Accepted Date:
26 August 2019 7 October 2019 8 October 2019
Please cite this article as: G. Ma, C. Liu, J. Hashim, G. Conley, N. Morriss, W.P. Meehan, J. Qiu, R. Mannix, Memantine mitigates oligodendrocyte damage after repetitive mild traumatic brain injury, Neuroscience (2019), doi: https://doi.org/10.1016/j.neuroscience.2019.10.016
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Memantine mitigates oligodendrocyte damage after repetitive mild traumatic brain injury Guixian Ma MD, PhD1,2,3*, Changyun Liu MD2,3,4*, Jumana Hashim BA2, Grace Conley BA2, Nicholas Morriss BA2, William P. Meehan III MD 2,3,5,6, Jianhua Qiu MD, PhD2,3*, Rebekah Mannix MD, MPH2,3*# 1. Department of Neurology, Guangdong Provincial People’s Hospital & Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China 2. Division of Emergency Medicine, Boston Children’s Hospital, Boston Massachusetts, United States of America; 3. Harvard Medical School, Boston Massachusetts, United States of America 4. Department of Neurology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China, 5. Sports Concussion Clinic, Division of Sports Medicine, Boston Massachusetts, United States of America; 6. The Micheli Center for Sports Injury Prevention, Waltham Massachusetts, United States of America Running Head: Excitotoxicity and oligodendrocytes after mild repetitive TBI * Equal contribution # Corresponding author Rebekah Mannix Boston Children’s Hospital, Division of Emergency Medicine 300 Longwood Avenue Boston, Massachusetts 02115 Phone: 617-355-6624 Email: [email protected] Key words: mild traumatic brain injury, excitoxicity, oligodendrocytes Portions of this work were presented in abstract form/in poster form at the National Neurotrauma Symposium, Toronto Canada, August 2018.
Abstract Repetitive mild traumatic brain injury (rmTBI; e.g., sports concussions) is common and results in significant cognitive impairment, white matter injury and increased risk of neurodegeneration. Targeted therapies for rmTBI are lacking, though evidence from other injury models indicates that targeting N-methyl-d-aspartate (NMDA) receptor (NMDAR)-mediated glutamatergic toxicity might mitigate rmTBI-induced injury. We have previously shown that the NMDAR antagonist memantine lessens axonal injury and restores long term potentiation after rmTBI. Here, we evaluated whether the protective effects of memantine include oligodendrocyte specific mechanisms, as prior studies suggest that oligodendrocytes are particularly vulnerable to glutamatergic toxicity. Mice were subjected to rmTBI injury (5 injuries in 5 days) and randomized to treatment with memantine or with vehicle (n=32/group). At the molecular level, oligodendrocyte counts and function (myelin basic protein, MBP) were assessed by immunohistochemistry and western blot at days 3, 7 and 28 days after the last injury. Axon integrity was assessed by neurofilament light chain (NF-l) expression and axonal ultrastructure was evaluated by electron microscopy. Compared to vehicle-treated mice, memantine-treated mice were protected against oligodendrocyte loss and decreased MBP expression at subacute time points after injury. Memantine treatment also protected against axon damage assessed by NF-l expression. These data suggest that the therapeutic effects of post-concussive NMDAR antagonism may in part work through oligodendrocyte specific mechanisms, which may have implications for long term neurodegenerative sequelae after multiple concussions.
Keywords: Traumatic brain injury; oligodendrocytes; white matter injury; NMDAR
Introduction Repetitive mild traumatic brain injury (rmTBI) is a significant public health problem, with as many as 25% of nonprofessional athletes reporting multiple concussions (Collins et al. 1999). Clinically, rmTBI has been associated with long-term neurological impairment including memory disturbances, behavioral abnormalities, personality changes, speech irregularities, and gait abnormalities (Critchley 1957, Grahmann and Ule 1957, Plassman et al. 2000, Bower et al. 2003), symptoms that correlate with disruption of the myelinated pathways crucial for major neural circuits. Gross pathological changes of rmTBI have also been reported, including longterm persistent white matter volume loss, as well as long-term neurodegenerative changes (Omalu et al. 2005, Omalu et al. 2006, McKee et al. 2009, Stern et al. 2011, McKee et al. 2013). The causal pathways linking rmTBI to persistently disrupt neural circuitry, demyelination and neurodegenerative disease have not been established.
Preclinical and clinical studies have demonstrated that axonal injury, particularly in the white matter, is one of the earliest pathologic features seen early after even a single instance of mTBI (Johnson et al. 2013). In severe cases, axonal injury manifests as an initial disruption of axonal transport and axonal swelling within the first 2 – 3 hours after injury followed by axotomy and Wallerian degeneration observed within hours to days (Povlishock and Christman 1995, Armstrong et al. 2016). In addition to primary, secondary axotomy has been observed, characterized by ionic permeability, cytoskeletal compaction and mitochondrial dysfunction (Povlishock and Christman 1995, Armstrong et al. 2016).
Myelin, a major component of white matter, plays a vital role in axon vulnerability after TBI. Myelin not only provides a physical barrier to axons but is also required for normal metabolic support of axons. TBI-induced traumatic axonal injury (TAI) can result in demyelination of intact axons, in the absence of overt cell death (Sullivan et al.
2013). Demyelination alone can result in axon dysfunction and degeneration (Dutta et al. 2011, Friese et al. 2014). Indeed, even in healthy adult mice, myelin modifications are sufficient to modulate neural circuit timing and are required for motor skill learning (McKenzie et al. 2014, Tomassy et al. 2014). Therefore, demyelination after TBI may slow processing speed or desynchronize communication within neural circuits, resulting in overt functional deficits (Mierzwa et al. 2015, Armstrong et al. 2016).
Oligodendrocytes, the myelinating cells of the central nervous system, are critical for maintaining axonal integrity and axonal metabolism (Bradl and Lassmann 2010). Preclinical and clinical studies have demonstrated the detrimental effects of demyelination due to oligodendrocyte death in a variety of neurological conditions (Fields 2008). Oligodendrocytes are particularly vulnerable to cell death via glutamatergic excitotoxicity mechanisms (McDonald et al. 1998, Karadottir et al. 2005, Salter and Fern 2005, Micu et al. 2006, Manning et al. 2008), though few studies have explored the effects of mild TBI-induced excitotoxicity on oligodendrocyte function. Oligodendrocyte death may also play an important role in the pathophysiology of TBI-induced demyelination and axon degeneration. The limited preclinical data on oligodendrocyte dynamics and white matter pathology after TBI have mainly employed models that result in hemorrhagic lesions which result in immediate neuron cell body death and axonal loss (Lotocki et al. 2011, Flygt et al. 2013, Sullivan et al. 2013) or focal traumatic axonal injury associated with skull fracture (Mierzwa et al. 2015) or craniotomy (Donovan et al. 2014). These data may not be relevant to rmTBI, which does not result in skull fracture, gross pathology or neuron cell death at acute time points. More recently, several studies have demonstrated changing in myelin ultrastructure and axonal integrity after rmTBI (Yates et al. 2017, Fehily et al. 2019) though these studies did not specifically assess the temporal interaction of these findings with oligodendrocyte dynamics at various time points after injury. Understanding oligodendrocyte function and vulnerability after rmTBI may offer new insights into
the pathophysiology of rmTBI including disrupted neural circuitry, demyelination and neurodegenerative disease.
We have developed a repetitive mild closed head injury mouse model that results in impact and rotational acceleration injuries in the absence of gross structural brain damage, though accompanied by marked and persistent behavioral deficits in balance, memory, and impulse control and progressive tau pathology (Mannix et al. 2014, Kondo et al. 2015). Our previous study shows that memantine, an N-methyl-d-aspartate (NMDA) receptor (NMDAR) antagonist, improved functional and histopathologic outcomes after rmTBI, focusing on neuronal mechanisms. In the current study, we have expanded our research scope and investigated whether NMDAR antagonism also protected oligodendrocytes after rmTBI since oligodendrocytes play a crucial role in maintaining axonal metabolisms and facilitating axonal repair (Coman et al. 2005, Guerriero et al. 2015). We hypothesized that oligodendrocytes would be vulnerable to NMDAR mediated toxicity after rmTBI, which could be mitigated by NMDAR antagonist such as memantine.
Methods All experiments were approved by the Boston Children's Hospital institutional animal care and use committee and complied with the NIH Guide for the Care and Use of Laboratory Animals. A total of 81 2-3 months old male C57Bl/6 mice were used for these experiments. Mice were obtained from the Jackson Laboratories (Bar Harbor, ME).
Repetitive Mild TBI and treatment The rmTBI was performed as previously described (Meehan et al. 2012, Mannix et al. 2014). Briefly, mice were randomized to three groups: sham (n= 32/group), rmTBI treated with vehicle (n= 32/group) and rmTBI treated with memantine (n=32/group). Mice were anesthetized using
4% isoflurane in oxygen for 45 seconds. Anesthetized mice were placed on a delicate tissue (Kimwipe, Irving, TX) and the head was placed directly under a copper guide tube centered over an area 0.9cm caudal to the interaural line, to approximate the bregma. Mice were held by the tail as an impact was delivered to the dorsal skull by dropping a 54 g metal bolt (12.75 mm diameter) from a 71 cm height, resulting in a rotational acceleration of the head through the Kimwipe. Mice underwent a single closed head injury daily for 5 consecutive days (Liu et al. 2017, Mei et al. 2017). Within 30 minutes after the last injury, mice received either intraperitoneal injection of memantine (10mg/kg, Tocris, Minneapolis, MN)(Ma et al. 2015) or vehicle (saline), each a volume of 0.2 ml as previous reported (Mei et al. 2017). A separate cohort underwent sham injury only, which consisted of 5 daily isoflurane exposures only. All mice recovered in room air after injury or sham injury. Duration of loss of consciousness, defined as the time from removal of anesthesia to spontaneous ambulation, was recorded. Mice were sacrificed at days 3 (n= 10/group), 7 (n= 12/group) and 28 (n= 10/group) after injury for molecular experiments. All molecular analyses were conducted by investigators blinded to injury status, using color coding stored in a password protected computer.
Electron microscopy Mice (n= 2/group) were perfused transcardially using a mixture of 2% formaldehyde and 2.5 % glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.4. The extracted brains were then drop fixed in the same solution for additional 2 weeks. The brains were processed at the Harvard Medical School Electron Microscopy Facility. The corpus callosum was dissected out (Fig. 1A), and the sections were embedded in Epon resin for 48 hours. Ultrathin sections (60nm) were cut and placed on copper grids stained with lead citrate. The sections were imaged using a JEOL 1200EX Transmission electron microscope. Images were taken from similar sections of the corpus callosum using an AMT 2k CCD camera. Photos were taken at 4000X (160m2 /picture).
Immunohistochemistry Mice were perfused with saline transcardially and pre-fixed with 4% paraformaldehyde at time points 3 (n= 10/group), 7 (n= 12/group) and 28 (n= 10/group) days after injury. To evaluate whether rmTBI results in loss of mature oligodendrocytes as reported in more severe injury models (Lotocki et al. 2011, Flygt et al. 2013), we examined the temporal expression of glutathione S-transferase pi (GSTpi), a marker of oligodendroglia (mature, myelinating oligodendrocytes and pre-mature oligodendrocytes) (Tansey and Cammer 1991, Tamura et al. 2007). Briefly, twenty micrometer thick coronal sections with interval of 240 micrometer between sections were stained with anti-GSTpi antibody (1:300 MBL Life Science, Japan, RRID: AB_591792). The sections were further incubated with an appropriate biotinylated secondary antibody followed by Elite ABC HRP kit and 3,3′-Diaminobenzidine (DAB) (Vector, CA). The GSTpi positive cells were counted in the corpus callosum from 3 sections at bregma 0.98mm, 0.74mm, 0.5mm from each mouse. The thickness of each section was 20m. The volume of corpus callosum was calculated as area multiplied by the interval between sections. The total numbers of the GSTpi positive cells from these 3 sections were divided by the volume of corpus callosum within these 3 sections. The results were presented as fold changes compared to sham controls. The operator who counted the cells was blinded to animal identification.
Immunoblotting Cortical tissues with white matter were collected from the frontal cortex at days 3 (n= 10/group), 7 (n= 12/group) and 28 (n= 10/group) days after injury. Due to technique difficulty, we were unable to isolate pure corpus callosum for western blot. We examined: 1) MBP expression for evaluation of myelination; 2) GSK3 as a key negative regulator of oligodendrocyte differentiation that contributes to diminished myelin repair in demyelinating injuries; and 3) neurofilament light chain expression as a biomarker for neurodegeneration and axonal injury.
Briefly, brain tissues were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 1% NP-40, 1% Sodium Deoxycholic acid, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Roche Life Science, IN) with phosphatase inhibitor cocktail (Santa Cruz Biotechnology, Dallas, TX). Proteins (30-50μg) were separated by electrophoresis on 4-15% SDS polyacrylamide gels (Bio-Rad, CA), then transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% fat free milk or 5% bovine serum albumin (for phosphorylated protein western blot) for 1 h and then incubated with primary antibodies: myelin basic protein (1:500, Chemicon International, CA, RRID: AB_94971), phospho GSK-3 (Tyr279/Tyr216), 1:1000, Santa Cruz Biotechnology, TX, RRID: AB_2115228), β-actin (1:5000, Abcam, MA, RRID: AB_445482) and neurofilament light chain (1:1000, Cell Signaling Technologies, MA, RRID: AB_823575). The blots were further incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity was detected using a chemiluminescence system (SuperSignalTM West Pico PLUS, Thermo Scientific, IL) and ImageQuant LAS 4000 (GE, PA) according to the manufacturer's protocol. Band signal intensity was quantified using NIH Image J software. The densitometry of each expression was normalized to the densitometry of β-actin. Each western blot was technically repeated at least once to validate the results.
Statistics The data are presented as median IQR and analyzed using the Kruskal Wallis test followed by Dunn’s multiple comparisons test. The significance is considered when the p-value is less than 0.05. As the purpose of the study was to find whether oligodendrocyte specific mechanisms relevant to NMDAR antagonism might warrant further exploration in future trials, we did not
additionally correct for multiple comparisons (outside the Dunn’s multiple comparison test) in these analyses thereby minimizing type 2 error at the risk of increasing type 1 error.
Results There was no mortality in this study. There were no difference in loss of consciousness between memantine and vehicle-treated injured mice after each injury (data not shown).
Altered axonal structure after rmTBI There were marked alteration in the appearance of myelin sheaths and axons in corpus callosum of injured mice compared to sham control. Numerous smaller unmyelinated axons (diameter less than 0.3m) appeared in the injured corpus callosum 3 and 7 days after last injury (Fig. 1). Normal axons predominantly appeared in sham control, while injured mice demonstrated damaged axons (splitting myelin sheaths, dark cytoplasmic inclusions in axons, axons with swollen mitochondria and excessive myelin figures) 3 days after the last injury, which appeared consistently on day 7 after the last injury. Hypertrophic myelin sheaths were seen in injured but not sham mice.
Repetitive mild TBI results in decline of oligodendrocytes and MBP and memantine diminishes these alterations at subacute time points after TBI Three days after the last injury, GSTpi positive cells were reduced in the corpus callosum in injured vehicle-treated but not injured memantine-treated mice [chi squared dF (2)=12.96 and p<0.01; p<0.01 (sham vs. veh), p>0.9999 (sham vs. mem) and p=0.033 (veh vs. mem), Fig. 2A and B] but there was no significant difference of MBP expression in cortex at the same time point [dF (2)=1.03 and p>0.05; p>0.9999 in all the comparisons (sham vs. veh; sham vs. mem; and veh vs. mem, Fig. 2C and D]. Seven days after the last injury there was still a significant reduction of the number of GSTpi positive cells in corpus callosum in vehicle-treated injury mice
while the protective effect on GSTpi positive cells in memantine-treated injury mice remained consistent [dF (2)=16.9 and p<0.01, p=0.012 (sham vs. veh), p=0.83 (sham vs. mem) and p<0.01 (veh vs. mem), Fig. 2E and F]. Importantly, there was also a statistically reduced MBP expression in the cortex in the vehicle-treated mice compared to sham controls 7 days after last injury [dF (2)=11.2 and p<0.01; p=0.046 (sham vs. veh), p>0.9999 (sham vs. mem) and p<0.01 (veh vs. mem)]. Early administration of memantine ameliorated decreased MBP expression in the cortex compared to vehicle-treated injured mice at this time point (Fig. 2G and H).
Increased phosphorylated GSK3 (Try216) expression after rmTBI is mitigated by memantine treatment There was no difference in GSK3 expression between groups at 3 days after the last injury (data not shown). Seven days after the last injury, phosphorylated GSK3 (Try216) was increased in vehicle-treated injured mice compared to sham mice, though memantine treatment significantly suppressed this injury-induced increase [dF (2)=20.6 and p<0.01; p=0.044 (sham vs. veh), p=0.12 (sham vs. mem) and p<0.0001 (veh vs. mem), Fig. 3].
Memantine protects against injury associated reduction of neurofilament light chain expression after rmTBI. There was no cortical NF-l expression changes observed 3 days after last injury among all the groups [dF (2)=1.87 and p>0.05; p=0.76 (sham vs. veh), p=0.67(sham vs. mem) and p>0.999 (veh vs. mem), Fig. 4A]. However, NF-l expression in cortex was significantly lower on day 7 after the last injury in vehicle-treated mice compared to sham controls, an effect that was attenuated by memantine treatment [dF (2)=16.3 and p<0.01; p<0.01 (sham v. veh), p=0.35 (sham vs. mem) and p=0.044 (veh vs. mem), Fig.4B].
No deficits of MBP expression and GSTpi positive oligodendroglia one month after last injury. Twenty-eight days after the last injury, there were no significant difference in cortical MBP expression between groups [dF (2)=1.95 and p>0.05; p>0.999 (sham vs. veh), p=0.49 (sham vs. mem) and p>0.999 (veh vs. mem)] and no detectable difference of GSTpi positive cell counts in corpus callosum in injured versus sham mice [dF (2)=1.32 and p>0.05; p>0.999 (sham vs. veh), p=0.89 (sham vs. mem) and p>0.999 (veh vs. mem), Fig. 5]. There was also no significant difference in NF-l expression in cortex between sham and injured mice (data not shown).
Discussion We found that administration of memantine immediately after rmTBI protects against injury induced white matter damage, demyelination (MBP expression) and oligodendrocyte loss. We have previously shown that memantine improves anxiety-like symptoms (open field and elevated plus maze tasks) as well partially prevents gliosis, tau hyper-phosphorylation and decrement of NMDA receptor subunit NR2b after rmTBI(Mei et al. 2017). Here we show an additional mechanism of protective effects of memantine, using the same rmTBI model. We found that memantine lessens white matter changes after rmTBI, demonstrated by western blot and immunostaining techniques. It is important to note that while memantine has been wellstudied in other mouse models (Sukhanov et al. 2004, Beconi et al. 2011, Saab and Roder 2011) it has not been specifically studied in vivo in the context of rmTBI. This is important because there are no drugs approved by the FDA to prevent acute and long-term effects of rmTBI. Given that memantine is already approved by the FDA for treatment of Alzheimer’s, further preclinical studies investigating timing and dosing of memantine in the context of rmTBI could result in rapid clinical translation.
Early administration of memantine resulted in protection against injury-induced changes in oligodendrocyte cell loss and myelin sheath loss and neurofilament light chain decline. These data offer an important extension of our prior work focusing on neuronal mechanisms of protection with memantine treatment (Mei et al. 2017), and suggest a new potential mechanism of therapeutic benefit for memantine in the setting of rmTBI. Mitigation of oligodendrocyte loss after rmTBI could be a vital pathway to prevent demyelinating pathology, preserve normal axon metabolism, improve axonal repair and protect against chronic white matter loss after TBI. Indeed, apoptotic oligodendrocytes have been observed acutely and chronically following clinical TBI (Shaw et al. 2001). Examination of the time course and mechanistic basis of demyelination following rmTBI will be an important future consideration for designing treatment regimens. Here, we delivered memantine after the last rmTBI injury, at which point excitotoxic injury mechanisms presumably had already been activated. Interestingly, delivery of memantine at both acute and subacute times after ischemic stroke resulted in improved functional and molecular outcomes (Lopez-Valdes et al. 2014, Wang et al. 2017). Future studies exploring the optimal timing of treatment would provide an important extension of the current study.
To our knowledge, this is the first study to evaluate the potential protective effects of NMDAR blockade on oligodendrocytes after rmTBI in vivo. NMDA receptor is expressed in mature oligodendrocytes and myelin (Karadottir et al. 2005, Burzomato et al. 2010, Pina-Crespo et al. 2010). Neurodegeneration is one of the sequelae of rmTBI and it has been speculated that multiple mechanisms and neural cell types may be involved in the pathological processes. Increase of extracellular glutamate has been reported in patients as well (Vespa et al. 1998, Guerriero et al. 2015). Prior studies in lateral fluid percussion TBI have suggested that early glutamate release after injury is a proximal event in the cascade of post-injury intracellular Ca2+ accumulation, axonopathy and metabolic crisis that characterizes neuronal secondary injury mechanisms (Katayama et al. 1990, Giza et al. 2006). Little is known about secondary injury
mechanisms after rmTBI relevant to oligodendrocytes. However, several lines of evidence have shown that oligodendrocytes express glutamate receptors including α-Amino-3-hydroxy-5methyl-4-isoxazole Propionic-Acid (AMPA) receptor and N-methyl-D-aspartate (NMDA) receptors and they are particularly vulnerable to glutamatergic excitotoxicity (McDonald et al. 1998, Karadottir et al. 2005, Salter and Fern 2005, Micu et al. 2006, Manning et al. 2008).
Although this mild TBI model induced by rotational acceleration does not cause acute massive cell death, it results in significant long-term behavioral deficits (Mannix et al. 2013, Liu et al. 2017). Axonal and myelin sheath damage were detected at ultrastructural level by electron microscopy several days after injury in this model, suggesting active demyelination. This is consistent with other reports in diverse injury mechanisms suggesting common pathological demyelinating mechanisms that are involved in secondary brain damage. Since electron microscopy is not the appropriate method for the quantification due to limited specimen sampling in this study, we applied other methods of molecular quantification in this study. Notably, no differences of MBP expression and number of GSTpi positive cell number were detected between injured mice and their sham controls 4 weeks after last injury, revealing remyelination may have occurred. This findings are consistent to other reports after close head injury (Mierzwa et al. 2015), suggesting demyelination and remyelination may be a common process after axonal injury.
We chose GSTpi positive oligodendrocytes since we aimed to examine relatively mature oligodendrocyte population that express myelin proteins. GSTpi has been shown to be expressed in mature oligodendrocytes related to myelin expression (Tansey and Cammer 1991, Mason et al. 2004, Tamura et al. 2007, Mierzwa et al. 2015) and has been used for histology in other studies to examine myelin damage (Mierzwa et al. 2015, Bonfanti et al. 2017) although other reports have shown it might be expressed in both mature and pre-mature
oligodendrocytes (Tamura et al. 2007). MBP is one of the major myelin components which reflex myelin sheath integrity. Our results showed that GSTpi positive cell number reduction correlated with changes in axon integrity seen on electron microscopy, though well before decreases in MBP expression were detected. The early mismatch of oligodendrocytes loss with preserved MBP expression may be due to the slow degradation of collapsed myelin in the central nerve system (Vargas and Barres 2007, Armstrong et al. 2016). It is also likely that regional differences in GSTpi positive oligodendrocytes in corpus callosum versus the MBP expressing mature oligodendrocyte population in cortex contributed to these findings. The sensitivity of various methods of detection may also play a role in such disparity.
It is notable that a similar delayed phenomenon was observed when neurofilament light chain expression in the cortex was examined, such that the loss of neurofilament light chain expression on western blot was only detectable by 7 days after last injury, despite the evident loss of axonal integrity on electron microscopy 3 days after last injury. Neurofilament light chain is one of the crucial cytoskeleton proteins that play a vital role in maintaining neuronal forms and functions. Numerous studies demonstrate that concussive TBI results in cytoskeleton protein damage with destabilization of neurofilaments and microtubules (Johnson et al. 2013, Fournier et al. 2015, Kirkcaldie and Collins 2016), with dramatic impact on neuronal transport and metabolisms. Neurofilament light chain expression in peripheral blood has been used as a biomarker for neurodegeneration and axonal injury (Byrne et al. 2017, Ljungqvist et al. 2017, Weston et al. 2017). Our data showed that 7 days after injury, cortical neurofilament expression is markedly reduced, an effect mitigated by memantine treatment.
Our study also demonstrated an increase in phosphorylated GSK3 (Tyr 216) in cortex and white matter after rmTBI, although we did not assess whether increase of GSK3 was in
oligodendrocytes or in other neural cells including neurons. Phosphorylation on Tyr216 of GSK3β is required for an increase of its activity (Bax et al. 2001, Noel et al. 2016). Upregulation of phosphorylated GSK3 has been reported after multiple forms of TBI (Dash et al. 2011, Swiatkowski et al. 2017), though glutamate alone, in the absence of mechanical injury, can sufficiently activate GSK3β (Campana et al. 2017). Our unpublished data show that extracellular glutamate is increased acutely after rmTBI and the results from the current study show that memantine, a glutamate receptor antagonist, partially suppressed post-injury elevation of GSK3 phosphorylation. This suggests that an increase of extracellular glutamate plays a role in pathological changes after rmTBI and early memantine treatment can suppress GSK3 activation, probably in oligodendrocytes and other neural cells. GSK3 has been implicated in the pathologic cascade of neurodegenerative diseases, including promoting beta amyloid accumulation and hyperphosphorylation of tau (Phiel et al. 2003). In addition, GSK3 has previously been shown to be a key negative regulator of oligodendrocyte differentiation that contributes to diminished myelin repair in demyelinating injuries (Azim and Butt 2011). Thus, multiple lines of evidence strongly suggest that the inhibition of GSK3 activation is a potential target in the treatment of TBI. Further studies are warranted to determine whether GSK3 activation is increased in oligodendrocytes and its precursor cells.
While our previous study showed that cognitive deficits persist more than 6 months after rmTBI (Mannix et al. 2013), we did not find persistent changes in the number of oligodendrocytes, MBP or neurofilament expression one month after the last injury. It will be important to further confirm this apparent “normalization” with more sensitive techniques, including electron microscopy, to evaluate whether microstructural changes persist despite relative stabilization of structural protein expression.
Taken together, our results from the current study and our prior study(Mei et al. 2017) demonstrate that memantine treatment protects against reduction of MBP expression and secondary axonal injury, suggesting that NMDAR antagonism may be a therapeutic target for rmTBI via both neuronal and oligodendrocyte specific mechanisms. Whether early treatment with memantine can prevent long-term sequelae of white matter disease associated with rmTBI was not shown in the current study. Recent other studies highlight the importance of white matter integrity in long-term recovery after TBI (Matute et al. 1997, Pham et al. 2012, Semple et al. 2013); preventing white matter injury and promoting white matter recovery are rational therapeutic targets. These data may have further implications for the progressive white matter disease and neurodegenerative sequelae of rmTBI.
Acknowledgments This work was supported in part by funding from the CHB IDDRC and by philanthropic support from the National Hockey League Alumni Association through the Corey C. Griffin Pro-Am Tournament and the National Football League
Disclosures Dr. Meehan receives royalties from ABC-Clio publishing for the sale of his book, Kids, Sports, and Concussion: A guide for coaches and parents, and from Springer International for the book Head and Neck Injuries in Young Athlete and from royalties from Wolters Kluwer for working as an author for UpToDate. He is under contract with ABC-Clio publishing for a future book entitled, Concussions.
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Repetitive mild TBI results in subacute loss of oligodendrocytes and decreased expression of myelin basic protein NMDAR-mediated excitotoxicity is proposed to mediate these post-injury sequelae Treatment with an NMDAR antagonist partially prevented these sequelae of injury
Figure 1. Axon and myelin damage after rmTBI. Top left cartoon shows the location of corpus callosum segments that were examined using electron microscopy. Representative electron microscope images from sham mice, vehicle-treated (VEH) injured mice 3 days and 7 days after last injury, damaged axons, demyelinated axons and some excessive myelin sheaths were found in the corpus callosum after rmTBI. Figure 2. Memantine attenuates reduction of GSTpi positive cells in corpus callosum and decline of MBP expression in cortex after rmTBI at early phase. The number of GSTpi positive cells and MBP expression were examined in sham (SHAM), vehicle treated (VEH) and memantine (MEM) treated injured mouse brains 3 days (A to D) and 7 days (E to H) after last injury. A and E show representative images of GSTpi immunostaining and B and F shows the quantitative result of fold changes of the number of GSTpi positive cells in corpus callosum. The scale bar is 500 µm. C and G show representative images of MBP western blot and D and H show densitometry result of MBP expression. The fold changes compared to sham control are presented as median with interquartile range (box) and minimum to maximum (whiskers). *p<0.05 and **p<0.01). Figure 3. Memantine suppresses phosphorylated GSK3 upregulation after rmTBI. Phosphorylated GSK3 at Tyr216 expression in the cortex was examined in sham (SHAM), vehicle (VEH) and memantine (MEM) treated injured mice. The left figure shows the representative image of western blot and the right graph is the densitometry result of the GSK3 bands (lower bands) at 7 days after last injury. The fold changes compared to sham control are presented as median with interquartile range (box) and minimum to maximum (whiskers). *p<0.05 and ***p<0.0001. Figure 4. Memantine protects neurofilament light chain decline after rmTBI. Neurofilament light chain (NF-l) expression in cortex was examined in sham, vehicle (VEH) and memantine treated (MEM) injured mice 3 days (A) and 7 days (B) after last injury. The upper figures in both A and B show the representative images of western blot and the lower graphs are the quantitative densitometry result of neurofilament light chain expression. The fold changes compared to sham control are presented as median with interquartile range (box) and minimum to maximum (whiskers). *p<0.05 and ** p<0.01. Figure 5. No differences in GSTpi positive cells in corpus callosum or MBP expression in cortex 28 days after rmTBI. A is the result of GSTpi positive cells in corpus callosum. The left figure shows the representative images of GSTpi immunostaining. The graph shows the results of the number of GSTpi positive cells per mm3. The scale bar is 500m. B is the result of MBP expression in cortex. The left figure shows the representative image of western blot and the
right graph is the densitometry result of MBP expression in cortex. The fold changes compared to sham control are presented as median with interquartile range (box) and minimum to maximum (whiskers).
S0306-4522(19)30703-1 https://doi.org/10.1016/j.neuroscience.2019.10.016 NSC 19319
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Neuroscience
Received Date: Revised Date: Accepted Date:
26 August 2019 7 October 2019 8 October 2019
Please cite this article as: G. Ma, C. Liu, J. Hashim, G. Conley, N. Morriss, W.P. Meehan, J. Qiu, R. Mannix, Memantine mitigates oligodendrocyte damage after repetitive mild traumatic brain injury, Neuroscience (2019), doi: https://doi.org/10.1016/j.neuroscience.2019.10.016
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Memantine mitigates oligodendrocyte damage after repetitive mild traumatic brain injury Guixian Ma MD, PhD1,2,3*, Changyun Liu MD2,3,4*, Jumana Hashim BA2, Grace Conley BA2, Nicholas Morriss BA2, William P. Meehan III MD 2,3,5,6, Jianhua Qiu MD, PhD2,3*, Rebekah Mannix MD, MPH2,3*# 1. Department of Neurology, Guangdong Provincial People’s Hospital & Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China 2. Division of Emergency Medicine, Boston Children’s Hospital, Boston Massachusetts, United States of America; 3. Harvard Medical School, Boston Massachusetts, United States of America 4. Department of Neurology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China, 5. Sports Concussion Clinic, Division of Sports Medicine, Boston Massachusetts, United States of America; 6. The Micheli Center for Sports Injury Prevention, Waltham Massachusetts, United States of America Running Head: Excitotoxicity and oligodendrocytes after mild repetitive TBI * Equal contribution # Corresponding author Rebekah Mannix Boston Children’s Hospital, Division of Emergency Medicine 300 Longwood Avenue Boston, Massachusetts 02115 Phone: 617-355-6624 Email: [email protected] Key words: mild traumatic brain injury, excitoxicity, oligodendrocytes Portions of this work were presented in abstract form/in poster form at the National Neurotrauma Symposium, Toronto Canada, August 2018.
Abstract Repetitive mild traumatic brain injury (rmTBI; e.g., sports concussions) is common and results in significant cognitive impairment, white matter injury and increased risk of neurodegeneration. Targeted therapies for rmTBI are lacking, though evidence from other injury models indicates that targeting N-methyl-d-aspartate (NMDA) receptor (NMDAR)-mediated glutamatergic toxicity might mitigate rmTBI-induced injury. We have previously shown that the NMDAR antagonist memantine lessens axonal injury and restores long term potentiation after rmTBI. Here, we evaluated whether the protective effects of memantine include oligodendrocyte specific mechanisms, as prior studies suggest that oligodendrocytes are particularly vulnerable to glutamatergic toxicity. Mice were subjected to rmTBI injury (5 injuries in 5 days) and randomized to treatment with memantine or with vehicle (n=32/group). At the molecular level, oligodendrocyte counts and function (myelin basic protein, MBP) were assessed by immunohistochemistry and western blot at days 3, 7 and 28 days after the last injury. Axon integrity was assessed by neurofilament light chain (NF-l) expression and axonal ultrastructure was evaluated by electron microscopy. Compared to vehicle-treated mice, memantine-treated mice were protected against oligodendrocyte loss and decreased MBP expression at subacute time points after injury. Memantine treatment also protected against axon damage assessed by NF-l expression. These data suggest that the therapeutic effects of post-concussive NMDAR antagonism may in part work through oligodendrocyte specific mechanisms, which may have implications for long term neurodegenerative sequelae after multiple concussions.
Keywords: Traumatic brain injury; oligodendrocytes; white matter injury; NMDAR
Introduction Repetitive mild traumatic brain injury (rmTBI) is a significant public health problem, with as many as 25% of nonprofessional athletes reporting multiple concussions (Collins et al. 1999). Clinically, rmTBI has been associated with long-term neurological impairment including memory disturbances, behavioral abnormalities, personality changes, speech irregularities, and gait abnormalities (Critchley 1957, Grahmann and Ule 1957, Plassman et al. 2000, Bower et al. 2003), symptoms that correlate with disruption of the myelinated pathways crucial for major neural circuits. Gross pathological changes of rmTBI have also been reported, including longterm persistent white matter volume loss, as well as long-term neurodegenerative changes (Omalu et al. 2005, Omalu et al. 2006, McKee et al. 2009, Stern et al. 2011, McKee et al. 2013). The causal pathways linking rmTBI to persistently disrupt neural circuitry, demyelination and neurodegenerative disease have not been established.
Preclinical and clinical studies have demonstrated that axonal injury, particularly in the white matter, is one of the earliest pathologic features seen early after even a single instance of mTBI (Johnson et al. 2013). In severe cases, axonal injury manifests as an initial disruption of axonal transport and axonal swelling within the first 2 – 3 hours after injury followed by axotomy and Wallerian degeneration observed within hours to days (Povlishock and Christman 1995, Armstrong et al. 2016). In addition to primary, secondary axotomy has been observed, characterized by ionic permeability, cytoskeletal compaction and mitochondrial dysfunction (Povlishock and Christman 1995, Armstrong et al. 2016).
Myelin, a major component of white matter, plays a vital role in axon vulnerability after TBI. Myelin not only provides a physical barrier to axons but is also required for normal metabolic support of axons. TBI-induced traumatic axonal injury (TAI) can result in demyelination of intact axons, in the absence of overt cell death (Sullivan et al.
2013). Demyelination alone can result in axon dysfunction and degeneration (Dutta et al. 2011, Friese et al. 2014). Indeed, even in healthy adult mice, myelin modifications are sufficient to modulate neural circuit timing and are required for motor skill learning (McKenzie et al. 2014, Tomassy et al. 2014). Therefore, demyelination after TBI may slow processing speed or desynchronize communication within neural circuits, resulting in overt functional deficits (Mierzwa et al. 2015, Armstrong et al. 2016).
Oligodendrocytes, the myelinating cells of the central nervous system, are critical for maintaining axonal integrity and axonal metabolism (Bradl and Lassmann 2010). Preclinical and clinical studies have demonstrated the detrimental effects of demyelination due to oligodendrocyte death in a variety of neurological conditions (Fields 2008). Oligodendrocytes are particularly vulnerable to cell death via glutamatergic excitotoxicity mechanisms (McDonald et al. 1998, Karadottir et al. 2005, Salter and Fern 2005, Micu et al. 2006, Manning et al. 2008), though few studies have explored the effects of mild TBI-induced excitotoxicity on oligodendrocyte function. Oligodendrocyte death may also play an important role in the pathophysiology of TBI-induced demyelination and axon degeneration. The limited preclinical data on oligodendrocyte dynamics and white matter pathology after TBI have mainly employed models that result in hemorrhagic lesions which result in immediate neuron cell body death and axonal loss (Lotocki et al. 2011, Flygt et al. 2013, Sullivan et al. 2013) or focal traumatic axonal injury associated with skull fracture (Mierzwa et al. 2015) or craniotomy (Donovan et al. 2014). These data may not be relevant to rmTBI, which does not result in skull fracture, gross pathology or neuron cell death at acute time points. More recently, several studies have demonstrated changing in myelin ultrastructure and axonal integrity after rmTBI (Yates et al. 2017, Fehily et al. 2019) though these studies did not specifically assess the temporal interaction of these findings with oligodendrocyte dynamics at various time points after injury. Understanding oligodendrocyte function and vulnerability after rmTBI may offer new insights into
the pathophysiology of rmTBI including disrupted neural circuitry, demyelination and neurodegenerative disease.
We have developed a repetitive mild closed head injury mouse model that results in impact and rotational acceleration injuries in the absence of gross structural brain damage, though accompanied by marked and persistent behavioral deficits in balance, memory, and impulse control and progressive tau pathology (Mannix et al. 2014, Kondo et al. 2015). Our previous study shows that memantine, an N-methyl-d-aspartate (NMDA) receptor (NMDAR) antagonist, improved functional and histopathologic outcomes after rmTBI, focusing on neuronal mechanisms. In the current study, we have expanded our research scope and investigated whether NMDAR antagonism also protected oligodendrocytes after rmTBI since oligodendrocytes play a crucial role in maintaining axonal metabolisms and facilitating axonal repair (Coman et al. 2005, Guerriero et al. 2015). We hypothesized that oligodendrocytes would be vulnerable to NMDAR mediated toxicity after rmTBI, which could be mitigated by NMDAR antagonist such as memantine.
Methods All experiments were approved by the Boston Children's Hospital institutional animal care and use committee and complied with the NIH Guide for the Care and Use of Laboratory Animals. A total of 81 2-3 months old male C57Bl/6 mice were used for these experiments. Mice were obtained from the Jackson Laboratories (Bar Harbor, ME).
Repetitive Mild TBI and treatment The rmTBI was performed as previously described (Meehan et al. 2012, Mannix et al. 2014). Briefly, mice were randomized to three groups: sham (n= 32/group), rmTBI treated with vehicle (n= 32/group) and rmTBI treated with memantine (n=32/group). Mice were anesthetized using
4% isoflurane in oxygen for 45 seconds. Anesthetized mice were placed on a delicate tissue (Kimwipe, Irving, TX) and the head was placed directly under a copper guide tube centered over an area 0.9cm caudal to the interaural line, to approximate the bregma. Mice were held by the tail as an impact was delivered to the dorsal skull by dropping a 54 g metal bolt (12.75 mm diameter) from a 71 cm height, resulting in a rotational acceleration of the head through the Kimwipe. Mice underwent a single closed head injury daily for 5 consecutive days (Liu et al. 2017, Mei et al. 2017). Within 30 minutes after the last injury, mice received either intraperitoneal injection of memantine (10mg/kg, Tocris, Minneapolis, MN)(Ma et al. 2015) or vehicle (saline), each a volume of 0.2 ml as previous reported (Mei et al. 2017). A separate cohort underwent sham injury only, which consisted of 5 daily isoflurane exposures only. All mice recovered in room air after injury or sham injury. Duration of loss of consciousness, defined as the time from removal of anesthesia to spontaneous ambulation, was recorded. Mice were sacrificed at days 3 (n= 10/group), 7 (n= 12/group) and 28 (n= 10/group) after injury for molecular experiments. All molecular analyses were conducted by investigators blinded to injury status, using color coding stored in a password protected computer.
Electron microscopy Mice (n= 2/group) were perfused transcardially using a mixture of 2% formaldehyde and 2.5 % glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.4. The extracted brains were then drop fixed in the same solution for additional 2 weeks. The brains were processed at the Harvard Medical School Electron Microscopy Facility. The corpus callosum was dissected out (Fig. 1A), and the sections were embedded in Epon resin for 48 hours. Ultrathin sections (60nm) were cut and placed on copper grids stained with lead citrate. The sections were imaged using a JEOL 1200EX Transmission electron microscope. Images were taken from similar sections of the corpus callosum using an AMT 2k CCD camera. Photos were taken at 4000X (160m2 /picture).
Immunohistochemistry Mice were perfused with saline transcardially and pre-fixed with 4% paraformaldehyde at time points 3 (n= 10/group), 7 (n= 12/group) and 28 (n= 10/group) days after injury. To evaluate whether rmTBI results in loss of mature oligodendrocytes as reported in more severe injury models (Lotocki et al. 2011, Flygt et al. 2013), we examined the temporal expression of glutathione S-transferase pi (GSTpi), a marker of oligodendroglia (mature, myelinating oligodendrocytes and pre-mature oligodendrocytes) (Tansey and Cammer 1991, Tamura et al. 2007). Briefly, twenty micrometer thick coronal sections with interval of 240 micrometer between sections were stained with anti-GSTpi antibody (1:300 MBL Life Science, Japan, RRID: AB_591792). The sections were further incubated with an appropriate biotinylated secondary antibody followed by Elite ABC HRP kit and 3,3′-Diaminobenzidine (DAB) (Vector, CA). The GSTpi positive cells were counted in the corpus callosum from 3 sections at bregma 0.98mm, 0.74mm, 0.5mm from each mouse. The thickness of each section was 20m. The volume of corpus callosum was calculated as area multiplied by the interval between sections. The total numbers of the GSTpi positive cells from these 3 sections were divided by the volume of corpus callosum within these 3 sections. The results were presented as fold changes compared to sham controls. The operator who counted the cells was blinded to animal identification.
Immunoblotting Cortical tissues with white matter were collected from the frontal cortex at days 3 (n= 10/group), 7 (n= 12/group) and 28 (n= 10/group) days after injury. Due to technique difficulty, we were unable to isolate pure corpus callosum for western blot. We examined: 1) MBP expression for evaluation of myelination; 2) GSK3 as a key negative regulator of oligodendrocyte differentiation that contributes to diminished myelin repair in demyelinating injuries; and 3) neurofilament light chain expression as a biomarker for neurodegeneration and axonal injury.
Briefly, brain tissues were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 1% NP-40, 1% Sodium Deoxycholic acid, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Roche Life Science, IN) with phosphatase inhibitor cocktail (Santa Cruz Biotechnology, Dallas, TX). Proteins (30-50μg) were separated by electrophoresis on 4-15% SDS polyacrylamide gels (Bio-Rad, CA), then transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% fat free milk or 5% bovine serum albumin (for phosphorylated protein western blot) for 1 h and then incubated with primary antibodies: myelin basic protein (1:500, Chemicon International, CA, RRID: AB_94971), phospho GSK-3 (Tyr279/Tyr216), 1:1000, Santa Cruz Biotechnology, TX, RRID: AB_2115228), β-actin (1:5000, Abcam, MA, RRID: AB_445482) and neurofilament light chain (1:1000, Cell Signaling Technologies, MA, RRID: AB_823575). The blots were further incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity was detected using a chemiluminescence system (SuperSignalTM West Pico PLUS, Thermo Scientific, IL) and ImageQuant LAS 4000 (GE, PA) according to the manufacturer's protocol. Band signal intensity was quantified using NIH Image J software. The densitometry of each expression was normalized to the densitometry of β-actin. Each western blot was technically repeated at least once to validate the results.
Statistics The data are presented as median IQR and analyzed using the Kruskal Wallis test followed by Dunn’s multiple comparisons test. The significance is considered when the p-value is less than 0.05. As the purpose of the study was to find whether oligodendrocyte specific mechanisms relevant to NMDAR antagonism might warrant further exploration in future trials, we did not
additionally correct for multiple comparisons (outside the Dunn’s multiple comparison test) in these analyses thereby minimizing type 2 error at the risk of increasing type 1 error.
Results There was no mortality in this study. There were no difference in loss of consciousness between memantine and vehicle-treated injured mice after each injury (data not shown).
Altered axonal structure after rmTBI There were marked alteration in the appearance of myelin sheaths and axons in corpus callosum of injured mice compared to sham control. Numerous smaller unmyelinated axons (diameter less than 0.3m) appeared in the injured corpus callosum 3 and 7 days after last injury (Fig. 1). Normal axons predominantly appeared in sham control, while injured mice demonstrated damaged axons (splitting myelin sheaths, dark cytoplasmic inclusions in axons, axons with swollen mitochondria and excessive myelin figures) 3 days after the last injury, which appeared consistently on day 7 after the last injury. Hypertrophic myelin sheaths were seen in injured but not sham mice.
Repetitive mild TBI results in decline of oligodendrocytes and MBP and memantine diminishes these alterations at subacute time points after TBI Three days after the last injury, GSTpi positive cells were reduced in the corpus callosum in injured vehicle-treated but not injured memantine-treated mice [chi squared dF (2)=12.96 and p<0.01; p<0.01 (sham vs. veh), p>0.9999 (sham vs. mem) and p=0.033 (veh vs. mem), Fig. 2A and B] but there was no significant difference of MBP expression in cortex at the same time point [dF (2)=1.03 and p>0.05; p>0.9999 in all the comparisons (sham vs. veh; sham vs. mem; and veh vs. mem, Fig. 2C and D]. Seven days after the last injury there was still a significant reduction of the number of GSTpi positive cells in corpus callosum in vehicle-treated injury mice
while the protective effect on GSTpi positive cells in memantine-treated injury mice remained consistent [dF (2)=16.9 and p<0.01, p=0.012 (sham vs. veh), p=0.83 (sham vs. mem) and p<0.01 (veh vs. mem), Fig. 2E and F]. Importantly, there was also a statistically reduced MBP expression in the cortex in the vehicle-treated mice compared to sham controls 7 days after last injury [dF (2)=11.2 and p<0.01; p=0.046 (sham vs. veh), p>0.9999 (sham vs. mem) and p<0.01 (veh vs. mem)]. Early administration of memantine ameliorated decreased MBP expression in the cortex compared to vehicle-treated injured mice at this time point (Fig. 2G and H).
Increased phosphorylated GSK3 (Try216) expression after rmTBI is mitigated by memantine treatment There was no difference in GSK3 expression between groups at 3 days after the last injury (data not shown). Seven days after the last injury, phosphorylated GSK3 (Try216) was increased in vehicle-treated injured mice compared to sham mice, though memantine treatment significantly suppressed this injury-induced increase [dF (2)=20.6 and p<0.01; p=0.044 (sham vs. veh), p=0.12 (sham vs. mem) and p<0.0001 (veh vs. mem), Fig. 3].
Memantine protects against injury associated reduction of neurofilament light chain expression after rmTBI. There was no cortical NF-l expression changes observed 3 days after last injury among all the groups [dF (2)=1.87 and p>0.05; p=0.76 (sham vs. veh), p=0.67(sham vs. mem) and p>0.999 (veh vs. mem), Fig. 4A]. However, NF-l expression in cortex was significantly lower on day 7 after the last injury in vehicle-treated mice compared to sham controls, an effect that was attenuated by memantine treatment [dF (2)=16.3 and p<0.01; p<0.01 (sham v. veh), p=0.35 (sham vs. mem) and p=0.044 (veh vs. mem), Fig.4B].
No deficits of MBP expression and GSTpi positive oligodendroglia one month after last injury. Twenty-eight days after the last injury, there were no significant difference in cortical MBP expression between groups [dF (2)=1.95 and p>0.05; p>0.999 (sham vs. veh), p=0.49 (sham vs. mem) and p>0.999 (veh vs. mem)] and no detectable difference of GSTpi positive cell counts in corpus callosum in injured versus sham mice [dF (2)=1.32 and p>0.05; p>0.999 (sham vs. veh), p=0.89 (sham vs. mem) and p>0.999 (veh vs. mem), Fig. 5]. There was also no significant difference in NF-l expression in cortex between sham and injured mice (data not shown).
Discussion We found that administration of memantine immediately after rmTBI protects against injury induced white matter damage, demyelination (MBP expression) and oligodendrocyte loss. We have previously shown that memantine improves anxiety-like symptoms (open field and elevated plus maze tasks) as well partially prevents gliosis, tau hyper-phosphorylation and decrement of NMDA receptor subunit NR2b after rmTBI(Mei et al. 2017). Here we show an additional mechanism of protective effects of memantine, using the same rmTBI model. We found that memantine lessens white matter changes after rmTBI, demonstrated by western blot and immunostaining techniques. It is important to note that while memantine has been wellstudied in other mouse models (Sukhanov et al. 2004, Beconi et al. 2011, Saab and Roder 2011) it has not been specifically studied in vivo in the context of rmTBI. This is important because there are no drugs approved by the FDA to prevent acute and long-term effects of rmTBI. Given that memantine is already approved by the FDA for treatment of Alzheimer’s, further preclinical studies investigating timing and dosing of memantine in the context of rmTBI could result in rapid clinical translation.
Early administration of memantine resulted in protection against injury-induced changes in oligodendrocyte cell loss and myelin sheath loss and neurofilament light chain decline. These data offer an important extension of our prior work focusing on neuronal mechanisms of protection with memantine treatment (Mei et al. 2017), and suggest a new potential mechanism of therapeutic benefit for memantine in the setting of rmTBI. Mitigation of oligodendrocyte loss after rmTBI could be a vital pathway to prevent demyelinating pathology, preserve normal axon metabolism, improve axonal repair and protect against chronic white matter loss after TBI. Indeed, apoptotic oligodendrocytes have been observed acutely and chronically following clinical TBI (Shaw et al. 2001). Examination of the time course and mechanistic basis of demyelination following rmTBI will be an important future consideration for designing treatment regimens. Here, we delivered memantine after the last rmTBI injury, at which point excitotoxic injury mechanisms presumably had already been activated. Interestingly, delivery of memantine at both acute and subacute times after ischemic stroke resulted in improved functional and molecular outcomes (Lopez-Valdes et al. 2014, Wang et al. 2017). Future studies exploring the optimal timing of treatment would provide an important extension of the current study.
To our knowledge, this is the first study to evaluate the potential protective effects of NMDAR blockade on oligodendrocytes after rmTBI in vivo. NMDA receptor is expressed in mature oligodendrocytes and myelin (Karadottir et al. 2005, Burzomato et al. 2010, Pina-Crespo et al. 2010). Neurodegeneration is one of the sequelae of rmTBI and it has been speculated that multiple mechanisms and neural cell types may be involved in the pathological processes. Increase of extracellular glutamate has been reported in patients as well (Vespa et al. 1998, Guerriero et al. 2015). Prior studies in lateral fluid percussion TBI have suggested that early glutamate release after injury is a proximal event in the cascade of post-injury intracellular Ca2+ accumulation, axonopathy and metabolic crisis that characterizes neuronal secondary injury mechanisms (Katayama et al. 1990, Giza et al. 2006). Little is known about secondary injury
mechanisms after rmTBI relevant to oligodendrocytes. However, several lines of evidence have shown that oligodendrocytes express glutamate receptors including α-Amino-3-hydroxy-5methyl-4-isoxazole Propionic-Acid (AMPA) receptor and N-methyl-D-aspartate (NMDA) receptors and they are particularly vulnerable to glutamatergic excitotoxicity (McDonald et al. 1998, Karadottir et al. 2005, Salter and Fern 2005, Micu et al. 2006, Manning et al. 2008).
Although this mild TBI model induced by rotational acceleration does not cause acute massive cell death, it results in significant long-term behavioral deficits (Mannix et al. 2013, Liu et al. 2017). Axonal and myelin sheath damage were detected at ultrastructural level by electron microscopy several days after injury in this model, suggesting active demyelination. This is consistent with other reports in diverse injury mechanisms suggesting common pathological demyelinating mechanisms that are involved in secondary brain damage. Since electron microscopy is not the appropriate method for the quantification due to limited specimen sampling in this study, we applied other methods of molecular quantification in this study. Notably, no differences of MBP expression and number of GSTpi positive cell number were detected between injured mice and their sham controls 4 weeks after last injury, revealing remyelination may have occurred. This findings are consistent to other reports after close head injury (Mierzwa et al. 2015), suggesting demyelination and remyelination may be a common process after axonal injury.
We chose GSTpi positive oligodendrocytes since we aimed to examine relatively mature oligodendrocyte population that express myelin proteins. GSTpi has been shown to be expressed in mature oligodendrocytes related to myelin expression (Tansey and Cammer 1991, Mason et al. 2004, Tamura et al. 2007, Mierzwa et al. 2015) and has been used for histology in other studies to examine myelin damage (Mierzwa et al. 2015, Bonfanti et al. 2017) although other reports have shown it might be expressed in both mature and pre-mature
oligodendrocytes (Tamura et al. 2007). MBP is one of the major myelin components which reflex myelin sheath integrity. Our results showed that GSTpi positive cell number reduction correlated with changes in axon integrity seen on electron microscopy, though well before decreases in MBP expression were detected. The early mismatch of oligodendrocytes loss with preserved MBP expression may be due to the slow degradation of collapsed myelin in the central nerve system (Vargas and Barres 2007, Armstrong et al. 2016). It is also likely that regional differences in GSTpi positive oligodendrocytes in corpus callosum versus the MBP expressing mature oligodendrocyte population in cortex contributed to these findings. The sensitivity of various methods of detection may also play a role in such disparity.
It is notable that a similar delayed phenomenon was observed when neurofilament light chain expression in the cortex was examined, such that the loss of neurofilament light chain expression on western blot was only detectable by 7 days after last injury, despite the evident loss of axonal integrity on electron microscopy 3 days after last injury. Neurofilament light chain is one of the crucial cytoskeleton proteins that play a vital role in maintaining neuronal forms and functions. Numerous studies demonstrate that concussive TBI results in cytoskeleton protein damage with destabilization of neurofilaments and microtubules (Johnson et al. 2013, Fournier et al. 2015, Kirkcaldie and Collins 2016), with dramatic impact on neuronal transport and metabolisms. Neurofilament light chain expression in peripheral blood has been used as a biomarker for neurodegeneration and axonal injury (Byrne et al. 2017, Ljungqvist et al. 2017, Weston et al. 2017). Our data showed that 7 days after injury, cortical neurofilament expression is markedly reduced, an effect mitigated by memantine treatment.
Our study also demonstrated an increase in phosphorylated GSK3 (Tyr 216) in cortex and white matter after rmTBI, although we did not assess whether increase of GSK3 was in
oligodendrocytes or in other neural cells including neurons. Phosphorylation on Tyr216 of GSK3β is required for an increase of its activity (Bax et al. 2001, Noel et al. 2016). Upregulation of phosphorylated GSK3 has been reported after multiple forms of TBI (Dash et al. 2011, Swiatkowski et al. 2017), though glutamate alone, in the absence of mechanical injury, can sufficiently activate GSK3β (Campana et al. 2017). Our unpublished data show that extracellular glutamate is increased acutely after rmTBI and the results from the current study show that memantine, a glutamate receptor antagonist, partially suppressed post-injury elevation of GSK3 phosphorylation. This suggests that an increase of extracellular glutamate plays a role in pathological changes after rmTBI and early memantine treatment can suppress GSK3 activation, probably in oligodendrocytes and other neural cells. GSK3 has been implicated in the pathologic cascade of neurodegenerative diseases, including promoting beta amyloid accumulation and hyperphosphorylation of tau (Phiel et al. 2003). In addition, GSK3 has previously been shown to be a key negative regulator of oligodendrocyte differentiation that contributes to diminished myelin repair in demyelinating injuries (Azim and Butt 2011). Thus, multiple lines of evidence strongly suggest that the inhibition of GSK3 activation is a potential target in the treatment of TBI. Further studies are warranted to determine whether GSK3 activation is increased in oligodendrocytes and its precursor cells.
While our previous study showed that cognitive deficits persist more than 6 months after rmTBI (Mannix et al. 2013), we did not find persistent changes in the number of oligodendrocytes, MBP or neurofilament expression one month after the last injury. It will be important to further confirm this apparent “normalization” with more sensitive techniques, including electron microscopy, to evaluate whether microstructural changes persist despite relative stabilization of structural protein expression.
Taken together, our results from the current study and our prior study(Mei et al. 2017) demonstrate that memantine treatment protects against reduction of MBP expression and secondary axonal injury, suggesting that NMDAR antagonism may be a therapeutic target for rmTBI via both neuronal and oligodendrocyte specific mechanisms. Whether early treatment with memantine can prevent long-term sequelae of white matter disease associated with rmTBI was not shown in the current study. Recent other studies highlight the importance of white matter integrity in long-term recovery after TBI (Matute et al. 1997, Pham et al. 2012, Semple et al. 2013); preventing white matter injury and promoting white matter recovery are rational therapeutic targets. These data may have further implications for the progressive white matter disease and neurodegenerative sequelae of rmTBI.
Acknowledgments This work was supported in part by funding from the CHB IDDRC and by philanthropic support from the National Hockey League Alumni Association through the Corey C. Griffin Pro-Am Tournament and the National Football League
Disclosures Dr. Meehan receives royalties from ABC-Clio publishing for the sale of his book, Kids, Sports, and Concussion: A guide for coaches and parents, and from Springer International for the book Head and Neck Injuries in Young Athlete and from royalties from Wolters Kluwer for working as an author for UpToDate. He is under contract with ABC-Clio publishing for a future book entitled, Concussions.
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Repetitive mild TBI results in subacute loss of oligodendrocytes and decreased expression of myelin basic protein NMDAR-mediated excitotoxicity is proposed to mediate these post-injury sequelae Treatment with an NMDAR antagonist partially prevented these sequelae of injury
Figure 1. Axon and myelin damage after rmTBI. Top left cartoon shows the location of corpus callosum segments that were examined using electron microscopy. Representative electron microscope images from sham mice, vehicle-treated (VEH) injured mice 3 days and 7 days after last injury, damaged axons, demyelinated axons and some excessive myelin sheaths were found in the corpus callosum after rmTBI. Figure 2. Memantine attenuates reduction of GSTpi positive cells in corpus callosum and decline of MBP expression in cortex after rmTBI at early phase. The number of GSTpi positive cells and MBP expression were examined in sham (SHAM), vehicle treated (VEH) and memantine (MEM) treated injured mouse brains 3 days (A to D) and 7 days (E to H) after last injury. A and E show representative images of GSTpi immunostaining and B and F shows the quantitative result of fold changes of the number of GSTpi positive cells in corpus callosum. The scale bar is 500 µm. C and G show representative images of MBP western blot and D and H show densitometry result of MBP expression. The fold changes compared to sham control are presented as median with interquartile range (box) and minimum to maximum (whiskers). *p<0.05 and **p<0.01). Figure 3. Memantine suppresses phosphorylated GSK3 upregulation after rmTBI. Phosphorylated GSK3 at Tyr216 expression in the cortex was examined in sham (SHAM), vehicle (VEH) and memantine (MEM) treated injured mice. The left figure shows the representative image of western blot and the right graph is the densitometry result of the GSK3 bands (lower bands) at 7 days after last injury. The fold changes compared to sham control are presented as median with interquartile range (box) and minimum to maximum (whiskers). *p<0.05 and ***p<0.0001. Figure 4. Memantine protects neurofilament light chain decline after rmTBI. Neurofilament light chain (NF-l) expression in cortex was examined in sham, vehicle (VEH) and memantine treated (MEM) injured mice 3 days (A) and 7 days (B) after last injury. The upper figures in both A and B show the representative images of western blot and the lower graphs are the quantitative densitometry result of neurofilament light chain expression. The fold changes compared to sham control are presented as median with interquartile range (box) and minimum to maximum (whiskers). *p<0.05 and ** p<0.01. Figure 5. No differences in GSTpi positive cells in corpus callosum or MBP expression in cortex 28 days after rmTBI. A is the result of GSTpi positive cells in corpus callosum. The left figure shows the representative images of GSTpi immunostaining. The graph shows the results of the number of GSTpi positive cells per mm3. The scale bar is 500m. B is the result of MBP expression in cortex. The left figure shows the representative image of western blot and the
right graph is the densitometry result of MBP expression in cortex. The fold changes compared to sham control are presented as median with interquartile range (box) and minimum to maximum (whiskers).