Introduction
In popular media, mild traumatic brain injury (mTBI) has been referred to as a ‘silent epidemic.’ Indeed, in a majority of mTBI cases there is distinct absence of clear structural damage alongside normal neuroimaging (Iverson, 2005, 2010; Belanger et al., 2007; Jagoda et al., 2009; Gao and Chen, 2011; Smith et al., 2013). Nevertheless, subtle perturbations in brain structure likely evoke an insidious cascade of evolving widespread damage to neural circuitry, thought to culminate in long-term and ongoing neurological impairment and associated problems (Büki and Povlishock, 2006; Farkas and Povlishock, 2007; Blyth and Bazarian, 2010; Iverson, 2010; Wang and Ma, 2010; Meaney and Smith, 2011; Johnson et al., 2012a; Hill et al., 2016). This typically incorporates widespread axonal perturbation, known as traumatic axonal injury, throughout the parenchyma and particularly in the white matter tracts. This injury may include changes in the somato-dendritic compartment such as somal atrophy, distorted dendritic arbor geometry and loss of complexity, and decreased dendritic spine density (Chen et al., 2003, 2010; Stone et al., 2004; Spain et al., 2010; Gao and Chen, 2011; Gao et al., 2011; Greer et al., 2011; Campbell et al., 2012).
Data from experimental models shows that mTBI characteristically generates sparse microscopic damage, whereby neural circuits are rendered dysfunctional but not destroyed (Iverson, 2005; DeKosky and Ikonomovic, 2010; Shultz et al., 2016). This highlights an important target for therapeutic intervention. Microtubule disruption and loss is a key ultrastructural hallmark of neuronal injury (Maxwell and Graham, 1997; Tang-Schomer et al., 2012). Moreover, microtubule dynamics are fundamental to a multitude of neuro-glial responses in the aftermath of injury (Chuckowree and Vickers, 2003; Tang-Schomer et al., 2012; Brizuela et al., 2015). Thus, manipulating microtubules provides a novel multi-target approach for intervening in these processes (Brunden et al., 2012; Baas and Ahmad, 2013; Dent, 2016). Of note, microtubule-stabilizing agents of the taxane and epothilone families are used chemotherapeutically at high doses to block the growth of cancerous cells (Goodin, 2004; Michaud, 2009; Zhao et al., 2009; Khrapunovich-Baine et al., 2011). Accumulating data derived from a variety of experimental neural injury and disease paradigms reveals that when used at low doses these drugs have a range of beneficial effects, including dampening detrimental gliotic responses, preventing synapse loss and enhancing adaptive neuronal alterations as well as preserving cognitive and motor functions (Moscarello et al., 2002; Zhang et al., 2005; Andrieux et al., 2006; Ertürk et al., 2007; Brunden et al., 2010, 2011, 2012; Hellal et al., 2011; Sengottuvel et al., 2011; Barten et al., 2012; Tang-Schomer et al., 2012; Baas and Ahmad, 2013; Cartelli et al., 2013; Hur and Lee, 2014; Popovich et al., 2014; Brizuela et al., 2015; Cross et al., 2015; Ruschel et al., 2015; Jang et al., 2016; Penazzi et al., 2016).
The epothilones promote microtubule formation and stabilization, and inhibit microtubule depolymerization (Altmann et al., 2000; Chen et al., 2008). With particular relevance to the brain, epothilones are more water soluble than their taxane counterparts, are blood–brain barrier penetrant, and are retained in the central nervous system for several days after administration (Andrieux et al., 2006; Browne et al., 2011). Importantly, the efficacy of epothilones as a therapeutic strategy in the context of brain injury remains to be elucidated. To address this shortfall we explored the effect of peripherally administered epothilone D following a single mTBI using the clinically relevant lateral fluid percussion brain injury (FPI) model (Thompson et al., 2005). To visualize discrete alterations in the somato-dendritic and axonal compartments of layer 5 cortical excitatory projections neurons we used the Thy1-YFPH mouse, which revealed exquisite neuronal sub-structure (Feng et al., 2000).
Materials and Methods
Breeding and Genotyping of Thy1-YFPH Transgenic Mice
All experimental procedures involving animals were approved by the Animal Ethics Committee of the University of Tasmania (ethics approval number A0011076) and are in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Animals were housed in standard conditions (20°C, 12 h/12 h light/dark cycle) with access to food and water ad libitum and monitored daily for signs of stress and illness. Thy1-YFPH line mice [B6.Cg-Tg(Thy1-YFP)HJrs/J, stock number 003782] were obtained from the Jackson Laboratory (Bar Harbor, ME, United States) and maintained as a heterozygous colony. In these animals YFP is expressed under the control of the neuron-specific Thy1 promoter in ∼80% of layer 5 and 2/3 neocortical pyramidal neurons (Feng et al., 2000). Ear punches were taken at weaning (4 weeks) to determine inheritance of the YFP transgene. Tissue was mounted on a glass slide and examined with the 488 nm filter on a Leica DM LB2 microscope (Leica Microsystems Pty Ltd., North Ryde, NSW, Australia). Animals carrying the YFP transgene were identified as possessing YFP-positive (YFP+) axons within their ear clips.
Surgical Preparation
Animals were prepared in groups of four per day. Two animals received FPI while two were sham-operated. Mice were subjected to lateral FPI using an established protocol (Carbonell et al., 1998; Lifshitz et al., 2007; Alder et al., 2011). Briefly, adult male YFP-H mice (10–12 weeks, 25–30 g; n = 12 FPI/brain-injured, n = 12 sham-operated) were anesthetised in a pre-charged induction chamber containing 5% isoflurane (Isoflo, Abbot Australasia Pty Ltd., Botany, NSW, Australia) in 100% O2. Mice were removed from the induction chamber, pre-emptive analgesia, temgesic (buprenorphine hydrochloride, 0.1 mg/kg; Reckitt Benckiser, West Ryde, NSW, Australia), was administered subcutaneously and the fur covering the scalp was removed. Mice were placed on a homeothermic blanket (Stoelting, Wood Dale, IL, United States) to maintain body temperature at 37°C during surgery and stabilized in a stereotaxic frame (Narishige, Tokyo, Japan) equipped with a nose cone to maintain anesthesia (1–2% isoflurane in 100% O2). The scalp was cleaned with betadine (Sanofi-aventis Consumer Healthcare, Virginia, QLD, Australia) and 70% ethanol and the topical anesthetic bupivicaine (Bupivicaine hydrochloride, 50 μl 0.25% in sterile saline; Pfizer, West Ryde, NSW, Australia) was administered under the scalp. A midline incision was made to expose the skull from bregma to lambda. The skin was retracted and the fascia covering the skull was removed. A 3.0 mm circular craniectomy was made 2.0 mm posterior and 2.5 mm lateral to bregma on the right hand side of the skull over the somatosensory cortex via manual trephination with a pin vice equipped with a 2.7 mm trephine drill bit (AgnTho’s, Lidingo, Sweden). The underlying dura was left intact. An injury-hub was constructed over the craniectomy – a sterile Luer-Loc syringe hub was cut from a 22-gauge needle, fixed over the craniectomy using Loctite cyanoacrylate (Henkel Australia, Sydney, NSW, Australia), secured to the skull using Paladur dental acrylic (Heraeus Dental Science, Villebon, France), filled with sterile saline and capped with a male Luer-Loc fitting. Pre-, peri-, and post-surgical monitoring was performed to determine respiratory rate, confirm absence of reflexes and monitor mucous membranes/capillary refill time. Following injury-hub placement animals were removed from the stereotaxic frame and allowed to recover in a warmed cage until fully ambulatory (30–60 min) and then placed back in their home cage.
Lateral Fluid Percussion Brain Injury and Drug Treatment
Two hours following application of the injury-hub, once mice had been fully ambulatory for over an hour, each animal was re-anesthetised in a pre-charged induction chamber containing 5% isoflurane in 100% O2. Following induction of anesthesia, the animal was removed from the induction chamber, the cap was removed from the injury hub and the hub was re-filled with sterile saline and attached to the FP302 Fluid Percussion Device (AmScien Instruments, Richmond, VA, United States) via a 30 cm spacing tube filled with sterile water. The animal was placed on a heated pad and once a normal pattern of breathing resumed, but prior sensitivity to stimulation, an injury of mild severity (1.5 ± 0.1 atmospheres) was delivered to the intact dura by releasing device’s pendulum onto a fluid filled piston, causing transient displacement and deformation of the dura and underlying brain. A transducer incorporated into the device measured the pulse pressure and the peak pressure was recorded within the software. Following injury, animals were placed on their back and visually monitored for recovery of spontaneous breathing. Additionally, the time taken for animals to recover the righting reflex was recorded as a measure of transient unconsciousness/loss of consciousness. Following injury, we did not record any convulsions, mortalities or other complications. Sham-operated animals underwent identical procedures to FPI animals, however, the pendulum was not released. Mice were re-anesthetised, the injury hub was removed and the incision sutured. Immediately following suturing, animals were administered with either epothilone D (2 mg/kg, i.p.; Anita Laboratories, Hangzhou, China; n = 6 FPI and n = 6 sham-operated) or vehicle (equivalent volume DMSO; n = 6 FPI and n = 6 sham-operated). Mice received drug/vehicle treatment within 20 min following completion application of the FPI/Sham-operation. Animals were placed in a heated cage and monitored during the recovery period until fully ambulatory (30–60 min), prior to return to their home cage.
Immunohistochemistry
At 1 week post-injury/sham-operation, mice were intraperitoneally injected with a terminal dose of sodium pentobarbital (300 mg/kg; Troy Laboratories Pty Ltd., Smithfield, NSW, Australia) and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were post-fixed in vivo for 24 h at 4°C. Each brain was removed from the skull, embedded in 5% agarose in 0.01 M phosphate buffered saline (PBS) and free-floating coronal sections (50 μm) were cut using a Leica VT1000S vibratome (Leica Biosystems Australia Pty Ltd., Mount Waverly, VIC, Australia) to incorporate the entire injury impact site as well as 0.5–1.0 mm anterior and posterior to this. Sections were serially collected into Costar 24-well culture plates (Corning Life Sciences, New York, NY, United States) containing 0.01M PBS and 0.02% sodium azide and stored until required.
To perform immunohistochemistry, every sixth section from each brain was moved into a fresh culture plate well to represent the injury site. Prior to immunohistochemistry, sections were rinsed in three washes of 0.01 M PBS. Sections then underwent immunofluorescence labeling for glial fibrillary acidic protein (GFAP). Briefly, sections were incubated in rabbit anti-GFAP (1:2000; DAKO, Z0334, Glostrup, Denmark) in diluent (0.01 M PBS with 0.03% Triton X-100) at 4°C for ∼20 h, washed, incubated in goat anti-rabbit Alexa 568 (1:1000; Invitrogen BRL, Life Technologies, Grand Island, NY, United States) and DAPI (1:6000; Invitrogen, D3571) in 0.01M PBS for 1.5 h, washed and mounted serially onto slides (Livingstone International Pty Ltd., Rosebery, NSW, Australia) with Permafluor mounting media (Thermo Scientific, Scoresbury, VIC, Australia).
Microscopy and Image Analysis
Images were collected with an UltraVIEW spinning disk confocal microscope running Volocity Software (PerkinElmer Pty Ltd., Glen Waverley, VIC, Australia), equipped with a 20×/0.5 air, 40×/0.95 air and Plan Apo 60×/1.20 water objective (Nikon, New York, NY, United States). For quantitation of cortical thickness, YFPH cell number and size and degenerated/dystrophic axonal bulb number and size (in the internal and external capsules) the microscope was configured to capture large stitched images of the upper hemispheric quadrant of each brain (20 μm z-stacks, 1 μm slices) with the 20× objective from three representative sections throughout the injury, designated middle, anterior, and posterior representing the middle section (first appearance of two blades of the dentate gyrus) as well as the section 300 μm anterior and 300 μm posterior to this. Using ImageJ freeware (Schneider et al., 2012) the cortex, external and internal capsule were traced in each of the three sections and within these anatomical boarders the individual YFPH+ cells in the cortex and axonal bulbs/dystrophic neurites in the external and internal capsules were traced for quantitation of size and density. To determine the axonal degeneration index (degenerating/beaded YFPH+ axons) single 40× magnification image stacks (20 μm z-stacks, 1 μm slices) were collected from the same three sections as used for the prior analysis. Images for the external capsule were captured from the white matter on the medio-lateral boarder of the lateral ventricle and those for the internal capsule were captured half way between the dorsal and ventral boarder of the internal capsule. For quantification of astrocyte activation (percent area occupied by GFAP expressing astrocytes), 20× single image stacks (20 μm z-stacks, 1 μm slices) were collected from the same regions used for analysis of axonal denegation, in addition to layer 2/3 of the cortex directly under the impact site. Analysis of percentage area GFAP and YFPH+ axonal degeneration was performed using ImageJ freeware. For dendritic spine analysis, image stacks were captured with the 60× water objective (0.2 μm slices). Image stacks were collected from layer 4/5 directly under the injury site. These included the image in the middle of the impact site as well as an image medial and lateral to this. All layer 5 apical dendrite obliquely projecting branches were traced from each stack and the spines contained on these dendrites were traced in Neurolucida (MBF Biosciences, Williston, VT, United States). Morphology data was generated by allocating each spine to one of three categories, mushroom (prominent head, thin neck), stubby (greater width than length), and thin (greater length than width). Changes in dendritic spine density, length, and morphology were determined by loading Neurolucida data files into Neurolucida ExplorerTM (MBF Biosciences).
Statistical Analysis
Data was analyzed (and graphs created) in GraphPad Prism (version 6.0, La Jolla, CA, United States) using unpaired t-tests with Welch’s correction, or one- or two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Averaged values were expressed as means ± standard error of the mean (SEM). A p-value of <0.05, designated ∗, was considered statistically significant. Figures were prepared in Adobe Illustrator CS6 (version 16.0.0, Adobe Systems, San Jose, CA, United States).
Discussion
Despite the absence of overt structural damage and the presence of normal neuroimaging in a majority of mTBI cases, subtle widespread and progressing damage within neural circuits is thought to underlie the development, and potentially ongoing evolution, of impairments in neurosensory, cognitive, psychosocial, and physical function (Iverson, 2005, 2010; Farkas and Povlishock, 2007; Dean and Sterr, 2013; McMahon et al., 2014; Hill et al., 2016; Shultz et al., 2016; Wang and Li, 2016; Wilde et al., 2016; de Koning et al., 2017). The cellular alterations contributing to pathology in the aftermath of mTBI are not fully understood and there exists no treatment to halt or reverse the damage to neural circuits. Thus, we investigated the discrete subcellular reactions of axonal and somato-dendritic compartments following transient structural brain injury and epothilone D-induced microtubule stabilization. We should note that this study represents a proof-of-concept investigation demonstrating that peripherally administered epothilone D can enter the brain and have an effect on neurons. In accordance with established protocols (Carbonell et al., 1998; Spain et al., 2010; Alder et al., 2011) we showed that a single episode of mild TBI generated significant loss of consciousness, as measured by injury-induced apnoea and delayed righting, in the absence of overt macroscopic change. At the cellular level there was no significant loss of YFP+ neurons, axons or dendrites or dendritic spines and no evidence of neuronal atrophy by 7 days post injury. Moreover, the dose of epothilone D was well tolerated and there were no overt indications of ill health or drug-induced cellular degeneration.
Dendritic spine loss is a characteristic feature of a variety of diseases and injuries of the nervous system (Fiala et al., 2002a; Campbell et al., 2012). Moreover, synapse loss is a likely key candidate for the emergence of functional deficits following TBI. Correct functioning of the neuronal cytoskeleton is crucial for the maintenance of neuronal integrity. Actin has a well-established and integral role in the function of dendritic spines (Matus, 2000; Hotulainen and Hoogenraad, 2010; Lei et al., 2016). More recently, microtubules have been implicated in spine formation and maintenance (Gu et al., 2008; Jaworski et al., 2009; Kapitein et al., 2010; Dent, 2016). To determine the effect of microtubule stabilization at the level of dendritic spines, and to avoid any potential confounding effects of the craniotomy, we examined spines on the radially projecting branches, residing in layer 4/5, of layer 5 YFP+ cortical projection neuron apical dendrites. Interestingly, while filopodia remained unaltered in terms of both length and density, epothilone D had dramatic effects on the spine population, causing an overall reduction in spine length, a shift to a greater proportion of short spines, and an increase in the formation of mushroom spines. The increase in spine density, specifically of mushroom spines, was an unanticipated response to epothilone D treatment and was observed both within the brain-injured and sham-operated cortex. This may indicate a more generalized effect of microtubule stabilization at the subtle level of the dendritic spine, rather than a neuroprotective effect of the drug evoked by injury. It is notable that the increase in spine density occurred specifically in the mushroom spines, which have previously been shown to be a relatively stable population likely to host synapses (Hering and Sheng, 2001; Fiala et al., 2002b). Although we were unable to trace axons to determine innervation interactions between degenerating axons and newly formed spines or determine the functionality of the newly formed spines using the current methodology, future studies quantifying colocalization of synaptic markers, ultrastructural and electorphysiological analysis as well as in vivo imaging of synaptic turnover could be used to determine the presense/absence and functionality of synapses on newly formed spines and validate the therapeutic potential of epothilone D.
Although the current study did not directly address the mechanism by which epothilone D increased spine density, it is plausibile that this may be due to a direct effect on the spines themselves, the axons innervating the spines or a more indirect effect. Nonetheless, in terms of therapeutic potential, microtubule stabilization may be useful for preserving spine and synapse integrity following injury. In accordance with this, we revealed a distinct increase in spine density following epothilone treatment. Moreover, spine restoration has been demonstrated following epothilone D treatment in an Alzheimer’s disease model (Penazzi et al., 2016). Although further investigations into the mechanisms of spine generation and maintenance by epothilone D are required, we postulate that microtubule stabilization and polymerization with epothilone D may prevent microtubule disassembly and catastrophe within both the dendrite shaft and spines, preserving spine integrity and reducing spine loss. If spine generation continues, this may contribute to an overall increase in spine density. Whether this is a detrimental or beneficial response in the aftermath of mTBI remains to be elucidated. Interestingly, since changes in spine morphology have been linked to alterations in synaptic strength and implicated in learning and memory (Bourne and Harris, 2007; Gu et al., 2008; Jaworski et al., 2009), manipulation of microtubule dynamics could potentially be used to modulate these parameters. As the epothilone D induced increase in spine density was unanticipated, based on previous literature future studies should determine whether these alterations evoke measureable functional responses in terms of electrophysiological and functional output.
Mild TBI has been described as a progressive disorder. Although we did not see a significant loss of axons, dendrites or spines by 7 days post-injury in the current study, there was certainly observable axonal damage throughout the ipsilateral cortex and white matter. Interestingly, the epothilone D treatment regime used in the current study did not have a protective effect on degenerating axons and our previous study using a mouse model of amyotrophic lateral sclerosis showed chronic epothilone D exposure may be detrimental to axons (Clark et al., 2018). However, using an in vitro model of structural axonal injury we have previously shown that low dose epothilone D has a protective effect on injured axons by increasing injury-induced axonal sprouting (Brizuela et al., 2015), indicating that microtubule stabilization within damaged axons may be partially responsible for the previously described neuroprotective effect of epothilone D and may have feed forward effects with regard to dendritic spine innervation. If followed to later post-injury time points, it is possible we may have observed evidence of neuronal atrophy, progressive axonal degeneration and spine loss following mild lateral FPI, as has been shown in other studies of brain injury (Fiala et al., 2002a; Chen et al., 2010; Gao and Chen, 2011; Gao et al., 2011; Greer et al., 2011; Campbell et al., 2012; Johnson et al., 2012b; Winston et al., 2013) and the previously described therapeutic effects of epothilone D may have been revealed. In our hands, and in accordance with previous studies, mild lateral FPI generated widespread traumatic axonal injury, which was particularly evident in the external and internal capsule white matter tracts (Smith and Meaney, 2000; Spain et al., 2010; Wang and Ma, 2010; Ekmark-Lewen et al., 2013; Smith et al., 2013; Hill et al., 2016). This included both beaded, degenerating axons and degenerated, dystrophic axonal fragments/bulbs. Both phenotypes have been reported in the literature and may represent stages of degeneration, differences in vulnerability between different axonal sub-classes or brain regions, or be specific to the mechanical forces sustained during injury (Reeves et al., 2005; Browne et al., 2011; Hånell et al., 2014).
Interestingly, axonal sprouting has been observed in certain classes of damaged axons in experimental models of brain injury (Salin et al., 1995; Batchelor et al., 2002; Deller et al., 2006; Dickson et al., 2007; Blizzard et al., 2011; Greer et al., 2011). Although not observed in the current study, it is possible that epothilone D could be used to modulate this response. Indeed, using an in vitro model, we have shown that epothilone D has dose-dependent effects on regenerating cortical neurons (Brizuela et al., 2015). This concept has important implications for dose-dependent modulation of axonal sprouting in vivo, for example enhancing adaptive regenerative attempts or dampening maladaptive sprouting, which may underlie the development of epileptic activity in the aftermath of injury (Santhakumar et al., 2001; Kharatishvili et al., 2006; Bolkvadze and Pitkänen, 2012).
In response to FPI we observed mild ipsilateral astroglial activation in the absence of glial scar formation. Contrary to previous studies, astroglial activation was not influenced by microtubule stabilization in the current study (Hellal et al., 2011; Popovich et al., 2014; Ruschel et al., 2015). This may have been due to a range of factors including the type of agent used (a taxane versus an epothilone), the concentration or timing of the epothilone D dose, the mild nature of the glial activation, or the context of the injury (for example, brain versus spinal cord). The absence of an observable injury-induced alteration in dendritic spine density in the current study may have been due to the spine population investigated, the proximity of the spines to the site of impact, the brain region assessed and its proximity to the injury, as well as the time point at which analysis was performed. Therefore, to elucidate the efficacy of microtubule stabilization as a therapeutic intervention for mTBI further investigations will be required to reveal the full repertoire of effects of microtubule stabilization in both the acute and chronic phases of the injury response. Future studies will use a range of dose regimes, including various concentrations and times of administration throughout the post-injury sequalae, to capture the dynamic aspects of injury-induced degeneration and remodeling.
In summary, our findings indicate that peripherally administered microtubule-stabilizing drugs alter synaptic plasticity at the level of the dendritic spine. This has important implications for controlling neuroplasticity in the aftermath of brain injury. More generally, microtubule-stabilizing agents may be useful for manipulating various aspects of the neuro-glial response to injury and disease, as well as providing a modulatory tool for investigating microtubule dynamics per se. Due to the ubiquity of microtubules it will be imperative to consider the full repertoire of roles in which they are involved, and take into consideration the interplay between intrinsic factors such as neuronal class and age and extrinsic factors such as the type and severity of injury.