Experimental Neurology
Mechanically-induced membrane poration causes axonal beading and localized cytoskeletal damage
Devrim Kilinc (a), Gianluca Gallo (b) and Kenneth A. Barbee (a)
(a) School of Biomedical Engineering, Science, and Health Systems, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104, USA
(b) Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA
Received 16 November 2007;
revised 14 April 2008;
accepted 20 April 2008.
Available online 3 May 2008.
doi:10.1016/j.expneurol.2008.04.025
Abstract
Diffuse axonal injury (DAI), a major component of traumatic brain injury, is a manifestation of microstructural cellular trauma and various ensuing neurochemical reactions that leads to secondary neuronal death. DAI is suggested to result from the initial increase in the membrane permeability caused by the mechanical forces acting on the axons. Permeability increases disturb ion balance and lead to cytoskeletal disruption resulting in the impairment of axonal transport. We present an in vitro model that reproduces important features of in vivo DAI such as membrane permeability changes, focal disruption of microtubules, impaired axonal transport, and focal accumulation of organelles. We induced fluid shear stress injury (FSSI) on cultured primary chick forebrain neurons and characterized the resulting structural and morphological changes. In addition, we tested the effect of Poloxamer 188 (P188), a tri-block co-polymer that is known to promote resealing membrane pores. We found that FSSI induces mechanoporation that leads to axonal bead formation, the “hallmark” morphology of DAI. Beads contained accumulated mitochondria and co-localized with focal microtubule disruptions, also a characteristic of DAI. Post-injury P188 treatment prevented FSSI-induced membrane permeability changes and reduced axonal beading to control levels. These results indicate that acute mechanoporation of axons in response to injury is a necessary condition for subsequent axonal pathology, suggesting that membrane integrity is a potential target for therapeutic interventions. P188 provides neuroprotection via resealing the plasma membrane following injury and prevents focal disruption of microtubules and axonal bead formation.
Keywords: Diffuse axonal injury; Mechanoporation; Neuroprotection; Poloxamer 188; Axonal bead formation; Fluid shear stress injury; Plasma membrane
Introduction
Traumatic brain injury (TBI) is a serious health concern constituting the majority of fatal injuries and may lead to serious neurological dysfunction in the case of survival (Schouten, 2007). Each year in the United States alone, more than 1.4 million people experience TBI, of which about 50,000 die and about 90,000 suffer from permanent impairment. There currently is no clinically proven therapy for TBI (Wang et al., 2006). The diffuse form of TBI, diffuse axonal injury (DAI), displays widespread damage in the white matter and has been suggested to result from inertial forces applied on the head (Maxwell et al., 1993; Gennarelli et al., 1998). DAI is the manifestation of microstructural cellular trauma, and various ensuing neurochemical reactions, and is accompanied by distinct morphological changes that evolve with time (McIntosh et al., 1996). Focal axonal swellings, also termed axonal beads, are the morphological hallmarks of DAI pathology (Povlishock and Christman, 1995). Axonal beading leads to secondary axotomy resulting in the disconnection of neurons from target tissues resulting in cell death (Strich, 1961; Gaetz, 2004). It has been suggested that DAI has a mechanical pathogenesis that is initiated at the time of injury (Adams et al., 1982). Understanding the neurobiological mechanisms by which the initial cellular damage leads to the clinical presentation of DAI is crucial for identifying potential therapeutic targets for in vivo intervention.DAI is suggested to result from mechanoporation, the generation of transient membrane pores due to mechanical deformation (Gennarelli, 1996; Buki and Povlishock, 2006). Membrane permeability increase has been demonstrated by influx of normally excluded molecules (Farkas et al., 2006; Geddes et al., 2003; Koob et al., 2005; Pettus et al., 1994; Povlishock et al., 1997; Stone et al., 2004; Whalen et al., 2007) and by efflux of normally confined molecules (LaPlaca et al., 1997; Serbest et al., 2005) through the membrane. In parallel with the increase in permeability, intracellular calcium ion concentration ([Ca2+]i) rises sharply following injury (Fineman et al., 1993; Cargill and Thibault, 1996; LaPlaca et al., 1997; LaPlaca an Thibault, 1998; Geddes-Kline et al., 2006). Permeability increases as well as peak [Ca2+]i depended on injury severity and loading rate (Geddes and Cargill, 2001; Geddes et al., 2003), suggesting a direct mechanical effect of the trauma causing membrane failure (Barbee, 2005). Calcium activates a variety of intracellular pathways and is therefore considered as a key factor in the neuropathology of TBI. Calpains, calcium-activated neutral proteases, are found in the neuronal cytoplasm and can degrade several cytoskeletal proteins including tubulin (reviewed in Gaetz, 2004). Though this sequence of events has not been definitively established, therapeutic interventions that target the initial mechanoporation of axons during injury could potentially prevent or mitigate the resulting calcium-activated cytoskeletal disruption.
Axonal beads are reflective of the accumulation of proteins and membrane-bound organelles, such as mitochondria, at multiple distinct locations along the axon due to impaired axonal transport (Maxwell, 1996; Maxwell and Graham, 1997). Immunostaining of amyloid precursor protein, which is known to undergo fast axonal transport, localizes to axonal swellings in traumatically injured human brain (Blumbergs et al., 1994). Axonal transport is conducted through motor protein driven movement of vesicles and organelles along axonal microtubules (Coleman, 2005). Therefore, a direct relationship between axonal microtubule loss and bead formation is suspected. After demonstrating the loss of microtubules at the nodes of Ranvier and later at the axonal swellings in the mechanically injured neurons, Maxwell (1996) speculated that Ca2+ influx and corresponding calpain activity could be responsible for changes in the cytoskeletal structure. Sites of high microtubule loss correlated with the sites of axonal swellings, as determined by ultrastructural studies (Maxwell and Graham, 1997).
The importance of mechanoporation for the subsequent development of axonal pathology has not been definitively established because of the experimental difficulty in demonstrating a causal relationship between membrane damage and the changes in cytoskeletal structure and axonal transport. Thus, identification of potential therapeutic agents to block the initial increase in membrane permeability provides a tool for demonstrating the role of mechanoporation in DAI and may lead to the development of a new treatment strategy for DAI. Poloxamer 188 (P188) is a nontoxic, nonionic, tri-block amphiphilic co-polymer (MW: ~ 8400) consisting of a central hydrophobic polyoxypropylene molecule that is flanked on both sides by two hydrophilic chains of polyoxyethylene. Having various clinical applications as a surfactant, P188 has been shown to be capable of sealing damaged cell membranes (Mascarinec et al., 2005). Two mechanisms have been suggested to explain how P188 promotes resealing: (1) by lowering the surface tension of the cell membrane (Sharma et al., 1996); and (2) by inserting into the bilayer (Baekmark et al., 1997). P188 has been shown to reseal electroporated (Lee et al., 1992), irradiated (Hannig and Lee, 2000), and thermally damaged (Padanilam et al., 1994) skeletal muscle cell membranes. Using hippocampal and cortical neuron cultures, P188 was shown to save neurons from excitotoxic or oxidative stress-related necrosis and from electroporation by inserting into the membrane and blocking membrane peroxidation (Marks et al., 2001). We previously demonstrated the ability of P188 to protect neuronal cells from trauma-induced necrotic and apoptotic death via membrane repair (Serbest et al., 2005; Serbest et al., 2006). The ability of P188 to seal permeabilized cell membranes has also been tested using in vivo models. Subcutaneous injection of P188 6 h after spinal cord compression injury to guinea pig promoted functional recovery (Borgens et al., 2004). In dystrophic mouse heart, P188 inhibited Ca2+ overloading due stretch-induced loss in membrane integrity and thereby blocked acute cardiac failure in response to stress (Yasuda et al., 2005). In excitotoxic injury to rat brain, P188 reduced neuronal loss when delivered immediately after the toxin (Curry et al., 2004). Collectively, these studies show the ability of P188 to repair damaged cell membranes and to rescue neurons following a variety of insults.
In this paper, we utilize a previously described in vitro neural injury model in which a fluid shear stress impulse causes mechanical injury of primary neurons (Kilinc et al., 2007). We now report that injured neurons recapitulate the hallmarks of in vivo DAI such as increase in membrane permeability, localized microtubule disruption, organelle accumulation and axonal bead formation. We also show that post-injury application of P188 reduces axonal beading by maintaining membrane integrity. These findings suggest that blocking increases in membrane permeability induced by injury may have therapeutic value in the treatment of TBI.
Materials and methods
Cell culture and reagents
Embryonic day 8 chick forebrain neurons were harvested, dissociated and plated on German glass coverslips (Bellco Glass, Vineland, NJ) at a concentration of 1.5 × 104 cells cm− 2 (Heidemann et al., 2003). Chick forebrain neurons are very similar to cultured mammalian hippocampal neurons and are commonly used as a cell model system to study axonal neurobiology (Heidemann et al., 2003). Coverslips were coated with 0.1 mg/ml poly-dl-lysine (Sigma, St. Louis, MO) in sodium borate buffer and rinsed 3× with phosphate buffered saline (PBS) (Mediatech, Herndon, VA). In experiments involving phase contrast microscopy and post hoc immunocytochemistry analysis, indexed coverslips (Bellco) were used to track individual neurons. Indexed coverslips were treated with 2-[2-(3-Trimethoxy-silyl-propyl-amino)-ethylamino]-ethylamine (Sigma) to promote attachment as described elsewhere (Geddes et al., 2003). Cultures were maintained in supplemented M199 medium (Invitrogen, Carlsbad, CA) and incubated (5% CO2; 37 °C) for 4–6 days before experimentation. Culture medium was changed every other day. HEPES buffered, supplemented Ham's F12 medium (Invitrogen) was used as experimental medium during live imaging experiments. The concentration of the microtubule depolymerizing drug vinblastine (Sigma) was 20 μM.
Experimental procedure
The controlled shear stress device is based on a cone-and-plate viscometer and applies uniform shear stress over the coverslip area through the controlled rotation of the cone (Blackman et al., 2000). Prior to the experiment, the coverslip is placed on the plate and held in position by vacuum suction. As the cone is slowly lowered towards the plate, the experiment medium fills the space between the cone and the plate. When used with high onset rates, the rotation of the cone induces fluid shear stress injury (FSSI) in the neural culture (Serbest et al., 2005). In our preliminary studies, we used shear impulse levels of 30, 45, 60, and 90 dyn cm− 2 and determined that a shear stress above 60 dyn cm− 2 caused significant detachment of the neurons from the substrate. 45 dyn cm− 2 with 20 ms onset time was used to induce FSSI since this level induced more pronounced axonal beading when compared to 30 dyn cm− 2. Sham (uninjured) controls underwent the exact protocol except without the cone rotation. A stage heater (NevTek, Burnsville, VA) maintained constant temperature (37 °C) during the experiment. Images were taken with an inverted Nikon Diaphot Eclipse TE300 microscope (Optical Apparatus, West Chester, PA). In experiments testing the effect of P188 treatment, P188 (Pluronic acid F-68, Sigma) was dissolved in the experimental medium and applied to neurons 5 min post-injury with a final concentration of 100 μM. 100 μM P188 was necessary and sufficient to ensure neuroprotection in mechanically-injured neuron-like PC2 cells (Serbest et al., 2005).
Measurement of axonal beading
Indexed coverslips allow tracing of individual neurons during the experiment, enabling us to image 7–8 neurons per coverslip. Phase contrast images were obtained before and 5, 20, and 60 min after the injury. To quantify axonal beading, individual beads that emerged following injury were counted and normalized by the length of the axon. To demonstrate that human influence in the counting process is negligible, we previously created a Matlab-based (MathWorks, Natick, MA) interactive image analysis program and showed that both manual and software-based methods produced similar results (Kilinc et al., 2007).
Visualization of cytoskeleton
Simultaneous fixation and extraction method was used to fix neurons at 20 and 60 min post-injury (Gallo and Letourneau, 1999). This method allows fixing microtubules while extracting free tubulin out of the axoplasm. Experimental cultures were fixed with 0.5% glutaraldehyde (Sigma) and 1% Triton X-100 (Sigma) in PHEM buffer (pH = 6.5) for 15 min at room temperature and stored in PBS at 4 °C until further processing. Fixed cultures were treated with 2 mg/ml sodium borohydride (Sigma) and stained to reveal tubulin with DM1A anti-tubulin (1:100, Sigma) and actin filaments with rhodamine phalloidin following the manufacturer's protocol (5:100, Invitrogen). Images were acquired using a Zeiss 200M microscope (Zeiss, Gottingen, Germany) and captured with AxioVision software (Zeiss). The variation in axonal microtubule mass along the axon, measured by tubulin staining intensity, was quantified with a Matlab-based, interactive image analysis program (Kilinc et al., 2007).
Staining of mitochondria
Neurons were stained with mitochondrion specific MitoTracker-Green dye (Invitrogen) to investigate whether axonal beads in vitro contained accumulated organelles as observed in in vivo beads. In separate experiments, neurons were exposed to 5 nM MitoTracker for 60 min before fluorescent imaging. Imaging was conducted at 30 and 60 min post-injury combining phase contrast and fluorescent microscopy.
Assessment of permeability
The plasma membrane is impermeable to Lucifer yellow ammonium salt (LY, MW = 480 Da, Invitrogen), enabling us to assess membrane permeability by determining the amount of LY trapped in the axoplasm following injury. In separate experiments, neurons were injured in LY-containing experimental medium (final concentration 20 μg/ml) at the same severity that successfully induced axonal beading and fixed with glutaraldehyde (5 μl/ml in PBS) 10 min after experimentation. Fixed cultures were first treated with LY-specific antibody (2:100, Invitrogen) followed by FITC-conjugated goat anti-rabbit (1:200; Sigma) as the secondary. Axoplasmic LY content was determined by averaging staining intensity along the axon and subtracting average background from this value.
Statistical analysis
In axonal beading and permeability measurements we analyzed 5–9 neurons per coverslip and used 3–8 coverslips per experimental condition. N for statistical calculations is the number of axons. Data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using single way analysis of variance (ANOVA), followed by Tukey's post hoc test to determine significance values between different experimental groups.
Results
Axonal beading gradually increases following injury
Fluid shear stress injury (FSSI) was applied to chick forebrain neurons at 4–6 days in vitro. Axonal bead formation was detected in injured neurons using live imaging (Fig. 1A). Axonal beads were usually present in low amounts in sham and incubator controls (0 min time point in Fig. 1A), likely reflecting packets of axonal transport observed in healthy neurons (Koenig et al., 1999). Bead formation started as early as 5 min post-injury throughout the length of the axon, whereas the majority of beads formed after 20 min. Images taken before injury and 5, 20 and 60 min after the injury were analyzed for quantifying bead formation. Axonal beading, expressed as the number of beads per 100 μm axon length, showed a significant increase over time for the injury group. Beading was significantly higher in injured neurons compared to sham controls at all time points (Fig. 1B).
 | Fig.
1. Mechanical injury results in axonal beading. A, Phase contrast
images of cultured chick forebrain neurons. Axonal beads (arrows)
formed on the injured axon during 60 minute post-injury, whereas
uninjured (sham) controls did not develop beads. Top row shows the
neurons at 1 h time point. Boxed regions are magnified 5×.
Bars = 20 μm. B, Graph of average axonal beading versus time. Dark and
light bars represent injury and sham control groups, respectively.
Beading is measured by the number of post-injury beads divided by axon
length. ANOVA is followed by Tukey's test. Number of axons analyzed is
displayed on bars. Error bars represent SEM.
|
Axonal beads contain membrane-bound organelles
In order to determine whether beads induced by FSSI are similar to axonal beads that form during DAI in vivo (Gaetz, 2004) we determined whether mitochondria accumulated in FSSI-induced axonal beads. Neurons were stained with MitoTracker Green, a mitochondrion-specific dye. MitoTracker revealed mitochondria distributed along axons of sham controls. However, in the injured neurons, mitochondria were concentrated at distinct locations that co-localized with axonal beads (Fig. 2).
 | Fig.
2. Axonal beads contain accumulated mitochondria. A,
Mitochondrion-specific dye MitoTracker Green-stained live cultures at
30 min post-injury. Control axons show a mixture of points and
elongated shapes, reflective of mitochondria, distributed throughout
the axon. In injured neurons, the mitochondria accumulate at axonal
beads. B, Boxed regions of panel A are magnified 2.5×. In the injured
axon the mitochondria localize to axonal beads (arrows). Bars = 10 μm.
|
Axonal beads co-localize with local cytoskeletal changes
Axonal transport of membrane-bound organelles is largely conducted over microtubules (Coleman, 2005). Accumulation of organelles at distinct locations along the axon during bead formation is likely to be indicative of an impairment of axonal transport. We investigated whether FSSI-induced axonal beading was related to changes in cytoskeletal organization by staining microtubules and actin filaments (F-actin) (Fig. 3). For these experiments, cultures were simultaneously fixed and extracted, an established protocol that removes soluble tubulin but preserves microtubules thereby allowing quantitative analysis of microtubule mass independent of soluble tubulin in axon (Gallo and Letourneau, 1999). In sham controls, microtubules exhibit the characteristic bundled appearance in the central shaft of the axon, whereas F-actin is found throughout the neuron (Fig. 3A). FSSI did not affect total microtubule levels during the post-injury period (data not shown; at 40 and 60 min post-injury; n = 15 neurons for injury, n = 21 neurons for sham; p = 0.49). However, examination of individual neurons that were fixed at 60 min post-injury revealed that the presence of beads was related to local decreases in axonal microtubule staining (Figs. 3C and E). Neurons that were fixed at 5 min post-injury did not exhibit focal microtubule disruptions except for occasional pre-existing bead locations. 85% of axonal beads co-localized with local microtubule discontinuities and this ratio was similar for uninjured (88%; n = 129 beads) and injured (88%; n = 118 beads) cultures. The observation that axonal beads form at sites along the axon that exhibit decreased microtubule content in both injured axons, and along control axons with naturally occurring beads, indicates that in these neurons bead formation correlates with the localized disruption of the microtubule cytoskeleton. We determined the effects of pharmacological microtubule depolymerization on axonal morphology by treating cultures with the microtubule depolymerizing drug vinblastine (20 μM) and imaging neurons before and 30 to 60 min following treatment. Vinblastine induced bead formation, indicating that decreases in microtubule mass results in axonal bead formation (Fig. 3D). In addition, sites of bead formation following FSSI correlated with increased levels of F-actin (Figs. 3B and E). These findings indicate that FSSI results in local disruption of microtubules, reorganization of the actin cytoskeleton, and co-localized axonal bead formation.
 | Fig.
3. Axonal cytoskeletal changes following mechanical injury. A, In sham
controls (60 minute post-experiment), microtubules exhibit the
characteristic bundled appearance, and actin filaments are found
throughout the axon. B, At 60 min following injury the regions of the
axon exhibiting beads contain decreased levels of microtubule staining
and F-actin accumulation (arrows). Only a subset of beads is labeled
for demonstration purposes. C, Boxed regions of panel B are magnified
3× and the microtubule image is pseudo-colored (bottom panel).
Microtubule staining intensity along the axon is not continuous and is
lower at locations where beads are present (arrowheads). D. Example of
axonal bead formation in neurons treated with the microtubule
depolymerizing drug vinblastine (20 μM). Before treatment the neuron
exhibits a normal axonal morphology characterized by a uniform
diameter. At 30 minute post treatment a few beads have become apparent
(arrows), while at 60 minute post treatment the axon exhibits numerous
beads (arrows). Only a subset of beads is labeled for demonstration
purposes. E, A high magnification image of an axonal bead formed in
response to mechanical injury. Microtubules appear typically bundled on
both sides of the bead. Disrupted microtubules and accumulated actin
filaments occupy the bead area. Bars = 20 μm (A, B, D) and 1 μm (E).
|
P188 restores membrane integrity and reduces axonal beading
An initial compromise in plasma membrane integrity due to trauma-induced mechanoporation is hypothesized to be a primary cause of DAI (Gaetz, 2004). However, this hypothesis has not been explicitly tested. We therefore sought to determine if FSSI induces mechanoporation and whether P188 would be able to block it. To detect changes in membrane permeability, we injured neurons in the presence of cell impermeable, aldehyde-fixable Lucifer yellow (LY) dye. Cells were fixed 10 min post-injury and immuno-stained with an antibody against LY to amplify the signal of internalized LY. A total of 172 neurons on 23 coverslips were analyzed. Injury groups had significantly higher cytoplasmic LY, compared to sham controls (Fig. 4), showing that FSSI increases membrane permeability.
 | Fig.
4. Injury-induced membrane permeability can be repaired by Poloxamer
188. A, Membrane permeability is higher in injured neurons than in
other groups as demonstrated by anti-Lucifer yellow staining (lower
panels). Bars = 20 μm. B, Membrane permeability, as quantified by
measuring the staining intensity of Lucifer yellow in axons 10 min
following injury. Lucifer yellow staining is higher in the injury group
compared to sham controls. Poloxamer 188 has no effect on sham controls
but reduces the permeability of injured neurons to control levels.
ANOVA is followed by Tukey's test. Number of axons analyzed is
displayed on bars. Error bars represent SEM.
|
We next tested whether P188 can block the increase in permeability. Neurons were injured in the presence of LY, while P188 was introduced at 5 min and the cultures were fixed at 10 min following injury. This timing of P188 treatment was chosen since most of the beads emerged later in the post-injury period (Fig 1B). Post-injury P188 treatment reduced internalization of LY by injured neurons (Fig. 4B), indicating a rapid reversal of the trauma-induced permeability increase.
In order to directly test the hypothesis that increased permeability is the cause of axonal beading, we investigated if P188 could block FSSI-induced axonal bead formation. Treatment with P188 blocked bead formation in response to FSSI (Fig 5A). In different experiments, a total of 145 neurons on 23 coverslips were analyzed. P188 treatment reduced the change in axonal beading to sham controls levels (Fig 5B). P188 treated neurons also maintained their microtubule structure and did not exhibit focal disruption (Fig. 5C). These results indicate that post-injury P188 treatment maintains membrane integrity and inhibits focal microtubule disruptions and axonal beading.
 | Fig.
5. Effect of post-injury Poloxamer 188 on axonal beading and
microtubule structure. A, Phase contrast images taken pre- and 60 min
post-injury show an increase in the number of beads (arrows) and
thinning of the axon caliber in the injured neurons compared to sham
controls and treated neurons. Bars = 20 μm. B, Beading is measured by
the number of post-injury beads divided by the axon length and is
higher in the injury group compared to sham controls. P188 has no
effect on sham controls but significantly reduces beading in the
injured neurons. ANOVA is followed by Tukey's test. Number of axons
analyzed is displayed on bars. Error bars represent SEM. C, Morphology
and microtubule structure of a P188-treated axon at 1 h post-injury.
P188 treatment prevented focal microtubule disruption and axonal bead
formation. Bar = 10 μm.
|
Discussion
The
mechanism of diffuse axonal injury (DAI) needs to be well understood
for the development of therapeutic interventions. Axonal beading has
been observed in postmortem human DAI (Adams et al., 1982; Blumbergs et al., 1994; Grady et al., 1993; Strich, 1961) and in in vivo injury models (Gennarelli et al., 1982; Maxwell et al., 1993; Maxwell, 1996; Saatman et al., 2003) making it the hallmark morphology of DAI. In vivo injury has been shown to increase membrane permeability and cause organelle accumulation in beads (Pettus et al., 1994; Gaetz, 2004). Our in vitro
injury model mimics this morphology by applying mechanical trauma on
cultured primary neurons. Therefore, our model provides a tool for
studying mechanically-induced DAI and for testing novel treatment
strategies of traumatic brain injury (TBI) at the cellular level.
Indeed, we demonstrate for the first time that preventing
injury-induced increase in membrane permeability blocks the formation
of axonal beads.
The controlled cell shearing device provides a uniform shear stress distribution over the entire coverslip (Blackman et al., 2000), reducing the complexity of the outcome due to heterogeneous loading. The mechanical loading conditions required to injure cells in culture depend on cell morphology and the strength of attachment to the substratum. In our earlier studies with neuron-like PC2 cells (Serbest et al., 2005; Serbest et al., 2006), we could apply a peak shear stress levels up to 100 dyn cm− 2. However, in the current study, a peak shear stress of 45 dyn cm− 2 was sufficient to cause injury while allowing the forebrain neurons to remain attached to the substrate during the injury. This value is similar to the peak shear stress applied to NT2-T cells in a parallel plate shear stress injury model, where axonal beading was reported at 24 h post-injury (LaPlaca et al., 1997). During TBI, the shear stress that is applied on the individual neuron is complex due to the spatial organization of the focal contacts with the extracellular matrix. Therefore, in vitro models aim to mimic the structural consequences of the injury, such as mechanoporation and axonal death, rather than mimicking the shear stress levels, per se.
Transport of membranous vesicles and organelles along axons is conducted via molecular motors associated with microtubules (Coleman, 2005). Therefore, post-traumatic impairment of axonal transport following TBI is likely related to changes in the axonal microtubule structure. Localized and rapid loss in microtubule mass, accompanied by organelle accumulation, has been shown at axonal beads along undisrupted axons following in vivo injury (Maxwell, 1996). Moreover, there was 70% loss in the number of microtubules in axonal swellings at 4 h post-injury (Maxwell and Graham, 1997). It is remarkable that our in vitro model mimics the localized disruption of the microtubule array observed in vivo; suggesting that mechanoporation of the plasma membrane is the primary cause of axonal bead formation in vivo and in vitro. Furthermore, we observed selective accumulation of organelles at sites of microtubule disruption, suggesting a direct link between deficits in the microtubule array and organelle accumulation.
Other elements of the axonal cytoskeleton are also susceptible to mechanically-induced injury. F-actin plays an important role in docking transported mitochondria at various sites along the axon (Hollenbeck and Saxton, 2005). We have shown the accumulation of F-actin at axonal beads suggesting that mitochondria might be trapped by the F-actin network at bead locations following their derailment from disrupted microtubule tracks. Neurofilaments, whose subunits have been shown to accumulate at axonal beads 6 h following traumatic injury to human brain (Christman et al., 1994; Grady et al., 1993]), could also be responsible from organelle accumulation at beads. However, no causal relationship has been detected between neurofilament disruption and organelle accumulation (Saatman et al., 2003). Furthermore, a recent study suggested that the axons that display neurofilament compaction were not the ones that undergo impaired axonal transport and subsequent axonal beading (Stone et al., 2001). Therefore, microtubules appear to be the main cytoskeletal elements whose disruption results in axonal transport impairment leading to organelle accumulation. Indeed we could induce axonal beading by globally applying microtubule-depolymerizing drug vinblastine.
The underlying mechanisms of heterogeneous microtubule disruption following traumatic injury are unknown. Membrane pump Ca2+-ATPase activity was shown to decrease heterogeneously following in vivo injury (Maxwell et al., 1995) suggesting non-uniform [Ca2+]i along the axon. Calpains, Ca2+-dependent non-specific proteases, are known to degrade tubulin, as well as other cytoskeletal proteins (Gaetz, 2004). Calpains might be heterogeneously activated in response to non-uniform [Ca2+]i leading to local microtubule disruption. Alternatively, certain locations of the microtubule array might be more vulnerable to degradation due to differences in the local structure, resulting in local microtubule loss. Our in vitro model is well suited for high temporal and spatial resolution cellular studies and will provide an excellent experimental system to investigate these issues in the future.
Traumatic injury increases membrane permeability (Farkas et al., 2006; Koob et al., 2005; LaPlaca et al., 1997; Pettus et al., 1994; Serbest et al., 2005; Whalen et al., 2007). The increase in permeability is acute and correlates with the loading rate and magnitude, as well as the size of the tracer (Geddes et al., 2003; ]Geddes-Kline et al., 2006; LaPlaca et al., 1997). We found that FSSI in our model also increased membrane permeability demonstrated by Lucifer yellow dye entry into the cytoplasm. Moreover, we show that the membrane sealant Poloxamer 188 (P188) blocks the increase in permeability, supporting the hypothesis that the increased permeability is due to nonspecific membrane wounding, or mechanoporation. The finding that P188 reduces axonal beading via reducing membrane permeability demonstrates that mechanoporation is the initiating event in the formation of axonal beads in the in vitro model system.
For a reagent to be useful in a therapeutic context, it has to be applied after the injury. In our in vitro model, majority of axonal beads form 20 min after the injury, restricting the application of P188 to the very beginning of the post-injury period. Hence, our timing of P188 treatment (5 min post-injury) is relevant for testing the therapeutic potential in such a fast progressing injury sequelae. Membrane permeability was shown to increase as early as 5 min after in vivo injury (Pettus et al., 1994) and to be more persistent compared to our post-injury period (Pettus and Povlishock, 1996; Singleton and Povlishock, 2004). In a rat model of diffuse TBI, membrane permeability was shown to persist for more than 4 h (Farkas et al., 2006). Furthermore, traumatic injury to the human brain (Grady et al., 1993) progresses remarkably slower when compared to experimental systems (see Gaetz, 2004 for a review), indicating a larger window of opportunity for therapeutic intervention. Repairing mechanically damaged cell membranes is a promising therapeutic approach in cases where the membrane disruption is the precipitating event that leads to cell death (see Barbee, 2005 for a review). Our in vitro results indicate that mechanoporation-induced cytoskeletal disruption and axonal beading can be blocked by repairing the cell membrane and suggest that P188 may be used in vivo to mitigate the effects of DAI.
In this study we applied mechanical trauma to primary central nervous system neurons and produced axonal bead formation and focal cytoskeletal damage, both hallmarks of DAI. Injury-induced mechanoporation could be blocked by post-injury application of membrane sealant P188, indicating a therapeutic effect via repair of membrane damage. Application of P188 also prevented focal microtubule disruption and axonal bead formation showing a strong causal relationship between membrane damage and subsequent axonal pathology. These findings establish the restoration of membrane integrity as a legitimate therapeutic target for acute intervention in DAI.
Acknowledgments
This research was supported in part by a grant from the State of Pennsylvania Tobacco Settlement Fund (K.A.B.); by the National Institutes Health (NS048090, G.G.); and by Drexel University Neuroengineering Major Research Initiative (K.A.B., D.K.). The authors thank Lee D. Silver for the technical help in vinblastine experiments. K.A. Barbee and G. Gallo share last authorship on this work.
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