Biophysical Stimulation Induces Demyelination via an Integrin-Dependent Mechanism

Objective: Chronic nerve compression (CNC) injuries occur when peripheral nerves are subjected to sustained mechanical forces, with increasing evidence implicating Schwann cells as key mediators. Integrins, a family of transmembrane adhesion molecules that are capable of intracellular signaling, have been implicated in a variety of biological processes such as myelination and nerve regeneration. In this study, we seek to define the physical stimuli mediating demyelination and to determine whether integrin plays a role in the demyelinating response. Methods: We used a previously described in vitro model of CNC injury where myelinating neuron–Schwann cell cocultures were subjected to independent manipulations of hydrostatic pressure, hypoxia, and glucose deprivation in a custom bioreactor. We assessed whether demyelination increased in response to applied manipulation and determined whether integrin-associated signaling cascades are upregulated. Results: Biophysical stimulation of neural tissue induced demyelination and Schwann cell proliferation without neuronal or glial cytotoxicity or apoptosis. Although glucose deprivation and hypoxia independently had minor effects on myelin stability, together they potentiated the demyelinating effects of hydrostatic compression, and in combination, significantly destabilized myelin. Biophysical stimuli transiently increased phosphorylation of the integrin-associated tyrosine kinase Src within Schwann cells. Silencing this integrin signaling cascade blocked Src activation and prevented pressure-induced demyelination. Colocalization analysis indicated that Src is localized within Schwann cells. Interpretation: These results indicate that myelin is sensitive to CNC injury and support the novel concept that myelinating cocultures respond directly to mechanical loading via activating an integrin

C hronic nerve compression (CNC) injuries such as car- pal tunnel syndrome, cubital tunnel syndrome, and spinal nerve root stenosis occur at fibro-osseous nerve tunnels where direct abutment against rigid structures leads to sustained mechanical insults, causing pain, paresthesia, and paralysis.Traditionally, research on CNC injury has focused on the secondary effects of compression on peripheral nerve microanatomy.Examples include disruption of neuronal microcirculation, 1,2 induction of intracellular hydrostatic pressure gradients, 3 and alteration of cellular substructures. 2 Recent evidence indicates that cells can directly respond to physiological mechanical loading, [4][5][6][7][8] suggesting the existence of a molecular sensor capable of trans-lating mechanical stresses into cellular responses.][13] Here we developed a custom bioreactor, where purified myelinating Schwann cell (SC)-dorsal root ganglion cocultures can be subjected to stimuli known to be associated with CNC injury.Candidate stimuli include oxygen tension, glucose deprivation, and hydrostatic pressure that we collectively termed biophysical stimuli.This in vitro injury model allows the determination of the effects of these stimuli on cellular behavior, and enabled the identification of the reactive cell and the molecular signals mediating the cellular response.
SCs were extracted from sciatic nerves of 3-day-old SD neonatal rats and purified using the modified Brockes technique. 15Cultures were plated on poly-D-lysine-coated Petri dishes (BD Biosciences, San Jose, CA) and expanded in supplemented Dulbecco modified Eagle/F12 media (Millipore).

Bioreactor
The previously described bioreactor 16 was pressurized with a diaphragm pump while a small, regulated amount of air was allowed to escape (0.5 standard cubic feet per hour) to maintain continual gas exchange.Sensors were used to monitor internal pressure, pH, and dissolved oxygen.A negative feedback control system regulated the flow of nitrogen and oxygen into the incubator, maintaining constant dissolved oxygen in the media during pressurization.Cells were exposed to biophysical stimuli for 24 hours, and then maintained at normal culture conditions (37 C, 5% CO2, 20% O2, 0mmHg) for 24 hours prior to assessment of demyelination.This period of delay ensures that all demyelinating response proceeds to completion, thus avoiding the possibility of underestimating demyelination.We applied 21mmHg of hydrostatic pressure in compression studies.Unless specified, experiments were performed in normal culture medium containing a total of 20mM glucose in atmospheric oxygen ($18%).Hypoxia studies were performed at 5% oxygen, and glucose deprivation was achieved by reducing the glucose media concentration to a total of 0.5mM or 5.0mM.

Quantifying Labeled Cells
Labeled cells were viewed and photographed under epifluorescence on an Olympus (Tokyo, Japan) IX71 microscope.Using Visiopharm (Hørsholm, Denmark) imaging software, we used Stereology method to obtain a uniformly random and unbiased estimate of the total number of labeled cells per coverslip. 17In each case, a percentage is calculated via dividing the total number of affected cell (estimated via Stereological technique) by the total number of cells (also estimated via Stereological technique).In the case of demyelination, the percentage is calculated by dividing the estimated number of axons with fragmented myelin by the estimated total number of myelinating axons.To eliminate interobserver variations, the same investigator, blinded to the experimental groups, performed all the quantification analysis.

Electron Microscopy
Cocultures were fixed in 2% paraformaldehyde and 2% glutaraldehyde and subsequently postfixed with 1% osmium tetroxide.Fixed specimen were dehydrated by sequential incubation in solutions of increasing ethanol concentration, and embedded in Embed 812 (Electron Microscopy Sciences, Hatfield, PA).Specimens were then polymerized overnight at 60 C and sectioned at 60nm using Leica Ultracut UCT ultramicrotome (Leica Microsystems, Bannockburn, IL).Ultrathin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and viewed using a JEOL 1400 transmission electron microscope (Jeol, Peabody, MA).Images were captured using a Gatan Orius camera (Gatan, Pleasanton, CA).

Assessment of Proliferation, Cytotoxicity, and Apoptosis
SC proliferation was evaluated by nuclear incorporation of 5 0bromo-2 0 -deoxy-uridine (BrdU), which was detected using a commercially available BrdU detection kit (Roche Applied Science, Indianapolis, IN).To access cytotoxicity, culture media was assayed for lactate dehydrogenase (LDH) using a Cytotoxicity Cell Death Detection kit (Roche Applied Science) according to the manufacturer's specifications.To analyze apoptosis, cocultures were doublelabeled for neuron-or SC-specific markers (NeuN and S100, respectively) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) with an In Situ Cell Death Detection Kit per the manufacturer's instructions (Roche Applied Science).

Western Blotting
Lysate protein concentration was determined using a commercially available BCA Protein Assay kit (Thermo Scientific, Rockford, IL).Seventy-five to 100lg of protein was separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (0.2lm; BioRad, Hercules, CA), blocked with 5% dry skimmed milk, and incubated overnight at 4 C with primary antibodies.
For detection, goat antirabbit secondary antibody conjugated with horseradish peroxidase (HRP; 1:10,000 dilution, AP132P from Chemicon) or donkey antimouse secondary antibody conjugated with HRP (1:10,000 dilution, AP192P from Chemicon) was used.Blot development utilized Immobilon Western Chemiluminescent HRP Substrate (Millipore).Films were digitized with a color scanner, and images were analyzed using Quantity One image analysis software (BioRad).Quantitative results from Western blots were obtained after normalization of the densitometric data obtained from each antibody with that of b-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the same blot.
Colocalization was assessed for each image using the colocalization module of IMARIS 7.1.1(Bitplane AG, Zurich, Switzerland; 64-bit version), which uses an iterative threshold calculator that allows an impartial calculation of the intensities in each channel. 18Mander coefficient was used to assess a proportion of each protein colocalized within the other.Each image was automatically thresholded in the Colocalization module, and then a new colocalized channel was created that provided a Mander coefficient for each protein.

Statistical Analysis
Student t tests were used for planned comparisons of experimental and control samples.In all cases, p < 0.05 was considered statistically significant.Results are reported as mean 6 standard error of the mean.

Biophysical Stimuli Induce Demyelination in Myelinated Cocultures
Initial experiments focused on determining whether hydrostatic compression induced demyelination in myelinated cocultures.As the magnitude of pressure exerted on cells in compressive neuropathy is unknown, we loaded the cocultures with a pressure near the range of normal intracarpal pressure previously shown to induce a biologic response without detectable injury (21mmHg). 16or hypoxia studies, we employed 5% oxygen, an oxygen tension that is well established in models of anoxia.Glucose deprivation was achieved by lowering the glucose concentration of culture media from the normal growing concentration of 20mM to 0.5 or 5.0mM.

Biophysical Stimuli Do Not Induce Cell Damage in DRG Neuron/SC Cocultures
Demyelination is known to occur secondary to neuronal or SC death. 19,20To ensure that this secondary demyelination did not confound our data interpretation, we first characterized the integrity of demyelinated axons with high-resolution confocal microscopy.Although biophysical stimulation induced fragmentation and myelin ovoids, it had no appreciable effect on axonal structure, as indicated by intact neurofilament immunostaining (red signals in Fig 2).We also performed MAG staining 21 of cocultures to confirm the identity of MBPstained internodes.Although slightly punctate, MAG- stained internodes are identical in appearance to those stained with MBP (compare Fig 2A vs 2D).These results confirm that myelin is readily identified with our immunohistochemical technique and that demyelination is reliably and unambiguously visualized.
We further assessed cell damage in our cultures via two other independent measures:

Ultrastructural Characterization of Biophysical Stimuli-Induced Demyelination
To further characterize demyelination induced by biophysical stimuli, we analyzed the myelin and axonal structures of control and stimulated cocultures at the ultrastructural level.As a positive control for demyelination, a set of coculture was exposed to dichloroacetate (DCA) 22 for comparison.Electron micrographs showed that myelinated cocultures formed compact myelin, which is seen ensheathed around axons.Careful examination of control cocultures showed that compact myelin is directly, uniformly, and continuously apposed around each axonal internode (Fig 4).With DCA treatment, there is characteristic demyelination along with axonal disruption that is visible at the ultrastructural level, with segmentation and unraveling of compact myelin as well as the formation of inclusion bodies and vacuoles within the compact myelin.Additionally, disrupted myelin is no longer closely apposed to axonal internodes.DCAinduced demyelination is clearly related to axonal injury, with the disruption of neurofilament architecture within the axonal internode.In contrast, samples exposed to the biophysical stimuli produced similar pattern of myelin disruption without detectable axonal pathology.This significant ultrastructural difference suggests that biophysical stimuli-induced demyelination occurs independent of axonal damage.

Hydrostatic Compression Induces SC Proliferation
Interestingly, hydrostatic pressure stimulated SC proliferation, indicating possible phenotypic switch. 23This phenomenon is similar to our in vivo observation that SCs increase in number within the first 4 weeks of CNC injury. 24Following 24 hours of hydrostatic compression, there was a 2.6-fold increase in BrdU-labeled SCs compared to control (Fig 5C; control, 1.89 6 0.54%, n ¼ 16; compression, 4.93 6 1.28%, n ¼ 14; p ¼ 0.041).
Because a small amount of gas is allowed to escape from the compression chamber during hydrostatic compression, a possible confounding effect of the chamber experiment is an increase in the osmolarity of the culture medium secondary to evaporation.Therefore, BrdU analysis was repeated in the presence of supplemental albumin to simulate increased osmolarity.The result indicates no increased apoptosis with increased osmolarity (see Fig 5C ; high serum, 0.41 6 0.15%, n ¼ 4). .Note the disruption of compact myelin (small arrows) and vacuole formation within the compact myelin (arrowheads).Also note that myelin is no longer tightly apposed to the axon (labeled with asterisks).(C) Biophysical stimuli-induced demyelination occurred without disruption of axonal ultrastructure.Shown is a representative image of myelinating coculture subjected to 24 hours of compression and hypoxia.The uniform, compact myelin structure (arrows) has been disrupted with segmentation, unraveling, and vacuole formation (arrowheads).Note that normal axonal ultrastructure is maintained (labeled with asterisk).One Schwann cell nuclei (N) is visible.Scale bar 5 5lm.
In Vitro Injury Alters Phosphorylation of Integrin-Associated Signaling and Scaffolding Proteins Biophysical Stimulation produces SC proliferation and demyelination, but what molecular pathways mediate this effect?Given the known role of the integrin signaling cascade in mediating cell-cell interactions, we explored whether hydrostatic compression and hypoxia would alter the phosphorylation state of different components of the integrin signaling pathway.Myelinated cocultures were exposed to hydrostatic pressure and hypoxia for varied durations, and Western blot analysis was used to assess the phosphorylation states of nonreceptor tyrosine kinase Src, focal adhesion kinase (FAK), the prolinerich tyrosine kinase 2 (Pyk2), as well as paxillin (Pax), a docking and actin cross-linking protein.To test if the observed increase in Src phosphorylation is the result of integrin signaling, we treated cultures with integrin function-blocking antisera 25,26 during the application of biophysical stimuli.Because the great majority of known integrin dimers include an aV or b1 subunit, and because myelin is known to also express functionally important levels of the a6/b4 dimer, 10,27 we treated cocultures with a cocktail of antisera that block the function of the aV, b1, and b4 integrin subunits.By including all 3 function-blocking antibodies, integrin signaling of at least 15 dimers is abolished, as inactivation of 1 integrin subunit is known to inactivate the entire integrin dimer (illustrated in

Functional Blockade of Integrin Signaling Alleviates Stimulation-Induced Demyelination
Having observed that functional blockade of integrin signaling abrogated the increased phosphorylation of pSrc in response to biophysical stimuli, we asked whether abolishing integrin signaling would also block biophysical stimuli-induced demyelination.Myelinated cocultures were subjected to 24 hours of hydrostatic compression and hypoxia in the presence of the integrin-neutralizing antisera cocktail.Quantification of demyelination via MBP staining consistently indicated a significant diminution of pressure-induced demyelination in cultures treated with the antibody cocktail (Fig 7C).Stimulation of cocultures in normal medium resulted in 2.4% demyelination.After the introduction of function-blocking antibodies in the culture medium, the observed demyelination decreased to 1.5% (n ¼ 5; t test, p ¼ 0.042, Fig 7D).Thus, although the antibody cocktail completely abolished Src phosphorylation and did markedly decrease biophysical stimuli-induced demyelination, it did not completely block this phenomenon.

Biophysical Stimulation and Functional Blockade of Integrin Signaling Does Not Induce SC Degeneration as Determined by Oct-6 Expression
Prior to myelination, immature SCs transition from a promyelinating state into a myelinating state, a process requiring the precise coordination of several transcriptional factors.Oct-6 is a key SC transcription factor found to have particular importance in cultured SCs because it is involved in the regulation of the expression of the Krox20/Egr2 gene, the product of which plays a key role in the maintenance of myelin in the peripheral nervous system.Upregulation of Oct-6 results in increased expression of myelination-associated genes, FIGURE 6: Src phosphorylation increased transiently with biophysical stimuli via an integrin-dependent mechanism.For each experiment, 5 to 6 coculture coverslips were subjected to the indicated duration of hydrostatic compression and hypoxia and immediately removed from the bioreactor and placed into an ice-cooled culture plate.The cocultures were extracted using sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 50mM Tris, pH 8.0) containing phosphatase inhibitor cocktail (PhosSTOP; Roche Applied Science).Lysates of cocultures (50-75lg/lane) were separated on Western blot and probed with polyclonal antibodies (Abs) to phosphorylated versions of integrin-associated signaling molecules Src, Pax, FAK, and Pyk2.(A) Biophysical stimuli of myelinated cocultures increased phosphorylation of Src.In normal culture medium, phosphorylation at tyrosine 418 of Src was upregulated with increasing duration of compression and hypoxia up to 60 minutes but subsequently decreased to baseline (blue solid line).To verify that the observed signaling response was integrin mediated, the experiment was repeated in a cocktail of function-blocking integrin antibodies to determine the effect of silencing integrin signaling.In the presence of function-blocking integrin antibodies, the upregulation of Src phosphorylation was abolished (green dotted line; n 5 3 at each time point).(B) Representative Western blots of coculture lysates probed with antibody recognizing pSrc (60kDa); biophysical stimuli were applied with the cocultures in normal (2 blocking Ab) and in media containing function-blocking anti-integrin antibodies (1 blocking Ab); a representative blot was subsequently stripped and reprobed for total Src; the blots were also reprobed with antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (37kDa) as a protein loading control (GAPDH).(C) Biophysical stimuli had minimal effect on tyrosine phosphorylation of Pax, FAK, and Pyk2 (n 5 3) each.(D) Representative Western blots of coculture lysates probed with an antibody recognizing phosphopaxillin at tyrosine 31 (68kDa) show no significant change in the extent of tyrosine phosphorylation in response to biophysical stimulation; the blot was reprobed with actin (43kDa) as loading control.(E) A schematic diagram shows known integrin dimer combinations; because the blockade of 1 subunit leads to inactivation of the integrin dimer, functional blockade of aV, b1, and b4 (red circles) leads to inactivation of 15 integrin dimer combinations; modified from Milner and Campbell (2002). 27Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]whereas deletion of Oct-6 leads to a delay in the switch to the myelinating phenotype. 28aving characterized the biophysical stimuli involved in demyelination, we wondered whether the observed demyelination in our cocultures system is associated with changes in Oct-6 expression.Myelinating neuronal-SC cocultures were subjected to 24 hours of hydrostatic pressure and hypoxia in normal medium or in medium containing function-blocking integrin antibodies.Immunohistochemistry and confocal images were then obtained to determine differential expression of Oct-6.There was no detectable change in Oct-6 expression in response to hydrostatic compression or to integrin silencing (see Fig 7E-G).
Nonreceptor Tyrosine Kinase Src Colocalizes with SCs Given the data above implicating Src phosphorylation as a critical initiating step, it was of significant interest to determine whether the hydrostatic compression-induced upregulation of Src phosphorylation occurred in SCs.Cocultures that had been stimulated for 60 minutes were triply immunostained with antisera to phospho-Src (Tyr 418), the pan SC marker S100, and axonal marker neurofilament (Fig 8).Colocalization analyses were then performed using IMARIS software to determine whether the pSrc signals colocalized with S100 or neurofilament signals.Colocalization analysis yielded colocalization coefficient of 59.94 for pSrc with S100, and 25.79 for pSrc with neurofilament.Surface reconstructions of high-resolution confocal images revealed the presence of pSrc in SCs (see Fig 8C).Taken together, these results indicate that the activated secondary messengers are located within SCs, implicating SCs as a mediator of the observed injury response.

Discussion
0][31][32] Mechanical stimuli are potent mitogens for mechanosensitive cells such as fibroblasts and osteoblasts. 33,34Increasing evidence suggests that neural tissues are also directly mechanically sensitive.For example, SCs respond to fluid shear stress in part by proliferating and downregulating myelin-associated genes. 6We show here that applied hydrostatic pressure triggers SC proliferation and Three axons are seen with fragmented myelin and myelin ovoids (arrowheads).(D) Blocking integrin signaling alleviated stimulation-induced demyelination by an average of 47%.Although 1 trial was not quite statistically significant (p 5 0.058, t test, 1-tailed), repeated experiment yielded a statistically significant difference in mean value between the control and blockade groups (*p 5 0.042, t test, 1-tailed); error bars represent standard error of the mean.To determine whether Schwann cells undergo degeneration in response to hydrostatic compression, we assessed Oct-6 expression in control samples (E), compressed samples (F), and in samples compressed in the presence of function-blocking integrin antibodies (G).Analysis with high-power confocal microscopy showed no difference in the pattern or intensity of Oct-6 staining regardless of treatment.
demyelination in a way that is similar to what occurs in vivo despite the maintenance of normal oxygen tension, pH, and available nutrients.These findings provide further evidence that neural tissues can respond directly to pressure.Moreover, because only 2 cell types exist in the cocultures, mechanosensitivity must exist within the neurons, the SCs, or possibly the interface between them.
We observed demyelination without detectable changes in axonal structure when investigated under highresolution confocal microscopy or electron microscopy.
Similarly, there was no detectable cell lysis or apoptosis as assayed by LDH release and TUNEL analysis in response to hydrostatic compression, hypoxia, or glucose deprivation applied individually or in combinations that induced demyelination.Taken together, these results suggest that biophysical stimuli cause demyelination independent of neuronal or SC death.In contrast to our observations, a recent study showed that pure DRG cultures exhibit cell death in response to mechanical stimuli and hypoxia. 35owever, with those experiments, neuronal cultures were exposed to supraphysiologic loading conditions of an alternative mechanical stimulus (ie, 20% tensile strain).
We demonstrate that demyelination is more severe when hydrostatic compression occurs under conditions of hypoxia, a result suggesting that cellular metabolism plays an important role in the maintenance of myelin structure.Myelin formation entails high metabolic demand due to the need for SCs to synthesize large quantities of myelinrelated lipids and proteins.Although little is known about the energy requirements of mature myelin, its highly specialized membrane domains may render it sensitive to metabolic insults. 36Experimental studies using a rat model of focal glucose deprivation showed that cerebral white matter is highly vulnerable to metabolic disruption, with oligodendrocyte death preceding the appearance of necrotic neurons. 37Similarly, exposure of rats to elemental tellurium, which inhibits the biosynthesis of cholesterol, results in reversible demyelination due to a preferential affect on myelinating SCs. 38Furthermore, the glycolysis inhibitor dichloroacetate also induces reversible demyelination in neuron/SC cocultures. 22Together, these results suggest that altering the delicate metabolic balance of SCs may predispose myelin to destabilization.This stringent metabolic requirement may explain why diabetes, which predisposes patients to vasculopathy, also increases a patient's susceptibility to developing CNC injuries.
Our findings are in keeping with other evidence that integrins are capable of acting as mechanoreceptors in mechanosensitive cells such as fibroblasts, osteoblasts, and chondrocytes.Hydrostatic pressure is a unique form of mechanical stimulation in that it has little affect on cellular morphology and volume, yet physiologic levels of hydrostatic pressures have been noted to alter cellular activity [39][40][41] and cytoskeleton polymerization. 42,43Cs are known to play critical roles in regulating the maturation, maintenance, and viability of axons. 44,45Compelling evidence now indicates that SCs play pivotal roles in the pathogenesis of CNC injury, as the CNC-induced SC proliferative response precedes the characteristic demyelination and remyelination response. 24,46Mechanical loading of SCs alters the expression of genes that permit axonal sprouting, a response observed in later stages of chronic compression injury. 6,47Our results reported here, colocalizing SCs with the messenger protein Src activated in response to demyelinating biophysical stimuli, further implicate SCs as the prime mediator of CNC injury.
Here we show, using a novel in vitro model, that hydrostatic compression of neuronal tissue directly triggers demyelination and SC proliferation.This injury response is associated with the activation of Src phosphorylation in SCs; blocking this response abrogates cellular responses to compression injury.We show that hypoxia and glucose deprivation, the secondary effects attributable to compression-induced disruption of peripheral nerve microcirculation, potentiate the injury response but in isolation have little effect on myelin integrity.Our results implicate the integrin pathway as a key mediator of the cellular changes underlying compression neuropathy, and point to SCs as the key player mediating the pathogenesis of CNC injury.

FIGURE 1 :
FIGURE 1: Biophysical stimuli induced demyelination in myelinating neuron-Schwann cell cocultures.Established myelinating cocultures were subjected to 24 hours of indicated biophysical stimuli in the custom bioreactor and subsequently immunostained for myelin basic protein.(A) A composite image of 144 sampling fields shows the typical myelination pattern of cocultures; scale bar 5 1mm.(B) Twenty-four hours of hydrostatic compression and hypoxia produced demyelination, which is visible as fragmented myelin basic protein-positive myelin segments or myelin ovoids (arrows); scale bar 5 50lm.Demyelination was assessed visually using an unbiased random stereology method as described by Larsen, 17 and summarized in the plot.(C) Biophysical Stimuli induces demyelination.Hypoxia (Hypox) or Glucose deprivation alone (Low Gluc) (2nd and 3rd bars from left) has negligible effect.Hydrostatic compression alone (Cmprss) produced statistically significantly more demyelination (4th bar from left, t test: *p 5 0.036, 1-tailed).Hypoxia combined with glucose deprivation also produced more demyelination than each stimulus alone (5th bar from left, **p 5 0.0076, ***p 5 0.0045, 1tailed).Hypoxia and glucose deprivation both potentiated the effect of compression, leading to more demyelination compared to compression alone (6th and 7th bars from left, ^p 5 0.0348, ^^p 5 0.0049, 1-tailed).Further combinations of biophysical stimuli produced only incrementally more demyelination (right-most bar).Cntrl 5 control.

FIGURE 2 :
FIGURE 2: Demyelination occurred without axonal damage.To further characterize observed demyelination, control and stimulated samples were immunostained for myelin basic protein (MBP; green) and neurofilament (red) and subjected to high-power, high-resolution confocal microscopy.Representative images of normal (A), and demyelinated samples (B, C) are shown.In our culture system, demyelination occurs via fragmentation (B) and the formation of myelin ovoids (C).Myelin fragmentation and myelin ovoid formation (green) occurred without disruption of neurofilament continuity or morphology (red), indicating no axonal damage despite profound myelin disruption.To confirm the identity of stained myelin structures, a separate sample was immunostained for myelin-associated glycoprotein (MAG; green) and neurofilament (red; D).Although relatively punctate compared to MBP staining, the MAG-positive structure was identical in appearance compared to MBP-positive structures.

FIGURE 3 :
FIGURE 3: Biophysical stimuli are without cytotoxic or apoptotic effects on neuron-Schwann cell cocultures.Established cocultures subjected to indicated biophysical stimuli for 24 hours were analyzed for cytotoxicity via 2 independent methods; cell membrane integrity was assessed with released lactate dehydrogenase (LDH), and apoptosis activity was assessed by terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay.(A) Biophysical stimuli had negligible effect on cell membrane integrity.Released LDH was measured after 24 hours of exposure to biophysical stimuli.The released LDH was expressed as a percentage of maximal releasable LDH, which was obtained according to the following formula: % maximum LDH release 5 (experimental value 2 low control)/(high control 2 low control).The high control value was obtained by lysing cells using 2% Triton X-100, whereas the low control was the LDH present in normal cultures.Hypoxia (Hypox) or hydrostatic compression (Cmprss) individually produced negligible LDH release (2nd and 3rd bars from left).Combinations of biophysical stimuli produced paradoxically but statistically insignificantly less LDH in response to compression (4th, 5th, and 6th bars from left).(B) Biophysical stimuli produced negligible apoptosis activity.In a separate set of experiments, cocultures were compressed for 24 hours and subsequently double-stained for TUNEL and either Schwann cell marker S100 (left 2 bars) or neuronal marker NeuN (right 2 bars).In either case, there was negligible difference in TUNEL activity compared to control cultures, indicating no increased apoptosis in either Schwann cells or neurons.(C) A sample image of compressed coculture subsequently triple-labeled for TUNEL (green), neuronal marker NeuN (red), and Schwann cell nuclei (blue) is shown.There were no TUNEL-positive cells.Scale bar 5 50lm.Gluc 5 glucose.[Colorfigure can be viewed in the online issue, which is available at www.annalsofneurology.org.]

FIGURE 4 :
FIGURE 4: Biophysical stimuli induced demyelination without disruption of axonal ultrastructure.(A) A representative transmission electron microscopy image of control myelinating cocultures.Normal compact myelin (arrows) is seen uniformly and tightly wrapped around the internode of each axon (labeled with asterisks).Three Schwann cell nuclei (N) are visible.(B) A representative transmission electron microscope image of myelinating cocultures exposed to dichloroacetate (DCA) as a positive control.DCA-induced demyelination is associated with disruption of axonal ultrastructure (large arrows).Note the disruption of compact myelin (small arrows) and vacuole formation within the compact myelin (arrowheads).Also note that myelin is no longer tightly apposed to the axon (labeled with asterisks).(C) Biophysical stimuli-induced demyelination occurred without disruption of axonal ultrastructure.Shown is a representative image of myelinating coculture subjected to 24 hours of compression and hypoxia.The uniform, compact myelin structure (arrows) has been disrupted with segmentation, unraveling, and vacuole formation (arrowheads).Note that normal axonal ultrastructure is maintained (labeled with asterisk).One Schwann cell nuclei (N) is visible.Scale bar 5 5lm.
Biophysical stimulation increased levels of Src phosphorylated at tyrosine 418, with levels rising with increasing duration of the biophysical stimulus up to 60 minutes (Fig 6A, blue curve).No significant differences in the phosphorylation state of FAK, Pyk2, and Pax were detected (see Fig 6C; n ¼ 3 each).
Fig 6E).Application of the function blocking antisera cocktail completely abolished the increases in phospho-Src otherwise induced by hydrostatic compression and hypoxia (see Fig 6A, green curve).

FIGURE 5 :
FIGURE 5: Hydrostatic compression induced proliferation of Schwann cells.Myelinated neuron-Schwann cell cocultures were compressed for 24 hours, and subsequently exposed to the nucleotide analogue 5 0 -bromo-2 0 -deoxyuridine (BrdU) for 1.5 hours.Cultures were then triple-immunostained for S100 (red), BrdU (green), and Schwann cell nuclei (blue).(A) An example image of control coculture shows the baseline amount of BrdU incorporation into Schwann cell nuclei.Scale bar 5 50lm.(B) An example image of compressed coculture reveals a markedly increased number of BrdU-positive Schwann cell nuclei compared to control cocultures.(C) Summary plot showing percentage BrdU-positive nuclei in control cultures (Cntrl; left column), compressed cultures (Cmprss; middle column), and cultures exposed to high osmolarity (right column); asterisk indicates significant difference (t test, 1-tailed, p 5 0.041).

FIGURE 7 :
FIGURE 7: Functional blockade of integrin signaling alleviated hydrostatic pressure-induced demyelination.Myelinating neuron-Schwann cell cocultures were subjected to 24 hours of hydrostatic pressure and hypoxia without (2 Blocking Ab) and with (1 Blocking Ab) function-blocking integrin antibodies (n 5 5).Subsequent to stimulation, coculture coverslips were assessed for demyelination (see Materials and Methods).Representative fluorescent images of coverslips are shown for comparison.(A) A noncompressed sample is shown for baseline comparison.(B) Samples subjected to 24 hours of hydrostatic compression and hypoxia are shown.Six axons are seen with fragmented myelin (arrowheads).(C) Samples stimulated in the presence of integrin function-blocking antibodies had identical appearance but significantly less demyelination.Three axons are seen with fragmented myelin and myelin ovoids (arrowheads).(D) Blocking integrin signaling alleviated stimulation-induced demyelination by an average of 47%.Although 1 trial was not quite statistically significant (p 5 0.058, t test, 1-tailed), repeated experiment yielded a statistically significant difference in mean value between the control and blockade groups (*p 5 0.042, t test, 1-tailed); error bars represent standard error of the mean.To determine whether Schwann cells undergo degeneration in response to hydrostatic compression, we assessed Oct-6 expression in control samples (E), compressed samples (F), and in samples compressed in the presence of function-blocking integrin antibodies (G).Analysis with high-power confocal microscopy showed no difference in the pattern or intensity of Oct-6 staining regardless of treatment.

FIGURE 8 :
FIGURE 8: The nonreceptor tyrosine kinase Src colocalized with Schwann cells.Myelinated cocultures were subjected to hydrostatic compression and hypoxia for 60 minutes and triple-immunolabeled for S100, neurofilament, and pSrc.High-resolution confocal images were taken to determine whether pSrc colocalizes with Schwann cells or with neurons.(A) Transparent composite 3-dimensional (3D) Z-stack shows all 3 signals.S100 signal (green) shows the filamentous arrangement of Schwann cell cytoplasm wrapping around neurofilament signals (red).pSrc signals (yellow) are punctate, possibly reflecting the location of focal adhesions.The Schwann cell nuclei are DAPI labeled and are shown in blue.(B) Opaque 3D volumetric surface rendering overlay of inset in panel A is shown.Most of colocalized neurofilament and pSrc is now masked because it is located within the opaque reconstructed S100 surface.(C) Zoomed-in perspective shows an example of pSrc localization within a rendered 3D volumetric surface using IMARIS 3D visualization.Colocalized pSrc signals (yellow) are within the rendered Schwann cell surface (green) and outside of the rendered neurofilament surface (red).