Shear stress alters the expression of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) in Schwann cells

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Journal of Orthopaedic Research

Journal of Orthopaedic Research 23 (2005)1232-1239

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Shear stress alters the expression of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) in Schwann cells Ranjan Gupta Linh Truong a, David Bear a, Dara Chafik a, Edward Modafferi ’, Clark T. Hung Peripheral Nerve Research Lah, Department of Ortliopedic Surgery, University of California, Irvine, Med Sciences I Room B120. Irvine. CA 92697, United States Department of Anatomy and Neurohiology, Uniaersity of Californiu, Irvine, C A 92697, United States Cellular Engineering Lah, Department of Biomedical Engineering, Columbia University, New York, N Y 10027, United States a

Received 29 November 2004;accepted 22 December 2004

Abstract Schwann cells within a peripheral nerve respond robustly after a n axonal injury. Recent results have revealed that Schwann cells undergo concurrent proliferation and apoptosis after a chronic nerve injury that is independent of axonal pathology. Although the exact nature of the stimulus that produces this Schwann cell response remains unknown, we postulated that this response may be triggered directly by mechanical stimuli. Thus, we sought to determine how pure Schwann cells responded to a sustained shear stress in the form of laminar fluid flow by evaluating for proliferation, expression of S-100, myelin-associated glycoprotein (MAG), and myelin basic protein (MBP). Immunohistochemistry demonstrated that the Schwann cells were positive for S- 100, MAG, and MBP in greater than 99% of the experimental cells. Stimulated cells also revealed an increased rate of proliferation by as much as 100% (p < .001). The m R N A expression of MAG and MBP was down-regulated by 21% (p < .035) and 18% (p < .015), respectively, in experimental cells from RT-PCR assays. Furthermore, Western blot showed a down-regulation in M A G and MBP protein expression by 29% (p < .035) and 35% (p < .02), respectively. This study provides novel information regarding Schwann cell direct response to this physical stimulus that is not secondary to an axonal injury. 0 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Keywords: Schwann cell; Mechanical stimulation; Myelin; Myelin-associated glycoprotein (MAG); Myelin basic protein (MBP)

Introduction Mechanical perturbation of a peripheral nerve may produce an axonal injury which may result in axonal degeneration distal to the site of injury. This phenome* Corresponding author. Address: Peripheral Nerve Research Lab. Department of Orthopedic Surgery, University of California, Irvine, Med Sciences I Room B120, Irvine, CA 92697,United States. Tel.: + I 949 824 1405;fax: + I 949 824 1462. E-mail address: [email protected] (R. Gupta).

non activated by neurons is known as Wallerian degeneration and subsequently triggers a cascade of glial cell responses including marked Schwann cell proliferation. Although this secondary response by Schwann cells is well characterized, there remains limited information as to how Schwann cells would directly respond to mechanical stimulation alone. Salzer and Bunge have previously reported that Schwann cells may proliferate secondary to direct mechanical stimulation [36]. Data from an in vivo animal model of a chronic nerve compression (CNC) injury have supported the idea that

0736-0266/$ - see front matter 0 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

doi: 10.10 16/j.orthres.2004.12.010

R Gupta et ul. I Journal of' Orthopedic Rrseurch 23 (2005) 1232-1239

Schwann cells undergo concurrent Schwann cell proliferation and apoptosis relatively independent of axonal pathology [15]. The data indicate that Wallerian degeneration does not occur early with CNC injury, unlike acute crush or axonotomy injury models where axonal pathology initiates the cascade of events [13,14]. Rather, it appears as if the axonal pathology occurs secondarily to the Schwann cell changes. Since CNC injury has both an ischemic and direct mechanical effect [25], we sought to determine how Schwann cells responded to one form of mechanical stimulus alone. Therefore, we used an in vitro model in which pure Schwann cell cultures were subjected to shear stress in the form of laminar fluid flow to isolate the effects of this direct mechanical stimulation. Both myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) are key myelin specific protein constituents in the peripheral nervous system (PNS). Although MAG, a transmembrane protein of the immunoglobulin superfamily, is a minor constituent of the myelin sheath and represents approximately 0.1% of the total myelin protein in the PNS [28], MAG has been recognized for its bifunctionality whereby it plays a key role in the early stages of myelination as well as in the maintenance of stable axon-myelin interactions [7]. Furthermore, previous data support the central importance of MAG as a promyelinogenic marker in Schwann cells [32]. This protein has two isoforms, LMAG and S-MAG, which differ only by the c-terminal portion of their cytoplasmic domains [9,37]. Approximately 95% of the total MAG present in the human and rodent PNS exists as the S-MAG isoform [29,34]. MAG plays a critical role as an inhibitor of axonal regeneration and axonal sprouting in addition to serving as an integral cell adhesion molecule at the Schwann cell-axon interface [311. The myelin-specific protein, MBP, has been shown to be highly important in the myelination process. Existing in at least six functionally similar isoforms, MBP is essential for proper formation of myelin thickness and compactness in the CNS and PNS [28,311. Additionally, this pro-myelinating protein is also an important component of the major dense line (MDL) in the myelin sheath [28,31]. The purpose of this study was to evaluate how sustained shear stress directly affects Schwann cell function. By evaluating for the expression of the pan-specific Schwann cell marker S-100 with immunohistochemistry, we determined whether Schwann cells de-differentiated after mechanical stimulation. Schwann cell proliferation was explored with bromodeoxyuridine (BrdU) uptake assays to ascertain if direct mechanical stimulation in the form of laminar fluid flow is mitogenic for Schwann cells. Finally, we measured how sustained shear stress affects cell function by evaluating the mRNA and protein expression of MAG and MBP, two markers for pro-myelinating Schwann cells.

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Materials and methods Schwann cell culture

Sciatic nerves were harvested from 3-day-old neonatal Sprague Dawley rats using the modified Brockes technique [3]. Approval was obtained from the University's Institute Review Board (IRB). The tissue was digested with 0.25% trypsin (Sigma, St. Louis, Missouri) and expanded in D-MEM (Dulbecco's Modified Eagle's Medium) and Ham's F12 supplemented with 10% FBS (fetal bovine serum, Gibco, Grand Island, New York) and 1% ABAM, (antibiotic-anti-mycotic, Gibco BRL). To establish pure Schwann cell cultures and eliminate fibroblasts, the anti-mitotic agent, cytosine b-D arabinouranoside M, Sigma) was used and cells were sorted using rat anti-mouse Thy 1.2 antibody (lox concentrated supernatant of hybridoma30H12-a generous gift from the Salk Institute) and rabbit complement (Life Technologies, Grand Island, New York). Complete media consisting of D-MEMIF12 with 10% FBS, 1% ABAM, 10 nM heregulin and 5 pM forskolin (Calbiochem) was used to expand Schwann cell cultures. Heregulin and forskolin are mitotic agents that escalate Schwann cell proliferation from 7-10 days to 1-2 days. Immunohistochemistry confirmed that greater than 99% of the cells were positive for the pan-specific Schwann marker S-100. Direct mechanical stimulation

The previously described system consisted of Schwann cell-loaded Row chambers in parallel formation, a roller pump, and a complete media reservoir [1,12]. Schwann cells were plated on standard microscope slides with FlexiPERM silicone discs (Sartorius Group) at a cell density of 5000 cellsll.77 cm2 for the proliferation assays to allow counting of each individual Schwann cell [5]. Slides seeded with Schwann cells were then individually placed into custom-designed parallel-plate flow chambers and placed in the incubator. A computer-controlled (pulse-less) Ismatec 8-roller pump (Cole Palmer E-78001-22, Vernon, Illinois) regulated the laminar flow rate and time using the LabView software program (National Instruments). A complete media (DMEM F/12) reservoir was vented to a temperature-regulated incubator and connected via Tygon flexible tubing (Fisher) to the experimental Aow chambers and pump. Twelve chambers were loaded with Schwann cell-seeded slides and complete media. Six chambers were subjected to 2 h of laminar fluid Row at a flow rate of 35 mllmin, producing a chamber wall shear stress of 31.0 dynes/ cm2 or 3.1 Pa. Simultaneously, the remaining six chambers served as controls in the same environment and were subjected to exactly the same procedure as the experimental cells but without the flow. Of note, preliminary trials showed excellent Schwann cell adhesion to microscope slides in the absence of substrate after exposure to the amount and duration of shear stress used in our experiments [5]. Consequently, no substrate was used to seed slides during this study in order to minimize unnecessary confounding variables and to isolate as much as possible the direct, independent effect of shear stress on Schwann cell biological response. In vitro Schwann cell prolijerarion assay

After subjection to flow, slides were incubated at 37 "C with 5% COz in complete media for 12 h and supplemented with 10 pM bromodeoxyuridine (BrdU, Roche) for an additional 2 h after Row. The cultures were fixed in absolute methanol at 4 "C for 10min and permeabilized three times in I .25% Proteinase K in PBS for 5 min at a pH of 7.5. The slides were blocked for 1 h in a 4yo goat serum/ 0.25% Triton X-100 solution. Cells were immunostained with mouse anti-BrdU monoclonal antibody (1:100, Chemicon) for 1 h at room temperature and subsequently incubated in goat anti-mouse IgG Ruorescein isothiocyandte (F1TC)-labeled antibody ( 1 :200, Chemicon) for 1 h. DAPI (Vector) and cover slips were added and slides were visualized under a fluorescent microscope. Four random images within the circular Schwann cell-plated region on each slide were captured at a magnification of 1OX using a (Zeiss) fluorescent microscope-computer interface (Applied Imaging Pathyvision software). The total number of Schwann cells for each image was determined by counting the number

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of blue DAPI-counterstained nuclei with the total number of proliferating Schwann cells determined by counting the number of green BrdU-positive nuclei. The percent of proliferation was determined by the ratio of total BrdU-positive nuclei to total number of cells (DAPI-stained nuclei). Immunohistochemistry

After the flow, Schwann cells were allowed to recover for 12 h at 37 "C and fixed for 10 min with 4"/0 paraformaldehyde solution at room temperature. Cultures were blocked in 4% goat serum with 0.25% triton in PBS for 1 h. Slides were incubated at 4 "C overnight in blocking solution with either mouse anti-S100 protein monoclonal antibody (1:100, Chemicon), mouse anti-myelin basic protein (MBP) monoclonal antibody (1:100, Chemicon), or mouse anti-myelinassociated glycoprotein (MAG) primary antibodies (1 :50, Chemicon). Schwann cells were incubated in goat anti-mouse IgG FITC labeled secondary antibody (1:200, Chemicon) for 1 h at room temperature and subsequently counterstained with DAPI (Vector). Four random images within the circular Schwann cell-plated region on each slide were captured at a magnification of IOX using a (Zeiss) fluorescent microscopecomputer interface (Applied Imaging Pathyvision software). The number of cells that contained fluorescently labeled antibody (S100, MAG or MBP) was compared to the number of DAPI stained nuclei. Primer design

The computer program Primerselect (DNASTAR, Madison, Wisconsin) was used to identify potential primers for RT-PCR analysis. Several primer pairs were tested until one set for each gene was found to work consistently. The MAG primers (sense) 5'-CCCCACCCCGCGTCATITGT-3'. and (antisense) 5'-CCGCCCCCACCCCTACCACT-3' amplified a 350 bp product. The MBP primer sequences were obtained from a study by Itoh et al. [I9]-MBP (sense) 5'-ACTCACACACGAGAACTACCC-3'. and MBP (antisense) 5'-CCAGCTAAATCTGCTGAGGG-3', which amplified a 170 bp sequence. In addition, the GAPDH primer, which resulted in a 452 bp sequence, was used as an internal control (Clontech, Palo Alto, California). Semi-quantitative RT-PCR

To obtain adequate RNA for RT-PCR assays, Schwann cells were plated at a cell density of 100,000 cells/l .77 cm2. After flow, cells were incubated for 12 h at 37 "C to allow for gene transcription. Cells were then trypsinized, collected as pooled samples, and RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the protocol for isolation of total RNA from animal cells. RNA concentrations and purity were determined using a spectrophotometer. cDNA was prepared for experimental and control samples using 3 pg of RNA with Superscript I1 RNase reverse transcriptase (Invitrogen, CA) and specific primers for MAG, MBP and GAPDH. An RT- control, in which no Superscript I1 was added, was used to detect any genomic DNA contamination. Reaction mixtures were heated to 42 "C for 50 min and then 72 "C for 15 min to stop the reaction. The resulting cDNA product was then amplified in a PCR thermal cycler (RoboCycler Gradient 96, Stratagene) using Faststart Taq DNA polymerase (Roche). MBP DNA was amplified at a 62°C annealing temperature for 30 cycles, while MAG DNA was amplified at a 72 "C annealing temperature for 25 cycles. Each cycle consisted of 45 s of denaturation at 95 "C, I min of annealing and 90 s of elongation at 72 "C. A negative control in which no DNA was added in the PCR reaction was used to control for external DNA contamination. PCR products were electrophoresed on 1.5% agarose gel containing 1X TBE buffer and subsequently stained with Syber Green (Molecular Probes, Eugene, OR). A picture of the gel was taken with a digital camera (Sony DSC-S75 Cybershot) and ImageQuant analysis software (Molecular Dynamics, Sunnyvale, CA) was used to determine the densities of the MAG and MBP bands as compared to the GAPDH internal control for both experimental and control samples.

Western blot analysis

Flow experiments were performed at a cell density of 200,000 cells/ 1.77 cmz for Western blot. After flow, Schwann cells were incubated for 24 h at 37 "C to allow for protein synthesis. Seeded cells were lysed using radioimmunoprecipation assay (RIPA) buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCI, 0.01 M sodium phosphate, 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodium vanadate, 1 mM PMSF, lOpg/ml leupeptin, IOpglml aprotonin, and 2 pglml pepstatin A) and centrifuged at 12,000 rpm for 5 min. The supernatant was removed and analyzed for content and purity with mass spectrophotometry. Extracts were loaded onto a 7.5% TrisHCI gel for MAG and a 15% TrisHCl gel for MBP and ran at 120 V for 1.5 h. Proteins were transferred to a PDVF membrane at 60 V for 2 h and incubated in either mouse anti-myelin-associated glycoprotein (MAG) monoclonal antibody (1:200, Chemicon) or rabbit anti-myelin basic protein (MBP) polyclonal antibody (1:200, Chemicon) at 4 "C overnight. Blots were incubated in biotin-conjugated secondaries the following day and proteins were visualized using Vectastain ABC and Vector VIP Substrate Kit (Vector Laboratories, Burlingame, CA) following the manufacturer's instructions. A digital picture was taken using the Sony DSC-S75 Cybershot and band intensities were determined using ImageQuunt analysis software (Molecular Dynamics, Sunnyvale, CA). Stutisticul analysiJ

Data from the proliferation assays, RT-PCR and Western blot experiments were analyzed for statistical significance using the Student's paired r-test. Significancebetween control and experimental values was determined with a set significance value of p < .05.

Results Proliferation assay

Control Schwann cells stained positive for the panspecific Schwann cell marker S-100 in greater than 99% of the evaluated cells. Schwann cells subjected to mechanical stimulation in the form of fluid-induced shear stress for a period of 2 h also retained the expression of S-100 (data not shown). Furthermore, Schwann cells that were subjected to this mechanical stress consistently demonstrated an increase in cell proliferation. Fig. 1 shows a fluorescent microscope picture demonstrating the increase in the percent of BrdU-positive cells in experimental and control cells. There was a statistically significant difference between experimental and control proliferation levels for all groups when the baseline (control) levels of BrdU positive cells were 9.5% (p < .01 l), 11.9% (p < .049), 13.8% (p < .001), 23.2% (p < .005), 24.7% (p < .005) and 26.1% (p C .001) (Fig. 2). The percent increase in proliferation varied depending upon the baseline levels of BrdU uptake (Fig. 2). For example, when the baseline level of proliferation was 13.9%, there was a 100%increase in proliferation in experimental cells (p < .001). Yet when there was a 26% baseline proliferation level, the experimental cells exhibited a 50% increase in proliferation (p < .001). It is possible that the higher levels of baseline proliferation may mask any measurable stress-induced increase in the proliferation rate. The variation in baseline proliferation is not secondary to

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Experimental

Control

Fig. 1. Mechanical stress induces increased mitogenesis of in vitro cultured Schwann cells. Fluorescent images depicting the increase in the ratio of the number of BrdU stained nuclei (green) to DAPI stained nuclei (blue) in experimental or control cells. Note the increased number of BrdU positive cells in experimental cells versus the control cells. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

Immunohistochemistry Immunostaining for MAG and MBP, both specific markers for pro-myelinating Schwann cells, was positive in greater than %%o of the evaluated cells in both experimental and control slides. Additionally, the percent of positively stained cells was uniform for both the inner and outer areas of the circular plated region. Therefore, the data support the idea that shear stress in the form of laminar fluid flow does not change the phenotype of the stimulated Schwann cells.

Trials =Ol

RT-PCR

45

(B)

Baseline IBrdU Uptake

Fig. 2. Increase in proliferation of cultured Schwann cells in response to mechanical stress. Results are shown as percent of BrdU positive cells (A) and percent increase in Schwann cell proliferation as a function of baseline control BrdU uptake (B). It is evident that the increase in proliferation is dependent on the baseline BrdU uptake. Statistical significance levels of p < .05 are denoted by * in the graph.

changes in media or culture media additive. Rather, the data suggest that Schwann cells are more responsive to this mechanical stimulus at different times of their cell cycle. No statistical significance was observed between experimental and control groups when baseline levels of proliferation were greater than 26.1%.

Schwann cells that were subjected to sustained shear stress exhibited a decrease in MAG mRNA expression (Fig. 3). Specifically, the average ratio of MAG/ GAPDH RT-PCR products was 21% lower in cells that were subjected to flow compared to control cell levels. Similarly, the average ratio of MBP/GAPDH products decreased by 18% in the experimental samples (Fig. 4). Using the Student’s paired t-test, differences in MAG and MBP products for experimental and control groups were found to be statistically significant (p < .035, n = 5 and p < .015, n = 6, respectively). Reverse transcriptase controls with no reverse transcriptase enzyme confirmed that there was no genomic DNA contamination. Furthermore, the negative control for the PCR reaction demonstrated that there was no external DNA contamination. Western blot Western blot analysis demonstrated a downregulation of both MAG and MBP expression after exposure to shear stress (Fig. 5). The ratio of the

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Protein Expression of MAG and MBP o control

GAPDH

experimental

MAG

MAG

GAPDH

MBP

MAG

66 kDa

MBP

control

Fig. 3. Expression of MAG and MBP in cultured Schwann cells was evaluated by semi-quantitative RT-PCR. The left lane represents the control mRNA expression and the right lane corresponds to the experimental mRNA expression. The GAPDH in each lane served as an internal control. Negative controls (not shown here) did not have any detectable amplification products.

mRNA Expression of MAG and MBP

T

r

E

0.04

2

0.4

I

I

*

m

T

0 wntrol experimental

'a

;

0.2 ~~

MAG

21.5 kDa

MBP

experimental

MBP

Fig. 4. Results from RT-PCR analysis are expressed relative to GAPDH mRNA expression. Averages for MAG and MBP mRNA expression in control and experimental cultures with standard errors of the mean (SEM). There was a 21% decrease in MAG mRNA expression ('p < ,035)and an 18% decrease in MBP mRNA expression ('p < .015), by Schwann cells subjected to mechanical stress.

experimental MAG protein band to the control values indicated that MAG expression decreased 29%. Likewise, MBP was also down-regulated in experimental cells, decreasing 35% from control levels (Fig. 5). Statistical analysis indicated that differences in MAG and MBP protein expression for the control and experimental groups were statistically significant (p < .035, n = 3 and p < .02, n = 4, respectively). Discussion We have developed an in vivo animal model for CNC injury where a non-constrictive silastic tube (ID 1.33 mm) is placed around a rat sciatic nerve [13-151. Data from this model demonstrated that mechanical injury induces Schwann cell proliferation and concurrent

97.4 kcJa

c _

control

experimental

Fig. 5. Western blot results for MAG and MBP are expressed relative to control protein levels with standard errors of the mean (SEM). MAG was down-regulated 29% (*p < ,035) and MBP was downregulated 35% ('p < .02) in mechanically stimulated cells. The left lane represents the control band, while the right lane corresponds to the experimental protein band.

apoptosis as well as a significant increase in unmyelinated axonal sprouting and demyelination in the compressed nerve [14,15]. However, CNC injury did not alter axonal number and integrity despite the observed functional and morphological responses post-compression [ 151. As these responses occurred in the absence of axonal injury, we proposed that they may be Schwann cell mediated, independent of axonal pathology [15]. Therefore, we investigated Schwann cell function by evaluating the proliferative response as well as MAG and MBP expression after the exposure to a quantifiable mechanical stimulus in the form of shear stress. Proliferation assays indicated that mechanical stimulation in the form of a laminar fluid flow induced an increase in Schwann cell proliferation, indicating that mechanical stress is mitogenic for Schwann cells in vitro. This increase in proliferation provides parallel results with previous in vivo data, which proposes that Schwann cells may be mitogenic in response to direct mechanical injury [36]. Furthermore, this data lends credence to the possibility that a low-level mechanical stimulus may directly trigger Schwann cell proliferation. Although previous studies have shown that shear stress in the form of laminar fluid flow induces a significant cellular response from vascular endothelial cells, osteoblasts, chondrocytes, and fibroblasts [1,4,10,12,16, 17,26,40,41], little is known about Schwann cell response to direct mechanical stimulation. Previous work with human NTera-2 neurons has demonstrated that the magnitude and duration of shear stress has a direct effect on whether this stimulus has a positive or negative effect

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on cellular function [21-231. Yet, there are currently no data about the actual types and levels of the forces for the cells within the peripheral nerve. Although using similar values from studies on chondrocytes, endothelial cells, and osteoblasts would allow direct comparison between different cell lines, these values may not have any significance to neural tissue based upon the different physiologic demands of each tissue type. Thus, we chose the magnitude and duration of stimulation for these experiments based on our previous work which demonstrated that shear stress induced a biological response in Schwann cells [5]. In addition to causing an increase in proliferation, mechanical loading of in vitro cell cultures has previously been demonstrated to induce an alteration of cellular phenotype [2,20,27,33]. While an alteration in phenotype has been observed in cells such as chondrocytes [20], ligament precursor cells [2,27], and osteoblasts [33], little is known about the effects of mechanical stimulation on Schwann cell phenotype. Thus, our initial experiments were directed at determining whether shear stress in the form of laminar fluid flow induced an alteration of the Schwann cell phenotype. Positive immunostaining for S-100, both before and after flow, confirmed that Schwann cells do not dedifferentiate into another cell type following mechanical stress. Furthermore, the expression of the cell surface proteins, MAG and MBP, before and after mechanical stimulation also revealed that Schwann cells retain the ability to myelinate. Mechanical stimulation of cultured Schwann cells induced an alteration in cell function as demonstrated by a down-regulation in mRNA and protein expression of MAG and MBP, which is consistent with the demyelination that has been observed in our in vivo animal model for CNC injury [14]. Previous studies supporting our results have demonstrated the role of MAG in promoting myelinogenesis and the maintenance of myelin stability through axo-glial contact in the PNS [18,24,30]. Consistent with our data and providing additional support to our findings, Fruttiger observed myelin degeneration in MAG knockout mice [8]. In addition to the role of MAG in myelination, MAG has also been shown to be a neurite outgrowth inhibitor [28,31]. Furthermore, Tang demonstrated that migrating factors from damaged CNS tissue displayed an inhibitory effect on neurite outgrowth [39]. Rigorous analysis of EM and immunohistochemistry data from the CNC model revealed significant axonal sprouting at the periphery of the nerve at the early time points [14]. The mechanical stress-induced decrease in MAG expression, as demonstrated with this in vitro model, may create an environment that is permissive for axonal sprouting. Future experimentation is essential in order to provide a more accurate description of MAG in the CNC environment and explore its effects on axonal sprouting.

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The down-regulation of MBP mRNA and protein expression after sustained shear stress is consistent with findings from our CNC model [14,15]. Previous studies using the shiverer mutant mouse lesion with a deletion in part of the MBP gene have examined the role of MBP in the peripheral nervous system [11,35,38]. In these studies, peripheral nerves lacking MBP exhibited decreased myelin thickness, yet axonal integrity was maintained. Similarly, our animal model has demonstrated a significant decrease in myelin thickness after CNC injury, while the health of the axons was preserved [14]. As myelin basic protein plays a critical role in modulating primary myelination, myelin maintenance, and remyelination [6], it is possible that the observed altered MBP gene expression secondary to mechanical stimulation may in part be responsible for the myelin changes after chronic nerve injury. Furthermore, a study of MBP in the PNS showed that mice with the shiverer mutation exhibited an increased number of Schmidt-Lanterman incisures (SLI), which are cytoplasmic channels along the myelinated axon that facilitate axon-Schwann cell signaling [l 11. A decrease in MBP expression might lead to an increase in the number of SLI in the axons. Further investigation is necessary to determine if there is a significant increase in SLI after CNC injury as suggested by the data presented here. Schwann cell response mechanisms may be independent of the reciprocal regulation between the glial cells and neurons. Normally during development and axonal injury, this reciprocal relationship between the glial cells and neurons causes a response in the glial cells, occurring secondary to the neuron; however, data from our in vivo model would indicate otherwise. Schwann cells responded robustly in the absence of axonal injury, suggesting that the Schwann cell responses may be directly elicited by the physical stimuli rather than by the axon. There are several examples of myelin pathologies where axonal pathology occurs secondarily to myelin dysfunction, such as Type I Charcot-Marie tooth disease and multiple sclerosis. Our data indicate that shear stress directly induces an alteration in Schwann cell function as demonstrated by increased proliferation and down-regulation of two pro-myelinating proteins, MAG and MBP. These findings provide new information regarding the direct effects of mechanical stimulation in the form of laminar fluid flow on Schwann cell function. As it becomes increasingly evident that Schwann cells can respond to nerve injury independent of axonal pathology, this data yields new insight into the Schwann cell mediated response within the in vitro environment. By identifying Schwann cell changes resulting from mechanical stimulation, we may better appreciate and understand the pathogenesis of clinical neuropathies.

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Acknowledgement

This work was supported by the AIRCAST Orthopaedic Research Foundation (R.G.) and the NIH/ NINDS NS02221-04 (R.G.).

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