AIM To investigate the directive importance of nanophysical properties on the morphological and protein expression responses of dibutyryladenosine cyclic monophosphate (dBcAMP)-treated cerebral cortical astrocytes in vitro. MATERIALS & METHODS Elasticity and work of adhesion characterizations of culture surfaces were performed using atomic force microscopy and combined with previous surface roughness and polarity results. The morphological and biochemical differentiation of dBcAMP-treated astrocytes cultured on promising nanofibrillar scaffolds and comparative culture surfaces were investigated by immunocytochemistry, colocalization, super resolution microscopy and atomic force microscopy. The dBcAMP-treated astrocyte responses were further compared with untreated astrocyte responses. RESULTS & CONCLUSION Nanofibrillar scaffold properties were shown to reduce immunoreactivity responses while poly-L-lysine-functionalized Aclar® (Ted Pella Inc., CA, USA) properties were shown to induce responses reminiscent of glial scar formation. The comparison study indicated that directive cues may differ in wound-healing versus quiescent situations.
Repair of traumatic injury to the CNS remains a challenging problem. One of the hurdles that must be overcome is the glial scar, which is established after traumatic injury in mammalian systems and creates a barrier to regeneration of axons [1, 2] . A glial scar consists mainly of reactive astrocytes and proteoglycans. Reactive astrocytes at a wound site undergo a morphological change, extending interwoven processes that form chain-like clusters  . Furthermore, they express tenascins  , and inhibitory chondroitin (C)-and keratan (K)-sulfate proteoglycans  . The glial scar biomechanically and biochemically blocks axonal elongation and reconnection  .
Recent research by our group has identified an implantable scaffold composed of electrospun polyamide nanofibers that appears to have promising wound-healing properties for CNS neural cell systems, including mitigation of astrocytic scarring. In vivo, when the scaffolds were introduced into spinal cord wound sites (rat model) accelerated hindlimb recovery, measured by standardized observational scoring (Beattie, Basso, Breshnahan [BBB] score), was observed, with aligned and fasciculated axon development and revascularization throughout wound sites [7, 8] . Moreover, low levels of astrocytic scarring were observed at 3 and 5 weeks after injury in comparison to injuryonly controls  . The promising in vivo results motivated a series of further in vitro investigations. Results from our group and from other groups have demonstrated that electrospun nanofibrillar scaffolds encourage biomimetic astrocyte stellation    , glial fibrillary acidic protein (GFAP) downregulation [10, 11] , growth factor upregulation  and neurite outgrowth by cocultured neurons  . Therefore, in vitro studies also Determination of how the physical properties of the nanofibrillar scaffolds influence the morphological and protein expressions is under investigation by our group and other groups. The effect of a physical environment on cell adhesion and differentiation has recently been recognized, especially in neural cells. Collectively, studies of cell responses to nano-patterning       , elasticity       , surface roughness [12, 24, 25] and surface polarity [12, 26] suggest that controlling aspects of the physical environment holds potential for inducing preferential differentiation of reactive astrocytes into noninhibitory pathways. This potential, combined with the promising in vitro and in vivo results, motivated the present investigation of dibutyryladenosine cyclic monophosphate (dBcAMP)-treated astrocyte differentiation in response to the external physical cues provided by the nano fibrillar scaffolds.
Cerebral cortical astrocytes, a relatively uniform population, were treated with dBcAMP, and used to investigate the responses of reactive-like astrocytes to external physical cues. Astrocytes treated with dBcAMP develop morphologies that resemble the reactive astrocytes  . As only external cues were used to trigger cell responses, astrocyte responses to nanofibrillar surfaces were studied in comparison with their responses to three additional culture surfaces: poly-l-lysine-functionalized planar glass (PLL glass), unfunctionalized planar Aclar (Aclar ® , Ted Pella Inc., CA, USA), and PLL-functionalized planar Aclar (PLL Aclar). PLL glass is a standard astrocyte culture surface, and astrocyte responses to it are well characterized, making it useful for identifying differences in astrocyte responses to other surfaces. The polyamide nanofibrillar scaffolds were electrospun on Aclar substrates; therefore, astrocyte responses to Aclar surfaces were investigated to distinguish responses to the nanofibrillar scaffolds from possible responses to the underlying Aclar substrate, particularly with respect to the elasticity property. Astrocyte responses to PLL Aclar surfaces were studied to clarify the role of the underlying substrate versus surface functionalization, particularly with respect to the elasticity and surface polarity properties. As these were the same comparative culture surfaces used in our recent investigation of untreated cerebral cortical astrocytes responses to external physical cues  , investigations of the differences between the untreated and dBcAMP-treated astrocyte responses to the same culture environments and properties were also performed.
In the present study, the local elasticity and work of adhesion of culture surfaces were investigated using atomic force microscopy (AFM) force curves and the results were added to the previous investigations of surface polarity and surface roughness. Reactive protein expressions for GFAP and tubulin, and chondroitin sulfate proteoglycan (CSPG), a neurite outgrowthinhibitory proteoglycan, were investigated for dBcAMPtreated and untreated astrocytes. Cytoskeletal protein expressions for the Rho GTPases Cdc42 (filopodia), Rac1 (lamellipodia) and RhoA (elevated: stress fibers and depressed: stellation) were investigated for the dBcAMP-treated astrocytes and the results were compared with previous results  for untreated astrocytes. The physical property trends were compared with the morphological and protein expression responses. The present work continues to explore the hypothesis that external physical cues of the nanofibrillar scaffolds can trigger the initiation of specific signaling cascades with morphological and reactivity consequences.
The morphological responses of cerebral cortical astrocytes were investigated at high-resolution using AFM and super-resolution microscopy (SRM). The three dimensional capability of AFM was also used to characterize cell spreading. The studies of the corresponding activations of the reactive proteins GFAP and tubulin, and the inhibitory proteoglycan CSPG, were performed using immunocytochemistry. GFAP, tubulin and CSPG expressions are associated with the astrocytic scarring response. Additionally, Rho GTPase proteins associated with morphological responses were investigated. Quantification of protein expressions were performed using confocal laser scanning microscopy (CLSM) z-series.
This article is organized as follows. Investigations of the physical properties of each culture environment, assessed in terms of local elasticity, work of adhesion, RMS surface roughness, and surface polarity, are presented first. The results indicated that the culture surfaces presented statistically significant property differences to astrocytes. Investigations of the astrocyte responses, assessed in terms of morphology and protein expressions, are presented next. Quantitative measures of the morphological responses for dBcAMP-treated astrocytes were compared with those of untreated astrocytes  . All of the protein expression investigations were performed for untreated and dBcAMP-treated astrocytes, with comparison of results.
One important finding of the present study was that dBcAMP-treated astrocytes cultured on nanofibrillar scaffolds showed a unique non-response, meaning that the reactivity proteins GFAP and tubulin, inhibitory proteoglycan CSPG, and cytoskeletal Rho GTPase expressions were little changed from their untreated expressions. A second important finding was that dBcAMP-treated astrocytes cultured on the PLL Aclar surfaces exhibited responses that were reminiscent of glial scar formation.
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Differentiation Of Reactive-Like Astrocytes Cultured On Nanofibrillar & Comparative Surfaces Preliminary Communication
The comparison of dBcAMP-treated and untreated astrocyte responses suggested that different nanophysical cues may have more directive importance in a woundhealing situation. The elasticity property was indicated as potentially more directive for dBcAMP-treated astrocytes than for untreated astrocytes.
Nanofibrillar Scaffolds & Comparative Culture Surfaces
Nanofibrillar scaffolds and comparative culture surfaces: PLL glass, PLL Aclar and Aclar were supplied/prepared as described in  . Scanning electron microscope (SEM) images (not shown) confirmed uniform coverage of nanofibers with no exposure of the Aclar coverslips. The culture surfaces used for the physical properties investigations were immersed in culture media only (no cells) for 24 h followed by fixative wash and dry.
Primary Untreated & Dbcamp-Treated Astrocyte Cultures
Primary untreated astrocyte culture preparation was identical to our previous work  . Primary dBcAMPtreated astrocyte culture preparation was as follows [27, 28] . Cerebral hemispheres of new born Sprague Dawley rats (postnatal day 1 or 2) were isolated aseptically. Cerebral cortices were dissected out, freed of meninges, and collected in HBSS. They were minced with sterile scissors and digested in 0.1% trypsin and 0.02% DNase for 20 min at 37°C. The softened tissue clumps were then triturated by passing several times through a fine bore glass pipette to obtain a cell suspension. The cell suspension was washed twice with culture media (DMEM + 10% FBS) and filtered through a 40-μm nylon mesh. For culturing, the cell suspension was placed in 75-cm 2 flasks (one brain/flask in 10 ml growth medium) and incubated at 37°C in a humidified CO 2 incubator. After 3 days of incubation the growth media was removed, cell debris was washed off and fresh medium was added. The medium was changed every 3-4 days. After reaching confluency (∼7 days), the cultures were shaken to remove macrophages and other loosely adherent cells. To obtain reactive-like astrocytes, 0.25 mM dBcAMP was added to the culture medium and the serum concentration was reduced to 1%. The cultures in dBcAMP containing medium were incubated for 7-8 more days with a media change every 3-4 days. The morphology of the cells was observed on alternate days under a phase contrast microscope. In the control cultures, the cells were fed with DMEM + 1% FBS (without dBcAMP). All procedures were approved by the Rutgers Animal Care and Facilities Committee (IACUC Protocol #02-004).
After completing dBcAMP treatment, reactivelike astrocytes were then harvested with 0.25% Trypsin/EDTA (Sigma-Aldrich) and re-seeded at a density of 30,000 cells per well directly on 12-mm Aclar or PLL Aclar coverslips, PLL glass coverslips, or on Aclar coverslips coated with nanofibers in 24-well plates in astrocyte medium containing dBcAMP (0.5 ml). After culturing the dBcAMP-treated astrocytes on the aforementioned substrates for 24 h, they were fixed with paraformaldehyde. Parallel cultures were immunostained with GFAP, an identification marker for astrocytes, and more than 95% were found to be GFAP-positive. AFM AFM images of astrocytes were captured using a Veeco Instruments Nanoscope IIIA (Bruker AXS Inc., WI, USA; formerly Veeco Metrology) operated in ambient air. A J scanner with 125 μm × 125 μm × 5.548 μm x-y-z scan range was used. The AFM was operated in contact mode silicon nitride tips with a nominal tip radius of 25 nm and cantilever spring constant k = 0.58 N/m (Nanoprobe SPM tips, Bruker AXS Inc.; formerly Veeco Metrology). Frequency domain Gaussian high pass filtering (GHPF) was used to segment the astrocytes from the substrate backgrounds in AFM height images  .
Astrocytes cultures prepared for AFM imaging were unstained. Four samples of astrocytes on each substrate were prepared. The uniformity of the four samples on each substrate was assessed by phase contrast microscopy to ensure that the cultures were representative. AFM images of at least 20 astrocytes for each culture surface were evaluated for process length, soma height, and cell shape index (CSI). Variations in astrocyte process length, soma height and CSI data among the culture surfaces were analyzed using ANOVA followed by pairwise post hoc comparisons with Tukey’s test  . Significance levels were set at p < 0.05.
Astrocyte Process Length Measurement
Untreated and dBcAMP-treated astrocyte cell process lengths were measured based on GHPF  AFM height images. An extension longer than the diameter of a cell soma was considered to be a process. Untreated and dBcAMP-treated astrocyte maximum soma height values were measured using the section analysis of Nanoscope Software version 5.30r3.sr3 and NanoScope Analysis 1.10 (Bruker AXS Inc.; formerly Veeco Metrology).
Astrocyte Cell Shape Index
The CSI was defined as the ratio of the perimeter squared to the two-dimensional cell area  :
2 CSI A P 2 r = (Equation 1)
Where P is the cell perimeter and A is the cell area. This definition describes stellation as a departure from CSI = 1 for a circular cell [32, 33] . The cell peri meter and area were calculated following manual segmentation of cells from GHPF AFM height images. The procedure was implemented in MATLAB version 7.7.0 (R2008b) Image Processing Toolbox (The Math-Works Inc., MA, USA).
Afm Force Curves Of Culture Surfaces
Twenty force versus z-piezo displacement curves from each culture surfaces were collected using the force mode analysis in Nanoscope Software version 5.30r3.sr3. The deflection sensitivity was determined on a silicon wafer, which is known to be harder than the culture surfaces  . The force versus z-piezo displacement data was converted to force versus distance (F-d) curve. Using the cantilever stiffness of the AFM provided by the manufacturer (0.6 N/m), the elasticity of tissue cultures was calculated from the retraction curve according to the Derjaguin, Muller and Toporov (DMT) model  :
R 2 d a = (Equation 2) (( ) ) a F F K R 1/3 n a d = + (Equation 3) 2 F W R 132 ad r = (Equation 4) (Equation 5)
Where δ is the indentation, a is the contact radius, R is the tip radius, F n is the applied force, F ad is the adhesion force, K is the total elastic modulus of the tip-substrate, W 132 is the work of adhesion required to separate AFM tip (subscript 2 ) from cell substrate (subscript 1 ) in air (subscript 3 )  , ν tip is the Poisson’s ratio of silicon nitride AFM tip, ν sample is the Poisson’s ratio for sample, E tip is the Young’s modulus of silicon nitride AFM tip set, and E sample is the Young’s modulus of sample. The AFM tip radius was measured to be 51 nm using field emission SEM images (not shown). F n was limited to approximately 25 nN by using the relative trigger threshold mode in the Nanoscope software. Poisson’s ratio values ν equal to 0.27 for silicon nitride  , 0.39 for polyamide  , 0.22 for glass  , and 0.33 for Aclar  were used. E tip was set equal to 310 GPa  and E sample was calculated by using a least-squares fit of F-d curves to the DMT model, and implemented using the MATLAB lsqcurvefit command  . The adhesive component of the AFM force curves was extracted, and used to investigate the work of adhesion between the AFM tip and the culture surfaces. Variations in elasticity and work of adhesion data among the culture surfaces were analyzed using ANOVA followed by pairwise post hoc comparisons with Tukey’s test  . Significance levels were set at p < 0.05.
Immunolabeling For Gfap & Tubulin
Astrocytes cultured on coverslips were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 5 min, and blocked with 10% normal goat serum for 30 min. After removing the normal goat serum, the cells were double stained with one of the primary antibodies and phalloidin. Rabbit anti-GFAP (Dako, CA, USA) was diluted 1:500 while mouse anti-β-tubulin (Sigma-Aldrich, MO, USA) was diluted 1:200. The incubation in the primary antibodies was done overnight at room temperature in a humidity chamber. Coverslips were then washed three times with PBS and stained appropriate secondary antibody either goat anti-rabbit Alexa 568 IgG (Life Technologies, CA, USA) or goat anti-mouse Alexa 568 IgG (Life Technologies). Both secondary antibodies were diluted 1:500 and incubation was for 1 h at room temperature. Secondary antibody staining coverslips were rinsed three times with PBS. Following PBS rinse, the coverslips were stained with Alexa Fluor ® 488 Phalloidin (Life Technologies) diluted 1:100 for 1 h, rinsed again with PBS as above. After staining, coverslips were mounted on microscopic slides with GelMount (Biomeda, CA, USA), and observed under an Olympus FluoView 1000 Laser Scanning Confocal Microscope system attached to an Olympus IX81 automated inverted microscope platform.
Quantitative Gfap & Tubulin Expression Estimate
A special data acquisition protocol was designed for the protein quantification studies using CLSM. First, immunofluorescence slides were always kept in dark. Any sample that was exposed to visible light was discarded and not used for protein quantification analysis. Confocal z-series images were captured using an Olympus FluoView 1000 CLSM system attached to an Olympus IX81 automated inverted microscope platform equipped with a 40× oil immersion objective (NA = 1.3). The z-series were acquired under identical imaging conditions, including the same high voltage, offset and gain settings, same objective, and same resolution (1024 × 1024 pixels). The intensity data were collected from samples in three dimensions. The z-step size was set to a level such that the intensity from a fluorophore was only recorded once, and determined by considering the axial resolution of the microscope system. The axial resolution is defined as  :
1 4 3 ( 1 1 ) K E v E v
1.4 / AR NA 2 em m = (Equation 6)
Where AR = axial resolution, λ em is the emission wavelength, and NA is the numerical aperture of the objective. In these experiments, the maximum λ em was 576 nm, NA was 1.3, and AR was equal to 477 nm. The z-step size was set to 1.13 μm to avoid oversampling. The upper and lower focal planes were determined with care as planes of complete darkness above and below any cell. The maximum pixel intensity was set to a level so that none of the pixels were saturated. This was important for both accuracy of quantification and reduction of photobleaching.
Images were analyzed using MATLAB version 7.7.0 (R2008b) Image Processing Toolbox (The Math-Works) and ImageJ version 1.46r (National Institutes of Health). The maximum intensity projection images were obtained from the z-series, the boundaries of the cells were determined manually, and then the total intensity and the number pixels were calculated by using the z-series and cell boundaries. The cell segmentation was done manually to avoid any error from the software settings since currently there is no robust algorithm for image segmentation. The background intensity was also calculated from slides in three dimensions. The average background intensity values were calculated for each z-series because the autofluorescence was different on different culture surfaces. The average background intensity was then multiplied by the number of pixels of z-series, and subtracted from the total intensity. Finally, the total amount of GFAP or tubulin expression/cell was determined. At least 50 cells were analyzed for each culture surface. Variations in GFAP and tubulin expression estimation data among the culture surfaces were analyzed using ANOVA followed by pairwise post hoc comparisons with Tukey’s test  . Significance levels were set at p < 0.05.
Colocalization Of Gfap & Tubulin
Colocalization analysis was based on three-dimensional z-series GFAP and tubulin immunofluorescence images of ten different regions per each culture. All images had 12 bit gray level depth and 1024 × 1024 pixels. Olympus software FV10-ASW, version 03.01.02.02 (Olympus) was used for the colocalization analysis. The threshold was set manually to a level that minimized background noise. The codependence of GFAP and tubulin expression of untreated and dBcAMP-treated astrocytes was quantitatively investigated by calculating the Pearson’s correlation coefficient and the overlap of GFAP and tubulin was quantitatively investigated by calculating the Mander’s coefficients M1 and M2 [12, 42] . M1 shows the portion of the tubulin intensity that coincides with some intensity in the GFAP image, and M2 shows the reverse. Variations in Pearson correlation coefficient (PC), M1 and M2 among the culture surfaces were analyzed using ANOVA followed by pairwise post hoc comparisons with Tukey’s test  . Significance levels were set at p < 0.05.
Quantitative Cspg Expression Estimate
Astrocytes cultured on coverslips of four different substrates were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 5 min, and blocked with 10% normal goat serum for 30 min. After removing the normal goat serum, cells were stained overnight at room temperature in a humidity chamber with monoclonal anti-chondrotin sulfate IGM antibody (Sigma-Aldrich) diluted 1:200. Coverslips were then washed three times with PBS and stained for 1 h with Alexa 488 goat anti-mouse IgM secondary antibody (Life Technologies) diluted 1:500. After secondary antibody incubation the coverslips were rinsed three times with PBS and mounted on microscopic slides with GelMount (Biomeda) and observed under a fluorescence microscope.
The 24-h untreated and dBcAMP-treated astrocyte cultures were used for CSPG quantification. Twelve images were captured from each substrate under identical imaging conditions: 1024 × 1024 pixels, 40× oil objective, same high voltage, gain and offset. The high voltage and offset levels were set to a level so that the pixels were not saturated. Total CSPG intensities of culture surfaces were analyzed using MATLAB version 7.7.0 (R2008b) Image Processing Toolbox (The MathWorks).
Three different sets of control cultures were prepared to ensure the accuracy of the quantification. In the first set, the autofluorescence of primary antibody was measured. Its intensity level was shown to be ignorable. In the second set, the nonspecific binding of the secondary antibody was measured, and the intensity measurements future science group Preliminary Communication Tiryaki, Ayres, Ahmed & Shreiber showed this was also ignorable. In the final control set, cell substrates were treated with primary and secondary antibodies without culturing cells to check if the primary antibody binds to the substrate. As intensities were different on different substrates, each of them was subtracted from the corresponding CSPG intensity. The total CSPG intensity data was then measured. Variations in CSPG expression estimation data among the culture surfaces were analyzed using ANOVA followed by pairwise post hoc comparisons with Tukey’s test  . Significance levels were set at p < 0.05.
Quantitative Cdc42, Rac1 & Rhoa Expression Estimate
The dBcAMP-treated astrocytes cultured on the substrates were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, and blocked with 10% normal goat serum for 30 min. The immunolabeling for Cdc42, Rac1 and RhoA, and the maximum intensity level image capture conditions, were identical to those described in our previous work for untreated astrocytes  to enable comparison of results. A total of 240 dBcAMPtreated astrocytes were investigated. Variations in Rho GTPase expression estimation data among the culture surfaces were then analyzed using ANOVA followed by pairwise post hoc comparisons with Tukey’s test. Significance levels were set at p < 0.05.
Local Elasticity & Surface Energy Investigation
Reported investigations    have identified local elasticity as potentially directive. Local elasticity along individual nanofibers was characterized in the present work. All force curves exhibited characteristics of interaction with a relatively firm material with an adhesive component and were therefore interpreted using the DMT elasticity interpretative model for the Young’s modulus that included adhesion forces  .
Analysis of the Young’s modulus results using median-value box plots ( Figure 1A) indicated that PLL glass surfaces presented the hardest and nanofibrillar scaffolds presented the softest culture environments to astrocytes. The Young’s moduli for Aclar and PLL Aclar were similar to each other, and higher in value to those of the nanofibrillar scaffolds. The results indicated that substrate elasticity dominated over the PLL chemical functionalization effect since unfunctionalized and PLL functionalized Aclar had similar elasticity median and variances while PLL functionalized glass and PLL functionalized Aclar had different results for both.
Use of the DMT interpretive model further enabled investigation of the surface energies of the cell substrates by measuring the work of adhesion, W 132 , between the AFM tip and each culture surface  . Analysis of the work of adhesion using median-value box plots ( Figure 1B) showed that despite the similar functionalization of PLL glass and PLL Aclar, the work of adhesion median value was higher for PLL Aclar. The combination of chemical functionalization with surface roughness may result in higher work of adhesion, possibly through PLL conformation changes. The highest variance was observed for nanofibrillar scaffolds.
The nanoscopic elasticity, work of adhesion, and surface roughness  , and the macroscopic surface polarity measured by contact angle  of culture surfaces, are summarized as mean values with standard error of the mean (SE) in Figure 2 . The work of adhesion and contact angle can be both used for measuring surface energy. Surface energy increases as work of adhesion increases, and work of adhesion decreases as contact angle increases. Therefore, the work of adhesion and the contact angle measurements are consistent Astrocyte responses: morphology investigation
|Cells||PLL glass||Nanofibrillar scaffolds||Aclar||PLL aclar|
|dBcAMP-treated||189 + 24||206 + 14||177 + 7||202 + 17|
|Untreated||243 + 26||186 + 12||173 + 12||252 + 19|
The initial cell plating density on all surfaces was 66 cells/mm 2 . Astrocyte density at 24 h was measured and the results for untreated and dBcAMP-treated astrocytes for each substrate are given in Table 1 .
Astrocyte density can be influenced by many factors. The dBcAMP-treated astrocyte density on Aclar was low compared with other substrates, indicating that the cell adhesion and/or proliferation on Aclar surfaces were negatively influenced by the properties of this substrate. The highest dBcAMP-treated astrocyte cell density was observed on the nanofibrillar scaffolds, which indicated that these surfaces were able to modulate cell adhesion and/or proliferation without requiring PLL functionalization.