2.1.

Multiphoton Image Setup and Acquisition

The following microscopy apparatus was used for the assessment of SHG F/B:

Multiphoton laser scanning microscopy (MPLSM) was conducted using a Ti:Sapphire excitation laser controlled through a BX61WI upright microscope (Olympus, Shinjuku, Tokyo), with beam scanning and image acquisition controlled by an Olympus Fluoview FV300 scanning system. For all imaging, the excitation light was circularly polarized with 100 fs pulses at 80 MHz, where circular polarization at the sample was achieved by passing the excitation light through a Berek compensator (Model 5540, New Focus, Irvine, CA) before entering the scan box. The light was focused through an Olympus UMPLFL20XW water immersion lens ($20\times$, 0.95 N.A.), which was also used to collect the backward propagating SHG and fluorescent signal. Backscattered signals were separated from the excitation beam using a 670 nm short-pass dichroic mirror. The backscattered collagen SHG signal was generated with an excitation wavelength of 810 nm, and the emission was filtered by a 405 nm band-pass filter (HQ405/30m-2P, Chroma, Rockingham, VT). The backward-scattered fluorescence signal, used to image tdTomato-labelled tumor cells, was excited using 740 nm light and the emission signal was filtered using a 580 nm band-pass (HQ580/180m-2P, Chroma) and a 700 nm short-pass filter (E700SP-2P, Chroma). Only SHG was captured in the forward-scattered direction, using an Olympus 0.9 N.A. optical condenser, reflected by a 565 nm long-pass dichroic mirror (565 DCSX, Chroma), and filtered by a 405 nm band-pass filter (HQ405/30m-2P, Chroma). All signals were captured by Hamamatsu HC125-02 photomultiplier tubes.

2.2.

In Vivo Model of Collagen Microstructural Changes

In order to study the relationship between collagen gel F/B ratio and tumor cell motility, we first identified a tumor cell line that recapitulated in vivo the relationship between F/B and metastatic output observed in patients:

Step 1: Mouse preparation. 40 female BALB/cByJ mice (Jackson Laboratories, Bar Harbor, ME) were anesthetized using ketamine/xylazine ($90/9\mathrm{mg}/\mathrm{kg}$ body weight). A cohort of 30 mice received an injection of $5\times {10}^5$ tdTomato-labelled 4T1 mouse mammary carcinoma cells in the right inguinal mammary fat pad; the remaining mice were injected with saline. Step 2: Tissue collection. Following 14 days of tumor development and metastasis, the animals were sacrificed using intraperitoneal injection of sodium pentobarbital and subsequent cervical dislocation. Tumors and the draining inguinal lymph node were dissected free of the surrounding tissue and immediately snap-frozen in dry ice. These experiments were performed in accordance with University Committee for Animal Resources regulations. Both tissues were sectioned into $20\text{-}\mu \mathrm{m}$ slices using a cryostat (Reichert-Jung, Depew, NY), mounted onto positively charged slides (VWR) and stored at $-20°\mathrm{C}$ until imaged. Step 3: Imaging of tumor sections. Before the start of each imaging session, a standard fluorescein isothiocyanate (FITC) sample was imaged as a calibration sample using 810 nm excitation and 535 nm emission filters (HQ535/40m-2P, Chroma) for both detection systems. Free FITC emits isotropically, but the measured F/B ratio was different from 1.0 each day due to slight variations in the system alignment. Therefore, a calibration factor was determined from the observed F/B ratio of FITC, which was applied to all SHG F/B ratios for that day. In each tumor section, one region was imaged from the center of the tumor, and then four additional regions were imaged by moving the field of view (FOV) in one of four directions from the center (i.e., up, down, left, and right) until the closest visible tumor edge was $\sim 300\mu \mathrm{m}$ distant. At each imaged region, both the forward and backward-scattered SHG were acquired in a series of 11 image pairs spaced $3\mu \mathrm{m}$ apart to create a $30\text{-}\mu \mathrm{m}$ thick z-stack.

Step 4: Image Analysis: All image analyses was performed with ImageJ software.¹⁶ A maximum intensity projection of each of the two stacks (F and B) reduced the image stacks to a single image pair for each region. On thin ($20\mu \mathrm{m}$) tissue sections, these 11 image stacks serve as an autofocus for the thin collagen layer. Each image was background subtracted using the average background value for the corresponding PMT determined by generating an F and B image pair with the laser shutter closed. Next, two masks of both the forward and backward-scattered SHG signals were created in which collagen pixels were set to 1 and background pixels were set to 0 by applying a threshold to each of the two images. The same threshold was applied to all images. This single threshold was chosen by a blinded observer to highlight collagen and to reject background by first surveying a random sampling of images. An example forward-scattered SHG image and the corresponding threshold are shown in Fig. 1(a). After trying different thresholds ranging over $\pm 10$ a.u., we found that the F/B ratio of the sample does not significantly differ as a result of different thresholds [Fig. 1(b), ANOVA $p=0.31$]. The two masks were multiplied together to create one mask that represents pixels with SHG emission above the threshold in both the forward and backward directions for that imaged region. A single F/B ratio image for that region was calculated from the F and B image pair. This F/B image was multiplied by the mask in order to set the value of all nonfiber pixels equal to zero, thereby excluding background (i.e., nonfiber) pixels from consideration. The average pixel value of fiber (i.e., nonbackground) pixels for that region was then calculated for each masked F/B ratio image. The five values of F/B for each tumor section were then averaged to determine the average F/B for that section. Note that, as in previous studies,¹⁰ tissue samples were mounted on full thickness glass slides under coverslips, not between two coverslips. While this does not affect the comparisons between identically mounted samples as performed here, the measured F/B of these tissue sections is an underestimate of the true F/B ratio due to the presence of optical aberrations produced by the underlying glass slide.

Fig. 1

A threshold is applied to second-harmonic generation (SHG) images in order to highlight the collagen regions. (a) Example backward-scattered (top) and forward-scattered (bottom) SHG image and corresponding thresholded images. (b) Comparison of F/B ratios at different chosen thresholds demonstrates that a 20 a.u. range causes no significant differences in F/B ratio (ANOVA, $p=0.31$).

Step 5: Quantify metastases. To quantify metastasis to the corresponding inguinal lymph nodes, clusters of tdTomato fluorescent cells were counted by a blinded observer on the MPLSM from one section taken from $400\mu \mathrm{m}$ within each lymph node. Step 6: Analysis and statistics. All tumors were divided into three groups based on the number of associated metastasis, the “Low” metastasis group had less than four metastases and the “High” group had greater than six metastases. The F/B ratios of the primary tumors were compared between the three groups. Statistical differences were tested using a one-way ANOVA, followed by a Newman–Keuls comparison of all groups. A $p$-value of $p<0.05$ was significantly considered.

2.3.

tdTomato Transfected 4T1 Cell Culture

Once the 4T1 cell line was chosen, it was cultured and eventually seeded on collagen gels:

The tdTomato transfected 4T1 mouse mammary adenocarcinoma cell line (Caliper Life Sciences, Hopkinton, MA) was frozen after three initial passages. After being thawed, each aliquot was used for less than 3 months then replaced from frozen stock. Cells were maintained in RPMI (Gibco, Invitrogen Inc., Carlsbad, CA) containing penicillin/streptomycin and 10% fetal calf serum. All cells were added to the surface of the gels in this media.

2.4.

Preparation of Control Collagen Gels

Step 1: Dish Preparation. Mattek petri dishes (Ashland, MA) with 14-mm diameter coverslip-bottomed wells were coated with 1% BSA and incubated for 30 min at 37°C and 5% ${\mathrm{CO}}_2$. Within a sterile hood, the BSA solution was removed and the petri dishes were allowed to dry while preparing the collagen gel solution. Step 2: Making the gel. Gels were made of human type I collagen solution at a concentration of $3\mathrm{mg}/\mathrm{mL}$, obtained from advanced biomatrix (San Diego, CA). With temperatures maintained at 4°C to minimize collagen polymerization, $810\mu \mathrm{l}$ of collagen solution was combined with $108\mu \mathrm{l}$ of RPMI media and $50\mu \mathrm{l}$ of distilled ${\mathrm{H}}_2\mathrm{O}$. Solutions were gently pipetted to avoid bubble formation in the solution. The pH of the total gel solution was increased between 7.5 and 7.7, using 2.5 M NaOH. $100\mu \mathrm{l}$ of solution was aliquoted into each of a maximum of six petri dishes. Step 3: Incubation and imaging. The gels were incubated for 2 h at 37°C. After this period, the gels were covered in phosphate-buffered saline (PBS) in order to avoid dehydration and a subsequent decrease in overall gel volume. Before imaging the PBS was removed from the top of the gel, media containing the appropriate number of cells was added, the gel was cover slipped, and then imaging proceeded immediately.

2.5.

Determining the Effect of Cell Seeding on F/B

In preliminary experiments, we applied large numbers of cells to the gels and incubated them for 3 days to explore the effects that the cells themselves can have on gel F/B:

Step 1: Cell/Gel preparation. To analyze the effect of cells on the local F/B in a gel, two sets of gels were created: gels with 4T1 cells added to the surface, and identically prepared gels with PBS added to the surface. To seed a gel, $\mathrm{30,000}\text{cells}/\mathrm{mL}$ were added to the top of gels in $200\mu \mathrm{L}$ of RPMI media and incubated for 3 days, with additional media provided as necessary to prevent drying. Gels without cells were covered in $200\mu \mathrm{L}$ of PBS and incubated for 3 days, with addition of PBS if needed to prevent drying. Step 2: Imaging. After removal of gels from the incubator, excess media or PBS was carefully discarded, and the gels were immediately cover slipped and imaged. To quantify the effect of the presence of cells on the local F/B ratio, F/B was determined at one location per gel, chosen to have a high tumor cell density if the gel contained cells, and chosen from the center of the gel if the gel did not have cells. The images were taken as $660\text{-}\mu \mathrm{m}$ FOV stacks of 11 images spaced $3\mu \mathrm{m}$ apart, in order to capture the region of fibers affected by the collagen reorganization. Step 3: Image analysis. These images were maximum intensity projected to produce a single F and B image pair, and then the F/B ratio for the imaged region was determined as described above. Step 4: Comparison: The average F/B ratios of these images were compared between the two gel conditions. Statistical difference was detected using a two-tailed Student’s $t$-test, $p$-value $<0.05$.

2.6.

Determining the F/B Ratio of a Collagen Gel

Next, lower concentrations of cells were applied to gels, and imaged within 3 h to minimize gel remodeling by the cells. Polymerization conditions of the gels were manipulated to vary F/B ratio, and F/B ratio of the gels was determined and evaluated as a function of imaging depth. Finally, the motility of cells within gels of varying F/B ratio was assessed:

Step 1: Calibration. Calibration using a standard FITC sample, and determination of background, were performed as described above. Step 2: Imaging through the gel. For F/B ratio analysis of gels, two stacks of simultaneously collected forward and backward images were taken at the geometric center of each gel, consisting of image pairs taken at $10\mu \mathrm{m}$ steps through the depth of the gel. Images were $660\mu \mathrm{m}$ across. Step 3: Image analysis. The F/B of each imaged depth was determined as described above. Step 4: Analysis as a function of depth. In order to determine how the measured F/B ratio varied as a function of imaging depth, the F/B ratio was plotted as a function of depth within the gel, in 10% increments by depth. This provides 10 values of F/B ratio for each gel, while allowing for slight differences in the overall depth of the gels (ranging from 450 to $550\mu \mathrm{m}$). A linear fit was applied to each plot to ensure that the slopes of the lines were not significantly different than zero, therefore, ensuring that the measured F/B was not affected by depth into the gel. Step 5: Producing one F/B value. A single value of the average F/B to represent the entire gel was then calculated by taking the average of the F/B ratios taken at each of the 10% depth increments. This value was used in all subsequent comparisons of different gels. All gel comparisons are conducted using a one-way ANOVA, followed by a Newman–Keuls comparison to determine the statistical significance between groups, where $p<0.05$ was considered statistically significant. Statistical analysis was performed using Prism 5 software (GraphPad, La Jolla, CA).

2.7.

F/B Ratio Manipulation

The F/B ratio of the gels was altered by manipulating one of three factors within the gel protocol during the previous “Step 2: Making the Gel”. The pH was altered by using increasing amounts of NaOH in the final gel solution to bring the total pH to 8.5 to 8.7 or 9.5 to 9.7, measured with a Mettler Toledo Micro Pro pH meter (Columbus, OH). The ionic strength was changed by incorporating potassium chloride (KCl) into the $50\mu \mathrm{l}$ ${\mathrm{dH}}_2\mathrm{O}$ added to the collagen gel solution at either 100 mM or 200 mM concentrations. The type of collagen forming the matrix was changed by adding increasing amounts of collagen type III by weight, varying from control gels made of 100% collagen type I to experimental conditions of 90% type 1: 10% type III, or 80% type I: 20% type III.

2.8.

Analysis of Tumor Cell Motility

Step 1: Make gel and add cells. Control gels and F/B ratio manipulated gels were made as previously stated. To dilute out any remaining differences in pH, KCl, or collagen I/collagen III from the polymerization mixture, gels were incubated in 5 mL of PBS for at least 30 min or until each gel was ready to be imaged (maximum of 12 h). Further preparation and imaging was performed one gel at a time. Immediately before imaging, the incubating PBS was removed and $1\times {10}^4$ tumor cells resuspended in RPMI media were added to the surface of a single collagen gel in a $200\mu \mathrm{L}$ droplet. After allowing the cells to settle onto the gel, the excess media were removed in order to minimize motion during the imaging period. Step 2: Image cells. Live cell images were taken on a Nikon Eclipse Ti, using a Nikon MRH20101 air lens ($10x$, 0.3 N.A.). Cells were incubated on the microscope stage throughout imaging using a live cell microscope incubator (Pathology Devices, Westminster, MD), at 37°C, 5% ${\mathrm{CO}}_2$, and 85% relative humidity. One brightfield image was taken every 2 min for 3 h, and saved as a tiff file.

Step 3: Image Analysis. Using the ImageJ¹⁶ “Template Matching” plugin, stacks were aligned through each time point based upon the SHG channel to minimize slight motion artifacts. For each gel, 10 cells (or the maximum number of cells visible) were tracked using the “Manual Tracking” plugin, which tabulates the XY location of each cell at each time point. From these values, the total distance traveled (sum of the distances traveled between each time point), the average velocity (the average of the distance traveled between each time point, divided by the time interval) and the maximum velocity each cell reached over the time course (the largest distance traveled between any two consecutive time points, divided by the time interval) was calculated for each cell and values were averaged across all cells to represent the values of that gel. Note that this is a two-dimensional analysis of tumor cell motility because over the timescales imaged the tumor cells to not penetrate significantly into the gel and their $xy$ motility is significantly greater than their $z$ motility. Allowing the cells sufficient time to significantly penetrate into the gel increases the chances that the cells will alter the F/B of the gel, as discussed above. Step 4: Statistical analysis. Significant differences between the various conditions were determined by comparing the average for each gel using a one-way ANOVA, followed by Newman–Keuls post-hoc comparison of all groups, where $p<0.05$ was considered statistically significant. Additionally, distance and velocity changes were graphed as a function of the average F/B ratio of the gels and the relationship between the two factors was tested using a Pearson’s correlation analysis.

3.1.

Animal Model

It has previously been shown that the average F/B ratio of the primary tumor of patients with IDC varies with the extent of lymph node involvement (i.e., the “N stage”) upon clinical presentation.¹⁰ The primary motivation of this study is to understand the relationship between F/B ratio and tumor cell motility in a relatively “clean” in vitro collagen cell system, in which confounding upstream effectors are not present. In order to accomplish this, we must first identify a tumor cell line that recapitulates the observed relationship between F/B and lymph node metastasis. This was accomplished by injecting tdTomato-labeled 4T1 murine mammary adenocarcinoma cells into the mammary fat pad of BALB/c mice. After 2 weeks of growth, tumors were removed along with the draining of the inguinal lymph node [Figure 2(a)], and the F/B ratio of the primary tumor as well as the number of metastases in the lymph node were assessed as described above, to model previous analyses of human samples. As seen in Fig. 2(b), healthy mammary fat pad tissue has a high F/B ratio, which was significantly decreased in a tumor that has a low or medium number of metastasis to the LN. The F/B ratio was not significantly different from healthy mammary fat pad tissue in tumors that had a high number of lymph node metastases. These results exactly match the pattern in human patients with IDC, as shown previously, wherein weakly and moderately metastatic tumors (N0, N1, and N2) exhibited a significantly lower F/B than healthy breast tissue, while highly metastatic tumors (N3) did not.¹⁰. This suggests that the 4T1 murine mammary adenocarcinoma is a useful model for understanding the relationship between SHG F/B and metastatic output.

Fig. 2

The relationship between metastasis to LN and SHG F/B in the 4T1 murine mammary adenocarcinoma model matches human data. (a) Diagram of the mouse torso revealing the location of the mammary fat pad where tumor cells were injected and the draining inguinal lymph node. (b) F/B ratio as a function of tumor metastasis to the lymph nodes. The samples are split into four groups: Healthy mammary fat pad (F/B ratio: $5.22\pm 0.79$, $n=10$), Low (1 to 3 mets, F/B ratio: $1.90\pm 0.17$, $n=9$) Medium (4 to 6 m, F/B ratio: $2.24\pm 0.39$, $n=10$), and High (7 to 12 m, F/B ratio: $3.78\pm 0.80$, $n=11$). * represents difference of $p<0.05$. Results expressed as $\text{mean}\pm \mathrm{SEM}$.

3.2.

Creation of Consistent and Optically Thin Collagen Gels

To evaluate the effect of gel F/B on tumor cell motility, we must next verify that we can produce optically thin gels with consistent F/B ratios. Figure 3(a) shows that the average F/B ratios of control gels were consistent between three different sessions of synthesis and imaging on three different days, demonstrating that the gel formation protocol produces collagen fibers of consistent microstructure. Figure 3(b) shows the average F/B ratio of standard control gels as a function of percent depth into the gel, with the total depths ranging from 450 to $550\mu \mathrm{m}$. These lines were characterized by a slope of zero within the 95% confidence interval, signifying that the measured F/B ratio was not significantly affected by depth, and demonstrating that this method creates optically thin gels, in which the SHG signal was not significantly affected by the thickness of the gel.

Fig. 3

Control gels revealing consistent collagen gel structure between (a) different synthesis sessions ($\mathrm{F}/\mathrm{B}=5.52\pm 0.59$, $6.27\pm 0.21$, and $6.15\pm 0.14$) and (b) throughout the depth of the gel. For all plots control $n=6$. Results expressed as $\text{mean}\pm \mathrm{SEM}$.

3.3.

Manipulation of the Average F/B ratio

Gels of significantly different F/B ratios were created by changing properties of the collagen gel during fibrillogenesis, including the ionic strength (concentration of KCl), the pH, and the collagen composition (ratio of type I: type III). Figure 4 shows the significant changes in the average F/B ratio of gels resulting from each of the different manipulations, with a significant decrease in F/B ratio as a function of increasing collagen type III and a significant increase in F/B ratio as a function of increasing pH during fibrillogenesis. The relationship between F/B and ionic strength during fibrillogenesis was biphasic, with a peak F/B at 100 mM KCl. The F/B ratio as a function of depth into the gel was a straight line with a slope of 0 within the 95% confidence interval for all gels in all of these conditions (data not shown). Using three methods of changing the F/B ratio allowed us to test how these changes in the collagen microstructure affected the ability of the tumor cells to move along the fibers, while minimizing the chance that this was due to a direct effect of the fibrillogenesis manipulation itself on cell motility, rather than via physical properties of the resulting fibers.

Fig. 4

Methods for significantly changing the F/B ratio. There were significant changes in the average F/B ratio of gels as a function of changes in (a) percent of the collagen in solution that is collagen type I, relative to type III ($n=\mathrm{6,6,6}$), (b) pH of the gel solution ($n=\mathrm{6,6,6}$) and (c) KCl concentration ($n=\mathrm{6,5,5}$). Results expressed as $\text{mean}\pm \mathrm{SEM}$. * represents difference of $p<0.05$, ** represents difference of $p<0.01$, and *** represents difference of $p<0.001$.

3.4.

Cell Manipulation of Collagen Organization

The overall motivation for this project was to determine the effects of collagen fiber F/B ratio on the motility rates of tumor cells. However, it has previously been shown that adding tumor cells to collagen gels induces reorganization of the overall collagen fiber morphology.⁹ This suggests that tumor cells may also alter the F/B ratio of collagen gels. We found that by adding a large number of cells ($3\times {10}^5\text{cells}/\mathrm{mL}$ of media) and allowing a significant amount of time (3 days), the tumor cells were able to induce a significant increase in the F/B ratio of fibers immediately surrounding cell clusters. Figure 5(a) confirms that in regions of high cellular density, the tumor cells alter the local fiber morphology, with fibers in between and surrounding the two clusters of cells becoming straightened and protruding perpendicularly. The regions adjacent to cellular clusters were also characterized by a higher F/B ratio [Figure 5(b)]. This was significant for the design of subsequent experiments because in order to determine the effect of gel F/B on cell motility, we must prevent the cells themselves from significantly altering the F/B of the gel. For this reason, in the experiments performed subsequently, the cells were added to the surface of the gels in lower concentrations (1000 versus $\mathrm{6,000}\text{cells}/\mathrm{gel}$) and then promptly imaged (3 h versus 3 days), to minimize significant collagen restructuring by the cells.

Fig. 5

Collagen restructuring from mouse mammary adenocarcinoma cells. (a) Over 3 days tumor cell (left, red) clusters result in morphological changes in collagen (left, blue) organization in areas surrounding tumor cells, leading to aligned fibers. This also causes changes in the collagen microstructure demonstrated in a heat map of the F/B ratio in the same area (right). (b) Tumor cell clusters can alter local fiber microstructure, depicted by increases in F/B ratio of those fibers (Control $n=10$, Cell gel $n=11$). Results expressed as $\text{mean}\pm \mathrm{SEM}$. *represents $p<0.05$.

3.5.

Cell Motility

Using the methods described above, gels of different average F/B ratio were used to determine how fibers of different microstructure affect the motility of 4T1 mouse mammary tumor cells. Based upon our previous results, we generated gels with four different F/B values using four different methods: gels generated under control conditions had an F/B of $6.20\pm 0.14$, gels generated with a collagen I/collagen III ratio of 80% had an F/B of $2.81\pm 0.29$, gels generated with 200 mM KCL had an F/B of $8.13\pm 0.16$, while gels generated in a pH of 9.5 had an F/B of $10.60\pm 1.08$. Note that these alterations are present within the polymerization media during gel fibrillogenesis: during the subsequent seeding and locomotion of tumor cells, which is performed well after the gels are polymerized, the components of the media are the same in all cases. As shown in Figs. 6(a) and 6(b), tumor cells were tracked for 3 h during which the total distance traveled, average velocity, and maximum velocity were measured. There was a significant main effect of the F/B category on total distance traveled (ANOVA $p<0.05$), and post-hoc analysis revealed that the total distance traveled by cells on gels of the two highest F/B categories was significantly greater than the total distance traveled by cells on gels of the two lowest F/B categories [$p<0.05$, Fig. 6(c)]. The mechanism by which these tumor cells covered more distance may be related to how efficiently the tumor cells pull themselves along the fiber, which would be revealed through differences in cell velocity. Quantification of the average velocity of cells on gels with different F/B ratios revealed a significant main effect of F/B on average velocity [$p<0.05$, Fig. 6(d)] while post-hoc analysis revealed that the average velocity of cells on the two highest F/B category gels was significantly higher than the average velocity of cells on the lowest F/B ratio condition. There was also a trend toward increased maximum velocity with increasing F/B ratio [ANOVA $p=0.06$, Fig. 6(e)]. To quantify the degree to which cell motility and gel F/B ratio are related, a Pearson’s correlation analysis and a linear regression were conducted. In these analyses, the F/B groups were formed by collapsing the F/B ratios across treatment conditions to form four distinct F/B ratio groupings, which were compared to the motility data shown in Figs. 5(c) to 5(e). The Pearson analysis revealed a significant positive relationship between F/B ratio and average velocity as well as maximum velocity (Figs. 5(g) and 5(h) $p<0.05$ for both) and a nearly significant relationship between F/B ratio and total distance travelled [Fig. 5(f); $p=0.06$]. A subsequent regression analysis reveals that these data are closely modeled by a linear fit with a positive slope for the average and maximum velocity ($R^2=0.94$ and 0.97, correspondingly). This suggests that these three motility parameters are highly correlated with, and are linearly related to, the F/B ratio.

Fig. 6

Effect of synthesis method and F/B ratio on tumor cell motility in collagen gels. Representative tracks of tumor cells over time on low F/B ratio (a) and high F/B ratio (b) gels, qualitatively revealing differences in tumor cell motility. (c) Total distance traveled versus F/B category; ANOVA $p<0.01$. (d) Average velocity versus F/B category, ANOVA $p<0.05$. (e) Maximum velocity versus F/B category; ANOVA $p=0.06$. (f–g) Correlation analyses: (f) Total distance traveled versus F/B value. ($p=0.06$; $R^2=0.88$). (g) Average velocity versus F/B value ($p<0.05$ and $R^2=0.94$). (h) Maximum velocity versus F/B value ($p<0.05$ and $R^2=0.97$). Results expressed as $\text{mean}\pm \mathrm{SEM}$. For all plots control $n=6$, for all other conditions $n=5$. *represents $p<0.05$ based on Newman–Keuls post-hoc test.