Regional and fiber-type percentages and sizes in the hamster diaphragm after swim training.

[Reid WD, Shanks J, Samrai B. Regional and fiber-type percentages and sizes in the hamster diaphragm after swim training. Phys Ther. 1997;77:178-186.]

Key Words: Exertion, Muscle fiber type, Myofibrillar adenosine triphosphatase, Respiratory muscles.

The diaphragm appears functionally and structurally to be similar to other skeletal muscles, and it can be conditioned to improve performance using both specific[1-8] and nonspecific exercise programs.[3-10] Respiratory muscle training may be an important therapeutic approach to improving respiratory muscle endurance in patients with chronic respiratory conditions in an effort to decrease dyspnea, increase overall exercise tolerance, and possibly prevent respiratory muscle fatigue.[11] Overloading the respiratory muscles by increased ventilation induced during endurance exercise involving the extremities rather than respiratory muscle training would be advantageous because of the widespread effects on other systems in the body such as enhanced cardiovascular function, improved endurance of the exercised limb muscles, and improved sense of well-being.[12]

Based on the origin of the muscle fibers, the diaphragm can be divided into three anatomic regions: sternal, costar, and crural.[13] De Troyer et al[14] demonstrated that based on the actions of the rib cage, the diaphragm consists of two major regions: costar and crural. Histologically, we found three distinct regions in the hamster diaphragm when examining variability of staining for myofibrillar adenosine triphosphatase (M-ATPase) after preincubation at various pHs. The regions were distinct, based on fiber proportions and sizes,[15] and in part they coincide with the anatomical regions.

The sternal and costar regions were similar and showed a very even mosaic distribution of type I, IIa, and IIb fibers. The crural region differed from the costar region. Each half of the crural region also differed from the other half. The half of the crural region closest to the thoracic cavity showed a higher percentage of type I fibers and a lower percentage of type IIb fibers compared with the rest of the diaphragm, and the half of the crural region closest to the abdominal cavity showed a very low percentage of type I fibers and a higher percentage of type IIb fibers compared with the rest of the diaphragm.[15] These three histologic regions also demonstrated variability in oxidative enzymes and glycogen content within the same M-ATPase fiber type.[16] These differences imply that the three histologic regions of the hamster diaphragm may have differing functions, which would likely be reflected by variations in energy metabolism and recruitment patterns.

Endurance training induces several metabolic and structural changes within skeletal muscle. These changes include such aspects as an increase in content of mitochondria, an increase in oxidative enzymes, an increase in skeletal muscle myoglobin, increased capillarization, and an increased ability to store and oxidize fat and carbohydrates.[17] Endurance training has been shown to increase[18] and decrease[19] the cross-sectional area of muscle fibers. Atrophy of muscle fibers in response to endurance training is thought to shorten the diffusion distance for oxygen transport to all parts of the muscle during exercise. One of the last features to transform in response to an endurance training stimulus, requiring the strongest stimulus, is the fiber-type proportion.[20]

Skeletal muscle fibers can be typed into two major categories: type I and type II. Physiologically, type I fibers are slower-contracting, fatigue-resistant fibers, and type II fibers are faster-contracting, fatigable fibers.[17] Using the histochemical techniques to examine the differing responses of the different kinds of M-ATPases to acid or alkaline preincubation, muscle fibers can be categorized as types I, IIa, IIb, and IIc.[21] Many investigators have subdivided the type II fibers into oxidative and nonoxidative fibers by examining serial sections with an M-ATPase technique and an oxidative stain such as succinic dehydrogenase. Hence, fibers have been named slow-twitch-oxidative, fast-twitch-oxidative-glycolytic, and fast-twitch-glycolytic. Although there is a general correspondence between fibers typed using physiologic and different histochemical techniques, none of these fiber-typing schemes is entirely compatible. In our study, histochemical techniques examining different types of M-ATPase were used to type muscle fibers. Thus, for the purposes of this report, we will use the nomenclature put forward by Brooke and Kaiser categorizing fibers as types I, IIa, and IIb.[21]

Inspiratory resistive loading applied intermittently[22] or continuously[23,24] increases the proportion of M-ATPase type I fibers in the costar diaphragm. The M-ATPase fiber-type proportions change in limb muscles[20] and in the diaphragm of rats[25,26] in response to running. The cross-sectional area of type I fibers[23] and growth-corrected slow-twitch-oxidative fibers[27] have been shown to increase in response to resistive loading in the diaphragm. Another form of endurance exercise involving the extremities that increases ventilation, and thus may overload the respiratory muscles, is swimming.

We hypothesized that type I or IIa fiber percentages and the size of type I fibers would increase in the diaphragm in response to swimming endurance training. We also hypothesized that because the fiber-type characteristics vary among different diaphragm regions, these regions would adapt differently to the swim training stimulus. The purpose of this study was to determine the changes in fiber-type percentages and sizes in different regions of the hamster diaphragm after 13 weeks of a swimming endurance training program.

Materials and Methods

Housing

Nineteen adult male golden Syrian hamsters(*) were obtained and cared for in accordance with the principles outlined in the Guide to the Care and Use of Experimental Animals.[28,29] Hamsters were housed in wire-mesh cages individually or in pairs, fed a diet of Purina Laboratory Chow([dagger]) and water, and maintained on a constant timed light-dark cycle (0900:2100) such that the light was on from 9 am until 9 pm; the lights were turned off for the other 12 hours.

Exercise Protocol

Animals were randomly assigned to either a control group (n=9) or a swimming group (n= 10). Hamsters in the swimming group swam for 80 minutes per session, 5 days per week, for 13 weeks. Each hamster swam in a separate container with vertical sides (diameter: 26.5 cm, height: 24 cm) filled with clean tap water (water depth of 18-20 cm) held at a temperature of 37 [degree] C. All animals in the swimming group were supervised closely to encourage constant swimming and to prevent drowning. If the hamster s nose dipped below the water, it was rescued immediately, allowed to rest for 10 to 15 seconds, and then returned to the water. No hamster in the swimming group required more than one rescue. Hamsters in the control group performed no exercise except for their normal movements in their cages. After the 13-week swimming or control period, and within 48 hours of the last training session, diaphragm muscle biopsies were obtained. Hamsters were weighed at the beginning and end of the study and at 2-week intervals throughout the study.

Regional Muscle Biopsies

The hamsters were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 6.5 mg/100 g of body weight. Through a midline abdominal incision, the aorta was transected and the entire diaphragm was quickly excised and placed on a phosphate-buffered (pH 7.4) saline-soaked gauze. Costal and crural biopsies were cut from the left hemidiaphragm. Biopsies were mounted in gum tragacanth, quickly frozen in isopentane cooled to the temperature of liquid nitrogen, and then stored at-700C until they were sectioned and stained. Fiber-type percentages and sizes were determined in three major regions of the diaphragm: (1) costar region, (2) thoracic surface of the crural region (thoracic/crural region), and (3) abdominal surface of the crural region (abdominal/cruel region).[15] The thoracic/crural region was defined as the area from the midline of the crural biopsy to the edge of the cross section facing the thoracic cavity. The abdominal/ crural region was defined as the remaining one half of the cross section facing the abdominal cavity.[15]

Muscle Histochemistry

Transverse sections from each biopsy were cut to a 10-[Micro]m thickness using a cryostat-microtome[double dagger] and processed for M-ATPase fiber types at a preincubation pH of 4.5 for acid-stable M-ATPase activity.[15] This staining procedure enabled three fiber types to be discerned, which could be categorized as (1) type I, dark, (2) type IIa, pale, and (3) type IIb, intermediate.[15] A previous investigation[15] showed that using the M-ATPase technique, type I and type II fibers at a preincubation pH of 4.5 matched those fiber types processed at a preincubation pH of 9.4, and a neglible number of type IIc fibers were found. All sections from the same batch were stained on the same day to minimize any interassay variation.

Percentages and Cross-sectional Areas of Muscle Fiber Types

Measurements of muscle fiber-type percentages and sizes were performed in a blinded fashion such that the observer did not know from which group the muscle cross sections were obtained. Fiber-type percentages were determined by typing all fibers in a region (approximately 600-800 fibers) and then dividing the number of fibers in a particular fiber-type category by the total number of fibers in a region. Cross-sectional areas were determined using a light microscope equipped with a camera lucida[section] and a digitizing pad([parallel]) connected to a personal computer(#) that used a morphometric software package(**). The image from the light microscope was projected onto the digitizing pad. Before each session, the system was calibrated across the entire surface of the digitizing pad to ensure that the appropriate magnification was being used and that the camera lucida was oriented squarely relative to the digitizing pad. Next, 50 fibers randomly selected from each fiber-type category and from each region were outlined using the cursor on the digitizing pad, and the cross-sectional area was determined. A total magnification of x 250 was used (de, objective x 25 and eyepieces x 10).

Data Analysis

Mean muscle fiber sizes and percentages for each animal in each region and type were determined. A three-way analysis of variance (ANOVA) blocking on animals nested within groups was performed to examine for differences between the dependent variables, which were fiber sizes and percentages, and the independent variables, which were group (control and swimming), region (costar, thoracic/crural, and abdominal/cruel) and fiber type (I, IIa, and IIb). Interactions were considered significant at P [is less than] .10. If interactions were present, appropriate ANOVAs were performed. The sequential rejective Bonferroni's procedure was used to correct for multiple comparisons.[30] Differences in weight between groups and over time compared with baseline (week 0) were examined using a two-way repeated-measures ANOVA with time as the repeated measure, followed by the Bonferroni multiple-comparisons procedure to examine for differences between weights at baseline and subsequent time intervals. Differences were considered to be significant at a corrected probability value of P [is less than] .05.

Results

Mean values and raw data for fiber-type sizes in each animal and region are shown in Figure 1 and Table 1, respectively. A summary of the results of the three-way ANOVA examining for differences in fiber-type areas is shown in Table 2. The two-way ANOVAs showed significant interactions for group x region and for region x fiber type. Thus, one-way ANOVAs were performed. A one-way ANOVA showed that muscle fiber sizes in the thoracic/crural region were smaller in the hamsters in the swimming group than in the hamsters in the control group. Another one-way ANOVA showed that muscle fiber sizes in the control and swimming groups were not different among regions. A third one-way ANOVA showed that type IIb fibers were larger than type I fibers in the costar and thoracic/crural regions and that type IIb fibers were larger than type IIa fibers in all three regions. The absence of data in Figure 1 for cross-sectional area of type II fibers in the abdominal/cruel region was due to the small number of fibers in this region. Thus, there were insufficient data for cross-sectional area of type I fibers in the abdominal/cruel region to be included for statistical analysis.

[Figure 1 ILLUSTRATION OMITTED] Table 1. Means, Standard Deviations, and Raw Data (in Square Micrometers) by Muscle Fiber Type and Region(a) for Control and Swimming Groups

        Animal           Costal
Group   No.
                  I       IIa    IIb

Control   1     1,776     2,398    3,584
          2     1,655     1,491    3,112
          3     1,598     1,368    2,190
          4     1,236     1,389    2,198
          5     1,633     1,693    2,990
          6     1,637     1,770    3,583
          7     2,169     1,866    3,571
          8     2,137     2,333    3,759
          9     1,709     1,639    2,787
                1,728
X                 284     1,772    3,086
SD                          375     599
Swimming  1     1,364     1,836    2,168
          2     1,691     1,468    3,197
          3     1,628     1,993    3,250
          4     2,004     1,720    3,130
          5     2,007     1,862    3,119
          6     1,804     1,883    2,525
          7     2,043     2,231    3,865
          9     1,575     1,587    3,075
         10     2,080     2,280    4,359

X               1,800     1,873    3,187
SD                251       269      646

                        Thoracic/Crural
Group
                       I         IIa      IIb

Control              2,478      2,317     3,516
                     1,622      1,542     1,752
                     2,728      2,272     2,989
                     1,960      1,981     2,908
                     2,583      2,818     4,081
                     1,793      2,192     2,387
                     2,147      2,016     2,748
                     2,470      2,671     3,274


X                    2,223      2,226     2,957
SD                     402        402       707
Swimming             1,820      1,977     2,481
                     1,807      1,693     2,518
                     1,867      1,717     2,206
                     2,731      2,407     3,407
                     2,373      2,788     2,767
                     2,287      1,687     1,974
                     1,658      1,764     3,057
                     1,728      1,451     1,888
                     1,678      1,458     1,922

X                    1,994      1,882     2,469
SD                     377        445       533

                   Abdominal/Crural
Group              IIa        IIb

Control            1,591      3,267
                   1,218      1,662
                   1,699      2,810
                   1,562      2,442
                   1,797      4,076
                   1,061      1,613
                   1,424      2,534
                   1,288      2,213


X                  1,455      2,577
SD                   253        819
Swimming           1,174      1,756
                   1,070      2,256
                     979      2,146
                   1,274      2,437
                   1,383      2,572
                   1,138      1,956
                   1,482      3,888
                   1,185      1,697
                   1,070      2,339

X                  1,195      2,339
SD                 160          652

(a) Thoracic/crural=the half of the crural region closest to the thoracic cavity; abdominal/crural=the half of the crural region closest to the abdominal cavity.

Table 2. Results of Three-Way Analysis of Variance for Fiber-Type Areas

Source                       df       SS            MS

Group                         1    6.12E + 05   6.12E + 05
Region                        1    1.44E + 05   1.44E + 05
Fiber type                    2    2.27E + 07   1.13E + 07
Group X region                1    1.55E + 06   1.55E + 06
Group X fiber type            2    5.91E + 04   2.96E + 04
Region X fiber type           2    3.01E + 06   1.50E + 06
Group X region X fiber type   2    9.14E + 04   4.57E + 04

Source                              F       P

Group                             3.91   .0514
Region                            0.92   .3401
Fiber type                       72.55   .0000
Group X region                    9.90   .0024
Group X fiber type                0.19   .8282
Region X fiber type               9.60   .0002
Group X region X fiber type       0.30   .7475

(a) Interaction significant at P [is less than] .10.

Mean values and raw data for fiber-type percentages in each animal and region are shown in Figure 2 and Table 3, respectively. A summary of the results of the three-way ANOVA examining for differences in fiber-type percentages is shown in Table 4. The two-way ANOVAs showed significant interactions for group X fiber type and for region X fiber type. Thus, one-way ANOVAs were performed. A one-way ANOVA showed that the percentage of fiber types in the three different regions of the diaphragm did not differ between the control and swimming groups. A second one-way ANOVA showed that the percentages of all fiber types in the control and swimming groups were different from each other. A third one-way ANOVA showed that the percentages of all fiber types in the costar and abdominal/crural regions were different from each other but were similar in the thoracic/crural region. A fourth one-way ANOVA showed that the percentages of type I and type IIb fibers differed among regions but that the percentages of type IIa fibers were similar among regions.

[Figure 2 ILLUSTRATION OMITTED]

Table 3.
Fiber-Type(a) Percentages in Control
and Swimming Groups

               Animal                 Costal
Group           No.             I       IIa     IIb

Control           1            25.0       33.9    41.1
                  2            20.9       34.8    44.3
                  3            25.7       29.4    44.9
                  4            19.2       25.9    55.0
                  5            25.1       35.7    39.2
                  6            24.2       29.6    46.2
                  7            23.6       28.1    48.3
                  8            22.3       29.4    48.3
                  9            22.3       37.2    40.5
X                              23.1       31.6    45.3
SD                              2.2        3.9     4.9

Swimming          1            26.7       27.7    45.5
                  2            27.4       26.6    46.0
                  3            25.7       31.5    42.8
                  4            25.7       23.3    51.0
                  5            26.8       31.5    41.6
                  6            22.0       38.9    39.2
                  7            24.7       36.6    38.7
                  8            22.1       38.7    39.2
                  9            30.0       31.2    38.9
X                              25.7       31.8    42.5
SD                              2.5        5.5     4.2

                        Thorocic/Crural

Group                   I       IIa      IIb

Control                28.3       32.3    39.4
                       36.0       39.1    24.9
                       34.2       31.3    34.6
                       27.4       36.4    36.2
                       42.3       32.0    25.7
                       50.0       28.4    21.6
                       20.4       40.3    39.2
                       26.7       34.5    38.8

                       33.2       34.3    32.6
                        9.5        4.1     7.3

Swimming               59.4       20.1    20.5
                       32.7       29.8    37.6
                       36.5       24.7    38.8
                       24.0       47.9    28.1
                       37.5       35.1    27.5
                       32.6       29.8    37.6
                       27.3       34.5    38.1
                       44.9       27.3    27.8
                       26.0       34.9    39.1
X                      35.7       31.6    32.8
SD                     11.0        7.9     6.9

                        Abdominal/Crumel

Group                    I      IIa     IIb

Control                 8.6       30.4    60.9
                        3.9       27.0    69.1
                        4.3       47.7    48.0
                        2.4       34.1    63.5
                       10.2       28.9    60.9
                        2.6       37.8    59.6
                        3.3       32.2    64.5
                        3.1       30.3    66.7

                        4.8       33.6    61.7
                        2.9        6.6     6.4

Swimming                7.4       26.9    65.7
                        5.2       30.1    64.7
                       13.4       27.4    59.2
                       12.4       32.1    55.5
                        6.7       38.8    54.4
                        7.5       30.2    62.3
                       14.2       28.3    57.5
                        9.3       29.8    60.9
                        4.9       31.4    63.7
X                       9.0       30.6    60.4
SD                      3.5        3.5     4.0

(a) Thoracic/crural=the half of the crural region closest to the thoracic cavity; abdominal/crural=the half of the crural region closest to the abdominal cavity.

Table 4. Results of Three-Way Analysis of Variance for Fiber-Type Percentages

Source                      df          SS

Group                         1        0.02371
Region                        2        0.00003
Fibertype                     2    14838.00
Group X region                2        0.00068
Group X fiber type            2      184.240
Region X fiber type           4    13256.10
Group X region X fiber type   4       55.7934

Source                           MS          F            P

Group                            0.02371      0.00061    .9804
Region                           0.00001      0.00000   1.0000
Fibertype                     7418.99       189.539      .0000
Group X region                   0.00034      0.00001    .9999
Group X fiber type              92.1198       2.35346    .0994(a)
Region X fiber type           3314.03        84.6661     .0000(a)
Group X region X fiber type     13.9473       0.35635    .8392

(a) Interaction significant at P [is less than] .10.

Body weight did not differ between the control and swimming groups during the 13-week training period (Fig. 3). Increases in body weight occurred in both groups, as shown by measurements obtained at 6, 8, 10, 12, and 13 weeks compared with baseline (week 0) values.

[Figure 3 ILLUSTRATION OMITTED]

Discussion

Our results showed that increased ventilation induced by swimming during a 13-week endurance training program decreased the size of muscle fibers in the thoracic/ crural region of the diaphragm but failed to cause any change in fiber-type percentages. Although a decrease in muscle fiber size is often considered to be indicative of disuse atrophy,[17,31] it may also be a response to over-use.[19] From the data obtained in our study, it is not possible to determine what induced the decrease in fiber size. Our findings, however, are consistent with the hypothesis that different regions of the diaphragm respond differently to an exercise stimulus.

Although muscle fiber sizes may increase in response to endurance training,[18] fiber atrophy may also occur.[19] A decrease in the cross-sectional area of muscle fibers in response to endurance training may facilitate diffusion by decreasing the diffusion distance for oxygen to be transported from the capillaries to all parts of the fiber during exercise. Reduction in food intake could induce a reduction in body weight and has also been associated with a reduction in the cross-sectional area of fibers in the diaphragm.[32] Although we did not measure food intake in our hamsters, we measured their body weight and found no differences in body weight between the control and swimming groups. Thus, it is likely that some aspect of swim training and not differences in body weight contributed to the reduction in fiber size in the thoracic/crural region of the diaphragm in the hamsters in the swimming group.

A power analysis[33] showed that to achieve statistical significance in the most prominent trend toward a greater percentage of type I fibers in the abdominal/ crural region at P [is less than] .05, 22 or 30 animals in each group needed to be studied for a power of 0.8 or 0.9, respectively. The effect size of swimming on diaphragm fiber-type percentages is smaller than that of other protocols such as treadmill running[25,26] or inspiratory resistive loading.[22-24] In a study by Green et al,[25] treadmill running produced a 34% reduction in the proportion of IIa fibers and a 15% increase in the proportion of IIb fibers in the costar diaphragm after a 13-week endurance training program. Inspiratory resistive loading resulted in a 23% increase in the type I fibers of the diaphragm after a 3-week intermittent program[22] and a 40% increase in type I fibers after a 24- to 28-week period of continuous loading induced by tracheal banding.[23] These differences in fiber-type percentages were achieved after treadmill running[25] or inspiratory resistive loading,[23] even though the numbers of animals used in these studies were similar to or smaller than the numbers of animals in our groups.

Swimming, though a form of whole-body endurance exercise, may not be the best stimulus to induce muscle fiber-type transitions in the diaphragm compared with other nonaquatic endurance training programs such as treadmill running. Maximal oxygen consumption is lower during swimming[34-36] than during running[34,35,37] in people and rodents. Furthermore, minute ventilation at a similar rate of oxygen consumption during swimming and running is lower during swimming.[33,34] Thus, the ventilatory overload induced by swimming may not be sufficient to induce as great a training response to the diaphragm. In addition, it is also difficult to control and monitor the degree of exercise being performed while the animal is in the water compared with treadmill running. Even though the swimming activity was supervised and encouraged, the pace of swimming may have been less than maximal for the duration of all swimming sessions.

In contrast to previous training studies using treadmill running or inspiratory resistive loading, swimming did not change muscle fiber-type percentages in the diaphragms of the hamsters in our study. Inspiratory resistive loading applied intermittently[22] or continuously[23,24] for long periods increased the percentage of type I fibers stained for M-ATPase in the costar region of the diaphragm. Treadmill running has produced conflicting data. It increased the percentage of type IIb fibers determined histochemically[25] but decreased the percentage of myosin heavy-chain isoform IIB[26] in the costar region of the diaphragm of rats. In another study,[38] no change in fiber-type percentages of the diaphragm was found after treadmill running.

Although swim training did not change fiber-type percentages, other changes characteristic of endurance training may have occurred in the diaphragm. Increased oxidative metabolism is an early change often seen in skeletal muscle during an endurance training program.[31] There is considerable evidence demonstrating that a continuous moderate-intensity training program using treadmill training[25,39,40] or swim training[41] increases the oxidative potential of the diaphragm. We did not measure any markers of oxidative metabolism in our study, so we are unable to confirm these findings.

Conclusion

Swim training decreased the size of muscle fibers in the thoracic/crural region of the diaphragm but did not change muscle fiber-type percentages in the diaphragm. Thus, swim training may have improved the endurance of this region of the diaphragm by decreasing the cross-sectional area and thus decreasing the distance for oxygen to diffuse to the internal regions of the muscle fiber. Another potential mechanism that may have occurred in this study is an increase in oxidative potential, as shown by previous work.[14] A third potential mechanism of improving endurance is increasing the pool of type I fibers,[31] thereby creating a larger pool of type I fibers that can be alternately recruited during the relatively low-intensity forces required during breathing. We did not find this to occur in response to swim training. Inspiratory resistive training is the only technique that consistently increases the percentage of type I fibers in the costar region of the diaphragm, in addition to increasing other typical training responses such as oxidative metabolism.[27]

Swimming may have many therapeutic benefits for patients receiving physical therapy; however, its impact on the diaphragm appears to be less strong than that of other interventions such as treadmill running or inspiratory resistive loading. These benefits should be carefully considered before encouraging a patient with respiratory dysfunction to perform swimming, other activities recruiting the upper or lower extremities, or a specific respiratory muscle training technique.

(*) Charles River Laboratories, La Prairie, Quebec, Canada.

([dagger]) PMI Feeds Inc, 1401 Hanley Rd, St Louis, MO 63144.

([double dagger]) Ames Cryostat II, Ames, Div of Miles Laboratories Inc, Elkhart, IN 46514.

([section]) Labophot, Nikon Inc, 1300 Walt Whitman Rd, Melville, NY 11756.

([parallel]) SummaSketch II, model MMII 1201, Summagraphics Corp, 60 Silvermine Rd, Seymour, CT 06483-3907.

(#) Packard Bell/NEC Canada Inc, 1024 Tristar Dr, Mississauga, Ontario, Canada L5T IV5.

(**) Bioquant System PV, R & M Biometrics, 5611 Ohio Ave, Nashville, TN 37209.

The study protocol received ethics approval from the University of British Columbia Animal Care Committee.

This project was supported by the Medical Research Council of Canada and the Canadian Lung Association.

This article was submitted October 5, 1995, and was accepted September 23, 1996.

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WD Reid, PhD, BMR(PT), is Associate Professor, School of Rehabilitation Sciences, University of British Columbia, Vancouver, British Columbia, Canada V6T 2B5. Address all correspondence to Dr Reid at School of Rehabilitation Sciences, University Hospital UBC Site, T325-2211 Wesbrook Mall, Vancouver, British Columbia, Canada V6T 2B5 ([e-mail]).

J Shanks, BSc(PT), is Physiotherapist, Enderby and District Memorial Hospital, Enderby, British Columbia, Canada VOE 1VO.

B Samrai, BSc, is a student in the Master of Science Program in Experimental Medicine, University of British Columbia.