@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix dc: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Medicine, Department of"@en, "Experimental Medicine, Division of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Naghshin, Jahanbakhsh"@en ; dcterms:issued "2009-09-28T23:04:53Z"@en, "2002"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "Airway smooth muscle (ASM) is known to adapt to large changes in length by restructuring its contractile apparatus. The temporary loss of the ability to generate maximal isometric force due to an acute length change usually recovers in 20-30 min when the muscle is stimulated periodically. ASM is capable of adapting to length changes even in the absence of repeated stimulation, although at a much slower rate. It has been previously shown that by setting the relaxed ASM at different lengths at 4 °C in physiological saline solution (PSS) for 24 hrs, the length-tension (L-T) relationship of the muscle could be plastically and reversibly altered [53]. In this study, I examined the time course of length adaptation at 4 °C to determine the minimum time needed for this process to happen. By measuring changes in the length associated with maximal force generation (L[sub max]), I found that under the same conditions used by Wang et al [53], the length adaptation became statistically significant after 6-12 hours of passive length change. I hypothesized that a similar length adaptation can also happen at 37°C when the ASM is maintained unstimulated at different lengths. The reversibility of active and passive length adaptations following prolonged length change was also examined. Rabbit tracheal muscle explants were passively maintained at shortened or in situ length for 3 and 7 days in culture media enriched with 5% fetal bovine serum. Using high K⁺ PSS to elicit contraction, the L-T relationship of control (CTL) and passively shortened (PS) preparations was examined and active tension recovery was measured on PS preparations. Formalin fixation, paraffin embedding, and morphometric analysis were used to measure the cross sectional area (CSA) of preparations, normalize the maximal active force (F[sub max]) and calculate the maximal stress (σ[sub max]. To examine the effect of muscle activation on passive tension recovery, using the same tissue preparation and culturing techniques, the recovery test was performed at Day 7 (or 8) PS preparations in the presence vs. absence of stimulations. Furthermore, the active recovery test was done on freshly isolated A SM preparations (Control Day 0; n = 5) and the active and passive force recovery rates were compared to those of the chronically length-adapted preparations (i.e. PS Day 7 or 8). Following 3 and 7 days of passive shortening (without stimulation), L[sub max] decreased by 10.6%±9.4 and 35.9%±15.9 (mean ±SD) respectively compared to the control preparations. There was also no significant change in σ[sub max] of PS smooth muscles compared to the control. After 7 days of passive shortening, it was either impossible to stretch the ASM preparations to their control lengths and/or they could not generate the same active force they did at their pre-stretched lengths. Following such a stretch, both active and passive force recoveries were incomplete. However, the passive tension recovery rate was not different in the presence vs. absence of muscle stimulations. It was observed that the active tension recovery could be initiated and progressed passively after stretching the PS preparations. Although the passive force recovery was greater than the active force recovery, these two processes were strongly correlated. Furthermore, the active force recovery was significantly slower in length adapted (Day 7 or 8) preparations compared to the CTL Day 0 group. However, the passive force recovery rate was not different between these two groups. I conclude that rabbit ASM is able to adapt to chronic shortening at body temperature even when it is not stimulated and the magnitude of the shift in L-T curve increases with time of length adaptation. By stretching a 7-day length adapted preparation to its original length, neither the active, nor the passive force could recover completely. Although muscle activation accelerates the active force recovery [14], it did not affect the passive force recovery. This result may have implications in asthma and COPD where chronic shortening of ASM could make the patients highly resistant to conventional bronchodilating therapies or to mechanical bronchodilating influences such as tidal breathing or deep inspiration."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/13291?expand=metadata"@en ; dcterms:extent "5157016 bytes"@en ; dc:format "application/pdf"@en ; skos:note "The Time Course of Length Adaptation and Tension Recovery in Airway Smooth Muscle by JAHANBAKHSH NAGHSHIN M.D, Isfahan University of Medical Sciences & Health Services, Isfahan, Iran, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in FACULTY OF GRADUATE STUDIES (EXPERIMENTAL MEDICINE PROGRAM) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 2002 © Jahanbakhsh Naghshin, 2002 In presenting t h i s thesis i n p a r t i a l f u l f i l l m e n t of the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of th i s thesis f o r sc h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of _EXPERIMENTAL MEDICINE, The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada ABSTRACT Airway smooth muscle (ASM) is known to adapt to large changes in length by restructuring its contractile apparatus. The temporary loss of the ability to generate maximal isometric force due to an acute length change usually recovers in 20-30 min when the muscle is stimulated periodically. A S M is capable of adapting to length changes even in the absence of repeated stimulation, although at a much slower rate. It has been previously shown that by setting the relaxed A S M at different lengths at 4 °C in physiological saline solution (PSS) for 24 hrs, the length-tension (L-T) relationship of the muscle could be plastically and reversibly altered [53]. In this study, I examined the time course of length adaptation at 4 °C to determine the minimum time needed for this process to happen. By measuring changes in the length associated with maximal force generation (Lmax), I found that under the same conditions used by Wang et al [53], the length adaptation became statistically significant after 6-12 hours of passive length change. I hypothesized that a similar length adaptation can also happen at 37°C when the A S M is maintained unstimulated at different lengths. The reversibility of active and passive length adaptations following prolonged length change was also examined. Rabbit tracheal muscle explants were passively maintained at shortened or in situ length for 3 and 7 days in culture media enriched with 5% fetal bovine serum. Using high K + PSS to elicit contraction, the L-T relationship of control (CTL) and passively shortened (PS) preparations was examined and active tension recovery was measured on PS preparations. Formalin fixation, paraffin embedding, and morphometric analysis were used to measure the cross sectional area (CSA) of preparations, normalize the maximal active force (Fmax) and calculate the maximal stress (Omax)- To examine the effect of muscle activation on passive tension recovery, using the same tissue preparation and culturing techniques, the recovery test was performed at Day 7 (or 8) PS preparations in the presence vs. absence of stimulations. Furthermore, the active recovery test was done on freshly isolated A S M preparations (Control Day 0; n - 5) and the active and passive force recovery rates were compared to those of the chronically length-adapted preparations (i.e. PS Day 7 or 8). Following 3 and 7 days of passive shortening (without stimulation), Lmax decreased by 10.6%±9.4 and 35.9%±15.9 (mean ±SD) respectively compared to the control ii preparations. There was also no significant change in amax of PS smooth muscles compared to the control. After 7 days of passive shortening, it was either impossible to stretch the A S M preparations to their control lengths and/or they could not generate the same active force they did at their pre-stretched lengths. Following such a stretch, both active and passive force recoveries were incomplete. However, the passive tension recovery rate was not different in the presence vs. absence of muscle stimulations. It was observed that the active tension recovery could be initiated and progressed passively after stretching the PS preparations. Although the passive force recovery was greater than the active force recovery, these two processes were strongly correlated. Furthermore, the active force recovery was significantly slower in length adapted (Day 7 or 8) preparations compared to the CTL Day 0 group. However, the passive force recovery rate was not different between these two groups. I conclude that rabbit A S M is able to adapt to chronic shortening at body temperature even when it is not stimulated and the magnitude of the shift in L-T curve increases with time of length adaptation. By stretching a 7-day length adapted preparation to its original length, neither the active, nor the passive force could recover completely. Although muscle activation accelerates the active force recovery [14], it did not affect the passive force recovery. This result may have implications in asthma and COPD where chronic shortening of A S M could make the patients highly resistant to conventional bronchodilating therapies or to mechanical bronchodilating influences such as tidal breathing or deep inspiration. iii TABLE OF CONTENTS Page Abstract ii Table of Contents iv List of Tables vi List of Figures vii List of Abbreviations viii Acknowledgements ix CHAPTER 1 Introduction and Background 1.1. Airway Smooth Muscle and Its in vivo Function 1 1.2. Length-Tension Relationship 3 1.3. Length Adaptation of Muscle 4 1.3.1. Skeletal Muscle 4 1.3.2. Smooth Muscle 5 1.4. Relation to Previous Studies 6 1.5. Specific Aims 9 1.5.1. The time course of length adaptation in A S M 9 1.5.2. Understanding the length-adaptation reversibility 9 1.5.3. The passive tension recovery process and the importance of stimulations in this process 10 1.5.4. The contribution of passive force in active force recovery 10 CHAPTER 2 The Time Course of Length Adaptation at 4°C 2.1. Methods 17 2.1.1. Adaptation to length change after 12 hours 18 2.1.2. Adaptation to length change after 6 hours 19 2.2. Data Analysis 20 2.3. Results 20 2.3.1. Length-tension measurements after 12 hours 20 2.3.2. Length-tension measurements after 6 hours 21 CHAPTER 3 The Airway Smooth Muscle Length-Tension Properties at Body Temperature 3.1. Length Adaptation at 37°C and Tension Recovery Test 29 3.1.1. Methods 29 - Tissue Culturing 30 - Mechanical Measurements 31 - Morphometric Studies 34 3.1.2. Data Analysis 35 3.1.3. Results 36 iv Page 3.2. Active vs. Passive Tension Recovery Test 39 3.2.1. Methods 39 3.2.1.1. Active Tension Recovery 40 3.2.1.2. Passive Tension Recovery 40 3.2.2. Data Analysis 41 3.2.3. Results 42 3.3. Active Recovery in Day 0 Preparations 44 3.3.1. Methods 44 3.3.2. Data Analysis 45 3.3.3. Results 45 CHAPTER 4 Discussion • Discussion 74 • Shift in L-T Relationship 74 • Tension Recovery 77 • The Rationale for the Methods 82 - Culturing Conditions 82 - Type of Stimulant 83 - The Frequency of Stimulations by High K + PSS 84 • Summary and Clinical Implications 86 BIBLIOGRAPHY 97 Appendix (I) Equations 105 v List of Tables No. Title Page 2.1 The effect of chronic passive lengthening on rabbit A S M after 12 hours at 4°C; the Lin s i t u , Lmax, and Fmax values 27 2.2 The effect of chronic passive lengthening on rabbit A S M after 6 hours at 4°C; the Lin s i t u , Lmax, and Fmax values 28 3.1 The Lm situ values of rabbit A S M preparations, Day 0, 3, and 7 55 3.2 Lmax values of rabbit A S M preparations, Day 0, 3, and 7 56 3.3 Maximal Force (Fmax) values of A S M preparations, Day 0, 3, and 7 57 3.4 The Maximal Stress values ( 0.05 was not considered statistically significant. In each case, n is the number of muscle preparations used. 2.3. RESULTS 2.3.1. Length-tension measurements after 12 hours: The Fmax values for the control and passively lengthened preparations were 11.6 ±3.8 mN and 13.5 ±3 .0 mN respectively (mean ± SD). The Fmax values were not normalized by the cross sectional areas in this experiment. No significant change was found in absolute Fmax values after 12 hours of chronic length change (P > 0.05). It was observed that the Lmax of passively lengthened preparations was shifted to lengths longer than the Lmax of control smooth muscles from the same animal (P = 0.035). The mean value of Lmax was 19.3% longer in passively lengthened compared to the control group. Figure 2-5 shows an example of the observed changes in L-T curves. As was observed in the case of lengthening for 24 hours 20 [53], both the active and passive force curves shifted to longer lengths due to chronic lengthening. 2.3.2. Length-tension measurements after 6 hours: The mean and SD of F O T a x for the control and passively lengthened preparations were 9.33 ± 3.16 mN and 9.56 ± 3.48 mN respectively. In this part of the experiment, no significant difference was found in mean values of Fmax (P = 0.80) and Lmax (P = 0.65). The mean value of Lmax of passively lengthened preparations was 4% longer than the control group, but it was not statistically significant. 21 Figure 2-1: A : A piece of rabbit trachea before starting the dissection (each small division on the ruler is 1.0 mm. The tissue is placed in cold PSS at room temperature. B: The same piece after removing superficial layer of connective tissue, vasculature, and fat. Dissecting pins are used to immobilize the tissue during the microscopic dissection. 22 • 1 | I 2 | CENTIMETRES Figure 2-2: A: Shows the way the length of smooth muscle (Lin situ) was measured before cutting the tracheal cartilage. Each division on the ruler is 0.25 mm. The arrow marks an area where smooth muscle bundles are more visible in this picture. B: Shows the lateral view of the same piece of trachea after the cartilage has been cut. The smooth muscle tissue is located at the middle part. 23 Figure 2-3: A : Demonstrates the outside view of the tracheal tissue before starting the dissection. B: The inside view of the same piece showing the endothelium, small blood vessels and a thin layer of connective tissue which covers the smooth muscle layer. 24 Figure 2-4: A : Shows a piece of the same trachea including two rings (outside view) after finishing the microscopic dissection. B: Demonstrates the final preparation. Two aluminum clips are holding the cartilage tissue at both sides. The entire length of smooth muscle (in the relaxed state) can be seen between two clips. 25 14 -, P a s s . Len, AF Pass. Len, PF Control, AF Control, PF Passively Lengthened o 2 3 4 Length (mm) Figure 2-5: This figure shows L-T curves (absolute values) from a pair of muscle preparations which were adjacent to each other in the distal part of the same trachea. The preparations were maintained at predetermined lengths for 12 hours. The upper curves demonstrate the active and lower curves the passive forces. One preparation was used as reference and set at its relaxed length ( V and • ) and the other (O and • ) was passively stretched to its Lin situ. Both the active and passive force curves shifted to longer lengths following 12 hours of passive lengthening. 26 CONTROL PASSIVELY LENGTHENED Animal # Li„ situ (mm) Lmax (mm) Fmax (mN) Lm situ (mm) Lmax (mm) Fmax (mN) 1 2.25 1.80 13.01 2.25 2.11 13.57 2 2.5 2.17 7.52 2.5 2.26 11.96 3 3.0 1.36 13.78 3.0 2.53 15.37 4 4.0 4.00 17.80 4.0 3.58 16.03 5 3.5 3.44 14.55 3.5 3.42 17.59 6 4.0 2.58 13.66 4.0 3.67 14.18 7 3.0 2.37 6.95 3.0 2.46 11.11 8 3.5 1.75 11.45 3.75 3.39 7.56 9 3.5 2.70 14.87 3.5 2.82 17.37 10 2.75 1.71 6.96 2.75 2.19 11.80 11 2.5 1.79 7.66 2.5 2.16 11.66 Mean ± SD 3.14 ±0.6 2.33 ±0.8 11.65 ±3.8 3.16 ±0.6 2.78 ±0.6* 13.47 ±3.0 Table 2-1: The effect of chronic passive lengthening on rabbit A S M after 12 hours at 4°C. Data include Lin situ, Lmax (both in mm), and Fmax (in mN) values of both control and passively lengthened groups. Except in one animal, the in situ lengths were equal for both groups. The Weibull 4-parameter equation of peak function (Sigma Plot 5.0) was used to obtain Fmax and Im c Kdata. [*: Statistically significant change; P = 0.035] 27 CONTROL PASSIVELY LENGTHENED Animal # Lm situ (mm) Lmax (mm) Fmax (mN) Lin situ (mm) Lmax (mm) Fmax (mN) 1 2.25 1.91 9.10 2.25 1.93 12.24 2 2.5 2.09 5.68 2.5 2.08 9.07 3 3.0 2.81 9.27 3.0 2.24 14.48 4 3.25 2.73 10.01 3.5 3.21 11.92 5 2.5 2.11 15.17 2.5 2.39 12.87 6 3.25 2.48 13.59 3.0 2.69 12.80 7 2.25 1.45 6.68 2.25 1.53 6.00 8 2.25 2.37 5.98 2.25 1.93 5.03 9 2.5 2.17 9.77 2.5 2.21 5.06 10 2.5 1.77 5.99 2.5 2.17 6.60 11 2.5 1.83 11.38 2.5 1.81 9.07 Mean ± SD 2.61 ± 0.4 2.16 ±0.4 9.33 ±3.2 2.61 ± 0.4 2.20 ±0.4 9.56 ±3.5 Table 2-2: The effect of chronic passive lengthening on rabbit A S M after 6 hours at 4°C. Data include Lin Situ, Lmax (both in mm), and Fmax (in mN) values of control and passively lengthened groups. The Weibull 4-parameter equation of peak function (Sigma Plot 5.0) was used to obtain Fmax and L m a x data. Except in two animals, the Lin s i t u values were identical for CTL and passively lengthened preparations. The changes in Lmax and Fmax between the two groups were not statistically significant (P = 0.65 and 0.80 respectively). 28 Chapter Three: The A S M L-T Properties at Body Temperature In this chapter three different experiments are discussed. 3.1. Length Adaptation at 37°C and Tension Recovery Test In order to have a closer look at the length adaptation process, previously described in Chapter 2, a similar model was used at 37°C. 3.1.1. METHODS Tissue Preparation: For this part of the experiment a total of 6 rabbits (New Zealand White, 2-4 mo, 2.0-2.5 kg) were used. The rabbits were temporarily housed and sacrificed in the animal facility of St. Paul's Hospital according to the procedure approved by the U B C Committee on Animal Care. The animals were sacrificed by an intravenous injection of Euthanyl (which contains sodium pentobarbital 240 mg/ml). The anterior part of the upper chest and the lower neck was opened. The trachea was removed immediately and placed in sterile 4°C Leibovitz's L-15 medium (GIBCO BRL). Penicillin (10,000 unit/ml) and Streptomycin (10,000 ug/ml; both from GIBCO BRL) were previously added to Leibovitz's L-15 medium. Under aseptic conditions and using microscopic dissection, 5 strips were dissected and prepared from each trachea as described in Chapter 2. The in situ length was measured for all the samples individually. Each preparation contained two tracheal rings 29 and was about 2.2-2.5 mm wide. Tissue preparations were assigned to different categories: Control Day Zero (CTLO), Control Day 3 (CTL3), Control Day 7 (CTL7), Passively Shortened Day 3 (PS3), and Passively Shortened Day 7 (PS7). For each trachea, the above-mentioned strips were selected randomly. However, for Day 3 and Day 7 preparations, each pair was dissected from adjacent tracheal rings with comparable in situ lengths. The L-T curve of the CTLO preparation was determined on day zero immediately after finishing the dissection. The rest of the preparations were placed on sterilized dental wax. Under a dissecting microscope, the control preparations, CTL3 and CTL7, were carefully stretched and set at their in situ lengths by securing them with sterilized dissecting pins. The passively shortened preparations, PS3 and PS7, were stimulated for 3-5 seconds using the platinum electrodes of an A C stimulator (60 Hz frequency). Following the single contraction, their lengths were set using the same method as that used for the CTL3 and CTL7 preparations. On average, the PS preparations were set at 27.6% of the CTL preparations' length. Tissue Culturing: The preparations were immediately transferred to the culture room, where they were placed in individual wells of a six-well culture plate (handled at room temperature). Using 4°C C M R L medium-1066 (GIBCO BRL) mixed with penicillin (10,000 unit/ml) and streptomycin (10,000 ug/ml), the explant muscle preparations were washed three times. The preparations were then covered with 8ml of C M R L Medium-1066 at 4°C, enriched with 5% fetal bovine serum (FBS), bovine insulin (4mg/ml), L -glutamine-200mM (10 ul/ml), amphotericin B (250 ug/ml), penicillin (10,000 unit/ml), 30 and streptomycin (10,000 |ig/ml). A l l the supplements were purchased from GIBCO B R L Company (Figure 3-1). At this stage, the culture plates were transferred to a controlled atmosphere gas chamber (BELLCO GLASS INC.). The chamber was immediately filled with a gas mixture of 4.85% C 0 2 , 44.5% 0 2 , and balanced Nitrogen (PRAXAIR Company) for 5-10 min, at room temperature. By doing this, the pressure inside the chamber was able to reach 3 times atmospheric pressure. In order to humidify and disinfect the chamber, an open dish filled with 10-15 ml of 2% copper sulfate in water was placed on the lower shelf of the gas chamber. The chamber was then transferred to an incubator where it was placed on a rocker platform (BELLCO GLASS INC.) and agitated at 10-cycles/ minute at 37°C. CTL3, PS3, CTL7, and PS7 (Day 3 and Day 7) preparations were kept under these conditions for 3 and 7 days respectively before being subjected to the mechanical measurements. The culture medium was changed with freshly prepared solution and the chamber was refilled with the above-mentioned gas mixture every 24 hours. Mechanical Measurements: Except for the Day 0 experiment, the protocol for mechanical measurements done on Day 3 and Day 7 was as follows. The control preparation was removed from the gas chamber and the chamber was refilled and returned to the incubator. The preparation was then transferred to a tissue bath and mounted as mentioned in Chapter 2. The tissue bath contained physiological saline solution (PSS) at 37°C that was bubbled with a 95% 0 2-5% C 0 2 mixture. 31 A high potassium PSS (80 mM) was used to induce smooth muscle contraction. The rationale behind using the high K + PSS can be found in Chapter 4. To produce an isometric contraction the PSS was replaced with high K + PSS. The maximal active force was calculated by averaging force recorded over a 10-second period in the plateau of contraction minus the resting tension. With each high K + PSS stimulation, the muscle was allowed to achieve a maximum force at a prolonged sustained plateau or tonic contraction following a phasic contraction. The tonic part was chosen over the phasic part of contraction because during the preliminary experiments the former proved to be more constant and reproducible than the latter. The average time to reach the plateau was different between the preparations. On average it was between 450 to 800 seconds. However, this time was almost constant for individual A S M strips during repetitive contractions. The second peak (tonic component) was either absent or smaller than the first peak (phasic contraction) in some preparations (mostly in CTLO experiments). In such situations, the first peak was chosen as it was found more reliable for the measurements. After reaching the plateau, the high K + PSS was replaced by oxygenated 37°C PSS. Washing with PSS was repeated 1-2 more times before the next contraction. The equilibration length (Lref) was chosen as discussed in Chapter 2. On average, the equilibration period took about 1.5-2 hours. An 8-minute resting period was allowed in between two consecutive contractions. During the equilibration, the A S M preparations were intermittently activated with 37°C high K + PSS. After being equilibrated at Lref, the muscle preparations were stretched incrementally by 8% of their own Lt„ w-m (i.e. the step size) following each contraction. The absolute step size was different among preparations 32 due to the normal variation of in situ lengths. However, the step size was identical for both the control and the passively shortened muscle strips of either Day 3 or 7 experiments. As described in Chapter 2, in order to allow the passive force to stabilize, the muscle preparations were stretched 60 seconds before the next contraction. Both passive and plateau (tonic phase) active forces were measured during each contraction. The stretching of control preparation was carried out intermittently until the active force passed its peak value by at least two further increments. After the completion of measurements, the control preparation was removed from the tissue bath and was mounted on a C-shaped (Q) piece of stainless steel wire under the dissecting microscope. The length was set at its own Lmax (absolute value) by manipulating the wire. The preparation was then fixed in 10% formalin. The same protocol was applied to the passively shortened preparations. Following the equilibration period, the Lmax was determined by using the same step size of increment as that described for the control preparation. After passing the Lmax by one increment and 60 seconds before the next contraction, the PS preparation was stretched to reach either the C T L Lmax or until the monitored passive force exceeded the linear range of the force transducer (15-20mN). The PS preparation was then intermittently stimulated at the new length by using the same method and intervals that were used during its equilibration period. For each contraction, the passive and active forces were measured. This was continued until the active force reached a plateau or started to decline. In many cases, due to the large increase in the passive force, it was impossible to stretch the PS preparation to the C T L 33 Lmax without exceeding the linear range of the force transducer. At the end of the experiments, the PS preparations were fixed at their own Lmax in 10% formalin similar to the C T L strips. The mechanical measurement and tissue fixation were carried out for Day 3 and Day 7 for all the cultured specimens obtained from 6 rabbits. Lmax, Fmax and recovery data were gathered for data analysis. For Day 0 experiments, the L-T relationship was measured and the L m a x was determined after the muscle had been equilibrated. At the end of the experiment, the muscle preparation was fixed at L m a x with formalin. 3.1.1-M. Morphometric Studies were performed to determine the cross sectional areas (CSA) of muscle preparations. A l l the preparations used in section 3.1 were previously fixed in 10% formalin. After removing the stainless steel wires and aluminum clips, they were embedded in paraffin and sectioned. Three cross sections were then obtained from each muscle preparation, representing cross-sections from three evenly divided segments of the preparation. Hematoxylin and eosin (H&E) staining was performed. Digital images were captured by using a Nikon Digital Spot Camera and saved on a computer. Image Pro-Plus 3.0 software was then used to measure the total cross sectional area (TCSA) as well as smooth muscle area (SMA) of each slide (Figure 3-2). The captured images were processed and the average TCSA and S M A were calculated for each preparation. A shrinkage factor of 10% due to dehydration during fixation and embedding processes was taken into consideration and added to the measured values 34 [55]. The S M A values were then used individually to normalize the measured Fmax values. 3.1.2. DATA ANALYSIS The measured active and passive forces were used to determine the L-T relationship of both the control and the passively shortened groups. As in Chapter 2, the Weibull 4-parameter equation of peak function (Sigma Plot 5.0; Equation 1 in Appendix I) was employed to fit the active force data and measure the Fmax and Lmax. The obtained values of Lin sou, Lmax, and Fmax are shown in Tables 3-1, 3-2, and 3-3 respectively. Paired Mests were used to compare the above-mentioned values between control and passively shortened preparations. The smooth muscle cross sectional area (SMA) was measured and used to normalize the Fmax values and calculate the maximal stress (Omax)- Gmax is the maximal force (mN) per unit of cross sectional area (mm ) and is measured in kPa (mN/mm2) (Table 3-4). To examine the possible effect of the tissue culturing technique used in the current study on the A S M tissues, the smooth muscle areas of C T L Day 0, C T L and PS preparations of Days 3 and 7 were compared. A paired /-test was used to compare the S M A values (Table 3-5). For tension recovery test (performed only on PS preparations), two indices were used to demonstrate the results: 1) L / C T L Lmax (%)'• this index demonstrates that how much a PS preparation (after it passed its own Lmax) could be stretched as the percentage of C T L Lmax- In other words, it shows at what percentage of C T L Lmax the recovery test was performed on the PS preparation. 2) F/PS Fmax (%)' this index shows the amount of 35 maximal force obtained during the recovery test (at the above-mentioned length). In other words, it demonstrates whether the PS preparation (which is now stretched to a longer length) is able to generate the same amount of force that it could generate before stretching. Table 3-6 shows the data. In addition, the maximal active force generated by PS preparation at the new length (post-stretching) was compared to the PS Fmax (pre-stretching) (Table 3-7). The active force values during the recovery test were plotted against time. A single 3-parameter exponential rise to maximum function: y -yO + a (1 -e ~bx) was used as it provided the best fit for the curves (Equation 4 in Appendix I). In this equation, the b value represents the slope of the recovery curve. Table 3-6 lists the calculated b values for the Day 3 and 7 tension recovery tests. Paired /-test was also used to compare the b values of both groups. Since the recovery rate and F/PS Fmax do not depend on the amount of generated force, the calculated CSAs were not used to normalize the values. Finally, a single 3-parameter exponential decay function (y - yO + ae ~bx) was used to fit the passive force recovery curves in this experiment, in which b value represents the slope of the curve or the recovery rate (Equation 3 in Appendix I). A paired /-test was used to compare the data (see Table 3-9 for the data). For all the statistical tests, P > 0.05 was not considered statistically significant. In each case, n is the number of muscle preparations used. 3.1.3. R E S U L T S The Lin situ values of Day 0, Day 3, and Day 7 groups (Table 3-1, Page 55) were not significantly different in individual paired /-tests (P > 0.2). However, the mean value of 36 Lmax (2.77 ± 0.34mm) (mean ± SD) for C T L Day 3 was significantly greater than the Lmax for PS Day 3 preparations (2.48 ± 0.41mm) (P = 0.025; Table 3-2, Page 56). The Lmax values of C T L Day 7 and PS Day 7 preparations were 2.49 ± 0.38mm and 1.60 ± 0.50mm respectively. Again, the paired /-test showed that the two groups were significantly different with a P value of 0.0039 (Figure 3-3 and Table 3-2). In brief, after 3 and 7 days of passive shortening, the Lmax decreased by 10.6% ± 9.4 and 35.9% ± 15.9 (mean ± SD) respectively compared to the control preparations. The average smooth muscle cross-sectional area (SMA) for all the preparations (« = 74; including preparations used in sections 3.1, 3.2, and 3.3) was 0.2400 ± 0.073mm2 (mean ± SD). Moreover, the ratio of SMA/ TCSA was 56.34 ± 7.75% (mean ± SD) for all the A S M preparations (see section 3.1.1-M, Page 34 for methods). The measured SMAs were then used to normalize the individual F w a x values shown in Table 3-3 and calculate the related Omax (maximal stress) values (Table 3-4). Paired t-tests were then employed to compare the CTmax values between different groups (Figure 3-4). Despite the high variance in Omax, there was no significant difference in amax mean values between CTL and PS preparations after 3 or 7 days (P values of 0.61 and 0.15 respectively) and also between C T L preparations of Day 0, 3, and 7. In other words, after 3 and 7 days, the 0.05). These data suggest that the applied tissue culturing protocol did not significantly change the amount of smooth muscle present in the tissue preparations (i.e. for up to 7 days)(Table 3-5). After 7 days, the mean value of post-stretching maximal adapted active force of PS preparations was significantly lower than their pre-stretching Fmax mean value (paired /-test: P = 0.039; n - 6). However, the same values were not different after 3 days of passive shortening (paired /-test: P = 0.688; n = 6) (Table 3-7). To enhance the sample size, Day 7 (or 8) active recovery data obtained from 8 animals in section 3.2 were added to the Day 7 recovery data of section 3.1 (Table 3-8). The mean and standard deviation values of L / C T L Z m < z c (%) for Day 3 (n - 6) and Day 7 (n - 14) active recoveries of PS preparations were 99.9 ± 3.0 and 87.2 ± 13.7 respectively. A /-test (P < 0.05) showed a significant difference between the two groups. It was observed that at Day 3, by the time the tension recovery test was started, on average, the recovery was almost complete (n - 6). In other words, following stretching the PS preparations to the pre-determined length (i.e. the C T L Lmax), the generated active force of the first contraction, was very close to the maximal adapted active force. This observation could be due to the closeness of Lmax values of C T L and PS preparations. However, it was found that in most cases of Day 7 recovery tests, PS preparations were either too stiff to be stretched to their CTL Lmax, or unlike in Day 3 experiments, at the new length the active force could not reach the pre-stretching Fmax (n = 14). As a result, it was impossible to obtain the recovery rate (b value) from Day 3 PS preparations to compare them with Day 7 strips. 38 The obtained passive force recovery rates were compared between Day 3 and Day 7 PS preparations. Calculated P values of 0.381 in paired /-test (n = 6) and 0.361 in /-test (Day 3: n - 6 and Day 7: n= 14) did not confirm any significant difference in passive force recovery rates between Day 3 and 7 preparations. 3.2. Active vs. Passive Tension Recovery Test In this part of the experiment, both active and passive force recoveries of A S M were examined in the presence vs. absence of periodic stimulations. 3.2.1. METHODS Eight rabbits (New Zealand White, 2-4 mo, and 2.0-2.5 kg) were used for this study. The same animal protocol, tissue preparation, and tissue culturing techniques were used as discussed in section 3.1. The mechanical study was done after maintaining the A S M preparations at pre-determined lengths for 7-8 days. As shown in section 3.1, at this time the smooth muscle is adapted to the new length. Five strips were dissected from each trachea in a similar way as in the previous experiments. One preparation was examined on Day 0 (CTLO) and the rest of them were cultured individually at their own Lin situ (CTL7 and CTL8) or at shortened lengths (PS7 and PS8). The tissues were removed from the culture medium and subjected to the mechanical measurements on Day 7 and 8. Since both the active and passive recovery tests were relatively long mechanical experiments, they had to be done in two consecutive days for each animal. To exclude the 39 possible effect of 24 hours time difference on the results, the active and passive recovery tests were performed on Day 7 and 8 preparations randomly. In other words, from a total number of 8 tension recovery tests, in a group of 4 experiments, the active recovery test was carried out at Day 7 and was then followed by the related passive recovery test at Day 8. In another group of 4 experiments, the passive recovery test was performed at Day 7 and prior to the active recovery test which was done at Day 8. Mechanical Measurements: 3.2.1.1. Active Recovery: A similar experiment was described in section 3.1. The CTL Lmax and PS Z w a x were determined from the L-T measurements. After the PS preparation was stretched beyond its L m a x by 1-2 step increments, it was stretched further by using the same criteria as in section 3.1. At this time, the PS strip was stimulated intermittently by using High K + PSS and the changes in passive and active force were measured until the active force reached a plateau or started to decline. Since the same protocol was applied in both sections, the Day 7 active recovery data from section 3.1 were added to section 3.2. 3.2.1.2. Passive Recovery: In this experiment, the L-T measurement and stretching were performed as above. However, following the sudden length change, no stimulation was applied to the PS preparation. At this point, the smooth muscle tissue was washed every 10 minutes with oxygenated 37°C PSS. The passive force decline was recorded over a prolonged period (between 100 to 200 minutes) comparable to the active recovery 40 recoding time. The passive force data were then compared with the related data obtained from section 3.2.1.1. At the end of the above-mentioned period, the A S M preparation was stimulated intermittently and the active and passive forces were recorded. During this period of time (1-1.5 hours), the A S M was allowed to recover until the active force reached its plateau or started to decline. The data were then used to compare with the active force recovery data obtained immediately following the stretching as mentioned in section 3.2.1.1. The A S M preparations mentioned in the above sections were fixed immediately in 10% formalin at their own Lmax after the mechanical measurements. To determine the cross-sectional areas, morphometric study was then performed on these preparations as described in section 3.1.1-M. 3.2.2. DATA ANALYSIS Similar to section 3.1, in this part of the experiment Lmax, Fmax, and amax values were obtained (Tables 3-10, 3-11 and 3-12). Paired /-tests were employed to compare these values between PS and control groups on Day 0, Day 7, and Day 8. To test whether the preparations were subjected to the similar stretch, the absolute amount of passive force at the beginning of the passive recovery was compared between two groups (Table 3-13, Page 67). A Single 3-Parameter Exponential Decay function: (y = yO + ae~bx) was used to fit the passive force recovery curves (Equation 3 in Appendix I). In this equation, b value represents the rate of decline, which was considered as the passive force recovery rate (see Figure 3-7 and Table 3-14). Using a /-test, the b values were compared between the two groups. A /-test was used to analyze the data. As mentioned above, since the 41 same protocol was used for the active recovery experiments in sections 3.1 and 3.2, in order to increase the sample size, the Day 7 recovery data from section 3.1 have been used in both sections 3.2 and 3.3. Furthermore, a comparison was made between the fractional active force values of the active recovery group (immediately after stretching) and those of passively recovered preparations after 123.7 ± 37.0min (mean ± SD) of continuous decline in passive force (n - 8) (Table 3-15). For all the /-tests and paired /-tests used in this section, P > 0.05 was not considered statistically significant. In each case, n is the number of muscle preparations that were used. 3 . 2 . 3 . R E S U L T S Tables 3-10 to 3-12 show the Lmax, Fmax, and 0.05). However, the difference between the CTLO and CTL8 groups was statistically significant (P < 0.05) (Table 3-12 and Figure 3-6). Using a /-test, the absolute amount of passive force at the beginning of the recovery test was compared between two groups (n = 8, Table 3-13). A P value of 0.25 showed that there was no significant difference between the starting passive force of active and passive recovery groups. The passive force recovery rate (b value) was 0.063 ± 0.02 (mean ± SD) during active recovery and 0.074 ± 0.04 for the passive recovery group (Table 3-14). A P value of 0.55 in /-test (n = 8) demonstrates that there is no significant statistical difference between the two groups. The active force recovery was compared between the \"early\" group (i.e. immediately after stretching) and \"late\" group (i.e. those who have been recovered passively for prolonged period of time before being stimulated). The fractional active force values calculated for the first 4-5 contractions of each group are shown in Table 3-15. The starting point of active force adaptation for the late recovery group was at the fractional value of 0.934 ± 0.04 (also see Figure 4-1). This average starting value had been achieved in early group by the 4 contraction (see Chapter 4 for more explanation). 43 3.3. Active Recovery in Day 0 Preparations This part of the study was done to compare the active and passive force recovery rates (during active recovery test) following length adaptation to the one obtained from freshly dissected A S M preparations. 3.3.1. METHODS Five rabbits (New Zealand White, 2-4 mo, 2.0-2.5 kg) were used for this experiment. From the middle part of each trachea only one smooth muscle preparation was dissected as described in Chapter 2. The mechanical measurement was then performed immediately. Following the L-T measurement and determining the Lmax, the A S M strip was stretched by 1-2 step size increments (each step size was 8% of £,„ J i m ) or until the passive force exceeded the linear range of the force transducer (10-15mN). The preparations were then stimulated intermittently by using oxygenated 37°C High K + PSS. The stimulation times were 11.1 min for the 1 s t contraction, 14.1 min for the 2 n d , and 16.0 min for 3 r d to 6 t h contractions (described in more details in Chapter 4). The resting periods between consecutive contractions and the washing protocol were as described in section 3.1.1. The tension recovery was carried out until the active force reached the plateau or started to decline. The tissues were then removed from the tissue bath and were fixed at their own Lmax as discussed in section 3.1.1. To measure the smooth muscle areas, preparations were then subjected to morphometric studies as described in section 3.1.1-M. 44 3.3.2. DATA ANALYSIS Similar to section 3.2, the force recovery curves were plotted against time and fitted. The recovery rates (b values) for both active and passive forces were then calculated (Tables 3-16 and 3-17). Two /-tests were then used to compare the above-mentioned recovery rates to those of Day 7 or 8 experiments in sections 3.1 and 3.2. Moreover, the passive force values of C T L Day 0 and PS Day 7 (or 8) at the beginning of the active recovery were obtained as described in section 3.2 (Table 3-18). A /-test was employed to compare these two groups. Furthermore, to study the S M tissue response to sudden length changes at different levels of length adaptation, the amount of active force decrease (%) following sudden length increase for both CTLO and PS7 (or 8) preparations was calculated (Table 3-19). A /-test was employed to compare the decrease in active force in the two groups. For all the above-mentioned /-tests, P > 0.05 was not considered statistically significant. In each case, n is the number of muscle preparations used. 3.3.3. RESULTS The active force recovery rate was 0.081 ± 0.027 on Day 7 (or 8) (n = 13) after length adaptation and 0.176 ± 0.058 (n = 5) on Day 0 during the active force recovery. The presence of a statistically significant difference was supported by the P value of 0.00019 in the /-test (Figure 3-8 and Table 3-16). The passive force recovery rate was 0.0682 ± 0.030 (mean ± SD) and 0.0464 ± 0.011 during active recovery for PS7 (or 8) (« = 14) and CTLO (n = 5) preparations respectively (Table 3-17). The P value of 0.16 in /-test did not show any significant difference between the two groups (Figure 3-8). Moreover, the passive force at the 45 beginning of active recovery experiment was 13.42 ± 5.9 (mean ± SD) on Day 7 (or 8) and 13.82 ± 2.8 on Day 0 (Table 3-18). The P value of 0.89 in /-test showed that there was no significant difference between the starting passive forces in the two groups. The active force decreased by 89.4 ± 22.8% (mean ± SD) at Day 7 (or 8) after stretching. However, using the same protocol, the amount of decrease in active force was 60.8 ± 11.8% (mean ± SD) at Day 0 (Table 3-19). The P value of 0.017 in /-test showed that the above-mentioned decrease in active force was significantly different between the two groups. 46 Figure 3-1: Two rabbit A S M preparations that were maintained in C M R L culture medium for 3 days. A 'TI-shaped\" aluminum wire was used to inhibit the wax from floating. A : Shows a strip (CTL Day 3), which is stretched and set at its Lin Situ (3 mm). The length of smooth muscle is marked in this picture. B: A similar preparation (PS Day 3) set at a shortened length (0.75 mm) following a single electrical stimulation. 4 7 C No Sort C Sort Op <\"* Sort3own On J A , M IDCU.*J W :.oc«:n»oy«w P SsfdUo object ] t^g>aacooi-wcro«^c»f...j J«)Aflrjoca-we^toftoff... Figure 3-2: Shows the cross section of a rabbit A S M preparation (H & E staining X 100). Image-Pro Plus software has been used to process the image. The outside line (red: 1) determines the Total Cross Sectional Area (TCSA) and the inside line {green: 2) traces the Smooth Muscle Area (SMA). These areas (in mm2) are measured and SMAs were then used to normalize the Fmax values (in mN) and calculate the Maximal Stress (a m a x; in kPa) (section 3.1.1-M). 4 8 CTLO CTL3 PS3 CTL7 PS7 Figure 3-3: Lmax mean values of preparations described in section 3.1 (n = 6). The paired /-test was used to compare the CTL3 to PS3 (n) and CTL7 to PS7 (*). The P values were 0.025 and 0.0039 respectively. [Bars show Mean and SD values] 49 80 A \"CC\" 60 H 20 H 0 J I I I i 1 1 1 1 1 CTLO CTL3 PS3 CTL7 PS7 Figure 3-4: The mean and standard deviations of Maximal Stress values (Gmax) of preparations described in section 3.1 (n - 6) (same data as in Table 3-4). Paired /-test was used to compare the CTL3 to PS3 and CTL7 to PS7 separately. Despite a trend for lower <3max at PS7, there was no significant difference between these groups and all the P values were > 0.05. [Unit: kPa (mN/mm2); Bars show Mean and SD values] 50 0 CTLO CTL7 PS7 CTL8 PS8 Figure 3-5: Lmax mean values and standard deviations of preparations described in section 3.2 (n - 8). Paired /-test was used to compare the CTL7 to PS7 (¥) and CTL8 to PS8 (*) separately. The P values were 0.00012 and 0.00011 respectively. The Lmax mean values were decreased by 34 ± 12% on Day 7 and by 42 ± 14% on Day 8 (mean ± SD) in C T L compared to PS preparations. [Bars show Mean and SD values] 51 Figure 3-6: Maximal Stress (amax) mean values and standard deviations of preparations described in section 3.2 (n = 8). To compare CTL7 and PS7 (#) preparations, a paired /-test was employed and the P value was 0.0223. Furthermore, CTL8 and PS8 groups (*) were compared and the P value of 0.0225 in paired /-test confirmed the presence of a significant difference between the two groups. The Gmax mean values were decreased by 40% on Day 7 and by 45% on Day 8 in PS preparations compared to their controls. Moreover, the difference between CTL0 and CTL8 groups Q¥) was statistically significant (P < 0.05). [Unit: kPa (mN/mm2); Bars show mean and SD values]. 52 12! 0 J 1 1 1 1 1 1 1 1 0 20 40 60 80 100 120 140 Time (min) Figure 3-7: A sample of passive force recovery curves, active ( • ) vs. passive (O). The absolute values of passive force are plotted against time. A Single 3-Parameter Exponential Decay function of Sigma Plot 5.0 (y = y0 + ae 'bx) is used to fit the curves. The starting passive forces for both active and passive recovery are similar (Table 3-13). 53 w 0.25 0.20 £ 0.15 0.10 0.05 A 0.00 Control Day 0 PS Day 7 (or 8) n = 13 n = 14 PS7AF Recovery CTLOAF Recovery PS7PF Recovery CTLO PF Recovery Figure 3-8: The active and passive Recovery Rates (b values) obtained during active tension recovery tests of the passively shortened Day 7 or 8 (PS7 or 8: black bars) and the freshly dissected A S M tissues (CTLO: white bars) (same data as in Tables 3-16 and 3-17). The P value of 0.00019 in Mest confirmed a significant difference between AF recovery rates (<¥). However, there was no statistically significant difference between the passive force recovery rates of PS7 and CTLO groups (Mest: P = 0.16). [AF: Active Force; PF: Passive Force; Bars show Mean and SD values]. 54 Animal # CTLO Lin situ (mm) CTL3 Lin situ (mm) PS3 L/n situ (mm) CTL7 L/n situ (mm) PS7 Lin situ (mm) 1 2.00 3.00 3.00 2.00 2.00 2 2.00 3.00 3.00 2.50 2.50 3 3.00 3.00 3.00 3.00 3.00 4 3.00 2.75 2.75 3.00 3.00 5 2.75 2.50 2.50 2.75 2.75 6 2.75 3.00 3.00 3.00 3.00 Mean ± SD 2.58 ± 0.46 2.87 ±0.21 2.87 ±0.21 2.71 ±0.40 2.71 ±0.40 Table 3-1: The 1;,,^ values of rabbit A S M preparations (in mm) used for section 3.1. The Lin situ values were equal for each pair of preparations assigned for Day 3 or 7 experiments. However, they were often different between Day 0, Day 3, and Day 7 groups. 55 Animal # CTLO Lmax (mm) CTL3 Lmax (mm) PS3 Lmax (mm) CTL7 Lmax (mm) PS7 Lmax (mm) 1 1.72 2.54 2.43 1.71 1.03 2 1.83 2.97 2.71 2.66 1.80 3 2.61 2.56 2.54 2.60 1.29 4 3.23 2.34 1.68 2.55 2.16 5 2.76 2.93 2.66 2.75 2.16 6 2.15 3.27 2.85 2.65 1.16 Mean ± SD 2.38 ± 0.58 2.77 ±0.34 2.48 ± 0.42 * 2.49 ± 0.39 1.60 ±0.50* Table 3-2: The values of optimum length (Lmax) calculated for the A S M preparations used in section 3.1 in - 6). A Weibull 4-parameter equation of peak function (Sigma Plot 5.0) was used to fit the active force curves and obtain the Lmax values. The Lmax values were significantly smaller in both Day 3 and 7 PS preparations compared to the control groups: Paired Mest: P = 0.025 and 0.0039 respectively. [*: Statistically significant change] 56 Animal # CTLO Fmax (mN) CTL3 Fmax (mN) PS3 Fmax (mN) CTL7 Fmax (mN) PS7 Fmax (mN) 1 11.73 7.69 4.98 4.51 4.98 2 18.61 12.29 10.06 14.81 9.79 3 12.67 14.78 10.52 18.82 11.57 4 13.03 18.05 16.85 15.94 6.41 5 11.30 12.78 15.78 11.91 12.11 6 13.54 9.83 9.87 20.32 8.48 Mean ± SD 13.48 ±2.6 12.57 ±3.6 11.34 ±4.4 14.38 ±5.7 8.89 ±2.8* Table 3-3: The values of Maximal Force (Fmax) obtained from A S M preparations used in section 3.1. A Weibull 4-parameter equation of peak function (Sigma Plot 5.0) was employed to fit the active force curves and find the Fmax values. These values are not normalized by the cross sectional areas of the A S M preparations. However, there was a significant difference between the mean Fmax of C T L and PS preparation at Day 7 (Paired Mest: P < 0.05). [#: Statistically significant change] 57 Animal # CTLO Omm (kPa) CTL3 Gmax (kPa) PS3 Omax (kPa) CTL7 Omax (kPa) PS7 Omax (kPa) 1 57.67 32.46 36.71 10.21 20.12 2 45.00 77.42 67.27 59.32 51.02 3 97.17 98.18 60.57 67.82 42.92 4 47.78 65.0 79.33 60.69 16.11 5 39.95 58.96 64.94 35.15 44.79 6 70.30 64.37 62.92 93.85 34.50 Meant SD 59.65 ±21.3 66.06 ±21.6 61.96 ±14.0 54.51 ±28.7 34.91 ± 14.1 Table 3-4: The Maximal Stress values ( 0.05. [Unit: mN/mm 2 (kPa)] 58 Animal # CTL DayO CTL Day3 PS Day3 CTL Day7 PS Day7 1 0.183 0.213 0.122 0.398 0.223 2 0.372 0.143 0.135 0.225 0.173 3 0.117 0.136 0.156 0.250 0.243 4 0.246 0.250 0.191 0.236 0.358 5 0.255 0.195 0.219 0.305 0.243 6 0.173 0.138 0.141 0.195 0.221 Mean ± SD 0.224 ± 0.09 0.179 ±0.05 0.161 ±0.04 0.268 ± 0.07 0.243 ±0.06 Table 3-5: The Smooth Muscle Area (SMA) of tissue preparations measured by using morphometric techniques described in section 3.1.1-M. Mean values and standard deviations are also shown in this table. The same dissecting technique was used for all the preparations as described in section 2.1. The paired t-test applied to compare these groups did not show any significant change in the S M A following 0-7 days of tissue culturing in the current study. These data suggest that the applied tissue culturing protocol did not change the amount of smooth muscle present in the tissue preparations. 59 No. Day 3 Day 7 u CTL Lmax (%) F/PS Fmax (%) b u CTL Lmax (%) F/PS Fmax (%) R b 1 102.8 81.8 0.032 100 91.3 0.998 0.057 2 100 108.6 0.065 89.3 70.3 0.996 0.083 3 102.2 112.5 N/A 77.9 67.9 0.998 0.103 4 94.2 49.9 0.066 100 104.0 N/A N/A 5 100 101.2 N/A 100 88.5 0.999 0.109 6 100 108.2 N/A 70.9 31.0 0.998 0.058 Mean ± SD 99.86 ±3.0 93.7 ±24.1 0.054 ± 0.019 89.7 ±12.7 75.5 ±25.7 0.998 ± 0.001 0.082 ± 0.024 Table 3-6: Day 3 and Day 7 active force recovery data. L /CTL Lmax (%) demonstrates that how much a PS preparation (after it passed its own Lmax) could be stretched as the percentage of C T L Lmax. F/PS Fmax index (%) shows the amount of maximal force obtained during the recovery test (at the above-mentioned length). For more description regarding these two indices see section 3.1.2. A Single 3-Parameter Exponential Rise to Maximum function from Sigma Plot 5.0: y = y0 + a (1 - e ~bx) was used to fit the active force recovery curves. In this table, R value demonstrates the suitability of the fit and b value shows the slope of the recovery curve. In 3/6 of the Day 3 and one of the Day 7 experiments, due to the small difference between PS Lmax and C T L Z O T a x , the recovery was almost complete after stretching the PS preparation. In these situations, the peak active force was achieved at the 1 s t or 2 n d contraction of the recovery period. As a result, the above-mentioned Exponential Rise to Maximum function was not applicable to these data (shown as N / A in the table). In these cases, no b or R value could be measured. 60 Animal # PSFmax Day3 (mN) PS MAAF Day 3 (mN) PS Fmax Day7 (mN) PS MAAF Day 7 (mN) 1 5.06 6.26 4.91 4.48 2 10.37 11.26 9.73 6.84 3 10.60 11.93 11.94 8.10 4 16.51 8.24 6.42 6.68 5 15.98 16.18 12.09 10.70 6 9.87 10.61 8.46 6.30 Mean ± SD 11.4 ±4.3 10.75 ±3.4 8.92 ±2.9 7.18 ±2.1 * Table 3-7: Shows the active force values calculated from 6 experiments discussed in section 3.1. This table compares the Fmax values of PS preparations (PS Fmax) on Day 3 and 7 to the related Maximal Adapted Active Force ( M A A F ) values that were measured after stretching the PS preparations to/toward their C T L Lmax. The data describe after stretching at Day 3 or 7, how capable were the PS preparations to fully recover (i.e. whether they could generate the same amount of force as they did at their pre-stretched lengths). After 7 days, the mean value of post-stretching maximal adapted active force of PS preparations was significantly lower than their pre-stretching Fmax mean value (paired /-test: P = 0.039). However, the same values were not different after 3 days of passive shortening (paired /-test: P = 0.69). [#: Statistically significant change] 61 No. Day 3 Day 7 (or 8) u CTL Lmax (%) F/ PS Fmax (%) b u CTL Lmax (%) Fl PS Fmax (%) R b 1 102.8 81.8 0.032 100 91.3 0.998 0.057 2 100 108.6 0.065 89.3 70.3 0.996 0.083 3 102.2 112.5 N/A 77.9 67.9 0.998 0.103 4 94.2 49.9 0.066 100 104.0 N/A N/A 5 100 101.2 N/A 100 88.5 0.999 0.109 6 100 108.2 N/A 70.9 31.0 0.998 0.058 7 - - - 100 68.5 0.997 0.044 8 - - - 76.2 55.1 0.985 0.022 9 - - - 100 69.0 0.999 0.084 10 - - - 85.3 121.4 0.999 0.098 11 - - - 54.2 61.2 0.996 0.097 12 - - - 86.1 122.2 0.999 0.110 13 - - - 93.97 134.8 0.999 0.078 14 - - - 89.50 101.7 0.998 0.107 Mean ± SD 99.9 ± 3.0 93.7 ±24.1 0.054 ± 0.02 87.4 ±13.7* 84.8 ±29.5 0.997 ± 0.004 0.081 ± 0.028 Table 3-8: Day 3 and Day 7 (or 8) active force recovery data. The data from section 3.2 (n - 8) were added to the data shown in Table 3-6. See Table 3-6 and Chapter 3 (Page 37) for the definition and more description of the data. [#: Statistically significant change; /-test: P < 0.05] 62 Animal # Day 3 Day 7 1 0.076 0.011 2 0.077 0.112 3 0.049 0.126 4 0.029 0.092 5 0.069 0.072 6 0.028 0.034 Mean ± SD 0.055 ± 0.022 0.075 ±0.045 Table 3-9: Passive force recovery rates on Day 3 and Day 7 active recovery test. A Single 3-Parameter Exponential Decay function of Sigma Plot 5.0 (y =y0 + ae ~bx) was used to fit the curves. Mean and SD values are also shown in this table. The difference between the two groups was not statistically significant (Paired /-test: P = 0.381). 63 Animal # CTLO Lmax (mm) CTL7 Lmax (mm) PS7 Lmax (mm) CTL8 Lmax (mm) PS8 Lmax (mm) 7 1.27 2.39 0.97 2.59 1.43 8 2.92 2.34 1.38 2.56 1.20 9 2.60 2.93 2.12 2.34 1.12 10 1.07 2.47 1.40 2.15 1.44 11 1.76 2.21 1.63 2.51 0.88 12 3.63 2.16 1.70 2.38 1.66 13 1.90 2.63 1.98 2.21 1.63 14 2.27 2.28 1.56 2.90 2.08 Mean ± SD 2.18 ±0.85 2.43 ± 0.25 1.59 ± 0.36 * 2.45 ± 0.24 1.43 ± 0.37 * Table 3-10: The values of optimum lengths (Lmax) calculated for the A S M preparations used in section 3.2 (n = 8). A Weibull 4-parameter equation of peak function (Sigma Plot 5.0) provided the best fit for the active force curves. P values of 0.00012 and 0.00011 in paired /-tests confirmed the presence of significant difference between Lmax mean values of C T L and PS preparations of both Day7 and 8 groups respectively. [#: Statistically significant change] 64 Animal # CTLO Fmax (mN) CTL7 Fmax (mN) PS7Fmax (mN) CTL8Fmax (mN) PS8 Fmax (mN) 7 8.85 7.08 4.85 5.33 4.44 8 7.35 11.46 6.89 4.44 3.70 9 18.03 9.27 6.23 8.50 6.55 10 9.82 6.18 4.70 8.20 7.07 11 18.11 3.64 2.79 5.66 2.99 12 15.03 16.02 7.23 13.84 3.42 13 10.22 6.79 5.49 8.09 6.23 14 9.77 12.85 6.16 7.93 5.99 Mean ± SD 12.15 ±4.26 9.16 ±4.06 5.54 ± 143 * 7.75 ±2.91* 5.05 ± 1.59 * Table 3-11: The values of Maximal Force (Fmax) obtained from A S M preparations studied in section 3.2 (n = 8). A Weibull 4-parameter equation of peak function (Sigma Plot 5.0) was employed to fit the active force curves and find the Fmax values. These values are not normalized by the cross sectional areas of A S M preparations. However, the Fmax values of PS Day 3 and 7 preparations were found to be significantly smaller (#) than their controls (Paired /-test: P = 0.008 and 0.047 for Day 3 and 7 respectively). Furthermore, there was a significant difference (*) between Fmax values of CTLO and CTL8 smooth muscles (Paired /-test: P = 0.03). These data suggest that other than passive shortening, the culturing conditions could have negatively affected the force generation of the PS preparations (see Chapter 4 for more explanation). [# and * : Statistically significant changes] 65 Animal # CTLO 0.05). As a result, we assumed that the culturing conditions did not significantly affect the contractility of A S M preparations. Type of Stimulation: For experiments described in Chapter 2, Electrical Field Stimulation (EFS) was used to stimulate the smooth muscle. Although EFS does not usually induce the maximal contractile response in A S M , it is an acceptable method for experiments that involve freshly isolated smooth muscle tissues. However, following 1-2 days of tissue culturing, EFS is not adequate to elicit S M contraction due to degeneration of nerve endings in the tissue. Acetylcholine (ACh) was instead used to stimulate smooth muscle during preliminary experiments on Day 3 and 7. Freshly dissected A S M preparations were subjected to length oscillation for 5 minutes and two concentrations of A C h ( lO^M andlO\"5M) were then tested separately to examine the active force recovery rate following this oscillation: 1) 10\"4M ACh induced a recovery rate comparable to the one by EFS. However, to allow the muscle to relax following each contraction, it required several washings with PSS, which made this protocol practically inapplicable for our study. 2) 10\"5M ACh did not induce a contractile response comparable to EFS and that by l O ^ M ACh. Moreover, using ACh as the stimulant is associated with desensitization of the smooth muscle cells to ACh. Therefore, to elicit a similar contractile response during a 4 to 8-hour mechanical experiment it would require a step-by-step increase in the concentration of ACh [57-59]. 83 A High K + PSS (contained 80mM/L potassium) was therefore tested and used for all the experiments discussed in Chapter 3. Membrane depolarization by high extracellular K + causes contraction in airway smooth muscle by allowing Ca influx through voltage-operated Ca channels [60, 61]. However, at least in uterine smooth muscle it has been shown that high K + PSS, in addition to its effect on C a 2 + influx, activates other cellular processes that lead to an increase in the C a 2 + sensitivity of the contractile machinery by a mechanism independent of extracellular C a 2 + [62]. It has also been observed that during the proliferation of A S M cells in culture, there are considerable changes in the expression of ionic channels, which could have a profound functional significance. In particular, these changes would tend to make the tissue more excitable compared to the freshly isolated A S M cells [63]. However, to our knowledge there is no evidence showing that this happens in tracheal explants. Moreover, it was assumed that the possible functional differences due to this phenomenon would affect both the control and passively shortened preparations equally. Furthermore, no significant increase in force generation was observed in control A S M tissues maintained for 0-8 days in culture media in the current experiment. The Frequency of Stimulations by High PSS: As in the experiments described in Chapter 2, when EFS is used to stimulate rabbit A S M preparations, a relatively narrow range of stimulation-time (6-8 seconds) is needed to achieve the active force plateau. However, when High K + PSS is used to elicit the contractions, the A S M response to the stimulation varies considerably between different preparations (i.e. the stimulation time needed to achieve the plateau varied between 60-1200 seconds in the experiments 84 described in sections 3.1 and 3.2). This has changed the protocol of how frequent the muscle was stimulated in our study. The experiments described in section 3.3 were designed to compare the active and passive tension recovery rates of freshly dissected (Day 0) smooth muscle and those of chronically length-adapted A S M preparations (Day 7 or 8). For Day 3 and 7 (or 8) active tension recovery tests (sections 3.1 and 3.2), the frequency of A S M contractions was determined by the individual stimulation time required for the muscle preparation to achieve a tonic plateau. However, as mentioned in section 3.1.1, in some preparations (mostly in CTL Day 0 experiments) the tonic component was either absent or smaller than the first peak (phasic contraction). In other words, in Day 0 experiments (section 3.3) the A S M preparation would often require only 60-240 seconds of stimulation with High K + PSS to reach the active force plateau. This could increase the frequency of A S M contractions by decreasing the applied stimulation time. Wang and colleagues (2000) showed that following a length oscillation, the recovery rate of an A S M preparation was affected by the frequency of applied stimulations [14]. As a result, in order to have comparable data, the average stimulation time of Day 7 (or 8) active recovery experiments were calculated and applied to the freshly dissected Day 0 preparations (n = 5) described in section 3.3 as follows. The average stimulation times were measured for all the induced contractions during active tension recovery tests (sections 3.1 and 3.2) performed on Day 7 (or 8) (Table 4-1; n- 14). Individual /-tests were then employed to compare the stimulation times during different contractions of all these experiments (Table 4-1). The stimulation time was different between 1 s t and 2 n d and 3 r d-6 t h contractions. This stimulation time was 11.16 ± 85 2.4 (mean ± SD) min for the first contraction of the recovery phase, 14.11 ±2 .4 min for the second one, and 15.99 ± 0.52 min for the 3 r d to 6 t h contractions (the stimulation time in 3 r d , 4 t h, 5 t h, and 6 t h contractions were not statistically different). In order to have the same frequency of stimulation, the above-mentioned mean values of stimulation time were applied to the freshly isolated A S M preparations (CTLOs) and their active and passive recovery rates were obtained as described in section 3.3 (Table 4-1). Summary and the Clinical Implications In these studies we found that it was relatively easy to recondition airway smooth muscle after periods of shortening of up to 3 days. At 3 days, the passively shortened smooth muscle preparations only showed a 10.6% decrease in Lmax and it was easy to stretch the shortened muscle back to the original Lmax. In addition, at the original Lmax, the maximal force for that preparation could again be achieved indicating that there was a complete adaptation. Presumably then, smooth muscle in vivo shortened for less than 3 days could be reconditioned and permanent airway narrowing and/or a shift of the length-tension curve would not occur. On the other hand, we noted that preparations which were passively shortened for 7 or 8 days were more resistant to a correction of their length-tension relationship. Not only was the Lmax considerably (35.9%) reduced by the prolonged passive shortening but it was impossible to completely recondition the muscle at its original length. This was evidenced by the fact that it was impossible to stretch the muscle to the original Lmax without causing a permanent disruption. In addition, even though the extended length was less than the original Lmax, repeated stimulation was not able to result in a return of isometric force to that achieved by that muscle at its shortened 86 Lmax- This suggests that the remodeling that occurs within the muscle involving the cytoskeleton and/or the contractile apparatus is not as malleable after a 7 or 8-day alteration in length as it is after 3 days or less. In skeletal muscle there can be profound changes in the shape and position of the length-tension curve after prolonged periods of lengthening or shortening of muscle. It has been shown that these alterations in length-tension curve are due to subtraction or addition of sarcomeres rather than a change in the operating length of the individual sarcomeres [44, 45, 47]. The mechanism of chronic adaptation in smooth muscle is not understood. Ford et al [51] and Gunst et al [52] have proposed a hypothesis based on a rearrangement of the contractile elements or of the cytoskeletal elements, respectively. These hypotheses are not mutually exclusive and indeed one would have to have reorganization of the cytoskeletal network [52, 54] to allow the reorganization of the contractile elements [48, 51] that Ford et al have proposed. The acute and subacute changes in the L-T curve (up to 3 days) appear to be relatively impermanent in that the smooth muscle length-tension curve appears to be plastic over this time period. Our finding of more striking and less reversible changes after prolonged periods of length change may be very important in disease. This phenomenon may represent an analog to the drop out of sarcomeres that occurs in skeletal muscle. Such a phenomenon has the capacity to contribute to irreversible airway narrowing in disease. The results of these studies could have profound implications for in vivo airway smooth muscle function and its ability to narrow the airways in disease. Airway smooth 87 muscle may be shortened for prolonged periods of time during acute attacks of asthma for a variety of reasons. Firstly, the high levels of contractile agonists produced during acute asthmatic inflammatory episodes could cause active smooth muscle shortening. Secondly, airway edema, located in the peribronchial space between the smooth muscle layer and the surrounding parenchyma, could cause passive shortening of airway smooth muscle. If airway smooth muscle is shortened due to a combination of active contraction and passive unloading it may adapt to the shorter length by optimizing the contractile apparatus for force generation and shortening. This could have catastrophic consequences. In addition, if the chronic passive or active shortening results in passive stiffening of the airway smooth muscle layer such that the stretch provided by deep inspirations or tidal breathing is ineffective in re-adapting the muscle to longer lengths then there is potential for even more shortening of the smooth muscle and a worsening of the disease. 88 1 1 1 1 1 1 0 50 100 150 200 250 Time (min) I .8 c o •6 1.00 A 0.75 A 0.50 A 0.25 • -• B 0.00 • 20 40 60 Time (min) 80 100 Figure 4-1: Fractional Active Force Recovery data obtained from active tension recovery tests (described in section 3.2). The force is expressed as fraction of maximal active force achieved at the pre-determined length at which the recovery test is performed. Fractional values of active force are plotted against time (n - 8). For each experiment, the absolute active force values are divided by the maximal active force of the preparation. Black circles (•) represent individual fractional active force values which are shown together. A : Shows the values measured during early active recovery tests (section 3.2.1.1) of PS preparations on Day 7 (or 8), when high values of passive were present. B: Shows the fractional active force values of late active recovery (section 3.2.1.2) The late recovery was performed on Day 8 (or 7), after a prolonged period of passive recovery (without periodic stimulations). The preparations were then stimulated intermittently to reach the maximal adapted active force at the pre-determined lengths. On average, the active recovery process in plot B starts (at time 0) from higher fractional AF values: 0.934 ± 0.04 (mean ± SD) compared to plot A (virtually 0). In plot B, after the period of passive force recovery, the active force recovery is already close to be complete. These data clearly show that the active force recovery process can start and proceed in the absence of smooth muscle periodic stimulations (see Table 4-2 and 3-15 for the data). 89 T-0 Fractional PF 1 Figure 4-2: Active Recovery Test; the fractional values of active and passive force for the individual experiments have been plotted (« = 8). A: Shows the individual experiments. Each regression line shows the fitted values for one experiment. The tension recovery in each case starts at passive force fractional value of 1 {right bottom). This value then decreases with time and reaches a minimum at fractional active force value of 1 {left top). B: Shows the pooled data. These data show a close correlation between the passive and active force recoveries. [AF: active force; PF: passive force] 90 Contraction Number Figure 4-3: The Fractional values of Total Force (TF«), Active Force (AF V ) , and Passive Force (PFU) for the first 4 contractions of active recovery tests performed on the passively shortened preparations (section 3.2) on Day 7 (or 8) (n = 8). Each set of force values (including TF, A F , and PF) were normalized by the total force measured when the M A A F is achieved by the muscle preparation. A 3-Pararmeter Exponential Decay function was used to fit the TF and PF curves. The A F recovery curve was fitted by using a 3-Parameter Exponential Rise to Maximum function (both equations from Sigma Plot 5.0). For all the curves the R value of fitting was > 0.998. As described in section 3.2.1, the active recovery process was started at high values of passive force. As a result, the mean value of active force measured for the first contractions was virtually zero. These data show that during the active tension recovery test, the decline in the passive force is greater than the rate of increase in the active force (see Chapter 4 for more discussion). [MAAF: Maximal Adapted Active Force] 91 —tw-I— —SfiftO-——| —-^ IRW 1—-UBtf — — — 1 — W '—W — S U W -— —~^5T£FS* -TJ&S^? —&Qft& -tftBtf—|| -^ 5 0, max (y), min (y)] b - fwhm [x,abs(y),.5] c = 1.5 x0 = iffpeaksign (y) > 0, xatymax (x,y), xatymin (x,y)J Variables: x = col (1) y — col (2) Automatic Initial Parameter Estimate Functions: peaksign (q) - if (total (q) > qfl], 1, -1) xatymin (q,r) = xatymax (q, max (r) - r) Constrains: b>0 c> 1.001 105 Equation 2: Exponential Growth / Single 2-Parameter Function (Sigma Plot 5.0) bx Equation: y — ae This could be also shown as: / = a*exp (b*x) Fit ftoy Initial Parameters: a = exp (F (0) fl]) b=F(0)[2] Variables: x = col (I) y = col (2) Automatic Initial Parameter Estimate Functions: F(q) = ape[x,ln(y), 1,0,1] Constrains: b> 0 Equation 3: Exponential Decay / Single 3-Parameter Function (Sigma Plot 5.0) Equation: y = yO + a (1 e~bx) This could be also shown as: /-y0+a*exp (-b*x) Fit ftoy Fit ftoy with weight reciprocal^ Fit ftoy with weight reciprocal_ysquare Initial Parameters: yO =yhat(y) a - max (y) - yhat (y) b = - In (.5)/x50[x,y -yhat (y)] Variables: x - col (I) y - col (2) reciprocal^ =l/abs(y) reciprocal_ysquare =l/yA2 106 c Automatic Initial Parameter Estimates: yhat (q) = q [size(q)] Constrains: b>0 E q u a t i o n 4: Exponential Rise to Maximum / Single 3-Parameter Function (Sigma Plot 5.0) Equation: y - yO + a (1 - e bx) This could be also shown as: f=y0 + a*[l-exp (-b*x)J Fit/toy Initial Parameters: yO = yatxnearO (y,x) a = [ylast (y) -yatxnear 0(y,x)J b--ln (.5)/[x50(x,y,.5) - min (x)] Variables: x = col (1) y = col (2) Automatic Initial Parameter Estimate Functions: First (q) = if (size (q) < 10, size (q)-l, int (0.9*size (q))) ylast (q) - mean (qfdata (first(q), size(q))]) xnear 0 (q) = max (abs (q))- abs (q) yatxnear 0 (q,r) = xatymax (q, xnear 0 (r)) Constrains: b>0 107 "@en ; edm:hasType "Thesis/Dissertation"@en ; vivo:dateIssued "2002-05"@en ; edm:isShownAt "10.14288/1.0090641"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Experimental Medicine"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "The time course of length adaptation and tension recovery in airway smooth muscle"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/13291"@en .