Articles in PresS. Am J Physiol Lung Cell Mol Physiol (December 5, 2014). doi:10.1152/ajplung.00218.2014
1 2
RELAXANT EFFECT OF SUPERIMPOSED LENGTH OSCILLATION ON SENSITIZED AIRWAY SMOOTH MUSCLE
3 4 5 6 7
Miguel Jo-Avila, Ahmed M. Al-Jumaily, Jun Lu. Institute of Biomedical Technologies Auckland University of Technology, Auckland, New Zealand.
8
9
10 11 12 13 14 15 16 17 18
Contact Author Ahmed Al-Jumaily, PhD, MSc, BSc Professor of Biomechanical Engineering Director, Institute of Biomedical Technologies Auckland University of Technology Private Bag 92006, City Campus, WD306 Auckland, New Zealand Tel: (649) 921 9777; Fax:(649) 921 9973 Email:[email protected] Website:www.ibtec.org.nz
19
20
21
22
23
24
25 1
Copyright © 2014 by the American Physiological Society.
26
Abstract
27
Asthma is associated with reductions in the airway lumen and breathing difficulties which
28
are attributed to airway smooth muscles (ASM) hyperconstriction. Pharmaceutical
29
bronchodilators such as salbutamol and Isoproterenol (ISO) are normally used to alleviate
30
this constriction. Deep inspirations (DI) and tidal oscillations (TO) have also been reported to
31
relax ASM in healthy airways with less response in asthmatics. Little information is available
32
on the effect of other forms of oscillation on asthmatic airways. This study investigates the
33
effect of length oscillations (LO), with amplitude 1% and 1.5% in the frequency range 5-20
34
Hz superimposed on breathing equivalent LO, on contracted ASM dissected from sensitized
35
mice. These mice are believed to show some symptoms such as airway hyperreactivity (AH)
36
similar to those associated with asthma in humans. In the frequency range used in this work,
37
this study shows an increase in ASM relaxation of an average of 10% for 1.5% amplitude
38
when compared with TO, ISO or the combination of both. No similar finding is observed with
39
1% amplitude. This suggests that superimposed length oscillation (SILO) acting over the
40
interaction of myosin and actin during contraction may lead to temporal rearrangement and
41
disturbance of the cross-bridge process in asthmatic airways.
42 43 44 45 46 47 48 2
49
1. Introduction
50
Asthma is primarily characterized by reversible airway hyper-constriction, hyper-
51
responsiveness and inflammation. It is believed that the key effector of airway
52
bronchoconstriction in asthma is attributed to ASM contraction (14). This contraction is
53
normally relieved by using a pharmaceutical relaxant such as Isoproterenol (ISO).
54
Unfortunately, some relaxants are often associated with various degrees of side effects (7,
55
25). Searching for alternative treatments with less-harmful side effects has been the main
56
objective of many investigators in this field of study (1, 3, 8, 9, 11, 12, 17, 20, 26, 29).
57
58
At the cellular level, ASM contraction and relaxation are determined by the extent of the
59
actin-myosin cross-bridge cycling process. With the activation of ASM, this biophysical
60
process may be initiated by hormonal or neural stimuli followed by biochemical activities
61
which result in relatively restricted movement between the myosin and actin filaments to
62
produce muscle contraction. Pharmacological treatments are normally used to alleviate this
63
contraction by either relaxing the constricted airways or reducing the inflammation
64
observed. Alternatively, several in vitro studies have demonstrated that LO equivalent to
65
those occurring during tidal breathing or with some SILO do disturb the cross-bridge cyclic
66
process and produce some relaxation in pre-contracted ASM obtained from healthy airways
67
(11, 12, 14, 32). Further, it has been demonstrated that combining LO with ISO enhances the
68
relaxation for contracted tissues obtained from healthy airways (17); particularly, it was
69
indicated that ISO of 10-7 to 10-5 M caused the same force inhibition as TO. It was also
70
observed that the dose response curve of ISO when examined with and without TO, the
71
amount of relaxation between the two was equivalent on all but the highest concentration 3
72
of ISO (10-5M), suggesting that the mechanisms acting on the relaxing effects of ISO and
73
tidal oscillations are largely independent, i.e. deactivation vs. disruption of the myosin cross-
74
bridge (17).
75
76
The above cited studies were conducted on tissues isolated from healthy airways. However,
77
recent studies such as Chin et al (10) and Noble et al (28) have focused on asthmatic
78
airways, with the latter focusing on DI and TO effect. To the best of our knowledge other LO
79
different to those occurring during breathing and deep inspiration have not been tested on
80
asthmatic airways as a therapeutic alternative. Thus our interest focuses on testing the
81
effect of different amplitudes and frequencies of SILO on contracted ASM from sensitized
82
mice. It is expected that this would help to fill the knowledge gap and will allow us to learn
83
more about the behavior of sensitized airways when SILO are applied during breathing.
84
85
In spite of the fact that no animal model can fully resemble human asthma, it has been
86
observed that some features like inflammation, AHR and remodeling of the airways can be
87
reproduced in animal models (16, 22-24). Mouse model presents advantages for
88
physiological studies of asthma because of their size, short cycles of breeding, and existing
89
data regarding sensitized models resembling various features of asthma. The recent
90
advances in transgenic technology and the development of species-specific probes, have
91
allowed detailed mechanistic studies to be conducted on mouse models (4,5, 33). The main
92
purpose of using mice in this study was to generate an acute sensitized model capable of
93
resembling closely airway hyperreactivity (AH) observed during an asthma attack. This will
4
94
be significant in understanding how airways from healthy and sensitized mice respond in
95
vitro to external agents such as ISO, LO and SILO during an induced asthmatic attack.
96
97
2. Material and methods
98
All experimental procedures were approved by the University of Auckland Animal Ethics
99
Committee according to the Code of Ethical Conduct and performed in accordance with the
100
Animal Welfare Act 1999 New Zealand.
101
102
2.1. Animal model and sensitization
103
We designed and used an ovalbumin (OVA) sensitization model (acute model) for this study,
104
following a protocol similar to that of Kumar et al 2008 (24). The mouse strain used during
105
the study was Balb/c, female, between 8-12 weeks old. They were selected due to the fact
106
that this strain is highly reactive in respiratory studies and hence commonly used for
107
sensitization protocols (27). The model included two methods of administration of the
108
allergen (Fig 1). First, intraperitoneal injections of OVA at 10g diluted in sodium chloride
109
0.9% (saline) + 1mg aluminum hydroxide mixture were administered to the animals on days
110
0, 7 and 14, in order to induce immunological response. Second, after 10 days of the last
111
intraperitoneal injection, aerial nebulization of OVA at 5%, diluted in saline, was used to
112
expose the airways to the allergen for 20 min, on day 24, 28 and 32. After the last
113
nebulization the mice were allowed to rest for 24 hrs and were culled on day 33 to obtain
114
the tissue (tracheal rings) for testing.
115
2.2. Tissue preparation and experimental apparatus 5
116
Tracheal rings 2mm wide were obtained from healthy and sensitized mice, and were
117
mounted in a 5mL computerized organ bath system (800A in vitro test system, Aurora
118
Scientific Inc., Ontario, Canada) as previously described (21). Each tracheal ring was
119
suspended vertically to a servo-controlled lever arm (Model 300C, Cambridge Technology,
120
Aurora Scientific Inc.) using steel wire and non-penetrating hooks. The bath was filled with
121
physiological Krebs solution (composition in mM: 110 NaCl, 0.82 MgSO4, 1.2 KH2PO4, 3.39
122
KCL, 2.4 CaCl2, 25.7 NaHCO3 and 5.6 Glucose) and bubbled with a gas mix of 95% O2/5% CO2
123
to maintain the pH at 7.4. A constant temperature of 37°C was maintained by using a
124
surrounding water jacket for the buffer. Force and length were controlled using a dual-
125
mode system (Dual Mode Servomotor model 300B, Cambridge Technology, Aurora Scientific
126
Inc. ON, Canada). Signal data was acquired and sent via a National Instruments DAQ board to
127
a LabVIEW program (NI-DAQ7/LabVIEW6) which was developed for this purpose. Reference
128
diameter (D0) was determined with repeated cycles of stretching, contraction (ACh 10-4M)
129
and rinsing, until the maximum contractile force (the force during contraction – also termed
130
passive force) was reached. Subsequently the airway was allowed to stabilize at D0 for 30
131
minutes.
132 133
2.3. Experimental protocols
134
For each testing protocol, the tissue was first constricted with ACh 10-4M until a stabilized
135
plateau was reached. Subsequently 1) ISO 10-6M, 2) oscillations or 3) ISO 10-6M + oscillations
136
were applied to the tracheal ring (Fig 2). The concentration used for ACh and ISO were
137
previously determined and selected using dose response curves for each drug. Following
6
138
this, each tracheal ring was rinsed repeatedly with Krebs solution and allowed to recover
139
prior to a subsequent test.
140
141
Assuming that airway compliance is similar to total lung compliance, the ASM length
142
oscillation can be derived from the cube root of the lung volume changes (∛(∆V )) (13, 18).
143
Values were calculated to be about 4% for normal breathing and about 25-30% for deep
144
inspiration (19). The following length oscillations were tested: 1) breathing oscillations with
145
amplitude/frequency of 4%/2.7Hz (determined using mice breathing patterns as reference),
146
2) SILO with amplitude/frequency of 1% and 1.5%/5, 10, 15 and 20 Hz. The combined ISO-
147
oscillations protocol was performed with 10-6M ISO. All oscillation amplitudes were
148
calculated as percentage of D0.
149
150
2.4. Evaluation of the model
151
The following procedures were used to assess the model:
152
AH plethysmography
153
Airway hyperreactivity was measured and evaluated in spontaneously tracheotomized mice
154
through plethysmography, using lung resistance (RL = tracheal pressure / Flow rate) as a
155
reference value. The objective of this technique was to evaluate bronchoconstriction in the
156
control, and sensitized animal model.
157
158 7
159
ELISA
160
In asthmatic models the levels of the antibody IgE are normally increased. In order to
161
confirm that our model showed some asthmatic symptoms, the level of this antibody was
162
tested using a direct sandwich Enzyme Linked Immunosorbent Assay (ELISA) method in
163
duplicate.
164
experimental protocols and using coagulation and centrifugation, samples of serum were
165
prepared from it and then stored at -80°C. Then IgE levels were quantified using the kit
166
Mouse OVA-IgE ELISA from mdbioproducts (division of mdbiosciences, Zúrich, Switzerland)
167
and compared between controls and sensitized.
Blood samples from the mice heart were obtained right after the
168 169
Broncho alveolar lavage (BAL)
170
BAL was used to recover an airway sample to evaluate and compare the cellularity of the
171
healthy and sensitized mice. This technique involves successive lavages of the airways with
172
physiological solutions. After the experimental protocols, the lungs were cannulated in situ
173
and washed with 1ml of saline (NaCl 0.9%) several times before collecting a representative
174
sample of the airways. Slides were prepared from the BAL and stained with hematoxylin and
175
eosin staining. White cells (WC) were counted and differentiated in a blinded fashion by
176
counting 100 cells by light microscopy. The number of eosinophies were expressed as a % of
177
the total WC.
178
179
8
180
3. Results
181
3.1 Evaluation of the model:
182
Respiratory plethysmography was used to diagnose sensitized models (15). Table 1 shows
183
RL, ELISA and BAL results for both tested groups, healthy and sensitized. It shows an increase
184
in airway response to Ach 10-4M in sensitized mice when compared to healthy ones (n=6 in
185
each group). RL increased from 1.62 ± 0.14 to 2.28 ± 0.16 cmH2O/ml/sec (Mean ± Standard
186
Error). This is a typical feature of AH associated with asthma. Other parameters such as the
187
levels of anti-OVA Immunoglobulin E (IgE) and analyses of cellularity in broncho-alveolar
188
lavage (BAL) were also analyzed and found to increase in the sensitized model when
189
compared with the healthy mice. ELISA showed a significant increase of IgE with a p < 0.05.
190
The BAL showed increased presence of white, red and epithelial cells in all slides from
191
sensitized slides. In fact the healthy slides showed poor presence or complete absence of
192
the same cellularity. A significant increase in eosinophils was observed (21 ± 6% compared
193
to 3 ± 1%.
194 195
3.2 In vitro testing:
196
Figure 3 shows the percentage relaxation (%R) for various conditions. %R was calculated as
197
the total reaction force b (see Fig 2) observed 5 minutes after the application of ISO and/or
198
oscillations relative to the force observed prior to these agents, which corresponds to the
199
plateau (a) (%R = ((a-b)/a) 100). The statistical analysis was performed considering a normal
200
distribution of the data and using a paired t-test for each dose of treatment (% amplitude,
201
Hz and ISO). The figure shows that combining ISO with oscillations increases relaxation in
202
healthy airways (as previously observed), compared to either ISO or breathing oscillations
9
203
alone (~43 ± 8.3, 47 ± 9 and 57 ± 9.30% respectively). However, this effect is missing in
204
sensitized airways. In fact, the effectiveness of ISO, breathing oscillations and both agents
205
combined significantly reduced in sensitized airways when compared to the effect of the
206
same agents in healthy airways (~3 ± 1.85, 5 ± 1.3 and 6 ± 3.2% respectively).
207 208
3.3 Breathing, ISO and both agents combined
209
Figure 4 compares the effect of SILO of 1% amplitude and frequencies of 5, 10, 15 and 20 Hz
210
with the effect of breathing oscillations and ISO alone as well as combined together in
211
sensitized airways. ANOVA and Wilcoxon signed ranked test was also used in these analyses.
212
The figure shows various responses can be observed but without any statistical significance
213
except a significant increase in relaxation is observed at 5 Hz (p value < 0.05 through t-test).
214
Figure 5, on the other hand, shows that SILO with an amplitude of 1.5% and at the four
215
frequencies tested in this work significantly increases relaxation (ANOVA and t-test p
1 2
RELAXANT EFFECT OF SUPERIMPOSED LENGTH OSCILLATION ON SENSITIZED AIRWAY SMOOTH MUSCLE
3 4 5 6 7
Miguel Jo-Avila, Ahmed M. Al-Jumaily, Jun Lu. Institute of Biomedical Technologies Auckland University of Technology, Auckland, New Zealand.
8
9
10 11 12 13 14 15 16 17 18
Contact Author Ahmed Al-Jumaily, PhD, MSc, BSc Professor of Biomechanical Engineering Director, Institute of Biomedical Technologies Auckland University of Technology Private Bag 92006, City Campus, WD306 Auckland, New Zealand Tel: (649) 921 9777; Fax:(649) 921 9973 Email:[email protected] Website:www.ibtec.org.nz
19
20
21
22
23
24
25 1
Copyright © 2014 by the American Physiological Society.
26
Abstract
27
Asthma is associated with reductions in the airway lumen and breathing difficulties which
28
are attributed to airway smooth muscles (ASM) hyperconstriction. Pharmaceutical
29
bronchodilators such as salbutamol and Isoproterenol (ISO) are normally used to alleviate
30
this constriction. Deep inspirations (DI) and tidal oscillations (TO) have also been reported to
31
relax ASM in healthy airways with less response in asthmatics. Little information is available
32
on the effect of other forms of oscillation on asthmatic airways. This study investigates the
33
effect of length oscillations (LO), with amplitude 1% and 1.5% in the frequency range 5-20
34
Hz superimposed on breathing equivalent LO, on contracted ASM dissected from sensitized
35
mice. These mice are believed to show some symptoms such as airway hyperreactivity (AH)
36
similar to those associated with asthma in humans. In the frequency range used in this work,
37
this study shows an increase in ASM relaxation of an average of 10% for 1.5% amplitude
38
when compared with TO, ISO or the combination of both. No similar finding is observed with
39
1% amplitude. This suggests that superimposed length oscillation (SILO) acting over the
40
interaction of myosin and actin during contraction may lead to temporal rearrangement and
41
disturbance of the cross-bridge process in asthmatic airways.
42 43 44 45 46 47 48 2
49
1. Introduction
50
Asthma is primarily characterized by reversible airway hyper-constriction, hyper-
51
responsiveness and inflammation. It is believed that the key effector of airway
52
bronchoconstriction in asthma is attributed to ASM contraction (14). This contraction is
53
normally relieved by using a pharmaceutical relaxant such as Isoproterenol (ISO).
54
Unfortunately, some relaxants are often associated with various degrees of side effects (7,
55
25). Searching for alternative treatments with less-harmful side effects has been the main
56
objective of many investigators in this field of study (1, 3, 8, 9, 11, 12, 17, 20, 26, 29).
57
58
At the cellular level, ASM contraction and relaxation are determined by the extent of the
59
actin-myosin cross-bridge cycling process. With the activation of ASM, this biophysical
60
process may be initiated by hormonal or neural stimuli followed by biochemical activities
61
which result in relatively restricted movement between the myosin and actin filaments to
62
produce muscle contraction. Pharmacological treatments are normally used to alleviate this
63
contraction by either relaxing the constricted airways or reducing the inflammation
64
observed. Alternatively, several in vitro studies have demonstrated that LO equivalent to
65
those occurring during tidal breathing or with some SILO do disturb the cross-bridge cyclic
66
process and produce some relaxation in pre-contracted ASM obtained from healthy airways
67
(11, 12, 14, 32). Further, it has been demonstrated that combining LO with ISO enhances the
68
relaxation for contracted tissues obtained from healthy airways (17); particularly, it was
69
indicated that ISO of 10-7 to 10-5 M caused the same force inhibition as TO. It was also
70
observed that the dose response curve of ISO when examined with and without TO, the
71
amount of relaxation between the two was equivalent on all but the highest concentration 3
72
of ISO (10-5M), suggesting that the mechanisms acting on the relaxing effects of ISO and
73
tidal oscillations are largely independent, i.e. deactivation vs. disruption of the myosin cross-
74
bridge (17).
75
76
The above cited studies were conducted on tissues isolated from healthy airways. However,
77
recent studies such as Chin et al (10) and Noble et al (28) have focused on asthmatic
78
airways, with the latter focusing on DI and TO effect. To the best of our knowledge other LO
79
different to those occurring during breathing and deep inspiration have not been tested on
80
asthmatic airways as a therapeutic alternative. Thus our interest focuses on testing the
81
effect of different amplitudes and frequencies of SILO on contracted ASM from sensitized
82
mice. It is expected that this would help to fill the knowledge gap and will allow us to learn
83
more about the behavior of sensitized airways when SILO are applied during breathing.
84
85
In spite of the fact that no animal model can fully resemble human asthma, it has been
86
observed that some features like inflammation, AHR and remodeling of the airways can be
87
reproduced in animal models (16, 22-24). Mouse model presents advantages for
88
physiological studies of asthma because of their size, short cycles of breeding, and existing
89
data regarding sensitized models resembling various features of asthma. The recent
90
advances in transgenic technology and the development of species-specific probes, have
91
allowed detailed mechanistic studies to be conducted on mouse models (4,5, 33). The main
92
purpose of using mice in this study was to generate an acute sensitized model capable of
93
resembling closely airway hyperreactivity (AH) observed during an asthma attack. This will
4
94
be significant in understanding how airways from healthy and sensitized mice respond in
95
vitro to external agents such as ISO, LO and SILO during an induced asthmatic attack.
96
97
2. Material and methods
98
All experimental procedures were approved by the University of Auckland Animal Ethics
99
Committee according to the Code of Ethical Conduct and performed in accordance with the
100
Animal Welfare Act 1999 New Zealand.
101
102
2.1. Animal model and sensitization
103
We designed and used an ovalbumin (OVA) sensitization model (acute model) for this study,
104
following a protocol similar to that of Kumar et al 2008 (24). The mouse strain used during
105
the study was Balb/c, female, between 8-12 weeks old. They were selected due to the fact
106
that this strain is highly reactive in respiratory studies and hence commonly used for
107
sensitization protocols (27). The model included two methods of administration of the
108
allergen (Fig 1). First, intraperitoneal injections of OVA at 10g diluted in sodium chloride
109
0.9% (saline) + 1mg aluminum hydroxide mixture were administered to the animals on days
110
0, 7 and 14, in order to induce immunological response. Second, after 10 days of the last
111
intraperitoneal injection, aerial nebulization of OVA at 5%, diluted in saline, was used to
112
expose the airways to the allergen for 20 min, on day 24, 28 and 32. After the last
113
nebulization the mice were allowed to rest for 24 hrs and were culled on day 33 to obtain
114
the tissue (tracheal rings) for testing.
115
2.2. Tissue preparation and experimental apparatus 5
116
Tracheal rings 2mm wide were obtained from healthy and sensitized mice, and were
117
mounted in a 5mL computerized organ bath system (800A in vitro test system, Aurora
118
Scientific Inc., Ontario, Canada) as previously described (21). Each tracheal ring was
119
suspended vertically to a servo-controlled lever arm (Model 300C, Cambridge Technology,
120
Aurora Scientific Inc.) using steel wire and non-penetrating hooks. The bath was filled with
121
physiological Krebs solution (composition in mM: 110 NaCl, 0.82 MgSO4, 1.2 KH2PO4, 3.39
122
KCL, 2.4 CaCl2, 25.7 NaHCO3 and 5.6 Glucose) and bubbled with a gas mix of 95% O2/5% CO2
123
to maintain the pH at 7.4. A constant temperature of 37°C was maintained by using a
124
surrounding water jacket for the buffer. Force and length were controlled using a dual-
125
mode system (Dual Mode Servomotor model 300B, Cambridge Technology, Aurora Scientific
126
Inc. ON, Canada). Signal data was acquired and sent via a National Instruments DAQ board to
127
a LabVIEW program (NI-DAQ7/LabVIEW6) which was developed for this purpose. Reference
128
diameter (D0) was determined with repeated cycles of stretching, contraction (ACh 10-4M)
129
and rinsing, until the maximum contractile force (the force during contraction – also termed
130
passive force) was reached. Subsequently the airway was allowed to stabilize at D0 for 30
131
minutes.
132 133
2.3. Experimental protocols
134
For each testing protocol, the tissue was first constricted with ACh 10-4M until a stabilized
135
plateau was reached. Subsequently 1) ISO 10-6M, 2) oscillations or 3) ISO 10-6M + oscillations
136
were applied to the tracheal ring (Fig 2). The concentration used for ACh and ISO were
137
previously determined and selected using dose response curves for each drug. Following
6
138
this, each tracheal ring was rinsed repeatedly with Krebs solution and allowed to recover
139
prior to a subsequent test.
140
141
Assuming that airway compliance is similar to total lung compliance, the ASM length
142
oscillation can be derived from the cube root of the lung volume changes (∛(∆V )) (13, 18).
143
Values were calculated to be about 4% for normal breathing and about 25-30% for deep
144
inspiration (19). The following length oscillations were tested: 1) breathing oscillations with
145
amplitude/frequency of 4%/2.7Hz (determined using mice breathing patterns as reference),
146
2) SILO with amplitude/frequency of 1% and 1.5%/5, 10, 15 and 20 Hz. The combined ISO-
147
oscillations protocol was performed with 10-6M ISO. All oscillation amplitudes were
148
calculated as percentage of D0.
149
150
2.4. Evaluation of the model
151
The following procedures were used to assess the model:
152
AH plethysmography
153
Airway hyperreactivity was measured and evaluated in spontaneously tracheotomized mice
154
through plethysmography, using lung resistance (RL = tracheal pressure / Flow rate) as a
155
reference value. The objective of this technique was to evaluate bronchoconstriction in the
156
control, and sensitized animal model.
157
158 7
159
ELISA
160
In asthmatic models the levels of the antibody IgE are normally increased. In order to
161
confirm that our model showed some asthmatic symptoms, the level of this antibody was
162
tested using a direct sandwich Enzyme Linked Immunosorbent Assay (ELISA) method in
163
duplicate.
164
experimental protocols and using coagulation and centrifugation, samples of serum were
165
prepared from it and then stored at -80°C. Then IgE levels were quantified using the kit
166
Mouse OVA-IgE ELISA from mdbioproducts (division of mdbiosciences, Zúrich, Switzerland)
167
and compared between controls and sensitized.
Blood samples from the mice heart were obtained right after the
168 169
Broncho alveolar lavage (BAL)
170
BAL was used to recover an airway sample to evaluate and compare the cellularity of the
171
healthy and sensitized mice. This technique involves successive lavages of the airways with
172
physiological solutions. After the experimental protocols, the lungs were cannulated in situ
173
and washed with 1ml of saline (NaCl 0.9%) several times before collecting a representative
174
sample of the airways. Slides were prepared from the BAL and stained with hematoxylin and
175
eosin staining. White cells (WC) were counted and differentiated in a blinded fashion by
176
counting 100 cells by light microscopy. The number of eosinophies were expressed as a % of
177
the total WC.
178
179
8
180
3. Results
181
3.1 Evaluation of the model:
182
Respiratory plethysmography was used to diagnose sensitized models (15). Table 1 shows
183
RL, ELISA and BAL results for both tested groups, healthy and sensitized. It shows an increase
184
in airway response to Ach 10-4M in sensitized mice when compared to healthy ones (n=6 in
185
each group). RL increased from 1.62 ± 0.14 to 2.28 ± 0.16 cmH2O/ml/sec (Mean ± Standard
186
Error). This is a typical feature of AH associated with asthma. Other parameters such as the
187
levels of anti-OVA Immunoglobulin E (IgE) and analyses of cellularity in broncho-alveolar
188
lavage (BAL) were also analyzed and found to increase in the sensitized model when
189
compared with the healthy mice. ELISA showed a significant increase of IgE with a p < 0.05.
190
The BAL showed increased presence of white, red and epithelial cells in all slides from
191
sensitized slides. In fact the healthy slides showed poor presence or complete absence of
192
the same cellularity. A significant increase in eosinophils was observed (21 ± 6% compared
193
to 3 ± 1%.
194 195
3.2 In vitro testing:
196
Figure 3 shows the percentage relaxation (%R) for various conditions. %R was calculated as
197
the total reaction force b (see Fig 2) observed 5 minutes after the application of ISO and/or
198
oscillations relative to the force observed prior to these agents, which corresponds to the
199
plateau (a) (%R = ((a-b)/a) 100). The statistical analysis was performed considering a normal
200
distribution of the data and using a paired t-test for each dose of treatment (% amplitude,
201
Hz and ISO). The figure shows that combining ISO with oscillations increases relaxation in
202
healthy airways (as previously observed), compared to either ISO or breathing oscillations
9
203
alone (~43 ± 8.3, 47 ± 9 and 57 ± 9.30% respectively). However, this effect is missing in
204
sensitized airways. In fact, the effectiveness of ISO, breathing oscillations and both agents
205
combined significantly reduced in sensitized airways when compared to the effect of the
206
same agents in healthy airways (~3 ± 1.85, 5 ± 1.3 and 6 ± 3.2% respectively).
207 208
3.3 Breathing, ISO and both agents combined
209
Figure 4 compares the effect of SILO of 1% amplitude and frequencies of 5, 10, 15 and 20 Hz
210
with the effect of breathing oscillations and ISO alone as well as combined together in
211
sensitized airways. ANOVA and Wilcoxon signed ranked test was also used in these analyses.
212
The figure shows various responses can be observed but without any statistical significance
213
except a significant increase in relaxation is observed at 5 Hz (p value < 0.05 through t-test).
214
Figure 5, on the other hand, shows that SILO with an amplitude of 1.5% and at the four
215
frequencies tested in this work significantly increases relaxation (ANOVA and t-test p
