Force adaptation through the intravenous route in naïve mice

Abstract Aim of the study: Force adaptation is a process whereby the contractile capacity of the airway smooth muscle increases during a sustained contraction (aka tone). Tone also increases the response to a nebulized challenge with methacholine in vivo, presumably through force adaptation. Yet, due to its patchy pattern of deposition, nebulized methacholine often spurs small airway narrowing heterogeneity and closure, two important enhancers of the methacholine response. This raises the possibility that the potentiating effect of tone on the methacholine response is not due to force adaptation but by furthering heterogeneity and closure. Herein, methacholine was delivered homogenously through the intravenous (i.v.) route. Materials and Methods: Female and male BALB/c mice were subjected to one of two i.v. methacholine challenges, each of the same cumulative dose but starting by a 20-min period either with or without tone induced by serial i.v. boluses. Changes in respiratory mechanics were monitored throughout by oscillometry, and the response after the final dose was compared between the two challenges to assess the effect of tone. Results: For the elastance of the respiratory system (Ers), tone potentiated the methacholine response by 64 and 405% in females (37.4 ± 10.7 vs. 61.5 ± 15.1 cmH2O/mL; p = 0.01) and males (33.0 ± 14.3 vs. 166.7 ± 60.6 cmH2O/mL; p = 0.0004), respectively. For the resistance of the respiratory system (Rrs), tone potentiated the methacholine response by 129 and 225% in females (9.7 ± 3.5 vs. 22.2 ± 4.3 cmH2O·s/mL; p = 0.0003) and males (10.7 ± 3.1 vs. 34.7 ± 7.9 cmH2O·s/mL; p < 0.0001), respectively. Conclusions: As previously reported with nebulized challenges, tone increases the response to i.v. methacholine in both sexes; albeit sexual dimorphisms were obvious regarding the relative resistive versus elastic nature of this potentiation. This represents further support that tone increases the lung response to methacholine through force adaptation.


Introduction
The contractile lability of the airway smooth muscle (ASM) is well documented 1,2 and likely contributes to airway hyperresponsiveness in inflammatory lung disorders, such as asthma. 24][5][6][7][8][9] This phenomenon was coined 'force adaptation' .There have been fewer reports investigating the impact of force adaptation on the response to methacholine in vivo.Yet, in both mice 6,7 and humans, 8 a methacholine dose staggered over 20-30 min always leads to a markedly greater response compared to the same dose delivered in one bolus.7][18] Together, it implies that the potentiating effect of tone on the methacholine response in vivo may occur through mechanisms other than force adaptation.
7][8] This amplifies the above concerns for at least two reasons.First, although the deposition pattern of certain nebulized nanoparticles, such as melamine resin, can be impressively widespread and homogeneous throughout the lung 19 using specific ventilation profile, 19,20 nebulized methacholine is typically thought to accumulate predominantly in large airways in a patchily manner; alike tantalum dust, 21 technetium-99m-labeled pentetic acid, 22 and iodine. 23Early after the nebulization, this means that some conducting airways are constricting while the surrounding peripheral airways are still compliant, an imbalance furthering airway narrowing heterogeneity 24,25 and small airway closure. 26,279][30][31][32] The latter promotes heterogeneity and closure, chiefly by increasing the propensity of plugging, 33 either by directly drowning small airways or by increasing surface tension and the formation of stretchable menisci across the luminal area that also functionally blocks airflow in subtending airways.
Sidestepping the confounding effect of narrowing heterogeneity and closure would thus be important to reinforce previous interpretations suggesting that force adaptation is the underlying mechanism whereby tone increases the methacholine response.7][8] This strategy should minimize the confounding effect of narrowing heterogeneity and closure.First, because it ensures a homogenous distribution of methacholine in peripheral airways and, this, irrespective of the constricted state of the lung prior to the delivery of the final dose. 23,34And second, because it limits mucus secretion, since methacholine is then stimulating ASM without first passing through the epithelium.[37]

Mice
Seven-week-old female and male BALB/c mice were purchased from Charles River Laboratory (Bar Harbor, ME).Mice were provided food and water ad libitum and were housed one week before experiments.They were thus tested at 8 wk.All protocols were approved by the Committee of Animal Care of Université Laval in accordance with guidelines of the Canadian Council on Animal Care (2022-977).

Changes in respiratory mechanics during the methacholine challenge
Mice were weighted, anesthetized, tracheotomized, mechanically ventilated and paralyzed as previously described. 38The outline is depicted in Figure 1.Within each sex, the degree of airway responsiveness was compared between two groups of mice subjected to a methacholine challenge that started with a ~20-min period either with or without tone.There was no deep inspiration before and throughout the methacholine challenge.The 20-min duration was chosen based on a previous in vitro study with excised mouse tracheas, showing that the gain in ASM's contractile capacity induced by tone is significant from 20 min post-exposure and beyond. 6The approach was very similar to our previous studies, 6,7 except that the methacholine was delivered i.v.instead of nebulized into the trachea.For injecting the i.v.boluses, a 25-gauge cannula was inserted into the right jugular vein.The injected doses were twice higher in females than males owing to their inherently lower degree of responsiveness. 7,39ince the effect of methacholine is waning more rapidly when administered i.v.than by nebulization, 40 it was added more frequently during the period of tone.More precisely, a bolus (5 µL) of methacholine was injected every 65 s (Figure 1).The dose delivered over ~20 min (in a total volume of 100 µL) was 0.5 and 1 mg/kg for males and females, respectively.The group without tone was receiving an equivalent amount of saline using the same dosing regimen.The degree of responsiveness was then assessed by injecting a high dose of methacholine.This final high dose was adjusted so that each mouse, exposed or not to tone, received the same cumulative dose.In females, the final dose was either 3 or 4 mg/kg in mice exposed or not to tone, respectively, for a total cumulative dose of 4 mg/kg.In males, the final dose was either 1.5 or 2 mg/kg in mice exposed or not to tone, respectively, for a total cumulative dose of 2 mg/kg.
Respiratory mechanics was monitored before and throughout the methacholine challenge with the flexiVent (FX Module 2, SCIREQ, Montreal, Canada) using an oscillometric perturbation called the Snapshot-150.This perturbation was actuated twice at baseline and at 13-s intervals throughout the methacholine challenge.Parameters obtained from the Snapshot-150 include the elastance of the respiratory system (E rs ) and the resistance of the respiratory system (R rs ). 41Only changes from baseline (Δ) are shown in figures, representing the methacholine response.
Five minutes after the administration of the final dose of methacholine, isoproterenol (10 mg/mL) was nebulized to pharmacologically relax the ASM.The nebulizer for small particle size (volume median diameter between 2.5 and 4 µm; Aeroneb Lab; Aerogen Inc, Ireland) was operating for a duration of 10 s at a duty cycle of 50% under regular ventilation.One deep inflation (DI) was then imposed 5 min afterwards to mechanically relax the ASM.The DI maneuver consisted of inflating the lung by increasing the pressure at a constant rate until it reaches 30 cmH 2 O. Respiratory mechanics were still monitored at regular intervals (38 s) after these interventions.[44][45][46][47][48][49]

Statistical analyses
Data shown are means and SD.Unpaired t tests were used to compare weight, as well as baseline R rs and E rs between sexes.Unpaired t tests were also used to compare ΔR rs and ΔE rs between challenges with and without tone at specific time-points (more precisely, at the very end of the period with or without tone, at the peak response after the final dose, 5 min after the final dose of methacholine, 5 min after the administration of isoproterenol, and immediately after the DI).3][44][45][46][47][48][49] Values of ΔE rs and ΔR rs before these interventions were taken at the peak methacholine response, and values after these interventions were taken immediately after the DI.For each readout (ΔE rs and ΔR rs ), this repeated measures two-way ANOVA assessed the effect of tone, the combined effect of isoproterenol + the DI, as well as their interaction on the methacholine response.A significant interaction means that the effect of tone on the methacholine response is different before versus after the inhibition of ASM, thereby implying that the potentiating effect of tone on the methacholine response is mediated through a mechanism relying on ASM.All statistical analyses and graphs were performed using Prism 9 (version 9.1.1,GraphPad, San Diego, CA).p < 0.05 was considered significant to reject the null hypothesis.
The new dosing regimen through the i.v.route was successful to maintain tone.The mean traces of ΔE rs in males during the first ~20 min for both methacholine challenges are depicted in Figure 2. Compared to mice receiving saline (i.e., not exposed to tone), the ΔE rs in mice exposed to tone was obvious after the first dose of methacholine.The effect of each dose was transient, as can be seen from the 20 clearly distinguished peaks in ΔE rs .Yet, the consistent rate of increase over a longer time-scale (Figure 2) indicated that the next dose was usually delivered before the effect of the previous dose had completely vanished.E rs was also creeping up in the challenge without tone, presumably due to a progressive stiffening of the lung tissue and/or accretion of small airway closure.However, the total rise after ~20 min without tone was nowhere near the one caused by tone (4.0 ± 1.6 vs. 16.1 ± 8.3 cmH 2 O/ mL, respectively; p = 0.01).
ΔE rs during the whole protocol is depicted in Figure 3, showing the responses to the i.v.methacholine challenge, the nebulized isoproterenol, and the DI.Tone potentiated the response to methacholine in both sexes.The potentiating effect was markedly greater in males than females (note the different scales on the y-axis).In fact, tone increased the methacholine response (i.e., the peak response) by a mean of 405% in males (t test, p = 0.0004) and 64% in females (t test, p = 0.01).As observed for each dose for inducing tone, the response to the final (and higher) dose was transient, chiefly reflecting the quick clearance of methacholine.Yet, ΔE rs was still higher in mice exposed to tone than in mice not exposed to tone 5 min after the final dose (p = 0.008 and 0.003 in females and males, respectively).Isoproterenol then slightly accelerated the rate of recovery.Yet, even after 5 min of isoproterenol exposure, ΔE rs was still clearly visible (i.e., E rs was still above baseline in both sexes).Notably, the difference between mice with and without tone was mostly preserved after isoproterenol in males (p = 0.004), while it was gone in females (p = 0.87).The DI then completely abolished the methacholine-induced ΔE rs in both sexes.In fact, ΔE rs became negative post-DI, presumably reflecting recruitment and/or a transient increase in Only the first 3 doses and the 20 th one prior to the final higher dose are indicated for clarity.the red vertical dotted line indicates when the higher final i.v.dose was delivered.these results are also shown in Figure 3 (first ~20 min of the upper left graph), but on a different scale.Data are means + or -sD for a n = 6. the dosing regimen was identical in females, except that all doses were twice lower.
lung compliance post-DI.Interestingly, the difference between mice with and without tone was completely abrogated by DI in males (t test, p = 0.80), while it was back observed in females (t test, p = 0.004).Note also the significant interaction in the repeated two-way ANOVA (Figure 3, underneath the bottom graph) between the effect of tone and the combined effect of the two interventions inhibiting ASM (i.e., isoproterenol + the DI) in males, but not in females.
ΔR rs during the whole protocol is depicted in Figure 4.As per E rs , tone potentiated the response of R rs to methacholine in both sexes.The potentiating effect was again greater in males than females (note the different scales on the y-axis).In fact, tone increased the methacholine response Figure 3. the effect of tone on the methacholine-induced changes in respiratory system elastance relative to baseline (ΔE rs ). the upper left and right graphs demonstrate the actual changes in E rs throughout the whole protocol in both males and females, respectively.note that the scale on the y-axis is smaller in females than males.the three vertical red dotted lines from left to right indicate when: 1-the final i.v.dose of methacholine was administered; 2-the nebulized dose of isoproterenol (iso) was administered; and 3-the deep inspiration (Di) was imposed. in the bottom graph, the max E rs after the final dose (i.e., peak response), as well as the minimal E rs after iso and the Di, are compared between challenges with and without tone in each sex.results of two-way anOVas are shown below the graph, which were used to evaluate the effect of tone, the combined effect of iso and the Di, and their interaction within each sex.asterisks are from post hoc analyses, showing significant differences between challenges with and without tone for the max response (**** is p < 0.0001).Data are means + or -sD for a n = 6.
(i.e., the peak response) by a mean of 225% in males (t test, p < 0.0001) and 129% in females (t test, p = 0.0003).The effect of methacholine on ΔR rs was peaking earlier (between 65 and 91s) than for ΔE rs (between 130 and 195 s).It was also receding more quickly, reaching nearly baseline values 5 min after the administration of the final dose.Isoproterenol still slightly hastened the recovery and the DI completely abolished the small remaining effect of methacholine on ΔR rs .In contrast to ΔE rs , the kinetics of changes for ΔR rs was qualitatively similar between sexes and, most relevant to the present study, the difference between mice with and without tone was .the effect of tone on the methacholine-induced changes in respiratory system resistance relative to baseline (Δr rs ). the upper left and right graphs demonstrate the actual changes in r rs throughout the whole protocol in both males and females, respectively.note that the scale on the y-axis is smaller in females than males.the three vertical red dotted lines from left to right indicate when: 1-the last i.v.dose of methacholine was administered; 2-the nebulized dose of isoproterenol (iso) was administered; and 3-the deep inspiration (Di) was imposed. in the bottom graph, the max r rs after the final dose (i.e., peak response), as well as the minimal r rs after iso and the Di, are compared between challenges with and without tone in each sex.results of two-way anOVas are shown below the graph, which were used to evaluate the effect of tone, the combined effect of iso and the Di, and their interaction within each sex.asterisks are from post hoc analyses, showing significant differences between challenges with and without tone for the max response (**** is p < 0.0001).Data are means + or -sD for a n = 6.
completely abrogated before the administration of isoproterenol (p = 0.33 and 0.83 in females and males, respectively).

Discussion
6][37] As previously reported with nebulized challenges, [6][7][8] tone still markedly potentiated the response to methacholine.The potentiating effect of tone on the methacholine response was observed by monitoring changes in both elastance and resistance of the respiratory system.It was also observed in both sexes, although the magnitude of the potentiated response was greater in males.The nature of the response also differed between sexes, being predominantly elastic in males, but rather resistive in females.The potentiating effect was also quickly reversed, at least to a great extent, by interventions inhibiting the ASM.Together, the results lend further support to the notion that tone potentiates the methacholine response in vivo by an ASM-mediated mechanism, likely force adaptation, and not by furthering small airway narrowing heterogeneity and closure.
On isolated ASM preparations in vitro, the gain in ASM's contractile capacity induced by force adaptation is quickly reversible by abating tone through spasmogen removal. 6,50Controlling tone in vivo is more difficult because of the inevitable clearance of methacholine, due to the washing effect of circulating blood and/or methacholine degradation.Yet, owing to the reversible nature of force adaptation, 6,50 the maintenance of tone was absolutely required in order to assess its impact on respiratory mechanics.One way to keep the level of ASM activation above baseline is to deliver serial doses of methacholine.][8] Preliminary experiments (not shown) clearly established that this was not the case for an i.v.challenge.In fact, the kinetics of changes in oscillometric readouts following methacholine delivery is strikingly different between nebulized and i.v.challenges. 40During a nebulized challenge, the effects of methacholine are manifested slower, and also last longer.Based on a computational model, this was predicted to be entirely due to the epithelium, which the inhaled methacholine has to traverse before reaching the ASM 40 .The effect of i.v.methacholine is more transient, acting more rapidly and also vanishing more quickly.For this reason, the dosing regimen during the period of tone was changed.Instead of delivering methacholine every 5 min, it was delivered at 65-s intervals.Figure 2 showed that tone was maintained throughout the period of tone.Elastance was actually slightly rising over time.This was also reassuring because the dwindling effect of methacholine on respiratory mechanics outlasts, sometimes by a substantial margin, the vanishing rate of methacholine concentration acting on the ASM. 51The slow progressive rise (Figure 2) thus suggests that the dosing regimen was successful to maintain a degree of ASM activation above baseline throughout the period of tone, rendering the protocol conducive to force adaptation.
Irrespective of whether the response was measured by monitoring the changes in elastance or resistance, tone significantly potentiated the response to i.v.methacholine in both sexes.Therefore, an i.v.challenge -a strategy curbing small airway heterogeneity and closure by delivering methacholine homogenously throughout the lung, as well as by limiting mucus secretion by acting first on the ASM before reaching the epithelium -was just as effective as a nebulized challenge to demonstrate the potentiating effect of tone on the methacholine response.This represents further evidence that the underlying mechanism whereby tone potentiates the methacholine response is force adaptation.
The potentiating effect of tone on the methacholine response was short-lived.This is mainly due to the quick clearance of methacholine, and the resulting transient nature of its effect.Yet, the proposal is that when tone is mediated by the production and release of endogenous spasmogens, such histamine and leukotrienes, which are typically overexpressed in asthma, 52 the phenomenon may contribute to airway hyperresponsiveness.In other words, the fleeting effect of tone on the methacholine response observed in the present may persist in diseased lungs as long as those inflammation-derived spasmogens are overexpressed.
Differences between sexes were observed though.The potentiating effect of tone on the methacholine response was greater in magnitude for males than females.This happened despite lower methacholine doses in the former.The nature of the response also varied markedly, being predominantly elastic in males but rather resistive in females.The reason for these sexual dimorphisms is uncertain.We have recently demonstrated in this mouse strain (BALB/c) that males have more ASM than females. 53We have also recently confirmed findings of others, 46,[54][55][56][57] showing that the ASM undergoes an elastic transition during a sustained contraction. 43e previously argued that 'force adaptation' and this 'elastic transition' are two monikers used to describe two phenomena in different experimental contexts but that probably rely on the same underlying mechanism. 7,43We therefore speculate that this elastic transition impacts lung mechanics to a greater extent in males merely because their airways are more muscular than females.
The responses to a bronchodilator drug and a DI were also different between sexes, at least for the changes in elastance.While elastance in mice exposed to tone was back to the level seen in mice not exposed to tone 5 min after the delivery of isoproterenol in females, it was still elevated by tone at that time-point in males.This can be due to the fact that the potentiating effect of tone was greater in males than females, implying a longer time to subside after relaxing the ASM with isoproterenol.The greater level of constriction in males may have also decreased the amount of nebulized isoproterenol entering the lung 23 and/ or foster a more proximal deposition pattern, 34 which may have then attenuated its effect in males compared to females.Contrastingly, after the DI, elastance in males exposed to tone settled to the level seen in males not exposed to tone, while it was then elevated anew by tone in females.The complete disappearance of the effect of tone post-DI in males reinforced the proposal that the potentiating effect of tone on the methacholine response occurs through an ASM-mediated mechanism.The remaining effect of tone after the DI in females is more perplexing.It can be due to several things.Perhaps two DIs instead of one would have been required to completely eliminate the mechanical effect of tone on the ASM in females.The different methacholine doses between sexes may also have played a role.Since the doses were twice higher in females, the effect of methacholine may have lingered or may not have been completely inhibited by the used dose of isoproterenol.It is also possible that this higher dose may have triggered mucus secretion.Indeed, the i.v.route may limit mucus secretion by acting first on the ASM, but it may still hit the epithelium after traversing the ASM and the lamina propria if delivered at sufficiently high concentrations.Obviously, the enhancing effect of an excess of mucus on lung elastance should not disappear after a DI, perhaps explaining why elastance was again higher in female mice exposed to tone post-DI.
It is also understood that the effect of tone on the methacholine response may be very different in a context where inflammation is prominent.1][12][13][14][15][16][17][18] Whether tone modifies asthmatic hyperresponsiveness to methacholine, and whether it occurs through a gain in ASM's contractile capacity caused by force adaptation or by altering other determinants of the methacholine response, such as heterogeneity and closure, will require further studies.

Conclusion
The present study dug deeper into the underlying mechanisms whereby tone potentiates the methacholine response in vivo.More precisely, it evaluated whether the potentiating effect of tone on the methacholine response occurs in an experimental setting where small airway narrowing heterogeneity and closure are limited by using an i.v.challenge instead of a nebulized challenge.As previously reported with nebulized challenges, [6][7][8] tone markedly increases the methacholine response to an i.v.challenge in both sexes.The mechanism whereby tone potentiates the methacholine response is thus unlikely due to small airway narrowing heterogeneity and

Figure 2 .
Figure 2. tone (i.e., a sustained activation of the airway smooth muscle) induced by methacholine.changes in respiratory system elastance relative to baseline (ΔE rs ) caused by 20 serial i.v.doses of saline (black) or methacholine (gray).Vertical black dotted lines, 65 s apart, indicate when doses were delivered.Only the first 3 doses and the 20 th one prior to the final higher dose are indicated for clarity.the red vertical dotted line indicates when the higher final i.v.dose was delivered.these results are also shown in Figure3(first ~20 min of the upper left graph), but on a different scale.Data are means + or -sD for a n = 6. the dosing regimen was identical in females, except that all doses were twice lower.

Figure 4
Figure 4. the effect of tone on the methacholine-induced changes in respiratory system resistance relative to baseline (Δr rs ). the upper left and right graphs demonstrate the actual changes in r rs throughout the whole protocol in both males and females, respectively.note that the scale on the y-axis is smaller in females than males.the three vertical red dotted lines from left to right indicate when: 1-the last i.v.dose of methacholine was administered; 2-the nebulized dose of isoproterenol (iso) was administered; and 3-the deep inspiration (Di) was imposed. in the bottom graph, the max r rs after the final dose (i.e., peak response), as well as the minimal r rs after iso and the Di, are compared between challenges with and without tone in each sex.results of two-way anOVas are shown below the graph, which were used to evaluate the effect of tone, the combined effect of iso and the Di, and their interaction within each sex.asterisks are from post hoc analyses, showing significant differences between challenges with and without tone for the max response (**** is p < 0.0001).Data are means + or -sD for a n = 6.