I. INTRODUCTION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTION <<II. RESULTSIII. DISCUSSIONIV. METHODSSUPPLEMENTARY MATERIALThe airway epithelial cells lining the luminal surface of the respiratory tract collectively generate protective barrier functions, including mucociliary (MCC) clearance,11. M. R. Knowles and R. C. Boucher, J. Clin. Invest. 109(5), 571 (2002). https://doi.org/10.1172/JCI0215217 intercellular junction formation,22. C. Pohl, M. I. Hermanns, C. Uboldi, M. Bock, S. Fuchs, J. Dei-Anang, E. Mayer, K. Kehe, W. Kummer, and C. J. Kirkpatrick, Eur. J. Pharm. Biopharm. 72(2), 339 (2009). https://doi.org/10.1016/j.ejpb.2008.07.012 and antimicrobial secretion.33. R. Bals and P. S. Hiemstra, Eur. Respir. J. 23(2), 327 (2004). https://doi.org/10.1183/09031936.03.00098803 In particular, the mucociliary flow is a unidirectional surface flow that moves across the airway lumen to ensure clearance of the inhaled materials.44. K. H. Kilburn, Am. Rev. Respir. Dis. 98(3), 449 (1968). https://doi.org/10.1164/arrd.1968.98.3.449 This unidirectional flow is produced in the direction from the distal (D) to proximal (P) lung regions by coordinated, repeated, and polarized beating of cilia of multiciliated cells [Fig. 1(a)].5,65. A. E. Tilley, M. S. Walters, R. Shaykhiev, and R. G. Crystal, Annu. Rev. Physiol. 77, 379 (2015). https://doi.org/10.1146/annurev-physiol-021014-0719316. E. K. Vladar, J. V. Nayak, C. E. Milla, and J. D. Axelrod, JCI Insight 1(13), 88027 (2016). https://doi.org/10.1172/jci.insight.88027 Polarized arrangement of these cells along the P–D axis, which is known as planar cell polarity (PCP), ensures generation of the unidirectional flow.6,76. E. K. Vladar, J. V. Nayak, C. E. Milla, and J. D. Axelrod, JCI Insight 1(13), 88027 (2016). https://doi.org/10.1172/jci.insight.880277. E. K. Vladar, R. D. Bayly, A. M. Sangoram, M. P. Scott, and J. D. Axelrod, Curr. Biol. 22(23), 2203 (2012). https://doi.org/10.1016/j.cub.2012.09.046 Tissue-level PCP is established and maintained by Frizzled (Fzd)/Dishevelled (Dvl) and Van Gogh-like (Vangl)/Prickle (Pk) protein complexes that are arranged at the proximal and distal cell membrane domains, respectively [Fig. 1(b)].6–86. E. K. Vladar, J. V. Nayak, C. E. Milla, and J. D. Axelrod, JCI Insight 1(13), 88027 (2016). https://doi.org/10.1172/jci.insight.880277. E. K. Vladar, R. D. Bayly, A. M. Sangoram, M. P. Scott, and J. D. Axelrod, Curr. Biol. 22(23), 2203 (2012). https://doi.org/10.1016/j.cub.2012.09.0468. E. K. Vladar and M. Konigshoff, Biochem. Soc. Trans. 48(1), 231 (2020). https://doi.org/10.1042/BST20190597 Furthermore, tight and adherens junctions formed between epithelial cells play an important role in the generation of the defensive epithelial barrier.99. S. Koch and A. Nusrat, Ann. N. Y. Acad. Sci. 1165(1), 220 (2009). https://doi.org/10.1111/j.1749-6632.2009.04025.x Transmembrane proteins, such as Zonula occludens-1 (ZO-1) and E-cadherin, promote tight connection between the membranes of adjacent epithelial cells, regulating paracellular permeability.1010. R. Invernizzi, C. M. Lloyd, and P. L. Molyneaux, Immunology 160(2), 171 (2020). https://doi.org/10.1111/imm.13195As the first defensive mechanism of the lung, the epithelial barrier is frequently challenged and injured by inhaled harmful particles, gases, or pathogens,1111. N. V. Dorrello, B. A. Guenthart, J. D. O'Neill, J. Kim, K. Cunningham, Y. W. Chen, M. Biscotti, T. Swayne, H. M. Wobma, S. X. L. Huang, H. W. Snoeck, M. Bacchetta, and G. Vunjak-Novakovic, Sci. Adv. 3(8), e1700521 (2017). https://doi.org/10.1126/sciadv.1700521 or through physical trauma that occurs during clinical procedures, such as intubation and anastomosis [Fig. 1(c)].1212. M. B. Brodsky, M. J. Levy, E. Jedlanek, V. Pandian, B. Blackford, C. Price, G. Cole, A. T. Hillel, S. R. Best, and L. M. Akst, Crit. Care Med. 46(12), 2010 (2018). https://doi.org/10.1097/CCM.0000000000003368 While a minor injury can be resolved quickly by intrinsic repair mechanisms of the healthy airway tissue, severely or repeatedly injured epithelium (EP) can cause prolonged or permanent failure in repair or reduced level of restoration relative to normal tissue integrity and functions.1313. T. Iosifidis, L. W. Garratt, D. R. Coombe, D. A. Knight, S. M. Stick, and A. Kicic, Respirology 21(3), 438 (2016). https://doi.org/10.1111/resp.12731 Disruption in the tissue repair and regeneration pathways and loss of the epithelial cells' ability to restore the defensive barrier could result in aberrant remodeling and structural damage that can further impair epithelial functions.1414. J. Tang, J. Liu, and X. Zhang, J. Healthc Eng. 2022, 7099097. https://doi.org/10.1155/2022/7099097 Acute or persistent structural damage induced to the airway tissue is known to degrade the epithelial barrier functions. However, the correlation between the structure and function of the airway epithelium remains largely elusive.

This study aimed to elucidate the impact of acute airway injury on epithelial barrier functions, including mucociliary clearance and tight/adherens junction formation. To this end, freshly harvested rat tracheal tissues have been studied ex vivo to assess the relationships between the epithelial structure and barrier functions of both healthy and acutely injured airway tissues. The ex vivo airway tissues retained the intrinsic epithelial barrier functions and structure, cellular composition, and underlying extracellular matrix (ECM) components, allowing accurate assessments of the airway tissues. To quantitatively evaluate the epithelial barrier dysfunction, we induced both localized and global injury with varying degrees to the isolated rat tracheas that recapitulates the structural and functional disruption in the injured airway epithelium. The impact of the injury on the mucociliary clearance function of the epithelium was assessed via real-time tracking of microparticles (MPs) being transported over the surface of the airway lumen. Furthermore, we confirmed that the epithelial injury can be diagnosed in situ by measuring electrical properties of the local airway tissue directly at the injury site. To rationalize the acquired experimental data and identified epithelial structure–function relationships, we created computational models that can numerically simulate and predict the impairment of the epithelial barrier functions.

Overall, this study has established both experimentally and computationally the correlations between the structural integrity and functional output of the airway epithelial barrier. The results of the study can contribute to the development of an innovative opto-electromechanically enabled diagnostic modalities coupled with computational models that can accurately quantify and predict initiation, progression, and resolution of the epithelial injury.

II. RESULTS

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. RESULTS <<III. DISCUSSIONIV. METHODSSUPPLEMENTARY MATERIAL

A. Generation of localized focal injury in the airway epithelium

To recapitulate localized mucociliary disruption caused by a focal epithelial injury, we generated an in vitro airway injury model (Fig. 2). Immunostaining analysis of healthy rat airway tissues revealed abundant cilia of multiciliated cells, intercellular ZO-1 tight junctions between the epithelial cells, and polarized localization of Vangl-1 PCP protein along the P–D axis [Fig. 2(a)]. Similarly, scanning electron microscopy (SEM) images of the healthy airway tissues showed densely populated multiciliated and secretory cells across the entire surface of the airway lumen [Fig. 2(b)]. We then generated a localized physical injury in the epithelium of isolated rat trachea tissue using a metal rod that was mounted on a micropositioner [Fig. 2(c)]. Real-time visualization of the epithelial integrity and cilia function was achieved by staining the cilia of freshly isolated airway tissues with fluorescein-labeled wheat germ agglutinin (FITC-WGA) prior to experiment. Furthermore, structural integrity of the airway tissue surface was monitored via SEM imaging and H&E staining following all experiments [Figs. 2(d) and 2(e); supplementary material Figs. 1 and 2)]. While native airway tissue showed abundant cilia that continuously beat along the P–D axis [Fig. 2(d); supplementary material Video 1), fibrous connective tissue that was stained with Evans blue (EB) was observed at the injured tissue region [Fig. 2(e)]. Post-analysis of the airway tissue with SEM imaging, immunostaining, and H&E staining showed localized damage on the epithelial layer and absence of tight/adherens junctions at the injured tissue region, while other tracheal layers were preserved [supplementary material Figs. 1(a), 1(b), and 2].

B. Quantification of mucociliary flow disruption caused by acute epithelial injury

We investigated the extent of the mucociliary flow affected by the localized airway epithelial injury (Fig. 3). To this end, the speed and direction of the mucociliary flow at both acutely injured and native tissue regions were monitored by tracking 1-μm microparticles using a fluorescent microscope [Figs. 3(a)–3(e)]. The acquired videos were processed to determine the trajectory of each microparticle. The obtained trajectories revealed that the microparticles at the injured epithelial surface moved shorter distances [Figs. 3(b) and 3(c); supplementary material Video 2] than the microparticles at the ciliated tissue surface [Figs. 3(d) and 3(e); supplementary material Video 3] over a given amount of time (e.g., 10 s). The average speed of the microparticles measured at the injured region of damaged airway tissue (2.93 ± 1.15 μm/s) was approximately 60% lower than those measured from the native tissue (7.13 ± 1.07 μm/s) (***p Fig. 3(f)]. We were able to continuously trace some of the microparticles as they passed through the injury site, and the travel distance of the traced particles were plotted as a function of time [Fig. 3(g)]. The speed of particles, which is indicated by the slope of the curve, decreased as the particles approached the injury site and increased back to nearly normal range after passing the injury site. In general, the average speed of microparticles just before entering the injury site was approximately 4.26 ± 1.80 μm/s, which was lower than the particle speed measured from the native tissue (7.13 ± 1.07 μm/s). The speed of microparticles decreased to 2.23 ± 1.30 μm/s within the injured region and increased to 7.29 ± 2.78 μm/s, which was similar to the normal speed, just after passing the injury site [Fig. 3(h)].We then studied how the traveling direction of the mucociliary flow was affected by the tissue injury by measuring the angle between each microparticle trajectory and the P–D axis of the airway [Figs. 3(i) and 3(j)]. Measured angles were plotted in a polar histogram, in which the angular direction of each pie-shaped wedge represents the microparticle moving direction (e.g., microparticles traveling toward the proximal direction along the P–D axis was set to 90°). The size of a wedge corresponds to the number of microparticles moving in that direction. Results showed that all microparticles tend to move toward the proximal direction in both injured and native tissues. In the injured tissue, only 22.41% of the traced microparticles moved between 85° and 95° of the angular direction [Fig. 3(i)], whereas 62.50% of the microparticles in the native tissue traveled between 85° and 95° [Fig. 3(j)]. The mean microparticle traveling direction was calculated to be 89.06° and 92.50°, respectively, in injured and native tissues. To further quantify the degree of unidirectionality based on the observations, we calculated the index of orientation of the traveling microparticles (see Sec. ). The index of orientation for injured and native tissues were determined to be 0.90 and 0.99, respectively, suggesting approximately 10% reduction in the unidirectional movement of the mucociliary flow in injured airway tissue [Fig. 3(k)]. We occasionally observed a lateral movement of the surface fluid at the boundary parallel to the P–D axis between the injured and intact tissue regions (supplementary material Fig. 3; supplementary material Video 4).

C. Computer simulation of the injury-induced mucociliary flow disruption

To rationalize the experimental results, we simulated the mucociliary flow near the injured airway luminal surface (Fig. 4). Mucociliary flow was modeled as a thin surface flow (thickness: 500 μm) that passed over the injured airway lumen [Fig. 4(a)]. The flow entered the simulated region through the inlet surface at a normal speed of 8 μm/s and exited through the outlet. The intact ciliated airway surface was modeled as a unidirectional sliding wall (speed: 8 μm/s) to recapitulate the flow of the thin airway surface liquid whose movement is driven by the continuous polarized ciliary motion. No-slip boundary condition (BC) was applied to the injured tissue region where the coordinated ciliary activity is lacking [Fig. 4(b)].1515. R. S. Juan, R. Guillermina, A. J. Mathijssen, M. He, L. Jan, W. Marshall, and M. Prakash, Nat. Phys. 16(9), 958 (2020). https://doi.org/10.1038/s41567-020-0923-8 The simulated flow, which was initially traveling at 8.00 μm/s, traveled slower in the region adjacent to the damaged area [Fig. 4(c)], and its speed further declined to 3.75 μm/s at the central region of the injury site [Figs. 4(d) and 4(e)]. The flow speed returned to the normal level (8.00 μm/s) after passing the injury site. The pattern of the flow speed change near the injury site is consistent with our experimentally observed results. In our experiments, the speed of particles at the normal tissue, injury site, and downstream region was 7.13 ± 1.07, 2.23 ± 1.30, and 7.29 ± 2.78 μm/s, respectively (Fig. 3). Furthermore, as the flow approached the injury site, the hydrodynamic pressure within the fluid increased from 0 Pa to 53.5 μPa because of the reduced flow speed. As the flow passed the injury site, the pressure reduced back to 0 Pa at the center of the injury site. Then, the pressure further decreased to −53.5 μPa and returned to 0 Pa as the flow continued to travel across the injury site [Figs. 4(f) and 4(g)].

D. Quantification of the effects of acute tissue injury on alteration of tissue bioimpedance

We investigated whether the localized damage created in the airway epithelium can be detected via measurement and analysis of the electrical property at the injury site (Fig. 5). To this end, we created a measurement platform that allowed us to quantify the bioelectrical impedance (i.e., bioimpedance) of the airway tissue [Fig. 5(a)]. This platform utilizes the four-point measurement approach, in which four electrode probes directly contact the airway tissue to measure the electrical properties of the tissue. For the experiment, we created a focal epithelial injury using a metal rod, and the four electrodes were placed near the injury site [Fig. 5(b)]. During the measurements, alternating electrical current (AC) with various frequencies (range: 250 Hz–135 kHz) and 100 μA of its maximum amplitude were injected into the tissue using two current carrying electrodes (CC1 and CC2), while two voltage pickup electrodes (PU1 and PU2) measured the potential distribution at the measurement site [Fig. 5(c)]. The data acquired were then analyzed using the Cole model that describes the biological tissue as a combination of two resistors (R1 = R∞ and R2 = R0–R∞) and a constant phase element (CPE)16,1716. T. J. Freeborn and S. Critcher, Fractal Fract. 5(1), 13 (2021). https://doi.org/10.3390/fractalfract501001317. A. Roy, S. Bhattacharjee, S. Podder, and A. Ghosh, AIMS Biophys. 7(4), 362 (2020). https://doi.org/10.3934/biophy.2020025 (see Sec. for details) [Figs. 5(d) and 5(e)].We then tested the hypothesis that disruption of the airway epithelium leads to reduced bioimpedance of the airway tissue measured at the airway lumen. The measurement results showed that the magnitude of both resistance (R) and reactance (Xc) measured at the injured tissue was noticeably lower than that measured at the native tissue region [Figs. 5(f) and 5(g)]. The magnitude of the reactance increased with increased AC frequency (from 0 to ∼500 Ω in the injured tissue; from 0 to ∼1000 Ω in the native tissue) and then gradually decreased as the frequency further increased after a certain threshold frequency (fc: ∼10 kHz in the injured tissue; ∼15 kHz in the native tissue) [Fig. 5(g)]. The bioimpedance (Z) of both injured and native airway tissues reduced gradually as the AC frequency increased [Fig. 5(h)]. Each dataset acquired from the injured and native tissues was fitted to a curve by least absolute deviation (LAD) method, from which the Cole parameters were extracted18,1918. Y. Yang, W. Ni, Q. Sun, H. Wen, and Z. Teng, Physiol. Meas. 34(10), 1239 (2013). https://doi.org/10.1088/0967-3334/34/10/123919. K. Chen, Z. Ying, H. Zhang, and L. Zhao, Biometrika 95(1), 107 (2008). https://doi.org/10.1093/biomet/asm082 (see Sec. ) [Fig. 5(i); supplementary material Table 1]. Notably, the resistance values measured at DC (R0) and infinite AC (R∞) and the time constant (τ) acquired from the injured tissue were smaller than those acquired from the native tissue, which is likely due to damaged airway epithelial barrier at the injury site.

E. Computer simulation of bioimpedance of injured airway tissue

To rationalize the contribution of the airway epithelial injury in the alteration of the electrical property of the airway tissue, we numerically simulated the electrical current propagation through both injured and native airway tissues (Fig. 6). The airway tissues were modeled as a composite of tissue layers that consisted of epithelium, submucosa, and cartilage. Furthermore, the computer model included a thin layer of pulmonary surface liquid (thickness: 15 μm) deposited on top of the epithelial layer [Fig. 6(a); supplementary material Fig. 4].2020. K. R. Atanasova and L. R. Reznikov, Respir. Res. 20(1), 261 (2019). https://doi.org/10.1186/s12931-019-1239-z The computational simulation results showed a similar trend observed in the experiments where the potential difference (ΔV) (supplementary material Fig. 5) and normalized bioimpedance (Ẑ) [Fig. 6(b)] between the voltage pickup electrodes (PU1 and PU2) decreased with increased AC frequency. Notably, the impedance values measured at the injured tissue were generally smaller than those measured from intact regions, which were consistent with our experimental results (Fig. 5). Contour plots of the simulated results showed that the distribution of the electric field (E) (supplementary material Fig. 6) and current density (J) (supplementary material Fig. 7) throughout the tissues were substantially affected by the AC frequency supplied to the tissue. While the electrical current propagates through the superficial tissue near the epithelium at lower AC frequency, the current can penetrate deeper tissue regions as it travels through the tissue at higher AC frequency.To investigate relative contribution of each tissue layer in the electrical measurement outcomes, we computed volume impedance density (VID) of different tissue layers in both injured and native airway tissues [Figs. 6(c)–6(f)]. Since VID is defined as the magnitude of the bioimpedance per unit tissue volume, it can provide information about the relative role of different tissue layers in resistance to the electrical current. At low AC frequency (e.g., 10 Hz), the VID value in the surface liquid was three to four orders of magnitude greater than that in the submucosa or cartilage layer in both injured and native tissues [Fig. 6(e)]. VID value of the surface liquid in the injured tissue (6.58 × 1012 Ω/m3) at 10 Hz was much smaller than that in the native tissue (3.36 × 1013 Ω/m3) due to easier penetration of electrical current through the damaged epithelium than intact epithelium. The difference of VID between the superficial and distal tissue regions became less significant at higher AC frequency (e.g., 135 kHz), suggesting low AC frequency could generate more robust and reliable electrical readouts when characterizing the tissue injury that occurs at the superficial airway tissue layers [Fig. 6(f)].

F. Opto-electromechanical quantification of airway barrier function disruption caused by inhalation-induced acute tissue injury

Following the thorough experimental and computational validations of our opto-electromechanical based methods using the airway tissue with focal injury, we then investigated whether less severe and more clinically relevant airway tissue injury can be assessed using this approach. To this end, we created an inhalation-induced airway injury model by intratracheally instilling either hypertonic saline or acidic solution (Fig. 7). Three percent sodium chloride (3% NaCl) solution was used as a hypertonic solution because its high osmotic pressure can disrupt the tight junctions of the airway epithelium, generating mild tissue injury with increased epithelium permeability.2121. M. Hogman, A. C. Mork, and G. M. Roomans, Eur. Respir. J. 20(6), 1444 (2002). https://doi.org/10.1183/09031936.02.00017202 Furthermore, hydrogen chloride (HCl) with low pH level (pH 1.5) was used to generate moderate injury in the epithelium that mimics gastric acid aspiration injury.2222. B. A. Davidson and R. Alluri, Bio-Protocol 3(22), e968 (2013). https://doi.org/10.21769/BioProtoc.968H&E and immunofluorescence staining were performed to confirm the establishment of epithelium injury with varying degrees [Figs. 7(a) and 7(b)]. H&E staining images showed that the native tissue contained intact epithelial layer, while the airway tissues exposed to 3% NaCl or HCl appeared to be considerably damaged. Notably, the severity of the injury in HCl-treated tissues was greater than NaCl-treated tissues as the H&E staining showed many of the epithelial cells that appeared to be dissociated from the tissue surface [Fig. 7(a)]. Similarly, immunostaining images clearly showed the establishment of inhalation-induced epithelium injury. Disruption of the ciliated cells (green) and tight junctions (red) were confirmed by decreased intensity or diffused pattern of associated fluorescence signals [Fig. 7(b)]. To more accurately quantify the tissue injury using the immunostaining images, we calculated the surface area of airway lumen covered with ciliated cells (green) in the native and injured tissues using ImageJ. In the native tissue, 44.43% of the luminal surface was covered with ciliated cells, while less than 22.91% and 13.03% of the airway lumen contained ciliated cells in the tissue treated with NaCl and HCl solutions, respectively (supplementary material Fig. 8).With the inhalation-induced injury model established, we investigated whether the mild and moderate epithelium injury can be probed via the particle tracing and impedance recording methods [Figs. 7(c) and 7(d)]. Results showed that the average speeds of the microparticles measured from 3% NaCl-treated tissues (8.13 μm/s) and HCl-treated tissues (4.29 μm/s) were approximately 36% and 66% lower than those determined from the native airway tissue (12.72 μm/s) (***p Fig. 7(c)]. Similarly, the magnitudes of the electrical impedance measured from the injured airway tissues were generally lower than those of the native tissues, most likely due to the considerable disruptions of the tight junctions in the injured tissues [Fig. 7(d)]. These results confirmed that our opto-electromechanical based method can provide quantitative information about airway epithelium with mild and moderate injury.

III. DISCUSSION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. RESULTSIII. DISCUSSION <<IV. METHODSSUPPLEMENTARY MATERIALIn this study, we investigated how acute physical injury generated in the airway epithelium affects the intrinsic protective functions of the epithelial barrier and whether the functional disruption can be measured directly at the injured tissue regions. Specifically, we quantified the impact of injury-induced degradation of the mucociliary clearance and tight/adherens junction formation, which are the two most essential barrier functions of the airway epithelium. To quantitatively investigate the changes of these epithelial functions following acute injury, we used in situ particle tracking and bioimpedance measurement techniques (Figs. 3, 5, and 7), combined with mathematical modeling and computer simulation (Figs. 4 and 6). To enhance reliability of the study outcomes, we utilized freshly isolated rat tracheal tissues that retained the intrinsic airway tissue architecture and functions, including unidirectional ciliary activities, physiologic cell diversity and distribution, and the native tissue matrix components. Localized acute physical injury was created in a selected region of the isolated tracheal tissues to determine the correlation between structure and functions of the airway epithelium with improved spatiotemporal resolution of the measurements. Similarly, inhalation-induced epithelium injury was generated to evaluate the utility of this study in broader clinical conditions. The numerical analyses conducted in this study facilitated elucidation of the structure–function correlation of the airway epithelium by allowing interpretation of the experimentally obtained results.Airway epithelial injury and subsequent impairment of the epithelial barrier functions can be caused by various reasons, such as inhalation injury, trauma, and surgery. The extent of the tissue damage and functional impairment can drastically vary depending on the cause, type, and severity of the injury. Accordingly, we focused on analyzing the tissue structure and function following different degrees of damages induced to the isolated airway mucosal tissues by either physical or chemical treatment. This injury model recapitulates the structural and functional disruption in the epithelium that nearly all pathologies of airway injury have in common with varying degrees.12,2312. M. B. Brodsky, M. J. Levy, E. Jedlanek, V. Pandian, B. Blackford, C. Price, G. Cole, A. T. Hillel, S. R. Best, and L. M. Akst, Crit. Care Med. 46(12), 2010 (2018). https://doi.org/10.1097/CCM.000000000000336823. E. Puchelle, J. M. Zahm, J. M. Tournier, and C. Coraux, Proc. Am. Thorac. Soc. 3(8), 726 (2006). https://doi.org/10.1513/pats.200605-126SFThe tissue-wise particle tracking revealed that the localized tissue injury substantially degraded the ability of the airway mucosa to transport its surface fluid as the speed of the flow reduced by nearly 60% at the injured tissue regions. Furthermore, the injury resulted in disorientation of the surface flow as the unidirectionality of the flow reduced by at least 10% near the central location of the injury site (Fig. 3). Computer simulation of the injury-induced fluid movements showed that the spatial variation of the flow speed near the injury site is likely due to the heterogeneous pressure distribution resulting from the absence of the functioning cilia at the injured tissue (Fig. 4). These findings are consistent with a pathological condition where airway tissue regions with substantially impaired mucociliary clearance are venerable to subsequent injury and infection due to increased time for the inhaled particles or pathogens to be removed from the respiratory tract.2424. Z. Zhou-Suckow, J. Duerr, M. Hagner, R. Agrawal, and M. A. Mall, Cell Tissue Res. 367(3), 537 (2017). https://doi.org/10.1007/s00441-016-2562-zWhile the flow speed and directionality reduced, transport of the surface fluid could persist at the tissue regions devoid of ciliated cells. This is because the fluid moves as a continuous medium due to intermolecular attractive forces between the fluid molecules.2525. B. Widom, Science 157(3787), 375 (1967). https://doi.org/10.1126/science.157.3787.375 Because of the physical connection between the fluid molecules across the airway surface, the hydrodynamic energy produced by repeated cilia beating at the neighboring intact tissue region can propagate through the fluid due to energy conservation, leading to fluid movement at the injured region. The hydrodynamic crosstalk between the fluids at different locations was clearly visualized via particle tracking at the interface between the intact and injured tissue regions (supplementary material Fig. 3; supplementary material Video 4). Due to substantial differences between these two regions in their flow speed and directionality, the surface flow moving over the intact tissue surface caused movement of the fluid from the injury site toward the intact region in the direction perpendicular to the P–D axis of the airway. Propagation of hydrodynamic energy within a fluid is heavily influenced by the fluid properties, such as viscosity, surface tension, and density.2626. J. Kim, B. Guenthart, J. D. O'Neill, N. V. Dorrello, M. Bacchetta, and G. Vunjak-Novakovic, Sci. Rep. 7(1), 13082 (2017). https://doi.org/10.1038/s41598-017-13280-9 Therefore, the maximum size of the injured tissue surface that neighboring cilia can exert their effects is expected to be dependent on the physical characteristics of the airway surface fluid.Visualization of the mucociliary flow and cilia motion can be achieved via advanced imaging modalities, in particular, optical coherence tomography (OCT).2727. U. A. Gamm, B. K. Huang, E. K. Mis, M. K. Khokha, and M. A. Choma, Sci. Rep. 7(1), 1 (2017). https://doi.org/10.1038/s41598-017-14670-9 OCT imaging, however, can only provide the cross-sectional views of tissues or fluids and thus is not suitable for monitoring the two-dimensional (2D) mucociliary flow across large airway surface. On the contrary, the current study employed a microparticle tracking method, which has been extensively used in the mucociliary-related studies,15,2815. R. S. Juan, R. Guillermina, A. J. Mathijssen, M. He, L. Jan, W. Marshall, and M. Prakash, Nat. Phys. 16(9), 958 (2020). https://doi.org/10.1038/s41567-020-0923-828. N. Sone, S. Konishi, K. Igura, K. Tamai, S. Ikeo, Y. Korogi, S. Kanagaki, T. Namba, Y. Yamamoto, Y. Xu, K. Takeuchi, Y. Adachi, T. F. Chen-Yoshikawa, H. Date, M. Hagiwara, S. Tsukita, T. Hirai, Y. S. Torisawa, and S. Gotoh, Sci. Transl. Med. 13(601), eabb1298 (2021). https://doi.org/10.1126/scitranslmed.abb1298 because of its proven utility in visualization of fluid movements with high spatial and temporal resolution. Tracking the movement of individual microparticles simultaneously along the 2D surface of the injured airway lumen allowed us to rapidly assess the impact of localized tissue damage on the mucociliary flow. Furthermore, assessment of the fluid interactions between the injured and intact tissue regions, which can be difficult to visualize using OCT imaging, was realized via the in situ particle tracking approach implemented in this study.We further showed that disruption of the tight/adherens junctions between the epithelial cells caused by the physical tissue damage can be quantified by measuring the electrical properties of the local airway tissue (Fig. 5). Notably, we determined that the acute tissue injury resulted in reduced bioimpedance using our custom-built measurement platform in combination of the mathematical analysis and computer simulation (Figs. 5 and 6). Loss of the physically protective and electrically resistant epithelial barrier is likely the main contributor of the lowered magnitude of the bioimpedance of the injured airway tissues compared with that of native tissues.29,3029. P. Patel and G. H. Markx, Enzyme Microb. Technol. 43(7), 463 (2008). https://doi.org/10.1016/j.enzmictec.2008.09.00530. P. W. Weijenborg, W. O. Rohof, L. M. Akkermans, J. Verheij, A. J. Smout, and A. J. Bredenoord, Neurogastroenterol. Motil. 25(7), 574 (2013). https://doi.org/10.1111/nmo.12106 Consistent with prior studies, our experimental and simulation results showed that the magnitude of the electrical resistance of both native and injured tissues decreased with increased AC frequency because the dielectric property of the cell membrane hampered propagation of AC with lower frequency across the cells.31,3231. S. L. Swisher, M. C. Lin, A. Liao, E. J. Leeflang, Y. Khan, F. J. Pavinatto, K. Mann, A. Naujokas, D. Young, S. Roy, M. R. Harrison, A. C. Arias, V. Subramanian, and M. M. Maharbiz, Nat. Commun. 6(1), 6575 (2015). https://doi.org/10.1038/ncomms757532. G. H. Markx and C. L. Davey, Enzyme Microb. Technol. 25(3–5), 161 (1999). https://doi.org/10.1016/S0141-0229(99)00008-3 In particular, the simulation results showed that the electrical current can propagate into deeper tissue regions when the resistant epithelial layer was removed, suggesting the reduced bioimpedance measured at the injured airway surface is likely due to diffusive distribution of the electrical potential across the larger volume of the airway tissue. Since measuring the electrical characteristics of each tissue layer is very challenging because of their thin geometry (supplementary material Fig. 5), the computer model developed can mathematically predict the electrical response of individual tissue components, providing additional quantitative information about the structural integrity of the airway tissue.Following the rigorous experimental and computational validations of our epithelium assessment modality using the focal injury model, we generated inhalation-induced mild and moderate injuries by intratracheally instilling 3% NaCl and HCl (pH 1.5), respectively. The injured tissues were analyzed via histology, immunostaining, particle tracing, and impedance recording. Results indicated that the severity of the tissue injury was correlated with the degree of the structural and functional disruption created in the injured airway mucosa (Fig. 7; supplementary material Fig. 8). Furthermore, the study confirmed that our opto-electromechanical based method can accurately detect and quantify minor tissue damage, demonstrating its potential utility in the diagnosis of airway tissue injury.Different types of bioimpedance measurement techniques have been developed and used extensively to evaluate the structure, content, and function of biological tissues and organs.17,30,3117. A. Roy, S. Bhattacharjee, S. Podder, and A. Ghosh, AIMS Biophys. 7(4), 362 (2020). https://doi.org/10.3934/biophy.202002530. P. W. Weijenborg, W. O. Rohof, L. M. Akkermans, J. Verheij, A. J. Smout, and A. J. Bredenoord, Neurogastroenterol. Motil. 25(7), 574 (2013). https://doi.org/10.1111/nmo.1210631. S. L. Swisher, M. C. Lin, A. Liao, E. J. Leeflang, Y. Khan, F. J. Pavinatto, K. Mann, A. Naujokas, D. Young, S. Roy, M. R. Harrison, A. C. Arias, V. Subramanian, and M. M. Maharbiz, Nat. Commun. 6(1), 6575 (2015). https://doi.org/10.1038/ncomms7575 For example, electrical impedance tomography (EIT) is an imaging modality that can visualize internal organs, such as lung, by mapping electrical properties of the patient's body in a noninvasive and radiation-f