Functional morphology of the bronchial mucociliary transport system in rats during postnatal ontogenesis

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BACKGROUND

The pseudostratified columnar ciliated (respiratory) epithelium ensures the effective functioning of the mucociliary transport system (MCTS) of the airways. The formation and clearance of mucus along the luminal surface of the bronchi and trachea are achieved through the coordinated activity of the ciliary apparatus of ciliated cells and the secretory activity of glandular elements [1, 2]. Age-related remodeling of the epithelial layer, accompanied by alterations in its barrier and transport functions, may play an important role in the pathogenesis of respiratory diseases [3, 4]. The microscopic structure and motor activity of the airway epithelium in the upper airways at different stages of postnatal development have been studied in detail mainly [2, 5]; however, the studies in the bronchial tree are much less extensive [6]. Typically, studies of bronchi are limited by the use of a few control time points, and the methodological approaches utilized to assess ciliary activity vary significantly, complicating cross-study comparisons. Furthermore, there are no experimental studies describing the dynamics in quantitative structural and functional parameters of the bronchial epithelial lining throughout postnatal ontogeny under standardized conditions with uniform methodological approaches.

This study aimed to investigate the age-related patterns of postnatal morphogenesis of the mucociliary transport system in the bronchi of rats based on structural and functional analysis of the respiratory epithelium.

METHODS

Study Design

An observational, single-center, cross-sectional, uncontrolled study was performed on laboratory animals.

Eligibility Criteria

The study included intact male Wistar rats with known birth dates. Animals reaching the required age for analysis were randomly assigned to study groups. The rats were bred under vivarium conditions in compliance with GOST 33215-2014 (January 7, 2016) and GOST 33216-2014 standards for this species. The animals were housed with free access to water and a standardized dry feed (Stoylenskaya Niva Agro-Industrial Complex, Russia).

Study Setting

The experiments, histological, and immunohistochemical studies were conducted at the Department of Histology, Cytology, and Embryology, Yaroslavl State Medical University, Ministry of Health of the Russian Federation. Transmission electron microscopy (TEM) was performed at the Shared Research Facility for Electron Microscopy, Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences. Low-vacuum scanning electron microscopy (SEM) was conducted at the Yaroslavl Branch of the Institute of Physics and Technology, Russian Academy of Sciences.

Study Duration

The study was conducted between 2017 and 2024. During the first three years, the rats were bred, and biological material was collected at different stages of postnatal development (1, 8, and 14 days; 1, 3, 6, 14, 20, and 26 months after birth). Histological and morphometric processing of samples was also performed during this period. In 2023, TEM and SEM studies of bronchial samples were carried out, followed by final data analysis. In 2024, the manuscript was prepared.

Intervention

The lung tissue for analysis was obtained following intramuscular administration of a combination of tiletamine and zolazepam (Zoletil 100^®, Virbac, France) at a dose of 10 mg/kg. The right lung was used for in vivo analysis, whereas fragments of the left lung were processed for histological examination. Euthanasia was performed before recovery from anesthesia by decapitation. Morphological and functional analyses were conducted separately for the main bronchi (MB), lobar bronchi (LB), and segmental bronchi (SB).

In vivo assessment. The motor activity of the ciliary apparatus was evaluated on thin (≤1 mm) fragments of lung tissue using a modified method previously described in detail [7]. A hardware–software complex (Azimut-4 Research and Production Association, Russia) was used, consisting of a Biomed-2 var. 3 microscope (LOMO JSC, Russia) equipped with an electronic thermostatic control unit, a high-frequency digital video camera Grasshopper 3 2.3 MP Color USB3 Vision (FLIR Integrated Imaging Solutions Inc., Canada), and a personal computer with specialized MOSFRO software (v.4; Azimut-4 Research and Production Association, Russia).

Light microscopy. Lung fragments were fixed in 10% neutral buffered formalin (Element, Russia) and embedded in paraffin (Deltalab, Spain). Sections 4–5 µm thick were stained with hematoxylin and eosin (Emmonya Biotech Ltd, Bulgaria) and Schiff’s reagent (Labiko, Russia) with counterstaining by hematoxylin.

Detection of the proliferation marker Ki-67 protein was performed on paraffin sections using rabbit monoclonal antibodies (Ventana Medical Systems, USA; catalog no. 790-4286; dilution 1:100) and the ultraView Universal DAB Detection Kit (Ventana Medical Systems, USA).

Electron microscopy. For TEM, samples were fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences, USA), postfixed in 1% osmium tetroxide (Aurat, Russia), dehydrated in acetone, and embedded in Epon (Sigma-Aldrich, USA). Ultrathin sections were prepared using an ultramicrotome Leica-EMU6C (Leica Microsystems, Germany), contrasted with uranyl acetate (Sigma-Aldrich, USA), and examined with a JEM-1011 transmission electron microscope (JEOL Ltd, Japan). The images were digitized. For SEM, lung fragments were fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences, USA) and analyzed using a Quanta 3D 200i scanning electron microscope (FEI Company, USA) under low-vacuum conditions at a water vapor pressure of 70–130 Pa.

Main Study Outcome

The primary endpoint of the study was the determination of morphometric and functional characteristics of the bronchial epithelial cells of rats throughout postnatal ontogeny.

Subgroup Analysis

Animals were allocated into age groups according to the postnatal development periodization of rats proposed by Zapadnyuk et al. [8]: day 1, newborns; days 8 and 14, suckling period; 1 month, infantile; 3 months, juvenile; 6 months, young; 14 months, adult; 20 months, aging; and 26 months, old rats.

Outcomes Registration

In vivo assessment. Quantitative processing of video recordings was performed using the MOSFRO software (v.4), which provided automatic calculation of ciliary beat frequency (CBF, Hz) as well as the duration of effective and recovery stroke phases of the ciliary beat (ms) [7].

Morphometry. The percentage of the main morphological types of epithelial cells was determined by counting 1000 cells per animal. A single animal served as the unit of observation for subsequent statistical analyses. The relative number of Ki-67–positive cells was determined by counting 500–1000 cells per animal. Frequency parameters were calculated using the Universal Histological Counter 2.0 application.¹ Ciliary length was measured on digital images using ImageJ software [9] at a total magnification of ×1000, with 100 ciliated cells assessed per animal.

Statistical Analysis

Sample size calculation principles. The sample size was not calculated in advance.

Statistical analysis methods. Statistical processing was performed using Microsoft Office Excel 2019 (Microsoft, USA) and Statistica 8.0 (StatSoft Inc., USA). The normality of distribution was verified using the Shapiro–Wilk test, and homogeneity of variances was confirmed with the F-test. Quantitative data are presented as mean (M) and standard error of the mean (m). Statistical significance between independent samples was assessed using Student t-test, with differences considered significant at p < 0.05.

RESULTS

Study Objects

The work was performed on 76 male Wistar rats without external signs of disease and with precisely known dates of birth. For in vivo and histological examinations, fragments of the main, lobar, and segmental bronchi were obtained from animals of different ages: 1 day (n = 9), 14 days (n = 6), 1 month (n=6), 3 months (n = 6), 6 months (n = 9), 14 months (n = 8), 20 months (n = 6), and 26 months (n = 5). SEM was performed on 3 samples of bronchi of different calibers from rats aged 1, 8, and 14 days and 1 month (12 animals in total). TEM was conducted on 3 samples of bronchi of different calibers from rats aged 1 day, 6 months, and 26 months (9 animals in total).

Primary Results

A characteristic feature of the histological structure of the rat bronchial tree is the absence of the submucosal glands, which makes it a convenient model for studying the subpopulations of motor (ciliated epithelial cells), secretory (goblet cells), and progenitor (basal cells) elements of the MCTS within a single epithelial layer.

In newborn rats, the bronchial tree in all regions is lined with a simple epithelium composed predominantly of undifferentiated (intermediate) cells, whereas ciliated cells are relatively scarce (Fig. 1, a–c). During the first two weeks after birth, the proportion of ciliated cells in the epithelial layer increases markedly because of active differentiation processes (ciliogenesis), accompanied by a proportional decrease in the content of poorly differentiated elements. Various stages of ciliary formation can be observed at the apical poles of differentiating ciliated cells (Fig. 1, d, e). The maturation of ciliated cells occurs in clusters, which is clearly visible under scanning electron microscopy (Fig. 1, f). By the end of the first month after birth, the typical structure of a pseudostratified ciliated epithelium is established.

Fig. 1. Bronchial epithelial lining in rats: a, main bronchus; b, lobar bronchus; c–e, segmental bronchi in newborns; f, segmental bronchus in 8-day-old rats. 1, ciliated cells; 2, brush (non-ciliated) cells; 3, basal cells. Transmission electron microscopy: a–c, ×5000; d, e, ×8000; f, scanning electron microscopy, ×4500.

In the definitive epithelial layer of sexually mature animals (6 months), ciliated epithelial cells predominate, whereas the subpopulations of goblet and basal cells are less numerous, and nonciliated (intermediate) cells are also relatively few (Fig. 2, a, b). Numerous fully differentiated cilia are clearly visible on the apical surface of ciliated epithelial cells (Fig. 2, c). In aging and old animals (20 and 26 months), the epithelium retains its differentiated structure with a well-developed ciliary apparatus (Fig. 2, d, e). However, in rats of these age groups, hypertrophied ciliated cells are consistently present, along with elements showing ultrastructural signs of damage: disruption of organelle architecture and cytoplasmic vacuolization, as well as areas of intercellular edema (Fig. 2, f).

Fig. 2. Bronchial epithelial lining in rats: at 6 months of age, a, main bronchus; b, segmental bronchus; at 26 months of age, c, segmental bronchus; d–f, main bronchi. 1, ciliated cells; 2, goblet cells; 3, brush (non-ciliated) cells; 4, basal cells; 5, hypertrophic ciliated cell; 6, areas of intercellular edema; 7, epithelial cell with morphological signs of damage. Transmission electron microscopy: a, b, e, f, ×5000; c, d, ×10000.

Age-related dynamics of the quantitative characteristics of the bronchial epithelium across bronchi of different calibers are summarized in Tables 1 and 2. Based on light microscopy, the epithelial lining was classified into ciliated, goblet, basal, and intermediate (intercalated) cells.

Table 1. Quantitative parameters of the structure and function of the bronchial mucociliary apparatus in rats during postnatal ontogenesis: ciliated cells

Note: Data are presented as M ± m, where M is the arithmetic mean and m is the standard error of the mean; ¹ p < 0.05; ² p < 0.01; ³ p < 0.001 vs the preceding age group for bronchi of the same type; * p < 0.05; ^& p < 0.01; ^# p < 0.001 vs the main bronchi.

Table 2. Quantitative parameters of the structure and function of the bronchial mucociliary apparatus in rats during postnatal ontogenesis: composition of goblet, basal, and intermediate cells as the main epithelial cell types in the epithelial layer

Note: Data are presented as M ± m, where M is the arithmetic mean and m is the standard error of the mean; ¹ p < 0.05, ² p < 0.01, ³ p < 0.001 vs the preceding age group for bronchi of the same type; * p < 0.05,^& p < 0.01, ^# p < 0.001 vs the main bronchi.

Ciliated epithelial cells. In newborn rats, the proportion of ciliated elements was minimal: 21% in the main and segmental (small) bronchi, and 26% in the lobar bronchi. During the subsequent two weeks, the number of ciliated epithelial cells in the layer increased most intensively: 2.2-fold in MB, 2.3-fold in LB, and 2.7-fold in SB. By day 30 after birth, the density of ciliated elements increased 2.5–2.7 times in the main and lobar bronchi and 3.3 times in the segmental bronchi compared with newborns (p < 0.001). These values remained stable throughout the subsequent observation period. At the same time, the proportion of ciliated cells in the lobar and segmental bronchi was, on average, 25%–30% higher than in the main bronchi.

The analysis of the ciliary apparatus of ciliated cells demonstrated that in newborn animals, the mean ciliary length was comparable across all bronchial segments (range: 2.3–2.6 µm, p > 0.05). After 2 weeks, this parameter increased 1.2-fold in MB and LB and 1.4-fold in SB. Between 6 and 20 months, the mean ciliary length stabilized at 3.8–3.9 µm in MB, 2.8–3.1 µm in LB, and 2.5–2.6 µm in SB. In old rats, the mean ciliary length in MB and SB was reduced by 18% and 27%, respectively, compared with 20-month-old animals.

The CBF in all bronchi was highest in newborns (25–26 Hz) but decreased by 37%–40% by day 14. Beginning at 3 months, and throughout the remaining observation period, CBF stabilized within 14.1–16.2 Hz. In 1-month-old rats, CBF in the lobar and segmental bronchi was 21%–25% lower than in the main bronchi; however, at later time points no significant inter-bronchial differences in ciliary activity were found.

The frame-by-frame analysis of video recordings revealed that in newborn rats, the rapid beating of short cilia had a poorly defined wave-like pattern and was insufficiently synchronized (Fig. 3, b–d). The appearance of typical ciliary beating cycles with distinct effective and recovery phases occurred by day 14 of postnatal development. Between 14 and 26 months, the wave-like character of long ciliary movement in the recovery phase was most pronounced (Fig. 3, f–h). These observations were confirmed by the phase analysis of ciliary beating. In newborn rats, the effective stroke phase was minimal (16.5–16.6 ms) across all bronchial regions. By day 14 and at all subsequent ages, this parameter stabilized at 26–32 ms. Regardless of CBF, the fundamental cycle structure was preserved: the ratio of the effective stroke phase to the total cycle duration (normalized to 1.0) remained stable across all time points (0.38–0.41).

Fig. 3. Phases of ciliary beating in epithelial cells of the main bronchi in rats: a, e, diagram; b–d, f–h , frames from intravital microvideo recording; b–d, newborn rats; f–h, 26-month-old rats; a, b, f, g, recovery stroke phase, bending direction indicated by arrows; c–e, h, effective stroke phase. Objective lens, ×40.

Goblet cells. The first isolated glandular elements appeared in the epithelial layer on day 14. By 6 months of age, their proportion in the main and lobar bronchi increased 10-fold (p < 0.001), after which it stabilized at 14%–17%. Throughout the observation period, the proportion of goblet cells in the segmental bronchi was consistently lower than in the main bronchi. In segmental bronchi, the number of goblet cells increased less intensively and reached the maximum (11.2%) only at 26 months.

Basal and intermediate cells. The proportion of basal cells in MB peaked at 25% in 14-day-old rats. Between 1 and 20 months, the proportion of progenitor elements remained stable at 20%–21%, with a decrease to 15.5% observed only in old rats. In smaller bronchi (lobar and segmental), the age-related dynamics of basal epithelial cell proportions followed a similar pattern; however, their numbers were statistically significantly lower than in MB in most observations.

Intermediate (nonciliated) cells constituted 59%–61% of epithelial cells in all types of bronchi in newborn rats. By day 14, their numbers had decreased 2.1-fold in MB and 2.5–2.7-fold in LB and SB. This trend persisted at 1 and 3 months. By 6 months, the proportion of intermediate cells stabilized at 9%–11% across all bronchial regions. In pre-senescent and senescent rats, against the background of a high proportion of differentiated elements, the content of intermediate cells reached its lowest values (3.4%–4.6%).

Cell proliferation. The results of quantifying Ki-67–expressing cells in the bronchial epithelium across bronchi of different calibers are presented in Table 3. The highest proportion of proliferating cells was observed during the first two weeks after birth, peaking on day 14 (8.2%–8.7%). Beginning at 6 months, the Ki-67 index in the main bronchi stabilized at 2.5%–3.9%. In LB and SB, the proportion of proliferating epithelial cells was substantially lower, averaging 66% and 51% of the values in MB, respectively. At all studied time points, Ki-67 expression was detected exclusively in the nuclei of basal and intermediate (intercalated) epithelial cells.

Table 3. Ki-67-positive epithelial cells in the bronchi of rats during postnatal ontogenesis

Note: Data are presented as M ± m, where M is the arithmetic mean and m is the standard error of the mean; ¹ p < 0.05, ² p < 0.01, ³ p < 0.001 vs the preceding age group for bronchi of the same type; * p < 0.05,^& p < 0.01 vs the main bronchi.

DISCUSSION

Summary of Primary Results

Morphogenetic processes in the respiratory epithelium of the bronchi are most active during the first postnatal month in rats and proceed asynchronously. During the first 2 weeks after birth, accelerated differentiation of ciliated cells predominates, and the formation of the goblet cell subpopulation becomes more prominent starting from day 14. Development of the ciliary apparatus progresses more rapidly in SB, whereas glandular differentiation is more pronounced in MB.

The establishment of the typical structure of the respiratory epithelium is completed during puberty (3 months). During the reproductive period (6–14 months), the quantitative and structural parameters stabilize at levels typical of each bronchial caliber. At the same time, the principal parameters of ciliary activity of ciliated epithelial cells remain consistent across all bronchial regions. In aging, preservation of high functional activity of MCTS is accompanied by morphological signs of cellular injury and reduced proliferative turnover of the epithelial layer.

Discussion of Primary Results

At all levels of bronchial tree branching in rats, structural and functional differentiation of the respiratory epithelium proceeds at the highest rate during the first postnatal month. Our observations are generally consistent with published data describing the early stages of airway development in laboratory animals and humans [1, 5, 10]. The principal manifestations of morphogenesis aimed at establishing the MCTS include active proliferation of undifferentiated cells and the differentiation of ciliated and goblet cell subpopulations. These differentiation processes in rats occur asynchronously: ciliogenesis is most pronounced in the first two weeks after birth, whereas the system of secretory elements develops by the end of the first postnatal month. Moreover, the development of the ciliary apparatus progresses more rapidly in the distal regions of the bronchial tree (SB), whereas glandular elements differentiate predominantly in the main bronchi. The intensive differentiation of the epithelial lining during the first month (a period of maximal growth rates in rats [8]) is supported by high proliferative activity across all bronchial regions.

The CBF in newborn rats is higher than in any subsequent age period. This phenomenon, characteristic of the airways of both laboratory animals and humans [11, 12], when combined with the underdeveloped ciliary apparatus and the absence of differentiated secretory elements at this stage, indicates the immaturity of the MCTS at birth.

Completion of the structurally and functionally differentiated epithelial layer occurs during puberty. In 3-month-old rats, the quantitative parameters of ciliated cell subpopulations approach those of sexually mature animals. The analysis of ciliary motility demonstrated that starting at 3 months, CBF stabilizes and remains unchanged at all subsequent ages. Moreover, no inter-bronchial differences in ciliary activity were detected. Temporal parameters of ciliary beating cycles also remain stable throughout life. These observations are consistent with measurements of mucus transport velocity in the rat trachea, showing that the establishment of a stable level of mucociliary clearance (0.10–0.13 mm/s) is completed by 3 months of age [5]. Comparable results for CBF across specific age periods have been reported for both the bronchial tree of rodents and in human studies [13, 14].

In animals of reproductive age (6 and 14 months), MCTS of the bronchial tree achieves full structural and functional maturity. Across bronchi of different calibers, a distal gradient can be traced in the proportion of differentiated cell types (ciliated and goblet cells) and proliferating cells within the epithelial lining.

In aging (20 months) and old (26 months) rats, the previously established level of structural and functional differentiation of the epithelial layer is preserved: the proportion of ciliated and goblet cells reaches its peak values, and the ciliary motility remains maintained. Similar findings were previously obtained in our laboratory when examining age-related remodeling of the tracheal epithelium [5]. These results differ from reports of several authors who observed a decline in mucociliary transport with aging in the trachea of mice, guinea pigs, and in the human nasal cavity [15, 16]. In addition to interspecies differences, such discrepancies may be explained by variations in in vivo methods used to assess ciliary activity. Furthermore, in human studies, the influence of comorbid airway conditions cannot be excluded.

Importantly, the processes described in aging and old rats occur against the background of a marked decline in the proportion of epithelial cells entering the mitotic cycle. With reduced proliferative turnover, previously differentiated ciliated cells are forced to maintain viability and function under increased workload. Morphologically, this is manifested by a higher proportion of hypertrophied ciliated cells and the appearance of ultrastructural features of epithelial injury, including disrupted organelle architecture and cytoplasmic vacuolization. The age-related changes identified in this study complement published evidence on involutional remodeling of the respiratory epithelium with aging: accumulation of ultrastructural defects in ciliary microtubules [15]; altered synthesis of proteins regulating epithelial barrier permeability in the areas of intercellular junctions [3]; weakening of adhesion to the basal lamina and enhanced desquamation of epithelial cells [17].

Study Limitations

No factors were identified that could substantially influence the study outcomes. However, it should be considered that the histological structure of the bronchial tree in rats differs significantly from that of humans and other mammals.

CONCLUSION

Postnatal morphogenesis of the bronchial respiratory epithelium in rats continues throughout life, with histogenetic processes being most pronounced during the first month after birth. The structural and temporal dynamics in differentiation of the motor (ciliated cells) and secretory (goblet cells) components of the MCTS demonstrate distinct regional features across the tracheobronchial system.

One hallmark of structural and functional maturity of the respiratory epithelium during reproductive age is the establishment of a gradient in the proportions of differentiated and proliferating epithelial cells across bronchi of different calibers, with their relative abundance decreasing in the distal direction. The preservation of high functional activity of the MCTS during aging is achieved at the expense of increased workload on ciliated and secretory elements, leading to cumulative cellular injury and reduced compensatory capacity of the epithelial lining.

Overall, the respiratory epithelium in all regions of the rat airways demonstrates uniform transport properties and functions as an integrated tissue system, ensuring a stable level of mucociliary clearance throughout the animal’s lifespan.

ADDITIONAL INFORMATION

Author contributions: A.V. Pavlov: conceptualization, methodology, formal analysis, writing—original draft, writing—review & editing, visualization, search for sources; N.A. Tyumina: investigation, data curation, formal analysis, visualization, collection and analysis of sources, writing—original draft. All the authors approved the version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgements: The authors express their gratitude to K.G. Kemoklidze, Cand. Sci. (Biology), Associate Professor at Yaroslavl State Medical University, for assistance in obtaining the raw data.

Ethics approval: Animal handling was carried out in accordance with current national and international regulations (GOST 33215-2014 dated January 7, 2016; GOST 33216-2014; Directive 2010/63/EU of the European Parliament and of the Council dated September 22, 2010; Recommendation of the EEC Board No. 33 dated November 14, 2023). The study protocol was approved by the Ethics Committee of Yaroslavl State Medical University (Protocol No. 07 dated February 16, 2016).

Funding sources: The authors declare no external funding was received for conducting the study.

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Data availability: All data obtained in the present study are presented in the article.

Generative artificial intelligence: No generative artificial intelligence technologies were used in the creation of this article.

Review and peer review: This work was submitted to the journal on an unsolicited basis and reviewed through the standard procedure. The review process involved two external peer reviewers, a member of the editorial board, and the journal's scientific editor.

¹ Kemoklidze KG. Universal histological counter. Version 2.0. Certificate of state registration of computer programs No. 2012617618 Russian Federation. Application No. 2012615714; application date 09.07.2012; registration date 23.08.2012.

About the authors

Alexey V. Pavlov

Author for correspondence.
Email: pavlov@ysmu.ru
ORCID iD: 0000-0001-8227-9997
SPIN-code: 8854-7083

MD, Dr. Sci. (Medicine), Professor

Natalya A. Tyumina

Email: Natellamail@mail.ru
ORCID iD: 0000-0001-7001-0851
SPIN-code: 8839-8300
Russian Federation, Yaroslavl

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2. Fig. 1. Bronchial epithelial lining in rats: a, main bronchus; b, lobar bronchus; c–e, segmental bronchi in newborns; f, segmental bronchus in 8-day-old rats. 1, ciliated cells; 2, brush (non-ciliated) cells; 3, basal cells. Transmission electron microscopy: a–c, ×5000; d, e, ×8000; f, scanning electron microscopy, ×4500.

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3. Fig. 2. Bronchial epithelial lining in rats: at 6 months of age, a, main bronchus; b, segmental bronchus; at 26 months of age, c, segmental bronchus; d–f, main bronchi. 1, ciliated cells; 2, goblet cells; 3, brush (non-ciliated) cells; 4, basal cells; 5, hypertrophic ciliated cell; 6, areas of intercellular edema; 7, epithelial cell with morphological signs of damage. Transmission electron microscopy: a, b, e, f, ×5000; c, d, ×10000.

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4. Fig. 3. Phases of ciliary beating in epithelial cells of the main bronchi in rats: a, e, diagram; b–d, f–h , frames from intravital microvideo recording; b–d, newborn rats; f–h, 26-month-old rats; a, b, f, g, recovery stroke phase, bending direction indicated by arrows; c–e, h, effective stroke phase. Objective lens, ×40.

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