CHANGES IN THE TRACHEAL EPITHELIUM DURING 24 HOURS AFTER INHALATION OF MINERAL WATER

Konrádová, V. , L. Vajner , J . Uhl ík , J . Zocová: Changes in the Tracheal Epithelium during 24 Hours after Inhalation of Mineral Water. Acta Vet. Brno 2001, 70: 167-172. Inhalation of aerosol of mineral water is frequently used in therapy of various respiratory disorders. Ultrastructure of the airway epithelium in rabbits and character of glycoconjugates produced by secretory cells were studied immediately and 24 h after 10-min inhalation of this aerosol. Goblet cells were overstimulated and the mechanism of mucus evacuation was accelerated. The exhausted goblet cells mostly took part in further secretory cycles, but the number of degenerated secretory elements gradually increased in the course of 24 h. Cells not entirely filled with secretory granules prevailed in the epithelium. Massive differentiation of goblet cells and development of intraepithelial mucous glands were noticed. Compared with controls, significant (α = 0.01) decrease in the acid sulphated glycoconjugates in the secretion of the goblet cells was accompanied by an increase of the acid sialylated ones immediately after inhalation. Twenty-four hours post exposure, significant (α = 0.05) decrease in total sialylated glycoconjugates was ascertained. The ciliated cells revealed only mild pathological alteration. Significant (α = 0.01) decrease in number of kinocilia/μm2 was accompanied by an increase in percentage of altered cilia. During 24 h post exposure, signs of ciliary border regeneration were noticed. Morphological signs of impaired self-cleaning ability of the airway epithelium were encountered during the whole experiment. Single 10-min inhalation of mineral water aerosol caused changes in the ultrastructure of the airway epithelium and influenced the chemical composition of the goblet cells’ secretion. These changes did not disappear completely during 24 h post exposure. Airways, ultrastructure, regeneration, lectin histochemistry, glycoconjugates, rabbit In our previous study, we demonstrated that 10-min inhalation of saline affected the ultrastructure of the airway epithelium (Konrádová et al. in press). We therefore decided to study also the effect of aerosol of mineral water, frequently used in therapy of various respiratory disorders, and to follow the process of regeneration of this epithelium in the course of 24 h. Materials and Methods In our experiments, 19 SPF New Zealand White rabbits (body weight 1,500-3,000 g, Charles River Deutschland, Sulzfeld, Germany) were used. Seven of them served as untreated controls. The remaining animals were placed successively for 10 min into a plastic cage connected with the inhalation device PARI Master and nebuliser PARI LL (Pari GmbH, Starnberg, Germany, medium diameter of produced droplets 3.1 μm, total output 0.6 g/min). The rabbits inhaled an aerosol of mineral water for 10 min. Chemical composition of this spring water is given in Tab. 1. Under general anaesthesia, the material for electron microscopic and histochemical examinations was collected from six animals immediately and 24 h post exposure, respectively. Tiny fragments of the tracheal mucous membrane were processed using standard methods for electron microscopy (Konrádová 1991). The ciliary border and the functional state of the goblet cells were evaluated quantitatively. To evaluate the distribution of goblet cells in the epithelium, the isolated elements and the goblet cells arranged in groups were distinguished. Kinocilia were classified into four categories: intact 9+2 cilia, slightly damaged pathological cilia with local swellings of the ciliary membrane or with tiny vacuoles situated in their shafts, degenerating cilia, represented by axonemes incorporated into cytoplasmic blebs or by isolated axonemes, and malformed cilia with either abnormal arrangement or number of microtubules in their axonemes. ACTA VET. BRNO 2001, 70: 167–172 Address for correspondence: Prof. MUDr. V. Konrádová, DrSc. Institute of Histology and Embryology 2nd Medical Faculty, Charles University V Úvalu 84, CZ-150 06 Prague 5, Czech Republic Phone: +420 2 24435980 Fax: +420 2 24435820 E-mail: vaclava.konradova@lfmotol.cuni.cz http://www.vfu.cz/acta-vet/actavet.htm

In our previous study, we demonstrated that 10-min inhalation of saline affected the ultrastructure of the airway epithelium (Konrádová et al. in press).We therefore decided to study also the effect of aerosol of mineral water, frequently used in therapy of various respiratory disorders, and to follow the process of regeneration of this epithelium in the course of 24 h.

Materials and Methods
In our experiments, 19 SPF New Zealand White rabbits (body weight 1,500-3,000 g, Charles River Deutschland, Sulzfeld, Germany) were used.Seven of them served as untreated controls.The remaining animals were placed successively for 10 min into a plastic cage connected with the inhalation device PARI Master and nebuliser PARI LL (Pari GmbH, Starnberg, Germany, medium diameter of produced droplets 3.1 µm, total output 0.6 g/min).The rabbits inhaled an aerosol of mineral water for 10 min.Chemical composition of this spring water is given in Tab. 1.Under general anaesthesia, the material for electron microscopic and histochemical examinations was collected from six animals immediately and 24 h post exposure, respectively.
Tiny fragments of the tracheal mucous membrane were processed using standard methods for electron microscopy (Konrádová 1991).The ciliary border and the functional state of the goblet cells were evaluated quantitatively.To evaluate the distribution of goblet cells in the epithelium, the isolated elements and the goblet cells arranged in groups were distinguished.Kinocilia were classified into four categories: intact 9+2 cilia, slightly damaged pathological cilia with local swellings of the ciliary membrane or with tiny vacuoles situated in their shafts, degenerating cilia, represented by axonemes incorporated into cytoplasmic blebs or by isolated axonemes, and malformed cilia with either abnormal arrangement or number of microtubules in their axonemes.
In controls, immediately and 24 h after inhalation of mineral water 1.058 µm 2 , 2,057 µm 2 and 1,300.25 µm 2 of ciliary border with 10,252, 13,040 and 9,294 kinocilia were evaluated, respectively.In those experimental groups also a total of 186, 567 and 344 goblet cells using electron microscopy and 398, 379 and 422 goblet cells in histochemical studies were studied, respectively.For statistical evaluation of the ultrastructural findings, relative values of 2 categories of goblet cells and 4 categories of cilia were evaluated by the chi-square test of homogeneity in frequency tables.To specify categories causing deviations from the hypothesis of homogeneity, adjusted standardised deviations were used.In histochemical studies, relative values of six categories of goblet cells, revealed by individual methods, were evaluated in similar manner described above.The Yates' correction in low frequencies was used when appropriate.The equivalency of the sialylated glycoconjugate detecting methods was tested by the paired t-test, median (sign) test and Wilcoxon's paired test.Means of cilia/µm 2 were compared by the one-way analysis of variance (ANOVA).The differences between groups were assessed by the Tukey's test or Bonferroni's method for multiple comparison.The Levene's test for equal variances was also performed.As a nonparametric analogy of the ANOVA, the Kruskal-Wallis test was used.

Control Rabbits
In control rabbits, the tracheae were lined with a pseudostratified columnar ciliated epithelium.The ciliated cells of standard ultrastructure were the most numerous in the epithelium.
Goblet cells mostly filled with mucus were scattered among the ciliated ones.Only 6 ± 3% of them were arranged in tiny groups.By means of gradual evacuation of the individual apical mucous granules, 3% of secretory elements discharged their secretion.The conventional histochemistry revealed the dominance of goblet cells containing acid GCs.The proportion of sulphated GCs, detected by classic histochemistry, and the total percentage of goblet cells containing sialylated GCs visualised by the reactions with lectins were given in Tab. 2.

Immediately after 10-min inhalation of mineral water
Tracheae of the rabbits exposed to mineral water aerosol were lined with an altered pseudostratified ciliated epithelium with narrow intercellular spaces and intact apical junctional complexes (Plate IV, Fig. 2).In the epithelium, 34 ± 1% of goblet cells took part in the formation of small intraepithelial mucous glands.Less than 10% of the goblet cells were filled with large light mucous granules, 4% of them were completely exhausted, their cytoplasm was highly electron dense.
Cells not entirely filled with mucus prevailed in the epithelium.These cells were equipped with short irregular microvilli and only a few small, highly electron dense granules or more numerous granules of various electron density were observed in their cytoplasm (Figs. 2, 3).In some smaller granules with typical fibrogranular matrix, electron dense cores were revealed (Plate IV,Figs. 3,4).Also cells rich in smaller typical mucous granules separated by voluminous cytoplasmic septa were encountered (Plate IV, Fig. 5).
Mucus filled cells as well as cells containing only isolated secretory granules often discharged secretion.Evacuation of apical mucous granules, detachment of whole packets of granules and also chain fusion of the adjacent mucous granules' membranes were noticed.
Compared with controls, significant (α = 0.01) decrease in number of goblet cells containing acid sulphated GCs was detected using methods of conventional histochemistry.Lectin histochemistry revealed slight, but significant increase in total number of goblet cells containing sialylated GCs (Tab.2).
The ciliated cells were less damaged compared to the secretory ones.On their apical portions, only isolated small cytoplasmic blebs were developed.In the deeper portions of ciliated cells' cytoplasm, a slight increase in the number of small vacuoles, secondary lysosomes, dilatation of the cisternae of granular endoplasmic reticulum and Golgi complex and altered mitochondria were observed (Plate V, Fig. 6).
The regular arrangement of the ciliary border was slightly impaired.The mean number of cilia was 6.3 ± 0.4/µm 2 .The altered elements represented 4.0 ± 0.7%.The proportions of the individual types of altered cilia were given in Fig. 1.In the area among the kinocilia, clumps or layers of inspissated secretion were observed (Plate V, Fig. 7).24 h after 10-min inhalation of mineral water All the time, the tracheae of the experimental animals were lined with an altered pseudostratified ciliated epithelium.The intercellular spaces remained narrow, the apical junctional complexes were intact.
Tiny groups were formed by 41 ± 3% of goblet cells.The goblet cells filled with large light mucous granules represented 7.5%, the exhausted degenerated elements amounted to 15%.Remnants of their degenerated, highly electron dense cytoplasm appeared infrequently above the epithelium.
Cells containing only a few secretory granules were most numerous in the epithelium.In their cytoplasm, medium sized granules of various electron density were encountered, but cells containing smaller mucous granules with typical fibrogranular matrix prevailed in the epithelium (Plate V, Fig. 8).Mucus filled cells as well as cells containing only isolated secretory granules often discharged secretion.Evacuation of apical mucous granules was mostly noticed.
Compared with previous experimental group, significant (α = 0.01) increase in number of goblet cells containing acid sulphated GCs accompanied by a decrease of total number of goblet cells containing sialylated GCs was recorded (Tab.2).
The ciliated cells revealed only slight signs of alteration of their cytoplasm.Compared with previous experimental group, insignificant increase in mean number of cilia to 7.1 ± 0.4/µm 2 and decrease of altered cilia to 3.5 ± 0.9% were noticed (Fig. 1).The amount of condensed mucus found in the area of the slightly altered ciliary border was also evidently reduced (Plate V, Fig. 9).

Discussion
The target cells for the function of mineral water aerosol were the goblet ones.Due to the 10-min inhalation of mineral water, about 90% of goblet cells were stimulated to discharge their mucus.Immediately post exposure, also the mechanism of mucus evacuation was accelerated.Signs of apocrine type of secretion and of compound exocytosis were encountered (Specian and Neutra 1980;Roumagnac and Laboisse 1987;Specian and Oliver 1991;Konrádová et al. 1996;Newman et al. 1996).After 24 h, only gradual evacuation of individual mucous granules was noticed.Immediately after aerosol administration, only a few exhausted secretory cells degenerated.The goblet cells mostly took part in further secretory cycles.The number of degenerated secretory elements gradually increased.Twenty-four h post exposure, a threefold number of degenerated cells was encountered in the epithelium.
During the whole experiment, the cells not entirely filled with secretory granules prevailed in the epithelium.Immediately post exposure, numerous cells containing small to medium sized electron dense granules were revealed.After 24 h, the smaller mucous granules prevailed in the cytoplasm of secretory cells.
The high level of stimulation of secretory cells also induced a massive differentiation of new secretory elements (Konrádová et al. 1990(Konrádová et al. , 1996)).As the differentiating goblet cells retained the ability to divide (Becci et al. 1978), the result of this process were changes in their distribution in the epithelium.(Konrádová et al. 1990(Konrádová et al. , 1996)).In controls, less than 10% of secretory elements formed small groups in the epithelium, while more than 30% of *2.7Ȁ0.9 Fig. 1 them participated in the formation of intraepithelial mucous glands immediately after inhalation.The increase was highly significant (α = 0.01).Their number even increased significantly (α = 0.05) during the next 24 h.In accordance with other authors (Castells et al. 1990;Mandal and Mandal 1990;Jeffery et al. 1992), the presence of neutral, acid sulphated and sialylated GCs, the majority of acid GCs to neutral ones, and the dominance of sulphated GCs in the secretion of the goblet cells in healthy rabbits were ascertained by both conventional and lectinhistochemistry methods.The proportions of total sialylated GCs revealed by two methods of lectin histochemistry did not differ significantly.
Administration of the mineral water aerosol induced changes in the composition of GCs in the secretion of the goblet cells.Percentage of neutral GCs containing goblet cells varied non-significantly, but immediately after inhalation, a significant (α = 0.01) decrease in the acid sulphated GCs was accompanied by an increase of the acid sialylated ones.The α(2-6) sialylated GCs disappeared completely from the epithelium.In our previous studies, we demonstrated that the disappearance of the α(2-6) sialylated GCs was one of the first signs of goblet cells overstimulation (Vajner 1998;Konrádová et al. 2000;Vajner et al. 2000).After administration of some bronchospasmolytic drugs, high level of goblet cells injury resulted in absolute predominance of acid sulphated GCs (Konrádová et al. 1997(Konrádová et al. , 1998;;Vajner 1998).Twenty-four h post exposure, significant decrease in total sialylated GCs was found, but the α(2-6) sialylated GCs reappeared in the goblet cells' secretion.
Due to the administration of mineral water aerosol, the ciliated cells were less damaged compared with the goblet ones.Slight apical blebbing and mild signs of pathological alteration were noticed in the deeper portions of the ciliated cells' cytoplasm.The tiny apical cytoplasmic protrusions were not revealed 24 h post exposure, but mild alteration of the cytoplasm was all the time present.
The alteration of the ciliated cells was reflected in the impairment of the ciliary border.Immediately after administration of mineral water aerosol, significant decrease (α = 0.01) in the mean number of kinocilia/µm 2 was accompanied by slight, but significant (α = 0.01) increase in percentage of altered cilia.During the next 24 h, signs of ciliary border regeneration were noticed, but the average number of kinocilia/µm 2 and also the percentage of altered elements still differed significantly compared with controls (α = 0.01).
After mineral water inhalation, condensed mucus was discovered in the area of the ciliary border.In agreement with our previous studies (Konrádová 1991) and also with other authors who studied the relation of the cilia to the layer of mucus in the airways (Stratmann et al. 1991;Wanner et al. 1996;Geiser et al. 1997), we regarded the appearance of inspissated secretion embedding free kinocilia as a morphological sign of impaired self-cleaning ability of the airway epithelium.The signs of mucus flow impairment were most apparent immediately after aerosol administration, but to a lesser degree were still present 24 h post exposure.
We arrived at the conclusion that even single 10-min inhalation of mineral water aerosol caused significant changes in the ultrastructure of the airway epithelium and influenced also the chemical composition of the goblet cells' secretion and the self-cleaning ability of the epithelium.These changes did not disappear completely during 24 h post exposure.
Altered cilia in the tracheal ciliary border of rabbits immediately and 24 hours after 10-min.inhalation of mineral water

Fig. 2 :Fig. 6 :
Fig. 2: Intact junctional complex in the apical portion of the epithelium.Tracheal epithelium, rabbit, immediately after 10-min inhalation of an aerosol of mineral water; bar represents 1 µm.Fig. 3: Isolated small highly electron dense granule in the apical portion of a secretory cell.Tracheal epithelium, rabbit, immediately after 10-min inhalation of an aerosol of mineral water; bar represents 1 µm.Fig. 4 Secretory granules of different electron density in the cytoplasm of a secretory cell.Tracheal epithelium, rabbit, immediately after 10-min inhalation of an aerosol of mineral water; bar represents 1 µm.Fig. 5: Portion of the cytoplasm of a secretory cell rich in smaller mucous granules.Tracheal epithelium, rabbit, immediately after 10-min inhalation of an aerosol of mineral water; bar represents 1 µm.

Table 2
Quantitative evaluation of the chemical composition of glycoconjugates (GCs) in goblet cells of the tracheal epithelium in control rabbits and in rabbits immediately and 24 h after 10-min inhalation of aerosol of mineral water (relative values)