Figure 4.
Time-course of OM onset and ET malformation. H&E histology over a time course revealed occurrence of OM inflammation and ET developmental malformation in LmnaDhe/+ mutant mice (Dhe/+), compared with the littermate control wild-type mice (+/+). n = 4 for each genotype at each time point. In each row, the center and right panels show magnified images corresponding to the two boxed regions in the left panel (excluding only U and X). A–F: Representative images of mice at P6. Sections exhibited no middle ear inflammation and no significant developmental disparity between the wild-type controls (A) and the mutant mice (D). Mesenchymal cells (asterisks in A, B, D, and E) were spread throughout the MEC. C and F: The ET was opened slightly and covered with pseudostratified ciliated columnar epithelium in both wild-type mice and mutants. G–L: Representative images of mice at P12. Onset of OM and dysplasia of ET occurred in the mutant mice. Mesenchymal cells still remain around the malleus in both groups (asterisks in G, H, J, and K). However, acute inflammatory response cells began to perfuse in the MEC of the mutant mice. Red blood cells (black arrows in MEC in K) and monocytes (open arrows in K) infiltrated in the mesenchymal cells. H and K: EACs were not fully open in mutants or controls. The TM of wild-type mice had developed to the normal three layers by this time, but was not fully developed in the mutant mice. The ET was enlarged in mutant mice (L), and debris of acute inflammatory response cells emerged (asterisks), compared with the narrow tubular ET shape and clear cavity in the wild-type mice (I). Red blood cells (shortest arrow in L) and monocyte masses (longer arrows in L) blocked the inner opening of the ET. M–R: Representative images of mice at P21. Ears of mutant mice continued to display OM and ET malformation. M and P: Mesenchymal cells disappeared, and middle ear cavitation was almost complete in both mutant and control mice. M and N: Wild-type mice (N) exhibited a clear MEC and healthy MEC mucosa (arrows), but mutant mice (P) exhibited inflammatory cell infiltration in MEC. Q: Detail of the MEC with thickened mucosa (black arrows) and inflammation (asterisk) in mutant mice; neutrophils infiltrated the MEC with exudates (open arrows). O and R: ET distortion was even more pronounced at P21. Dilation of ET interior diameter (double-ended arrows) was evident in mutant mice (R), compared with wild-type mice (O), and in the mutant mice (R) inflammatory exudate partially blocked the ET (asterisk). S–X: Representative images of mice at 8 weeks. S and V: Panoramic view of the bullae. In wild-type mice, neither middle ear inflammation nor TM retraction was detected (S), and the middle ear mucosa remained a thin layer and the TM retained normal position without retracting to the MEC (T). V: In mutant mice (V), inflammatory infiltration (asterisks) and debris (short arrow) pervaded the MEC, and TM retraction and thickness were accompanied by fibroblastic exudate (asterisk) and chronic inflammatory debris accumulation (short black solid arrow in MEC) with capillary hyperplasia (open arrows) in MEC (W). U and X: In ET at 8 weeks, wild-type mice (U) exhibited orderly, aligned, pseudostratified ciliated columnar epithelium (arrows). In mutant mice (X), the ET exhibited inflammatory cells (asterisk), epithelium with poor alignment, and shortened or obsolescent cilia (black arrows). Scale bars: 200 μm (A, D, G, J, M, P, S, and V); 100 μm (T, U, and W); 50 μm (B, E, H, K, L, O, R, and X); and 25 μm (C, F, I, N, and Q). EAC, external auditory canal; ET, Eustachian tube; IE, inner ear; M, malleus; MEC, middle ear cavity; TM, tympanic membrane.