Early and late immunohistochemical and ultrastructural changes associated with functional impairment of the lachrymal gland following external beam radiation

Abstract

The aim of this study was to investigate scintigraphic, immunohistological and ultrastructural changes associated with radiation-induced dysfunction of the lachrymal gland in an established experimental animal model. Ten rabbits were randomized into two groups and used for the study; in the control as well as experimental group, the Schirmer-test, lachrymal gland scintigraphy, and immunohistological and ultrastructural investigations were carried out prior to irradiation and 72 h as well as 1 month after single-dose irradiation with 15 Gy. Seventy-two hours after irradiation, secretion reduction evaluated by the Schirmer-test was evident. At this phase, we could observe a decrease in the expression of alpha-SMA and a re-distribution of tenascin-C matrix. Ultrastructural changes of acinar and myoepithelial cells were noticed; simultaneously, disturbance in the primary (99m)TcO(4)- uptake as well as significant reduction of the lachrymal ejection fraction was assessed scintigraphically. These changes were still evident 1 month following irradiation but became less intensive. Single-dose irradiation with 15 Gy implicates a functional impairment of the lachrymal gland, which is associated with early immunohistological and ultrastructural alterations. These changes may represent objective surrogate parameters for radiogenic dysfunction and prerequisites for further investigations on radioprotection of lachrymal glands during radiotherapy of the periorbital region.

Figure 1

Conventional haematoxylin-and-eosin-staining of the control lachrymal gland (a) shows the typical histological tubulo-acinar structure of the inferior lachrymal gland and the cubic regular shape of acinar cells with basally located nuclei (arrows). Three days after irradiation with 15 Gy, vacuolopathy and secretory retention were evident in the end-piece epithelium (b; arrows) while ductal cells stayed unaffected.

Figure 2

(a) Immunohistochemical expression pattern of α-SMA in the control lachrymal glands showing a basket-like pattern in the acinar region (arrows). (b) The inferior lachrymal gland 72 h after irradiation with 15 Gy shows a marked loss of α-SMA reaction in the acinar region, the labelling displayed an irregular pattern and is limited to scattered acini. (c) Tenascin-C expression is observed in the duct system and blood vessels of control lachrymal glands (arrows); the acinar cells themselves are not labelled. (d) Seventy-two hours following irradiation, the acinar cells display an excessive intracellular expression of tenascin-C (arrows). Bar = 100 µm.

Figure 3

(a) Electron microscopic appearance of the lachrymal acinar cells (AC) 72 h after irradiation showing granula retention and peripheral displacement of the cell nuclei (arrows). Bar = 2.5 µm. (b) Thickening of the basal membrane (arrows) as well as condensation of the myofilaments in the myoepithelial cells (MECs) noticed 72 h after irradiation (asterisks). Bar = 0.6 µm.