Transmission Electron Microscopic Findings of Lacrimal Gland Acinar Cells Induced by In Vivo Dry Eye

Yu Ri Seo; A Reum Yeo; Hye Mi Noh; Dong Yong Chung; Tae Im Kim; Kyoung Yul Seo; Eung Kweon Kim; Hyung Keun Lee

doi:10.3341/jkos.2014.55.8.1187

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Seo, Yeo, Noh, Chung, Kim, Seo, Kim, and Lee: Transmission Electron Microscopic Findings of Lacrimal Gland Acinar Cells Induced by In Vivo Dry Eye

Original Article

J Korean Ophthalmol Soc 2014;55(8):1187-1194.

Published online: 31 August 2014

DOI: https://doi.org/10.3341/jkos.2014.55.8.1187

Transmission Electron Microscopic Findings of Lacrimal Gland Acinar Cells Induced by In Vivo Dry Eye

Yu Ri Seo, MD¹, A Reum Yeo, MD, PhD¹, Hye Mi Noh, MD, PhD¹, Dong Yong Chung², Tae Im Kim, MD^1,³, Kyoung Yul Seo, MD^1,³, Eung Kweon Kim, MD^1,³, Hyung Keun Lee, MD^1,³

¹Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea

²Morphology Lab., Yonsei Biomedical Research Institute, Seoul, Korea

³Institute of Corneal Dystrophy Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea

Address reprint requests to Hyung Keun Lee, MD, Department of Ophthalmology, Gangnam Severance Hospital, #211 Eonju-ro, Gangnam-gu, Seoul 135-720, Korea, Tel: 82-2-2019-3440, Fax: 82-2-3463-1049 E-mail: shadik@yuhs.ac

Received 28 December 2013 Revised 1 February 2014 Accepted 12 July 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

To determine the change in lacrimal gland (LG) acinar cells induced by in vivo dry eye (DE).

Methods

Six to 8-week-old (C57BL/6) mice were placed in a controlled environment chamber at <20% humidity for 2 weeks, and a control group was bred in a normal environment. After these 2 weeks of dry eye (DE) induction, the mice were sacrificed and their LGs were collected. Lacrimal gland acinar cell organelle structures were observed with Transmission Electron Microscopy (TEM). TEM images were analyzed using the Image J program.

Results

The size of the LGs of DE-induced mice decreased compared to those of normal mice. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) staining was negative in DE-induced LGs. Under the TEM, the endoplasmic reticulum (ER) lumen was dilated and the lumen density increased in DE-induced mice. Additionally, cell organelles were surrounded by elongated ER lumens. The mitochondrial structure was destroyed and the number of vacuoles increased in the LGs of DE-induced mice.

Conclusions

Structural changes of the LG developed due to DE induction. This suggests that the detailed mechanisms of these changes were ER stress and autophagy. However, there were no definite signs of apoptosis in the acinar cells of the DE-induced LGs. These findings are regarded as an important clue of the pathogenesis of non-Sjogren-type dry eye.

Keywords: Autophagy, Dry eye, ER stress, Lacrimal gland

References

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Figure 1.

Dissection of lacrimal gland in mice. (A) After making an incision as V-shaped from the inferolateral side of the ear to ramus of mandibule on the skin, we dissected a subcutaneous tissue along the incision line. We flipped over the skin and subcutaneous tissue. (B) Then, submandibular gland (3) was noted at just inferolateral side of the ear. We observed that the lacrimal gland (1) and parotid gland (2) were in front of the submandibular gland (3). The lacrimal and parotid glands were very close to each other. They could be separated along the fissure. (C) After separation, the lacrimal gland was little bit smaller than the parotid gland.

Figure 2.

Comparison of lacrimal gland size between normal and dry eye (DE) induced mice. The size of the lacrimal gland of DE induced mice was decreased significantly comparing to the normal mouse lacrimal gland (LG). The blood vessels were decreased on surface taking on pallor in the DE induced LG comparing to the normal mouse LG. Width and height were measured for determination of LG size. The width by height was averaged and compared (Table 1).

Figure 3.

Determination of apoptosis in dry eye (DE) induced lacrimal gland (LG) with Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) staining. TUNEL staining of lacrimal gland in the DE induced mice showing no difference compared to the lacrimal gland of normal mice (left: ×40, right: × 100).

Figure 4.

Comparison of lacrimal gland cellular structure between normal mice and dry eye (DE) induced mice under Transmission Electron Microscopy (TEM). [Normal (A) (× 10⁴), normal (B) (× 2 × 10⁴), dry eye (A) (× 10⁴), dry eye (B) (× 2 × 10⁴)]. Arrow shows normal mitochondrial structure and arrow head shows normal the endoplasmic reticulum (ER) lumen in normal (A) and normal (B). In dry eye induced lacrimal glands, cristae structure of the mitochondria was destructed (arrow) and vacuoles were increased (arrow head) in dry eye (A). In dry eye (B), ER lumen was elongated and surrounding cell organelles (arrow) and ER lumen density was increased and the lumen was dilated (arrow head).

Figure 5.

Comparison of the endoplasmic reticulum (ER) lumen density between normal mice and dry eye (DE) induced mice using gray scale measure of image J program. Five spots were selected randomly in ER lumen (black stars). The darkest spot was selected in the nucleus (yellow star). Gray scales were measured at each spots by using Image J. Table 2 shows ratio between average ER lumen gray scale and nucleus gray scale.

Table 1.

Comparison of lacrimal gland size between normal mice and dry eye induced mice

	Normal	Dry eye	p-value
Average of size (mm²)	8.42 ± 2.06	4.70 ± 0.97	<0.001^*

Values are presented as mean ± SD.

^* Independent samples t-test.

Table 2.

Comparison of gray scale ratio between normal mice and dry eye induced mice with image J

	Normal	Dry eye	p-value
Ratio the endoplasmic reticulum (ER)/Nucleus	7.43 ± 0.64	5.76 ± 0.30	<0.001^*

Values are presented as mean ± SD.

^* Independent samples t-test.

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