NADPH-diaphorase expression in the meibomian glands of rat palpebra in postnatal development

Abstract

In the current study, we aimed at investigating the presence of nitric oxide synthase (NOS) positive nerve fibers in rat meibomian glands (MGs) at various stages of development. There is good evidence to suggest that nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) is a surrogate for neuronal nitric oxide synthase (NOS). Sections of the central, upper eyelids of Wistar rats were processed histochemically for NADPH-d to investigate the presence and distribution of NOS-positive nerve fibers at the following time points: day 1 and weeks 1, 2 and 3 post partum, and in adult controls. At day 1, MG acini were lightly stained and located at a distance from the mucosal border. Vessels were accompanied by intensely stained NADPH-d positive nerve fibers. At the week 1 time point, both the vessels and the NADPH-d positive fibers were still present, but less numerous. MGs were now closer to the mucosa, so that the submucosa was thinner. The acini were mostly pale but occasionally darker. At week 3, there were fewer blood vessels in both the submucosa and within the septa. Darker acini were more common than lightly stained acini. NADPH-d positive dots were observed in the vicinity of the MGs. At the week 3 time point, MGs were adjacent to the mucosal border and stained more intensely than at earlier times; almost all acini were stained. The microscopic appearances were almost identical with those of adult palpebra. Submucosal and septal blood vessels and NADPH-d positive nerve fibers were less numerous. NADPH-d histochemical staining confirmed differences in the density of stained nerve fibers at different developmental stages. The greatest density of NADPH-d -positive nerve fibers occurred in 1-day-old rats whereas they were less numerous in adult rat eyelids. Nerves innervating MGs utilize nitric oxide (NO) as a neurotransmitter mostly in early developmental stages and this need thereafter decreases and stabilizes at 3 weeks postnatally.

Figure 1

Parasagittal section through the upper rat eyelid at postnatal day #1. (a) Meibomian glands (MG) are barely NADPH-d stained and are located at a distance from the tarsal conjunctival surface (C). Deep to the skin (S) there are numerous hair bulbs (hb); scale bar = 500 µm. (b) Inset from previous figure: Non-uniformly stained blood vessels (arrows) with accompanying nerve fibers. The submucous layer is relatively wide and heavily vascularised, scale bar = 100 µm. (c) Developing Meibomian glands (MG) stained with haematoxylin and eosin. Orbicularis oculi muscle fibers (m) are visible on the right. Arrows point towards the conjunctiva (C) or skin (S) respectively; scale bar = 100 µm.

Figure 2

Parasagittal section through the upper rat eyelid at postnatal week #1. (a) NADPH-d stain. Acini of Meibomian glands are mostly pale (MG). Deep to the skin (S) there are many hair bulbs and hairs (hb, h); scale bar = 500 µm. (b) Inset from previous figure: NADPH-d positive nerve fibers were visualised, running along the blood vessels (arrows) and in addition, single nerve fibers in the submucosa under the conjunctival epithelium (C); scale bar = 100 µm. (c) Contours of the Meibomian acini (MG) and Meibomian duct (d) shown with haematoxylin and eosin staining. A hair bulb (hb) is visible at the centre of picture and orbicularis oculi muscle fibers (m) are visible to the right; scale bar = 100 µm.

Figure 3

Parasagittal section through the rat upper eyelid at postnatal week #2. (a) Darker NADPH-d stained acini (MG) are clearly seen at this period of development close to the conjunctival surface (C); hairs (h) are brown; scale bar = 500 µm. (b) Inset from previous figure: Blood vessels, accompanied by nerve fibers (arrows), are running in the septa between the Meibomian glands and at their surface and are moderately NADPH-d positive in both the sub-mucosa and within the septa. A blood vessel lumen is visible centrally (v); scale bar = 100 µm. (c) Almost mature Meibomian glands (MGs) with septa between acini (s) are seen with haematoxylin and eosin staining; scale bar = 100 µm.

Figure 4

Parasagittal section through the upper rat eyelid at postnatal week 3. The microscopic appearances were almost identical with those of adult lids. (a) NADPH-d stain. Meibomian glands (MGs) are adjacent to the mucosal border (C) and like adult glands, are intensely NADPH-d positive, except the ducts (d) which stain yellow; scale bar = 500 µm. (b) Inset from the previous figure: NADPH-d positive nerve fibers (arrows) and submucosal and septal blood vessels are less numerous. Nerves are visible as fine fibers running in the septa (s) between the acini; scale bar = 100 µm. (c) Fully developed Meibomian gland acini (MGs) and septa (s). Orbicularis oculi muscle fibers (m) are visible on the right. Haematoxylin and eosin staining; scale bar = 100 µm.