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. 2023 Jul 28;9(5):2078–2084. doi: 10.1002/vms3.1229

Morphological and histological study of the third eyelid in hedgehogs

Erfan Shakibapour 1, Mohammadreza Paryani 1,✉
PMCID: PMC10508502  PMID: 37503954

Abstract

Background

The third eyelid in animals plays an important role in maintaining eye health. Like other organs of the body, the third eyelid can be afflicted with minor or deep injuries, inflammations or even tumours in different species.

Objective

The current study investigates the morphological and histological characteristics of the third eyelid in hedgehogs.

Methods

Eight healthy adult hedgehogs (male and female) weighing 500–700 g were included in this study. Deceased animals were used for this study. Few incisions were applied around the eye and eye socket. The eye was removed, and the samples were fixated in 10% formalin solution to prepare for the histological study. To evaluate the morphological characteristics, the third eyelid was placed in 2% and 4% formalin solutions.

Results

The histological study revealed that the third eyelid cartilage is an elastic cartilage and includes chondroblasts and chondrocytes. The cells of this cartilage were either distributed individually or in isogenic groups. The bulbar and palpebral surfaces of the eyelids were devoid of any glands and were covered with a non‐keratinized stratified squamous epithelium. The anatomical examination also showed that the third eyelid had an oval‐shaped cartilage.

Conclusions

Comparing the results of this study with reports on other rodents showed that the morphological and histological structure of the cartilage in the hedgehog's third eyelid is mostly like the structure of this tissue in Indian mongoose (Herpestes javanicus).

Keywords: hedgehog, histology, morphology, third eyelid cartilage

Graphical Abstract: The cartilage of the third eyelid in the long‐eared hedgehog has a very delicate structure and is of elastic type. The bulbar and palpebral surfaces of the eyelids were devoid of any glands and were covered by a non‐keratinized stratified squamous epithelium.

graphic file with name VMS3-9-2078-g001.jpg

1. INTRODUCTION

The hedgehog is a small, thorny and nocturnal animal that has become a popular pet in many parts of the world (Banks et al., 2010). The etymology of hedgehog reveals that this word is derived from ‘hedge’, which means a boundary formed by abundant bushes or shrubs that defines its habitat, and ‘hog’ which refers to the hedgehog's pig‐like snout and the similarities in hedgehog and pig's skulls. The adult European hedgehog is 12–14 inches long. In general, among the five senses in hedgehogs, the senses of smell and touch are highly developed. Their visual acuity is low and their ability to differentiate colours is underdeveloped. Hedgehogs use a variety of sounds to communicate with each other (Bradley et al., 2006).

In most animals, the structure of the third eyelid, also known as the nictitating membrane, consists of a T‐shaped cartilaginous skeleton, the Harderian gland and the conjunctiva covering both the bulbar and palpebral surfaces. The cartilage curves at the nictitating membrane and becomes convex to match the eyeball. The shape of the cartilage varies in different domestic animals; for example in the horse, it is stretched, in ruminants, it is leafy or ladle‐like, in pigs, it is hook‐formed, and in dogs and cats, it has a crescent‐shape with a straight body (Heine, 1909).

The third eyelid plays a very important role in maintaining the health of the eye surface. It is also capable of protecting the cornea of some predators such as cats from injuries when they move among the vegetation as well as during predation (Kamali et al., 2015). The third eyelid in the dromedary has a wide anterior part and its palpebral surface is convex. The skeleton of this eyelid is cartilaginous and masses of glandular glands or the glands of the third eyelid are located at its posterior part (Yousefi et al., 2003). In a study by Mohammadpour (2009) on the structure of the glands of the third eyelid of the dromedary, the cartilage material of the third eyelid was reported as a type of hyaline cartilage. In birds, the upper eyelid is thick and short, whereas the lower eyelid is thinner and highly mobile. The third eyelid in the birds is known to be an elastic membrane where its free edge is thick and complex, allowing easier release of particles and liquids in the cornea (Klećkowska‐Nawrot et al., 2016).

Due to the fact that hedgehogs have recently been considered pets, a relatively high number of cases are referred to veterinary clinics for different reasons including eye problems. It seems that the results of the current study can be helpful in the diagnosis and treatment of eye diseases by providing accurate information on the anatomical features of the third eyelid in this animal. According to our knowledge, the anatomical features of the third eyelid in hedgehogs, especially the species in Iran that belong to the European type (Erinaceus europaeus) or the long‐eared type (Hemiechinus auritus), were not described before the present study. The purpose of this study was to describe the morphological and histological features of third eyelid gland of hedgehog.

2. MATERIALS AND METHODS

In this study, eight hedgehogs (adult H. auritus from both sexes) that were died in a veterinary clinic in Tehran due to severe traumatic incidence. After signing a consent form by the owner, the animal was dissected. Eyeballs were removed from the eye sockets post euthanasia, and after disconnecting the third eyelid connections, this part was completely separated, removed and categorized as the left or right nictitating membrane. Four membranes from each category were sent for morphological analysis while the remaining four were prepared for histological investigation. In the morphological examination, the third eyelid cartilage was separated via the modified maceration process (Eurell & Frappier, 2006). In this procedure, the third eyelid was rinsed in 2% KOH at a temperature of 40°C for a maximum duration of 18 h.

After separating the cartilage, it was examined and photographed under a stereomicroscope. The stereomicroscope is an optical device utilized for examining samples, offering a three‐dimensional representation of the observed specimen. This particular instrument is also referred to as a stereoscopic microscope or dissecting microscope. For the histological examination, after placing the third eyelid in 4% formalin buffer and fixating it, it was dehydrated, cleared and finally embedded in paraffin. Each sample was cut longitudinally into 5‐μm specimens and then stained with haematoxylin and eosin (H&E). For some sections, orcein staining was used to specify the elastic strands in the cartilage specimen. Periodic acid–Schiff (PAS) staining was also employed to determine possible glandular cells in the eyelid, whereas Masson's trichrome staining was used to measure the amount of collagen fibres in the cartilage and lamina propria around it. The stained sections were evaluated at magnifications of ×4, ×10, ×40 and ×100 under light microscopy (Eurell & Frappier, 2006). In contrast to a light microscope, the illumination method commonly employed in a stereo microscope predominantly relies on reflected illumination instead of transmitted illumination. This means that the light used originates from the object's surface rather than passing through it.

3. RESULTS

3.1. Anatomical results

The anatomical examination of the third eyelid cartilage in the hedgehog revealed its oval shape as well as the horn‐shaped elongation at both ends (Figure 1). Examining the morphology of cartilage in males and females did not show any significant difference.

FIGURE 1.

FIGURE 1

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Cartilage separated from the third eyelid of a hedgehog. (a) Rostral view; (b) caudal view; (c) lateral view. The cartilage is almost oval‐shaped with horn‐shaped elongation on both extremities while its crescent‐shape is apparent in the lateral view.

In the lateral view, the cartilage is convex, and its concave surface is in contact with the eyeball. The medial portion of this cartilage is oval and is characterized by a short protrusion. It starts as a narrow strip from the free edge of the cartilage (the side conforming to the cornea) and gradually thickens. It reaches its thickest point at the middle section and is again reduced in thickness from there on. The cartilage becomes thin at its stem and then forms a bulge. Eventually, the cartilage shows a sharp end in the lateral view (Figure 1C).

3.2. Histological results

3.2.1. Haematoxylin and eosin (H&E) staining

Examination of the third eyelid of the hedgehog using H&E staining revealed that the crescent‐shaped cartilage forms the central portion of the third eyelid. This cartilage is enveloped by the perichondrium. The cartilage is wrapped by loose connective tissue (lamina propria) containing fibres and connective cells. Non‐keratinized stratified squamous epithelium covers both surfaces except that on the palpebral surface this epithelium is pigmented (Figure 2).

FIGURE 2.

FIGURE 2

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Third eyelid in Hemiechinus auritus. Cross‐section of the central cartilage, lamina propria and epithelium. Haematoxylin–eosin (H&E) staining magnified at ×40 and ×400. (1) Epithelial tissue of the palpebral surface; (2) epithelial tissue of the bulbar surface; (3) central cartilage.

3.2.2. Orcein staining

Orcein staining was used in the histological examination of the tissue to identify the elastic cartilage as well as chondroblasts and chondrocytes, which were observed individually or as isogenic groups. As depicted in Figure 3, the cartilage matrix is filled with elastic fibres and actively reacts to the orcein stain. Thus, the cartilage in the third eyelid of the hedgehog is a type of elastic cartilage. There are also some elastic fibres visible in the lamina propria (Figure 3).

FIGURE 3.

FIGURE 3

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The third eyelid in the long‐eared hedgehog. The nictitating membrane cartilage is of elastic type. Orcein staining, scale bar = 400 μm.

3.2.3. Masson's trichrome staining

The histological examination of the third eyelid of the hedgehog using Masson's trichrome staining revealed the collagenous structure of perichondrium in the nictitating membrane cartilage. The collagen fibres reacted positively to Masson's trichrome and were dyed blue. These collagen fibres were also visible in the lamina propria in conjunction with blood and lymph vessels (Figure 4).

FIGURE 4.

FIGURE 4

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The third eyelid in the long‐eared hedgehog. The cartilage matrix lacks collagen fibres and does not react positively to Masson's trichrome staining, whereas dense collagen fibres in perichondrium and lamina propria are easily detectable. Masson's trichrome staining.

3.2.4. PAS staining

The histological examination of the third eyelid sections of the hedgehog using PAS staining revealed an abundant carbohydrate content in the cartilage matrix, which reacts positively to this stain. No gland was observed in the vicinity of the cartilage. Although the superficial gland of the third eyelid could not be detected, the tarsal gland was visible, which was like a sebaceous gland (Figure 5).

FIGURE 5.

FIGURE 5

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Third eyelid in the long‐eared hedgehog. Periodic acid–Schiff (PAS) staining.

4. DISCUSSION

Results of this study suggested that the third eyelid in the long‐eared hedgehog (H. auritus) consists of central elastic cartilage. The outermost layer in this cartilage is the perichondrium. The cartilage bends at the junction to the eyeball and adopts a convex shape along the way to conform the eyeball. The cartilage is composed of a loose connective tissue called lamina propria, which includes the fibres and cells corresponding to the connective tissue. Non‐keratinized stratified squamous epithelium covers both the bulbar and palpebral surfaces of the third eyelid and there are no intraepithelial glands visible in its structure.

There are apparent anatomical and morphological differences in the nictitating membrane of domestic animals. Many previous studies have reported the shape of the cartilage of the third eyelid as T‐shaped or anchor‐shaped (Dobberstein & Hoffmann, 1964; Franz, 1967; Heine, 1909; Mitchell & Tully, 2008; Sisson et al., 1975).

Schlegel et al. (2001) stated that despite the similarities, there are significant differences, especially regarding the appendages, in the cartilage of the third eyelid of domestic animals. The cartilage of the third eyelid in canines is conical in shape, which is almost sharp at the top while at the base it spreads wide and yields a triangular plate. This cartilage's shape in felines and canines is similar except that in canines the proximal end is shorter. The shape of the third eyelid's cartilage in pigs and cows is, however, anchor‐like while in small ruminants it starts as a thin rod and extends in a slightly curved form, eventually ending in an oval plate (Schlegel et al., 2001).

In the current study, the shape of the cartilage of the third eyelid in the hedgehog was observed to be an elongated oval, which was consistent with the results of studies on small ruminants. However, these results were different from the reported shape of nictitating membrane's cartilage in horses, which has a stretched structure, in large ruminants, which is leafy or ladle‐like, in pigs, which is hook‐formed, or in felines and canines, which is reported to be crescent‐shaped with a straight body (Heine, 1909). Some researchers suggest that this cartilage in horses, pigs and felines is elastic while in canines and ruminants it is of hyaline nature (Mitchell & Tully, 2008; Smollich & Michel, 1992). Other studies have proposed a mixed character with some elastic parts in felines and pigs (Czuberka, 1923).

The third eyelid in the dromedary has a wide anterior segment and its palpebral surface is convex. The skeleton of this eyelid is cartilaginous and the masses of Harderian glands or the glands of the third eyelid are located at the proximal end (Yousefi et al., 2003). In Mohammadpour's (2009) study on the structure of the glands of the third eyelid in the dromedary, the cartilage of this animal was described as a type of transparent cartilage. Our results were consistent with previous reports on the nature of cartilage in horses, pigs and cats, although it differs from the reported cartilage structure in dogs and ruminants.

Some studies have investigated the presence and distribution of elastic fibres in the nictitating membrane's cartilage of domestic animals. Among the species studied, only in cats and horses, the elastic fibres of the connective tissue surrounding the cartilage were found. It was also reported that thick elastic fibres are mainly concentrated in the central parts of the cartilage (Grau & Walter, 1967; Riley & Chomel, 2005; Schechter et al., 2010; Schlegel et al., 2001). In our study, we identified elastic fibres in the cartilage of the third eye, which is like the reported structure for felines and horses. The Harderian gland does not exist in all species. For example, this gland is not present in species, such as carnivores, primates and humans. However, this gland has been developed in many laboratory animals, amphibians, reptiles and birds (Sabry & Al‐Ghaith, 2000; Seely, 1987). The lacrimal gland and the superficial gland of the third eyelid in pigs, horses, guinea pigs, rabbits and mammals have a tubulo‐acinar structure (Schechter et al., 2010).

In terms of the cartilage type, epithelial tissue, the presence or absence of glands, the results of the present study were most consistent with the results provided by Kamali et al. (2015) who studied the nictitating membrane in the small Indian mongoose. The Harderian gland does not exist in all species. For example, this gland is not present in species, such as carnivores, primates and humans. However, this gland has been developed in many laboratory animals, amphibians, reptiles and birds (Sabry & Al‐Ghaith, 2000; Seely, 1987). The lacrimal gland and the superficial gland of the third eyelid in pigs, horses, guinea pigs, rabbits and mammals have a tubulo‐acinar structure (Schechter et al., 2010).

The authors did not observe surface secretory glands in any of the studied samples, and it seems that these glands are not present at least in this breed of hedgehog. To determine the condition of surface secretory glands in the third eyelid of the hedgehog, this is necessary that glands in other breeds of hedgehogs should be studied carefully with specific dyes.

In conclusion, the cartilage of the third eyelid in the long‐eared hedgehog has a very delicate structure and is of elastic type. The bulbar and palpebral surfaces of the eyelids were devoid of any glands and were covered by a non‐keratinized stratified squamous epithelium. It seems that application of electron microscopy as well as histochemical methods to study the structure of cartilage cells can provide useful additional information.

AUTHORS CONTRIBUTION

Data curation; investigation; project administration; writing – original draft: Erfan Shakibapour. Investigation; methodology; supervision; writing – review and editing; conceptualization; methodology; project administration; resources; visualization; formal analysis; methodology; software; validation: Mohammadreza Paryani.

CONFLICT OF INTEREST STATEMENT

The authors do not have any potential conflicts of interest to declare.

FUNDING INFORMATION

The authors received no financial support for the research, authorship and/or publication of this article

ETHICS STATEMENT

This study was conducted based on Iranian ethical codes for studies on animals and approved by the Ethical Review Committee of the Islamic Azad University (IAU), Karaj Branch.

PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.1229.

INFORMED CONSENT

Informed consent (verbal or written) was obtained from the owner or legal custodian of all animals described in this work (experimental or non‐experimental animals, including cadavers) for all procedure(s) undertaken (prospective or retrospective studies). For any animals or people individually identifiable within this publication, informed consent (verbal or written) for their use in the publication was obtained from the people involved.

Shakibapour, E. , & Paryani, M. (2023). Morphological and histological study of the third eyelid in hedgehogs. Veterinary Medicine and Science, 9, 2078–2084. 10.1002/vms3.1229

DATA AVAILABILITY STATEMENT

Data is available upon request from the corresponding author.

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Associated Data

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Data Availability Statement

Data is available upon request from the corresponding author.

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