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Keywords:

  • cornea;
  • ecdysis;
  • OCT ;
  • SLO ;
  • snake;
  • spectacle

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References

Purpose

One of the singularities of the eyes of snakes is the presence of the spectacle, a transparent and vascularized integument covering the cornea. The spectacle is completely renewed during ecdysis. Combined scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), and conventional macrophotography were used to image this phenomenon.

Material and Methods

A spectral OCT/SLO examination and macrophotography were performed in four healthy adult corn snakes (Pantherophis guttatus) and one healthy adult California king snake (Lampropeltis getulus californiae) the day before the start of ecdysis and then daily during ecdysis.

Results

In all animals, ecdysis lasted 5 days. The spectacle was hardly visible at baseline, but became obvious at day one, while the subspectacular space became larger and the superficial cornea presented a hyperechoic band. At day two, eye surface became translucent, and at the same time, vascularization of the spectacle was visible using SLO. At day 3, the vascularization was no longer visible, while the subspectacular space increased and the eye surface remained translucent. At day 4, the eye surface was transparent and the superficial hyperechoic band started to become less bright. At day 5, the old spectacle was shed and all the parameters returned to baseline.

Conclusion

We hypothesize that the echogenicity modifications of the anterior cornea correspond to major metabolic activity associated with new spectacle formation. This increased metabolic activity may contribute to the neovascularization and play an important role in the accumulation of fluid in the subspectacular space, facilitating the shedding of the old spectacle.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References

Snakes, some burrowing lizards, and most geckoes possess a fixed transparent integument covering the cornea.[1-5] This membrane, also called the spectacle or brille, is thought to have evolved from fusion of the eyelids.[6-8] Its primary functions are to provide mechanical protection to the cornea,[9-12] minimize evaporative water loss,[13] and protect the eye against solar radiation.[14] The spectacle's anatomy is homologous with that of the skin consisting of a stratum corneum (the spectacle scale), a complex epidermis and a dermis. It differs in possessing a subdermal lining of conjunctiva similar to that of eyelids.[11, 15]Unlike skin and most eyelids, the spectacle is transparent and suited to vision despite an extensive, discrete vascular network,[15, 16] with a subspecies-specific pattern.[17-22] The vessels are transparent; their presence has been demonstrated by fluoroangiography on living snakes, and by intra-arterial injection of micro-silicone on recently euthanatized specimens.[22]

The space between the spectacle and the cornea (also called subspectacular space) is filled with Harderian gland secretions.[3] These secretions are drained by the nasolacrimal duct, which passes from the medial canthus and enters the roof of the buccal cavity, immediately posterior to the vomeronasal organ.[3]

The spectacle, like the skin, is periodically shed and replaced during ecdysis.[3] During ecdysis, the ocular surface becomes translucent and fluid accumulates between the old and the new spectacle until the old spectacle is shed.[4] The new spectacle becomes transparent prior to elimination of the old spectacle.[3]

The singularity of the spectacle within the animal kingdom has aroused the interest of biologists for centuries.[23] From direct observation to more complex investigative procedures,[24] many techniques have been used to describe spectacular form and function. One of the most recent reports documents spectacular ecdysis in the royal python (Python regius) using optical coherence tomography (OCT).[25] OCT has become a useful tool both in human and veterinary ophthalmology as a safe noninvasive procedure that enables high-resolution evaluation of anterior segment structures without the need for sedation. It allows for precise biometry and cross-section visualization of the cornea, anterior lens capsule, iridocorneal angle, and anterior chamber.[26-28] OCT can complement other ocular imaging tools including scanning laser ophthalmoscopy (SLO), which utilizes confocal laser scanning microscopy for diagnostic imaging of the cornea. This study describes spectacular ecdysis (SE) in the corn snake and the California king snake using a combined OCT-SLO device and macrophotography.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References

Animals examined

Four adult corn snakes (Pantherophis guttatus) and one adult California king snake (Lampropeltis getulus californiae) were included in the study. Subjects were free of ocular disease as determined by biomicroscopy (SL15 Kowa Company, Tokyo, Japan). OCT-SLO imaging and macrophotography were performed the day before the start of ecdysis and then daily throughout the process, until complete elimination of the shed structures and baseline recovery. As the snakes were kept in a vivarium at the Centre Hospitalier Vétérinaire Saint-Martin for several months before the start of the study, an approximate date for the next impending ecdysis was calculated. The study was started a few days prior to this. The first manifestations of ecdysis were considered as a drab and bluish aspect of the skin.

Optical coherence tomography and scanning laser ophthalmoscopy

Instrumentation for this study consisted of a spectral OCT-SLO (OCT-SLO, Group OTI/USA; EDC Vet, Carvin, France) with a specific corneal module. The OCT-SLO device provided both OCT and SLO images simultaneously. A veterinarian, qualified in exotic animal care, restrained each snake. In the interests of animal welfare, one eye only was examined. The left eye was chosen for all the animals in our study. The animal was positioned with the left eye 5 cm from the lens of the OCT-SLO, such that the horizontal scanning line was positioned centrally on the eye, crossing the central portion of the pupillary area. A video sequence was recorded, and one image that accurately depicted the spectacle, the cornea, the iris, the pupillary area, and the lens anterior capsule was selected from this sequence. Similarly, one image was selected for each day throughout the ecdysis period. Four parameters were evaluated for each image: (i) subspectacular space in the region of the corneal vertex, (ii) central corneal thickness, (iii) peripheral corneal thickness, and (iv) anterior chamber depth (Fig. 1). The subspectacular space was determined as the distance between the old spectacle and the underlying new spectacle. The central corneal thickness was determined as the distance between the spectacle and the corneal endothelium in the region of the corneal vertex (excluding the old spectacle when both the old and the new spectacles were present). The peripheral corneal thickness was determined as for the central corneal thickness except that the measurements were made at the corneal periphery, close to the limbus. The anterior chamber depth was determined as the distance between the corneal endothelium in the region of the corneal vertex and the anterior pole of the lens. All these measurements were made using the software provided with the OCT equipment.

image

Figure 1. Schematic aspect of the anterior segment of the eye of the snake and biometric parameters as measured by OCT. a: Subspectacular space; b: central corneal thickness; c: anterior chamber depth; and d: peripheral corneal thickness. The asterisk (*) represents the peripheral scale.

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Macrophotography

Macrophotography was made with a DSL-R camera (A700; Sony, Tokyo, Japan), a 105-mm macro lens (105 mm f/2.8; Sigma, Kawasaki, Japan), and three extension tubes (12, 20, and 36 mm; Kenko, Tokyo, Japan). A Finoff transilluminator provided illumination.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References

In both species (Pantherophis guttata and Lampropeltis getulus californae), spectacular ecdysis followed the same chronology and lasted 5 days.

The day before ecdysis, at baseline, the spectacle was barely distinguishable from the underlying cornea by OCT and the subspectacular space was not evident. The reflectivity of the cornea was homogeneous with a lamellar organization. The central region of the cornea (old spectacle excluded) was thicker in the corn snakes (mean value of 330 μm) than in the king snake (240 μm) (Fig. 2a,b). The anterior chamber depth in the region of the anterior pole of the lens was smaller in the corn snakes (mean value of 330 μm) than in the king snake (500 μm). The peripheral scale was covered by a homogeneous, thin echoic band at its periphery. SLO imaging and macrophotography gave similar results and showed a normal, transparent cornea in both species (Figs 3a,b and 4a,b). The spectacle was not discernible from the underlying cornea.

image

Figure 2. OCT appearance of the spectacular ecdysis in the Pantherophis guttatus number 1 (on the left) and in the Lampropeltis getulus californiae (on the right) at baseline (a, b), day 1 (c, d), day 2 (e, f), day 3 (g, h), day 4 (i, j), and day 5 (k, l).

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image

Figure 3. SLO appearance of the left eye of the Pantherophis guttatus number 1 (on the left) and the Lampropeltis getulus californiae (on the right) at baseline (a, b) and at day 2 (c, d). Spectacular vascularization was only visible at day 2. The appearance of the eye at day 1, day 3, day 4, and day 5 was the same as at baseline for both species.

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image

Figure 4. Macrophotographic appearance of the left eye of the Pantherophis guttatus number 1 (on the left) and the Lampropeltis getulus californiae (on the right) at baseline (a, b), day 1 (c, d), day 2 (e, f), and day 3 (g, h). The appearance of the eye at day 4 and day 5 was the same as at day 3.

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On the first day of ecdysis, the spectacle was visible as a curvilinear hyperechoic structure more clearly defined in the vertex region than at the periphery. An extremely thin subspectacular space narrowed toward the periphery and was nonechoic. The cornea became thicker centrally with a maximum mean thickness of 400 μm in the corn snakes and 380 μm in the king snake, old spectacle excluded. Its echogenicity was increased toward the limbus in both species. The peripheral scale was seen as two hyperechoic bands, separated by a nonechoic space (Fig. 2c,d). SLO imaging of the eye was unchanged, while the cornea was translucent by macrophotography (Fig. 4c,d).

During the second day of ecdysis, the spectacle appeared thicker and the subspectacular space had increased in both species, reaching 30 μm at its center. The echogenicity of the underlying cornea was more intense. Corneal thickness was unchanged from day 1 for both the corn snakes and the king snake. The appearance of the peripheral scale did not change (Fig. 2e,f). SLO imaging clearly demonstrated spectacular vascularization; the spectacular vessels were vertically oriented in a rectilinear pattern in both species (Fig. 3c,d). The cornea appeared transparent by SLO but remained translucent by macrophotography (Fig. 4e,f).

On the third day of ecdysis, the subspectacular space was wider, reaching a mean of 40 μm in the corn snakes and 140 μm in the king snake. The echogenicity of the superficial portion of the cornea had increased slightly. Corneal thickness and the appearance of the peripheral scale remained unchanged in both species (Fig. 2g,h). The cornea was transparent both with SLO and conventional macrophotography (Fig. 4g,h). Spectacular vascularization was no longer visible with SLO.

On the fourth day of ecdysis, the subspectacular space was wider (mean of 90 μm in the corn snakes and 200 μm in the king snake) and the echogenicity of the superficial portion of the cornea had decreased. Corneal thickness started to decrease in both species (mean of 340 μm in the corn snakes and 250 μm in the king snake) (Fig. 2i,j). The anechoic space between the two echoic bands over the peripheral scale was greater. Both SLO and macrophotography aspect of the eye were unremarkable at this time.

By the fifth day of ecdysis, all the structures had returned to baseline. The superficial spectacle was shed, as was the superficial peripheral scale. The echogenicity of the superficial portion of the cornea had decreased to match the deeper corneal layers, and the spectacle was undistinguishable from the underlying corneal epithelium (Fig. 2k,l). The sloughed skin of all animals was found in the vivarium. The spectacle was creased, but still transparent and attached to the peripheral scales.

An overview of the biometric parameters measured using OCT is presented in Table 1.

Table 1. Biometric parameters during spectacular ecdysis as measured by optical coherence tomography in Pantherophis guttatus and Lampropeltis getulus californiae
 Day of ecdysisAnimal numberSubspectacular space (μm)Central corneal thickness (μm)Peripheral corneal thickness (μm)Anterior chamber depth (μm)
Corn Snake (Pantherophis guttatus)Baseline1Not measurable360200320
2Not measurable270220270
3Not measurable350190380
4Not measurable340210360
Day 11Not measurable430200330
2Not measurable330230270
3Not measurable420200400
4Not measurable410210380
Day 2130430230290
220340250260
330430220360
430410240350
Day 3140430240310
230340250260
340400230380
440400240350
Day 4190360200330
280280230260
3100370220390
4100350220360
Day 51Not measurable300190420
2Not measurable260220360
3Not measurable350190450
4Not measurable330200450
California King Snake (Lampropeltis getulus californiae)Baseline5Not measurable240150500
Day 15Not measurable380260590
Day 2530350230390
Day 35140330210370
Day 45200250170590
Day 55Not measurable270150350

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References

As with the skin, the spectacle undergoes ecdysis, a physiological process that results in the periodic elimination of worn epidermis. The frequency of skin ecdysis is highly variable and depends on humidity, ambient temperature, hormonal balance, state of nutrition, reproductive status, parasites load, concurrent skin disease, species, and age.[3] Normal ecdysis is a complex phenomenon during which cells of the upper stratum germinativum replicate and form a new three-layered epidermis. Once the new surface is complete, lymph diffuses into the area and local enzymatic action results in the formation of a cleavage zone which precedes the elimination of the former structures.[29]

Our study demonstrated that SE mechanisms are comparable to that of the skin, but present some variations. At baseline, the subspectacular space was not visible by OCT, contrary to the recent report describing SE in the royal python.[25] This difference may reflect the size of the animals, the royal python being larger than the snakes of our study.

In the early stage of SE, the subspectacular space progressively widened. Simultaneously, the superficial band of the cornea became thicker and hyperechoic, as seen by OCT, before returning to baseline just before elimination of the ‘old’ spectacle. We believe that the superficial band changes represent physiologic and anatomic alterations to the cornea and precursors to the new spectacle.

The corneal translucency demonstrated by macrophotography was present for the two first days of spectacular ecdysis and corresponded to the spectacular vascularization as observed by SLO. The vascular lumens were not distinguishable by OCT at any time. In recent studies, van Doorn demonstrated that spectacular blood flow is low and variable in resting and undisturbed coachwhip snakes (Masticophis flagellum, Colubridae), and becomes much increased and continuous during the integument renewal phase.[23, 30, 31] This is concordant with our observations. In the same reports, van Doorn has also highlighted that the visual perception of a threat induces a reduction in the proportion of time during which blood flow occurs in the spectacle[23, 30, 31] and that sympathetic innervation and thermoregulation may provide an explanation to the varying spectacular blood flow. He also suggests that constant spectacular blood flow during the integument renewal phase is necessary to support the cellular proliferation involved in the generation of a new stratum corneum.[23, 30, 31] However, van Doorn did not conclude whether the visualized vascular meshwork at the moment of SE was associated with the old or the new spectacle. As the observation of the spectacular vascularization was at its maximum in the early stage of the spectacular ecdysis, concurrent with translucency, we hypothesize that the vascularization is associated with the new spectacle. Vascularization of the old spectacle may cease prior to the beginning of ecdysis. With the increased metabolic activity of the superficial cornea necessary for the generation of the new spectacle, we believe that the vessels of the new spectacle are perfused for the first time. This transient vascular perfusion of the new spectacle might explain the accumulation of fluid between the old and the new, facilitating the progressive elimination of the former. This maximal spectacular blood flow correlates with the presence of translucency of the ocular surface. Once spectacular vascularization (as observed by SLO) subsides, transparency returns.

Spectacular ecdysis in Panterophis guttatus and Lampropeltis getulus californiae, both belonging to the Colubridae family, progressed with the same chronology and the same vascular meshwork orientation with vertically oriented vessels, as described by Mead.[22]

It should be noted again that our study included only four corn snakes (Pantherophis gutattus) and only one California king snake (Lampropeltis getulus californiae). While we may draw general conclusions from our study, the number of subjects from each species is limited and further studies are required to definitively describe SE for these two species.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References

SLO-OCT and macrophotographic imaging of the ocular surface during ecdysis suggests that spectacular vascularization plays an integral role in the process of SE. In our study, only two species were examined, both belonging to the Colubridae family. The dynamics of the process in other species remains to be determined.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
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