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Keywords:

  • anophthalmia;
  • eye;
  • microphthalmia;
  • python;
  • unilateral

Abstract

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

Objective

To provide morphological descriptions of microphthalmia or anophthalmia in eight pythons using microcomputerized tomography (μCT), magnetic resonance imaging (MRI), and histopathology.

Animals studied

Seven Burmese pythons (Python bivittatus) and one ball python (P. regius) with clinically normal right eyes and an abnormal or missing left eye.

Procedure

At the time of euthanasia, four of the eight snakes underwent necropsy. Hereafter, the heads of two Burmese pythons and one ball python were examined using μCT, and another Burmese python was subjected to MRI. Following these procedures, the heads of these four pythons along with the heads of an additional three Burmese pythons were prepared for histology.

Results

All eight snakes had left ocular openings seen as dermal invaginations between 0.2 and 2.0 mm in diameter. They also had varying degrees of malformations of the orbital bones and a limited presence of nervous, glandular, and muscle tissue in the posterior orbit. Two individuals had small but identifiable eyes. Furthermore, remnants of the pigmented embryonic framework of the hyaloid vessels were found in the anophthalmic snakes. Necropsies revealed no other macroscopic anomalies.

Conclusions

Eight pythons with unilateral left-sided microphthalmia or anophthalmia had one normal eye and a left orbit with malformed or incompletely developed ocular structures along with remnants of fetal structures. These cases lend further information to a condition that is often seen in snakes, but infrequently described.


Introduction

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

Although congenital malformations are common in snakes where cranial abnormalities often include shortening of the upper jaw, cleft palate as well as defects of the eyes,[1] very little is known about developmental ophthalmic conditions in snakes.[2, 3] Both microphthalmia where one or both eyes are smaller than normal and anophthalmia where one or both eyes are missing[4] are reported conditions in snakes and may be either unilateral or bilateral.[3, 5] The etiology of microphthalmia or anophthalmia is unknown, but is thought to be of either a genetic or an environmental nature.[1, 6]

Pythons are oviparous snakes that lay their eggs 3–4 months after mating. At the time of ovipositioning, the embryos are well into organogenesis and the eyes are visible.[7] Females gather the eggs into a pyramid-shaped pile and coil themselves around the eggs to provide protection and to ensure an incubation temperature of 30–32 °C by periodic contraction of body muscles.[8] Temperature is even more critical for embryonic development in pythons than other snakes, and slight drops in temperature can result in poor or abnormal development.[8, 9] The embryos will die at 23 °C.[8, 9]

The aim of this study is to provide a detailed morphological account of microphthalmia or anophthalmia in seven Burmese pythons (Python bivittatus) and one ball python (P. regius) with the aid of microcomputerized tomography (μCT), magnetic resonance imaging (MRI), and histopathology. These findings will add details to a condition that has been reported by several authors, yet remains poorly understood.

Materials and Methods

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

Seven Burmese pythons and one ball python purchased from the same dealer had either a clinically abnormal or a missing eye. All came from large, artificially incubated clutches with many clinically normal siblings, and there was no indication that the malformation influenced their health as they fed normally and exhibited normal behavior. In the group of seven Burmese pythons, there were two sets of three siblings. Three of the Burmese pythons were albinos, and all others had normal coloring. The ball python was a male measuring 86 cm, estimated to be 8 months old. The length of each Burmese python was around 100 cm, indicating an age of approximately 1 year. Two of the Burmese pythons were male, one was female, and the gender of the remaining was not noted. The snakes were anaesthetized with sevoflurane and euthanized with an overdose of pentobarbital. The head, and especially the eye region, was examined visually, and the eye opening measured with a slide caliper. Thereafter, the heads were fixed in 4% phosphate-buffered formaldehyde, and the bodies of four individuals underwent necropsy. After 24 h of formaldehyde fixation, the snake heads underwent preparation for various examination techniques: μCT (n = 3 snakes), MRI (n = 1 snake), and histologic examination (n = 7). One of the albino Burmese pythons was accidentally frozen following euthanasia and was considered unsuitable for histological analysis.

μCT

Traditionally, μCT is a valuable technique in imaging calcified structures; however, it provides only little information about different soft tissue structures in a sample due to the narrow range of X-ray attenuation of noncalcified tissues. Nevertheless, by staining soft tissue structures with heavier elements such as I, Hg, Os, and Ag that are assimilated to varying degrees in soft tissues, μCT can be applied for high-resolution visualization of soft tissues.[10] In the present study, we applied an iodine-based staining protocol to reveal soft tissue structures.[11] Samples were washed for 1 week in phosphate buffer to remove any residual formaldehyde, thereafter immersed for two weeks in diluted Lugol's solution (0.33% I2 and 0.67% KI). Specimens underwent μCT before and after staining with Lugol's solution. μCT was performed on two different systems: a Scanco Medical XtremeCT and a Scanco Medical μCT-35 (Brüttisellen, Switzerland). Imaging parameters of the XtremeCT system were as follows: 63.63 × 28.54 mm2 field of view; 776 × 348 image resolution; 0.082 mm slice thickness; 59.4 kVp tube voltage; 119 μAs tube current; and 41-μm pixel pitch, resulting in an acquisition time of 35 min. Higher resolution was acquired (15 μm3) using the μCT-35 system with parameters: 21.04 × 23.56 mm2 field of view; 1403 × 1571 image resolution; 0.015 mm slice thickness; 55 kVp tube voltage; 116 μA tube current; and 24-μm pixel pitch, resulting in an acquisition time of 10 h. The XtremeCT system was used on the heads of two Burmese pythons, and due to limited availability, the μCT-35 system was used only on the head of the ball python. Except from a slightly lower image resolution produced by the XtremeCT system vs. the μCT-35 system, no differences in imaging results are to be expected from the two systems with nearly identical X-ray sources (59.4 vs. 55 kVp). Images of interrelated samples were registered, and 3D representations were generated using the commercially available OsiriX software (Pixmeo, Bernex, Switzerland).

MRI

MRI was performed on a 16.7 Tesla Bruker BioSpin (Ettlingen, Germany) system. Images were acquired with a T1-weighted 3D-Flash protocol with the following parameters: 20.19 × 18.11 mm2 field of view; 512 × 400 image resolution; 0.094 mm slice thickness; 10 ms echo time; 22.293 ms repetition time; 15° flip angle; and 16 averages, resulting in an acquisition time of 59 h. MRI was performed on the head of one Burmese python.

Histology

Following the imaging procedures, seven of the eight heads were decalcified with either a solution of formic acid (85%), formaldehyde, and water (0.2:0.088:0.71) for four to six weeks or Decalc decalcifying fluid (Histolab, Västra Frölunda, Sweden) for three to four days before being prepared routinely for histology. The heads were embedded in paraffin and serially sectioned sagittally at intervals of 200 μm at the level of the normal eye to facilitate comparison with the contralateral eye. Sections of 4 μm thickness were stained with hematoxylin and eosin (HE).

Results

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

All eight snakes had one normal eye and one abnormal eye. In all individuals, the abnormal eye was on the left side (Fig. 1). The head shapes were otherwise symmetrical, and necropsies did not reveal further macroscopic abnormalities. Table 1 summarizes the findings in the examined snakes. Snakes subsequently determined to be anophthalmic or microphthalmic could not be differentiated clinically; however, data are organized and presented here according to their final diagnosis.

Table 1. Summary of studied snakes, examination methods and histological findings in the left ocular region of eight pythons with an abnormal or missing left eye. Snake ID refers to images in Fig. 1
Snake ID (n = 8)SpeciesSkin colorExamination methodTissues detected histologicallyDiagnosis
BoneNerveMuscleGlandPCTRet.Lens
  1. Bone = malformation of the orbital bones; PCT = pigmented connective tissue in the posterior orbit; Ret.  = rudiments of the pigmented retina; + = present; − = absent; A = anophthalmia; M = microphthalmia; N/A = diagnosis unavailable.

aBall pythonNormalµCT++++++A
bBurmese pythonNormalNecropsy++++++A
cBurmese pythonAlbinoNecropsy++++++M
dBurmese pythonNormalNecropsy+++++A
eBurmese pythonNormalNecropsy and MRI++++++A
fBurmese pythonAlbinoµCT++++++M
gBurmese pythonNormalµCT++++++A
hBurmese pythonAlbino Histology not performedN/A
image

Figure 1. Macroscopic images of eight snakes examined in this study. Note that all malformed eyes are on the left side of the head. Image (a) is a ball python, images (b–h) are Burmese pythons. Macroscopically, it is not possible to evaluate whether the snakes were microphthalmic or anophthalmic. Image letters are aligned with data in Table 1.

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Microphthalmia

In the two albino Burmese pythons with microphthalmia, periocular scales surrounded an eye opening on the left side of 1.5–2 mm in diameter which was smaller than on the right side. The opening revealed only a seemingly opaque structure, possibly the spectacle or other skin structure, and it was not possible to grossly evaluate whether a globe was present. μCT revealed slight malformation of the prefrontal, frontal, and postorbital bones on the left side of the head (Fig. 2a,b). The orbital opening was reduced in size compared with the right side.

image

Figure 2. A microphthalmic Burmese python. (a) μCT image showing the normal appearance of the postorbital (po), frontal (fr), and prefrontal (pf) bones on the right side of the head. (b) μCT image showing malformations of the orbital bones on the left side of the head. The left orbit is smaller than normal. (c) Photomicrograph of the left orbit displaying a smaller than normal globe with a lens (L) with a disrupted capsule and extracapsular lens material. In the retrobulbar space is muscle (M), nerve (N), and glandular tissue (HG). Hematoxylin–eosin. Bar = 1 mm.

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Histology showed that the left globes were visibly smaller than the right eyes with the diameters measuring 3 and 4 mm, respectively, in comparison with 5.5 and 5 mm of the right eyes. All ocular structures were identifiable in the specimens, including the lens. In one of the snakes, the lens was half the size of the normal lens, and in the other case, the lens was about two-thirds of the normal lens and had a disrupted lens capsule with extracapsular lens material (Fig. 2c), which included Morgagnian globules. No perilenticular inflammation was present. In one of the two microphthalmic snakes, a layer of muscle interposed the orbital bone structures and the skin, while the other individual had no muscle or connective tissue between the malformed bone and the skin. Apart from the eye, the left orbits contained muscle, nerve, glandular tissue, and connective tissue. Optic nerve tissue was seen in the posterior orbit and was surrounded by muscle tissue which in turn was enveloped by connective tissue that lined the bony orbit. Glandular tissue was less abundant than on the normal side.

Anophthalmia

Five snakes were classified as anophthalmic. One of them had periocular scales, whereas the remaining individuals had scales in the left eye area that surrounded a very small opening of 0.2–0.3 mm in diameter. It was not possible to macroscopically identify any spectacle or ocular structure through this opening. μCT showed a very small left orbit, and the cranial skeleton was maldeveloped to such an extent that the orbital opening was minute (Fig. 3a). MRI revealed a circular structure in place of the left globe (Fig. 3b). It was not possible to identify the nature of this structure from the MRI images. On the right side, all normal ocular structures were identifiable.

image

Figure 3. An anophthalmic Burmese python. (a) μCT image showing malformations of the cranial periocular bones. (b) Cross-sectional MRI at the level of the eye. A circular area in place of the left globe was identified as rudiments of the pigmented retina (arrow). This area is surrounded by connective tissue (CT). On the right side, the globe (G) containing a lens (L) is clearly visible. The Harderian gland (HG), muscle (M), and nerve (N) tissues are also identifiable in the retrobulbar space. (c) Photomicrograph of the left orbit. Pigmented retina (arrow) in place of the left globe. Pigmented connective tissue (CT) surrounding nerve (N) and muscle tissue (M). Hematoxylin–eosin. Bar = 200 μm.

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In three of the five anophthalmic snakes, histology showed a layer of muscle between the orbital bone structures and the skin, while one snake had a thick layer of connective tissue underneath the integument. In one snake, the skin lay directly over the prefrontal, frontal, and postorbital bones with no presence of muscle or connective tissue. The left orbits displayed muscle, nerve, glandular tissue, and connective tissue as described in the microphthalmic snakes. The connective tissue that lined the bony orbit was pigmented and contained occasional blood vessels (Fig. 3c). The pigment of this connective tissue was black and finely granulated. Glandular tissue was less abundant than on the normal side. One snake lacked Harderian gland tissue, but had nerve, muscle, and connective tissue. In place of the left globe, these five snakes displayed a circular pigmented structure. The cells of this structure contained many large brown melanin granules.

Discussion

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

Although the eight snakes in this study showed varying degrees of ocular malformation, they consistently displayed certain features, such as abnormal bone formation and presence of optic nerve and muscle tissue. All snakes but two also had Harderian gland tissue. The five anophthalmic snakes displayed a circular area of pigmentation with an appearance similar to the pigmented epithelium of the retina of the normal eye. This was seen as rudiments of the retina. Furthermore, they had connective tissue in the posterior of the orbit with pigment that resembled the melanophores of the dermal layer of the skin. The eyes of the albino snakes were more developed than the colored snakes and lacked connective tissue in the orbit. Albinism can be caused by any one of several different mutations, and the overall color of the skin may vary.[12] It is not known whether albino appearance may be correlated with ocular disorders; however, due to the fact that albino appearance is seen more often than monocular snakes, we consider it incidental that the microphthalmic snakes were albinos and the anophthalmic snakes normally colored.

The formation of the eye takes place in early organogenesis, at around two weeks in the dog and four weeks in humans,[4, 13] where the optic vesicle extends toward the surface ectoderm.[4, 13, 14] Upon contact with the optic vesicle, the surface ectoderm thickens and forms the lens placode.[4, 13, 14] This contact between the optic vesicle and the surface ectoderm serves as an induction for the optic vesicle to start invaginating and forming the double-layered optic cup.[4] The lens placode subsequently invaginates and eventually embeds in the cavity of the optic cup.[4, 13, 14]

In the early embryological stages of snakes, pigmented connective tissue enters the optic cup fissure together with the hyaloid vessels and optic nerve.[15, 16] This tissue regresses as the snake grows and is often absent in adults.[16] The pigmented connective tissue that surrounded the nerve and muscle in the orbit of the examined snakes is thought to be part of this framework, suggesting an early arrest of embryological development. Following the initial development of the globe, once the python egg has been laid, the skull bones become ossified[7, 17] and the eyelids form and fuse over the eye to form the spectacle.[7, 17] In the cases investigated, it was not always possible to macroscopically identify the spectacle, especially in snakes with very small eye openings. All snakes, however, had an invagination of the skin, demonstrated as a hole between the scales in the eye area.

Microphthalmia in humans refers to a normal but small eye (International Clearinghouse for Birth Defects, http://www.icbdsr.org/page.asp?n=WebGuide#Anophthalmos/microphthalmos:). Microphthalmia in animals may also occur in eyes with multiple ocular anomalies.[4] The two snakes with lenses also had the most developed eyes, albeit malformed. A globe with adnexal structures was present but smaller and less developed than on the normal side. In one snake, the lens capsule was disrupted and lens material had leaked. It was not possible to determine whether the left eyes of these two snakes would have been functional. However, these two snakes were classified as microphthalmic, whereas the other individuals examined by histology were anophthalmic due to lack of discernible globe.

All the examined snakes displayed some degree of eye formation, indicating that during embryonic development, the optic vesicle had contact with the surface ectoderm and induced the creation of the optic cup and the lens. Ofri[4] suggested that microphthalmia or anophthalmia is caused by suppression of the optic primordial during the development of the forebrain, occurring either by abnormal development of the forebrain or by degeneration of the optic vesicles after they have already formed as a result of teratogenic insult. Interestingly, all individuals in the present study were affected on the left side. Likewise, in a previous case series,[18] three congenitally monocular Burmese pythons and one amethystine python (Morelia amethystinus) also were all reported to have one normal right eye and an abnormal left eye. A different article by the same authors[19] describes three monocular Burmese pythons as each having a normal left eye and an abnormal right eye according to the text of their publication; however, in both publications,[18, 19] the figures are identical and show the abnormal eye to be on the left side and are thus in contradiction with the text of the second study.[18] We can only speculate as to whether the location of the abnormal eye on the left side of all the studied snakes is of importance or merely a coincidence. In future studies involving monocular snakes, it would be relevant to note the affected side.

In humans, microphthalmia and anophthalmia have very varied and complex etiologies with identified chromosomal, monogenic, and environmental causes such as gestational infections, maternal vitamin A deficiency, and exposure to radiographic radiation.[20, 21] In two-thirds of all cases, the condition occurs without associated malformations, whereas in the remaining third of the cases, microphthalmia or anophthalmia occurs concomitantly with anomalies such as blocked esophagus, tracheoesophageal fistula, genital abnormalities in males, CNS malformations, and seizures.[20-22] The Pax6 gene plays a significant role in the development of the human eye and brain and regulates the expression of Sox2 and Otx2 genes, both identified as major causative genes of microphthalmia or anophthalmia.[20-22] The genes that regulate reptilian eye development remain to be characterized.

The exact etiologies of microphthalmia and anophthalmia are difficult to ascertain in snakes,[1, 23] but are typically ascribed to environmental influences, such as gestational conditions and abnormal incubation.[6, 23, 24] In oviparous pythons, the eyes are developed before the eggs are laid,[7] indicating an early gestational rather than incubational condition.

All examined heads were fixed in formalin prior to examination. Formalin causes shrinkage of tissues; however, as this was a common procedure for all, it was possible to compare the individual snakes. μCT gave an excellent view of the malformation of the cranial bones but did not provide much information of the structures within the orbit. MRI displayed the soft tissue structures, but histology was needed to identify the structures in detail.

In summary, this examination included eight pythons with an abnormal or missing left eye. μCT (n = 3), MRI (n = 1), and histology (n = 7) were used to examine the snakes. The microphthalmic snakes had smaller than normal ocular structures on the left side, whereas the left orbits of the anophthalmic snakes had incompletely developed eyes and remnants of fetal structures indicating a gestational etiology. To our knowledge, this is the first specific description of this unusual feature.

Acknowledgments

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

The research project was partly funded by the Ministry of Science, Technology and Innovation, Grant Number 10-091595. The Center for Zoo and Wild Animal Health is supported by the Alfred Benzon Foundation. The authors appreciate the assistance of animal care technician Heidi Meldgaard Jensen and biologist Rasmus Buchanan for care of the animals and the technicians of the Eye Pathology Institute for preparing the specimens.

References

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