Gross, Microscopic and Ultra structural Pathology of Ocular Abnormalities in Farmed Halibut

This report documents the gross pathological appearance, histopathology and ultra-structural studies of the ocular abnormalities previously reported in farmed Atlantic halibut (Hippoglossus hippoglossus). 37% of eyes investigated were normal on gross and histopathological examination while 32% showed formation of an intraocular cyst in the choroid resulting in anterior movement of the retina. 27% showed scleral ectasia in which the posterior portion of the globe was enlarged, either generally or in a focal ectatic focus. In a small number of eyes the cystic posterior segment lesions contained fi brous and infl ammatory tissue but most affected eyes showed cystic structures fi lled with gas or fl uid. Although morphology was somewhat compromised in the majority of samples, histopathological and electron microscopic ultrastructural examination showed an endothelial lining to these posterior segment cysts suggesting that they have developed following gas bubble formation in vessels of the choroidal rete. This fi nding concurs with our previous research showing increased intraocular oxygen concentrations in the eyes of affected fi sh and suggests that the pathology seen here is predominantly associated with the formation of oxygen bubbles in the countercurrent multiplier system of the choroidal rete. Research Article Gross, Microscopic and Ultra structural Pathology of Ocular Abnormalities in Farmed Halibut David L Williams1* and W Mary Brancker2 1Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, UK 238 Streetley Lane, Sutton Coldfi eld, UK Dates: Received: 13 December, 2017; Accepted: 11 January, 2018; Published: 13 January, 2018 *Corresponding author: David L Williams, MA, Vet, MD, Ph.D, CertVOphthal FRCVS, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, UK, E-mail:


Introduction
We have previously reported ocular abnormalities in farmed halibut [1], and documented studies showing an association between the gas-fi lled intraocular and retrobulbar cysts characteristic of ophthalmic disease in these fi sh and increased choroidal carbonic anhydrase activity [2], high intraocular oxygen tensions [3], and aggravating factors such as handling trauma [4]. Here we report pathological fi ndings in the eyes of these fi sh at a gross, histological and ultra-structural level, providing further evidence showing that the majority of ocular lesions in these fi sh are associated with choroidal gas bubble formation.

Animals
Halibut from a group of broodstock at the Marine Farming Unit, Ardtoe, examined and reported previously [1], were culled to provide a sample of fi sh with normal eyes, exophthalmic eyes, eyes with peribulbar cysts and eyes with intraocular infl ammatory change. While not fully random in nature the selection of eyes taken gave numbers of globes refl ecting the proportions of ocular lesions in the broodstock population. Fish were euthanased by rapid decapitation followed by pithing.
Eyes were removed and fi xed in Davison's solution (glacial acetic acid, ethyl alcohol, formol saline) for gross pathological examination and histopathology. A small proportion of these eyes were post-fi xed in 2.5% gluteraldehyde in cacodylate buffer for electron microscopy.
Ongrowing fi sh maintained in commercial aquaculture setting by Marine Harvest McConnell were randomly culled to investigate ocular and systemic pathology. Fish were culled by a blow to the back of the head avoiding ocular damage. Eyes were removed and fi xed in 10% formol saline.
Eyes were bisected and the cut surface photographed using a standard technique with the specimen submerged as previously reported [5]. Histological processing of globe calottes to wax was performed in a routine manner and 6 micron sections stained with haematoxylin and eosin, again in a routine manner. Processing for electron microscopy involved refi xation of small samples of normal and affected choroidal gland with 4% gluteraldehyde in 0.13M phosphate buffered saline, staining with 1% osmium tetroxide in 0.13M phosphate buffered saline, dehydration in acetone, immersion in 10% hexamethyldisilazine and fi nally air-drying in a dessicator when mounted on aluminium stubs using epoxy araldyte gel. Samples were gold coated using a Polaron E5105 splutter coater and then viewed in a Hitachi S2300 scanning electron 001-005. DOI: http://doi.org/10.17352/2455-8400.000034 microscope. Processing of sections taken from samples previously evaluated by scanning electron microscopy involved immersion in propylene oxide transferred to a 50:50 mixture of propylene oxide and then embedded in TAAB resin with dodecenyl succinic anhydride and methyl nadic anhydride as hardener as previously described [6]. 80nm sections were cut on a Reichert ultratome using a Diatome diamond knife and double stained with aqueous 3% uranyl acetate and lead citrate and viewed on a Hitachi H607 transmission electron microscope.
The study was reviewed and accepted by the ethics and welfare committee of the department of veterinary medicine, university of Cambridge.

Results
Eighty two eyes from 41 broodstock halibut were removed post-mortem, fi xed and examined grossly. Histopathological sections were taken from 20 representative eyes and in four of these, higher power ultrastructural investigations were undertaken with scanning and transmission electron microscopy. Eighty eyes from 40 ongrowing halibut of ages from 4 months to one year were similarly removed, fi xed and subjected to gross and histopathological examination.

Gross pathology
Thirty of the 82 broodstock eyes (37%) were normal on gross examination or had intraocular changes such as vitreal traction bands and debris or anterior segment debris which were considered likely to be agonal or post-mortem change. Twenty six eyes (32%) showed formation of posterior segment cystic structures whereby the retina was displaced anteriorly by a gas-fi lled cyst. Twenty two eyes (27%) conversely were affected by scleral ectasia, where the posterior segment gasfi lled cyst had caused posterior expansion of the sclera either over a wide expanse of the posterior pole or as an extrusion over a more focal area of posterior sclera. One hundred and three eyes from on growing fi sh were apparently normal on gross inspection. The three remaining eyes showed posterior segment disruption which may be attributed to trauma or gasbubble-associated pathology as seen in broodstock eyes, but distinguishing between these two aetiopathogenic possibilities was not possible on gross examination alone.

Histopathology
The choroidal rete seen in the eyes considered normal on gross examination consisted of numerous tightly packed blood vessels of similar if not identical diameter, positioned near the posterior pole of the eye (Figures 8,9). In some older broodstock with eyes which appeared normal on gross inspection there was a degree of fi brosis between some choroidal rete vessels, this also being evident at the boundaries of the rete structure in some fi sh (Figure 10).
In broodstock with cystic changes in the posterior segment and also in the smaller number of ongrowing fi sh with similar       though less pronounced signs, the vessels were dramatically distended ( Figure 11). Although after histological processing it was impossible to determine whether these were fi lled with gas or fl uid, the fi nding that many of these eyes fl oated, as noted above, suggested that the former was the case.
Ongrowing halibut were rarely noted to have the ocular pathology common in broodstock, but in three out of the 102 fi sh examined histopathologically, choroidal vessel enlargement was noted, postulated here to be related to choroidal gas bubble formation.
Cysts were on occasion evidently lined with endothelium, although often this was not easy to identify in histological sections. More often it was the endothelial basement membrane which was evident and cellular detail was impossible to evaluate in these histological sections. Between cysts fi brous septae fi lled the intervening space in the majority of cases (Figure 11).
In the three cases noted above in which the posterior segment was tissue-fi lled, the cystic spaces were occupied by dense infl ammatory tissue ( Figure 12).

Scanning electron microscopy
Scanning electron microscopy of the normal choroidal rete showed a matrix of connective tissue septae which, although vessel wall detail had often been lost in fi xation and processing, were taken, in conjunction with the histological fi ndings, to indicate a large number of blood vessels arranged in a tightly packed manner (Figure 13), with equally sized vessels forming the centre of the rete while large vessels occupied the periphery.
Scanning electron microscopy of the pathological cystic structures in abnormal eyes suggested them to be contiguous with the smaller blood vessels of the choroidal rete ( Figure   13), formed as a pathological expansion of one vessel. The connective tissue septae surrounding the cyst appeared to be compressed together but neighbouring blood vessel lumina are still evident.

Transmission electron microscopy
Preservation of ultrastructural detail was poor in the samples subjected to transmission electron microscopy processing but suffi cient morphological characteristics were present to demonstrate that endothelial cells were present lining both the blood vessels of the choroidal rete (Figure 14), and the choroidal cysts (Figure 15). Nuclear and cytoplasmic detail was very sparse but suffi cient to indicate the presence of fl attened cells lining the blood vessels and cysts. Endothelial cells lining the retial blood vessels appeared more plump than the more squamiform cells lining the cyst wall.

Discussion
The choroids of many species of fi sh is characterized by a vascular rete acting as a countercurrent system [7], through which high oxygen tensions are produced to supply the retina with suffi cient oxygen to satisfy its high metablic rate in deep water environments where otherwise relatively hypoxic conditions apply [8]. At substantial depths this oxygen remains in solution, but in shallow tanks and with additional factors such as handling trauma, the gas can come out of solution as gas bubbles [4]. Our hypothesis is that the majority of ocular lesions noted in these fi sh are the result of gas bubble formation in the vessels of the choroidal rete with subsequent alteration of intraocular contents such as cystic change in the posterior segment, anterior displacement of the retina, generation of gross infl ammatory or fi brotic change within the posterior segment, globe enlargement and/or focal scleral ectasia.
If the origin of these cystic structures is indeed gas bubble formation in the vascular rete of the choroidal body in these fi sh, the position of the cystic lesions should be in the posterior     At a histological level it was often diffi cult to ascertain whether the lining of the cysts was the same as that of the blood vessels of the choroidal rete. Ultrastructurally the evaluation of similarity between the retial vessel wall and the cyst wall was complicated by the poor preservation of the tissue. Fixation and processing artefacts may be responsible for the poor preservation of cells in the cyst wall, since the time to full fi xation of cells liing a gas-fi lled space within an eye immersed in fi xative is necessarily slow. Conceivably it may be that cell death was caused by gas pressure or oxygen toxicity, resulting in the loss of endothelial cell viability.
Even given these problems, there is suffi cient evidence from gross and histopathological examination and from both scannng and electron microscopy to indicate that the cysts are formed from dilation of gas-bubble-induced choroidal vessels and, we propose, to formulate a pathogenic mechanism by which gas bubble formation in the retial vasculature results in vessel enlargement and subsequent cyst formation.