Neonatal and juvenile ocular development in Göttingen Minipigs

The use of juvenile animals in preclinical toxicity studies conducted for drug development has generated substantial interest over the last decade (11,15). Regulatory agencies, such as the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), are now considering the use of juvenile animals essential in order to perform a proper risk and safety assessment of new xenobiotics intended for the pediatric population (5,6).

It is known that children or immature animals do not always respond to xenobiotics in the same manner as adults do when it comes to drug efficacy and/or toxicity (1,3,4,8). The evaluation of tissue samples from juvenile animals brings additional challenges for pathologists involved in preclinical toxicity studies. Indeed, they must not only identify “standard” drug-related effects, but must also detect any developmental abnormality/delay and not misinterpret physiological developmental events as drug-related changes. Thus, an excellent understanding of the normal histology of developing organs in various species is crucial. Yet, as age-matched controls are not always available in juvenile toxicity studies based on the study design, pathologists often rely on published literature to find reference information on the normal histology of structures collected during the postnatal phase of development; however, such data is still limited and incomplete, particularly for Göttingen Minipigs. Comprehensive histological descriptions of immature tissues from juvenile animals become even more critical for organs composed of highly sophisticated and complex structures, such as the eye.

Study objectives and design

This study aimed to 1) characterize the normal postanal histomorphological ocular development in Göttingen Minipigs from birth until adulthood, 2) establish the age timepoints when each structure of the eye reaches histomorphological maturity, i.e. when the histology is similar to that of the adult mature eye, and 3) compare the histology of developing eyes from Göttingen Minipigs with the eyes of age-matched domestic pigs.

To conduct this study, 16 Göttingen Minipigs divided into groups based on age (postnatal day [PND] 1, 7, 14, 21 and 28; 2 months, 3 months and 6 months) were donated by Marshall BioResources, New York, USA. Twenty five (25) age-matched F2 domestic pigs were obtained via the Diagnostic Service of the Faculty of Veterinary Medicine of the Université de Montréal, Quebec, Canada. 

For all animals, a thorough histological evaluation of the eyes was performed by an American College of Veterinary Pathologists (ACVP) board certified pathologist using standard hematoxylin and eosin staining, and immunohistochemistry labeling was done to detect ki-67, caspase-3, GFAP, calbindin, synaptophysin and rhodopsin.

What did we learn from this study?

Despite the more advanced ocular developmental stage in neonatal Göttingen Minipigs compared to other commonly used laboratory animals such as rodents and dogs, (16,19) histomorphological signs of immaturity were observed in every structure of the eye of Göttingen Minipigs at birth, and the eyes continued to develop until 6 months of age. 

Examples of noteworthy histological and immunolabeling features highlighting the immaturity of the eye of Göttingen Minipigs at birth, and for variable time periods thereafter depending on the structures, are listed in the table below and illustrated in figure 1.

Furthermore, this study was the first to report histomorphological and immunolabeling pattern differences between the area centralis (or visual streak) region of the retina and other retinal regions, in juvenile and adult Göttingen Minipigs. Notably, this study has shown obvious variations in the distribution of a new subset of cone photoreceptors positive for calbindin between the different regions of the retina. 

Overall, when compared to the adult mature eye, the eye of Göttingen Minipigs was considered fully developed histologically at 6 months of age. The age timepoints when specific structures reached histomorphological maturity are included in figure 1. 

Readers are invited to refer to the published article of this research for the complete results and further details (18 ).

Is the postnatal ocular development in Göttingen Minipigs different from that of domestic pigs?

Histologically speaking, the eyes of adult Göttingen Minipigs and domestic pigs are very similar. This study reported few subtle histological and/or immunohistochemical variations between these 2 breeds suggesting that some structures, such as the cornea and lens, would be slightly more developed at birth in domestic pigs compared to Göttingen Minipigs. Nevertheless, every structure of the eye reached histomorphological maturity at the same age timepoints, in both pig breeds.

Are Göttingen Minipigs a good model for ocular (juvenile) research?

Minipigs are increasingly considered a relevant animal model for ocular research in the scientific field and for preclinical toxicity studies as they share several histological and anatomical similarities with the human eye (9,10,13,17 ). Most importantly, minipigs and domestic pigs are particularly relevant for retinal research as the swine retina contains a region called the area centralis (or visual streak) which mimics to some extent the macula in the human eye, a structure associated with sight-threatening conditions in people, such as macular degeneration, which are widely studied (2,7,17,18 ). The current study has taught us that the developmental stage of the eye of Göttingen Minipigs at birth, and particularly of the retina, more closely resembles that of neonatal human and non-human primates, compared to other commonly used laboratory animals, such as rodents and dogs, which are born with markedly underdeveloped eyes (12,14,16,18 ).

Take-home message

Overall, this study showed that the eyes of Göttingen Minipigs are not fully developed at birth and still undergo histological and immunohistological developmental changes until 6 months of life, when the eyes are considered histomorphologically mature. Compared to other commonly used non-primate laboratory animals, such as rodents and dogs, the developmental stage of the eyes of Göttingen Minipigs at birth more closely resemble that of neonatal human, making the minipig a promising model to study pediatric ocular diseases or for the development of ophthalmic drugs intended for use in children. 

ACKNOWLEDGEMENTS
We are thankful to Michelle Salerno and Nicole Navratil from Marshall BioResources, North Rose, New York, for donating all Göttingen minipigs and to the technical staff from Charles River Laboratories Montreal ULC for the eye collection and preparation of histological and immunohistochemical slides.

FUNDING
The study was supported through grants from Mitacs, the Fonds du Centenaire de la Faculté de médecine vétérinaire of the Université de Montréal, and from Charles River Laboratories Montreal ULC.

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