Project description: 
Background and aims:
Aim of the project is the development of an alternative method for the Draize rabbit eye irritation test on the basis of artificial human cornea equivalents.

Although different ex vivo models like the BCOP-test, isolated eye tests or the HET-CAM assay, are accepted as alternative test methods in some EU member states, none of these methods meets all the requirements to fully replace the Draize rabbit eye test.

It has been shown that artificial corneas (reconstructed epithelia or whole corneal constructs) are proper models for the prediction of eye irritation and it has often been supposed that these models offer the potential to fully replace the Draize eye irritation test. However, up to now, these techniques do not cover the range for the prediction of mild and moderate irritancy. Therefore valid methods for the prediction of less severe chemical irritants are still outstanding– To approach this issue, we intend to profile Cytokine production in whole corneal constructs.

Interactions between epithelial stromal and endothelial cells maintain a healthy cornea and its ability to respond to an insult. Following chemical insults, we are going to assess cytokine production, as an early indication of disturbed intercellular communication.

Subject and goals of the project
Aim of the project is to evaluate organotypic, three-dimensional (3-dim) human corneal equivalents as tools for eye irritation testing. Up to now, alternative methods to the Draize rabbit eye test do not cover the range for the prediction of mild/moderate irritancy and reversibility of corneal injury. To approach this issue, we intend to profile Cytokine production in 3-dim corneal constructs. Thereby we hope to develop an alternative method which offers the potential to fully replace the Draize eye irritation test.

Methods
Immortalized human corneal epithelial cell models are built and stratified at the air-liquid interface in a fully defined medium. Models are also built with the epithelium stratified on top of a collagen gel seeded with immortalized human keratocytes and endothelial cells. The stratified constructs are treated with reference substances selected according to ICCVAM-proposed reference substances (for Validation Studies of In Vitro Test Methods for the Identification of Ocular Irritants). Concentration dependent effects on cellular viability are evaluated by MTT. Cytokine profiles of the supernatant medium and cell lysates are assessed qualitatively by Cytokine Antibody Arrays and quantitative by ELISA techniques. Re-evaluation of array data is performed using RT-PCR to compare the gene and protein expression patterns in the presence and absence of different chemical substances. 

Laboratory/Institute: Animal Welfare Academy (Akademie für Tierschutz), Neubiberg, Germany

Laboratory and scientific management:     Dr. Michaela Zorn-Kruppa

Scientific staff:                                                Dipl.-Biol. Heike Scholz

Address of applicant:
Dr. Brigitte Rusche
Animal Welfare Academy (Akademie für Tierschutz)
Spechtstrasse 1
85579 Neubiberg, Germany
Brigitte.Rusche(ät)tierschutzakademie(dot)de