Limitations of our study may be
Limitations of our study may be mentioned. First, as for any experimental study, caution has to be applied when data measured and conclusions drawn are transferred on humans. Second, the question is unresolved, whether the actions of amphiregulin observed in this study were specific to amphiregulin, since the preparation of the antibody to amphiregulin occurred in goats by immunization with the mouse amphiregulin-precursor sequence (i.e. Ser94 à Lys191). The manufacture stated however that amphiregulin antibody neutralized the biological activity of amphiregulin and that the amino order clemastine fumarate sequences of mouse amphiregulin and guinea pig amphiregulin were similar. Future studies using guinea pigs may apply an amphiregulin antibody produced in guinea pigs. To strengthen the conclusion that the observed results were due to amphiregulin itself, future studies may therefore be performed applying an injection of non-immune goat IgG, at the same doses as those of anti-amphiregulin IgG, into eyes with lens-induced myopia to control for nonspecific effects of intravitreal goat IgG. Other studies may examine a potential preabsorption of the antibody with the amphiregulin peptide at concentrations in the 10–10M range to determine whether this may abolish the immunolabeling of the amphiregulin-like material in ciliary epithelium, retina and RPE. It may control for the specificity of the antibody binding to endogenous amphiregulin. Third, when assessing the tissue dimensions in the histological sections, one has to take into account that dehydration, formaline treatment and paraffin embedding might have altered the volume of the cells and extracellular matrix during the preparation of the histologic sections. Frozen-sectioning or embedding in an aqueous medium would have given tissue measurements closer to the real, in vivo, measurements. Fourth, the blockade of amphiregulin by its antibody resulted in a significant decrease in further axial elongation. The intraocular application of amphiregulin, however, did not result in an increased axial elongation or myopization. The reason for the discrepancy may be that for the induction of axial elongation more than one factor may be necessary while the blockade of one of them, i.e., amphiregulin, was sufficient to reduce the process of axial elongation, or that a higher dose of amphiregulin or a higher frequency of its applications or a longer follow-up might have bene necessary to detect an axial length elongating effect of amphiregulin applied intraocularly. One may also discuss that a blockade of amphiregulin by the antibody affected only one amphiregulin-specific pathway, whereas amphiregulin itself could have affected multiple pathways (e.g., by penetrating through barriers that excluded the antibody). These aspects may be addressed in future studies. It could open up the possibility of a medical prevention of hyperopia. Fifth, instead of the use of Ringer\'s solution for intraocular application in the eyes of the control groups, one might have used an irrelevant antibody. We chose Ringer\'s solution since it was assumed that Ringer\'s solution had least likely an effect on the intraocular structures, while any, even irrelevant, protein might have changed the intraocular homeostasis. Correspondingly, the immunohistochemistry revealed that labelling for amphiregulin in the control groups with intraocular injection of Ringer\'s solution did not markedly differ from the labelling for amphiregulin in the groups without any intraocular injection (Fig. 4). It may suggest that the intraocular injection with Ringer\'s solution as procedure as such did not have a major influence on the findings obtained in the study. The strengths of the study are its novelty with investigating the role of amphiregulin in an environmental animal model of myopia, the application of biometry before and after experimental manipulations allowing the assessment of axial length changes over time, and the inclusion of a range of experimental groups including groups without intraocular injections, groups with intraocular injection of Ringer\'s solution, and groups with intraocular injection of amphiregulin antibody and with intraocular injection of amphiregulin. Sixth, at start of the study the guinea pigs had an age of 2–3weeks and the investigation was conducted over a period about three times longer than the age of the guinea pigs at study baseline. Although one group of animals did not undergo lens-induced myopization or any other intervention and served as untouched control group, there was the possibility that the natural process of emmetropization physiologically undergoing in guinea pigs of that age might have mingled with the process of lens-induced myopization additionally influenced by the intraocular application of amphiregulin antibody or amphiregulin.