Anat Cell Biol 2022; 55(3): 311-319
Published online September 30, 2022
Copyright © Korean Association of ANATOMISTS.
1Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, 2Chulabhorn International College of Medicine, Thammasat University, Rangsit Campus, Pathumthani, 3Department of Physical Therapy, Walailak University, Nakhon Si Thammarat, Thailand
Correspondence to:Somluk Asuvapongpatana
Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
It has been reported that overconsumption of caffeine during pregnancy leads to a deleterious effect within the nervous tissues during embryonic development. In this study, we further extrapolated the effect of caffeine in the developing retinas, which is known to be one of the most sensitive tissues in chick embryos. Morphological changes of retinal thickness and organization of neuroretinal epithelium were monitored using three gene markers, Atoh7, FoxN4, and Lim1. Upon treating with a single dose of caffeine (15 µmol at embryonic day 1 [E1]), relative thicknesses of developing retinas (particularly of E7 and E9) were significantly altered. Among the three genes studied, the expression pattern of Atoh7 was notably altered while those of FoxN4, and Lim1 mRNA showed only a slight change in these developing retinas. Quantitative polymerase chain reaction results supported the most notable changes of Atoh7 but not FoxN4, and Lim1 gene in the developing retinas, particularly at E7. The effect of caffeine towards other organs during development should be extrapolated and the awareness of its intensive consumption should be raised.
Keywords: Caffeine, Chick embryo, Retina, Epithelium, Retinal ganglion cells
Retinogenesis is a complex event that is involved in cellular organization in the definitive period which has been well studied in vertebrates, including chick and mouse models. Based on the periods of differentiation, retina progenitor cells (RPCs) have been divided into 2 subgroups, early-born group (retinal ganglion cell [RGC], horizontal cell [HC], and amacrine cell [AC]) and late-born group (cone photoreceptor, bipolar cell, rod photoreceptor, and Müller cell). The molecular controls of retinogenesis have been extensively documented, and many well-characterized molecules have been anticipated as the universal biomarkers for the key steps of retinal development [1, 2]. Our focus in this study is on the early-born population and their markers since they are the targets for many teratogens.
One of the common teratogens is caffeine, a white crystalline xanthine alkaloid, which exerts both positive and negative effects on the central nervous system (CNS). Its beneficial effects on recovering signs and symptoms of the CNS disorders have been demonstrated in the cases of Parkinson’s disease, Alzheimer’s disease, and glaucoma [3-5]. Alternatively, caffeine consumption in pregnant women has raised concern as a result of its increasing evidence in its interruption of organogenesis during the early development of embryos. The adverse effect of caffeine has been reported in many highly suspectable organs including heart, skeleton, gonad, neural tissue; brain, spinal cord, and retina [6-12]. Due to the structural homology of caffein and adenosine nucleoside, caffeine is known to interact with adenosine receptors (ARs) which antagonistically modulates an inhibition of phosphodiesterase enzyme and the release of intracellular calcium ions through the G-protein and cyclic adenosine monophosphate (cAMP)-independent signaling cascade [3, 13]. Effect of caffeine during retinal development has also been reported to be involved in 2 types of ARs, AR1 and AR2A, expressed in the inner and outer plexiform layers (IPL and OPL) from E10 until E17 of chick retinas . The exposure to caffeine subsequently leads to down-regulation of both AR1 and AR2A in the developing retinas . Apart from ARs, there are also a number of caffeine-responsive receptors such as GABAA-, ryanodine-, glycine-, gustatory-, and dopaminergic- receptors that are found in
During early retinal development, the orchestrated molecular controls to synthesize cellular organization in developing retinas has been well documented [1, 2]. In this study, we focused on the 3 marker genes which are spaciotemporal expressed in the 3 population of neuroepithelial cells,
Fertilized chick eggs,
Administered chick embryos at E3–E9 were harvested by cracking the shell and dissected free from yolk matter. The eyes of embryos were carefully removed from the heads and fixed in 4% paraformaldehyde in 0.12 M phosphate buffer for either 6 hours (E3) or overnight (E5–E9) at 4°C. For paraffin embedding, the tissue was further dehydrated in the increasing percentage of ethanol, infiltrated in xylene, and finally embedded in paraffin. The 5 µm-thick sections were cut and routinely processed for hematoxylin and eosin staining. The images were acquired by a Nikon E600 light microscope (Nikon Instruments Inc., NY, USA) and Panoramic Digital slide Scanner (3D-Histech; 3DHISTECH Kft., Budapest, Hungary). They were further processed and analyzed by Adobe Photoshop CS6 software (Peachpit Press, Berkeley, CA, USA) to measure the retinal thickness. The measurement of retinal thickness was conducted at the retinal area which is opposite the mid-center point of the posterior lens surface. Statistical analysis was performed by paired
For cryo-sectioning, they were transferred into 10% sucrose in phosphate buffer overnight at 4°C and further submersed and embedded in gelatin-sucrose (14% gelatin, 0.1% agarose, 10% sucrose solution) and kept at –80°C. Sections were cut by a Leica RM2235 rotatory cryo-microtome at –20°C (Leica, Buffalo Grove, IL, USA) at a thickness of 25 µm and adhered onto Matsunami platinum microscopic slides (Matsunami Glass, Bellingham, WA, USA). All images were acquired, processed, and analyzed as mentioned above.
Riboprobes of 400 to 500 nucleotides were designed and cloned in pBluescript II SK+ plasmids (General Biosystem, Durham, NC, USA) for constructing
Caffeine treated- and controlled- chick retinas were isolated, and the retinal pigment epithelium and lens were carefully removed. Isolated retinas were then subjected to a total RNA extraction using a Trizol reagent (Life Technology, Carlsbad, CA, USA) and Direct-zol RNA Mini Prep (ZYMO Research, Tustin, CA, USA) and resuspended in a DNase/RNase-free water. Approximately, 1 µg of RNA was reversely transcribed by an iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, Hercules, CA, USA). Complementary DNA (cDNA) was amplified using a Luna Universal qPCR master mix (BioLabs, Ipswich, MA, USA) containing 10 µM primers (Table 1) and 0.5 µg of cDNA, in a total volume of 10 µl. Thermal cycle conditions were 95°C for 1 minute, 53.9°C for 30 seconds and 72°C for 30 seconds run at 44 cycles. The data were presented as mean±standard error of the mean. The differences between groups were analyzed by one-way ANOVA followed by the Bonferroni post-test by using a Graphpad Prism 9 software (Graphpad Inc.). A
The histological structures and their morphometric parameters (thickness and cellular organization) between caffeine-treated and control chick embryos (E3–E9) were compared and shown in Table 2. The measured thicknesses of retinas in both groups were 47.49±4.62 (n=8) vs. 62.18±10.53 for E3 (n=5); 70.85±9.78 (n=7) vs. 80.38±6.50 for E5 (n=6); 203.27±18.21 (n=5) vs. 140.09±9.24 for E7 (n=6); and 254.81±27.52 (n=9) vs. 215.30±23.83 for E9 (n=7). Interestingly, significant differences in the epithelial thickness were noted in E7 (
Among the three markers studied, expression of
Two other markers that were studied herein were
We also quantitated the expression levels of the three marker genes collected from E7 and E9 embryos (n=3 each) using real-time PCR analysis (Fig. 3). We had to confess that in our handling method, the retinas of E3 and E5 were too small to be collected and it was difficult obtaining enough RNA, therefore, only data of E7 and E9 were presented here. The results of qPCR well agreed with those of
During retinogenesis, many teratogens are known to cause a change of the retinal formation and maturation. Since caffeine is the most consumable bioactive alkaloid, its effects could potentiate both beneficial and adverse outcomes. It has been reported in human that caffeine can pass freely through the placenta and blood-brain barrier and it can potentially affect development by lowering body length and weight of newborn [30-33]. Similar effect has been proven in the chick embryo model in which exposure to caffeine at concentrations of 5, 10, and 15 µmol/egg at the early stage-embryos (the highest dose) directly disrupts the expression and function of
One of the caffeine-responsive receptors whose effect on many developing tissues have been most well studied is the AR which may be, due to the structural homolog between caffeine and adenosine molecules. In this regard, the effect of caffeine on developing retinas may not be out of expectation as many previous studies have reported that ARs were localized in the retinal of chicken, rabbit, mice, rats, monkeys, and human [15, 34-36]. However, it has also been reported that the expression of both AR receptors in the developing chick retinas starts to be observed at E12 to E15, the timing which does not well correspond with the expression of the three marker genes studied herein. Other receptors expressed during the early embryonic period and have been reported to have interfered with caffeine should thus be more considerable. As earlier mentioned, DR1A is expressed as early as E7 embryos, its interruption by caffeine antagonistic binding would interfere with developing genes or transcription factors expressed in this given period . The best example earlier mentioned is the expression of Pax6 transcriptional factor which is also known to be interrupted by caffeine treatment . In fact, Pax6 (as well as Sox2) are the upstream regulators of
This study was supported by a grant from the Development and Promotion of Science and Technology Talents Project (DPST), Thailand. We also thank the Center of Nanoimaging (CNI) and the Central Instrument Facility (CIF), Faculty of Science, Mahidol University, for providing instrumental support throughout this work.
Conceptualization: TL, WW, S Asuvapongpatana. Data acquisition: TL, AB, S Ahi. Data analysis or interpretation: TL, AB, NC. Drafting of the manuscript: TL, S Asuvapongpatana. Critical revision of the manuscript: WW, S Asuvapongpatana. Approval of the final version of the manuscript: all authors.
No potential conflict of interest relevant to this article was reported.