@2024 Afarand., IRAN

---

ISSN: 2252-0805    The Horizon of Medical Sciences 2014;20(2):101-107

Background
Cell reactions, leading to organ evolution during embryo evolution, play an important role in procedures relating to evolution, like cell differentiations, and they are effective on retina evolution as a specific nerve structure [1-7].
Previous Studies
There are some researches on the effects of lectins on ganglionic cells in the vertebrates [7-10].
Aim(s)
The aim of this study was to investigate the distribution of glycoconjugates terminal sugars and their changes on ganglionic cells surface of rat’s retina using histochemichal lectin technique during eye morphogenesis.
Research type
Non-declared
Research society, place and time
Two-month female Wistar rats with male Wistar rats were obtained from Animal Home of Mashhad University of Medical Sciences.
Sampling method and number
20 female and 10 male rats were studied.
Used devices&materials
The rats were kept in standard conditions of Animal Home with free access to food and water, 12-hour light/dark cycle, appropriate wet, and 18-24℃. After adaptation in especial cages, the rats were brought into intercourse (one male by two females); and providing vaginal smear, zero day of pregnancy was determined. The pregnant rats were anesthetized with chloroform at every 14th to 16th days of gestation, successively. Branches of rat uterus were removed with cesarean section, and immediately, the embryos were separated from the membrane in physiology serum and fixed in formalin solution in room temperature [11]. Passaged via common methods of histology, the samples were molded in paraffin blocks, and 5μm-thickness sections from each sample were serially prepared with Rotary Microtome 1512 (Leit; Germany) in both sagittal and coronal directions. To stain with lectin histochemistry methods, and using desired lectins (Sigma, Aldrich; USA), the sections were prepared (Table 1). At first stage, the histological sections were hydrated via routine histological method. At second stage, according to histological hydration routine methods, the sections were made transparent with xylenol and then, they were stuck. At every stage, at least three samples were stained with each lectin as ‘experiment group’, with one section, exposed to HRP, DAB, and hydrogen peroxide (no lectin), as ‘control’. In addition, as positive control and alongside each lectin at every embryo stage, one combined sample was stained. The slides were checked out with an ordinary light microscope; and Likert scale was used to evaluate staining intensity. Intensity of different reactions to the lectins was scored and graded as follows [12]: Zero (-), no reaction or negative reaction 1 (+), mild or weak reaction 2(++), mean reaction 3(+++), severe reaction Based on observations done by 3 persons, staining intensity was determined in Blind manner; and based on the reaction intensity to each lectin at different embryo days (14th to 16th days), the samples were separately classified.
Findings by Text
Since the retina and ganglionic cells were in different evolutionary stages at different embryo days and after, responds of the cells to the specific lectins showed a diverse spectrum of reaction intensity (Table 2). There was a mild reaction (+) in ganglionic cells of the retina in the stained tissue sections of 14th embryo day put by DBA lectin. However, there was no impressive reaction in the nerve fibers scattered throughout the retina (Fig. 1, A). No layer of the eyeball, especially from the retina, had reaction to DBA lectin, in the tissue sections obtained from 15th day embryo stained by DBA lectin (Fig. 1, B). There was no reaction to DBA lectin in 16th day embryo ganglionic cells (Table 2). The ganglionic cells of the retia in the tissue sections of 14th evolutionary day had mild reaction (+) to GSA1-B4 lectin. In addition, some connecting fibers of the ganglionic cells, expanded towards other retinal cells, responded to GSA1-B4 lectin; however, their reactions to cell body and combinations of the surfaces of the ganglionic cells were more severe (Fig. 1, C). 15th and 16th days embryo sections had no reaction to GSA1-B4 lectin (Table 2). 14th day, in contrast to 15th day, ganglionic cells of the retina put by MPA lectin had mild reaction (+), while the 15th day cells had the most severe reaction (+++). In addition, many nerve connecting branches of ganglionic cells had severe reaction to other retina layers with MPA (Fig. 1, A). 16th day ganglionic cells put by MPA lectin had mean intensity (++). Yet, there were severe scattered reactions among the nerve fibers in the retina cells (Fig. 2, A; Table 2). There was mild reaction (++) on the surface of ganglionic layer in 14th day tissue sections put by OFA lectin. In addition, nerve fibers out of ganglionic layer had mean reaction (Fig. 2, B). A comparison between 14th and 15th days tissue sections, put by OFA lectin, revealed an increase in the reaction intensity (+++) in ganglionic cells, as well as their nerve fibers, at 15th embryo day, than the used lectin did (Fig. 2, C). In 16th day ganglionic layer cells, there was mid reaction (+) to OFA lectin (Table 2).
Main comparison to the similar studies
… [13-16] The results confirmed many previous studies, showing the effect of sugar compounds on changes in the ganglionic cells of the retina during embryo evolution. In spite of the proved key roles of revealed sugars by the lectins [6, 17-19], they could not have any determined role in formation and differentiation of the desired cells at the said days. Maybe for the first time, the results show much more reaction intensity of the ganglionic cells to MPA lectin at 15th day, than similar samples at 14th and 16th days do.
Suggestions
Histochemical lectin studies can be the base of the researches on the retina diseases.
Limitations
The lack of accurate determination of different embryo stages was one of the limitations for the present study.
Conclusions
In differentiations of the retina ganglionic cells during eye evolution, the expression different pattern of glycoconjugate terminal sugars is temporally fully set. In addition, including fucose, α-D-Gal, β-D-Gal, and N-acetylgalactoseamin terminal sugars, the glycoconjugates of cell surface play an important role in the evolution of the retina ganglionic cells.
Acknowledgments
The researchers feel grateful to Ms. Mojadded, who provided laboratory services.
Conflict of interest
Non-declared
Ethical Permissions
The animals were kept according to National Institutions of Health (NIH) for human use of the laboratory animals.
Funding Sources
The presented results here are of an MSc thesis approved as research project and numbered as 911216. Research Deputy of Mashhad University of Medical Sciences founded the research.

TABLES and CHARTS

Show attach file