Design

Parallel study of cytology.

Time and setting

The experiments were performed at the Central Laboratory of Third Xiangya Hospital, Central South University, China from October 2009 to October 2010.

Materials

A total of 20 Wistar neonatal rats, within 1–3 days of birth, were provided by the Department of Experimental Animals, Xiangya Medical College, Central South University, China [license No. SCXK (Xiang) 2006-0002]. All protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[37].

Methods

Isolation and primary culture of retinal neurons

The neonatal rats were sacrificed by drowning in 75% alcohol. After disinfection for 5 minutes, the eyeballs were harvested in a sterile manner, and washed twice in D-Hanks solution. The eyeballs were cut open at 0.5 mm posterior to corneal limbus using a dissecting microscope (Olympus, Tokyo, Japan). The retina was dissociated in D-Hanks solution and cut into pieces, triturated into a single cell suspension, and digested with 0.05% trypsin (Gibco, Carlsbad, CA, USA) at 37°C for  15 minutes, which was terminated by Dulbecco’s modified Eagle’s medium/F-12 culture solution (Gibco) containing fetal bovine serum. The suspension was filtrated through a stainless steel mesh, centrifuged at 800–1 000 r/min for 5 minutes, resuspended in medium, and centrifuged. This process was repeated twice. The upper suspension was harvested, and the number of cells was quantified using a light microscope (Olympus). Cells at a density of 1 × 10⁶/mL were added to 12 mm × 12 mm coverslips and incubated in polylysine-coated 6-well culture plates with 80% Dulbecco’s modified Eagle’s medium/F-12 in 5% CO₂ at 37°C. Maintenance medium 1 [Neurobasal (Gibco), B27 supplement,    0.06 g/L glutamine, 100 U/mL penicillin, and 50 μg/mL streptomycin] was used after 24 hours, and maintenance medium 2 (Neurobasal, B27 supplement, 100 U/mL penicillin, and 50 μg/mL streptomycin) was used at    72 hours. One third of the medium was renewed every 1–2 days. Cell morphology was observed daily using an inverted microscope (Olympus) to identify quantity and length of processes and cell adherence.

Identification of retinal neurons

For Nissl staining, cell slices of 5–6 day primary cultures of retinal neurons were fixed in 4% paraformaldehyde for 25 minutes, washed with double distilled water three times, 5 minutes each, mixed with 10 g/L toluidine blue solution (Invitrogen, Carlsbad, CA, USA), incubated at 54°C for 30 minutes, cooled, and washed twice with double distilled water with a wash of 5 minutes each, rapidly washed with 95% alcohol, dehydrated with absolute alcohol, cleared with xylene, and mounted in neutral gum. Five high power fields (400 ×) from each coverslip were randomly observed, and the number of positive neurons in every 100 cells in every field of view was quantified using a light microscope. The positive neuron amount in each field of view was calculated using the formula: percentage of neurons (%) = number of neurons/total number of cells × 100%^[1]. Measurements were conducted in triplicate, and the mean value was calculated.

For immunocytochemical staining, retinal neuronal and glial cells were observed using anti-neurofilament and anti-glial fibrillary acidic protein immunocytochemical staining methods^[2]. Briefly, cells were harvested from the coverslips, fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed with PBS three times, 3 minutes each, blocked with 0.3% methanol in H₂O₂ at room temperature for 20 minutes, incubated with PBS containing 1% fetal bovine serum and 0.5%   Triton X-100 for 20 minutes, followed by PBS containing 5–10% normal goat serum for 20 minutes. The cells were incubated with rabbit anti-rat neurofilament (1:100; Maixin-Bio, Fujian, China) and glial fibrillary acidic protein (1:50; Sigma, St. Louis, MO, USA) monoclonal antibody at room temperature for 2 hours, then overnight at 4°C, then washed with PBS three times, for 3 minutes each. The negative control was treated with PBS rather than primary antibody. Cells were incubated with biotinylated goat anti-rabbit IgG (1:200; Sigma) and peroxide-labeled streptavidin (Gibco), followed by coloration with 0.01 M Tris-HCl (pH 7.4) containing 0.05% diaminobenzidine and 0.03% H₂O₂. Coloration time was controlled using a microscope to terminate the reaction time. Cells were dehydrated and photographed. Glial cells were identified as follows: after nuclei were hematoxylin counterstained for 25 seconds, cells were washed with tap water, dehydrated with gradient alcohol, cleared with xylene, and mounted with neutral gum. Cells stained brownish-yellow were neuron-positive staining, cells with brownish-yellow or brown stained particles in the cytoplasm and membrane were glial cell-positive staining. Five high power fields (400 ×) from each coverslip were randomly observed, and the number of neurofilament and glial fibrillary acidic protein positive neurons in every 100 cells in every field of view was quantified. The percentage of positive cells out of the total number of cells was calculated. Measurements were conducted in triplicate, and the mean value was calculated.

Establishment of a retinal neuron model of high glucose

According to a previously described method, cells were treated with 0 mM (normal control), 5.5, 15, 25, and    35 mM glucose (Sigma) for 24, 48, and 72 hours. Cell survival rate was determined using the MTT assay, and the apoptosis rate detected was determined using flow cytometry. The optimal glucose concentration and intervention duration for establishing the high glucose model were confirmed according to experimental results.

MTT assay for retinal neuron survival

Primary cultures of retinal neurons for 3–4 days were trypsinized (0.25%). A single cell suspension was prepared using serum-free culture medium, and cells at a concentration of 1–3 × 10⁵ cells/mL were seeded into 96-well culture plates, with 100 μL in each well. The culture media containing different concentrations of glucose was replaced at a cell confluency of 80%. After culture for 24, 48, and 72 hours, 20 μL MTT solution  (0.5 mg/mL, Sigma) was added to each well and cultured for 4 hours, treated with 100 μL dimethyl sulfoxide after the supernatant was discarded, and shaken at room temperature for 10 minutes to completely dissolve crystals. Absorbance at 570 nm was detected using an enzyme-linked immunosorbent assay reader (Perkin Elmer, Turku, Finland). The experiment was conducted in triplicate, and the mean value was calculated. Higher absorbance value represented more surviving cells.

Flow cytometry for apoptosis

Cells treated with different concentrations of glucose for 24, 48, and 72 hours were harvested, and digested with 0.25% trypsin (200 μL) in each well. The reaction was terminated by adding 10% fetal bovine serum after the cells shrunk. Cells were detached by trituration, centrifuged at 1 000 r/min for 5 minutes, washed with PBS after the supernatant was discarded, and centrifuged. The process was conducted in triplicate. A single cell suspension was prepared using PBS, diluted with deionized water at a ratio of 1:4, and washed with PBS twice. Cells were resuspended using 250 μL binding buffer, and cell concentration was adjusted to 1 × 10⁶ cells/ mL. The cell suspension (100 μL) was placed in a 5 μL flow tube, incubated with 1 μL annexin/fluorescein isothiocyanate (Bender MedSystems, Vienna, Austria) and 5 μL propidium iodide (20 μg/mL) for 15 minutes in the dark, followed by 400 μL PBS. Cell apoptosis was analyzed using flow cytometry (Becton Dickinson, San Jose, CA, USA).

Statistical analysis

Data were expressed as mean ± SD and analyzed using SPSS 13.0 software (SPSS, Chicago, IL, USA). Comparison of the multiple group mean was conducted using one-way analysis of variance, and multiple comparisons among groups were analyzed using the Least Significant Difference test. A value of P < 0.05 was considered statistically significant.

1 Retinal neurons from neonatal Wistar rats (1–3 days) were cultured with plating medium and maintenance medium to establish a reliable and stable primary culture system using neurons as the predominant cells.
2 The retinal neurons were treated with glucose at different concentrations for different time, and 35 mM for 72 hours was identified as the optimal concentration and intervention time.
3 A retinal neuron model of high glucose was successfully established, which is significant for further studies of structure characteristics and biological functions of cell components in the retina, retinal pathological changes, and drug reactions.
1 实验采用接种培养基和维持培养基培养出生后1～3d Wistar乳鼠视网膜，建立了一种稳定可靠、以神经元为主体的视网膜神经细胞原代培养体系。
2 实验采用不同浓度的葡萄糖作用于原代培养的Wistar大鼠视网膜神经元，最终确定葡萄糖最适干预浓度为35mM，干预时间为72h。
3 实验成功建立了视网膜神经元高糖模型，对于深入研究视网膜内各种细胞成分的结构特征与生物学功能、病理改变过程与机制以及药物反应等具有重要意义。

1 实验设计构思介绍

实验采用接种培养基和维持培养基培养出生后1～3d Wistar乳鼠视网膜，建立了一种稳定可靠、以神经元为主体的视网膜神经细胞原代培养体系。采用不同浓度的葡萄糖作用于原代培养的Wistar大鼠视网膜神经元不同时间，最终确定葡萄糖最适干预浓度为35mM，干预时间为72h,成功建立了视网膜神经元高糖模型。

2 国内外同类研究水平的介绍

与国内外同类研究水平相比较：目前国内尚未见体外培养视网膜神经元的高糖损伤模型的报道，与同类国外研究水平相当。

3 与其他同类研究的比较

国外各种视网膜神经元髙糖模型的建立中，针对不同的研究内容所给予的葡萄糖干预浓度及时间均不同，这些研究中高糖浓度及干预时间的选择，仅参考以往文献，并未连续性观察不同浓度葡萄糖干预不同时间点视网膜神经元的凋亡的变化情况，而且所选择的培养基也并非针对神经元培养的专用培养基。

4 文章创新性性

首次通过建立视网膜神经元的最优培养体系，选择最适的神经元高糖模型，为后续的药物保护髙糖状态下视网膜神经元的机制研究奠定基础。

5 专家意见与答疑

专家意见：结果中缺少高糖环境下细胞的形态变化，最好补充图片。

作者答疑：高糖环境下总体上神经元的数量及形态是有变化的，但由于神经元是聚集生长，每组的单个视野照相可能看不出明显的差异，因此很难挑出每组有明显差异对比的组织学图片。