Experimental Datasets on the Histopathological Assessment of Purkinje cells in the Cerebellum of Japanese Medaka (Oryzias latipes) Fish Exposed to Graphene Oxide

The datasets of this article present the experimental data resulted from the assessment of Purkinje cells (PKCs) in the cerebellum of Japanese medaka (Oryzias latipes) brain, considering PKCs as a potential target of graphene oxide (GO), a nanocarbon, extensively used in industries. The GO we used in these experiments was obtained either from a commercial source or synthesized in the laboratory. Before experimental use, GO was sonicated for 5 min in ice temperature. The reproductively active adult male and female fish used in the experiments were obtained from the Japanese medaka culture facilities of the Jackson State University, Jackson, MS, USA, and were maintained as a breeding pair (one male and one female) in 500 ml balanced salt solution (BSS). The fish were exposed to GO in vivo either by immersion (IMR) (20 mg/L in BSS) or by a single intraperitoneal (IP) injection (100 µg/g), injected into the peritoneal cavity of both male and female fish. Parallel controls were maintained in BSS only (IMR experiment) or injected with nanopure water (IP experiment). To avoid movement during injections, the fish were anesthetized in MS 222 (IMR), however, for IMR experiments, fish did not require anaesthesia. The injected volume (0.5µL/10 mg fish) never exceeds 50µl/fish. After injection, the injected fish were allowed for recovery in clean BSS and after recovery both partners were transferred to a 1L glass jars with 500 mL BSS (no GO). In IMR experiments, the GO was dissolved in BSS (20 mg/L) and replaced every 24 h for 96 h. During depuration, the media of the breeders refreshed once every 24h and the reproductive activity of the experimental fish were assessed by evaluating the eggs (fertilized, unfertilized, damaged) collected from the experimental fish. After 21 days of depuration, the survived fish were anaesthetized, and the head and trunk regions were preserved in 4% paraformaldehyde in PBS (20 mM) containing 0.05% Tween 20. The phenotypic sex of the fish was assessed externally by secondary sex characters (fin features) and confirmed by gonad histology (testis and ovary). The location of cerebellum in the brain was determined in paraffin sections after haematoxylin/eosin (HE) staining, and the images of cerebellum were captured using an Olympus CKX53 inverted microscope with DP22 camera and CellSens software. Using imagej software, a minimum 2 images of cerebellum/fish were assessed. The larger size, water droplet-like shape, and the distribution of the PKCs in specific regions of cerebellum (Purkinje layers), enabled us to separate them from other cellular layers (molecular and granular layers) and neurons found in the cerebellum of Japanese medaka and count them to the extent possible. The nuclear area (µm2), the cell area (soma) (µm2), and the ratio of nuclear area: cell area of the PKCs were also considered for assessment. The data were expressed as number of PKCs/mm2. Numerical data were analysed by Kruskal-Walli’s test followed by Mann-Whitney’s test as post hoc test and presented as means ± SEM. Statistically significant differences were considered for p ≤ 0.05.

本文数据集呈现了针对日本青鳉（Oryzias latipes）小脑浦肯野细胞（Purkinje Cells, PKCs）的实验评估数据，该研究将PKCs作为工业中广泛应用的纳米碳材料——氧化石墨烯（graphene oxide, GO）的潜在作用靶点。本实验所用GO既可从商业渠道购得，也可在实验室合成。实验使用前，GO需在冰浴条件下超声处理5分钟。
实验所用生殖活性成熟的雌雄成鱼均取自美国密西西比州杰克逊市杰克逊州立大学的青鳉养殖设施，实验期间以1雄1雌为一组，饲养于500mL平衡盐溶液（balanced salt solution, BSS）中。
实验鱼通过两种方式体内暴露于GO：一是浸浴法（immersion, IMR）（将GO以20mg/L浓度溶解于BSS中），二是单次腹腔注射（intraperitoneal, IP）（剂量为100μg/g体重），注射部位为雌雄鱼的腹膜腔。平行对照组分别设置为仅用BSS饲养（浸浴组实验）、注射超纯水（腹腔注射组实验）。
为避免注射时鱼体移动，腹腔注射组实验鱼需经MS222麻醉；但浸浴组实验无需麻醉。单鱼注射体积为0.5μL/10mg鱼体重，且不超过50μL。注射后，实验鱼需在新鲜BSS中恢复，恢复后将雌雄配对转移至盛有500mL无GO的BSS的1L玻璃缸中。
浸浴组实验中，GO以20mg/L浓度溶解于BSS，每24小时更换一次药液，持续暴露96小时。净化期内，每24小时更换一次养殖水体，通过收集实验鱼所产卵的状态（受精卵、未受精卵、受损卵）评估其生殖活性。
净化21天后，将存活鱼麻醉，取头部和躯干组织，置于含0.05%吐温20（Tween 20）的20mM磷酸盐缓冲液（phosphate buffered saline, PBS）配制的4%多聚甲醛中固定保存。实验鱼的表型性别通过第二性征（鳍部形态）进行外部判定，并通过性腺组织学（睾丸和卵巢）验证。
脑组织石蜡切片经苏木精-伊红（haematoxylin/eosin staining, HE）染色后，确定小脑的位置；使用奥林巴斯CKX53倒置显微镜搭配DP22相机及CellSens软件采集小脑图像。采用ImageJ软件，每条鱼至少分析2张小脑图像。
浦肯野细胞体积较大、呈水滴状，且定位于小脑特定区域（浦肯野细胞层），据此可将其与日本青鳉小脑中的其他细胞层（分子层和颗粒层）及其他神经元区分开来，并尽可能完成计数。本研究同时评估了PKCs的核面积（μm²）、胞体面积（μm²）以及核面积与胞体面积的比值。数据以每mm²内的浦肯野细胞数量表示。
数值数据采用Kruskal-Wallis检验，随后以Mann-Whitney检验作为事后检验进行统计学分析，结果以平均值±标准误（standard error of the mean, SEM）表示；当p≤0.05时认为差异具有统计学意义。