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GALT 缺失经典半乳糖血症模型(SD 大鼠)

GALT 活性在纯合仔鼠肝中不可检出;仔鼠出生后体重低于野生型,出现双侧白内障;成年表现运动协调与隐藏平台水迷宫学习记忆缺陷;肝、脑等组织半乳糖、半乳糖醇及 Gal-1P 随年龄和饮食变化。

Outcome measures

获得可供光学显微镜观察的 H&E 染色组织切片:细胞核蓝紫色、细胞质与结缔组织纤维粉红色,用于观察组织中细胞的整体结构与形态细节。本方案本身不产出病理评分数值,也不提供特定分子标志物的定量结果。

记录每次定位试验的到台潜伏期和游泳轨迹,比较相继试验中的空间定位表现。到达平台与达到时限未到台的情况按对应条件路径分别处理。

Experimental animals

Hematoxylin and Eosin (H&E) staining for histopathology
Age
8–60 日龄(来源研究取材时点)
Sex
Mixed
Tissue
liver and brain
Hematoxylin and Eosin (H&E) staining for histopathology
Age
8–60 日龄(来源研究取材时点)
Sex
Mixed
Tissue
liver and brain
大鼠 Morris 水迷宫空间定位与再学习测定
Age
成年 4-5 月龄
Sex
Male
Tissue
brain (behavior)
Hematoxylin and Eosin (H&E) staining for histopathology
Age
未记载周龄,终点取材组织切片 HE 判读(离体)
Sex
Mixed
Tissue
liver and brain

Experimental procedures

脱蜡(Dewaxing) 石蜡切片置于二甲苯中脱蜡 3 次,每次 00:03:00。 复水(Rehydration) 石蜡切片依次置于 100% 乙醇中 3 次、每次 00:03:00,再置于 95% 乙醇中 1 次、00:03:00,随后用水漂洗。 苏木素核染色(Hematoxylin staining) 石蜡切片以苏木素染液染色 00:00:20,随后在流水下冲洗 00:01:00。 分化与返蓝(Differentiation and bluing) 石蜡切片置于返蓝液中 00:01:00,随后在流水下冲洗 00:01:00;此步使细胞核呈现蓝色。 伊红染色(Eosin staining) 石蜡切片以伊红复染 00:00:10;此步使细胞质成分与结缔组织纤维呈现粉红色。 脱水与透明(Dehydration and Clearing) 石蜡切片依次置于 95% 乙醇中 00:02:00、100% 乙醇中 2 次、每次 00:02:00,随后置于二甲苯中 2 次、每次 00:02:00。 封片(Mounting) 石蜡切片以 Permount 封片介质加盖玻片封固。

检查摄像机设置 检查摄像机设置。 将 Atlantis 自动升降平台置于水池,确认升降运动正常,避免平台上下移动时产生水波 将 Atlantis 自动升降平台置于水池,确认升降运动正常,避免平台上下移动时产生水波。 将平台放置在与前一天不同的位置 将平台放置在与前一天不同的位置。 实验第一天不执行与前一天位置比较的要求 实验第一天不执行与前一天位置比较的要求。 启动 Bonsai 工作流,确认以 30 fps 记录视频 启动 Bonsai 工作流,确认以 30 fps 记录视频。 降下平台,使动物暂时无法使用平台 降下平台,使动物暂时无法使用平台。 将动物放在所选起点,面向水池中心 将动物放在所选起点,面向水池中心。 轻放动物入水,避免抛掷和溅水 轻放动物入水,避免抛掷和溅水。 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序。 经过 1 分钟 后,按下按钮升起平台 经过 1 分钟 后,按下按钮升起平台。 动物到达并接触平台时停止计时;本次试验时限为 2 分钟 Choose the matching condition: Platform — Rats will find the platform. They will do this in two minutes or less; Not platform — If the rat does not find the platform within 2 minutes. 动物到达并接触平台时停止计时;本次试验时限为 2 分钟。 返回取出动物,放入笼内擦干 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 返回取出动物,放入笼内擦干。 间隔 30 分钟 后开始第 2 次试验 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 间隔 30 分钟 后开始第 2 次试验。 升起平台,使动物能够登台,但平台仍不可直接看见 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 升起平台,使动物能够登台,但平台仍不可直接看见。 将动物放在所选起点,面向水池中心;每次试验随机选择不同的起点 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 将动物放在所选起点,面向水池中心;每次试验随机选择不同的起点。 轻放动物入水,避免抛掷和溅水 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 轻放动物入水,避免抛掷和溅水。 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序。 动物到达并接触平台,或经过 1 分钟 时停止计时 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Choose the matching condition: Platform — Rats will find the platform. They will do this in 1 minute or less; Not platform — If the rat does not find the platform within 1 minute 动物到达并接触平台,或经过 1 分钟 时停止计时。 返回取出动物,放入笼内擦干 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Platform — Rats will find the platform. They will do this in 1 minute or less 返回取出动物,放入笼内擦干。 间隔 5 分钟 后进行下一次试验 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Platform — Rats will find the platform. They will do this in 1 minute or less 间隔 5 分钟 后进行下一次试验。 将大鼠放在平台上 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Not platform — If the rat does not find the platform within 1 minute 将大鼠放在平台上。 离开实验区域 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Not platform — If the rat does not find the platform within 1 minute 离开实验区域。 等待 10 秒 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Not platform — If the rat does not find the platform within 1 minute 等待 10 秒。 返回取出动物,放入笼内擦干并等待 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Not platform — If the rat does not find the platform within 1 minute 返回取出动物,放入笼内擦干并等待。 间隔 5 分钟 后进行下一次试验 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Not platform — If the rat does not find the platform within 1 minute 间隔 5 分钟 后进行下一次试验。 将大鼠放在平台上 Condition: Not platform — If the rat does not find the platform within 2 minutes. 将大鼠放在平台上。 离开实验区域 Condition: Not platform — If the rat does not find the platform within 2 minutes. 离开实验区域。 等待 10 秒 Condition: Not platform — If the rat does not find the platform within 2 minutes. 等待 10 秒。 返回取出动物,放入笼内擦干并等待 Condition: Not platform — If the rat does not find the platform within 2 minutes. 返回取出动物,放入笼内擦干并等待。 间隔 30 分钟 后开始第 2 次试验 Condition: Not platform — If the rat does not find the platform within 2 minutes. 间隔 30 分钟 后开始第 2 次试验。 升起平台,使动物能够登台,但平台仍不可直接看见 Condition: Not platform — If the rat does not find the platform within 2 minutes. 升起平台,使动物能够登台,但平台仍不可直接看见。 将动物放在所选起点,面向水池中心;每次试验使用不同的起点 Condition: Not platform — If the rat does not find the platform within 2 minutes. 将动物放在所选起点,面向水池中心;每次试验使用不同的起点。 轻放动物入水,避免抛掷和溅水 Condition: Not platform — If the rat does not find the platform within 2 minutes. 轻放动物入水,避免抛掷和溅水。 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序 Condition: Not platform — If the rat does not find the platform within 2 minutes. 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序。 动物到达并接触平台时停止计时;本次试验时限为 1 分钟 Condition: Not platform — If the rat does not find the platform within 2 minutes. 动物到达并接触平台时停止计时;本次试验时限为 1 分钟。

Model identity

Model name
GALT 缺失经典半乳糖血症模型(SD 大鼠)
Species
rat
Strain
SD-Galt M3
Genetic background
远交 Sprague-Dawley 背景,CRISPR-Cas9 构建的 Galt 无效等位基因纯合。
Genotype / zygosity
Galt M3/M3
Allele
Galt M3(第 6 外显子 2 bp 插入)

Biological characteristics

Phenotype stage
新生至断奶期出现生长延迟与白内障;4–6 月龄评估运动与认知;组织代谢物从新生至 12–16 月龄纵向观察。
Research questions
GALT 缺失如何导致组织半乳糖代谢物蓄积及白内障、生长和神经行为表型;出生后基因替代能否纠正代谢与表型。
Limitations
远交 SD 背景与特定 Galt M3 无效等位基因构成该模型;饮食半乳糖含量影响代谢物水平,断奶后限制半乳糖并不能使组织代谢物完全正常化;大鼠表型不能直接等同于人类患者的长期并发症谱。

Experimental methods

Experimental protocols

  • Hematoxylin and Eosin (H&E) staining for histopathology

    Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.

  • Hematoxylin and Eosin (H&E) staining for histopathology

    Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.

  • 大鼠 Morris 水迷宫空间定位与再学习测定

    A rat water-maze procedure using an automated Atlantis platform and distal cues, with a new platform location each day. Preserve the separate platform-found and platform-not-found paths, and record escape latency and swim trajectories across the successive trials.

  • Hematoxylin and Eosin (H&E) staining for histopathology

    Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.

Reagents and materials

References

  1. A galactose-1-phosphate uridylyltransferase-null rat model of classic galactosemia mimics relevant patient outcomes and reveals tissue-specific and longitudinal differences in galactose metabolism. Journal of Inherited Metabolic Disease (2020), PMID 31845342.
  2. Neonatal GALT gene replacement offers metabolic and phenotypic correction through early adulthood in a rat model of classic galactosemia. Journal of Inherited Metabolic Disease (2022), PMID 34964137.
  3. Nathalia L. Oliveira; Samantha Gruenheid. Hematoxylin and Eosin (H&E) staining for histopathology. protocols.io, v1, 2026-07-31. DOI: 10.17504/protocols.io.kxygxr9jdg8j
  4. Epredia Signature Series Hematoxylin (7221), 产品信息页, Fisher Scientific
  5. Epredia Signature Series Bluing Reagent (7301), 产品信息页, Fisher Scientific
  6. Epredia Signature Series Eosin Y (7111), 产品信息页, Fisher Scientific
  7. Xylene, Fisherbrand HistoPrep (HC7001GAL), 产品信息页, Fisher Scientific
  8. Fisher Chemical Permount Mounting Medium (SP15 产品族), 产品信息页, Fisher Scientific
  9. Ethanol (CID 702, CAS 64-17-5), PubChem 化合物标准记录
  10. Maroto AS, Ugarte Perez E, Cerdán Cerdá A, De Santis S. Morris Water Maze. protocols.io, published 2026-03-26, v1. DOI:10.17504/protocols.io.e6nvwn832vmk/v1.
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  14. Schisandrin ameliorates cognitive deficits, endoplasmic reticulum stress and neuroinflammation in streptozotocin (STZ)-induced Alzheimer’s disease rats. PMID:32336744; DOI:10.1538/expanim.19-0146. Model/assay applicability; not exact replication of the separate protocols.io procedure.
  15. ARRIVE 2.0 guidelines