Lateral ventricular wall whole mount immunofluorescence from brains of Ara-C-infused Lrig1T2A-iCreERT2/+; Rosa26Ai14/+ mice

Dataset from the Ara-C infusion experiment presented in Nam and Capecchi, 2020.The ZIP files can be opened directly with Fiji.✶Ara-C is a classical nucleoside chemotherapeutic that kills dividing cells.Ara-C was slowly infused over six days into one of the two lateral ventricles using a cannula and an osmotic pump, in contrast to infusion into the cortex as in Doetsch et al., 1999 and Doetsch et al., 1999.Recall that Ara-C doesn't directly induce cell cycle entry of quiescent stem cells. It does so indirectly by killing nearby proliferating cells, and their absence induces the quiescent stem cells into cell cycle. As such, the cell cycle entry does not necessarily happen exactly when the infusion is stopped, but rather when a sufficient number of cells is killed by the Ara-C. Indeed, in my implementation of this paradigm, it would seem that the cell cycle entry of the quiescent stem cells had started before the cessation of the Ara-C infusion because at that time point, the RFP+ quiescent stem cells were already KI-67+, i.e, activated. A key difference indicating that the Ara-C infusion (indirectly) caused the cell cycle entry is the greatly increased number of these RFP+ KI-67+ activated stem cells in the infused mice compared to uninfused mice.There were some neuroblasts that had not been killed by the Ara-C. Subsequently, I noticed there are KI-67+ DCX+ and KI-67- DCX+ neuroblasts (in Nam and Capecchi, 2023). Perhaps the KI-67- DCX+ neuroblasts are post-mitotic and cannot be killed by the Ara-C, but they will migrate rostrally and out of the ventricular wall during the chase after the infusion.One of the mice in the first try was not infused correctly because the tubing that connects the osmotic pump and the infusion cannula came loose after surgery. This could be considered an incidental sham surgery control. Note the presence of RFP+ proliferating clusters and RFP+ neuroblasts in the uninfused mouse. They are absent in the Ara-C-infused mice at early time points because they were killed by the Ara-C. That and the dramatic reduction of DCX+ neuroblast number indicate a successful Ara-C infusion.One of the two ventricular walls in one of the mice in the first try was damaged. At the time, I assumed I damaged the tissue during dissection and didn't think much more about it, but the damage could be due to the Ara-C infusion (as recently reported in Thanou et al., 2025). Perhaps in this mouse the infusion cannula was placed too close to the ventricular wall, and the high concentration Ara-C flowing out of the cannula was too toxic to the nearby cells and damaged the tissue. In the first try, I analyzed both ventricular walls, and the damage was observed in only one of the two successful infusions. In the second try, I unfortunately did not analyze both ventricular walls. I randomly chose one of the two, then analyzed only one. Nevertheless, none in this set were damaged. In the third try, I analyzed both ventricular walls from one mouse, and neither were damaged. So, I will not know for sure from this dataset, but the damage doesn't seem to happen in every mouse using this particular setup.These were done very early on in my post-doctoral training, so the immunostaining quality is not as good as the ones done later.

本数据集源自Nam与Capecchi于2020年发表的阿糖胞苷（Ara-C）灌注实验。压缩包可直接使用Fiji软件打开。
阿糖胞苷是经典的核苷类化疗药物，可杀伤增殖期细胞。与Doetsch et al. (1999)及Doetsch et al. (1999)研究中向皮层灌注的方式不同，本实验通过套管（cannula）与渗透泵（osmotic pump），将阿糖胞苷缓慢灌注至侧脑室之一，灌注时长共计六天。
值得注意的是，阿糖胞苷并不会直接诱导静息干细胞进入细胞周期，而是通过间接途径发挥作用：先杀伤邻近的增殖细胞，当增殖细胞大量缺失后，静息干细胞才会被激活并进入细胞周期。因此，静息干细胞的细胞周期启动未必恰好发生在灌注停止之时，而是取决于阿糖胞苷杀伤细胞的数量是否达到阈值。在本实验的实施过程中，可观察到静息干细胞的细胞周期启动早于阿糖胞苷灌注结束，因为在灌注终止前，红色荧光蛋白（RFP）标记的静息干细胞已呈现KI-67阳性，即已被激活。与未灌注组小鼠相比，灌注组小鼠体内RFP阳性且KI-67阳性的激活干细胞数量显著增多，这一关键差异证明阿糖胞苷灌注间接触发了干细胞的细胞周期激活。
本实验中仍有部分神经母细胞未被阿糖胞苷杀伤。后续研究（Nam与Capecchi, 2023）中观察到KI-67阳性DCX阳性与KI-67阴性DCX阳性的两类神经母细胞。推测KI-67阴性DCX阳性的神经母细胞属于有丝分裂后细胞，无法被阿糖胞苷杀伤，但在灌注结束后的追踪阶段，它们会向吻侧迁移并离开脑室壁。
首批实验中的一只小鼠未完成正确灌注：手术完成后，连接渗透泵与套管的管路发生脱落。该样本可视为偶然的假手术对照。需注意，未灌注组小鼠体内存在RFP阳性增殖簇与RFP阳性神经母细胞，而早期时间点的阿糖胞苷灌注组小鼠中并无此类结构——这是因为它们被阿糖胞苷杀伤。结合DCX阳性神经母细胞数量的显著减少，可确认阿糖胞苷灌注实验成功。
首批实验中的一只小鼠的双侧脑室壁之一出现损伤。当时我推测是解剖过程中造成的组织损伤，未过多深究，但该损伤也可能由阿糖胞苷灌注导致（正如Thanou et al., 2025年近期报道的结论）。推测该小鼠的套管放置位置过于贴近脑室壁，从套管流出的高浓度阿糖胞苷对邻近细胞毒性过强，进而造成组织损伤。首批实验中我分析了双侧脑室壁，仅在一例成功灌注的样本中观察到损伤。第二批实验中，遗憾的是我未对双侧脑室壁均进行分析，仅随机选取一侧进行检测，且该批次样本均未出现组织损伤。第三批实验中，我对一只小鼠的双侧脑室壁均进行了分析，两侧均未出现损伤。因此，尽管无法通过本数据集完全确认，但采用该特定实验装置时，并非所有小鼠都会出现组织损伤。
本批实验均为我博士后训练早期完成的工作，因此免疫染色的质量不及后续实验。