Adaptive changes of the ground squirrel retina during hibernation

冬眠的哺乳动物在深度低温下存活而没有受伤，这是一项细胞保存的非凡壮举，具有潜在的医学应用意义。我们着手研究冬眠的十三线地松鼠（TLGS）是否对神经损伤的反应不同于清醒的。我们选择视神经挤压（ONC）损伤模型，因为它是一种经典的轴突损伤模型，没有血管损伤的并发症，并且它是一种可以在冬眠的TLGS中进行而不干扰冬眠状态的小手术。当完全压碎时，大多数RGC在损伤后14天死亡。然而，对于TLGs在迟钝，大多数RGCs存活的损伤。然后，我们专注于部分ONC损伤模型，以便我们可以通过计算损伤后不同时间点的鼻（受伤侧）/颞（未受伤侧）RGC比率来量化存活率。对于活性TLGS，RGC群体的下降在第3天开始，并以指数方式持续，直到在第21天达到平台期，保留约20%的RGC，类似于小鼠和大鼠ONC模型中报道的细胞死亡动力学。与此形成鲜明对比的是，多达90%的RGCs在冬眠的TLGSs中存活到第21天的ONC损伤。而且，这些残存的RGC，并不是没有被清理的死细胞体。当我们使用多电极阵列（MEA）记录活跃TLGS在视网膜鼻侧半部分的自发RGC放电活动时，正如预期的那样，我们观察到与颞侧半部分相比，RGC活动急剧减少。然而，对于迟钝的TLGSs，鼻侧视网膜的RGC活动保持在与颞侧视网膜相当的水平，这表明它们仍然活跃。 为了理解为什么活跃和迟钝的TLGS中的RGC对相同的轴突损伤的反应如此不同，我们从活跃和迟钝的条件下收集了受损的视神经样本，并将它们进行RNAseq。 我们首先鉴定了响应于ONC的差异表达基因（DEG），然后基于细胞类型对其进行分类（McKenzie等人，2018年）。有趣的是，在来自活动性TLGS（ONC后3天）的样品中，超过一半的上调DEG（136个中的80个）与小胶质细胞相关。相比之下，小胶质细胞相关基因仅占来自迟钝动物的样品中上调的DEG的不到10%。在上调的小胶质细胞相关DEG中，典型的小胶质细胞活化基因如CD 68（Hendrickx et al.，2017）、CD 74（Wang等人，2014）、C1 QB（Stephan等人，2012）和TREM 2（Wang等人，2015），所有这些事实上在迟钝动物中下调（图2B和数据S1）。这种活跃和迟钝动物中小胶质细胞相关基因的极化转录组模式促使我们直接检查挤压部位附近的小胶质细胞反应。在来自活性TLGSs的样品中，我们确实观察到Iba 1+阳性小胶质细胞在损伤部位的大量聚集。这种小胶质细胞的积累早在ONC后第1天就开始了，并持续到我们采样时（第21天）。此外，这些Iba 1+细胞大多数对CD 68标记和其它巨噬细胞样标志物如F4/80呈阳性（卡森等人，1998）和MFGE 8（Liu等人，2013），表明它们是活化的小胶质细胞。相比之下，这种小胶质细胞聚集是完全不存在的样品从迟钝的TLGSs。相反，在挤压部位的细胞稀疏区域是明显的，如通过细胞核的DAPI标记所揭示的。这些结果证实了转录组学分析，即挤压部位的小胶质细胞反应在活动性和迟钝性TLGSs之间存在显着差异。但RGC索马体位于视网膜内，离挤压部位有一定距离。因此，我们进一步研究了视网膜中小胶质细胞在长达21天的不同时间点对ONC的响应动力学。在来自患有ONC的活动性TLGSs的视网膜样品中，沿着进行性RGC损失，存在小胶质细胞的持续积累，以及小胶质细胞中CD 68表达的逐渐增加。相反，在来自迟钝动物的样品中，几乎没有RGC损失，并且在ONC后21天内既没有小胶质细胞聚集也没有CD 68过表达。从活跃的TLGS观察到的另一个突出特征是ONC后神经纤维层中Iba 1阳性小胶质细胞突起的显著增加。这些过程中的许多与星形胶质细胞的树突平行运行，并进行多次接触，表明小胶质细胞和星形胶质细胞之间可能的相互作用，可能导致神经元死亡。 这些结果揭示了一个显着不同的先天免疫反应的轴突损伤冬眠动物。我们将进一步研究这种神经存活的机制。这将有助于开发治疗神经损伤和变性的治疗策略。

Hibernating mammals survive profound hypothermia without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. We set out to examine whether hibernating thirteen-lined ground squirrel (TLGS) respond to neural injury differently from awake ones. We chose optic nerve crush (ONC) injury model as it is a classic model of axonal injury without the complication of blood vessel damage, and it is a minor surgery that can be performed in hibernating TLGSs without disturbing the hibernation status. When crushed totally, majority of RGCs died 14 days after the injury in active TLGSs. For TLGSs in torpid, however, most of RGCs survived the injury. We then focused on the partial ONC injury model so that we can quantify the survival rate by calculating the nasal (injured side)/ temporal (uninjured side) RGC ratio at the different time points after the injury. For active TLGSs, the decline in RGC population started at day 3 and went on exponentially until reaching a plateau at day 21 with about 20% of RGCs remained, similar to the cell death kinetics reported in the mouse and rat ONC models. In stark contrast, as much as 90% of RGCs survived the ONC injury by day 21 in hibernating TLGSs. Moreover, these surviving RGCs were not merely dead cell bodies yet to be cleared away. When we used multi-electrode array (MEA) to record the spontaneous RGC firing activities in the nasal half of the retina from active TLGSs, as expected, we observed a sharp reduction of RGC activities compared to that of the temporal half. For torpid TLGSs, however, the RGC activities in the nasal half of retina remained at a level comparable to that of the temporal half, suggesting that they remain active. To understand why RGCs in active and torpid TLGSs respond so differently to the same axonal injury, we collected injured optic nerve samples from both active and torpid conditions and subjected them to RNAseq. We first identified differentially expressed genes (DEGs) in response to ONC and then categorized them based on cell types (McKenzie et al., 2018). Interestingly, in samples from active TLGSs (3 days after ONC), over half of the up-regulated DEGs (80 out of 136) are microglia-related. In contrast, microglia-related genes only account for less than 10% of the upregulated DEGs in samples from torpid animals. Among the up-regulated microglia-related DEGs, typical microglial activation genes such as CD68 (Hendrickx et al., 2017), CD74 (Wang et al., 2014) , C1QB (Stephan et al., 2012) and TREM2 (Wang et al., 2015) were identified, all of which are in fact downregulated in torpid animals (Figure 2B and Data S1). This polarized transcriptome pattern of microglia-related genes in active and torpid animals prompted us to directly examine the microglial reaction near the crush site. In samples from active TLGSs, we indeed observed massive aggregation of Iba1+ positive microglial cells at the injury site. Such microglial accumulation stared as early as day 1 after ONC and last as long as we sampled (day 21). Moreover, these Iba1+ cells are mostly positive for CD68 labeling, and other macrophage-like markers, such as F4/80 (Carson et al., 1998) and MFGE8 (Liu et al., 2013), indicating that they are activated microglial cells. In contrast, such microglial aggregation was completely absent in samples from torpid TLGSs. Instead, a cell-sparse region at the crush site is apparent as revealed by DAPI labeling of nuclei. These results confirmed the transcriptomic analysis that microglial response at the crush site is a significant difference between active and torpid TLGSs. However, RGC soma situate in the retina, some distance away from the crush site. Therefore, we further examine the dynamics of microglial in the retina in response to the ONC at different time points up to 21 days. In retina samples from the active TLGSs with ONC, along with the progressive RGC loss, there is a continuing accumulation of microglial cells, as well as gradually increase of CD68 expression in microglial cells. In contrast, in samples from torpid animals, there is little RGC loss, and neither microglia aggregation nor CD68 overexpression occurs up to 21 days post ONC. Another prominent feature observed from the active TLGSs is a substantial increase of Iba1 positive microglial processes in the nerve fiber layer after ONC. Many of these processes run parallel with dendrites of astrocytes and make numerous contacts, suggesting possible interactions between microglia and astrocytes that may lead to neuronal death. These results revealed a remarkably different innate immune response to axonal injury in hibernating animals. We will further investigate the mechanism(s) of such neural survival after injury in the hibernating condition. This will help to develop therapeutic strategies for treating neural injury and degeneration.