Results

Ultrastructural Features of Autophagic Maturation after Traumatic Brain Injury

To determine whether autophagic activity is altered after TBI, we first examined brain sections by transmission EM. The EM manifestation of bubblelike vacuoles that enclose recognizable cytoplasmic structures still represents the gold standard for identifying APs (Brunk and Terman, 2002). Preexisting APs were occasionally seen in sham-operated control cortical neurons (Figure 1A). Neurons subjected to TBI followed by recovery, however, displayed a striking increase in the density of APs (Figures 1B to 1F). Neocortical neurons from shamoperated control rats contained polyribosomes (arrows), nucleus (N), rough endoplasmic reticulum (ER), and mitochondria (M) (Figure 1A). At 4 h post-TBI (Figure 1B), the dominant ultrastructural changes were (i) mild accumulation of APs and autolysosomes (ALs) and (ii) mild mitochondrial swelling (M). During 24 h to 15 days post-TBI (Figures 1C to 1F), APs and ALs accumulated markedly, whereas polyribosomes (arrows), mitochondria (M), the endoplasmic reticulum (ER) and Golgi apparatus (G) appeared normal in TBI neurons. Several morphologic features of AP maturation were clearly observed in neurons at different periods of recovery after TBI (Figure 2). Autophagosome formation began with isolation membranes, i.e., the formation of double-membraned cisterns in the cytoplasm after TBI (Figure 2A, arrows). The double-membraned cisterns enveloped cytoplasmic contents or whole organelles to form APs (Figure 2A, AP). The formation of double membranes and APs was seen as early as 4 h after TBI (Figure 2A) and was an ongoing process during the post-TBI phase (see biochemical analysis below). Autophagosome clusters containing membrane whorls, probably representing damaged mitochondria (M), were frequently seen in dendritic trunks (Figure 2C) and neuronal process-rich regions (neuropil) (Figure 2B) after TBI. Autophagosomes eventually merged with lysosomes to become ALs (Figure 2D). Partially degraded cargo contents within ALs were then manifested as unevenly distributed dense (dark) masses (Figure 2D, AL) and eventually as evenly distributed dense (dark) materials (Figure 2A, AL).

外伤性脑损伤后自噬成熟的超结构特征

决定TBI后的自噬活性是否变化，首先在透射电镜下观察脑切片。

透射电镜可以识别胞质结构附近的泡沫样液泡,是鉴别自噬小体APs的金标准(Brunk and Terman, 2002)。在假手术对照组皮质神经元里偶尔能见到先前存在的自噬小体 (Figure 1A)。在接下来的TBI后恢复组的脑神经元里,显示自噬小体密度的明显增高(Figures 1B to 1F)。假手术对照组鼠的脑皮层神经元里包含有多核糖体(箭头所指)，核(N)，粗面内质网(ER)和线粒体(M)。图一。

在TBI后4小时，明显的超微结构变化是：一，少量的自噬小体和自体溶酶体产生，二，少量的线粒体产生(M)。在TBI后24小时到15天（图1C到1F）神经元内自噬小体和自噬溶酶体明显积聚，而多核糖体(箭头所指)，核(N)，高尔基复合体(G)和线粒体(M)显示正常。

在TBI后不同时期的神经元里，能清楚的观察到个别成熟的自噬小体形态特征(Figure 2)。

自噬体形成始于隔离膜片，也就是说，在TBI后的细胞质里形成双膜泡(Figure 2A, rrows)。

双膜泡围住包膜或整个细胞器形成自噬小体(Figure 2A, AP)。

双膜泡和自噬小体的形成最早在TBI后4小时可见(Figure 2A)，并且持续到TBI之后的整个阶段。(see biochemical analysis below)。

在TBI后，在树枝状中继线(Figure 2C)和充足的神经区域(Figure 2B)中常见到提示线粒体损伤的包含膜螺纹的自噬体群。最后自噬体与溶酶体结合形成自噬溶酶体，(图2D)

自噬溶酶体内的部分降解物分布为密集的不均匀的黑斑，最后成为均匀密集分布的黑斑。(图2A)