3.1 Ischemia-induced changes of postsynaptic components in the CA1 subfield
Relative to sham-operated control rats, the protein levels of MAP2 and PSD95 were significantly reduced in the CA1 subfield in ischemic animals at day 2 and 7 after I/R (Fig. 1A and 1B). Similarly, the ratio of F/G in the CA1 subfield was reduced from 0.84 in sham-operated controls to 0.61 and 0.37 in ischemic animals at day 2 and 7 after I/R, respectively (Fig. 1C).
Under the conventional fluorescence microscope, the positive signals for MAP2 or F-actin were distributed throughout the hippocampus except for the layers containing neuronal cell bodies in sham-operated control rats (Fig. 2A and 2F). After I/R, MAP2 and F-actin were both selectively lost in the CA1 subfield in ischemic animals from day 2 (Fig. 2C and 2G), and became progressively severe over time (Fig. 2D-2E, and 3C-3D).
Under the laser scanning confocal microscope, MAP was distributed in the proximal and distal dendrites of CA1 pyramidal cells (Fig. 2A and 2E), while F-actin was located predominantly in the dendritic spines of CA1 pyramidal cells in sham-operated control rats (Fig. 2A1). Double fluorescence labeling revealed little overlapping between F-actin and MAP2 (Fig. 3A2).
After I/R, MAP2 first disappeared from the distal branches, and finally from the proximal branches of the dendrites (Fig. 2B, 2C, and 2D), and few fluorescence signals for MAP2 were detected in the CA1 stratum radiatum in the later stage of I/R (Fig. 3D). As previously described by Guo et al. (2019), global cerebral ischemia induced progressive decrease in F-actin in the dendritic spines, but the F-actin in the dendrites was dramatically increased at day 1.5 and 2 (Fig. 2B1, 2C1, 3B2 and 3C2). The F-actin-positive dendrites eventually disappeared in CA1 stratum radiatum, but some tubular or circular structures, which were positively labeled for both F-actin and microglial Iba1 (Fig. 4), became prominent in this region (Fig. 3D1).
The content of F-actin in microglia was very low in the sham-operated control and ischemic animals in the early stage of I/R, probably due to the small number and size of rest microglia (Fig. 4A, 4A1–4A2, and 4B1). From day 3 to 7 after I/R, microglia were dramatically increased in both number and size, and F-actin was concomitantly increased in the microglia (Fig. 4C − 4D, and 4C1-4D1). F-actin was localized predominantly in the peripheral cytoplasm of activated microglia, showing circular or tubular shapes (Fig. 4C2-4D2).
Quantitative analysis showed that the puncta number of F-actin per unit area was significantly decreased in CA1 stratum radiatum in ischemic animals at day 1.5 after I/R (Figs. 5B, 5B1, and 5F), but the area fraction of F-actin was not significantly changed at this time point (Fig. 5C), relative to sham-operated control rats (Fig. 5A and 5A1). Due to the aggregation of F-actin into threadlike structures, the mean size of F-actin-positive structures became much larger in ischemic animals (Figs. 5B, 5B, and 5G).
The puncta number per unit area and the area fraction of F-actin became progressively reduced in the CA1 stratum radiatum in ischemic animals from day 2 to 7 after I/R (Fig. 5C-5E, and 5C1-5E1), while the mean size of F-actin-positive structures became significantly increased from day 2 to 7 (Fig. 5G). Since F-actin in microglia was not excluded from the quantitative analysis, the number of the remaining dendritic spines was much lower than that measured by counting the F-actin puncta, especially in the later stage of I/R.
3.2 Ischemia-induced change of presynaptic components in CA1 stratum radiatum
Relative to sham-operated control rats, no significant change was detected for synapsin I at the protein level in the CA1 subfield in ischemic animals (Fig. 1D).
Under the conventional fluorescence microscope, synapsin I was evenly distributed throughout the hippocampus except for the pyramidal cell bodies and their dendrites (Fig. 2K). Relative to sham-operated control rats, the conventional fluorescence microscopy did not reveal any remarkable changes of synaspin I in either fluorescence intensity or staining pattern within the first week following ischemia (Fig. 2L -2O).
Under the laser scanning confocal microscope, the positive signals for synapsin I was observed as discrete dots in CA1 stratum radiatum (Fig. 3E1). The dendrites, which contained no synapsin I in their cytoplasm, exhibited “silhouettes” due to negative staining (Fig. 3E, 3E1, and 3E2).
In ischemic rats, the punctate labeling of synapsin I became slightly sparse in CA1 stratum radiatum from day 2 after I/R, while the sizes of synapsin I-positive puncta were remarkably increased from day 2 after I/R (Fig. 3F1-3H1). At day 1.5 after I/R, quantitative analysis showed that synapsin I was not significantly changed in puncta number in ischemic animals (Figs. 5J, 5J1, and 5N), relative to the sham-operated control (Fig. 5I and 5I1). However, the puncta size of synapsin I became significantly reduced at this time point (Fig. 5O) so that the area fraction of synapsin I became significantly decreased (Fig. 5P).
From day 2 to 7 after I/R, the puncta number of synapsin I per unit area became significantly reduced in CA1 stratum radiatum in ischemic animals (Figs. 5K-5M, 5K1-5M1, and 5O). However, due to the increased puncta size, the mean area fraction of synaspin I was not significantly changed in CA1 stratum radiatum in ischemic animals (Fig. 5P), relative to the sham-operated control. Due to the more significant decrease of F-actin in puncta number, the ratio of synapsin I/F-actin puncta number became significantly increased from day 1.5 to 7 after I/R (Fig. 5G).
Since some dendrites could still be identified due to the uneven labeling for MAP2 in their shafts (Fig. 3F and 3G), many large puncta of synapsin I were found terminating onto the shafts of these dendrites (arrows in Fig. 3F2-G2).