Morphological and Phagocytic Profile of Microglia in the Developing Rat Cerebellum

Morphological profile of microglia in the postnatal developing cerebellum. A, The frequency of round/amoeboid microglia significantly decreased after the first postnatal week (B), as well as stout microglia. C, Conversely, the density of microglia with thick processes increased only during the second postnatal week followed by a decrease in the third postnatal week. D, The density of microglia with thin processes gradually increased after the first postnatal week doubling their density by the third postnatal week. E, Sagittal views of the midvermis, labeled with Iba1, across the first 3 postnatal weeks. All data are expressed as mean ± SEM (n = 4, 2 males + 2 females for each group). Significant differences are detonated by *p < 0.05, **p < 0.01, and ***p < 0.000 compared with P5. Insets depict a higher magnification of selected microglia (red squares) in each panel. Scale bars: gray scale images, 100 µm; color images (inset), 25 µm; E, 500 µm (from P5 to P21). Images in A, B, C, and D depict the morphology of microglia at two different postnatal ages: P7 (A, B) and P12 (C, D).

Microglial phagocytosis in the postnatal developing cerebellum. A, The highest density of phagocytic cups was observed during the third postnatal week at P17 (***p < 0.000 compared with P5, P7, P10, P12, P14, and P21; n = 4, 2 males + 2 females for each group). Data are expressed as mean ± SEM. B, Phagocytic cups exhibited by microglia (red arrows) in the developing cerebellum at P17. Scale bar, 100 µm. C, Microglia with phagocytic cups (top) or microglia without phagocytic cups (bottom row) at different time points during postnatal development. Scale bars, 25 µm.

Microglia location in the cerebellar cortex based on morphological classification. A, Total microglia were significantly higher in the GL than the ML during the second and third postnatal week in the cerebellum (*p < 0.05, **p < 0.01, ***p < 0.000). B, The density of round/amoeboid microglia was very low and did not differ between the ML and the GL from P12 to P21. C, The density of stout microglia was significantly higher in the ML than the GL at all days examined except P14 (**p < 0.01, ***p < 0.000). D, Microglia with thick processes were the most abundant but did not differ between the ML and the GL. E, There were significantly more microglia with thin processes in the GL than the ML at P17 and P21 but not at younger ages examined (*p < 0.05, ***p < 0.000). All data are expressed as mean ± SEM (n = 6, 3 males + 3 females for each group).

Frequency of phagocytosis by microglia changes by location in the cerebellar cortex across development. A, The density of phagocytic cups was higher in the ML than the GL at P12 (**p < 0.01), and P14 (*p < 0.05), but switched at P17 (***p < 0.000) and P21 (*p < 0.05), so that the GL exhibited more phagocytic cups than the ML. The highest density of phagocytic cups was found in the GL at P17 compared with P12, P14, and P21 (^@p < 0.000). Scale bar, 100 µm. B, Proportion of microglia that exhibited phagocytic cups in the GL at P17: 67% of all phagocytic microglia had thick processes and 33% had thin processes. No round/amoeboid or stout microglia showed phagocytic cups. C, A difference in the density of phagocytic cups was found at younger ages (P15, **p = 0.003; P16, *p = 0.05) compared with P17, but no significant differences were found at older ages (P18, p = 0.583; P19, p = 0.615). In contrast, in the ML, the density of phagocytic cups was lower only at P19 (^p = 0.043) compared with P17. Additionally, a difference in the density of phagocytic cups between the GL and ML was found from P16 to P19 (P16, ^#p < 0.000; P17; ⁺p < 0.000; P18, ^&p < 0.000; P19, ^@p < 0.000) but not at P15 (p = 0.467) (n = 6, 3 males + 3 females for each group for A, B, and C). In this experiment the density of phagocytic cups was not counted in animals at P21 but the dashed lines depict the pattern previously observed at the end of the third postnatal week in both the GL and ML (Fig. 5A ). D, The diameter of microglial phagocytic cups was bigger on P17 compared with P10 (^@p = 0.06; see effect size estimation in Table 2), P14 (*p < 0.000) and P21 (^#p = 0.003). All data are expressed as mean ± SEM (^n = 4, 2 males + 2 females; *n = 8, 4 males + 4 females: ^P10, *P14, *P17, and *P21).

Identification of pyknotic bodies by Nissl staining in the postnatal developing cerebellum. A, The density of pyknotic bodies (red arrows) decreased only in the GL after the first postnatal week at P14, P17 and P21 (***p < 0.000), but not at P7 (p = 0.302), compared with P5. No changes in the density of pyknotic bodies were detected in the ML across the developmental time points analyzed when compared with P7 (P5, p = 0.199; P14, p = 0.688; P17, p = 0.487; P21, p = 0.375). The GL exhibited more pyknotic bodies than the ML only during the first postnatal week at P5 (^#p < 0.000) and P7 (^p < 0.000). Scale bar, 25 µm. Data are expressed as mean ± SEM (*n = 6, 3 males + 3 females; ^n = 4, 2 males + 2 females: *P5, ^P7, *P14, *P17, and *P21). B, P7 cerebellar sagittal section stained with cresyl violet showing pyknotic bodies pointed out by red arrows. Pk, Purkinje layer; EGL, external granular layer. C, Confocal colocalization of a pyknotic body (fragmented nucleus in yellow) and a phagocytic cup (red) in the cerebellar cortex at P17. Scale bars, 15 µm. D, 3D confocal image depicting a colocalization of a microglial phagocytic cup (red) and a cleaved caspase-3-positive cell (green) at the tip of a microglia process (white arrow).