Lengthening the G(1) phase of neural progenitor cells is concurrent with an increase of symmetric neuron generating division after stroke

Abstract

The proportion of neural progenitors that remain in (P fraction) and exit from (Q fraction) the cell cycle determines the degree of neurogenesis. Using S-phase labeling with 5-bromo-2'-deoxyuridine and a double nucleoside analog-labeling scheme, we measured the cell-cycle kinetics of neural progenitors and estimated the proportion of P and Q fractions in the subventricular zone (SVZ) of adult rats subjected to stroke. Stroke increased SVZ cell proliferation, starting 2 days, reaching a maximum 4 and 7 days after stroke. The cell-cycle length (T(C)) of SVZ cells changed dynamically over a period of 2 to 14 days after stroke, with the shortest length of 11 h at 2 days after stroke. The reduction of the T(C) resulted from a decrease of the G(1) phase because the G(2), M, and S phases were unchanged. In addition, during this period, reduction of the G(1) phase was concomitant with an increase in the P fraction, whereas an augmentation of the Q fraction was associated with lengthening of the G(1) phase. Furthermore, approximately 90% of cells that exited the cell cycle were neurons and the population of a pair of dividing daughter cells with a neuronal marker increased from 9% at 2 days to 26% at 14 days after stroke. These data suggest that stroke triggers early expansion of the progenitor pool via shortening the cell-cycle length and retaining daughter cells within the cell cycle, and the lengthening of G(1) leads to daughter cells exiting the cell cycle and differentiating into neurons.

Figure 1

Stroke changes of BrdU-labeling index (LI) determined by cumulative labeling with BrdU in SVZ cells. (A to D) Linear ascending of LI toward the plateau in normal rats and rats subjected to stroke. (A to D) The time at which BrdU reached a maximum (T_C-T_S) was 14 h in rats without stroke. However, the maximum (T_C-T_S) was (A)9,(A) 10, (C) 11, and (D) 15 h at (A)2,(B)4,(C)7, and (D) 14 days after stroke, respectively. The percentage of cells proliferating (GF) was 20% in non-stroke and (A) 24% (2 days), (B) 30% (4 days), (C) 31% (7 days), and (D) 24% (14 days) in stroke.

Figure 2

CldU- and IdU-positive cells in the SVZ were used for determining the combined Q + P fractions (protocol 1) and the Q fraction (protocol 2). (A and B) CldU- and ldU-positive SVZ cells in rats after the protocol 1 and 2 labeling, respectively. (A) CldU-positive cells (green, arrows) indicate cells that remained or left the cell cycle (P + Q fractions, N_P+Q) during the interval between injection of IdU and killing time. (A) IdU-positive cells (red, arrows) represent cells that entered S phase during 2 h interinjection interval. (A) CldU- and IdU-positive cells (yellow, arrows) are cells that remained in the S phase during the interval. In (B) protocol 2, rats were injected with additional IdU within a time, which was longer than that the duration of the cell cycle minus the duration of S phase. Therefore, (B) CldU-positive cells (green, arrows) are cells that exit the cell cycle (Q fraction, N_Q), whereas (B) CldU- and IdU-positive cells (yellow, arrows) are cells that re-entered the S phase. Images were acquired from representative rats without stroke. Bar = 20 μm. LV, lateral ventricle.

Figure 3

CldU- and IdU-positive cells are evenly distributed in the expanded SVZ. (A) An increase in the number of CldU-(green) and IdU-(red) positive cells increased in the SVZ 7 days after (A) stroke compared with the number in the SVZ of (B) non-stroke rat. The area of the ipsilateral SVZ expanded 7 days after (C) stroke compared with the area of the SVZ in (D) non-stroke rat. (E) Changes of P and Q fractions over a period of 2 to 14 days after stroke. Bar = 100 μm. CC, corpus callosum; LV, lateral ventricle and Str., striatum.

Figure 4

Cells that exited the cell cycle exhibit the neuronal phenotype. Microscopic images of triple immunofluorescent staining were obtained from a representative rat subjected to dual S-phase labeling with protocol 2 at 14 days after stroke. (A and D) CldU-positive cells (arrows) were (C and D) DCX-positive (arrows) but not (B) IdU-positive. Bar = 10 μm. LV, lateral ventricle.

Figure 5

P and Q in relation to proliferative fate predicted by the mathematical model over a period of 2 to 14 days after stroke.

Figure 6

Dividing daughter cells exhibit neuronal phenotype. (A and D) Telophase HH3⁺ cells in the ipsilateral SVZ (red, double arrows) were (B and D) DCX⁺ (green, arrows). An arrowhead in A indicates that a dividing HH3⁺ cell was not (D) DCX⁺ (arrowhead). (C) 4′,6′-Diamidino-2-phenylindole staining revealed that HH3⁺ cells were in telophase of the cell cycle (double arrows). An asterisk in A, B, and D shows a HH3⁺ cell that was not counted because it was not in telophase. Bar = 20 μm. LV, lateral ventricle.