BrdU-positive cells in the neonatal mouse hippocampus following hypoxic-ischemic brain injury

Abstract

Background: Mechanisms that affect recovery from fetal and neonatal hypoxic-ischemic (H-I) brain injury have not been fully elucidated. The incidence of intrapartum asphyxia is approximately 2.5%, but the occurrence of adverse clinical outcome is much lower. One of the factors which may account for this relatively good outcome is the process of neurogenesis, which has been described in adult animals. We used a neonatal mouse model to assess new cells in the hippocampus after H-I injury.

Results: Neonatal mice underwent permanent unilateral carotid ligation on the seventh postnatal day followed by exposure to 8% hypoxia for 75 minutes. The presence of new cells was determined by bromodeoxyuridine (BrdU) incorporation into cells with sacrifice of the animals at intervals. Brain sections were stained for BrdU in combination with neuronal, glial, endothelial and microglial stains. We found a significant increase in BrdU-positive cells in the neonatal mouse hippocampus in the injured area compared to the non-injured area, most prominent in the dentate gyrus (DG) (154.5 +/- 59.6 v. 92.9 +/- 32.7 at 3 days after injury; 68.9 +/- 23.4 v. 52.4 +/- 17.1 at 35 days after injury, p < 0.0011). Among the cells which showed differentiation, those which were stained as either microglial or endothelial cells showed a peak increase at three days after the injury in the DG, injured versus non-injured side (30.5 +/- 17.8 v. 2.7 +/- 2.6, p < 0.0002). As in the adult animal, neurogenesis was significantly increased in the DG with injury (15.0 +/- 4.6 v. 5.2 +/- 1.6 at 35 days after injury, p < 0.0002), and this increase was subsequent to the appearance of the other dividing cells. Numbers of new oligodendrocytes were significantly higher in the DG on the non-injured side (7.0 +/- 24.2 v. 0.1 +/- 0.3, p < 0.0002), suggesting that oligodendrocyte synthesis was reduced in the injured hippocampus.

Conclusion: These findings demonstrate that the neonatal animal responds to brain injury with neurogenesis, much like the adult animal. In addition, H-I insult leads to more neurogenesis than hypoxia alone. This process may play a role in the recovery of the neonatal animal from H-I insult, and if so, enhancement of the process may improve recovery.

Figure 1

Schematic diagram showing BrdU injection schedule

Figure 2

Hippocampal injury following hypoxic-ischemic injury in a P17 mouse. A – HI injury (small arrows), triple filter 100X; B – colocalization with NeuN and BrdU, triple filter, 400X; C – BrdU label alone, Cy3 filter, 400X; D – NeuN label alone, FITC filter, 400X; and E – Hoechst counterstain, DAPI filter, 400X. B through E shows the same field. The large arrows show a neuron with BrdU and NeuN staining, demonstrating that the cell has recently divided. The circles show the three areas where the counting was performed.

Figure 3

Three dimensional reconstructed confocal microscopy demonstrating colabeling BrdU positive and NeuN positive cell in granule cell layer of HI injured hippocampus (P42). Orthogonal view. The neurons are labeled with NeuN (green), and the BrdU positive cells are shown in red. The square is a the Z slice, the superior rectangle is the X slice, and the right rectangle of the Y slice. A and B show two different cells, both co-labeled with BrdU and NeuN.

Figure 4

200× magnification of TUNEL staining developed with stable DAB in the injured hippocampus of a ten day old animal (P10), showing a small amount of label. Brown coloration indicates 3'OH DNA termini (apoptotic or necrotic cells).

Figure 5

200× magnification of TUNEL staining developed with stable DAB in the injured hippocampus of a 42 day old animal (P42), showing no labeling of apoptotic or necrotic cells. Brown coloration indicates 3'OH DNA termini (apoptotic or necrotic cells).