We report that chronic sublethal hypoxia in newborn mice produces an initial 30% deficit in cortical neurons, two thirds of which are excitatory neurons expressing the transcription factor Tbr1. Over the ensuing four weeks in normoxic conditions, the deficit in neuron number recovers, such that neither NeuN+, Tbr1+ nor SMI-32+ neurons are decreased in the cortex of hypoxic-reared mice at P48. However, there is an enduring loss in PV+ and CR+ inhibitory interneurons in the hypoxic mice, which creates an imbalance between excitatory and inhibitory neurons in the hypoxic cortex. Disrupting the Fgfr1 gene in GFAP+ cells of the developing dorsal telencephalon (including cortical radial glial cells and all their progeny) does not alter the initial loss of cortical neurons but precludes the recovery of NeuN+, Tbr1+ and SMI-32+ neuron number in the cerebral cortex. Hypoxic exposure increases cell proliferation and the generation of Tbr1+ cortical excitatory neurons and that of OB granular neurons, processes that are absent or greatly attenuated in Fgfr1 cKO mice. Thus the juvenile cerebral cortex possesses an unsuspected capacity to recover from a postnatal hypoxic insult, which depends on the functioning of critical signaling systems, i.e., FGFR-1. As rodents are born earlier in the developmental program compared to primates, this hypoxic protocol may model injury during late human gestation and the neonatal period, as it typically occurs in prematurely born infants, as well as ensuing recovery processes in human childhood.
The reconstitution of excitatory cortical neuron number after the hypoxic insult is surprising, as excitatory neurons were previously thought to be generated only during embryogenesis. It is possible that the initial loss may be less profound than what is revealed by NeuN and Tbr1 neuron counts, since some of the damaged neurons may not express neuronal markers and may subsequently recover. We cannot exclude that our assessment of the initial extent of neuron loss may be an over-estimate due to such a process. Nevertheless, we consistently observed corresponding decreases in neuron number, brain weight and cortical thickness in the hypoxia-reared mice, in at least two genetic backgrounds () (Fagel et al., 2006
). Furthermore, we show that cell death is two fold higher the hypoxic brain, indicating probable cell losses.
We noticed that in the current mixed strain of mice, the total number of cortical neurons increases by almost 6 million cells between P10 and P48 in normoxic mice (compare and ), suggesting a high degree of cellular plasticity even in the normal juvenile mouse cerebral cortex. This is consistent with recent unbiased stereological studies suggesting that total neuron number progressively increases in the early postnatal rodent cerebral cortex, albeit to varying degrees in different mouse strains, and that there is delayed acquisition of cortical neuron features over the postnatal period (Lyck et al., 2007
). Thus, part of the postnatal increase in the number of cortical neurons may be attributable to a progressive accumulation of neuronal antigens, such as NeuN and SMI-32. Nonetheless, using BrdU birthdating assays, we report that part of the increase in cortical neuron number is attributable to the addition of new cortical neurons in the third postnatal week after birth. These BrdU+ neurons are unlikely to be the result of neuronal repair or impending neuronal death (Kuan et al., 2004
), because apoptotic cells would have been already eliminated 4 weeks after BrdU incorporation. Furthermore, we have previously shown (Zheng et al., 2004
) that neurons strongly immunolabeled with BrdU do not reflect ongoing DNA repair at these dosages of the nucleotide. Also supporting the idea of a hypoxia-induced proliferative effect as opposed to DNA repair are the increase in total number of cells and volume of the SVZ observed by us (Fagel et al., 2006
) and others (Plane et al., 2004
) and the absence of a correlation between apoptotic cell number and BrdU+ cells in the SVZ at different survival times after the hypoxic insult (Fagel et al., 2006
). Finally, we have detected postnatal addition of neurons to the immature cerebral cortex using genetic fate mapping, which allowed us to mark with β-galactosidase GFAP+ neural stem cells in vivo
(Ganat et al., 2006
In wild type hypoxic mice, BrdU/NeuN and BrdU/Tbr1 double positive neurons increased two-fold and three-fold, respectively, compared to age-matched normoxic mice. This can be attributed to hypoxia increasing the proliferation or survival of progenitors, particularly those committed to a Tbr1+ phenotype. Alternatively, hypoxia may increase the capacity of newly born, post-mitotic neurons to differentiate and survive in the cortical environment. The first hypothesis is supported by a nearly twofold increase in cell proliferation in the SVZ in hypoxia-exposed mice as compared to age-matched controls (this study, and Fagel et al., 2006
), and by previous reports suggesting that hypoxia-ischemia increases cell proliferation in the brain (Plane et al., 2004
; Ong et al., 2005
; Park et al., 2006
; Zhao et al., 2008
). Furthermore, neural precursor cells expressing the neurogenic transcription factors Sox2, Pax6 and Tbr2 in the SVZ appear to increase after hypoxia. In contrast, in the hypoxic Fgfr1 cKO mice, the SVZ proliferative response is absent, and the increase in Sox2-, Pax6- and Tbr2-immunoreactive cells is attenuated. These data highlight the central role of FGFR-1,
whose expression is restricted to glial cells (Belluardo et al., 1997
; Vaccarino et al., 2001
; Ganat et al., 2002
) (Gensat Project, http://www.gensat.org/index.html
) in promoting neuronal progenitor proliferation after postnatal hypoxic injury.
Dividing precursors in the SVZ and rostral migratory stream generate neurons for the OB (Luskin, 1993
; Alvarez-Buylla and Garcia-Verdugo, 2002
; Aguirre and Gallo, 2004
; Hack et al., 2005
) and FGF-2 (a ligand for FGFR1) is required for the maintenance of this proliferating cell pool (Zheng et al., 2004
). Consistently, Fgfr1 cKO mice are unable to properly upregulate OB neurogenesis in response to hypoxia. The failure to observe a statistically significant increase in density of NeuN+/BrdU+ cells in the OB of Fgfr1 cKO mice may be attributable in part to the large variability between animals; however, the mean increase in NeuN+/BrdU+ cells from normoxic to hypoxic cKO (70%) is much less than that observed wild type mice (236%), suggesting that if there was an increase in OB neurogenesis in the hypoxic cKO, it was more modest. Thus, the absence of a post-hypoxic proliferative response in the SVZ of Fgfr1 cKO mice likely underlies their decreased ability to regenerate OB neurons, but other events unrelated to Fgfr1
, such as cell survival, may play a compensatory role in regulating OB neurogenesis.
Fgfr1 cKO mice exposed to hypoxia suffer the same level of apoptosis and initial cortical cell loss as wild type mice, but fail to recover, exhibiting a persistent 31–33% deficit in total neurons and excitatory cortical neurons 5 weeks later. Furthermore, in the absence of Fgfr1
, the generation of cortical neurons from BrdU-labeled cells is not up-regulated in the recovery period. These data suggest that Fgfr1
is not involved in neuroprotection after injury, but rather, in the regenerative and reactive response. At present we do not understand whether the unresponsiveness of SVZ progenitors underlies the failure to boost cortical neurogenesis in the Fgfr1 cKO mice, as BrdU birthdating cannot reveal whether the progenitors that generate cortical neurons arise from the SVZ. It is possible that increased generation of NeuN+
cortical neurons occurs from proliferative progenitors in the cortex itself, as mature cortical astrocytes may re-enter the cell cycle and acquire multipotency in vitro
after cortical injury (Buffo et al., 2008
). Further, although Fgfr1
is clearly required for the hypoxia-induced increase in proliferation in the SVZ, it may play an additional role in fostering neuronal repair in the brain parenchyma, and thus contribute to the recovery of neurons damaged by hypoxia. For example, the loss of Fgfr1 may alter the ability of astroglial cells to regulate the exchange of molecules involved in energy regulation, or secrete trophic factors that might affect neurons or neuronal progenitors in a paracrine fashion.
In contrast to the recovery of Tbr1+ neuron number, hypoxia caused an enduring deficit in inhibitory cortical PV+ and CR+ interneurons (32% and 43%, respectively). We could not estimate the extent of the initial loss of PV+ and CR+ inhibitory neurons after hypoxia, since at P11 the maturation of calcium-binding protein immunoreactivities is not yet complete. Hence, we cannot ascertain whether the significant decreases in PV+ and CR+ cells present in hypoxia-reared mice represent an enduring loss, a failure to properly mature, or a progressive demise.
In conclusion, chronic neonatal hypoxia induces a chain of linked events, beginning with the upregulation of FGFR-1 expression in neural stem cells, which in turn may increase the generation of Tbr2+
proliferative neuronal progenitors. This work provides the first demonstration that, following neonatal chronic hypoxic injury, the initial loss of SMI-32- and Tbr1-expressing excitatory cortical neurons spontaneously recovers in the juvenile cerebral cortex. Further, glial FGFR-1 plays a central role in driving this recovery, as both the increased neurogenesis and reconstitution of normal cortical neuron number are not seen in mice that lack Fgfr1
cells. This reconstitution neuron number is stable 9 weeks after neonatal hypoxic injury (Fagel et al., 2006
). In contrast, we show that PV and CR inhibitory interneurons do not show a comparable degree of recovery, resulting in marked imbalance in excitatory/inhibitory neurons in the hypoxic cerebral cortex.