Evidence for stroke-induced neurogenesis in the human brain

Results

Sections were obtained from human brain biopsies performed for the diagnosis of cerebral lesions that proved to be ischemic strokes (Table 1). The sections were stained with antibodies against markers of cell proliferation (proliferation-related Ki-67 antigen, Ki-67; Saccharomyces cereviseae minichromosome maintenance 2 homolog, MCM2; and proliferating cell nuclear antigen, PCNA) and neuronal lineage (doublecortin, DCX; TOAD/Ulip/CRMP family protein 4, TUC-4; embryonic nerve cell adhesion molecule, ENCAM; and βIII tubulin). Control specimens (n = 6), from the cerebral cortex of autopsied patients who died without brain pathology, contained ≤1 Ki-67-positive and no Ki-67/DCX-positive cells per mm². In stroke patients (n = 6), however, the cortical region adjacent to the infarct core (ischemic penumbra) contained 63 ± 16 Ki-67-positive cells and 10 ± 2 Ki-67/DCX-positive cells per mm² (mean ± SEM) (Table 1 and Fig. 1a). To confirm that Ki-67 labeling reflected recent cell proliferation, some sections were also stained for MCM2 (Fig. 1b) or PCNA (Fig. 1c), which were co-expressed in Ki-67-positive cells. Although dying neurons may aberrantly express cell-cycle proteins, fewer than 3% of Ki-67-positive cells in the ischemic penumbra showed evidence of caspase-3 cleavage, and these had distinctly abnormal nuclear morphology (Fig. 1d).

Because cell-cycle markers do not discriminate cells of different lineages, some sections were stained with lineage-specific markers. Markers expressed in new neurons, including DCX (Fig. 2a) and βIII tubulin (Fig. 2b), were localized to the ischemic penumbra. Moreover, DCX (Fig. 2c), βIII tubulin (Fig. 2d), and TUC-4 (Fig. 2e) were detected in cells that also expressed Ki-67, and multiple immature neuronal markers were expressed in a given cell (Fig. 2f), confirming that these were newborn neurons. Consistent with this interpretation, some Ki-67/DCX-positive cells exhibited a migratory phenotype consisting of an elongated cellular profile with a leading edge and trailing nucleus (Fig. 2g).

In the hippocampal dentate gyrus (which, together with the subventricular zone, accounts for most neurogenesis in the adult brain), new neurons arise in proximity to blood vessels, which are thought to provide a “vascular niche” that supports their proliferation (10). A similar relationship was observed in stroke brain specimens, in which DCX-positive cells tended to cluster near blood vessels labeled with antibodies against nestin (Fig. 3a), von Willebrand factor (Fig. 3b), or vascular smooth muscle actin (Fig. 3c). This finding may relate to observations that vascular endothelial cells promote neurogenesis in vitro (11) and that vascular endothelial growth factor, which stimulates the proliferation of neurons as well as endothelial cells (12), is induced in the brain after stroke (13). However, whether these new neurons arise locally or migrate from canonical neuroproliferative regions such as the subventricular zone cannot be resolved on the basis of biopsies restricted to the ischemic brain area.

Discussion

Neurogenesis persists in the adult mammalian brain, where it can be stimulated by physiological factors, such as growth factors and environmental enrichment, and by pathological processes, including ischemia and neurodegeneration. Although these influences are more difficult to demonstrate in humans than in experimental animals, evidence for injury-induced human neurogenesis has been presented previously in Huntington’s disease (14) and Alzheimer’s disease (15). Here, we report similar evidence for stroke-induced neurogenesis in human brain. Standard methods used to detect neurogenesis in animals, such as the administration of BrdU or GFP-expressing viral vectors, cannot be used in humans. Therefore, we relied on the expression of endogenous-cell-proliferation markers and neuronal-lineage markers to identify putative newborn neurons in the cortical ischemic penumbra of patients with stroke. These markers have limitations in that their relationship to neurogenesis has been characterized in most detail in rodents, and some of them may also be expressed in injured cells. However, we observed cells that stained for multiple early neuronal markers, showed normal nuclear morphology, did not stain for caspase-cleavage products, had features consistent with a migratory cell phenotype, were present in the ischemic penumbra but not in the ischemic core (where injured cells would be expected to be found), and were situated preferentially in the vicinity of patent (erythrocyte-containing) blood vessels (which is not where ischemic cells would be anticipated). It is possible that all of these attributes are associated with ischemic injury rather than neurogenesis, but it seems unlikely.

In rodent models of stroke, the source of new neurons that migrate to ischemic brain areas appears to be the subventricular zone (6–9). In human biopsy specimens, the source of these cells is unclear, but their perivascular location may provide clues. One possibility is that newborn neurons migrate from elsewhere (such as the subventricular zone), using blood vessels as scaffolds for migration or as destination markers. Alternatively, these neurons could arise locally, although whether neurogenesis can occur in situ in primate cerebral neocortex is controversial (16, 17). The extent to which stroke-induced neurogenesis in human brain results in the production of functional neurons with the capacity to integrate into brain circuitry is also uncertain and is not addressed by our findings.

Several drugs can stimulate neurogenesis in the dentate gyrus or subventricular zone of ischemic rodent brain, including statins (18) and medications used to treat erectile dysfunction (19). Because none of the patients we studied was known to have taken any of these drugs, the increased neurogenesis we observed is unlikely to have been drug-induced. On the other hand, the ability of drugs and environmental alterations to promote neurogenesis suggests clinical interventions that might be used to enhance this process. Although its functional impact is as yet unknown, postischemic neurogenesis in the human brain may represent both an endogenous repair mechanism and a therapeutic target for stroke.

Materials and Methods