Noninvasive imaging of endogenous neural stem cell mobilization in vivo using positron emission tomography

Abstract

Neural stem cells reside in two major niches in the adult brain [i.e., the subventricular zone (SVZ) and the dentate gyrus of the hippocampus]. Insults to the brain such as cerebral ischemia result in a physiological mobilization of endogenous neural stem cells. Since recent studies showed that pharmacological stimulation can be used to expand the endogenous neural stem cell niche, hope has been raised to enhance the brain's own regenerative capacity. For the evaluation of such novel therapeutic approaches, longitudinal and intraindividual monitoring of the endogenous neural stem cell niche would be required. However, to date no conclusive imaging technique has been established. We used positron emission tomography (PET) and the radiotracer 3'-deoxy-3'-[(18)F]fluoro-l-thymidine ([(18)F]FLT) that enables imaging and measuring of proliferation to noninvasively detect endogenous neural stem cells in the normal and diseased adult rat brain in vivo. This method indeed visualized neural stem cell niches in the living rat brain, identified as increased [(18)F]FLT-binding in the SVZ and the hippocampus. Focal cerebral ischemia and subsequent damage of the blood-brain barrier did not interfere with the capability of [(18)F]FLT-PET to visualize neural stem cell mobilization. Moreover, [(18)F]FLT-PET allowed for an in vivo quantification of increased neural stem cell mobilization caused by pharmacological stimulation or by focal cerebral ischemia. The data suggest that noninvasive longitudinal monitoring and quantification of endogenous neural stem cell activation in the brain is feasible and that [(18)F]FLT-PET could be used to monitor the effects of drugs aimed at expanding the neural stem cell niche.

Figure 1.

[¹⁸F]FLT labels proliferating NSCs in vitro. A, Fetal rat cortical NSCs grown in monolayer cultures expressed the transcription factor SOX2, verifying their undifferentiated state. B, Rat hippocampal neurons expressed the neuron-specific cytoskeletal protein MAP2. C, Left, Proliferating NSCs incorporated the thymidine analog BrdU, whereas nonproliferating neurons did not. Right, The radiolabeled thymidine [¹⁸F]FLT was significantly better incorporated into proliferating NSCs than into nonproliferating neurons (all values displayed as means ± SEM).

Figure 2.

[¹⁸F]FLT-PET visualizes endogenous NSCs in vivo. A, Adult rats injected with [¹⁸F]FLT showed elevated tracer binding in the SVZ in vivo as a major NSC niche ([¹⁸F]FLT-PET matched on MRI-atlas of rat brain; white box in upper image is magnified in A′ below; scale bars, 1 mm). B, BrdU accumulation in proliferating cells in the SVZ corresponded with the [¹⁸F]FLT signal (objectives ×1, ×10; scale bar (in overview image), 1 mm). C, Elevated [¹⁸F]FLT binding was detected in the hippocampus of untreated rats in vivo ([¹⁸F]FLT-PET matched on MRI-atlas of rat brain; scale bar, 1 mm). D, Proliferation in both adult NSC niches, SVZ and hippocampus, was quantified in vivo by [¹⁸F]FLT-binding, with significantly increased SUVs for [¹⁸F]FLT in both SVZ and hippocampus compared to background binding (values displayed as means ± SEM).

Figure 3.

[¹⁸F]FLT-PET quantifies the expansion of the SVZ induced by pharmacological manipulations in vivo. A, Single intracerebroventricular injection of a cocktail containing Dll4 and insulin, with or without addition of FGF2, mobilized NSCs and expanded the SVZ as quantified by BrdU-incorporation. B, Histological quantitation of SVZ expansion. The width of SVZ was measured perpendicular to the ependymal surface as indicated in A 9 d after intracerebroventricular injection of vehicle, FGF2+Dll4+insulin, or Dll4 and insulin alone (p < 0.001 and p < 0.05, respectively, compared to the pool of all control animals; values displayed as means ± SEM). C, Single intracerebroventricular injection of a cocktail containing Dll4 and insulin, with or without addition of FGF-2, mobilized NSCs and expanded the SVZ as visualized by [¹⁸F]FLT-PET. Please note the different pseudocolor scaling of SUV compared to Figure 2. The subtle [¹⁸F]FLT accumulation in naive animal (right), as displayed in Figure 2, is missed after adjusting the pseudocolor to higher SUV in pharmacologically stimulated animals (left, middle). D, Quantitation of SVZ expansion by [¹⁸F]FLT binding, 9 d after intracerebroventricular injection of vehicle, FGF2+Dll4+insulin, or Dll4 and insulin alone (both p < 0.01, compared to the pool of control animals; values displayed as means ± SEM).

Figure 4.

Stroke leads to enhanced proliferation within the ischemic lesion. A, Focal cerebral ischemia caused characteristic signal changes in T₂-weighted MRI 1 week after induction of ischemia (brain outlined in white, infarct outlined in purple in A, C, E, F). B, Ischemic tissue damage verified histologically by H&E staining. C, Gadolinium-enhanced MRI 1 week after stroke outlined the disruption of the BBB. D, Histological verification of BBB damage by IgG-staining. E, Local elevation of [¹⁸F]FLT binding 1 week after stroke. F, Coregistration of MRI and PET illustrates that at 1 week after stroke, most [¹⁸F]FLT binding was localized in the ischemic lesion. G, Permanent ischemia (pMCAO) as well as transient ischemia (tMCAO) showed significantly elevated [¹⁸F]FLT binding compared to the respective contralateral side (values displayed as means ± SEM). H, BrdU staining revealed an accumulation of proliferating cells within the infarct as the immunohistochemical correlate of [¹⁸F]FLT binding (objectives ×10, ×100).

Figure 5.

Stroke-induced expansion of the NSC niches can be distinguished with PET. A, Coregistration of [¹⁸F]FLT-PET and gadolinium-enhanced MRI 1 week after stroke (infarct outlined in purple, for schematic overview see inset). [¹⁸F]FLT binding within the infarct core was repressed by image processing to emphasize comparatively weak tracer binding in the SVZ. B, Coregistration of [¹⁸F]FLT-PET and gadolinium-enhanced MRI 1 week after stroke shows [¹⁸F]FLT binding within the hippocampus region (for schematic overview see inset). C, After stroke, [¹⁸F]FLT binding was upregulated in both SVZ and hippocampus compared to control animals (values displayed as means ± SEM). D, Infarct sizes determined volumetrically in vivo correlated well with the proliferative activity in both NSC niches as assessed by [¹⁸F]FLT-PET.