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(1) Background: Neurogenesis is considered to be a potential brain repair mechanism and is enhanced in stroke. It is difficult to reconstruct the neurogenesis process only from the histological sections taken from different animals at different stages of brain damage and restoration. Study of neurogenesis would greatly benefit from development of tissue-specific visualization probes. (2) Purpose: The study aimed to explore if overexpression of ferritin, a nontoxic iron-binding protein, under a doublecortin promoter can be used for non-invasive visualization of neurogenesis using magnetic resonance imaging (MRI). (3) Methods: Ferritin heavy chain (FerrH) was expressed in the adeno-associated viral backbone (AAV) under the doublecortin promoter (pDCX), specific for young neurons, in the viral construct AAV-pDCX-FerrH. Expression of the enhanced green fluorescent protein (eGFP) was used as an expression control (AAV-pDCX-eGFP). The viral vectors or phosphate-buffered saline (PBS) were injected intracerebrally into 18 adult male Sprague-Dawley rats. Three days before injection, rats underwent transient middle-cerebral-artery occlusion or sham operation. Animals were subjected to In vivo MRI study before surgery and on days 7, 14, 21, and 28 days after injection using a Bruker BioSpec 11.7 T scanner. Brain sections obtained on day 28 after injection were immunostained for ferritin, young (DCX) and mature (NeuN) neurons, and activated microglia/macrophages (CD68). Additionally, RT-PCR was performed to confirm ferritin expression. (4) Results: T2* images in post-ischemic brains of animals injected with AAV-pDCX-FerrH showed two distinct zones of MRI signal hypointensity in the ipsilesioned hemisphere starting from 14 days after viral injection-in the ischemic lesion and near the lateral ventricle and subventricular zone (SVZ). In sham-operated animals, only one zone of hypointensity near the lateral ventricle and SVZ was revealed. Immunochemistry showed that ferritin-expressing cells in ischemic lesions were macrophages (88.1%), while ferritin-expressing cells near the lateral ventricle in animals both after ischemia and sham operation were mostly mature (55.7% and 61.8%, respectively) and young (30.6% and 7.1%, respectively) neurons. RT-PCR confirmed upregulated expression of ferritin in the caudoputamen and corpus callosum. Surprisingly, in animals injected with AAV-pDCX-eGFP we similarly observed two zones of hypointensity on T2* images. Cellular studies also showed the presence of mature (81.5%) and young neurons (6.1%) near the lateral ventricle in both postischemic and sham-operated animals, while macrophages in ischemic lesions were ferritin-positive (98.2%). (5) Conclusion: Ferritin overexpression induced by injection of AAV-pDCX-FerrH was detected by MRI using T2*-weighted images, which was confirmed by immunochemistry showing ferritin in young and mature neurons. Expression of eGFP also caused a comparable reduced MR signal intensity in T2*-weighted images. Additional studies are needed to investigate the potential and tissue-specific features of the use of eGFP and ferritin expression in MRI studies.

Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain