Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain.
Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain.
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DOI：
10.3390/ijms21238951
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发表时间：
2020-11-25
影响因子：
5.6
通讯作者：
Naumova AV
中科院分区：
文献类型：
--
作者：
Khodanovich MY;Akulov AE;Anan'ina TV;Kudabaeva MS;Pishchelko AO;Krutenkova EP;Nemirovich-Danchenko NM;Svetlik MV;Tumentceva YA;Van den Haute C;Gijsbers R;Daniëls V;Thiry I;Pershina AG;Shadrina MM;Naumova AV
(1)背景：神经发生被认为是一种潜在的脑修复机制，在卒中中增强。仅从不同动物脑损伤和恢复不同阶段的组织切片重建神经发生过程是困难的。组织特异性可视化探针的发展将使神经发生的研究受益匪浅。(2)目的：该研究旨在探索在doublecortin启动子下铁蛋白（一种无毒的铁结合蛋白）的过表达是否可用于使用磁共振成像（MRI）进行神经发生的非侵入性可视化。(3)研究方法：铁蛋白重链（FerrH）在病毒构建体AAV-pDCX-FerrH中在双皮质素启动子（pDCX）下在腺相关病毒骨架（AAV）中表达，其对年轻神经元特异。增强型绿色荧光蛋白（eGFP）的表达用作表达对照（AAV-pDCX-eGFP）。将病毒载体或磷酸盐缓冲盐水（PBS）脑内注射到18只成年雄性Sprague-Dawley大鼠中。注射前3天，大鼠进行短暂的大脑中动脉闭塞或假手术。在手术前和注射后第7、14、21和28天，使用Bruker BioSpec 11.7 T扫描仪对动物进行体内MRI研究。对注射后第28天获得的脑切片进行铁蛋白、年轻（DCX）和成熟（NeuN）神经元以及活化的小胶质细胞/巨噬细胞（CD 68）的免疫染色。此外，进行RT-PCR以确认铁蛋白表达。(4)结果如下：注射AAV-pDCX-FerrH的动物的缺血后脑中的T2* 图像显示，从病毒注射后14天开始，在缺血性病变中以及侧脑室和脑室下区（SVZ）附近的病变半球中存在两个不同的MRI信号低强度区。在假手术动物，只有一个低信号区附近的侧脑室和SVZ显示。免疫组化结果显示，缺血灶铁蛋白表达细胞为巨噬细胞（88.1%），而缺血和假手术后侧脑室铁蛋白表达细胞多为成熟神经元（分别为55.7%和61.8%）和年轻神经元（分别为30.6%和7.1%）。RT-PCR证实尾壳核和胼胝体中铁蛋白的表达上调。令人惊讶的是，在注射AAV-pDCX-eGFP的动物中，我们在T2* 图像上类似地观察到两个低信号区。细胞研究还显示，在缺血后和假手术动物中，侧脑室附近存在成熟（81.5%）和年轻神经元（6.1%），而缺血性病变中的巨噬细胞铁蛋白阳性（98.2%）。(5)结论：通过使用T2* 加权图像的MRI检测由注射AAV-pDCX-FerrH诱导的铁蛋白过表达，这通过免疫化学证实，显示年轻和成熟神经元中的铁蛋白。eGFP的表达也导致了T2* 加权图像中MR信号强度的相当降低。需要进一步的研究来调查在MRI研究中使用eGFP和铁蛋白表达的潜在和组织特异性特征。
(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.
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DOI：
10.1073/pnas.0510419103
发表时间：
2006-01-31
影响因子：
11.1
作者：
Ge, WH;He, F;Sun, YE
通讯作者：
Sun, YE
影响因子：
3.7
作者：
Elvira G;García I;Benito M;Gallo J;Desco M;Penadés S;Garcia-Sanz JA;Silva A
通讯作者：
Silva A
影响因子：
3
作者：
Li, Xuan;Wolf, Marina E.
通讯作者：
Wolf, Marina E.
影响因子：
16.6
作者：
Marschallinger J;Schäffner I;Klein B;Gelfert R;Rivera FJ;Illes S;Grassner L;Janssen M;Rotheneichner P;Schmuckermair C;Coras R;Boccazzi M;Chishty M;Lagler FB;Renic M;Bauer HC;Singewald N;Blümcke I;Bogdahn U;Couillard-Despres S;Lie DC;Abbracchio MP;Aigner L
通讯作者：
Aigner L
影响因子：
8.3
作者：
BEDERSON, JB;PITTS, LH;BARTKOWSKI, H
通讯作者：
BARTKOWSKI, H