A new minimally-invasive method for microinjection into the mouse spinal dorsal horn.
A new minimally-invasive method for microinjection into the mouse spinal dorsal horn.
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DOI：
10.1038/srep14306
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发表时间：
2015-09-21
影响因子：
4.6
通讯作者：
Tsuda M
中科院分区：
文献类型：
--
作者：
Kohro Y;Sakaguchi E;Tashima R;Tozaki-Saitoh H;Okano H;Inoue K;Tsuda M
非侵入性基因递送到脊髓背角（SDH）仍然具有挑战性，因为现有的直接显微注射载体的方法需要椎板切除术，这导致组织损伤和炎症。这种反应可能会妨碍准确读出引入基因的细胞和行为效应。在这里，我们开发了一种新的微创SDH显微注射技术，而不需要椎板切除术，其中微毛细管插入到SDH实质通过椎间隙。使用这种方法，我们显微注射腺相关病毒与星形胶质细胞启动子到SDH，并实现了有效的基因表达的星形胶质细胞特异性的方式没有神经胶质细胞增生，神经元丢失或炎症。此外，通过分别表达STAT3的显性负性形式和组成型活性形式的转录因子STAT3的星形胶质细胞功能丧失和获得，证明了星形胶质细胞STAT3在维持周围神经损伤后的神经性疼痛中的必要性和充分性，这是一种使人衰弱的慢性疼痛状态，其中目前可用的治疗通常无效。因此，我们的技术能够操纵基因表达的细胞类型和空间特异性的方式没有不良影响，并可能是有用的SDH生理和病理学的研究。
Noninvasive gene delivery to the spinal dorsal horn (SDH) remains challenging because existing methods to directly microinject vectors require laminectomy, which leads to tissue damage and inflammation. Such responses might hamper accurate readouts of cellular and behavioural effects of an introduced gene. Here we develop a new minimally-invasive SDH microinjection technique without the need of laminectomy in which a microcapillary is inserted into the SDH parenchyma through an intervertebral space. Using this method, we microinjected adeno-associated virus with an astrocytic promoter into the SDH and achieved efficient gene expression in an astrocyte-specific manner without gliosis, neuronal loss or inflammation. Furthermore, astrocytic loss- and gain-of-function of the transcription factor STAT3 by expressing a dominant-negative form and a constitutive-active form of STAT3, respectively, demonstrated the necessity and sufficiency of astrocytic STAT3 in the maintenance of neuropathic pain following peripheral nerve injury, a debilitating chronic pain state in which currently available treatments are frequently ineffective. Thus, our technique enables manipulation of gene expression in cell type- and spatial-specific manners without adverse effects, and may be useful for research in SDH physiology and pathology.