Yaida Y, Nowak T S
Department of Neurology, University of Tennessee, Memphis, USA.
Regul Pept. 1995 Oct 20;59(2):193-9. doi: 10.1016/0167-0115(95)00100-p.
The distribution and stability of exogenously administered oligonucleotides (oligos) are important variables determining the potential utility of antisense oligos as agents for modifying gene expression within a given brain region in vivo. In the present study, phosphodiester (PO) and phosphorothioate (PS) oligos antisense with respect to a recently cloned rat hsp70 sequence were localized in rat brain following intraventricular and intrahippocampal administration using an in situ hybridization detection method. Unlabeled PO and PS oligos were dissolved in artificial cerebrospinal fluid and infused under stereotaxic control using a syringe pump. At various intervals after administration frozen brain sections were collected on gelatin-coated slides and hybridized with 35S-labeled probe consisting of the corresponding phosphodiester sense sequence. After intraventricular administration the unmodified PO oligo exhibited a limited and strictly periventricular distribution. In contrast the PS oligo showed significant penetration into and accumulation within brain, with extensive uptake in ipsilateral striatum and dorsal hippocampus, as well as in midline periventricular structures. Both oligos remained detectable for at least two days after administration. Following intrahippocampal injection the PO oligo was rapidly lost from the injection site, with detectable signal persisting only along the hippocampal fissure at 24 h. The PS oligo exhibited a more diffuse initial distribution as well as greater stability. While there was no indication of specific accumulation in the major hippocampal neuron layers through 24 h, there was some indication of selective localization in neuronal soma by 48 h. These results confirm that the relative instability of unmodified oligos may severely limit their utility as antisense reagents in brain in vivo. While PS oligos show more widespread distribution than PO oligos after intraventricular infusion, even these do not detectably accumulate in cortex and other structures without immediate access to the ventricular space under the dosing conditions employed here. The hybridization approach used in these studies should prove to be of general use in verifying the targeting of specific brain structures with antisense oligos by various routes of administration.
外源性施用的寡核苷酸（oligos）的分布和稳定性是决定反义寡核苷酸作为体内特定脑区基因表达修饰剂潜在效用的重要变量。在本研究中，针对最近克隆的大鼠hsp70序列的磷酸二酯（PO）和硫代磷酸酯（PS）反义寡核苷酸，在脑室内和海马内给药后，使用原位杂交检测方法在大鼠脑中进行定位。未标记的PO和PS寡核苷酸溶解在人工脑脊液中，并使用注射泵在立体定位控制下注入。给药后不同时间间隔，收集冷冻脑切片，置于涂有明胶的载玻片上，并用由相应磷酸二酯有义序列组成的35S标记探针进行杂交。脑室内给药后，未修饰的PO寡核苷酸表现出有限且严格的脑室周围分布。相比之下，PS寡核苷酸显示出显著穿透并积聚在脑内，在同侧纹状体和背侧海马以及中线脑室周围结构中有大量摄取。两种寡核苷酸在给药后至少两天内均可检测到。海马内注射后，PO寡核苷酸迅速从注射部位消失，仅在24小时时沿海马裂有可检测到的信号持续存在。PS寡核苷酸表现出更弥散的初始分布以及更高的稳定性。虽然在24小时内没有迹象表明在主要海马神经元层中有特异性积聚，但在48小时时有一些迹象表明在神经元胞体中有选择性定位。这些结果证实，未修饰寡核苷酸的相对不稳定性可能严重限制其作为体内脑反义试剂的效用。虽然PS寡核苷酸在脑室内注入后比PO寡核苷酸分布更广泛，但即使是这些寡核苷酸，在本文采用的给药条件下，在没有直接进入脑室空间的情况下，在皮质和其他结构中也没有可检测到的积聚。这些研究中使用的杂交方法应证明在通过各种给药途径用反义寡核苷酸验证特定脑结构的靶向方面具有普遍用途。
