厦门免费医学论文发表-中风后结合伽马神经调控和机器人康复可恢复小白蛋白中间神经元动力学并改善小鼠运动恢复 利维亚·维尼奥齐 ，弗朗西斯卡·马奇 ，埃琳娜·蒙塔尼 ，玛丽亚·帕斯奎尼，亚历山德拉·马泰洛，安蒂亚·米内蒂，埃莱亚·库仑，安娜·莱蒂齐亚·阿莱格拉·马斯卡罗，西尔维斯特罗·米塞拉，马泰奥·卡莱奥 ，克里斯蒂娜·斯帕莱蒂 抽象 中风是长期残疾的主要原因，通常与持续的运动缺陷有关。由小白蛋白阳性中间神经元 （PV-IN） 同步放电产生的伽马能带振荡在人类和动物中风后受到严重影响。伽马波段和 PV-IN 在运动功能中都起着关键作用，因此是中风后神经康复的一个有前途的靶点。无创神经调节方法被认为是一种安全的干预措施，可用于此目的。在这里，我们提出了一种新型的、临床相关的、无创的、耐受性良好的亚急性治疗，将机器人康复与先进的神经调控技术相结合，在缺血性损伤的小鼠模型中得到了验证。在亚急性中风后阶段，我们发现病灶周围皮层中与运动相关的伽马波段活动存在严重缺陷。这些缺陷伴随着 PV-IN 放电率的降低和病灶周围和整个皮层水平的功能连接的增加。因此，我们测试了在运动训练期间的亚急性中风后阶段将机器人康复与光遗传学 PV-IN 驱动的伽马波段刺激相结合的治疗潜力，以增强治疗效果。特定频率运动相关的伽马波段刺激与体能训练相结合，可显着改善前肢运动功能。更重要的是，通过将机器人康复与类似临床的无创 40 Hz 经颅交流电刺激相结合，我们实现了类似的运动改善，这些改善是通过有效恢复运动相关的伽马波段功率、改善 PV-IN 适应不良网络动力学以及增加运动前皮层中的 PV-IN 连接。我们的研究引入了对小白蛋白-中间神经元在中风后损伤和恢复中的作用的新认识。这些结果凸显了将病灶周围伽马波段刺激与机器人康复相结合作为中风患者一种有前途且现实的治疗方法的协同潜力。 作者总结 中风引起的运动缺陷伴随着伽马调制和 PV-IN 网络的改变;所有这些参数都通过无创伽马刺激和机器人疗法恢复。 数字 Fig 7Fig 1Fig 2Fig 3Fig 4Fig 5Fig 6Fig 7Fig 1Fig 2Fig 3 引文： Vignozzi L， Macchi F， Montagni E， Pasquini M， Martello A， Minetti A， et al. （2025） 中风后结合伽马神经调控和机器人康复可恢复小白蛋白中间神经元动力学并改善小鼠运动恢复。公共科学图书馆生物学 23（10）： 电子3002806号。 https://doi.org/10.1371/journal.pbio.3002806 学术编辑： 理查德·丹尼曼，加州大学圣地亚哥分校，美利坚合众国 收到： 2024 年 8 月 5 日;接受： 2025 年 9 月 9 日;发表： 10月 14， 2025 版权所有： © 2025 Vignozzi 等人。这是一篇根据知识共享署名许可条款分发的开放获取文章，允许在任何媒体上不受限制地使用、分发和复制，前提是注明原作者和来源。 数据可用性： 所有相关数据均可从以下存储库获得：Spalletti， Cristina （2024），“通过小鼠伽马刺激和机器人康复恢复中风后前肢功能”，Mendeley Data，V1，https://doi.org/10.17632/mw82tzp4rx.1。 资金： 该研究由以下机构资助：托斯卡纳地区，PERSONA 项目，bando Salute 2018，https://www.regione.toscana.it/-/bando-ricerca-salute-2018，SM; H2020 优秀科学-欧洲研究委员会 （ERC） 根据资助协议 692943， https://cordis.europa.eu/programme/id/H2020-EU.1.1.， MC;托斯卡纳地区，NIMBLE 项目 （20RSVP），bando Salute 2018，https://www.regione.toscana.it/-/bando-ricerca-salute-2018，（ALAM）; 托斯卡纳健康生态系统 ECS_00000017 MUR_ PNRR、https://www.mur.gov.it/it/pnrr/missione-istruzione-e-ricerca、ALAM;格兰特巴罗尼基金会 2021，https://fondazionebaroni.it/bandi/，CS。 资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。 利益争夺： 提交人声明不存在竞争利益。 缩写： 终审 法院 尾前肢区;FC， 功能连接;GSR， 全局信号回归;PBS， 磷酸盐缓冲盐水;PFA， 多聚甲醛;PSD， 功率谱密度;PV-INs， 小白蛋白阳性中间神经元;RFA， 喙前肢区域;扫描电镜， 均值标准误差;tACS， 经颅交流电刺激 介绍 中风引起的运动皮层功能缺陷是全世界残疾的主要原因之一。最近，新的康复策略丰富了中风后物理治疗，例如机器人工具与神经调节技术的结合[1\u20123]。机器人疗法是一种很有前途的工具，可以收集有关运动表现的高精度动力学和运动学数据并提供可定制的练习。此外，人们普遍认为物理治疗对于中风康复至关重要，但应与神经调节策略相结合，以最大限度地发挥其有效性。这些策略旨在增强可塑性和“启动”保留的病变周围组织，增强其对“活动依赖性可塑性”的敏感性。此类策略可能涉及塑化药物（即氟西汀）或无创脑刺激（noninvaistive brain stimulation， NIBS）技术，以精确和受控地调节皮质活动[4,5]。塑性环境与适当的体育锻炼之间的最佳相互作用对于运动恢复至关重要[6]。 在过去的几十年里，人们越来越了解振荡神经活动在健康和受伤受试者运动功能中的重要性。具体而言，在较高伽马频率范围（60-90 Hz）的同步振荡导致伽马功率增加，与运动的启动和执行（称为运动相关伽马同步）同时发生，并反映了参与运动的初级运动神经元的初始激活[7,8]。对人类的研究表明，驱动高伽马振荡可以增强运动性能[9]，证明这些神经节律在可塑性和运动控制中具有因果作用[10,11]。相反，较低的伽马波段振荡（30-60 Hz）与控制更强肌肉力量产生的策略有关[12]。 在人类中，大脑中动脉区域的缺血性发作会引起受累和未受影响的半球静息神经节律活动的急性改变[13]。值得注意的是，受影响半球的 delta （2-3.5 Hz） 和 theta （4-7.5 Hz） 频段功率的不对称扩大伴随着缺血事件，而 gamma 活性降低与手部功能受损相关。一致地，上肢运动恢复不完全且持续缺陷的中风幸存者在使用肩/肘肌肉执行伸展任务时表现出明显较低的γ波段皮质-肌肉连贯性[14]。这种连贯性下降可能反映了大脑-肌肉交流不良或运动动作期间来自两个来源的信号整合不良。这种较差的脑电图-肌电图相干性可能反映了导致中风患者伸展性能受损的潜在机制[14]。相反，受影响半球的伽马功率增加与更好的恢复前景相关[13]。最近在动物模型和人体研究中发现，γ波段的振荡活动与初级运动皮层内GABA能中间神经元（IN）和锥体细胞相互连接的网络内的兴奋性/抑制性平衡之间存在直接联系[15\u201217]。事实上，Nowak及其同事[18]证明，通过NIBS技术驱动伽马频率振荡是可行的，并且对健康受试者的运动功能和GABA介导的抑制产生显着影响。 在这种情况下，一个明确定义的IN亚组，即快速加标的小白蛋白阳性（PV）GABA能IN，其特定的放电特征已证明在产生和调节γ节律中具有因果作用[16,19,20]。最近，在中风的啮齿动物模型中，PV-IN活性与中风后功能障碍以及运动恢复有关[21]。因此，快速尖峰IN光遗传学调节已被用于改善动物模型中的运动恢复[22,23]。重要的是，Wang及其同事证明，在中风后急性期，病灶周围光遗传学40 Hz刺激抑制性IN可增加神经元存活率和可塑性[22,24]。康复似乎直接参与 PV-IN，加强它们的突触连接并促进神经元网络的重组，可能是通过增加伽马活性。这些发现强调了靶向PV-INs以促进卒中后功能恢复的治疗潜力[25]。 这些证据可能为开发涉及与物理治疗相关的大脑节律调节的新治疗策略提供良好的基础。尽管取得了这些进步，但将这些新范式整合和调整到人类康复后策略中的连贯框架仍然难以捉摸。光遗传学方法的时间非常有限，必须正确地转化为临床现实，才能成功地应用于人体。病变体积、梗死位置和临床方法之间的高变异性也增加了这一差距[26]。此外，功能损伤和中风后治疗影响的精确神经生理学机制仍然定义不明确。因此，尽管进行了康复治疗，但仍有相当大比例的中风患者在日常生活活动方面表现出持续的障碍。 本研究探讨了缺血性病变对缺血性中风小鼠模型中 PV-IN 和运动相关伽马波段活性的纵向影响。此外，我们利用这些数据验证了在保留的前肢运动前皮层中诱导伽马频率振荡的转化治疗潜力，并结合机器人引导的运动康复。我们采用光遗传学诱导PV-IN引导的伽马节律，并结合专为小鼠前肢训练而设计的定制设备上的日常锻炼[27]。然后，我们使用临床相关的 NIBS 方法结合机器人康复确认了治疗的疗效。我们的数据为推动康复的神经机制提供了新颖的重要见解，并为开发更有效的中风后疗法提供了可靠的支持。 结果 缺血性病变诱发伽马波段同步运动相关功能障碍 我们首先评估了尾前肢区 （CFA） 缺血性病变对随意运动期间保留的病灶周围前运动皮层（喙前肢区，RFA）伽马波段调节的影响。小鼠在CFA中接受了光血栓病变或假手术（图1A）。中风病变的代表性图像如图1B所示（黄色虚线）。中风前后通过Gridwalk测试进行运动功能评估，如S1A图所示。当小鼠在M-Platform上进行自愿回缩任务时，从RFA获取局部场电位记录，M-Platform是一种用于小鼠前肢功能评估和神经康复的定制设备（图1C和1D）。为了充分取样神经胶质瘢痕形成之前的“早期亚急性期”[28]，这对于康复方案的开始至关重要，在中风后2天和5天进行记录。我们量化了特定时间窗口内的伽马波段功率，描绘了基线（即，从运动开始到-1.5秒），发病前阶段（PRE，从-500毫秒到发作）和发作后阶段（POST，从发病到+500毫秒），如图1E所示。在假动物中，我们观察到所有皮质层在发病前阶段运动前皮层的伽马波段功率显着增加（图1F和S1C，灰色条形图）。在整个运动执行（POST）过程中，伽马功率在表层（线性多探针的通道1-8）中保持升高，而伽马调制在皮层的深层（线性多探针的通道9-16，图1G和S1D，灰色条形图）中几乎不明显。相比之下，在病变后2天（D2）和5天（D5）评估的中风动物在运动开始前没有表现出伽马波段上调（图1F，分别为浅蓝色和蓝色），并且与对照组相比，运动期间的伽马功率显着降低。一致地，在运动执行过程中，中风动物的伽马波段功率在所有皮质层都下降，并且与上层的对照组显着不同，上层的伽马波段调节更为明显（图1G和S1D）。假小鼠和中风小鼠之间的基线伽马功率没有差异（S1B图）。 thumbnail 下载： PPT的PowerPoint 幻灯片 巴布亚新几内亚大图 蒂夫原图 图1. 中风后运动相关伽马波段调节受损。 病灶周围前运动皮层随意运动期间的电生理记录显示，运动皮层缺血性中风后伽马波段调节存在显着缺陷。A，右侧 CFA 和进行电生理记录的病灶周围区域 （RFA） 中风病变的示意图。B，用Hoechst核染色标记的中风动物的代表性冠状脑切片，显示病变（由黄色虚线勾勒）。比例尺 = 2 mm。C，实验方案的示意图。D、M平台上退缩任务的两个不同阶段的示意图。小鼠被头部束缚，手腕闭合在连接到称重传感器的手柄中，以进行力检测。在被动阶段（左），线性执行器向前推动手柄和鼠标前肢。在活动阶段（右），小鼠被训练通过克服定义的摩擦力将手柄返回到原位置，以获得奖励。E，任务期间与运动开始相关的分析窗口。黑线代表整个任务中力跟踪的示例;运动的开始以红色突出显示;紫色窗口标识未检测到移动的基线周期;粉红色窗口标识发病前阶段（即运动准备），绿色窗口表示发病后阶段（即运动）。F 和 G，分别在发病前和发病后窗口中跨越所有皮质层（通道 1 ≃ 50 μm，通道 16 ≃ 800 μm）的所有 16 个通道的伽马波段功率的量化。该图显示了四个通道组的平均数据;有关单通道的详细信息，请参阅 S1C 和 S1D 图。健康小鼠（假，灰色，n = 9），中风后2天记录的动物（D2，浅蓝色，n = 6），中风后5天记录的小鼠（D5，深蓝色，n = 8）。在亚急性中风中，发病前和发病后阶段较高层的正伽马波段调制被消除甚至逆转。双向方差分析后进行 Tukey 检验，* P < 0.05，*** P < 0.001。数据显示为SEM±平均值。在BioRender中创建。维尼奥齐，L. （2025） https://BioRender.com/md8zh2c。该数字背后的数据可以在 https://data.mendeley.com/datasets/mw82tzp4rx/1 中找到。 https://doi.org/10.1371/journal.pbio.3002806.g001 缺血性中风在受伤后长达 1 个月内深刻改变 PV-IN 的静息状态功能连接 我们推断，伽马波段调节的损害可能与PV-IN功能的局部和广泛变化有关。为了检验这一假设，我们在PV-IN中表达GCaMP7f的清醒头部固定小鼠中进行了纵向宽视场钙成像（图2A，S2G和S2H）。我们记录了同一只小鼠中风前一天（中风前）和中风后2、5、8、14、21和28天的自发PV-IN活性，监测病变从急性期到慢性期的演变（即小鼠30天）。根据电生理实验，对中风后的“早期亚急性期”进行采样，特别强调作为康复方案开始的敏感时间窗口的重要性。梗死周围皮层的自发性PV-IN活性急剧降低，涉及两个半球的很大一部分（图2C）。因此，我们假设病变后 PV-IN 功能连接 （FC） 发生重大变化。因此，通过计算血流动力学校正后所有配对皮质区域之间的皮尔逊相关性（费舍尔的z变换）来评估整个背侧皮层的FC（图2B）。在健康小鼠中，静息态FC在各半球之间是均匀和对称的。值得注意的是，前初级运动皮层显示出较低的相关值，唯一的例外是同位连接。在中风后的亚急性期，明显的改变是明显的，对半球内和半球间FC产生相反的后果（图2C）。尽管远非中风前状态，但平均 FC 在接下来的几周内变得更加均匀。通过计算中风前后FC之间不同时间点的差异（S2A图）并使用基于网络的统计量[29,30]测试相关性的显着变化（图2D），证实了这一定性评估。有趣的是，一个低连接的半球间网络很早就出现了，并在中风后持续了长达 28 天。相比之下，病变侧的半球内 FC 在亚急性期显示梗死周围区域短暂增加，随后在慢性期逐渐减少。中风后一个月，低相关网络扩展到整个背侧皮层，也涉及视觉区域等远处区域（图2D）。通过量化随时间变化的全皮层FC，我们确认中风后28天整体减少没有恢复（图2E）。然后，分别评估了半球间和半球内 FC 对全球减少的贡献。有趣的是，半球间连通性以相关强度的降低为主（图2F）。值得注意的是，同侧次级运动皮层 （MOsa） 的前部是中风后唯一显示 FC 显着减少的运动区域（图 2G）。相反，半球内FC在损伤部位的同侧显示出大幅但短暂的增加（图2H），梗死周围初级和次级运动皮层的前部都有很大的贡献（图2I）。相反，对侧半球内FC没有显着变化（图2J和2K）。总体而言，这些结果表明，全球强度的下降主要是由半球间的变化引起的。因此，我们专门剖析了同位不同步的贡献：同位连通性的大幅减少主要影响亚急性期的梗死围和结合区域（M2、M1、SSp.tr、RSP），随后从损伤开始一周开始部分恢复（图2L）。Gridwalk 中缺血性病变体积和运动表现的量化如 S2F 和 S2G 图所示。我们的结果表明，PV-IN 连接发生了深刻的变化，这些变化不仅限于受伤部位，而是延伸到整个背侧大脑皮层，主要影响半球间强度。 thumbnail Download: PPTPowerPoint slide PNGlarger image TIFForiginal image Fig 2. Enduring reduction of resting state functional connectivity (FC) of parvalbumin-positive interneurons (PV-INs) after stroke. A, Experimental timeline, with the cranial optical window implanted one week before the first imaging session. Imaging time point at −1 (PRE), 2, 5, 8, 14, 21, and 28 days after stroke. B, Left, representative image sequence of cortical PV-IN activity before stroke. The black dot indicates bregma. L: lateral; M: medial; R: rostral; C: caudal (Scale bar, 1 mm). Right, wide-field calcium imaging field-of-view aligned with the surface of the Allen Institute Mouse Brain atlas. The green area on the left hemisphere locates the damaged region. Yellow squares represent cortical areas defined in both left (L, contralesional) and right (R, ipsilesional) hemispheres. Red dot indicates bregma (Scale bar, 1 mm). C, Pairwise Pearson’s correlation coefficients of cortical activity were visualized as averaged correlation matrices for each imaging time point after hemodynamic correction. D, Network diagrams of statistically significant FC alterations after 2, 5, 8, 14, 21, or 28 days from injury. Blue and red lines denote significant hyper-correlation and hypo-correlation compared to prestroke values, respectively. The bar plots (bottom) indicate the number of significant FC alterations for each cortical area. E, Box chart illustrating FC averaged over the whole cortex. F, Box chart illustrating the averaged inter-hemispheric FC. G, Box charts showing inter-hemispheric FC of ipsilesional and contralesional secondary and primary motor cortices in the anterior regions (MOsa and MOpa, respectively). H, Intra-hemispheric FC of ipsi-lesional areas. I, Box charts displaying intra-hemispheric FC of the secondary (left) and primary (right) ipsilesional motor cortices in the anterior region. J, Intra-hemispheric FC of contralesional areas. K, the box charts displaying intra-hemispheric FC of the secondary (left) and primary (right) contralesional motor cortices in the anterior region. L, Homotopic FC changes from prestroke to 28 days after injury (MOsa, anterior secondary motor cortex; MOsp, anterior primary motor cortex; SSp.tr, primary somatosensory cortex-trunk; RSP, dorsal part of the retrosplenial cortex; VISp, primary visual cortex). One-way ANOVA followed by Tukey test, * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± SEM. Each color indicates a single subject, n = 5. Created in BioRender. Vignozzi, L. (2025) https://BioRender.com/md8zh2c. The data underlying this figure can be found in https://data.mendeley.com/datasets/mw82tzp4rx/1. https://doi.org/10.1371/journal.pbio.3002806.g002 To assess the potential influence of global activity patterns on FC, we performed the same analysis after applying global signal regression (GSR, S4 Fig). As expected by removing widespread global components, GSR shifted the overall FC values toward lower correlations across the cortex. However, global network alterations after stroke were consistent with those observed without GSR (S4C Fig). We observed a persistent reduction of inter-hemispheric FC (S4D and S4E Fig) and a transient increase of intra-hemispheric FC ipsilateral to the lesion (S4F Fig). Similarly, homotopic connectivity across peri-infarct and associative regions showed an early decrease with partial recovery at later stages (S4J Fig). Notably, GSR revealed a persistent impairment of intra-hemispheric connectivity between peri-infarct regions (S4G Fig), which remained significantly impaired across all poststroke time points. Our findings highlight that while FC measured without GSR may suggest widespread compensatory processes, the recovery of local functional networks remains severely limited in the peri-infarct cortex, even 1 month after injury by indicating a failure of spontaneous recovery in the chronic phase. Furthermore, to explore the relationship between PV-based FC and hemodynamic signaling, we performed a parallel FC analysis using the 530 nm reflectance signal, which primarily reflects changes in hemoglobin concentration. Interestingly, we observed a strong correspondence between calcium-based PV FC maps (Fig 2C) and those derived from hemodynamic signals (S7D Fig). This similarity is consistent across both healthy and poststroke conditions. These results are in line with previous findings showing that GCaMP signals from excitatory neurons can recapitulate hemodynamic-based FC maps [31,32] and extend this concept to PV+ inhibitory neurons. Importantly, this observation suggests that PV-based calcium imaging may capture network-level dynamics that are ultimately reflected in the slower hemodynamic fluctuations measured by techniques like fMRI and Intrinsic Optical Signal Imaging. PV-INs in perilesional premotor cortex involved in motor execution can be engaged by optogenetic stimulation Once the significant impact of ischemic injury in the CFA on PV-IN activity in perilesional tissue was established, we investigated whether the PV-IN circuitry involved in voluntary movement could still be engaged by external stimulation. We induced either an ischemic or a sham lesion in CFA of B6;129P2-Pvalb tm1(cre)Arbr/J (PV-CRE) mice previously injected with a dflox.hChR2 AAV in RFA. Motor function was evaluated with Gridwalk test before and D2 after lesion in order to assess motor impairment in stroke mice (Fig 3B). Subsequently, recordings from the spared perilesional premotor cortex were conducted 5–7 days after ischemic or sham lesion in awake, head-restrained mice. The time window was chosen considering the beginning of a neuromodulation phase starting from day 5. First, we used 200 ms blue-light pulses to identify putative PV-IN that exhibited increased firing rate lasting for the entire duration of the stimulation and putative neurons connected to the stimulated PV-IN, whose spontaneous discharge was inhibited during the stimulation. Next, we monitored the identified neurons during the execution of the forelimb retraction task on the M-Platform (Fig 3A). Out of 35 PV-INs identified in 6 healthy animals, 19 correlated positively or negatively with movement onset. In 4 stroke animals, 17 out of 34 putative PV-INs were movement-related (Fig 3C and 3D). PV-INs in stroke animals exhibited a significantly reduced firing rate during stimulation (Fig 3E). These findings demonstrated that, despite the profound impairment of PV-IN network and cell responsiveness resulting from the ischemic lesion, individual cells retain their reactivity, their involvement in forelimb movement execution and could be recruited by external stimulation. thumbnail Download: PPT的PowerPoint 幻灯片 巴布亚新几内亚大图 蒂夫原图 图3. 病变周围组织中的小白蛋白阳性中间神经元 （PV-IN） 对光遗传学刺激有反应并参与随意运动，但放电率降低。 应用光遗传学刺激和单单位记录来识别健康和缺血动物运动前皮层中的PV-IN，并评估它们的放电特性及其对自主运动的参与。A，报告了实验程序的示意图。B、假动物（n = 5，灰色条形图）和中风（n = 5，紫色条形图）动物中风诱导前和后 2 天的 Gridwalk 测试中的运动功能评估，圆圈代表单个动物（双向方差分析，然后是 Tukey 检验 *** P < 0.001）。C，假动物和中风动物已识别的PV-IN的代表性栅格图。上图显示了PV-IN响应光遗传学刺激（左）的栅格图，并在时间上与假动物的运动开始（右）对齐。下部面板显示描边动物的相同栅格图。D，假动物和中风动物记录的PV-IN的平均波形。E，量化记录的PV-IN响应光遗传学刺激的平均放电率。中风动物的放电活动显着降低（双尾 T 检验 *** P < 0.001）。数据显示为SEM±平均值。在BioRender中创建。维尼奥齐，L. （2025） https://BioRender.com/md8zh2c。该数字背后的数据可以在 https://data.mendeley.com/datasets/mw82tzp4rx/1 中找到。 https://doi.org/10.1371/journal.pbio.3002806.g003 选择性 40 Hz 光遗传学 PV-IN 刺激结合机器人康复可恢复前肢功能 为了确定PV-IN驱动的伽马波段刺激的治疗潜力，我们在先前在RFA中注射dflox.hChR2 AAV的PV-CRE小鼠中使用了光遗传学刺激。我们首先证明，40 Hz光遗传学刺激（3秒脉冲）会增加PV-CRE小鼠的伽马波段功率，但在注射dflox.hChR2 AAV的WT小鼠中不会增加伽马波段功率，从而证实了PV-IN激活直接参与伽马波段生成（图4A）。随后，我们研究了一种联合神经康复方法，其中注射了dflox.hChR2 AAV的PV-CRE小鼠在CFA中进行光血栓性中风。将小鼠分为3个实验组：Robot组接受假刺激，Robot 8 Hz组在回缩任务执行过程中接受RFA中的8 Hz光遗传学刺激，Robot 40 Hz组接受40 Hz刺激。所有组均在 M 平台上接受每日机器人康复。所有治疗均从中风后第 5 天开始应用，一直应用到中风后 37 天（慢性期）。使用 Gridwalk 和 Schallert Cylinder 测试评估治疗效果，该测试在基线、中风后 2 天（治疗前）、每周一次直至中风后 37 天以及无治疗随访 1 周后进行。实验方案示意图如图4B所示。通过两种行为运动测试检测到的初始缺陷在各组之间是一致的（图 4C 和 4D）。即使经过 5 周的治疗，单独的机器人康复（灰条图）也没有产生治疗效果。值得注意的是，将 8 Hz 刺激与机器人康复相结合（黄色条形图）并没有导致额外的运动改善，这表明在不同的振荡模式下激活 PV-IN 无法恢复运动功能。相反，40 Hz 刺激与机器人康复相结合（蓝色条形图）产生了高度的治疗效果，这在 Gridwalk 测试的对侧前肢足部故障的数量和 Schallert Cylinder 测试的对侧前肢使用的百分比中都很明显。在两项功能性运动测试中，参数在受伤后 37 天恢复到基线水平。重要的是，在治疗结束后一周进行的随访评估期间，改善保持稳定。尸检分析显示，PV感染细胞百分比在各组之间没有显著差异（图4E）。在图4F中，机器人40Hz组的病灶周围RFA的放大倍数被报告为AAV感染细胞（红色）和免疫组织化学标记的PV-IN（绿色）双重标记的示例。 thumbnail Download: PPTPowerPoint slide PNGlarger image TIFForiginal image Fig 4. Robotic rehabilitation combined with optogenetic parvalbumin-positive interneuron (PV-IN) stimulation at 40 but not 8 Hz improves motor function after stroke. A, Average Gamma power response across all the cortical layers (i.e., the average of the values detected by the 16 channels of the linear electrode) to a 40 Hz optogenetic stimulation in PV-CRE mice (green, n = 5) but not in WT mice (pink, n = 5) infected with dflox.hChR2 AAV (Student t test, ** P < 0.01). B, Schematic of the experimental protocol. C and D, Forelimb motor function assessment in Gridwalk and Schallert cylinder test respectively. Robotic rehabilitation alone (gray bar plots, n = 6) is not able to restore forelimb motor function as expected. Similarly, 8 Hz stimulation (yellow bar plot, n = 5) fails to provide any improvement to motor rehabilitation. On the contrary, 40 Hz stimulation (blue bar plots, n = 6) significantly improved motor function. Two-way RM ANOVA followed by Tukey test, * P < 0.05 ** P < 0.01, *** P < 0.001. E, Quantification of PV-infected cells in the perilesional premotor cortex in the three experimental groups, revealing no significant differences among them (Robotic rehabilitation alone, n = 5; 8 Hz stimulation, n = 5; 40 Hz stimulation, n = 5). One-way ANOVA followed by Dunnett’s test, P > 0.05. F, Representative image of PV-INs (in green), AAV-infected cells (in red). Scale bar: 100 µm. Data are shown as mean ± SEM, circles represent single animals. Created in BioRender. Vignozzi, L. (2025) https://BioRender.com/md8zh2c. The data underlying this figure can be found in https://data.mendeley.com/datasets/mw82tzp4rx/1. https://doi.org/10.1371/journal.pbio.3002806.g004 病灶周围前运动皮层的无创伽马波段刺激联合机器人康复可改善中风后前肢运动功能 然后，我们评估了通过 PV-IN 的特异性光遗传学激活获得的治疗效果是否可以使用更具转化性的方法，即经颅交流电刺激 （tACS） 来复制。为此，在CFA中诱导了总共12只C57Bl6 / J小鼠的光血栓性中风，随后将其分为两个实验组。所有动物都接受了为期 5 周的每日机器人康复，与之前的实验一致。机器人tACS组接受40 Hz tACS应用于病灶周围RFA，而机器人组则接受假刺激，需要电极插入和连接，而没有实际的电流传输（图5A）。使用 Gridwalk 和 Schallert Cylinder 测试评估的运动功能在缺血性病变诱导后 2 天显示显着缺陷，而 Robot tACS 组和 Robot 组在病变前和 D2 病变后没有观察到差异。仅靠机器人康复并不能导致康复。然而，机器人tACS组表现出显着的改善，在两项测试中都恢复到基线，并在随访期间保持这些收益（图5B和5C）。 thumbnail Download: PPTPowerPoint slide PNGlarger image TIFForiginal image Fig 5. Forelimb motor improvements can be achieved by coupling robotic rehabilitation with noninvasive 40 Hz transcranial Alternating Current Stimulation (tACS). A, Schematic of the experimental protocol. B and C, Motor function assessment in mice treated with robotic rehabilitation alone (gray bar plots, n = 5) and coupled with noninvasive 40 Hz tACS (red bar plots, n = 6) on the perilesional premotor cortex evaluated with Gridwalk test and Schallert Cylinder test, respectively. The use of 40 Hz stimulation succeeded in restoring forelimb motor function and maintained this improvement in a follow-up measure. D and E, Quantification of gamma band power in pre- and postonset windows, respectively, across all 16 channels spanning the entire cortical layers at the follow-up time point (channel 1 ≃ 50 μm, channel 16 ≃ 800 μm). The graph presents data averaged over groups of four channels; for single-channel details, refer to S3C and S3D Fig. Gray bar plots n = 7, red bar plots n = 6. Two-way RM ANOVA followed by Tukey test, * P < 0.05 ** P < 0.01, *** P < 0.001. Data are shown as mean ± SEM, circles represent single animals. Created in BioRender. Vignozzi, L. (2025) https://BioRender.com/md8zh2c. The data underlying this figure can be found in https://data.mendeley.com/datasets/mw82tzp4rx/1. https://doi.org/10.1371/journal.pbio.3002806.g005 Next, we evaluated whether tACS treatment could induce a recovery in gamma band oscillations in a new cohort of C57Bl6/J mice subjected to photothrombotic stroke in the CFA. Mice were divided into two experimental groups: the Robot-tACS group underwent 40 Hz tACS combined with robotic rehabilitation, while the Robot group received robotic rehabilitation alone. At the follow-up time point, the Robot-tACS group exhibited a significant increase in gamma band power in both the pre- and postonset windows (Figs 5D, 5E, S3C, and S3D), suggesting that the combined treatment approach not only improves motor function but also has a substantial impact on power within the gamma frequency range. Quantification of lesion volume showed no differences among animals untreated and treated with robot or robot + tACS (S3A Fig). Motor performance assessed with Gridwalk test confirmed motor recovery for this robot + tACS group with respect to both untreated and robot-only groups (S3B Fig). These findings underline the potential benefits of integrating robotic therapy with tACS in order to promote neural recovery and rehabilitation. Adaptive contralesional reorganization of PV-INs FC drives motor recovery after stroke in rehabilitated mice To further investigate the mesoscale mechanisms underlying motor recovery, we next analyzed large-scale cortical FC following combined rehabilitation. One week before stroke induction, mice were implanted with an optical window. A small opening was left in the cranial implant over the RFA to allow daily transcranial stimulation. Baseline wide-field calcium imaging was performed one day before stroke induction. Starting from day 2 after stroke, animals underwent daily sessions of robotic rehabilitation on the M-Platform, coupled with 40 Hz tACS. After 1 month of rehabilitation, 2 weeks of stimulation-free follow-up were executed (Figs 6A and S8). thumbnail Download: PPTPowerPoint slide PNGlarger image TIFForiginal image Fig 6. Combined rehabilitation improves maladaptive network dynamics. A, Experimental timeline, with the cranial optical window implanted one week before the first imaging session. Imaging time point at −1 (PRE), 2, 5, 8, 14, 21, and 28 days after stroke. tACS was performed from day 2 to day 28 followed by two time points of follow-up (day 35 and day 42). B, Pairwise Pearson’s correlation coefficients of cortical activity were visualized as averaged correlation matrices for each imaging time point prestroke and 2, 5, 8, 14, 21, or 28 days after injury. C, Network diagrams of statistically significant FC alterations after 2, 5, 8, 14, 21, 28, 35, and 42 days from injury. Blue and red lines denote significant hyper-correlation and hypo-correlation compared to prestroke values, respectively. The bar plots (on the right) indicate the number of significant FC alterations for each cortical area. D, Box chart illustrating FC averaged over the whole cortex. E, Box chart illustrating the averaged inter-hemispheric FC. F, Intra-hemispheric FC of ipsi-lesional areas. G, Box charts displaying intra-hemispheric FC of the secondary (left) and primary (right) ipsilesional motor cortices in the anterior region. H, Homotopic FC changes from prestroke to 28 days after injury (MOsa, anterior secondary motor cortex; MOsp, anterior primary motor cortex; SSp.tr, primary somatosensory cortex-trunk; RSP, dorsal part of the retrosplenial cortex; VISp, primary visual cortex). One-way ANOVA followed by Tukey test, * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± SEM. The gray dashed line indicates the beginning of the follow-up period after the end of tACS treatment. Each color indicates a single subject, n = 5. I-L Comparison of changes in functional connectivity (ΔFC) relative to baseline (prestroke) between Stroke (gray) and Robot + tACS (dark red) groups across different time points after stroke (D2–D28) in terms of whole-cortex (I), inter-hemispheric connectivity (J), intra-hemispheric connectivity of ipsilesional hemisphere (K), and intra-hemispheric connectivity of the contralesional hemisphere (L). Data are shown as box plots (mean ± SE). *p < 0.05, main effect of treatment (two-sample t test). Blue and red arrows indicate the directions of hyper- and hypo-connectivity compared to the prestroke FC, respectively. Created in BioRender. Vignozzi, L. (2025) https://BioRender.com/md8zh2c. The data underlying this figure can be found in https://data.mendeley.com/datasets/mw82tzp4rx/1. https://doi.org/10.1371/journal.pbio.3002806.g006 We evaluated alteration in FC over weeks (Figs 6B and S2B) by computing differences between pre- and poststroke FC across timepoints after hemodynamic correction (S2C Fig). Interestingly, we observed a different network reorganization compared to spontaneous recovery in the late phase after combined rehabilitation. Initially (at 2 and 5 days after stroke), a hypo-connected network was still evident, similar to the untreated condition (S2C Fig). From day 8, the extent and strength of hypo-connectivity progressively diminished (S2C Fig). By day 28, before the end of the rehabilitation protocol, the hypo-connected network was largely attenuated, and after cessation of tACS (day 35 and 42), network alterations were further normalized (Fig 2C), with values continuing to rise during the follow-up period. Notably, whole-cortex FC was no longer significantly different from prestroke levels at Day 42, suggesting a sustained and consolidating effect of the combined intervention (Fig 6D). However, inter-hemispheric FC remains strongly altered (Figs 6E and S2D). Intra-hemispheric connectivity within the ipsilesional and contralesional hemisphere instead did not show significant alterations over time except for poststroke day 2 and poststroke day 5, respectively (Figs 6F, S2E, and S2F). Nevertheless, FC within the ipsilesional primary motor area (MOpa-ipsi, Fig 6G) remained persistently impaired up to 42 days poststroke, with no significant recovery even after the cessation of tACS stimulation, in contrast to the partial restoration observed in untreated animals. Despite the overall improvement in global FC, homotopic FC did not exhibit a comparable recovery after combined robotic rehabilitation and tACS (Fig 6H). In particular, connectivity between MOsa remained significantly impaired throughout the observation period (Fig 6H). This result is in contrast with spontaneous stroke recovery, where a partial normalization of homotopic FC was observed over time. To further explore the impact of rehabilitation, we performed a direct comparison between animals undergoing combined rehabilitation and stroke controls. Animals receiving robot rehabilitation combined with tACS showed a significant reduction in abnormal FC across the whole cortex (Fig 6I), in inter-hemispheric FC (Fig 6J), and in the intra-hemispheric contralesional FC (Fig 6L) 28 days after the stroke. In contrast, no major differences were observed between groups in the intra-hemispheric ipsilesional FC (Fig 6K), which was the direct target of the stimulation. This lack of modulation might reflect the severe disruption of ipsilesional circuits after stroke, which limits their capacity for functional recovery. Previous studies have shown that removing the global component of functional signals can help uncover network alterations that might otherwise be obscured by widespread activity patterns. Interestingly, after removing the global component, FC alterations remained largely consistent with those shown without GSR (S5 Fig), with one notable exception. Intra-hemispheric FC within the contralesional hemisphere remained persistently elevated and failed to normalize over time (S5K, S6D, and S6E Figs). These results indicate that the functional motor recovery promoted by robotic rehabilitation and tACS (S2J Fig) does not correspond to a restoration of the prestroke network organization but instead relies on a persistent reorganization within the contralesional hemisphere to compensate for deficits caused by the infarct (S2I Fig). Combined neurorehabilitation increases PV-INs connections and modulates GABAergic system Following the assessment of motor function, brain tissues from both Robot and Robot-tACS not implanted groups were collected for immunohistochemical analysis in order to explore structural and biochemical impacts of the treatments. Focusing on the spared perilesional premotor cortex, we investigated changes in PV-IN synaptic connections by examining PV expression in “puncta rings” surrounding nonPV neuronal somas. The Robot-tACS group exhibited increased mean fluorescence compared to the Robot group, indicating enhanced PV-IN connectivity (Fig 7A). Furthermore, we assessed whether the 40 Hz stimulation and the consequent increase in PV connections induces homeostatic changes in the GABAergic transporters in the perilesional cortex. We quantified the mean fluorescence of inhibitory terminals impinging on the soma of identified neurons. The expression of Vesicular GABA Transporter (VGAT) in the Robot-tACS group was significantly increased compared to Robot mice (Fig 7B). These findings reveal that 40 Hz tACS stimulation significantly impacts the PV-IN network and the GABAergic inhibitory system posttherapy. thumbnail Download: PPTPowerPoint slide PNGlarger image TIFForiginal image Fig 7. Increased parvalbumin-positive interneuron (PV-IN) connections and modulation of GABAergic transporters after combined neurorehabilitation. Immunohistochemical characterization of the consequences of combined neurorehabilitation on the density of PV-IN connections and the expression of GABAergic transporters. A, Magnification of the perilesional premotor cortex immunostained with anti-PV for robot-only and robot + tACS groups together with the quantification of mean anti-PV fluorescence calculated in puncta-rings around cell bodies of nonPV positive neurons (unpaired Student t test, * P < 0.05). Scale bar: 20 µm. B, Magnification of the perilesional premotor cortex immunostained with anti-VGAT antibody for robot-only and robot+tACS groups together with the quantification of mean anti-PV fluorescence calculated in puncta-rings around cell bodies of VGAT positive neurons (unpaired Student t test, * P < 0.05). Scale bar: 20 µm. Data are shown as mean ± SEM. The data underlying this figure can be found in https://data.mendeley.com/datasets/mw82tzp4rx/1. https://doi.org/10.1371/journal.pbio.3002806.g007 Discussion 在人类和非人灵长类动物中，初级运动皮层的病变会诱导梗死邻邻区域的广泛重组，例如运动前区域[33,34]，并被认为与运动（正向或负向）代偿有关。尽管小鼠初级运动皮层和前运动区的细分比灵长类动物更为微妙[35,36]，但已经确定了两个前肢特异性运动区：CFA和RFA[37,38]。因此，RFA被认为是小鼠中风后恢复的一个有希望的靶点，作为人类的前运动区域，正如先前的研究所证明的那样[39,40]。 在这项工作中，我们探讨了PV-IN网络和运动相关伽马调制在小鼠中风后运动损伤和康复中的作用，提供了可以显着促进中风后治疗的新见解。 我们的研究结果表明，在 CFA 中风后，在随意运动之前和期间，备用前运动皮层 （RFA） 的伽马调节都发生了显着变化。此外，缺血性病变会导致半球内和半球间 PV-IN 连接的明显损害，并随着时间的推移而恶化。 首先，我们记录了前肢回缩任务期间病灶周围RFA的皮质活动，并观察到与人类研究一致的伽马功率调节[41\u201243]。振荡伽马活动在认知任务期间有效地调动大脑区域，最近与运动控制和执行有关[41\u201246]。 这支持运动前皮层在运动计划和执行中的作用。我们的结果表明，在运动前窗口期间，所有皮质层的伽马功率增加，反映了前运动皮层在准备和计划运动时的募集。在运动执行过程中，高层的伽马活动保持升高，而与非运动条件相比，低层没有表现出显着的调制。这种层特异性模式表明，可能参与计算处理的颗粒上层在功能上与深层不同，后者包含较大的锥体神经元，负责将运动信号传递到皮质下区域[47,48]。在视觉系统中也描述了类似的层特异性模式，其中伽马波段功率主要局限于第4层，有助于编码视觉对比度[49,50]。 In general, layer-specific electrophysiological recordings indicate that gamma synchronization underlies feed-forward communication between cortical areas—primarily through supragranular layers, which are the main source of feed-forward projections [51]. Importantly, gamma band synchronization seems far from being a purely sensory-driven phenomenon, but it reflects a general aspect of cortical function [52], involving also inter-regional processing in motor areas. After stroke, gamma band modulation in the motor cortex was significantly impaired, both before and during movement execution. Remarkably, no distinctions were observed between stroke and sham mice in the baseline window, underscoring that the deficits in frequency modulation only emerge during active movement. To correlate these findings with PV-IN connectivity alterations, we employed longitudinal Wide-Field imaging in PV-CRE mice infected with a viral vector expressing a floxed calcium indicator. Our results indicated significant disruption in PV activity and connectivity during the acute phase, primarily in peri-infarct cortical regions, with global hypoconnectivity persisting into the chronic phase of spontaneous recovery. Previous works using wide-field calcium imaging have underscored the crucial role of PV-INs in motor learning [44,45]. Despite their sparsity, inhibitory cells play a predominant role in many key brain functions, including neural network coordination [53] and memory formation [54]. Recent studies demonstrated a loss of homotopic connectivity in stroke animals [55–57]. Here, we reveal the role of IN in intra- and inter-hemispheric diaschisis by showing that PV-IN FC over the entire cortex is strongly compromised by a focal lesion, primarily on peri-infarct and homotopic regions. Stroke-induced de-differentiation of cortical activation may explain the transient increase in ipsilesional connectivity in the sub-acute phase. The reduced specificity in functional activation [58] might be advantageous in stroke subjects to activate brain regions nearby the lesion when executing functions that would normally require the damaged area. Therefore, hyperconnectivity could result from synchronous activation of the entire lesioned cortex. Dedifferentiation and synchronous activation of the entire cortex have already been shown to occur in excitatory cortical neurons during both acute and chronic phases in spontaneously recovering stroke mice [1]. Collectively, these findings suggest that the excitatory/inhibitory balance is significantly disrupted at both local and distal levels, beginning in the acute phase and stabilizing in the chronic phase. Our group previously demonstrated that poststroke rehabilitation can alter the FC of excitatory neurons [1]. Furthermore, recent work has highlighted the cortex-wide properties of PV-IN networks in a rat model of epilepsy [59], and our current research contributes further to this knowledge by addressing large-scale activity of PV-INs poststroke. Given the crucial role of inhibitory neurons in other neurological conditions, including autism and Alzheimer’s disease [60,61], we foresee that the same approach could be applied to study large-scale alterations of inhibitory circuits in these and other neuropathologies. We explored whether impairments in PV-IN connectivity could contribute to the observed deficits in gamma band modulation. It has been demonstrated that PV-INs play an active role in voluntary movement execution through inhibition of pyramidal neurons [62,63], and dysfunction in cortical PV-IN networks has been reported in motor pathologies [64–67]. Combined with our wide-field imaging results, these findings suggest an early and long-lasting PV-IN network dysfunction, potentially affecting their recruitment as targets for neurostimulation. Using optogenetic stimulation and single-unit recordings in PV-CRE head-fixed mice, we identified PV-IN activity during voluntary movement. Isomura and colleagues [62] identified representative fast-spiking IN as PV-INs correlated with lever pulling in mice. More recently, Giordano and colleagues [68] demonstrated that fast-spiking IN in the motor cortex and RFA display unique discharge properties, firing earlier and longer during movement compared to pyramidal neurons. Accordingly, we found many cells responding to optogenetic stimulation manifesting spiking activity aligned with movement onset in both control and stroke animals. This confirms active, movement-related PV-INs in the perilesional premotor cortex that can be recruited for movement-related gamma band stimulation shortly after stroke. However, the ischemic lesion led to a significant reduction in the physiological spiking activity of these cells, as evidenced by a decrease of firing rate identified in our recordings. This aligns with our previous findings of decreased numbers of PV-INs and their connections impinging on pyramidal neurons in the perilesional cortex of chronic stroke animals [2,3,69,70]. This underscores the importance of timing in interventions, confirming a critical window for cortical plasticity [71–73] that has to be exploited for rehabilitative purposes. Notably, despite the reduced firing rate in perilesional tissue, the surviving PV-INs remain responsive to optogenetic stimulation, with their discharge reliably correlated with voluntary movement. These findings suggest that even compromised PV-IN networks can still be recruited to trigger gamma oscillatory activity tuned with movement control, if properly stimulated during physical rehabilitation. In line with this hypothesis, the therapeutic potential of targeting gamma oscillations is increasingly explored [23,24,74]. Building on these observations, we first explored a novel neurorehabilitative approach, combining physical robotic therapy with 40 Hz optogenetic stimulation of PV-INs in spared perilesional tissue. Our previous studies, in line with several pieces of evidence from the literature on human patients, already demonstrated that robotic rehabilitation alone was able to improve forelimb motor function after stroke in mice, but only if we considered the retraction task on the platform. These improvements were not generalized to other forelimb functions [3] indicating that motor rehabilitation alone, even when guided by robotic devices that help to increase the number and the repeatability of the task, is not sufficient to guarantee a true recovery instead of a motor compensation strategy. For these reasons, we implemented combined treatment approaches [1–3] where a plasticizing treatment is applied to prime the spared motor tissue and to make it more responsible for activity-dependent plasticity. Thanks to this approach, we’ve been able to validate several possible rehabilitation strategies to offer to the clinical practice for possible translations on human patients. Here, our hypothesis was that local 40 Hz stimulation, synchronized with forelimb motor training, could effectively engage the surviving PV-INs in perilesional tissue, thereby enhancing movement-related gamma band oscillations and promoting motor recovery. We first combined daily robotic rehabilitation with gamma band stimulation via direct PV-IN activation using optogenetics. Initiating treatment 5 days poststroke, during the subacute phase, allowed us to exploit the plastic critical period and accommodate the physiological delay necessary for engaging in rehabilitative exercises, mandatory for human patients. This clinically relevant approach resulted in significant improvements of forelimb function, with benefits sustained even after a period without intervention. This successful protocol leverages the consistent action potential output of PV-INs evoked by 40 Hz light pulses -which align with the kinetics of the ChR2 channel [20,75–77] to restore forelimb function in motor tests. Notably, robotic rehabilitation alone, or combined with 8 Hz stimulation, did not yield similar improvements, reinforcing our previous findings [1–3]. Indeed, robotic-guided motor rehabilitation alone—despite enhancing task repetition and consistency—is insufficient for true recovery and instead promotes task-specific motor compensation strategies. Considering these results, we focused on noninvasive neuromodulation techniques—particularly tACS—for their translational potential [78]. These methods have been validated both clinically [79,80] and in animal models [81]. By leveraging noninvasive techniques like tACS, which can entrain specific brain rhythms either at rest or during task performance while requiring minimal current to achieve desired effect [82], this strategy offers accessible and effective rehabilitation solutions for human patients, eliminating the need for invasive procedures such as optogenetics. Moreover, it is well-established that tACS is more effective when applied during tasks aligned with the target frequency of brain activity [83,84]. This makes tACS protocols fit well with the crucial necessity of physical rehabilitation for stroke patients. 我们始终保持 40 Hz 的刺激频率，因为高频刺激优先同步快速尖峰 IN，而低频主要同步常规尖峰神经元。已经证明，γ tACS促进了健康个体的运动表现[85\u201287]。遗憾的是，目前对于脑卒中治疗的最佳tACS方案尚未达成共识[88]。在这里，我们证明了通过在病灶周围RFA上采用侵入性较小的γ tACS来复制通过PV-IN的40 Hz光遗传学刺激获得的积极结果的可行性。这种多节程康复方案不仅有效地增加了主动运动期间的伽马功率，而且即使在一段时间没有康复后也能持续改善运动。 有趣的是，我们发现功能改进与中风前对称网络架构的归一化无关。相反，运动恢复依赖于病灶对侧半球内持续的适应性 FC。尽管 tACS 通过同侧半球输送，但我们没有观察到该区域内半球内连通性的显着变化。这可能是由于影响局部电路的广泛损坏，限制了它们的塑性重排。然而，对病灶周围皮层的刺激足以诱导远程效应，选择性地减少病灶对侧半球和半球之间的连通性低下。这些发现强调了梗塞周围区域的神经刺激如何通过参与完整的远程回路来促进恢复，支持中风后功能补偿依赖于替代网络募集而不是受损回路的恢复的观点。 我们进一步检查了病灶周围 RFA 内联合康复引起的形态和功能变化。我们的研究结果表明，γ 波段刺激显着影响 PV-IN 连接，支持伽马波段振荡与初级运动皮层中 GABA 能 IN 和锥体细胞网络内的兴奋/抑制平衡 [19\u201220] 之间的密切关系 [15\u201217]值得注意的是，γ 振荡似乎会诱导抑制系统的稳态变化，我们的免疫组织化学分析显示治疗后GABA能转运蛋白的表达。这表明神经康复后细胞对 PV-IN 回路加强的反应性增强，这也可能减轻急性期的进一步神经元损失。然而，在慢性阶段，GABA 的作用变得更加复杂。中风后 GABA 释放的改变可以显着改变 GABA-A 受体的表达，从而改变突触（阶段性）和突触外（强直）抑制。这导致了文献中的相互矛盾的发现。最近的研究，如Clarkson及其同事[89]，专注于纵强直抑制，表明中风后减少强直抑制可以改善小鼠的运动表现。这些结果也在我们的实验室中得到了证实，在小鼠模型中风后第一周调节突触前GABA信号传导可改善长期运动功能[69]。相比之下，文献报道了调节阶段性抑制时相互矛盾的效果[90]，报告了在中风小鼠模型皮质可塑性的关键窗口期间病变周围区域的阶段性抑制增加。通过用唑吡坦正向调节这种相位抑制，获得了动物运动性能的改善。最近，在对缺血性病变的小鼠进行“连续θ爆发模拟”治疗并相应的功能改善后，已经证明了阶段性抑制的选择性增加。这些结果表明，增加阶段性抑制在缺血性损伤的急性后期具有积极作用，但该主题仍有争议[91]。 In summary, our study demonstrates the effectiveness of a novel, clinically valuable, neurorehabilitation approach that combines physical therapy with movement-related gamma band stimulation. Our results highlight that gamma band stimulation can significantly enhance motor function by engaging and strengthening neural circuits within the perilesional and distal cortices. The integration of robotic therapy further enhances these beneficial effects, providing a practical and scalable tool for neurorehabilitation. Given the robust restoration of PV-IN-mediated gamma modulation and the significant motor recovery observed in our mouse model, our findings offer significant proof-of-concept for this integrated approach. Future clinical trials will be critical to confirm its translational potential and facilitate its widespread adoption, ultimately improving the quality of life and long-term outcomes for stroke survivors. Limitations of the study This study raises several important questions and highlights issues that warrant further investigation with different techniques. First, while animal models provide valuable insights, differences in nervous system complexity and brain size between rodents and humans may affect the precision and applicability of tACS treatment in clinical settings. Furthermore, the precise molecular and electrophysiological mechanisms of tACS are not entirely understood and the effect observed at a molecular point of view requires further investigations. Electrical stimulation with tACS protocol has a totally different mechanism of stimulation of brain circuitry with respect to optogenetics, so long as it lacks specificity for a single cell population. From our experiments, we can argue that tACS is able to synchronize brain oscillations on gamma rhythm across all the cortical layers, as reported in Fig 6, even if we cannot report cellular or molecular mechanisms underlying this effect. The evaluation of tACS as a standalone intervention is beyond the scope of this work as our focus was on rehabilitative outcomes and physical rehabilitation is a standard therapy for poststroke patients. Moreover, the photothrombotic lesion method used in this study, although advantageous in terms of reproducibility and spatial specificity, does not perfectly mimic human ischemic pathology due to its lack of a penumbra and highly focal nature. Additionally, because of the relatively slow temporal kinetics of the calcium indicator GCaMP7f, our widefield experiment was not suitable for detecting gamma-band activity. Although PV-INs are critically involved in gamma-band oscillations, the calcium signals we measured do not directly reflect gamma-frequency fluctuations. These limitations underscore the need for further refinement of these methods and their adaptation for clinical use. Nevertheless, the high translatability of the results obtained with tACS -combined with its well-tolerated, noninvasive nature- and the rapidly expanding integration of robotic devices in poststroke rehabilitation strongly support the prompt initiation of clinical trials to validate and potentially implement this integrated rehabilitative approach. Materials and methods Study design 本研究包括 49 个 C57BL6/J 和 37 个 B6;129P2-Pvalb-tm1（cre）Arbr/J（PV：：Cre，Jackson Laboratories，JAX stock #017320）成年小鼠，2-3个月大。动物在标准条件下饲养，光/暗循环为 12 小时，可以自由获取食物和水。所有实验程序均遵守 ARRIVE 指南和欧洲共同体理事会指令 #86/609/EEC，已获得意大利卫生部的批准（协议 #684/2020-PR，于 2023 年 1 月 19 日集成，2015 年 7 月 27 日的 753/2015-PR，723/2019-PR）。根据 3R 原则，特别是减少原则，动物数量被最小化，以确保动物的道德使用，同时保持足够的统计功效。在行为任务期间，在具有慢性植入物的清醒头固定小鼠中使用电生理记录和宽视场钙成像需要技术要求高的纵向方案。因此，采用受试者内实验设计和重复采集会话来最大限度地提高每只动物的数据产量并减少个体间变异性。样本量根据先前的研究和试点数据提供信息，以平衡科学有效性与伦理考虑，并使用 G Power 软件 （v3.1.5） 进行功效计算。动物被随机分配到组中。 Ischemic lesion Cortical ischemic damage in the CFA was induced using the photothrombosis method, as previously reported [3]. Briefly, animals were anesthetized with ketamine/xylazine (100/10 mg/kg i.p.) and placed in a stereotaxic apparatus. After a midline scalp incision, the skull was cleaned and dried. Subsequently, Rose Bengal (0.2 ml 10 mg/ml in phosphate-buffered saline (PBS); Sigma Aldrich) was intraperitoneally injected. After 5 min, the brain was illuminated through the intact skull for 15 min using a cold light source (ZEISS CL 6000, Germany) linked to a 20× objective positioned over the CFA of the right hemisphere (0.5 mm anterior and 1.75 mm lateral from Bregma, using a motorized micromanipulator (Sutter Instruments, USA). Sham animals underwent scalp incision and Rose Bengal injection but were not subjected to light irradiation. Following the photothrombotic procedure, animals underwent a head-restraining implantation surgery, consisting in a metal L-shaped bar posted on the occipital bone using dental cement (Super Bond C&B, Sun Medical Company, Japan). For electrophysiology, a metal screw connected to a ground electrode was implanted in the occipital bone, and a craniotomy was performed to expose the RFA (2.0 mm anterior and 1.25 mm lateral to Bregma [36]). The recording chamber was created by encircling the craniotomy hole with dental cement, then covered with agarose (1% in physiological solution) and silicon (Kwik-Cast Sealant, World Precision Instruments, USA) to protect the underlying tissues. For optogenetics, an optical fiber was positioned over the craniotomy. For tACS, a plastic tube was cemented over the RFA. Time points evaluation. In mice, the 2–5 day poststroke period is considered an “early subacute phase” that is crucial for the initiation of the rehabilitation treatment. Accordingly, for both electrophysiology and wide-field imaging, data were collected at day 2 and day 5 poststroke, and subsequently once per week during the subacute phase. Additionally, if no plasticizing treatment is applied, 30 days poststroke is considered chronic. Therefore, in the wide-field experiments, the imaging was performed until day 28 (also considering a possible decrease in window clarity and signal quality). Conversely, all treatments were applied for 37 days poststroke, with 1 week of follow-up without treatment; and for consistency, the postrehabilitation wide-field experiment was carried out for 44 days. Behavioral assessment was conducted before the induction of the stroke (baseline) and tracking the evolution of the lesion from the acute to the chronic phase at 2, 9, 16, 23, 30, and 37 days postlesion. Viral injection PV::Cre transgenic mice received a stereotactic injection of the AAV vector (AAV1.EF1.dflox.hChR2(H134R)-mCherry.WPRE.hGH (Addgene, USA) to induce a Cre-dependent expression of Channelrhodopsin-2 (ChR2). Briefly, a craniotomy was performed in RFA and 600 µl of AAV was injected in RFA 800 µm below the dura, with a flow rate of 0.1 µl/min with a Legato 130 syringe pump (kdScientific) and a 10 µl Hamilton syringe (Hamilton Company). PV-Cre mice express Cre recombinase in parvalbumin-expressing neurons. The term PV-ChR2 denotes PV-Cre mice injected with the AAV. The surgery was performed under a cocktail of ketamine/xylazine (100/10 mg/kg i.p.). For PV-INs labeling with GCaMP7f, the viral construct ssAAV-PHP.eB/2-hSyn1-chl-dlox-jGCaMPf(rev)-dlox-WPRE-SV40p(A) (Viral Vector Facility, CH) was intravenously injected in the retro-orbital sinus of PV-Cre mice under isoflurane anesthesia. A representative image of GCaMP7f expressing cells with quantification of viral efficiency and specificity is shown in S2G and S2H Fig. Wide-field calcium imaging WF setup. Imaging was performed through the intact skull using a custom-made microscope. The microscope consisted of back-to-back 50 mm f/1.2 camera lenses (Nikon). To excite the GCaMP7f indicator, a 470 nm light source (LED, M470L3, Thorlabs) filtered by a bandpass filter (469/17.5 nm, Thorlabs) was deflected by a dichroic filter (MD498, Thorlabs) on the objective (TL2X-SAP 2× Super Apochromatic Microscope Objective, 0.1NA, 56.3 mm WD, Thorlabs). Reflectance images were acquired using a light source positioned at 45° incident to the brain surface (530 nm LED light, M530L4; Thorlabs, New Jersey, USA). Stroboscopic illumination (25 Hz/LED) was used. The fluorescence and reflectance signals were selected by a bandpass filter (525 ± 19.5 nm Thorlabs) and collected by a CMOS camera (Orca 4.0 v2, Hamamatsu). Images were acquired at 50 Hz, with a resolution of 512 × 512 pixels with a FOV of 11.5 × 11.5 mm. Surgery. One week after AAV injection, mice were implanted with an intact skull preparation to allow free optical access to the cortex (modified from [2,92]). The skin and the periosteum were removed. Bregma was marked for stereotactic reference. A custom-made aluminum head-bar placed behind lambda was glued to the skull using transparent dental cement (Super Bond C&B—Sun Medical). The exposed cortex was then covered with the same cement. One week after the surgery, mice were habituated to head fixation under the wide-field microscope before the first imaging session. Calcium imaging was performed on awake, head-restrained PV-CRE mice during resting state the week before and 5–30 days after stroke. Each animal was imaged for approximately 30 min, including head fixation, five consecutive imaging blocks of 120 s, data storage between blocks, and head-fixation release. Habituation and imaging protocol. 从手术中恢复至少1周后，小鼠习惯于实验设置3-4天（每天20分钟/小鼠），以逐渐减少成像前的焦虑和突然运动。然后，将小鼠轻轻置于显微镜物镜下并固定。在清醒、头部受约束的小鼠中记录了皮质活动，这些小鼠可以自由移动四肢，并且不从事任何不同的任务。每个会话至少包含5个块，每个块持续120 s。 图像处理和数据分析。 使用定制软件注册从不同会话中收集的每只动物的图像堆栈，并考虑 bregma 和 λ 位置。使用动物特定的视野模板每天手动调整成像场。为了剖析每个皮质区域的贡献，我们将皮层记录到投影到我们的成像平面上的艾伦研究所小鼠脑图谱 （www.brain-map.org） 的表面。对于每个块，处理图像堆栈以获得 ΔF/F0 的估计值。计算每个像素的 ΔF/F，其中 ΔF 是该像素在特定时间点的强度值，F 是信号随时间的平均荧光强度。血流动力学校正是按照Scott及其同事的描述进行的[93]。简而言之，使用比例方法： 其中 F/F0是给定像素的最终校正 GCaMP7f 时间序列，I482指检测到的荧光信号，I525是反射率信号。然后，应用GSR。 “Inter-hem”是指半球间 FC。“下摆内”是指半球内 FC。“ipsi”是指同侧半球，而“contra”是指对侧半球。通过对相关矩阵中的所有值进行平均来计算整个皮层连接强度。通过对所有左半球（对侧）或所有右半球（同侧）内的相关值（Fisher z 变换）进行半球内 FC 随时间变化的计算。通过平均所有右半球和左半球区域对之间的相关值获得半球间 FC。同位FC是通过对半球相同大脑区域之间的相关值进行平均而获得的。 然后总共选择了 22 个 ROI（每个半球 11 个 ROI，20 × 20 个像素）。通过计算从每个 ROI 中提取的平均信号与其他 ROI 之间的 Pearson 相关系数，对每个受试者进行相关映射。然后使用 Fisher 的 r 到 z 变换对单受试者相关图进行转换，然后对所有动物进行平均。将平均映射重新转换为相关值（r 分数）。对于每只小鼠，计算 r（中风前）− r（中风后 x 天）并在小鼠之间进行平均，以可视化中风前条件和中风后所有时间点之间的差异矩阵。在FC分析之前，未进行时间滤波。 M平台上的前缩任务 M-Platform是一种用于小鼠前肢功能评估和神经康复的机器人设备[1,27]，由一个线性执行器（Micro Cylinder RCL，IAI，德国）、一个称重传感器（Nano 17，ATI Industrial Automation，美国）和一个放置在精密线性IKO滑块上的定制手柄（IKO BWU 25-75， USA）固定在动物的左手腕上。手柄连接到称重传感器上，可无损地将力传递到传感器，同时为动物的手腕提供支撑。这只动物被关在一个 U 形约束器中，它的头部固定在整个水泥柱上。每天进行电生理记录和康复实验训练。每个会话涉及每只小鼠的 15 次前肢回缩，结合了被动（设备伸展 10 毫米）和主动（动物缩回）运动。克服力阈值，小鼠每次成功完成任务都会获得液体奖励。小鼠通常掌握了这项任务，并在2-3天内提高了性能[27]。康复在病变后 5 天开始，一直持续到第 37 天，每周连续 4 天。根据每只动物的功能缺陷对任务摩擦进行调整。下面提供了有关联合假手术或神经调节治疗的详细信息。 Optogenetic stimulation 光遗传学刺激用于在电生理记录期间识别 PV-IN，并作为康复的神经调节治疗。刺激装置包括一个 PlexBright 光遗传学刺激系统，带有一个 LD-1 单通道 LED 驱动器和一个 456 nm LED 模块（PlexonInc，美国），连接到 200 μm 核心 0.39 NA 光纤（ThorlabsInc，美国）。每次实验前，使用 PlexBright 光测量套件 （~10， 79.55 mW/mm ） 测量光纤的最大发射功率2).基于 ~75 mW/mm 的安全范围2对于短脉冲（0.5-50 ms）[94]，我们将光遗传学刺激限制在31.82 mW/mm2（最大值的 40%）。我们可以假设，根据已经使用特定模拟器执行的有机组织对光纤实际辐照度的计算，具有我们的刺激参数的蓝光实际上可以到达下层（https://web.stanford.edu/group/dlab/cgi-bin/graph/chart.php）。刺激参数的控制通过LabWindows/CVI中的自定义软件进行管理，并通过NI USB-6212BNC DAQ板（National Instruments，美国）连接。对于PV-IN的光遗传学鉴定，我们以0.2 Hz的频率传递单个200毫秒光脉冲[95]，并增加光功率，以确保记录的活性是由于刺激而不是细胞的内在活性。对于伽马波段诱导，在整个任务过程中（从M平台上的被动阶段到回缩阶段）连续施用更多的生理1毫秒光脉冲（40或8 Hz），任务持续时间在5到8分钟之间变化，具体取决于每只动物的表现。 电生理记录 在 C57Bl6/J 和 PV-ChR2 小鼠中，使用 16 通道线性探针（NeuroNexus，美国）评估了 RFA 中的伽马功率调制和 PV-INs 放电特性。将小鼠头部固定在 M 平台上，左前爪连接到称重传感器，并将电极立体定向插入右半球 RFA 的 850 μm 深度。 使用 DigiAmp（Plexon，美国）获取和放大神经信号，在小脑中接地。在休息和回缩任务期间进行记录。使用 NeuroExplorer（Plexon，美国）和 Matlab 中的自定义算法执行的离线分析涉及计算能力谱密度 （PSD） 并将神经信号与来自平台的力信号同步。选择孤立的力峰值（运动开始前后 2.5 秒内没有运动的力峰值）。围事件频谱图分析用于计算 31−49 Hz 频段内相对于力峰值开始的三个不同时间窗口，间隔为 0.5 秒的功率：基线（-2 至 -1.5 秒）、发病前（-0.5 至 0 秒）和发病后（0 至 0.5 秒）。计算发病前/基线和发病后/基线的比率，以评估运动期间 CFA 相对于无运动状态的伽马波段功率变化。伽马功率变化被量化为总PSD的百分比，不包括力峰值和运动开始前后的时间窗口。 Optogenetic identification of PV-INs during resting states involved light pulses administered near the recording electrode. After the optogenetic stimulation protocol, neural activity was recorded during the retraction task without moving the recording electrode. To identify putative PV-INs, whose firing rate increased selectively during optogenetic stimulation. Spike Sorting analysis for single units was performed offline using Offline Sorter Software (Plexon, USA) and was used to refine the principal component analysis (PCA) to exclude potential spikes originating from neighboring neurons. Once identified, putative PV-INs peri-event rate histogram was referred to the movement onset to verify firing rate alignment with voluntary movement. Recordings spanned 3 days to enhance identification of PV-INs involved in motor tasks. Behavioral motor tests Functional assessment of forelimb motor performances was used to evaluate the impact of the ischemic damage and the applied neurorehabilitative protocols. Mice performances were assessed in baseline condition and then once a week at days 2, 9, 16, 23, 30, 37, and 44 postlesion, as reported in Lai and colleagues [96] Two behavioral tests have been used, Gridwalk and Schallert Cylinder test. In the Gridwalk test, mice were allowed to move freely on an elevated grid (32 × 20 cm, with 11 × 11 mm large openings, Micromecc, Italy) for 5 min and the task was video recorded (SMXF50BP/EDC, Samsung, Seoul, South Korea) by a camera positioned in front of the Gridwalk apparatus. Off-line analysis of the videos involved a custom-designed Graphical User Interface implemented in Matlab [96], to assess correct steps and foot-faults, namely, steps not providing body support, with the foot falling into a grid hole. The percentage of foot faults for each limb was then calculated. In the Schallert Cylinder test, animals were placed in a custom-made Plexiglas cylinder (8 cm diameter, 15 cm height) recorded for 5 min by a video-camera (SMXF50BP/EDC, Samsung, Seoul, South Korea) placed below the cylinder. Videos were analyzed frame by frame and the spontaneous use of both forelimbs was assessed during exploration of the walls, by counting the number of contacts performed by the paws of the animal. For each wall exploration, the last paw that left and the first paw that contacted the wall or the ground were assessed. In order to quantify forelimb-use, percentage of contralesional forelimb contacts over the total number of single-paw contacts was calculated as % of Contralateral Forelimb = (C_contra/C_contra + C_ipsi) * 100 where C_ipsi and C_contra correspond to the number of touching performed with the limb ipsilateral and contralateral to the lesioned hemisphere when the mouse was leaning at the vertical walls. The experimenters were blind to all the experimental groups in both motor tests. Transcranial Alternating Current Stimulation (tACS) tACS was administered using AnimaltES Model 2101 (Soterix Medical, USA) at a frequency of 40 Hz. The cathode was positioned in a saline-filled tube cemented to the skull, and the anode was placed under the abdomen against a saline-soaked sponge. Stimulation began 5 min prior to task onset, with current gradually increasing to a maximum of 0.2 mA, remaining below the movement threshold for mice. The stimulation continued for the task duration (5–8 min), with current ramp-down starting 10 min posttask initiation. Sham stimulation involved electrode placement without current flow. No signs of discomfort or freezing were observed in the animals during tACS. Histology Mice underwent anesthesia with Chloral hydrate followed by cardiac perfusion using 0.01 M PBS (Sigma Aldrich) and 4% paraformaldehyde (PFA, Electron Microscopy Sciences) in 0.1 M Phosphate Buffer. Brains were postfixed with 4% PFA for 2 h, rinsed with 30% sucrose (Sigma Aldrich) in Phosphate Buffer at 4 °C, and sectioned coronally using a sliding microtome (Leica, Germany) to obtain 50 µm thick slices maintained free-floating in PBS for additional treating. For immunostaining, brain slices were incubated in a blocking solution for 1 h (10% donkey serum; 0.3% Triton X-100 in PBS) and treated with primary antibodies overnight at 4 °C. As primary antibodies, Guinea Pig a-Parva (1:300, SynapticSystem) was used for PV-INs staining and a-VGAT (1:1,000, Synaptic System), for staining vesicular transporters of GABA and Glutamate, respectively. Following three washes in PBS, the sections were incubated for 2 h at room temperature with the specific secondary antibodies; therefore, a-GP-Alexa488 (1:200, Synaptic System) for the PV, a-GP-RRX (1:400, Synaptic System) for labeling the transporters of GABA and Glutamate. The VGAT and PV signals were acquired by airyscan confocal microscope (Zeiss, Germany) with 63× objective and 1.3 digital zoom, in the medial-superficial/deep and lateral-superficial/ deep regions of the peri-wound tissue. Three fields (77 μm × 77 μm) were acquired for each location (three sections per animal). The acquired images were processed using ImageJ (National Institutes of Health, USA) software to analyze the mean fluorescence in puncta-rings around cell bodies of VGAT and nonPV positive neurons. To minimize the variations due to the different quality of immunostaining in the individual mice/sections, the fluorescence in the periwound areas was normalized to values calculated in three reference fields taken in the basal cortices of each coronal section analyzed. To quantify the lesion volume, one out of every six sections was stained with Hoechst 33258 (Sigma-Aldrich, USA). The ischemic region was imaged with Apotome fluorescence microscopy (Zeiss, Germany) with a 10× objective and its area measured using ImageJ (National Institutes of Health, USA) software. The lesion volume for each animal was calculated by summing up all damaged areas and multiplying the number by section thickness and by 6 (the spacing factor). A total infarction volume in mm3 is given as the mean ± standard error of all analyzed animals. Statistical considerations Statistical analyses were conducted on raw data using SigmaPlot 11.0 (Systat Software, USA), Matlab (R2019a), and OriginLab (2018), with a significance threshold set at alpha = 0.05. For behavioral tests (Gridwalk and Schallert Cylinder), two-way Repeated Measures ANOVA with Tukey posthoc tests were applied. Group comparisons utilized one- or two-way ANOVA, depending on the data structure, followed by either Dunnett’s or Tukey’s posthoc tests. T tests were used for immunohistochemical analyses and firing rate comparisons. Group-level ROI-based FC differences pre- and poststroke were analyzed using one-way repeated measure ANOVA with Tukey correction. Network-Based Statistic (NBS) Toolbox in MATLAB assessed functional network connectivity [29,30]. Significance was determined at p < 0.05, and errors are expressed as Standard Error of Means (SEM), with significance markers (* P < 0.05, ** P < 0.01, *** P < 0.001). Data visualization Data visualization was performed using OriginPro, while figure editing was performed with Affinity Designer 2 (Serif (Europe), Version 2.6.3). Supporting information Referred to Fig 1. Showing 1/8: pbio.3002806.s001.tiff Skip to figshare navigation Sorry we could not load your data. 1 / 8 Download figshare S1 图。 参见图1。 A，在病变诱导前（基线）和2天（D2）对假（灰色条形图，n = 9）和中风（浅蓝色条形图，n = 8）小鼠进行Gridwalk测试的运动性能评估。（双向 RM 方差分析，然后是 Holm-Sidak 检验，*** = P < 0.001。B，诱导病变后2天假（灰色条形图）和中风（蓝色条形图）小鼠伽马功率调节的量化。（双向 RM 方差分析，然后是 Holm-Sidak 检验。C 和 D，分别在发病前和发病后窗口中跨越所有皮质层（通道 1 ≃ 50 μm，通道 16 ≃ 800 μm）的所有 16 个通道的伽马波段功率的量化。健康小鼠（假，灰色，n = 9），中风后2天记录的动物（D2，浅蓝色，n = 6），中风后5天记录的小鼠（D5，深蓝色，n = 8）。双向方差分析后进行 Tukey 检验，* P < 0.05，** P < 0.01，*** P < 0.001。每个点代表一只动物。数据显示为SEM±平均值。该数字背后的数据可以在 https://data.mendeley.com/datasets/mw82tzp4rx/1 中找到。 https://doi.org/10.1371/journal.pbio.3002806.s001 （TIFF） S2 图。 参见图2。 A，从中风前 FC 中减去中风后 FC（受伤后 2、5、8、14 或 30 天）得出的平均差相关矩阵。红色方块表示卒中后低连通性，蓝色方块表示卒中后 PV-IN 超连通性。B、中风前（左）和中风后2天（右）的平均相关矩阵，无血流动力学校正。C，机器人 tACS 组的平均差相关矩阵。D，显示机器人 tACS 组前部区域（分别为 MOsa 和 MOpa）同侧和对侧次级和初级运动皮层的半球间 FC 的框图。E，机器人 tACS 组对侧区域的半球内 FC。F，显示机器人 tACS 组前部区域次要（左）和初级（右）对侧运动皮层的半球内 FC 的框图。G，冠状脑切片中表达GCaMP7f的细胞（绿色）、PV-IN（红色）的代表性图像;在左侧，为单通道和合并（300 × 1,000 μm）提供了蓝色突出显示区域的放大倍数。比例尺：500 μm。H，顶部，PV-Cre小鼠模型中病毒感染的病毒效率（90.61±2.56%）和底部，特异性（77.72±3.97%）的量化（n = 4）。我在中风后 30 天（未进行任何治疗）和机器人 + tACS（ 红色，n = 6）（t 测试，p = 0.57）中的平均病变体积。J，通过网格步行测试评估运动性能。动物在 D2 病变后表现出显着的运动障碍，而在随访 （FU） 时，表现与基线相当。（单因素 RM 方差分析，然后是 Tukey 检验，** = P < 0.01，*** = P < 0.001。每个点代表一只动物。数据显示为SEM±平均值。该数字背后的数据可以在 https://data.mendeley.com/datasets/mw82tzp4rx/1 中找到。 https://doi.org/10.1371/journal.pbio.3002806.s002 （TIF） S3 图。 参见图5。 A，未接受干预（无治疗，深灰色，n = 6）、单独机器人康复（机器人，浅灰色，n = 6）和机器人康复与 tACS 相结合的中风动物的病变体积量化（机器人 + tACS，红色，n = 6）。三组病灶体积差异无统计学意义。B、通过网格步行测试评估运动性能。动物在 D2 病变后表现出显着的运动障碍。然而，只有机器人-tACS 组在随访期 （FU） 表现出与基线相当的性能。（双向 RM 方差分析，然后是 Tukey 检验，* = P < 0.05，** = P < 0.01，*** = P < 0.001。C 和 D，分别在发病前和发病后窗口中跨越所有皮质层（通道 1 ≃ 50 μm，通道 16 ≃ 800 μm）的所有 16 个通道的伽马波段功率的量化。单独接受康复治疗的小鼠（灰色条形图，n = 7）并与无创40 Hz tACS（红色条形图，n = 6）相结合。双向方差分析后进行 Tukey 检验，* P < 0.05，*** P < 0.001。每个点代表一只动物。数据显示为SEM±平均值。该数字背后的数据可以在 https://data.mendeley.com/datasets/mw82tzp4rx/1 中找到。 https://doi.org/10.1371/journal.pbio.3002806.s003 （TIFF） S4 图。 参见图2。 A、全局信号回归后脑卒中组各成像时间点皮质活动的成对Pearson相关系数。B，受伤后 2、5、8、14、21 或 28 天后具有统计学意义的 FC 改变的网络图。蓝线和红线分别表示与中风前值相比显着的高相关性和低相关性。条形图（底部）表示每个皮质区域的显着 FC 改变的数量。C，显示整个皮层平均 FC 的箱形图。D，说明平均半球间 FC 的箱形图。E，显示前部区域（分别为 MOsa 和 MOpa）的同侧和对侧次级和初级运动皮层的半球间 FC 的框图。F，同侧病变区域的半球内 FC。G，显示前部区域次级（左）和初级（右）同侧运动皮层的半球内 FC 的框图。H，病灶对半球半球内 FC。I，框图显示前部区域次要（左）和初级（右）对侧运动皮层的半球内 FC。J，同位FC从中风前到损伤后28天的变化（MOsa，前次级运动皮层;MOsp，前初级运动皮层;SSp.tr，初级体感皮层-躯干;RSP，脾后皮层的背侧部分;VISp，初级视觉皮层）。单因素方差分析，然后是 Tukey 检验，* P < 0.05，** P < 0.01，*** P < 0.001。数据显示为SEM±平均值。每种颜色表示单个主题，n = 5。该数字背后的数据可以在 https://data.mendeley.com/datasets/mw82tzp4rx/1 中找到。 https://doi.org/10.1371/journal.pbio.3002806.s004 （TIF） S5 图。 参见图6。 A、全局信号回归后Stroke + tACS组各成像时间点皮质活动的成对Pearson相关系数。B，受伤后 2、5、8、14、21 或 28 天后具有统计学意义的 FC 改变的网络图。蓝线和红线分别表示与中风前值相比显着的高相关性和低相关性。条形图（底部）表示每个皮质区域的显着 FC 改变的数量。C，显示整个皮层平均 FC 的箱形图。D，说明平均半球间 FC 的箱形图。E，同病变区域的半球内 FC。F， 显示前部区域次级（左）和初级（右）同侧运动皮层的半球内 FC 的箱形图。G，同位FC从中风前到损伤后28天的变化（MOsa，前次级运动皮层;MOsp，前初级运动皮层;SSp.tr，初级体感皮层-躯干;RSP，脾后皮层的背侧部分;VISp，初级视觉皮层）。单因素方差分析后进行 Tukey 检验，* P < 0.05，** P < 0.01，*** P < 0.001。数据显示为SEM±平均值。每种颜色表示一个主题，n = 6。H-K 中风后不同时间点（D2-D28）中风（灰色）和机器人 + tACS（深红色）组相对于基线（中风前）功能连接 （ΔFC） 在全皮层 （H）、半球间连通性 （I）、同侧半球半球内连通性 （J） 和对侧半球半球内连通性 （K） 方面的变化比较。数据显示为箱线图（均值 ± SE）。*p < 0.05，处理主效应（二样本t检验）。蓝色和红色箭头分别表示与中风前FC相比的超连接和低连接方向（中风n = 5小鼠，机器人+ tACS n = 6小鼠）。该数字背后的数据可以在 https://data.mendeley.com/datasets/mw82tzp4rx/1 中找到。 https://doi.org/10.1371/journal.pbio.3002806.s005 (TIF) S6 Fig. Referred to Fig 6. A, the averaged difference correlation matrices, produced by subtracting the poststroke FC (2, 5, 8, 14, or 30 days after injury) from prestroke FC in the stroke group after global signal regression. Red squares indicate poststroke hypo-connectivity, blue squares indicate poststroke hyper-connectivity of PV-IN. B, the averaged difference correlation matrices, produced by subtracting the poststroke FC (2, 5, 8, 14, or 30 days after injury) from prestroke FC in the Robot + tACS group after global signal regression. Red squares indicate poststroke hypo-connectivity, blue squares indicate poststroke hyper-connectivity of PV-IN. C, Box charts showing inter-hemispheric FC of ipsilesional and contralesional secondary and primary motor cortices in the anterior regions (MOsa and MOpa, respectively) for the Robot tACS group. D, Intra-hemispheric FC of contralesional areas for the Robot tACS group. E, The box charts displaying intra-hemispheric FC of the secondary (left) and primary (right) contralesional motor cortices in the anterior region for the Robot tACS group. One-way ANOVA followed by Tukey test, * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± SEM. Each color indicates a single subject, n = 6. The data underlying this figure can be found in https://data.mendeley.com/datasets/mw82tzp4rx/1. https://doi.org/10.1371/journal.pbio.3002806.s006 (TIF) S7 Fig. Referred to Fig 2. A, Power spectral density (PSD) of GCaMP7f fluorescence and 530 nm reflectance signals from left CFA region (in red) and left SSp-bfd region (in blue) in wake resting-state condition prestroke (ROI: 5 × 5 pixels, FOV: 128 × 128 pixels). These regions are clearly indicated in the inset of the figure (scale bar: 1 mm, white dot indicates bregma). For each region, we report the raw (uncorrected) GCaMP7f signal (darker color), the signal after correction for hemodynamic contamination (palet tone), and the corresponding 530 nm reflectance trace (lighter color). Data are plotted on a log–log scale to highlight frequency-dependent power and the presence of 1/f dynamics (n = 1). B, The upper plot shows representative time courses of raw GCaMP7f fluorescence (ΔF/F, dark red) and 530 nm reflectance (ΔI/I, light red) signals extracted from the left CFA region (CFA L) during a 120-s widefield optical imaging (WFOI) session. Corrected GCaMP7f fluorescence (bright red) was obtained by regressing out the 530 nm reflectance signal from the raw GCaMP7f signal, to reduce contamination from hemoglobin absorption. The bottom panel presents a magnified view of the time window highlighted in green in the upper plot (10–30 s), allowing clearer visualization of the temporal relationships between the raw and corrected GCaMP7f signals and the hemodynamic component. C, The upper plot shows representative time courses of raw GCaMP7f fluorescence (ΔF/F, dark red) and 530 nm reflectance (ΔI/I, light red) signals extracted from the left barrelfield region (SSp-bfd L) during a 120-s widefield optical imaging (WFOI) session. Corrected GCaMP7f fluorescence (bright red) was obtained by regressing out the 530 nm reflectance signal from the raw GCaMP7f signal, to reduce contamination from hemoglobin absorption. The bottom panel presents a magnified view of the time window highlighted in green in the upper plot (approximately 10–30 s), allowing clearer visualization of the temporal relationships between the raw and corrected GCaMP7f signals and the hemodynamic component. D, Functional connectivity matrices derived from hemodynamic signals before (PRE) and at multiple time points after stroke (2, 5, 8, 14, 21, and 28 days poststroke). Color scale represents Pearson’s correlation coefficient (r), ranging from −0.6 to 1 for hemodynamic data (n = 5 mice). The data underlying this figure can be found in https://data.mendeley.com/datasets/mw82tzp4rx/1. https://doi.org/10.1371/journal.pbio.3002806.s007 (TIF) S8 Fig. Referred to Fig 6. Representative PV-Cre::GCaMP7f resting state cortical activity maps in a mouse at baseline (A) and after 2 (B), 5 (C), 8 (D), 14 (E), 21 (F), 28 (G), 36 (H), and 43 (I) days after stroke. From day 5 to day 28, the mouse received daily tACS. L: lateral; M: medial; R: rostral; C: caudal. Scale bar: 1 mm. The data underlying this figure can be found in https://data.mendeley.com/datasets/mw82tzp4rx/1. https://doi.org/10.1371/journal.pbio.3002806.s008 (TIF) Acknowledgments We thank Francesca Biondi (CNR Pisa) for the excellent animal care and Renzo di Renzo and Elena Novelli (CNR Pisa) for their technical support in microscopy and informatics. References 1.Allegra Mascaro AL, Conti E, Lai S, Di Giovanna AP, Spalletti C, Alia C, et al. Combined rehabilitation promotes the recovery of structural and functional features of healthy neuronal networks after stroke. Cell Rep. 2019;28(13):3474–85.e6. pmid:31553915 View ArticlePubMed/NCBIGoogle Scholar 2.Conti S, Spalletti C, Pasquini M, Giordano N, Barsotti N, Mainardi M, et al. Combining robotics with enhanced serotonin-driven cortical plasticity improves post-stroke motor recovery. Prog Neurobiol. 2021;203:102073. View ArticleGoogle Scholar 3.Spalletti C, Alia C, Lai S, Panarese A, Conti S, Micera S, et al. Combining robotic training and inactivation of the healthy hemisphere restores pre-stroke motor patterns in mice. Elife. 2017;6:e28662. pmid:29280732 View ArticlePubMed/NCBIGoogle Scholar 4.Di Pino G, Pellegrino G, Assenza G, Capone F, Ferreri F, Formica D, et al. Modulation of brain plasticity in stroke: a novel model for neurorehabilitation. Nat Rev Neurol. 2014;10:597–608. View ArticleGoogle Scholar 5.Koganemaru S, Kitatani R, Fukushima-Maeda A, Mikami Y, Okita Y, Matsuhashi M, et al. Gait-synchronized rhythmic brain stimulation improves poststroke gait disturbance: a pilot study. Stroke. 2019;50(11):3205–12. pmid:31500557 View ArticlePubMed/NCBIGoogle Scholar 6.Alia C、Spalletti C、Lai S、Panarese A、Lamola G、Bertolucci F 等。脑缺血后的神经可塑性变化及其对中风恢复的贡献：神经康复的新方法。前细胞神经科学。2017;11:76. 查看文章谷歌学术 7.Cheyne D， Ferrari P. MEG 运动皮层伽马振荡的研究：大脑中伽马“指纹”的证据？前嗡嗡声神经科学。2013;7:575.PMID：24062675 查看文章考研/NCBI谷歌学术 8.盖茨 W、刘 C、朱 H、布洛伊 L、罗伯茨 TPL。控制响应干扰的运动伽马波段网络的证据。神经影像。2013;74:245–53.PMID：23454050 查看文章考研/NCBI谷歌学术 9.Joundi RA， Jenkinson N， Brittain J-S， Aziz TZ， Brown P. 驱动人类皮层的振荡活动可以增强运动性能。Curr Biol. 2012;22(5):403–7.PMID：22305755 查看文章考研/NCBI谷歌学术 10.乌尔哈斯 PJ、琵琶 G、诺伊恩施万德 S、威布拉尔 M、辛格 W.伽马的新面貌？皮质网络中的高 （>60 Hz） γ 波段活动：功能、机制和损伤。Prog Biophys Mol Biol. 2011 年;105(1–2):14–28.PMID：21034768 查看文章考研/NCBI谷歌学术 11.阿莫 C、德圣地亚哥 L、扎尔扎·卢西亚涅斯 D、莱昂·阿隆索-科尔特斯 JM、阿隆索-阿隆索 M、巴雷亚 R 等。脑电图诱导的伽马波段活动是运动任务中与训练相关的大脑可塑性的可能指标。公共科学图书馆一号。2017;12（10）：e0186008。PMID：28982173 View ArticlePubMed/NCBIGoogle Scholar 12.Brown P、Salenius S、Rothwell JC、Hari R. 人类 Piper 节律的皮质相关性。神经生理学杂志。1998;80(6):2911–7.PMID：9862895 查看文章考研/NCBI谷歌学术 13.Tecchio F、Pasqualetti P、Zappasodi F、Tombini M、Lupoi D、Vernieri F 等。通过脑磁图预测急性单半球中风的结果。神经学杂志 2007 年;254(3):296–305.PMID：17345051 查看文章考研/NCBI谷歌学术 14.方 Y、戴利 JJ、孙 J、赫沃拉特 K、弗雷德里克森 E、庞迪克 S 等。中风后，到达过程中的功能性皮质肌肉连接减弱。临床神经生理学。2009;120:994. 查看文章谷歌学术 15.Hall SD、Stanford IM、Yamawaki N、Mcallister CJ、Rönnqvist KC、Woodhall GL 等。GABA能调节在运动功能相关神经元网络活动中的作用。神经图像。2011. 查看文章谷歌学术 16.布萨基 G，王 XJ。伽马振荡的机制。安努牧师神经科学。2012;35:203–25.PMID：22443509 查看文章考研/NCBI谷歌学术 17.盖茨 W、埃德加 JC、王 DJ、罗伯茨 TPL。将 MEG 测量的运动皮层振荡与静息 γ-氨基丁酸 （GABA） 浓度联系起来。神经影像。2011;55(2):616–21.PMID：21215806 查看文章考研/NCBI谷歌学术 18.诺瓦克 DA、格雷夫克斯 C、达福塔基斯 M、艾克霍夫 S、库斯特 J、卡贝 H 等。低频重复经颅磁刺激对病灶对侧初级运动皮层对皮质下中风运动运动学和神经活动的影响。Arch Neurol. 2008;65(6):741–7.PMID：18541794 查看文章考研/NCBI谷歌学术 19.Sohal VS、Zhang F、Yizhar O、Deisseroth K. 小白蛋白神经元和伽马节律可增强皮质回路性能。自然界。2009;459(7247):698–702.PMID：19396159 查看文章考研/NCBI谷歌学术 20.Cardin JA、Carlén M、Meletis K、Knoblich U、Zhang F、Deisseroth K 等。驱动快速尖峰细胞可诱导伽马节律并控制感觉反应。自然界。2009;459(7247):663–7.PMID：19396156 查看文章考研/NCBI谷歌学术 21.唐 X、Shi J、Lin S、He Z、Cui S、Di W 等。锥体和小白蛋白神经元调节中风康复的电针刺激过程。iScience。2024;27(5):109695.PMID：38680657 查看文章考研/NCBI谷歌学术 22.刘 W、何 X、林 H、杨 M、戴 Y、陈 L 等。通过对侧运动皮层小白蛋白神经元的光遗传学调节进行缺血性中风康复。Exp Neurol. 2023;360：114289。PMID：36471512 查看文章考研/NCBI谷歌学术 23.巴尔比 M、肖 D、贾蒂瓦·维加 M、胡 H、万尼 MP、伯尼尔 LP 等。中风后急性期抑制性神经元的伽马频率激活可减轻血管和行为功能障碍。细胞代表 2021;34：108696。 查看文章谷歌学术 24.Wang C、Lin C、Zhao Y、Samantzis M、Sedlak P、Sah P 等人 40 Hz 光遗传学刺激可挽救中风后的功能性突触可塑性。细胞代表 2023 年;42(12):113475.PMID：37979173 查看文章考研/NCBI谷歌学术 25.冈部 N、Wei X、Abumeri F、Batac J、Hovanesyan M、Dai W 等。小白蛋白中间神经元调节中风后康复诱导的功能恢复，并确定康复药物。纳特公社。2025;16(1):2556.PMID：40089466 查看文章考研/NCBI谷歌学术 26.Ovadia-Caro S、Khalil AA、Sehm B、Villringer A、Nikulin VV、Nazarova M. 预测中风对无创脑刺激的反应。前神经学 2019;10：302。PMID：31001190 查看文章考研/NCBI谷歌学术 27.Spalletti C、Lai S、Mainardi M、Panarese A、Ghionzoli A、Alia C 等。一种用于小鼠前肢回缩定量评估和中风后训练的机器人系统。神经康复神经修复。2014;28(2):188–96.PMID：24213954 查看文章考研/NCBI谷歌学术 28.李 H、张 N、林 HY、Yu Y、蔡 Q-Y、马 L 等。成年小鼠光血栓形成诱导的缺血后中风结果的组织学、细胞和行为评估。BMC 神经科学。2014;15:58.PMID：24886391 查看文章考研/NCBI谷歌学术 29.扎莱斯基 A、福尼托 A、布尔莫尔 ET。基于网络的统计：识别大脑网络中的差异。神经影像。2010;53(4):1197–207.PMID：20600983 View ArticlePubMed/NCBIGoogle Scholar 30.Nakai N, Sato M, Yamashita O, Sekine Y, Fu X, Nakai J, et al. Virtual reality-based real-time imaging reveals abnormal cortical dynamics during behavioral transitions in a mouse model of autism. Cell Rep. 2023;42(4):112258. pmid:36990094 View ArticlePubMed/NCBIGoogle Scholar 31.Ma Y, Shaik MA, Kozberg MG, Kim SH, Portes JP, Timerman D, et al. Resting-state hemodynamics are spatiotemporally coupled to synchronized and symmetric neural activity in excitatory neurons. Proc Natl Acad Sci U S A. 2016;113(52):E8463–71. pmid:27974609 View ArticlePubMed/NCBIGoogle Scholar 32.Wright PW, Brier LM, Bauer AQ, Baxter GA, Kraft AW, Reisman MD, et al. Functional connectivity structure of cortical calcium dynamics in anesthetized and awake mice. PLoS One. 2017;12:e0185759. View ArticleGoogle Scholar 33.Dancause N. Vicarious function of remote cortex following stroke: recent evidence from human and animal studies. Neuroscientist. 2006;12(6):489–99. pmid:17079515 View ArticlePubMed/NCBIGoogle Scholar 34.Silasi G, Murphy TH. Stroke and the connectome: how connectivity guides therapeutic intervention. Neuron. 2014;83(6):1354–68. pmid:25233317 View ArticlePubMed/NCBIGoogle Scholar 35.Zilles K、Schlaug G、Geyer S、Luppino G、Matelli M、Qü M 等。人类和非人类灵长类动物大脑中辅助运动区域的解剖学和递质受体。Adv Neurol. 1996;70:29–43.PMID：8615210 查看文章考研/NCBI谷歌学术 36.Tennant KA、Adkins DL、Donlan NA、Asay AL、Thomas N、Kleim JA 等。C57BL/6 小鼠运动皮层前肢表示的组织，由皮质内微刺激和细胞结构定义。大脑皮层。2011;21(4):865–76.PMID：20739477 查看文章考研/NCBI谷歌学术 37.西部 M、巴贝 S、古根莫斯 D、努多 RJ。控制大鼠皮质冲击后运动皮层的重组及其对功能恢复的影响。J 神经创伤。2010;27(12):2221–32.PMID：20873958 查看文章考研/NCBI谷歌学术 38.Umeda T， Isa T. 大鼠新生儿半皮质化后喙部和尾部额叶前肢区域对代偿过程的不同贡献。欧洲神经科学杂志。2011;34(9):1453–60.PMID：22034976 查看文章考研/NCBI谷歌学术 39.Dancause N、Touvykine B、Mansoori BK。中风后对侧半球的抑制：回顾一些以动物模型为重点的构建块。Prog Brain Res. 2015 年;218:361–87.PMID：25890146 查看文章考研/NCBI谷歌学术 40.Zeiler SR、Gibson EM、Hoesch RE、Li MY、Worley PF、O'Brien RJ 等。内侧运动前皮层显示抑制标志物减少，并在局灶性中风小鼠模型中介导恢复。中风。2013;44(2):483–9.PMID：23321442 查看文章考研/NCBI谷歌学术 41.Darvas F、Scherer R、Ojemann JG、Rao RP、Miller KJ、Sorensen LB. 使用脑电图进行高伽马映射。神经影像。2010;49(1):930–8.PMID：19715762 查看文章考研/NCBI谷歌学术 42.Ball T、Demandt E、Mutschler I、Neitzel E、Mehring C、Vogt K 等。人类脑电图高伽马范围内的运动相关活动。神经影像。2008;41(2):302–10.PMID：18424182 查看文章考研/NCBI谷歌学术 43.Kim T、Thankachan S、McKenna JT、McNally JM、Yang C、Choi JH 等。皮质投射的基底前脑小白蛋白神经元调节皮质伽马带振荡。Proc Natl Acad Sci U S A. 2015;112(11):3535–40.PMID：25733878 查看文章考研/NCBI谷歌学术 44.Allen WE、Kauvar IV、Chen MZ、Richman EB、Yang SJ、Chan K 等。小鼠新皮质不同细胞类型中目标导向行为的全局表示。神经元。2017;94（4）：891-907.e6。PMID：28521139 查看文章考研/NCBI谷歌学术 45.任 C、Peng K、Yang R、Liu W、Liu C、Komiyama T. 运动学习过程中乙酰胆碱调节的皮质抑制神经元的全局和亚型特异性调节。神经元。2022;110（14）：2334–50.e8。PMID：35584693 查看文章考研/NCBI谷歌学术 46.撒哈拉 S、柳川 Y、奥利里 DDM、史蒂文斯 CF。皮质 GABA 能神经元的分数从皮质神经发生开始到成年期是恒定的。神经科学杂志。2012;32:4755–61. 查看文章谷歌学术 47.贝克 A、卡尔姆巴赫 B、森岛 M、金 J、胡维内特 A、李 N 等。新皮质深层锥体神经元的特殊亚群：将细胞特性与功能后果联系起来。神经科学杂志。2018;38(24):5441–55.PMID：29798890 查看文章考研/NCBI谷歌学术 48.Park J、Phillips JW、Guo J-Z、Martin KA、Hantman AW、Dudman JT。熟练前肢运动的运动皮层输出选择性地分布在投射神经元类别中。科学高级 2022;8（10）：eabj5167。PMID：35263129 查看文章考研/NCBI谷歌学术 49.Welle CG， Contreras D. 感觉驱动和自发伽马振荡涉及不同的皮质回路。神经生理学杂志。2016;115(4):1821–35.PMID：26719085 查看文章考研/NCBI谷歌学术 50.Meneghetti N、Cerri C、Tantillo E、Vannini E、Caleo M、Mazzoni A. 窄带和宽γ带处理小鼠初级视觉皮层中的互补视觉信息。电子神经。2021;8（6）：ENEURO.0106-21.2021。PMID：34663617 查看文章考研/NCBI谷歌学术 51.Buffalo EA、Fries P、Landman R、Buschman TJ、Desimone R. 腹侧流中 γ 和 α 相干性的层流差异。Proc Natl Acad Sci U S A. 2011;108(27):11262–7.PMID：21690410 查看文章考研/NCBI谷歌学术 52.Scheeringa R， Fries P. 皮质层、节奏和 BOLD 信号。神经影像。2019;197:689–98.PMID：29108940 查看文章考研/NCBI谷歌学术 53.卡丹 JA。抑制性中间神经元调节皮质回路中的时间精度和相关性。趋势神经科学。2018;41(10):689–700.PMID：30274604 查看文章考研/NCBI谷歌学术 54.Kamigaki T， Dan Y. 特定前额叶中间神经元亚型的延迟活动调节记忆引导行为。纳特神经科学。2017;20(6):854–63.PMID：28436982 查看文章考研/NCBI谷歌学术 55.鲍尔 AQ、卡夫 AW、赖特 PW、斯奈德 AZ、李 JM、卡尔弗 JP。小鼠缺血性中风后功能连接中断的光学成像。神经影像。2014;99:388–401. 查看文章谷歌学术 56.Lim DH, LeDue JM, Mohajerani MH, Murphy TH. Optogenetic mapping after stroke reveals network-wide scaling of functional connections and heterogeneous recovery of the peri-infarct. J Neurosci. 2014;34:16455–66. View ArticleGoogle Scholar 57.Cramer JV, Gesierich B, Roth S, Dichgans M, Düring M, Liesz A. In vivo widefield calcium imaging of the mouse cortex for analysis of network connectivity in health and brain disease. Neuroimage. 2019;199:570–84. pmid:31181333 View ArticlePubMed/NCBIGoogle Scholar 58.Purcell JJ, Wiley RW, Rapp B. Re-learning to be different: increased neural differentiation supports post-stroke language recovery. Neuroimage. 2019;202:116145. pmid:31479754 View ArticlePubMed/NCBIGoogle Scholar 59.Liou J-Y, Ma H, Wenzel M, Zhao M, Baird-Daniel E, Smith EH, et al. Role of inhibitory control in modulating focal seizure spread. Brain. 2018;141(7):2083–97. pmid:29757347 View ArticlePubMed/NCBIGoogle Scholar 60.Contractor A, Ethell IM, Portera-Cailliau C. Cortical interneurons in autism. Nat Neurosci. 2021;24:1648–59. View ArticleGoogle Scholar 61.Xu Y, Zhao M, Han Y, Zhang H. GABAergic inhibitory interneuron deficits in Alzheimer’s disease: implications for treatment. Front Neurosci. 2020;14:660. pmid:32714136 View ArticlePubMed/NCBIGoogle Scholar 62.Isomura Y, Harukuni R, Takekawa T, Aizawa H, Fukai T. Microcircuitry coordination of cortical motor information in self-initiation of voluntary movements. Nat Neurosci. 2009;12(12):1586–93. pmid:19898469 View ArticlePubMed/NCBIGoogle Scholar 63.Estebanez L, Hoffmann D, Voigt BC, Poulet JFA. Parvalbumin-expressing GABAergic neurons in primary motor cortex signal reaching. Cell Rep. 2017;20:308–18. View ArticleGoogle Scholar 64.Magliozzi R, Pitteri M, Ziccardi S, Pisani AI, Montibeller L, Marastoni D, et al. CSF parvalbumin levels reflect interneuron loss linked with cortical pathology in multiple sclerosis. Ann Clin Transl Neurol. 2021;8(3):534–47. pmid:33484486 View ArticlePubMed/NCBIGoogle Scholar 65.Ince P, Stout N, Shaw P, Slade J, Hunziker W, Heizmann CW, et al. Parvalbumin and calbindin D-28k in the human motor system and in motor neuron disease. Neuropathol Appl Neurobiol. 1993;19:291–9. View ArticleGoogle Scholar 66.Hsieh T-H, Lee HHC, Hameed MQ, Pascual-Leone A, Hensch TK, Rotenberg A. Trajectory of parvalbumin cell impairment and loss of cortical inhibition in traumatic brain injury. Cereb Cortex. 2017;27(12):5509–24. pmid:27909008 View ArticlePubMed/NCBIGoogle Scholar 67.Nihei K, McKee AC, Kowall NW. Patterns of neuronal degeneration in the motor cortex of amyotrophic lateral sclerosis patients. Acta Neuropathol. 1993;86(1):55–64. pmid:8396837 View ArticlePubMed/NCBIGoogle Scholar 68.Giordano N, Alia C, Fruzzetti L, Pasquini M, Palla G, Mazzoni A, et al. Fast-spiking interneurons of the premotor cortex contribute to initiation and execution of spontaneous actions. J Neurosci. 2023;43(23):4234–50. pmid:37197980 View ArticlePubMed/NCBIGoogle Scholar 69.Alia C, Spalletti C, Lai S, Panarese A, Micera S, Caleo M. Reducing GABAA-mediated inhibition improves forelimb motor function after focal cortical stroke in mice. Sci Rep. 2016;6:37823. pmid:27897203 View ArticlePubMed/NCBIGoogle Scholar 70.Conti E, Scaglione A, de Vito G, Calugi F, Pasquini M, Pizzorusso T, et al. Combining optogenetic stimulation and motor training improves functional recovery and perilesional cortical activity. Neurorehabil Neural Repair. 2022;36:107–18. View ArticleGoogle Scholar 71.Liu Y, Yin J-H, Lee J-T, Peng G-S, Yang F-C. Early rehabilitation after acute stroke: the golden recovery period. acta neurol Taiwan; 2022. Available from: https://www.ncbi.nlm.nih.gov/pubmed/34918304 72.Dromerick AW, Geed S, Barth J, Brady K, Giannetti ML, Mitchell A, et al. Critical Period After Stroke Study (CPASS): a phase II clinical trial testing an optimal time for motor recovery after stroke in humans. Proc Natl Acad Sci U S A. 2021;118(39):e2026676118. pmid:34544853 View ArticlePubMed/NCBIGoogle Scholar 73.海沃德 KS、克莱默 SF、道尔顿 EJ、休斯 GR、布罗德曼 A、丘里洛夫 L 等。中风后上肢运动干预的时间和剂量：系统评价。中风。2021;52(11):3706–17.PMID：34601901 查看文章考研/NCBI谷歌学术 74.Iaccarino HF、Singer AC、Martorell AJ、Rudenko A、Gao F、Gillingham TZ 等。伽马频率夹带减弱淀粉样蛋白负荷并修饰小胶质细胞。自然界。2016;540(7632):230–5.PMID：27929004 查看文章考研/NCBI谷歌学术 75.Malyshev A、Goz R、LoTurco JJ、Volgushev M. 在研究快速尖峰编码中使用光遗传学方法的优势和局限性。公共科学图书馆一号。2015;10：e0122286。 查看文章谷歌学术 76.Boyden ES、Zhang F、Bamberg E、Nagel G、Deisseroth K. 毫秒时间尺度，神经活动的基因靶向光学控制。纳特神经科学。2005;8:1263–8. 查看文章谷歌学术 77.林 JY、林 MZ、斯坦巴赫 P、钱 RY。具有改进特性和动力学的工程通道视紫红质变体的表征。生物物理学杂志 2009 年;96(5):1803–14.PMID：19254539 查看文章考研/NCBI谷歌学术 78.迪皮诺 G、佩莱格里诺 G、阿森扎 G、卡彭 F、费雷里 F、福米卡 D 等。中风大脑可塑性的调节：一种神经康复的新模型。Nat Rev Neurol. 2014 年;10(10):597–608.PMID：25201238 查看文章考研/NCBI谷歌学术 79.车 THH，黄 HS。康复干预联合无创脑刺激对中风患者上肢运动功能的影响。脑科学 2022;12(8):994.PMID：35892435 查看文章考研/NCBI谷歌学术 80.Naro A、Billeri L、Manuli A、Balletta T、Cannavò A、Portaro S 等。打破僵局以改善慢性中风患者的运动结果：一项关于神经调控加机器人技术的回顾性临床研究。神经科学 2021;42:2785–93. 查看文章谷歌学术 81.Zeiler SR，克拉考尔 JW。中风后大脑中训练与可塑性之间的相互作用。Curr Opin Neurol. 2013 年;26(6):609–16.PMID：24136129 查看文章考研/NCBI谷歌学术 82.Elyamany O、Leicht G、Herrmann CS、Mulert C. 经颅交流电刺激 （tACS）：从基本机制到精神病学的首次应用。Eur Arch 精神病学临床神经科学。2021;271(1):135–56.PMID：33211157 查看文章考研/NCBI谷歌学术 83.莱因哈特 RMG，阮 JA。通过同步有节奏的大脑回路，老年人的工作记忆得以恢复。纳特神经科学。2019;22(5):820–7.PMID：30962628 查看文章考研/NCBI谷歌学术 84.Vosskuhl J、Huster RJ、Herrmann CS. α 范围内经颅交流电刺激 （tACS） 的大胆信号效应：一项同步 tACS-fMRI 研究。神经影像。2016;140:118–25.PMID：26458516 查看文章考研/NCBI谷歌学术 85.Moisa M、Polania R、Grueschow M、Ruff CC. 通过经颅夹带伽马振荡增强运动的脑网络机制。神经科学杂志。2016;36(47):12053–65.PMID：27881788 查看文章考研/NCBI谷歌学术 86.Rostami M、Lee A、Frazer AK、Akalu Y、Siddique U、Pearce AJ 等。确定经颅交流电刺激对皮质运动兴奋性和运动性能的影响：四个频率的假对照比较。神经。2025;568:12–26. 查看文章谷歌学术 87.Naro A、Bramanti A、Leo A、Manuli A、Sciarrone F、Russo M 等。小脑经颅交流电刺激对运动皮层兴奋性和运动功能的影响。大脑结构功能。2017;222:2891–906. 查看文章谷歌学术 88.丁X， 周Y， 刘Y， Yao X-L， Wang J-X， Xie Q. 不同频率tACS在卒中康复中的应用与研究进展：系统评价.大脑研究 2025;1852：149521。PMID：39983809 查看文章考研/NCBI谷歌学术 89.Clarkson AN、Huang BS、Macisaac SE、Mody I、Carmichael ST. 减少过度的 GABA 介导的强直抑制可促进中风后的功能恢复。自然界。2010;468(7321):305–9.PMID：21048709 查看文章考研/NCBI谷歌学术 90.Hiu T、Farzampour Z、Paz JT、Wang EHJ、Badgely C、Olson A 等。中风修复阶段增强阶段性 GABA 抑制：一种新的治疗靶点。脑。2016;139（第 2 部分）：468–80。PMID：26685158 查看文章考研/NCBI谷歌学术 91.Alia C、Cangi D、Massa V、Salluzzo M、Vignozzi L、Caleo M 等。介导中风后功能恢复的细胞间相互作用。细胞。2021;10(11):3050.PMID：34831273 查看文章考研/NCBI谷歌学术 92.Resta F、Montagni E、De Vito G、Scaglione A、Letizia A、Mascaro A 等。运动皮层连接的大规模全光学解剖揭示了小鼠前肢表征的分离功能组织。生物Rxiv。2021. 查看文章谷歌学术 93.Scott BB、Thiberge SY、Guo C、Tervo DGR、Brody CD、Karpova AY 等。使用头戴式宽视场宏观镜对GCaMP转基因大鼠的皮质动力学进行成像。神经元。2018;100（5）：1045-1058.e5。PMID：30482694 查看文章考研/NCBI谷歌学术 94.Cardin JA、Carlén M、Meletis K、Knoblich U、Zhang F、Deisseroth K 等。使用通道视紫红质-2 的细胞类型特异性表达在体内靶向光遗传学刺激和记录神经元。国家协议。2010;5(2):247–54.PMID：20134425 查看文章考研/NCBI谷歌学术 95.Sessolo M、Marcon I、Bovetti S、Losi G、Cammarota M、Ratto GM 等。小白蛋白阳性抑制性中间神经元反对传播，但有利于局灶性癫痫样活性的产生。神经科学杂志。2015;35(26):9544–57.PMID：26134638 查看文章考研/NCBI谷歌学术 96.Lai S、Panarese A、Spalletti C、Alia C、Ghionzoli A、Caleo M 等。中风后小鼠到达损伤的定量运动学特征。神经康复神经修复。2015;29(4):382–92.PMID：25323462 查看文章考研/NCBI谷歌学术