Precise Modulation of Gold Nanorods for Protecting against Malignant Ventricular Arrhythmias via Near-Infrared Neuromodulation
通过近红外神经调节精确调节金纳米棒预防恶性室性心律失常

1 Introduction 1 简介

Malignant ventricular arrhythmias (VAs) induced by ischemia are considered the major cause of sudden cardiac death.1, 2 Hyperactivity of the cardiac sympathetic nerves, particularly the left stellate ganglion (LSG), appears to directly trigger VAs in ischemic hearts.3-7 Strategies that decrease LSG function and neural activity have been shown to stabilize ventricular electrophysiology and reduce the occurrence of ischemia-induced VAs in animals.8-11 In addition, clinical interventions applied to the LSG, such as left cardiac sympathetic denervation (LCSD) and stellate ganglion blockade (SGB), have been used to treat patients with drug-refractory VAs, catecholaminergic polymorphic ventricular tachycardia or long QT syndrome (LQTS), showing significant effects in protecting against VAs.12-15 However, stellate ganglion ablation surgery has some side effects, such as Horner's syndrome, unilateral hand dryness, unexpected hemorrhage, abnormal sweating, chest pain, and incomplete denervation, which have impeded its wide use in the clinic.16 Moreover, recent study demonstrated that sympathetic nerves denervation significantly compromised the compensatory hemodynamic response to hypotensive hemorrhage.17 These studies indicate that complete sympathetic denervation may cause unavoidable side-effects and attenuate sympathetic protection during hemorrhage. Therefore, we hypothesized that neuromodulation might become an optimized strategy for treating autonomic activity-related diseases rather than complete ablation. Our previous study has found that optogenetics, combining gene modified LSG neurons with light stimuli, could reversely inhibit the LSG activity and protect against ischemia-induced VAs in dogs with myocardial ischemia (MI).18 Although optogenetics enables precise control of neural activity, adeno-associated viral (AAV) transfection might induce potential toxic effects or immune response and light-mediated neuromodulation for deep tissue has yet to be achieved.19 Therefore, it is necessary to find a safer and effective method to modulate the LSG neural activity, thus preventing ischemia-induced VAs.
由缺血引起的恶性室性心律失常（VA）被认为是心源性猝死的主要原因。 1, 2 心脏交感神经（尤其是左星状神经节 (LSG)）的过度活跃似乎会直接触发缺血心脏中的 VA。 3- 7 减少 LSG 功能和神经活动的策略已被证明可以稳定心室电生理并减少动物缺血引起的 VA 的发生。 8- 11 此外，应用于 LSG 的临床干预措施，如左心交感神经去神经术 (LCSD) 和星状神经节阻滞 (SGB)，已用于治疗药物难治性 VA、儿茶酚胺能多形性室性心动过速或长 QT 综合征的患者(LQTS)，在预防 VA 方面显示出显着效果。 12- 15 然而，星状神经节消融手术存在一些副作用，如霍纳综合征、单侧手部干燥、意外出血、出汗异常、胸痛、去神经不完全等，这些都阻碍了其在临床的广泛应用。 16 此外，最近的研究表明，交感神经去神经支配显着损害了对低血压出血的代偿性血流动力学反应。 17 这些研究表明，完全去交感神经支配可能会导致不可避免的副作用，并削弱出血期间的交感神经保护作用。因此，我们假设神经调节可能成为治疗自主活动相关疾病的优化策略，而不是完全消融。我们之前的研究发现，光遗传学，将基因修饰的LSG神经元与光刺激相结合，可以反向抑制LSG活性，并保护患有心肌缺血（MI）的狗免受缺血诱导的VA。 18 尽管光遗传学能够精确控制神经活动，但腺相关病毒 (AAV) 转染可能会引起潜在的毒性作用或免疫反应，并且光介导的深层组织神经调节尚未实现。 19 因此，有必要找到一种更安全、有效的方法来调节 LSG 神经活动，从而预防缺血引起的 VA。

In recent years, optical neuromodulation based on nanotechnology has revealed great advantages in vitro and in vivo, enabling the precise control of neural activity.20-22 Near-infrared (NIR) light, due to its deepest tissue penetration compared to visible and UV light, has been widely applied in biological tissues.23-25 NIR-sensitive gold nanorods (AuNRs), as a good biocompatibility and high photothermal conversion agent, have been investigated to modulate neural activity due to their ability to convert NIR light energy to heat energy through the localized surface plasmonic response (LSPR). The local heating mediated by AuNRs upon NIR irradiation has been reported to suppress neural activity through the photothermal effects of AuNRs.21, 22, 26, 27 Therefore, in the present study, we aimed to determine the capacity of NIR-sensitive AuNRs to photothermally inhibit LSG function and neural activity and their effects on the occurrence of ischemia-induced VAs in an acute ischemic canine model (Figure 1).
近年来，基于纳米技术的光学神经调节在体外和体内表现出巨大的优势，能够精确控制神经活动。 20- 22 与可见光和紫外光相比，近红外 (NIR) 光具有最深的组织穿透力，已广泛应用于生物组织。 23- 25 近红外敏感金纳米棒 (AuNR) 作为一种良好的生物相容性和高光热转化剂，由于其能够通过局域表面等离子体响应 (LSPR) 将近红外光能转化为热能，因此已被研究用于调节神经活动。据报道，近红外辐射下 AuNR 介导的局部加热可通过 AuNR 的光热效应抑制神经活动。 21, 22, 26, 27 因此，在本研究中，我们的目的是确定近红外敏感的 AuNRs 光热抑制 LSG 功能和神经活动的能力及其对急性缺血犬模型中缺血诱导的 VAs 发生的影响（图1）。

2 Results and Discussion
2 结果与讨论

Since biological tissues have minimal absorption in the medical spectral window (650–900 nm), light in this window can penetrate deeper into biological tissues and avoid collateral thermal damage.24, 28 First, to optimize the response of the AuNRs to the 808 nm NIR laser, the longitudinal absorption peak of the AuNRs should be close to 808 nm for maximum absorption. The longitudinal absorption peak of the AuNRs can be tuned by varying the amount of Ag ions and the volume of the seed solution. AuNRs with different absorption peaks were prepared and showed longitudinal surface plasmon resonance (SPR) peaks at 794, 810, and 834 nm (Figure S1a, Supporting Information). The absorption peak at 810 nm is very close to the wavelength of the NIR laser. The AuNRs were examined by transmission electron microscopy (TEM) (Figure 2a), which were captured and analyzed using Nano Measurer to establish the diameter and length of the AuNRs, which were 21.1 ± 0.2 and 74.0 ± 0.5 nm, respectively (Figure S2a,b, Supporting Information). To verify that the absorption peak of the gold nanorods of this size prepared by us is around 808 nm, we further simulated the absorption spectra of the AuNRs using the finite-difference time-domain method, and the results are shown in Figure S1b (Supporting Information). When the diameter and length of the AuNRs were set as 21.1 and 74.0 nm, the simulated longitudinal absorption peak at 802 nm was largely consistent with the above experimental result. To explore the relationship between the absorption peak and the photothermal effect of the AuNRs, we further studied the photothermal curves of the AuNRs with different absorption peaks under the 808 nm NIR laser. AuNRs at 80 µg mL⁻¹ were investigated with 5 min of irradiation at 1.5 W cm⁻². All the AuNRs exhibited an obvious photothermal effect, and the effect of the AuNRs with a longitudinal SPR peak at 810 nm was much larger than that of the other AuNRs (Figure S3, Supporting Information). These results show that the photothermal effect is stronger the closer the absorption peak of the AuNRs is to the excitation light wavelength. Thus, the AuNRs with an absorption peak at 810 nm were used for further research.
由于生物组织在医学光谱窗口（650-900 nm）内的吸收最小，因此该窗口中的光可以更深入地穿透生物组织并避免附带的热损伤。 24, 28 首先，为了优化 AuNR 对 808 nm NIR 激光的响应，AuNR 的纵向吸收峰应接近 808 nm，以获得最大吸收。 AuNR 的纵向吸收峰可以通过改变 Ag 离子的量和种子溶液的体积来调节。制备了具有不同吸收峰的 AuNR，并在 794、810 和 834 nm 处显示纵向表面等离子共振 (SPR) 峰（图 S1a，支持信息）。 810 nm 处的吸收峰非常接近 NIR 激光的波长。通过透射电子显微镜 (TEM) 检查 AuNR（图 2a），使用纳米测量器捕获并分析 AuNR，以确定 AuNR 的直径和长度，分别为 21.1 ± 0.2 和 74.0 ± 0.5 nm（图 S2a， b，支持信息）。为了验证我们制备的这种尺寸的金纳米棒的吸收峰在808 nm左右，我们利用时域有限差分法进一步模拟了AuNRs的吸收光谱，结果如图S1b所示（支持信息）。当AuNRs的直径和长度设置为21.1和74.0 nm时，模拟的802 nm处的纵向吸收峰与上述实验结果基本一致。为了探讨AuNRs的吸收峰与光热效应之间的关系，我们进一步研究了808 nm近红外激光下具有不同吸收峰的AuNRs的光热曲线。在 1.5 W cm ⁻² 下照射 5 分钟，研究了 80 µg mL ⁻¹ 的 AuNR。 所有AuNRs都表现出明显的光热效应，并且在810 nm处具有纵向SPR峰值的AuNRs的效应远大于其他AuNRs（图S3，支持信息）。这些结果表明，AuNRs的吸收峰越接近激发光波长，光热效应越强。因此，吸收峰在 810 nm 的 AuNR 可用于进一步的研究。

After the AuNRs were synthesized, cetyltrimethylammonium bromide (CTAB, a cationic micellar surfactant) capping molecules remained on the surface. However, CTAB is known for its cytotoxicity; hence, it is important to mask the CTAB layer for future biomedical applications.29 Here, we replaced CTAB with mPEG-SH to improve the biocompatibility, and the PEG-AuNRs became more stable and dispersed in aqueous media (Figure S4, Supporting Information).30-32 The UV–vis absorbance spectrum indicates that the AuNRs have a longitudinal SPR peak at 810 nm; after mPEG-SH conjugation, an 8 nm blueshift was observed. The modification of AuNRs by PEG-SH did not cause a significant spectral shift in plasmon resonance (Figure 2b). In the present study, the laser source used for LSG neural stimulation has a wavelength of 808 nm, which overlaps with the longitudinal absorption peak of the PEG-AuNRs. The zeta potential of the AuNRs was +46.55 ± 5.97 mV due to remaining CTAB molecules. After modification with mPEG-SH, the zeta potential decreased to +4.34 ± 1.85 mV (Figure S5, Supporting Information). The Raman spectra in Figure S6a (Supporting Information) show that the Au–Br peak33 at 180 cm⁻¹ present for the raw NR sample is absent for the PEG-AuNRs, suggesting that CTAB is no longer present at the NR surface. The curves in Figure S6b (Supporting Information) show the IR spectra of AuNRs before and after modification. The adsorption at 1105 cm⁻¹ clearly indicated the stretching and antisymmetric stretching of C−O−C in mPEG-SH. These results clearly indicate the chemical conjugation of mPEG-SH to the AuNRs.
AuNRs 合成后，十六烷基三甲基溴化铵（CTAB，一种阳离子胶束表面活性剂）封端分子保留在表面。然而，CTAB 因其细胞毒性而闻名。因此，掩蔽 CTAB 层对于未来的生物医学应用非常重要。 29 在这里，我们用 mPEG-SH 代替 CTAB 以提高生物相容性，并且 PEG-AuNR 在水介质中变得更加稳定和分散（图 S4，支持信息）。 30- 32 紫外可见吸收光谱表明 AuNR 在 810 nm 处有一个纵向 SPR 峰； mPEG-SH 缀合后，观察到 8 nm 蓝移。 PEG-SH 对 AuNR 的修饰并未导致等离子共振光谱发生显着变化（图 2b）。在本研究中，用于LSG神经刺激的激光源的波长为808 nm，与PEG-AuNRs的纵向吸收峰重叠。由于残留的 CTAB 分子，AuNR 的 zeta 电位为 +46.55 ± 5.97 mV。使用 mPEG-SH 修饰后，zeta 电位降低至 +4.34 ± 1.85 mV（图 S5，支持信息）。图 S6a（支持信息）中的拉曼光谱表明，原始 NR 样品中存在的 180 cm 处的 Au-Br 峰 33 ⁻¹ 在 PEG-AuNR 中不存在，这表明 CTAB 不再存在于原始 NR 样品中。 NR 表面。图 S6b（支持信息）中的曲线显示了修饰前后 AuNR 的红外光谱。 1105 cm ⁻¹ 处的吸附清楚地表明了 mPEG-SH 中 C−O−C 的拉伸和反对称拉伸。这些结果清楚地表明 mPEG-SH 与 AuNR 的化学缀合。

The photothermal effect of the PEG-AuNRs was investigated by NIR irradiation in vitro. First, different concentrations of PEG-AuNRs (5, 10, 20, 40, 80, and 160 µg mL⁻¹) were exposed to 808 nm NIR laser irradiation at 1.5 W cm⁻² for 5 min. The temperature of the PEG-AuNR solution increased with irradiation time, and the temperature increased more rapidly with increasing PEG-AuNR concentration (Figure 2c). Moreover, the AuNR solution (80 µg mL⁻¹) enabled the temperature of suspension increasing from 27.5 to 56.5 °C after irradiation for 5 min. In comparison, the temperature of PBS increased by only 4 °C under the same conditions. These results indicate that the PEG-AuNRs have potential for application as an effective hyperthermia agent for neuromodulation. When the concentration of PEG-AuNR solution reaches 80 µg mL⁻¹, by further increasing the concentration, the photothermal effect has no significant increase. Therefore, the PEG-AuNRs (80 µg mL⁻¹) were chosen for further photothermal treatment experiments in vitro and in vivo.
通过近红外辐射体外研究了 PEG-AuNRs 的光热效应。首先，将不同浓度的 PEG-AuNR（5、10、20、40、80 和 160 µg mL ⁻¹ ）暴露于 1.5 W cm ⁻² 808 nm NIR 激光照射下5分钟。 PEG-AuNR 溶液的温度随着照射时间的增加而升高，并且随着 PEG-AuNR 浓度的增加，温度升高得更快（图 2c）。此外，AuNR溶液（80 µg mL ⁻¹ ）在照射5分钟后使悬浮液的温度从27.5°C升高到56.5°C。相比之下，相同条件下PBS的温度仅升高4℃。这些结果表明 PEG-AuNRs 有潜力作为神经调节的有效热疗剂。当PEG-AuNR溶液浓度达到80 µg mL ⁻¹ 时，进一步提高浓度，光热效应没有明显增加。因此，选择PEG-AuNRs（80 µg mL ⁻¹ ）进行进一步的体外和体内光热处理实验。

To detect the photothermal effect of PEG-AuNRs under 808 nm NIR laser irradiation with different power densities, 80 µg mL⁻¹ PEG-AuNRs was irradiated at 0.5, 0.75, 1.0, 1.5, and 2.0 W cm⁻² for 5 min. The results show that the temperature of the PEG-AuNRs increased significantly as the laser power density increases (Figure 2d); a thermal image is shown in Figure S7a (Supporting Information). The action potential of unmodified neurons can be inhibited by a temperature increase of ≈8.5 to 10 °C.21 Considering the vulnerability of nervous tissue, a laser power of 0.75 W cm⁻² was chosen for further photothermal treatment experiments in vivo. Photothermal stability is another prerequisite for hyperthermia agents; the photothermal stability of the PEG-AuNRs was tested next. Treatment with repeated irradiation cycles (0.5, 0.75, and 2.0 W cm⁻²) did not significantly affect the thermal generation ability of the PEG-AuNRs (Figure S7b, Supporting Information).
为了检测 PEG-AuNRs 在不同功率密度的 808 nm NIR 激光照射下的光热效应，80 µg mL ⁻¹ PEG-AuNRs 分别以 0.5、0.75、1.0、1.5 和 2.0 W cm ⁻² 5 分钟。结果表明，随着激光功率密度的增加，PEG-AuNRs的温度显着升高（图2d）；热图像如图 S7a（支持信息）所示。未修饰神经元的动作电位可因温度升高约 8.5 至 10 °C 而受到抑制。 21 考虑到神经组织的脆弱性，选择0.75 W cm ⁻² 激光功率进行进一步的体内光热治疗实验。光热稳定性是热疗剂的另一个先决条件；接下来测试了 PEG-AuNRs 的光热稳定性。重复照射周期（0.5、0.75 和 2.0 W cm ⁻² ）处理并没有显着影响 PEG-AuNR 的热生成能力（图 S7b，支持信息）。

The location of the LSG is shown in Figure 3a. A volume of 0.1 mL of NIR-sensitive PEG-AuNRs (80 µg mL⁻¹) or PBS was slowly injected into the LSG. In the NIR-sensitive PEG-AuNR group, the LSG was irradiated by the 808 nm NIR laser at 0.75 W cm⁻² (Figure 3b). When visualized by a photothermal camera (Figure 3c), the local temperature of the LSG in beagles treated with PEG-AuNRs was increased from 36.6 to 46.6 °C after 5 min of NIR exposure, which is sufficient for a hyperthermal effect to inhibit LSG.
LSG 的位置如图 3a 所示。将 0.1 mL 体积的 NIR 敏感 PEG-AuNR（80 µg mL ⁻¹ ）或 PBS 缓慢注入 LSG 中。在近红外敏感的 PEG-AuNR 组中，LSG 由 808 nm 近红外激光以 0.75 W cm ⁻² 照射（图 3b）。当通过光热相机观察时（图3c），经过 PEG-AuNR 处理的比格犬中 LSG 的局部温度在 NIR 暴露 5 分钟后从 36.6 °C 增加到 46.6 °C，这足以产生抑制 LSG 的超热效应。

The study protocol shown in Figure S8 (Supporting Information) was implemented to evaluate the photothermal effect of NIR-sensitive PEG-AuNRs on LSG function, neural activity and acute myocardial ischemia (AMI)-induced VAs. Thirteen healthy adult male beagles (8–15 kg, 8–12 months) were anesthetized, and unilateral thoracotomy was performed at the left fourth intercostal space. All dogs were randomly divided into two groups: the control group (n = 7, PBS microinjection into the LSG) and the NIR-sensitive PEG-AuNR group (n = 6, PEG-AuNR microinjection into the LSG followed by 5 min of NIR irradiation).
图 S8（支持信息）所示的研究方案用于评估近红外敏感 PEG-AuNR 对 LSG 功能、神经活动和急性心肌缺血 (AMI) 诱导的 VA 的光热效应。将13只健康成年雄性比格犬（8-15公斤，8-12个月）麻醉，在左侧第四肋间进行单侧开胸手术。所有狗被随机分为两组：对照组（n = 7，将 PBS 显微注射到 LSG 中）和 NIR 敏感 PEG-AuNR 组（n = 6，将 PEG-AuNR 显微注射到 LSG 中，然后进行 5 分钟的 NIR辐照）。

LSG function was defined as the maximal change in systolic blood pressure (SBP) in response to direct electrical stimulation of the LSG.18 Briefly, high-frequency stimulation (HFS) released by a stimulator was applied to the LSG. However, a significant variation in the maximal SBP increase in response to HFS was observed, as shown in Figure S9 (Supporting Information); therefore, four incremental voltage levels were applied to the LSG of each dog. LSG stimulation was categorized as level 1 (0–4 V), level 2 (4–8 V), level 3 (8–12 V), and level 4 (12–16 V) (the maximum but not the minimum was included in each level). LSG function was measured at baseline and after the LSG intervention. Then, a voltage level/SBP response curve was constructed.34
LSG 功能定义为响应 LSG 直接电刺激的收缩压 (SBP) 的最大变化。 18 简而言之，将刺激器释放的高频刺激 (HFS) 应用于 LSG。然而，如图 S9（支持信息）所示，观察到 HFS 引起的最大 SBP 增加存在显着变化；因此，对每只狗的 LSG 施加四个增量电压水平。 LSG 刺激分为 1 级（0-4 V）、2 级（4-8 V）、3 级（8-12 V）和 4 级（12-16 V）（包括最大值但不包括最小值）在每个级别）。在基线时和 LSG 干预后测量 LSG 功能。然后，构建了电压水平/SBP响应曲线。 34

As shown in Figure 4a,b, LSG function significantly decreased following the microinjection of NIR-sensitive PEG-AuNRs and 5 min of laser irradiation at the same stimulating voltage levels. For example, the maximal SBP change at level 4 decreased from 41.1 ± 7.6% to 19.8 ± 2.5% after NIR irradiation (P < 0.05). However, in the control group, no significant difference in the maximal change in SBP was observed between the baseline and after the LSG intervention. In order to determine whether the photothermal inhibition of NIR-sensitive PEG-AuNRs on LSG function was reversible, we measured the LSG function at different time points after NIR was turned off. As shown in Figure 4c–e, the LSG function returned to baseline (43.6 ± 4.4%) within 3 h after NIR was turned off.
如图 4a、b 所示，在相同刺激电压水平下显微注射近红外敏感 PEG-AuNR 和激光照射 5 分钟后，LSG 功能显着下降。例如，NIR 照射后，4 级最大 SBP 变化从 41.1 ± 7.6% 降至 19.8 ± 2.5%（P < 0.05）。然而，在对照组中，基线和 LSG 干预后的收缩压最大变化没有观察到显着差异。为了确定NIR敏感的PEG-AuNRs对LSG功能的光热抑制是否可逆，我们测量了NIR关闭后不同时间点的LSG功能。如图 4c-e 所示，关闭 NIR 后 3 小时内，LSG 函数返回到基线 (43.6 ± 4.4%)。

The neural activity of the LSG was recorded for 1 min at three time points (baseline, after the LSG intervention and after acute myocardial infarction) and analyzed as described in our previous studies.18, 35 Figure 4f shows representative recordings of LSG neural activity at different time points in the two groups. As shown in Figure 4g, no significant differences in the LSG neural activity recordings (frequency: 32.7 ± 6.7 vs 34.7 ± 4.7, compared to the control group, P > 0.05) were observed between the two groups at baseline. However, after treatment with the NIR-sensitive PEG-AuNRs, the LSG neural activity (frequency: 13.0 ± 1.5 vs 40.1 ± 3.0, P < 0.01) significantly decreased in the NIR-sensitive PEG-AuNRs group, in contrast with the control group, which did not exhibit significant changes in the neural activity. The inhibited LSG neural activity recovered to baseline within 3 h after NIR was turned off (frequency: 39.0 ± 14.1 impulses min⁻¹). After left anterior descending artery occlusion (LADO), the LSG neural activity of the control group was significantly higher than that of the NIR-sensitive PEG-AuNRs group (frequency: 105.3 ± 17.6 vs 60.2 ± 9.7, P < 0.01). The results indicate that the LSG neural activity could be suppressed and increased LSG neural activity induced by myocardial infarction also significantly decreased.
在三个时间点（基线、LSG 干预后和急性心肌梗死后）记录 1 分钟的 LSG 神经活动，并按照我们之前的研究中所述进行分析。 18, 35 图 4f 显示了两组不同时间点 LSG 神经活动的代表性记录。如图4g所示，基线时两组之间的LSG神经活动记录没有显着差异（频率：32.7±6.7 vs 34.7±4.7，与对照组相比，P>0.05）。然而，在使用 NIR 敏感 PEG-AuNRs 治疗后，与对照组相比，NIR 敏感 PEG-AuNRs 组的 LSG 神经活动（频率：13.0 ± 1.5 vs 40.1 ± 3.0，P < 0.01）显着降低，其神经活动没有表现出显着变化。 NIR 关闭后 3 小时内，受抑制的 LSG 神经活动恢复至基线（频率：39.0 ± 14.1 脉冲分钟 ⁻¹ ）。左前降支闭塞（LADO）后，对照组的LSG神经活性显着高于NIR敏感的PEG-AuNRs组（频率：105.3±17.6 vs 60.2±9.7，P < 0.01）。结果表明，LSG神经活动可以被抑制，并且心肌梗塞引起的LSG神经活动增加也显着减少。

AMI was induced by occluding the proximal left anterior descending coronary artery. Electrocardiograms were continuously recorded in dogs for one hour to evaluate the incidence of VAs. VAs were classified as ventricular premature beats (VPBs), salvos (two consecutive VPBs), ventricular tachycardia (VT, three or more consecutive VPBs, including sustained VT (persisting for 30 s) and nonsustained VT (spontaneously terminating within 30 s)), and ventricular fibrillation (VF) according to the Lambeth conventions.18, 36 Typical images of various kinds of VAs induced by acute ischemia are presented in Figure 5a. The numbers of VPBs (14.0 ± 5.2 vs 87.3 ± 20.3, P < 0.05), salvo beats (7.7 ± 4.8 vs 35.0 ± 10.0, P < 0.05), and VT/VF episodes (3.8 ± 2.1 vs 15.1 ± 4.3, P < 0.05) in the NIR-sensitive PEG-AuNR group were significantly lower than those in the control group (Figure 5b–d). In addition, 4 of 7 dogs in the control group died of VF, whereas 1 of 6 dogs in the NIR-sensitive PEG-AuNR group died of VF. Therefore, irradiation of the PEG-AuNRs with 0.75 W cm⁻² NIR following their injection into the LSG decreased the occurrence of acute ischemia-induced VAs.
AMI是通过闭塞冠状动脉左前降支近端诱发的。连续记录狗一小时的心电图以评估VA的发生率。 VAs分为室性早搏（VPBs）、齐发性（连续两次VPB）、室性心动过速（VT，连续3次或以上VPB，包括持续性VT（持续30秒）和非持续性VT（30秒内自发终止）），和根据兰贝斯公约的心室颤动（VF）。 18, 36 急性缺血引起的各种 VA 的典型图像如图 5a 所示。 VPB 次数（14.0 ± 5.2 vs 87.3 ± 20.3，P < 0.05）、齐射次数（7.7 ± 4.8 vs 35.0 ± 10.0，P < 0.05）和 VT/VF 发作次数（3.8 ± 2.1 vs 15.1 ± 4.3，P < 0.05）在近红外敏感的 PEG-AuNR 组中显着低于对照组（图 5b-d）。此外，对照组的 7 只狗中有 4 只死于心室颤动，而近红外敏感 PEG-AuNR 组的 6 只狗中有 1 只死于心室颤动。因此，将 PEG-AuNRs 注射到 LSG 中后用 0.75 W cm ⁻² NIR 照射可减少急性缺血诱导的 VAs 的发生。

To evaluate the ultrastructure of the LSG after treatment with the NIR-sensitive PEG-AuNRs, the LSG tissues were quickly excised at the end of the experiments, sectioned, and prepared for observation by TEM. Figure S10 (Supporting Information) clearly shows PEG-AuNRs in the ultrathin tissue sections, as observed by TEM, and that the PEG-AuNRs were located among cells of the LSG. In addition, we investigated the time remaining of these nanoparticles in the LSG tissue. Proximal nerve fibers around LSG were also quickly excised and prepared for observation by TEM. We found that there were no PEG-AuNRs observed in these sections (Figure S11, Supporting Information). The results indicated that the nanoparticles remained in the LSG tissue at least 5 h.
为了评估用近红外敏感的 PEG-AuNR 处理后 LSG 的超微结构，在实验结束时快速切除 LSG 组织，切片并准备用于 TEM 观察。图 S10（支持信息）清楚地显示了 TEM 观察到的超薄组织切片中的 PEG-AuNR，并且 PEG-AuNR 位于 LSG 的细胞之间。此外，我们还研究了这些纳米颗粒在 LSG 组织中的剩余时间。 LSG 周围的近端神经纤维也被快速切除并准备用于 TEM 观察。我们发现在这些部分中没有观察到 PEG-AuNR（图 S11，支持信息）。结果表明纳米粒子在LSG组织中保留至少5小时。

To evaluate the therapeutic efficacy and the potential risk of treating the LSG with NIR-sensitive PEG-AuNRs and NIR light, the LSG tissues were quickly excised for histopathological staining. Briefly, LSG tissues were placed in 4% paraformaldehyde and embedded in paraffin. The blocked tissue was sectioned into 4 µm thick slices, and the sections were stained with hematoxylin and eosin (HE). In addition, double immunofluorescence staining for c-fos and tyrosine hydroxylase (TH) was performed. The nuclei of LSG neurons were stained with 4,6-diamidino-2-phenylindole (DAPI). As shown in Figure S12 (Supporting Information), in the control group and the PEG-AuNR group, HE staining revealed no signs of damage to LSG neurons. We have added additional experiment to investigate the histopathology of LSG tissues treated with PEG-AuNRs alone, no significant damage was observed. Concerning about the biotoxicity of the synthesized PEG-AuNRs in vivo, we clarified that the equate PEG-AuNRs injected intravenously caused no damage in major organs (heart, spleen, liver, lung, and kidney) from canines 6, 12, or 24 h after intravenous injection (Figure S13, Supporting Information). Figure 5e shows representative images of immunofluorescence staining for c-fos and TH in the two groups. Quantitative analysis (Figure S14, Supporting Information) showed that c-fos expression was significantly lower (50.6 ± 3.3% vs 66.2 ± 2.8%, P < 0.01) in LSG neurons in the NIR-sensitive PEG-AuNR group than in the control group, indicating that the PEG-AuNRs induced photothermal inhibition of LSG neurons upon NIR irradiation.
为了评估用近红外敏感的 PEG-AuNR 和近红外光治疗 LSG 的治疗效果和潜在风险，快速切除 LSG 组织进行组织病理学染色。简而言之，将LSG组织置于4%多聚甲醛中并包埋在石蜡中。将封闭的组织切成4μm厚的切片，并用苏木精和伊红(HE)对切片进行染色。此外，还对 c-fos 和酪氨酸羟化酶 (TH) 进行了双重免疫荧光染色。 LSG 神经元的细胞核用 4,6-二脒基-2-苯基吲哚 (DAPI) 染色。如图S12（支持信息）所示，在对照组和PEG-AuNR组中，HE染色显示LSG神经元没有损伤的迹象。我们增加了额外的实验来研究单独使用 PEG-AuNRs 处理的 LSG 组织的组织病理学，没有观察到明显的损伤。关于合成的 PEG-AuNRs 的体内生物毒性，我们澄清静脉注射等量的 PEG-AuNRs 在 6、12 或 24 小时内不会对犬的主要器官（心、脾、肝、肺和肾）造成损害。静脉注射后（图S13，支持信息）。图5e显示了两组中c-fos和TH的免疫荧光染色的代表性图像。定量分析（图 S14，支持信息）显示，NIR 敏感 PEG-AuNR 组 LSG 神经元中 c-fos 表达显着低于对照组（50.6 ± 3.3% vs 66.2 ± 2.8%，P < 0.01） ，表明 PEG-AuNRs 在近红外辐射下诱导 LSG 神经元的光热抑制。

In this study, we demonstrated that NIR-sensitive PEG-AuNRs could reversely suppress the LSG function and neural activity through their photothermal effect. Moreover, the increased LSG neural activity and the occurrence of ischemia-induced VAs induced by myocardial infarction were also significantly decreased because of the NIR-sensitive PEG-AuNRs. Additionally, c-fos protein expression in LSG neurons was downregulated by the photothermal effect of NIR-sensitive PEG-AuNRs, which may underlie the photothermal inhibitory effect of NIR-sensitive PEG-AuNRs on LSG neural activity. To the best of our knowledge, this is the first study in which NIR-sensitive PEG-AuNRs were applied to modulate cardiac autonomic neurons in vivo.
在这项研究中，我们证明了近红外敏感的 PEG-AuNRs 可以通过其光热效应反向抑制 LSG 功能和神经活动。此外，由于近红外敏感的PEG-AuNRs，LSG神经活动的增加和心肌梗塞引起的缺血引起的VAs的发生也显着减少。此外，近红外敏感的PEG-AuNRs的光热效应下调了LSG神经元中的c-fos蛋白表达，这可能是近红外敏感的PEG-AuNRs对LSG神经活动的光热抑制作用的基础。据我们所知，这是第一项将近红外敏感的 PEG-AuNR 用于调节体内心脏自主神经元的研究。

Currently, NIR-sensitive AuNRs are being investigated in neuromodulation due to their efficient photothermal conversion properties. Upon NIR irradiation, AuNRs produce heat locally by converting light energy to heat energy. Moreover, this light-mediated nanotechnology exhibits high spatial and temporal resolution in modulating neural activity. Recent studies have shown that NIR-sensitive PEG-AuNR-mediated photothermal heating could significantly increase the local temperature, thereby inhibiting the electrical activity of cultured hippocampal neurons.21 These results reveal the potential for the photothermal inhibition of neurons by NIR-sensitive PEG-AuNRs.
目前，近红外敏感金纳米棒因其高效的光热转换特性而在神经调节中得到研究。在近红外辐射下，AuNRs 通过将光能转化为热能来局部产生热量。此外，这种光介导的纳米技术在调节神经活动方面表现出高空间和时间分辨率。最近的研究表明，近红外敏感的PEG-AuNR介导的光热加热可以显着升高局部温度，从而抑制培养的海马神经元的电活动。 21 这些结果揭示了近红外敏感 PEG-AuNR 对神经元进行光热抑制的潜力。

Since LSG hyperactivity plays an important role in the genesis and maintenance of ischemia-induced VAs, suppressing LSG activity by ablation or blockade is beneficial for stabilizing cardiac electrophysiological properties and reducing VAs.37 In the present study, we investigated the photothermal effect of NIR-sensitive PEG-AuNRs on LSG function and neural activity in a canine model. The results show that the NIR-sensitive PEG-AuNRs inhibited LSG function and neural activity by increasing the temperature by 9.6 ± 0.5 °C in vivo; additionally, the PEG-AuNRs reduced the occurrence of ischemia-induced VAs.
由于LSG过度活跃在缺血引起的VAs的发生和维持中起着重要作用，因此通过消融或阻断来抑制LSG活性有利于稳定心脏电生理特性并减少VAs。 37 在本研究中，我们研究了近红外敏感 PEG-AuNR 对犬模型中 LSG 功能和神经活动的光热效应。结果表明，近红外敏感的PEG-AuNRs通过将体内温度升高9.6±0.5℃来抑制LSG功能和神经活动；此外，PEG-AuNR 还减少了缺血引起的 VA 的发生。

Mechanically, NIR-sensitive PEG-AuNR-mediated photothermal neural inhibition may be controlled by short- and long-term regulation mechanisms. A transmembrane thermosensitive potassium channel called TREK-1 drives the cellular membrane potential closer to the equilibrium potential of K⁺ through hyperpolarization, thus tending to reduce excitability.38 TREK-1 activation by thermal stimuli may enhance the K⁺ currents against the membrane depolarization. A recent study showed that PEG-AuNR-treated hippocampal neurons were immediately inhibited by NIR laser irradiation and restored after the NIR light was removed.21 In addition, by blocking the thermosensitive TREK-1 in hippocampal neurons, the photothermal neural inhibition induced by the NIR-sensitive PEG-AuNRs was eliminated. This finding suggests that hyperpolarization through TREK-1 activation might contribute to the transient photothermal neural inhibition induced by NIR-sensitive PEG-AuNRs. In terms of long-term regulation, our previous study demonstrated that the hyperpolarization of LSG neurons mediated by optogenetic technology induced neural inhibition and downregulated the expression levels of c-fos and nerve growth factor in LSG neurons.18 In the present study, we found that the increase in local temperature mediated by the NIR-sensitive PEG-AuNRs significantly reduced LSG function and neural activity and downregulated c-fos expression, suggesting that neural hyperpolarization might induce the downregulation of c-fos, which may in turn underlie the long-term photothermal inhibition of LSG neural activity. Moreover, the shrinkage of some LSG neurons may also contribute to the long-term photothermal neural inhibition induced by NIR-sensitive PEG-AuNRs.
从机械角度来看，近红外敏感的 PEG-AuNR 介导的光热神经抑制可能受到短期和长期调节机制的控制。称为 TREK-1 的跨膜热敏钾通道通过超极化驱动细胞膜电位接近 K ⁺ 的平衡电位，从而倾向于降低兴奋性。 38 热刺激激活 TREK-1 可能会增强 K ⁺ 电流以抵抗膜去极化。最近的一项研究表明，经 PEG-AuNR 处理的海马神经元立即受到近红外激光照射的抑制，并在近红外光移除后恢复。 21 此外，通过阻断海马神经元中的热敏 TREK-1，近红外敏感 PEG-AuNR 诱导的光热神经抑制被消除。这一发现表明，TREK-1 激活引起的超极化可能有助于近红外敏感 PEG-AuNR 诱导的瞬时光热神经抑制。在长期调控方面，我们前期的研究表明，光遗传学技术介导的LSG神经元超极化可诱导神经抑制，下调LSG神经元中c-fos和神经生长因子的表达水平。 18 在本研究中，我们发现近红外敏感 PEG-AuNR 介导的局部温度升高显着降低了 LSG 功能和神经活动，并下调了 c-fos 表达，这表明神经超极化可能会导致 c-fos 下调，这可能反过来是 LSG 神经活动长期光热抑制的基础。此外，一些LSG神经元的收缩也可能导致近红外敏感的PEG-AuNRs诱导的长期光热神经抑制。

3 Conclusion 3 结论

In summary, we demonstrated that the photothermal effect of PEG-AuNRs combined with NIR irradiation could reversibly modulate LSG function and neural activity and decreased the occurrence of VAs in a canine model of acute ischemia without genetic transfection. To the best of our knowledge, this is the first report of a nanoparticle-based neuromodulation technique that can inhibit LSG function and neural activity photothermally. Concerning tissue-penetrating NIR light and the effective photothermal neural inhibition of AuNRs, this nanotechnological approach has potential as a valuable, minimally invasive therapeutic strategy to remotely manipulate LSG neural activity with high spatiotemporal resolution, thereby ameliorating ischemia-induced VAs.
总之，我们证明，在未进行基因转染的急性缺血犬模型中，PEG-AuNRs 的光热效应与近红外辐射相结合可以可逆地调节 LSG 功能和神经活动，并减少 VA 的发生。据我们所知，这是基于纳米颗粒的神经调节技术的第一份报告，该技术可以通过光热抑制 LSG 功能和神经活动。关于组织穿透性近红外光和 AuNR 的有效光热神经抑制，这种纳米技术方法有潜力作为一种有价值的微创治疗策略，以高时空分辨率远程操纵 LSG 神经活动，从而改善缺血引起的 VA。

4 Experimental Section 4 实验部分

Synthesis and Modification of AuNRs: The well-established seeded growth synthesis approach was used to synthesize AuNRs.39 Briefly, the seed solution was prepared by adding an aqueous tetrachloroaurate trihydrate (HAuCl₄·3H₂O) (Shanghai Chemical Reagent Co., Ltd., Shanghai, China) solution (50 µL, 0.05 m) to cetyltrimethylammonium bromide (CTAB) (Shanghai Chemical Reagent Co., Ltd., Shanghai, China) solution (10 mL, 0.1 m) in a glass tube. Then, 0.6 mL of ice-cold 0.01 m sodium borohydride was added to the solution and magnetically stirred at 1200 rpm for 2 min to produce a brownish-yellow seed solution. Then, the seed solution was incubated at room temperature for 2 h before use.
AuNR 的合成和修饰：采用成熟的晶种生长合成方法来合成 AuNR。 39 简而言之，通过添加四氯金酸盐三水合物 (HAuCl ₄ ·3H ₂ O)（上海化学试剂有限公司，中国上海）水溶液来制备种子溶液（将 50 µL，0.05 m）加入到玻璃管中的十六烷基三甲基溴化铵 (CTAB)（上海化学试剂有限公司，中国上海）溶液（10 mL，0.1 m）中。然后，将 0.6 mL 冰冷的 0.01 m 硼氢化钠添加到溶液中，并以 1200 rpm 磁力搅拌 2 分钟，产生棕黄色种子溶液。然后，使用前将种子溶液在室温下孵育2小时。

The growth solution was prepared by mixing HAuCl₄ (200 µL, 0.05 m), silver nitrate (200 µL, 0.01 m), CTAB (20 mL, 0.1 m) and HCl (400 µL, 1.0 m) together in a glass tube. A freshly prepared l-ascorbic acid solution (160 µL, 0.1 m) was added; after mild stirring, the color of the growth solution changed from yellowish-orange to colorless. Then, the seed solution (30 µL) was added, and the resultant mixture was stirred for 30 s. Finally, it was left undisturbed at room temperature for 12 h. The AuNRs were then purified by centrifugation (9000 rpm, 10 min). The supernatant was discarded, and the precipitate was resuspended in 25 mL of deionized water before use. To prepare PEG-coated AuNRs (PEG-AuNRs), 4 mL of the as-synthesized AuNRs solution was centrifuged at 9000 rpm for 10 min. The precipitate were redispersed in 4 mL of deionized water and decanted. Then, 50 µL of 1 × 10⁻³ m thiol-terminated methoxypolyethylene glycol (mPEG-SH, M_W 5000 g mol⁻¹) (Ziqi Biotechnology Co., Ltd., Shanghai, China) was added to the AuNR solution. The mixture was incubated at room temperature overnight, centrifuged, decanted, and resuspended in phosphate-buffered saline (PBS) (HyClone, USA).
通过混合 HAuCl ₄ (200 µL, 0.05 m)、硝酸银 (200 µL, 0.01 m)、CTAB (20 mL, 0.1 m) 和 HCl (400 µL, 1.0 m) 制备生长溶液一起装在玻璃管中。加入新配制的l-抗坏血酸溶液（160 µL，0.1 m）；温和搅拌后，生长溶液的颜色从黄橙色变为无色。然后，加入种子溶液（30μL），并将所得混合物搅拌30秒。最后在室温下静置12小时。然后通过离心（9000 rpm，10 分钟）纯化 AuNR。弃去上清液，使用前将沉淀重悬于25mL去离子水中。为了制备 PEG 包被的 AuNR (PEG-AuNR)，将 4 mL 合成的 AuNRs 溶液在 9000 rpm 下离心 10 分钟。将沉淀物重新分散在4mL去离子水中并倾析。然后，50 µL 1 × 10 ⁻³ m 硫醇封端的甲氧基聚乙二醇（mPEG-SH，M _W 5000 g mol ⁻¹ ）（紫气生物技术有限公司）。 ，有限公司，上海，中国）被添加到AuNR溶液中。将混合物在室温下孵育过夜，离心，倾析，并重悬于磷酸盐缓冲盐水（PBS）（HyClone，美国）中。

Characterization: The morphology and size of the PEG-AuNRs were determined by TEM (JEM-2010, JEOL, Japan). The samples were analyzed at an operating voltage of 200 kV. The average size and size distribution were obtained from more than 100 particles observed by TEM. The UV–vis–NIR absorption spectra of CTAB-AuNRs and PEG-AuNRs were recorded on a UV–vis spectrophotometer (UV-2500, Shimadzu, Japan) at wavelengths ranging from 400 to 1000 nm. Zeta potentials were measured using a nanoparticle potentiometer (Zetasizer Nano S, Malvern Instruments, UK).
表征：通过 TEM（JEM-2010，JEOL，日本）测定 PEG-AuNR 的形态和尺寸。样品在 200 kV 的工作电压下进行分析。通过 TEM 观察 100 多个颗粒，获得平均尺寸和尺寸分布。 CTAB-AuNR 和 PEG-AuNR 的 UV-vis-NIR 吸收光谱在 UV-vis 分光光度计（UV-2500，Shimadzu，日本）上记录，波长范围为 400 至 1000 nm。使用纳米颗粒电位计（Zetasizer Nano S，马尔文仪器公司，英国）测量 Zeta 电位。

Photothermal Effects: A 0.5 mL volume of different PEG-AuNR concentrations (5, 10, 20, 40, 80, and 160 µg mL⁻¹) was placed in a quartz cell (1 mL) and then irradiated with an 808 nm NIR laser (Changchun New Industries Optoelectronics Technology Co., Ltd., Changchun, China) at 1.5 W cm⁻² for 5 min to measure the influence of concentration on the photothermal effect. The same volume of PEG-AuNRs at 80 µg mL⁻¹ was placed in a quartz cell and irradiated with different power densities for 5 min to measure the influence of power density on the photothermal effect in vitro. The temperature of the solutions was measured using a digital thermometer (1319A-K type, TES, Taiwan) at different time points. Further in vivo experiments, the LSG was then microinjected with 0.1 mL of 80 µg mL⁻¹ PEG-AuNRs, and the NIR laser was subsequently applied to the LSG tissue in a vertical position for 5 min. In vivo thermal images were obtained using a thermal imaging camera (FLIR C2, USA).
光热效应：将 0.5 mL 体积的不同 PEG-AuNR 浓度（5、10、20、40、80 和 160 µg mL ⁻¹ ）置于石英池 (1 mL) 中，然后用808 nm NIR 激光器（长春新产业光电科技有限公司，中国长春）1.5 W cm ⁻² 5 分钟，测量浓度对光热效应的影响。将相同体积的80 µg mL ⁻¹ 的PEG-AuNRs放入石英池中，用不同功率密度照射5 min，测量功率密度对体外光热效应的影响。使用数字温度计（1319A-K型，TES，台湾）在不同时间点测量溶液的温度。进一步的体内实验，然后将 0.1 mL 的 80 µg mL ⁻¹ PEG-AuNRs 显微注射到 LSG，随后将 NIR 激光垂直应用于 LSG 组织 5 分钟。使用热像仪（FLIR C2，美国）获得体内热图像。

Animal Preparation: Thirteen healthy male beagles (8–15 kg) were included in the present study. All experimental procedures were conducted in compliance with the Animal Care and Use Committees of the Renmin Hospital of Wuhan University and followed the guidelines outlined by the National Institutes of Health. Sodium pentobarbital (3%) was used for anesthetization at an initial dose of 1 mL kg⁻¹ and an additional maintenance dose of 2 mL h⁻¹. A body surface electrocardiogram and the blood pressure of each canine were recorded throughout the experiments using the Lead 7000 Lab System (Jinjiang, Inc., Chengdu, China). The core body temperature of the dogs was maintained at 36.5 ± 1.5 °C. A unilateral thoracotomy was performed at the left fourth intercostal space.
动物准备：本研究包括 13 只健康雄性比格犬（8-15 公斤）。所有实验程序均按照武汉大学人民医院动物护理和使用委员会的规定进行，并遵循美国国立卫生研究院概述的指南。使用戊巴比妥钠 (3%) 进行麻醉，初始剂量为 1 mL·kg ⁻¹ ，追加维持剂量为 2 mL·h ⁻¹ 。在整个实验过程中使用 Lead 7000 实验室系统（中国成都锦江公司）记录每只犬的体表心电图和血压。犬的核心体温维持在36.5±1.5℃。在左侧第四肋间进行单侧开胸手术。

A left thoracotomy was conducted to expose and dissect the LSG. Then, 0.1 mL of NIR-sensitive PEG-AuNRs (80 µg mL⁻¹) or PBS was slowly injected into four sites of the LSG under direct visual control to achieve optimal injection. NIR laser irradiation at 0.75 W cm⁻² was performed vertically on the surface of the LSG. The NIR laser irradiation was kept constant, and the laser spot was maintained at 1.0 cm².
进行左胸廓切开术以暴露和解剖 LSG。然后，在直接视觉控制下将0.1 mL近红外敏感的PEG-AuNR（80 µg mL ⁻¹ ）或PBS缓慢注射到LSG的四个位点以实现最佳注射。 0.75 W cm ⁻² 的近红外激光照射在 LSG 表面垂直进行。近红外激光照射保持恒定，激光光斑保持在1.0cm ² 。

Evaluation of LSG Function and Neural Activity: Briefly, HFS (20 Hz, 0.1 ms duration) released by a Grass-S88 stimulator (Astro-Med, West Warwick, Rhode Island, USA) was applied to the LSG of each dog at four incremental voltage levels. LSG stimulation was categorized as level 1 (0–4 V), level 2 (4–8 V), level 3 (8–12 V), and level 4 (12–16 V) (the maximum but not the minimum was included in each level). The maximal change of systolic blood pressure in response to direct LSG stimulation represents the LSG function. LSG function was measured at baseline and immediately after removing the NIR light. Then, a voltage level/SBP response curve was constructed. The voltages reported this study represent the voltage readings of the stimulator, not the voltages delivered to the tissue. The neural activity of the LSG was recorded for 1 min at three time points (baseline, after the LSG intervention and after AMI) and analyzed as described in our previous studies.
LSG 功能和神经活动的评估：简而言之，将 Grass-S88 刺激器（Astro-Med，West Warwick，罗德岛州，美国）释放的 HFS（20 Hz，0.1 ms 持续时间）以四个增量应用于每只狗的 LSG电压水平。 LSG 刺激分为 1 级（0-4 V）、2 级（4-8 V）、3 级（8-12 V）和 4 级（12-16 V）（包括最大值但不包括最小值）在每个级别）。响应直接 LSG 刺激的收缩压的最大变化代表 LSG 功能。在基线和移除近红外光后立即测量 LSG 功能。然后，构建了电压水平/SBP响应曲线。本研究报告的电压代表刺激器的电压读数，而不是传递到组织的电压。在三个时间点（基线、LSG 干预后和 AMI 后）记录 1 分钟的 LSG 神经活动，并按照我们之前的研究中所述进行分析。

Histopathological Staining of Laser-Irradiated LSG Tissues: At the end of the experiment, the LSG tissues were quickly excised for histopathological staining. Briefly, LSG tissues were placed in 4% paraformaldehyde and embedded in paraffin. The blocked tissue was sectioned into 4 µm thick slices, and the sections were stained with HE. In addition, double immunofluorescence staining for c-fos and TH was performed. The nuclei of LSG neurons were stained with DAPI. To evaluate the ultrastructure of the LSG treated with NIR-sensitive PEG-AuNRs LSG, the LSG tissues were quickly excised at the end of the experiments, immediately fixed with Karnovsky fixative (primary fixation) for 2–4 h, and then washed with PBS (0.1 m, pH 7.4) three times prior to obtaining electron micrographs. Next, 1% osmic acid in 0.1 m PBS was used to postfix the tissues at room temperature for 2 h, followed by three washes with PBS (0.1 m, pH 7.4). Then, the tissues were dehydrated with ethyl alcohol and acetone. After dehydration, the samples were placed in an embedding plate with acetone and embedding medium for permeation, followed by embedding at 60 °C for 48 h. The blocked tissues were cut into 60–80 nm thick sections and double stained with uranium and lead. Finally, Finally, a transmission electron microscope (HT7700, HITACHI, Japan) was used to review the slices and collect images.
激光照射的LSG组织的组织病理学染色：在实验结束时，快速切除LSG组织进行组织病理学染色。简而言之，将LSG组织置于4%多聚甲醛中并包埋在石蜡中。将封闭的组织切成4μm厚的切片，并用HE染色。此外，对c-fos和TH进行双重免疫荧光染色。 LSG 神经元的细胞核用 DAPI 染色。为了评估用近红外敏感的 PEG-AuNRs LSG 处理的 LSG 的超微结构，在实验结束时快速切除 LSG 组织，立即用卡诺夫斯基固定剂（初级固定）固定 2-4 小时，然后用 PBS 清洗（0.1 m，pH 7.4）在获得电子显微照片之前三次。接下来，使用 0.1 m PBS 中的 1% 锇酸在室温下后固定组织 2 小时，然后用 PBS（0.1 m，pH 7.4）洗涤 3 次。然后，用乙醇和丙酮将组织脱水。脱水后，将样品置于装有丙酮和包埋剂的包埋板中进行渗透，然后在60℃下包埋48h。将阻塞的组织切成60-80 nm厚的切片并用铀和铅双重染色。最后，最后，使用透射电子显微镜（HT7700，日立，日本）检查切片并收集图像。

Statistical Analysis: All continuous variables are expressed as the means ± SEM and were tested for a normal distribution utilizing the Shapiro–Wilk normality test. Two-way repeated-measure analysis of variance followed by Bonferroni posttests was used to compare LSG function and neural activity in the two groups. An unpaired t-test was used to compare the temperature changes of NIR on LSG tissues with or without PEG-AuNRs in vivo and histologic changes between the two groups. To analyze VAs (VPB, Salvo, VT/VF), an unpaired t-test with Welch's correction was used for statistical comparison. GraphPad Prism software 7.0 (GraphPad Software, Inc., La Jolla, CA, USA) and Origin 8.0 (OriginLab, USA) were used to analyze the data and generate graphs. Statistical significance was set to P < 0.05.
统计分析：所有连续变量均表示为平均值±SEM，并利用夏皮罗-威尔克正态性检验进行正态分布测试。采用双向重复测量方差分析和 Bonferroni 后测来比较两组的 LSG 功能和神经活动。使用未配对的 t 检验来比较体内有或没有 PEG-AuNRs 的 LSG 组织上 NIR 的温度变化以及两组之间的组织学变化。为了分析 VA（VPB、Salvo、VT/VF），使用带有 Welch 校正的未配对 t 检验进行统计比较。使用 GraphPad Prism 软件 7.0（GraphPad Software, Inc.，La Jolla，CA，USA）和 Origin 8.0（OriginLab，美国）分析数据并生成图表。统计显着性设置为 P < 0.05。

Conflict of Interest 利益冲突

The authors declare no conflict of interest.
作者声明不存在利益冲突。