Preclinical high intensity focused ultrasound: mechanisms and applications

脉冲FUS（pFUS）采用非连续暴露，降低能量沉积速率并允许在脉冲之间发生冷却，从而最大限度地减少热效应并强调FUS的非热机制（即声辐射力和声空化）产生的效应。然而，很少有人了解这些暴露对组织的FUS影响，特别是关于细胞促归巢因子（生长因子，细胞因子，细胞粘附分子）。我们研究了pFUS或cFUS后小鼠腘绳肌的变化，并证明pFUS与cFUS不同，对肌肉的组织学完整性影响不大，不会诱导细胞死亡。在pFUS或cFUS暴露后3天和8天观察到巨噬细胞浸润。pFUS增加了几种细胞因子（例如IL-1 α、IL-1 β、TNF α、INF γ、MIP-1 α、MCP-1、GMCSF）的表达，在pFUS后第0天和第1天产生局部细胞因子梯度，该梯度在pFUS后第3天恢复到基线水平。pFUS暴露诱导肌肉脉管系统上其它信号分子（例如VEGF、FGF、PlGF、HGF、SDF-1 α）和细胞粘附分子（例如ICAM-1和VCAM-1）的上调。pFUS后肌肉中观察到的分子变化可用于靶向细胞治疗，通过增加对病理区域的归巢。骨髓基质细胞（BMSC）在疾病治疗中显示出显著的前景，但是它们的治疗功效通常受到全身施用的细胞的低效归巢的限制，这导致在病理部位处积累的细胞数量少。BMSC归巢于存在整合素和趋化因子梯度的局部表达的炎症区域。我们证明，强调超声-组织相互作用的机械效应的pFUS暴露诱导了化学引诱物的局部和瞬时升高（即，细胞因子、整联蛋白和生长因子）。pFUS诱导的细胞因子上调发生在治疗后约1天，并在第3天恢复到对侧肾脏水平。细胞因子表达显著增加的这一窗口伴随着其他营养因子和整合素的局部增加，这些营养因子和整合素已被证明可促进BMSC归巢。当在pFUS处理后将BMSC静脉内施用至单个肾脏时，在处理的肾脏中相对于对侧肾脏观察到BMSC的增强的归巢、渗透性和保留。组织学分析显示，在治疗后第1天和第3天，在经治疗的肾脏的小管周围区域中的BMSC高达8倍。此外，发现BMSC施用后pFUS处理的肾脏中的细胞因子水平与对照相似，表明BMSC对细胞因子水平的调节。这些结果表明，pFUS可以潜在地改善基于细胞的治疗作为一种非侵入性的方式，通过建立局部趋化梯度和增加细胞粘附分子的表达，以增强细胞对治疗组织的向性，以靶向归巢。

Pulsed FUS (pFUS) employs non-continuous exposures, that lower the rate of energy deposition and allow cooling to occur between pulses, thereby minimizing thermal effects and emphasizing the effects created by non-thermal mechanisms of FUS (i.e. acoustic radiation forces and acoustic cavitation). However, little is understood about the FUS effects these exposures have on tissue, especially with regard to cellular pro-homing factors (growth factors, cytokines, cell adhesion molecules). We examined changes in mice hamstring muscle following pFUS or cFUS and demonstrate that pFUS, unlike cFUS, has little effect on the histological integrity of muscle and does not induce cell death. Infiltration of macrophages was observed 3 and 8 days following pFUS or cFUS exposures. pFUS increased expression of several cytokines (e.g. IL-1alpha;, IL-1beta;, TNFalpha;, INFgamma;, MIP-1alpha;, MCP-1, GMCSF) creating a local cytokine gradient on days 0 and 1 following pFUS that returns to baseline levels by day 3 post-pFUS. pFUS exposures induced up-regulation of other signaling molecules (e.g. VEGF, FGF, PlGF, HGF, SDF-1alpha;) and cell adhesion molecules (e.g. ICAM-1 and VCAM-1) on muscle vasculature. The observed molecular changes in muscle following pFUS may be utilized to target cellular therapies, by increasing homing to areas of pathology. Bone marrow stromal cells (BMSCs) have shown significant promise in the treatment of disease, but their therapeutic efficacy is often limited by inefficient homing of systemically administered cells, which results in low number of cells accumulating at sites of pathology. BMSC home to areas of inflammation where local expression of integrins and chemokine gradients is present. We demonstrated that pFUS exposures that emphasize the mechanical effects of ultrasound-tissue interactions induced local and transient elevations of chemoattractants (i.e., cytokines, integrins, and growth factors) in the murine kidney. pFUS-induced upregulation of cytokines occurred through approximately 1 day post-treatment and returned to contralateral kidney levels by day 3. This window of significant increases in cytokine expression was accompanied by local increases of other trophic factors and integrins that have been shown to promote BMSC homing. When BMSCs were intravenously administered following pFUS treatment to a single kidney, enhanced homing, permeability, and retention of BMSC was observed in the treated kidney versus the contralateral kidney. Histological analysis revealed up to eight times more BMSC in the peritubular regions of the treated kidneys on days 1 and 3 post-treatment. Furthermore, cytokine levels in pFUS-treated kidneys following BMSC administration were found to be similar to controls, suggesting modulation of cytokine levels by BMSC. These results suggest that pFUS could potentially improve cell-based therapies as a noninvasive modality to target homing by establishing local chemoattractant gradients and increasing expression of cell adhesion molecules to enhance tropism of cells toward treated tissues.