Preclinical high intensity focused ultrasound: mechanisms and applications

骨髓基质细胞(BMSCs)在疾病的治疗中显示出巨大的前景，但其治疗效果往往受到系统给药细胞低效归巢的限制，导致在病理部位聚集的细胞数量较少。BMSC位于炎症区域，局部表达细胞黏附分子和趋化因子梯度。脉冲聚焦超声(PFUS)采用非连续曝光，降低了能量沉积的速度，允许在脉冲之间进行冷却，从而最大限度地减少了热效应，并强调了FUS的非热机制(即声辐射力和声空化)造成的影响。我们检查了pFUS后肾脏的变化，对组织的组织完整性几乎没有影响，也不会导致细胞死亡。PFUS增加了几种趋化物质的表达，在pFUS后的第0天和第1天产生了一个瞬时的分子邮政编码，在pFUS后的第3天恢复到基线水平。PFUS暴露诱导肌肉血管上的细胞黏附分子上调。我们诱导机械转导反应，启动在很大程度上抗炎的M2型巨噬细胞环境。我们证明，在pFUS治疗的肾脏中，化学诱导剂的局部上调导致骨髓间充质干细胞的归巢、通透性和保留性增强。我们还证明，pFUS后引起的肾脏分子变化可以被环氧合酶2抑制剂布洛芬或肿瘤坏死因子α受体结合蛋白依那西普阻断，这表明机械转导效应是通过组织中的NFkB和COX 2途径发挥作用的。在pFUS和干细胞输注之前给予布洛芬或依那西普均可阻断干细胞对靶肌肉的归巢，这表明使用这种方法，我们可以使用pFUS来询问药物与宿主组织的相互作用及其对归巢的影响。 我们还证明，在急性肾损伤模型中，pFUS联合骨髓间充质干细胞可以在小鼠肾脏(AKI)中诱导机械转导效应。为了检验pFUS增强的细胞归巢在疾病中的有效性，我们将pFUS靶向肾脏，以增强顺铂诱导的AKI后的BMSC归巢。我们发现，与单独使用BMSCs相比，pFUS促进了顺铂后1天的BMSC归巢，而在顺铂后5天，与单独使用BMSCs相比，增强归巢改善了肾功能、肾小管细胞死亡和再生的结果。在观察了早期AKI期间改善的归巢和AKI结果后，我们调查了pFUS+BMSC治疗是否可以挽救已建立的AKI。在顺铂后3天单独应用BMSC，肾功能受损明显后，动物的7天存活率显著提高。使用pFUS+BMSC可进一步提高存活率。骨髓间充质干细胞单独或与pFUS共同作用时，肾巨噬细胞表型由M1变为M2。这项研究表明，pFUS是一种新的辅助治疗方法，可以改善MSC对病变器官的归巢。我们还表明，pFUS增加了组织中的干扰素伽马，通过刺激输注的细胞释放白细胞介素10，增强了输注的骨髓间充质干细胞的局部效力。我们在严重肢体缺血模型中也有类似的结果，在该模型中，我们已经能够证明，与仅接受pFUS或BMSC的动物相比，pFUS+BMSC联合使用导致了更大的缺血肢体再灌注。对组织的评估表明，与只接受骨髓间充质干细胞注射的动物相比，pFUS预处理后的骨髓间充质干细胞输注缺血肌肉的微环境中人血管内皮生长因子和白介素10的数量增加。这些结果表明，pFUS预调节了组织，从而提高了定位于靶组织的骨髓间充质干细胞的效力，并对转化为临床试验具有重要意义。 磁共振成像(MRI)引导的脉冲聚焦超声(PFUS)结合全身注射超声造影剂微泡(MB)可引起局部血脑屏障(BBB)的破坏，如增强MRI所证明的那样，导致立即的损伤相关分子模式(DAMP)反应，包括热休克蛋白70、IL-1、IL-18和指示实质内无菌炎症反应(SIR)的肿瘤坏死因子的升高。PFUS+MB的作用是由于MB稳定空化产生的血管内冲击波，导致TUNEL+神经元、小胶质细胞和星形胶质细胞的激活，以及与轻度脑损伤或脑缺血相适应的先天免疫反应。

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 cell adhesion molecules and chemokine gradients are present. Pulsed focused ultrasound (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). We examined changes in the kidney following pFUS has little effect on the histological integrity of the tissue and does not induce cell death. pFUS increased expression of several chemoattractants creating a transient molecular zip code on days 0 and 1 following pFUS that returns to baseline levels by day 3 post-pFUS. pFUS exposures induced up-regulation of cell adhesion molecules on muscle vasculature. We induce a mechanotransductive response that initiates a largely an anti-inflammatory M2-type macrophage environment. We demonstrated local upregulation of chemoattractants in pFUS-treated kidney leads to enhance homing, permeability, and retention of BMSC. We also demonstrate that the induced molecular changes following pFUS to the kidney can be block by ibuprofen, a cyclooxygenase 2 inhibitor, or TNF alpha receptor binding protein, etanercept, indicating that the mechanotransductive effects are acting through a NFkB and COX 2 pathway in the tissue. Both ibuprofen or etanercept administered prior to pFUS and stem cell infusion block the homing of stem cells to targeted muscle which indicated that using this approach we can use pFUS to interrogate drug-host tissue interactions and their effect on homing. We also demonstrated that pFUS exposures in combination with BMSC in an acute kidney injury model induce mechanotransductive effects in the murine kidney (AKI). To examine the efficacy of pFUS-enhanced cell homing in disease, we targeted pFUS to kidneys to enhance BMSC homing after cisplatin-induced AKI. We found that pFUS enhanced BMSC homing at 1 day post-cisplatin, prior to renal functional deficits, and that enhanced homing improved outcomes of renal function, tubular cell death, and regeneration at 5 days post-cisplatin compared to BMSCs alone. After observing improved homing and AKI outcomes during early AKI, we investigated whether pFUS+BMSC therapy could rescue established AKI. BMSC administration alone at 3 days post-cisplatin, after renal functional deficits become obvious, significantly improved 7-day survival of animals. Survival was further improved using pFUS+BMSC. BMSCs, alone or with pFUS, shifted the kidney macrophage phenotype from M1 to M2. This study shows that pFUS serves as a neoadjuvant treatment to improve MSC homing to diseased organs. We have also shown that pFUS increases interferon gamma in tissue that augments the local potency of the infused BMSC that home to the tissue by stimulating release of interleukin (IL) 10 from infused cells.. We also have similar results in critical limb ischemia model in which we have been able to demonstrate that the combination of pFUS+BMSC resulted in greater reperfusion of the ischemic limb compared to animal receiving pFUS or BMSC alone. Evaluation of the tissue demonstrated pFUS pretreatment followed by BMSC infusion of the ischemic muscle increased the amount of human vascular endothelia growth factor and interleukin 10 present in the microenvironment compared to animals only receiving BMSC injections. These results indicate that pFUS preconditions the tissues that results in increased potency of the BMSC that homed to targeted tissues and has important implication for translation to clinical trials. Magnetic resonance imaging (MRI)-guided pulsed focused ultrasound (pFUS) combined with systemic infusion of ultrasound contrast agent microbubbles (MB) causes localized bloodbrain barrier (BBB) disruption as evidenced by contrast-enhanced MRI, resulted in an immediate damage-associated molecular pattern (DAMP) response including elevations in heat-shock protein 70, IL-1, IL-18, and tumor necrosis factor indicative of a sterile inflammatory response (SIR) in the parenchyma. Concurrent with DAMP presentation, significant elevations in proinflammatory, antiinflammatory, and trophic factors along with neurotrophic and neurogenesis factors were detected that lasted 24 h. The effect of pFUS+MB was due to an intravascular shock wave from stable cavitation of the MB and resulted in TUNEL+ neurons, activated microglia and astrocytes and an innate immune response that would be compatible with mild trauma or ischemia in the brain.