Neuroinflammation associated with ultrasound-mediated permeabilization of the blood-brain barrier.

The blood-brain barrier (BBB) continues to represent one of the most significant challenges for successful drug-based treatments of neurological disease. Mechanical modulation of the BBB using focused ultrasound (FUS) and microbubbles (MBs) has shown considerable promise in enhancing the delivery of therapeutics to the brain, but questions remain regarding possible long-term effects of such forced disruption. This review examines the evidence for inflammation associated with ultrasound-induced BBB disruption and potential strategies for managing such inflammatory effects to improve both the efficacy and safety of therapeutic ultrasound in neurological applications.

Erratum: Focused ultrasound activates voltage-gated calcium channels through depolarizing TRPC1 sodium currents in kidney and skeletal muscle.

[This corrects the article DOI: 10.7150/thno.33876.].

Pulsed-Focused Ultrasound Slows B16 Melanoma and 4T1 Breast Tumor Growth through Differential Tumor Microenvironmental Changes.

Focused ultrasound (FUS) has shown promise as a non-invasive treatment modality for solid malignancies. FUS targeting to tumors has been shown to initiate pro-inflammatory immune responses within the tumor microenvironment. Pulsed FUS (pFUS) can alter the expression of cytokines, chemokines, trophic factors, cell adhesion molecules, and immune cell phenotypes within tissues. Here, we investigated the molecular and immune cell effects of pFUS on murine B16 melanoma and 4T1 breast cancer flank tumors. Temporal changes following sonication were evaluated by proteomics, RNA-seq, flow-cytometry, and histological analyses. Proteomic profiling revealed molecular changes occurring over 24 h post-pFUS that were consistent with a shift toward inflamed tumor microenvironment. Over 5 days post-pFUS, tumor growth rates were significantly decreased while flow cytometric analysis revealed differences in the temporal migration of immune cells. Transcriptomic analyses following sonication identified differences in gene expression patterns between the two tumor types. Histological analyses further demonstrated reduction of proliferation marker, Ki-67 in 4T1, but not in B16 tumors, and activated cleaved-caspase 3 for apoptosis remained elevated up to 3 days post-pFUS in both tumor types. This study revealed diverse biological mechanisms following pFUS treatment and supports its use as a possible adjuvant to ablative tumor treatment to elicit enhanced anti-tumor responses and slow tumor growth.

Blood-brain barrier opening by intracarotid artery hyperosmolar mannitol induces sterile inflammatory and innate immune responses.

Intracarotid arterial hyperosmolar mannitol (ICAHM) blood-brain barrier disruption (BBBD) is effective and safe for delivery of therapeutics for central nervous system malignancies. ICAHM osmotically alters endothelial cells and tight junction integrity to achieve BBBD. However, occurrence of neuroinflammation following hemispheric BBBD by ICAHM remains unknown. Temporal proteomic changes in rat brains following ICAHM included increased damage-associated molecular patterns, cytokines, chemokines, trophic factors, and cell adhesion molecules, indicative of a sterile inflammatory response (SIR). Proteomic changes occurred within 5 min of ICAHM infusion and returned to baseline by 96 h. Transcriptomic analyses following ICAHM BBBD further supported an SIR. Immunohistochemistry revealed activated astrocytes, microglia, and macrophages. Moreover, proinflammatory proteins were elevated in serum, and proteomic and histological findings from the contralateral hemisphere demonstrated a less pronounced SIR, suggesting neuroinflammation beyond regions of ICAHM infusion. Collectively, these results demonstrate ICAHM induces a transient SIR that could potentially be harnessed for neuroimmunomodulation.

Temporally distinct myeloid cell responses mediate damage and repair after cerebrovascular injury.

Cerebrovascular injuries can cause severe edema and inflammation that adversely affect human health. Here, we observed that recanalization after successful endovascular thrombectomy for acute large vessel occlusion was associated with cerebral edema and poor clinical outcomes in patients who experienced hemorrhagic transformation. To understand this process, we developed a cerebrovascular injury model using transcranial ultrasound that enabled spatiotemporal evaluation of resident and peripheral myeloid cells. We discovered that injurious and reparative responses diverged based on time and cellular origin. Resident microglia initially stabilized damaged vessels in a purinergic receptor-dependent manner, which was followed by an influx of myelomonocytic cells that caused severe edema. Prolonged blockade of myeloid cell recruitment with anti-adhesion molecule therapy prevented severe edema but also promoted neuronal destruction and fibrosis by interfering with vascular repair subsequently orchestrated by proinflammatory monocytes and proangiogenic repair-associated microglia (RAM). These data demonstrate how temporally distinct myeloid cell responses can contain, exacerbate and ultimately repair a cerebrovascular injury.

Cytosolic Ca2+ transients during pulsed focused ultrasound generate reactive oxygen species and cause DNA damage in tumor cells.

Mechanical forces from non-ablative pulsed focused ultrasound (pFUS) generate pro-inflammatory tumor microenvironments (TME), marked by increased cytokines, chemokines, and trophic factors, as well as immune cell infiltration and reduced tumor growth. pFUS also causes DNA damage within tumors, which is a potent activator of immunity and could contribute to changes in the TME. This study investigated mechanisms behind the mechanotransductive effects of pFUS causing DNA damage in several tumor cell types. Methods: 4T1 (murine breast tumor), B16 (murine melanoma), C6 (rat glioma), or MDA-MB-231 (human breast tumor) cells were sonicated in vitro (1.1MHz; 6MPa PNP; 10ms pulses; 10% duty cycle; 300 pulses). DNA damage was detected by TUNEL, apoptosis was measured by immunocytochemistry for cleaved caspase-3. Calcium, superoxide, and H2O2 were detected by fluorescent indicators and modulated by BAPTA-AM, mtTEMPOL, or Trolox, respectively. Results: pFUS increased TUNEL reactivity (range = 1.6-2.7-fold) in all cell types except C6 and did not induce apoptosis in any cell line. All lines displayed cytosolic Ca2+ transients during sonication. pFUS increased superoxide (range = 1.6-2.0-fold) and H2O2 (range = 2.3-2.8-fold) in all cell types except C6. BAPTA-AM blocked increased TUNEL reactivity, superoxide and H2O2 formation, while Trolox also blocked increased TUNEL reactivity increased after pFUS. mtTEMPOL allowed H2O2 formation and did not block increased TUNEL reactivity after pFUS. Unsonicated C6 cells had higher baseline concentrations of cytosolic Ca2+, superoxide, and H2O2, which were not associated with greater baseline TUNEL reactivity than the other cell lines. Conclusions: Mechanotransduction of pFUS directly induces DNA damage in tumor cells by cytosolic Ca2+ transients causing formation of superoxide and subsequently, H2O2. These results further suggest potential clinical utility for pFUS. However, the lack of pFUS-induced DNA damage in C6 cells demonstrates a range of potential tumor responses that may arise from physiological differences such as Ca2+ or redox homeostasis.

MR-guided pulsed focused ultrasound improves mesenchymal stromal cell homing to the myocardium.

Image-guided pulsed focused ultrasound (pFUS) is a non-invasive technique that can increase tropism of intravenously (IV)-infused mesenchymal stromal cells (MSC) to sonicated tissues. MSC have shown promise for cardiac regenerative medicine strategies but can be hampered by inefficient homing to the myocardium. This study sonicated the left ventricles (LV) in rats with magnetic resonance imaging (MRI)-guided pFUS and examined both proteomic responses and subsequent MSC tropism to treated myocardium. T2-weighted MRI was used for pFUS targeting of the entire LV. pFUS increased numerous pro- and anti-inflammatory cytokines, chemokines, and trophic factors and cell adhesion molecules in the myocardial microenvironment for up to 48 hours post-sonication. Cardiac troponin I and N-terminal pro-B-type natriuretic peptide were elevated in the serum and myocardium. Immunohistochemistry revealed transient hypoxia and immune cell infiltration in pFUS-targeted regions. Myocardial tropism of IV-infused human MSC following pFUS increased twofold-threefold compared with controls. Proteomic and histological changes in myocardium following pFUS suggested a reversible inflammatory and hypoxic response leading to increased tropism of MSC. MR-guided pFUS could represent a non-invasive modality to improve MSC therapies for cardiac regenerative medicine approaches.

Acoustic Radiation or Cavitation Forces From Therapeutic Ultrasound Generate Prostaglandins and Increase Mesenchymal Stromal Cell Homing to Murine Muscle.

Non-ablative ultrasound (US)-based techniques to improve targeted tropism of systemically infused cell therapies, particularly mesenchymal stromal cell (MSC), have gained attention in recent years. Mechanotransduction following targeted US sonications have been shown to modulate tissue microenvironments by upregulating cytokines, chemokines, and trophic factors in addition to vascular cell adhesion molecules (CAM) that are necessary to promote tropism of MSC. While numerous US treatment parameters have demonstrated increased MSC homing, it remains unclear how the different mechanical US forces [i.e., acoustic radiation forces (ARF) or cavitation forces] influence tissue microenvironments. This study sonicated murine muscle tissue with pulsed focused ultrasound (pFUS) at 0.5 or 1.15 MHz each over a range of US intensities. Following sonication, tissue was assayed for the prostaglandins (PG) PGH2 and PGE2 as indicators of microenvironmental changes that would support MSC tropism. PGH2 and PGE2 levels were correlated to physical pFUS parameters and acoustic emissions measured by hydrophone. While ARF (pFUS with absence of cavitation signatures) was sufficient to increase PGH2 and PGE2, non-linear curve fitting revealed a frequency-independent relationship between prostaglandin production and mechanical index (MI), which accounts for increased cavitation probabilities of lower frequencies. The prostaglandin data suggested molecular changes in muscle would be particularly sensitive to cavitation. Therefore, low-intensity pulsed ultrasound (LIPUS) at 1 MHz was administered with low ARF (MI = 0.2) in combination with intravenous (IV) infusions of microbubble (MB) contrast agents. This combination upregulated prostaglandins and CAM without ultrasound-mediated microbubble destruction and ultimately promoted tropism of IV-infused MSC. This study revealed that accentuating non-destructive MB cavitation by US using parameters similar to diagnostic US contrast imaging increased MSC homing. Such approaches are particularly attractive to overcome clinical translation barriers of many still-experimental US parameters used in previous stem cell tropism studies.

The Impact of Focused Ultrasound in Two Tumor Models: Temporal Alterations in the Natural History on Tumor Microenvironment and Immune Cell Response.

Image-guided focused ultrasound (FUS) has been successfully employed as an ablative treatment for solid malignancies by exposing immune cells to tumor debris/antigens, consequently inducing an immune response within the tumor microenvironment (TME). To date, immunomodulation effects of non-ablative pulsed-FUS (pFUS) on the TME are poorly understood. In this study, the temporal differences of cytokines, chemokines, and trophic factors (CCTFs) and immune cell populations induced by pFUS were interrogated in murine B16 melanoma or 4T1 breast cancer cells subcutaneously inoculated into C57BL/6 or BALB/c mice. Natural history growth characteristics during the course of 11 days showed a progressive increase in size for both tumors, and proteomic analysis revealed a shift toward an immunosuppressive TME. With respect to tumor natural growth, pFUS applied to tumors on days 1, 5, or 9 demonstrated a decrease in the growth rate 24 h post-sonication. Flow cytometry analysis of tumors, LNs, and Sp, as well as CCTF profiles, relative DNA damage, and adaptive T-cell localization within tumors, demonstrated dynamic innate and adaptive immune-modulation following pFUS in early time points of B16 tumors and in advanced 4T1 tumors. These results provide insight into the temporal dynamics in the treatment-associated TME, which could be used to evaluate an immunomodulatory approach in different tumor types.

Focused ultrasound activates voltage-gated calcium channels through depolarizing TRPC1 sodium currents in kidney and skeletal muscle.

Pulsed focused ultrasound (pFUS) technology is being developed for clinical neuro/immune modulation and regenerative medicine. Biological signal transduction of pFUS forces can require mechanosensitive or voltage-gated plasma membrane ion channels. Previous studies suggested pFUS is capable of activating either channel type, but their mechanistic relationship remains ambiguous. We demonstrated pFUS bioeffects increased mesenchymal stem cell tropism (MSC) by altering molecular microenvironments through cyclooxygenase-2 (COX2)-dependent pathways. This study explored specific relationships between mechanosensitive and voltage-gated Ca2+ channels (VGCC) to initiate pFUS bioeffects that increase stem cell tropism. Methods: Murine kidneys and hamstring were given pFUS (1.15 or 1.125 MHz; 4MPa peak rarefactional pressure) under ultrasound or magnetic resonance imaging guidance. Cavitation and tissue displacement were measure by hydrophone and ultrasound radiofrequency data, respectively. Elastic modeling was performed from displacement measurements. COX2 expression and MSC tropism were evaluated in the presence of pharmacological ion channel inhibitors or in transient-receptor-potential-channel-1 (TRPC1)-deficient mice. Immunohistochemistry and co-immunoprecipitation examined physical channel relationships. Fluorescent ionophore imaging of cultured C2C12 muscle cells or TCMK1 kidney cells probed physiological interactions. Results: pFUS induced tissue deformations resulting in kPa-scale forces suggesting mechanical activation of pFUS-induced bioeffects. Inhibiting VGCC or TRPC1 in vivo blocked pFUS-induced COX2 upregulation and MSC tropism to kidneys and muscle. A TRPC1/VGCC complex was observed in plasma membranes. VGCC or TRPC1 suppression blocked pFUS-induced Ca2+ transients in TCMK1 and C2C12 cells. Additionally, Ca2+ transients were blocked by reducing transmembrane Na+ potentials and observed Na+ transients were diminished by genetic TRPC1 suppression. Conclusion: This study suggests that pFUS acoustic radiation forces mechanically activate a Na+-containing TRPC1 current upstream of VGCC rather than directly opening VGCC. The electrogenic function of TRPC1 provides potential mechanistic insight into other pFUS techniques for physiological modulation and optimization strategies for clinical implementation.

The Proteomic Effects of Pulsed Focused Ultrasound on Tumor Microenvironments of Murine Melanoma and Breast Cancer Models.

Non-ablative pulsed focused ultrasound (pFUS) targets non-thermal forces that activate local molecular and cellular immune responses. Optimal parameters to stimulate immunotherapeutic tumor microenvironments (TME) and responses in different tumor types remain uninvestigated. Flank B16 murine melanoma and 4T1 breast tumors received 1 MHz pFUS at 1-8 MPa peak negative pressures (PNP) and were analyzed 24 hr post-sonication. Necrosis or hemorrhage were unaltered in both tumors, but pFUS induced DNA strand breaks in tumor cells at PNP ≥6 MPa. pFUS at >4 MPa suppressed anti-inflammatory cytokines in B16 tumors. pFUS to 4T1 tumors decreased anti-inflammatory cytokines and increased pro-inflammatory cytokines and cell adhesion molecules. pFUS at 6 MPa increased calreticulin and alterations in check-point proteins along with tumoral and splenic immune cell changes that could be consistent with a shift towards an anti-TME. pFUS-induced TME alterations shows promise in generating anti-tumor immune responses, but non-uniform responses between tumor types require additional investigation to assess pFUS as a suitable anti-tumor therapy.

Chlorotoxin-A Multimodal Imaging Platform for Targeting Glioma Tumors.

Chlorotoxin (CTX) is a 36-amino-acid disulfide-containing peptide derived from the venom of the scorpion Leiurus quinquestriatus. CTX alters physiology in numerous ways. It interacts with voltage gated chloride channels, Annexin-2, and matrix metalloproteinase-2 (MMP-2). CTX-based bioconjugates have been widely subjected to phase I/II clinical trials and have shown substantial promise. Many studies have demonstrated that CTX preferentially binds to neuroectodermal tumors, such as glioblastoma, without cross-reactivity to normal brain cells. With its ability to penetrate the blood-brain-barrier (BBB) and its tyrosine residue allows covalent conjugation with functional moieties, CTX is an attractive platform to explore development of diagnostic and therapeutic agents for gliomas. In this review, we outline CTX structure and its molecular targets, summarize molecular variations of CTX developed for glioma imaging, and discuss future trends and perspectives for CTX conjugates as a theranostic agent.

MRI and histological evaluation of pulsed focused ultrasound and microbubbles treatment effects in the brain.

Magnetic resonance imaging (MRI)-guided pulsed focused ultrasound (pFUS) combined with microbubbles (MB) contrast agent infusion has been shown to transiently disrupt the blood-brain barrier (BBBD), increasing the delivery of neurotherapeutics to treat central nervous system (CNS) diseases. pFUS interaction with the intravascular MB results in acoustic cavitation forces passing through the neurovascular unit (NVU), inducing BBBD detected on contrast-enhanced MRI. Multiple pFUS+MB exposures in Alzheimer's disease (AD) models are being investigated as a method to clear amyloid plaques by activated microglia or infiltrating immune cells. Since it has been reported that pFUS+MB can induce a sterile inflammatory response (SIR) [1-5] in the rat, the goal of this study was to investigate the potential long-term effects of SIR in the brain following single and six weekly sonications by serial high-resolution MRI and pathology. Methods: Female Sprague Dawley rats weighing 217±16.6 g prior to sonication received bromo-deoxyuridine (BrdU) to tag proliferating cells in the brain. pFUS was performed at 548 kHz, ultrasound burst 10 ms and initial peak negative pressure of 0.3 MPa (in water) for 120 s coupled with a slow infusion of ~460 µL/kg (5-8×107 MB) that started 30 s before and 30 s during sonication. Nine 2 mm focal regions in the left cortex and four regions over the right hippocampus were treated with pFUS+MB. Serial high-resolution brain MRIs at 3 T and 9.4 T were obtained following a single or during the course of six weekly pFUS+MB resulting in BBBD in the left cortex and the right hippocampus. Animals were monitored over 7 to 13 weeks and imaging results were compared to histology. Results: Fewer than half of the rats receiving a single pFUS+MB exposure displayed hypointense voxels on T2*-weighted (w) MRI at week 7 or 13 in the cortex or hippocampus without differences compared to the contralateral side on histograms of T2* maps. Single sonicated rats had evidence of limited microglia activation on pathology compared to the contralateral hemisphere. Six weekly pFUS+MB treatments resulted in pathological changes on T2*w images with multiple hypointense regions, cortical atrophy, along with 50% of rats having persistent BBBD and astrogliosis by MRI. Pathologic analysis of the multiple sonicated animals demonstrated the presence of metallophagocytic Prussian blue-positive cells in the parenchyma with significantly (p<0.05) increased areas of activated astrocytes and microglia, and high numbers of systemic infiltrating CD68+ macrophages along with BrdU+ cells compared to contralateral brain. In addition, multiple treatments caused an increase in the number of hyperphosphorylated Tau (pTau)-positive neurons containing neurofibrillary tangles (NFT) in the sonicated cortex but not in the hippocampus when compared to contralateral brain, which was confirmed by Western blot (WB) (p<0.04). Conclusions: The repeated SIR following multiple pFUS+MB treatments could contribute to changes on MR imaging including persistent BBBD, cortical atrophy, and hypointense voxels on T2w and T2*w images consistent with pathological injury. Moreover, areas of astrogliosis, activated microglia, along with higher numbers of CD68+ infiltrating macrophages and BrdU+ cells were detected in multiple sonicated areas of the cortex and hippocampus. Elevations in pTau and NFT were detected in neurons of the multiple sonicated cortex. Minimal changes on MRI and histology were observed in single pFUS+MB-treated rats at 7 and 13 weeks post sonication. In comparison, animals that received 6 weekly sonications demonstrated evidence on MRI and histology of vascular damage, inflammation and neurodegeneration associated with the NVU commonly observed in trauma. Further investigation is recommended of the long-term effects of multiple pFUS+MB in clinical trials.

Mesenchymal stromal cell potency to treat acute kidney injury increased by ultrasound-activated interferon-γ/interleukin-10 axis.

Mesenchymal stromal cell (MSC) therapies combined with renal pulsed focused ultrasound (pFUS) pretreatment increase MSC homing and improve cisplatin-induced acute kidney injury (AKI) better than MSC alone. However, mechanisms underlying improved outcomes remain unknown. We hypothesize pFUS up-regulates renal interferon-γ (IFNγ) and stimulates MSC to produce interleukin-10 (IL-10) after migrating to kidneys. To demonstrate initially, MSC cultured with IFNγ up-regulated IL-10. More MSC-derived IL-10 was detected in kidneys when IFNγ-stimulated MSC were infused and they improved AKI better than unstimulated MSC. Next, IFNγ-knockout mice with AKI received pFUS+MSC, but MSC-derived IL-10 expression and AKI were similar to using MSC alone. AKI in wild-type mice receiving pFUS and IL-10-deficient MSC was also unimproved compared to administering IL-10-deficient MSC alone. Indoleamine 2,3-dioxygenase (IDO), an anti-inflammatory enzyme up-regulated in MSC by IFNγ, was up-regulated during AKI, but was not further elevated in MSC from pFUS-treated kidneys, suggesting that IDO is not involved in improved AKI healing by pFUS+MSC. These data suggest IFNγ is up-regulated by pFUS and after i.v.-infused MSC home to pFUS-treated kidneys, IFNγ stimulates additional IL-10 production by MSC to improve AKI. Analogous mechanisms of ultrasound-treated tissue microenvironments stimulating therapeutic MSC may exist in other pathologies where adjuvant ultrasound techniques are successful.

Focused ultrasound with microbubbles induces sterile inflammatory response proportional to the blood brain barrier opening: Attention to experimental conditions.

This editorial highlights the findings of McMahon [1] and demonstrates the need for careful attention to experimental conditions that influence microbubble concentration and pharmacokinetics contributed to focused ultrasound-induced blood brain barrier opening and sterile inflammation.

Molecular and histological effects of MR-guided pulsed focused ultrasound to the rat heart.

BACKGROUND: Image-guided high intensity focused ultrasound has been used as an extracorporeal cardiac pacing tool and to enhance homing of stem cells to targeted tissues. However, molecular changes in the myocardium after sonication have not been widely investigated. Magnetic-resonance (MR)-guided pulsed focused ultrasound (pFUS) was targeted to the rat myocardium over a range of pressures and the microenvironmental and histological effects were evaluated over time. METHODS: Eight-to-ten-week-old Sprague-Dawley rats received T2-weighted MR images to target pFUS to the left ventricular and septum without cardiac or respiratory gating. Rats were sonicated through the thoracic wall at peak negative pressures (PNP) from 1 to 8 MPa at a center frequency of 1 MHz, 10 ms pulse duration and 1 Hz pulse repetition frequency for 100 pulses per focal target. Following pFUS, myocardium was harvested over 24 h and subjected to imaging, proteomic, and histological measurements. RESULTS: pFUS to the myocardium increased expression of cytokines, chemokines, and trophic factors characterized by an initial increase in tumor necrosis factor (TNF)-α followed by increases in pro- and anti-inflammatory factors that returned to baseline by 24 h. Immediately after pFUS, there was a transient (< 1 h) increase in N-terminal pro b-type natriuretic peptide (NT-proBNP) without elevation of other cardiac injury markers. A relationship between PNP and expression of TNF-α and NT-proBNP was observed with significant changes (p < 0.05 ANOVA) ≥ 4 MPa compared to untreated controls. Contrast-enhanced ex vivo T1-weighted MRI revealed vascular leakage in sonicated myocardium that was accompanied by the presence of albumin upon immunohistochemistry. Histology revealed infiltration of neutrophils and macrophages without morphological myofibril changes in sonicated tissue accompanied by pulmonary hemorrhage at PNP > 4 MPa. CONCLUSIONS: MR-guided pFUS to myocardium induced transient proteomic and histological changes. The temporal proteomic changes in the myocardium indicate a short-lived sterile inflammatory response consistent with ischemia or contusion. Further study of myocardial function and strain is needed to determine if pFUS could be developed as an experimental model of cardiac injury and chest trauma.

Anti-inflammatory drugs suppress ultrasound-mediated mesenchymal stromal cell tropism to kidneys.

Mesenchymal stromal cells (MSC) are potential renal therapeutics. Clinically, results are mixed partly because MSC tropism to kidneys is minimal following infusion. Ultrasound augmentation of the renal microenvironment is becoming increasingly-important in renal MSC therapies. We demonstrated pulsed-focused-ultrasound (pFUS) increases enhanced homing permeability and retention of MSC in mouse kidneys. Here, we characterized the temporal proteomic response to pFUS in mouse kidneys and its association with MSC tropism. pFUS induced molecular cascades of initial increases in tumor necrosis factor-α (TNFα) and interleukin (IL)-1α, that activated nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and cyclooxygenase-2 (COX2) pathways without cell death. This was followed by a 24-48 hour-long response of increased cell adhesion molecules (CAM), trophic and anti-inflammatory factors. Pretreating animals with anti-inflammatory drugs etanercept (TNFα inhibitor), anakinra (IL-1 receptor antagonist), prednisone (NFκB translocation inhibitor), or ibuprofen (COX inhibitor) suppressed molecular changes and inhibited renal MSC tropism. We further examined the role of COX2 using a COX2-knock-out mouse where pFUS was unable to increase MSC tropism. These results demonstrate that renal micro-environmental changes induce MSC tropism and could influence the therapeutic efficacy of MSC. Optimizing the microenvironment and understanding drug effects will enable improvements in MSC therapies for renal disease.

Improving the therapeutic efficacy of mesenchymal stromal cells to restore perfusion in critical limb ischemia through pulsed focused ultrasound.

Mesenchymal stem cells (MSC) are promising therapeutics for critical limb ischemia (CLI). Mechanotransduction from pulsed focused ultrasound (pFUS) upregulates local chemoattractants to enhance homing of intravenously (IV)-infused MSC and improve outcomes. This study investigated whether pFUS exposures to skeletal muscle would improve local homing of iv-infused MSCs and their therapeutic efficacy compared to iv-infused MSCs alone. CLI was induced by external iliac arterial cauterization in 10-12-month-old mice. pFUS/MSC treatments were delayed 14 days, when surgical inflammation subsided. Mice were treated with iv-saline, pFUS alone, IV-MSC, or pFUS and IV-MSC. Proteomic analyses revealed pFUS upregulated local chemoattractants and increased MSC tropism to CLI muscle. By 7 weeks post-treatment, pFUS + MSC significantly increased perfusion and CD31 expression, while reducing fibrosis compared to saline. pFUS or MSC alone reduced fibrosis, but did not increase perfusion or CD31. Furthermore, MSCs homing to pFUS-treated CLI muscle expressed more vascular endothelial growth factor (VEGF) and interleukin-10 (IL-10) than MSCs homing to non-pFUS-treated muscle. pFUS + MSC improved perfusion and vascular density in this clinically-relevant CLI model. The molecular effects of pFUS increased both MSC homing and MSC production of VEGF and IL-10, suggesting microenvironmental changes from pFUS also increased potency of MSCs in situ to further enhance their efficacy.

Disrupting the blood-brain barrier by focused ultrasound induces sterile inflammation.

MRI-guided pulsed focused ultrasound (pFUS) combined with systemic infusion of ultrasound contrast agent microbubbles (MB) causes localized blood-brain barrier (BBB) disruption that is currently being advocated for increasing drug or gene delivery in neurological diseases. The mechanical acoustic cavitation effects of opening the BBB by low-intensity pFUS+MB, 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 TNFα 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; these elevations lasted 24 h. Transcriptomic analysis of sonicated brain supported the proteomic findings and indicated that the SIR was facilitated through the induction of the NFκB pathway. Histological evaluation demonstrated increased albumin in the parenchyma that cleared by 24 h along with TUNEL+ neurons, activated astrocytes, microglia, and increased cell adhesion molecules in the vasculature. Infusion of fluorescent beads 3 d before pFUS+MB revealed the infiltration of CD68+ macrophages at 6 d postsonication, as is consistent with an innate immune response. pFUS+MB is being considered as part of a noninvasive adjuvant treatment for malignancy or neurodegenerative diseases. These results demonstrate that pFUS+MB induces an SIR compatible with ischemia or mild traumatic brain injury. Further investigation will be required before this approach can be widely implemented in clinical trials.