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.

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.

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.

In vivo imaging of sterile microglial activation in rat brain after disrupting the blood-brain barrier with pulsed focused ultrasound: [18F]DPA-714 PET study.

BACKGROUND: Magnetic resonance imaging (MRI)-guided pulsed focused ultrasound combined with the infusion of microbubbles (pFUS+MB) induces transient blood-brain barrier opening (BBBO) in targeted regions. pFUS+MB, through the facilitation of neurotherapeutics' delivery, has been advocated as an adjuvant treatment for neurodegenerative diseases and malignancies. Sterile neuroinflammation has been recently described following pFUS+MB BBBO. In this study, we used PET imaging with [18F]-DPA714, a biomarker of translocator protein (TSPO), to assess for neuroinflammatory changes following single and multiple pFUS+MB sessions. METHODS: Three groups of Sprague-Dawley female rats received MRI-guided pFUS+MB (Optison™; 5-8 × 107 MB/rat) treatments to the left frontal cortex and right hippocampus. Group A rats were sonicated once. Group B rats were sonicated twice and group C rats were sonicated six times on weekly basis. Passive cavitation detection feedback (PCD) controlled the peak negative pressure during sonication. We performed T1-weighted scans immediately after sonication to assess efficiency of BBBO and T2*-weighted scans to evaluate for hypointense voxels. [18F]DPA-714 PET/CT scans were acquired after the BBB had closed, 24 h after sonication in group A and within an average of 10 days from the last sonication in groups B and C. Ratios of T1 enhancement, T2* values, and [18F]DPA-714 percent injected dose/cc (%ID/cc) values in the targeted areas to the contralateral brain were calculated. Histological assessment for microglial activation/astrocytosis was performed. RESULTS: In all groups, [18F]DPA-714 binding was increased at the sonicated compared to non-sonicated brain (%ID/cc ratios > 1). Immunohistopathology showed increased staining for microglial and astrocytic markers in the sonicated frontal cortex compared to contralateral brain and to a lesser extent in the sonicated hippocampus. Using MRI, we documented BBB disruption immediately after sonication with resolution of BBBO 24 h later. We found more T2* hypointense voxels with increasing number of sonications. In a longitudinal group of animals imaged after two and after six sonications, there was no cumulative increase of neuroinflammation on PET. CONCLUSION: Using [18F]DPA-714 PET, we documented in vivo neuroinflammatory changes in association with pFUS+MB. Our protocol (utilizing PCD feedback to minimize damage) resulted in neuroinflammation visualized 24 h post one sonication. Our findings were supported by immunohistochemistry showing microglial activation and astrocytosis. Experimental sonication parameters intended for BBB disruption should be evaluated for neuroinflammatory sequelae prior to implementation 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.

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.

Radiological-pathological correlation of diffusion tensor and magnetization transfer imaging in a closed head traumatic brain injury model.

OBJECTIVE: Metrics of diffusion tensor imaging (DTI) and magnetization transfer imaging (MTI) can detect diffuse axonal injury in traumatic brain injury (TBI). The relationship between the changes in these imaging measures and the underlying pathologies is still relatively unknown. This study investigated the radiological-pathological correlation between these imaging techniques and immunohistochemistry using a closed head rat model of TBI. METHODS: TBI was performed on female rats followed longitudinally by magnetic resonance imaging (MRI) out to 30 days postinjury, with a subset of animals selected for histopathological analyses. A MRI-based finite element analysis was generated to characterize the pattern of the mechanical insult and estimate the extent of brain injury to direct the pathological correlation with imaging findings. RESULTS: DTI axial diffusivity and fractional anisotropy (FA) were sensitive to axonal integrity, whereas radial diffusivity showed significant correlation to the myelin compactness. FA was correlated with astrogliosis in the gray matter, whereas mean diffusivity was correlated with increased cellularity. Secondary inflammatory responses also partly affected the changes of these DTI metrics. The magnetization transfer ratio (MTR) at 3.5ppm demonstrated a strong correlation with both axon and myelin integrity. Decrease in MTR at 20ppm correlated with the extent of astrogliosis in both gray and white matter. INTERPRETATION: Although conventional T2-weighted MRI did not detect abnormalities following TBI, DTI and MTI afforded complementary insight into the underlying pathologies reflecting varying injury states over time, and thus may substitute for histology to reveal diffusive axonal injury pathologies in vivo. This correlation of MRI and histology furthers understanding of the microscopic pathology underlying DTI and MTI changes in TBI. Ann Neurol 2016;79:907-920.

Physicochemical characterization of ferumoxytol, heparin and protamine nanocomplexes for improved magnetic labeling of stem cells.

Stem cell-based therapies have become a major focus in regenerative medicine and to treat diseases. A straightforward approach combining three drugs, heparin (H), protamine (P) with ferumoxytol (F) in the form of nanocomplexes (NCs) effectively labeled stem cells for cellular MRI. We report on the physicochemical characteristics for optimizing the H, P, and F components in different ratios, and mixing sequences, producing NCs that varied in hydrodynamic size. NC size depended on the order in which drugs were mixed in media. Electron microscopy of HPF or FHP showed that F was located on the surface of spheroidal shaped HP complexes. Human stem cells incubated with FHP NCs resulted in a significantly greater iron concentration per cell compared to that found in HPF NCs with the same concentration of F. These results indicate that FHP could be useful for labeling stem cells in translational studies in the clinic.

Cyclooxygenase-2 or tumor necrosis factor-α inhibitors attenuate the mechanotransductive effects of pulsed focused ultrasound to suppress mesenchymal stromal cell homing to healthy and dystrophic muscle.

Maximal homing of infused stem cells to diseased tissue is critical for regenerative medicine. Pulsed focused ultrasound (pFUS) is a clinically relevant platform to direct stem cell migration. Through mechanotransduction, pFUS establishes local gradients of cytokines, chemokines, trophic factors (CCTF) and cell adhesion molecules (CAM) in treated skeletal muscle that subsequently infused mesenchymal stromal cells (MSC) can capitalize to migrate into the parenchyma. Characterizing molecular responses to mechanical pFUS effects revealed tumor necrosis factor-alpha (TNFα) drives cyclooxygenase-2 (COX2) signaling to locally increase CCTF/CAM that are necessary for MSC homing. pFUS failed to increase chemoattractants and induce MSC homing to treated muscle in mice pretreated with ibuprofen (nonspecific COX inhibitor) or etanercept (TNFα inhibitor). pFUS-induced MSC homing was also suppressed in COX2-knockout mice, demonstrating ibuprofen blocked the mechanically induced CCTF/CAM by acting on COX2. Anti-inflammatory drugs, including ibuprofen, are administered to muscular dystrophy (MD) patients, and ibuprofen also suppressed pFUS-induced homing to muscle in a mouse model of MD. Drug interactions with cell therapies remain unexplored and are not controlled for during clinical cell therapy trials. This study highlights potentially negative drug-host interactions that suppress stem cell homing and could undermine cell-based approaches for regenerative medicine.

Pulsed focused ultrasound pretreatment improves mesenchymal stromal cell efficacy in preventing and rescuing established acute kidney injury in mice.

Animal studies have shown that mesenchymal stromal cell (MSC) infusions improve acute kidney injury (AKI) outcomes when administered early after ischemic/reperfusion injury or within 24 hours after cisplatin administration. These findings have spurred several human clinical trials to prevent AKI. However, no specific therapy effectively treats clinically obvious AKI or rescues renal function once advanced injury is established. We investigated if noninvasive image-guided pulsed focused ultrasound (pFUS) could alter the kidney microenvironment to enhance homing of subsequently infused MSC. To examine the efficacy of pFUS-enhanced cell homing in disease, we targeted pFUS to kidneys to enhance MSC homing after cisplatin-induced AKI. We found that pFUS enhanced MSC 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 MSC alone. We then investigated whether pFUS+MSC therapy could rescue established AKI. MSC alone at 3 days post-cisplatin, after renal functional deficits were obvious, significantly improved 7-day survival of animals. Survival was further improved by pFUS and MSC. pFUS prior to MSC injections increased IL-10 production by MSC that homed to kidneys and generated an anti-inflammatory immune cell profile in treated kidneys. This study shows pFUS is a neoadjuvant approach to improve MSC homing to diseased organs. pFUS with MSC better prevents AKI than MSC alone and allows rescue therapy in established AKI, which currently has no meaningful therapeutic options.

Manganese G8 dendrimers targeted to oxidation-specific epitopes: in vivo MR imaging of atherosclerosis.

PURPOSE: To determine if manganese (Mn) G8 dendrimers targeted to oxidation-specific epitopes (OSE) allow for in vivo detection of atherosclerotic lesions. MATERIALS AND METHODS: OSE have been identified as key factors in atherosclerotic plaque progression and destabilization. Mn offers a potentially clinically translatable alternative to gadolinium-based agents when bioretention and potential toxicity of gadolinium is anticipated. However, to be effective, high payloads of Mn must accumulate intracellularly in macrophages. It was hypothesized that G8 dendrimers targeted to OSE may allow delivery of high Mn payloads, thereby enabling in vivo detection of macrophage-rich plaques. G8 dendrimers were modified to allow conjugation with MnDTPA (758 Mn ion) and the antibody MDA2 that is targeted to malondialdehyde (MDA)-lysine epitopes. Both the untargeted and targeted G8 dendrimers were characterized and their in vivo efficacy evaluated in apoE(-/-) mice over a 96-hour time period after bolus administration of a 0.05 mmol Mn/kg dose using a clinical MR system (3T). RESULTS: Significant enhancement (normalized enhancement >60%, P = 0.0013) of atherosclerotic lesions was observed within a 72-hour time period following administration of the targeted dendrimers. The presence of Mn within atherosclerotic lesions was confirmed using spectroscopic methods (>8 µg Mn/g). Limited signal attenuation (<18%) and Mn deposition (<1 µg Mn/g) was observed in the arterial wall following injection of the untargeted material. CONCLUSION: This study demonstrates that manganese-labeled dendrimers, allowing a high Mn payload, targeted to OSE may allow in vivo image of atherosclerotic lesions.

Synthesis and characterization of gadolinium-Peptidomimetic complex as an αvß3 integrin targeted MR contrast agent.

There is growing interest in small and rigid peptidomimetic αvß3 integrin antagonists that are readily synthesized and characterized and amenable to physiological conditions. Peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-ß-alanine (IAC) was successfully conjugated to DOTA, complexed with Gd(III) and radiolabeled with (153)Gd. Radioassay results demonstrated specificity of the labeled conjugate by blocking ∼95% binding with the addition of a 50-fold molar excess of cold IAC to the reaction solution. Relaxometry was used to support the hypothesis that the specificity of the Gd-peptidomimetic targeting αvß3 integrin would increase the contrast and therefore enhance the sensitivity of an MRI scan of αvß3 integrin positive tissues. Magnetic resonance imaging of cell pellets (M21 human melanoma) was also performed, and the images clearly show that cells reacted with Gd(III)-DOTA-IAC display a brighter image than cells without the Gd(III)-DOTA-IAC contrast agent. In addition, Gd(III)-DOTA-IAC and IAC, with IC50 of 300nM and 230nM, respectively, are 2.1 and 2.7 times more potent than c(RGDfK) whose IC50 is 625nM. This promising preliminary data fuels further investigation of DOTA-IAC conjugates for targeting tumor associated angiogenesis and αvß3 integrin positive tumors using magnetic resonance imaging.

Macrophagic and microglial responses after focal traumatic brain injury in the female rat.

BACKGROUND: After central nervous system injury, inflammatory macrophages (M1) predominate over anti-inflammatory macrophages (M2). The temporal profile of M1/M2 phenotypes in macrophages and microglia after traumatic brain injury (TBI) in rats is unknown. We subjected female rats to severe controlled cortical impact (CCI) and examined the postinjury M1/M2 time course in their brains. METHODS: The motor cortex (2.5 mm left laterally and 1.0 mm anteriorly from the bregma) of anesthetized female Wistar rats (ages 8 to 10 weeks; N = 72) underwent histologically moderate to severe CCI with a 5-mm impactor tip. Separate cohorts of rats had their brains dissociated into cells for flow cytometry, perfusion-fixed for immunohistochemistry (IHC) and ex vivo magnetic resonance imaging or flash-frozen for RNA and protein analysis. For each analytical method used, separate postinjury times were included for 24 hours; 3 or 5 days; or 1, 2, 4 or 8 weeks. RESULTS: By IHC, we found that the macrophagic and microglial responses peaked at 5 to 7 days post-TBI with characteristics of mixed populations of M1 and M2 phenotypes. Upon flow cytometry examination of immunological cells isolated from brain tissue, we observed that peak M2-associated staining occurred at 5 days post-TBI. Chemokine analysis by multiplex assay showed statistically significant increases in macrophage inflammatory protein 1α and keratinocyte chemoattractant/growth-related oncogene on the ipsilateral side within the first 24 hours after injury relative to controls and to the contralateral side. Quantitative RT-PCR analysis demonstrated expression of both M1- and M2-associated markers, which peaked at 5 days post-TBI. CONCLUSIONS: The responses of macrophagic and microglial cells to histologically severe CCI in the female rat are maximal between days 3 and 7 postinjury. The response to injury is a mixture of M1 and M2 phenotypes.

Noninvasive pulsed focused ultrasound allows spatiotemporal control of targeted homing for multiple stem cell types in murine skeletal muscle and the magnitude of cell homing can be increased through repeated applications.

Stem cells are promising therapeutics for cardiovascular diseases, and i.v. injection is the most desirable route of administration clinically. Subsequent homing of exogenous stem cells to pathological loci is frequently required for therapeutic efficacy and is mediated by chemoattractants (cell adhesion molecules, cytokines, and growth factors). Homing processes are inefficient and depend on short-lived pathological inflammation that limits the window of opportunity for cell injections. Noninvasive pulsed focused ultrasound (pFUS), which emphasizes mechanical ultrasound-tissue interactions, can be precisely targeted in the body and is a promising approach to target and maximize stem cell delivery by stimulating chemoattractant expression in pFUS-treated tissue prior to cell infusions. We demonstrate that pFUS is nondestructive to murine skeletal muscle tissue (no necrosis, hemorrhage, or muscle stem cell activation) and initiates a largely M2-type macrophage response. We also demonstrate that local upregulation of chemoattractants in pFUS-treated skeletal muscle leads to enhance homing, permeability, and retention of human mesenchymal stem cells (MSC) and human endothelial precursor cells (EPC). Furthermore, the magnitude of MSC or EPC homing was increased when pFUS treatments and cell infusions were repeated daily. This study demonstrates that pFUS defines transient "molecular zip codes" of elevated chemoattractants in targeted muscle tissue, which effectively provides spatiotemporal control and tunability of the homing process for multiple stem cell types. pFUS is a clinically translatable modality that may ultimately improve homing efficiency and flexibility of cell therapies for cardiovascular diseases.

Subcurative radiation significantly increases cell proliferation, invasion, and migration of primary glioblastoma multiforme in vivo.

Tumor cell proliferation, infiltration, migration, and neovascularization are known causes of treatment resistance in glioblastoma multiforme (GBM). The purpose of this study was to determine the effect of radiation on the growth characteristics of primary human GBM developed in a nude rat. Primary GBM cells grown from explanted GBM tissues were implanted orthotopically in nude rats. Tumor growth was confirmed by magnetic resonance imaging on day 77 (baseline) after implantation. The rats underwent irradiation to a dose of 50 Gy delivered subcuratively on day 84 postimplantation (n = 8), or underwent no radiation (n = 8). Brain tissues were obtained on day 112 (nonirradiated) or day 133 (irradiated). Immunohistochemistry was performed to determine tumor cell proliferation (Ki-67) and to assess the expression of infiltration marker (matrix metalloproteinase-2, MMP-2) and cell migration marker (CD44). Tumor neovascularization was assessed by microvessel density using von-Willebrand factor (vWF) staining. Magnetic resonance imaging showed well-developed, infiltrative tumors in 11 weeks postimplantation. The proportion of Ki-67-positive cells in tumors undergoing radiation was (71 +/- 15)% compared with (25 +/- 12)% in the nonirradiated group (P = 0.02). The number of MMP-2-positive areas and proportion of CD44-positive cells were also high in tumors receiving radiation, indicating great invasion and infiltration. Microvessel density analysis did not show a significant difference between nonirradiated and irradiated tumors. Taken together, we found that subcurative radiation significantly increased proliferation, invasion, and migration of primary GBM. Our study provides insights into possible mechanisms of treatment resistance following radiation therapy for GBM.

Ultrasound Pressure-Dependent Cytokine and Immune Cell Response Lost in Aged Muscle.

OBJECTIVE: Therapeutic ultrasound remains a highly discussed topic in physical therapy due to uncertainty between treatment regimens and biological benefits. Its impact on aged populations, who are vulnerable to insufficient healing after muscle injury because of sarcopenia, is understudied. Despite the coupling between muscle inflammation and regeneration, research on the immune response after therapeutic ultrasound is limited. The objective of this study was to evaluate structure, inflammatory cytokine signaling and immune cell infiltration after therapeutic ultrasound in young and aging murine muscle. METHODS: Young (6-week-old) and Adult (52-week-old) male and female mouse non-injured gastrocnemii were treated with either low-intensity pulsed ultrasound at 2 W/cm2 (∼0.243 MPa) or high-intensity pulsed focused ultrasound at 554 W/cm2 (∼5.96 MPa). Cytokine expression was evaluated at 1, 8 and 24 hours, cell infiltration was measured via flow cytometry at 1 and 24 hours and immunofluorescence assessed muscle fiber area, fibrosis and satellite cells at 24 hours after sonication. RESULTS: Low-intensity pulsed ultrasound induced an early, transient inflammatory response where interleukin (IL)-15 and macrophages (M2 > M1) were increased 1 hour post-sonication. High-intensity pulsed focused ultrasound caused a late, extended immune response where monocyte chemoattractant protein 1 (MCP-1), neutrophils, monocytes and macrophages (M1 > M2) were increased 24 hours post-sonication. Notably, these changes manifested solely in Young gastrocnemius. The Adult gastrocnemius exhibited decreased cytokine expression (IL-1α, IL-6, IL-15, macrophage colony-stimulating factor [M-CSF]) and no alteration in immune cell recruitment post-sonication. There was no damage to muscle structure. CONCLUSION: Therapeutic ultrasound induced a pressure-dependent inflammatory response that can augment or mitigate intrinsic muscle cytokine signaling and cell recruitment in adolescent or aged muscle, respectively.

The evolution of traumatic brain injury in a rat focal contusion model.

Serial MRI facilitates the in vivo analysis of the intra- and intersubject evolution of traumatic brain injury lesions. Despite the availability of MRI, the natural history of experimental focal contusion lesions in the controlled cortical impact (CCI) rat model has not been well described. We performed CCI on rats and MRI during the acute to chronic stages of cerebral injury to investigate the time course of changes in the brain. Female Wistar rats underwent CCI of their left motor cortex with a flat impact tip driven by an electromagnetic piston. In vivo MRI was performed at 7 T serially over 6 weeks post-CCI. The appearances of CCI-induced lesions and lesion-associated cortical volumes were variable on MRI, with the percentage change in cortical volume of the CCI ipsilateral side relative to the contralateral side ranging from 18% within 2 h of injury on day 0 to a peak of 35% on day 1, and a trough of -28% by week 5/6, with an average standard deviation of ± 14% at any given time point. In contrast, the percentage change in cortical volume of the ipsilateral side relative to the contralateral side in control rats was not significant (1 ± 2%). Hemorrhagic conversion within and surrounding the CCI lesion occurred between days 2 and 9 in 45% of rats, with no hemorrhage noted on the initial scan. Furthermore, hemorrhage and hemosiderin within the lesion were positive for Prussian blue and highly autofluorescent on histological examination. Although some variation in injuries may be technique related, the divergence of similar lesions between initial and final scans demonstrates the inherent biological variability of the CCI rat model.

Enhanced homing permeability and retention of bone marrow stromal cells by noninvasive pulsed focused ultrasound.

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 nondestructive pulsed focused ultrasound (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. pFUS could potentially improve cell-based therapies as a noninvasive modality to target homing by establishing local chemoattractant gradients and increasing expression of integrins to enhance tropism of cells toward treated tissues.

P22 viral capsids as nanocomposite high-relaxivity MRI contrast agents.

Attachment of multiple chelated Gd(3+) ions to the interior of bacteriophage P22 viral capsids affords nanoscale MRI contrast agents with extremely high relaxivity values. Highly fenestrated "wiffleball" morphology is unique to P22 and assures water exchange between the environment and interior cavity of the capsid. The cavity of P22 "wiffleball" was functionalized with a branched oligomer comprising multiple DTPA-Gd complexes resulting in an impressive payload of 1,900 Gd(3+) ions inside each 64 nm capsid. High relaxivities of r(1,ionic) = 21.7 mM(-1) s(-1) and r(1,particle) = 41,300 mM(-1) s(-1) at 298 K, 0.65 T (28 MHz) are reported, with r(1)/r(2) ratio of 0.80 and optimized rotational correlation time for this system. Specific design modifications are suggested for future improvements of viral capsid-based MRI contrast agents directed toward clinical translation.