What Are the Effects of Irreversible Electroporation on a Staphylococcus aureus Rabbit Model of Osteomyelitis?

BACKGROUND: The treatment of osteomyelitis can be challenging because of poor antibiotic penetration into the infected bone and toxicities associated with prolonged antibiotic regimens to control infection. Irreversible electroporation (IRE), a percutaneous image-guided ablation technology in which the targeted delivery of high-voltage electrical pulses permanently damages the cell membrane, has been shown to effectively control bacterial growth in various settings. However, IRE for the management of bone infections has yet to be evaluated. QUESTIONS/PURPOSES: We aimed to evaluate IRE for treating osteomyelitis by assessing (1) the efficacy of IRE to suppress the in vitro growth of a clinical isolate of S. aureus, alone or combined with cefazolin; and (2) the effects of IRE on the in vivo treatment of a rabbit model of osteomyelitis. METHODS: S. aureus strain UAMS-1 expanded in vitro to the log phase was subjected to an electric field of 2700 V/cm, which was delivered in increasing numbers of pulses. Immediately after electroporation, bacteria were plated on agar plates with or without cefazolin. The number of colony-forming units (CFUs) was scored the following day. ANOVA tests were used to analyze in vitro data. In a rabbit osteomyelitis model, we inoculated the same bacterial strain into the radius of adult male New Zealand White rabbits. Three weeks after inoculation, all animals (n = 32) underwent irrigation and débridement, as well as wound culture of the infected forelimb. Then, they were randomly assigned to one of four treatment groups (n = eight per group): untreated control, cefazolin only, IRE only, or combined IRE + cefazolin. Serial radiography was performed to assess disease progression using a semiquantitative grading scale. Bone and soft-tissue specimens from the infected and contralateral forelimbs were collected at 4 weeks after treatment for bacterial isolation and histologic assessment using a semiquantitative scale. RESULTS: The in vitro growth of S. aureus UAMS-1 was impaired by IRE in a pulse-dependent fashion; the number of CFUs/mL was different among seven pulse levels, namely 0, 10, 30, 60, 90, 120, and 150 pulses. With the number of CFUs/mL observed in untreated controls set as 100%, 10 pulses rendered a median of 50.2% (range 47.1% to 58.2%), 30 pulses rendered a median of 2.7% (range 2.5% to 2.8%), 60 pulses rendered a median of 0.014% (range 0.012% to 0.015%), 90 pulses rendered a median of 0.004% (range 0.002% to 0.004%), 120 pulses rendered a median of 0.001% (range 0.001% to 0.001%), and 150 pulses rendered a median of 0.001% (range 0.000% to 0.001%) (Kruskal-Wallis test: p = 0.003). There was an interaction between the effect of the number of pulses and the concentration of cefazolin (two-way ANOVA: F [8, 30] = 17.24; p < 0.001), indicating that combining IRE with cefazolin is more effective than either treatment alone at suppressing the growth of S. aureus UAMS-1. Likewise, the clinical response in the rabbit model (the percentage of animals without detectable residual bacteria in the bone and surrounding soft tissue after treatment) was better in the combination group than in the other groups: control, 12.5% (one of eight animals); IRE only, 12.5% (one of eight animals); cefazolin only, 25% (two of eight animals); and IRE + cefazolin, 75% (six of eight animals) (two-sided Fisher's exact test: p = 0.030). CONCLUSIONS: IRE effectively suppressed the growth of S. aureus UAMS-1 and enhanced the antibacterial effect of cefazolin in in vitro studies. When translated to a rabbit osteomyelitis model, the addition of IRE to conventional parenteral antibiotic treatment produced the strongest response, which supports the in vitro findings. CLINICAL RELEVANCE: Our results show that IRE may improve the results of standard parenteral antibiotic treatment, thus setting the stage for models with larger animals and perhaps trials in humans for validation.

Safety and Efficacy of an Absorbable Filter in the Inferior Vena Cava to Prevent Pulmonary Embolism in Swine.

Purpose To evaluate the immediate and long-term safety as well as thrombus-capturing efficacy for 5 weeks after implantation of an absorbable inferior vena cava (IVC) filter in a swine model. Materials and Methods This study was approved by the institutional animal care and use committee. Eleven absorbable IVC filters made from polydioxanone suture were deployed via a catheter in the IVC of 11 swine. Filters remained in situ for 2 weeks (n = 2), 5 weeks (n = 2), 12 weeks (n = 2), 24 weeks (n = 2), and 32 weeks (n = 3). Autologous thrombus was administered from below the filter in seven swine from 0 to 35 days after filter placement. Fluoroscopy and computed tomography follow-up was performed after filter deployment from weeks 1-6 (weekly), weeks 7-20 (biweekly), and weeks 21-32 (monthly). The infrarenal IVC, lungs, heart, liver, kidneys, and spleen were harvested at necropsy. Continuous variables were evaluated with a Student t test. Results There was no evidence of IVC thrombosis, device migration, caval penetration, or pulmonary embolism. Gross pathologic analysis showed gradual device resorption until 32 weeks after deployment. Histologic assessment demonstrated neointimal hyperplasia around the IVC filter within 2 weeks after IVC filter deployment with residual microscopic fragments of polydioxanone suture within the caval wall at 32 weeks. Each iatrogenic-administered thrombus was successfully captured by the filter until resorbed (range, 1-4 weeks). Conclusion An absorbable IVC filter can be safely deployed in swine and resorbs gradually over the 32-week testing period. The device is effective for the prevention of pulmonary embolism for at least 5 weeks after placement in swine. © RSNA, 2017.

Interaction between vascularized lymph node transfer and recipient lymphatics after lymph node dissection-a pilot study in a canine model.

BACKGROUND: Vascularized lymph node transfer (VLNT) has become more widespread for surgical treatment of lymphedema. However, interaction between a transferred lymph node and the recipient lymphatic system in relieving lymphedema has not been identified. The aims of this study were to investigate anatomic changes in the lymphatic system in the forelimb of a canine after lymph node dissection and irradiation and to clarify the interaction between the transferred lymph node and recipient lymphatics. MATERIALS AND METHODS: Two adult female mongrel canines were used for this exploratory study. The unilateral axillary and lower neck node dissections were performed, and 15-Gy irradiation was applied on postoperative day 3. After 1 y, a VLNT flap was harvested from the lower abdominal region and inset in the axilla with vascular anastomoses. The girth of each forelimb was determined with a tape measure at different time points. Indocyanine green fluorescence lymphography and lymphangiography were performed before and after each surgery to evaluate morphologic changes in the lymphatics. RESULTS: Both canines revealed identical changes in the lymphatic system, but only one canine developed lymphedema. After lymph node dissection, a collateral lymphatic pathway formed a connection to the contralateral cervical node. After VLNT, an additional collateral pathway formed a connection to the internal mammary node via the transferred node in the axilla. CONCLUSIONS: The findings suggest that the lymphatic system has a homing mechanism, which allows the severed lymphatic vessels to detect and connect to adjacent lymph nodes. VLNT may create new collateral pathways to relieve lymphedema.

Effects of Near-infrared Laser Irradiation of Biodegradable Microspheres Containing Hollow Gold Nanospheres and Paclitaxel Administered Intraarterially in a Rabbit Liver Tumor Model.

PURPOSE: To evaluate the effects of near-infrared (NIR) laser irradiation of microspheres (MS) containing hollow gold nanospheres (HAuNS) and paclitaxel (PTX) administered intraarterially in an animal model. MATERIALS AND METHODS: For the ex vivo experiments, VX2 tumor-bearing rabbits underwent administration of MS-HAuNS or MS via the hepatic artery (HA). The animals were killed, the liver tumors were subjected to NIR irradiation, and temperature changes were estimated with magnetic resonance (MR) imaging. For the in vivo study, VX2 tumor-bearing rabbits were randomly assigned to three groups: MS-HAuNS-PTX-plus-NIR, MS-HAuNS-PTX, and saline-plus-NIR. Laser irradiation was delivered at 1 hour and at 3 days after administration of saline or MS-HAuNS-PTX via the HA. Animals were euthanized, and tumors were analyzed for necrosis and apoptosis. Plasma samples were collected from the MS-HAuNS-PTX-plus-NIR animals for PTX analysis. RESULTS: Ex vivo experiments showed intratumoral heating in animals that received MS-HAuNS but no temperature change in animals that received MS. Animals treated with MS-HAuNS-PTX-plus-NIR showed a transient increase in plasma PTX levels after each NIR irradiation and significantly greater tumor necrosis than animals that received MS-HAuNS-PTX or saline-plus-NIR (44.9% vs 13.8% or 23.7%; P < .0001). The mean apoptotic index in the MS-HAuNS-PTX-plus-NIR group (5.01 ± 1.66) was significantly higher than the mean apoptotic index in the MS-HAuNS-PTX (2.99 ± 0.97) or saline-plus-NIR (1.96 ± 0.40) groups (P = .0013). CONCLUSIONS: NIR laser irradiation after MS-HAuNS-PTX administration results in intratumoral heating and increases the efficacy of treatment. Further studies are required to evaluate the optimal laser settings to maximize therapeutic efficacy.

Bismuth Nanoparticle and Polyhydroxybutyrate Coatings Enhance the Radiopacity of Absorbable Inferior Vena Cava Filters for Fluoroscopy-Guided Placement and Longitudinal Computed Tomography Monitoring in Pigs.

Inferior vena cava filters (IVCFs) constructed with poly-p-dioxanone (PPDO) are promising alternatives to metallic filters and their associated risks and complications. Incorporating high-Z nanoparticles (NPs) improves PPDO IVCFs' radiopacity without adversely affecting their safety or performance. However, increased radiopacity from these studies are insufficient for filter visualization during fluoroscopy-guided PPDO IVCF deployment. This study focuses on the use of bismuth nanoparticles (BiNPs) as radiopacifiers to render sufficient signal intensity for the fluoroscopy-guided deployment and long-term CT monitoring of PPDO IVCFs. The use of polyhydroxybutyate (PHB) as an additional layer to increase the surface adsorption of NPs resulted in a 2-fold increase in BiNP coating (BiNP-PPDO IVCFs, 3.8%; BiNP-PPDO + PHB IVCFs, 6.2%), enabling complete filter visualization during fluoroscopy-guided IVCF deployment and, 1 week later, clot deployment. The biocompatibility, clot-trapping efficacy, and mechanical strength of the control PPDO (load-at-break, 6.23 ± 0.13 kg), BiNP-PPDO (6.10 ± 0.09 kg), and BiNP-PPDO + PHB (6.15 ± 0.13 kg) IVCFs did not differ significantly over a 12-week monitoring period in pigs. These results indicate that BiNP-PPDO + PHB can increase the radiodensity of a novel absorbable IVCF without compromising device strength. Visualizing the device under conventional radiographic imaging is key to allow safe and effective clinical translation of the device.

Perfusion CT assessment of tissue hemodynamics following hepatic arterial infusion of increasing doses of angiotensin II in a rabbit liver tumor model.

PURPOSE: To investigate the effects of increasing doses of angiotensin II on hepatic hemodynamics in the normal rabbit liver and in hepatic VX2 tumors by using dynamic contrast material-enhanced perfusion computed tomography (CT). MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Solitary hepatic VX2 tumors were implanted into 12 rabbits. In each animal, perfusion CT of the liver was performed before (at baseline) and after hepatic arterial infusion of varying doses (0.1-50.0 µg/mL) of angiotensin II. Images were acquired continuously for 80 seconds after the start of the intravenous contrast material administration. Blood flow (BF), blood volume (BV), mean transit time (MTT), and capillary permeability-surface area product were calculated for the tumor and the adjacent and distant normal liver tissue. Generalized linear mixed models were used to estimate the effects of angiotensin II dose on outcome measures. RESULTS: Angiotensin II infusion increased contrast enhancement of the tumor and distal liver vessels. Tumor BF increased in a dose-dependent manner after administration of 0.5-25.0 µg/mL angiotensin II, but only the 2.5 µg/mL dose induced a significant increase in tumor BF compared with BF in the adjacent (68.0 vs 26.3 mL/min/100 g, P < .0001) and distant (68.0 vs 28.3 mL/min/100 g, P = .02) normal liver tissue. Tumor BV varied with angiotensin II dose but was greater than the BV of the adjacent and distant liver tissue at only the 2.5 µg/mL (4.8 vs 3.5 mL/100 g for adjacent liver [P < .0001], 4.8 vs 3.3 mL/100 g for distant liver [P = .0006]) and 10.0 µg/mL (4.9 vs 4.4 mL/100 g for adjacent liver [P = .007], 4.9 vs 4.3 mL/100 g for distant liver [P = .04]) doses. Tumor MTT was significantly shorter than the adjacent liver tissue MTT at angiotensin II doses of 2.5 µg/mL (9.7 vs 15.8 sec, P = .001) and 10.0 µg/mL (5.1 vs 13.2 sec, P = .007) and significantly shorter than the distant liver tissue MTT at 2.5 µg/mL only (9.7 vs 15.3 sec, P = .0006). The capillary permeability-surface area product for the tumor was higher than that for the adjacent liver tissue at the 2.5 µg/mL angiotensin II dose only (11.5 vs 8.1 mL/min/100 g, P = .01). CONCLUSION: Perfusion CT enables a mechanistic understanding of angiotensin II infusion in the liver and derivation of the optimal effective dose. The 2.5 µg/mL angiotensin II dose increases perfusion in hepatic VX2 tumors versus that in adjacent and distant normal liver tissue primarily by constricting normal distal liver vessels and in turn increasing tumor BF and BV.

Endobronchial ultrasound-guided injection of NBTXR3 radio-enhancing nanoparticles into mediastinal and hilar lymph nodes: a swine model to evaluate feasibility, injection technique, safety, nanoparticle retention and dispersion.

BACKGROUND: Loco-regionally advanced lung cancer is typically treated with a combination of chemotherapy and radiation therapy, but overall survival and local control remain poor. Radio-enhancing nanoparticles such as NBTXR3 activated by radiotherapy results in increased cell death and potentially an anti-tumor immune response. The goal of this study was to assess the feasibility and safety of endobronchial ultrasound (EBUS)-guided injection of NBTXR3 into mediastinal and hilar lymph nodes (LN), as well as assess nanoparticle retention in the LN post-injection. METHODS: Animals underwent bronchoscopy under general anesthesia with EBUS-guided injection of NBTXR3 into hilar and mediastinal LN. LN and injection volumes were calculated based on pre-injection computed tomography (CT) scans. CT scans were repeated at 5 min, 30 min, and 8 days post-injection. Blood-draws were also obtained at baseline and post-injection. Animals were then housed, monitored, and sacrificed 8 days post-injection. Necropsy was then performed with gross and histologic analysis of LN. RESULTS: A total of 20 LN were injected in 5 pigs (4 LN per animal). Nanoparticles were retained in 100% of LN at 30 min, and 90% of LN at 8 days. Extravasation of nanoparticles was seen in 4 out of the 20 LN. There were no cases of nanoparticle embolization visible by CT in distant organs. Small air-bubbles were introduced in the targets and surrounding tissue in 3 out of 20 LN. Of note, at 8 days, none of these air-bubbles were present on CT scan. There were no intra-procedural or post-procedural complications in either CT scans or necropsy findings. Pigs remained clinically stable and neither laboratory values nor necropsy showed evidence of inflammation. CONCLUSIONS: EBUS-guided injection of NBTXR3 radio-enhancing nanoparticles can be safely performed achieving a high rate of nanoparticle retention, low extravasation, and no visible nanoparticle embolization.

In vivo performance of gold nanoparticle-loaded absorbable inferior vena cava filters in a swine model.

Absorbable inferior vena cava filters (IVCFs) offer a promising alternative to metallic retrievable filters in providing protection against pulmonary embolism (PE) for patients contraindicated for anticoagulant therapy. However, because absorbable filters are not radiopaque, monitoring of the filter using conventional X-ray imaging modalities (e.g. plain film radiographs, computed tomography [CT] and fluoroscopy) during deployment and follow-up is not possible and represents a potential obstacle to widespread clinical integration of the device. Here, we demonstrate that gold nanoparticles (AuNPs) infused into biodegradable filters made up of poly-p-dioxanone (PPDO) may improve device radiopacity without untoward effects on device efficacy and safety, as assessed in swine models for 12 weeks. The absorbable AuNP-infused filters demonstrated significantly improved visualization using CT without affecting tensile strength, in vitro degradation, in vivo resorption, or thrombus-capturing efficacy, as compared to similar non-AuNPs infused resorbable IVCFs. This study presents a significant advancement to the development of imaging enhancers for absorbable IVCFs.

Angiotensin II increases gene expression after selective intra-arterial adenovirus delivery in a rabbit model assessed using in vivo SSTR2-based reporter imaging.

BACKGROUND: Gene therapy has been hampered by low expression upon in vivo delivery. Using a somatostatin receptor type 2 (SSTR2)-based reporter, we assessed whether angiotensin II (AII) can improve gene expression by adenovirus upon intra-arterial (IA) delivery in a large animal model. METHODS: A SSTR2-based reporter that can be imaged by a clinically approved radiopharmaceutical was used to assess gene expression. Eight rabbits bearing VX2 tumors in each thigh were randomly injected IA with adenovirus containing a human SSTR2 (Ad-CMV-HA-SSTR2) gene chimera ± AII or control adenovirus containing green fluorescent protein (Ad-CMV-GFP). Three days later, (111)In-octreotide was given IV after computed tomography (CT) imaging using a clinical CT scanner and intravenous contrast. Tumor uptake of (111)In-octreotide was evaluated the next day using a clinical gamma camera. Gene expression was normalized to tumor weight and morphology from CT to obtain in vivo biodistribution. RESULTS: SSTR2-based expression was readily visualized. VX2 tumors infected with Ad-CMV-HA-SSTR2 upon intra-arterial delivery with AII had greater in vivo biodistribution, thus greater gene expression, than those without AII (p < 0.01, n = 6). VX2 tumors infected with Ad-CMV-HA-SSTR2 upon IA delivery had greater biodistribution, thus greater gene expression, than those with the negative control Ad-CMV-GFP (p < 0.02). Similarly, VX2 tumors infected with Ad-CMV-HA-SSTR2 upon IA delivery with AII had greater biodistribution, thus greater gene expression, than those with the negative control Ad-CMV-GFP (p < 0.01). CONCLUSIONS: Angiotensin II improves in vivo gene expression by adenovirus upon intra-arterial delivery and thus may improve gene therapy efficacy. In vivo SSTR2-based reporter imaging can be used to compare methodologies for improving gene expression.

Tumor uptake of hollow gold nanospheres after intravenous and intra-arterial injection: PET/CT study in a rabbit VX2 liver cancer model.

PURPOSE: This study was designed to investigate the intratumoral uptake of hollow gold nanospheres (HAuNS) after hepatic intra-arterial (IA) and intravenous (IV) injection in a liver tumor model. MATERIALS AND METHODS: Fifteen VX2 tumor-bearing rabbits were randomized into five groups (n = 3 in each group) that received either IV (64)Cu-labeled PEG-HAuNS (IV-PEG-HAuNS), IA (64)Cu-labeled PEG-HAuNS (IA-PEG-HAuNS), IV cyclic peptide (RGD)-conjugated (64)Cu-labeled PEG-HAuNS (IV-RGD-PEG-HAuNS), IA RGD-conjugated (64)Cu-labeled PEG-HAuNS (IA-RGD-PEG-HAuNS), or IA (64)Cu-labeled PEG-HAuNS with lipiodol (IA-PEG-HAuNS-lipiodol). The animals underwent PET/CT 1 h after injection, and uptake expressed as percentage of injected dose per gram of tissue (%ID/g) was measured in tumor and major organs. The animals were euthanized 24 h after injection, and tissues were evaluated for radioactivity. RESULTS: At 1 h after injection, animals in the IA-PEG-HAuNS-lipiodol group showed significantly higher tumor uptake (P < 0.001) and higher ratios of tumor-to-normal liver uptake (P < 0.001) than those in all other groups. The biodistribution of radioactivity 24 h after injection showed that IA delivery of PEG-HAuNS with lipiodol resulted in the highest tumor uptake (0.33 %ID/g; P < 0.001) and tumor-to-normal liver ratio (P < 0.001) among all delivery methods. At 24 h, the IA-RGD-PEG-HAuNS group showed higher tumor uptake than the IA-PEG-HAuNS group (0.20 vs. 0.099 %ID/g; P < 0.001). CONCLUSION: Adding iodized oil to IA-PEG-HAuNS maximizes nanoparticle delivery to hepatic tumors and therefore may be useful in targeted chemotherapy and photoablative therapy. PET/CT can be used to noninvasively monitor the biodistribution of radiolabeled HAuNS after IV or IA injection.

Hepatic arterial embolization with doxorubicin-loaded superabsorbent polymer microspheres in a rabbit liver tumor model.

OBJECTIVES: The pharmacokinetic profile after hepatic arterial embolization with superabsorbent microspheres (QuadraSpheres) loaded with doxorubicin was studied. METHODS: Rabbits with hepatic VX2 tumors were treated with intra-arterial administration of QuadraSpheres loaded with doxorubicin, or transarterial chemoembolization (TACE) using doxorubicin, Lipiodol and Embospheres, or hepatic arterial infusion (HAI) of doxorubicin. Tumor specimens were evaluated by fluorescence microscopy, and plasma and tumor concentrations of doxorubicin were measured. RESULTS: The peak plasma concentration of doxorubicin was lower in the QuadraSphere group (309.9 ng/ml) than in the HAI (673.4 ng/ml) or TACE (360.5 ng/ml) groups, suggesting higher tumor retention in the QuadraSphere group. Intratumoral doxorubicin levels declined to negligible levels at 1 and 3 days after treatment, respectively, in the HAI and TACE groups. In the QuadraSphere groups, intratumoral doxorubicin level declined after day 1, but was still detectable at 14 days after treatment and was higher than that in the other groups at 1, 3, and 7 days. Intratumoral doxorubicin fluorescence was detected at all time points in the QuadraSphere group, but only at 1 day after treatment in the TACE group. CONCLUSIONS: Hepatic arterial administration of doxorubicin-loaded QuadraSpheres enables the sustained release of doxorubicin to hepatic tumors.