Dorsal Skinfold Window Chamber Research Articles

Hyperspectral imaging of 4T1 mammary carcinomas grown in dorsal skinfold window chambers: spectral renormalization and fluorescence modeling.

Hyperspectral imaging (HSI) of murine tumor models grown in dorsal skinfold window chambers (DSWCs) offers invaluable insight into the tumor microenvironment. However, light loss in a glass coverslip is often overlooked, and particular tissue characteristics are improperly modeled, leading to errors in tissue properties extracted from hyperspectral images. We highlight the significance of spectral renormalization in HSI of DSWC models and demonstrate the benefit of incorporating enhanced green fluorescent protein (EGFP) excitation and emission in the skin tissue model for tumors expressing genes to produce EGFP. We employed an HSI system for intravital imaging of mice with 4T1 mammary carcinoma in a DSWC over 14 days. We performed spectral renormalization of hyperspectral images based on the measured reflectance spectra of glass coverslips and utilized an inverse adding-doubling (IAD) algorithm with a two-layer murine skin model, to extract tissue parameters, such as total hemoglobin concentration and tissue oxygenation ( ). The model was upgraded to consider EGFP fluorescence excitation and emission. Moreover, we conducted additional experiments involving tissue phantoms, human forearm skin imaging, and numerical simulations. Hyperspectral image renormalization and the addition of EGFP fluorescence in the murine skin model reduced the mean absolute percentage errors (MAPEs) of fitted and measured spectra by up to 10% in tissue phantoms, 0.55% to 1.5% in the human forearm experiment and numerical simulations, and up to 0.7% in 4T1 tumors. Similarly, the MAPEs for tissue parameters extracted by IAD were reduced by up to 3% in human forearms and numerical simulations. For some parameters, statistically significant differences ( ) were observed in 4T1 tumors. Ultimately, we have shown that fluorescence emission could be helpful for 4T1 tumor segmentation. The results contribute to improving intravital monitoring of DWSC models using HSI and pave the way for more accurate and precise quantitative imaging.

A Hypovolemic Infusion of Human Methemoglobin in Golden Syrian Hamsters Provides Insight on its effect on the Microcirculation

Introduction: Hemoglobin (HbFe(II)) plays a crucial role in transporting oxygen to tissues for metabolic needs. However, this protein isn't just an oxygen carrier; it also regulates vascular tone and inflammation by its redox coupling with methemoglobin (HbFe(III)). Methemoglobin's oxidized form, Fe (III), possesses the potential to produce water from oxygen as a free energy reaction. It functions as a nitrite reductase, regulating the oxygen level in the blood and controlling vascular tone by generating nitric oxide from nitrite, thereby increasing blood flow when oxygenated hemoglobin decreases. If methemoglobin can't be reduced, it leads to the dissociation of tetrameric hemoglobin into αβ dimers, releasing heme and iron into circulation. Methods: The study involved Golden Syrian Hamsters with dorsal skin fold window chambers, observing acute methemoglobin effects via hypovolemic infusion (10% of the animal’s blood volume) compared to human hemoglobin and a lactated ringer's control. Microhemodynamics, mean arterial pressure, heart rate, blood gases, and blood properties were measured. Results: Methemoglobin did not significantly affect MAP and HR but increased arteriole diameters by 5% and venule diameter by 15% compared to baseline. However, it caused a 50% reduction in functional capillary density compared to baseline, alongside significant cardiac and kidney inflammation. Conclusion: The study concludes that a hypovolemic infusion of methemoglobin induces vasoreactivity but results in poor microcirculatory pressure and inflammation in vital organs. This demonstrates the delicate balance and potential adverse effects associated with alterations in the hemoglobin redox state and highlights the importance of maintaining its balance for vascular and organ health. Project number: R01HL159862 Project number: R01HL162120. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

A mathematical model of tissue axial and radial diffusion in the microvasculature for intravascular microscopy and phosphorescence quenching data

This study aims to extend earlier Krogh Cylinder Models of an oxygen profile by considering axial diffusion and analytically solving Fick's Law Partial Differential Equation with novel boundary conditions via the separation of variables. We next prospectively collected a total of 20 animals, which were randomly assigned to receive either fresh or two-week-old stored red blood cell (RBC) transfusions and PQM oxygen data were measured acutely (90 min) or chronically (24 h). Transfusion effects were evaluated in vivo using intravital microscopy of the dorsal skinfold window chamber in Golden Syrian Hamsters. Hamsters were initially hemorrhaged by 50% of total blood volume and resuscitated 1-h post hemorrhage. PQM data were subsequently collected and fit the derived 2D Krogh cylinder model. Systemic hemodynamics (mean arterial pressure, heart rate) were similar in both pre and post-transfusion with either stored or fresh cells. Transfusion with stored cells was found to impair axial and radial oxygen gradients as quantified by our model and consistent with previous studies. Specifically, we observed a statistically significant decrease in the arteriolar tissue radial oxygen gradient after transfusion with stored RBCs at 24 h compared with fresh RBCs (0.33 ± 0.17 mmHg μ m-1 vs, 0.14 ± 0.12 mmHg μ m-1; p = 0.0280). We also observed a deficit in the arteriolar tissue oxygen gradient (0.03 ± 0.01 mmHg μ m-1 fresh vs. 0.018 ± 0.007 mmHg μ m-1 stored; p = 0.0185). We successfully derived and validated an analytical 2D Krogh cylinder model in an animal model of microhemodynamic oxygen diffusion aberration secondary to storage lesions.

Imaging of Extravasation of Splenocytes in the Dorsal Skinfold Window Chamber.

Infiltration of immune cells into the tumor is one of the major drivers of antitumor immune response, which can direct the outcome of anticancer therapies. In mice, implantation of dorsal skinfold window chamber (DSWC) combined with intravital confocal fluorescence microscopy allows real-time observation of splenocyte extravasation and infiltration into tumors. Here, we describe a detailed procedure of the DSWC implantation, splenocyte isolation and fluorescent labeling, intravenous injection of labeled splenocytes, and imaging of splenocyte extravasation into tumors using confocal fluorescence microscopy.

CD133-targeted multifunctional nanomicelles for dual-modality imaging and synergistic high-intensity focus ultrasound (HIFU) ablation on pancreatic cancer in nude mice.

Pancreatic cancer is an aggressive and highly fatal malignant tumor. Recent studies have shown that cancer stem cells (CSCs) play an important role in resisting current therapeutic modalities. Furthermore, CD133 is highly expressed in CSCs. High-intensity focused ultrasound (HIFU) is a promising non-invasive therapeutic strategy for unresectable pancreatic cancers. In our study, we synthesized targeted CD133 organosilane nanomicelles by encapsulating perfluorohexane (PFH). The CD133 antibody on the surface could specifically bind to CD133-positive pancreatic cancer cells and selectively concentrate in pancreatic cancer tumor tissues. PFH was introduced to improve the ablation effect of HIFU due to its liquid-gas phase transition properties. By combining with the dorsal skinfold window chamber model (DSWC) of pancreatic cancer in nude mice, multiphoton fluorescence microscopy was used to evaluate the targeting effect of nanomicelles on pancreatic cancer tumor tissue. These multifunctional nanomicelles synergistically affected HIFU treatment of pancreatic cancer, providing an integrated research platform for diagnosing and treating pancreatic cancer with HIFU.

High-molecular-weight linear polymers improve microvascular perfusion after extracorporeal circulation.

High-molecular-weight linear polymers (HMWLPs) have earned the name "drag-reducing polymers" because of their ability to reduce drag in turbulent flows. Recently, these polymers have become popular in bioengineering applications. This study investigated whether the addition of HMWLP in a venoarterial extracorporeal circulation (ECC) model could improve microvascular perfusion and oxygenation. Golden Syrian hamsters were instrumented with a dorsal skinfold window chamber and subjected to ECC using a circuit comprised of a peristaltic pump and a bubble trap. The circuit was primed with lactated Ringer solution (LR) containing either 5 ppm of polyethylene glycol (PEG) with a low molecular weight of 500 kDa (PEG500k) or 5 ppm of PEG with a high molecular weight of 3,500 kDa (PEG3500k). After 90 min of ECC at 15% of the animal's cardiac output, the results showed that the addition of PEG3500k to LR improved microvascular blood flow in arterioles and venules acutely (2 h after ECC), whereas functional capillary density showed improvement up to 24 h after ECC. Similarly, PEG3500k improved venular hemoglobin O2 saturation on the following day after ECC. The serum and various excised organs all displayed reduced inflammation with the addition of PEG3500k, and several of these organs also had a reduction in markers of damage with the HMWLPs compared to LR alone. These promising results suggest that the addition of small amounts of PEG3500k can help mitigate the loss of microcirculatory function and reduce the inflammatory response from ECC procedures.NEW & NOTEWORTHY High-molecular-weight linear polymers have gained traction in bioengineering applications. The addition of PEG3500k to lactated Ringer solution (LR) improved microvascular blood flow in arterioles and venules acutely after extracorporeal circulation (ECC) in a hamster model and improved functional capillary density up to 24 h after ECC. PEG3500k improved venular hemoglobin O2 saturation and oxygen delivery acutely after ECC and reduced inflammation in various organs compared to LR alone.

BIO9: The Addition of High Molecular Weight Linear Polymers Improves Microvascular Perfusion in an Animal Model of Veno-arterial Extracorporeal Circulation.

High molecular weight linear polymers have been used in turbulent flow applications such as crude oil transfer to reduce drag. These polymers have branched into bioengineering applications to improve pressure changes within the macrocirculation and improve RBC distribution within the microcirculation. The current study aims to determine whether the addition of these polymers to priming fluids used for veno-arterial extracorporeal circulation (VA-ECC) procedures improve microvascular flow and tissue oxygenation. To assess the microcirculation, male Golden Syrian hamsters were instrumented with a dorsal skinfold window chamber, and an exteriorized carotid artery catheter was implanted for arterial blood and blood pressure access. After baseline (BL) measurements were taken, the hamster underwent surgery under isoflurane to insert a drainage catheter into the jugular vein. A heparin bolus (0.05 IU/g) was then provided, and the animal was connected to the VA-ECC circuit. The circuit, consisting of a peristaltic pump and bubble trap, was primed with lactated ringers (LR, control), LR with 5 ppm of 500 kDa polyethylene glycol (PEG500k) or LR with 5 ppm of 3500kDa polyethylene glycol (PEG3500k). Blood flow was ramped up over 15 minutes, maintained at 15% of the animal’s cardiac output for 60 minutes, then ramped down over 15 minutes. The animal was then surgically closed and placed on a heating pad and allowed at least 30 minutes to wake up from the anesthetic plane before taking measurements at 1, 2, and 24 hours after VA-ECC. Capillary perfusion (Figure 1) within the skinfold preparation showed a significant decrease immediately post-ECC for all three groups. However, the PEG3500k group consistently showed significant increase in functional capillary density (FCD) as compared to both other groups after ECC. 24 hrs after ECC, FCD was recovered close to BL levels for both PEG groups, while the LR control did not. Arteriole and venule velocity (Figure 2) was significantly elevated at both 1 and 2 hrs post-ECC for the PEG3500k group when compared to the other two groups. Arteriole flow for the PEG3500k was significantly improved over the LR control at 24 hrs post-ECC, and was significantly higher than BL levels. Hemoglobin saturation within arterioles and venules (Figure 3) revealed that PEG3500k had higher venule saturation at 1 and 2 hrs post-ECC compared to both other groups. The results indicate that the use of PEG3500k improved microvascular perfusion acutely post-ECC, and continued to up to 24 hrs afterwards. The most important changes were in arteriole perfusion and FCD, since that directly impacts oxygen delivery and metabolic washout respectively. Ultimately, this aids in maintaining organ function and preventing the development of ischemic injury and inflammatory cascades. Future studies could optimize the molecular weight, concentration, and type of polymer, along with testing in a clinical ECC circuit to determine hemodynamic changes.Figure 1. Functional capillary density (FCD), normalized to baseline (BL, represented by the dotted line). † P < 0.05 as compared to BL, within each group. * P < 0.05; ** P < 0.01; *** P < 0.001 between groups, at each timepoint.Figure 2. Perfusion within the microcirculation. From top to bottom: diameter, velocity, blood flow. Arterioles are on the left, while venules are on the right. Note that all values were normalized to baseline (BL, represented by the dashed line) and that blood flow was calculated from the diameter and velocity measurements of each vessel. Bar plots represent mean and standard deviation. † P < 0.05 as compared to BL, within each group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 between groups, at each timepoint.Figure 3. Hemoglobin oxygen saturation within the microcirculation. Top = arterioles, bottom = venules. Bar plots show mean and standard deviation. † P < 0.05 as compared to BL, within each group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 between groups, at each timepoint.

A Novel Preclinical Murine Model to Monitor Inflammatory Breast Cancer Tumor Growth and Lymphovascular Invasion

Inflammatory breast cancer (IBC), an understudied and lethal breast cancer, is often misdiagnosed due to its unique presentation of diffuse tumor cell clusters in the skin and dermal lymphatics. Here, we describe a window chamber technique in combination with a novel transgenic mouse model that has red fluorescent lymphatics (ProxTom RFP Nu/Nu) to simulate IBC clinicopathological hallmarks. Various breast cancer cells stably transfected to express green or red fluorescent reporters were transplanted into mice bearing dorsal skinfold window chambers. Intravital fluorescence microscopy and the in vivo imaging system (IVIS) were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0-140 h. This short-term, longitudinal imaging time frame in studying transient or dynamic events of diffuse and collectively migrating tumor cells in the local environment and quantitative analysis of the tumor area, motility, and vessel characteristics can be expanded to investigate other cancer cell types exhibiting lymphovascular invasion, a key step in metastatic dissemination. It was found that these models were able to effectively track tumor cluster migration and dissemination, which is a hallmark of IBC clinically, and was recapitulated in these mouse models.

TGF-β1 promotes human breast cancer angiogenesis and malignant behavior by regulating endothelial-mesenchymal transition.

Endothelial-mesenchymal transition (EndMT) is an important process of angiogenesis, which plays a significant role in in tumor invasion and metastasis, while its regulatory mechanisms in breast cancer remain to be fully elucidated. We previously demonstrated that tumor-associated macrophages (TAMs) can induce EndMT in endothelial cells by secreting CCL18 through the activation of the TGF-β and Notch signaling pathways in breast cancer. This study was designed to study the role of EndMT in breast cancer angiogenesis and progression in order to explore the underlying mechanism. Immunohistochemistry (IHC) was used to evaluate the expression of microvascular density (MVD) and EndMT markers in breast cancer. TGF-β1 was used to induce EndMT models of differentiated-endothelial breast cancer stem-like cells (BCSLCs). In vitro cell migration, proliferation and matrigel tube-formation assays, as well as in vivo nude mouse tumor-bearing model and nude mouse dorsal skinfold window chamber (DSWC) model, were utilized to investigate the effects in order to explore the mechanism of EndMT induced by TGF-β1 on breast cancer progression. In this study, we demonstrated that the EndMT markers were positively associated with MVD indicating unfavorable prognosis of invasive ductal carcinoma (IDC) patients. Functionally, TGF-β1 promoted migration, proliferation and angiogenesis of differentiated-endothelial BCSLCs by inducing EndMT in vitro and promoted tumor growth and angiogenesis in vivo. Mechanically, we revealed TGF-β1 induced EndMT by activation of TGF-β and Notch signaling pathways with increase of p-Smad2/3 and Notch1 expression. Moreover, we found Snail and Slug were key factors of TGF-β and Notch signaling pathways. Our findings elucidated the mechanism of TGF-β1 in the promotion of angiogenesis and progression by EndMT in breast cancer.

Intravital optical imaging for immune cell tracking after photoimmunotherapy with plasmonic gold nanostars

Bladder cancer has been ranked as one of the most commonly occurring cancers in men and women with approximately half of the diagnoses being the late stage and/or metastatic diseases. We have developed a novel cancer treatment by combining gold nanostar-mediated photothermal therapy with checkpoint inhibitor immunotherapy to treat bladder cancer. Experiment results with a murine animal model demonstrated that our developed photoimmunotherapy therapy is more efficacious than any individual studied treatment. In addition, we used intravital optical imaging with a dorsal skinfold window chamber animal model to study immune responses and immune cell accumulation in a distant tumor following our photoimmunotherapy. The mice used have the CX3CR1-GFP receptor on monocytes, natural killer cells, and dendritic cells allowing us to dynamically track their presence by fluorescence imaging. Our proof-of-principle study results showed that the photoimmunotherapy triggered anti-cancer immune responses to generate anti-cancer immune cells which accumulate in metastatic tumors. Our study results illustrate that intravital optical imaging is an efficient and versatile tool to investigate immune responses and mechanisms of photoimmunotherapy in future studies.

Drag Reducing Polymers Improves Microvascular Perfusion After Extracorporeal Circulation

Drag reducing polymers (DRPs) have been used in various engineering applications, such as the transfer of crude oil or seawater in pipelines. The use of DRPs has crossed into the bioengineering sphere, with previous ex‐vivo and in‐vivo studies suggesting that the addition of DRPs decreases the erythrocyte cell free layer (CFL) or cell depleted layer near the vessel wall and increases microvascular blood flow. The objective of this study was to investigate whether the addition of DRPs would improve microvascular perfusion in a rodent model of extracorporeal circulation (ECC).Male Golden Syrian hamsters were instrumented with a dorsal skinfold window chamber, along with an arterial catheter into the right carotid artery to monitor blood pressure and heart rate. During ECC, animals were anesthetized with isoflurane, and a drainage cannula was inserted through the left jugular. The animal was connected to the circuit, primed with either lactate ringers (LR group) or 5 PPM 500 kDa polyethylene glycol in lactate ringers (DRP group). Blood was drained from the jugular cannula, run through a peristaltic pump and bubble trap, before entering the carotid catheter for reinfusion. The circuit was run for 90 minutes in total, with 60 minutes at a flowrate equivalent to 15% of the animal’s cardiac output. Measurements were taken before ECC (baseline, BL) and after 1, 2, and 24 hrs post‐ECC. At each timepoint, microvessel (arterioles and venules) diameter, red blood cell velocity and functional capillary density (FCD) were measured using intravital microscopy.Arteriole and venule hemodynamics are displayed in Figure 1. The arteriole results showed an interesting contrast between effects at 1 hr and 24 hrs post‐ECC. At 1 hr post‐ECC, the DRP group displayed a statistically lower arteriole diameter compared to the LR group, but no statistical difference in blood flow between the two groups. At 24 hrs post‐ECC, no difference in arteriole diameter was observed between groups, while arteriole blood flow was statistically higher in the DRP group compared to the LR group. In venules, no changes were observed between groups at 1 hr post‐ECC. However, at 2 and 24 hrs post‐ECC, the DRP group had a statistically significant higher diameter compared to the LR group. Venular blood flow was statistically elevated in the DRP group at 24 hrs post‐ECC compared to the LR group. Figure 2 shows the changes in FCD. FCD was lower for both DRP and LR groups at 1 and 2 hrs post‐ECC when compared to BL levels. The DRP group displayed a higher FCD at 2 hrs post‐ECC compared to the LR group. At 24 hrs however, while the DRP group was able to recover back to BL levels, the LR group was statistically lower than BL.From these results we conclude that the addition of DRPs to priming fluids for ECC procedures may be beneficial in preserving microvascular perfusion 24 hrs post‐ECC. This may help reduce the risk of ischemic injury, production of pro‐inflammatory markers, and development of multiorgan dysfunction. Further research is warranted to optimize the DRPs parameters, such as concentration and molecular weight.

Multi-step deep neural network for identifying subfascial vessels in a dorsal skinfold window chamber model.

Automatic segmentation of blood vessels in the dorsal skinfold window chamber (DWSC) model is a prerequisite for the evaluation of vascular-targeted photodynamic therapy (V-PDT) biological response. Recently, deep learning methods have been widely applied in blood vessel segmentation, but they have difficulty precisely identifying the subfascial vessels. This study proposed a multi-step deep neural network, named the global attention-Xnet (GA-Xnet) model, to precisely segment subfascial vessels in the DSWC model. We first used Hough transform combined with a U-Net model to extract circular regions of interest for image processing. GA step was then employed to obtain global feature learning followed by coarse segmentation for the entire blood vessel image. Secondly, the coarse segmentation of blood vessel images from the GA step and the same number of retinal images from the DRIVE datasets were combined as the mixing sample, inputted into the Xnet step to learn the multiscale feature predicting fine segmentation maps of blood vessels. The data show that the accuracy, sensitivity, and specificity for the segmentation of multiscale blood vessels in the DSWC model are 96.00%, 86.27%, 96.47%, respectively. As a result, the subfascial vessels could be accurately identified, and the connectedness of the vessel skeleton is well preserved. These findings suggest that the proposed multi-step deep neural network helps evaluate the short-term vascular responses in V-PDT.

Evaluation of Tumor Development Using Hemoglobin Saturation Profile on Rodent Dorsal Window Chamber.

Tumor development can be indirectly evaluated using features of the tumor microenvironment (TME), such as hemoglobin saturation (HbSat), blood vessel dilation, and formation of new vessels. High values of HbSat and other features of the TME could indicate high metabolic activity and could precede the formation of angiogenic tumors; therefore, changes in HbSat profile can be used as a biomarker for tumor progression. One methodology to evaluate HbSat profile over time, and correlate it with tumor development in vivo in a preclinical model, is through a dorsal skin-fold window chamber. In this chapter, we provide a detailed description of this methodology to evaluate hemoglobin saturation profile and to predict tumor development. We will cover the surgical preparation of the mouse, the installation/maintenance of the dorsal window chamber, and the imaging processing and evaluation to the HbSat profile to predict new development of new tumor areas over time. We included, in this chapter, step by step examples of the imaging processing method to obtain pixel level HbSat values from raw pixels data, the computational method to determine the HbSat profile, and the steps for the classification of the areas into tumor and no-tumor.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Dual delivery of IL-10 and AT-RvD1 from PEG hydrogels polarize immune cells towards pro-regenerative phenotypes

Inflammation after traumatic injury or surgical intervention is both a protective tissue response leading to regeneration and a potential cause of wound complications. One potentially successful strategy to harness to pro-regenerative roles of host inflammation is the localized delivery of bioactive materials to induce immune suppressive cellular responses by cells responding to injury. In this study, we designed a fully synthetic poly (ethylene) glycol (PEG)-based hydrogel to release the specialized pro-resolving lipid mediator aspirin-triggered resolvin-D1 (AT-RvD1) and recombinant human interleukin 10 (IL-10). We utilized a unique side-by-side internally controlled implant design wherein bioactive hydrogels were implanted adjacent to control hydrogels devoid of immune modulatory factors in the dorsal skinfold window chamber. We also explored single-immune cell data with unsupervised approaches such as SPADE. First, we show that RGD-presenting hydrogel delivery results in enhanced immune cell recruitment to the site of injury. We then use intra-vital imaging to assess cellular recruitment and microvascular remodeling to show an increase in the caliber and density of local microvessels. Finally, we show that the recruitment and re-education of mononuclear phagocytes by combined delivery IL-10 and AT-RvD1 localizes immune suppressive subsets to the hydrogel, including CD206+ macrophages (M2a/c) and IL-10 expressing dendritic cells in the context of chronic inflammation following surgical tissue disruption. These data demonstrate the potential of combined delivery on the recruitment of regenerative cell subsets involved in wound healing complications.

Targeted delivery of siRNAs against hepatocellular carcinoma-related genes by a galactosylated polyaspartamide copolymer

Given the lack of effective treatments for Hepatocellular carcinoma (HCC), the development of novel therapeutic approaches is very urgent. Here, siRNAs were delivered to HCC cells by a synthetic polymer containing α,β-poly-(N-2-hydroxyethyl)-D,L-aspartamide-(PHEA) derivatized with diethylene triamine (DETA) and bearing in the side chain galactose (GAL) linked via a polyethylene glycol (PEG) to obtain (PHEA-DETA-PEG-GAL, PDPG). The GAL residue allows the targeting to the asialo-glycoprotein receptor (ASGPR), overexpressed in HCC cells compared to normal hepatocytes.Uptake studies performed using a model siRNA or a siRNA targeted against the enhanced green fluorescence protein, demonstrated the PDPG specific delivery of siRNA to HuH7 cells, a human cellular model of HCC. GAL-free copolymer (PHEA-DETA-PEG-NH2, PDP) or the chemical block of ASGPR, impaired PDPG targeting effectiveness in vitro. The specificity of PDPG delivery was confirmed in vivo in a mouse dorsal skinfold window chamber assay.Functional studies using siRNAs targeting the mRNAs of HCC-related genes (eEF1A1, eEF1A2 and E2F1) delivered by PDPG, significantly decreased HuH7 vitality/number and down regulated the expression of the target genes. Only minor effectiveness was in contrast observed for PDP. In IHH, a human model of normal hepatocytes with reduced ASGPR expression, PDPG barely reduced cell vitality. In a subcutaneous xenograft mouse model of HCC, PDPG-siRNAs reduced HCC tumor growth compared to controls without significant toxic effects.In conclusion, our study demonstrates the valuable potentials of PDPG for the specific delivery of siRNAs targeting HCC-related genes.

Automatic protocol for quantifying the vasoconstriction in blood vessel images.

Vascular targeted photodynamic therapy (V-PDT) has been successfully utilized for various vascular-related diseases. To optimize the PDT dose and treatment protocols for clinical treatments and to elucidate the biological mechanisms for V-PDT, blood vessels in the dorsal skin-fold window chamber (DSWC) of nude mice are often chosen to perform in vivo studies. In this study, a new automatic protocol to quantify the vasoconstriction of blood vessels in the DSWC model is proposed, which focused on tracking the pixels of blood vessels in pre- V-PDT images that disappear after V-PDT. The disappearing pixels indicate that the blood vessels were constricted, and thus, the vasoconstriction image for pixel distribution can be constructed. For this, the image of the circular region of interest was automatically extracted using the Hough transform. In addition, the U-Net model is employed to segment the image, and the Speeded-Up Robust Features algorithm to automatically register the segmented pre- and post- V-PDT images. The vasoconstriction of blood vessels in the DSWC model after V-PDT is directly quantified, which can avoid by the potential of generating new capillaries. The accuracy, sensitivity and specificity of the U-Net model for image segmentation are 90.64%, 80.12% and 92.83%, respectively. A significant difference in vasoconstriction between a control and a V-PDT group was observed. This new automatic protocol is well suitable for quantifying vasoconstriction in blood vessel image, which holds the potential application in V-PDT studies.

Hypo-fractionation radiotherapy normalizes tumor vasculature in non-small cell lung cancer xenografts through the p-STAT3/HIF-1 alpha signaling pathway.

Background:Hypo-fractionation radiotherapy (HFRT) was considered to be an important treatment for non-small cell lung cancer (NSCLC), but the radiobiological effects of HFRT on NSCLC remain unclear. The aim of this study was to investigate specific biological effect of HFRT on tumor angiogenesis, compared with conventional radiotherapy (CRT).Methods:The subcutaneous xenograft models and the dorsal skinfold window chamber (DSWC) models of nude mice bearing H460 and HCC827 NSCLC cells were irradiated with doses of 0 Gy (sham group), 22 Gy delivered into 11 fractions (CRT group) or 12 Gy delivered into 1 fraction (HFRT group). At certain time-points after irradiation, the volumes, hypoxic area, coverage rate of pericyte and micro-vessel density (MVD) of the subcutaneous xenograft models were detected, and the tumor vasculature was visualized in the DSMC model. The expressions of phosphorylated signal transducer and activator of transcription (p-STAT3), hypoxia-inducible factor 1-α (HIF-1α), CXCL12 and VEGFA were detected.Results:Compared with the CRT groups, HFRT showed more-efficient tumor growth-suppression, accompanied by a HFRT-induced window-period, during which vasculature was normalized, tumor hypoxia was improved and MVD was decreased. Moreover, during the window-period, the signal levels of p-STAT3/HIF-1α pathway and the expressions of its downstream angiogenic factors (VEGFA and CXCL12) were inhibited by HFRT.Conclusion:Compared with CRT, HFRT induced tumor vasculature normalization by rendering the remaining vessels less tortuous and increasing pericyte coverage of tumor blood vessels, thereby ameliorating tumor hypoxia and enhancing the tumor-killing effect. Moreover, HFRT might exert the aforementioned effects through p-STAT3/HIF-1α signaling pathway.