Vessel_Density_commsbio.m

A MATLAB-based computational code to calculate the functional vessel density in fluorescent images. <br

Multifunctional Albumin–MnO₂ Nanoparticles Modulate Solid Tumor Microenvironment by Attenuating Hypoxia, Acidosis, Vascular Endothelial Growth Factor and Enhance Radiation Response

Insufficient oxygenation (hypoxia), acidic pH (acidosis), and elevated levels of reactive oxygen species (ROS), such as H₂O_2, are characteristic abnormalities of the tumor microenvironment (TME). These abnormalities promote tumor aggressiveness, metastasis, and resistance to therapies. To date, there is no treatment available for comprehensive modulation of the TME. Approaches so far have been limited to regulating hypoxia, acidosis, or ROS individually, without accounting for their interdependent effects on tumor progression and response to treatments. Hence we have engineered multifunctional and colloidally stable bioinorganic nanoparticles composed of polyelectrolyte–albumin complex and MnO₂ nanoparticles (A-MnO₂ NPs) and utilized the reactivity of MnO₂ toward peroxides for regulation of the TME with simultaneous oxygen generation and pH increase. <i>In vitro</i> studies showed that these NPs can generate oxygen by reacting with H₂O₂ produced by cancer cells under hypoxic conditions. A-MnO₂ NPs simultaneously increased tumor oxygenation by 45% while increasing tumor pH from pH 6.7 to pH 7.2 by reacting with endogenous H₂O₂ produced within the tumor in a murine breast tumor model. Intratumoral treatment with NPs also led to the downregulation of two major regulators in tumor progression and aggressiveness, that is, hypoxia-inducible factor-1 alpha and vascular endothelial growth factor in the tumor. Combination treatment of the tumors with NPs and ionizing radiation significantly inhibited breast tumor growth, increased DNA double strand breaks and cancer cell death as compared to radiation therapy alone. These results suggest great potential of A-MnO₂ NPs for modulation of the TME and enhancement of radiation response in the treatment of cancer

A Spinal Cord Window Chamber Model for <i>In Vivo</i> Longitudinal Multimodal Optical and Acoustic Imaging in a Murine Model

<div><p><i>In vivo</i> and direct imaging of the murine spinal cord and its vasculature using multimodal (optical and acoustic) imaging techniques could significantly advance preclinical studies of the spinal cord. Such intrinsically high resolution and complementary imaging technologies could provide a powerful means of quantitatively monitoring changes in anatomy, structure, physiology and function of the living cord over time after traumatic injury, onset of disease, or therapeutic intervention. However, longitudinal <i>in vivo</i> imaging of the intact spinal cord in rodent models has been challenging, requiring repeated surgeries to expose the cord for imaging or sacrifice of animals at various time points for <i>ex vivo</i> tissue analysis. To address these limitations, we have developed an implantable spinal cord window chamber (SCWC) device and procedures in mice for repeated multimodal intravital microscopic imaging of the cord and its vasculature <i>in situ</i>. We present methodology for using our SCWC to achieve spatially co-registered optical-acoustic imaging performed serially for up to four weeks, without damaging the cord or induction of locomotor deficits in implanted animals. To demonstrate the feasibility, we used the SCWC model to study the response of the normal spinal cord vasculature to ionizing radiation over time using white light and fluorescence microscopy combined with optical coherence tomography (OCT) <i>in vivo</i>. <i>In vivo</i> power Doppler ultrasound and photoacoustics were used to directly visualize the cord and vascular structures and to measure hemoglobin oxygen saturation through the complete spinal cord, respectively. The model was also used for intravital imaging of spinal micrometastases resulting from primary brain tumor using fluorescence and bioluminescence imaging. Our SCWC model overcomes previous <i>in vivo</i> imaging challenges, and our data provide evidence of the broader utility of hybridized optical-acoustic imaging methods for obtaining multiparametric and rich imaging data sets, including over extended periods, for preclinical <i>in vivo</i> spinal cord research.</p></div

SCWC model permits X-ray microirradiation of the spinal cord <i>in situ</i>.

<p>(A) Anaesthetized mice were placed directly under the micro-irradiation collimator of the small animal irradiator for delivery of X-rays to the spinal cord through the coverglass of the window chamber. (B, C) <i>In situ</i> fluoroscopic imaging was used for image-guided delivery of the X-ray beam (centered on the crosshairs) to the spinal cord. (D) Custom fit radiochromic film was used to confirm the location of the irradiation beam which had a 3 mm diameter (as seen by the dark blue circle). Scale bar = 1 cm. SCWC = spinal cord window chamber.</p

Hemoglobin oxygen saturation (sO₂) measurement made using <i>in vivo</i> photoacoustic imaging.

<p>Baseline vascular sO₂ was measured <i>in situ</i> for 1 minute while the animal breathed 100% oxygen mixed with 2% isoflurane. The animal was shifted to breathing 7% oxygen mixed with 2% isoflurane for an additional 1 minute. The oxygen concentration was then returned to 100%.</p

Intravital multispectral fluorescence microscopic imaging of medulloblastoma tumor metastasis to the spinal cord.

<p>(A) <i>In vivo</i> bioluminescence images of mice 7 days following intracranial tumor implantation of human WW426 medulloblastoma tumor cells, demonstrating local tumor growth. (B) SCWC was implanted 27 days after tumor implantation, when metastatic GFP+ tumor cells to the spinal cord could be seen using both BLI and intravital two-photon images (color bar indicates bioluminescence signal intensity; BLI units are photons/s/cm²/Sr). The head of the mouse was covered in “B” to reduce the bioluminescence signal from the brain in order to detect lower bioluminescence from the tumor micrometastases. (C) Wide-field fluorescence imaging and (D) confocal fluorescence microscopy of the SCWC-bearing mouse 28 days after initial tumor implantation (1 day post-SCWC installation). The outline of the spinal cord is highlighted with the orange dotted line in “C”. TRITC-dextran shows the posterior spinal cord vein. The arrows in “C” and “D” indicate the location of multiple tumor micrometastases in close proximity to the spinal cord vasculature. Scale bars = 1 mm. SCWC = spinal cord window chamber. BLI = bioluminescence imaging.</p

Longitudinal optical imaging of radiation response of the normal spinal cord and its vasculature through the SCWC.

<p>White light, wide-field fluorescence, and svOCT images were taken at (A) 1 day before, (B) 1 hour, (C) 24 hours, and (D) 48 hours following a single 30 Gy radiation dose to the cord. White light images revealed significant radiation-induced hematoma in the spinal cord two days after irradiation (arrows). FITC-dextran was injected intravenously prior to acquiring the fluorescence images at each time point. Fiducial markers consisting of India ink (black dots shown by arrows) on the white light images were used to as spatial landmarks to allow identification and long-term imaging of vascular structures. Compared with vascular function, corresponding en-face projected svOCT images revealed that the posterior spinal cord vein and other vasculature did not suffer significant short-term radiation-induced structural damage. Scale bar = 1 mm. SCWC = spinal cord window chamber. svOCT = speckle variance optical coherence tomography.</p

Visual and histological confirmation that SCWC implantation does not damage the spinal cord structure or cause significant inflammation or infection.

<p>(A) White light images following SCWC implantation at 0, 1, 3 and 7 days showed no signs of local infection, excessive bleeding around the installation site, or device rejection. SCWC remained optically clear for 29 days, permitting long-term high-resolution imaging of cord and vascular structures. Yellow arrows indicate the location of the spinal cord. (B–E) Histological analysis and quantification of spinal cord tissue cross-sections cut directly below the caudal edge of the implanted SCWC. (B) H&E staining confirmed tissue morphology was intact after WC implantation. (C) Representative Iba-1 immunohistochemistry images from spinal cords 24, 48 and 72 hours following SCWC implantation. Sham and spinal cord injury (SCI) (Iba-1 positive control) animals are also shown for comparison. SCI positive control animals showed a significant increase in Iba-1 expression, * p<0.001. No notable changes in Iba-1 expression in <i>ex vivo</i> spinal cord were observed between the SCWC implanted groups). (D) Western blot for Iba-1 prior to SCWC implant (sham), and at 24, 48, and 72 hours post-implantation in athymic nude mice. (E) Bar graph representing the fluorescent intensity quantification of Iba-1, and no significant increase in Iba-1 was observed; n = 3 per group (p-values >0.212 for comparisons between all groups). (F) Bar graph showing quantification of Western blot data. Increases in Iba-1 protein were observed in animals receiving SCWC implantation, although these increases were insignificant (p = 0.15); n = 3 per group. (G) Western blot for Iba-1 prior to SCWC implant (sham) (n = 3), and at 24 hours (n = 4), 3 days (n = 3), 10 days (n = 3) and 28 days (n = 3) post-implantation in C57BL6 mice. (H) Bar graph showing quantification of Western blot Iba-1 data (C57BL6 mice). No significant increases in Iba-1 protein were observed (p = 0.405). It is anticipated that the slight increases in Iba-1 in the athymic nude mice may be due to the laminectomy and surgical procedures performed. β-Actin was used as a protein loading control. Scale bars = 400 µm. Data was transformed (square-root transformation) and analyzed using a one-way ANOVA; Tukey post-hoc analysis. SCWC = spinal cord window chamber.</p

Mouse spinal cord window chamber (SCWC) device and surgical implantation procedures.

<p>(A) The SCWC device design and dimensions are shown. An 8 mm diameter glass coverslip was inserted into the SCWC device and held in place by a metal ring clamp once the device is surgically implanted into the mouse (shown in panel “F” and “G”). Four radial extension arms have been built into the device in order to immobilize the animal during imaging sessions. (B-E) Photographs showing <i>step-by-step</i> surgical procedures for implanting the SCWC in the mouse exposing the spinal cord at the L2–L3 vertebrae. (E) Artificial dura was placed on the dorsal surface of exposed spinal cord below the coverslip to prevent scar tissue formation. Two separate devices were manufactured from either durable (F) polycarbonate or (G) light-weight surgical steel. Polycarbonate was used to allow photoacoustic imaging <i>in vivo</i>. (H) X-ray images were taken following SCWC implantation to confirm the device had been placed over L2–L3 and demonstrate that the spinal cord and vertebrae remain structurally sound after implantation of the device. Scale bars = 1 cm.</p

SCWC permits structural, functional and oxygenation imaging of the intact spinal cord vasculature <i>in situ</i>.

<p>(A) Power Doppler ultrasound (color) overlaid on a B-mode structural ultrasound (gray-scale) image obtained through the polycarbonate SCWC along a longitudinal section of the normal spinal cord <i>in vivo</i> (device is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058081#pone-0058081-g001" target="_blank">Figure 1F</a>). The power Doppler depicts vascular architecture in several vessels of the spinal cord. The color bar represents the signal intensity. (B) Corresponding multispectral photoacoustic imaging of the same cross section of normal spinal cord permitted <i>in situ</i> measurement of hemoglobin oxygen saturation in the anterior spinal artery and posterior spinal vein. It demonstrated that the cord is well oxygenated. The color bar represents the relative hemoglobin oxygen saturation level. (C) Cross-sectional Doppler OCT image demonstrated significant blood flow in the posterior spinal vein. The color bar represents the phase-shift of the backscattered light in radians which is proportional to the velocity of the red blood cells in the axial direction. (D) Corresponding structural OCT image of the spinal cord permitted visualization of key spinal cord features, including the glass coverslip (1), anterior spinal vein (2), white matter (3), and grey matter (4) of the intact cord. Scale Bars = 500 µm (A–D). SCWC = spinal cord window chamber. OCT = optical coherence tomography.</p