This work was partially supported by grants from NSFC (82273796, 82111530209), Special Funds for Guiding Local Science and Technology Development of Central Government (XZ202202YD0021C, XZ202102YD0033C, XZ202001YD0028C), Hubei Province Scientific and Technical Innovation Key Project (2020BAB058), the Fundamental Research Funds for the Central Universities, and the Tibet Autonomous Region COVID-19 Prevention and Control Programs for Science and Technology Development.

Animal experiments for NIR-II imaging studies were conducted at the Animal Experiment Center of Wuhan University, which has been awarded the International Association for Experimental Animal Care (AALAC). All animal studies were conducted following the China Animal Welfare Commission Guidelines for the Care and Use of Experimental Animals and approved by the Animal Care and Use Committee (IACUC) of the Animal Experimental Center of Wuhan University.
Female BALB/c nude mice (~20 g) at 6 weeks of age were used for the present study.
1. NIR-II imaging preparation
<ol>
	<li>Place commercially available black cardboard (see Table of Materials) in the center of the carrier. Then, place the sample on top of the black cardboard, so that the sample is in the center of the carrier (a stage located in the imaging device). 
	NOTE: Compared with white cardboard, black cardboard has less background interference during NIR-II imaging.</li>
	<li>Select a suitable filter based on the wavelength of the NIR-II probe. Press long (&#62;2 s) to control the box area (such as 900 LP) corresponding to the filter model in the screen interface when the system moves the filter into the optical imaging path.</li>
	<li>Long press platform up on the touch screen interface of the carrier console control area so that the carrier consoles up; long press platform down so the carrier consoles down.</li>
	<li>Adjust the platform height to &#34;0 mm&#34; (height adjustment) and use automatic focusing to make the NIR-II image clear.</li>
</ol>
2. Synthesis of NIR-II dye (HLY1)
<ol>
	<li>Weigh the raw materials required for the synthesis experiment. Ensure they do not deteriorate.</li>
	<li>Add Compound 1 (200 mg, 0.18 mmol), PdCl2(dppf)2CH2Cl2 (28 mg, 0.04 mmol), N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-2-amine&#160;(170 mg, 0.4 mmol), and K2CO3 (46 mg, 0.34 mmol) to tetrahydrofuran (THF) solution in a 25 mLround-bottom flask. Stir the mixture for 4 h at 75 &#176;C under N2 atmosphere (Figure 1A). 
	NOTE: For the synthesis procedure of Compound 1 and N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-2-amine, refer to Li et al21. The chemical structures are shown in Figure 1A.</li>
	<li>After cooling to ambient temperature, quench the reaction with distilled (DI) water (80 mL), and extract the mixture with DCM (dichloromethane)/H2O (30 mL) (three times). Purify the crude product by column chromatography16 (petroleum ether:DCM = 10:1) to make HLY1 a green solid (78 mg, 30% yield).</li>
	<li>Place the dye HLY1 under the protection of nitrogen in the refrigerator for later use. This can be stored for up to 6 months.</li>
</ol>
3. Preparation of water-suspensible nanoprobe
<ol>
	<li>Weigh HLY1 (1 mg) and amphipathic encapsulation materials, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2k (DSPE-PEG2k, 10 mg; see Table of Materials).</li>
	<li>Prepare HLY1 dots20 by employing DSPE-PEG2k as an encapsulation matrix (nanoprecipitation method12) (Figure 1C). Dissolve HLY1 in THF (1 mL) and slowly add into a beaker containing DSPE-PEG2k aqueous solution (9 mL) with sonication at 25 &#176;C. Subsequently, remove THF from the mixture by dialysis20.</li>
	<li>Concentrate the above solution centrifugally with ultrafiltration18 (7100 x g for 10 min) and then place it in a 4 &#176;C refrigerator for future use. This can be stored for up to 1 month. 
	NOTE: The nanoprobe aqueous solution loaded by DSPE-PEG2k should be stored above 0 &#176;C and used as soon as possible.</li>
</ol>
4. Construction of tumor-bearing mice
<ol>
	<li>Culture 4T1 mouse breast cancer cells (4T1) in Dulbecco&#39;s Modified Eagle Medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (see Table of Materials), and maintain in a humidified incubator with 5% CO2 at 37 &#176;C.</li>
	<li>For the NIR-II fluorescent imaging experiment, culture 4T1 cells (5 x 107) for 24 h, digest with trypsin (1 mL), and wash twice with serum-free DMEM (4 mL).</li>
	<li>Anesthetize the mice by treating with isoflurane (2%). Confirm adequate anesthetization by stimulating the toes or the soles of the feet of the mice, and observe whether the mice respond. If there is no response, it means that the anesthesia is sufficient32.</li>
	<li>Then, using an insulin injection needle, inject the 4T1 cell mixture into the mice through subcutaneous injection (100 &#181;L). 
	&#8203;NOTE: NIR-II imaging studies were performed ~2 weeks after inoculation, when the tumor had grown to a volume of ~100 mm3. Before NIR-II tumor imaging, please confirm the tumor size. The tumor size was estimated by an electronic vernier caliper for the present study11.</li>
</ol>
5. In vivo NIR-II fluorescence imaging
<ol>
	<li>Anesthetize the mice by treating with isoflurane (2%) and perform NIR-II imaging of the whole body of the mice using an optical NIR-II imaging system (see Table of Materials). 
	NOTE: Pay attention to the dosage of anesthetic to avoid mice death. Generally, anesthesia lasts for 5-10 min. Stimulate the toes or the soles of the feet of the mice, and observe whether the mice respond. If there is no response, it means that the anesthesia is sufficient.</li>
	<li>Take a solution of HLY1 dots (0.8 mg/mL, 200 &#181;L). Inject the HLY1 dots intravenously into the anesthetized mice, and 3 min later, perform NIR-II fluorescence imaging of the blood vessels of the whole body of mice using an NIR-II imaging system. Focus further on the mouse&#39;s head to collect brain vascular imaging. 
	NOTE: Use clean experimental gloves during imaging, which will help to obtain clean NIR-II images.</li>
	<li>Collect the images 5 min after the injection of HLY1 dots in mice, and process the data using ImageJ software. The instrument parameters of the optical NIR-II imaging system are 90 mW/cm2 (808 nm laser).</li>
	<li>On completion of the experiment, euthanize the animals following institutionally approved protocols. 
	NOTE: For the present study, the animals were euthanized by exposing them to excess isoflurane32.</li>
</ol>

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The authors have nothing to disclose.

NIR-I fluorescent imaging can be used to some extent for tumor and vascular imaging, but due to the limited maximum emission wavelength of NIR-I fluorophores (&#60;900 nm), it results in poor tissue penetration and tumor signal background ratio33,34. Poor and low imaging resolution may cause a deviation between the outcome of the imaging feedback treatment and the actual therapeutic effect. In addition, most NIR-I fluorophores have poor optical stability and extremely fast metabolism, resulting in instability in the imaging process. Because of the low tissue penetration and instability of NIR-I fluorophores, the application in tumor and vascular imaging is greatly limited35. Compared with NIR-I light, NIR-II fluorescence imaging has the advantages of significantly reduced photon scattering and absorption, lower tissue auto-fluorescence, stronger body tissue penetration, and better imaging spacetime resolution36.
This article describes a bright AIE dye based on a D-A-D skeleton, which has excellent stability. An effective nanoprecipitation method was utilized to prepare a nanoprobe for multi-purpose bioimaging, including vascular diseases and tumor imaging. The high quantum yield in the aqueous solution is due to the luminescent properties induced by aggregation, which can achieve high-definition NIR-II imaging with low dose and high biosecurity. The brightness of the NIR-II probe and the water solubility determine the quality of the imaging. Additionally, when injecting a probe into a mouse, it is necessary to avoid leaking the probe into the mouse&#39;s tail, which affects the accuracy of the imaging results. The current method of administration is only limited to intravenous injection, and cannot use multiple injection methods, which is a limitation of the current method. In addition, the NIR-II nanoprobe of this method can only be accumulated to the target by passive targeting, and cannot identify specific targets by active targeting.
In the process of implementing NIR-II imaging, the operation of the NIR-II device is also important for the acquisition of images. To obtain high-resolution vascular imaging, the InGaAs camera needs to be focused on the mouse and positioned close to the mouse, making it easy to observe the tiny blood vessels. For tumor imaging, probes need to be effectively accumulated into the tumor, and NIR-II fluorescence should be emitted by the probes accumulated in the tumor, effectively distinguishing the boundary between the tumor and the surrounding tissue. Because of the high sensitivity of NIR-II fluorescence imaging, images can be observed dynamically during imaging, which is lacking in many other imaging techniques.
In this study, the preparation of a fluorescent probe is introduced. At the same time, high-resolution vascular and tumor imaging is realized by an NIR-II fluorescent nanoprobe, which provides an accurate and effective method for the detection of vascular diseases and cancer.

Fluorescence imaging is the commonly used molecular imaging tool in basic research, and is also often used to guide surgical tumor resection in clinics1. The essential principle of fluorescence imaging is to employ a camera to receive fluorescence emitted by a laser after the irradiation of samples (tissues, organs, etc.)2. The process is completed within a few milliseconds3. The fluorescence imaging wavelengths can be divided into ultraviolet (200-400 nm), visible region (400-700 nm), near-infrared I (NIR-I, 700-900 nm), and near-infrared II (NIR-II, 1000-1700 nm)4,5,6. Because the endogenous molecules such as hemoglobin, melanin, deoxyhemoglobin, and bilirubin in biological tissues have strong absorption and a scattering effect on the light in visible regions, the penetration and sensitivity of light are greatly reduced, and the fluorescence imaging in visible light wavelengths is adversely affected7,8,9.
NIR-II fluorescence imaging has low photon absorption and scattering, high imaging speed, and high image contrast (or sensitivity)10,11. As the fluorescence wavelength increases, the absorption and scattering of fluorescence in biological tissues decrease gradually, and the auto-fluorescence in the NIR-II region is extremely low12. Thus, the NIR-II window significantly increases the penetration depth of tissues and obtains a higher resolution and signal-to-noise ratio13,14,15. The NIR-II window can be further subdivided into the NIR-IIa (1300-1400 nm) and NIR-lIb (1500-1700 nm) windows16. To date, several milestone NIR-II materials have been reported, including inorganic material single-walled carbon nanotubes, rare earth nanoparticles, quantum dots, and organic material semiconductor polymer nanoparticles, small-molecule dyes, aggregation-induced luminescent materials, etc.1,17,18,19,20,21,22. Inorganic nanomaterials are easily accumulated in the liver, spleen, etc., and have potential long-term biotoxicity23. Organic small-molecule fluorophore has the advantages of rapid metabolism, low toxicity, easy modification, and a clear structure, which is the most promising probe for clinical use24.
The NIR-II optics imaging system is also a critical component of fluorescence bioimaging because it can efficaciously collect NIR-II fluorescence signals from the NIR-II probe, thus rendering precise functional, anatomical, and molecular images25,26. The NIR-II imaging system mainly comprises shortwave infrared cameras, long-pass (LP) filters, lasers, and computer processors. In vivo NIR-II fluorescent imaging is considered one of the most feasible imaging approaches for elucidating the mechanisms of diseases and the nature of life27,28,29. NIR-II imaging technology has been widely used in biomedical fields such as cancer cell detection, dynamic imaging, in vivo targeted tracing, and targeted therapy, especially in oncology research30,31. However, considering the high technical requirements of NIR-II imaging technology on imaging probes and instruments, it also puzzles and restricts the practical use of researchers in different fields. Therefore, the preparation of NIR-II imaging probes and the applications of NIR-II imaging are introduced in detail in this article.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anhydrous pyridine</td>
			<td>Perimed&#160;</td>
			<td>110-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous sodium sulfate</td>
			<td>China national medicines Co.,Ltd</td>
			<td>SY006376</td>
			<td></td>
		</tr>
		<tr>
			<td>Black cardboard</td>
			<td>Suzhou Yingrui Optical Technology Co., Ltd</td>
			<td>AO00158</td>
			<td></td>
		</tr>
		<tr>
			<td>Column chromatography</td>
			<td>Energy Chemical</td>
			<td>E080498</td>
			<td></td>
		</tr>
		<tr>
			<td>Diphenylphosphine palladium dichloride</td>
			<td>Sigma-Aldrich</td>
			<td>B2161-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>DSPE-PEG2000</td>
			<td>Ponsure</td>
			<td>PS-E1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium&#160;</td>
			<td>Gibco</td>
			<td>8121587</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Biofroxx</td>
			<td>EZ6789D115</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>2166090RP</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>GLPBIO</td>
			<td>GC45487-1</td>
			<td></td>
		</tr>
		<tr>
			<td>K2CO3</td>
			<td>Macklin</td>
			<td>P816305-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>N. N &#39;- dimethylformamide</td>
			<td>China national medicines Co.,Ltd</td>
			<td>02-12-1968</td>
			<td></td>
		</tr>
		<tr>
			<td>NIR-II imaging instrument</td>
			<td>Suzhou Yingrui Optical Technology Co., Ltd</td>
			<td>16011109</td>
			<td></td>
		</tr>
		<tr>
			<td>N-sulfenanilide</td>
			<td>Enerry chemical&#160;</td>
			<td>1250030-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>PdCl2(dppf)2CH2Cl2</td>
			<td>TCI&#160;</td>
			<td>B2064-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran</td>
			<td>China national medicines Co.,Ltd</td>
			<td>M005197</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetratriphenylphosphine palladium</td>
			<td>Immochem</td>
			<td>1021232-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetratriphenylphosphine palladium</td>
			<td>Sigma-Aldrich</td>
			<td>1021232-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Tributyltin chloride</td>
			<td>Immochem</td>
			<td>QH004335</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimethylchlorosilane</td>
			<td>China national medicines Co.,Ltd</td>
			<td>40060560</td>
			<td></td>
		</tr>
	</tbody>
</table>

a bright nir-ii fluorescence probe for vascular and tumor imaging

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing to its high sensitivity, deep tissue penetration, and superior imaging with spatial and temporal resolution. However, the method to facilitate the implementation of NIR-II fluorescence imaging for some urgently needed fields, such as medical science and pharmacy, has puzzled relevant researchers. This protocol describes in detail the construction and bioimaging applications of a NIR-II fluorescence molecular probe, HLY1, with a D-A-D (donor-acceptor-donor) skeleton. HLY1 showed good optical properties and biocompatibility. Furthermore, NIR-II vascular and tumor imaging in mice was performed using a NIR-II optics imaging device. Real-time high-resolution NIR-II fluorescence images were acquired to guide the detection of tumors and vascular diseases. From probe preparation to data acquisition, the imaging quality is greatly improved, and the authenticity of the NIR-II molecular probes for data recording in intravital imaging is ensured.

The fluorescent intensity and brightness of water-suspensible HLY1 dots were determined by an NIR-II imaging instrument. The fluorescent intensity of HLY1 in the 90% fwTHF/H2O mixture was five times that in the THF solution, which indicated a prominent AIE feature of HLY1 (Figure 1B). Moreover, HLY1 dots emitted strong fluorescent signals under a 1,500 nm LP filter, showing that HLY1 dots can be used for NIR-IIb imaging (Figure 1D). The maximum absorption and maximum emission wavelength of HLY1 dots were 740 nm and 1,040 nm, respectively (Figure 2A). Moreover, the hydrodynamic size of HLY1 dots was determined to be 145 nm by dynamic light scattering (DLS) (Figure 2B). HLY1 dots (0.2 mL, 0.8 mg/mL) were administered into normal Balb/c mice via tail vein injection for vascular imaging (Supplementary Figure 1). The micro-vessels in the hindlimb were identified clearly under a 1,500 nm LP filter (Figure 3B). In addition, the cerebral vessels were also clearly identified under a 1,500 nm LP filter (Figure 3A). The NIR-II imaging performance of the HLY1 dots in 4T1 tumor-bearing mice was also evaluated through the NIR-II imaging system. HLY1 dots (0.2 mL, 0.8 mg/mL) were intravenously injected into 4T1 mice through the tail vein. The 4T1 tumor of the tumor-bearing mice was clearly visible by NIR-II imaging (Figure 3C), indicating the EPR effect of HLY1 dots. All these results suggest that HLY1 dots are a bright NIR-II fluorescence probe, which is applicable for vascular and tumor imaging.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64875/64875fig01.jpg" /> 
Figure 1: Synthesis of dye molecules and preparation of water-suspensible probes. (A) The synthetic path of HLY1&#160;(a: Pd(dppf)Cl2 CH2Cl2, K2CO3, 75 &#176;C). (B) The NIR-II images of HLY1 in THF and 90% fw THF/H2O (1,000 nm LP, 2 ms). (C) A schematic diagram of the preparation of HLY1 dots. (D) The NIR-IIb fluorescent intensity of HLY1 dots in aqueous solution (1,500 nm LP, 200 ms). <a href="https://www.jove.com/files/ftp_upload/64875/64875fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64875/64875fig02.jpg" /> 
Figure 2: The optical properties and hydrodynamic size of HLY1 dots. (A) The absorption and emission spectra of HLY1 dots in aqueous solution. (B) The DLS of HLY1 dots. <a href="https://www.jove.com/files/ftp_upload/64875/64875fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64875/64875fig03.jpg" /> 
Figure 3: NIR-II fluorescence imaging using HLY1 dots. (A) Brain vascular imaging in mice (1,500 nm LP, 300 ms exposure time). Scale bar: 2 cm. (B) Whole body vascular imaging in mice (1,500 nm LP, 300 ms). (C) 4T1 tumor imaging (1,250 nm LP, 30 ms). Scale bar: 1 cm. <a href="https://www.jove.com/files/ftp_upload/64875/64875fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1:&#160;NIR-II imaging setup. (A) Schematic diagram of injection&#160;of&#160;HLY1 dots into mice. (B) The photograph of the NIR-II imaging device. <a href="https://www.jove.com/files/ftp_upload/64875/Supplementary Figure 1.png" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging at JoVE.com

A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging

The present protocol describes a detailed, real-time NIR-II fluorescence imaging operation of a mouse using a NIR-II optics imaging device.

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing ...

a-bright-nir-ii-fluorescence-probe-for-vascular-and-tumor-imaging

Universidad San Sebastian

Research

JoVE Journal

Medicine

1.6K Views.  Tibet University. The present protocol describes a detailed, real-time NIR-II fluorescence imaging operation of a mouse using a NIR-II optics imaging device.

Video: A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...