KEYWORDS: Lymphatic system, Luminescence, Brain, Imaging systems, In vivo imaging, Near infrared, Medicine, Health sciences, Indocyanine green, Fluid dynamics
Cerebrospinal fluid (CSF) is known to be reabsorbed by the lymphatic vessels and drain into the lymph nodes (LNs) through peripheral lymphatic vessels. In the peripheral lymphatics, the contractile pumping action of lymphangions mediates lymph drainage; yet it is unknown whether lymphatic vessels draining cranial and spinal CSF show similar function. Herein, we used non-invasive near-infrared fluorescence imaging (NIRFI) to image (i) indocyanine green (ICG) distribution along the neuraxis and (ii) routes of ICG-laden CSF outflow into the lymphatics following intrathecal lumbar administration. For intrathecal injection, a 31-gauge needle was inserted between L5 and L6 vertebrae and a tail flick response was referenced as indication of correct position of the needle into the intradural space. Imaging agents were injected immediately after a tail flick. A volume of 10 to 30μl of 645μΜ of ICG was injected. We demonstrate lymphatic contractile function in peripheral lymphatics draining from the nasal lymphatics to the mandibular LNs. In addition, we observed afferent sciatic lymphatic vessels, which also show contractile activity and transport spinal CSF into the sciatic LNs. NIRFI could be used as a tool to probe CSF pathology including neurological disorders by imaging CSF outflow dynamics to lymphatics.
Cardiovascular diseases remain the greatest cause of death in the US and gene therapy has the potential to be an effective
therapy. In this study, we demonstrated MMP-9 based protease-activatable virus (PAV) for selective infection of
myocardial infarct (MI) that is associated with active MMP-9 expression. To test the specificity of PAV, we used
expression of a far-red fluorescence protein (iRFP) delivered by the PAV together with a dual PET/NIRF imaging agent
specific for active MMP-9 activity at the site of MI in a murine model. Calibrated fluorescence imaging employed a
highly-sensitive intensified camera, laser diode excitation sources, and filtration schemes based upon the spectra of iRFP
and the NIRF agent. One to two days after ligation of the left anterior descending artery, the PAV or WT AAV9 virus
encoding for iRFP (5x1010 genomic particles) and radiolabeled MMP-9 imaging agent (3 nmol) were injected
intravenously (i.v.). PET imaging showed MMP activity was associated with adverse tissue remodeling at the site of the
MI. One week after, animals were again injected i.v. with the MMP-9 agent (3 nmol) and 18-24 h later, the animals
were euthanized and the hearts were harvested, sliced, and imaged for congruent iRFP transgene expression and NIRF
signals associated with MMP-9 tissue activity. The fluorescent margins of iRFP and NIRF contrasted tissues were
quantified in terms Standard International units of mW/cm2/sr. The sensitivity, specificity, and accuracy of PAV and
WT targeting to sites of MI was determined from these calibrated fluorescence measurements. The PAV demonstrated
significantly higher delivery performance than that of the WT AAV9 virus.
The lymphatic system plays an important role in maintaining the fluid homeostasis between the blood vascular and interstitial tissue compartment and there is recent evidence that its transport capabilities may regulate blood pressure in salt-induced hypertension. Yet, there is little known how the lymphatic contractile function and architecture responds to dietary salt-intake. Thus, we longitudinally characterized lymphatic contractile function and vessel remodeling noninvasively using dynamic near-infrared fluorescence imaging in animal models of salt-induced hypertension. The lymphatics of mice and rats were imaged following intradermal injection of indocyanine green to the ear tip or the base of the tail before and during two weeks of either a high salt diet (HSD) or normal chow. Our noninvasive imaging data demonstrated dilated lymphatic vessels in the skin of mice and rats on a HSD as compared to their baseline levels. In addition, our dynamic imaging results showed increased lymphatic contraction frequency in HSD-fed mice and rats. Lymphatic contractile function and vessel remodeling occurs in response to salt-induced hypertension suggesting a possible role for the lymphatics in the regulation of vascular blood pressure.
The lymphatic system plays an important role in cancer cell dissemination; however whether lymphatic drainage
pathways and function change during tumor progression and metastasis remains to be elucidated. In this report, we
employed a non-invasive, dynamic near-infrared (NIR) fluorescence imaging technique for functional lymphatic
imaging. Indocyanine green (ICG) was intradermally injected into tumor-free mice and mice bearing C6/LacZ rat
glioma tumors in the tail or hindlimb. Our imaging data showed abnormal lymphatic drainage pathways and
reduction/loss of lymphatic contractile function in mice with lymph node (LN) metastasis, indicating that cancer
metastasis to the draining LNs is accompanied by transient changes of the lymphatic architectural network and its
function. Therefore, functional lymphatic imaging may provide a role in the clinical staging of cancer.
Targeted fluorescent molecular imaging probes may provide an optimal means of detecting disease. Stable, organic fluorophores can be repeatedly excited in vivo by propagated light and consequentially can provide large signal-to-noise ratios (SNRs) for image detection of target tissues. In the literature, many small animal imaging studies are performed with a red excitable dye, Cy5.5, conjugated to the targeting component. We report the comparison of the in vivo fluorescent imaging performance of a near-IR (NIR) and a red-excitable dye. Epidermal growth factor (EGF) was conjugated with Cy5.5 [excitation/emission (ex/em), 660/710 nm] or IRDye® 800CW (ex/em: 785/830 nm) for imaging EGF receptor (EGFr) positive (MDA-MB-468) and/or negative (MDA-MB-435) human breast cancer cell lines in subcutaneous xenograft models. The conjugates were injected intravenously at 1-nmol-dye equivalent with and without anti-EGFr monoclonal antibody C225, preadministered 24 h prior as a competitive ligand to EGFr. Our images show that while both agents target EGFr, the EGF-IRDye® 800CW evidenced a significantly reduced background and enhanced the tumor-to-background ratio (TBR) compared to the EGF-Cy5.5. Immunohistochemistry shows that EGF causes activation of the EGFr signaling pathway, suggesting that prior to use as a targeting, diagnostic agent, potential deleterious effects should be considered.
Dynamic multi-wavelength fluorescence imaging was accomplished using a liquid crystal tunable filter (LCTF).
Since several different emission wavelengths can be selected by tuning the LCTF, two wavelength dynamic
fluorescence imaging was conducted in mice bearing human melanoma M21 and M21L after injection of a mixture
of (i) RGD peptide conjugated with a near-infrared (NIR) dye that targeted integrin &agr;v&bgr;3 and (ii) non-specific dye,
Cy5.5. Dynamic multi-wavelength imaging with LCTF can differentiate the uptake of the two different fluorescent
contrast agents between tumor and normal tissue ROIs in the M21 and M21L xenograft models. Although the LCTF
attenuated fluorescence signals by a factor of two when compared to holographic and bandpass filter sets used
previously, Tumor to background ratio (TBR) from NIR fluorescence images with a bandpass and holographic filter
were not statistically different from those acquired with the LCTF. Therefore, the benefit of spectral information as
well as dynamic multi-wavelength may outweigh the impact of the lower transmission efficiencies, and could enable
in vivo small animal imaging.
The dose dependence of near-infrared (NIR) fluorescent labeled RGD peptide targeted to the αvβ3 integrin was assessed from xenografts bearing a subcutaneous human Kaposi’s sarcoma (KS1767) with dynamic NIR fluorescence optical imaging. The three-compartment pharmacokinetic (PK) model was used to determine PK parameters from fluorescence images acquired with an intensified charge-coupled device (ICCD) system. Dynamic imaging of Kaposi’s sarcoma bearing animals was conducted with i.v. administration of Cy5.5-c(KRGDf) at doses of 0.75 to 6 nmol/animal and at the doses of 300 or 600 nmol of c(KRGDf) administered 1 hour before the injection of 3 nmol dose of the conjugate. The results show early and rapid uptake of Cy5.5-c(KRGDf), which was mediated by the administration of c(KRGDf) 1 hour before administration at the conjugate agent. From the results we found a linear increase in PK uptake rates at doses of 0.75 to 1.5 nmol, reflecting unsaturated binding to the integrin receptor. However, the results show the dose independence at large dose amounts from 3 to 6 nmol per animal. The effects of cancer treatments as well as diagnostics may be evaluated by in vivo PK analysis with NIR fluorescence optical imaging.
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