Imaging demyelination and remyelination in multiple sclerosis and spinal cord injury.
Myelin sheath is an extended plasma membrane wrapping around an axon and is crucial for high-speed impulse conduction. Demyelination, the loss of normal myelin sheath, accounts for long-term neurologic disability. As a primary inventor of coherent anti-Stokes Raman scattering (CARS) microscopy that permits high-speed vibrational imaging of molecules (J Phys Chem, Feature Article 04), Cheng and his Purdue group first applied this technique to visualize intact myelin sheath under physiological conditions (Biophys J 05) and in vivo (J Microscopy 07). CARS microscopy permits high-speed vibrational imaging due to coherent addition of the scattering signal and spectral focusing on single Raman bands. The myelin membranes contain about 70% lipid by weight, and the high-density CH2 groups produce a large CARS signal. The 3D submicron resolution of CARS microscopy enables real time imaging of detailed myelin structure around a Node of Ranvier at a speed of a frame per second. Such capability has allowed us to examine enzymatic and cellular activities involved in demyelination.
Using the resonant CARS signal from symmetric CH2 stretch vibration, we have characterized lysolecithin-induced myelin degradation. In this most widely used experimental demyelination model, we show that the breakdown of myelin membrane was featured by decrease of CARS intensity and loss of dependence excitation polarization. By coupling CARS imaging with compound action potential recording, we have identified a Ca2+ dependent pathway which involves the activation of cPLA2 and calpain for lysolecithin-induced myelin degradation (J Neurosci 07), reported by National Science Foundation. With the demonstrated capabilities, we have recently evaluated the effect of glutamate excitotoxicity on myelin. Our data show that exposure of white matter to glutamate leads to paranodal myelin splitting and retraction (PLoS ONE 2009). Since elevated glutamate level was found in cerebral spinal fluid of multiple sclerosis patients, our study shows that paranodal myelin degradation is a possible onset of demyelination in multiple sclerosis.
Towards the goal of elucidating the initiation and progression of multiple sclerosis, we are now investigating experimental autoimmune encephalomyelitis (EAE) lesions in SJL/J mice via collaboration with Professor Stephen Miller in Northwest University Medical School. EAE is the most frequently studied animal model of multiple sclerosis. In this model, rodents that are immunized with myelin-derived peptides develop a paralytic disease with similarities to the human condition. Our ex vivo CARS images of spinal cord white matter extracted from EAE mice are able to show demyelinating lesions as small as 10 mm in diameter. Confocal Raman analysis of the normal and degraded myelins has revealed significant changes of lipids (unpublished data).
Using Long-Evans Rats with survival surgery, we are developing in vivo CARS imaging of spinal cord white matter on an upright microscope. This technique enables us to follow the progression of white matter injury or disease in the same animal, so that causalities of different events including T cell infiltration, microglial cell activation, and demyelination can be clarified. Using cultured brain slices, we are developing a lysolecithin-induced demyelination and remyelination model for high throughput screening of agents that promote or inhibit remyelination.
Chasing lipid bodies in health and disease.
Obesity is an established risk factor for type II diabetes, hypertension, strokes, many types of cancer, atherosclerosis, and other diseases. A central goal of our lipid metabolism study is to understand how cells store excess energy in the form of cytoplasmic lipid droplets (LDs) and how excess lipid affects cancer incidence and progression (BMC Cancer 2009). Until recently the studies of LDs have been relying on non-specific, invasive, or population measurements. Traditionally, intracellular LDs are visualized based on the fluorescence of lipophilic dyes such as Oil red O (ORO) or Nile red. Nonetheless, the fluorescence signals from ORO or Nile red contain no information regarding lipid composition or organization. Recent advances in vibrational imaging are opening up exciting opportunities for dynamic, non-invasive, and compositional analysis of single LDs.
Our lab has made two significant advances in understanding the formation of LDs. In collaboration with Dr. Kimberly Buhman’s lab at Purdue University, we observed for the first time a highly dynamic triacylclycerol (TG) pool in mouse enterocytes by ex vivo and in vivo CARS imaging of small intestine during dietary fat absorption (J Lipid Res 2009). The finding of such a TG pool highlights new possibilities for regulation of the efficiency and/or rate of dietary fat absorption by controlling the rate of hydrolysis by TG lipases or the transport rate of TG hydrolyzed products in enterocytes. In another study published in PLoS One (2009), we revealed the source of heterogeneity in fat storage in 3T3-L1 cells. Researchers have been using this cell line to study the molecular control of adipogenesis for the past 35 years. Tremendous cell-to-cell variability is observed in the amount of fat stored in cells despite the fact that all cells have the same genes. However, no one knows the cause for such heterogeneity. We find that variability in fat storage is dependent on how cells process insulin, a hormone normally secreted by the pancreas after meals to trigger the uptake of glucose in blood into liver, muscle, or fat cells. This mechanistic insight from our study will assist in the understanding of the roles of insulin in obesity or type II diabetes, and the design of effective intervention strategies.
In order to probe the composition of LDs, our lab has recently developed a compound Raman microscope capable of both high-speed chemical imaging and quantitative spectral analysis on the same platform (J Phys Chem 2009). We use a picosecond laser source to perform coherent Raman scattering imaging of a biological sample and confocal Raman spectral analysis at points of interest. We are currently employing this tool to study the function of specific genes in lipid metabolism in C. elegans.
Imaging Cellular Activities in Tissue Microenvironment by Multimodal Nonlinear Optical Microscopy
Since the invention of first optical microscope in 17th century, biological science has been largely advanced via a reductionist approach. However, knowledge from individual cells cultured in a dish may not represent their behavior in vivo. Understanding the interactions between cells, extracellular matrix, and stromal molecules in a tissue environment is an emerging frontier of biology. Tissues are usually studied by histology and electron microscopy, in which fixation and staining inhibit real-time monitoring of cellular processes. NLO microscopy opened up a new window for imaging live tissues with inherent 3D submicron resolution, relatively large penetration depth, and real-time data acquisition. Major NLO imaging modalities include two-photon excited fluorescence (TPEF), second harmonic generation (SHG) or sum-frequency generation (SFG), and coherent anti-Stokes Raman scattering (CARS). In a CARS process, the interaction of a pump field Ep(ωp) and a Stokes field Es(ωs) with a sample generates an anti-Stokes field Eas at frequency 2ωp-ωs. A unique advantage of CARS is that TPEF and SFG/SHG modalities can be naturally incorporated into the same platform, enabling real-time visualization of various components in a tissue.
Our lab pioneers the development of multimodal NLO microscopy and its application to tissue biology. Our first version implements CARS/SHG/TPEF imaging of atherosclerotic lesions (J Biomed Opt 2007) using a combination of ps and fs lasers. Our second version implements CARS/SFG/TPEF imaging of the central nervous system (Biophys J 2007) and arteries (Opt Comm 2008) using a picosecond laser source. In the third version we have demonstrated multimodal NLO microscopy using a fs laser source and imaging of fresh liver tissues (Opt Express 2009). Multimodal imaging has enabled use to evaluate the impact of obesity on mammary tumor stroma composition (Mol Imaging 2007) and single cell profiling (PLoS ONE 2009).
In Vivo Detection of Circulating Tumor Cells
CTCs represent a potential alternative to invasive biopsies of tumor tissue for detection, characterization and monitoring of non-haematologic cancers. Although various in vitro assays have been developed, these methods are not sufficient for detection of rare CTCs in early stage cancer due to the limited sample volume. However, in vivo detection of single CTCs in the whole blood is far beyond the sensitivity of current medical imaging technologies such as MRI.
Our lab has recently demonstrated the proof-of-concept of intravital flow cytometry that noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature (PNAS 2007). The method involved i.v. injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs on a laser-scanning microscope. Folate dye conjugates were chosen because many human carcinomas over-express the folate receptor and because the folate conjugate can be quickly removed from circulation if uncaptured by CTCs. Our intravital flow cytometry studies in mice with metastatic tumors demonstrate that CTCs can be quantitated weeks before metastatic disease is detected by other means such as histology.
Via collaboration with Philip Low’s group (providing tumor-targeting ligands) in Department of Chemistry, Debbie Knapp’s group (providing dogs with bladder cancers) in School of Veterinary Medicine, and Dr. Wael Harb in Horizon Oncology Center, we are developing a catheter-based fiber-optic probe for intravascular detection of CTCs in large animals and eventually in human patients. Meanwhile, our lab has demonstrated microfluidic CARS flow cytometry for in vitro quantitation of adipocytes (Opt. Express 2008). Development of in vivo CARS flow cytometry is under progress in our lab with the goal of achieving label-free detection of CTCs based on the CARS signal from intracellular lipid bodies.
Gold Nanorod as a Theranostic Agent
Gold nanorods produce strong optical responses as a result of plasmon resonance. Being different from nanoparticles, gold nanorods have a transverse and a longitudinal mode, with resonance wavelengths in the visible and near IR regions, respectively. Our lab demonstrated a bright two-photon luminescence (TPL) from CTAB-coated nanorods prepared by a seed growth method (PNAS 2005). The TPL was found to arise from enhanced two-photon absorption by the longitudinal plasmon resonance. Compared to quantum dots, gold nanorods are less susceptible to photobleaching and are chemically inert under physiological conditions. These properties make gold nanorods an appealing imaging agent for in vivo applications. We are investigating second harmonic generation, third harmonic generation, and coherent anti-Stokes scattering signals from nanorods and their applications to bioimaging.
A unique advantage of gold nanorods is the ease of surface modification. Bio-conjugation allowed us to control the cellular uptake efficiency of gold nanorods (Langmuir 2007) and to target tumor cells over-expressing folate receptors (Ad Mater 2007). Recently we have shown that nanorods conjugated with arginine-rich peptides can be effectively internalized by activated macrophages in live animals (Nanomedicine 2009). By exchanging the CTAB on the nanorod surface with polyethylene glycol (PEG) of various molecular weights, we have achieved a circulation half-life time of 5.0 h. Delivery of homing peptide conjugated and PEG-coated nanorods to macrophages in atherosclerotic plaques in ApoE-/- mice is under investigation.
Importantly, gold nanorods are also efficient converters of light energy into heat. We have applied folate-conjugated nanorods to optical hyperthermia of malignant KB cells (Adv Mater 2007). Moreover, we have revealed that gold nanorods mediate acute membrane blebbing and subsequent cell death through compromising the membrane integrity and influx of extracellular Ca2+. These studies pave the way to in vivo applications of nanorods for photothermolysis of cancer cells and macrophages in inflammatory diseases.
Polymer Micelle based Nanomedicine for Spinal Cord Injury Repair
Polymeric micelles are spherical assemblies of di-block copolymers, containing a hydrophilic PEG shell and a hydrophobic inner core. These micelles have unique properties such as biocompatibility and long blood circulation, and have been widely used as carriers of water insoluble drugs. By recognizing the high-density coronal PEG in a polymeric micelle, we realized the potential of using micelles to fuse membrane breaches created in spinal cord injury. As a proof of principle we have prepared and applied monomethoxy poly(ethylene glycol)-poly(D,L-lactide acid) (mPEG-PDLLA) micelles to compression-injured ventral white matter isolated from adult guinea pigs. With this approach we could achieve a rapid, significant restoration of impulse conduction and a simultaneous reduction of Ca2+ entry into axons in injured spinal cords. With a grant from Indiana State of Health, the in vivo therapeutic potential of polymeric micelles is under evaluation using a rat spinal cord injury model.
To evaluate the potential of using the micelles to deliver anti-inflammatory drug to suppress the secondary injury, we have revisited the conventional wisdom that polymeric micelles systemically carry drug molecules until they are taken up into cells followed by intracellular release. We employed Forster resonant energy transfer (FRET) imaging to monitor in real time the release of hydrophobic molecules from circulating poly(ethylene glycol)-poly(D,L-Lactic acid) micelles. A lipophilic FRET pair, DiIC18 and DiOC18, was physically entrapped into the micelle core by mimicking the loading of hydrophobic drugs. By monitoring the hetero- and homo-FRET efficiency, release of core-loaded probes to model membrane and cell membrane was demonstrated, indicating a membrane mediated pathway for cellular uptake of hydrophobic molecules pre-loaded in polymeric micelles (PNAS 2008). Furthermore, intravital imaging of blood stream showed that FRET efficiency significantly reduced within 15 min after intravenous injection of FRET micelles into BALB/c mice, implying quick escape of the probes from circulating micelles (Langmuir 2008). These studies have prompted us to make shell-crosslinked micelles to ensure systemic delivery of loaded drug to the injured site.