Monitoring Lipids Uptake in Neurodegenerative Disorders By SECARS Microscopy

In this study, we investigated a novel application of Surface-Enhanced Coherent anti-Stokes Raman Scattering (SECARS) microscopy for imaging intravenously injected ultra small paramagnetic iron oxide nanoparticles (USPION) in Amyotrophic Lateral Sclerosis (ALS) experimental model. Experiments were performed on transgenic rat model, expressing multiple copies of mutant (G93A) human SOD-1 gene (hSOD-1G93A), treated with antibodies against CD4+ T cells magnetically labelled with USPION. Marked intensity enhancements have been observed in specific pathological regions of the ALS brain as compared to the wild-type model. The results obtained correlated SECARS enhancements to selective association of lipids to up-taken USPION which shows high accumulation in the brainstem and midbrain region. The work presented shows the promising potential of SECARS microscopy in investigating neurodegenerative disorders in experimental systems using USPION as molecular nanoprobes. [DOI: 10.1380/ejssnt.2010.362]


I. INTRODUCTION
Nanoparticles have drawn a large degree of interest as targeted imaging and therapeutic agents [1,2].Over the past few decades, biodegradable nanoparticles have created considerable interest as effective drug carrier devices.Nanomedicine specifically employs nanomaterials for invivo imaging, diagnostics and therapeutics, to gain better understanding of the origin of diseases on the nanometer scale.Recently, a novel application method using Surface Enhanced Coherent anti-Stokes Raman Scattering (SECARS) microscopy is taking a large step towards becoming a medical diagnostic tool.The potential of this techniqe is highlighted by its ability to chemically and selectively detect aggregations or accumulations of nanoparticles in living cells.Apart from its chemical selectivity, the main strength of the technique lies in the fact that the sample or the nanoparticles do not have to be treated (e.g.labeled with a fluorescent marker), but are imaged in their native, unaltered state.This is important, since labeling might change the behavior of the (metallic/organic, soluble/insoluble, functionalized etc.) nanoparticles in the cells, and thus influence the outcome of investigations on the fate of the particles.The ultra small paramagnetic iron oxide nanoparticles (US-PION) a potent new class of magnetic resonance imaging (MRI) contrast agents have recently drawn much attention for its wide diagnostic and potential therapeutic applications, specifically in the central nervous system (CNS).Recent findings reported USPION used to monitor the inflammatory responses in various CNS diseases including cerebral ischemia, multiple sclerosis and acute disseminated encephalomyelitis through evaluating areas of blood-brain barrier (BBB) dysfunction related to tumors and other neuroinflammatory pathologies [3].There have been many attempts to find the best imaging modality and to develop in vivo diagnostic techniques to detect the histopathological hallmarks of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's and Parkinson's diseases.ALS, also called Lou Gehrig's disease, is a progressive fatal neurological disease affecting upper and lower motoneurons.The neuropathology of ALS is mostly confined to motor neurons in the cerebral cortex, some motor nuclei of the brainstem, and anterior horns of the spinal cord.The important discovery in the study of the disease was the identification of mutations in the copper/zinc superoxide dismutase (SOD1) gene in some families with hereditary ALS [4].To date, more than 100 mutations scattered throughout the SOD1 protein have been identified and it has been established that mutant SOD1 causes ALS through a gain-of-function mechanisms [5].Many hypotheses have been proposed of how mutant SOD1 could cause neurodegeneration, including aberrant redox chemistry, mitochondrial damage, excitotoxicity, microglial activation and inflammation, as well as SOD1 aggregation [6].Recent studies have reported significant elevated levels of ceramides, cholesterol esters and several other lipids in the spinal cords of people with ALS.To test whether these elevated levels of ceramides, a cell wall component, and cholesterol esters, a form of cholesterol, cause motor neuron degeneration associated with ALS, several investigations were carried on mice baring multiple copies of a mutated human gene for SOD1 [7].As in humans, analysis of the spinal cords of these animals revealed increased levels of ceramides and cholesterol esters.What is unclear is whether these elevated levels are the result of cell death processes or are themselves directly contributing to it.Our recent studies on the ALS rat model, using clinical magnetic resonance imaging (MRI), revealed T 2 -weighted hyperintensities in the brainstem, rubrospinal tract and vagus motor nuclei with prominent lateral ventricle and cerebral aqueduct enlargements [8,9].Notably, with magnetically labeled antibodies (against CD4 receptor) MRI revealed infiltrations of helper T cells in the interbrain regions.In this work our main goal is to explore the potential of SECARS microscopy in investigating neurodegenerative disorders by monitoring more closely the distribution of USPION in the brain of the ALS animal model.

II. EXPERIMENTAL
The animal experiments were surveyed by the Committee for Animal Experimentation treated in accordance with the European Community Council Directive (Ref.Nr. 86/609/EEC) and the NIH Guidelines, with approval of the Ethical Committee of the Faculty of Biology University of Belgrade.The experiments were performed on Sprague-Dawley rats expressing mutated (G93A) human SOD-1 gene (Taconic Farms, NY), and wild type (WT-standard Sprague-Dawley rats).Details of the preparation of the ALS animal model is described elsewhere [8].Commercially available antibodies against CD4+ T cells, magnetically labeled with ultra small particles of iron oxide (USPIO; MACS R ⃝, Miltenyi Biotec) were i.p.or i.v.injected into rats (200 µ L of original solution in 1 mL of physiological saline).Rats were sacrificed 24 h after and brains were isolated and postfixed in 4% paraformaldehyde for 48 h at +4 • C. The brain tissues samples were extracted from three animal models; wide type rat model, USPION-treated ALS and untreated ALS rat model served as a control sample.Additional control experiments were performed on murine adipocytes, grown on gelatin coated glass-coverslips and exposed to 5 ng/mL of iron oxides nanoparticles (in total: 15 ng in 3 mL of fresh medium).After incubation for 24 h, adipocytes were fixed at room temperature with 4% paraformaldehyd for 30 min and stored in PBS until further preparation for microscopic measurements.SECARS experiments were performed using combination of two laser systems; NIR excitation of mode-locked Nd:YVO 4 (1064 nm,7 ps, 76 MHz) and Ti:sapphire laser (Coherent Mira HP, 3.5 W 3 ps, 700-1000 nm) for the Stokes beam.A tunable optical parametric oscillator (OPO) was used for the pump and probe that covers the vibrational frequency range (200-3600 cm −1 ).The beams were scanned over the sample using specially designed laser scanning microscope and focused by water immersion objective lens with 1.2 numerical apertures.Both beams have a power of several tens of milliwatts at the sample.The SECARS signal is collected in the backward direction using standard confocal microscope connected with a photomultiplier tube.FT-Raman spectra were measured using a Bruker RFS 100/S spectrometer (Bruker Optics Inc, Billerica, MA, USA).Spectra of all samples were measured at room temperature in a 180 • backscattering geometry in a microscope.The absorption measurements were performed using a Hitachi U-3410 Spectrophotometer.Microscopic Raman mapping images were recorded using an alpha 300R instrument WITec, Inc. (Ulm, Germany) equipped with a back illuminated deep-depletion CCD camera.The samples were irradiated by a He-Ne laser at 632.8 nm, coupled into a confocal Raman microscope through a wavelength specific single mode optical fiber, and focused by a 40×0.65 NA microscope objective lens.

III. RESULTS AND DISCUSSIONS
Ex vivo SECARS measurements on unstrained brain tissues taken from ALS transgenic rat model i.v.injected with USPION showed marked increase in signal enhancement in specific pathological regions particularly in the midbrain and the brainstem known to be infiltrated by helper T cells [8][9][10].The measurements were performed by tuning the pump beam in the wavelength range of 2840-3000 cm −1 and in the low frequency region (fingerprint region 1400-1800 cm −1 ).The prominent bands that show significant enhancement in the high frequency region were around 2845 and 2875 cm −1 (Figs.1(A) and  (B)).Significant enhancements were also observed in the fingerprint region around 1660-1675 cm −1 (Fig. 2).Experiments performed in the same tuning range on samples not treated with USPION and on brain tissues taken from the wild-type rat model, have shown no significant indication of enhancement around these bands.According to Raman assignments, the high frequency wavelength region between 2800 and 3100 cm −1 is dominated by valence vibrations of C-H 2 groups which are typical bands for fatty acids.Particularly the band around 2850 cm −1 is assigned to C-H 2 symmetric stretch, and from 2700 to 3500 cm −1 are correlated to cholesterol ester (cholesteryl palmitate), triacylglyceride (glyceryl palmitate), phosphatidic acid, and sphingomyelin.The region between 1000 and 1800 cm −1 is dominated by deformation vibration of the C-H 2 particularly 1130, 1299 and 1440, and around 1669 cm −1 which is correlated to the steroid ring of cholesterol.The resonance band observed around 1660 cm −1 is correlated to unsaturated fatty acids, a typical lipids band accumulated in the gray matter.The observed enhancement in the ALS brain can be correlated as well to accumulation of iron that might cause lipid peroxidation and degeneration in these regions.To investigate the metabolic effect of iron particles and the possibility of iron lipid binding activity, we performed several control experiments on adipocytes treated with iron oxides nanoparticles and extracted lipids from biological tissues, incubated in vitro with or without US-PION.An intensive signal enhancement could be found in the vibrational resonance wavelengths range around 2850 cm −1 from USPION-incubated lipids and particular regions highlighting accumulation of iron oxides nanoparticles in adipocytes (Figs.3(A) and (B)).Similar measurements on control tissues have shown no indication of any enhancements.The signal enhancement has been further investigated on all tissues by Raman maps chemical imaging (Fig. 4).Clear contrast enhancement is observed, in USPION treated ALS tissues, indicated by http://www.sssj.org/ejssnt(J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) the bright regions highlighting accumulation of iron oxide nanoparticles.To support the SERS activity of iron oxide nanoparticles, additional control microscopic Raman measurements have been performed on iron oxide nanoparticles dissolved in a specific heterocyclic organic compound (C 5 H 5 N; Pyridine).Clear intensity enhancement could be detected from iron oxides nanoparticles in Pyridine compared with the pure solution (Fig. 5).
Several hypotheses have been advanced to explain the pathogenic mechanisms underlying ALS, including oxidative stress, over activation of glutamate receptors, and apoptosis.Motor neuron death induced with oxidative stress has been associated with increased production of ceramides and alternated subcellular cholesterol metabolism which lead to apoptosis.Recent findings reported abnormalities in sphingolipid and cholesterol metabolism in the spinal cords of ALS patients and in a transgenic mouse model (Cu/ZnS OD mutant mice), which manifest increased levels of sphingomyelin, ceramides, and cholesterol esters [11].Increased oxidative stress in cultured motor neurons also leads to lipid alterations, and accumulation of the same lipid species.Namely, increased sphingolipid metabolism is implicated in the death of motor neurons and this involves increased formation of the membrane lipid peroxidation product 4-hydroxynonenal [12].Moreover, studies in which sphingomyelinases were manipulated indicated that ceramide production can induce accumulation of cholesterol esters in another neurodegenerative disease, Alzheimer's, leading to increased production of amyloid peptide in cultured cells.Together, these studies implied that the neurodegenerative cascade in ALS involves an early increase in levels of oxidative stress (induced by genetic and/or environmental factors) causing disturbances in membrane lipid metabolism and resulting in the accumulation of ceramides and cholesterol esters.Nitric oxide (NO) also plays a crucial role in the activation of caspases and apoptosis, and recent studies demonstrated that its increased production is correlated with mitochondrial damage and lipid peroxidation [13].According to our results, and Raman assignments [14], the observed strong lipid signal enhancement indicated high accumulation of lipids and iron particles in the brainstem and midbrain region of the ALS brain.iron-binding in filtration assays, with highest affinity at 5 to 10-fold on a molar basis with oleic acid [15].
In line with our results, previous studies supported the hypothesis that Iron (Fe 3+ ) binds to membrane lipids generating free radicals at the binding site.Iron-induced lipid peroxidation has previously been demonstrated for iron more often than any other transition metal.For example, an in vivo study has shown direct evidence of lipid peroxidation, in both mitochondrial and microsomal membrane lipids [16].The binding of Fe 3+ to cell membranes has been investigated in a system in which lipid peroxidation was proportional to Fe 3+ concentration; the results indicated that 95% of the Fe 3+ was membrane bound when evaluated by labeling with 59 FeCl 3 combined to measurement of nuclear magnetic resonance of water-proton relaxation times.Both spin-lattice (T 1 ) and spin-spin (T 2 ) relaxation times decreased with increasing Fe 3+ concentration.This study suggested that the charge transfer to Fe 3+ may occur at the membrane binding-site, leading to reduced Fe 3+ effect on water-proton relaxation times.
The iron uptake by cortex neurons has been previously demonstrated by using immunocytochemical techniques that shows the presence of transferring receptors on neurons [17].Recent studies on ceruloplasmin (CP) of brain iron homoestasis have shown that solubale CP has a role in iron uptake by iron-deficient brain neurons [18].The iron deposits in amyotrophic lateral sclerosis (ALS) have been found to be restricted to the precentral gyruses of gray matter (PGGM) [19].According to recent findings by MRI, performed with anti-CD4 antibodies tagged with magnetic nanoparticles in the transgenic ALS rat model, hypointensities were observed in particular regions that show accumulation of inflammatory cells in the vicinity of dilated ventricles [8,9].From our results we could correlate the SECARS enhancement to the lipid-iron accumulation in the inflammatory cells or from regions with perturbed sphingolipid metabolism resulting in ceramide and cholesterol ester accumulation in the ALS brain.

IV. CONCLUSIONS
We observed marked intensity enhancements in USPION-treated ALS brain rat model using Surface-Enhanced Coherent anti-Stokes Raman Scattering (SE-CARS) microscopy.The observed signal enhancement is correlated to a selective iron-binding enhancement from surrounding lipid molecules adsorbed to the surface of iron-oxides nanoparticles observed near the Raman resonance of 2850 cm −1 and 2875 cm −1 , and in the fingerprint region around 1660-1675 cm −1 .The results obtained correlate SECARS enhancements to selective association of lipids to up-taken USPION which shows high accumulation in the brainstem and midbrain region.The SERS-based optical properties of iron oxide nanoparticles is shown to be promising for future magnetic and optical probes for cell imaging applications and investigating neurodegenerative disorder model systems.
FIG. 4: Raman spectrum extracted from the Raman map image of USPION-treated brain tissue, in the range of 200-3100 cm −1 .