Comparative in vitro cytotoxicity study on uncoated magnetic nanoparticles: effects on cell viability, cell morphology, and cellular uptake.

Magnetic iron oxide nanoparticles (MIONPs) must be biocompatible, and a thorough knowledge on their potential cytotoxicity is crucial for their biomedical applications. However, the detailed study about the effects of iron oxide nanoparticles on cell viability, cell morphology, and cellular uptake of different mammalian cells is still insufficient. In this paper, comparative cytotoxicity study of uncoated magnetite nanoparticles at different concentrations was performed on human cervical cancer cell line (HeLa) and immortalized normal human retinal pigment epithelial cell line (RPE). The size, structure, and magnetic behavior of the MIONPs were characterized using transmission electron microscopy (TEM), X-ray diffractometry (XRD), and vibrating sample magnetometry (VSM) respectively. After 24-hour incubation with the MIONPs, the cell viability was determined by live/dead assay, the cell morphology at high magnification was observed under scanning electron microscopy (SEM), and the cellular uptake of MIONPs was measured under TEM and verified by energy-dispersive X-ray spectroscopy (EDX) analysis. Our results indicate that the uncoated MIONPs at a high concentration (0.40 mg/ml) were toxic to both HeLa and RPE cells. However, the cytotoxicity of uncoated MIONPs at low concentrations was cell-type specific, and RPE cells were more susceptible to these MIONPs than HeLa cells. The effects of the MIONPs on cell morphology and the nanoparticles uptake also showed different features between these two cell lines. Hence cell type should be taken into consideration in the in vitro cytotoxicity study of uncoated MIONPs. Additionally, it should be noticed that the cell morphological changes and the uptake of nanoparticles can take place even though no toxic effect of these MIONPs at low concentrations was reflected in the traditional cell viability assay.

Non-viral Smad7 gene delivery and attenuation of postoperative peritoneal adhesion in an experimental model.

BACKGROUND: Postoperative intra-abdominal adhesion is associated with high morbidity and mortality. Smad7, a protein that occupies a strategic position in fibrogenesis, inhibits the transforming growth factor (TGF) beta/Smad signalling pathway. In this study the therapeutic potential of exogenous Smad7 in preventing fibrogenesis in postoperative intra-abdominal adhesion was investigated. METHODS: Intra-abdominal adhesion was induced in a rodent model by peritoneal abrasion. Smad7 was delivered into the peritoneal cavity by a non-viral ultrasound-microbubble-mediated naked gene transfection system. The effect of Smad7 transgene on adhesion formation was studied by measuring changes in TGF-beta, fibrogenic factors, alpha-SMA and Smad2/3 activation in the anterior abdominal wall. RESULTS: Four weeks after surgical abrasion, all rats developed significant peritoneal adhesion with enhanced TGF-beta expression, increased levels of extracellular matrix components and activated myofibroblasts, accompanied by decreased Smad7 expression and increased Smad2/3 activation. In rats treated with the Smad7 transgene, the incidence and severity of peritoneal adhesion were significantly reduced, with biochemical downregulation of fibrogenic factors and inhibition of Smad2/3 activation. Serial quantitation using magnetic resonance imaging revealed a significant reduction in adhesion areas from day 14 onwards. CONCLUSION: Ultrasound-microbubble-mediated gene transfection provides timely targeted gene delivery for the treatment of postoperative peritoneal adhesions.

Imaging physiologic dysfunction of individual hippocampal subregions in humans and genetically modified mice.

We have developed a variant of functional magnetic resonance imaging (fMRI) designed to be sensitive to static neuronal function. This method is based on resting instead of dynamic changes in oxygen-dependent signal and therefore allows for a spatial resolution that can detect signal from different hippocampal subregions in human subjects as well as in mice. We found that hippocampal signal was significantly diminished in elderly subjects with memory decline compared to age-matched controls, and different subjects showed dysfunction in different subregions. Among healthy elders, signal intensity from the subiculum was correlated selectively with memory performance. This method does not require an activation task; it can be used in anesthetized normal and in genetically modified and cognitively impaired mice. In mice the signal was found to be sufficiently sensitive to detect functional changes in the absence of underlying anatomical changes.

Age-related degeneration of lumbar intervertebral discs in rabbits revealed by deuterium oxide-assisted MRI.

OBJECTIVES: Intervertebral disc (IVD) degeneration is associated with a loss of disc water content and change in biochemical composition of the disc. Rabbit is a frequently used model to evaluate the efficacy of therapeutics for disc degeneration. This study addresses whether rabbits undergo age-related disc degeneration, assessed using deuterium oxide-assisted magnetic resonance imaging (MRI) of the lumbar IVDs. MATERIALS AND METHODS: The lumbar spines of adolescent, adult, and aged rabbits (6-36 months) were subjected to T2-weighted/short-tau inversion recovery (STIR) MRI scan along with water-deuterium oxide (H(2)O:D(2)O) dilutions. The total and maximum H(2)O:D(2)O index (HDi) of the lumbar IVDs were determined and compared between disc levels at different ages. RESULTS: Adolescent rabbit lumbar discs had similar total HDi, suggesting the hydration and biochemical composition was similar among the lumbar levels. With the use of H(2)O:D(2)O reference, the discs were shown to undergo continual decrease in signal with aging which non-calibrated measurement method could not reveal. The HDi decrease rate was higher at the caudal than cranial levels. CONCLUSION: This study provided in vivo evidence of age-related progressive disc degenerative change in rabbit lumbar discs, suggesting aged rabbits can be considered as a natural disc degeneration model in disc regeneration studies. However, it is important to select proper disc levels as intra-subject controls due to different rates of degenerative changes between caudal and cranial levels.

Hypoxic-ischemic brain injury in the neonatal rat model: relationship between lesion size at early MR imaging and irreversible infarction.

BACKGROUND AND PURPOSE: By using a neonatal rat hypoxia-ischemia (HI) model, we studied the relationship between lesion volume-measured by diffusion-weighted imaging (DWI) and T2-weighted imaging (T2WI) at an early time point-and irreversible infarct volume. We also evaluated the optimal apparent diffusion coefficient (ADC) threshold that provides the best correlation with irreversible infarct size. MATERIALS AND METHODS: Twenty-three neonatal rats underwent right common carotid artery ligation and hypoxia. MR imaging was performed 1-2 hours post-HI by using DWI and T2WI and at day 4 post-HI by using T2WI. Lesion volumes relative to whole brain (%LV) were measured on ADC maps by using different relative ADC thresholds 60%-80% of mean contralateral ADC and T2WI. Pearson correlation and multiple linear regression analysis were used to study the relationships between ln(%LV) at MR imaging and %LV at histopathology. RESULTS: At 1-2 hours post-HI, all lesion volume measurements on DWI were significantly correlated with the infarct volume on histopathology, with the best correlation attained at the 80% ADC threshold (r = 0.738; P < .001). The estimated regression formula was %LV on histopathology = 20.60 + 3.33 ln(%LV on 80% ADC threshold) (adjusted R(2) = 0.523; P < .001). Lesion volume at 1-2 hours post-HI tended to underestimate the final infarct volume. CONCLUSION: Early post-HI MR imaging by using DWI correlates moderately well with the size of irreversible infarct, especially when measured by using a relative ADC threshold of 80% mean contralateral ADC.

Rapid in vivo monitoring of chemotherapeutic response using weighted sodium magnetic resonance imaging.

A novel pulse sequence strategy uses sodium magnetic resonance imaging to monitor the response to chemotherapy of mouse xenograft tumors propagated from human prostate cancer cell lines. An inversion pulse suppresses sodium with long longitudinal relaxation times, weighting the image toward intracellular sodium nuclei. Comparing these weighted sodium images before and 24 h after administration of antineoplastics, we measured a 36 +/- 4% (P < 0.001; n = 16) increase in signal intensity. Experiments with these same drugs and cells, treated in culture, detected a significant intracellular sodium elevation (10-20 mM) using a ratiometric fluorescent dye. Flow cytometry studies showed that this elevation preceded cell death by apoptosis, as determined by fluorescent end-labeling of apoptotic nuclei or Annexin V binding. Histopathology on formalin-fixed sections of explanted tumors confirmed that drug administration reduces proliferation (2.2 versus 8.6 mitotic figures per high power field; P < 0.0001), an effect that inversely correlates with the sodium magnetic resonance image response on a tumor-to-tumor basis (P < 0.02; n = 10). Morphological features, such as central zones of nonviable cells, rims of active apoptosis, and areas of viable tumor, could be distinguished by comparing weighted and unweighted images. Advantages of this sodium imaging technique include rapid determination of drug efficacy, improved diagnosis of lesions, ease of coregistration with high resolution proton magnetic resonance imaging, and absence of costly or toxic reagents.

Dietary supplementation with n-3 fatty acids from weaning limits brain biochemistry and behavioural changes elicited by prenatal exposure to maternal inflammation in the mouse model.

Prenatal exposure to maternal immune activation (MIA) increases the risk of schizophrenia and autism in the offspring. The MIA rodent model provides a valuable tool to directly test the postnatal consequences of exposure to an early inflammatory insult; and examine novel preventative strategies. Here we tested the hypotheses that behavioural differences in the MIA mouse model are accompanied by in vivo and ex vivo alterations in brain biochemistry; and that these can be prevented by a post-weaning diet enriched with n-3 polyunsaturated fatty acid (PUFA). The viral analogue PolyI:C (POL) or saline (SAL) was administered to pregnant mice on gestation day 9. Half the resulting male offspring (POL=21; SAL=17) were weaned onto a conventional lab diet (n-6 PUFA); half were weaned onto n-3 PUFA-enriched diet. In vivo magnetic resonance spectroscopy measures were acquired prior to behavioural tests; glutamic acid decarboxylase 67 (GAD67) and tyrosine hydroxylase protein levels were measured ex vivo. The main findings were: (i) Adult MIA-exposed mice fed a standard diet had greater N-acetylaspartate/creatine (Cr) and lower myo-inositol/Cr levels in the cingulate cortex in vivo. (ii) The extent of these metabolite differences was correlated with impairment in prepulse inhibition. (iii) MIA-exposed mice on the control diet also had higher levels of anxiety and altered levels of GAD67 ex vivo. (iv) An n-3 PUFA diet prevented all the in vivo and ex vivo effects of MIA observed. Thus, n-3 PUFA dietary enrichment from early life may offer a relatively safe and non-toxic approach to limit the otherwise persistent behavioural and biochemical consequences of prenatal exposure to inflammation. This result may have translational importance.

Sonographic and computed tomography characteristics of liver ablation lesions induced by high-intensity focussed ultrasound.

RATIONALE AND OBJECTIVES: The authors have previously demonstrated the ability of high-intensity focused ultrasound (HIFU) to extracorporeally induce selective tissue destruction in the liver without causing damage to the intervening abdominal wall. The potential usefulness of HIFU as a noninvasive therapy for liver cancer has been suggested. This study observes sonographic and computed tomography (CT) characteristics of HIFU-ablated liver tissue in an attempt to assess the possibility of using these imaging methods to monitor the therapeutic results. METHODS: A sonoablated lesion was induced in the liver in each of 20 rabbits with a HIFU therapeutic system. Sequential imaging of the hepatic sonolesions with sonography and CT was performed up to 8 days after treatment, and the imaging patterns were correlated with the histopathology. RESULTS: Hepatic sonoablated tissue could be clearly visualized by sonography as a hypoechoic lesion. On contrast-enhanced CT, the sonolesions were depicted as nonenhanced low-density regions. There was good correlation among the sizes of sonography- and CT-depicted lesions and pathologic specimens. CONCLUSION: In this model, sonography and contrast-enhanced CT were useful imaging modalities for monitoring sonolesion evolution after HIFU treatment.

Intracerebral clysis in a rat glioma model.

OBJECTIVE: Intracerebral clysis (ICC) is a new term we use to describe convection-enhanced microinfusion into the brain. This study establishes baseline parameters for preclinical, in vivo, drug investigations using ICC in a rat glioma model. METHODS: Intracranial pressure was measured, with an intraparenchymal fiber-optic catheter, in male Fischer rats 10, 15, 20, and 25 days after implantation of C6 glioma cells in the right frontal lobe (n = 80) and in control rats without tumor (n = 20), before and during ICC. A 25% albumin solution (100 microl) was infused through an intratumoral catheter at 0.5, 1.0, 2.0, 3.0, and 4.0 microl/min. Infusate distribution was assessed by infusion of fluorescein isothiocyanate-dextran (Mr 20,000), using the aforementioned parameters (n = 36). Brains were sectioned and photographed under ultraviolet light, and distribution was calculated by computer analysis (NIH Image for Macintosh). Safe effective drug distribution was demonstrated by measuring tumor sizes and apoptosis in animals treated with N,N'-bis(2-chloroethyl)-N-nitrosourea via ICC, compared with untreated controls. Magnetic resonance imaging noninvasively confirmed tumor growth before treatment. RESULTS: Intracranial pressure increased with tumor progression, from 5.5 mm Hg at baseline to 12.95 mm Hg on Day 25 after tumor cell implantation. Intracranial pressure during ICC ranged from 5 to 21 mm Hg and was correlated with increasing infusion volumes and increasing rates of infusion. No toxicity was observed, except at the higher ends of the tumor size and volume ranges. Fluorescein isothiocyanate-dextran distribution was greater with larger infusion volumes (30 microl versus 10 microl, n = 8, P < 0.05). No significant differences in distribution were observed when different infusion rates were compared while the volume was kept constant. At tolerated flow rates, the volumes of distribution were sufficient to promote adequate drug delivery to tumors. N,N'-Bis(2-chloroethyl)-N-nitrosourea treatment resulted in significant decreases in tumor size, compared with untreated controls. CONCLUSION: The C6 glioma model can be easily modified to study aspects of interstitial delivery via ICC and the application of ICC to the screening of potential antitumor agents for safety and efficacy.

Realizations of fast 2-D/3-D image filtering and enhancement.

This paper proposes a novel algorithm for multidimensional image enhancement based on a fuzzy domain enhancement method, and an implementation of a recursive and separable low-pass filter. Considering a smoothed image as a fuzzy data set, each pixel in an image is processed independently, using fuzzy domain transformation and enhancement of both the dynamic range and the local gray level variations. The algorithm has the advantages of being fast and adaptive, so it can be used in real-time image processing applications and for multidimensional data with low computational cost. It also has the ability to reduce noise and unwanted background that may affect the visualization quality of two-dimensional (2-D)/three-dimensional (3-D) data. Examples for the applications of the algorithm are given for mammograms, ultrasound 3-D images, and photographic images.

Weighted backprojection approach to cone beam 3D projection reconstruction for truncated spherical detection geometry.

A new analytical three-dimensional cone beam reconstruction algorithm is presented for truncated spherical detection geometry. The basic idea of the proposed algorithm is the formation of spatially invariant 3D blurred back-projected volumetric image by the use of the weighted backprojection of cone beam projection data and subsequent 3D filtering using an acceptance angle dependent rho filter. The backprojection weighting function is calculated on the basis of each given geometrical condition, i.e. detection geometry or degree of truncation, position of cone beam apex, and backprojection point. The proposed algorithm is derived analytically and is computationally efficient. Performance of the algorithm is evaluated by the reconstruction of 3D volumetric images using simulated data from arbitrarily truncated spherical detector geometries.

Experimental high-frequency ultrasound can detect graft rejection after small bowel transplantation.

Early diagnosis of graft rejection after small bowel transplantation (SBT) can allow prompt institution of vigorous immunosuppressive therapy, with resultant reversal of the rejection process. The current method for graft monitoring is random mucosal biopsy from a stomal site or through an endoscope. However, because early rejection often has a patchy distribution, it could be missed by random biopsy. We hypothesized that the pathological process of rejection would alter acoustic impedance of the tissue and thus change the ultrasonic patterns of the graft intestinal wall. If this hypothesis is correct, then high-frequency endoscopic ultrasound (US) could be used to monitor the entire transplanted bowel and guide the biopsy, with improved yields. This hypothesis was tested in a rat orthotopic SBT model. Sixty-two intestinal specimens (9 isografts, 12 allografts treated with cyclosporine A [CsA], 22 untreated allografts, and 19 intestines from normal rats) were collected for in vitro transluminal US imaging (30 MHz) and histopathologic study. The echo pattern of normal rat intestinal wall consisted of five echo layers that correlated spatially with the histological layers: the innermost hyperechoic layer 1, plus hypoechoic layer 2, corresponded to the mucosa; hyperechoic layer 3, the submucosa; anechoic layer 4, the muscularis propria; and hyperechoic layer 5, the serosa. The isografts and CsA-treated allografts were identical histologically and ultrasonically to normal intestine. However, the echo patterns of the untreated allografts had progressive loss of architectural stratification, with worsening rejection. The change began with patchy indistinctness and disruption of hyperechoic layers 1, 3 and 5, and progressed to total obliteration of the layers, with the intestinal wall becoming a nonstratified hypoechoic structure.(ABSTRACT TRUNCATED AT 250 WORDS)

MRI brain image segmentation by multi-resolution edge detection and region selection.

Combining both spatial and intensity information in image, we present an MRI brain image segmentation approach based on multi-resolution edge detection, region selection, and intensity threshold methods. The detection of white matter structure in brain is emphasized in this paper. First, a multi-resolution brain image representation and segmentation procedure based on a multi-scale image filtering method is presented. Given the nature of the structural connectivity and intensity homogeneity of brain tissues, region-based methods such as region growing and subtraction to segment the brain tissue structure from the multi-resolution images are utilized. From the segmented structure, the region-of-interest (ROI) image in the structure region is derived, and then a modified segmentation of the ROI based on an automatic threshold method using our threshold selection criterion is presented. Examples on both T1 and T2 weighted MRI brain image segmentation is presented, showing finer brain tissue structures.

MR imaging assessment of lumbar intervertebral disk degeneration and age-related changes: apparent diffusion coefficient versus T2 quantitation.

BACKGROUND AND PURPOSE: T2 and ADC mappings are 2 quantitative MR imaging tools for assessing IVDD. This study aimed to compare these 2 measures in detecting IVDD and its age-related changes. MATERIALS AND METHODS: Thirty-seven asymptomatic volunteers and 28 patients with back pain or sciatica were examined, and their lumbar disk T2 and ADC maps were quantified via sagittal imaging protocols at 1.5T. For all participants, the Pfirrmann system was used by 2 radiologists for grading disks. T2 and ADC values in the inner portion of disks were measured, and their variances in different grades were analyzed by 1-way ANOVA testing. The ability of T2 and ADC measures to differentiate IVDD grades was compared on the basis of their ROC curves. For asymptomatic subjects, the correlations between age and the 2 MR imaging measures were assessed by the Pearson correlation test. RESULTS: Both T2 and ADC values were found to decrease with the increasing Pfirrmann grades except T2 in grade V. Significant T2 differences were seen among grades I-IV, but not between grades IV and V. There were no significant ADC differences among grades I-III. Moreover, the areas under the ROC curves differed significantly (0.95 and 0.67 for T2 and ADC, respectively). Linear regression analysis revealed that T2 yielded more significant correlation with age (r = -0.77) than ADC did (r = -0.37). CONCLUSIONS: T2 quantitation provides a more sensitive and robust approach for detecting and characterizing the early stage of IVDD and age-related disk changes.

Mild hypoxic-ischemic injury in the neonatal rat brain: longitudinal evaluation of white matter using diffusion tensor MR imaging.

BACKGROUND AND PURPOSE: Selective white matter (WM) damage is a known sequela of mild hypoxic-ischemic (HI) injury in the neonatal rat model. The aim of this study was to evaluate longitudinally mild HI-induced WM damage (represented by the external capsule [EC]) by diffusion tensor MR imaging (DTI) and to correlate the findings with histology. MATERIALS AND METHODS: Seven-day-old Sprague-Dawley rats (n = 19) underwent unilateral ligation of the left common carotid artery followed by hypoxia for 50 minutes to create mild HI injury. DTI was performed longitudinally at 5 time points from day 1 to day 90 postinjury (n = 19, 16, 13, 11, 9, respectively), and fractional anisotropy (FA), trace, radial diffusivity (lambda( perpendicular)), and axial diffusivity (lambda(//)) of the injury and control contralateral ECs were quantified. Rats were randomly sacrificed (n = 15, in total), and the corresponding ECs were stained with hematoxylin-eosin, Luxol fast blue (LFB), and neurofilament (NF) to evaluate morphologic changes, amount of myelin, and axonal count at every time point. A paired t test was applied to evaluate statistical differences between both ECs, and the Pearson correlation test was used to evaluate the relationships between DTI indices and histologic evaluations. In addition, longitudinal changes in DTI indices and histologic evaluations were analyzed by a linear mixed model and an analysis of variance test, respectively. RESULTS: We demonstrated significantly decreased FA, increased lambda( perpendicular), and similar lambda(//) in the injury compared with the control EC, which was persistent through all time points. Histologic evaluation by LFB and NF staining showed reduced myelin stain intensity in the injury EC and similar axonal counts in both ECs. Longitudinally, there was an increase in FA, a decrease in lambda( perpendicular) and trace, and stability in lambda(//) in both ECs. Also, there was progressive reduction in the differences in FA, trace, and lambda( perpendicular) between the injury and control EC, especially between day 1 and day 7 postinjury and in tandem with changes in myelin stain. FA was significantly correlated with myelin stain (r = 0.681, P < .01) and axonal count (r = 0.673, P < .01), whereas lambda( perpendicular) was significantly correlated with myelin stain only (r = -0.528, P < .01), and lambda(//), with axonal count only (r = 0.372, P = .043). CONCLUSIONS: Diffusion indices can reflect dysmyelination in mild HI injury, continual myelination of both injury and control ECs with growth, and the partial recovery of myelin postinjury. We propose that diffusion indices may be used as biomarkers to monitor noninvasively the longitudinal changes of mild HI-induced WM damage.

Identifying rodent olfactory bulb structures with micro-DTI.

Olfactory bulb (OB) is one of the most developed systems in rodent models with complex neuronal organization and anatomical structures. MR diffusion tensor imaging (DTI) is a non-invasive technique to probe tissue microstructures by examining the diffusion characteristics of water molecules. This paper presents how different OB layers can be identified and quantitatively characterized by micro-DTI using a specially constructed micro-imaging radio frequency (RF) coil. High spatial resolution and high signal to noise ratio (SNR) DTI images of ex vivo rat OBs were obtained. Distinct contrasts were observed between various olfactory bulb layers in trace map, fractional anisotropy (FA) map and FA color map, all in consistency with the known OB neuroanatomy. These experimental results demonstrate the utility of micro-DTI in investigation of complex OB organization.

Diffusion tensor MRI study of myocardium structural remodeling after infarction in porcine model.

Investigation of infarct myocardium structure will lead to better understanding of functional adaptation and remodeling. Diffusion tensor magnetic resonance imaging (DTI) provides a means for rapid and nondestructive characterization of the three-dimensional fiber architecture of myocardium. DTI studies were performed on 10 excised, formalin-fixed hearts of both infarct (two months after left anterior descending coronary artery (LAD) occlusion surgery, n=4) and control (n=6) porcine. Each slice was divided into eight segments, and fractional anisotropy (FA) value and helix angle were measured in multiples short-axis slices, respectively. Infarct myocardium exhibited decreased FA value, flatter helix angle courses fluctuating around small helix angle with greater standard error of the mean (SEM) and smaller range of helix angle. The results provide structure information of infarct myocardium.

Myocardial Fiber Length Mapping with MR Diffusion Tensor Imaging.

Diffusion tensor MRI is emerging as a rapid, nondestructive method to map myocardial fiber organization. A precise biological description of myocardial fiber performance requires knowledge of four variables: length, force, velocity and time. However, study of quantification of myocardial fiber length is lacking. The current study aims to show myocardial fiber length maps of formalin-fixed heats. Diffusion tensor MRI with medium diffusion resolution (15 directions) was performed in one isolated pig heart. Fiber length maps were investigated in multiple short-axis slices. The results provide supplementary information of myocardial fiber organization. To our knowledge, the present study is the first report of the myocardial fiber length mapping.

Microscopic mechanism of attenuation of compressional ultrasonic waves in tissue-mimicking phantom materials.

An investigation was performed to determine whether the sound-attenuation-insuspensions theory of Allegra and Hawley can be used to explain the compressional (longitudinal wave) attenuation of ultrasonically tissue-mimicking materials commonly used in phantoms for testing the performance of medical ultrasound systems. These materials are composed of microscopic graphite particles suspended in a gel. The theory was first tested using materials containing spherical glass beads instead of graphite particles because these materials more closely fit the geometric conditions assumed in the theory. For the glass bead type materials as well as the graphite particle type materials, the attenuation coefficients predicted using the Allegra and Hawley model agreed rather well with experimental measurements over the diagnostic frequency range.