VEGF and angiogenesis in acute and chronic MOG((35-55)) peptide induced EAE.

An increased expression of vascular endothelial growth factor (VEGF) is associated with demyelinated lesions in both multiple sclerosis (MS) and its model (EAE), implicating changes in vasculature as a potential component of CNS plaque formation. The purpose of this study was to investigate the vascular changes in acute and chronic EAE in C57BL/6 mice induced with myelin oligodendrocyte glycoprotein (MOG ((35-55))) peptide. We investigated the functional contribution of VEGF to acute and chronic EAE by treating immunized mice with SU5416 (Semaxinib), a potent and selective inhibitor of VEGF receptor 2 (VEGFR2). Animals received seven daily injections of SU5416 (50 mg/kg) or vehicle beginning on the day after disease onset (acute study) or on day 45 post-immunization (chronic study). Spinal cord sections were collected on the day of sacrifice. Modulation of angiogenic gene expression was determined using RNA isolated from 4 acute and 4 non-immunized controls. MOG peptide induction produced extensive demyelination, immune cell infiltration, tissue laminin deposits, and axonal loss in lesions. VEGF expression was extensively increased in the acute mice, which correlated positively with clinical score. In the acute study, SU5416 treatment produced a significant clinical improvement versus vehicle controls (p<0.001), with less demyelination (-37%) and cellular infiltration (-23%) in the spinal cord (p<0.05). Treated animals also had significantly fewer blood vessels per section than controls (56.1+/-6.1 v. 81.6+/-11.5, p<0.05), and significantly reduced laminin abnormalities (28.9% of lesion area v. 46.8%, p<0.05). There was no improvement in clinical score or tissue pathology, and no difference in vessel number or lesion laminin expression, when SU5416 was administered during the chronic disease (all p>0.05). In the acute study only, VEGF staining correlated with demyelination and the extent of cellular infiltration in both control (r=0.723, r=0.665) and treated (r=0.681, r=0.487) animals (all p<0.05). Laminin staining in lesion areas was strongly correlated with tissue pathology for all animals in both the acute and chronic study (all p<0.001). Vascular alterations in MOG peptide-induced EAE in the mouse are accompanied by increased lesion-specific levels of VEGF, extensive laminin deposits in the tissue and altered transcription of numerous angiogenic factors. In the microarray studies, acute mice showed a significant increase in several angiogenic RNA transcripts, six of which were verified by RT-PCR, alanyl aminopeptidase, caspase 8, Hif1a, MMP-19, plasminogen activator inhibitor, and thrombospondin1.

Experimental allergic encephalomyelitis in connexin 43-heterozygous mice.

Alterations in the expression of gap junction proteins (connexins) have previously been observed in experimental allergic encephalomyelitis (EAE). Demyelinating lesions have significantly decreased Cx43, while recovering lesions have greatly increased Cx43 and increased glial fibrillary acidic protein-expressing astrocytes. This suggests an important role for gap-junctional intercellular communication in astrocytes in the recovery from CNS inflammation. To study the effects of decreased Cx43 expression during acute disease (21 days post-immunization) and in recovering spinal cord tissue (55 days post-immunization) we induced EAE in Cx43 heterozygous and wild-type mice. Mice showed signs of disease by day 10, and signs of recovery by day 25. There were no clinical or pathological differences between heterozygous and wild-type mice in the acute disease stage, except that wild-type male mice had fewer clinical signs of disease. Male mice that were heterozygous for Cx43, and therefore had decreased expression of Cx43, had increased EAE disease severity. All demyelinating lesions had reduced numbers of reactive astrocytes and a significant decrease in Cx43 expression. In the 55-day study, all heterozygous and wild-type mice were clinically improved, showed decreased pathological signs, and showed increased laminin expression, indicative of CNS recovery. Furthermore, all heterozygous mice showed a striking increase in Cx43 expression during recovery, suggesting that the regulatory factors affecting Cx43 expression are still present in mice that have only one wild-type Cx43 allele.

Connexin 43 gap junction proteins are up-regulated in remyelinating spinal cord.

Alterations in the expression of gap junction proteins have previously been observed in several diseases affecting the central nervous system; however, the status of connexin 43 (Cx43) has not yet been reported in spinal cord remyelination. We studied Cx43 expression in demyelination and remyelination by using a chronic guinea pig model of experimental allergic encephalomyelitis (EAE). Hartley guinea pigs were immunized with homogenized whole CNS and complete Freund's adjuvant. Animals became chronically ill by day 40 postimmunization, and animals with paralysis were entered into the study. Animals were treated on days 40-60 postimmunization with either saline or drugs that promote remyelination: an adenosine amine congener (100 mug/kg), an anti-alpha4-integrin blocker (CT301; ELN 69299; 30 mg/kg), or a combination of both drugs. Remyelination was induced in all drug-treated groups. Cx43 expression was virtually absent in demyelinated lesions of saline-treated controls compared with healthy tissue and normal appearing white matter (P < 0.001), whereas Cx43 was considerably increased (300-500%) in remyelinating lesions of all treatment groups (P < 0.001), most notably in CT301-treated animals. These changes in Cx43 expression indicate that Cx43 may beimportant for recovery from neuroinflammation.

Suppression of acute experimental allergic encephalomyelitis with a small molecule inhibitor of alpha4 integrin.

PURPOSE: To determine the efficacy of a small molecule inhibitor of alpha4 integrin (CT301) at reversing the clinical, pathological and MR-detectable deficits associated with the acute phase of experimental allergic encephalomyelitis (EAE). MATERIALS AND METHODS: EAE was induced in 36 female Hartley guinea pigs, and the treatment period was from day 11 to day 17 post-immunization. Animals received either saline (n = 12), anti-alpha4 integrin antibody (AN100226m; n = 12) or CT301 (n = 12). T2-weighted fast spin echo and T1-weighted pre- and post-contrast scans were performed at the beginning (day 11) and end (day 18) of the treatment period, and scored for cerebral inflammation and gadolinium enhancement. T1-weighted images were further analyzed to quantify this enhancement as a measure of blood-brain barrier integrity. Dissected CNS was evaluated for inflammation and demyelination. RESULTS: CT301 successfully reversed two clinical indicators of disease over the course of the treatment period. These animals showed decreased T2-weighted abnormalities, as well as a reduction in gadolinium leakage on T1-weighted images. Meningeal and perivascular inflammation was decreased by anti-alpha4 integrin treatments. CONCLUSION: CT301 effectively reverses the clinical, pathological and MR-detectable deficits of acute EAE, and may therefore be a promising therapeutic agent in multiple sclerosis (MS).

Spontaneous remyelination following prolonged inhibition of alpha4 integrin in chronic EAE.

Inhibition of alpha(4)beta(1) integrin blocks immune cell influx into the CNS providing benefit to patients with multiple sclerosis and in animal model systems. We have used this mechanism to examine whether the presence of inflammatory cells suppresses spontaneous myelin repair in experimental autoimmune encephalomyelitis. We observed (1) 87% of plaques showed remyelination after 40 days of treatment; (2) myelin repair occurred in half of the total lesion area; (3) half of the animals regained motor function. There was no significant repair or gain of motor function in vehicle-treated animals. Therefore, prolonged inhibition of CNS inflammation, in the absence of targeted myelin repair, facilitates mechanisms of spontaneous remyelination.

Resolution-dependent estimates of multiple sclerosis lesion loads.

BACKGROUND: Changes in brain lesion loads assessed with magnetic resonance imaging obtained at 1.5 Telsa (T) are used as a measure of disease evolution in natural history studies and treatment trials of multiple sclerosis. METHODS: A comparison was made between the total lesion volume and individual lesions observed on 1.5 T images and on high-resolution 4 T images. Lesions were quantified using a computer-assisted segmentation tool. RESULTS: There was a 46% increase in the total number of lesions detected with 4 T versus 1.5 T imaging (p < 0.005). The 4 T also showed a 60% increase in total lesion volume when compared with the 1.5 T (p < 0.005). In several instances, the 1.5 T scans showed individual lesions that coalesced into larger areas of abnormality in the 4 T scans. The relationship between individual lesion volumes was linear (slope 1.231) showing that the lesion volume observed at 4 T increased with the size of the lesion detected at 1.5 T. The 4 T voxels were less than one quarter the size of those used at 1.5 T and there were no consistent differences between their signal-to-noise ratios. CONCLUSIONS: The increase in signal strength that accompanied the increase in field strength compensated for the loss in signal amplitude produced by the use of smaller voxels. This enabled the acquisition of images with improved resolution, resulting in increased lesion detection at 4 T and larger lesion volumes.

VEGF and vascular changes in chronic neuroinflammation.

A vascular component has long been associated with the pathological changes in multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE). Despite the codependence of angiogenesis and many chronic inflammatory disorders, only circumstantial evidence is available to support the existence of angiogenesis in MS or EAE. To determine if angiogenesis occurs in conjunction with clinical and pathological signs of CNS inflammatory disease we evaluated temporal and spatial blood vessel counts, VEGF immunoreactivity, and histopathological changes in the spinal cord of guinea pigs with chronic-progressive (CP)-EAE (day 0-90 post-immunization, n=64) and controls (n=17). The number of vessels per section increased in infiltrated and demyelinated lesions by day 15 post-immunization and remained significantly higher than controls throughout the course of the disease. The number of vessels correlated with both clinical and pathological scores for inflammation, infiltration and demyelination. Vascular endothelial growth factor (VEGF) expression increased during acute disease peaking at day 26, which was the transition from the acute-inflammatory to chronic-demyelinating phase, before gradually returning to baseline levels. These observations implicate angiogenesis as a component of chronic neuroinflammation and demyelination and may suggest alternative therapeutic strategies for multiple sclerosis.

Prolonged reversal of chronic experimental allergic encephalomyelitis using a small molecule inhibitor of alpha4 integrin.

CNS leukocytic invasion in experimental allergic encephalomyelitis (EAE) depends on alpha4beta1 integrin/vascular cell adhesion molecule-1 (VCAM-1) interactions. A small molecule inhibitor of alpha4beta1 integrin (CT301) was administered to guinea pigs in the chronic phase (>d40) of EAE for 10, 20, 30 or 40 days. CT301 elicited a rapid, significant improvement in the clinical and pathological scores that was maintained throughout the treatment period. A progressive loss of cells in the spinal cord of treated animals confirmed the resolution of inflammation associated with clinical recovery. Therefore, prolonged inhibition of alpha4beta1 integrin caused a sustained reversal of disease pathology in chronic EAE and may be similarly useful in MS.

Stereotactic core biopsy of breast microcalcifications: comparison of film versus digital mammography, both using an add-on unit.

OBJECTIVE: The goal of this study was to assess the accuracy of an add-on stereotactic unit for core biopsy of indeterminate breast microcalcifications and to compare digital with conventional stereotactic guidance. MATERIALS AND METHODS: We conducted a retrospective review of 232 lesions with indeterminate microcalcifications in 218 women who underwent stereotactically guided breast biopsies. All biopsies were performed using a standard mammography machine with an add-on unit, 121 with conventional and 111 with digital stereotactic guidance. Successful sampling of the lesion was determined by the detection of microcalcifications on specimen radiography or at pathology. RESULTS: Using the add-on unit, 219 (94.4%) of the 232 targeted lesions were successfully sampled. The size, location, number of cores per lesion, and histology of the lesions were not different between the conventional and digital stereotactic biopsy groups (p > 0.1). Indeterminate microcalcifications were missed on biopsy in nine (7.4%) of 121 cases using conventional radiography and in only four (3.6%) of 111 cases using digital imaging. Digital stereotactic guidance allowed sampling of lesions with fewer calcifications per square centimeter (p < 0.001). CONCLUSION: Sampling of indeterminate microcalcifications using a standard mammography machine and an add-on unit has a high accuracy, similar to rates reported for dedicated prone biopsy tables. An add-on unit offers the advantage of considerable cost and space savings. Relative to conventional radiography, digital stereotactic guidance allows lesions with fewer calcifications to be sampled and achieves a greater biopsy success rate. Immediate digital images in the biopsy room also permit rapid adjustment of alignment and minimize patient movement.

Magnetization transfer and multicomponent T2 relaxation measurements with histopathologic correlation in an experimental model of MS.

Magnetization transfer and multicomponent T2 imaging techniques were implemented to study guinea pig in vivo. A chronic-progressive model of experimental allergic encephalomyelitis (EAE) was produced, and the inflammatory component of the disease was manipulated using antibodies against integrin. The magnetization transfer ratio (MTR) and T2 relaxation properties were measured in normal-appearing white matter (NAWM) with histological comparisons. Significant reductions in both the mean MTR and the myelin water percentage were measured in NAWM of EAE guinea pig brain. However, the MTR and myelin water percentage appear to measure different aspects of pathology in NAWM in EAE. Reductions in the MTR were prevented or reversed with suppression of inflammation. However, modulation of inflammatory activity was not reflected in the measurement of the myelin water percentage. Since the amount of myelin is not expected to vary with inflammatory-related changes, these observations support our hypothesis that the MTR is sensitive to physiological changes to myelin induced by inflammation, while the short T2 component is a more specific indicator of myelin content in tissue. Pathologic features other than demyelination may be important in the determination of the MTR.

Computational blood flow modeling based on in vivo measurements.

Study of the relationship between hemodynamics and atherogenesis requires accurate three-dimensional descriptions of in vivo arterial geometries. Common methods for obtaining such geometries include in vivo medical imaging and postmortem preparations (vessel casts, pressure-fixed vessels). We sought to determine the relative accuracy of these methods. The aorto-iliac (A/I) region of six rabbits was imaged in vivo using contrast-enhanced magnetic resonance imaging (MRI). After sacrifice, the geometry of the A/I region was preserved via vascular casts in four animals, and ex situ pressure fixation (while preserving dimensions) in the remaining two animals. The MR images and postmortem preparations were used to build computer representations of the A/I bifurcations, which were then used as input for computational blood flow analyses. Substantial differences were seen between MRI-based models and postmortem preparations. Bifurcation angles were consistently larger in postmortem specimens, and vessel dimensions were consistently smaller in pressure-fixed specimens. In vivo MRI-based models underpredicted aortic dimensions immediately proximal to the bifurcation, causing appreciable variation in the aorto-iliac parent/child area ratio. This had an important effect on wall shear stress and separation patterns on the "hips" of the bifurcation, with mean wall shear stress differences ranging from 15% to 35%, depending on the model. The above results, as well as consideration of known and probable sources of error, suggests that in vivo MRI best replicates overall vessel geometry (vessel paths and bifurcation angle). However, vascular casting seems to better capture detailed vessel cross-sectional dimensions and shape. It is important to accurately characterize the local aorto-iliac area ratio when studying in vivo bifurcation hemodynamics.

In vivo measurements of multi-component T2 relaxation behaviour in guinea pig brain.

Multi-echo Carr-Purcell-Meiboom-Gill (CPMG) imaging sequences were implemented on 1.5 T and 4.0 T imaging systems to test their ability to measure in vivo multi-component T2 relaxation behavior in normal guinea pig brain. The known dependence of accurate T2 measurements on the signal-to-noise ratio (SNR) was explored in vivo by comparing T2 decay data obtained using three methods to increase SNR (improved RF coil design, signal averaging and increased magnetic field strength). Good agreement between T2 values of nickel-doped agarose phantoms was found between imaging and spectroscopic methods. T2 values were determined for gray matter (GM) and white matter (WM) locations from images of guinea pig brain in vivo. T2 measurements of GM were found to be monoexponential at both field strengths. The mean T2 times for GM were 71 ms at 1.5 T, and 53 ms at 4.0T. The highest average SNR was achieved using an improved RF coil at 4.0T. In this case, two peaks were extracted in WM, a "short" T2 peak at approximately 6 ms, and a "medium" T2 peak at approximately 48 ms. T2 values in GM and the major component of WM were significantly decreased at 4.0T compared to 1.5 T. The improved SNR attained with this optimized imaging protocol at 4.0T has allowed for the first time extraction of the myelin-sensitive T2 component of WM in animal brain in vivo.

Reduced visual evoked responses in multiple sclerosis patients with optic neuritis: comparison of functional magnetic resonance imaging and visual evoked potentials.

The limited application of functional magnetic resonance imaging (fMRI) for investigations of multiple sclerosis (MS) patients has already shown that deficits of the motor, cognitive and visual systems may be identified by differences in the patterns of activation in response to a suitable stimulus. In MS patients with unilateral optic neuritis, the area of activation in the primary visual cortex, measured by fMRI techniques, is dramatically reduced in response to stimulation of the affected eye. The latency of the major positive component of the visual evoked potential (VEP) recorded upon stimulation of the affected eye is significantly increased in these patients, as compared to the unaffected eye and normal volunteers. We have found a correlation between the neural response measured using fMRI and the latency of the VEP. fMRI signal responses have the potential to provide more detailed topographic information relating to functional deficits in MS.

Correlation between MRI and clinico-pathological manifestations in Lewis rats protected from experimental allergic encephalomyelitis by acylated synthetic peptide of myelin basic protein.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system which constitutes an accepted animal model for multiple sclerosis (MS). The disease can take an acute or chronic form depending on the injection route, animal strain and nature of the disease-inducing antigen administered. The neuroinflammation associated with the acute form can be detected with T2-weighted, T1-weighted and diffusion MRI, and blood-brain barrier changes can be investigated with Gd-DTPA-enhanced T1-weighted imaging, similar to that of MS patients. A synthetic peptide of myelin basic protein (MBP) encephalitogenic for the Lewis rat (MBP 68-86) was acylated by the attachment of a palmitoyl residue (PAL68-86), and was shown to confer almost complete protection against EAE, when administered to rats before and after an encephalitogenic challenge. In this study, treatment of Lewis rats with PAL68-86 prevented the appearance of clinical signs (p < 0.0001) after challenge with the native peptide (p68-86) in complete Freund's adjuvant (CFA), and reduced considerably the MRI and histopathological signs of the disease (p < 0.0001). Measurement of the gadolinium leakage due to neuroinflammation revealed a significant decrease in permeability from 4.09 +/- 2.1 to 2.95 +/- 1.79% pixels > mean + 2 SD (p = 0.011). Therefore, quantitative MRI measurements correlate very well with the reduced cellular infiltration in the CNS and the absence of clinical signs in the EAE-protected animal.

Radiology residents' research in Canada: status of infrastructure.

OBJECTIVE: To survey all Canadian radiology residency training programs concerning their programs in research and critical inquiry, in order to assess the status of the research infrastructure for residents. METHODS: A questionnaire was developed through consensus among the radiology research directors in Canadian training programs. RESULTS: Fourteen of the 16 universities have a research and critical inquiry program, although there is considerable variation in the components and structure of the programs. Many programs do not have personnel, funds or written guidelines for their operation. Didactic aspects of research, such as biostatistics, ethics and quality assurance, are widely taught and thought to be important. Similarly, there is widespread agreement on which methods qualify for research projects, such as departmental audits or retrospective chart reviews. However, except for requiring a presentation at a research day, very few residency programs view research work as essential to the completion of training. The commitment to research programs is strong, as evidenced by the time devoted by residents and mentors and the funds made available for presentations. CONCLUSION: This survey provides information to assist in the development of individual programs and national guidelines for radiology residency programs in critical inquiry and research.

Apoptotic cells are present in the CNS throughout acute and chronic-progressive EAE in the absence of clinical recovery.

Experimental allergic encephalomyelitis (EAE) is an autoimmune, demyelinating disorder of the central nervous system induced in susceptible animals as a model for the human disease multiple sclerosis. Antibodies against the leukocyte adhesion molecule alpha4 integrin have been shown to prevent and reverse acute and chronic EAE of the guinea pig. The results presented in this paper implicate apoptosis as the mechanism of reversal of EAE following treatment with anti-alpha4 integrin antibody. Apoptotic cells were observed in the central nervous system (CNS) throughout chronic-progressive EAE of the guinea pig in the absence of clinical recovery. Many of the apoptotic cells were identified as T cells using immunohistochemistry. Similarly, apoptotic cells were present in the CNS of animals during anti-alpha4 integrin-mediated recovery from acute and chronic disease. Therefore, anti-alpha4 integrin-mediated recovery from EAE is due to the prevention of the influx of new inflammatory cells into the CNS that are required to replace those undergoing apoptosis.

Genetically regulated response to intravesical bacillus Calmette Guerin immunotherapy of orthotopic murine bladder tumor.

PURPOSE: Genetically regulated host response to intravesical Bacillus Calmette Guerin (BCG) immunotherapy was assessed using the murine bladder tumor MM45T in Bcgr and Bcgs inbred congenic strains of mice. MATERIALS AND METHODS: Tumor detection and monitoring of treatment response to BCG was carried out using magnetic resonance imaging (MRI) of BALB/c (Bcgs allele) and BALB/c. CD2 (CD2) (Bcgr allele) mice implanted orthotopically with MM45T tumor cells. Intravesical BCG instillation (3 doses per week for 3 weeks) was used as prophylaxis against tumor implantation in both Bcgr and Bcgs strains and as definitive treatment against MRI-confirmed established tumors. Tumors implanted in both strains of untreated mice served as controls. Intravesical injection of BCG was also performed in established heterotopic subcutaneous tumors in both strains. Immunologic response in all groups was assessed by flow cytometric analysis of the bladder irrigation fluid cell composition, measuring CD4+ (helper/inducer) and CD8+ (cytotoxic/ suppressor) cell subsets. RESULTS: Intralesional injection of BCG into established heterotopic tumors showed growth inhibition in the Bcgs strain but not in the Bcgr strain. Intravesical BCG treatment against established orthotopic tumors showed significant tumor regression in the Bcgs strain compared to control but there was no effect in the Bcgr strain. CONCLUSION: The differential anti-tumor activity of BCG in the Bcgs and Bcgr congenic murine strains supports the notion that Bcg gene-controlled responsiveness to BCG innoculation determines, at least partially, the host response to immunotherapy. These results have potential clinical significance in patient selection for intravesical therapy for bladder cancer.

A magnetic resonance imaging study of double elevator palsy.

OBJECTIVE: The pathophysiology of double elevator palsy is poorly understood. We assessed two patients with this condition using magnetic resonance imaging (MRI) to evaluate the appearance of the extraocular muscles. DESIGN: Cross-sectional study. SETTING: Radiology department of a university-affiliated hospital in London, Ont. PATIENTS: Two patients from a private ophthalmology practice who had undergone complete transpositions of the horizontal rectus muscles to treat hypotropia associated with double elevator palsy. INTERVENTION: MRI. A volume scanning technique was used to obtain maximum information about the muscles. OUTCOME MEASURE: Appearance of the extraocular muscles. RESULTS: In both patients MRI showed decreased volume of the superior rectus muscle on the affected side. The other rectus muscles were normal. This suggested either congenital hypoplasia or paresis of the involved superior rectus muscle. In addition, the full tendon transpositions of the medial and lateral recti did not appreciably change the middle and deep orbital pathways of the transposed horizontal rectus muscles. CONCLUSIONS: MRI may be a useful adjunct to saccadic velocity assessments in differentiating between primary inferior rectus restriction, primary superior rectus paresis and congenital supranuclear elevator deficiency.