Gene Therapy with Single-Subunit Yeast NADH-Ubiquinone Oxidoreductase (NDI1) Improves the Visual Function in Experimental Autoimmune Encephalomyelitis (EAE) Mice Model of Multiple Sclerosis (MS).

Mitochondrial dysfunction mediated loss of respiration, oxidative stress, and loss of cellular homeostasis contributes to the neuronal and axonal degenerations permanent loss of function in experimental autoimmune encephalomyelitis model (EAE) of multiple sclerosis (MS). To address the mitochondrial dysfunction mediated visual loss in EAE mice, self-complementary adeno-associated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene was intravitreally injected into the mice after the onset of visual defects. Visual function assessed by pattern electroretinogram (PERGs) showed progressive loss of function in EAE mice were improved significantly in NDI1 gene therapy-treated mice. Serial optical coherence tomography (OCT) revealed that progressive thinning of inner retinal layers in EAE mice was prevented upon NDI1 expression. The 45% optic nerve axonal and 33% retinal ganglion cell (RGC) loss contributed to the permanent loss of visual function in EAE mice were ameliorated by NDI1-mediated prevention of mitochondrial cristae dissolution and improved mitochondrial homeostasis. In conclusion, targeting the dysfunctional complex I using NDI1 gene can be an approach to address axonal and neuronal loss responsible for permanent disability in MS that is unaltered by current disease modifying drugs.

Folate receptor-targeted positron emission tomography of experimental autoimmune encephalomyelitis in rats.

BACKGROUND: Folate receptor-ß (FR-ß) is a cell surface receptor that is significantly upregulated on activated macrophages during inflammation and provides a potential target for folate-based therapeutic and diagnostic agents. FR-ß expression in central nervous system inflammation remains relatively unexplored. Therefore, we used focally induced acute and chronic phases of experimental autoimmune encephalomyelitis (EAE) to study patterns of FR-ß expression and evaluated its potential as an in vivo imaging target. METHODS: Focal EAE was induced in rats using heat-killed Bacillus Calmette-Guérin followed by activation with complete Freund's adjuvant supplemented with Mycobacterium tuberculosis. The rats were assessed with magnetic resonance imaging and positron emission tomography/computed tomography (PET/CT) at acute (14 days) and chronic (90 days) phases of inflammation. The animals were finally sacrificed for ex vivo autoradiography of their brains. PET studies were performed using FR-ß-targeting aluminum [18F]fluoride-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate ([18F]AlF-NOTA-folate, 18F-FOL) and 18 kDa translocator protein (TSPO)-targeting N-acetyl-N-(2-[11C]methoxybenzyl)-2-phenoxy-5-pyridinamine (11C-PBR28). Post-mortem immunohistochemistry was performed using anti-FR-ß, anti-cluster of differentiation 68 (anti-CD68), anti-inducible nitric oxide synthase (anti-iNOS), and anti-mannose receptor C-type 1 (anti-MRC-1) antibodies. The specificity of 18F-FOL binding was verified using in vitro brain sections with folate glucosamine used as a blocking agent. RESULTS: Immunohistochemical evaluation of focal EAE lesions demonstrated anti-FR-ß positive cells at the lesion border in both acute and chronic phases of inflammation. We found that anti-FR-ß correlated with anti-CD68 and anti-MRC-1 immunohistochemistry; for MRC-1, the correlation was most prominent in the chronic phase of inflammation. Both 18F-FOL and 11C-PBR28 radiotracers bound to the EAE lesions. Autoradiography studies verified that this binding took place in areas of anti-FR-ß positivity. A blocking assay using folate glucosamine further verified the tracer's specificity. In the chronic phase of EAE, the lesion-to-background ratio of 18F-FOL was significantly higher than that of 11C-PBR28 (P = 0.016). CONCLUSION: Our EAE results imply that FR-ß may be a useful target for in vivo imaging of multiple sclerosis-related immunopathology. FR-ß-targeted PET imaging with 18F-FOL may facilitate the monitoring of lesion development and complement the information obtained from TSPO imaging by bringing more specificity to the PET imaging armamentarium for neuroinflammation.

Imaging B Cells in a Mouse Model of Multiple Sclerosis Using 64Cu-Rituximab PET.

B lymphocytes are a key pathologic feature of multiple sclerosis (MS) and are becoming an important therapeutic target for this condition. Currently, there is no approved technique to noninvasively visualize B cells in the central nervous system (CNS) to monitor MS disease progression and response to therapies. Here, we evaluated 64Cu-rituximab, a radiolabeled antibody specifically targeting the human B cell marker CD20, for its ability to image B cells in a mouse model of MS using PET. Methods: To model CNS infiltration by B cells, experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice that express human CD20 on B cells. EAE mice were given subcutaneous injections of myelin oligodendrocyte glycoprotein fragment1-125 emulsified in complete Freund adjuvant. Control mice received complete Freund adjuvant alone. PET imaging of EAE and control mice was performed 1, 4, and 19 h after 64Cu-rituximab administration. Mice were perfused and sacrificed after the final PET scan, and radioactivity in dissected tissues was measured with a γ-counter. CNS tissues from these mice were immunostained to quantify B cells or were further analyzed via digital autoradiography. Results: Lumbar spinal cord PET signal was significantly higher in EAE mice than in controls at all evaluated time points (e.g., 1 h after injection: 5.44 ± 0.37 vs. 3.33 ± 0.20 percentage injected dose [%ID]/g, P < 0.05). 64Cu-rituximab PET signal in brain regions ranged between 1.74 ± 0.11 and 2.93 ± 0.15 %ID/g for EAE mice, compared with 1.25 ± 0.08 and 2.24 ± 0.11 %ID/g for controls (P < 0.05 for all regions except striatum and thalamus at 1 h after injection). Similarly, ex vivo biodistribution results revealed notably higher 64Cu-rituximab uptake in the brain and spinal cord of huCD20tg EAE, and B220 immunostaining verified that increased 64Cu-rituximab uptake in CNS tissues corresponded with elevated B cells. Conclusion: B cells can be detected in the CNS of EAE mice using 64Cu-rituximab PET. Results from these studies warrant further investigation of 64Cu-rituximab in EAE models and consideration of use in MS patients to evaluate its potential for detecting and monitoring B cells in the progression and treatment of this disease. These results represent an initial step toward generating a platform to evaluate B cell-targeted therapeutics en route to the clinic.

Widespread cortical demyelination of both hemispheres can be induced by injection of pro-inflammatory cytokines via an implanted catheter in the cortex of MOG-immunized rats.

Cortical demyelination is a common finding in patients with chronic multiple sclerosis (MS) and contributes to disease progression and overall disability. The exact pathomechanism that leads to cortical lesions is not clear. Research is limited by the fact that standard animal models of multiple sclerosis do not commonly affect the cortex, or if they do in some variants, the cortical demyelination is rather sparse and already remyelinated within a few days. In an attempt to overcome these limitations we implanted a tissue-compatible catheter into the cortex of Dark Agouti rats. After 14days the rats were immunized with 5µg myelin oligodendrocyte glycoprotein (MOG) in incomplete Freund's Adjuvant, which did not cause any clinical signs but animals developed a stable anti-MOG antibody titer. Then the animals received an injection of proinflammatory cytokines through the catheter. This led to a demyelination of cortical and subcortical areas starting from day 1 in a cone-like pattern spreading from the catheter area towards the subarachnoid space. On day 3 cortical demyelination already expanded to the contralateral hemisphere and reached its peak between days 9-15 after cytokine injection with a widespread demyelination of cortical and subcortical areas of both hemispheres. Clinically the animals showed only discrete signs of fatigue and recovered completely after day 15. Even on day 30 we still were able to detect demyelination in subpial and intracortical areas along with areas of partial and complete remyelination. Loss of cortical myelin was accompanied with marked microglia activation. A second injection of cytokines through the catheter on day 30 led to a second demyelination phase with the same symptoms, but again no detectable motor dysfunction. Suffering of the animals appeared minor compared to standard Experimental Autoimmune Encephalomyelitis and therefore, even long-term observation and repeated demyelination phases seem ethically acceptable.

Evaluation of a radiolabelled peripheral benzodiazepine receptor ligand in the central nervous system inflammation of experimental autoimmune encephalomyelitis: a possible probe for imaging multiple sclerosis.

PURPOSE: Peripheral benzodiazepine receptors (PBRs) are upregulated on macrophages and activated microglia, and radioligands for the PBRs can be used to detect in vivo neuroinflammatory changes in a variety of neurological insults, including multiple sclerosis. Substituted 2-phenyl imidazopyridine-3-acetamides with high affinity and selectivity for PBRs have been prepared that are suitable for radiolabelling with a number of positron emission tomography and single-photon emission computed tomography (SPECT) isotopes. In this investigation, the newly developed high-affinity PBR ligand 6-chloro-2-(4'-iodophenyl)-3-(N,N-diethyl)imidazo[1,2-a]pyridine-3-acetamide, or CLINDE, was radiolabelled with 123I and its biodistribution in the central nervous system (CNS) of rats with experimental autoimmune encephalomyelitis (EAE) evaluated. METHODS: EAE was induced in male Lewis rats by injection of an emulsion of myelin basic protein and incomplete Freund's adjuvant containing Mycobacterium butyricum. Biodistribution studies with 123I-CLINDE were undertaken on EAE rats exhibiting different clinical disease severity and compared with results in controls. Disease severity was confirmed by histopathology in the spinal cord of rats. The relationship between inflammatory lesions and PBR ligand binding was investigated using ex vivo autoradiography and immunohistochemistry on rats with various clinical scores. RESULTS: 123I-CLINDE uptake was enhanced in the CNS of all rats exhibiting EAE when compared to controls. Binding reflected the ascending nature of EAE inflammation, with lumbar/sacral cord>thoracic cord>cervical cord>medulla. The amount of ligand binding also reflected the clinical severity of disease. Ex vivo autoradiography and immunohistochemistry revealed a good spatial correspondence between radioligand signal and foci of inflammation and in particular ED-1+ cells representing macrophages and microglia. CONCLUSION: These results demonstrate the ability of 123I-CLINDE to measure in vivo inflammatory events represented by increased density of PBRs and suggest that 123I-CLINDE warrants further investigation as a potential SPECT marker for imaging of CNS inflammation.