Oxidative damage and chemokine production dominate days before immune cell infiltration and EAE disease debut

BACKGROUND: Multiple sclerosis is widely accepted as an inflammatory disease. However, studies indicate that degenerative processes in the CNS occur prior to inflammation. In the widely used animal model experimental autoimmune encephalomyelitis (EAE), we investigated the significance of degenerative processes from mitochondrial membrane potentials, reactive oxidative species, cell death markers, chemokines, and inflammatory cell types in brain, spinal cord, and optic nerve tissue during the effector phase of the disease, before clinical disease was evident. METHODS: Sixty-two rats were placed in eight groups, n = 6 to 10. Four groups were immunized with spinal cord homogenate emulsified in complete Freund’s adjuvant (one served as EAE group), three groups were immunized with complete Freund’s adjuvant only, and a control group was injected with phosphate buffered saline only. Groups were sacrificed 3, 5, 7, or 12–13 days after the intervention and analyzed for early signs of CNS degeneration. RESULTS: Loss of mitochondrial membrane potential and oxidative changes was observed days before clinical disease debut at day 9.75 ± 0.89. The early mitochondrial changes were not associated with cytochrome C release, cleavage of caspases 9 (38/40 kDa) and 3 (17/19 kDa), and cleavage of PARP (89 kDa) or spectrin (120/150 kDa), and apoptosis was not initiated. Axonal degeneration was only present at disease onset. Increases in a range of cytokines and chemokines were observed systemically as a consequence of immunization with complete Freund’s adjuvant, whereas the encephalitogenic emulsion induced an upregulation of the chemokines Ccl2, Ccl20, and Cxcl1, specifically in brain tissue, 7 days after immunization. CONCLUSION: Five to seven days after immunization, subtle decreases in the mitochondrial membrane potential and an increased reactive oxygen species burden in brain tissue were observed. No cell death was detected at these time-points, but a specific expression pattern of chemokines indicates activity in the CNS, several days before clinical disease debut

RhoA Drives T-Cell Activation and Encephalitogenic Potential in an Animal Model of Multiple Sclerosis

T-cells are known to be intimately involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). T-cell activation is controlled by a range of intracellular signaling pathways regulating cellular responses such as proliferation, cytokine production, integrin expression, and migration. These processes are crucial for the T-cells’ ability to mediate inflammatory processes in autoimmune diseases such as MS. RhoA is a ubiquitously expressed small GTPase well described as a regulator of the actin cytoskeleton. It is essential for embryonic development and together with other Rho GTPases controls various cellular processes such as cell development, shaping, proliferation, and locomotion. However, the specific contribution of RhoA to these processes in T-cells in general, and in autoreactive T-cells in particular, has not been fully characterized. Using mice with a T-cell specific deletion of the RhoA gene (RhoAfl/flLckCre+), we investigated the role of RhoA in T-cell development, functionality, and encephalitogenic potential in EAE. We show that lack of RhoA specifically in T-cells results in reduced numbers of mature T-cells in thymus and spleen but normal counts in peripheral blood. EAE induction in RhoAfl/flLckCre+ mice results in significantly reduced disease incidence and severity, which coincides with a reduced CNS T-cell infiltration. Besides presenting reduced migratory capacity, both naïve and autoreactive effector T-cells from RhoAfl/flLckCre+ mice show decreased viability, proliferative capacity, and an activation profile associated with reduced production of Th1 pro-inflammatory cytokines. Our study demonstrates that RhoA is a central regulator of several archetypical T-cell responses, and furthermore points toward RhoA as a new potential therapeutic target in diseases such as MS, where T-cell activity plays a central role

Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials

Oxidative damage and chemokine production dominate days before immune cell infiltration and EAE disease debut

Data files on which the results presented in the manuscript '<a>Oxidative damage and chemokine production dominate days before immune cell infiltration and EAE disease debut' are based.</a

N-acetyl aspartate levels early after ischemic stroke accurately reflect long-term brain damage

Background: Estimation of brain damage following an ischemic stroke is most often performed within the first few days after the insult, where large amounts of oedematous fluid have accumulated. This can potentially hamper correct measurement of infarcted area, since oedema formation poorly reflects infarct size. This study presents a non-invasive, easily applicable and reliable method to accurately predict long-term evolution and late-stage infarction. Objective: We performed a longitudinal analysis of brain infarct evolution after MCAO in mice, in order to determine whether water-compensated N-Acetylaspartate (NAA) levels in the infarct area, measured 24 h after the insult, is a suitable marker for late-stage infarction and thereby prognosis. Methods: Twenty mice were divided into 4 groups and scanned longitudinally at different time-points after MCAO, followed by euthanisation for histology: Group 1) MRI/MRS at day 1 after MCAO (n = 4), Group 2) MRI/MRS at days 1 and 7 after MCAO (n = 5), Group 3) MRI/MRS at days 1, 7, and 14 after MCAO (n = 3), and Group 4) MRI/MRS at days 1, 7, 14, and 28 after MCAO (n = 4). At days 1, 7, 14, and 28, NAA levels were correlated with histological determination of neuronal death based on Nissl and H&E stainings. Results: Twenty-four hours after the insult, NAA levels in the infarcted area decreased by 35 %, but steadily returned to normal after 28 days. In the acute phases, NAA levels strongly correlated with loss of Nissl substance (r2 = −0.874, p = 0.002), whereas NAA levels in later stages reflect glial metabolism and tissue reorganisation. Most importantly, NAA levels 24 h after MCAO was highly correlated with late stage infarction at days 14 and 28 (r2 = 0.73, p = 0.01), in contrast to T2 (r2 = 0.06, p = 0.59). Conclusions: By using a fixed voxel, which is easily positioned in the affected area, it is possible to obtain reliable measures of the extent of neuronal loss at early time points independent of oedema and brain deformation. Importantly, NAA levels 24 h after MCAO accurately reflects late-stage infarction, suggesting that NAA is a useful prognostic biomarker early after an ischemic stroke