Lack of Neuronal IFN-ß-IFNAR Causes Lewy Body- and Parkinson's Disease-like Dementia.

Neurodegenerative diseases have been linked to inflammation, but whether altered immunomodulation plays a causative role in neurodegeneration is not clear. We show that lack of cytokine interferon-ß (IFN-ß) signaling causes spontaneous neurodegeneration in the absence of neurodegenerative disease-causing mutant proteins. Mice lacking Ifnb function exhibited motor and cognitive learning impairments with accompanying α-synuclein-containing Lewy bodies in the brain, as well as a reduction in dopaminergic neurons and defective dopamine signaling in the nigrostriatal region. Lack of IFN-ß signaling caused defects in neuronal autophagy prior to α-synucleinopathy, which was associated with accumulation of senescent mitochondria. Recombinant IFN-ß promoted neurite growth and branching, autophagy flux, and α-synuclein degradation in neurons. In addition, lentiviral IFN-ß overexpression prevented dopaminergic neuron loss in a familial Parkinson's disease model. These results indicate a protective role for IFN-ß in neuronal homeostasis and validate Ifnb mutant mice as a model for sporadic Lewy body and Parkinson's disease dementia.

IFN-ß rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1.

Mitochondrial homeostasis is essential for providing cellular energy, particularly in resource-demanding neurons, defects in which cause neurodegeneration, but the function of interferons (IFNs) in regulating neuronal mitochondrial homeostasis is unknown. We found that neuronal IFN-ß is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN-ß induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN-ß signaling then recruits Drp1 to mitochondria, oligomerizes it, and engages INF2 to stabilize mitochondria-endoplasmic reticulum (ER) platforms. This process tethers damaged mitochondria to the ER to separate them via fission. Lack of neuronal IFN-ß in the Ifnb-/- model of Parkinson disease (PD) disrupts STAT5-PGAM5-Drp1 signaling, impairing fission and causing large multibranched, damaged mitochondria with insufficient ATP production and excessive oxidative stress to accumulate. In other PD models, IFN-ß rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN-ß activates mitochondrial fission in neurons through the pSTAT5/PGAM5/S622 Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism.

Inhibiting metabotropic glutamate receptor 5 after stroke restores brain function and connectivity.

Stroke results in local neural disconnection and brain-wide neuronal network dysfunction leading to neurological deficits. Beyond the hyper-acute phase of ischaemic stroke, there is no clinically-approved pharmacological treatment that alleviates sensorimotor impairments. Functional recovery after stroke involves the formation of new or alternative neuronal circuits including existing neural connections. The type-5 metabotropic glutamate receptor (mGluR5) has been shown to modulate brain plasticity and function and is a therapeutic target in neurological diseases outside of stroke. We investigated whether mGluR5 influences functional recovery and network reorganization rodent models of focal ischaemia. Using multiple behavioural tests, we observed that treatment with negative allosteric modulators (NAMs) of mGluR5 (MTEP, fenobam and AFQ056) for 12 days, starting 2 or 10 days after stroke, restored lost sensorimotor functions, without diminishing infarct size. Recovery was evident within hours after initiation of treatment and progressed over the subsequent 12 days. Recovery was prevented by activation of mGluR5 with the positive allosteric modulator VU0360172 and accelerated in mGluR5 knock-out mice compared with wild-type mice. After stroke, multisensory stimulation by enriched environments enhanced recovery, a result prevented by VU0360172, implying a role of mGluR5 in enriched environment-mediated recovery. Additionally, MTEP treatment in conjunction with enriched environment housing provided an additive recovery enhancement compared to either MTEP or enriched environment alone. Using optical intrinsic signal imaging, we observed brain-wide disruptions in resting-state functional connectivity after stroke that were prevented by mGluR5 inhibition in distinct areas of contralesional sensorimotor and bilateral visual cortices. The levels of mGluR5 protein in mice and in tissue samples of stroke patients were unchanged after stroke. We conclude that neuronal circuitry subserving sensorimotor function after stroke is depressed by a mGluR5-dependent maladaptive plasticity mechanism that can be restored by mGluR5 inhibition. Post-acute stroke treatment with mGluR5 NAMs combined with rehabilitative training may represent a novel post-acute stroke therapy.

Impaired oligodendrogenesis in the white matter of aged mice following diffuse traumatic brain injury.

Senescence is a negative prognostic factor for outcome and recovery following traumatic brain injury (TBI). TBI-induced white matter injury may be partially due to oligodendrocyte demise. We hypothesized that the regenerative capacity of oligodendrocyte precursor cells (OPCs) declines with age. To test this hypothesis, the regenerative capability of OPCs in young [(10 weeks ±2 (SD)] and aged [(62 weeks ±10 (SD)] mice was studied in mice subjected to central fluid percussion injury (cFPI), a TBI model causing widespread white matter injury. Proliferating OPCs were assessed by immunohistochemistry for the proliferating cell nuclear antigen (PCNA) marker and labeled by 5-ethynyl-2'-deoxyuridine (EdU) administered daily through intraperitoneal injections (50 mg/kg) from day 2 to day 6 after cFPI. Proliferating OPCs were quantified in the corpus callosum and external capsule on day 2 and 7 post-injury (dpi). The number of PCNA/Olig2-positive and EdU/Olig2-positive cells were increased at 2dpi (p < .01) and 7dpi (p < .01), respectively, in young mice subjected to cFPI, changes not observed in aged mice. Proliferating Olig2+/Nestin+ cells were less common (p < .05) in the white matter of brain-injured aged mice, without difference in proliferating Olig2+/PDGFRα+ cells, indicating a diminished proliferation of progenitors with different spatial origin. Following TBI, co-staining for EdU/CC1/Olig2 revealed a reduced number of newly generated mature oligodendrocytes in the white matter of aged mice when compared to the young, brain-injured mice (p < .05). We observed an age-related decline of oligodendrogenesis following experimental TBI that may contribute to the worse outcome of elderly patients following TBI.

Cerebrospinal fluid levels of neuroinflammatory biomarkers are increased in athletes with persistent post-concussive symptoms following sports-related concussion.

A sports-related concussion (SRC) is often caused by rapid head rotation at impact, leading to shearing and stretching of axons in the white matter and initiation of secondary inflammatory processes that may exacerbate the initial injury. We hypothesized that athletes with persistent post-concussive symptoms (PPCS) display signs of ongoing neuroinflammation, as reflected by altered profiles of cerebrospinal fluid (CSF) biomarkers, in turn relating to symptom severity. We recruited athletes with PPCS preventing sports participation as well as limiting work, school and/or social activities for ≥ 6 months for symptom rating using the Sport Concussion Assessment Tool, version 5 (SCAT-5) and for cognitive assessment using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). Following a spinal tap, we analysed 27 CSF inflammatory biomarkers (pro-inflammatory chemokines and cytokine panels) by a multiplex immunoassay using antibodies as electrochemiluminescent labels to quantify concentrations in PPCS athletes, and in healthy age- and sex-matched controls exercising ≤ 2 times/week at low-to-moderate intensity. Thirty-six subjects were included, 24 athletes with PPCS and 12 controls. The SRC athletes had sustained a median of five concussions, the most recent at a median of 17 months prior to the investigation. CSF cytokines and chemokines levels were significantly increased in eight (IL-2, TNF-α, IL-15, TNF-ß, VEGF, Eotaxin, IP-10, and TARC), significantly decreased in one (Eotaxin-3), and unaltered in 16 in SRC athletes when compared to controls, and two were un-detectable. The SRC athletes reported many and severe post-concussive symptoms on SCAT5, and 10 out of 24 athletes performed in the impaired range (Z < - 1.5) on cognitive testing. Individual biomarker concentrations did not strongly correlate with symptom rating or cognitive function. Limitations include evaluation at a single post-injury time point in relatively small cohorts, and no control group of concussed athletes without persisting symptoms was included. Based on CSF inflammatory marker profiling we find signs of ongoing neuroinflammation persisting months to years after the last SRC in athletes with persistent post-concussive symptoms. Since an ongoing inflammatory response may exacerbate the brain injury these results encourage studies of treatments targeting the post-injury inflammatory response in sports-related concussion.

Intracerebral Proinflammatory Cytokine Increase in Surgically Evacuated Intracerebral Hemorrhage: A Microdialysis Study.

BACKGROUND: Treatment options for spontaneous intracerebral hemorrhage (ICH) are limited. A possible inflammatory response in the brain tissue surrounding an ICH may exacerbate the initial injury and could be a target for treatment of subsequent secondary brain injury. The study objective was to compare levels of inflammatory mediators in the interstitial fluid of the perihemorrhagic zone (PHZ) and in seemingly normal cortex (SNX) in the acute phase after surgical evacuation of ICH, with the hypothesis being that a difference could be demonstrated between the PHZ and the SNX. METHODS: In this observational study, ten patients needing surgical evacuation of supratentorial ICH received two cerebral microdialysis catheters: one in the PHZ and one in the SNX that is remote from the ICH. The microdialysate was analyzed for energy metabolites (including lactate pyruvate ratio and glucose) and for inflammatory mediators by using a multiplex immunoassay of 27 cytokines and chemokines at 6-10 h, 20-26 h, and 44-50 h after surgery. RESULTS: A metabolic crisis, indicated by altered energy metabolic markers, that persisted throughout the observation period was observed in the PHZ when compared with the SNX. Proinflammatory cytokines interleukin (IL) 8, tumor necrosis factor α, IL-2, IL-1ß, IL-6 and interferon γ, anti-inflammatory cytokine IL-13, IL-4, and vascular endothelial growth factor A were significantly higher in PHZ compared with SNX and were most prominent at 20-26 h following ICH evacuation. CONCLUSIONS: Higher levels of both proinflammatory and anti-inflammatory cytokines in the perihemorrhagic brain tissue implies a complex role for inflammatory mediators in the secondary injury cascades following ICH surgery, suggesting a need for targeted pharmacological interventions.

A Novel In Vivo Model for Multiplexed Analysis of Callosal Connections upon Cortical Damage.

Brain damage is the major cause of permanent disability and it is particularly relevant in the elderly. While most studies focused on the immediate phase of neuronal loss upon injury, much less is known about the process of axonal regeneration after damage. The development of new refined preclinical models to investigate neuronal regeneration and the recovery of brain tissue upon injury is a major unmet challenge. Here, we present a novel experimental paradigm in mice that entails the (i) tracing of cortico-callosal connections, (ii) a mechanical lesion of the motor cortex, (iii) the stereological and histological analysis of the damaged tissue, and (iv) the functional characterization of motor deficits. By combining conventional microscopy with semi-automated 3D reconstruction, this approach allows the analysis of fine subcellular structures, such as axonal terminals, with the tridimensional overview of the connectivity and tissue integrity around the lesioned area. Since this 3D reconstruction is performed in serial sections, multiple labeling can be performed by combining diverse histological markers. We provide an example of how this methodology can be used to study cellular interactions. Namely, we show the correlation between active microglial cells and the perineuronal nets that envelop parvalbumin interneurons. In conclusion, this novel experimental paradigm will contribute to a better understanding of the molecular and cellular interactions underpinning the process of cortical regeneration upon brain damage.

Plasticity-Enhancing Effects of Levodopa Treatment after Stroke.

Dopaminergic treatment in combination with rehabilitative training enhances long-term recovery after stroke. However, the underlying mechanisms on structural plasticity are unknown. Here, we show an increased dopaminergic innervation of the ischemic territory during the first week after stroke induced in Wistar rats subjected to transient occlusion of the middle cerebral artery (tMCAO) for 120 min. This response was also found in rats subjected to permanent focal ischemia induced by photothrombosis (PT) and mice subjected to PT or tMCAO. Dopaminergic branches were detected in the infarct core of mice and rats in both stroke models. In addition, the Nogo A pathway was significantly downregulated in rats treated with levodopa (LD) compared to vehicle-treated animals subjected to tMCAO. Specifically, the number of Nogo A positive oligodendrocytes as well as the levels of Nogo A and the Nogo A receptor were significantly downregulated in the peri-infarct area of LD-treated animals, while the number of Oligodendrocyte transcription factor 2 positive cells increased in this region after treatment. In addition, we observed lower protein levels of Growth Associated Protein 43 in the peri-infarct area compared to sham-operated animals without treatment effect. The results provide the first evidence of the plasticity-promoting actions of dopaminergic treatment following stroke.

Interleukin-1 Beta Neutralization Attenuates Traumatic Brain Injury-Induced Microglia Activation and Neuronal Changes in the Globus Pallidus.

Traumatic brain injury (TBI) increases the risk of delayed neurodegenerative processes, including Parkinson's disease (PD). Interleukin-1beta (IL-1ß), a key pro-inflammatory cytokine, may promote secondary injury development after TBI. Conversely, neutralizing IL-1ß was found to improve functional recovery following experimental TBI. However, the mechanisms underlying the behavioral improvements observed by IL-1ß neutralization are still poorly understood. The present study investigated the role of IL-1ß on the microglia response and neuronal changes in the globus pallidus in response to diffuse TBI. Mice were subjected to sham injury or the central fluid percussion injury (cFPI) (a model of traumatic axonal injury), and were randomly administered an IL-1ß neutralizing or a control antibody at 30 min post-injury. The animals were analyzed at 2, 7, or 14 days post-injury. When compared to controls, mice subjected to cFPI TBI had increased microglia activation and dopaminergic innervation in the globus pallidus, and a decreased number of parvalbumin (PV) positive interneurons in the globus pallidus. Neutralization of IL-1ß attenuated the microglia activation, prevented the loss of PV+ interneurons and normalized dopaminergic fiber density in the globus pallidus of brain-injured animals. These findings argue for an important role for neuro-inflammation in the PD-like pathology observed in TBI.

Neuroprotective dobutamine treatment upregulates superoxide dismutase 3, anti-oxidant and survival genes and attenuates genes mediating inflammation.

BACKGROUND: Labor subjects the fetus to an hypoxic episode and concomitant adrenomodullary catecholamine surge that may provide protection against the hypoxic insult. The beta1-adrenergic agonist dobutamine protects against hypoxia/aglycemia induced neuronal damage. We aimed to identify the associated protective biological processes involved. RESULTS: Hippocampal slices from 6 days old mice showed significant changes of gene expression comparing slices with or without dobutamine (50 mM) in the following two experimental paradigms: (1) control conditions versus lipopolysacharide (LPS) stimulation and (2) oxygen-glucose deprivation (OGD), versus combined LPS/OGD. Dobutamine depressed the inflammatory response by modifying the toll-like receptor-4 signalling pathways, including interferon regulatory factors and nuclear factor κ B activation in experimental paradigm 1. The anti-oxidant defense genes superoxide dismutase 3 showed an upregulation in the OGD paradigm while thioredoxin reductase was upregulated in LPS paradigm. The survival genes Bag-3, Tinf2, and TMBIM-1, were up-regulated in paradigm 1. Moreover, increased levels of SOD3 were verified on the protein level 24 h after OGD and control stimulation in cultures with or without preconditioning with LPS and dobutamine, respectively. CONCLUSIONS: Neuroprotective treatment with dobutamine depresses expression of inflammatory mediators and promotes the defense against oxidative stress and depresses apoptotic genes in a model of neonatal brain hypoxia/ischemia interpreted as pharmacological preconditioning. We conclude that beta1-adrenoceptor activation might be an efficient strategy for identifying novel pharmacological targets for protection of the neonatal brain.

CX3C chemokine receptor 1 deficiency modulates microglia morphology but does not affect lesion size and short-term deficits after experimental stroke.

BACKGROUND: The fractalkine/CX3C chemokine receptor 1 (CX3CR1) pathway has been identified to play an essential role in the chemotaxis of microglia, leukocyte trafficking and microglia/macrophage recruitment. It has also been shown to be important in the regulation of the inflammatory response in the early phase after experimental stroke. The present study was performed to investigate if CX3CR1 deficiency affects microglia during the first 14 days with consequences for tissue damage after experimental stroke. RESULTS: CX3CR1 deficiency significantly increased the number of intersections of GFP positive microglia in the proximal peri-infarct area at 2, 7 and 14 days following tMCAO compared to heterozygous and wildtype littermates. In addition, the length of microglial branches increased until day 7 in CX3CR1 knockout mice while the presence of a functional CX3CR1 allele resulted in a gradual reduction of their length following tMCAO. After stroke, wildtype, heterozygous and CX3CR1 deficient mice did not show differences in the composite neuroscore and assessment of infarct volumes from CX3CR1 wildtype, heterozygous and deficient mice revealed no differences between the genotypes 7 and 14 days after stroke. CONCLUSION: Results demonstrate that CX3CR1 deficiency affects the morphology of GFP positive microglia located in the proximal peri-infarct region during the first 14 days after tMCAO. Our data also indicate that CX3CR1 deficiency does not affect definite infarct volumes. Modulation of the CX3CR1 pathway may have implication for microglia function contributing to mechanisms of tissue reorganization in the post-ischemic brain.

Treatment with AMD3100 attenuates the microglial response and improves outcome after experimental stroke.

BACKGROUND: Recovery of lost neurological function after stroke is limited and dependent on multiple mechanisms including inflammatory processes. Selective pharmacological modulation of inflammation might be a promising approach to improve stroke outcome. METHODS: We used 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (AMD3100), an antagonist to the C-X-C chemokine receptor type 4 (CXCR4) and potential allosteric agonist to CXCR7, administered to mice twice daily from day 2 after induction of photothrombosis (PT). In addition to functional outcome, the dynamics of post-stroke microglia response were monitored in vivo by 2-photon-laser-microscopy in heterozygous transgenic CX3CR1-green fluorescent protein (GFP) mice (CX3CR1(GFP/+)) and complemented with analyses for fractalkine (FKN) and pro-inflammatory cytokines. RESULTS: We found a significantly enhanced recovery and modified microglia activation without affecting infarct size in mice treated with AMD3100 after PT. AMD3100 treatment significantly reduced the number of microglia in the peri-infarct area accompanied by stabilization of soma size and ramified cell morphology. Within the ischemic infarct core of AMD3100 treated wild-type mice we obtained significantly reduced levels of the endogenous CX3CR1 ligand FKN and the pro-inflammatory cytokines interleukin (IL)-1ß and IL-6. Interestingly, in CX3CR1-deficient mice (homozygous transgenic CX3CR1-GFP mice) subjected to PT, the levels of FKN were significantly lower compared to their wild-type littermates. Moreover, AMD3100 treatment did not induce any relevant changes of cytokine levels in CX3CR1 deficient mice. CONCLUSION: After AMD3100 treatment, attenuation of microglia activation contributes to enhanced recovery of lost neurological function in experimental stroke possibly due to a depression of FKN levels in the brain. We further hypothesize that this mechanism is dependent on a functional receptor CX3CR1.

The involvement of the sigma-1 receptor in neurodegeneration and neurorestoration.

The sigma-1 receptor (Sig-1R) is a single 25 kD polypeptide and a chaperone protein immersed in lipid rafts of the endoplasmic reticulum (ER) where it interacts with mitochondria at the mitochondria-associated ER membrane domain (MAM). Upon activation, the Sig-1R binds to the inositol trisphosphate receptor (IP3R), and modulates cellular calcium (Ca(2+)) homeostasis. Also, the activated Sig-1R modulates plasma membrane receptor and ion channel functions, and may regulate cellular excitability. Further, the Sig-1R promotes trafficking of lipids and proteins essential for neurotransmission, cell growth and motility. Activation of the Sig-1R provides neuroprotection and is neurorestorative in cellular and animal models of neurodegenerative diseases and brain ischaemia. Neuroprotection appears to be due to inhibition of cellular Ca(2+) toxicity and/or inflammation, and neurorestoration may include balancing abberant neurotransmission or stimulation of synaptogenesis, thus remodelling brain connectivity. Single nucleotide polymorphisms and mutations of the SIGMAR1 gene worsen outcome in Alzheimer's disease and myotrophic lateral sclerosis supporting a role of Sig-1R in neurodegenerative disease. The combined neuroprotective and neurorestorative actions of the Sig-1R, provide a broad therapeutic time window of Sig-1R agonists. The Sig-1R is therefore a strong therapeutic target for the development of new treatments for neurodegenerative diseases and stroke.

Pharmacological stimulation of sigma-1 receptors has neurorestorative effects in experimental parkinsonism.

The sigma-1 receptor, an endoplasmic reticulum-associated molecular chaperone, is attracting great interest as a potential target for neuroprotective treatments. We provide the first evidence that pharmacological modulation of this protein produces functional neurorestoration in experimental parkinsonism. Mice with intrastriatal 6-hydroxydopamine lesions were treated daily with the selective sigma-1 receptor agonist, PRE-084, for 5 weeks. At the dose of 0.3 mg/kg/day, PRE-084 produced a gradual and significant improvement of spontaneous forelimb use. The behavioural recovery was paralleled by an increased density of dopaminergic fibres in the most denervated striatal regions, by a modest recovery of dopamine levels, and by an upregulation of neurotrophic factors (BDNF and GDNF) and their downstream effector pathways (extracellular signal regulated kinases 1/2 and Akt). No treatment-induced behavioural-histological restoration occurred in sigma-1 receptor knockout mice subjected to 6-hydroxydopamine lesions and treated with PRE-084. Immunoreactivity for the sigma-1 receptor protein was evident in both astrocytes and neurons in the substantia nigra and the striatum, and its intracellular distribution was modulated by PRE-084 (the treatment resulted in a wider intracellular distribution of the protein). Our results suggest that sigma-1 receptor regulates endogenous defence and plasticity mechanisms in experimental parkinsonism. Boosting the activity of this protein may have disease-modifying effects in Parkinson's disease.

Can diffusion kurtosis imaging improve the sensitivity and specificity of detecting microstructural alterations in brain tissue chronically after experimental stroke? Comparisons with diffusion tensor imaging and histology.

Imaging techniques that provide detailed insights into structural tissue changes after stroke can vitalize development of treatment strategies and diagnosis of disease. Diffusion-weighted MRI has been playing an important role in this regard. Diffusion kurtosis imaging (DKI), a recent addition to this repertoire, has opened up further possibilities in extending our knowledge about structural tissue changes related to injury as well as plasticity. In this study we sought to discern the microstructural alterations characterized by changes in diffusion tensor imaging (DTI) and DKI parameters at a chronic time point after experimental stroke. Of particular interest was the question of whether DKI parameters provide additional information in comparison to DTI parameters in understanding structural tissue changes, and if so, what their histological origins could be. Region-of-interest analysis and a data-driven approach to identify tissue abnormality were adopted to compare DTI- and DKI-based parameters in post mortem rat brain tissue, which were compared against immunohistochemistry of various cellular characteristics. The unilateral infarcted area encompassed the ventrolateral cortex and the lateral striatum. Results from region-of-interest analysis in the lesion borderzone and contralateral tissue revealed significant differences in DTI and DKI parameters between ipsi- and contralateral sensorimotor cortex, corpus callosum, internal capsule and striatum. This was reflected by a significant reduction in ipsilateral mean diffusivity (MD) and fractional anisotropy (FA) values, accompanied by significant increases in kurtosis parameters in these regions. Data-driven analysis to identify tissue abnormality revealed that the use of kurtosis-based parameters improved the detection of tissue changes in comparison with FA and MD, both in terms of dynamic range and in being able to detect changes to which DTI parameters were insensitive. This was observed in gray as well as white matter. Comparison against immunohistochemical stainings divulged no straightforward correlation between diffusion-based parameters and individual neuronal, glial or inflammatory tissue features. Our study demonstrates that DKI allows sensitive detection of structural tissue changes that reflect post-stroke tissue remodeling. However, our data also highlights the generic difficulty in unambiguously asserting specific causal relationships between tissue status and MR diffusion parameters.

Enriched housing down-regulates the Toll-like receptor 2 response in the mouse brain after experimental stroke.

Post-ischemic inflammation plays an important role in the evolution of brain injury, recovery and repair after stroke. Housing rodents in an enriched environment provides multisensory stimulation to the brain and enhances functional recovery after experimental stroke, also depressing the release of cytokines and chemokines in the peri-infarct. In order to identify targets for late stroke treatment, we studied the dynamics of inflammation and the contribution of resident Toll-like receptor 2 (TLR2) expressing microglia cells. We took advantage of the biophotonic/bioluminescent imaging technique using the reporter mouse-expressing luciferase and GFP reporter genes under transcriptional control of the murine TLR2 promoter (TLR2-luc/GFP mice) for non-invasive in vivo analysis of TLR2 activation/response in photothrombotic stroke after differential housing. Real-time imaging at 1day after stroke, revealed up-regulation of TLR2 in response to photothrombotic stroke that subsequently declined over time of recovery (14days). The inflammatory response was persistently down-regulated within days of enriched housing, enhancing recovery of lost sensori-motor function in TLR2-luc mice without affecting infarct size. The number of YM1-expressing microglia in the peri-infarct and areas remote from the infarct was also markedly attenuated. Using a live imaging approach, we demonstrate that multisensory stimulation rapidly, persistently and generally attenuates brain inflammation after experimental stroke, reducing the TLR2 response and leading to improved neurological outcome. TLR2-expressing microglia cells may provide targets for new stroke therapeutics.

Reduction of rat brain CD8+ T-cells by levodopa/benserazide treatment after experimental stroke.

The activation of inflammatory cascades in the ischemic hemisphere impairs mechanisms of tissue reorganization with consequences for recovery of lost neurological function. Recruitment of T-cell populations to the post-ischemic brain occurs and represents a significant part of the inflammatory response. This study was conducted to investigate if treatment with levodopa, potentially acting as an immunomodulator, affects the T-cell accumulation in the post-ischemic brain. Male Sprague-Dawley rats were subjected to transient occlusion of the middle cerebral artery (tMCAO) for 105 min followed by levodopa/benserazide treatment (20 mg/kg/15 mg/kg) for 5 days initiated on day 2 post-stroke. One week after tMCAO, T-cell populations were analysed from brains, and levels of interleukin (IL)-1ß, chemokine (C-X-C motif) ligand 1, IL-4, IL-5, interferon gamma and IL-13 were analysed. After levodopa/benserazide treatment, we found a significant reduction of cytotoxic T-cells (CD3+ CD8+ ) in the ischemic hemisphere together with reduced levels of T-cell-associated cytokine IL-5, while other T-cell populations (CD3+, CD3+ CD4+, CD3+ CD4+ CD25+) were unchanged compared with vehicle-treated rats. Moreover, a reduced number of cells was associated with reduced levels of intercellular adhesion molecule 1, expressed in endothelial cells, in the infarct core of levodopa/benserazide-treated animals. Together, we provide the first evidence that dopamine can act as a potential immunomodulator by attenuating inflammation in the post-ischemic brain.

Dynamics of major histocompatibility complex class II-positive cells in the postischemic brain--influence of levodopa treatment.

BACKGROUND: Cerebral ischemia activates both the innate and the adaptive immune response, the latter being activated within days after the stroke onset and triggered by the recognition of foreign antigens. METHODS: In this study we have investigated the phenotype of antigen presenting cells and the levels of associated major histocompatibility complex class II (MHC II) molecules in the postischemic brain after transient occlusion of the middle cerebral artery (tMCAO) followed by levodopa/benserazide treatment. Male Sprague Dawley rats were subjected to tMCAO for 105 minutes and received levodopa (20 mg/kg)/benserazide (15 mg/kg) for 5 days starting on day 2 after tMCAO. Thereafter, immune cells were isolated from the ischemic and contralateral hemisphere and analyzed by flow cytometry. Complementarily, the spatiotemporal profile of MHC II-positive (MHC II(+)) cells was studied in the ischemic brain during the first 30 days after tMCAO; protein levels of MHC II and the levels of inflammation associated cytokines were determined in the ischemic hemisphere. RESULTS: We found that microglia/macrophages represent the main MHC II expressing cell in the postischemic brain one week after tMCAO. No differences in absolute cell numbers were found between levodopa/benserazide and vehicle-treated animals. In contrast, MHC II protein levels were significant downregulated in the ischemic infarct core by levodopa/benserazide treatment. This reduction was accompanied by reduced levels of IFN-γ, TNF-α and IL-4 in the ischemic hemisphere. In the contralateral hemisphere, we exclusively detected MHC II(+) cells in the corpus callosum. Interestingly, the number of cells was increased by treatment with levodopa/benserazide independent from the infarct size 14 days after tMCAO. CONCLUSIONS: Results suggest that dopamine signaling is involved in the adaptive immune response after stroke and involves microglia/macrophages.

Non-toxic amyloid beta formed in the presence of glypican-1 or its deaminatively generated heparan sulfate degradation products.

The amyloid beta (Aß) peptides (mainly Aß40 and Aß42), which are derived from the amyloid precursor protein (APP), can oligomerize into antibody A11-positive, neurotoxic species, believed to be involved in Alzheimer's disease. Interestingly, APP binds strongly to the heparan sulfate (HS) proteoglycan (PG) glypican-1 (Gpc-1) in vitro and both proteins are colocalized inside cells. In endosomes, APP is proteolytically processed to yield Aß peptides. The HS chains of S-nitrosylated (SNO) Gpc-1 PG are cleaved into anhydromannose (anMan)-containing di- and oligosaccharides by an NO-dependent reaction in the same compartments. Here, we have studied the toxicity of oligomers/aggregates of Aß40 and Aß42, as well as Aß40/42 mixtures that were formed in the presence of immobilized Gpc-1 PG or immobilized HS oligosaccharides. Afterwards, Aß was displaced from the matrices, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for A11 immunoreactivity, for effects on growth of mouse N2a neuroblastoma cells and for membrane leakage in rat cortical neurons. HS generally promoted and accelerated Aß multimerization into oligomers as well as larger aggregates that were mostly A11 positive and showed toxic effects. However, non-toxic Aß was formed in the presence of Gpc-1 PG or when anMan-containing HS degradation products were simultaneously generated. Both toxic and non-toxic Aß peptides were taken up by the cells but toxic forms appeared to enter the nuclei to a larger extent. The protection afforded by the presence of HS degradation products may reflect a normal intracellular function for the Aß peptides.

Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation.

BACKGROUND: Cerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH. METHODS: Using a preterm rabbit pup model, we investigated the molecular mechanisms and events following IVH. We also characterized the concentrations of cell-free Hb metabolites and pro-inflammatory mediators in the cerebrospinal fluid (CSF) of preterm human infants and rabbit pups. Finally, Hb metabolites were evaluated as causal initiators of inflammation in primary rabbit astrocyte cell cultures. RESULTS: Following IVH in preterm rabbit pups, the intraventricular CSF concentration of cell-free methemoglobin (metHb) increased from 24 to 72 hours and was strongly correlated with the concentration of TNFα at 72 hours (r2 = 0.896, P <0.001). Also, the mRNA expression of TNFα, IL-1ß, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining). Furthermore, exposure of primary rabbit astrocyte cell cultures to metHb caused a dose-dependent increase in TNFα mRNA and protein levels, which was not observed following exposure to oxyhemoglobin (oxyHb) or hemin. Finally, a positive correlation (r2 = 0.237, P <0.03) between metHb and TNFα concentrations was observed in the CSF of preterm human infants following IVH. CONCLUSIONS: Following preterm IVH, increased metHb formation in the intraventricular space induces expression of pro-inflammatory cytokines. Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH. Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.