Adib Zendedel

Cuprizone-Induced Demyelination as a Tool to Study Remyelination and Axonal Protection

In the brain of multiple sclerosis (MS) patients, the conduction block of axons due to demyelination and inflammation underlies early neurological symptoms, whereas axonal transection accounts for permanent deficits occurring during later disease stages. The beneficial function of myelin for the protection of the axonal compartment and network stability between neurons has been shown in numerous studies. Thus, rapid and adequate remyelination is an important factor for axonal patronage during neuroinflammatory conditions. In this review article, we discuss frequently used experimental in vivo and in vitro animal models to examine remyelination and repair in MS. The focus of the discussion is the relevance of the toxin model ‘cuprizone’ to study the pathology of demyelination and the physiology of remyelination. This also includes recent findings in this animal model which implicate that axonal damage is an ongoing process independent of the initiation of endogenous remyelination.

Omega-3 polyunsaturated fatty acids ameliorate neuroinflammation and mitigate ischemic stroke damage through interactions with astrocytes and microglia

Omega-3 polyunsaturated fatty acids (PUFA n3) provide neuroprotection due to their anti-inflammatory and anti-apoptotic properties as well as their regulatory function on growth factors and neuronal plasticity. These qualities enable PUFA n3 to ameliorate stroke outcome and limit neuronal damage. Young adult male rats received transient middle cerebral artery occlusion (tMCAO). PUFA n3 were intravenously administered into the jugular vein immediately after stroke and 12h later. We analyzed stroke volume and behavioral performance as well as the regulation of functionally-relevant genes in the penumbra. The extent of ischemic damage was reduced and behavioral performance improved subject to applied PUFA n3. Expression of Tau and growth-associated protein-43 genes were likewise restored. Ischemia-induced increase of cytokine mRNA levels was abated by PUFA n3. Using an in vitro approach, we demonstrate that cultured astroglial and microglia directly respond to PUFA n3 administration by preventing ischemia-induced increase of cyclooxygenase 2, hypoxia-inducible factor 1alpha, inducible nitric oxide synthase, and interleukin 1beta. Cultured cortical neurons also appeared as direct targets, since PUFA n3 shifted the Bcl-2-like protein 4 (Bax)/B-cell lymphoma 2 (Bcl 2) ratio towards an anti-apoptotic constellation. Thus, PUFA n3 reveal a high neuroprotective and anti-inflammatory potential in an acute ischemic stroke model by targeting astroglial and microglial function as well as improving neuronal survival strategies. Our findings signify the potential clinical feasibility of PUFA n3 therapeutic treatment in stroke and other acute neurological diseases.

Regulation of Hypoxia-Induced Inflammatory Responses and M1-M2 Phenotype Switch of Primary Rat Microglia by Sex Steroids

Microglia cells are the primary mediators of the CNS immune defense system and crucial for the outcome of shaping inflammatory responses. They are highly dynamic, moving constantly, and become activated by neuronal signaling under pathological conditions. They fulfill a dual role by not only regulating local neuroinflammation but also conferring neuronal protection. Gonadal steroids are known to exert anti-inflammatory effects in the CNS. Recently, we have shown that the microglial-like cell line BV-2 is hypoxia-sensitive and regulated by gonadal steroids. The present study used primary rat cerebral cortex-derived microglia to analyze whether this cell type directly perceive and respond to acute hypoxia. Second, we investigated whether 17β-estradiol (E2) and progesterone (P) interfere with hypoxia-induced changes. Short-term hypoxia increased the expression of a subset of pro-inflammatory (TNFa, IL1b) and oxidative stress-related (Hif1a) genes. The induction of TNFa and IL1b was counteracted by P. Hypoxia shifted the primary microglia to the pro-inflammatory M1 phenotype. The administration of E2 and P favored the neuroprotective M2 phenotype. Our findings extend previous data obtained with BV-2 cells and show that the primary microglia directly perceive hypoxia which increase their inflammatory activity. Both steroid hormones directly and indirectly interact with the microglia cells by reducing the inflammatory scenario and stimulating neuroprotection.

Poststroke Inflammasome Expression and Regulation in the Peri-Infarct Area by Gonadal Steroids after Transient Focal Ischemia in the Rat Brain.

CNS ischemia results in locally confined and rapid tissue damage accompanied by a loss of neurons and their circuits. Early and time-delayed inflammatory responses are critical variables determining the extent of neural disintegration and regeneration. Inflammasomes are vital effectors in innate immunity. Their activation in brain-intrinsic immune cells contributes to ischemia-related brain damage. The steroids 17β-estradiol (E2) and progesterone (P) are neuroprotective and anti-inflammatory. Using a transient focal rat ischemic model, we evaluated the time response of different inflammasomes in the peri-infarct zone from the early to late phases after poststroke ischemia. We show that the different inflammasome complexes reveal a specific time-oriented sequential expression pattern with a maximum at approximately 24 h after the infarct. Within the limits of antibody availability, immunofluorescence labeling demonstrated that microglia and neurons are major sources of the locally activated inflammasomes NOD-like receptor protein-3 (NLRP3) and associated speck-like protein (ASC), respectively. E2 and P given for 24 h immediately after ischemia onset reduced hypoxia-induced mRNA expression of the inflammasomes NLRC4, AIM2 and ASC, and decreased the protein levels of ASC and NLRP3. In addition, mRNA protein levels of the cytokines interleukin-1β (IL1β), IL18 and TNFα were reduced by the steroids. The findings provide for the first time a detailed flow chart of hypoxia-driven inflammasome regulation in the peri-infarct cerebral cortex. Further, we demonstrate that E2 and P alleviate the expression of certain inflammasome components, sometimes in a hormone-specific way. Besides directly regulating other cellular neuroprotective pathways, the control of inflammasomes by these steroids might contribute to its neuroprotective potency.

Inflammasome: its role in traumatic brain and spinal cord injury†

Traumatic brain injury (TBI) and spinal cord injury (SCI) are pathological events that lead to neuropathological conditions which have in consequence the initiation of pro‐inflammatory cytokine production. Neuroinflammation plays a key role in the secondary phase of both TBI and SCI after initial cell death. Activation of cytoplasmic inflammasome complexes is regarded as the essential step of neuroinflammation and a key trigger for neuronal death called pyroptosis. Inflammasome complexes are involved in activation of caspase‐1 which catalyzes the cleavage of pro‐interleukins into their active forms (including interleukin‐18 [IL‐18] and IL‐1β). The focus of this article is to discuss the time‐course and regulation of inflammasome assembly and activation during TBI and SCI and their targeting in designing therapeutic approaches. We particularly focus on the inflammasomes NLRP1 and NLRP3 which play a pivotal function during TBI and SCI in the central nervous system (CNS).

Neurodegeneration Triggers Peripheral Immune Cell Recruitment into the Forebrain

Brain-intrinsic degenerative cascades have been proposed to be an initial factor driving lesion formation in multiple sclerosis (MS). Here, we identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the mouse forebrain. Female C57BL/6 mice were fed cuprizone for 3 weeks, followed by a period of 2 weeks on normal chow to induce the formation of lesion foci in the forebrain. Subsequent immunization with myelin oligodendrocyte glycoprotein 35–55 peptide, which induces myelin autoreactive T cells in the periphery, resulted in massive immune cell recruitment into the affected forebrain. Additional adoptive transfer experiments together with flow cytometry analysis underline the importance of brain-derived signals for immune cell recruitment. This study clearly illustrates the significance of brain-intrinsic degenerative cascades for immune cell recruitment and MS lesion formation. Additional studies have to address the signaling cascades and mechanistic processes that form the top-down communication between the affected brain area, neurovascular unit, and peripheral immune cells. SIGNIFICANCE STATEMENT We identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the forebrain. Thus, immune cell recruitment might be a second step during the formation of new inflammatory lesions in multiple sclerosis. A better understanding of factors regulating neurodegeneration-induced immune cell recruitment will pave the way for the development of novel therapeutic treatment strategies.

The neuroprotective effect of tropisetron on vincristine-induced neurotoxicity

Vincristine (VCR) peripheral neuropathy is a dose-limiting side effect. Several studies have shown that tropisetron, a 5-HT3 receptor antagonist, exerts anti-inflammatory and immunomodulatory properties. Current study was designed to investigate a suppressive effect of tropisetron on VCR-induced neuropathy and whether this effect exerts through the 5-HT3 receptor or not. Neuropathy was induced in rats by administration of vincristine (0.5mg/kg, 3 intraperitoneal injections on alternate days) and in treatment group, tropisetron (3mg/kg); m-chlorophenylbiguanide (mCPBG), a selective 5-HT3 receptor agonist (15mg/kg); tropisetron (3mg/kg) plus mCPBG (15mg/kg); granisetron, another selective 5-HT3 receptor antagonist (3mg/kg) were administered intraperitoneally 1h prior to vincristine injection. Hot plate, open field tests (total distance moved, mean velocity and percentage of total duration of the movement) and motor nerve conduction velocity (MNCV) were performed to evaluate the sensory and motor neuropathy. Further, plasma levels of tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) and the level of TNF-α in sciatic nerve were assessed as well as histological examination. In only VCR-treated rats hot plate latencies were significantly increased, total distance moved, mean velocity, total duration of the movement and sciatic MNCV significantly decreased compared with control. In tropisetron and tropisetron plus mCPBG groups, one injection of tropisetron prior to each VCR injection robustly diminished TNF-α and IL-2 levels, and also prevented mixed sensory-motor neuropathy, as indicated by less mortality rate, better general conditions, behavioral and electrophysiological studies. Moreover, pathological evidence confirmed the results obtained from other findings. But granisetron and mCPBG had no significant effect on the mentioned parameters. In conclusion, these studies demonstrate that tropisetron significantly suppressed VCR-induced neuropathy and could be a neuroprotective agent for prevention of VCR-induced neuropathy via a receptor-independent pathway.

Melatonin Pretreatment Enhances the Homing of Bone Marrow-derived Mesenchymal Stem Cells Following Transplantation in a Rat Model of Liver Fibrosis.

Background: Bone marrow-derived mesenchymal stem cells (BMMSCs) transplantation has been considered as a promising milestone in liver fibrosis treatment. However, low amounts of homing are a major obstacle. We aimed to investigate the role of melatonin pretreatment in BMMSC homing into experimental liver fibrosis. Methods: BMMSCs were obtained, grown, propagated and preconditioned with 5 µM melatonin and analyzed for multipotency and immunophenotypic features at passage three. The cells were labelled with CM-Dil and infused into the rats received the i.p. injection of carbon tetrachloride (CCl4) for five weeks to induce liver fibrosis. Animals were divided into two groups: One group received BMMSCs, whereas the other group received melatonin-pretreated BMMSCs (MT-BMMSCs). After cell injection at 72 h, animals were sacrificed, and the liver tissues were assessed for further evaluations: fibrosis using Masson’s trichrome and hematoxylin and eosin staining and homing using fluorescent microscopy and flow cytometry. Results: BMMSCs and MT-BMMSCs expressed a high level of CD44 but low levels of CD11b, CD45 and CD34 (for all P≤0.05) and were able to differentiate into adipocytes and Schwann cells. CCl4 induction resulted in extensive collagen deposition, tissue disruption and fatty accumulation with no obvious difference between the two groups. There was a significant increase in homing of MT-BMMSCs in both florescent microscopy (P≤0.001) and flow cytometry (P≤0.01) assays, as compared with non-treated BMMSCs. Conclusion: This study indicates the improved homing potential of BMMSCs in pretreatment with melatonin. Therefore, this strategy may represent an applied approach for improving the stem cell therapy of liver fibrosis.

Impact of 17beta-estradiol and progesterone on inflammatory and apoptotic microRNA expression after ischemia in a rat model

17β-estradiol (E2) and progesterone (P) are neuroprotective factors in the brain preventing neuronal death under different injury paradigms. In previous studies, we demonstrated that both steroids dampen neuronal damage, improve local energy metabolism and attenuate pro-inflammatory responses. MicroRNAs (miRNAs) are small regulators of distinct target genes on the RNA level. Their expression patterns are misbalanced in several neurological disorders. To explore the regulatory mechanisms of steroid hormones on selected miRNAs and their validated targets in ischemia, we used the transient middle cerebral artery occlusion (tMCAO) model. 12-week old male rats were subjected to 2h tMCAO and expression patterns of miR-223, miR-200c, miR-375, miR-199 and miR-214 (all -3p) were determined. Using semi-quantitative real time PCR, we examined the role of E2 or P as regulatory factors for miRNAs and theirs target genes. Besides miR-375, all mentioned miRNAs showed a steady increase with a peak at 72h post tMCAO, whereas highest levels of miR-375 were detected at 12h post tMCAO. E2 or P selectively dampened miR-223 and miR-214 but further boosted miR-375 levels. With respect to the miR-223 regulated target genes NR2B and GRIA2 which both decreased after tMCAO, E2 and P application reversed this effect. Further, steroid treatment inhibited the hypoxia-induced increase of the miR-375 target genes Bcl-2 and RAD1. These findings provide new insights into the regulatory role of neuroprotection mediated by sex steroids in the brain. Both hormones are capable of influencing the expression of miRNAs which are relevant during neuropathological processes. Thereby, E2 and P indirectly control pro-apoptotic and -inflammatory gene translation and provide a mechanism to dampen explosive tissue damage.

Effect of Intrastriatal 6-OHDA Lesions on Extrastriatal Brain Structures in the Mouse

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive loss of midbrain dopaminergic neurons, resulting in motor and non-motor symptoms. The underlying pathology of non-motor symptoms is poorly understood. Discussed are pathological changes of extrastriatal brain structures. In this study, we characterized histopathological alterations of extrastriatal brain structures in the 6-hydroxydopamine (6-OHDA) PD animal model. Lesions were induced by unilateral stereotactic injections of 6-OHDA into the striatum or medial forebrain bundle of adult male mice. Loss of tyrosine hydroxylase positive (TH+) fibers as well as glia activation was quantified following stereological principles. Loss of dopaminergic innervation was further investigated by western-blotting. As expected, 6-OHDA injection into the nigrostriatal route induced retrograde degeneration of dopaminergic neurons within the substantia nigra pars compacta (SNpc), less so within the ventral tegmental area. Furthermore, we observed a region-specific drop of TH+ projection fiber density in distinct cortical regions. This pathology was most pronounced in the cingulate- and motor cortex, whereas the piriform cortex was just modestly affected. Loss of cortical TH+ fibers was not paralleled by microglia or astrocyte activation. Our results demonstrate that the loss of dopaminergic neurons within the substantia nigra pars compacta is paralleled by a cortical dopaminergic denervation in the 6-OHDA model. This model serves as a valuable tool to investigate mechanisms operant during cortical pathology in PD patients. Further studies are needed to understand why cortical dopaminergic innervation is lost in this model, and what functional consequence is associated with the observed denervation.