In situ and transcriptomic identification of microglia in synapse-rich regions of the developing zebrafish brain.

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct microglial subset in the synapse rich regions of the zebrafish (Danio rerio) brain. We found that ramified microglia increased in synaptic regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions.

Single-cell RNA-seq landscape midbrain cell responses to red spotted grouper nervous necrosis virus infection.

Viral nervous necrosis (VNN) is an acute and serious fish disease caused by nervous necrosis virus (NNV) which has been reported massive mortality in more than fifty teleost species worldwide. VNN causes damage of necrosis and vacuolation to central nervous system (CNS) cells in fish. It is difficult to identify the specific type of cell targeted by NNV, and to decipher the host immune response because of the functional diversity and highly complex anatomical and cellular composition of the CNS. In this study, we found that the red spotted grouper NNV (RGNNV) mainly attacked the midbrain of orange-spotted grouper (Epinephelus coioides). We conducted single-cell RNA-seq analysis of the midbrain of healthy and RGNNV-infected fish and identified 35 transcriptionally distinct cell subtypes, including 28 neuronal and 7 non-neuronal cell types. An evaluation of the subpopulations of immune cells revealed that macrophages were enriched in RGNNV-infected fish, and the transcriptional profiles of macrophages indicated an acute cytokine and inflammatory response. Unsupervised pseudotime analysis of immune cells showed that microglia transformed into M1-type activated macrophages to produce cytokines to reduce the damage to nerve tissue caused by the virus. We also found that RGNNV targeted neuronal cell types was GLU1 and GLU3, and we found that the key genes and pathways by which causes cell cytoplasmic vacuoles and autophagy significant enrichment, this may be the major route viruses cause cell death. These data provided a comprehensive transcriptional perspective of the grouper midbrain and the basis for further research on how viruses infect the teleost CNS.

Overexpression of microRNA-202-3p in bone marrow mesenchymal stem cells improves cerebral ischemia-reperfusion injury by promoting angiogenesis and inhibiting inflammation.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) can cause brain tissue inflammation, neuronal degeneration, and apoptosis. There is increasing evidence that microRNAs (miRNA) exert neuroprotective effects by regulating the inflammatory process during cerebral ischemia-reperfusion injury. Additionally, it is increasingly acknowledged that neuroinflammation is regulated by Toll-like receptor 4 (TLR4). However, it is unclear whether miRNA can exert its neuroprotective effects by regulating TLR4-mediated inflammation. METHODS: The effects of BMSCs over-expressing miR-202-3p on CIRI, angiogenesis in midbrain tissue, and the release of inflammatory factors (IFs) in the serum were measured using in vivo rat models. We also used SH-SY5Y cells to establish an ischemia-reperfusion in vitro cell model. The interaction between miR-202-3p and TLR4 was analyzed by overexpressing miR-202-3p and knocking down TLR4. Knockdown of TLR4 was performed using siRNA. RESULTS: Overexpression of miR-202-3p in BMSCs could significantly improve brain function and reduce brain damage. Simultaneously, miR-202-3p could significantly promote angiogenesis, increase the expression of vWF and VEGF, and reduce the expression of IFs. When the expression of TLR4 was significantly reduced in SH-SY5Y cells, the expression of IFs increased. Therefore, miRNA-202-3p may interact with TLR4 to modulate inflammation. CONCLUSION: Our data indicated that miR-202-3p potentially exerts its neuroprotective effects and protects against CIRI by regulating TLR4-mediated inflammation.

Olfactory ensheathing cells improve the survival of porcine neural xenografts in a Parkinsonian rat model.

BACKGROUND: Parkinson's disease (PD) features the motor control deficits resulting from irreversible, progressive degeneration of dopaminergic (DA) neurons of the nigrostriatal pathway. Although intracerebral transplantation of human fetal ventral mesencephalon (hfVM) has been proven effective at reviving DA function in the PD patients, this treatment is clinically limited by availability of hfVM and the related ethical issues. Homologous tissues to hfVM, such as porcine fetal ventral mesencephalon (pfVM) thus present a strong clinical potential if immune response following xenotransplantation could be tamed. Olfactory ensheathing cells (OECs) are glial cells showing immunomodulatory properties. It is unclear but intriuging whether these properties can be applied to reducing immune response following neural xenotransplantation of PD. METHODS: To determine whether OECs may benefit neural xenografts for PD, different compositions of grafting cells were transplanted into striatum of the PD model rats. We used apomorphine-induced rotational behavior to evaluate effectiveness of the neural grafts on reviving DA function. Immunohistochemistry was applied to investigate the effect of OECs on the survival of neuroxenografts and underlying mechanisms of this effect. RESULTS: Four weeks following the xenotransplantation, we found that the PD rats receiving pfVM + OECs co-graft exhibited a better improvement in apomorphine-induced rotational behavior compared with those receiving only pfVM cells. This result can be explained by higher survival of DA neurons (tyrosine hydroxylase immunoreactivity) in grafted striatum of pfVM + OECs group. Furthermore, pfVM + OECs group has less immune response (CD3+ T cells and OX-6+ microglia) around the grafted area compared with pfVM only group. These results suggest that OECs may enhance the survival of the striatal xenografts via dampening the immune response at the grafted sites. CONCLUSIONS: Using allogeneic OECs as a co-graft material for xenogeneic neural grafts could be a feasible therapeutic strategy to enhance results and applicability of the cell replacement therapy for PD.

Midbrain and cerebral inflammatory and glutamatergic adaptations during chronic hypercapnia in goats.

Cognitive impairment is associated with multiple human diseases that have in common chronic hypercapnia. However, the mechanisms leading to chronic hypercapnia-induced cognitive decline are not known. We have previously shown chronic hypercapnia through exposure to increased inspired CO2 (6% InCO2) in conscious goats caused an immediate (within hours) and sustained decline in cognitive performance during a shape discrimination test. Herein, within the same goats, we assessed markers of neuroinflammation and glutamate receptor expression/phosphorylation within CNS regions important for cognitive function following 24 hours (h) or 30 days (d) of chronic hypercapnia. Within 24 h, chronic hypercapnia increased expression of the inflammatory cytokine IL-1ß in the orbitofrontal cortex and medial prefrontal cortex, but at 30d IL-1ß levels were not different relative to time-matched goats exposed to room-air. Additionally, Iba1 expression (a marker of microglial activation) was unaltered by chronic hypercapnia in all regions tested. Finally, levels of the total and phosphorylated AMPA receptor subunit GluR2 were reduced within the hippocampus at both 24 h and 30 d of hypercapnia, and reduced following 30 d within the anterior insular cortex. These data suggest that chronic hypercapnia leads to CNS site-dependent acute inflammatory responses and shifts in select glutamate receptor expression/phosphorylation in brain regions contributing to cognitive function. Such changes may be indicative of alterations in glutamatergic receptor-mediated signaling and neuronal dysfunction that contribute to declines in cognitive function associated with human diseases defined or marked by chronic CO2 retention.

Involvement of exosomes in dopaminergic neurodegeneration by microglial activation in midbrain slice cultures.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopamine neurons in the substantia nigra. Microglial activation is frequently observed in the brains of patients with PD and animal models. Interferon-γ (IFN-γ)/lipopolysaccharide (LPS) treatment triggers microglial activation and the reduction of dopamine neurons in midbrain slice cultures. We have previously reported that nitric oxide (NO) is mainly involved in this dopaminergic degeneration. However, this degeneration was not completely suppressed by the inhibition of NO synthesis, suggesting that factors other than NO also contribute to dopaminergic neurodegeneration. Exosomes are extracellular vesicles with diameters of 40-200 nm that contain various proteins and micro RNAs and are regarded as a novel factor that mediates cell-to-cell interactions. Previous studies have demonstrated that exosome release is enhanced by microglial stimulation and that microglia-derived exosomes increases neuronal apoptosis. In the present study, we investigated whether exosomes are involved in dopaminergic neurodegeneration triggered by microglial activation in midbrain slice cultures. IFN-γ/LPS treatment to the midbrain slice cultures activated microglia, increased exosomal release, and decreased dopamine neurons. GW4869, an inhibitor of a neutral sphingomyelinase 2, decreased exosomal release and significantly prevented dopaminergic neurodegeneration by IFN-γ/LPS without affecting NO production. In contrast, D609, an inhibitor of sphingomyelin synthase and NO synthase, did not affect dopaminergic neurodegeneration, although it strongly inhibited NO production. The protective effect mediated by inhibition of NO synthase would be counteracted by enhanced exosomal release caused by D609 treatment. In addition, dopaminergic neurodegeneration is triggered by the treatment of exosomes isolated from culture media of IFN-γ/LPS-treated slices. These results suggest that exosomes are involved in dopaminergic neurodegeneration by microglial activation.

Treg Cells Protect Dopaminergic Neurons against MPP+ Neurotoxicity via CD47-SIRPA Interaction.

BACKGROUND/AIMS: Regulatory T (Treg) cells have been associated with neuroprotection by inhibiting microglial activation in animal models of Parkinson's disease (PD), a progressive neurodegenerative disease characterized by dopaminergic neuronal loss in the nigrostriatal system. Herein, we show that Treg cells directly protect dopaminergic neurons against 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity via an interaction between the two transmembrane proteins CD47 and signal regulatory protein α (SIRPA). METHODS: Primary ventral mesencephalic (VM) cells or VM neurons were pretreated with Treg cells before MPP+ treatment. Transwell co-culture of Treg cells and VM neurons was used to assess the effects of the Treg cytokines transforming growth factor (TGF)-ß1 and interleukin (IL)-10 on dopaminergic neurons. Live cell imaging system detected a dynamic contact of Treg cells with VM neurons that were stained with CD47 and SIRPA, respectively. Dopaminergic neuronal loss, which was assessed by the number of tyrosine hydroxylase (TH)-immunoreactive cells, was examined after silencing CD47 in Treg cells or silencing SIRPA in VM neurons. RESULTS: Treg cells prevented MPP+-induced dopaminergic neuronal loss and glial inflammatory responses. TGF-ß1 and IL-10 secreted from Treg cells did not significantly prevent MPP+-induced dopaminergic neuronal loss in transwell co-culture of Treg cells and VM neurons. CD47 and SIRPA were expressed by Treg cells and VM neurons, respectively. CD47-labeled Treg cells dynamically contacted with SIRPA-labeled VM neurons. Silencing CD47 gene in Treg cells impaired the ability of Treg cells to protect dopaminergic neurons against MPP+ toxicity. Similarly, SIRPA knockdown in VM neurons reduced the ability of Treg cell neuroprotection. Rac1/Akt signaling pathway in VM neurons was activated by CD47-SIRPA interaction between Treg cells and the neurons. Inhibiting Rac1/Akt signaling in VM neurons compromised Treg cell neuroprotection. CONCLUSION: Treg cells protect dopaminergic neurons against MPP+ neurotoxicity by a cell-to-cell contact mechanism underlying CD47-SIRPA interaction and Rac1/Akt activation.

A requirement of serotonergic p38α mitogen-activated protein kinase for peripheral immune system activation of CNS serotonin uptake and serotonin-linked behaviors.

Alterations in central serotonin (5-hydroxytryptamine, 5-HT) neurotransmission and peripheral immune activation have been linked to multiple neuropsychiatric disorders, including depression, schizophrenia and autism. The antidepressant-sensitive 5-HT transporter (SERT, SLC6A4), a critical determinant of synaptic 5-HT inactivation, can be regulated by pro-inflammatory cytokine signaling. Systemic innate immune system activation via intraperitoneal lipopolysaccharide (LPS) injection rapidly elevates brain SERT activity and 5-HT clearance. Moreover, the pro-inflammatory cytokine interleukin (IL)-1ß rapidly stimulates SERT activity in raphe nerve terminal preparations ex vivo, effects that are attenuated by pharmacological p38 MAPK inhibition. To establish a role of serotonergic p38α MAPK signaling in LPS/IL-1ß-induced SERT regulation and attendant behavioral responses, we pursued studies in mice that afford conditional elimination of p38α MAPK in 5-HT neurons (p38α(5HT-)). We found p38α(5HT-) and control (p38α(5HT+)) littermates to be indistinguishable in viability and growth and to express equivalent levels of SERT protein and synaptosomal 5-HT transport activity. Consistent with pharmacological studies, however, IL-1ß fails to increase SERT activity in midbrain synaptosomes prepared from p38α(5HT-) animals. Moreover, although LPS elevated plasma corticosterone and central/peripheral pro-inflammatory cytokines in p38α(5HT-) animals, elevations in midbrain SERT activity were absent nor were changes in depressive and anxiety-like behaviors observed. Our studies support an obligate role of p38α MAPK signaling in 5-HT neurons for the translation of immune activation to SERT regulation and 5-HT-modulated behaviors.

Interleukin-10 Protection against Lipopolysaccharide-Induced Neuro-Inflammation and Neurotoxicity in Ventral Mesencephalic Cultures.

Interleukin (IL)-10, an anti-inflammatory cytokine, is expressed in the brain and can inhibit microglial activation. Herein, we utilized lipopolysaccharide (LPS)-induced inflammatory Parkinson's disease (PD) cell model to determine whether microglia and astrocytes are necessary targets for IL-10 neuroprotection. Primary ventral mesencephalic (VM) cultures with different composition of neurons, microglia and astrocytes were prepared. The cells were exposed to IL-10 (15, 50 or 150 ng/mL) 1 h prior to LPS (50 ng/mL) treatment. LPS induced dopaminergic and non-dopaminergic neuronal loss in VM cultures, VM neuron-enriched cultures, and neuron-microglia co-cultures, but not in neuron-astrocyte co-cultures. IL-10 reduced LPS-induced neuronal loss particularly in single VM neuron cultures. Pro-inflammatory mediators (TNF-α, IL-1ß, inducible nitric oxide synthase and cyclooxygenase-2) were upregulated in both neuron-microglia and neuron-astrocyte co-cultures by LPS. In contrast, neurotrophic factors (brain-derived neurotrophic factor, insulin-like growth factor-1 or glial cell-derived neurotrophic factor) were downregulated in neuron-microglia co-cultures, but upregulated in neuron-astrocyte co-cultures by LPS. IL-10 reduced both the increase in production of the pro-inflammatory mediators and the decrease in production of the neurotrophic factors induced by LPS. These results suggest that astrocytes can balance LPS neurotoxicity by releasing more neurotrophic factors and that IL-10 exerts neuroprotective property by an extensive action including direct on neurons and indirect via inhibiting microglial activation.

Brain leukocyte infiltration initiated by peripheral inflammation or experimental autoimmune encephalomyelitis occurs through pathways connected to the CSF-filled compartments of the forebrain and midbrain.

BACKGROUND: Cerebrospinal fluid (CSF) has been considered as a preferential pathway of circulation for immune cells during neuroimmune surveillance. In order to evaluate the involvement of CSF-filled spaces in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, we performed a time-course analysis of immune cell association with the CSF-containing ventricles, velae, and cisterns in two active models of this disease. METHODS: Guinea-pig spinal cord homogenate-induced EAE in rat and myelin oligodendrocyte glycoprotein-induced EAE in mouse were used. Leukocyte distribution and phenotypes were investigated by immunohistochemistry in serial sections of brain areas of interest, as well as in CSF withdrawn from rat. Immune cells associated with the choroid plexuses were quantified. RESULTS: Freund's adjuvant-induced peripheral inflammation in the absence of brain antigen led to a subtle but definite increase in the number of myeloid cells in the extraventricular CSF spaces. In both rats and mice, EAE was characterized by a sustained and initial infiltration of lymphocytes and monocytes within forebrain/midbrain fluid-filled compartments such as the velum interpositum and ambient cisterns, and certain basal cisterns. Leukocytes further infiltrated periventricular and pericisternal parenchymal areas, along perivascular spaces or following a downward CSF-to-tissue gradient. Cells quantified in CSF sampled from rats included lymphocytes and neutrophils. The distinctive pattern of cell distribution suggests that both the choroid plexus and the vessels lying in the velae and cisterns are gates for early leukocyte entry in the central nervous system. B-cell infiltration observed in the mouse model was restricted to CSF-filled extraventricular compartments. CONCLUSION: These results identified distinctive velae and cisterns of the forebrain and midbrain as preferential sites of immune cell homing following peripheral and early central inflammation and point to a role of CSF in directing brain invasion by immune cells during EAE.

Wall-eyed bilateral internuclear ophthalmoplegia (WEBINO) syndrome in a patient with neuromyelitis optica spectrum disorder and anti-aquaporin-4 antibody.

This report describes, for the first time, an occurrence of wall-eyed bilateral internuclear ophthalmoplegia (WEBINO) in a 19-year-old female with neuromyelitis optica (NMO) spectrum disorder, who had anti-aquaporin-4 (AQP4) antibody. A high signal intensity lesion on T2-weighted MRI was detected in the midbrain tegmentum adjacent to the aqueduct, and presumably involved the medial longitudinal fasciculus bilaterally at the caudal levels. Plasma exchange resolved both WEBINO syndrome and the midbrain lesion. Although WEBINO syndrome is occasionally reported in multiple sclerosis patients, diagnosis of NMO should not be excluded in patients with WEBINO syndrome, because AQP4 is expressed abundantly around the periaqueductal region.

A developmental characterization of mesolimbocortical serotonergic gene expression changes following early immune challenge.

An immunogenic challenge during early postnatal development leads to long-term changes in behavioural and physiological measures reflecting enhanced emotionality and anxiety. Altered CNS serotonin (5-HT) signalling during the third postnatal week is thought to modify the developing neurocircuitry governing anxiety-like behaviour. Changes in 5-HT signalling during this time window may underlie increased emotionality reported in early immune challenge rodents. Here we examine both the spatial and temporal profile of 5-HT related gene expression, including 5HT1A, 2A, 2C receptors, the 5-HT transporter (5HTT), and tryptophan hydroxylase 2 (TPH2) during early development (postnatal day [P]14, P17, P21, P28) in mice challenged with lipopolysaccharide (LPS) during the first postnatal week. Expression levels were measured using in situ hybridization in regions associated with mediating emotive behaviours: the dorsal raphe (DR), hippocampus, amygdala, and prefrontal cortex (PFC). Increased TPH2 and 5HTT expression in the ventrolateral region of the DR of LPS-mice accompanied decreased expression of ventral DR 5HT1A and dorsal DR 5HTT. In the forebrain, 5HT1A and 2A receptors were increased, whereas 5HT2C receptors were decreased in the hippocampus. Decreased mRNA expression of 5HT2C was detected in the amygdala and PFC of LPS-treated pups; 5HT1A was increased in the PFC. The majority of these changes were restricted to P14-21. These transient changes in 5-HT expression coincide with the critical time window in which 5-HT disturbance leads to permanent modification of anxiety-related behaviours. This suggests that alterations in CNS 5-HT during development may underlie the enhanced emotionality associated with an early immune challenge.

Differential numbers of foci of lymphocytes within the brains of Lewis rats exposed to weak complex nocturnal magnetic fields during development of experimental allergic encephalomyelitis.

To discern if specific structures of the rat brain contained more foci of lymphocytes following induction of experimental allergic encephalomyelitis and exposures to weak, amplitude-modulated magnetic fields for 6 min once per hour during the scotophase, the residuals between the observed and predicted values for the numbers of foci for 320 structures were obtained. Compared to the brains of sham-field exposed rats, the brains of rats exposed to 7-Hz 50 nT (0.5 mG) amplitude-modulated fields showed more foci within hippocampal structures and the dorsal central grey of the midbrain while those exposed to 7-Hz 500 nT (5 mG) fields showed greater densities within the hypothalamus and optic chiasm. The brains of rats exposed to either the 50 nT or 500 nT amplitude-modulated 40-Hz fields displayed greater densities of foci within the midbrain structures related to rapid eye movement. Most of the enhancements of infiltrations within the magnetic field-exposed rats occurred in structures within periventricular or periaqueductal regions and were both frequency- and intensity-dependent. The specificity and complexity of the configurations of the residuals of the numbers of infiltrated foci following exposures to the different fields suggest that the brain itself may be a "sensory organ" for the detection of these stimuli.

Lipopolysaccharide preconditioning induces protection against lipopolysaccharide-induced neurotoxicity in organotypic midbrain slice culture.

OBJECTIVE: To identify the protective effect of lipopolysaccharide (LPS) preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms. METHODS: After cultured in vitro for 14 d, the rat organotypic midbrain slices were pretreated with different concentrations (0, 1, 3, 6 or 10 ng/mL) of LPS for 24 h followed by treatment with 100 ng/mL LPS for 72 h. The whole slice viability was determined by measurement of the activity of lactic acid dehydrogenase (LDH). Tyrosine hydroxylase-immunoreactive (TH-IR) neurons and CD11b/c equivalent-immunoreactive (OX-42-IR) microglia in the slices were observed by immunohistochemical method, and tumor necrosis factor-alpha (TNF-alpha) levels in the culture media were detected by enzyme-linked immunosorbent assays (ELISA). RESULTS: In the slices treated with 100 ng/mL LPS for 72 h, the number of TH-IR neurons reduced from 191+/-12 in the control slices to 46+/-4, and the LDH activity elevated obviously (P < 0.01), along with remarkably increased number of OX-42-IR cells and production of TNF-alpha (P < 0.01). Preconditioning with 3 or 6 ng/mL LPS attenuated neuron loss (the number of TH-IR neurons increased to 126+/-12 and 180+/-13, respectively) and markedly reduced LDH levels (P < 0.05), accompanied by significant decreases of OX-42-IR microglia activation and TNF-alpha production (P < 0.05). CONCLUSION: Low-dose LPS preconditioning could protect dopaminergic neurons against inflammatory damage in rat midbrain slice culture, and inhibition of microglial activation and reduction of the proinflammatory factor TNF-alpha production may contribute to this protective effect. Further understanding the underlying mechanism of LPS preconditioning may open a new window for treatment of Parkinson's disease.

Brain-region-specific astroglial responses in vitro after LPS exposure.

Astroglia is well-known to be integrated in the complex regulation of neuroinflammation in the central nervous system. Astrocytes become activated and synthesize cytokines, chemokines, and prostanoids during degenerative and vulnerable processes and interact with other immune-competent cells. Degenerative disorders often occur in a brain-region-specific fashion suggesting differences in the activity and reactivity of innate immune cells. We have investigated the potency of lipopolysaccharides (LPS) to differently stimulate astrocytes from the cortex and midbrain. Astroglial cultures were prepared from Bagg albino/c mice and exposed to LPS. Astrocytes from both brain areas already differed in their capacity and profile of cytokine expression under basal unstimulated conditions. In response to LPS, we observed both a region-specific pattern of up-regulation of distinct cytokines and differences in the extent and time-course of activation. Our data demonstrate that astrocytes reveal a region-specific basal profile of cytokine expression and a selective area-specific regulation of cytokines upon LPS-induced inflammation. This makes astrocytes likely candidates to be responsible for region-specific incidence rates of neurological and neurodegenerative disorders.

Gene expression profiling reveals upregulation of Tlr4 receptors in Cckb receptor deficient mice.

The cholecystokinin B (2) receptor knockout (Cckbr KO) protects against allodynia induced by chronic constriction injury (CCI). The mechanism of this phenomenon is unknown, but must involve persistent changes in pain modulation and/or inflammatory pathways. We performed a gene expression study in two brain areas (midbrain and medulla) after surgical induction of CCI in Cckbr KO and wild-type (wt) control mice. The patterns of gene expression differences suggest that the immune system is activated in higher brain structures following CCI in the wt mice. The strongest differences include genes related to the MAPK pathway activation and cytokine production. In Cckbr KO mice this expressional pattern was absent. In addition, we found significant elevation of the Toll-like receptor 4 (Tlr4) in the supraspinal structures of the mice with deleted Cckbr compared to wt control mice. This up-regulation is most likely induced by the deletion of Cckbr. We suggest that there is a functional deficiency in the Tlr4 pathway which disables the development of neuropathic pain in Cckbr KO mice. Indeed, real time PCR analysis detected a CCI-induced upregulation of Tlr4 and Il1b expression in the lumbar region of wt but not Cckbr KO mice. Gene expression profiling indicates that elements of the immune response are not activated in Cckbr KO mice following CCI. Our findings suggest that there may be a role for CCK in the regulation of innate immunity.

Oestrogen and progesterone reduce lipopolysaccharide-induced expression of tumour necrosis factor-alpha and interleukin-18 in midbrain astrocytes.

Besides microglia, astrocytes exert an important regulatory function in the initiation and control of neuro-inflammatory processes in the central nervous system. Clinical and experimental data suggest that sex steroids are neuroprotective and that neurological/neurodegenerative disorders display sex-specific characteristics. Astroglia is known to respond to toxic stimuli by secretion of distinct pro-inflammatory/apoptotic cytokines. In the present study, we investigated the influence of oestrogen and progesterone on the expression of the cytokines tumour necrosis factor (TNF)-alpha and interleukin (IL)-18 in primary astrocytes obtained from neonatal mouse midbrain and cerebral cortex after the stimulation with lipopolysaccharides (LPS). LPS strongly induced the expression of TNF-alpha in astrocytes from both brain regions and IL-18 in those from midbrain. Oestrogen significantly attenuated LPS-induced TNF-alpha expression in the midbrain glia but not in the cortex glia. Combined treatment with oestrogen and progesterone together diminished LPS-induced IL-18 expression in the midbrain completely. Both steroid effects could be specifically antagonised by the steroid hormone receptor antagonists ICI 182 780 and mifepristone. We conclude that neuroprotective oestrogen and progesterone effects in the midbrain might be in part the consequence of a reduced pro-inflammatory response of astroglia.

Catalpol protects dopaminergic neurons from LPS-induced neurotoxicity in mesencephalic neuron-glia cultures.

Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Microglia, the resident immune cells in the central nervous system, are pivotal in the inflammatory reaction. Activated microglia can induce expression of inducible nitric-oxide synthase (iNOS) and release significant amounts of nitric oxide (NO) and TNF-alpha, which can damage the dopaminergic neurons. Catalpol, an iridoid glycoside, contained richly in the roots of Rehmannia glutinosa, was found to be neuroprotective in gerbils subjected to transient global cerebral ischemia. But the effect of catalpol on inflammation-mediated neurodegeneration has not been examined. In this study, microglia in mesencephalic neuron-glia cultures were activated with lipopolysaccharide (LPS) and the aim of the study was to examine whether catalpol could protect dopaminergic neurons from LPS-induced neurotoxicity. The results showed that catalpol significantly reduced the release of reactive oxygen species (ROS), TNF-alpha and NO after LPS-induced microglial activation. Further, catalpol attenuated LPS-induced the expression of iNOS. As determined by immunocytochemical analysis, pretreatment by catalpol dose-dependently protected dopaminergic neurons against LPS-induced neurotoxicity. These results suggest that catalpol exerts its protective effect on dopaminergic neurons by inhibiting microglial activation and reducing the production of proinflammatory factors. Thus, catalpol may possess therapeutic potential against inflammation-related neurodegenerative diseases.

Porcine fetal ventral mesencephalic cells are targets for primed xenoreactive human T cells.

Xenotransplantation of porcine fetal ventral mesencephalic (pfVM) cells to overcome the dopamine shortage in the striatum of patients with Parkinson's disease seems a viable alternative to allotransplantion of human fetal donor tissue, especially because the latter is complicated by both practical and ethical issues. There is, however, little known about the xenospecific immune responses involved in such an intracerebral xenotransplantation. The aim of our study was to investigate whether (1) naive human peripheral blood mononuclear cells (PMBC) display cytotoxicity against pfVM cells of E28 pig fetuses, and (2) priming of human PBMC by xenogeneic antigen presenting cells (APC) modulates pfVM-directed cellular cytotoxicity. For this purpose fresh PMBC from nine individual donors were primed by incubation with either irradiated pfVM cells or porcine spleen cells (PSC) as APC in the presence of IL-2 for 1 week before assessing cytotoxicity in a 51Cr release assay. Also, direct NK reactivity and antibody-dependent cellular cytotoxicity (ADCC) of fresh PMBC against pfVM cells was assessed. No direct cytotoxicity of naive cells (either NK reactivity or ADCC) against pfVM cells could be determined. Only PMBC primed with PSC were capable of lysing pfVM cells. PBMC primed with pfVM cells did not show cytolytic capacity towards pfVM. Interestingly, large differences in xenospecific T-cell responses exist between individual donor PBMC. Thus, human T cells are capable of killing pfVM cells in a xenoreactive response, but only after priming by donor APC. The large interindividual differences between human donors in their xenoreactive response may influence patient selection for xenotransplantation and chances of graft survival for individual patients.

Prior intraperitoneal injection of rat recombinant IL-6 increases hypothalamic IL-6 contents in subsequent forced swim stressor in rats.

BACKGROUND: Cytokines do not only mediate responses to infection, but are also involved in behavioral and physiological responses to psychological stressors. IL-6 has received special attention in relation to the development of posttraumatic stress disorders and depression. OBJECTIVE: We tested effects of prior injection of rat recombinant IL-6 (rrIL-6) on behaviors induced by the forced swim (FS) stressor, and central and peripheral responses of IL-6 to FS. METHODS: Rats were injected intraperitoneally with either rrIL-6 (250 ng/0.5 ml) or equal-volume sterile saline twice within an interval of 24 h. One hour after each injection, the rats were exposed to FS or remained at the home cage (control). RESULTS: Injection of rrIL-6 did not affect immobility, swimming or climbing behaviors during FS compared with the saline control. Although FS was not a significant factor for hypothalamic and midbrain IL-6 mRNA and plasma IL-6 responses, FS with prior administration of rrIL-6 significantly increased hypothalamic IL-6 contents in response to FS compared with the saline injection-FS condition. CONCLUSIONS: Our results suggested that stressor alone had no influence on plasma IL-6 levels and IL-6 mRNA expression levels in midbrain and hypothalamus, but administration of rrIL-6 followed by FS significantly increased hypothalamic IL-6. Our results support the notion that the interaction between IL-6 and stressor might have implications for the pathophysiology of IL-6-induced depressive symptoms.