Microglia polarization and inflammatory challenges

A wealth of recent evidence indicates that microglia activation is a polarized process, leading to a potentially neurotoxic M1 “classical activation” or potentially neuroprotective M2 “alternative activation”. The regulation of this process is, however, still largely unknown. We here investigated the induction of molecules which characterize M1 and M2 microglia responses after systemic (ip) and central (intracerebroventricular, icv) exposure to lipopolysaccharide (LPS). These challenges elicit different inflammatory responses of the brain parenchyma; in particular, leukocyte infiltration in the brain occurs after icv but not after ip LPS exposure. Young adult mice were subjected to ip or icv LPS injection, sacrificed at different time points from 2h up to 10 days, and compared with matched saline-treated control mice. Analyses with the pan-T cell marker CD3 confirmed the occurrence of T cells in the brain parenchyma after icv but not after ip LPS injections. Immunohistochemical phenotyping of microglia was pursued to reveal major histocompatibility complex class II (MHCII) antigen, a key molecule in M1 activation, and chitinase 3-like 3 (YM1), part of the M2 molecular repertoire. The M1 type of microglial response, revealed by upregulation of MHCII immunosignal, was very intense and occurred during the first 24h, with a peak at 2h after icv LPS, and at 6h after ip LPS. The M2 type of response, revealed by YM1 immunoreactivity in some microglial cells, followed the M1 type response evolving in the days which followed the first 24h, with an earlier peak (at 5 days) and more marked persistence (up to 10 days) after icv LPS, i.e., in the presence of T cell infiltration. Altogether the findings point out dynamic processes of microglia activation and its polarization over time, and a microglia – T cell dialogue with potential neuroprotective effects of T cell recruitment. Further investigation on the profiling of the M1 and M2 types of microglia activation, and of the possible molecules implicated in the microglia – T cell dialogue will further enlighten these crucial polarization mechanisms

Trypanosoma brucei brucei invasion and T-cell infiltration of the brain parenchyma in experimental sleeping sickness: timing and correlation with functional changes

Background: The timing of Trypanosoma brucei entry into the brain parenchyma to initiate the second, meningoencephalitic stage of human African trypanosomiasis or sleeping sickness is currently debated and even parasite invasion of the neuropil has been recently questioned. Furthermore, the relationship between neurological features and disease stage are unclear, despite the important diagnostic and therapeutic implications. Methodology: Using a rat model of chronic Trypanosoma brucei brucei infection we determined the timing of parasite and T-cell neuropil infiltration and its correlation with functional changes. Parasite DNA was detected using trypanosome-specific PCR. Body weight and sleep structure alterations represented by sleep-onset rapid eye movement (SOREM) periods, reported in human and experimental African trypanosomiasis, were monitored. The presence of parasites, as well as CD4+ and CD8+ T-cells in the neuropil was assessed over time in the brain of the same animals by immunocytochemistry and quantitative analyses. Principal findings: Trypanosome DNA was present in the brain at day 6 post-infection and increased more than 15-fold by day 21. Parasites and T-cells were observed in the parenchyma from day 9 onwards. Parasites traversing blood vessel walls were observed in the hypothalamus and other brain regions. Body weight gain was reduced from day 7 onwards. SOREM episodes started in most cases early after infection, with an increase in number and duration after parasite neuroinvasion. Conclusion: These findings demonstrate invasion of the neuropil over time, after an initial interval, by parasites and lymphocytes crossing the blood-brain barrier, and show that neurological features can precede this event. The data thus challenge the current clinical and cerebrospinal fluid criteria of disease staging

Expression of interferon-inducible chemokines and sleep/wake changes during early encephalitis in experimental African trypanosomiasis

Background: Human African trypanosomiasis or sleeping sickness, caused by the parasite Trypanosoma brucei, leads to neuroinflammation and characteristic sleep/wake alterations. The relationship between the onset of these alterations and the development of neuroinflammation is of high translational relevance, but remains unclear. This study investigates the expression of interferon (IFN)-γ and IFN-inducible chemokine genes in the brain, and the levels of CXCL10 in the serum and cerebrospinal fluid prior to and during the encephalitic stage of trypanosome infection, and correlates these with sleep/wake changes in a rat model of the disease. Methodology/Principal findings: The expression of genes encoding IFN-γ, CXCL9, CXCL10, and CXCL11 was assessed in the brain of rats infected with Trypanosoma brucei brucei and matched controls using semi-quantitative end-point RT-PCR. Levels of CXCL10 in the serum and cerebrospinal fluid were determined using ELISA. Sleep/wake states were monitored by telemetric recording. Using immunohistochemistry, parasites were found in the brain parenchyma at 14 days post-infection (dpi), but not at 6 dpi. Ifn-γ, Cxcl9, Cxcl10 and Cxcl11 mRNA levels showed moderate upregulation by 14 dpi followed by further increase between 14 and 21 dpi. CXCL10 concentration in the cerebrospinal fluid increased between 14 and 21 dpi, preceded by a rise in the serum CXCL10 level between 6 and 14 dpi. Sleep/wake pattern fragmentation was evident at 14 dpi, especially in the phase of wake predominance, with intrusion of sleep episodes into wakefulness. Conclusions/Significance: The results show a modest increase in Cxcl9 and Cxcl11 transcripts in the brain and the emergence of sleep/wake cycle fragmentation in the initial encephalitic stage, followed by increases in Ifn-γ and IFN-dependent chemokine transcripts in the brain and of CXCL10 in the cerebrospinal fluid. The latter parameter and sleep/wake alterations could provide combined humoral and functional biomarkers of the early encephalitic stage in African trypanosomiasis

Neonatal treatment with clomipramine induces morphological and cellular changes in the adult rat brain

Clomipramine (CLI) is a tricyclic serotonin reuptake blocker, widely used to treat depression, obsessive compulsive disorder (OCD), and other psychiatric conditions in human patients. Chronic CLI administration in the neonate rodent alters serotonergic circuits and serotonine levels in the brain, and has been reported to cause a complex pattern of behavioral changes in the adult life, including abnormalities of rapid eye movement sleep, decreased aggression and sexual behavior, anhaedonia and helplessness. Such symptoms suggest a parallel with humans endogenous depression and have been proposed as a novel animal model of OCD. The present study was aimed at identifying morphological and cellular changes after chronic neonatal treatment with clomipramine (daily i.p. injections, 20 mg/kg, from P5 to P21) in the brain of 5 month-old male Sprague-Dawley rats, compared to saline-treated littermates, using three distinct experimental approaches. 1) In vivo volumetric analyses based on structural MRI scans performed at 4.7T on 6 CLI-treated and 6 control rats revealed a significant reduction in total brain and hippocampal volume, as well as enlarged ventricles in CLI-treated rats, compared to saline-treated cohorts. 2) In order to investigate treatment-related developmental disorders, we studied the dendritic arborization of newly generated cells in the hippocampus of 7 CLI and 7 control rats. Two-dimensional dendritic tracing diagrams were reconstructed with Neurolucida, and quantitative analyses of total dendritic length and arborization indices in the two groups are still ongoing. 3) Brain-derived neurotrophic factor (BDNF) levels were assessed in the hippocampus and neocortex of 5 CLI and 5 control rats by ELISA assay. Interestingly, we found significant region-specific, between-group differences. In particular, BDNF levels, important for neurogenesis, differentiation and neuronal survival, and highly expressed in brain areas involved in cognitive and emotional behavior, were significantly decreased in the hippocampus of CLI rats compared to controls, whereas no differences were found in the cortex. Taken together, the data suggest that interfering with serotonergic regulation during early postnatal development can produce permanent brain changes. These resemble brain abnormalities repeatedly observed not only in human depression but also in schizophrenia. Further morphological analyses as well as experiments aimed at characterizing the behavioral correlates of early CLI administration are in progress

Brain metabolic DNA in memory processing and genome turnover

Sophisticated methods are currently used to investigate the properties of brain DNA and clarify its role under physiological conditions and in neurological and psychiatric disorders. Attention is now called on a DNA fraction present in the adult rat brain that is characterized by an elevated turnover and is not involved in cell division or DNA repair. The fraction, known as brain metabolic DNA (BMD), is modulated by strain, stress, circadian oscillations, exposure to enriched or impoverished environment, and notably by several training protocols and post-trial sleep. BMD is frequently localized in glial cells but is also present in neurons, often in the perinucleolar region. Its distribution in repetitive and non-repetitive DNA fractions shows that BMD differs from native DNA and that in learning rats its profile differs from that of control rats. More detailed knowledge of the molecular, cellular, and time-dependent BMD features will be necessary to define its role in memory acquisition and processing and in the pathogenesis of neurologic disorders

Brain dendritic cells

Dendritic cells (DCs) are a subset of leukocytes highly specialized in antigen-presentation to T cells, thus promoting the immune response. DCs occur in the meninges and choroid plexus. Brain DCs and brain-derived antigens are drained by cerebrospinal fluid in the afferent lymphatic vessels of cervical lymph nodes (cLNs) for antigen presentation. Information on the role of DCs in intracerebral immune response is still limited. We recently demonstrated (Laperchia et al., 2013) that in thy1GFP-M transgenic mice, engineered for the expression of green fluorescent protein (GFP) in a proportion of neurons, also myeloid DCs are GFP-tagged. Our in vivo analysis by two-photon microscopy on young (3-6 months) thy1GFP-M mice showed DCs floating in the cerebrospinal fluid or static at the pia mater/parenchyma interface. We are using this animal model to study brain DCs trafficking by two-photon microscopy in two different inflammatory conditions. The first concerns chronic encephalitis caused by the extracellular parasite Trypanosoma brucei. During the first, hemolymphatic stage of this infection, direct interactions between DCs and parasites were seen within meningeal and cortical microvessels. In the second stage, determined by parasite neuroinvasion, DCs invaded the brain parenchyma, exhibiting a random motion for target antigen recognition. With disease progression, intraparenchymal brain DCs were instead mainly arranged in static clusters which incorporated parasites for efficient antigen capture, and extravasated cytotoxic CD8+ T cells established contact with parasites. Ex vivo analysis on cLNs shown that the subcapsular zone was invaded by migratory DCs, and both migratory and resident DCs preferentially contacted CD8+ T cells. The second condition concerns normal aging (18-20 month old mice), which is known to be associated with low-grade chronic inflammation level and with functional impairments of the immune system, also known as immunosenescence. In these mice we observed that DCs infiltrate the brain parenchyma by transmigration from blood vessels and exhibit a motile behavior suggesting a scanning motion. Moreover, some DCs showed a progressive reduction of their motility until convergence, in about 30 minutes, into clusters whose functional significant has yet to be elucidated. Taken together, our studies enlightens key events of the intracerebral immune response both in presence of pathogens and in physiological aging

H1N1 influenza virus induces narcolepsy-like sleep disruption and targets sleep-wake regulatory neurons in mice

An increased incidence in the sleep-disorder narcolepsy has been associated with the 2009-2010 pandemic of H1N1 influenza virus in China and with mass vaccination campaigns against influenza during the pandemic in Finland and Sweden. Pathogenetic mechanisms of narcolepsy have so far mainly focused on autoimmunity. We here tested an alternative working hypothesis involving a direct role of influenza virus infection in the pathogenesis of narcolepsy in susceptible subjects. We show that infection with H1N1 influenza virus in mice that lack B and T cells (Recombinant activating gene 1-deficient mice) can lead to narcoleptic-like sleep-wake fragmentation and sleep structure alterations. Interestingly, the infection targeted brainstem and hypothalamic neurons, including orexin/hypocretin-producing neurons that regulate sleep-wake stability and are affected in narcolepsy. Because changes occurred in the absence of adaptive autoimmune responses, the findings show that brain infections with H1N1 virus have the potential to cause per se narcoleptic-like sleep disruption

Taqman PCR for determining parasite load.

<p><b>A:</b> Primer and probe sequences used in Taqman PCR to detect and quantify <i>T</i>. <i>b</i>. <i>brucei Pfr2</i> DNA. The amplification plot <b>(B)</b> and standard curve <b>(C)</b> obtained using a 10 fold dilution of standards containing 1x10⁶ to 1x10¹ copies of the <i>Pfr2</i> gene sequence indicate that the assay performs with high efficiency (102.9%) and returns a linear response (R² = 0.999) across a wide range of template concentrations. <b>D:</b> The interval plot represents the trypanosome load measured using this Taqman PCR assay within rat brains at 6, 14 and 21 day post-infection (dpi), <i>n</i> = 3 at each time-point; ♦ indicates the group mean; bars represent 95% confidence interval for the group mean; * significantly higher (P<0.001) than unmarked groups.</p