Roadmap for Postnatal Brain Maturation: Changes in Gray and White Matter Composition during Development Measured by Fourier Transformed Infrared Microspectroscopy.

Key events in postnatal brain development, such as neuronal migration, synaptogenesis, and myelination, shape the adult brain. These events are reflected in changes in gray and white matter (GM and WM) occurring during this period. Therefore, precise knowledge of GM and WM composition in perinatal brain development is crucial to characterizing brain formation as well as the neurodevelopmental disruption observed in diseases such as autism and schizophrenia. In this study, we combined histochemical and immunohistochemical staining with biochemical and biophysical analyses using Fourier transform infrared (IR) microspectroscopy (µFTIR) to better understand the chemical changes during postnatal developmental myelination. For this purpose, we analyzed the GM and WM in the mouse brain and cerebellum (strain C57BL/6) from postnatal day 0 (P0) to day P28 and established presumed correlations between staining and IR data. IR spectra allowed the (i) quantification of lipid and protein content through the CH2/amide I ratio, (ii) determination of chemical characteristics of lipids, such as the presence of unsaturated bonds in the carbonate chain or carbonyls from ester groups in the polar head, and (iii) determination of the protein secondary structure (α-helix and intramolecular ß-sheets). The results indicate that the increase in the CH2/amide I ratio calculated from the µFTIR data correlates well with lipid histochemical staining. IR data indicated a change in the lipid composition in WM since carbonyl and unsaturated olefinic groups do not increase when lipids accumulate during myelination. Our correlation analysis between IR data and immunohistochemical staining of myelin-associated proteins revealed that myelin oligodendrocyte protein correlated well with lipid accumulation, while myelin basic protein appeared before lipid modifications, which indicated that myelin-associated proteins and lipid deposition were not synchronic. These events were related to a decrease in the intramolecular ß/α protein ratio. Our results indicate that lipids and proteins in WM substantially change their composition due to primary myelination, and according to results obtained from staining, these modifications are better described by lipid histochemical staining than by immunohistochemistry against myelin-related proteins. In conclusion, µFTIR can be a useful technique to study WM during perinatal development and provide detailed information about alterations in the chemical composition related to neurodevelopmental diseases.

Astrocyte-targeted Overproduction of IL-10 Reduces Neurodegeneration after TBI.

Traumatic brain injury is the greatest cause of disability and death in young adults in the developed world. The outcome for a TBI patient is determined by the severity of the injury, not only from the initial insult but, especially, as a product of the secondary injury. It is proposed that this secondary injury is directly linked to neuro-inflammation, with the production of pro-inflammatory mediators, activation of resident glial cells and infiltration of peripheral immune cells. In this context, anti-inflammatory treatments are one of the most promising therapies to dampen the inflammatory response associated with TBI and to reduce secondary injury. In this sense, the main objective of the present study is to elucidate the effect of local production of IL-10 in the neurological outcome after TBI. For this purpose, a cryogenic lesion was caused in transgenic animals overproducing IL-10 under the GFAP promoter on astrocytes (GFAP-IL10Tg mice) and the neuro-protection, microglial activation and leukocyte recruitment were evaluated. Our results showed a protective effect of IL-10 on neurons at early time-points after TBI, in correlation with a shift in the microglial activation profile towards a down-regulating phenotype and lower production of pro-inflammatory cytokines. Concomitantly, we observed a reduction in the BBB leakage together with modifications in leukocyte infiltration into the affected area. In conclusion, local IL-10 production modifies the neuro-inflammatory response after TBI, shifting it to anti-inflammatory and neuro-protective conditions. These results point to IL-10 as a promising candidate to improve neuro-inflammation associated with TBI.

TRPV2: A Key Player in Myelination Disorders of the Central Nervous System.

Transient potential receptor vanilloid 2 (TRPV2) is widely expressed through the nervous system and specifically found in neuronal subpopulations and some glial cells. TRPV2 is known to be sensitized by methionine oxidation, which results from inflammation. Here we aim to characterize the expression and regulation of TRPV2 in myelination pathologies, such as hypomyelination and demyelination. We validated the interaction between TRPV2 and its putative interactor Opalin, an oligodendrocyte marker, in mixed glial cultures under pro- and anti-inflammatory conditions. Then, we characterized TRPV2 time-course expression in experimental animal models of hypomyelination (jimpy mice) and de-/remyelination (cuprizone intoxication and experimental autoimmune encephalomyelitis (EAE)). TRPV2 showed upregulation associated with remyelination, inflammation in cuprizone and EAE models, and downregulation in hypomyelinated jimpy mice. TRPV2 expression was altered in human samples of multiple sclerosis (MS) patients. Additionally, we analyzed the expression of methionine sulfoxide reductase A (MSRA), an enzyme that reduces oxidated methionines in TRPV2, which we found increased in inflammatory conditions. These results suggest that TRPV2 may be a key player in myelination in accordance with the recapitulation hypothesis, and that it may become an interesting clinical target in the treatment of demyelination disorders.

Chronic IL-10 overproduction disrupts microglia-neuron dialogue similar to aging, resulting in impaired hippocampal neurogenesis and spatial memory.

The subgranular zone of the dentate gyrus is an adult neurogenic niche where new neurons are continuously generated. A dramatic hippocampal neurogenesis decline occurs with increasing age, contributing to cognitive deficits. The process of neurogenesis is intimately regulated by the microenvironment, with inflammation being considered a strong negative factor for this process. Thus, we hypothesize that the reduction of new neurons in the aged brain could be attributed to the age-related microenvironmental changes towards a pro-inflammatory status. In this work, we evaluated whether an anti-inflammatory microenvironment could counteract the negative effect of age on promoting new hippocampal neurons. Surprisingly, our results show that transgenic animals chronically overexpressing IL-10 by astrocytes present a decreased hippocampal neurogenesis in adulthood. This results from an impairment in the survival of neural newborn cells without differences in cell proliferation. In parallel, hippocampal-dependent spatial learning and memory processes were affected by IL-10 overproduction as assessed by the Morris water maze test. Microglial cells, which are key players in the neurogenesis process, presented a different phenotype in transgenic animals characterized by high activation together with alterations in receptors involved in neuronal communication, such as CD200R and CX3CR1. Interestingly, the changes described in adult transgenic animals were similar to those observed by the effect of normal aging. Thus, our data suggest that chronic IL-10 overproduction mimics the physiological age-related disruption of the microglia-neuron dialogue, resulting in hippocampal neurogenesis decrease and spatial memory impairment.

Specific microglial phagocytic phenotype and decrease of lipid oxidation in white matter areas during aging: Implications of different microenvironments.

Physiological aging is characterized by an imbalance of pro-inflammatory and anti-inflammatory mediators leading to neuroinflammation. Microglial cells, which are highly regulated by the local microenvironment, undergo specific changes depending upon the brain area during aging. The aim of this study was to evaluate the influence of age over microglial cells along different brain areas and microenvironments. For this purpose, transgenic mice with overproduction of either the anti-inflammatory IL-10 cytokine or the pro-inflammatory IL-6 cytokine were used. Our results show that, during aging, microglial cells located in white matter (WM) areas maintain their phagocytic capacity but present a specific phagocytic phenotype with receptors involved in myelin recognition, arguing for aging-derived myelin damage. Whereas IL-10 overproduction anticipates the age-related microglial phagocytic phenotype, maintaining it over time, IL-6 overproduction exacerbates this phenotype in aging. These modifications were linked with a higher efficiency of myelin engulfment by microglia in aged transgenic animals. Moreover, we show, in a novel way, lower lipid oxidation during aging in WM areas, regardless of the genotype. The novelty of the insights presented in this study open a window to deeply investigate myelin lipid oxidation and the role of microglial cells in its regulation during physiological aging.

Evaluation of Myelin Phagocytosis by Microglia/Macrophages in Nervous Tissue Using Flow Cytometry.

Determination of microglial phagocytosis of myelin has acquired importance in the study of demyelinating diseases. One strategy to determine microglial phagocytosis capacity consists of assaying microglia with fluorescently labeled myelin; however, most approaches are performed in cell culture, where microglia usually show important phenotypic differences compared with in vivo conditions. In this article we describe an adapted flow cytometry protocol to assay myelin phagocytosis by microglia obtained directly from in vivo tissue of the central nervous system. Key steps for a first analysis of phagocytic microglia are provided. Additionally, we describe how to fluorescently label myelin using a pH-sensitive tag, pHrodo™ Green STP Ester. © 2021 Wiley Periodicals LLC. Basic Protocol: Assay for determination of myelin phagocytosis by microglia/macrophages using flow cytometry Support Protocol 1: Conjugation of isolated and purified myelin with pHrodo Green STP Ester Support Protocol 2: Quantification of phagocytic cell number by flow cytometry.

Chronic exposure to IL-6 induces a desensitized phenotype of the microglia.

BACKGROUND: When the homeostasis of the central nervous system (CNS) is altered, microglial cells become activated displaying a wide range of phenotypes that depend on the specific site, the nature of the activator, and particularly the microenvironment generated by the lesion. Cytokines are important signals involved in the modulation of the molecular microenvironment and hence play a pivotal role in orchestrating microglial activation. Among them, interleukin-6 (IL-6) is a pleiotropic cytokine described in a wide range of pathological conditions as a potent inducer and modulator of microglial activation, but with contradictory results regarding its detrimental or beneficial functions. The objective of the present study was to evaluate the effects of chronic IL-6 production on the immune response associated with CNS-axonal anterograde degeneration. METHODS: The perforant pathway transection (PPT) paradigm was used in transgenic mice with astrocyte-targeted IL6-production (GFAP-IL6Tg). At 2, 3, 7, 14, and 21 days post-lesion, the hippocampal areas were processed for immunohistochemistry, flow cytometry, and protein microarray. RESULTS: An increase in the microglia/macrophage density was observed in GFAP-IL6Tg animals in non-lesion conditions and at later time-points after PPT, associated with higher microglial proliferation and a major monocyte/macrophage cell infiltration. Besides, in homeostasis, GFAP-IL6Tg showed an environment usually linked with an innate immune response, with more perivascular CD11b+/CD45high/MHCII+/CD86+ macrophages, higher T cell infiltration, and higher IL-10, IL-13, IL-17, and IL-6 production. After PPT, WT animals show a change in microglia phenotype expressing MHCII and co-stimulatory molecules, whereas transgenic mice lack this shift. This lack of response in the GFAP-IL6Tg was associated with lower axonal sprouting. CONCLUSIONS: Chronic exposure to IL-6 induces a desensitized phenotype of the microglia.

Differential Roles of TREM2+ Microglia in Anterograde and Retrograde Axonal Injury Models.

Microglia are the main immune cells of the central nervous system (CNS), and they are devoted to the active surveillance of the CNS during homeostasis and disease. In the last years, the microglial receptor Triggering Receptor Expressed on Myeloid cells-2 (TREM2) has been defined to mediate several microglial functions, including phagocytosis, survival, proliferation, and migration, and to be a key regulator of a new common microglial signature induced under neurodegenerative conditions and aging, also known as disease-associated microglia (DAM). Although microglial TREM2 has been mainly studied in chronic neurodegenerative diseases, few studies address its regulation and functions in acute inflammatory injuries. In this context, the present work aims to study the regulation of TREM2 and its functions after reparative axonal injuries, using two-well established animal models of anterograde and retrograde neuronal degeneration: the perforant pathway transection (PPT) and the facial nerve axotomy (FNA). Our results indicate the appearance of a subpopulation of microglia expressing TREM2 after both anterograde and retrograde axonal injury. TREM2+ microglia were not directly related to proliferation, instead, they were associated with specific recognition and/or phagocytosis of myelin and degenerating neurons, as assessed by immunohistochemistry and flow cytometry. Characterization of TREM2+ microglia showed expression of CD16/32, CD68, and occasional Galectin-3. However, specific singularities within each model were observed in P2RY12 expression, which was only downregulated after PPT, and in ApoE, where de novo expression was detected only in TREM2+ microglia after FNA. Finally, we report that the pro-inflammatory or anti-inflammatory cytokine microenvironment, which may affect phagocytosis, did not directly modify the induction of TREM2+ subpopulation in any injury model, although it changed TREM2 levels due to modification of the microglial activation pattern. In conclusion, we describe a unique TREM2+ microglial subpopulation induced after axonal injury, which is directly associated with phagocytosis of specific cell remnants and show different phenotypes, depending on the microglial activation status and the degree of tissue injury.

From Mouse to Human: Comparative Analysis between Grey and White Matter by Synchrotron-Fourier Transformed Infrared Microspectroscopy.

Fourier Transform Infrared microspectroscopy (µFTIR) is a very useful method to analyze the biochemical properties of biological samples in situ. Many diseases affecting the central nervous system (CNS) have been studied using this method, to elucidate alterations in lipid oxidation or protein aggregation, among others. In this work, we describe in detail the characteristics between grey matter (GM) and white matter (WM) areas of the human brain by µFTIR, and we compare them with the mouse brain (strain C57BL/6), the most used animal model in neurological disorders. Our results show a clear different infrared profile between brain areas in the lipid region of both species. After applying a second derivative in the data, we established a 1.5 threshold value for the lipid/protein ratio to discriminate between GM and WM areas in non-pathological conditions. Furthermore, we demonstrated intrinsic differences of lipids and proteins by cerebral area. Lipids from GM present higher C=CH, C=O and CH3 functional groups compared to WM in humans and mice. Regarding proteins, GM present lower Amide II amounts and higher intramolecular ß-sheet structure amounts with respect to WM in both species. However, the presence of intermolecular ß-sheet structures, which is related to ß-aggregation, was only observed in the GM of some human individuals. The present study defines the relevant biochemical properties of non-pathological human and mouse brains by µFTIR as a benchmark for future studies involving CNS pathological samples.

The benefits of voluntary physical exercise after traumatic brain injury on rat's object recognition memory: A comparison of different temporal schedules.

Physical exercise can reduce the cognitive decline associated with traumatic brain injury, yet little is known about the optimal administration schedules. Here, different protocols of voluntary wheel running were evaluated for their effects on object recognition memory (ORM), neuroprotection (NeuN+ cells), microglial reactivity (Iba1 staining) and neurogenesis (DCX+ cells) after controlled cortical impact injury (CCI). CCI-lesioned rats were divided into a sedentary group and three exercise groups: early discontinued exercise (3 weeks of exercise initiated 4 days post-injury, followed by 4 weeks in a sedentary state); delayed exercise (3 weeks of exercise initiated 4 weeks post-injury), and early continuous exercise (7 weeks of exercise starting 4 days post-injury). The deficits induced by CCI in a 24 h ORM test were reversed in the delayed exercise group and reduced in the early discontinued and early continuous groups. The early discontinued protocol also reduced the loss of NeuN+ cells in the hilus, while attenuated microglial reactivity was found in the dorsal hippocampus of both the early exercising groups. Running at the end of the experiment increased the number of DCX+ cells in the early continuous and delayed groups, and an inverted U-shaped relationship was found between the mean daily exercise time and the amount of neurogenesis. Thus, exercise had benefits on memory both when it was commenced soon and later after injury, although the neural mechanisms implicated differed. Accordingly, the effects of exercise on memory and neurogenesis appear to not only depend on the specific temporal schedule but also, they may be influenced by the amount of daily exercise.

Role of the CD200-CD200R Axis During Homeostasis and Neuroinflammation.

Microglia are considered to be the resident macrophages of the CNS and main effector of immune brain function. Due to their essential role in the regulation of neuroinflammatory response, microglia constitute an important target for neurological diseases, such as multiple sclerosis, Alzheimer's or Parkinson's disease. The communication between neurons and microglia contributes to a proper maintenance of homeostasis in the CNS. Research developed in the last decade has demonstrated that this interaction is mediated by "Off-signals" - molecules exerting immune inhibition - and "On signals" - molecules triggering immune activation. Among "Off signals", molecular pair CD200 and its CD200R receptor, expressed mainly in the membrane of neurons and microglia, respectively, have centered our attention due to its unexplored and powerful immunoregulatory functions. In this review, we will offer an updated global view of the CD200-CD200R role in the microglia-neuron crosstalk during homeostasis and neuroinflammation. Specifically, the effects of CD200-CD200R in the inhibition of pro-inflammatory microglial activation will be explained, and their involvement in other functions such as homeostasis preservation, tissue repair, and brain aging, among others, will be pointed out. In addition, we will depict the effects of CD200-CD200R uncoupling in the etiopathogenesis of autoimmune and neurodegenerative diseases. Finally, we will explore how to translate the scientific evidence of CD200-CD200R interaction into possible clinical therapeutic strategies to tackle neuroinflammatory CNS diseases.

Astrocyte-targeted IL-10 production decreases proliferation and induces a downregulation of activated microglia/macrophages after PPT.

When central nervous system (CNS) homeostasis is altered, microglial cells become rapidly activated, proliferate and release a broad range of molecules. Among the plethora of molecules involved in the regulation of microglial activation, cytokines are considered crucial. Although production of interleukin-10 (IL-10) has been demonstrated after different types of CNS injuries and associated with protective functions, the specific role played by IL-10 modulating microglial cells remains unclear. Hence, the objective of this study was to evaluate the effects of transgenic astrocyte IL-10 production on microglial activation associated with axonal anterograde degeneration. To address it, the hippocampal area subjected to perforant pathway transection (PPT) was analyzed by immunohistochemistry (IHC), flow cytometry and protein microarray in transgenic (GFAP-IL10Tg) mice and their corresponding wild types (WT) littermates. Our results demonstrated increased microglial/macrophages density in nonlesioned and PPT-lesioned GFAP-IL10Tg animals when compared with nonlesioned and lesioned WT, respectively. This increase was not due to proliferation, as GFAP-IL10Tg mice showed a reduced proliferation of microglial cells, but was related to an increased population of CD11b+/CD45high monocyte/macrophages. Despite this higher number, the microglia/macrophage population in transgenic animals displayed a downregulated phenotype characterized by lower MHCII, ICOSL, and CD11c. Moreover, a sustained T-cell infiltration was found in transgenic animals. We strongly suggest these modifications must be associated with indirect effects derived from the influence of IL-10 on astrocytes and/or neurons, which express IL-10R. We finally suggested that TGF-ß produced by astrocytes, along with IL-2 and CXCL10 might be crucial molecules mediating the effects of transgenic IL-10.

Purine Signaling and Microglial Wrapping.

Microglial cells are highly dynamic cells with processes continuously moving to survey the surrounding territory. Microglia possess a broad variety of surface receptors and subtle changes in their microenvironment cause microglial cell processes to extend, retract, and interact with neuronal synaptic contacts. When the nervous system is disturbed, microglia activate, proliferate, and migrate to sites of injury in response to alert signals. Released nucleotides like ATP and UTP are among the wide range of molecules promoting microglial activation and guiding their migration and phagocytic function. The increased concentration of nucleotides in the extracellular space could be involved in the microglial wrapping found around injured neurons in various pathological conditions, especially after peripheral axotomy. Microglial wrappings isolate injured neurons from synaptic inputs and facilitate the molecular dialog between endangered or injured neurons and activated microglia. Astrocytes may also participate in neuronal ensheathment. Degradation of ATP by microglial ecto-nucleotidases and the expression of various purine receptors might be decisive in regulating the function of enwrapping glial cells and in determining the fate of damaged neurons, which may die or may regenerate their axons and survive.

Are Microglial Cells the Regulators of Lymphocyte Responses in the CNS?

The infiltration of immune cells in the central nervous system is a common hallmark in different neuroinflammatory conditions. Accumulating evidence indicates that resident glial cells can establish a cross-talk with infiltrated immune cells, including T-cells, regulating their recruitment, activation and function within the CNS. Although the healthy CNS has been thought to be devoid of professional dendritic cells (DCs), numerous studies have reported the presence of a population of DCs in specific locations such as the meninges, choroid plexuses and the perivascular space. Moreover, the infiltration of DC precursors during neuroinflammatory situations has been proposed, suggesting a putative role of these cells in the regulation of lymphocyte activity within the CNS. On the other hand, under specific circumstances, microglial cells are able to acquire a phenotype of DC expressing a wide range of molecules that equip these cells with all the necessary machinery for communication with T-cells. In this review, we summarize the current knowledge on the expression of molecules involved in the cross-talk with T-cells in both microglial cells and DCs and discuss the potential contribution of each of these cell populations on the control of lymphocyte function within the CNS.

Astrocyte-targeted production of IL-10 induces changes in microglial reactivity and reduces motor neuron death after facial nerve axotomy.

Interleukin-10 (IL-10) is a cytokine that plays a crucial role in regulating the inflammatory response and immune reactions. In the central nervous system (CNS), IL-10 is mainly produced by astrocytes and microglia and it is upregulated after various insults, such as experimental autoimmune encephalomyelitis, middle cerebral artery occlusion, excitotoxicity and traumatic brain injury. To better understand the effects of IL-10 in the normal and injured CNS, we generated transgenic mice (termed GFAP-IL-10Tg) that expressed the murine IL-10 gene under the transcriptional control of the glial fibrillary acidic protein (GFAP) promoter. Previous studies demonstrated marked changes in the microglial phenotype in these mice under basal conditions. The objective of the present study was to investigate the effects of local astrocyte-targeted IL-10 production on glial activation, neuronal degeneration and leukocyte recruitment after axotomy. GFAP-IL-10Tg mice had marked changes in the phenotype of activated microglial cells, as well as in the number of microglial clusters and in microglial cell density. These microglial changes are accompanied by a twofold increase in lymphocyte infiltration in GFAP-IL-10Tg mice and around twofold decrease in neuronal cell death at 21 dpi. Altogether, our findings suggested that astrocyte-targeted production of IL-10 impacted the microglial response and lymphocyte recruitment and culminated in a beneficial effect on neuronal survival.

Alterations in microglial phenotype and hippocampal neuronal function in transgenic mice with astrocyte-targeted production of interleukin-10.

Interleukin-10 (IL-10) is a cytokine classically linked with anti-inflammatory and protective functions in the central nervous system (CNS) in different neurodegenerative and neuroinflammatory conditions. In order to study the specific role of local CNS produced IL-10, we have created a new transgenic mouse line with astrocyte-targeted production of IL-10 (GFAP-IL10Tg). In the present study, the effects of local CNS IL-10 production on microglia, astrocytes and neuronal connectivity under basal conditions were investigated using immunohistochemistry, molecular biology techniques, electrophysiology and behavioural studies. Our results showed that, in GFAP-IL10Tg animals, microglia displayed an increase in density and a specific activated phenotype characterised by morphological changes in specific areas of the brain including the hippocampus, cortex and cerebellum that correlated with the level of transgene expressed IL-10 mRNA. Distinctively, in the hippocampus, microglial cells adopted an elongated morphology following the same direction as the dendrites of pyramidal neurons. Moreover, this IL-10-induced microglial phenotype showed increased expression of certain molecules including Iba1, CD11b, CD16/32 and F4/80 markers, "de novo" expression of CD150 and no detectable levels of either CD206 or MHCII. To evaluate whether this specific activated microglial phenotype was associated with changes in neuronal activity, the electrophysiological properties of pyramidal neurons of the hippocampus (CA3-CA1) were analysed in vivo. We found a lower excitability of the CA3-CA1 synapses and absence of long-term potentiation (LTP) in GFAP-IL10Tg mice. This study is the first description of a transgenic mouse with astrocyte-targeted production of the cytokine IL-10. The findings indicate that IL-10 induces a specific activated microglial phenotype concomitant with changes in hippocampal LTP responses. This transgenic animal will be a very useful tool to study IL-10 functions in the CNS, not only under basal conditions, but also after different experimental lesions or induced diseases.

Effects of astrocyte-targeted production of interleukin-6 in the mouse on the host response to nerve injury.

Interleukin-6 (IL-6) is a pleiotropic cytokine with a key role in the control of inflammatory/immune responses. In the central nervous system (CNS), an increase in IL-6 occurs in a wide range of pathological conditions such as excitotoxicity and traumatic brain injury. We evaluated the effects of astrocyte-targeted production of IL-6 in the CNS in the sterile-nerve injury model of facial nerve axotomy. To accomplish this, facial nerve transection was performed in transgenic mice (glial fibrillary acidic protein [GFAP]-IL6Tg) with IL-6 production under the GFAP promoter. Neuronal death, glial activation, lymphocyte recruitment, and integrin expression were evaluated by immunohistochemistry and flow cytometry from 3 to 28 days postinjury. Our findings revealed an increase in motor neuron cell death in GFAP-IL6Tg mice correlating with changes in the microglial activation pattern, characterized principally by less attachment to neurons and reduced expression of both CD11b and CD18. We also found a higher CD4(+) T-lymphocyte recruitment in GFAP-IL6Tg mice. In addition, changes in the expression pattern of different integrins and their receptors were observed in transgenic animals. Specifically, alterations in osteopontin expression in motor neurons and its receptors CD44 and CD49e in lymphocytes and microglia, respectively, which may account for the variations related to glial reactivity and lymphocyte infiltration. In conclusion, our results indicated that forced local production of IL-6 has a direct impact on the outcome of nerve injury in the CNS inducing an increase in neurodegeneration, changes in glial response, and lymphocyte recruitment as well as in the expression of different integrins and their receptors.

Microglia detection by enzymatic histochemistry.

Visualization of microglia by means of histochemistry has been for years a reliable method to demonstrate this population of cells in the central nervous system (CNS). Wide range of data on microglia has been published using lectin and enzymatic histochemistry. While at present, in most laboratories, the use of specific antibodies is the first choice, histochemical detection of microglia remains a powerful method as it has certain advantages upon immunohistochemical methods because it is faster, cheaper, and can be used in different species including human. In this chapter we want to present the detailed methodology for microglial staining using the histoenzymatic demonstration of the enzyme nucleoside-diphosphatase (NDPase), a phosphatase found in the plasma membrane of microglia that is absent in the plasma membrane of other glial cells and neurons. With this technique it is possible to visualize amoeboid microglia during development, ramified microglia in the adult brain, and also reactive microglia. As the technique also stains the blood vessels, it allows the analysis of the relationship between microglia and vasculature. This method can be performed in either histological sections or cell cultures for light microscopy analysis. Furthermore, we described how to combine this histochemical method with conventional immunohistochemistry for double labelling using other markers, and finally we give details to perform the procedure not only for optical microscopic studies but also for transmission electron microscopy (TEM).

Tomato lectin histochemistry for microglial visualization.

The use of different lectins for the study of microglial cells in the central nervous system (CNS) is a valuable tool that has been extensively used in the last years for the selective staining of this glial cell population, not only in normal physiological conditions, but also in a wide range of pathological situations where the normal homeostasis of the parenchyma is disturbed. In this chapter we accurately describe the methodology for the selective labelling of microglial cells by using the tomato lectin (TL), a protein lectin obtained from Lycopersicum esculentum with specific affinity for poly-N-acetyl lactosamine sugar residues which are found on the plasma membrane and in the cytoplasm of microglia. Here we describe how to perform this technique on vibratome, frozen, and paraffin sections for optical microscopy, as well as for transmission electron microscopy (TEM) studies. Using this methodology it is possible to visualize amoeboid microglia in the developing brain, ramified microglia in the adult, and activated/reactive microglia in the experimentally damaged brain. In addition, as TL also recognized sugar residues in endothelial cells, this technique is very useful for the study of the relationship established between microglia and the CNS vasculature.

Increase in Th17 and T-reg lymphocytes and decrease of IL22 correlate with the recovery phase of acute EAE in rat.

Experimental autoimmune encephalomyelitis (EAE), a well-established model of multiple sclerosis, is characterised by microglial activation and lymphocyte infiltration. Induction of EAE in Lewis rats produces an acute monophasic disease characterised by a single peak of disability followed by a spontaneous and complete recovery and a subsequent tolerance to further immunizations. In the current study we have performed a detailed analysis of the dynamics of different lymphocyte populations and cytokine profile along the induction, peak, recovery and post-recovery phases in this paradigm. MBP-injected rats were sacrificed attending exclusively to their clinical score, and the different populations of T-lymphocytes as well as the dynamics of different pro- and anti-inflammatory cytokines were analysed in the spinal cord by flow cytometry, immunohistochemistry and ELISA. Our results revealed that, during the induction and peak phases, in parallel to an increase in symptomatology, the number of CD3+ and CD4+ cells increased progressively, showing a Th1 phenotype, but unexpectedly during recovery, although clinical signs progressively decreased, the number and proportion of CD3+ and CD4+ populations remained unaltered. Interestingly, during this recovery phase, we observed a marked decrease of Th1 and an important increase in Th17 and T-reg cells. Moreover, our results indicate a specific cytokine expression profile along the EAE course characterized by no changes of IL10 and IL17 levels, decrease of IL21 on the peak, and high IL22 levels during the induction and peak phases that markedly decrease during recovery. In summary, these results revealed the existence of a specific pattern of lymphocyte infiltration and cytokine secretion along the different phases of the acute EAE model in Lewis rat that differs from those already described in chronic or relapsing-remitting mouse models, where Th17-cells were found mostly during the peak, suggesting a specific role of these lymphocytes and cytokines in the evolution of this acute EAE model.