Lentiviral transduction of microglial cells

Microglial cells are the resident immune cells of the central nervous system. Their function resembles that of tissue macrophages and, as such, they share many properties with both peripheral macrophages and monocytes. One striking similarity is the difficulty with which these cells can be genetically manipulated via transfection or transduction. We have sought to overcome this challenge and generate stably transduced microglial cell lines. Based on encouraging results from macrophages, we hypothesized that lentiviral vectors might provide a valuable tool in the transduction of microglial cells. Using a lentiviral-based vector system expressing enhanced green fluorescent protein (eGFP) under the control of the murine stem cell virus promoter (MSCV), we found that multiplicities of infection (MOIs) of 1, 10, and 100 transduce >70%, >88%, and >95% of the cells, respectively. From the pool of transduced cells, we established lines of N9 and BV-2 microglial cells with distinct fluorescence intensities. Using real time-polymerase chain reaction (PCR), we correlated the integrated eGFP copy numbers to eGFP fluorescence measured by flow cytometry. When mixed, up to three lines with different eGFP intensities could be separated by flow cytometry and fluorescence microscopy. Neither infection nor transgene expression influenced microglial activation as assessed by nitric oxide (NO) production, cytokine release, and surface antigen expression. Our findings that microglial cells are easily transduced by lentiviral based vectors will facilitate research depending on genetic manipulation and help generate transgenic cell lines. In addition, the availability of microglial cell lines with defined fluorescence properties could replace elaborate staining procedures for microglial identification in co-culture experiments.     

Automatic counting of microglial cell activation and its applications

Glaucoma is a multifactorial optic neuropathy characterized by the damage and death of the retinal gan-glion cells. This disease results in vision loss and blindness. Any vision loss resulting from the disease cannot be restored and nowadays there is no available cure for glaucoma;however an early detection and treatment, could offer neuronal protection and avoid later serious damages to the visual function. A full understanding of the etiology of the disease will still require the contribution of many scientiifc efforts. Glial activation has been observed in glaucoma, being microglial proliferation a hallmark in this neuro-degenerative disease. A typical project studying these cellular changes involved in glaucoma often needs thousands of images-from several animals-covering different layers and regions of the retina. The gold standard to evaluate them is the manual count. This method requires a large amount of time from special-ized personnel. It is a tedious process and prone to human error. We present here a new method to count microglial cells by using a computer algorithm. It counts in one hour the same number of images that a researcher counts in four weeks, with no loss of reliability.     

Hypersensitivity to ovalbumin induces chronic intestinal dysmotility and increases the number of intestinal mast cells

Undiagnosed food allergies have been proposed as possible causes of promoting and perpetuating irritable bowel syndrome . Our aim was to find out if sensitization could induce chronic functional motor disturbances in the intestine and the mechanisms implicated. Rats were sensitized to ovalbumin (OVA) following three hypersensitivity induction protocols, two parenteral and one oral. Rat mast cell protease II (RMCP II) release in response to OVA challenge and immunoglobulin E (IgE) concentration were measured in serum. At least 1 week after challenge, small intestinal motility was evaluated using strain gauges. Intestinal tissue samples from orally sensitized rats were checked for in vitro stimulation with OVA. Mucosal mast cells were counted from duodenum sections. All sensitized rats showed intestinal hypermotility. Only rats sensitized by parenteral procedure showed an increase in RMCP II after OVA challenge in serum. IgEs increased only in the Bordetella pertussis sensitized group. Small intestine sections from orally sensitized rats released more RMCP II than sections from control rats. All sensitized rats showed an increase in the number of mucosal mast cells in duodenum. In conclusion, hypersensitivity to food proteins induces chronic motor alteration that persists long after antigen challenge and an excited/activated state of sensitized mucosal mast cells.     

Novel insights in the role of peripheral corticotropin-releasing factor and mast cells in stress-induced visceral hypersensitivity

Visceral hypersensitivity is one of the hallmarks in irritable bowel syndrome (IBS) pathophysiology. Stress is well known to affect visceral sensitivity in humans and rodents, an effect which is associated in part with alterations of intestinal epithelial permeability in rodents. Although the pathophysiology of visceral hypersensitivity is still unclear, two key factors have been identified as playing a major role in its modulation, namely peripheral corticotropin-releasing factor (CRF) and mast cells. In a recent study in Neurogastroenterology and Motility, van den Wijngaard et al. demonstrate that the mast-cell dependent visceral hypersensitivity observed in maternally separated rats after an acute exposure to a psychological stress can be prevented but not reversed by the peripherally restricted CRF receptor antagonist, α-helical CRF(9-41). They further show that the preventive effect of the CRF receptor antagonist is linked to a stabilization of mast cells and maintenance of the epithelial barrier at the colonic level. These data suggest that post stress mast cell activation and subsequent visceral hypersensitivity are not targeted by peripheral CRF receptor antagonists. These novel insights in the role of peripheral CRF in the modulation of stress-induced visceral hypersensitivity add to our growing understanding of the mechanisms that may lie at the origin of visceral pain disturbances following stress and will contribute to enhance the development of drugs that may have potential therapeutic benefits for IBS patients.     

Factors inducing mast cell accumulation in skeletal muscle

It has been suggested that mast cells contribute to the phenotype of dystrophinopathies, but the mechanisms of their recruitment into the skeletal muscle remain hypothetical. The aim of this study is to quantify the presence of mast cells in muscle during the cellular events of myofibre degeneration and regeneration. For this purpose, we compare the mast cell profile in dystrophin-deficient mdx mice in which muscles exhibit spontaneous cycles of degeneration-regeneration from 3 weeks of age, with that in Swiss mice in which muscles were injured either by ischaemia or by notexin injection. Notexin is an A2-type phospholipase that rapidly disrupts myofibre plasma membranes, while ischaemia results in a slower process of degeneration. Both lesions are followed by a successful regeneration. In intact muscles, mast cell counts (mean±SEM/mm²) range from 1.8± 1 to 4.3 ± 1.6. The injection of notexin is far more potent in recruiting mast cells into damaged muscle than is ischaemia (118.5±13.0 vs 12.3 ± 1.8/mm²). Thus we conclude that the early disruption of the myofibre membrane could elicit mast cell accumulation in skeletal muscle. This may explain the elevated number of mast cells observed in mdx muscles, as dystrophin deficiency is thought to induce myofibre membrane leakage. On the other hand, mast cells are more numerous in muscles of young and adult mdx mice that are allowed to regenerate, than in muscles of older animals in which there is little regeneration and fibrosis develops. In injured muscles, the peak of mast cell number is at the onset of regeneration (by day 3 after notexin injection, and by day 11 after ischaemia), rather than during the phase of myofibre necrosis. Therefore, we suggest that the mast cells, through the effects of released mediators, could contribute to muscle regeneration.     

Ultrastructural injury to interstitial cells of Cajal and communication with mast cells in Crohn''s disease

Crohn's disease associated dysmotility has been attributed to fibrosis and damage to enteric nerves but injury to interstitial cells of Cajal (ICC) could also be involved. We assessed ICC in specimens obtained from patients with Crohn's disease and determined the relation between ICC and the inflammatory infiltrate, particularly mast cells (MC) using quantitative immunohistochemistry and electron microscopy. Ultrastructural injury to ICC was patchy in all ICC subtypes but ICC-Auerbach's plexus (AP) showed damage more frequently, i.e. swelling of mitochondria, decreased electron density, autophagosomes and partial depletion of the cytoplasm. Light microscopy confirmed a significant decrease in c-kit immunoreactivity for ICC-AP and an increased number of MC in the muscularis externa. Electron microscopy showed MC exhibiting piecemeal degranulation and making frequent and selective membrane-to-membrane contact with all types of injured ICC which suggests chronic release of granule content to affect ICC. Extent of ICC injury was not associated with duration of the disease. In conclusion, ultrastructural injury and loss of ICC-AP is evident in Crohn's disease. Epidemiological and morphological data suggest that ICC have the capacity to regenerate in spite of the chronic insult. The muscularis hosts a marked number of MC that exhibit piecemeal degranulation associated with ICC and may facilitate ICC maintenance.     

Transient expression of endothelins in the amoeboid microglial cells in the developing rat brain

Amoeboid microglial cells (AMC) which transiently exist in the corpus callosum in the postnatal rat brain expressed endothelins (ETs), specifically endothelin-1 (ET-1) and ET3 as revealed by real time RT-PCR. ET immunoreactive AMC occurred in large numbers at birth, but were progressively reduced with age and were undetected in 14 days. In rats subjected to hypoxia exposure, ET immunoexpression in AMC was reduced but the incidence of apoptotic cells was not increased when compared with the control suggesting that this was due to its downregulation that may help regulate the constriction of blood vessels bearing ET-A receptor. AMC were endowed ET-B receptor indicating that ET released by the cells may also act via an autocrine manner. In microglia activated by lipopolysaccharide (LPS), ET-1 mNA expression coupled with that of monocyte chemoattractant protein (MCP-1) and stromal derived factor-1 (SDF-1) was markedly increased; ET-3 mRNA, however, remained unaffected. AMC exposed to oxygen glucose deprivation (OGD) in vitro resulted in increase in both ET-1 and ET-3 mRNA expression. It is suggested that the downregulated ETs expression in vivo of AMC subjected to hypoxia as opposed to its upregulated expression in vitro may be due to the complexity of the brain tissue. Furthermore, the differential ET-1 and ET-3 mRNA expression in LPS and OGD treatments may be due to different signaling pathways independently regulating the two isoforms. The present novel finding has added microglia as a new cellular source of ET that may take part in multiple functions including regulating vascular constriction and chemokines release.     

In vivo observation of the locomotion of microglial cells in the retina

Microglial cells (MCs) are active sensors and reactive phagocytes of neural tissues. They are known to migrate and accumulate in areas of neuronal damage. Thus, microglial locomotion is an essential feature of the inflammatory reaction in neural tissue. Yet, to our knowledge there has been no report of direct in vivo observation of the migration of MCs. Here, we show that intravitreally injected cyanine dyes (DiO, DiI, and indocyanine green) are sequestrated in MCs during several months, and subsequently in vivo images of these fluorescent MCs can be obtained by confocal scanning laser ophthalmoscopy. This enabled noninvasive, time‐lapse observation of the migrating behavior of MCs, both in the basal state and following laser damage. In the basal state, a slow, intermittent, random‐like locomotion was observed. Following focal laser damage, MCs promptly (i.e., within 1 h) initiated centripetal, convergent migration. MCs up to 400 μm away migrated into the scar at velocities up to 7 μm/min. This early phase of centripetal migration was followed by a more prolonged phase of nontargeted locomotion around and within injured sites during at least 24 h. Cyanine‐positive cells persisted within the scar during several weeks. To our knowledge, this is the first in vivo observation of the locomotion of individual MCs. Our results show that the locomotion of MCs is not limited to translocation to acutely damaged area, but may also be observed in the basal state and after completion of the recruitment of MCs into scars. © 2010 Wiley‐Liss, Inc.     

Intramuscular interstitial cells of Cajal associated with mast cells survive nitrergic nerves in achalasia

Achalasia is dominated by injury to inhibitory nerves. As intramuscular interstitial cells of Cajal (ICC-IM) are proposed to form functional units with nitrergic nerves, their fate in achalasia may be critically important. We studied the relationship between loss of nitrergic nerves and injury to ICC-IM in patients with achalasia and determined associations between ICC-IM and mast cells (MC), using quantitative immunohistochemistry and electron microscopy. Loss of neuronal nitric oxide synthase (nNOS) immunoreactivity was completed within 3 years of acquiring achalasia. Thereafter, progressive ultrastructural injury to remaining nerve structures was evident. Within the first 2 years, the number of ICC-IM did not decline although ultrastructural injury was already present. Thereafter, loss of ICC-IM occurred unrelated to duration of disease. Damage to ICC-IM appeared unrelated to nerve injury. A significant MC infiltration was observed in the musculature; the number of MC was positively related to the persistent number of ICC-IM. Mast cell formed close contacts with ICC-IM and piecemeal-degranulation occurred towards ICC-IM. In conclusion, injury to ICC-IM in achalasia is variable, but not related to duration of disease and injury to nitrergic nerves. MC are prominent and form close functional contact with ICC-IM which may be responsible for their relatively long survival.     

Involvement of P2X4 receptors in hippocampal microglial activation after status epilepticus

Within the central nervous system, functions of the ATP‐gated receptor‐channel P2X4 (P2X4R) are still poorly understood, yet P2X4R activation in neurons and microglia coincides with high or pathological neuronal activities. In this study, we investigated the potential involvement of P2X4R in microglial functions in a model of kainate (KA)‐induced status epilepticus (SE). We found that SE was associated with an induction of P2X4R expression in the hippocampus, mostly localized in activated microglial cells. In P2X4R‐deficient mice, behavioral responses during KA‐induced SE were unaltered. However, 48h post SE specific features of microglial activation, such as cell recruitment and upregulation of voltage‐dependent potassium channels were impaired in P2X4R‐deficient mice, whereas the expression and function of other microglial purinergic receptors remained unaffected. Consistent with the role of P2X4R in activity‐dependent degenerative processes, the CA1 area was partially protected from SE‐induced neuronal death in P2X4R‐deficient mice compared with wild‐type animals. Our findings demonstrate that P2X4Rs are brought into play during neuronal hyperexcitability and that they control specific aspects of microglial activation. Our results also suggest that P2X4Rs contribute to excitotoxic damages by regulating microglial activation. GLIA 2013;61:1306–1319     

Radial migration of developing microglial cells in quail retina: a confocal microscopy study

Microglial cells spread within the nervous system by tangential and radial migration. The cellular mechanism of tangential migration of microglia has been described in the quail retina but the mechanism of their radial migration has not been studied. In this work, we clarify some aspects of this mechanism by analyzing morphological features of microglial cells at different steps of their radial migration in the quail retina. Microglial cells migrate in the vitreal half of the retina by successive jumps from the vitreal border to progressively more scleral levels located at the vitreal border, intermediate regions, and scleral border of the inner plexiform layer (IPL). The cellular mechanism used for each jump consists of the emission of a leading thin radial process that ramifies at a more scleral level before retraction of the rear of the cell. Hence, radial migration and ramification of microglial cells are simultaneous events. Once at the scleral border of the IPL, microglial cells migrate through the inner nuclear layer to the outer plexiform layer by another mechanism: they retract cell processes, become round, and squeeze through neuronal bodies. Microglial cells use radial processes of s-laminin-expressing Müller cells as substratum for radial migration. Levels where microglial cells stop and ramify at each jump are always interfaces between retinal strata with strong tenascin immunostaining and strata showing weak or no tenascin immunoreactivity. When microglial cell radial migration ends, tenascin immunostaining is no longer present in the retina. These findings suggest that tenascin plays a role in the stopping and ramification of radially migrating microglial cells.     

Functional gastrointestinal disorders and mast cells: implications for therapy

The pathophysiology of functional gastrointestinal disorders is poorly understood. Accepted common mechanisms include psychosocial factors, abnormal gastrointestinal motility and disturbed visceral sensory perception, but the underlying causes remain unclear. Mast cells (MCs) are immunocytes widely distributed throughout the gastrointestinal tract. Several stimuli (e.g. allergens, neuropeptides and stress) lead to MC activation with consequent mediator release (e.g. histamine, tryptase and prostanoids). The MC mediators interact with nerves supplying the gut leading to altered gut physiology and increased sensory perception. The intestinal mucosa of irritable bowel syndrome patients contains on average an increased number of MCs. These cells release an increased amount of mediators in close vicinity to mucosal innervation. The MC activation and their close proximity to nerve fibres is correlated with the severity of perceived abdominal painful sensations. These data provide a strong basis for considering MCs as important participants in visceral hypersensitivity and pain perception in irritable bowel syndrome. Inhibition of MC function may ameliorate irritable bowel symptoms. Novel drugs with an increased potential in the control of MC function (e.g., anti-IgE antibodies, the intracellular protein tyrosine kinase inhibitor Syk) and mediator release (e.g., second generation antihistamines, proteinase-activated receptor antagonists) may be useful pharmacological tools for these common disorders.     

Identification and distribution of microglial cells in the cerebral cortex of the lizard: a histochemical study.

The histochemical demonstration of nucleoside diphosphatase as a specific microglial marker was used to study the distribution of this glial cell type in the cerebral cortex of Podarcis muralis and Podarcis hispanica. Our results showed that in both species, NDPase staining was specific for the microglial cell population and that microglial cells displayed a specific localization pattern in the different cortical areas. In the medal cortex, microglial cells were principally found in the outer and inner plexiform layers in the strata adjacent to the granular layer. Moreover, some microglial cells were found near the ependymal layer, but no microglial cells were normally present near the brain surface and never in the deep inner plexiform layer. In the dorsomedial cortex, microglial cells were found near the brain surface in the outer plexiform layer, in the upper part of the granular layer, and near the ependymal layer. No microglial cells were found, however, in the outer and inner plexiform layers adjacent to the granular layer. Finally, in the dorsolateral cortex, microglial cells were located in the upper part of the outer plexiform layer, in and bordering the granular layer, and scattered in the inner plexiform layer. This layered-pattern distribution of microglial cell population in the cerebral cortex of the lizard differs from the apparently homogeneous distribution of microglia in the brain of mammals.     

Neonatal handling in eae-susceptible rats altersNGFlevels and mast cell distribution in the brain

Maternal separation in neonatal rodents causes a wide range of behaviouralandmetabolic alterations, affecting the physiological response of theneuro-immune-endocrinesystem. For example, interference with the normal mother-infantinteractions leads to anincreased susceptibility to experimentally-induced allergicencephalomyelitis (EAE) in adult life.Since it has been reported that mast cells (MCs) participatein the pathophysiology of theautoimmune inflammatory disease multiple sclerosis (MS) and alsoEAE and that brain nervegrowth factor (NGF) levels are altered in EAE, we studied whethermaternal separation andgentle manipulation (gentling) of neonatal Lewis rats perturb NGF levelsor MC distribution inthe brain. EAE-induction susceptibility in adult life was also evaluated andNGF levels and mastcell distribution within the hippocampus and thalamus were measured at 0,10, 20 and 60postnatal days. Our results show an exacerbation of clinical signs in rats separatedfrom motherswhere EAE was induced, a general decrease in NGF protein levels and MC numberin thehippocampus during the first developmental period and a significant increase in the numberofMC in the hippocampus and the thalamus at young-adulthood (60 days of age). Theseresultsindicate that disruption of the maternal bond during early infancy may producelong-lastingalterations in the brain cellular and molecular environment, leading to increasedsusceptibility toEAE in adult life.     

NGF primed spleen cells injected in brain of developing rats differentiate into mast cells

The aim of this work was to study the topographic distribution and the morphological behaviour of nerve growth factor (NGF) primed spleen cells injected into the lateral ventricles of developing rat brain. Serial coronal brain sections showed that these transplanted cells acquire phenotypical characteristics similar to those of mast cells (MCs) and that they enhance local neovascularization. These results, together with the observation that these cells are located in proximity to the hippocampus, a brain tissue which contains one of the highest levels of NGF, provide a model for studying the relationship between NGF and MC differentiation and secretion.