Loss of Gas6 and Axl signaling results in extensive axonal damage,motor deficits,prolonged neuroinflammation,and less remyelination following cuprizone exposure

The TAM (Tyro3, Axl, and MerTK) family of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and ProS1, are important for innate immune responses and central nervous system (CNS) homeostasis. While only Gas6 directly activates Axl, ProS1 activation of Tyro3/MerTK can indirectly activate Axl through receptor heterodimerization. Therefore, we generated Gas6^–/–Axl^–/– double knockout (DKO) mice to specifically examine the contribution of this signaling axis while retaining ProS1 signaling through Tyro3 and MerTK. We found that naïve young adult DKO and WT mice have comparable myelination and equal numbers of axons and oligodendrocytes in the corpus callosum. Using the cuprizone model of demyelination/remyelination, transmission electron microscopy revealed extensive axonal swellings containing autophagolysosomes and multivesicular bodies, and fewer myelinated axons in brains of DKO mice at 3‐weeks recovery from a 6‐week cuprizone diet. Analysis of immunofluorescent staining demonstrated more SMI32+ and APP+ axons and less myelin in the DKO mice. There were no significant differences in the number of GFAP+ astrocytes or Iba1+ microglia/macrophages between the groups of mice. However, at 6‐weeks cuprizone and recovery, DKO mice had increased proinflammatory cytokine and altered suppressor of cytokine signaling (SOCS) mRNA expression supporting a role for Gas6‐Axl signaling in proinflammatory cytokine suppression. Significant motor deficits in DKO mice relative to WT mice on cuprizone were also observed. These data suggest that Gas6‐Axl signaling plays an important role in maintaining axonal integrity and regulating and reducing CNS inflammation that cannot be compensated for by ProS1/Tyro3/MerTK signaling.     

Age increases axon loss associated with primary demyelination in cuprizone-induced demyelination in C57BL/6 mice

Axon loss is recognised as a significant contributor to the progression of the disability associated with multiple sclerosis. Although evidence of axon damage is found in areas of chronic demyelination it is more frequently seen in association with acute demyelination. This study compares the incidence of axon degeneration associated with the areas undergoing demyelination in young adult (8-10 weeks) and aged (6-7 months) C57BL/6 mice in cuprizone intoxication; a widely used model of demyelination. The incidence of axon transection, as indicated by the presence of SMI 32 positive axonal spheroids, and evidence of axon loss in the medial corpus callosum, were significantly greater in aged mice, as was the magnitude of the macrophage and astrocyte response to demyelination. Aged C57BL/6 mice are thus more prone to axon degeneration in association with demyelination than young adult mice. A retrospective study indicated that the incidence of axon degeneration was much higher in C57BL/6 mice than in the Swiss albino mice used in the early cuprizone intoxication studies which were fed much higher doses of cuprizone. These results indicate both a genetic and age susceptibility to demyelination-associated axon transection.     

Lesion response of long-term and recently immigrated resident endoneurial macrophages in peripheral nerve explant cultures from bone marrow chimeric mice

Resident macrophages of the peripheral nervous system have recently been shown to respond rapidly to Wallerian degeneration before the influx of blood-derived macrophages. Because resident endoneurial macrophages are slowly but incompletely exchanged from the blood within 3 months, they could potentially comprise a heterogenous cell population consisting of long-term resident cells and more mobile cells undergoing turnover. We used bone marrow chimeric mice created by transplanting bone marrow from green fluorescent protein-transgenic mice into irradiated wildtype recipients to selectively analyse the response of these two resident macrophage populations to Wallerian degeneration in sciatic nerve explant cultures. In such nerves, recently immigrated macrophages exhibit green fluorescence whereas long-term resident macrophages do not. Studies in cultures from wildtype controls revealed rapid morphological changes of resident macrophages towards a bloated phenotype, a proliferative response resulting in a 3.7-fold increase of macrophage numbers over 2 weeks, and phagocytosis of myelin basic protein-immunoreactive myelin debris. When chimeric mice were analysed, both populations of resident endoneurial macrophages participated in morphological transformation, proliferation and phagocytosis. Quantitative studies revealed a stronger proliferative and phagocytic response in long-term resident endoneurial macrophages compared with recently immigrated macrophages. Our results point towards subtle, but not principal, differences between the two macrophage populations, which might indicate different stages of macrophage differentiation rather than the existence of entirely distinct endoneurial macrophage populations. The results further underline the versatility of resident endoneurial macrophages following peripheral nerve injury, which is reminiscent of the lesion response of microglial cells within the brain.     

On the longevity of resident endoneurial macrophages in the peripheral nervous system: a study of physiological macrophage turnover in bone marrow chimeric mice

Macrophages are intimately involved in the pathogenesis of peripheral nervous system (PNS) disorders. Recently, we characterized a resident endoneurial macrophage population, which contributes rapidly to the endoneurial macrophage response in PNS diseases. Unlike microglial cells, resident macrophages undergo a physiological turnover of 50% in the sciatic nerve and 80% in dorsal root ganglia (DRG) within 12 weeks. Further information about the dynamics of this turnover is not available. This study examined the macrophage turnover in the sciatic nerve and DRGs over a longer period and addresses the question whether the turnover of resident macrophages is complete or whether there is a truly resident endoneurial macrophage population. We used chimeric mice carrying GFP(+) bone marrow and immunohistochemistry to detect hematogenous (GFP(+)) endoneurial macrophages after turnover. Non-exchanged, resident macrophages were GFP(-). Quantification of GFP(+) and GFP(-) macrophages revealed a maximal turnover of 75%, reached in DRGs after 12 weeks and in sciatic nerves after 36 weeks. GFP(-) long-term resident macrophages were further characterized after sciatic nerve injury, where they participated in the early macrophage response of Wallerian degeneration. Our results point toward a small but truly resident PNS macrophage population. These macrophages are an interesting target for further characterization and might have a distinct role in peripheral nerve disease.     

Neuroinflammatory responses after experimental diffuse traumatic brain injury

Little is known about microglial activation and macrophage localization after diffuse brain injury (DBI). DBI-mediated perisomatic traumatic axonal injury (TAI) was recently identified within the neocortex, hippocampus, and thalamus, providing an opportunity to characterize immune cell responses within diffusely injured brain loci uncomplicated by contusion. By using moderate midline/central fluid percussion injury, microglial/macrophage responses were examined with antibodies targeting immune cell phenotypes and amyloid precursor protein, a marker of TAI. Parallel assessments of blood-brain barrier alterations were also performed. Within 6 to 48 hours postinjury, microglial activation within injured loci was observed, whereas microglia within non-TAI-containing regions maintained a resting phenotype. Microglial activation shared a spatiotemporal relationship with TAI though no clear interactions were observed. By 7 to 28 days postinjury, activated microglia contained myelin debris, yet revealed limited aggregation. Immunophenotypic macrophages were also localized to injured loci. Select macrophages approximated somatic membranes of perisomatically axotomized neurons with evidence of bouton disruption. No causality was established between blood-brain barrier alterations and these inflammatory responses. These findings indicate rapid, yet initially nonspecific, and persistent microglial/macrophage responses to DBI. DBI-mediated inflammatory responses suggest further expansion of traumatic brain injury histopathologic evaluations to identify neuroinflammation indicative of diffuse pathology.     

Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre

Exposure of young adult C57BL/6 mice to cuprizone in the diet initiated profound and synchronous demyelination of the corpus callosum, which was virtually complete by 4 weeks of exposure. Interestingly, even in the face of a continued exposure to cuprizone, there was spontaneous remyelination 2 weeks later. This remyelination preferentially involved smaller calibre axons. There was a suggestion of yet another cycle of demyelination (at 10 weeks) and remyelination (at 12 weeks), but by 16 weeks of exposure, the regenerative capacity was exhausted and the animals were near death. The relapsing-remitting pattern suggests this may be a useful model for certain human demyelinating disorders. In contrast to the above chronic model, the corpus callosum from mice exposed to cuprizone for only 6 weeks continued to remyelinate, with 67% of the axons being myelinated or remyelinated at 10 weeks. Interestingly, a significant reduction in the mean value for axonal diameter was observed during acute demyelination. Upon remyelination, however, the axonal calibre distribution returned to near-normal. In contrast, when mice were maintained on a cuprizone diet for 16 weeks, the mean value for axonal diameter was reduced to 60% of normal. These results provide further evidence that the interactions between oligodendrocytes and axons alter axonal calibre.     

Abbreviated exposure to cuprizone is sufficient to induce demyelination and oligodendrocyte loss

Cuprizone intoxication is one of several animal models used to study demyelination and remyelination. Early treatment protocols exposed mice to cuprizone for 6 weeks to induce demyelination; however, more recent reports have varied exposure times from 4 to 5 weeks. The goal of this study was to determine the minimal exposure of cuprizone in C57BL/6 mice that would induce a pathology of robust demyelination and gliosis similar to that described for a 5‐ or 6‐week treatment. We found that an abbreviated insult of only 2 weeks of exposure to cuprizone induced significant demyelination 3 weeks later (5‐week time point) but was somewhat variable. Three weeks of exposure to cuprizone produced extensive demyelination by week 5, equivalent to that observed with 5 weeks of exposure. The depletion of mature oligodendrocytes, as well as microglia and astrocyte accumulation, showed trends similar to those with 5‐week exposure to cuprizone. Once mature oligodendrocytes are perturbed after a 3‐week treatment, the progression to demyelination occurs without requiring further exposure. Furthermore, the early removal of cuprizone did not accelerate remyelination, suggesting that other sequences of events must follow before repair can occur. Thus, a short, “hit and run” CNS insult triggers a cascade of events leading to demyelination 2–3 weeks later. © 2012 Wiley Periodicals, Inc.     

The role of CD36 in peripheral nerve remyelination after crush injury

We previously demonstrated that the deficiency of class A macrophage scavenger receptor type I/II was involved in the delayed phagocytosis of degraded myelin by macrophages in class A macrophage scavenger receptor type I/II knockout mice after crush injury of the sciatic nerve [Naba et al. (2000) Exp. Neurol., 166, 83-89]. In order to elucidate the role of CD36, one of the scavenger receptors, here we inflicted crush injury to the sciatic nerves of CD36 knockout mice and investigated the remyelination after crush injury in comparison with that of class A macrophage scavenger receptor type I/II knockout mice. Although we previously reported a lot of onion-bulbs in class A macrophage scavenger receptor type I/II knockout mice at 3 weeks, the number of onion-bulbs was limited both in CD36 knockout mice and wild-type mice. In the morphometry, the remyelination was seriously delayed, and the infiltrating macrophages into the nerve fascicles were quite frequent in CD36 knockout mice compared with wild-type mice at 3 and 6 weeks postinjury. The immunohistochemistry with the monoclonal antibody reacted with oxidized phosphatidylcholine and oil red O staining were positive in wild-type mice, but were negative in CD36 knockout mice, suggesting that the oxidation of phosphatidylcholine and the generation of neutral lipids in macrophages were disturbed in CD36 knockout mice. We hypothesize that the delayed phagocytosis by macrophages and the defect in reuse of lipids from degraded myelin are related to seriously delayed remyelination and a small number of onion-bulbs in CD36 knockout mice.     

Persistent immune activation associated with a mouse model of Staphylococcus aureus-induced experimental brain abscess

We have established a mouse experimental brain abscess model using Staphylococcus aureus where lesion sites are greatly exaggerated compared to the localized area of initial infection, reminiscent of an overactive immune response. Here we demonstrate the prolonged expression of IL-1 beta, TNF-alpha, and macrophage inflammatory protein-2 (MIP-2/CXCL2), concomitant with a chronic disruption of the blood-brain barrier (BBB) in mice with S. aureus-induced brain abscess. These changes correlated with the continued presence of infiltrating neutrophils and macrophages/microglia. Collectively these findings suggest that the excessive tissue damage that often results from brain abscess may be mediated, in part, by the perpetuation of antibacterial immune responses that are not downregulated in a timely manner.     

Lysophosphatidylcholine induces rapid recruitment and activation of macrophages in the adult mouse spinal cord

Lysophosphatidylcholine (LPC) can induce rapid breakdown and removal of myelin from the adult mammalian CNS. In this paper we report the detailed characterization of the immune cell response as well as changes in the expression of cell adhesion molecules and the permeability of the blood-brain barrier after microinjection of LPC into the adult mouse spinal cord. T cells and neutrophils were seen in the spinal cord 6-12 h after LPC injection, but not in PBS-injected mice. Mac-1+ monocytes were also seen at 6 h and 12 h in the white and gray matter of mice injected with LPC and PBS but were significantly greater in the white matter after LPC injections. At later time points LPC induced an increase in the number of activated Mac-1+ macrophages that displayed a variety of morphologies in the white and gray matter. These cells were not present in PBS-injected control mice. LPC also induced widespread microglial activation in the white and gray matter. The number of these Mac-1+ microglia reduced drastically at 96 h after LPC injection suggesting that they may have transformed into Mac-1+ phagocytic cells with a different morphology. These LPC-induced changes in immune cells were accompanied by significant increases in VCAM-1+ and ICAM-1+ blood vessels in the spinal cord. In addition, LPC induced a rapid and widespread disruption of the blood-brain barrier, as compared to PBS injected mice. Therefore, LPC can induce an early and transient T cell and neutrophil response in the CNS. These cells likely promote the rapid influx of monocytes followed by widespread and effective activation of macrophages that mediate rapid phagocytosis of myelin debris.     

Altered transition metal homeostasis in the cuprizone model of demyelination

In the cuprizone model of demyelination, the neurotoxin cuprizone is fed to mice to induce a reproducible pattern of demyelination in the brain. Cuprizone is a copper chelator and it has been hypothesized that it induces a copper deficiency in the brain, which leads to demyelination. To test this hypothesis and investigate the possible role of other transition metals in the model, we fed C57Bl/6 mice a standard dose of cuprizone (0.2% dry chemical to dry food weight) for 6 weeks then measured levels of copper, manganese, iron, and zinc in regions of the brain and visceral organs. As expected, this treatment induced demyelination in the mice. We found, however, that while the treatment significantly reduced copper concentrations in the blood and liver in treated animals, there was no significant difference in concentrations in brain regions relative to control. Interestingly, cuprizone disrupted concentrations of the other transition metals in the visceral organs, with the most notable changes being decreased manganese and increased iron in the liver. In the brain, manganese concentrations were also significantly reduced in the cerebellum and striatum. These data suggest a possible role of manganese deficiency in the brain in the cuprizone model.     

Cocaine opens the blood-brain barrier to HIV-1 invasion

Cocaine abuse has been associated with vasculitis and stroke, and is suspected to influence the progression of AIDS dementia. Cocaine may enhance HIV-1 neuroinvasion by actions directed at the blood-brain barrier. HIV-1 appears to penetrate the human brain microvascular endothelial cell barrier by a paracellular route breached by tumor necrosis factor-alpha (TNF-alpha). Cocaine's effects on the blood-brain barrier were investigated using human brain microvascular endothelial cells and peripheral blood monocytes. Cocaine (10(-5) M and 10(-6) M) increased molecular permeability of the barrier and viral invasion by the macrophage-tropic HIV-1(JR-FL) into the brain chamber. Cocaine also augmented apoptosis of brain endothelial cells and monocytes, increased secretion of four chemokines (interleukin-8, interferon-inducible protein-10, macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1) and the cytokine, TNF-alpha, by human monocytes. TNF-alpha enhanced invasion of the brain compartment by macrophage-tropic, lymphotropic, and bitropic HIV-1 strains. These data indicate that HIV-1 neuroinvasion can be increased by (a) cocaine's direct effects on brain microvascular endothelial cells and (b) paracrine effects of cocaine-induced pro-inflammatory cytokines and chemokines on the blood-brain barrier.     

Investigation of Cuprizone Inactivation by Temperature

Animal models, such as cuprizone (bis-cyclohexanone oxaldihydrazone) feeding, are helpful to study experimental demyelination and remyelination in the context of diseases like multiple sclerosis. Cuprizone is a copper chelator, which when supplemented to the normal food of C57BL/6J mice in a concentration of 0.2% leads to oligodendroglial loss, subsequent microglia and astrocyte activation, resulting in demyelination. Termination of the cuprizone diet results in remyelination, promoted by newly formed mature oligodendrocytes. The exact mode of cuprizone’s action is not well understood, and information about its inactivation and cleavage are still not available. The knowledge of these processes could lead to a better understanding of cuprizone’s mode of action, as well as a safer handling of this toxin. We therefore performed experiments with the aim to inactivate cuprizone by thermal heating, since it was suggested in the past that cuprizone is heat sensitive. C57BL/6J mice were fed for 4 weeks with 0.2% cuprizone, either thermally pretreated (60, 80, 105, 121 °C) or not heated. In addition, primary rat oligodendrocytes, as a known selective toxic target of cuprizone, were incubated with 350 μM cuprizone solutions, which were either thermally pretreated or not. Our results demonstrate that none of the tested thermal pretreatment conditions could abrogate or restrict the toxic and demyelinating effects of cuprizone, neither in vitro nor in vivo. In conclusion, the current study rebuts the hypothesis of cuprizone as a heat-sensitive compound, as well as the assumption that heat exposure is a reason for an insufficient demyelination of cuprizone-containing pellets.     

Monocyte infiltration is highly associated with loss of the tight junction protein zonula occludens in HIV-1-associated dementia

In human immunodeficiency virus (HIV)-1-associated dementia (HAD), consequences of interactions between infiltrating monocytes and brain endothelial cells are not yet fully understood. This study investigated whether the blood-brain barrier is affected in brain tissue of patients suffering from HAD and whether it was possible to find a correlation with the presence or absence of monocytic cells, which have been suggested to play a major role in HAD. Immunohistochemical analysis for zonula occludens 1, a tight junction protein, and CD68, a macrophage marker, revealed that loss of tight junction immunoreactivity was highly correlated with monocyte infiltration and with HAD. This suggests that the presence of perivascular macrophages cells is associated with breakdown of the blood-brain barrier thereby facilitating infiltration of more monocytic cells hence enhancing disease progression.     

Astrocyte-specific expression of a soluble form of the murine complement control protein Crry confers demyelination protection in the cuprizone model

Complement has been implicated as a potential effector mechanism in neurodegeneration; yet the precise role of complement in this process remains elusive. In this report, we have utilized the cuprizone model of demyelination-remyelination to examine the contribution of complement to disease. C1q deposition was observed in the corpus callosum of C57BL/6 mice during demyelination, suggesting complement activation by apoptotic oligodendrocyte debris. Simultaneously, these mice lost expression of the rodent complement regulatory protein, Crry. A soluble CNS-specific form of the Crry protein (sCrry) expressed in a transgenic mouse under the control of an astrocyte-specific promoter was induced in the corpus callosum during cuprizone treatment. Expression of this protein completely protected the mice from demyelination. Interestingly, sCrry mice had low levels of demyelination at later times when control mice were remyelinating. Although the sCrry transgenic mice had lower levels of demyelination, there was no decrease in overall cellularity, however there were decreased numbers of microglia in the sCrry mice relative to controls. Strikingly, sCrry mice had early recovery of mature oligodendrocytes, although they later disappeared. TUNEL staining suggested that production of the sCrry protein in the transgenic mice protected from a late apoptosis event at 3 weeks of cuprizone treatment. Our data suggest complement provides some protection of mature oligodendrocytes during cuprizone treatment but may be critical for subsequent remyelination events. These data suggest that temporal restriction of complement inhibition may be required in some disease settings.     

Immunization-induced inflammatory infiltration of the central nervous system in transgenic mice expressing a microbial antigen in astrocytes

Transgenic mice expressing a defined microbial antigen from central nervous system (CNS) cell type-specific promoters can be utilized to investigate the consequences of induction of peripheral immune responses to foreign antigens produced by different CNS cell types. Immunization of mice expressing β-galactosidase (β-gal) in astrocytes with this protein resulted in antigen-dependent infiltration of the CNS by mononuclear cells, principally CD4⁺ T lymphocytes and monocyte/macrophages. The perivascular and intraparenchymal infiltrates, which were located predominantly in the hippocampal formation and cerebellum, the areas of highest β-gal expression, were associated with astrocytosis, microgliosis, and a generalized increase in blood-brain barrier permeability. The resemblance of these pathological changes to aspects of human immune inflammatory CNS disorders e.g. multiple sclerosis, suggests that an initiating step in the process by which such complex diseases are produced could be the induction of peripheral immune responses to antigens expressed in astrocytes.     

IL-6 deficiency leads to reduced metallothionein-I+II expression and increased oxidative stress in the brain stem after 6-aminonicotinamide treatment

We examined the effects of interleukin-6 (IL-6) deficiency on brain inflammation and the accompanying bone marrow (BM) leukopoiesis and spleen immune reaction after systemic administration of a niacin antagonist, 6-aminonicotinamide (6-AN), which causes both astroglial degeneration/cell death in brain stem gray matter areas and BM toxicity. In both normal and genetically IL-6-deficient mice (IL-6 knockout (IL-6KO) mice), the extent of astroglial degeneration/cell death in the brain stem was similar as determined from disappearance of GFAP immunoreactivity. In 6-AN-injected normal mice reactive astrocytosis encircled gray matter areas containing astroglial degeneration/cell death, which were infiltrated by several macrophages and some T-lymphocytes. Reactive astrocytes and a few macrophages increased significantly the antioxidants metallothionein-I+II (MT-I+II) and moderately the MT-III isoform. In 6-AN-injected IL-6KO mice reactive astrocytosis and recruitment of macrophages and T-lymphocytes were clearly reduced, as were BM leukopoiesis and spleen immune reaction. Expression of MT-I+II was significantly reduced while MT-III was increased. Oxidative stress, as determined by measuring nitrated tyrosine and malondialdehyde, was increased by 6-AN to a greater extent in IL-6KO mice. The blood-brain barrier to albumin was only disrupted in 6-AN-injected normal mice, which likely is due to the substantial migration of blood-derived inflammatory cells into the CNS. The present results demonstrate that inflammation in CNS is clearly reduced during IL-6 deficiency and this effect is likely due to significant inhibition of BM leukopoiesis. We also show that IL-6 deficiency reduces the levels of neuroprotective antioxidants MT-I+II followed by an increased oxidative stress during CNS inflammation.     

In vivo monitoring of macrophage infiltration in experimental ischemic brain lesions by magnetic resonance imaging.

Although macrophages represent the major inflammatory cells in cerebral ischemia, the kinetics of macrophage infiltration are largely unknown. To address this issue, we injected superparamagnetic iron oxide (SPIO) particles into the circulation of rats at different time points after focal photothrombotic cerebral infarction and performed magnetic resonance imaging (MRI) 24 hours later. Infarcts appeared as hyperintense lesions on T2-w and CISS MR images during all stages. At days 5.5 and 6, an additional rim of signal loss indicative of local accumulation of SPIO particles appeared at the outer margin of the hyperintense ischemic lesions, which was not present at days 1 to 5. Areas of signal loss corresponded to local accumulation of iron-loaded macrophages in histologic sections. At day 8, signal loss became restricted to the inner core of the lesions and ceased thereafter. Macrophages, however, were still present in late ischemic brain lesions, but they were iron-negative. Thus SPIO-induced signal loss indicates active macrophage transmigration into ischemic infarcts but not their mere presence. SPIO-induced signal loss was independent from the disturbance of the blood-brain barrier. In conclusion, we have shown by in vivo monitoring that macrophages enter photothrombotic infarcts at late stages of infarct development, suggesting a role in tissue remodeling rather than neuronal injury.     

Concentration-dependent effects of the esterase inhibitor BNPP on macrophage migration and myelin phagocytosis

Wallerian degeneration of a peripheral nerve is mainly characterized by axon and myelin degradation and is paralleled by a massive invasion of peripheral macrophages into the nerve. These cells enter the nerve attracted by a cascade of chemokines and cytokines but require proteolytic and enzymatic factors which enables them to cross the blood-nerve barrier. Here we investigated whether alpha-naphthyl (alpha-NA) esterases -- which have been shown to be exclusively expressed in human monocytes -- play a role during Wallerian degeneration. These enzymes were blocked by the specific inhibitor bis(4-nitrophenyl)-phosphate (BNPP) in an established in vitro model of Wallerian degeneration. Sciatic nerve segments of mice were co-cultured with peritoneal macrophages and BNPP was added to the cultures in various concentrations and at different timepoints. The macrophage numbers and myelin density in the nerve segments and the myelin load of macrophages were evaluated. After BNPP treatment the macrophage number within the nerve was significantly diminished and the myelin load within the macrophages was decreased, resulting in elevated levels of preserved myelin within the nerves. These experiments clearly showed a double effect of the alphaNA esterase inhibitor BNPP on macrophages. First, it suggests a role for alphaNA esterases on the migratory potential of macrophages since their invasion into the nerves was diminished. Second, the reduced myelin uptake is due to the inhibition of phagocytic capacity of these cells by BNPP. The therapeutical use of this inhibitor for treatment of autoimmune diseases such as multiple sclerosis or Guillain-Barré syndrome remains to be investigated.