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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 15, 2003 as doi:10.1096/fj.02-1029fje. |
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,2
* Department of Biological Sciences, Rutgers University, Newark, New Jersey, USA; and
Instituto de Parasitologia y Biomedicina "Lopez-Neyra," CSIC, Granada 18001, Spain
2Correspondence: Instituto de Parasitologia y Biomedicina "Lopez-Neyra," CSIC, Granada 18001, Spain. E-mail: mdelgado{at}ipb.csic.es
SPECIFIC AIMS
In most neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and AIDS dementia, massive neuronal cell death occurs as a consequence of an uncontrolled inflammatory response, where activated microglia and its cytotoxic agents seem to play a crucial pathologic role. Because current treatments for these diseases are not effective, a number of regulatory molecules termed microglia-deactivating factors have been the focus of considerable research lately. Vasoactive intestinal peptide (VIP) is a potent anti-inflammatory neuropeptide that protects from some inflammatory disorders, such as endotoxic shock and rheumatoid arthritis. In the present study, we investigate the effect of VIP on inflammation-mediated neurodegeneration in vitro and in vivo, and the putative neuroprotective effect of VIP on pathological conditions where inflammation of CNS is involved, such as brain trauma. The involvement of activated microglia and their derived cytotoxic products is also studied.
PRINCIPAL FINDINGS
1. VIP protects from inflammation-induced neurodegeneration in vitro by inhibiting microglial activation
We first investigated the effect of VIP on neuronal death induced by the bacterial endotoxin LPS in vitro in mixed neuron-microglia cultures. Mouse embryonic neurons were cocultured with neonate mesencephalic microglial cells and stimulated with LPS in the presence or absence of VIP. LPS treatment of cocultures resulted in a profound neuronal cell loss (determined by immunostaining against the neuronal cytoskeletal protein MAP-2); most of the few remaining cells displayed shortened neurites compared with the control cultures (Fig. 1
A). VIP addition to cocultures drastically inhibited LPS-induced neural mortality and change in morphology (Fig. 1A
). A similar neuroprotective effect of VIP was observed in primary mixed glia-neuron cultures established using embryonic mouse brain regions from mesencephalon, where VIP inhibited in a dose-dependent manner the LPS-induced LDH release into the medium (Fig. 1B
). The VIP neuroprotective effect was also confirmed by immunostaining of MAP-2+ neurons in these cultures (not shown), and was especially relevant when dopaminergic neurons (tyrosine hydroxylase TH+ neurons) were analyzed in these cultures instead of total neuronal populations. LPS caused a dramatic loss of TH+ neurons in mesencephalic mixed neuronal-glial cultures (Fig. 1C
), confirmed by a decrease in the [3H]-dopamine uptake (Fig. 1D
). VIP protected dopaminergic cells from LPS-induced inflammation (Fig. 1C, D
). VIP-mediated neuroprotection of dopaminergic neurons has important therapeutic perspectives, because the loss of these neurons in substantia nigra is one cause of Parkinson’s disease. In fact, VIP has recently been described as a neuroprotective agent in an experimental model of Parkinson’s disease.
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LPS-induced neurodegeneration was associated with an increased microglial activation, characterized by changes in morphology from a ramified resting state to an amoeboid appearance, with an increase in the expression of Mac-1+ microglial cells, in free radical production and in the production of the proinflammatory mediators TNF-
and NO (Fig. 1E
). Involvement of activated microglia and their derived cytotoxic factors in inflammation-induced neurodegeneration is suggested by the facts that LPS failed to induce cell loss in cultures with neurons alone and that cell-free supernatants from LPS-stimulated microglia cultures induced significant cell death in neurons cultured alone (Fig. 1F
). Furthermore, addition of neutralizing antibodies against TNF- and IL-1ß to microglial-neuronal cocultures significantly reduced LPS-induced MAP-2+ cell loss (% inhibition of cell loss: 46, 24, and 68 for anti-TNF, anti-IL-1, or both, respectively). Treatment with VIP inhibited in a dose-dependent manner all these hallmarks of LPS-induced microglial activation and the production of TNF- and NO (Fig. 1E
). A similar "microglial-deactivating" action of VIP was observed in mixed neuron-glial cultures prepared from embryonic mesencephalic tissue (not shown). The VIP dose-response curve observed for the neuroprotective effect correlates with that for the microglial deactivation, suggesting that VIP could mediate its protective effect through its action on microglia activation. This is correlated with the fact that treatment of neuronal cultures with conditioned medium from LPS-activated microglia treated with VIP did not result in significant neuronal death (Fig. 1F
). The neuroprotective effect of VIP in LPS-treated neuronal-microglial cocultures was partially reversed by the addition of TNF- and IL-1ß or of conditioned medium from LPS-stimulated microglial cultures (Fig. 1G
), suggesting that the neuroprotective effect of VIP against endotoxin could be mainly mediated through the inhibition on the production of microglia-derived cytotoxic factors.
Next we investigated whether the inhibitory effect of VIP could be related to occupancy of specific receptors. VIP belongs to the family of peptides that includes pituitary adenylate cyclase-activating polypeptide (PACAP). Both peptides share several functions, exerted through a family of receptors consisting of VPAC1, VPAC2, and PAC1. PACAP and a VPAC1 agonist, but not a VPAC2 or a PAC1 agonist, inhibited LPS-induced neurodegeneration and microglia activation, with a potency similar to that of VIP (not shown), suggesting that both neuropeptides exert their action primarily through VPAC1.
2. VIP prevents neurodegeneration and microglial activation in vivo
We next investigated whether the neuroprotective effect of VIP under inflammatory conditions is also observed in vivo. LPS was injected in the third ventricle (i.c.v.) of adult mouse brains. Different doses of VIP were administered concomitantly with LPS. Neurodegeneration in different mesencephalic areas was determined by immunostaining of brain sections with an antibody against the neuron-specific nuclear protein (NeuN). LPS administration caused a dramatic NeuN+ cell loss in certain brain areas, especially in the periventricular nucleus (PVN), caudate putamen (CP), and substantia nigra (SN), which was accompanied by massive microglia activation and an increase in the expression of TNF-, IL-1ß, and iNOS mRNA in the same areas (not shown). Phenotypic analysis by double in situ hybridization and immunocytochemical examination showed that the majority of cells expressing these inflammatory mediators are microglia. Local VIP administration (i.c.v. injection) prevented in a dose-dependent manner LPS-induced neurodegeneration, microglia activation, and TNF-, IL-1ß, and iNOS expression in PVN, CP, and SN. Systemic VIP injection (i.p.) was also neuroprotective, although VIP doses 15-fold higher than for local VIP administration were needed to obtain similar results.
3. Neuroprotective role of VIP following brain trauma
We wanted to study the possible therapeutic effect of VIP in brain trauma, a pathological condition associated with inflammation. The increase in the levels of proinflammatory cytokines is a normal and early feature of the CNS response to trauma. However, it is controversial whether inflammation in the injured CNS serves a beneficial or detrimental purpose. We used a model of adult mice stab wound brain trauma in the periventricular area. Medium (controls) or VIP was administered at the injury site through a micropipette at the time of brain trauma induction. At different times after injury, neuronal cell loss, inflammatory cell invasion, and microglia activation were determined. After mechanical injury, several events occurred at the injury site: dramatic neurodegeneration, recruitment of mononuclear phagocytes, a significant increase in activated microglia, and increased production of TNF- and IL-1ß (Fig. 2
). Treatment with VIP significantly reduced all these events (Fig. 2)
. The source of inflammatory cytokines in the CNS following trauma remains unresolved. Although infiltrating leukocytes are obvious candidates, CNS mounts an early and intrinsic inflammatory response upon trauma, before leukocyte infiltration occurs. Microglia have been proposed as the origin of the early increase in CNS inflammatory cytokines after injury. We propose that VIP reduces neuronal cell loss in the vicinity of the lesion site through the inhibition of microglial-derived proinflammatory mediators such as TNF- and IL-1ß and of chemokines responsible for the inhibition of the influx of blood-derived leukocytes into the parenchyma surrounding the injured region. Although it is unlikely that the reduction of LPS-induced microglia activation by VIP is secondary to the attenuation of neuronal loss rather than the reverse (Fig. 1)
, a direct action of VIP on neurons cannot be ruled out. Although minimal, the involvement of astrocytes in the mediation of VIP should not be discounted, because VIP slightly reduced the trauma-induced astrogliosis (not shown). Neurotrophic effects mediated through astrocytes cannot be excluded. In this sense, a neuroprotective action of VIP in response to different neuronal insults, by inducing the production of neuroprotective factors (i.e., ADNF) by astrocytes, has been described.
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CONCLUSIONS AND SIGNIFICANCE
The administration of VIP following inflammation or acute trauma of the CNS could be beneficial, at least in certain regions of the brain. Our study suggests possible therapeutic uses of VIP in certain brain disorders, such as multiple sclerosis, Parkinson’ and Alzheimer’s diseases, and AIDS dementia, where inflammatory responses play a major role.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1029fje; doi: 10.1096/fj.02-1029fje 
This article has been cited by other articles:
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D. POZO and M. DELGADO The many faces of VIP in neuroimmunology: a cytokine rather a neuropeptide? FASEB J, September 1, 2004; 18(12): 1325 - 1334. [Abstract] [Full Text] [PDF]
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 M. Delgado, D. Pozo, and D. Ganea The Significance of Vasoactive Intestinal Peptide in Immunomodulation Pharmacol. Rev., June 1, 2004; 56(2): 249 - 290. [Abstract] [Full Text] [PDF]
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