A. Machado

Lipopolysaccharide intranigral injection induces inflammatory reaction and damage in nigrostriatal dopaminergic system.

Abstract: The pathogenesis of Parkinsons disease is still poorly understood. To address the hypothesis that immunemediated events, such as microglial activation, may be involved in the dopaminergic neurodegeneration, we have studied the effect that intranigral injection of the immunostimulant lipopolysaccharide has on monoaminergic neurotransmitters in rats. Activation of microglial cells, visualized by immunohistochemistry with a specific monoclonal antibody, was already obvious 2 days after injection. In relation to the biochemical parameters studied, we found a significant decrease of dopamine levels in both the substantia nigra and striatum up to at least 21 days after intranigral injection of lipopolysaccharide. This result was supported by the decrease in tyrosine hydroxylase activity and the loss of tyrosine hydroxylase‐positive neuronal bodies, shown by immunohistochemistry. These alterations of the dopaminergic system did not reverse during the interval studied (21 days); conversely, the serotoninergic system suffered only transient damage. In addition, we found that the neurotoxic effect of lipopolysaccharide was not mediated by nitric oxide. Based on our results we suggest that the nigrostriatal dopaminergic system is susceptible to damage by inflammatory events and that these may be implicated in neurodegeneration processes such as Parkinsons disease.

The Single Intranigral Injection of LPS as a New Model for Studying the Selective Effects of Inflammatory Reactions on Dopaminergic System

We have injected lipopolysaccharide (LPS) into the nigrostriatal pathway of rats in order to address the role of inflammation in Parkinsons disease (PD). LPS induced a strong macrophage/microglial reaction in Substantia nigra (SN), with a characteristic clustering of macrophage cells around blood-vessels. The SN was far more sensitive than the striatum to the inflammatory stimulus. Moreover, only the dopaminergic neurons of the SN were affected, with no detectable damage to either the GABAergic or the serotoninergic neurons. The damage to the DA neurons in the SN was permanent, as observed 1 year postinjection. Unlike the direct death of dopaminergic neurons caused by agents as MPP(+) or 6-OHDA, LPS seems to cause indirect death due to inflammatory reaction. Therefore, we suggest that the injection of a single dose of LPS within the SN is an interesting model for studying the selective effects of inflammatory reaction on dopaminergic system and also potentially useful for studying PD.

The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone, and not mimicked by rh‐TNF‐α, IL‐1β and IFN‐γ

It is becoming widely accepted that the inflammatory response is involved in neurodegenerative disease. In this context, we have developed an animal model of dopaminergic system degeneration by the intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation. To address the importance of the inflammatory response in the LPS‐induced degeneration of nigral dopaminergic neurones, we carried out two different kinds of studies: (i) the possible protective effect of an anti‐inflammatory compound, and (ii) the effect of the intranigral injection of inflammatory cytokines (TNF‐α, IL‐1β and IFN‐γ) on dopaminergic neurones viability. Present results show that dexamethasone, a potent anti‐inflammatory drug that interferes with many of the features characterizing pro‐inflammatory glial activation, prevented the loss of catecholamine content, Tyrosine hydroxylase (TH) activity and TH immunostaining induced by LPS‐injection and also the bulk activation of microglia/macrophages. Surprisingly, injection of the pro‐inflammatory cytokines failed to reproduce the LPS effect. Taken together, our results suggest that inflammatory response is implicated in LPS‐induced neurodegeneration. This damage may be due, at least in part, to a cascade of events independent of that described for TNF‐α/IL‐1β/IFN‐γ.

Stress Increases Vulnerability to Inflammation in the Rat Prefrontal Cortex

Inflammation could be involved in some neurodegenerative disorders that accompany signs of inflammation. However, because sensitivity to inflammation is not equal in all brain structures, a direct relationship is not clear. Our aim was to test whether some physiological circumstances, such as stress, could enhance susceptibility to inflammation in the prefrontal cortex (PFC), which shows a relative resistance to inflammation. PFC is important in many brain functions and is a target for some neurodegenerative diseases. We induced an inflammatory process by a single intracortical injection of 2 μg of lipopolysaccharide (LPS), a potent proinflammogen, in nonstressed and stressed rats. We evaluated the effect of our treatment on inflammatory markers, neuronal populations, BDNF expression, and behavior of several mitogen-activated protein (MAP) kinases and the transcription factor cAMP response element-binding protein. Stress strengthens the changes induced by LPS injection: microglial activation and proliferation with an increase in the levels of the proinflammatory cytokine tumor necrosis factor-α; loss of cells such as astroglia, seen as loss of glial fibrillary acidic protein immunoreactivity, and neurons, studied by neuronal-specific nuclear protein immunohistochemistry and GAD67 and NMDA receptor 1A mRNAs expression by in situ hybridization. A significant increase in the BDNF mRNA expression and modifications in the levels of MAP kinase phosphorylation were also found. In addition, we observed a protective effect from RU486 [mifepristone (11β-[p-(dimethylamino)phenyl]-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one)], a potent inhibitor of the glucocorticoid receptor activation. All of these data show a synergistic effect between inflammation and stress, which could explain the relationship described between stress and some neurodegenerative pathologies.

Stress is critical for LPS-induced activation of microglia and damage in the rat hippocampus.

The hippocampus is insensitive to strong inflammatory stimulus under normal conditions and one of the most severely affected areas in Alzheimers disease. We have analyzed the effect of chronic stress for 9 days in the hippocampus unilaterally injected with LPS. In non-stressed rats, LPS injection failed to activate microglia although a subset of degenerating cells in the CA1 area was evident. This effect was not accompanied by loss of Neu-N positive neurons in the CA1 area. In stressed rats, LPS injection had a dramatic effect in activating microglia along with astrogliosis and BDNF mRNA induction. NeuN immunostaining demonstrated a loss of about 50% of CA1 pyramidal neurons under these conditions. Fluoro jade B histochemistry demonstrated the presence of degenerating cells in most of CA1 area. Mechanistically, combination of chronic stress and LPS resulted in prominent activation of MAPKs including JNK, p38 and ERK clearly different from LPS injection in controls. Further, LPS+stress induced a dramatic decrease in phosphorylated levels of both Akt and CREB, which fully supports a consistent deleterious state in the hippocampal system under these conditions. Treatment with RU486, a potent inhibitor of glucocorticoid receptor activation, significantly protected animals against the deleterious effects observed in LPS-stressed animals.

Blood-brain barrier disruption highly induces aquaporin-4 mRNA and protein in perivascular and parenchymal astrocytes : Protective effect by estradiol treatment in ovariectomized animals

Strong evidence involves aquaporin‐4 (AQP4) in the physiopathology of brain edema. Two major points remain unsolved: (1) the capacity of perivascular glial cells to regulate AQP4 in response to disruption of the blood–brain barrier (BBB); and (2) the potential beneficial role of AQP4 in the clearance of brain edema. We used intraparenchymal injection of lipopolysaccharide (LPS) as an efficient model to induce BBB disruption. This was monitored by IgG extravasation and AQP4 was studied at the mRNA and protein level. The first signs of BBB disruption coincided with strong induction of AQP4 mRNA in perivascular glial cells. At the early phase, estradiol treatment highly prevented the LPS‐induced disruption of the BBB and the induction of AQP4. Efficient clearance of vasogenic edema is supposed to occur once BBB is restored. This phase coincided with high induction of AQP4 mRNA in parenchymal reactive astrocytes and perivascular glial processes. High levels of AQP4 mRNA may be beneficial under these conditions. Our data may clarify why estradiol treatment reduces mortality in conditions typically associated with edema formation, like stroke.

Inflammatory process as a determinant factor for the degeneration of substantia nigra dopaminergic neurons

Summary.The specific degeneration of dopaminergic neurons in the substantia nigra (SN) is a pathological hallmark of Parkinson’s disease (PD). Although the cause of chronic nigral cell death in PD and its underlying mechanisms remain elusive, substantial involvement of inflammatory events has been postulated since inflammatory features have been described in parkinsonians CNS tissue. We have developed an animal model of dopaminergic neurons degeneration by the single intranigral injection of lipopolysaccharide (LPS), an inflammatory compound. This single injection produced the induction of inflammatory process with the activation of microglia along with the specific degeneration of dopaminergic neurons in the SN without affecting neither other neurotransmitter systems nor other structures of the CNS. Dexamethasone, a potent anti-inflammatory drug preventing many of the features characterizing pro-inflammatory glial activation, prevented the loss of dopaminergic cells. We also discuss other inductors of inflammatory process in relationship to the dopaminergic degeneration in the SN.

Histamine Infusion Induces a Selective Dopaminergic Neuronal Death Along with an Inflammatory Reaction in Rat Substantia Nigra

We have evaluated the effects of a direct infusion of histamine, as mediator of inflammatory response, in substantia nigra, striatum, medial septum, and medial lemniscus. Injection of 100 and 250 nmol of histamine in substantia nigra produced a selective damage in dopaminergic neurons evidenced by the loss of tyrosine hydroxylase mRNA‐expressing cells, tyrosine hydroxylase‐immunolabeled‐positive cell bodies, and dopamine and 3,4‐dihydroxyphenylacetic acid levels. In parallel we found an acute inflammatory response manifested by a loss of glial fibrillary acidic protein‐immunolabeled astrocytes and, at precisely the same area, an activation of microglia. In the striatum, only high doses (500 nmol) produced an evident terminal degeneration. The selective neurotoxicity of histamine for dopaminergic cells was demonstrated by the unaltered transcription of glutamic acid decarboxylase mRNA in substantia nigra. Moreover, intraseptal injection of 100 nmol of histamine failed to alter the pattern of choline acetyltransferase mRNA‐expressing cells, and intraparenchymal injection of histamine in medial lemniscus failed to alter the pattern of serotonin‐immunolabeled cells. We conclude that the substantia nigra is highly sensitive to histamine‐derived neurotoxicity, where inflammatory processes mediated by histamine could be important in the pathological changes that lead to dopaminergic neuronal damage after histamine infusion.

Expression of 5-HT7 receptor mRNA in rat brain during postnatal development.

The present study is the first one to demonstrate the expression of 5-HT7 receptor mRNA by in situ hybridization during postnatal development. No quantitative developmental changes in the 5-HT7 gene expression was observed in neocortex, pyramidal layers of CA1 and CA2, dentate gyrus, most of thalamic nuclei, mammillary region, superior colliculus and central gray. However, in retrosplenial cortex, subiculum and medial habenula an increase of labeling is observed between postnatal days (PN) PN15 and PN21. Striatum showed a transient expression during the first stages of development to be undetectable in adults. CA3 pyramidal cell layer, intramediodorsal thalamic nucleus and lateral habenula displayed a high mRNA expression at PN5 and PN8 which decreased throughout development but it was still present in adults. A possible non-neurotransmitter trophic function of 5-HT mediated through 5-HT7 receptors could be suggested.

Comparative study of the neuroprotective effect of dehydroepiandrosterone and 17β-estradiol against 1-methyl-4-phenylpyridium toxicity on rat striatum

The effects of dehydroepiandrosterone, estradiol and testosterone on 1-methyl-4-phenylpyridium (MPP+)-induced neurotoxicity of the nigrostriatal dopaminergic system were examined in rat. They were subjected to a unilateral intrastriatal infusion of the following treatment conditions: MPP+ alone or co-injection of MPP+ plus each hormone. Four days after injection, concentrations of dopamine and their metabolites were determined from the corpus striatum. To corroborate the neurochemical data an immunohistochemical analysis of tyrosine hydroxylase-immunoreactive fibers and acetylcholinesterase histochemistry in the striatum was performed. Moreover, we performed a dose-response study of the three hormones on the high-affinity dopamine transport system in rat striatal synaptosomes. Rats co-injected within the striatum with MPP+ and either dehydroepiandrosterone or estradiol had significantly greater concentrations of dopamine and less tyrosine hydroxylase-immunoreactive fibers and acetylcholinesterase fiber density loss compared with their respective controls. In addition, 4 days after injection, the brain was fixed and cut into coronal sections, and was immunostained with major histocompatibility complex class II antigens for activated microglia, and glial fibrillary acidic protein for activated astrocytes. Dehydroepiandrosterone also attenuated microglial cell activation. In contrast, testosterone showed reductions in dopamine concentrations similar to those obtained by MPP+. The protective effect of dehydroepiandrosterone against the MPP+ neurotoxic dopaminergic system may be produced by its partial prevention of MPP+ inhibition of NADH oxidase activity, whereas the estradiol may function as a neuroprotectant by reducing the uptake of MPP+ into dopaminergic neurons. Our findings we suggest indicate that dehydroepiandrosterone and estradiol by a non-genomic effect may have an important modulatory action, capable of attenuating degeneration within the striatum, and in this way serve as neuroprotectants of the nigrostriatal dopaminergic system.