Transfection of the BDNF Gene in the Surviving Dopamine Neurons in Conjunction with Continuous Administration of Pramipexole Restores Normal Motor Behavior in a Bilateral Rat Model of Parkinson's Disease.

In Parkinson's disease (PD), progressive degeneration of nigrostriatal innervation leads to atrophy and loss of dendritic spines of striatal medium spiny neurons (MSNs). The loss disrupts corticostriatal transmission, impairs motor behavior, and produces nonmotor symptoms. Nigral neurons express brain-derived neurotropic factor (BDNF) and dopamine D3 receptors, both protecting the dopamine neurons and the spines of MSNs. To restore motor and nonmotor symptoms to normality, we assessed a combined therapy in a bilateral rat Parkinson's model, with only 30% of surviving neurons. The preferential D3 agonist pramipexole (PPX) was infused for four ½ months via mini-osmotic pumps and one month after PPX initiation; the BDNF-gene was transfected into the surviving nigral cells using the nonviral transfection NTS-polyplex vector. Overexpression of the BDNF-gene associated with continuous PPX infusion restored motor coordination, balance, normal gait, and working memory. Recovery was also related to the restoration of the average number of dendritic spines of the striatal projection neurons and the number of TH-positive neurons of the substantia nigra and ventral tegmental area. These positive results could pave the way for further clinical research into this promising therapy.

Changes in the Proliferation of the Neural Progenitor Cells of Adult Mice Chronically Infected with Toxoplasma gondii.

During Toxoplasma gondii chronic infection, certain internal factors that trigger the proliferation of neural progenitor cells (NPCs), such as brain inflammation, cell death, and changes in cytokine levels, are observed. NPCs give rise to neuronal cell types in the adult brain of some mammals. NPCs are capable of dividing and differentiating into a restricted repertoire of neuronal and glial cell types. In this study, the proliferation of NPCs was evaluated in CD-1 adult male mice chronically infected with the T. gondii ME49 strain. Histological brain sections from the infected mice were evaluated in order to observe T. gondii tissue cysts. Sagittal and coronal sections from the subventricular zone of the lateral ventricles and from the subgranular zone of the hippocampal dentate gyrus, as well as sagittal sections from the rostral migratory stream, were obtained from infected and non-infected mice previously injected with bromodeoxyuridine (BrdU). A flotation immunofluorescence technique was used to identify BrdU+ NPC. The scanning of BrdU+ cells was conducted using a confocal microscope, and the counting was performed with ImageJ® software (version 1.48q). In all the evaluated zones from the infected mice, a significant proliferation of the NPCs was observed when compared with that of the control group. We concluded that chronic infection with T. gondii increased the proliferation of NPCs in the three evaluated zones. Regardless of the role these cells are playing, our results could be useful to better understand the pathogenesis of chronic toxoplasmosis.

Schwann Cell Autophagy and Necrosis as Mechanisms of Cell Death by Acanthamoeba.

Amoebae of the genus Acanthamoeba are etiological agents of granulomatous amoebic encephalitis (GAE). Recently, through an in vivo GAE model, Acanthamoeba trophozoites were immunolocalized in contact with the peripheral nervous system (PNS) cells-Schwann cells (SC). In this study, we analyzed in greater detail the in vitro early morphological events (1, 2, 3, and 4 h) during the interaction of A. culbertsoni trophozoites (ATCC 30171) with SC from Rattus norvegicus (ATCC CRL-2941). Samples were processed for scanning and transmission electron microscopy as well as confocal microscopy. After 1 h of interaction, amoebae were observed to be adhered to the SC cultures, emitting sucker-like structures associated with micro-phagocytic channels. In addition, evidence of necrosis was identified since edematous organelles as well as multivesicular and multilamellar bodies characteristics of autophagy were detected. At 2 h, trophozoites migrated beneath the SC culture in which necrosis and autophagy persisted. By 3 and 4 h, extensive lytic zones were observed. SC necrosis was confirmed by confocal microscopy. We reported for the first time the induction of autophagic and necrotic processes in PNS cells, associated in part with the contact-dependent pathogenic mechanisms of A. culbertsoni trophozoites.

Alteration of Fermentative Metabolism Enhances Mucor circinelloides Virulence.

The fungus Mucor circinelloides undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in M. circinelloides virulence. A mutant in the adh1 gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-adh1+) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-adh1+ strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H2O2, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by M. circinelloides contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in M. circinelloides, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.

Heterotrimeric G-alpha subunits Gpa11 and Gpa12 define a transduction pathway that control spore size and virulence in Mucor circinelloides.

Mucor circinelloides is one of the causal agents of mucormycosis, an emerging and high mortality rate fungal infection produced by asexual spores (sporangiospores) of fungi that belong to the order Mucorales. M. circinelloides has served as a model genetic system to understand the virulence mechanism of this infection. Although the G-protein signaling cascade plays crucial roles in virulence in many pathogenic fungi, its roles in Mucorales are yet to be elucidated. Previous study found that sporangiospore size and calcineurin are related to the virulence in Mucor, in which larger spores are more virulent in an animal mucormycosis model and loss of a calcineurin A catalytic subunit CnaA results in larger spore production and virulent phenotype. The M. circinelloides genome is known to harbor twelve gpa (gpa1 to gpa12) encoding G-protein alpha subunits and the transcripts of the gpa11 and gpa12 comprise nearly 72% of all twelve gpa genes transcript in spores. In this study we demonstrated that loss of function of Gpa11 and Gpa12 led to larger spore size associated with reduced activation of the calcineurin pathway. Interestingly, we found lower levels of the cnaA mRNAs in sporangiospores from the Δgpa12 and double Δgpa11/Δgpa12 mutant strains compared to wild-type and the ΔcnaA mutant had significantly lower gpa11 and gpa12 mRNA levels compared to wild-type. However, in contrast to the high virulence showed by the large spores of ΔcnaA, the spores from Δgpa11/Δgpa12 were avirulent and produced lower tissue invasion and cellular damage, suggesting that the gpa11 and gpa12 define a signal pathway with two branches. One of the branches controls spore size through regulation of calcineurin pathway, whereas virulences is controlled by an independent pathway. This virulence-related regulatory pathway could control the expression of genes involved in cellular responses important for virulence, since sporangiospores of Δgpa11/Δgpa12 were less resistant to oxidative stress and phagocytosis by macrophages than the ΔcnaA and wild-type strains. The characterization of this pathway could contribute to decipher the signals and mechanism used by Mucorales to produce mucormycosis.

The transfection of BDNF to dopamine neurons potentiates the effect of dopamine D3 receptor agonist recovering the striatal innervation, dendritic spines and motor behavior in an aged rat model of Parkinson's disease.

The progressive degeneration of the dopamine neurons of the pars compacta of substantia nigra and the consequent loss of the dopamine innervation of the striatum leads to the impairment of motor behavior in Parkinson's disease. Accordingly, an efficient therapy of the disease should protect and regenerate the dopamine neurons of the substantia nigra and the dopamine innervation of the striatum. Nigral neurons express Brain Derived Neurotropic Factor (BDNF) and dopamine D3 receptors, both of which protect the dopamine neurons. The chronic activation of dopamine D3 receptors by their agonists, in addition, restores, in part, the dopamine innervation of the striatum. Here we explored whether the over-expression of BDNF by dopamine neurons potentiates the effect of the activation of D3 receptors restoring nigrostriatal innervation. Twelve-month old Wistar rats were unilaterally injected with 6-hydroxydopamine into the striatum. Five months later, rats were treated with the D3 agonist 7-hydroxy-N,N-di-n-propy1-2-aminotetralin (7-OH-DPAT) administered i.p. during 4½ months via osmotic pumps and the BDNF gene transfection into nigral cells using the neurotensin-polyplex nanovector (a non-viral transfection) that selectively transfect the dopamine neurons via the high-affinity neurotensin receptor expressed by these neurons. Two months after the withdrawal of 7-OH-DPAT when rats were aged (24 months old), immunohistochemistry assays were made. The over-expression of BDNF in rats receiving the D3 agonist normalized gait and motor coordination; in addition, it eliminated the muscle rigidity produced by the loss of dopamine. The recovery of motor behavior was associated with the recovery of the nigral neurons, the dopamine innervation of the striatum and of the number of dendritic spines of the striatal neurons. Thus, the over-expression of BDNF in dopamine neurons associated with the chronic activation of the D3 receptors appears to be a promising strategy for restoring dopamine neurons in Parkinson's disease.

The presence of perforated synapses in the striatum after dopamine depletion, is this a sign of maladaptive brain plasticity?

Synaptic plasticity is the process by which long-lasting changes take place at synaptic connections. The phenomenon itself is complex and can involve many levels of organization. Some authors separate forms into adaptations that have positive or negative consequences for the individual. It has been hypothesized that an increase in the number of synapses may represent a structural basis for the enduring expression of synaptic plasticity during some events that involve memory and learning; also, it has been suggested that perforated synapses increase in number after some diseases and experimental situations. The aim of this study was to analyze whether dopamine depletion induces changes in the synaptology of the corpus striatum of rats after the unilateral injection of 6-OHDA. The findings suggest that after the lesion, both contralateral and ipsilateral striata exhibit an increased length of the synaptic ending in ipsilateral (since third day) and contralateral striatum (since Day 20), loss of axospinous synapses in ipsilateral striatum and a significant increment in the number of perforated synapses, suggesting brain plasticity that might be deleterious for the spines, because this type of synaptic contacts are presumably excitatory, and in the absence of the modulatory effects of dopamine, the neuron could die through excitotoxic mechanisms. Thus, we can conclude that the presence of perforated synapses after striatal dopamine depletion might be a form of maladaptive synaptic plasticity.

The combination of oral L-DOPA/rimonabant for effective dyskinesia treatment and cytological preservation in a rat model of Parkinson's disease and L-DOPA-induced dyskinesia.

Parkinson's disease is the second most prevalent neurodegenerative disease in the world. Its treatment is limited so far to the management of parkinsonian symptoms with L-DOPA (LD). The long-term use of LD is limited by the development of L-DOPA-induced dyskinesias and dystonia. However, recent studies have suggested that pharmacological targeting of the endocannabinoid system may potentially provide a valuable therapeutic tool to suppress these motor alterations. In the present study, we have explored the behavioral (L-DOPA-induced dyskinesias severity) and cytological (substantia nigra compacta neurons and striatum neuropil preservation) effects of the oral coadministration of LD and rimonabant, a selective antagonist of CB1 receptors, in the 6-hydroxydopamine rat model of Parkinson's disease. Oral coadministration of LD (30 mg/kg) and rimonabant (1 mg/kg) significantly decreased abnormal involuntary movements and dystonia, possibly through the conservation of some functional tyrosine hydroxylase-immunoreactive dopaminergic cells, which in turn translates into a well-preserved neuropil of a less denervated striatum. Our results provide anatomical evidence that long-term coadministration of LD with cannabinoid antagonist-based therapy may not only alleviate specific motor symptoms but also delay/arrest the degeneration of striatal and substantia nigra compacta cells.

Manganese mixture inhalation is a reliable Parkinson disease model in rats.

Manganese (Mn) is an essential trace metal. Regardless of its essentiality, it has been reported that the overexposure causes neurotoxicity manifested as extrapyramidal symptoms similar to those observed in Parkinson disease (PD). Recently, our group reported that mice that inhaled for 5 months the mixture of manganese chloride (MnCl(2)) and manganese acetate Mn(OAc)(3) developed movement abnormalities, significant loss of substantia nigra compacta (SNc) dopaminergic neurons, dopamine depletion and improved behavior with l-DOPA treatment. However, this model has only been characterized in mice. In order to have a well-supported and generalizable model in rodents, we used male Wistar rats that inhaled a mixture of 0.04 M MnCl(2) and 0.02 M Mn(OAc)(3), 1h three times a week for 6 months. Before Mn exposure, animals were trained to perform motor tests (Beam-walking and Single-pellet reaching tasks) and were evaluated each week after the exposure. The mixture of MnCl(2)/Mn(OAc)(3) caused alterations in the motor tests, 75.95% loss of SNc dopaminergic neurons, and no cell alterations in Globus Pallidus or striatum. With these results we conclude that the inhalation of the mixture of Mn compounds is a useful model in rodents for the study of PD.

Effect of chronic L-dopa or melatonin treatments after dopamine deafferentation in rats: dyskinesia, motor performance, and cytological analysis.

The present study examines the ability of melatonin to protect striatal dopaminergic loss induced by 6-OHDA in a rat model of Parkinson's disease, comparing the results with L-DOPA-treated rats. The drugs were administered orally daily for a month, their therapeutic or dyskinetic effects were assessed by means of abnormal involuntary movements (AIMs) and stepping ability. At the cellular level, the response was evaluated using tyrosine hydroxylase immunoreactivity and striatal ultrastructural changes to compare between L-DOPA-induced AIMs and Melatonin-treated rats. Our findings demonstrated that chronic oral administration of Melatonin improved the alterations caused by the neurotoxin 6-OHDA. Melatonin-treated animals perform better in the motor tasks and had no dyskinetic alterations compared to L-DOPA-treated group. At the cellular level, we found that Melatonin-treated rats showed more TH-positive neurons and their striatal ultrastructure was well preserved. Thus, Melatonin is a useful treatment to delay the cellular and behavioral alterations observed in Parkinson's disease.

L-DOPA treatment reverses the motor alterations induced by manganese exposure as a Parkinson disease experimental model.

This investigation was designed to determine whether l-DOPA treatment improves the motor alterations observed after divalent and trivalent manganese (Mn) mixture inhalation on mice to ensure that the alterations are of dopaminergic origin. CD-1 male mice inhaled a mixture of 0.04 M manganese chloride (MnCl(2)) and manganese acetate (Mn(OAc)(3)), 1h twice a week for 5 months. Before Mn exposure, animals were trained to perform motor function tests and were evaluated each week after the exposure. Overall behavior was assessed by ratings and by videotaped analyses; by the end of Mn exposure period, 10 mice were orally treated with 7.5mg/kg L-DOPA. After 5 months of Mn-mixture inhalation striatal dopamine content decreased 71%, mice developed evident deficits in motor performance manifested as akinesia, postural instability and action tremor; these alterations were reverted with L-DOPA treatment. Our results suggest that the motor alterations induced by the inhalation of the combination of MnCl(2)/Mn(OAc)(3) are related to nigrostriatal dopaminergic function providing new light on the understanding of manganese neurotoxicity as a suitable Parkinson disease experimental model.

Manganese inhalation as a Parkinson disease model.

The present study examines the effects of divalent and trivalent Manganese (Mn(2+)/Mn(3+)) mixture inhalation on mice to obtain a novel animal model of Parkinson disease (PD) inducing bilateral and progressive dopaminergic cell death, correlate those alterations with motor disturbances, and determine whether L-DOPA treatment improves the behavior, to ensure that the alterations are of dopaminergic origin. CD-1 male mice inhaled a mixture of Manganese chloride and Manganese acetate, one hour twice a week for five months. Before Mn exposure, animals were trained to perform motor function tests and were evaluated each week after the exposure. By the end of Mn exposure, 10 mice were orally treated with 7.5 mg/kg L-DOPA. After 5 months of Mn mixture inhalation, striatal dopamine content decreased 71%, the SNc showed important reduction in the number of TH-immunopositive neurons, mice developed akinesia, postural instability, and action tremor; these motor alterations were reverted with L-DOPA treatment. Our data provide evidence that Mn(2+)/Mn(3+) mixture inhalation produces similar morphological, neurochemical, and behavioral alterations to those observed in PD providing a useful experimental model for the study of this neurodegenerative disease.

6-Hydroxydopamine lesion in thalamic reticular nucleus reduces anxiety behaviour in the rat.

We have studied the effect of the lesion of the dopaminergic innervation of the thalamic reticular nucleus (TRn) on anxiety and motor behaviour. The lesion of the dopamine innervation was produced by the injection of 6-hydroxydopamine into the dorsal part of the thalamic reticular nucleus. The lesion decreased the number of TH (+) cells of the pars compacta of substantia nigra by 33%, without modifying the number of TH (+) cells in ventral tegmental area. The lesion increased the time spent by the rats on the open arms of the elevated plus maze and decreased the duration of burying in the shock-probe test. Both results suggest reduced anxiety. The loss of the dopamine innervation to the TRn decreased the number of rearings but did not significantly affect total motor activity, gait or motor coordination, as evidenced by rotarod performance. These findings suggest that dopamine in the TRn plays a role in fear-related behaviour.

Neurotensin polyplex as an efficient carrier for delivering the human GDNF gene into nigral dopamine neurons of hemiparkinsonian rats.

Recently we showed that the neurotensin polyplex is a nanoparticle carrier system that targets reporter genes in nigral dopamine neurons in vivo. Herein, we report its first practical application in experimental parkinsonism, which consisted of transfecting dopamine neurons with the gene coding for human glial cell line-derived neurotrophic factor (hGDNF). Hemiparkinsonism was induced in rats by a single dose of 6-hydroxydopamine (30 microg) into the ventrolateral part of the striatum. We showed that transfection of the hGDNF gene into the substantia nigra of rats 1 week after the neurotoxin injection produced biochemical, anatomical, and functional recovery from hemiparkinsonism. RT-PCR analysis showed mRNA expression of exogenous hGDNF in the transfected substantia nigra. Western blot analysis verified transgene expression by recognizing the flag epitope added at the C-terminus of the hGDNF polypeptide, which was found mainly in dopamine neurons by double immunofluorescence techniques. These data indicate that the neurotensin polyplex holds great promise for the neuroprotective therapy of Parkinson disease.

Hippocampal cell alterations induced by the inhalation of vanadium pentoxide (V(2)O(5)) promote memory deterioration.

Spatial memory may be severely impaired as a consequence of ageing and neurodegenerative diseases, conditions that include neuronal damage. Vanadium (V) is a metalloid widely distributed in the environment and exerts severe toxic effects on a wide variety of biological systems. Reports about V inhalation toxicity on the CNS are limited, thus the purpose of this study is to determine the effects of Vanadium pentoxide (V(2)O(5)) inhalation (0.02M) on the memory and its correlation with the cytology of the hippocampus CA1. Forty eight CD-1 male mice were trained in spatial memory tasks and inhaled 1h twice a week; after each inhalation animals were evaluated and sacrificed from 1 to 4 weeks, perfused and processed for Golgi method and for ultrastructure evaluation. The cytological analysis consisted in counting the number of dendritic spines of 20 pyramidal neurons of hippocampus CA1, as well as ultrastructural characteristics. Results show that V inhalation produces a time dependent loss of dendritic spines, necrotic-like cell death, and notorious alterations of the hippocampus CA1 neuropile, which correlate with spatial memory impairment. Our data suggest that V induces important cellular and functional alterations, fact that deserves special attention since the concentration's trend of this element in the atmosphere is increasing.

Ependymal epithelium disruption after vanadium pentoxide inhalation. A mice experimental model.

The blood-brain barrier (BBB) protects the CNS against chemical insults. Regulation of blood-brain tissue exchange is accomplished by ependymal cells, which possess intercellular tight junctions. Loss of BBB function is an etiologic component of many neurological disorders. Vanadium (V) is a metalloid widely distributed in the environment and exerts potent toxic effects on a wide variety of biological systems. The current study examines the effects of Vanadium pentoxide (V2O5) inhalation in mice ependymal epithelium, through the analysis of the brain metal concentrations and the morphological modifications in the ependymal cells identified by scanning and transmission electron microscopy after 8 weeks of inhalation, in order to obtain a possible explanation about the mechanisms that V uses to enter and alter the CNS. Our results showed that V2O5 concentrations increase from the first week of study, stabilizing its values during the rest of the experiment. The morphological effects included cilia loss, cell sloughing and ependymal cell layer detachment. This damage can allow toxicants to modify the permeability of the epithelium and promote access of inflammatory mediators to the underlying neuronal tissue causing injury and neuronal death. Thus, understanding the mechanisms of BBB disruption would allow planning strategies to protect the brain from toxicants such as metals, which have increased in the atmosphere during the last decades and constitute an important health problem.