Tissue clarification has been recently proposed to allow deep tissue imaging without light scatte... more Tissue clarification has been recently proposed to allow deep tissue imaging without light scattering. The clarification parameters are somewhat arbitrary and dependent on tissue type, source and dimension: every laboratory has its own protocol, but a quantitative approach to determine the optimum clearing time is still lacking. Since the use of transgenic mouse lines that express fluorescent proteins to visualize specific cell populations is widespread, a quantitative approach to determine the optimum clearing time for genetically labeled neurons from thick murine brain slices using CLARITY2 is described. In particular, as the main objective of the delipidation treatment is to clarify tissues, while limiting loss of fluorescent signal, the "goodness" of clarification was evaluated by considering the bulk tissue clarification index (BTCi) and the fraction of the fluorescent marker retained in the slice as easily quantifiable macroscale parameters. Here we describe the appr...
Autism spectrum disorder (ASD) is a congenital neurodevelopmental behavioral disorder that appear... more Autism spectrum disorder (ASD) is a congenital neurodevelopmental behavioral disorder that appears in early childhood. Recent human genetic studies identified the homeobox transcription factor, Engrailed 2 (EN2), as a possible ASD susceptibility gene. En2 knockout mice (En2-/-) display subtle cerebellar neuropathological changes and reduced levels of tyrosine hydroxylase, noradrenaline and serotonin in the hippocampus and cerebral cortex similar to those ones which have been observed in the ASD brain. Furthermore other similarities link En2 knockout mice to ASD patients. Several lines of evidence suggest that serotonin may play an important role in the pathophysiology of the disease. In the present study we measured, by using an HPLC, the 5-HT levels in different brain areas and at different ages in En2-/- mice. In the frontal and occipital cortex, the content of 5HT was reduced in En2-/- 1 and 3 months old mice; in 6 month old mice, the difference was still present, but it was not statistically significant. The 5-HT content of cerebellar cortex was significantly reduced at 1 month old but significantly high when the KO mice reached 3 months of age. The increase was present even at 6 months of age. A similar trend was highlighted by SERT immunolabeling in En2-/- mice compared to control in the same areas and age analyzed. Our findings, in agreement with the current knowledge on the 5-HT system alterations in ASD, confirm the early neurotransmitter deficit with a late compensatory recovery in En2 KO-mice further suggesting that this experimental animal may be considered a good predictive model for the human disease.
Journal of neural transmission. Supplementum, 2007
In order to reach a deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enh... more In order to reach a deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enhancement of MPTP toxicity in mice, we showed that CYP450 (2E1) inhibitors, such as diallyl sulfide (DAS) or phenylethylisothiocyanate (PIC), also potentiate the selective DA neuron degeneration in C57/bl mice. Furthermore we showed that CYP 2E1 is present in the brain and in the basal ganglia of mice (Vaglini et al., 2004). However, because DAS and PIC are not selective CYP 2E1 inhibitors and in order to provide direct evidence for CYP 2E1 involvement in the enhancement of MPTP toxicity, CYP 2E1 knockout mice (GONZ) and wild type animals (SVI) of the same genetic background were treated with MPTP or the combined DDC + MPTP treatment. In CYP 2E1 knockout mice, DDC pretreatment completely fails to enhance MPTP toxicity, although enhancement of MPTP toxicity was regularly present in the SVI control animals. The immunohistochemical study confirms our results and suggests that CYP 2E1 may hav...
The mechanism by which the dopamine neurons of the substantia nigra pars compacta degenerate in P... more The mechanism by which the dopamine neurons of the substantia nigra pars compacta degenerate in Parkinson's disease, is partly unknown. Dopamine could be implicated in this phenomenon, and in order to explain its toxicity several hypotheses have been suggested. The similarity between apomorphine and dopamine as regards their chemical, pharmacological and toxicological properties provided a basis for investigating the nature of the toxicity of the former agent. In this study we describe some effects of apomorphine on mouse mesencephalic cell cultures at relatively low concentrations (from 0.5 to 2.5microM), apomorphine produced a neurotrophic effect, consisting of a 60% increase in dopaminergic neuron survival as measured by [(3)H] dopamine uptake. At high concentrations (over 20microM), however, apomorphine induced an increasing cytotoxic effect, as measured by the marked decrease in [(3)H] dopamine uptake, and by the direct observation of the dopaminergic neurons after TH immunostaining. This study may offer a new strategy for investigating the mechanisms underlying DA neuron vulnerability.
Evidence for involvement of cytochrome P450 2E1 in the MPTP-induced mouse model of PD has been re... more Evidence for involvement of cytochrome P450 2E1 in the MPTP-induced mouse model of PD has been reported [Vaglini, F., Pardini, C., Viaggi, C., Bartoli, C., Dinucci, D., Corsini, G.U., 2004. Involvement of cytochrome P450 2E1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease. J. Neurochem. 91, 285-298]. We studied the sensitivity of Cyp2e1(-/-) mice to the acute administration of MPTP in comparison with their wild-type counterparts. In Cyp2e1(-/-) mice, the reduction of striatal DA content was less pronounced 7 days after MPTP treatment compared to treated wild-type mice. Similarly, TH immunoreactivity analysis of the substantia nigra of Cyp2e1(-/-) mice did not show any neuronal lesions after MPTP treatment. In contrast to this, wild-type animals showed a minimal but significant lesioning by the toxin as evaluated also by means of non-stereologic computerized assisted analysis of this brain area. Striatal levels of DA metabolites after 7 days were variably affected by the toxin, but consistent differences between the two animal strains were not observed. We evaluated short-term changes in the levels of striatal DA and its metabolites, and we monitored striatal MPP(+) levels. Striatal MPP(+) was cleared more rapidly in Cyp2e1(-/-) mice than in wild-type animals and, consistently, striatal DA content decreased faster in Cyp2e1(-/-) mice than in wild-type animals, and 3-methoxytyramine and HVA levels showed an early and sharp rise. Our findings suggest that Cyp2e1(-/-) mice are weakly sensitive to MPTP-induced brain lesions, markedly in contrast with a protective role of the enzyme as suggested previously. The differences observed between the knockout mice and their wild-type counterparts are modest and may be due to an efficient compensatory mechanism or genetic drift in the colonies.
Tissue clarification has been recently proposed to allow deep tissue imaging without light scatte... more Tissue clarification has been recently proposed to allow deep tissue imaging without light scattering. The clarification parameters are somewhat arbitrary and dependent on tissue type, source and dimension: every laboratory has its own protocol, but a quantitative approach to determine the optimum clearing time is still lacking. Since the use of transgenic mouse lines that express fluorescent proteins to visualize specific cell populations is widespread, a quantitative approach to determine the optimum clearing time for genetically labeled neurons from thick murine brain slices using CLARITY2 is described. In particular, as the main objective of the delipidation treatment is to clarify tissues, while limiting loss of fluorescent signal, the "goodness" of clarification was evaluated by considering the bulk tissue clarification index (BTCi) and the fraction of the fluorescent marker retained in the slice as easily quantifiable macroscale parameters. Here we describe the appr...
Autism spectrum disorder (ASD) is a congenital neurodevelopmental behavioral disorder that appear... more Autism spectrum disorder (ASD) is a congenital neurodevelopmental behavioral disorder that appears in early childhood. Recent human genetic studies identified the homeobox transcription factor, Engrailed 2 (EN2), as a possible ASD susceptibility gene. En2 knockout mice (En2-/-) display subtle cerebellar neuropathological changes and reduced levels of tyrosine hydroxylase, noradrenaline and serotonin in the hippocampus and cerebral cortex similar to those ones which have been observed in the ASD brain. Furthermore other similarities link En2 knockout mice to ASD patients. Several lines of evidence suggest that serotonin may play an important role in the pathophysiology of the disease. In the present study we measured, by using an HPLC, the 5-HT levels in different brain areas and at different ages in En2-/- mice. In the frontal and occipital cortex, the content of 5HT was reduced in En2-/- 1 and 3 months old mice; in 6 month old mice, the difference was still present, but it was not statistically significant. The 5-HT content of cerebellar cortex was significantly reduced at 1 month old but significantly high when the KO mice reached 3 months of age. The increase was present even at 6 months of age. A similar trend was highlighted by SERT immunolabeling in En2-/- mice compared to control in the same areas and age analyzed. Our findings, in agreement with the current knowledge on the 5-HT system alterations in ASD, confirm the early neurotransmitter deficit with a late compensatory recovery in En2 KO-mice further suggesting that this experimental animal may be considered a good predictive model for the human disease.
Journal of neural transmission. Supplementum, 2007
In order to reach a deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enh... more In order to reach a deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enhancement of MPTP toxicity in mice, we showed that CYP450 (2E1) inhibitors, such as diallyl sulfide (DAS) or phenylethylisothiocyanate (PIC), also potentiate the selective DA neuron degeneration in C57/bl mice. Furthermore we showed that CYP 2E1 is present in the brain and in the basal ganglia of mice (Vaglini et al., 2004). However, because DAS and PIC are not selective CYP 2E1 inhibitors and in order to provide direct evidence for CYP 2E1 involvement in the enhancement of MPTP toxicity, CYP 2E1 knockout mice (GONZ) and wild type animals (SVI) of the same genetic background were treated with MPTP or the combined DDC + MPTP treatment. In CYP 2E1 knockout mice, DDC pretreatment completely fails to enhance MPTP toxicity, although enhancement of MPTP toxicity was regularly present in the SVI control animals. The immunohistochemical study confirms our results and suggests that CYP 2E1 may hav...
The mechanism by which the dopamine neurons of the substantia nigra pars compacta degenerate in P... more The mechanism by which the dopamine neurons of the substantia nigra pars compacta degenerate in Parkinson's disease, is partly unknown. Dopamine could be implicated in this phenomenon, and in order to explain its toxicity several hypotheses have been suggested. The similarity between apomorphine and dopamine as regards their chemical, pharmacological and toxicological properties provided a basis for investigating the nature of the toxicity of the former agent. In this study we describe some effects of apomorphine on mouse mesencephalic cell cultures at relatively low concentrations (from 0.5 to 2.5microM), apomorphine produced a neurotrophic effect, consisting of a 60% increase in dopaminergic neuron survival as measured by [(3)H] dopamine uptake. At high concentrations (over 20microM), however, apomorphine induced an increasing cytotoxic effect, as measured by the marked decrease in [(3)H] dopamine uptake, and by the direct observation of the dopaminergic neurons after TH immunostaining. This study may offer a new strategy for investigating the mechanisms underlying DA neuron vulnerability.
Evidence for involvement of cytochrome P450 2E1 in the MPTP-induced mouse model of PD has been re... more Evidence for involvement of cytochrome P450 2E1 in the MPTP-induced mouse model of PD has been reported [Vaglini, F., Pardini, C., Viaggi, C., Bartoli, C., Dinucci, D., Corsini, G.U., 2004. Involvement of cytochrome P450 2E1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease. J. Neurochem. 91, 285-298]. We studied the sensitivity of Cyp2e1(-/-) mice to the acute administration of MPTP in comparison with their wild-type counterparts. In Cyp2e1(-/-) mice, the reduction of striatal DA content was less pronounced 7 days after MPTP treatment compared to treated wild-type mice. Similarly, TH immunoreactivity analysis of the substantia nigra of Cyp2e1(-/-) mice did not show any neuronal lesions after MPTP treatment. In contrast to this, wild-type animals showed a minimal but significant lesioning by the toxin as evaluated also by means of non-stereologic computerized assisted analysis of this brain area. Striatal levels of DA metabolites after 7 days were variably affected by the toxin, but consistent differences between the two animal strains were not observed. We evaluated short-term changes in the levels of striatal DA and its metabolites, and we monitored striatal MPP(+) levels. Striatal MPP(+) was cleared more rapidly in Cyp2e1(-/-) mice than in wild-type animals and, consistently, striatal DA content decreased faster in Cyp2e1(-/-) mice than in wild-type animals, and 3-methoxytyramine and HVA levels showed an early and sharp rise. Our findings suggest that Cyp2e1(-/-) mice are weakly sensitive to MPTP-induced brain lesions, markedly in contrast with a protective role of the enzyme as suggested previously. The differences observed between the knockout mice and their wild-type counterparts are modest and may be due to an efficient compensatory mechanism or genetic drift in the colonies.
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Papers by Cristina Viaggi