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The Progressive BSSG Rat Model of Parkinson's: Recapitulating Multiple Key Features of the Human Disease.

Van Kampen JM, Baranowski DC, Robertson HA, Shaw CA, Kay DG - PLoS ONE (2015)

Bottom Line: Locomotor deficits developed gradually over time, initially appearing as locomotor asymmetry and developing into akinesia/bradykinesia, which was reversed by levodopa treatment.In addition to the progressive loss of TH+ cells in the substantia nigra, the appearance of proteinase K-resistant intracellular α-synuclein aggregates was also observed to develop progressively, appearing first in the olfactory bulb, then the striatum, the substantia nigra and, finally, hippocampal and cortical regions.The slowly progressive nature of this model, together with its construct, face and predictive validity, make it ideal for the screening of potential neuroprotective therapies for the treatment of PD.

View Article: PubMed Central - PubMed

Affiliation: Neurodyn Inc., 550 University Ave, Charlottetown, Prince Edward Island, C1A 4P3, Canada; Dept. Biomedical Science, University of Prince Edward Island, 550 University Ave, Charlottetown, Prince Edward Island, C1A 4P3, Canada.

ABSTRACT
The development of effective neuroprotective therapies for Parkinson's disease (PD) has been severely hindered by the notable lack of an appropriate animal model for preclinical screening. Indeed, most models currently available are either acute in nature or fail to recapitulate all characteristic features of the disease. Here, we present a novel progressive model of PD, with behavioural and cellular features that closely approximate those observed in patients. Chronic exposure to dietary phytosterol glucosides has been found to be neurotoxic. When fed to rats, β-sitosterol β-d-glucoside (BSSG) triggers the progressive development of parkinsonism, with clinical signs and histopathology beginning to appear following cessation of exposure to the neurotoxic insult and continuing to develop over several months. Here, we characterize the progressive nature of this model, its non-motor features, the anatomical spread of synucleinopathy, and response to levodopa administration. In Sprague Dawley rats, chronic BSSG feeding for 4 months triggered the progressive development of a parkinsonian phenotype and pathological events that evolved slowly over time, with neuronal loss beginning only after toxin exposure was terminated. At approximately 3 months following initiation of BSSG exposure, animals displayed the early emergence of an olfactory deficit, in the absence of significant dopaminergic nigral cell loss or locomotor deficits. Locomotor deficits developed gradually over time, initially appearing as locomotor asymmetry and developing into akinesia/bradykinesia, which was reversed by levodopa treatment. Late-stage cognitive impairment was observed in the form of spatial working memory deficits, as assessed by the radial arm maze. In addition to the progressive loss of TH+ cells in the substantia nigra, the appearance of proteinase K-resistant intracellular α-synuclein aggregates was also observed to develop progressively, appearing first in the olfactory bulb, then the striatum, the substantia nigra and, finally, hippocampal and cortical regions. The slowly progressive nature of this model, together with its construct, face and predictive validity, make it ideal for the screening of potential neuroprotective therapies for the treatment of PD.

No MeSH data available.


Related in: MedlinePlus

BSSG increases indices of apoptosis.(A) Representative photomicrographs of TH (red) and activated caspase–3 (green) immunostaining in the SNc 4, 6, 8, and 10 months following initial BSSG exposure. (B) Exposure to BSSG triggered the progressive increase in the percentage of TH+ cells expressing activated caspase–3, an indicator of apoptosis. Each bar represents the mean (± S.E.M., n = 9–10) percentage of TH+ cells labeled for activated caspase-3-positive cells in the SNc. (C) BSSG similarly triggered time-dependent elevations in TUNEL labeling within the SNc. Each bar represents the mean (± S.E.M., n = 9–10) number of TUNEL-positive cells counted in the SNc. ** sig. diff. from flour control, p < 0.001; * p < 0.05; ++ sig. diff. from 4 months, p < 0.001; + p < 0.05; ## sig. diff. from 6 months, p < 0.001.
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pone.0139694.g005: BSSG increases indices of apoptosis.(A) Representative photomicrographs of TH (red) and activated caspase–3 (green) immunostaining in the SNc 4, 6, 8, and 10 months following initial BSSG exposure. (B) Exposure to BSSG triggered the progressive increase in the percentage of TH+ cells expressing activated caspase–3, an indicator of apoptosis. Each bar represents the mean (± S.E.M., n = 9–10) percentage of TH+ cells labeled for activated caspase-3-positive cells in the SNc. (C) BSSG similarly triggered time-dependent elevations in TUNEL labeling within the SNc. Each bar represents the mean (± S.E.M., n = 9–10) number of TUNEL-positive cells counted in the SNc. ** sig. diff. from flour control, p < 0.001; * p < 0.05; ++ sig. diff. from 4 months, p < 0.001; + p < 0.05; ## sig. diff. from 6 months, p < 0.001.

Mentions: Animals were sacrificed by perfusion at 4, 6, 8, and 10 months following initial exposure to BSSG and their brains harvested for histopathological assessment of markers for cell death, nigrostriatal integrity, inflammation, and proteinase K-resistant synuclein deposits. Immunolabeling for activated caspase–3 and TUNEL, both apoptotic markers, progressively increased with time in the SNc of those animals exposed to BSSG, beginning at 6 months following initial exposure to the toxin (caspase: F1,71 = 233.44, p<0.0001, BSSG main effect; F3,71 = 65.53, p<0.0001, time main effect; F3,71 = 33.69, p<0.0001, interaction effect) (TUNEL: F1,71 = 252.34, p<0.0001, BSSG main effect; F3,71 = 56.21, p<0.0001, time main effect; F3,71 = 37.31, p<0.0001, interaction effect) (Fig 5). The persistence of this response, as opposed to the early rise and decline observed in many acute models, may be reflective of ongoing degeneration. In order to determine nigro-striatal integrity, sections were assessed for TH and dopamine transporter (DAT) immunolabeling in the SNc and striatum. As TH+ cell counts are only a marker of dopaminergic phenotype and do not necessarily imply cell death/survival, lesion severity was determined by assessment of both TH+ and Nissl cell counts. Stereological analyses of TH+ and Nissl+ neurons in the SNc revealed a significant loss of both following BSSG intoxication (TH: F1,71 = 185.56, p<0.0001, BSSG main effect; F3,71 = 16.15, p<0.0001, time main effect; F3,71 = 8.81, p<0.0001, interaction effect) (Nissl: F1,71 = 186.54, p<0.0001, BSSG main effect; F3,71 = 59.78, p<0.0001, time main effect; F3,71 = 29.96, p<0.0001, interaction effect) (Fig 6). However, significant nigral cell loss was not observed until after BSSG exposure was terminated. Thus, at 4 months following initial exposure to BSSG, when toxin feeding was terminated, there was no significant loss of either TH+ or Nissl+ cells in the SNc. However, a time-dependent, progressive loss of these cells was observed in the ensuing months, with nearly 70% fewer TH+ cells observed in the SNc by 10 months following initial BSSG exposure, compared to controls. Although the average loss of TH+ cells was not significant at 4 months, hemispheric differences in TH+ cell counts were evident at this time point. When hemispheric comparisons of TH+ and Nissl+ cell counts were made, hemispheric asymmetry was found to be significantly higher in BSSG-fed animals at 4 months following initial exposure, peaked at 6 months, and significantly dropped at 8 months (TH: F1,71 = 166.46, p<0.0001, BSSG main effect; F3,71 = 33.91, p<0.0001, time main effect; F3,71 = 39.61, p<0.0001, interaction effect) (Nissl: F1,71 = 124.51, p<0.0001, BSSG main effect; F3,71 = 30.12, p<0.0001, time main effect; F3,71 = 23.99, p<0.0001, interaction effect) (Fig 6C and 6E). No significant hemispheric asymmetry was evident at 10 months following initial BSSG exposure.


The Progressive BSSG Rat Model of Parkinson's: Recapitulating Multiple Key Features of the Human Disease.

Van Kampen JM, Baranowski DC, Robertson HA, Shaw CA, Kay DG - PLoS ONE (2015)

BSSG increases indices of apoptosis.(A) Representative photomicrographs of TH (red) and activated caspase–3 (green) immunostaining in the SNc 4, 6, 8, and 10 months following initial BSSG exposure. (B) Exposure to BSSG triggered the progressive increase in the percentage of TH+ cells expressing activated caspase–3, an indicator of apoptosis. Each bar represents the mean (± S.E.M., n = 9–10) percentage of TH+ cells labeled for activated caspase-3-positive cells in the SNc. (C) BSSG similarly triggered time-dependent elevations in TUNEL labeling within the SNc. Each bar represents the mean (± S.E.M., n = 9–10) number of TUNEL-positive cells counted in the SNc. ** sig. diff. from flour control, p < 0.001; * p < 0.05; ++ sig. diff. from 4 months, p < 0.001; + p < 0.05; ## sig. diff. from 6 months, p < 0.001.
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Related In: Results  -  Collection

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pone.0139694.g005: BSSG increases indices of apoptosis.(A) Representative photomicrographs of TH (red) and activated caspase–3 (green) immunostaining in the SNc 4, 6, 8, and 10 months following initial BSSG exposure. (B) Exposure to BSSG triggered the progressive increase in the percentage of TH+ cells expressing activated caspase–3, an indicator of apoptosis. Each bar represents the mean (± S.E.M., n = 9–10) percentage of TH+ cells labeled for activated caspase-3-positive cells in the SNc. (C) BSSG similarly triggered time-dependent elevations in TUNEL labeling within the SNc. Each bar represents the mean (± S.E.M., n = 9–10) number of TUNEL-positive cells counted in the SNc. ** sig. diff. from flour control, p < 0.001; * p < 0.05; ++ sig. diff. from 4 months, p < 0.001; + p < 0.05; ## sig. diff. from 6 months, p < 0.001.
Mentions: Animals were sacrificed by perfusion at 4, 6, 8, and 10 months following initial exposure to BSSG and their brains harvested for histopathological assessment of markers for cell death, nigrostriatal integrity, inflammation, and proteinase K-resistant synuclein deposits. Immunolabeling for activated caspase–3 and TUNEL, both apoptotic markers, progressively increased with time in the SNc of those animals exposed to BSSG, beginning at 6 months following initial exposure to the toxin (caspase: F1,71 = 233.44, p<0.0001, BSSG main effect; F3,71 = 65.53, p<0.0001, time main effect; F3,71 = 33.69, p<0.0001, interaction effect) (TUNEL: F1,71 = 252.34, p<0.0001, BSSG main effect; F3,71 = 56.21, p<0.0001, time main effect; F3,71 = 37.31, p<0.0001, interaction effect) (Fig 5). The persistence of this response, as opposed to the early rise and decline observed in many acute models, may be reflective of ongoing degeneration. In order to determine nigro-striatal integrity, sections were assessed for TH and dopamine transporter (DAT) immunolabeling in the SNc and striatum. As TH+ cell counts are only a marker of dopaminergic phenotype and do not necessarily imply cell death/survival, lesion severity was determined by assessment of both TH+ and Nissl cell counts. Stereological analyses of TH+ and Nissl+ neurons in the SNc revealed a significant loss of both following BSSG intoxication (TH: F1,71 = 185.56, p<0.0001, BSSG main effect; F3,71 = 16.15, p<0.0001, time main effect; F3,71 = 8.81, p<0.0001, interaction effect) (Nissl: F1,71 = 186.54, p<0.0001, BSSG main effect; F3,71 = 59.78, p<0.0001, time main effect; F3,71 = 29.96, p<0.0001, interaction effect) (Fig 6). However, significant nigral cell loss was not observed until after BSSG exposure was terminated. Thus, at 4 months following initial exposure to BSSG, when toxin feeding was terminated, there was no significant loss of either TH+ or Nissl+ cells in the SNc. However, a time-dependent, progressive loss of these cells was observed in the ensuing months, with nearly 70% fewer TH+ cells observed in the SNc by 10 months following initial BSSG exposure, compared to controls. Although the average loss of TH+ cells was not significant at 4 months, hemispheric differences in TH+ cell counts were evident at this time point. When hemispheric comparisons of TH+ and Nissl+ cell counts were made, hemispheric asymmetry was found to be significantly higher in BSSG-fed animals at 4 months following initial exposure, peaked at 6 months, and significantly dropped at 8 months (TH: F1,71 = 166.46, p<0.0001, BSSG main effect; F3,71 = 33.91, p<0.0001, time main effect; F3,71 = 39.61, p<0.0001, interaction effect) (Nissl: F1,71 = 124.51, p<0.0001, BSSG main effect; F3,71 = 30.12, p<0.0001, time main effect; F3,71 = 23.99, p<0.0001, interaction effect) (Fig 6C and 6E). No significant hemispheric asymmetry was evident at 10 months following initial BSSG exposure.

Bottom Line: Locomotor deficits developed gradually over time, initially appearing as locomotor asymmetry and developing into akinesia/bradykinesia, which was reversed by levodopa treatment.In addition to the progressive loss of TH+ cells in the substantia nigra, the appearance of proteinase K-resistant intracellular α-synuclein aggregates was also observed to develop progressively, appearing first in the olfactory bulb, then the striatum, the substantia nigra and, finally, hippocampal and cortical regions.The slowly progressive nature of this model, together with its construct, face and predictive validity, make it ideal for the screening of potential neuroprotective therapies for the treatment of PD.

View Article: PubMed Central - PubMed

Affiliation: Neurodyn Inc., 550 University Ave, Charlottetown, Prince Edward Island, C1A 4P3, Canada; Dept. Biomedical Science, University of Prince Edward Island, 550 University Ave, Charlottetown, Prince Edward Island, C1A 4P3, Canada.

ABSTRACT
The development of effective neuroprotective therapies for Parkinson's disease (PD) has been severely hindered by the notable lack of an appropriate animal model for preclinical screening. Indeed, most models currently available are either acute in nature or fail to recapitulate all characteristic features of the disease. Here, we present a novel progressive model of PD, with behavioural and cellular features that closely approximate those observed in patients. Chronic exposure to dietary phytosterol glucosides has been found to be neurotoxic. When fed to rats, β-sitosterol β-d-glucoside (BSSG) triggers the progressive development of parkinsonism, with clinical signs and histopathology beginning to appear following cessation of exposure to the neurotoxic insult and continuing to develop over several months. Here, we characterize the progressive nature of this model, its non-motor features, the anatomical spread of synucleinopathy, and response to levodopa administration. In Sprague Dawley rats, chronic BSSG feeding for 4 months triggered the progressive development of a parkinsonian phenotype and pathological events that evolved slowly over time, with neuronal loss beginning only after toxin exposure was terminated. At approximately 3 months following initiation of BSSG exposure, animals displayed the early emergence of an olfactory deficit, in the absence of significant dopaminergic nigral cell loss or locomotor deficits. Locomotor deficits developed gradually over time, initially appearing as locomotor asymmetry and developing into akinesia/bradykinesia, which was reversed by levodopa treatment. Late-stage cognitive impairment was observed in the form of spatial working memory deficits, as assessed by the radial arm maze. In addition to the progressive loss of TH+ cells in the substantia nigra, the appearance of proteinase K-resistant intracellular α-synuclein aggregates was also observed to develop progressively, appearing first in the olfactory bulb, then the striatum, the substantia nigra and, finally, hippocampal and cortical regions. The slowly progressive nature of this model, together with its construct, face and predictive validity, make it ideal for the screening of potential neuroprotective therapies for the treatment of PD.

No MeSH data available.


Related in: MedlinePlus