title: Quantification of MPTP-induced dopaminergic neurodegeneration in the mouse substantia nigra by laser capture microdissection.
abstract: The neurotoxin MPTP is widely used to cause damage to the dopaminergic system in rodents and non-human primates to model various aspects of Parkinson's disease. In mice, depletion of striatal dopamine is the commonly used endpoint to assess neuronal damage. However, it has proved technically challenging to quantify dopaminergic cell bodies as an index of neuronal integrity. To meet this challenge, we applied laser pressure catapult microdissection (LCM) of the substantia nigra in combination with quantitative Western blot to provide an index of dopamine neurodegeneration in mice treated with MPTP. Seven days following initiation of MPTP treatment, striatal dopamine depletion was maximal and there was histological evidence of neuronal degeneration in the substantia nigra. To index the integrity of dopamine cell bodies, tyrosine hydroxylase (TH) and beta-actin were quantified by Western blot in LCM extracts. In untreated mice, TH was detected in LCM extracts of substantia nigra but was undetectable in equivalently sized extracts of cortex from the same animals. In MPTP-treated mice, there was a significant 70% reduction in TH relative to beta-actin in LCM extracts as compared to vehicle-injected controls. This reduction corresponded to decreases in striatal dopamine and loss of immunocytochemically detected TH but not beta-actin in the substantia nigra (SN). Thus, this method provides a quantitative means to measure dopamine neuron toxicity in the substantia nigra and, as such has potential application in evaluating regimens that may be neuroprotective or neurorestorative for dopaminergic neurons.
authors.affiliation: Pfizer Global Research and Development, Worldwide Safety Sciences and CNS Discovery, MS8274-1348, Eastern Point Road, Groton, CT 06340, United States. Diane.t.Stephenson@pfizer.com
authors.forename: Diane
authors.initials: D
authors.lastname: Stephenson
authors.forename: Andres
authors.initials: A
authors.lastname: Ramirez
authors.forename: Jill
authors.initials: J
authors.lastname: Long
authors.forename: Nestor
authors.initials: N
authors.lastname: Barrezueta
authors.forename: Eva
authors.initials: E
authors.lastname: Hajos-Korcsok
authors.forename: Curt
authors.initials: C
authors.lastname: Matherne
authors.forename: Dan
authors.initials: D
authors.lastname: Gallagher
authors.forename: Anne
authors.initials: A
authors.lastname: Ryan
authors.forename: Ricardo
authors.initials: R
authors.lastname: Ochoa
authors.forename: Frank
authors.initials: F
authors.lastname: Menniti
authors.forename: James
authors.initials: J
authors.lastname: Yan
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods; D004298:Dopamine / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007834:Lasers; D020267:MPTP Poisoning / Q000473:pathology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D042282:Microdissection / Q000295:instrumentation* / Q000379:methods*; D009410:Nerve Degeneration / Q000473:pathology; D013378:Substantia Nigra / Q000473:pathology*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
chemical_list: D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine