An immunohistochemical and stereological analysis of PSI‐induced nigral neuronal degeneration in the rat

Abstract

Systemic administration of the proteasomal inhibitor I (PSI) to rats was reported to cause progressive nigral dopaminergic neuronal loss but this is disputed. A major controversy centres over the use of manual counting of tyrosine hydroxylase (TH) positive neurons at the level of third cranial nerve as opposed to employing systematic stereological analysis of cell loss in the entire substantia nigra (SN). To provide a method of marking SN neurones independent of protein expression, fluorogold äÔ (FG) was stereotaxically injected bilaterally into the striatum of male Wistar rats to retrogradely label nigral dopaminergic neurons. After 1 week, animals were treated with six doses of PSI (8 mg/kg, s.c.) or its vehicle (dimethyl sulphoxide) on alternate days over a 2-week period. Five weeks after the last treatment, PSI-treated animals showed decreased spontaneous locomotor activity and reduced TH positive SN cell number at the level of the third cranial nerve compared to control rats. Manual cell counting showed loss of FG-labelled SN neurones at this level, with a subpopulation of surviving neurons displaying abnormal morphology. Manual counting of all FG-labelled cells in the entire SN also showed regional PSI-induced loss of neurones with both normal and compromised morphology. Stereological optical fractionator estimates of total FG-labelled cell number confirmed the manual cell counting data both at the level of the third cranial nerve and throughout the entire SN. These findings confirm that PSI does cause a persistent nigral dopaminergic neuronal loss. The reason for the lack of reproducibility between laboratories requires further investigation. We suggest that a failure to distinguish between TH-positive neurones with normal and abnormal morphology following PSI administration contributes to equivocal results.

Magnetic resonance imaging and tensor-based morphometry in the MPTP non-human primate model of Parkinson’s disease

“…Sections were observed and photographed using a Zeiss microscope equipped with a Zeiss Axiovision digital camera. Nigral TH-immunoreactive neurons were then counted according to previously published protocol for manual nigral neuronal counting validated against a stereology-based method (optical fractionator) [54]. …”

Altered detrusor contractility in MPTP-treated common marmosets with bladder hyperreflexia

“…We have already shown that there is a close correlation between manual counting in this manner and unbiased stereology using the dissector method [25]. Based on the counts of dopaminergic neurones throughout the SN at regular 100 μm intervals, we have previously shown that the third nerve rootlets provide a reliable anatomical landmark at which the extent of cell loss can be accurately assessed and the extent of cell loss at this point is reflective of cell loss throughout the entire structure [25]. The extent of dopamine neuronal loss was estimated by counting the number of TH-ir SN neurones at the level of the third nerve rootlets on the lesioned side compared with the control side of the SN.…”

Neuroprotection and Functional Recovery Associated with Decreased Microglial Activation Following Selective Activation of mGluR2/3 Receptors in a Rodent Model of Parkinson's Disease

“…The number of TH positive cells was counted in the contralateral and ipsilateral hemispheres at the level of third cranial nerve, within a 100  μ m × 100  μ m counting area at high magnification (×40) only within this defined ROI. Importantly, the level of the third cranial nerve provides a robust anatomical landmark where the SNc can be reliably delineated from the VTA, as previously described [31, 32]. Lesion size was then calculated as a percentage of the unlesioned contralateral hemisphere.…”

“…Although not a stereological procedure, previous studies have shown that the 3rd nerve rootlets provide a reliable anatomical landmark at which the extent of cell loss is reflective of cell loss throughout the entire substantia nigra [32]. Further, manual cell counts assessed at the level of the third cranial nerve have been demonstrated to give equivalent results, not significantly different to that obtained from unbiased stereological estimates at the same level using an optical fractionator probe design [31]. These data strongly suggest that manual cell counting at the level of the third nerve is a viable method of determining cell loss and neuroprotection in this model [31].…”