A comparison of model-based (2D) and design-based (3D) stereological methods for estimating cell number in the substantia nigra pars compacta (SNpc) of the C57BL/6J mouse

Abstract

The substantia nigra pars compacta (SNpc) is a compact brain structure that contains a variable distribution of cells in both medial to lateral and rostral to caudal dimensions. The SNpc is the primary brain structure affected in Parkinson's disease, where loss of dopaminergic neurons is one of the major hallmarks of the disorder. Neurotoxic and genetic models of Parkinson's disease, as well as mechanisms to treat this disorder, are modeled in the mouse. To accurately assess the validity of a model, one needs to be assured that the method(s) of analysis is accurate. Here, we determine the total number of dopaminergic neurons in the SNpc of the C57BL/6J mouse by serial reconstruction then compared that value to estimates derived using model-based stereology and design-based stereology. Serial reconstruction of the SNpc revealed the total number of SNpc dopaminergic neurons to be 8305±540 (SEM). We compared this empirically derived neuron number to model based and design-based stereological estimates. We found that model based estimates gave a value of 8002±91 (SEM) while design-based estimates were 8716±338 (SEM). Statistical analysis showed no significant difference between estimates generated using model-or design-based stereological methods compared to empirically-derived counts using serial reconstruction.

Cytoprotective effects of growth factors: BDNF more potent than GDNF in an organotypic culture model of Parkinson's disease

“…Using this sampling procedure, we estimated a total number of ~3000 TH positive neurons/hemisphere of WT control hemispheres with Gundersen's coefficients of errors <10%, indicating low variance. These numbers are in line with the literature, which shows a range between 5200-23,670 TH+ neurons bilaterally, with a mean value of 12,000 neurons (408). This wide range indicates that the underlying method highly affects the quantifications, and that the ratio between the two hemispheres is a more accurate measurement.…”

Neurodegeneration by Activation of the Microglial Complement-Phagosome Pathway

“…For neuronal quantification, slides were double-stained with antibodies directed against the dopaminergic neuronal marker TH (1:1000; Sigma) and with neuronal neuclei marker NeuN (1:500; Millipore). SN pars compacta (SNpc) was demarcated based on TH-positive immunostaining as previously described (Baquet et al, 2009). Optical sections of four SNpc matched levels per animal were used for cell counting (McCormack et al, 2002).…”

“…The TH-positive neurons were only counted if the whole nucleus was visible within the section. SNpc neurons were distinguished from the ones of the ventral tegmental area based on size and orientation (Baquet et al, 2009). For microglia evaluation, TH immunostaining (1:1000; Millipore) was used for positioning on the slice, together with an antibody directed against the microglial marker ionized calcium binding adaptor molecule1 (Iba1; 1:1000; Wako).…”

The Colayer Method as an Efficient Way to Genetically Modify Mesencephalic Progenitor Cells Transplanted into 6-OHDA Rat Model of Parkinson's Disease

“…On the fourth day in vitro (DIV4) 7.8, tooth bar −2.4 and second 3 µl of 6-OHDA at AP cells were transfected (see below) and medium was −4, LAT −0.8, VERT −8, tooth bar +3. 4 or 400,000 cells per 6 well (for grafting experiments). On Drug-Induced Rotational Behavior DIV4 sister cultures were detached and total cell number estimated.…”