2.9. Stereological cell counting

To measure the effects of ANA-12 on TH-positive neuron survival, we calculated the number of TH-positive neurons in the LC (ROI outline: Figure 3A) and SN (ROI outline: Figure 4J) using unbiased, stereological cell counting [20,29]. Cell counts for the LC included the neurons on the lateral edges of the fourth ventricle using the laterodorsal tegmentum as the anterior border and the parvicellular portion of the medial vestibular nucleus as the posterior border. For the SN, counts excluded the SN pars reticulata and ventral tegmental area, so that only the SN pars compacta was counted. Images were taken using Stereoinvestigator software (MicroBrightField) and a BX-61 microscope (Olympus) fitted with an automated headstage for the optical fractionator counting method. A low magnification lense (10x) was used to outline the LC (every 3^rd section) and SN (every 6^th section). Cells were counted in a rostral to caudal manner at 20x with a systematic random sampling of dissector frames (100×100 μm) from 4-6 sections per animal. Average cells per counting frame were 3.50 cells per frame for the LC, and 1.65 cells per frame for the SN. The Stereoinvestigator software then computed an estimated population of cells for each animal.

Photomicrographs of LC (A-H, scale = 100 μm, quantified in I), measurement outline shown in A. TH-positive cells were significantly lower in the LC of lesion nonVNS vehicle rats compared to saline nonVNS vehicle rats (A, C, ****p < 0.0001). Saline vehicle treated rats had increased TH-positive cells after VNS (A-B, **p < 0.01), and lesion +VNS vehicle rats had greater TH-positive cells than lesion nonVNS vehicle rats (C–D, ****p < 0.0001). Administration of ANA-12 resulted in fewer TH-positive neurons for the lesion +VNS group (D, H, ****p < 0.0001), although numbers were still higher for the lesion +VNS ANA-12 rats compared to the lesion nonVNS ANA-12 rats (G-H, **p < 0.01). ANA-12 did not alter LC TH-positive neurons for the saline nonVNS, saline +VNS, or lesion nonVNS groups (A–C, E–G).