and 640-nm diode lasers. Full thickness, tiled, confocal image
stacks with a 2- to 3- mm interval in the Z-axis were obtained
through a 203dry lens (0.8 NA) with the confocal aperture set to 1
Airy unit or less. All image capture was performed using Zen Blue
Edition software (Carl Zeiss Microscopy GmbH, Jena, Germany),
and analyses were performed using Zen Blue or FIJI. 45
2.5. Image analysis
During all image quantification, the experimenter was blind to the
experimental groups. For quantification of the total number of cells
within the DRG, a modified optical dissector stereological method
was used 11,18,47 (Fig. S1, http://links.lww.com/PAIN/C84). To
account for tissue shrinkage duringprocessing, the mean thickness
(t) of each section on one slide (ie, 1 in 5 sections) was calculated by
taking the mean of the thickest and thinnest cell-containing regions
(ie, not fiber tract-containing regions) of the section (NB: no optical
correction to thickness was applied; given the use of a dry lens, this
value will not reflect actual section thickness, though this was kept
consistent throughout the study). The cell-containing, cross-
sectional area ( a) was then calculated, using the middle optical
section from the series and drawi ng around the cell-containing
regions. Section volume (Vsec) was then calculated:
Vsec ¼ t 3a
Using the Cavalieri principle, the cell-containing volume of the
DRG was calculated 11:
VDRG ¼ /C22a 3/C22t 3l
where /C22a 5 mean cell-containing cross-sectional area, /C22t 5
mean section thickness, and l 5“length” of the DRG (determined
from the total number of sections collected). The number of
neurons per section ( Nsec) was quantified in all immunostained
sections. This included only neurons with a visible nucleus (in the
NeuN channel), excluded cells with a nucleus visible within the
top frame of the Z-stack, and included any neurons with a nucleus
visible in any other field within Z-stack, including the bottom frame
of Z-stack. The cell density or the number of cells per unit vol (Nv)
was then calculated:
Nv ¼ Nsec
Vsec
Finally, the total number of cells per DRG ( NDRG) was
calculated:
NDRG ¼ Nv 3VDRG
For quantification of the proportion of FB-labelled cells co-
labelled with afferent subpopulation markers, initially, the total
number of FB-filled neuronal cell profiles with a visible nucleus
anywhere within the section was counted, with the observer blind
to other channels. The other channel was then revealed, and
instances of co-labelling were quantified. No stereological
correction was applied, given that the similar size of neuronal
nuclei would prevent over-counts of large neurons and that no
comparisons of the total number of labelled cells were made. For
soma area analyses, the area of neuronal soma expressing the
appropriate marker was measured in the optical section within
the Z-stack in which that neuron was at its largest, by drawing
around the perimeter of the neuron in Fiji/ImageJ v2.14.0/1.54f.
2.6. Tissue clearing and 3D volumetric analyses
Dorsal root ganglia were extracted from animals 4 weeks post-
SNItrans for whole DRG analyses. In this study, tissue was extracted
from a combination of MrgD CreERT2;Ai14, Th CreERT2;Ai14, and
CalcaCreERT2;Ai14 lines (mixed sex). 3 One month after SNI trans,
animals were transcardially perfused with sterile saline followed by
a fixative containing 4% formaldehyde. Ipsilateral and contralateral
L4 DRG were removed and postfixed for 24 hours on a shaker at
room temperature before being washed in PBS and stored
at 280˚C in CI-VM1 (35% dimethyl sulfoxide, 35% ethylene glycol
in PBS) until clearing. Tissue clearing was then performed as
previously described. 67 In brief, the tissue was exposed to
a gradient of 1-propanol containing 0.3% triethylamine (30, 50,
75, 90, 95, 100, 100%) and washed in this solution at 37˚C for
24 hours. The tissue was then rehydrated in PBS and labelled with
primary antibodies for 1 week at 37˚C (mouse anti-TDP43 and 2x