Ceyhan et al.
Supplementary Figure 1: Methodology for quantitative analysis of the neural
immunoreactivity for each antigen on section from normal human pancreas (NP), chronic
pancreatitis (CP) and pancreatic cancer (PCa). All nerves on a section were identified and
subsequently photographed with the help of a consecutive PGP9.5-stained section and a
consecutive hematoxylin-eosin-stained section. Then, on each photo, the nerves were defined
as regions of interest. After conversion of the RGB image to an 8-bit image, thresholding
function was used to define a phase with the help of a pre-set threshold, and the software
automatically determined the per cent area of this phase inside each nerve (Figure 1). This
percentage corresponded to the proportion of immunostained area within each nerve (“neuroimmunoreactivity“). From each patient, two sections which were at least 30μm apart and
contained around 100 nerves were analyzed, and the average immunoreactivity from a patient
(“Mean of the Patient”) was calculated by taking the mean of the neural immunoreactivities
on the two examined sections. Finally, the mean in each group (“the average neural
immunoreactivity for an antigen” in normal pancreas, chronic pancreatitis or pancreatic
cancer) was calculated by determining the mean from all patients from that group (Figure 1).
In nerves invaded by cancer cells, the area occupied by cancer cells was not included into the
region of interest.
Supplementary Figure 2: (A) Note the remarkable “network-like” Sox10-immunostaining
spread in the intralobular septules of normal pancreatic tissue. (B) Note the impressive Nestin
neural immunoreactivity and additionally prominent immunoreactivities in desmoplastic areas
of PCa (see arrows).
Supplementary Figure 3: Sox10 and Nestin neural immunorectivities in the whole neural
area are related to (A, C) abdominal pain sensation and (B, D) the severity of pain in patients
with chronic pancreatitis [CP; 944 nerves from 11 patients with no pain (Pain I) and 876
nerves from 9 patients with pain; 348 nerves from 3 Pain II patients and 438 nerves from 8
Pain III patients), or pancreatic cancer (PCa; 906 nerves from 8 patients with no pain and 802
Ceyhan et al.
nerves from 12 patients with pain; 110 nerves from 3 Pain II patients and 692 nerves from 9
Pain III patients).
Supplementary Figure 4. Quantitative analysis of the neural immunoreacitivties for TH (A),
ChAT (B), Sox10 (C) and Nestin (D) in a total of 5828 nerves from normal human pancreas
(NP, 2132 nerves), from 4 tissues with early-onset idiopathic chronic pancreatitis (Early ICP,
255 nerves), 3 tissues late-onset idiopathic chronic pancreatitis (Late ICP, 186 nerves) and
from 13 tissues with CP due other etiologies [CP (other), i.e. alcoholic, biliary or
autoimmune; 1382 nerves].