Original Article

Cephalalgia
2017, Vol. 37(1) 36–48
Sex-, stress-, and sympathetic ! International Headache Society 2016
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DOI: 10.1177/0333102416637832
cep.sagepub.com
identity and proportions of immune
cells in the dura

Lisa A McIlvried1,2, J Agustin Cruz3, Lisa A Borghesi3 and

Michael S Gold1,2,4,5

Abstract
Aim of investigation: Due to compelling evidence in support of links between sex, stress, sympathetic post-ganglionic
innervation, dural immune cells, and migraine, our aim was to characterize the impacts of these factors on the type and
proportion of immune cells in the dura.
Methods: Dural immune cells were obtained from naı̈ve or stressed adult male and female Sprague Dawley rats for flow
cytometry. Rats with surgical denervation of sympathetic post-ganglionic neurons of the dura were also studied.
Results: Immune cells comprise 17% of all cells in the dura. These included: macrophages/granulocytes (‘‘Macs’’; 63.2%
of immune cells), dendritic cells (0.88%), T-cells (4.51%), natural killer T-cells (0.51%), natural killer cells (3.08%), and
B-cells (20.0%). There were significantly more Macs and fewer B- and natural killer T-cells in the dura of females
compared with males. Macs and dendritic cells were significantly increased by stress in males, but not females. In
contrast, T-cells were significantly increased in females with a 24-hour delay following stress. Lastly, Macs, dendritic
cells, and T-cells were significantly higher in sympathectomized-naı̈ve males, but not females.
Conclusions: It may not only be possible, but necessary to use different strategies for the most effective treatment of
migraine in men and women.

Keywords
Headache, meninges, inflammation, autonomic
Date received: 30 March 2015; accepted: 2 February 2016

Introduction
Evidence suggests that a ‘‘sterile’’ inflammation of the
dura is responsible for the activation and sensitization
of dural afferents, which is thought to be responsible
for migraine pain (1–3). Dural mast cells (4) and macro- 1
Center for Neuroscience at the University of Pittsburgh, Pittsburgh, PA,
phages (5) may play critical roles in this process. While USA
2
the extent to which other immune cell types may con- The Pittsburgh Center for Pain Research, University of Pittsburgh,
tribute to migraine remains to be determined, the clin- Pittsburgh, PA, USA
3
Department of Immunology, School of Medicine, University of
ical manifestations of migraine suggest several
Pittsburgh, Pittsburgh, PA, USA
additional reasons to focus on dural immune cells as 4
Department of Anesthesiology, School of Medicine, University of
underlying contributors to this neurological disorder. Pittsburgh, Pittsburgh, PA, USA
5
First, the prevalence of migraine is approximately Department of Neurobiology, School of Medicine, University of
three-times higher in women than in men (6), and Pittsburgh, Pittsburgh, PA, USA
there are sex differences in immune cell number, pheno-
Corresponding author:
type, and regulation (7–10). Second, approximately Michael S Gold, E1440 Biomedical Science Tower, 3500 Terrace Street,
80% of migraineurs report stress as a trigger for a Pittsburgh, PA 15213, USA.
migraine attack and 60% report it as their main trigger Email: msg22@pitt.edu
McIlvried et al.
(11), with attacks often occurring during the relaxation
phase after stress (‘‘stress–relaxation’’ model) (12).
That time-dependent changes in the regulation of
immune cells could account for the delay in the
migraine attack after the resolution of the stressor is
suggested by the observation that there are time-
dependent shifts (13,14) and sexually dimorphic expres-
sion (15–18) of adrenergic receptors (ARs) on immune
cells, which regulate pro- versus anti-inflammatory
cytokine production (14,19). Third, there is evidence
of sympathetic dysregulation in migraineurs (20) and
evidence that a-AR agonists and b-AR antagonists
can decrease the frequency of migraine attacks (21).
Finally, the link between these clinical features of
migraine and immune cells is suggested by observations
that stress is associated with the activation of both the
hypothalamic–pituitary–adrenal and the sympatho–
adrenal axes, the dura is heavily innervated by sympa-
thetic post-ganglionic neurons (SPGNs) (22,23), and
immune cells are under AR regulation.
Despite this compelling evidence in support of a link
between dural immune cells and migraine, there have
been no systematic analyses of immune cells in the
dura, let alone studies to address sex differences and/
or stress-induced changes in these immune cells that
would be consistent with a role in migraine. In order
to begin to address this issue, we characterized the
immune cells in the dura of male and female rats, as
well as the impact of stress and SPGN innervation on
the proportion of different immune cell types
present. Our prediction was that stress would drive
delayed SPGN-dependent changes in immune cell sub-
types in females that would be consistent with the pen-
ultimate step prior to the initiation of an attack in
migraineurs.
Methods
Animals
Pathogen-free adult (P50–70) male (200–300 g) and
female (150–250 g) Sprague Dawley rats (Harlan
Sprague Dawley, Indianapolis, IN) were used for all
experiments. All procedures were approved by the
University of Pittsburgh Institutional Animal Care
and Use Committee and performed in accordance
with National Institutes of Health Guide for the Care
and Use of Laboratory Animals.
Details of animal care and methods of the chronic
variable stress (CVS) paradigm, surgical sympathec-
tomy (SCGx), analysis of plasma corticosterone
(CORT), isolation of cells from the dura, flow cytome-
try, fluorescence-activated cell sorting (FACS), cell hist-
ology, and immunohistochemistry (IHC) are provided
in supplemental text.
37
Chronic variable stress
Animals were exposed to CVS, consisting of an
ongoing series of ‘‘mild’’ stressors applied for variable
durations over a period of 4 days (Supplemental
Figure 1). Stressors included: food or water depriv-
ation, overnight illumination, cage tilt, paired housing
with a stranger, damp bedding, white noise, strobe
light, and predator odor (supplemental text).
It is important to note we are not proposing that
CVS is a model of migraine, nor do we anticipate
that this stress paradigm causes migraine in rats.
Rather, we have proposed to study changes in response
to stress because we hypothesize that these changes set
the stage for the initiation of an attack in migraineurs.
Data analysis
Four related a priori hypotheses based on the clinical
features of migraine were tested. First, there are more
immune cells in the dura of females than males. Second,
stress increases the relative proportion of dural immune
cells with a delay following the cessation of stress that is
greater in females than males. Thus, we examined the
influence of both sex and stress on the proportion of
immune cell subtypes in the dura by comparing naı̈ve
rats with rats exposed to 4 days of CVS, assessed either
immediately following stress (CVS þ 0 h) or with a
24-hour delay following stress (CVS þ 24 h) (Figure 1a).
The choice of a 24-hour delay was based on results from
preliminary experiments also including a 12-hour delay
group (n ¼ 4 for both males and females). No significant
differences were detected between the 12- and 0-hour
groups (data not shown), and thus the 12-hour delay
group was not pursued further.
The third hypothesis was that the proportion of
dural immune cells is dependent on SPGN innervation,
and is more pronounced in females than males.
Changes in the proportion of dural immune cells in
rats that underwent SCGx by removal of the superior
cervical ganglion bilaterally (Figure 1b) were assessed.
Lastly, given that stress drives activity in the SPGN,
our fourth hypothesis was that the stress-induced
increase in dural immune cells in females is dependent
on the presence of SPGN innervation.
Immune cells from each animal were quantified with
flow cytometry and analyzed as a percentage of total
live cells so that changes in one subtype of immune
cell could be determined independently of changes in
another cell type. The percentage of total live cells was
normalized to the respective male naı̈ve group in order
to facilitate comparisons between groups.
Data were analyzed with t-tests for sex effects (male
versus female), two-way analyses of variance (ANOVAs)
for sex and stress effects (naı̈ve, 0, 12 or 24 hours follow-
ing CVS) and sex and SPGN effects (intact versus SCGx)
38 Cephalalgia 37(1)

(a) (b) TH
CGRP

CONTRA
le

h
es
ab

on h
24
iv
ic

cti – 0
na
pl

–
ap

co tion
n
if

io
am

ue llec
ct

lle
100µm

lle
sh

Ti e co
co
or

ue
x

u
G

ss
ss
ss
STRESS
SC

Ti
Ti

IPSI
2 3 4 5
1 week DAYS
100µm

(c) Sex: p>0.05

(d) Sex: p>0.05
Stress: p<0.01 Stress: p<0.001
800 Sex x stress: p>0.05 12 Sex x stress: p>0.05
plasma CORT (ng/mL)

Male Male
10

Weight gain (g/day)

600 Female Female
8
6
400
4
200 2
0
0 –2
8 7 6 6 8 8 4 4 N 7 6 25 26 15 18 N
naive CVS CVS 20-30min naive CVS CVS
+0h +24h restraint +0h +24h
stress

Figure 1. Experimental design. (a) Intact and SCGx male and female rats were either considered ‘‘naı̈ve’’ or stressed with the ‘‘CVS’’
paradigm. Tissue was collected from CVS rats immediately after the last day of stress (‘‘CVS þ 0 h’’) as a measure of the response to
chronic stress or following a 24-hour delay after stress (‘‘CVS þ 24 h’’) as a measure of the response to stress–relaxation. (b) To test
the impact of sympathetic post-ganglionic neuron innervation, prior to stress, superior cervical ganglia were surgically removed
bilaterally (SCGx) in the groups as indicated. On the left, a representative whole-mount dura from an adult rat 7 days after a unilateral
SCGx is shown, probed with anti-CGRP (green) or anti-TH (red) antibodies. Note the complete loss of TH-like immunoreactivity (a
marker for sympathetic fibers) ipsilateral to the SCGx, but no change in CGRP-like immunoreactivity (a marker for peptidergic
afferents). (c) CVS is a mild stress paradigm. There was a significant (p < 0.01) main effect of stress on plasma levels of CORT in rats.
CORT was significantly (p < 0.01) higher in rats exposed to CVS (naı̈ve versus CVS þ 0 h), and this remained significantly (p < 0.01)
elevated with a delay after stress (naı̈ve versus CVS þ 24 h). There was no significant (p > 0.05) main effect of sex or interaction with
stress. Note that the CVS paradigm produced a significantly (p < 0.001) smaller increase in CORT compared with the potent, acute
restraint stress paradigm. (d) In addition, there was a significant (p < 0.001) main effect of stress on daily weight gain in rats. Rats
gained significantly (p < 0.05) less weight per day during CVS (naı̈ve versus CVS þ 0 h), which recovered (p < 0.001) during the
relaxation phase after stress (CVS þ 0 h versus CVS þ 24 h).
CGRP: calcitonin gene-related peptide; CORT: corticosterone; CVS: chronic variable stress; SCGx: surgical sympathectomy; TH:
tyrosine hydroxylase.

in naı̈ve animals, and three-way ANOVAs for sex, stress, that are present in the dura and validated these data
and SPGN effects, with p < 0.05 considered significant. with complementary morphology following single-cell
The Tukey HSD test was used for post-hoc comparisons sorting and IHC of intact dura. Third, we used flow
if significant interactions were detected. An analysis of cytometry to determine the impact of sex, stress, and
covariance was used in order to assess the effect of SPGN innervation on the relative proportions of dural
estrus cycle with uterus weight as an approximate meas- immune cell subtypes.
ure of cycle stage (24).
CORT and weight changes associated with the
Results CVS model
This study was completed in three parts. First, we con- CVS was associated with a small but significant
firmed that the CVS model was stressful. Second, we (p < 0.01) elevation of plasma CORT in both male
used flow cytometry to identify the immune cell types and female rats that persisted for 24 hours after stress
McIlvried et al. 39

was terminated (Figure 1c). Furthermore, weight gain monocytes. The NK cell population consisted of
in both males and females was halted (p < 0.01) during 51.3% large and 48.7% small lymphocytes, of low
the CVS exposure, but returned to normal over the granularity. The T-cell population consisted of 79.7%
24-hour period following CVS (Figure 1d). small and 20.3% large non-granular lymphocytes.
In order to exclude the possibility that immune cell
estimates were confounded by incomplete perfusion of
Immune cell types present in the dura the dural vasculature, as well as to determine the dis-
Flow cytometry, with the gating strategy illustrated in tribution of major immune cell populations detected
Figure 2, was used to characterize immune cell types. within the dura, Macs, NK cells, and T-cells were
Within CD45þ leukocytes, six types of immune cells assessed in situ with ED-2-, CD161a-, and CD3-like
were detected (Figure 3), as assessed using antibodies immunoreactivity (-LI), respectively (Supplemental
previously validated in the rat (25–28). In the naı̈ve Figure 2). Together, these observations indicate that
male dura, 63.2  0.9% of immune cells were CD11bþ there is a large and diverse immune cell population in
macrophages/monocytes, granulocytes, and mast cells the dura.
(‘‘Macs’’); 0.9  0.2% were CD11b/cþ/CD11b den-
dritic cells (DCs); 20.0  0.1% were CD45Rþ B-cells; Sex, stress, and sympathetic innervation influence
3.1  0.6% were CD161aþ natural killer (NK) cells;
4.5  0.4% were CD3þ T-cells; and 0.5  0.1% were
dural immune cell proportions
CD161aþ/CD3þ NK T (NKT) cells. An average of Immune cells in the dura. While there was no detectable
7.8  0.3% of immune cells were not specifically identi- difference between naı̈ve male (n ¼ 7) and female (n ¼ 6)
fied in our panel (‘‘unidentified’’). This flow cytometry rats in the proportion of total immune cells (CD45þ) in
strategy was validated by FACS for the major subpo- the dura (Figure 4a, ‘‘Intact, naı̈ve’’ groups), there was
pulations of immune cells, confirming their identity a significant (p < 0.05) interaction between sex and
with morphological and histological analysis. Diff- stress (Figure 4a, ‘‘Intact’’ groups). This was due to a
Quik staining (Figure 3, insets) confirmed that Macs stress-induced increase in dural immune cells present in
consisted of 89.1% macrophages/monocytes with a males, but not females, which was significantly greater
characteristic and pronounced granularity, 5.1% neu- (p < 0.05; n ¼ 6) immediately after stress (0 hours) and
trophils, 3.6% mast cells (further identified with tolui- maintained (p < 0.05; n ¼ 8) for 24 hours after stress.
dine blue stain), and 2% contamination by There was a significant (p < 0.05) interaction
lymphocytes. The B-cell population consisted of between sex and SPGN innervation in naı̈ve animals
93.0% small lymphocytes (10 mm) and 5.1% large regarding the proportion of immune cells in the dura
lymphocytes (>10 mm), both of which were non-granu- (Figure 4a, ‘‘Intact, naive’’ and ‘‘SCGx, naive’’ groups).
lar, with <2% contamination by macrophages/ This was due to the significant (p < 0.05) decrease in

105 104 B cells 105 NK cells NKT cells

Immune DCs Macs
104
CD11b/c PEcy7

CD45R FITC

CD161a PE
CD45 APCcy7

cells 10 4
10 4
Minus debris 103
Minus dead cells 103
10 3 103
Minus doublets
0 102 0
0 0
-103 “unidentified” T cells
-102
0 50K 100K150K 200K 250K 0 103 104 0 103 104 0 103 104
FSC-A CD11b V450 CD3 APC CD3 APC

Figure 2. Gating strategy used for the isolation of immune cell subtypes from the dura. The data shown are from a naı̈ve male rat.
On the day of the experiment, dural cells were dissociated and stained with antibodies specific to markers of immune cells and/or
distinct immune cell subtypes. The gating strategy illustrated was used for all flow and fluorescence-activated cell sorting experiments.
CD45þ immune cells were first selected, and then sorted into CD11bþ macrophages/granulocytes/mast cells (‘‘Macs’’) and CD11cþ
DCs. CD11b/c immune cells were further sorted into CD45Rþ B-cells. CD11b/c and CD45R immune cells were lastly sorted
into either CD161aþ NK cells, CD3þ T-cells, or CD161aþ/CD3þ NKT cells. A small subset of immune cells was left unstained by any
of the immune subtype antibodies that were utilized in this strategy (‘‘unidentified’’). Viability was uniformly 80–85%, as determined by
propidium iodide and trypan blue staining. This resulted in an average of 1.56  106  0.18  106 live cells recovered per dura. Spleen
cells were used for compensation controls (51,52). Images are from FlowJo software, contour plots were used with 5% levels and
outliers displayed.
DC: dendritic cell; NK: natural killer; FSC-A: forward scatter.
40 Cephalalgia 37(1)

% of CD45+ immune cells in the dura

(=17% of total dural cells)
CD11b+ Macs 63.2%
CD11c + DCs 0.9%
CD161a + NK cells 3.1%
CD11b+ Macs CD3 + T-cells 4.5%
Macrophages: 63.2% CD161a+/CD3+ NKT cells 0.5%
CD45R + B-cells 20.0%
M M MC unidentified 7.8%

N Unid
entifi
ed: 7
.8%

B-c
: 4. %
ells : 3.1
0.5% %
ce .9%

ells
5
NK s: 0
lls

:2
DC

0.0
NKT:
T-c

%
CD45R+ B-cells
CD161a+ NK cells

CD3+ T-cells

Figure 3. Proportion of immune cell subtypes in the naı̈ve male dura as determined with flow cytometry using the gating strategy
shown in Figure 2. Of the live cells recovered from the dura, an average of 16.9  0.90% were CD45þ, and thus determined to be
immune cells. The pie chart and legend show the relative proportions of the six immune cell subtypes identified in the dura. The insets
show fluorescence-activated cell sorted, cyto-centrifuged preparations of immune cell subtypes, subsequently stained with Diff-Quik.
Macs consisted of 89.1% macrophages/monocytes (M), 5.1% neutrophils (N), 3.6% mast cells (MC; identified with toluidine blue stain),
and 2.2% lymphocytes. The NK cell population consisted of 51.3% large lymphocytes (>10 mm) and 48.7% small lymphocytes
(10 mm). The T-cell population consisted of 79.7% small lymphocytes and 20.3% large lymphocytes. Lastly, the B-cell population
consisted of 93.0% small lymphocytes, 5.1% lymphocytes, and 1.9% macrophages/monocytes. Scale bars: 10 mm.
DC: dendritic cell; Macs: macrophages/granulocytes; NK: natural killer.

immune cells with SCGx in naı̈ve females (n ¼ 6),
although this may have been a surgery effect, as it
was also seen in sham-operated rats (Figure 4b).
There was also a significant (p < 0.05) increase in
immune cells with SCGx in naı̈ve males (n ¼ 6), which
was likely due to SPGN effects, as it was not seen in
sham animals. Folding all additional groups into the
analysis revealed a significant (p < 0.05) interaction
between sex, stress, and SPGN innervation on dural
immune cells (Figure 4a). It is important to note, how-
ever, that the loss of the stress effect in males with
SCGx may have been a surgery effect based on sham
animals (Figure 4b).
Myeloid-derived dural immune cell subtypes
Macrophages/monocytes, granulocytes, and mast
cells. The proportion of Macs was significantly
(p < 0.05) higher in the dura from intact naı̈ve females
than intact naı̈ve males. There was also a significant
(p < 0.05) interaction between sex and stress on the pro-
portion of Macs in the dura (Figure 5a). This was due
to a stress-induced increase in Macs present in males,
but not females, which was significantly greater
immediately after stress (p < 0.05) and maintained
with a 24-hour delay after stress (p < 0.01) compared
with naı̈ve rats. In addition, there was a significant
(p < 0.05) interaction between sex and SPGN innerv-
ation in naı̈ve rats on the proportion of Macs in the
dura (Figure 5a). The increase in Macs in naı̈ve SCGx
males was significant (p < 0.05), while there was only a
trend (p ¼ 0.056) toward a decrease in Macs in naı̈ve
SCGx females. Notably, while the trend in the decrease
in naı̈ve SCGx females was observed in the sham sur-
gery group, the significant increase in Macs in naı̈ve
SCGx males was not, and is therefore likely due to
SPGN effects (Figure 5b). Lastly, there was a signifi-
cant (p < 0.05) interaction between sex, stress, and
SPGN innervation regarding Macs in the dura
(Figure 5a). However, the loss of a stress effect with
SCGx was also seen in sham-operated rats
(Figure 5b), and may be due to surgery effects.
Dendritic cells. There was a significant (p < 0.05)
interaction between sex and stress on the proportion of
dural DCs (Figure 5c). This was due to a stress-induced
increase in DCs present in males, but not females, which
was significant (p < 0.001) immediately after stress
McIlvried et al. 41

IMMUNE CELLS
H2 Sex x Stress: p<0.05
H3 Sex x SPGN (naïve): p<0.05
(a) H4 Sex x Stress x SPGN: p<0.05 (b)
80 Male 80
Female
*
% change of CD45+ cells to
60 60

* *
40 40
naïve males

20 20

0 0

-20 -20

-40 -40
7 6 6 4 7 8 6 6 6 4 6 6 N 6 6 4 4 4 4 N
-60 naive CVS CVS -60 naive CVS CVS
naive CVS CVS
+0h +24h +0h +24h +0h +24h

INTACT SCGx SHAM

Figure 4. Sex-, stress-, and SPGN-dependent changes in the percentage of CD45þ immune cells in the dura (relative to the total
number of live cells recovered). Data are presented as percentages of naı̈ve males in order to facilitate comparisons between groups.
(a) There was a significant interaction between sex and stress regarding the proportion of live immune cells in the dura (p < 0.05)
(‘‘Intact’’ groups). In addition, there was a significant (p < 0.05) interaction between sex and SPGN innervation in naı̈ve animals
regarding the proportions of immune cells in the dura (‘‘Intact, naı̈ve’’ and ‘‘SCGx, naı̈ve’’ groups). Lastly, there was an interaction
between sex, stress, and SPGN innervation (p < 0.05) regarding the proportion of immune cells in the dura. (b) Similar to male SCGx
groups, male sham-operated rats also showed the loss of a stress-induced increase in immune cells.
H1 ¼ a priori hypothesis 1: sex difference in ‘‘Intact, naı̈ve’’ groups, analyzed with a t-test; H2 ¼ a priori hypothesis 2: sex  stress
comparison in ‘‘Intact’’ groups, analyzed with a two-way analysis of variance (ANOVA); H3 ¼ a priori hypothesis 3: sex  SCGx in
‘‘Naı̈ve’’ groups (i.e. ‘‘Intact’’ versus ‘‘SCGx’’ naı̈ve), analyzed with a two-way ANOVA; H4 ¼ a priori hypothesis 4: sex  stress  SCGx
interaction between all groups, analyzed with a three-way ANOVA.
*p < 0.05.
CVS: chronic variable stress; SCGx: surgical sympathectomy; SPGN: sympathetic post-ganglionic neuron.

(Figure 5c). There was also a significantly (p < 0.05)
higher proportion of DCs in naı̈ve males than females
following SCGx (Figure 5c), which was not an effect of
surgery (Figure 5d). Lastly, there was a significant
(p < 0.05) interaction between sex, stress, and SCGx
regarding the proportion of dural DCs (Figure 5c). The
stress-induced increase in DCs in SCGx females was sig-
nificant (p < 0.001) immediately after stress and was also
not an effect of surgery, although the loss of the stress-
induced increase at 0 hours in SCGx male rats may have
been. Interestingly, in sham rats, the stress-induced
increase in DCs was only significant with a 24-hour
delay following stress (Figure 5d).
Lymphoid-derived dural immune cell subtypes
T-cells. There was a significant (p < 0.05) interaction
between sex and stress (Figure 6a) regarding the pro-
portion of dural T-cells. This was due to a stress-
induced increase in T-cells that was present in females,
but not males, which was significantly greater 24 hours
after stress than immediately after stress (p < 0.05) or in
the naı̈ve rats (p < 0.01). In addition, there was a sig-
nificant main effect (p < 0.05) of SPGN innervation
(Figure 6a) due to the larger proportion of T-cells in
the dura for SCGx than intact rats. Lastly, the sex and
stress interaction and SPGN main effect persisted with
all groups folded into the analysis. However, the overall
increase in T-cells with SCGx was also seen in sham-
operated rats (Figure 6b) and may be due to surgery
effects.
NKT cells. The variability was large for the minor
subgroup of T-cells – NKT cells – likely due to the over-
all low number of NKT cells in the dura. Nevertheless,
there was a significant (p < 0.05) main effect of sex on
the proportion of NKT cells in the ‘‘Intact’’ groups
(Figure 6c). This was due to a smaller proportion of
NKT cells in females than in males. This overall pattern
was maintained in SCGx rats, and as a result, SPGN
innervation had no significant effect on NKT cells.
NK cells. The variability was considerably larger for
NK cells than any other immune cell type studied.
42 Cephalalgia 37(1)

MACS
(a) (b)
H1 Sex: p <0.05
H2 Sex x Stress: p <0.05
H3 Sex x SPGN (naïve): p <0.05
H4 Sex x Stress x SPGN: p <0.05

Male
80
* Female
**
% change of CD11b+ cells to

60 60
*
40 * 40
naïve males

20 20

0 0

-20 -20

-40 -40

-60 -60
7 6 6 4 7 8 6 6 6 4 6 6 N 6 6 4 4 4 4 N
naive CVS CVS naive CVS CVS naive CVS CVS
+0h +24h +0h +24h +0h +24h

INTACT SCGx SHAM

DCs
(c) (d)
H2 Sex x Stress: p<0.05
H3 Sex x SPGN (naïve): Sex = p <0.05
H4 Sex x Stress x SPGN: p <0.05

**
120 ** 120
100
% change of CD11c+ cells to

100
80 80
naïve males

60 60
40 40
20 20
0 0
-20 -20
-40 -40
7 6 6 4 7 8 6 6 6 4 6 6 N 6 6 4 4 4 4 N
naive CVS CVS naive CVS CVS naive CVS CVS
+0h +24h +0h +24h +0h +24h

INTACT SCGx SHAM

Figure 5. Sex-, stress-, and SPGN-dependent changes in the percentage of myeloid-derived immune cells dissociated from the dura
(relative to the total number of live cells from each dura). Data are presented as percentages of naı̈ve males for comparison. (a) The
proportion of Macs was significantly (p < 0.05) higher in the dura from intact naı̈ve females than males. There was a significant
(p < 0.05) interaction between sex and stress regarding the proportion of Macs in the dura (‘‘Intact’’ groups). There was also a
significant (p < 0.05) interaction between sex and SPGN innervation in naı̈ve rats regarding the proportion of Macs in the dura (‘‘Intact,
naı̈ve’’ and ‘‘SCGx, naı̈ve’’ groups). Lastly, there was an interaction between sex, stress, and SPGN innervation (p < 0.05). (b) Similar to
male SCGx groups, male sham-operated rats also showed the loss of a stress-induced increase in Macs. (c) There was a significant
(p < 0.05) interaction between sex and stress on the proportion of dural DCs (‘‘Intact’’ groups). In addition, there was a significant
(p < 0.05) interaction between sex, stress, and SCGx. (d) Interestingly, in sham rats, the stress-induced increase in DCs was only seen
with a delay following stress. The groups and a priori hypotheses (H1–4) tested were the same as those in Figure 4.
*p < 0.05; **p < 0.01.
CVS: chronic variable stress; DC: dendritic cell; Macs: macrophages/granulocytes; SCGx: surgical sympathectomy; SPGN: sympathetic
post-ganglionic neuron.
McIlvried et al. 43

T- CELLS
(a) (b)
H2 Sex x Stress: p<0.05
H3 Sex x SPGN (naïve): SPGN= p<0.05
H4 Sex x Stress x SPGN: Sex x stress=
p<0.05 & SPGN= p<0.05
Male
Female
% change of CD3+ cells to 140 140
120 * 120
**
100 * 100
naïve males

**
80 80
60 60
40 40
20 20
0 0

7 6 6 4 7 8 6 6 6 4 6 6 N 6 6 4 4 4 4 N
naive CVS CVS naive CVS CVS naive CVS CVS
+0h +24h +0h +24h +0h +24h

INTACT SCGx SHAM

NKT CELLS
(c) H2 Sex x Stress: Sex= p<0.05 (d)
H3 Sex x SPGN (naïve): Sex= p<0.05
H4 Sex x Stress x SPGN: Sex= p<0.05

200 200
% change of CD3+/CD161a+

150 150
cells to naïve males

100 100

50 50

0 0

–50 –50
7 6 6 4 7 8 6 6 6 4 6 6 N 6 6 4 4 4 4 N
naive CVS CVS naive CVS CVS naive CVS CVS
+0h +24h +0h +24h +0h +24h

INTACT SCGx SHAM

Figure 6. Sex-, stress-, and SPGN-dependent changes in the percentage of lymphoid-derived immune cells dissociated from the dura
(relative to the total number of live cells from each dura). Data are presented as percentages of naı̈ve males for comparison. (a) There
was a significant (p < 0.05) interaction between sex and stress regarding the relative proportions of T-cells (‘‘Intact’’ groups). In
addition, there was a significant main effect (p < 0.05) of SPGN innervation in naı̈ve rats. Lastly, the sex and stress interaction and
SPGN main effect persisted with all groups folded into the analysis. (b) Comparable stress-induced changes in T-cells were observed in
sham surgery groups and in the SCGx groups. (c) There was a main effect of sex (p < 0.05) on the proportion of NKT cells. (d) Sham-
operated groups showed a stress effect in males, which may have been masked by SCGx. (e) There was a significant (p < 0.05) sex and
stress interaction when all groups were folded into the analysis of NK cells. (f) Sham surgery did not appear to affect NK cells. (g) The
proportion of B-cells was significantly (p < 0.001) lower in the dura from intact naı̈ve females than males, and this sex difference
persisted as a main effect (p < 0.05) after folding stress groups into the analysis (‘‘Intact’’ groups). In addition, when all groups were
folded in, there was a significant (p < 0.05) main effect of SPGN innervation. (h) Sham surgery did not appear to affect B-cells. The
groups and a priori hypotheses (H1–4) tested were the same as those in Figure 4.
*p < 0.05; **p < 0.01.
CVS: chronic variable stress; DC: dendritic cell; NK: natural killer; SCGx: surgical sympathectomy; SPGN: sympathetic post-ganglionic
neuron.
44 Cephalalgia 37(1)

NK CELLS
(e) (f)
H4 Sex x Stress x SPGN: Sex x stress= p<0.05
60 60

40 40

% change of CD161a+ cells 20 20

to naïve males
0 0

–20 –20

–40 –40

–60 –60

–80 –80
7 6 6 4 7 8 6 6 6 4 6 6 N 6 6 4 4 4 4 N
naive CVS CVS naive CVS CVS naive CVS CVS
+0h +24h +0h +24h +0h +24h

INTACT SCGx SHAM

B- CELLS
(g) (h)
H1 Sex: p<0.05
H2 Sex x Stress: Sex= p<0.05
H3 Sex x SPGN (naïve): Sex= p<0.05
H4 Sex x Stress x SPGN: Sex= p<0.05 &
SPGN= p<0.05
80 80
% change of CD45R+ cells to

60 60

40 40
naïve males

20 20

0 0

–20 –20

–40 –40
**
–60 –60
7 6 6 4 7 8 6 6 6 4 6 6 N 6 6 4 4 4 4 N
naive CVS CVS naive CVS CVS naive CVS CVS
+0h +24h +0h +24h +0h +24h

INTACT SCGx SHAM

Figure 6. Continued.

Nevertheless, we were still able to detect a significant ‘‘Unidentified’’ dural immune cell subtypes. When all groups
(p < 0.05) sex by stress interaction when all groups were were folded into the analysis, the proportion of
folded into the analysis (Figure 6e). This was due to a ‘‘unidentified’’ immune cells in females was significantly
significant (p < 0.001) increase in NK cells immediately (p < 0.05) greater than in males (Figure 7a).
after stress in females. There were no SCGx effects.
Discussion
B-cells. The relative proportion of B-cells was signifi-
cantly (p < 0.05) lower in the dura from intact naı̈ve The purposes of this study were twofold: 1) to charac-
females than males (Figure 6g). This sex difference per- terize the identity and proportions of immune cells pre-
sisted as a main effect (p < 0.05) after folding stress sent in the dura; and 2) to determine how the
groups into the analysis (Figure 6g). In addition, proportions of immune cells in the dura were influenced
when folding in all groups, there was a significant by sex, stress, and/or SPGN innervation.
(p < 0.05) main effect of SPGN innervation A combination of flow cytometry, IHC and FACS of
(Figure 6g). This was not likely due to surgery effects, rat dura indicated that 17% of the total cells in the
as the decrease in the proportion of B-cells across all dura were immune cells, generally comparable to the
SCGx groups was not detected in the sham surgery colon (20% in our preliminary studies; data not
groups (Figure 6h). shown), a highly immune-competent tissue (29,30).
McIlvried et al. 45

UNIDENTIFIED IMMUNE CELLS

H4 Sex x Stress x SPGN: Sex= p<0.05
(a) 80 Male (b)
80
Female
% change of unidentified 60 60
cells to naïve males
40 40

20 20

0 0

–20 –20

– 40 –40

–60 –60
7 6 6 4 7 8 6 6 6 4 6 6 N 6 6 4 4 4 4 N
naive CVS CVS naive CVS CVS naive CVS CVS
+0h +24h +0h +24h +0h +24h

INTACT SCGx SHAM

Figure 7. Sex-, stress-, and SPGN-dependent changes in the percentage of unidentified immune cells dissociated from the dura
(relative to the total number of live cells from each dura). Data are presented as percentages of naı̈ve males for comparison. (a) With
all groups folded into the analysis, there was a main effect of sex (p < 0.05) on the proportion of ‘‘unidentified’’ immune cells. (b) Sham
surgery did not appear to affect ‘‘unidentified’’ cells. The groups and a priori hypothesis (H4) tested were the same as those in Figure 4.
CVS: chronic variable stress; SCGx: surgical sympathectomy; SPGN: sympathetic post-ganglionic neuron.

Dural immune cells were composed of at least six
subtypes: myeloid-derived Macs and DCs and lym-
phoid-derived T-, NKT, NK, and B-cells, identified
by lineage-specific cell surface markers, morphology,
and histology. These six immune cell types accounted
for 92% of the total dural immune cells. The small
subset of ‘‘unidentified’’ immune cells may have been
comprised of CD11b DCs, CD11b mast cells, and/or
immune cell progenitors that had not yet differentiated/
matured into peripheral tissue leukocytes. Having con-
firmed that CVS produced a stress response, we found
that the relative proportions of these immune cell types
were influenced sex, stress, and SPGN innervation.
There were significantly more myeloid-derived Macs
and less lymphoid-derived B- and NKT cells in the
dura of females compared with males. Both Macs and
DCs were increased by stress in males but not females,
and this increase was maintained for at least 24 hours
following stress for the Macs. In contrast, lymphoid-
derived T-cells were significantly increased in females
with a 24-hour delay following stress.
Previous data indicate that the dura is enriched with
resident myeloid-derived mast cells (4,31–33) and
macrophages (5,34–36). Our observations extend these
previous results in three important ways. First, we
quantified the relative proportion of resident immune
cells in the dura. Second, we described the impacts of
sex, stress, and SPGN innervation on the proportions
of these cells. Third, our results clearly indicate that in
addition to the classical resident immune cells, immune
cells traditionally described as ‘‘recruited’’ are also pre-
sent. Previously, lymphoid-derived immune cells have
been described in the dura under pathological condi-
tions such as meningitis (37–39) or tumors (40), but
only recently also under ‘‘naı̈ve’’ conditions (41).
Because we minimized vascular contamination of
immune cells by removing blood prior to tissue collec-
tion and demonstrated the presence of NK and T-cells
in situ with IHC, our results strongly argue against the
possibility that the presence of lymphoid-derived
immune cells in the dura was an artifact.
Although it is still unknown whether the immune cell
subtypes vary within regions of the dura, the localiza-
tion of both lymphoid- and myeloid-derived immune
cells near the vasculature and large nerve bundles (not
shown) would enable these cells to play a role in the
initiation and the resolution of migraine pain. We sug-
gest that this is true for all the immune cell types
detected, despite the relatively small numbers in which
some types were present. Indeed, mast cell degranula-
tion drives dural afferent activity (4), despite the fact
that this subpopulation of immune cells appears to rep-
resent only 2.4% of immune cells in the dura, as esti-
mated by the results of our cytological analysis and
toluidine blue staining (Figure 3).
While performed as a control, the results from the
sham surgery groups were interesting in several
respects. The response to sham surgery was dependent
on immune cell type. If present, sham surgery was
associated with a decrease in the proportion of
46 Cephalalgia 37(1)

myeloid-derived subtypes and an increase in lymphoid-
derived subtypes. There are clear examples of a stressor
sensitizing or priming the immune system response
to a subsequent stress (42,43), and this may explain
the enhanced responses in the lymphoid-derived
immune cell subtypes with sham surgery. However,
surgery as a stressor is generally immunosuppressive
for less than 1 week (44–47). That any effect would
last for 12 days in the sham groups was unexpected.
We acknowledge the possibility of other compensatory
effects due to SCGx, although we (Figure 1b) and
others (48) have not detected any surgery-induced
changes in calcitonin gene-related peptide IHC. While
the relative impact of SPGN innervation on stress-
induced changes in dural immune cells was con-
founded by the influence of surgery, per se, there were
still cases of marked differences between SCGx and
sham surgery groups, implicating a role of SPGN in
the regulation of dural immune cells. This was most
clearly evident in the proportion of Macs, DCs, and
T-cells, which were significantly higher in SCGx-naı̈ve
males but not females.
Our observations suggest several new areas of investi-
gation concerning the mechanisms underlying the
impacts of sex, stress, and SPGN innervation on
immune cells in the dura. Given the evidence of gonadal
hormone regulation of immune cells (7,8,49), it will be
important to determine the extent to which the regulation
of immune cells in the dura is dependent on gonadal hor-
mones. In order to begin to address this issue, we analyzed
uterus weights as an indirect measure of cycling in the rats
(24). Although a subtype of migraine is correlated with
the menstrual cycle in humans, we did not find correl-
ations between any immune cell subtypes and the uterus
weights of rats, suggesting that hormone fluctuations
associated with the estrus cycle in female rats were not
likely to be responsible for the sex differences observed in
the dura. Second, with the additional evidence of adren-
ergic regulation of immune cells, it will be important to
determine whether norepinephrine is the mediator that is
primarily responsible for the impact of SPGN innervation
on immune cells. However, given our evidence of several
immune cell types being influenced by stress but not
SPGN innervation or in which there was an interaction
between stress and SPGN innervation, additional medi-
ators are likely to contribute to the regulation of dural
immune cells. For example, mediators released from the
primary afferents themselves are also likely to play a role.
Third, the full implications of the dynamic regula-
tion of dural immune cells will ultimately require func-
tional analysis. In order to begin to address this issue,
we recently reported (50) that there was a delayed
stress-induced shift from anti- to pro-inflammatory
mediator expression in female dural myeloid-derived
cells. This is suggestive of a shift to an M1 phenotype
in females. There was also a delayed stress-induced
increase in pro- and anti-inflammatory mediator
expression in female dural lymphoid-derived cells. In
combination with the changes in immune cell propor-
tion described in the present study, these observations
are consistent with changes that may set the stage for a
migraine attack. As we did not propose to create a
model of migraine or to have given a migraine to the
rats in this study, it will be important to determine
whether and how these changes in immune cells are
engaged by additional factor(s) that must be necessary
to tip the balance either towards the initiation of an
attack or towards the normalization of immune cells
in the dura in the absence of an attack.
Nevertheless, the changes observed in this study
have at least two important implications. First, the
observation that there is a sex difference in the
stress-induced increase in dural immune cells suggests
that it may not only be possible, but necessary to use
different strategies for the most effective treatment of
migraine in men and women. Second, our observa-
tions provide the first evidence implicating T-cells in
migraine pathogenesis. We suggest this because: 1)
migraine is associated with sterile inflammation of
the dura; 2) stress can trigger a migraine attack, but
only after a delay following the termination of the
stress; and 3) migraine is more prevalent in females.
Our observation that stress can drive an increase in T-
cells in the dura, but only after a delay and only in
females, indicates that there may be an association
between T-cells and migraine.

Article highlights
. The composition of immune cells in the dura in the absence of pathology is far more complex than originally
appreciated.
. The relative distribution of immune cell types within the dura is influenced by sex, stress, and sympathetic
post-ganglionic neuron innervation, as well as a significant interaction between these three factors.
. The presence of a sex difference in the stress-induced increase in dural immune cells suggests that it may not
only be possible, but necessary to use different strategies for the most effective treatment of migraine in men
and women.
. T-cells may not only account for the sex difference in the manifestation of migraine, but also the delay in the
initiation of a migraine attack following a period of stress.
McIlvried et al.
Acknowledgements
We thank the Unified Flow Core facilities at the University of
Pittsburgh and Dewayne Falkner and Ailing Liu for their
assistance with flow cytometry and FACS.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The authors declared the following potential conflicts of inter-
est with respect to the research, authorship, and/or publica-
tion of this article: NIH grants R01 NS083347 (MSG) and
R01 AI079047 (LAB).
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