PAINÒ 147 (2009) 287–298

www.elsevier.com/locate/pain

Clinical note

Absence of pain with hyperhidrosis: A new syndrome where vascular afferents

may mediate cutaneous sensation
David Bowsher a, C. Geoffrey Woods b, Adeline K. Nicholas b, Ofelia M. Carvalho b, Carol E. Haggett c,
Brian Tedman d, James M. Mackenzie e, Daniel Crooks d, Nasir Mahmood f, J. Aidan Twomey g,
Samantha Hann h, Dilwyn Jones i, James P. Wymer j, Phillip J. Albrecht k,m, Charles E. Argoff l,
Frank L. Rice k,m,*
a
Pain Research Institute, Liverpool L9 7AL, UK
b
Department of Medical Genetics, Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge CB2 0XY, UK
c
North Wales Probation Service, Wrexham LL13 7YX, UK
d
Neurophysiology and Neuropathology Departments, Walton Centre for Neurology and Neurosurgery NHS Trust, Liverpool L9 7LJ, UK
e
Department of Pathology, Aberdeen Royal Infirmary, Foresterhill, Aberdeen AB25 2ZD, UK
f
Department of Urology, Armed Forces Hospital Al-Hada, Taif, Saudi Arabia
g
Department of Clinical Neurophysiology, Pindersfields Hospital, Wakefield WF1 4DG, UK
h
The Welsh Institute of Dermatology, University Hospital of Wales, Cardiff CF14 4XW, UK
i
Biochemistry Department, Bronglais Hospital, Aberystwyth SY23 1ER, UK
j
Upstate Neurology Consultants, Albany, NY 12205, USA
k
Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY 12208, USA
l
Department of Neurology, Albany Medical College, Albany, NY 12208, USA
m
Integrated Tissue Dynamics, LLC, Renssalaer, NY 12144, USA

a r t i c l e i n f o a b s t r a c t

Article history: Congenital absence of pain perception is a rare phenotype. Here we report two unrelated adult individ-
Received 10 February 2009 uals who have a previously unreported neuropathy consisting of congenital absence of pain with hyper-
Received in revised form 1 September 2009 hidrosis (CAPH). Both subjects had normal intelligence and productive lives despite failure to experience
Accepted 9 September 2009
pain due to broken bones, severe cold or burns. Functional assessments revealed that both are generally
hypesthetic with thresholds greater than two standard deviations above normal for a several of modal-
ities in addition to noxious stimuli. Sweating was 3 to 8-fold greater than normal. Sural nerve biopsy
Keywords:
showed that all types of myelinated and unmyelinated fibers were severely reduced. Extensive multi-
Cholinergic vascular innervation
Congenital analgesia
antibody immunofluorescence analyses were conducted on several skin biopsies from the hands and back
Congenital hypesthesia of one CAPH subject and two normal subjects. The CAPH subject had all normal types of immunochem-
Cutaneous innervation ically and morphologically distinct sensory and autonomic innervation to the vasculature and sweat
glands, including a previously unknown cholinergic arterial innervation. Virtually all other types of nor-
mal cutaneous C, Ad and Ab-fiber endings were absent. This subject had no mutations in the genes SCN9A,
SCN10A, SCN11A, NGFB, TRKA, NRTN and GFRA2. Our findings suggest three hypotheses: (1) that develop-
ment or maintenance of sensory innervation to cutaneous vasculature and sweat glands may be under
separate genetic control from that of all other cutaneous sensory innervation, (2) the latter innervation
is preferentially vulnerable to some environmental factor, and (3) vascular and sweat gland afferents
may contribute to conscious cutaneous perception.
Ó 2009 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.

1. Introduction Type IV (HSAN IV) [11]. This condition is characterized by impaired

Congenital absence of, or insensitivity to, pain has been re-
ported mostly as individual case studies. Although rare, the most
reported form is hereditary sensory and autonomic neuropathy
* Corresponding author. Address: Integrated Tissue Dynamics LLC, 7 University
Place, Rensselaer, NY 12144, USA. Tel.: +1 518 505 7429.
pain and temperature perception and anhydrosis, but spared
vibratory sensation [3,47]. There is a severe reduction of unmyeli-
nated fibers in peripheral nerves, deficient C and Ad fiber endings
in the epidermis, and absent or hypoplastic denervated dermal
sweat glands [38]. These deficits can be due to an autosomal
recessive mutation of the genes for Nerve Growth Factor or its high
affinity tyrosine kinase receptor, TRKA [22,24].

0304-3959/$36.00 Ó 2009 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.
doi:10.1016/j.pain.2009.09.007
288 D. Bowsher et al. / PAINÒ 147 (2009) 287–298
Channelopathy-associated insensitivity to pain (CIP) is another
autosomal recessive disorder characterized by a congenital indif-
ference to pain but otherwise normal sensory responses [8]. The
nerves appear to be morphologically normal but the small caliber
fibers are functionally impaired due to a mutation in the SCN9A
gene. This gene encodes the voltage-gated sodium channel
Nav1.7 that is normally expressed at high levels particularly in
nociceptive small-diameter neurons in dorsal root ganglia
[29,48,52]. CIP can be distinguished from another pain impairment
condition, HSAN V, where afflicted subjects experience decreased
sweating and are anosmic [1,8,20].
Herein, we report two unrelated individuals, living productive
fairly normal lives, who were diagnosed with congenital analgesia
together with hyperhidrosis (CAPH). One subject was born in 1923,
had a life history of symptoms that were formally diagnosed clini-
cally in 1991 with additional benefit of a sural nerve biopsy, but died
without further genetic or skin biopsy analyses. The second subject
was born in 1968 and has been extensively evaluated clinically since
the late 1980s. This subject donated skin biopsies and blood samples
for an extensive evaluation of his cutaneous innervation and for tests
for known forms of genetic neuropathies. This report was prompted
by the unusual nature of his cutaneous innervation in which all
known types of sensory endings are absent except those affiliated
with his cutaneous blood vessels. Presumably, these remaining vas-
cular afferents may account for existing, albeit hypesthetic sensa-
tion. This subject also had intact autonomic innervation, which
included a previously unknown, normal cholinergic innervation to
small cutaneous arteries and arteriovenous shunts.
2. Methods
2.1. Clinical evaluations
Two adult males humans were diagnosed with CAPH as assessed
by subject histories, by thresholds for somatosensory perception on
the thenar eminence and dorsum of the foot and by sweating at rest
(Table 1). The assessments included: touch (von Frey filaments),
sharpness discrimination of weighted needles [7], skinfold pinch
(strain-gauge coupled forceps), thermal sensations [14] and vibra-
tion (Reidel–Seiffert tuning fork). To test autonomic functions, elec-
tronic skin thermometers and laser Doppler sensors were used to
assess skin temperature and blood flux before and after application
of an ice-pack to the back of the neck for 30 s (Table 1). Sweat produc-
tion was measured using weighed filter papers or the ServoMedR
evaporimeter. Data were compared to those in our data bank of indi-
viduals with no known sensory or autonomic disturbance of the
limbs. Nerve conduction studies and sural nerve biopsies analysed
by electron microscopy (Table 2) were performed according to stan-
dard protocols [28]. Due to the fact that one of the subjects was
examined very thoroughly on a number of different occasions while
the other was only seen briefly on a single occasion, not all tests were
carried out on both subjects at all sites.
2.2. Skin biopsy assessments
Cutaneous innervation in CAPH Subject 2 was assessed by
immunofluorescence in six 3 mm punch biopsies taken under local
lidocaine anesthesia: 1 from the hypothenar glabrous skin of each
hand, 1 from the ulnar side of each distal ventral forearm (about
2 cm proximal to the wrist), and 2 from the lower back. For
comparison, two normal subjects (males 45 and 53 years old) each
supplied two 3 mm punch biopsies from the hypothenar compart-
ment: 1 from the glabrous skin and 1 from the dorsal hairy skin. A
third normal subject (male 48 years old) supplied a biopsy from
the same location on the distal forearm as CAPH Subject 2. Each
biopsy was fixed by immersion for 4 h in 4% paraformaldehyde
in 0.1 M phosphate buffered saline (PBS) at pH7.4 and 4 °C. Biop-
sies were rinsed and stored in cold PBS, cryoprotected by immer-
sion in 30% sucrose in PBS and sectioned by cryostat.
Serial sections 14 lm thick were consecutively rotated and
thawed onto ten slides, so that approximately 15–20 sections taken
at equal intervals throughout each biopsy were obtained per slide.
One slide of sections was processed only with antibodies against
PGP9.5. Other slides were processed with various double combina-
tions of antibodies that discriminate specific subsets of sensory and
autonomic innervation (Table 3). Secondary antibodies were don-
key or goat made against the appropriate species of primary anti-
bodies and conjugated either with Cy3 (1:500 dilution, Jackson
ImmunoResearch Laboratories, West Grove PA) for red immunoflu-
orescence, or Alexa 488 (1:250–500, Molecular Probes Inc., Eugene
OR) for green immunofluorescence. Detailed methods are described
in Paré et al. [40]. The sections were also counterstained with DAPI
to facilitate the evaluation of the skin integrity. Every section on
each slide was evaluated for all antibody combinations.
2.3. DNA analysis
DNA was extracted from a peripheral blood sample by standard
methods and genes were bi-directionally sequenced as previously
described [8].
3. Results
3.1. CAPH subject histories
CAPH Subject 1 was a Caucasian male born in England in 1923,
and was the youngest of five siblings. He had three adult children
and three grandchildren. His parents, brothers, sisters, children,
and grandchildren are all reported to be able, or to have been able,
to feel pain normally. The subject himself said he has ‘‘never been
able to feel pain” for as long as he could remember and was also un-
able to appreciate water temperature. He reported experiencing
skeletal fractures and skin burns without pain. At age 62 he suffered
from hypothermia while working up to his waist in cold water, but
he was unaware of any discomfort when in the water. Both he and
his wife stated that he sweats excessively. General and neurological
examination revealed no motor abnormalities, and no deficits in the
special senses. Somatosensory perception was blunted (see Table 1).
CAPH Subject 2 was born in Wales in 1968 and is a right-handed
Caucasian male with scoliosis due to childhood injury. Purportedly,
his sensory abnormalities were evident shortly after birth. By the
age of eight he had suffered multiple fractures, including a leg on
which he walked for 3 days before it was visibly obvious as well
as several fractures in the left forearm, which has left him with
permanent motor and sensory deficits in the left forearm and hand.
He also has a history of second degree skin burns of which he was
unaware. Despite this, he said that he is able to feel itching, and
experienced ticklishness as a child. Family history is unremarkable.
He was first evaluated in 1991 for hyperhidrosis with attacks of
excessive sweating lasting 2–3 min occurring about 5 times every
day, mainly affecting the hands, feet, axilla, forehead, and natal
cleft. The sweating was greatly increased by emotional tension.
On physical examination, he was of small stature (1.58 m), weigh-
ing only 46.8 kg. General examination was unremarkable. Cranial
nerve exam showed the pupils contracted sluggishly to light, but
re-dilated rapidly when the light was removed. Fungiform papillae
were present on the tongue, and the senses of smell and taste were
normal. He had a 60% loss of hearing in the left ear. Clinically, he
could distinguish rough from smooth, and warm and cold. Muscle
power was normal for his size. Sensation was decreased but not
 D. Bowsher et al. / PAINÒ 147 (2009) 287–298 289

Table 1
Somatosensory thresholds and skin parameters in CAPH Subjects 1 and 2 compared to averages for comparably aged control subjects.

Thenar eminence Dorsum of Foot

Right Left Right Left
Somatosensory perception thresholds at rest (Values differing by more than two standard deviations from normal averages)
von Frey filaments (Log10.force mgX10)
CAPH Subject 1 4.93* 4.93* 6.1*
Avg. normals aged 50–70 ± SD 2.4 ± 0.3 3.3 ± 0.7
CAPH Subject 2 (1998) 2.83 3.84* 3.84*
Avg. normals aged 17–30 ± SD 2.34 ± 0.27 2.18 ± 0.36
Vibration (Rydel–Seiffer)
CAPH Subject 2 (2007) 6.5 8.0
Avg. normals aged 6 40 ± SD P6.5 P4.5
Skinfold pinch (kg)
CAPH Subject 2 (1998) 6.5* >10*
Avg. normals aged 17–30 ± SD 0.45 ± 0.2
Sharpness (g)
CAPH Subject 1 None None None
Avg. normals aged 50–70 ± SD 1.1 ± 0.4 1.2 ± 0.9
CAPH Subject 2 (1998) 3.2* >5.2* >5.2*
Avg. normals aged 17–30 ± SD 0.8 ± 0.3 0.8 ± 0.4
Warmth (°C)
CAPH Subject 1 47.6* 45.2* 45.9*
Avg. normals aged 50–70 ± SD 32.5 ± 1.2 35.4 ± 0.9
CAPH Subject 2
(1998) 32.6 48.8* 45.3*
(2007) 49.2* 49.5* 49.8*
Avg. normals aged 17–30 ± SD 31.1 ± 0.5 32.6 ± 0.9
Cold (°C)
CAPH Subject 1 <0* 14.1* 9.4*
Avg. normals aged 50–70 ± SD 28.7 ± 0.4 27.0 ± 1.2
CAPH Subject 2
(1998) 27.6* 16.2*
(2007) 15.7* 0.0* 0.0*
Avg. normals aged 17–30 ± SD 29.25 ± 0.2
Hot pain (°C)
CAPH Subject 1 >50* >50*
Avg. normals aged 50–70 ± SD 42.36 ± 3.6
CAPH Subject 2
(1998) 47.5 50* >50*
(2007) >50* >50* >50*
Avg. normals aged 17–30 ± SD 43.3 ± 2.6 41.4 ± 2.5

Skin parameters (Values differing by more than 2 standard deviations from normal averages)
Skin temperature (°C)
CAPH Subject 1 33.9* 35.3*
Avg. normals aged 50–70 ± SD 29.0 ± 1.9 25.0 ± 3.1
CAPH Subject 2 25.4* 23.8* 24.1
Avg. normals aged 17–30 ± SD 29.2 ± 1.65 28.6 ± 2.3
Cutaneous flux (arbitrary units)
CAPH Subject 1 23.0 26.5
Unchanged by autonomic challenge
CAPH Subject 2 16.6 15.8
Reduced by autonomic challenge (as are normals)
Sweat production (g/m2/h)
CAPH Subject 1 126* 138*
Avg. normals aged 50–70 ± SD
CAPH Subject 2 392* 254*
Avg. normals aged 17–30 ± SD 48 ± 14

Table 2
Fiber composition of a sural nerve biopsy in CAPH Subject 1 as compared to the averages for comparably aged normal subjects.

Total # of fibers Ratio C:Ad:Ab Area of nerve (mm2)**

Unmyelinated Myelinated
C fibers* Ad fibers Ab fibers
CAPH Subject 1 1178 234 36 33:6.5:1 0.744
Normal subjects (n = 6)  22,427 3201 1067 21:3:1   0.895
*
Unmyelinated fibers may be C fibers or sympathetic fibers.
**
Includes the nerve sheath which is greatly thickened in Subject 1.
 
From Jacobs and Love [26].
  
From Dyck et al. [12].
290 D. Bowsher et al. / PAINÒ 147 (2009) 287–298

Table 3
Types and sources of antibodies used for immunfluorescence.

Antigen (abrev.) Antigen Antibody species Dilution Source Innervation labeled

species
PGP9.5 (PGP) Human Rabbit polyclonal 1:800 UltraClone Limited, Isle of Wight, All innervation
England
Myelin basic protein (MBP) Human Mouse monoclonal 1:1000 Sternberger Monoclonals Myelin sheaths on Ab and Ad fibers
Incorporated, Lutherville MD
200kD neurofilament Bovine Rabbit polyclonal 1:800 Chemicon International, Temecula CA Mostly Ab and Ad fibers with perhaps a
protein (NF) Bovine Mouse monoclonal 1:400 Sigma, St. Louis MO small percentage of C fibers
Growth Associated Rat Mouse monoclonal 1:1000 Gift from Dr. David Schreyer [50] Most C fibers and sympathetic fibers
Protein-43 (GAP43)
a-Calcitonin gene-related Rat Rabbit polyclonal 1:800 Chemicon International, Temecula CA Subset of Ad and C fibers
peptide (CGRP) (synthetic)
f-Calcitonin Human Guinea pig polyclonal 1:400 Peninsula Laboratories, San Carlos CA
gene-related peptide (synthetic)
Neuropeptide Y (NPY) synthetic Sheep 1:800 Chemicon International, Temecula CA Adrenergic sympathetic fibers
Tyrosine hydroxylase (TH) Rat Rabbit polyclonal 1:800 Pel-Freez, Rogers AK Adrenergic sympathetic fibers
Vasoactive intestinal Porcine Rabbit polyclonal 1:2000 INCSTAR Corporation, Stillwater MN Cholinergic sympathetic (consistent).
peptide (VIP) Some Adrenergic sympathetic fibers and
CGRP-containing C fibers (variable)
Peripheral choline Rat Rabbit polyclonal 1:10,000 Gift from Dr. Hiroshi Kimura [53] Cholinergic sympathetic fibers.
acetyl transferase (pChAT)
Vesicular acetylcholine Human Goat 1:1000 Santa Cruz Biotechnology Cholinergic sympathetic terminals
transporter (VAChT) Incorporated, Santa Cruz CA

absent to all modalities. Tendon reflexes in the left upper limb
were less brisk than in the right. Both knee jerks were brisk, and
ankle jerks absent. Plantar reflexes were downgoing. Thyroid func-
tion tests, ESR, blood glucose, and full blood counts were all nor-
mal. Auto-antibodies for thyroid, thyroglobulin, microsomes,
acetylcholine receptor, striated muscle, smooth muscle, nuclei
and mitochondria were absent. MRI and CT scan revealed no obvi-
ous abnormalities.
3.2. Quantitative clinical evaluations
The results of the quantitative clinical evaluations conducted by
the same neurologist (DB) on both patients are summarized in
Table 1. Although they lacked pain sensation, both subjects lived
relatively normal lives and were able to say whether objects feel
warm or cold, or rough or smooth. However, clinical tests revealed
a general hypesthesia (Table 1).

"
Fig. 1. Except for the innervation to blood vessels, virtually no innervation can be detected in the epidermis (e), upper dermis (ud) and dermal papillae (dp) in the glabrous
hypothenar skin of CAPH Subject 2. Blood vessels are indicated by open chevrons. Insets in various panels are 2X enlargements of areas shown in small white frames. Scale bar
in Panel A = 50 lm. Panels A, B: Immunolabeling only with anti-PGP9.5 reveals all known types of innervation in normal skin. Endings in the epidermis (small straight arrows)
are supplied by C fibers and Ad fibers located in plexuses of small nerves (large straight arrows) in the upper dermis. Meissner corpuscles (curved arrows) are located in
dermal papillae and contain endings supplied by Ab fibers and two types of C fibers. Endings (large arrowheads) supplied by Ab fibers also terminate on Merkel cells (small
arrowheads) located in the deepest layer of the epidermis. C fiber and Ad fibers supply endings (broad arrowheads) on small blood vessels in the upper dermis and dermal
papillae. Smaller broad arrows indicate endings on likely capillaries. Larger broad arrows indicate endings on likely precapillary arterioles. Panels C, D: Anti-PGP
immunolabeling for CAPH Subject 2 only reveals endings on blood vessels (broad arrows). The right hand (C) has more vascular innervation than the left hand (D). Some
uninnervated presumptive Merkel cells label with anti-PGP (small arrowheads) Panels E–L: Different biochemically distinct types of innervation are shown in pairs of double-
labeled images using antibodies against the antigens indicated in the upper left corner of each image. The left images are only the immunolabeling for the antigens revealed
by red fluorescence (Cy3). The right images are the combined double labeling including the antigen revealed by green fluorescence (Alex-488). Red arrows and arrowheads
are for structures labeled only for the red-labeled antigen; green for only the green-labeled antigen; and yellow for double labeled. Yellow and green striped arrows indicate a
mix of single (green) and double labeled (yellow) fibers. Panel E: In normal skin, anti-GAP43 co-labels most C-fiber endings in the epidermis (straight yellow arrows), in
Meissner corpuscles (curved yellow arrows), and on blood vessels (broad yellow arrows). Based on fiber caliber and other co-labeling with anti-NF (see Panel G) and anti-MBP
(not shown), the innervation labeled only with anti-PGP consists of Ab and likely Ad fibers, which normally lack GAP43. Ab fibers supply endings to Meissner corpuscles
(curved green arrow) and endings (large green arrowheads) on Merkel cells (small green arrowheads). Likely Ad fibers supply endings on blood vessels (broad green arrow)
and in the epidermis (none shown). This image also shows an example of DAPI co-labeling of all cell nuclei (blue), which was used in all preparations to facilitate the
assessment of skin structure. Panel F: For CAPH Subject 2, the only innervation labeled with anti-GAP43 consists of likely C-fiber endings on blood vessels (broad yellow
arrows). Additional innervation labeled only with anti-PGP is also on blood vessels and consists of likely Ad fibers (broad green arrows). Panel G: Based on co-labeling with
anti-MBP (not shown), anti-NF only labels large caliber Ab fibers and small caliber Ad fibers in the skin of normal subjects. Large caliber Ab supply endings to Meissner
corpuscles (curved yellow arrows) and endings (yellow arrowheads) on Merkel cells. The Merkel cells only label with anti-PGP (green arrowheads). Thinner caliber fibers
labeled with anti-NF are likely Ad fibers that supply some endings in the epidermis (small straight yellow arrows) and on blood vessels (broad yellow arrows). Likely C fibers
only label with anti-PGP (green arrows) and terminate in the epidermis (small straight green arrows), in Meissner corpuscles (curved green arrow), and on blood vessels
(broad green arrow). The plexus of small nerves in the upper dermis contains a mix of Ad and C fibers (large straight yellow arrow and striped arrow). Panel H: The extremely
sparse innervation in the left hand of CAPH Subject 2 consists of likely Ad (yellow arrows) and C fibers (green arrow) that terminate among small blood vessels in the dermal
papillae. Panel I: In normal skin, anti-CGRP labels some endings in the epidermis (small straight yellow arrows), most endings in Meissner corpuscles (curved yellow arrows),
and some endings on blood vessels (broad yellow arrows). Anti-CGRP labeled axons are in the upper dermal plexus of small nerves (large straight yellow arrows). Only anti-
PGP labels other axons in the dermal plexus (large straight striped arrows) and endings in the epidermis (small straight green arrow). Innervation that only labels with anti-
PGP is also on blood vessels (small broad green arrow and large broad striped arrow). Panel J: In CAPH Subject 2, the innervation is only on the blood vessels and consists of
some endings that only label with anti-PGP (broad green arrow) and some that co-label with anti-CGRP (broad yellow arrows). Panel K: In normal skin, anti-CGRP co-labels
with anti-NF on Ab-fiber endings in Meissner corpuscles (yellow curved arrows). In the plexuses in the upper dermis, some likely Ad fibers co-label with anti-NF and anti-
CGRP (straight yellow arrows), some likely Ad fibers only label with anti-NF (straight green arrow), and some likely C fibers only label with anti-CGRP (straight red arrows). All
three types can supply endings to the epidermis: only an ending labeled with anti-CGRP is shown (small red arrows). Small blood vessels can also be innervated with all three
types of innervation (broad red, green and yellow arrows). Panel L: In CAPH Subject 2 skin, blood vessels also have innervation that only labels with anti-CGRP, others with
only anti-NF and others with both (broad red, green and yellow arrows). A sparse axon that co-colabels with anti-CGRP and anti-NF (medium straight yellow arrows) may be
the source of one of a few endings (small straight yellow arrows) observed in the epidermis in over 40 sections.
 D. Bowsher et al. / PAINÒ 147 (2009) 287–298
Testing of CAPH Subject 1 found elevation greater than two stan-
dard deviations for all modalities with skinfold pinch pain and heat
up to 50 °C thresholds impossible to elicit at non-damaging intensi-
ties. Naloxone challenge in CAPH Subject 1 produced mild changes,
but did not reverse the deficits (data not shown). CAPH Subject 2 was
extensively evaluated in 1998 and partially re-evaluated in 2007.
Skin biopsies were taken in 2005. Instrumental somatosensory
examinations of the right hand and foot from CAPH Subject 2 in
1998 showed slightly reduced sensory perception for skinfold pinch,
sharpness, and cold detection. Instrumental testing of the left hand
(the left arm had suffered multiple fractures in childhood) showed
deficits greater than two standard deviations in all modalities. In
2007, right hand thresholds for von Frey, warmth and heat pain
had increased in comparison with earlier studies.
In CAPH Subject 1 skin evaporation was measured with a Serv-
oMedR evaporimeter on the right thenar eminence at 125.9 g/m2/h
291
and the right foot dorsum at 138.2 g/m2/h. Normal age appropriate
subjects have resting values of 39.4 ± 9.9 g/m2/h in the thenar emi-
nence and 13.1 ± 1.5 g/m2/h in the dorsum of the foot. Following
the intravenous injection of 1.2 mg naloxone, CAPH Subject 1’s
evaporation increased slightly to 148.5 g/m2/h in the hand and
146.1 g/m2/h in the foot. Sweat analysis of CAPH Subject 1 identi-
fied low molar concentrations of sodium (17 mmol/l) and chloride
(15 mmol/l) against normal values of about 40 mmol/l for both.
Hyperhidrosis in CAPH Subject 2 was measured by re-weighing
pre-weighed Whatman filter papers placed on the thenar emi-
nences for 5 min, and yielded average values of 392 g/m2/h on
the right and 254 g/m2/h on the left, against a normal thenar value
in this age group of 48 ± 13.6 g/m2/h (Table 1). No significant
change occurred following autonomic challenge (ice-pack applied
to the back of the neck) and diagnostic stellate ganglion blockade
with 10 ml of 0.5% bupivicaine (data not shown). After unsuccess-
292 D. Bowsher et al. / PAINÒ 147 (2009) 287–298

ful therapeutic trial of several drugs, CAPH Subject 2 has been tak- small caliber are presumptive Ad fibers. C fibers and Ad fibers can
ing methixene for several years, and finds that this reduces his be non-peptidergic that label with anti-PGP but not anti-CGRP,
sweating to some extent. or peptidergic that also label with anti-CGRP (Figs. 1I, 1K, 2A, 3A,
In CAPH Subject 1, sensory nerve conduction studies were car- 3E).
ried out on the right and left median and ulnar nerves and the right
sural nerve. Motor nerves studied were the left median, right ulnar, 3.3.1.2. Autonomic innervation. Adrenergic innervation labels with
and left common peroneal nerves. Amplitude and conduction anti-TH and anti-NPY (Fig. 3G) [2,32,45]. Cholinergic innervation
velocity were found to be normal in all cases. Nerve conduction labels with anti-pChAT and anti-VAChT (Fig. 3I) [54] with pChAT
studies were carried out on CAPH Subject 2 on the right median, throughout the fibers and VAChT concentrated more in the termi-
ulnar, radial, peroneal, and tibial motor nerves and on the right nals [23]. VIP can be immunodetectable in subsets of cholinergic
median, ulnar, radial, superficial peroneal, and sural sensory (Fig. 4K) and adrenergic fibers (Fig. 3K) as well as in some peptider-
nerves. Motor function was essentially normal at all sites studied gic C fibers (Fig. 4I). Upregulation of VIP can occur especially under
while the right median and radial sensory responses had normal pathological conditions [31,32,35]. NPY can also upregulate and
latencies but reduced (median > 36% and radial > 53%) amplitudes. CGRP can downregulate among C fibers under pathological condi-
The ulnar and lower limb sensory responses were absent (data not tions [37].
shown). Upper limb somatosensory evoked responses in CAPH
Subject 2 were recorded at Erb’s point, over the cervical spine, 3.3.2. CAPH Subject 2 lacks all cutaneous innervation that is not
and from the scalp. While there was considerable reduction in affiliated blood vessels or sweat glands
amplitude at Erb’s point, it was essentially normal over the cervical The background fluorescence of the biopsy sections (Figs. 1–4)
spinal cord and postcentral cortex (data not shown). and DAPI co-labeling (Fig. 1E and F) revealed that the overall struc-
In CAPH Subject 1, the sheath of the sural nerve was greatly ture of the skin in CAPH Subject 2 is comparable to that in the nor-
thickened with electron microscopy finding the total area of the mal subjects. Based upon the results using a wide variety of
nerve at 0.744 mm2. Fibers in seven electron micrographs were immunolabels for different types of innervation, all biopsies from
then taken at known magnifications, counted and measured. A to- CAPH Subject 2 lacked virtually all glabrous and hairy skin innerva-
tal of 113 myelinated and 493 unmyelinated fibers were counted tion (Figs. 1–4) except for that affiliated with blood vessels and
(Table 2). Measurement of the myelinated fibers showed 15 to be sweat glands. The missing innervation was not detected with
large myelinated (Ab, >6 lm) and 98 small myelinated (Ad, anti-GAP43 immunolabeling (Fig. 1E and F), which we have previ-
<6 lm) fibers. Based on this the C:Ad:Ab ratio is 33: 6.5: 1 with a ously found could detect abnormal innervation that lacked labeling
normal ratio of 21:3:1 [26]. Sural nerve biopsy in CAPH Subject 2 with other antibodies used in the current study [2,39] .The missing
consisted of dense collagenous tissue with numerous blood ves- innervation includes:
sels, islands of fat, and a few nerve fascicles, containing very few
myelinated fibers. Six months later, biopsy was performed on the 1. All peptidergic and non-peptidegic C and Ad fibers that nor-
other side; no nerve tissue was found in the specimen. mally terminate in the epidermis, within dermal papillae, and
within or around the necks of hair follicles (Figs. 1 and 2).
3.3. Skin biopsy analyses 2. Ab fiber endings as well as peptidergic and non-peptidergic C-
fiber endings that normally terminate in Meissner corpuscles
3.3.1. General Immunochemical characteristics of cutaneous in dermal papillae of glabrous skin (Fig. 1) [27,40] and pilo-neu-
innervation ral complexes affiliated with hair follicles (Fig. 2) [2].
3.3.1.1. Sensory Innervation. All known cutaneous innervation la- 3. Ab-fiber Merkel endings that normally terminate as disks on
bels with anti-PGP under normal conditions (Figs. 1–4) [15,45]. Merkel cells in both glabrous and hairy skin (Fig. 1).
Thin caliber axons that lack NF immunolabeling (Figs. 1G, 2C, 2G, 4. All small nerves in the upper dermis that are not affiliated with
3C, 3D) consistently lack myelin basic protein and are likely C blood vessels and that are normally the source of the missing
fibers or autonomic fibers. Virtually all NF-positive axons co-label endings described above as well as potential free nerve endings
with anti-MBP (not shown): large caliber are likely Ab fibers and in the upper dermis (Figs. 1 and 2).
"
Fig. 2. Except for the innervation to blood vessels and pilo-erector muscles (pm), virtually no innervation can be detected in the epidermis (e), upper dermis (ud), dermal
papillae (dp) and hair follicles (hf) in the hairy forearm skin of CAPH Subject 2. Scale bar in Panel A = 50 lm. Panels A–H: Different biochemically distinct types of innervation
are shown in pairs of double-labeled images using antibodies against the antigens indicated in the upper left corner of each image. The left images are only the
immunolabeling for the antigens revealed by red fluorescence (Cy3). The right images are the combined double labeling including the antigen revealed by green fluorescence
(Alex-488). Red arrows and arrowheads are for structures labeled only for the red-labeled antigen; green for only the green-labeled antigen; and yellow for double labeled.
Yellow and green striped arrows indicate a mix of single (green) and double labeled fibers (yellow). Panel A: In normal hairy skin, anti-PGP labeling reveals endings in the
epidermis (small straight arrows) that are supplied by a plexus of small nerves (large straight arrows) in the upper dermis. Medium straight arrows indicate small nerves
entering dermal papillae. The small nerves contain a mix of axons that co-label with anti-CGRP (medium and large straight yellow arrows) and axons that only label with
anti-PGP (medium and large straight striped arrows). Some epidermal endings co-label with anti-CGRP (small straight yellow arrows) and others only label with anti-PGP
(small straight green arrows and green and yellow striped arrows). Axons with and without CGRP innervate the upper ends of the hair follicles (yellow and green broad
arrows respectively). Panel B: In CAPH Subject 2, the only innervation detected was a few axons in the upper dermis that co-label with anti-CGRP and anti-PGP (yellow
arrows) or only label with anti-PGP (green arrow). Virtually no innervation was present in the epidermis or the necks of hair follicles. Panel C: In normal skin, the small nerves
in the upper dermal plexus contain a mix of axons that co-label with anti-NF (medium and large straight yellow arrows) and that only label with anti-PGP (medium and large
straight striped arrows). Such NF-positive axons in the plexus are thin caliber and also co-label with anti-MBP on other sections (not shown) indicating that they are likely Ad
fibers. Those axons only labeling with anti-PGP are likely C fibers. The presumptive Ad fibers and C fibers supply endings to the epidermis (small straight yellow and green
arrows, respectively) and to the necks of hair follicles (broad yellow and green arrows, respectively). Double labeling with anti-CGRP and anti-NF on other sections (not
shown) revealed that the Ad fibers can be CGRP positive or negative and that C fibers can be CGRP positive or negative. Panel D: CAPH Subject 2 has rare NF-positive axons in
the upper dermis (yellow arrows). Panel E, G: In normal skin, anti-PGP labels pilo-neural complexes located at the mid-follicle level. Pilo-neural complexes are normally
composed of a palisade of longitudinally oriented lanceolate endings (arrowheads) that are surrounded by several varieties of circumferentially oriented endings (curved
arrows). The lanceolate endings are supplied by large caliber Ab fibers and co-label with anti-NF (G, yellow arrowheads). Some circumferentially oriented endings co-label
with anti-CGRP (E: yellow curved arrows) and others lack CGRP (E: curved green arrows). Some circumferentially oriented endings co-label with anti-NF (G: yellow curved
arrows) and others lack NF (G: curved green arrows). Other double label combinations reveal that the NF positive circumferential endings are supplied by Ab-fibers. Other
circumferential endings are suppled by C fibers and can be CGRP positive or negative. Panels F, H: In CAPH Subject 2, no pilo-neural complexes were detected on hair follicles.
Sympathetic innervation (green chevrons) to pilo-erector muscles is present. Panels I–L. Innervation was present on the sweat gland located adjacent to hair follicles in
normal subjects (I, J). This was also present in Subject 2 but was relatively depleted on the left (K, L).
 D. Bowsher et al. / PAINÒ 147 (2009) 287–298 293

5. Pacinian corpuscles were also not found, but these are rarely
obtained in comparable biopsies from normal subjects.
All normal types of sensory and autonomic vasculature innerva-
tion were present in all biopsies of CAPH Subject 2 (Figs. 1–3),
although they appeared to be reduced compared to normal espe-
cially in the left hand. The autonomic innervation is mostly located
in the tunica adventitia concentrated near the tunica media of
small arteries and arteriovenous shunts (AVS) located deep in the
dermis. An adrenergic innervation labeled with anti-TH (not
shown) and anti-NPY (Fig. 3G and H) was especially dense, accom-
panied by a substantial cholinergic innervation labeled with pChAT
and VAChT (Figs. 3I–L) as well as anti-VIP. Sensory innervation to
arteries and AV shunts was located more centripetally in the tunica
adventitia (Figs. 3G and H). One type consists of C-fibers that are
NF-negative/CGRP-positive, some likely with VIP (Fig. 3E, F, K, L).
294 D. Bowsher et al. / PAINÒ 147 (2009) 287–298

Two other types are NF-positive presumptive Ad fibers that can be Subject 2 glabrous skin lacked all innervation and had an abnormal
CGRP-positive or negative (Fig. 3C–F). Occasional arteries in CAPH multi-laminated appearance (insets in Fig. 3B and D).

Fig. 3. Although reduced compared to normal, all types of sensory and sympathetic innervation are present on small arteries (a) and arteriovenous shunts (avs) in the deep
dermis of CAPH Subject 2. Scale bar in A = 50 lm. Panels A–L: Different biochemically distinct types of innervation are shown in pairs of double-labeled images using
antibodies against the antigens indicated in the upper left corner of each image. The left image is only the immunolabeling for the antigen revealed by red fluorescence, the
right image is the combined double label including the antigen revealed by green fluorescence. Red arrows indicate innervation that is labeled only for the red-labeled
antigen, green arrows only for the green-labeled antigen, and yellow for double labeled. Long arrows indicate various types of innervation affiliated with the vessels. Broad
arrows indicate small nerves near the blood vessels. Panels A–F: Large arteriole and arteriovenous shunts in the normal subject and CAPH Subject 2 have extensive CGRP-
negative and positive innervation in the tunica adventitia (A, B: green and yellow arrows, respectively). The CGRP-positive innervation is likely sensory. NF-positive
innervation (C, D: yellow arrows) are likely Ad fibers, which can be CGRP-negative or positive (E, F: green and yellow arrows, respectively). Some CGRP-positive innervation is
NF-negative (E, F; red arrows) indicative of likely peptidergic C fibers. Panels G, H: CGRP-positive innervation (red arrows), which is likely sensory, is intermingled with NPY-
positive innervation (green arrows) which is likely adrenergic sympathetic innervation. The NPY innervation is more dense and more concentrated towards the tunica media
than the CGRP innervation. Panels I, J: Some innervation expresses immunoreactivity for pChAT in axons (I, J: green arrows) and, in the normal subject, VAChT in the terminals
(I: red arrows) indicative of cholinergic innervation. VAChT was not evident in CAPH Subject 2 (J). Panels K, L: Also indicative of cholinergic innervation, VIP immunolabeling
was detected on fibers (red arrows) that were not labeled with a cocktail of anti-CGRP and anti-NPY (green and yellow arrows). VIP labeling is also present on some sensory
and/or adrenergic fibers (yellow arrows) but not on others (green arrows). In general, all innervation was lower in CAPH Subject 2 than in normal subjects, and was lower in
the CAPH left hand (F, J) than the right hand (B, D, H, L). Some arteries in CAPH Subject 2 lacked any innervation (insets in B, D) and had an abnormally thickened wall.
 D. Bowsher et al. / PAINÒ 147 (2009) 287–298 295

The innervation of precapillary arterioles and some capillaries
in the upper dermis and dermal papillae normally consists of
mostly CGRP-positive/NF-negative C-fibers (Fig. 1I), some CGRP-
positive/NF-positive Ad fibers (Fig. 1K), and occasional NF-posi-
tive/CGRP-negative Ad fibers. Although reduced in number, all
these types of this innervation were present and were virtually
the only innervation in the upper dermis of CAPH Subject 2
(Fig. 1C). Even this innervation was nearly absent in his left hand
biopsy (Fig. 1D).
Normal sweat gland tubules typically have a dense cholinergic
sympathetic innervation that is positive for VIP, pChAT and VAChT
(Fig. 4I and K). Small vessels in the sweat glands had an NPY-posi-
tive presumptive adrenergic innervation (not shown). A sparse
sensory innervation consists of CGRP-positive C fibers (NF-nega-
tive) and Ad fibers (NF-positive) that sometimes may be VIP-posi-
tive (Fig. 4E–J). Most sweat glands in CAPH Subject 2 had most
types of innervation (Fig. 4J, 4L). Many sweat glands in all biopsies
lacked innervation (Figs. 2K, 2L, 4C, 4D).

Fig. 4. In CAPH Subject 2, the innervation to sweat glands in glabrous hypothenar skin is generally intact but may be more variable than normal. Scale bar in A = 50 lm.
Panels A, B: Anti-PGP reveals extensive, but variable innervation (arrows) among sweat tubules. A least (A) and best (B) example are shown. Panels C, D: In CAPH Subject 2,
some sweat glands have barely any innervation (C) compared to others (D). Panels E–L: Different biochemically distinct types of innervation are shown in pairs of double-
labeled images using antibodies against the antigens indicated in the upper left corner of each image. The left image is only the immunolabeling for the antigen revealed by
red fluorescence, the right image is the combined double label including the antigen revealed by green fluorescence. Red arrows indicate innervation labeled only for the red-
labeled antigen, green arrows only for the green-labeled antigen, and yellow arrows for double labeled. Panels E, F: In normal and CAPH skin, some innervation co-labels with
anti-CGRP (E-G: yellow arrows) indicative of sensory innervation, but most is only labeled with anti-PGP (green arrows). Panels G, H: In normal and CAPH skin, some
innervation labels with anti-NF (yellow and green arrows), indicative of likely Ad fibers, of which some co-label with anti-CGRP (yellow arrows). Some innervation labels only
with anti-CGRP (red arrows) indicative of likely C fibers. Panels I, J: In normal and CAPH skin, anti-CGRP labels with and without anti-VIP co-labeling (red and yellow arrows)
indicative of multiple types of sensory innervation. Much of the innervation labels with anti-VIP but not anti-CGRP indicative of likely cholinergic innervation. Panels K, L:
Consistent with cholinergic innervation, VAChT is expressed in the terminals of many VIP-positive fibers (K, yellow arrows). Other VIP-positive fibers lack VAChT (green
arrows) and are likely some of the CGRP-positive sensory innervation.
296 D. Bowsher et al. / PAINÒ 147 (2009) 287–298
3.4. DNA analysis
Blood was taken from CAPH Subject 2 and subjected to DNA
analysis of SCN9A, SCN10A, SCN11A, NGFB and TRKA, which are
genes which have been previously shown to be mutated in some
forms of pain related channelopathies and absence of C-fiber inner-
vation. In view of the absence of all types of cutaneous innervation
in CAPH Subject 2, except that to the vasculature and sweat glands,
the NRTN and GFRA2 genes were also assessed based on prelimin-
ary observations that neurturin/GFA2 may contribute to the devel-
opment of a wide variety of cutaneous innervation except that to
the vasculature in mice (Rice, unpublished). No mutations were
found, in any exon, splice site or polyadenylation signal. Therefore,
it seems likely that the subject’s genetic disorder is distinct and
non-allelic to channelopathy-associated insensitivity to pain,
HSAN IV and HSAN V.
4. Discussion
These two unrelated cases do not fit any of the described catego-
ries of hereditary sensory and autonomic neuropathy. Both had
significantly diminished responses to non-noxious stimuli, with
complete analgesia for noxious mechanical and thermal stimuli,
whereas other cases of congenital insensitivity to pain have purport-
edly normal sensory thresholds for other sensations [3,10,13] . CAPH
Subject 2 has scoliosis which is sometimes a feature of HSAN Type II
[42] and HSAN III; however, he lacks other features of these syn-
dromes (swallowing problems, gastroesophageal reflux disease,
Rombergism, and ulceration of toe and finger tips); reflexes are pres-
ent although some, particularly in the left upper limb, are depressed.
We have previously reported cases with reduced perception for
other somatosensory modalities, but this was in two siblings with
insensitivity to pain but with normal sweating [4,18,25]. With the
exception of the Ad-mediated modalities of sharpness discrimina-
tion and cold on the dominant thenar eminence in CAPH Subject 2,
all somatosensory perception in the CAPH subjects had significantly
raised thresholds. In CAPH Subject 1, tactile threshold is raised in
comparison with comparable-age normal subjects [5], but pinprick
(sharpness) sensation was ‘‘off-scale” at all sites tested, and mechan-
ical (skinfold pinch) and heat pain could not be induced at intensities
which did not damage tissue. Despite this, CAPH Subject 1 reported
experiencing sensation throughout his life, working in an occupation
demanding a great deal of physical exertion. Electrophysiologically,
his peripheral motor system was normal.
Consistent with his impaired cutaneous sensation, CAPH Sub-
ject 2 lacked all types of Ab, Ad and C fibers and endings terminat-
ing in and around the epidermis, dermal papillae and hair follicles.
We cannot rule out the possibility that the missing innervation
was still present but no longer expressed the wide variety of anti-
gens that are labeled by the antibodies used in our study. Indeed,
we have found that some chemotherapy can eliminate immunola-
beling of all the cutaneous innervation with most of these antibod-
ies including anti-PGP (Albrecht and Rice, unpublished). However,
this included all the vascular innervation, which was labeled by the
various antibodies in the skin of CAPH Subject 2. Moreover, we
found that the apparently missing innervation following chemo-
therapy was still detected with anti-GAP43 (Albrecht and Rice,
unpublished), whereas anti-GAP43 still failed to detect the missing
innervation in the skin of CAPH Subject 2.
Importantly, all normal immunochemical varieties of innerva-
tion were present, albeit apparently reduced on both the deep
and superficial cutaneous vasculature in CAPH Subject 2. This in-
cluded not only previously described peptidergic and non-pepti-
dergic varieties of C and Ad fibers and adrenergic sympathetic
fibers [2,32,41,45,46], but also a normal cholinergic innervation
of unknown origin to the arteries and AVS. Cholinergic innervation
of parasympathetic origin has been shown previously on cerebral
and facial arteries [19,36,43]. Although a vasodilatory cholinergic
innervation has been suspected on the vasculature to the limbs
[33], immunochemical evidence has been contradictory and lim-
ited to arteries among the musculature [21,49]. Sweat glands were
also present and generally well innervated, unlike the atrophy and
lack of innervation seen in subjects with anhidrosis.
Interestingly, most of the intact cutaneous vascular innervation
in CAPH Subject 2 is known to have the same neurotrophin depen-
dencies as many types of missing innervation, in particular a
dependency on NGF and TRKA signaling for survival during devel-
opment [9,16,17,44]. Consistent with the survival of these vascular
afferents we did not find a mutation of NGF or TrkA. Moreover, we
did not find evidence of mutations involving SCN9A, SCN10A or
SCN11A. In contrast to our CAPH subjects, those with SCN9A chan-
nelopathy do not experience increased sweating [1,8,20]. In the
case of CAPH Subject 2, it would appear that all of the non-vascu-
lar/non-sweat gland innervation has a common genetic progenitor
mechanism that does not involve NGF/TrkA signaling or NaV-re-
lated sodium channels. Based on some preliminary data in an
ongoing study which indicated that the missing innervation might
share a unique dependency on neurturin (Rice, unpublished), we
searched for, but did not find mutations in NRTN and GFRA2 genes.
Assessments for other receptors such as c-RET and VEGFR were
considered but mutations in these receptors would likely have
far more complex phenotypes. Future assessments remain possible
as warranted. Alternatively, the missing innervation may have an
environmental factor vulnerability, such as organophosphate and
acrylamide toxicity [30,51], which may not affect the vascular or
sweat gland innervation.
A particularly startling aspect of CAPH Subject 2, is that he
experiences mechanical and innocuous thermal sensations despite
lacking virtually all the types of innervation that are believed to be
responsible for such sensation. Conceivably, this residual sensory
capacity may be mediated by the multiple types of persisting sen-
sory innervation to the vasculature and sweat glands. Other studies
have shown that there are further subtypes of this innervation that
have additional immunochemical characteristics – not assessed in
this study – that are implicated in mechanical and thermal sensory
transduction [2,6,17,34,45,46]. Moreover, the vascular innervation
has been shown to be supplied by small closely spaced nerves that
are differentially targeted to morphologically distinct sites within
the vascular arborization [2,41,45], which indicates that there
may be a fairly high resolution vasculotopic organization that
may contribute to conscious cutaneous perception.
Although CAPH Subject 2 had grossly excessive sweating, the
sweat glands were not obviously larger or more densely innervated
than normal. All immunochemically distinct types of normal inner-
vation were present on at least many of the sweat glands. Thus, the
reason for his enormous hyperhidrosis is not clear. To some extent,
the sweating appears to be independent of neural regulation. One
intriguing possibility is that missing C and Ad fiber innervation in
the epidermis and upper dermis may include thermoreceptors nor-
mally essential for comparing core and surface temperature for the
purpose of thermoregulation. Since only the innervation to the
blood vessels and sweat glands is intact, the thermal detection
from deeper tissues and the blood may be misperceived as though
there is a continuously high surface temperature, thereby eliciting
excessive sweating.
5. Conclusion
These CAPH subjects represent a previously undescribed type of
sensory neuropathy characterized by a diminished sensitivity to
 D. Bowsher et al. / PAINÒ 147 (2009) 287–298
pain, touch, and temperature, with hyperhidrosis and no motor
deficit. CAPH Subject 2 lacked all types of cutaneous innervation
except the multiple varieties of sensory and autonomic innervation
to the blood vessels and sweat glands. These observations suggest
that the development or maintenance of the innervation to the
sweat glands and vasculature may be influenced by environmental
or genetic factors independent of those for all the other types of
cutaneous innervation regardless of their fiber types. The fact that
the CAPH subjects maintained some ability to detect several varie-
ties of cutaneous stimulation suggests that vascular afferents may
have the capacity to contribute to conscious cutaneous perception.
6. Disclosure
The authors report no conflicts of interest.
Acknowledgements
Thanks are due to Mr. J.B. Miles, FRCS, Miss R. Page, FRCS, and
Mr. D. Price, FRCS, Consultant Neurosurgeons, for undertaking sur-
al nerve biopsy; to Mr. M. Stringer, BSc., for invaluable help with
the estimations of sweat output by the filter paper method, and
to Marilyn Dockum for the histological preparation of the skin
biopsies. The costs of investigations have been defrayed by the Pain
Relief Foundation and the Rosenblatt Fund for Neurofibromatosis.
References
[1] Ahmad S, Dahllund L, Eriksson AB, Hellgren D, Karlsson U, Lund PE, Meijer IA,
Meury L, Mills T, Moody A, Morinville A, Morten J, O’Donnell D, Raynoschek C,
Salter H, Rouleau GA, Krupp JJ. A stop codon mutation in SCN9A causes lack of
pain sensation. Human Mole Genet 2007;16:2114–21.
[2] Albrecht PJ, Hines S, Eisenberg E, Pud D, Finlay DR, Connolly MK, Pare M, Davar
G, Rice FL. Pathologic alterations of cutaneous innervation and vasculature in
affected limbs from patients with complex regional pain syndrome. Pain
2006;120:244–66.
[3] Axelrod FB, Gold-von Simson G. Hereditary sensory and autonomic
neuropathies: types II, III, and IV. Orphanet J Rare Dis 2007;2:39.
[4] Bowsher D, Peach B, Venn D, Hayward M, Campbell JA, Mumford J, Haggett C.
Forme familiale d’insensibilité à la douleur avec asymétrie de la somesthésie:
anomalie centrale? Rev Neurol 2001;158:195–202.
[5] Campbell JA, Lahuerta J, Bowsher D. Quantitative assessment of sensory
function. Brit J Therap Rehab 1996;3:135–41.
[6] Cannon KE, Chazot PL, Hann V, Shenton F, Hough LB, Rice FL.
Immunohistochemical localization of histamine H3 receptors in rodent skin,
dorsal root ganglia, superior cervical ganglia, and spinal cord: potential
antinociceptive targets. Pain 2007;129:76–92.
[7] Chan AW, MacFarlane IA, Bowsher D, Campbell JA. Weighted needle pinprick
sensory thresholds: a simple test of sensory function in diabetic peripheral
neuropathy. J Neurol Neurosurg Psychiatry 1992;55:56–9.
[8] Cox JJ, Reimann F, Nicholas AK, Thornton G, Roberts E, Springell K, Karbani G,
Jafri H, Mannan J, Raashid Y, Al-Gazali L, Hamamy H, Valente EM, Gorman S,
Williams R, McHale DP, Wood JN, Gribble FM, Woods CG. An SCN9A
channelopathy causes congenital inability to experience pain. Nature
2006;444:894–8.
[9] Cronk KM, Wilkinson GA, Grimes R, Wheeler EF, Jhaveri S, Fundin BT, Silos-
Santiago I, Tessarollo L, Reichardt LF, Rice FL. Diverse dependencies of
developing Merkel innervation on the trkA and both full-length and
truncated isoforms of trkC. Development 2002;129:3739–50.
[10] Dearborn GV. Case of congenital pure analgesia. J Nerve Ment Dis
1932;75:612–5.
[11] Dehen H, Willer JC, Cambier J. Congenital insensitivity to pain and endogenous
morphine-like system. Adv Pain Res Therap 1979;3:553–7.
[12] Dyck PJ, Johnson WJ, Lambert EH, O’Brien PC. Segmental demyelination
secondary to axonal degeneration in uremic neuropathy. Mayo Clin Proc
1971;46:400–31.
[13] Dyck PJ, Mellinger JF, Reagan TJ, Horowitz SJ, McDonald JW, Litchy WJ, Daube
JR, Fealey RD, Go VL, Kao PC, Brimijoin WS, Lambert EH. Not, indifference to
pain’ but varieties of hereditary sensory and autonomic neuropathy. Brain
1983;106:373–90.
[14] Fruhstorfer H, Lindblom U, Schmidt WC. Method for quantitative estimation of
thermal thresholds in patients. J Neurol Neurosurg Psychiatry
1976;39:1071–5.
[15] Fundin BT, Arvidsson J, Aldskogius H, Johansson O, Rice SN, Rice FL.
Comprehensive immunofluorescence and lectin binding analysis of
intervibrissal fur innervation in the mystacial pad of the rat. J Comp Neurol
1997;385:185–206.
297
[16] Fundin BT, Pfaller K, Rice FL. Different distributions of the sensory and
autonomic innervation among the microvasculature of the rat mystacial pad. J
Comp Neurol 1997;389:545–68.
[17] Fünfschilling U, Ng YG, Zang K, Miyazaki J, Reichardt LF, Rice FL. TrkC kinase
expression in distinct subsets of cutaneous trigeminal innervation and non-
neuronal cells. J Comp Neurol 2004;480:392–414.
[18] Furioli J, Fesq G, Cesaro P, Ponsot G, Le Loc’h H. Indifference to pain secondary
to congenital sensory neuropathy. Apropos of a new case. Arch Fr Pediatr
1987;44:445–7.
[19] Gaw AJ, Aberdeen J, Humphrey PP, Wadsworth RM, Burnstock G. Relaxation of
sheep cerebral arteries by vasoactive intestinal polypeptide and neurogenic
stimulation: inhibition by L-NG-monomethyl arginine in endothelium-
denuded vessels. Br J Pharmacol 1991;102:567–72.
[20] Goldberg YP, MacFarlane J, MacDonald ML, Thompson J, Dube MP, Mattice M,
Fraser R, Young C, Hossain S, Pape T, Payne B, Radomski C, Donaldson G, Ives E,
Cox J, Younghusband HB, Green R, Duff A, Boltshauser E, Grinspan GA, Dimon
JH, Sibley BG, Andria G, Toscano E, Kerdraon J, Bowsher D, Pimstone SN,
Samuels ME, Sherrington R, Hayden MR. Loss-of-function mutations in the
Nav1.7 gene underlie congenital indifference to pain in multiple human
populations.. Clin Genet 2007;71:311–9.
[21] Guidry G, Landis SC. Absence of cholinergic sympathetic innervation from limb
muscle vasculature in rats and mice. Auton Neurosci 2000;82:97–108.
[22] Guo YC, Liao KK, Soong BW, Tsai CP, Niu DM, Lee HY, Lin KP. Congenital
insensitivity to pain with anhidrosis in Taiwan: a morphometric and genetic
study. Eur Neurol 2004;51:206–14.
[23] Ichikawa T, Ajiki K, Matsuura J, Misawa H. Localization of two cholinergic
markers, choline acetyltransferase and vesicular acetylcholine transporter in
the central nervous system of the rat: in situ hybridization histochemistry and
immunohistochemistry. J Chem Neuroanat 1997;13:23–39.
[24] Indo Y, Tsuruta M, Hayashida Y, Karim MA, Ohta K, Kawano T, Mitsubuchi H,
Tonoki H, Awaya Y, Matsuda I. Mutations in the TRKA/NGF receptor gene in
patients with congenital insensitivity to pain with anhidrosis. Nat Genet
1996;13:485–8.
[25] Itoh Y, Yagishita S, Nakajima S, Nakano T, Kawada H. Congenital insensitivity
to pain with anhidrosis: morphological and morphometrical studies on the
skin and peripheral nerves. Neuropediatrics 1986;17:103–10.
[26] Jacobs JM, Love S. Qualitative and quantitative morphology of human sural
nerve at different ages. Brain 1985;108:897–924.
[27] Johansson O, Fantini F, Hu H. Neuronal structural proteins, transmitters,
transmitter enzymes and neuropeptides in human Meissner’s corpuscles: a
reappraisal using immunohistochemistry. Arch Dermatol Res
1999;291:419–24.
[28] Kimura J. Electrodiagnosis in diseases of nerve and muscle: principles and
practice. FA Davis Company; 1989.
[29] Klugbauer N, Lacinova L, Flockerzi V, Hofmann F. Structure and functional
expression of a new member of the tetrodotoxin-sensitive voltage-activated
sodium channel family from human neuroendocrine cells. The EMBO J
1995;14:1084–90.
[30] Ko MH, Chen WP, Hsieh ST. Neuropathology of skin denervation in acrylamide-
induced neuropathy. Neurobiol Dis 2002;11:155–65.
[31] Leblanc GG, Trimmer BA, Landis SC. Neuropeptide Y-like immunoreactivity in
rat cranial parasympathetic neurons: coexistence with vasoactive intestinal
peptide and choline acetyltransferase. Proc Natl Acad Sci USA
1987;84:3511–5.
[32] Lindh B, Lundberg JM, Hokfelt T. NPY-, galanin-, VIP/PHI-, CGRP- and substance
P-immunoreactive neuronal subpopulations in cat autonomic and sensory
ganglia and their projections. Cell Tissue Res 1989;256:259–73.
[33] Matsukawa K, Shindo T, Shirai M, Ninomiya I. Direct observations of
sympathetic cholinergic vasodilatation of skeletal muscle small arteries in
the cat. J Physiol 1997;500:213–25.
[34] Molliver DC, Immke DC, Fierro L, Pare M, Rice FL, McCleskey EW. ASIC3, an
acid-sensing ion channel, is expressed in metaboreceptive sensory neurons.
Mol Pain 2005;1:35.
[35] Morales MA, Holmberg K, Xu ZQ, Cozzari C, Hartman BK, Emson P, Goldstein
M, Elfvin LG, Hokfelt T. Localization of choline acetyltransferase in rat
peripheral sympathetic neurons and its coexistence with nitric oxide
synthase and neuropeptides. Proc Natl Acad Sci USA 1995;92:11819–23.
[36] Morita-Tsuzuki Y, Hardebo JE, Bouskela E. Interaction between
cerebrovascular sympathetic, parasympathetic and sensory nerves in blood
flow regulation. J Vasc Res 1993;30:263–71.
[37] Noguchi K, De Leon M, Nahin RL, Senba E, Ruda MA. Quantification of
axotomy-induced alteration of neuropeptide mRNAs in dorsal root ganglion
neurons with special reference to neuropeptide Y mRNA and the effects of
neonatal capsaicin treatment. J Neurosci Res 1993;35:54–66.
[38] Nolano M, Crisci C, Santoro L, Barbieri F, Casale R, Kennedy WR,
Wendelschafer-Crabb G, Provitera V, Di Lorenzo N, Caruso G. Absent
innervation of skin and sweat glands in congenital insensitivity to pain with
anhidrosis. Clin Neurophysiol 2000;111:1596–601.
[39] Pare M, Albrecht PJ, Noto CJ, Bodkin NL, Pittenger GL, Schreyer DJ, Tigno XT,
Hansen BC, Rice FL. Differential hypertrophy and atrophy among all types of
cutaneous innervation in the glabrous skin of the monkey hand during
aging and naturally occurring type 2 diabetes. J Comp Neurol
2007;501:543–67.
[40] Paré M, Elde R, Mazurkiewicz JE, Smith AM, Rice FL. The Meissner corpuscle
revised: a multiafferented mechanoreceptor with nociceptor immunochemical
properties. J Neurosci 2001;21:7236–46.
298 D. Bowsher et al. / PAINÒ 147 (2009) 287–298
[41] Paré M, Smith AM, Rice FL. Distribution and terminal arborizations of
cutaneous mechanoreceptors in the glabrous finger pads of the monkey. J
Comp Neurol 2002;445:347–59.
[42] Piazza MR, Bassett GS, Bunnell WP. Neuropathic spinal arthropathy in
congenital insensitivity to pain. Clin Orthop Relat Res 1988:175–9.
[43] Ramien M, Ruocco I, Cuello AC, St-Louis M, Ribeiro-Da-Silva A.
Parasympathetic nerve fibers invade the upper dermis following sensory
denervation of the rat lower lip skin. J Comp Neurol 2004;469:83–95.
[44] Rice FL, Albers KM, Davis BM, Silos-Santiago I, Wilkinson GA, LeMaster AM,
Ernfors P, Smeyne RJ, Aldskogius H, Phillips HS, Barbacid M, DeChiara TM,
Yancopoulos GD, Dunne CE, Fundin BT. Differential dependency of
unmyelinated and a delta epidermal and upper dermal innervation on
neurotrophins, trk receptors, and p75LNGFR. Dev Biol 1998;198:57–81.
[45] Rice FL, Fundin BT, Arvidsson J, Aldskogius H, Johansson O. Comprehensive
immunofluorescence and lectin binding analysis of vibrissal follicle sinus
complex innervation in the mystacial pad of the rat. J Comp Neurol
1997;385:149–84.
[46] Rice FL, Rasmusson DD. Innervation of the digit on the forepaw of the raccoon.
J Comp Neurol 2000;417:467–90.
[47] Riley CM, Day RL, Greely DM, Langford WS. Central autonomic dysfunction
with defective lacrimation; report of five cases. Pediatrics 1949;3:468–78.
[48] Sangameswaran L, Fish LM, Koch BD, Rabert DK, Delgado SG, Ilnicka M,
Jakeman LB, Novakovic S, Wong K, Sze P, Tzoumaka E, Stewart GR, Herman RC,
Chan H, Eglen RM, Hunter JC. A novel tetrodotoxin-sensitive, voltage-gated
sodium channel expressed in rat and human dorsal root ganglia. J Biol Chem
1997;272:14805–9.
[49] Schäfer MK, Eiden LE, Weihe E. Cholinergic neurons and terminal fields
revealed by immunohistochemistry for the vesicular acetylcholine transporter
II. The peripheral nervous system. Neuroscience 1998;84:361–76.
[50] Schreyer DJ, Skene JH. Fate of GAP-43 in ascending spinal axons of DRG
neurons after peripheral nerve injury: delayed accumulation and correlation
with regenerative potential. J Neurosci 1991;11:3738–51.
[51] Stokes L, Stark A, Marshall E, Narang A. Neurotoxicity among pesticide
applicators exposed to organophosphates. Occup Environ Med
1995;52:648–53.
[52] Toledo-Aral JJ, Moss BL, He ZJ, Koszowski AG, Whisenand T, Levinson SR, Wolf
JJ, Silos-Santiago I, Halegoua S, Mandel G. Identification of PN1, a predominant
voltage-dependent sodium channel expressed principally in peripheral
neurons. Proc Nat Acad Sci USA 1997;94:1527–32.
[53] Tooyama I, Kimura H. A protein encoded by an alternative splice variant of
choline acetyltransferase mRNA is localized preferentially in peripheral nerve
cells and fibers. J Chem Neuroanat 2000;17:217–26.
[54] Yasuhara O, Matsuo A, Bellier JP, Aimi Y. Demonstration of choline
acetyltransferase of a peripheral type in the rat heart. J Histochem Cytochem
2007;55:287–99.