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Depletion of corticotrophin-releasing factor neurons in the pontine micturition area in multiple system atrophy.

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Depletion of Corticotrophin-Releasing Factor
Neurons in the Pontine Micturition Area in
Multiple System Atrophy
Eduardo E. Benarroch, MD, DSci, and Ann M. Schmeichel, AS
We sought to determine whether the putative pontine micturition center in the human dorsal pons contains
corticotrophin-releasing factor (CRF) neurons, and whether these neurons are depleted in patients with multiple system
atrophy and bladder dysfunction. Brains were obtained at autopsy from 4 control subjects and 4 patients with clinical
diagnosis of multiple system atrophy, confirmed neuropathologically. Serial 50␮m cryostat sections were obtained
throughout the rostral half of the pons, and every eighth section was processed for CRF immunocytochemistry (rabbit
polyclonal antibody). Consecutive sections were stained for nicotinamide adenine dinucleotide phosphate diaphorase
(NADPH-d) to identify neurons of the laterodorsal tegmental nucleus or for both CRF and NADPH. Locus ceruleus
neurons were identified by their neuromelanin content. Abundant CRF immunoreactive neurons were identified in the
dorsal pontine tegmentum just ventral to the locus ceruleus. CRF neurons were intermingled with, but distinct from, the
NADPH-d-reactive neurons of the laterodorsal tegmental nucleus. In all multiple system atrophy cases, there was a severe
depletion of these CRF-immunoreactive neurons (26.6 ⴞ 3 neurons/section in patients; 73.7 ⴞ 4 neurons/section in
controls). Our results suggest that depletion of CRF neurons in the putative pontine micturition center may contribute
to the severe bladder dysfunction that characterizes multiple system atrophy.
Ann Neurol 2001;50:640 – 645
Abundant experimental evidence indicates that a restricted area of the dorsal pons is critically involved in
control of the micturition reflex, allowing coordinated
contraction of the bladder detrusor and relaxation of
the external urethral sphincter.1 This region, referred
to as the M region,2 corresponds to the classic Barrington nucleus or pontine micturition center3 described in experimental animals. Neurons of this region
send direct excitatory projections to the sacral parasympathetic nucleus, and inhibit more laterally located
pontine neurons that activate the sphincter motor neurons of the Onuf nucleus.2 Neurons of the putative
pontine micturition center contain corticotrophinreleasing factor (CRF), as well as glutamate and other
cotransmitters.4 Positron emission tomography studies
in normal subjects5 and clinical consequences of lesions6 indicate that the dorsal pontine tegmentum in
humans contains a region corresponding to the pontine
micturition center. The presence of CRF neurons in
this region and their relationship with neighboring cell
groups has not yet been explored in humans.
Patients with multiple system atrophy (MSA) characteristically have early and prominent bladder dysfunction.7 Although this has been attributed to neuro-
nal loss in the sacral parasympathetic nucleus and the
Onuf nucleus,8 the possibility of involvement of supraspinal structures controlling micturition has not
been explored. We sought to determine whether (1)
the area of the dorsal pons containing the putative
pontine micturition center in humans contains CRF
neurons; and (2) these CRF immunoreactive neurons
are depleted in patients with MSA and impaired bladder function.
From the Department of Neurology, Mayo Clinic, Rochester, MN.
Received May 29, 2001, and in revised form Jul 27. Accepted for
publication Jul 30, 2001.
Address correspondence to Dr Benarroch, Department of Neurology, Mayo Clinic, 811 Guggenheim Building, 200 First Street SW,
Rochester, MN 55905. E-mail: benarroch.eduardo@mayo.edu
Published online Oct 9, 2001; DOI: 10.1002/ana.1258
640
© 2001 Wiley-Liss, Inc.
Patients and Methods
Brains were obtained at autopsy from 4 subjects (1 man and
3 women, age 65 ⫾ 5 years) with no history of neurological
disease, and 4 patients (all men, age 65 ⫾ 2 years) with
clinical diagnosis of MSA (Table). Three of these patients
had parkinsonism (MSA-P), and 1 had cerebellar ataxia
(MSA-C) as the presenting feature of their disease. All had
severe orthostatic hypotension, impotence, and urinary incontinence and retention requiring indwelling catheterization. Two patients had prominent gastrointestinal dysmotility. Postmortem delay was not significantly different between
controls (16 ⫾ 3 hours) and MSA patients (17 ⫾ 4 hours).
The brains were divided at the level of the pontomesencephalic junction. The forebrain and midbrain were
processed for routine neuropathological studies, including
Table. Subject Population
Age (yr)/
Gender
Postmortem
Delay (hr)
Control 1
Control 2
58/M
75/W
8.5
25.0
Control 3
Control 4
MSA 1
67/W
52/W
70/M
15.0
16.0
25.0
MSA 2
70/M
8.0
MSA-P
15
MSA 3
67/M
24.0
MSA-P
7
MSA 4
54/M
13.0
MSA-C
8
Case
Disease
Duration (yr)
Diagnosis
Lymphoma
Acute respiratory distress
syndrome
Gastrointestinal bleeding
Congestive heart failure
MSA-P
5
N/A
N/A
Lifelong
5
Autonomic Symptoms
None
None
None
None
OH, bladder incontinence, urinary
retention, impotence
OH, bladder incontinence, urinary
retention, impotence
OH, incontinence, urinary retention,
impotence, constipation, distal
anhidrosis
OH, incontinence, urinary retention,
impotence, upper GI dysmotility
MSA ⫽ multiple system atrophy; MSA-P ⫽ parkinsonism-predominant MSA; OH ⫽ orthostatic hypotension; MSA-C ⫽ cerebellarpredominant MSA; GI ⫽ gastrointestinal.
immunostaining for ␣-synuclein. In all cases the diagnosis of
MSA was confirmed neuropathologically, with evidence of
neuronal loss in the substantia nigra pars compacta, putamen, basis pontis, and cerebellum; and accumulation of
␣-synuclein-immunoreactive oligodendroglial cytoplasmic inclusions. In no cases were Lewy bodies identified in these
regions or in the cerebral cortex.
For the purpose of the present study, the pons and medulla were immersion fixed in 2% paraformaldehyde in 0.1%
phosphate buffer for 24 hours at 4°C, cryoprotected in a
buffered 30% sucrose solution for 24 hours, and frozen on
dry ice. Serial 50␮m cryostat sections were obtained
throughout the rostral half of the pons (22–32mm above the
obex). Every eighth section was processed for CRF immunocytochemistry using a rabbit polyclonal anti-CRF antibody
(1:1000; Peninsula Laboratories, San Carlos, CA). Diaminobenzidine/glucose oxidase solution with nickel enhancement
(Sigma, St. Louis, MO) was used for the substrate reaction.
Specificity was tested by omission of the primary antibody.
Under these conditions, no immunostaining was detected.
Nickel enhancement was used to obtain a black staining of
CRF-immunoreactive neurons to help distinguish them from
the neighboring neuromelanin-containing neurons of the locus ceruleus. Subsequent sections were histochemically
stained for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d),9 which is a marker of cholinergic
neurons of the laterodorsal tegmental nucleus (LDT). Some
sections were processed for both CRF and NADPH-d to assess the possibility and extent of colocalization of these two
markers. Neurons of the locus ceruleus were identified by
their neuromelanin content.
The sections were examined under bright field microscopy. In the 4 controls, the number of sections examined
were 19, 10, 10, and 12, respectively. In the 4 MSA patients
the number of sections were 20, 10, 19, and 17, respectively.
These sections were at comparable rostro–caudal levels, as
assessed by the distribution of neuromelanin-containing locus ceruleus and NADPH-d-reactive LDT neurons. Sections
were examined on an Axiophot microscope (Carl Zeiss,
Oberkochen, Germany) equipped with a 2.5⫻ (NA 0.075)
objective lens. Image analysis was performed using a Zeiss
KS400 image analysis system. Cell counts were performed
manually, including both sides of the section. No correction
for split size was used, as there was no significant difference
in size between the surviving CRF neurons in MSA (32 ⫾
1␮m; n ⫽ 6) and control (35 ⫾ 2␮m; n ⫽ 6) cases. Cell
numbers (mean ⫾ SEM) were compared among individual
cases using analysis of variance; comparison between the control and MSA groups was performed using unpaired t test
( p ⬍ 0.05 was considered significant).
Results
Abundant CRF-immunoreactive neurons were identified in the dorsal pontine tegmentum, in all segments
examined, between 22 and 32mm rostral to the obex
(Fig 1). These CRF-immunoreactive neurons were located just ventral to the neuromelanin-containing neurons of the locus ceruleus, and had a polygonal shape
and size of between 20 and 40␮m. These CRF neurons were intermingled with, but distinct from, the
NADPH-d-reactive neurons of the LDT. Processes for
NADPH-d neurons appeared to reach the CRF cells
(see Fig 1). Overall, the topographic distribution of the
CRF-immunoreactive neurons in relationship with
neurons of the LDT and locus ceruleus was similar to
that described in experimental animals (Fig 2), and
corresponded to the area of activation during micturition in humans, as assessed with functional neuroimaging.
In all cases with clinical and pathological diagnosis
of MSA, there was severe depletion of CRFimmunoreactive neurons in this putative micturition
area of the dorsal pons (26.6 ⫾ 3 neurons/section in
MSA; 73.7 ⫾ 4 neurons/section in controls; p ⬍ 0.01;
Fig 3). There was no significant difference in size be-
Benarroch and Schmeichel: CRF Depletion in the Pontine Micturition Area
641
bers among individual controls or MSA patients (Fig
4). There was no significant difference in the numbers
of neighboring neurons in the trigeminal motor nucleus used as a control.
Fig 1. Fifty-micrometer section of the rostral pons processed for
immunocytochemistry for corticotrophin releasing factor (CRF)
either alone (A) or together with histochemistry for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d;
B). The CRF-immunoreactive neurons had a typical polygonal
shape and a size of 20 to 40␮m, and were distinct from
neighboring NADPH-d neurons. NADPH-d processes appear
to surround the CRF neurons. Bar ⫽ 50␮m.
tween the surviving CRF neurons in MSA (32 ⫾
1␮m; n ⫽ 6) and control (35 ⫾ 2␮m; n ⫽ 6) cases.
There was little interindividual variation of cell num-
Discussion
Our results show that (1) there are CRF-immunoreactive neurons in the area of the dorsal pontine tegmentum corresponding to the putative pontine micturition center in humans; and (2) these CRF neurons
are depleted in patients with MSA. The presence of
CRF-immunoreactive neurons in the human pontine
micturition area and their topographic relationship
with neurons of the locus ceruleus and LDT nucleus
are consistent with findings in experimental animals.10
The overall distribution of the CRF neurons identified
in this study was distinct from that of the NADPH-dreactive neurons of the LDT, also consistent with experimental evidence.10 However, the coexistence of
both markers in some neurons cannot be absolutely excluded in 50␮m-thick sections. Double labeling for
CRF and nitric oxide synthase would have further supported our findings, but this would have required immunofluorescence techniques, which are difficult to apply to human brain tissue. The close topographic
relationship between the CRF-immunoreactive neurons, NADPH-d-reactive LDT neurons, and neuromelanin-containing neurons of the locus ceruleus suggests that in humans, as in experimental animals, there
is potential for interconnections and therefore functional interactions among these three cell groups.4 Although NADPH-d-reactive processes appeared to reach
Fig 2. Computer reconstruction of a 50␮m section obtained 26mm above the obex from a 67-year-old woman with no history of neurological disease. A plotted distribution of CRF neurons, NADPH-d neurons of the laterodorsal tegmental nucleus (LDT), and
neuromelanin-containing neurons of the locus ceruleus (LC) is shown. CRF neurons were located just ventral to the locus ceruleus and
intermingled with NADPH-d neurons of the LTD. There was little or no overlap between the distribution of CRF and NADPH-d
neurons.
642
Annals of Neurology
Vol 50
No 5
November 2001
Fig 3. (A) Fifty-micrometer sections of the
dorsal pons obtained 30mm above the
obex and processed for CRF immunoreactivity. (B and C) Composed images obtained by plotting the distribution of CRF
neurons in serial sections obtained 400␮m
apart. (B) Seventy-five-year-old man with
no history of neurological disease (postmortem delay, 25 hours). (C) Seventy-year-old
man with clinical and neuropathological
diagnosis of MSA and history of urinary
retention and incontinence (postmortem
delay, 8 hours). There was a severe depletion of CRF neurons in the dorsal pons of
the MSA patient. Bar ⫽ 50␮m.
and surround the CRF-immunoreactive cells, our study
on 50␮m-thick sections does not allow to definitely
establish whether these processes contact the CRF neurons.
Although our results do not provide direct evidence
that the dorsolateral pontine CRF neurons in humans
are involved in micturition and project to the sacral
cord, their similar distribution and relationship with
other neuronal groups in the region suggest that they
are likely to be akin to the CRF-containing pontine
micturition neurons of the M region, or Barrington’s
nucleus, which have been shown to project to the sacral parasympathetic nucleus in experimental animals.2,4 Positron emission tomography studies indicate
that the pontine region containing these neurons is activated during micturition,5 and lesions in this region
produce neurogenic bladder dysfunction, including
bladder hypotonia, in humans.6 Thus, it can be speculated that in humans, CRF-containing projections
from the dorsolateral pontine tegmentum neurons may
modulate the activity of sacral parasympathetic neurons
controlling the bladder, as well as rectal and sexual
function.4
Our results show that patients with MSA have a
profound depletion of CRF-immunoreactive neurons
in the putative pontine micturition center. Neuronal
depletion could not be attributed to the effects of age
or postmortem delay. One limitation of our study is
the small number of subjects, all of whom were men.
However, the consistency of our findings along all the
rostrocaudal levels analyzed, and the small variation in
cell number between subjects, indicate that loss of dorsolateral pontine CRF neurons is a consistent feature in
MSA. The main question that arises from our results is
whether depletion of these CRF neurons contributes to
bladder dysfunction in MSA. Early and prominent
bladder dysfunction, including detrusor hyperreflexia
followed by urinary incontinence and retention caused
by hypotonic bladder, is a typical feature of
MSA8,11–13 and was present in the 4 subjects analyzed
in this study. In MSA, urinary incontinence has been
attributed primarily to a combination of factors. These
include incomplete emptying, open bladder neck, and
a degree of detrusor hypererflexia. Denervation of the
external urethral sphincter reflects the loss of neurons
in the Onuf nucleus8,14,15; whereas hypotonic bladder
could be explained by loss of sacral parasympathetic
neurons.8 Given the involvement of these target neurons, the relative contribution of impairment of pontine as compared to sacral micturition mechanisms to
bladder dysfunction in MSA may be difficult to determine. Unfortunately, the sacral cord was not available
for this study, but we plan to examine both the pon-
Benarroch and Schmeichel: CRF Depletion in the Pontine Micturition Area
643
Fig 4. Number of CRF-immunoreactive neurons per section in 50␮m sections of the pontine micturition area obtained between 22
and 32mm above the obex in 4 controls and 4 subjects with MSA. There were no significant differences in the number of CRF
cells per section among individual controls or among MSA patients. There was a severe depletion of CRF neurons in the dorsal
pons in MSA. ***p ⬍ 0.001.
tine and sacral micturition regions of a larger number
of subjects, to obtain further insight into this problem.
A second question is, what is the mechanism by
which depletion of CRF neurons in the pontine micturition center could contribute to bladder dysfunction
of MSA. This neuropeptide is present in several central
autonomic pathways, such as projections from the
paraventricular nucleus and the extended amygdala to
autonomic nuclei of the brainstem and spinal cord. As
demonstrated in experimental animals, neurons of the
pontine micturition center activate the sacral parasympathetic neurons via glutamatergic mechanisms, and
CRF may be cotransmitter of these descending projections and modulate these excitatory effects.4 Lesions involving the pontine micturition center may impair detrusor contraction and produce urinary retention.6
Therefore, it is conceivable that interruption of the
micturition reflex at the level of the pons may deprive
the sacral preganglionic neurons from supraspinal modulation. In addition, loss of CRF neurons in the M
region may impair the inhibitory effect of this area on
neurons of the L region that activate the Onuf nucleus.
Our results provide several directions for further research. First, it is necessary to confirm these findings
by studying a larger group of MSA patients, ideally
comparing patients with prominent neurogenic bladder
644
Annals of Neurology
Vol 50
No 5
November 2001
dysfunction with those with less severe dysfunction.
This may prove difficult, as neurogenic bladder occurs
early in the disease, and most patients have had longstanding bladder dysfunction at the time of death. Second, we are currently collecting samples to study the
pontine CRF neurons in subjects with Parkinson’s disease. This would provide very useful information, as
bladder dysfunction in Parkinson’s disease typically differs from that in MSA. Parkinson’s disease does not
typically affect the Onuf nucleus, but can produce internal sphincter denervation caused by loss of sympathetic neurons.8 If pontine CRF neurons are also depleted in patients with Parkinson’s disease in the
absence of bladder hypotonia, these neurons may not
be critical for the pathophysiology of detrusor arreflexia
and urinary retention in MSA, but may contribute to
detrusor hyperreflexia in both disorders.
Regardless of its relative contribution to the pathophysiology of neurogenic bladder in MSA, depletion of
putative pontine micturition CRF neurons projecting
to and modulating excitatory responses in the sacral
parasympathetic nucleus provides further evidence to
suggest that MSA produces a “system” degeneration of
autonomic as well as basal ganglia and cerebellar circuits. This is consistent with the finding that MSA
produces depletion not only of preganglionic sympa-
thetic neurons but also of the C1 catecholaminergic
neurons of the rostral ventrolateral medulla.16 These
neurons monosynaptically project and modulate the response to the preganglionic sympathetic neurons to excitatory glutamatergic inputs. The concept of system
degeneration thus provides testable hypotheses on
mechanisms of neurodegeneration in central autonomic
pathways in MSA. For example, lack of supraspinal
modulation may lead to excessive depolarization of target preganglionic sympathetic or parasympathetic neurons in response to segmental or descending glutamatergic afferents. This would result in excitotoxic
neuronal injury and the loss of these neurons that is
characteristic of MSA.
This work was supported in part by the National Institute of Neurological Disorders and Stroke (PO1 NS32352-P2).
We appreciate the collaboration of Dr Joseph Parisi from the Department of Pathology at the Mayo Clinic, Rochester, MN, who
performed the neuropathological examination to confirm the diagnosis of MSA in our cases.
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