Increased tyrosine hydroxylase immunoreactivity in the
urinary bladder of cats with interstitial cystitis
Archivaldo Reche Jr. DVM, PhDa, C.A Tony Buffington DVM, PhDb, Mitika K.Hagiwara
DVM, PhDa, Alexandre G.T. Daniela
a. From the Department of Medical Clinics, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Brazil
b.From the Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Ohio State University, Columbus, Ohio – USA
Author to whom correspondence should be sent: Archivaldo Reche Junior
Address: Av. Prof. Dr. Orlando Marques de Paiva, 87 – Ci. Universitária – 05508-900 – São Paulo – Brazil
Phone and Fax # 55 11 3091-1287
e-mail: valdorec@usp.br
Supported by NIH – NIDDK grant DK 47538
ABSTRACT
Reche Jr. A, Buffington CAT, Hagiwara MK, Daniel AGT. Increased tyrosine
hydroxylase immunoreactivity in the urinary bladder of cats with interstitial
cystitis
Purpose: The goals of this study were to identify and quantify the sympathetic nervous
fibers in the urinary bladder of healthy cats and those with interstitial cystitis (IC), but
with no clinical signs for at least 6 months prior the study.
Materials and Methods: Six healthy adult cats and six cats with IC were studied. All
cats were euthanized and immediately perfused transcardially with Krebs-Ringer and
4% paraformaldehyde solutions. The urinary bladders were collected and frozen at 70C until analysis. The tissue slides were processed for immunohistochemistry using
the avidin-biotin method for localization of neurons containing the enzyme tyrosine
hydroxylase(TH).
Results: An increased number of sympathetic nervous fibers was observed in the
lamina propria and muscular layer of the urinary bladder of cats with IC compared to
controls.
Conclusions: The increased number of catecholaminergic fibers in the urinary bladder
of cats with IC might be related to the neurogenic inflammation of the urinary bladder.
KEY WORDS: interstitial cystitis, cats, tyrosine hydroxylase, urinary bladder
INTRODUCTION
Interstitial cystitis is a chronic painful bladder disease of human beings and domestic
cats (Buffington et al 1996 ; Ratner, 2001). The symptoms of IC follow a waxing and
waning course, and often are exacerbated by stressful circumstances in both human
beings and cats (Buffington et al 1996; Rothrock et al 2001).
Viral infections, mast cell-mediated disease and defect in the superficial layer of
glycosaminoglycan, are included among the theories proposed to explain the vesical
inflammation in IC human beings and cats (Buffington et al 1996; Elbadawi, 1997).
Recently, neurogenic inflammation of the urinary bladder has been postulated as the
most reasonable explanation for the increased number of substance P-containing fibers
found in the urinary bladder from human beings and cats with IC (Caito, 1995; Ratner,
2001). Moreover, studies of neurogenic inflammation have shown that sensory fibers
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seem to interact with efferent sympathetic fibers, which does not occur in normal
conditions (Coderre et al 1989).
The involvement of the sympathetic nervous system (SNS) in neurogenic inflammation
has been extensively studied (Coderre et al 1989). The findings of increased numbers
of sympathetic fibers in the urinary bladder of humans with IC and the evidence of a
hyperactivity of the central noradrenergic neurons from the pontine locus coeruleus in
cats with IC, suggest that the SNS also may be involved in the pathogenesis of IC
(Reche, 1998 ; Peeker et al 2000).
The goals of this study were to identify and quantify the sympathetic nervous fibers in
the urinary bladder of healthy cats and cats with IC, but showing no clinical signs of
the disease for at least 6 months prior the study.
MATERIALS AND METHODS
Six healthy adult cats (3 males, 3 females), and six cats with IC (3 males, 3 females)
were studied. Healthy cats were determined to be clinically normal based on history,
physical examination findings and results of serum biochemical analyses, urynalises,
negative urine culture, and cystoscopy. Cats with IC were obtained from clients for
whom the cat’s urinary problems made them unacceptable as a pet. Diagnosis of IC
was based on history of recurrent frequent, painful and/or inappropriate (outside the
litter box) urination, hematuria, exclusion of other diagnoses based on urinalysis and
negative bacterial culture of the urine, and observation of glomerulations during direct
evaluation of the bladder of all cats by cystoscopy performed at 80 cm water pressure.
All cats were housed in stainless steel metabolism cages in the animal colony of the
College of Veterinary Medicine at The Ohio State University until they were used in the
experiment.
For tissue collection, cats were anesthetized with 2.5% halothane in air, euthanized
with sodium pentobarbital (50 mg./Kg. i.p.), and immediately perfused transcadially
with Krebs-Ringer solution (2-3liters/cat), containing 3.3mg sodium nitroprusside and
333 units heparin per liter to wash blood from circulatory system, followed by 4%
paraformaldehyde at 4ºC . Then the urinary bladders from each cat was collected and
transferred to 0.1M phosphate buffer saline (PBS) containing 30% sucrose (pH 7.4) at
4ºC for 48 hours, and then frozen at –70ºC until analysis. Prior to staining, ten m.
sections were cut using a cryostat, collected on chrome-alum gelatin-coated slides and
air-dried. Sections were taken from three different regions of the bladder: apex, body
and neck.
Slide-mounted sections from all cats were coded to prevent identification of the animal
from which it had come, and processed in a single batch for immunohistochemical
localization of neurons containing the enzyme tyrosine hydroxylase (TH). Sections
were rinsed for 10 minutes (twice) in 0.1M PBS containing 0.3% Triton X-100 (PBST;
pH 7.4), and soaked in 0.01% trypsin and 0.1% CaCl 2 in PBST for 1 minute. To block
endogenous tissue peroxidase activity, sections were incubated for 30 minutes in 3.0%
hydrogen peroxide in methanol. Following another rinse with PBST, sections were
incubated for 1 hour with normal goat serum (BioGenex Laboratories, San Ramon,
CA), and then for 72 hours with either primary rabbit anti-TH (1:500) (Pel-Freez
Biologicals, Rodgers, AR) or non-immune rabbit serum, which served as negative
controls. Biotinylated anti-rabbit IgG and peroxidase-conjugated streptavidin
(BioGenex Laboratories) were then applied to sections and incubated for 20 minutes
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each. The peroxidase reaction was carried out with 3,3”-diaminobenzidine
tetrahydrochloride (0.05%) as chromogen and 0.01% hydrogen peroxide as oxidant,
after which sections were dehydrated and mounted (Permount  Fisher Scientific,
Pittsburgh, PA).
Coded sections from all cats were examined for TH-IR. Images were obtained during a
single session using a video camera attached to a microscope and digitized using
identical illumination parameters. Montages of 40X photomicrographs were constructed
and analyzed using the same threshold (Adobe Photoshop 3.0. Adobe Systems, Inc.
Seattle, WA). The TH-IR was measured in each section, and the mean value of each
group was used for statistical analysis. After analysis, the code was broken and results
obtained from control subjects were compared with those obtained from cats with IC
by Student’s unpaired t test using microcomputer based software (Sigma-Stat, Jandel
Scientific, San Rafael, CA). Differences between groups were considered significant
when the value of p was less than 0.05. Results are reported as mean  S.D.
RESULTS
The observed cell distribution and morphology corresponded well with previous
descriptions of catecholamine-containing neurons in the urinary bladder of cats
(Wakabayashi et al 1993).
The overall TH-IR was significantly greater (p<0,05) in sections from cats with
IC (28.9 ± 2.4%) than controls (19.8 ± 2.7%) (Figures 1 A and B).
Considering separately the three different regions of the urinary bladder the THIR was also significantly greater in the mucosa of cats with IC than controls: apex
(26,5 ± 2,7% vs 19,7 ± 1,4% p<0,05), body (28,6 ± 3,4% vs 20,6 ± 3,7% p<0,05)
and neck (31,8 ± 1,3% vs 25,2 ± 3,2% p<0,05). The same was observed in the
muscular layers: apex (11,2 ± 7,6% vs 7,6 ± 0,6% p<0,05), body (12,4 ± 1,6% vs
8,3 ± 2,1% p<0,05) and neck (15,5 ± 0,8% vs 11,8 ±1,6% p<0,05).
DISCUSSION
The main finding of this study was a significant increase in the density of TH-IR fibers
throughout the bladder of cats with IC. Tyrosine hydroxylase is the rate-limiting
enzyme in catecholamine synthesis, and serves as a marker of SNS activity. The
results of our study are consistent with others that have reported increased neuron
density in the bladder of human beings with IC (Peeker et al 2000).
These results might help explain the role of autonomous nervous system in the
pathogenesis of the neurogenic inflammation associated with IC. Normally, there is no
communication between post-ganglionic sympathetic neurons and sensory neurons,
i.e., sensory neurons are neither stimulated nor sensitized by the activation of
sympathetic neurons, except by few occasional indirect impulses, or during nonphysiological stimulation of sympathetic neurons (Jänig and Koltzenburg 1992).
Therefore, afferent input from the periphery to the spinal cord and efferent
sympathetic output from the spinal cord to the periphery are functionally independent
in normal physiological conditions. This distinction between peripheral afferents and
sympathetic efferents may not exist during inflammation, when efferent sympathetic
neurons interact with afferent fibers, possibly through release of prostaglandins, thus
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contributing either to the severity of the inflammation and/or the hyperalgesia (Levine
et al 1986; Coderre et al 1989).
Besides IC, SNS activation seems to aggravate other inflammatory diseases like
arthritis. Guanethidine, which induces an immune-mediated sympathectomy, or
reserpine, which depletes catecholamines both in the peripheral and central nervous
system, significantly reduces the severity of joint lesions in rats with arthritis induced
by the intra-articular injection of adjuvant. These observations may suggest that
efferent post-ganglionic sympathetic neurons contribute to the articular lesions in the
arthritis caused by adjuvant injection. Additionally, rats from a spontaneously
hypertensive lineage, which shows a more intense activity of the SNS, develop more
severe arthritis than those from non-hypertensive lineages (Levine et al 1986).
Stressful events can aggravate IC symptoms in both human beings and cats (Caston,
1973; Rothrock et al 2001). Stress also seems to be involved in the exacerbation of
the symtoms of other inflammatory diseases, such as rheumathoid arthritis (Baker,
1982). However, the mechanism by which stress could cause the symptoms of these
diseases to reoccur or exacerbate is unknown or merely speculative for the time being.
The effect of external stressors on bladder sensory fibers might be important for the
onset or the maintenance of the vesical inflammation in IC patients. It is believed that
2 adrenoceptors may mediate activation of sensory fibers in chronic inflammation
(Gold et al 1997).
Therefore, in stressful circumstances, central stimuli via efferent sympathetic neurons
might stimulate some sensory neurons to release inflammatory neurotransmitters.
However, a local release of neurotransmitters by efferent sympathetic endings could
also stimulate the sensory fibers to release their neurotransmitters. Sensory fibers
could also propagate a local axon reflex after activation by circulating catecholamines.
This reflex would result in release of neurotransmitters such as SP by nerve endings.
The interaction of SP with specific receptors in the wall of blood capillaries would
enhance the vascular permeability, which could be increased by the action of
histamine, released during mast cell degranulation induced by SP. Increased capillary
permeability could result in formation of the glomerulations usually observed during
cystoscopy, and in the histopathological lesions of the bladder observed both in cats
and human beings with IC (Buffington et al 1996; Messing et al 1997).
Studies using electronic microscopy have shown a likely connection between mast cells
and autonomic nervous fibers, and that the former could be involved in the nervous
proliferation (Wiesner-Menzel et al 1981). The observation that the bladder of both,
cats and human beings with IC show an increased number of mast cells, suggests that
these cells migth participate in the nervous proliferation, observed in the bladder of
these patients (Messing, 1987; Buffington et al 1996).
Additionally, the high levels of nervous growth factor (NGF) found in the bladder of
human beings with IC seem to participate directly in the vesical neuroplasticity,
observed in these patients (Lowe et al 1997). It is also known that an excessive
production of NGF could increase sympathetic innervation locally (Spitsbergen et al
1995), which would explain the fact that the bladder of human beings with IC shows
an increased number of efferent sympathetic fibers (Peeker et al 2000), as well as the
results of the present study that show the vesical sympathetic neuroplasticity in cats
with IC.
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Activation of sensory fibers likely results in damage to the urothelium. It is known that
both cats and human beings with IC show decreased renal excretion of
glycosaminoglycan, which represents an important layer of protection of the vesical
epithelium. Therefore, it is supposed that these patients, cats and human beings,
urothelial damage may result in failures of this protective glycosaminoglycan layer,
possibly as a consequence of increased local release of nitric oxide (Hassan et al
1998). This would permit the components of urine, such as protons and potassium ions
to reach deep layers of the bladder and to activate sensorial fibers thus initiating the
inflammation (Buffington et al 1996).
These results are consistent with those of other investigators who have evaluated the
role of the autonomic nervous system in patients with IC, like the report in which
women with IC had abnormal peripheral vasomotor tone, which might be indicative of
increased spinal sympathetic activity (Irwin et al 1993). Moreover, an increased plasma
and urinary norepinephrine concentration was found in cats with interstitial cystitis
(Buffington and Pacak 2001, Buffington et al 2002). However, one must still wonder if
the increase in the number of efferent catecholaminergic fibers observed in the
bladders of cats with IC is primary or secondary to the inflammation. It is possible that
the increased afferent sensory or the sympathetic efferent activity observed in the
bladder of both species with IC occurs secondarily to inflammation. In this case, the
nervous system would play an important role both in the exacerbation and
maintenance of the inflammation.
CONCLUSION
The demonstration that cats with IC show an increased number of catecholaminnergic
fibers in the urinary bladder supports the neurogenic theory of vesical inflammation in
IC. However, further studies must be carried out to attempt to clarify the mechanism
by which efferent sympathetic fibers might interact with sensory neurons, both
peripherically and centrally in the pathogenesis of IC.
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Figure 1: Photomicrographs of sections of urinary bladder (body) of an IC cat (A) and a
normal cat (B), immunostained for tyrosine hydroxylase. Note increased staining
density in the urinary bladder of the IC cat compared to normal cat.
FIGURE 1 A
FIGURE 1 B