INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY Vol. 21, no. 1, 0-0 (2008)

0394-6320 (2008)Copyright © by BIOLIFE, s.a.s.

This publication and/or article is for individual use only and may not be furtherreproduced without written permission from the copyright holder.

Unauthorized reproduction may result in financial and other penalties129

ACTIVITY AND EXPRESSION OF NITRIC OXIDE SYNTHASE IN RAT BLADDER AFTER SACRAL NEUROMODULATION

D. MINARDI, R. GHISELLI1, G. LUCARINI2, F. MOCCHEGIANI1, A. FILOSA3, A. ZIZZI2,O. SIMONETTI4, F. ORLANDO5, G. PELLICCIONI6, G. PARRI, V. SABA1, L. LO MUZIO7,

G. BIAGINI2, R. MONTIRONI3 and G. MUZZONIGRO

Institute of Urology, 1Institute of General Surgery (INRCA-IRRCS), 2Department of Molecular Pathology and Innovative Therapies–Histology, 3Institute of Pathology, 4Clinic of Dermatology,

Polytechnic University of the Marche Region, Ancona; 5Biotechnology Centre Research Department, 6Department of Neurology, INRCA-IRRCS, Ancona; 7Department of Surgical Science,

University of Foggia, Foggia, Italy

Received July 4, 2007 – Accepted October 25, 2007

The aim of our study is to investigate the effects of chronic sacral neuromodulation on Nitric Oxide (NO) metabolism in the rat bladder. 26 female Sprangue-Dawley rats were considered: group I, normal control rats; group II, a sham treatment, in whom catheters for electrical stimulation were placed in the S1 foramen bilaterally and left in place for 21 days, without performing neuromodulation; group III in whom electrical sacral neuromodulation was performed for 21 days. Finally a cystectomy was performed and the bladder biopsy specimens were sent for immunostaining with n-NOS and i-NOS. Morphological and immunohistochemical analysis was carried out, and evaluated in urothelial cells, endothelial cells and muscle fibers of the muscularis propria. Differences between the 3 groups were analyzed by Student Newman-Keuls test. We could observe that urothelial and endothelial i-NOS (37.00+4.69 and 59.00+7.42 respectively) and urothelial n-NOS (36.80+7.85) expression are significantly increased in neuromodulated rats, compared to groups 1 and 2 (p < 0.005). In conclusion, the increase of i-NOS expression on endothelial cells after sacral neuromodulation could be in some way related to angiogenetic responses in the microvascular structures; the increase of n-NOS and i-NOS expression on urothelial cells can suggest that NO is able to influence the plasticity of bladder response, inducing the release of messengers within the urothelium. This study can therefore improve our understanding of the mechanisms of sacral neuromodulation on chronic bladder dysfunction; further studies will need to better demonstrate the role of angiogenesis in the bladder after sacral neuromodulation and to investigate the effects of neuromodulation in rats with chronically induced bladder dysfunction.

Mailing address: Dr. Daniele Minardi,Institute of Urology,Polytechnic University of the Marche Region,A.O. Ospedali Riuniti,Via Conca 71, 60100 Ancona, ItalyTel: ++39 071 5963377 Fax: ++39 071 5963367e-mail d.minardi@univpm.it

Key words: nitric oxide, bladder, neuromodulation

Sacral neuromodulation for treating chronic functional voiding dysfunction has become established in Urology; but although it represents a valid treatment option for patients who do not show

improvement with conventional pharmacological and surgical treatment, very little is known regarding the underlying mechanism of electrical stimulation (1-2): some authors found that the blockade of C-

130 131Int. J. Immunopathol. Pharmacol.

afferent fiber activity is one of the mechanisms of action of sacral root neuromodulation (3); alterations in neuropeptide expression in lumbosacral bladder pathways can also be involved (4).

The role of Nitric Oxide (NO) has begun to be appreciated in the pathogenesis of bladder dysfunctions and some studies indicate that NO has an important role in functional neuroregulation of the lower urinary tract and is involved in acute and chronic inflammation (5); NO was discovered to be a potent vasodilator in 1979 (6) and later identified as an endothelium-relaxing factor (EDRF). This simple molecule is a short-lived free radical, which has a number of physiological functions including smooth muscle relaxation and nonadrenergic-noncolinergic neurotransmission and has emerged as a potent biological mediator and neurotransmitter in the central and peripheral nervous system, cardiovascular and immune system, and participates in the functioning of neurons, glia, macrophages and endothelial cells (7). NO is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS); there are three isoforms of NOS: endothelial NOS (e-NOS) and neuronal NOS (n-NOS) are constitutive, i.e. normal constituents of cells, and Ca2+-dependent for activity; the third isoform of NOS is inducible (i-NOS) and Ca2+-independent and is present in various cells including glomerular, tubular epithelial cells and urothelium.

The aim of our study is to investigate whether chronic sacral neuromodulation can have an effect on the bladder in an animal model; in particular we wanted to observe whether sacral neuromodulation is able to influence the expression of neuronal (n-NOS) and inducible (i-NOS) nitric oxide synthase in the bladder of rats.

MATERIALS AND METHODS

This study was conducted in accordance with the Italian law on the protection of animals, and approved by the Animal Research Ethics Committee of the INRCA, IRCCS (Istituto Nazionale di Ricovero e Cura Anziani, Istituto di Ricovero e Cura a Carattere Scientifico), Polytechnic University of the Marche Region, Ancona, Italy; it is a spontaneous investigation, not commissioned by Industry; the funds for the research came from a grant offered by the Polytechnic University of the Marche Region, after approval of the research project.

Twenty-six female Sprangue-Dawley rats weighing 220 to 250 g were examined in our study and divided into 3 groups: group I, normal control rats (n = 6); group II, a sham treatment (i.e. rats in whom catheters for electrical stimulation were placed in the S1 foramen bilaterally and left in place for 21 days without performing neuromodulation) (n = 10); group III in whom, through the catheters left in place for 21 days, electrical sacral neuromodulation was performed (n = 10).

Electrodes were implanted under general anesthesia using a combined subcutaneous injection with acepromazine 0.05 mg/kg, ketamine 50 mg/kg and xylasine 5mg/kg. A 2 cm dorsal midline incision was made over the upper sacrum; a 22 G spinal needle was used to probe into the S1 foramens bilaterally to stimulate the S1 dorsal root; the location was confirmed by the tail-tremor response to the electrical stimulation delivered by an external pulse generator, as previously described (2); two commonly available PNE wire electrodes were placed bilaterally; the other ends of the electrodes were extended from back skin through a subcutaneous tunnel, and anchored to the skin using a 3/0 non-absorbable suture; the subcutaneous fascia and skin were closed with 4/0 vicryl suture.

Electrical stimulation was started the day after electrode implantation in group III; it lasted for 6 hours daily and for 21 days; the stimulator units consisted of a 3245 A Universal Source generator (Hewlett-Packard, USA); bipolar pulses of 3 volts at a frequency of 20 Hz were delivered, and the stimulation amplitude was adjusted to 80% of the value at which a visible tail movement could be induced. The animals did not show any discomfort and consumed rat chow and water ad lib during the stimulation period.

At the end of the procedures the rats were sacrificed, cystectomy was performed and bladder biopsy specimens were sent to the laboratory for morphology and immunohistochemistry.

ImmunohistochemistryThe bladder biopsies obtained from the surgical

specimens of the 3 groups were frozen in liquid nitrogen and stored at –70°C. 6 µm-thick sections were air-dried overnight and then fixed in acetone for 10 min. Sections were then incubated overnight at 4°C with the following polyclonal antibodies: anti-n-NOS (dil. 1:50, BD Biosciences, US) and anti-i-NOS (dil. 1:150, Santa Cruz, CA). The reaction was revealed using the streptoavidin-biotin-peroxidase technique (Dako-LSAB peroxidase kit, Dako). Positive controls were obtained from frozen sections of rat liver biopsies, previously shown to react with primary antibodies. Negative controls were performed by substituting the primary

D. MINARDI ET AL.

130 131Int. J. Immunopathol. Pharmacol.

antibody with non immune serum. The number of n-Nos and i-Nos positive cells was counted among at least 500 cells in the more representative fields by using a light microscope at X200 magnification and expressed as mean value + standard deviation (SD). We observed the immunostaining in urothelial cells, endothelial cells of sub epithelial connective tissue vessels and muscle fibers of the muscularis propria. The count was performed by two investigators (G.L., F.A.) simultaneously, using a double-headed light microscope. Both had to agree on the count of the positive cells.

Statistical analysis All results were expressed as mean values ± SD.

Differences between the 3 groups were analyzed by Student Newman-Keuls test. Significance was set at p < 0.05.

RESULTS

The n-NOS and i-NOS immunoreactivity is shown in Table I.

n-NOS was highly represented in endothelial cells of the control group (62.33+2.52) (Fig. 1a), whilst in the sham treatment group and after neuromodulation its expression was slightly reduced (56.75+5.38 and 48.00+4.69 respectively) (Fig. 1b-c); the expression of n-NOS in muscle cells was equally low in the three groups of animals (16.67+4.16, 18.75+2.22, 17.00+4.69, respectively), whilst its expression is increased in urothelial cells of neuromodulated rats

(36.80+7.85) (Fig. 1c) compared to groups I and II (15.67+4.04 and 16.75+3.77, respectively) (Fig. 1 a-b).

Considering i-NOS expression, we could observe that the control group showed a low level of immunoreactivity (Fig. 1d); in detail, the muscle cells of the control group were the least immunoreactive (mean value 7.67±2.52), followed by the urothelial cells (mean value 8.33±3.51) and the endothelial cells (mean value 12.33±2.52); the endothelial cells of group II showed a slight increase of staining compared to group I (20.01+4.08) (Fig. 1e), while after neuromodulation group III exhibited high frequency of diffuse immunoreactivity (59.00+7.42) (Fig. 1f); in the muscle cells of groups II and III rats, the i-NOS expression was similar (9.25+3.50 and 10.20+3.70) to the control group (7.67+2.52); on urothelium, i-NOS expression showed a slight increase in the sham treatment group (10.25+3.50) and a further increase after neuromodulation (37.00+4.69).

No inflammatory cells were observed in the bladder biopsy specimens obtained from each group.

Comparing the immunohistochemical results for n-NOS staining, we did not find a statistically significant difference in endothelial cell expression in the three groups of animals (62.33±2.52 vs. 56.75±5.38 vs. 48.00+4.69 respectively in groups

Table I. Distribution of n-NOS and i-NOS expression in the different cell populations of rat bladders.

n-NOS

ENDOTHELIAL

CELLS

(MEAN�SD)

i-NOS

ENDOTHELIAL

CELLS

(MEAN�SD)

n-NOS

MUSCLE

CELLS

(MEAN�SD)

i-NOS

MUSCLE

CELLS

(MEAN�SD)

n-NOS

UROTHELIAL

CELLS

(MEAN�SD)

i-NOS

UROTHELIAL

CELLS

(MEAN�SD)

GROUP I:

(N=6)62.33±2.52 12.33±2.52 16.67±4.16 7.67±2.52 15.67±4.04 8.33±3.51

GROUP II:

(N=8)56.75±5.38 20.01±4.08 18.75±2.22 9.25±3.50 16.75±3.77 10.25±3.50

GROUP III:

(N=10)48.00±4.69 59.00±7.42 17.00±4.69 10.20±3.70 36.80±7.85 37.00±4.69

STATISTICAL

ANALYSIS

(P<0.05)

III VS I

III VS II

III VS I

III VS II

III VS I

III VS II

Table I. Distribution of n-NOS and i-NOS expression in the different cell populations of rat bladders.

132 133Int. J. Immunopathol. Pharmacol.

I, II and III); n-NOS immunoreactivity was not statistically different in muscle cells (16.67+4.16 vs. 18.75+2.22 vs. 17.00+4.69 respectively), while its expression in urothelial cells was significantly increased in group III (36.80±8.85) compared to groups I and II (15.67±4.04 and 16.75+3.77 respectively) (p < 0.05).

A statistically significant difference was observed in i-NOS expression on endothelial cells between group III (59.00±7.42) and groups I and II (20.01±4.08 and 12.33+2.52, respectively) (p<0.05); i-NOS expression on muscle cells was not statistically different between the three groups (7.67±2.52 vs. 9.25±3.50 vs. 10.20+3.70); i-NOS expression on urothelial cells was significantly increased in group III (37.00+4.69) compared to groups I and II

(8.33±3.51 vs. 10.25±3.50, respectively) (p<0.05).

DISCUSSION

Neurotransmitter release is a highly organized and regulated process that provides a large degree of plasticity and adaptability; NO is a very important second messenger known to regulate neurotransmitter release (8-12).

The release of NO from the urothelium, afferent nerves, endothelial cells and smooth muscle has been suggested to be the cause of detrusor relaxation; furthermore NO may modulate bladder reflexes by altering afferent nerve activity (13-19).

In the rat, intravesical administration of NO depressed bladder hyperactivity induced by

Fig. 1. Immunohistochemical expression of antibodies against n-nos (a-c) and i-nos (d-f) in bladder of rats of groups I (control), II (sham) and III (neuromodulated). nNOS was highly expressed in endothelial cells (↑) of rats of group I (a) and II (b) in respect to group III (c). Note the increased nNOS expression in urothelial cells (↑) in rats of group III (c) in respect to group I (a) and to group II (b). Increased i-NOS expression in endothelial (↑) and urothelial cells (↑) in neuromodulated rats of group III (f) compared to group I (d) and group II (e), (immunoperoxidase x200).

D. MINARDI ET AL.

132 133Int. J. Immunopathol. Pharmacol.

chemical irritants (20-25). After damage to the urinary bladder, NO generation is most likely caused by i-NOS, which in other tissues is up regulated by inflammatory stimuli and the activity of which is independent of calcium concentration (13).

Sacral neuromodulation has become a valid treatment option in voiding dysfunctions; many parameters have been investigated to improve its efficacy, but the underlying mechanism of neuromodulation is still unclear (3, 26-29).

To our knowledge this is the first report on the effects of neuromodulation on NO metabolism in an animal model; we think that our model is suitable for studying the effects of neuromodulation; in fact we did not adopt a model in which a pathologic condition was created before performing neuromodulation (for example chemically induced cystitis, spinalized rats), as this could allow us to observe the effect of neuromodulation under circumstances not modified by conditions that by themselves can influence the release of neuromediators;

Our results show that sacral neuromodulation has an effect on NO synthase, and in particular it is able to activate i-NOS expression on endothelial cells and n-NOS and i-NOS expression on urothelial cells.

Endothelial i-NOS expression is significantly increased in group III rats, and we may suggest it could be in some way related to the induction of angiogenesis in the microvascular structures, as observed by other authors (28-29); if this could be the case, we will need to demonstrate whether neuromodulation could activate a mechanism able to interfere with the regional hypoxia that can happen in circumstances of chronic detrusor impairment, as observed (30).

n-NOS and i-NOS expression is significantly increased on urothelial cells after chronic neuromodulation; localization of afferent nerves next to urothelium suggests that chemicals released by these cells may modulate afferent excitability (7); neuromediators released from the urothelium could in turn affect afferent nerves, playing a role in sensory mechanisms in the bladder (14-16) or by modulating the interstitial cell network; in our study no inflammatory cells were observed in the bladder biopsy specimens obtained from each group. The activated macrophages are one of the most important effector cells in the inflammatory

response, they secrete NO and numerous pro-inflammatory cytokines; NO release activates urothelial GMP production, ultimately leading to the induction of inflammatory response (5); it has therefore been supposed (5) that macrophages function as an inducible intracellular messenger within the urothelium. Since no inflammatory cells were observed in specimens, our observation supports the hypothesis that neuromodulation has a direct effect on urothelium and that NO released in the submucosal nitrergic nerve fibers is not mediated by inflammatory reactions; this could be consistent with the observation that neuromodulation is able to influence the plasticity of bladder response, inducing the release of messengers within the urothelium.

On the basis of our observation sacral neuromodulation is not able to influence directly the activity of the detrusor muscle, since the expression of n-NOS and i-NOS on muscle cells is not modified after neuromodulation.

This study can therefore improve our understanding of the mechanisms of sacral neuromodulation on chronic bladder dysfunction, and how it is able to influence NO synthesis; this mechanism can cooperate with those already described as important in the NMS; further studies will need to better demonstrate the role of angiogenesis in bladders after sacral neuromodulation and to investigate the effects of neuromodulation in rats with chronically induced bladder dysfunction; experiments can be done with inhibition of NO production by NOS-antagonists (such as L-NAME) to further understand the underlying mechanisms of neuromodulation.

REFERENCES

1. Wang Y. and M.M. Hassouna. 2000. Neuromodulation reduces C-fos gene expression in spinalized rats: a bouble-blind randomized study. J. Urol. 163:1966.

2. Wang Y., Y. Zhan, M.S. Mourad and M.M. Hassouna. 2000. Neuromodulation reduces urinary frequency in rats with hydrocloride acid-induced cystitis. Br. J. Urol. 86:726.

3. Shaker H., Y. Wang, D. Loung, L. Balbaa, M.G. Fehlings and M.M. Hassouna. 2000 Role of C-afferent fibres in the mechanism of action of sacral root neuromodulation in chronic spinal cord injuries.

134 135Int. J. Immunopathol. Pharmacol.

B.J.U. Int., 85:905. 4. Vizzard M.A. 2001. Alteration in neuropeptide

expression in lumbosacral bladder pathways following chronic cystitits. J. Chem. Neuroanat. 21:125.

5. Chertin B., U. Rolle, V. Solari, S. Cascio and P. Puri. 2004. The role of nitric oxide in bladder urothelial injury after bladder outlet obstruction. B.J.U. Int. 94:392.

6. Gruetter C.A., B.K. Barry, D.B. McNamara, D.Y. Gruetter, P.J. Kadowitz and L. Ignarro. 1979. Relaxation of bovine coronary artery and activation of coronary arterial guanylate cyclase by nitric oxide nitroprusside and a carcinogenic nitrosoamine. J. Cyclic Nucleotide Res. 5:211.

7. Xu X., L.X. Cubeddu and A. Malave. 2001. Expression of inducible nitric oxide synthetase in primary culture of rat bladder smooth muscle cells by plasma from cyclophosphamide-treated rats. Eur. J. Pharmacol. 416:1.

8. Thomas S. and R. Robitaille. 2001. Differential frequency-dependent regulation of transmitter release by endogenous nitric oxide at the amphibian neuromuscolar synapse. J. Neurosci. 21:1087.

9. Calabresi P., P. Gubellini, D. Centonze, G. Sancesario, M. Morello, M. Giorgi, A. Pisani and G. Bernardi. 1999. A critical role of the nitric oxide/cGMP pathway in corticostrial long-term depression. J. Neurosci. 19:2489.

10. Fossier P., B. Blanchard, C. Ducrocq, C. Leprince, L. Tauc and G. Baux. 1999. Nitric oxide transforms serotonin into an inactive form and this affects neuromodulation. Neuroscience 93:597.

11. Yoshimura N., S. Seki and W.C. de Groat. 2001. Nitric oxide nodulates Ca2+ dependent channels in dorsal root ganglion neurons innervating rat urinary bladder. J. Neurophysiol. 86:304.

12. Smith S.D., M.A. Wheeler, H.E. Foster and R.M. Weiss. 1996. Urinary nitric oxide synthase activity and cyclic GMP levels are decreased with interstitial cystitis and increased urinary tract infections. J. Urol. 155:1432.

13. Birder L.A., M.L. Nealen, S. Kiss, W.C. de Groat, M.J. Caterina, E. Wang, G. Apodaca and A.J. Kanai. 2002. β-adrenoceptor agonists stimulate endothelial nitric oxide synthetase in rat urinary

bladder urothelial cells. J. Neurosci. 22:8063.14. Gillespie J.I. 2004. Modulation of autonomous

contractile activity in the isolated whole bladder of the guinea pig. B.J.U. Int. 93: 393.

15. Gillespie J.I., M. Markerink-van Ittersum and J. de Vente. 2005. Expression of neuronal nitric oxide synthase (nNOS) and nitric-xide-induced changes in cGMP in the urothelial layer of the guinea pig bladder. Cell Tissue Res. 321:341.

16. Gillespie J.I., M. Markerink-van Ittersum and J. de Vente. 2006. Endogenous nitric oxide/cGMP signalling in the guinea pig bladder : evidence for distinct populations of sub-urothelial interstitial cells. Cell Tissue Res. 325:325.

17. Ho M.H., N.N. Bhatia and O. Khorram. 2004. Physiologic role of nitric oxide and nitric oxide synthase in female lower urinary tract. Curr. Opin. Obstr. Gynec. 16:423.

18. Saito M. and I. Miyagawa. 2002. N(G)-nitro-L-arginine methylester, a nitric oxide synthase inhibitor, diminishes aproptosis induced by ischemia-reperfusion in the rat bladder. Neurourol. Urodyn. 21:566.

19. Theobald R.J.jr. 2003. Differing effects of N(G)-monomehtyl L-arginine and 7-nitroidazole on detrusor activity. Neurourol. Urodyn. 22:62.

20. Ozawa H., M.B. Chancellor, S.K. Jung, T. Yokoyama, M.O. Fraser, Y. Yu, W.C. de Groat and N. Yoshimura. 1999. Effect of intravesical nitric oxide therapy on cyclophosphamide-induced cystitis. J. Urol. 162:2211.

21. Müntener M., B. Schurch, B. Wefer and A. Reitz. 2006. Systemic nitric oxide augmentation leads to a rapid decrease of the bladder outlet resistance in healthy men. Eur. Urol. 50:112.

22. Riazimand S.H. and S. Mense. 2005. Interaction between neurotransmitter antagonists and effects of sacral neuromodulation in rats with chronically hyperactive bladder. B.J.U. Int. 96:900.

23. De Groat W.C. 2006. Integrative control of the lower urinary tract: preclinical perspective. Br. J. Pharmacol. 147(S):25.

24. Lagou M., J. De Vente, T.B. Kirkwood, P. Hedlund, K.E. Andersson, J.I. Gillespie and M.J. Drake. 2006. Location of interstitial cells and neurotransmitters in the mouse bladder. B.J.U. Int.

D. MINARDI ET AL.

134 135Int. J. Immunopathol. Pharmacol.

97:1332.25. Kawano K., H. Masuda, M. Yano, K. Kihara,

A. Sugimoto and H. Azuma. 2006. Altered nitric oxide synthase, arginase and ornithine decarboxylase activities, and polyamine synthesis in response to ischemia of the rabbit detrusor. J. Urol. 176:387.

26. Riazimand S.H. and S. Mense. 2004. A rat model for studying effects of sacral neuromodulation on the contractile activity of a chronically inflamed bladder. B.J.U. Int. 94:158.

27. Zhaou Y., Y. Wang, M. Abdelhady, M.S. Mourad and M.M. Hassouna. 2002 Change in vanilloid receptor 1 following neuromodulation in rats with spinal cord injury. J. Surg. Res. 07:140.

28. Bai H., C.D. McCaig, J.V. Forrester and M. Zhao. 2004. DC electric fields induce distinct preangiogenic responses in microvascular and macrovascular cells. Arterioscl. Thromb. Vasc. Biol. 24: 1234.

29. Milkiewicz M., O. Hudliccka, M.D. Browa and H. Silgram. 2005. Nitric oxide, VEGF and VEGFR-2: interactions in activity-induced angiogenesis in rat skeletal muscle. Am. J. Physiol. Heart Circ. Physiol. 289:336.

30. Hu J., C.M. Chin, J.C. Png, Y.K. Ng and E.A. Ling. 2004. The effect of chronic bladder outlet obstruction on neuronal nitric oxide synthase expression in the intramural ganglia of the guinea pig bladder. J. Urol. 172:1160.