Neurourology and Urodynamics 34:586–591 (2015)
Bladder Dysfunction Induced by Cerebral Hypoperfusion after Bilateral Common Carotid Artery Occlusion in Rats 1
Ching-Chung Liang,1,2 Yi-Hao Lin,1,2 Ho-Ling Liu,3 and Tsong-Hai Lee2,4*
Division of Urogynecology, Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan 2 College of Medicine, Chang Gung University, Taoyuan, Taiwan 3 Department of Medical Imaging and Radiological Sciences, and Healthy Aging Research Center, Chang Gung University College of Medicine, Taoyuan, Taiwan 4 Stroke Section, Department of Neurology and Stroke Center, Chang Gung Memorial Hospital Linkou Medical Center, Taoyuan, Taiwan Aims: The role of forebrain in controlling micturition has been studied extensively using rat model with ischemic injury; however, the influence of cerebral hypoperfusion on voiding function remains unclear. The study was conducted to evaluate the bladder dysfunction and the temporal expression of bladder nerve growth factor (NGF) after cerebral hypoperfusion induced by bilateral common carotid artery occlusion (BCCAO). Materials and Methods: Forty female rats were subjected to either BCCAO or sham operation. Cerebral T2-weighted magnetic resonance images (MRI) and diffusion and perfusion change were studied to characterize the extent of the ischemic injury in the cortex and hippocampus. On 1, 7, and 28 days after BCCAO, the bladder dysfunction was assessed by cystometric studies, and the expressions of NGF in bladder muscle and urothelium were measured by immunohistochemistry and real-time polymerase chain reaction. Results: In the MRI study, cerebral blood flow in the cortex and hippocampus was significantly decreased from 1 day and subsequently returned to sham-operated level at 28 days after BCCAO. Compared with the sham-operated group, significant reduction in voided volume and intercontraction interval was found from 1 to 28 days after cerebral hypoperfusion. The NGF immunoreactivity and mRNA in the bladder muscle and urothelium were transiently increased at 1 day, and declined significantly at 28 days after BCCAO. Conclusions: Our results indicate that bladder dysfunction may be caused by cerebral hypoperfusion and is less likely related to bladder NGF expression. Neurourol. Urodynam. 34:586–591, 2015. # 2014 Wiley Periodicals, Inc. Key words: bladder dysfunction; cerebral hypoperfusion; cystometric study; nerve growth factor
INTRODUCTION
Voiding dysfunction is not an uncommon sequela from various brain diseases, including cerebral infarction and dementia.1–3 Previous studies using rats with ischemic injury revealed that forebrain plays an important role in controlling micturition.4–6 Damage to the neural circuitry in the forebrain induced by ischemic injury may produce bladder overactivity.5–7 In human, aging and dementia are accompanied by a reduced cerebral blood flow (CBF).8 Rats with a 25–50% reduction of CBF may result in impaired spatial cognitive function.9,10 In patients with Alzheimer’s disease, there is urinary urgency and incontinence due to uninhibited detrusor contraction,3,11 but its real pathophysiological mechanism remains unclear. Permanent occlusion of bilateral common carotid artery in rat can result in severe reduction of CBF and cause ischemic injury to cerebral cortex and hippocampus.12 This ischemic model has been established as a procedure to investigate the effects of cerebral hypoperfusion on cognitive function. Whether cerebral hypoperfusion may have influence on voiding function has rarely been discussed. Previous study has demonstrated that there was a significant reduction of brain nerve growth factor (NGF) after severe cerebral ischemia.13 Additionally NGF has an effect on bladder dysfunction by mediating inflammation, as well as morphological and functional changes in sensory neurons innervating the bladder.14 We hypothesized that the cerebral hypoperfusion after bilateral common carotid artery occlusion (BCCAO) may result in bladder overactivity, which might be related to #
2014 Wiley Periodicals, Inc.
the expressions of NGF in bladder muscle and urothelium. The purpose of this study was to evaluate the association between bladder function and the temporal expression of NGF in urinary bladder after cerebral hypoperfusion induced by BCCAO. MATERIALS AND METHODS Animal Model
All protocols were approved by our Institutional Ethics Committee for the Care and Use of Experimental Animals. Forty 12-week-old female Sprague-Dawley rats (250–280 g) from National Laboratory Animal Center in our country were allowed to live at a 228C constant room temperature and 47% humidity with 12 hr light-dark cycle and free access to standard laboratory chow and tap water. Rats were subjected to Lori Birder led the peer-review process as the Associate Editor responsible for the paper. Conflict of interest: none. Grant sponsor: Chang Gung Memorial Hospital under the Medical Research Project; Grant number: CMRPG371561 Correspondence to: Tsong-Hai Lee, M.D., Ph.D., Stroke Section, Department of Neurology and Stroke Center, Chang Gung Memorial Hospital Linkou Medical Center, Taoyuan, Taiwan No. 5, Fu-Hsing St., Kweishan, Taoyuan 333, Taiwan. E-mail: [email protected] Received 21 January 2014; Accepted 14 April 2014 Published online 13 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/nau.22628
Bladder Dysfunction after Cerebral Hypoperfusion either BCCAO or sham operation. Rats with BCCAO were divided into 3 time points: 1 day, 7 days, and 28 days after ischemia (10 rats in each time point). Brain magnetic resonance images (MRI) were done for all experimental rats at 1 day after BCCAO and three rats with infarcted brain lesions in T2-weighted image were excluded. Cystometric studies, immunohistochemistry, and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for bladder were examined at each time point. Cerebral Hypoperfusion Induction
Rats were anesthetized with isoflurane. Bilateral common carotid arteries were carefully exposed and dissected out. Then the mid-portion of both common carotid arteries was ligated with silk suture.8 All surgical procedures were performed within 20 min after anesthesia induction.
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performance of conscious cystometric studies at each time point according to the method used in previous study.16 Briefly, the suprapubic catheter was connected to both the syringe pump and the pressure transducer. Pressure and force transducer signals were amplified, recorded on a chart recorder and digitized for computer data collection. The bladder was then filled with room temperature 0.9% saline at 5 ml/hr through the bladder catheter, while bladder pressure was recorded. Urine was collected in a beaker on a balance placed beneath each cage. Changes in the weight of the collection were recorded. Saline infusion was continued until rhythmic bladder micturition contractions became stable. The data on five representative micturition cycles were collected for analyzing all of the cystometric parameters. The means of the collected data including peak voiding pressure, voided volume per micturition, and intercontraction interval were reported for analysis. Cystometry Analysis Version 1.05 (Catamount Research and Development) was used for cystometric analysis.
MRI Method
Twenty-four hours after BCCAO or sham operation, rats were anesthetized with intraperitoneal injection of 2.5 mg ketamine hyhrochloride and 1.16 mg xylazine per 100 g body weight before MRI examination. For the injection of contrast medium (0.15 mmol Dimeglumine gadopentetate contrast, Magnevist, Bayer Schering Pharma, Berlin, Germany), biomedical silicone catheter was inserted into the left femoral vein and then well fixed. In vivo MRI examination was performed on rat brain according to the method used in our previous study.15 Briefly, each rat was placed prone in a nonmagnetic cradle with the head fixed in a reproducible position by appropriate spacer. Images were acquired at a 3.0T Tim-Trio MRI system (Siemens, Erlangen, Germany). T2-weighted coronal images were acquired using a fast spin-echo sequence. Seven slices were carefully positioned by aligning the 4th slice along the anterior edge of the third ventricle, with 2 mm slice thickness and 2.2 mm slice gap. The diffusion-weighted images (DWI) used a spin-echo echo-planar imaging sequence with diffusion gradients applied in x, y, and z-axes. With the DWI, apparent diffusion coefficients (ADC) were measured for each of the three directions (x, y, and z) and averaged for the calculation of the isotropic ADC value. Three of the seven slice positions in the T2weighted image and the DWI were covered with the same orientation and slice thickness. During the dynamic imaging series of 128 measurements, the contrast media was manually injected as a bolus into the femoral vein started at the 11th measurement. Relative ADC and CBF maps were calculated by dividing the integral of concentration time course with the first moment using the Nordic ICE v2.2 software (Nordic Imaging Lab, AS, Norway).
Tissue Preparation
At each time point after cystometric studies, the rats were sacrificed with an overdose of pentobarbital (60 mg/kg, intraperitoneally) and bladders were transected at the level of the ureteral orifice. The dissected bladders were fixed in optimal cutting temperature compound and frozen in powdered dry ice and stored at 708C. Then, they were subjected to cryosections (10 mm) at 188C, with the sections transferred to glass microscope slides coated with saline (Muto Pure Chemical, Tokyo, Japan). Immunohistochemistry
The frozen bladder sections of 10-mm thickness from each animal were mounted on a slide glass, fixed with 4% of paraformaldehyde, and washed in phosphate buffered saline. After blocking the endogenous peroxidase activity, the nonspecific antibody binding was suppressed, and the slides were incubated overnight at room temperature with a rabbit polyclonal antibody directed against the NGF at a 1:250 dilution (Chemicon International, Temecula, CA). After washing with a buffer, the sections were immunostained by the avidinbiotin peroxidase method using the Vectastain Elite Kit (Vector Laboratories, Burlingame, CA) with 3–3-diaminobenzidine plus hydrogen peroxide as the chromogen. The negative control slides were prepared from the identical tissue blocks by omitting the specific primary antibodies and using normal, non-immune serum supernatant from the identical sources. The ratio of the optical density of BCCAO rats to that of shamoperated rats was determined in NGF. Image-Pro Plus Software (Media Cybernetics, Silver Spring, MD) was used for immunohistochemical calculations.
Suprapubic Tube Implantation
At each time point after MRI examination, all rats received suprapubic tube implantation 3 hr prior to cystometric studies. The abdomen of rats was opened through a midline incision to expose the bladder under general anesthesia with isoflurane. A polyethylene catheter (PE-50 tubing with a flared tip) was implanted in the bladder through the dome. The catheter was tunneled subcutaneously to the neck and plugged until used. Conscious Cystometric Studies
After suprapubic catheter implantation, rats were placed in the metabolic cages (Med Associates, Inc., St. Albans, VT) for Neurourology and Urodynamics DOI 10.1002/nau
Real-Time Quantitative RT-PCR
TaqMan real-time RT-PCR was carried out according to the manufacturer’s protocol. Total RNAs were prepared using an extraction kit (Amersham Pharmacia Biotech, Inc., Uppsala, Sweden) and incubated in reverse transcription mixture at 428C for 1 hr, then 708C for 10 min, and finally the tubes were cooled to 48C for 5 min. Transcripts encoding for NGF were measured by TaqMan real-time quantitative RT-PCR with the TaqMan Universal PCR Master Mix Kit (Applied Biosystems, Oster City, CA) and the ABI Prism 7900 Sequence Detection System (Applied Biosystems). PCR primers and TaqMan probe were obtained from Applied Biosystems and optimized according to
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Fig. 1. Temporal profile of CBF and ADC shows that CBF in the cortex and hippocampus (A) are significantly decreased at 1 day and return subsequently to the sham-operated level at 28 days after bilateral common carotid artery occlusion. The ADC in the cortex and hippocampus (B) does not show any significant difference at any time point from sham-operated level. Compared with sham-operated group, P < 0.05. CBF, cerebral blood flow; ADC, apparent diffusion coefficient.
the manufacturer’s protocol. The sequences of primer pair were NGF sense, 50 -CCCACCCAGTCTTCCACATG-30 ; antisense, 50 -CAACCCACACACTGACAC- TGT-30 ; probe sequences, 50 -CACACACTGAAAACTC-30 . PCR conditions were 508C for 2 min, 958C for 10 min, followed by 40 cycles at 958C for 15 sec, and 608C for 1 min. 18S rRNA transcript was used as internal control gene and was amplified in a separate tube to normalize for variance in input RNA. A ratio of the mRNA level of ischemic rats at each time point to that of sham-operated rats was determined. The data were calculated with 2[-Delta Delta C(T)].17 Statistical Analysis
The data were analyzed statistically using one-way analysis of variance test followed by Tukey test. All data used Prism 5 software for statistical analysis (GraphPad, San Diego, CA). Values were considered significant if P < 0.05. RESULTS
Data of 40 rats were collected including 30 in BCCAO group and 10 in sham-operated groups. In the MRI study, CBF in the cortex and hippocampus were significantly decreased at 1 day and subsequently returned to the level of sham-operated group at 28 days after BCCAO (Fig. 1). ADC in the cortex and hippocampus at any time point after BCCAO was not significantly different from that of sham-operated group.
Cystometric studies showed intercontraction intervals and voided volumes decreased significantly from 1 day after ischemia and did not return to the level of sham-operated group at 28 days after ischemic insult (Fig. 2). However, the ischemic rats had no significant changes in the peak voiding pressure at any time point. NGF immunoreactivity in both bladder muscle and urothelium increased transiently at 1 day, and declined significantly at 28 days after ischemic insult compared with the shamoperated group (Fig. 3). Likewise, NGF mRNA expressions in both bladder muscle and urothelium increased transiently at 1 day after ischemia, and subsequently decreased at 28 days after ischemic insult compared with the sham-operated group (Fig. 4). DISCUSSION
In recent years, magnetic resonance diffusion- and perfusionweighted imaging has been developed for the measurement of cerebral hemodynamic changes after ischemic injury.15,18 Previous studies have shown the regional CBF in selected brain structures of rats can be reduced immediately after permanent BCCAO.12,19–21 At 2 to 3 days following ischemic insult, the greatest reduction in CBF was recorded 35–45% of the control in the cortical area and 60% of the control in the hippocampus.12,19–21 The CBF can be reduced severely in the early stage after ischemic insult, creating a hypoxic-ischemic condition that may compromise the electrophysiological activity of the
Fig. 2. Cystometric results in the experimental rats (A–C) are presented. Ischemic rats have no significant changes in the peak voiding pressure at any time point (A), but have significant decrease of voided volumes (B), and intercontraction intervals (C) from 1 day to 28 days after bilateral common carotid artery occlusion when compared with sham-operated rats. Compared with sham-operated group, P < 0.05.
Neurourology and Urodynamics DOI 10.1002/nau
Bladder Dysfunction after Cerebral Hypoperfusion
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Fig. 4. The ratio of NGF mRNA signal intensity in ischemic bladder after normalization to that in sham-operated bladder is presented. The expressions of NGF mRNA in ischemic bladder increase transiently at 1 day and decrease subsequently at 28 days after bilateral common carotid artery occlusion compared with sham-operated group. Compared with sham-operated group, P < 0.05. NGF, nerve growth factor.
Fig. 3. The expressions of NGF immunoreactivity in both bladder urothelium (A–D) and smooth muscle (E–H) are transiently increased at 1 day and then decline significantly at 28 days after bilateral common carotid artery occlusion. Bar indicates 50 mm. The optical density ratio is the ratio of immunoreactivity in ischemic bladder to that in sham-operated bladder. Compared with sham-operated group, P < 0.05. NGF, nerve growth factor.
nervous tissue.22 Then, CBF may start to recover gradually at 7 days, but is still lower than the control value at 28 days after BCCAO.12,19–21 Our results are in agreement with the aforementioned studies demonstrating that CBF in the cerebral cortex and hippocampus is significantly decreased at 1 day and returned subsequently to the level of sham-operated group at 28 days after permanent BCCAO. The changes of ADC values in the animal model of permanent BCCAO have been reported rarely. Lee et al.15 found there was Neurourology and Urodynamics DOI 10.1002/nau
cerebral diffusion–perfusion disparity with increased ADC value and reduced CBF in the cortical area after permanent BCCAO in the aged hypertensive rat, which correlated to the infarct size in the brain tissue. Our study demonstrates that in the young non-hypertensive rat, ADC values decreased at 1 day in cortex after ischemic insult and recovered toward normal levels at 28 days, but had no significant difference between BCCAO and sham-operated groups. The most likely explanation for the reduction of ADC in the early stages of ischemia is the migration of extracellular water into intracellular space.23 In an experimental study of transient middle cerebral artery occlusion, Lin et al.18 reported that the ADC value in rats subjected to decreased to 60% of sham-operated level at 1 day after reperfusion, and then slowly reached a value higher than the sham-operated level during the period of 7–14 days after ischemic insult. The role of forebrain in controlling micturition has been studied extensively using rats with cerebral infarction.4–6 Bladder overactivity in rats after middle cerebral artery occlusion was attributed to the interruption of inhibitory neuronal pathways from forebrain to pontine micturition center.4–6 This overactivity has been found to involve receptors such as dopamine, glutamate, and gama-aminobutyric acid in the central nervous system.4,24,25 However, middle cerebral artery occlusion model causes focal brain injury and the mechanism to cause bladder dysfunction is probably different from chronic cerebral hypoperfusion which is associated with cognitive dysfunction and neurodegenerative process. Yotsuyanagi et al.7 used a rat model induced by anastomosis between right external jugular vein and right common carotid artery to evaluate the influence of brain ischemia without cerebral infarction on voiding function.7 They reported that mild forebrain ischemia without infarction may lead to impairment of memory and bladder overactivity.7 Their results also showed that voided volume per micturition in rats with cerebral hypoperfusion was significantly reduced from 14 to 28 days, but became no difference to sham-operated rats at 8 weeks. In our experiments, consistent reduction in voided volume and intercontraction interval was found in the rats with BCCAO from 1 to 28 days after ischemic insult; however, there was no
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significant change in the peak voiding pressure at any time point. In an experimental study of cerebral infarction in rat, peak voiding pressure was not significantly affected after left middle cerebral artery occlusion but bladder capacity had a significant reduction. 26 Accordingly, we speculate that the signs of bladder overactivity maybe result from reduction in bladder capacity in permanent BCCAO rats. NGF is normally present in the bladder muscle cells and urothelium.27 Clinical and experimental studies demonstrated that a direct link between increased NGF levels in the bladder tissue and urine and lower urinary tract dysfunction.28–30 NGF administered intramuscularly to the detrusor muscle can induce rat bladder hyperreflexia and neuronal hypersen sitivity.28 In rats with urethral obstruction, increased NGF production may induce detrusor overactivity via afferent nerve excitability resulting from altered ion channel expression.29 Increased NGF levels in urine have been reported in the patients with detrusor orveractivity.30 Liu et al.31 reported that urinary NGF levels in stroke patients were significantly higher than those in controls and were correlated with the severity of neurological impairment. They inferred that stroke patients with moderate to severe neurological impairment have increased circulating NGF and this causes an increase in urinary NGF level.30 This inference is accordant with the result of previous study that serum NGF levels were significantly associated with stroke severity and lesion volume of brain in the acute phases of stroke, indicating that stroke may modulate peripheral neurotrophin levels.32 In fact, experimental evidence indicates neurotrophins not only may be released from blood cells, but also may be produced in brain tissue after stroke.32 NGF and brain-derived neurotrophic factors are present at the highest level in the hippocampal formation and cerebral cortex.13 Both neurotrophic factors may interact with their high-affinity receptors and have been reported to protect the hippocampal CA1 neurons against ischemic cell damage.13 The reason why the transient change of cerebral hypoperfusion and the elevated (not significantly) NGF levels only lasted for 1 day. It is likely that the change of cerebral perfusion and NGF levels is related to the severity of cerebral ischemia.33 Our previous report in gerbil has shown following non-lethal cerebral ischemia, NGF and brain-derived neurotrophic factors can increase in the hippocampal neurons at the initial several hours, but both neurotrophic factors decreased to a significant level 1 day after lethal ischemia.13 Our present study found that there is significant reduction of CBF at 1 day after BCCAO, which possibly causes a transient increase in circulating NGF levels and then results in a transient increase of bladder NGF immunoreactivity and mRNA. Significant reduction of CBF was related to significant reduction of voided volume and intercontraction interval from 1 day after BCCAO. The increased NGF expression in bladder 1 day after cerebral hypoperfusion may be associated with bladder hyperactivity which can sustain for a certain period even after NGF expression is decreased. This finding suggests that bladder hyperactivity in rats after cerebral hypoperfusion may be due to alterations in brain function rather than a confined dysfunction in bladder. CONCLUSIONS
Our study suggests the cerebral hypoperfusion may cause bladder dysfunction, which could be possibly related to damage to the neural circuitry in the forebrain rather than changes in bladder NGF expression.
Neurourology and Urodynamics DOI 10.1002/nau
ACKNOWLEDGMENTS
This work was supported by the Chang Gung Memorial Hospital under the Medical Research Project (CMRPG371561). We thank the assistance from the Molecular Imaging Center, Chang Gung Memorial Hospital, Linkou Medical center. REFERENCES 1. Skelly J, Flint AJ. Urinary incontinence associated with dementia. J Am Geriatr Soc 1995;43:286–294. 2. Sakakibara R, Fowler CJ, Hattori T. Voiding and MRI analysis of the brain. Int Urogynecol J 1999;10:192–199. 3. Sugiyama T, Hashimoto K, Kiwamoto H, et al. Urinary incontinence in senile dementia of the Alzheimer type. Int J Urol 1994;1:337–340. 4. Yokoyama O, Yoshiyama M, Namiki M, et al. Glutamatergic and dopaminergic contributions to rat bladder hyperactivity after cerebral artery occlusion. Am J Physiol 1999;276:R935–R942. 5. Yokoyama O, Yoshiyama M, Namiki M, et al. Role of the forebrain in bladder overactivity following cerebral infarction in the rat. Exp Neurol 2000; 163:469–476. 6. Yokoyama O, Ootsuka N, Komatsu K, et al. Forebrain muscarinic control of micturition reflex in rats. Neuropharmacology 2001;41:629–638. 7. Yotsuyanagi S, Narimoto K, Namiki M. Mild brain ischemia produces bladder hyperactivity without brain damage in rats. Urol Int 2006;77:57–63. 8. Farkas E, Luiten PG. Cerebral microvascular pathology in aging and Alzheimer’s disease. Prog Neurobiol 2001;64:575–611. 9. Morgan MK, Anderson RE, Spence I, et al. A model of the pathophysiology of cerebral arteriovenous malformations by a carotid-jugular fistula in the rat. Brain Res 1989;496:241–250. 10. Sekhon LH, Morgan MK, Spence I, et al. Chronic cerebral hypoperfusion and impaired neuronal function in rats. Stroke 1994;25:1022–1027. 11. Schultz-Lampel D. Bladder disorders in dementia and Alzheimer’s disease. Rational diagnostic and therapeutic options. Urologe A 2003;42:1579–1587. 12. Farkas E, Luiten PGM, Bari F. Permanent, bilateral common carotid artery occlusion in the rat: A model for chronic cerebral hypoperfusion-related neurodegenerative diseases. Brain Res Rev 2007;54:162–180. 13. Lee TH, Yang JT, Ko YS. et al. Influence of ischemic preconditioning on levels of nerve growth factor, brain-derived neurotrophic factor and their high affinity receptors in hippocampus following forebrain ischemia. Brain Res 2008; 1187:1–11. 14. Ochodnicky P, Cruz CD, Yoshimura N, et al. Nerve growth factor in bladder dysfunction: Contributing factor, biomarker, and therapeutic target. Neurourol Urodyn 2011;30:1227–1241. 15. Lee TH, Liu HL, Yang ST, et al. Effects of aging and hypertension on cerebral ischemic susceptibility: Evidenced by MR diffusion-perfusion study in rat. Exp Neurol 2011;227:314–321. 16. Lin YH, Liu G, Kavran M, et al. Lower urinary tract phenotype of experimental autoimmune cystitis in mouse: A potential animal model for interstitial cystitis. BJU Int 2008;102:1724–1730. 17. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25:402–408. 18. Lin TN, Sun SW, Cheung WM, et al. Dynamic changes in cerebral blood flow and angiogenesis after transient focal cerebral ischemia in rats. Evaluation with serial magnetic resonance imaging. Stroke 2002;33:2985–2991. 19. Tsuchiya M, Sako K, Yura S, et al. Cerebral blood flow and histopathological changes following permanent bilateral carotid artery ligation in Wistar rats. Exp Brain Res 1992;89:87–92. 20. Schmidt-Kastner R, Truettner J, Lin B, et al. Transient changes of brain derived neurotrophic factor mRNA expression in hippocampus during moderate ischemia induced by chronic bilateral common carotid artery occlusion in the rat. Brain Res Mol Brain Res 2001;92:157–166. 21. Otori T, Katsumata T, Muramatsu H, et al. Long-term measurements of cerebral blood flow and metabolism in a rat chronic hypoperfusion model. Clin Exp Pharmacol Physiol 2003;30:266–272. 22. Marosi M, Rakos G, Robotka H, et al. Hippocampal (CA1) activities in Wistar rats from different vendors. Fundamental differences in acute ischemia. J Neurosci Methods 2006;156:231–235. 23. Matsumoto K, Lo EH, Pierce AR, et al. Role of vasogenic edema and tissue cavitation in ischemic evolution on diffusion-weighted imaging: Comparison with multiparameter MR and immunohistochemistry. AJNR Am J Neuroradiaol 1995;16:1107–1115. 24. Yokoyama O, Yoshiyama M, Namiki M, et al. Changes in dopaminergic and glutamatergic excitatory mechanisms of micturition reflex after middle cerebral artery occlusion in conscious rats. Exp Neurol 2002;173:129–135. 25. Kanie S, Yokoyama O, Komatsu K, et al. GABAergic contribution to rat bladder hyperactivity after middle cerebral artery occlusion. Am J Physiol 2000;279: R1230–R1238.
Bladder Dysfunction after Cerebral Hypoperfusion 26. Nakamura Y, Kontani H, Tanaka T, et al. Effects of adenosine triphosphate dependent potassium channel opener on bladder overactivity in rats with cerebral infarction. J Urol 2002;168:2275–2279. 27. Steers WD, Tuttle JB. Mechanisms of disease: The role of nerve growth factor in the pathophysiology of bladder disorders. Nat Clin Pract Urol 2006;3:101–110. 28. Yoshimura N, Bennett NE, Hayashi Y, et al. Bladder overactivity and hyperexcitability of bladder afferent neurons after intrathecal delivery of nerve growth factor in rats. J Neurosci 2006;26:10847–10855. 29. Lee SR, Hong CH, Choi YD, et al. Increased urinary nerve growth factor as a predictor of persistent detrusor overactivity after bladder outlet obstruction relief in a rat model. J Urol 2010;183:2440–2444. 30. Liu HT, Kuo HC. Urinary nerve growth factor levels are increased in patients with bladder outlet obstruction with overactive bladder
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symptoms and reduced after successful medical treatment. Urology 2008;72:104–108. 31. Liu HT, Liu AB, Chancellor MB, et al. Urinary nerve growth factor levels is correlated with the severity of neurological impairment in patients with cerebrovascular accident. BJU Int 2009;104:1158–1162. 32. Stanzani L, Zoia C, Sala G, et al. Nerve growth factor and transforming growth factor-beta serum levels in acute stroke patients. Possible involvement of neurotrophins in cerebrovascular disease. Cerebrovasc Dis 2001;12:240–244. 33. Lindvall O, Ernfors P, Bengzon J, et al. Differential regulation of mRNAs for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 in the adult rat brain following cerebral ischemia and hypoglycemic coma. Proc Natl Acad Sci USA 1992;89:648–652.
Bladder Dysfunction Induced by Cerebral Hypoperfusion after Bilateral Common Carotid Artery Occlusion in Rats 1
Ching-Chung Liang,1,2 Yi-Hao Lin,1,2 Ho-Ling Liu,3 and Tsong-Hai Lee2,4*
Division of Urogynecology, Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan 2 College of Medicine, Chang Gung University, Taoyuan, Taiwan 3 Department of Medical Imaging and Radiological Sciences, and Healthy Aging Research Center, Chang Gung University College of Medicine, Taoyuan, Taiwan 4 Stroke Section, Department of Neurology and Stroke Center, Chang Gung Memorial Hospital Linkou Medical Center, Taoyuan, Taiwan Aims: The role of forebrain in controlling micturition has been studied extensively using rat model with ischemic injury; however, the influence of cerebral hypoperfusion on voiding function remains unclear. The study was conducted to evaluate the bladder dysfunction and the temporal expression of bladder nerve growth factor (NGF) after cerebral hypoperfusion induced by bilateral common carotid artery occlusion (BCCAO). Materials and Methods: Forty female rats were subjected to either BCCAO or sham operation. Cerebral T2-weighted magnetic resonance images (MRI) and diffusion and perfusion change were studied to characterize the extent of the ischemic injury in the cortex and hippocampus. On 1, 7, and 28 days after BCCAO, the bladder dysfunction was assessed by cystometric studies, and the expressions of NGF in bladder muscle and urothelium were measured by immunohistochemistry and real-time polymerase chain reaction. Results: In the MRI study, cerebral blood flow in the cortex and hippocampus was significantly decreased from 1 day and subsequently returned to sham-operated level at 28 days after BCCAO. Compared with the sham-operated group, significant reduction in voided volume and intercontraction interval was found from 1 to 28 days after cerebral hypoperfusion. The NGF immunoreactivity and mRNA in the bladder muscle and urothelium were transiently increased at 1 day, and declined significantly at 28 days after BCCAO. Conclusions: Our results indicate that bladder dysfunction may be caused by cerebral hypoperfusion and is less likely related to bladder NGF expression. Neurourol. Urodynam. 34:586–591, 2015. # 2014 Wiley Periodicals, Inc. Key words: bladder dysfunction; cerebral hypoperfusion; cystometric study; nerve growth factor
INTRODUCTION
Voiding dysfunction is not an uncommon sequela from various brain diseases, including cerebral infarction and dementia.1–3 Previous studies using rats with ischemic injury revealed that forebrain plays an important role in controlling micturition.4–6 Damage to the neural circuitry in the forebrain induced by ischemic injury may produce bladder overactivity.5–7 In human, aging and dementia are accompanied by a reduced cerebral blood flow (CBF).8 Rats with a 25–50% reduction of CBF may result in impaired spatial cognitive function.9,10 In patients with Alzheimer’s disease, there is urinary urgency and incontinence due to uninhibited detrusor contraction,3,11 but its real pathophysiological mechanism remains unclear. Permanent occlusion of bilateral common carotid artery in rat can result in severe reduction of CBF and cause ischemic injury to cerebral cortex and hippocampus.12 This ischemic model has been established as a procedure to investigate the effects of cerebral hypoperfusion on cognitive function. Whether cerebral hypoperfusion may have influence on voiding function has rarely been discussed. Previous study has demonstrated that there was a significant reduction of brain nerve growth factor (NGF) after severe cerebral ischemia.13 Additionally NGF has an effect on bladder dysfunction by mediating inflammation, as well as morphological and functional changes in sensory neurons innervating the bladder.14 We hypothesized that the cerebral hypoperfusion after bilateral common carotid artery occlusion (BCCAO) may result in bladder overactivity, which might be related to #
2014 Wiley Periodicals, Inc.
the expressions of NGF in bladder muscle and urothelium. The purpose of this study was to evaluate the association between bladder function and the temporal expression of NGF in urinary bladder after cerebral hypoperfusion induced by BCCAO. MATERIALS AND METHODS Animal Model
All protocols were approved by our Institutional Ethics Committee for the Care and Use of Experimental Animals. Forty 12-week-old female Sprague-Dawley rats (250–280 g) from National Laboratory Animal Center in our country were allowed to live at a 228C constant room temperature and 47% humidity with 12 hr light-dark cycle and free access to standard laboratory chow and tap water. Rats were subjected to Lori Birder led the peer-review process as the Associate Editor responsible for the paper. Conflict of interest: none. Grant sponsor: Chang Gung Memorial Hospital under the Medical Research Project; Grant number: CMRPG371561 Correspondence to: Tsong-Hai Lee, M.D., Ph.D., Stroke Section, Department of Neurology and Stroke Center, Chang Gung Memorial Hospital Linkou Medical Center, Taoyuan, Taiwan No. 5, Fu-Hsing St., Kweishan, Taoyuan 333, Taiwan. E-mail: [email protected] Received 21 January 2014; Accepted 14 April 2014 Published online 13 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/nau.22628
Bladder Dysfunction after Cerebral Hypoperfusion either BCCAO or sham operation. Rats with BCCAO were divided into 3 time points: 1 day, 7 days, and 28 days after ischemia (10 rats in each time point). Brain magnetic resonance images (MRI) were done for all experimental rats at 1 day after BCCAO and three rats with infarcted brain lesions in T2-weighted image were excluded. Cystometric studies, immunohistochemistry, and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for bladder were examined at each time point. Cerebral Hypoperfusion Induction
Rats were anesthetized with isoflurane. Bilateral common carotid arteries were carefully exposed and dissected out. Then the mid-portion of both common carotid arteries was ligated with silk suture.8 All surgical procedures were performed within 20 min after anesthesia induction.
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performance of conscious cystometric studies at each time point according to the method used in previous study.16 Briefly, the suprapubic catheter was connected to both the syringe pump and the pressure transducer. Pressure and force transducer signals were amplified, recorded on a chart recorder and digitized for computer data collection. The bladder was then filled with room temperature 0.9% saline at 5 ml/hr through the bladder catheter, while bladder pressure was recorded. Urine was collected in a beaker on a balance placed beneath each cage. Changes in the weight of the collection were recorded. Saline infusion was continued until rhythmic bladder micturition contractions became stable. The data on five representative micturition cycles were collected for analyzing all of the cystometric parameters. The means of the collected data including peak voiding pressure, voided volume per micturition, and intercontraction interval were reported for analysis. Cystometry Analysis Version 1.05 (Catamount Research and Development) was used for cystometric analysis.
MRI Method
Twenty-four hours after BCCAO or sham operation, rats were anesthetized with intraperitoneal injection of 2.5 mg ketamine hyhrochloride and 1.16 mg xylazine per 100 g body weight before MRI examination. For the injection of contrast medium (0.15 mmol Dimeglumine gadopentetate contrast, Magnevist, Bayer Schering Pharma, Berlin, Germany), biomedical silicone catheter was inserted into the left femoral vein and then well fixed. In vivo MRI examination was performed on rat brain according to the method used in our previous study.15 Briefly, each rat was placed prone in a nonmagnetic cradle with the head fixed in a reproducible position by appropriate spacer. Images were acquired at a 3.0T Tim-Trio MRI system (Siemens, Erlangen, Germany). T2-weighted coronal images were acquired using a fast spin-echo sequence. Seven slices were carefully positioned by aligning the 4th slice along the anterior edge of the third ventricle, with 2 mm slice thickness and 2.2 mm slice gap. The diffusion-weighted images (DWI) used a spin-echo echo-planar imaging sequence with diffusion gradients applied in x, y, and z-axes. With the DWI, apparent diffusion coefficients (ADC) were measured for each of the three directions (x, y, and z) and averaged for the calculation of the isotropic ADC value. Three of the seven slice positions in the T2weighted image and the DWI were covered with the same orientation and slice thickness. During the dynamic imaging series of 128 measurements, the contrast media was manually injected as a bolus into the femoral vein started at the 11th measurement. Relative ADC and CBF maps were calculated by dividing the integral of concentration time course with the first moment using the Nordic ICE v2.2 software (Nordic Imaging Lab, AS, Norway).
Tissue Preparation
At each time point after cystometric studies, the rats were sacrificed with an overdose of pentobarbital (60 mg/kg, intraperitoneally) and bladders were transected at the level of the ureteral orifice. The dissected bladders were fixed in optimal cutting temperature compound and frozen in powdered dry ice and stored at 708C. Then, they were subjected to cryosections (10 mm) at 188C, with the sections transferred to glass microscope slides coated with saline (Muto Pure Chemical, Tokyo, Japan). Immunohistochemistry
The frozen bladder sections of 10-mm thickness from each animal were mounted on a slide glass, fixed with 4% of paraformaldehyde, and washed in phosphate buffered saline. After blocking the endogenous peroxidase activity, the nonspecific antibody binding was suppressed, and the slides were incubated overnight at room temperature with a rabbit polyclonal antibody directed against the NGF at a 1:250 dilution (Chemicon International, Temecula, CA). After washing with a buffer, the sections were immunostained by the avidinbiotin peroxidase method using the Vectastain Elite Kit (Vector Laboratories, Burlingame, CA) with 3–3-diaminobenzidine plus hydrogen peroxide as the chromogen. The negative control slides were prepared from the identical tissue blocks by omitting the specific primary antibodies and using normal, non-immune serum supernatant from the identical sources. The ratio of the optical density of BCCAO rats to that of shamoperated rats was determined in NGF. Image-Pro Plus Software (Media Cybernetics, Silver Spring, MD) was used for immunohistochemical calculations.
Suprapubic Tube Implantation
At each time point after MRI examination, all rats received suprapubic tube implantation 3 hr prior to cystometric studies. The abdomen of rats was opened through a midline incision to expose the bladder under general anesthesia with isoflurane. A polyethylene catheter (PE-50 tubing with a flared tip) was implanted in the bladder through the dome. The catheter was tunneled subcutaneously to the neck and plugged until used. Conscious Cystometric Studies
After suprapubic catheter implantation, rats were placed in the metabolic cages (Med Associates, Inc., St. Albans, VT) for Neurourology and Urodynamics DOI 10.1002/nau
Real-Time Quantitative RT-PCR
TaqMan real-time RT-PCR was carried out according to the manufacturer’s protocol. Total RNAs were prepared using an extraction kit (Amersham Pharmacia Biotech, Inc., Uppsala, Sweden) and incubated in reverse transcription mixture at 428C for 1 hr, then 708C for 10 min, and finally the tubes were cooled to 48C for 5 min. Transcripts encoding for NGF were measured by TaqMan real-time quantitative RT-PCR with the TaqMan Universal PCR Master Mix Kit (Applied Biosystems, Oster City, CA) and the ABI Prism 7900 Sequence Detection System (Applied Biosystems). PCR primers and TaqMan probe were obtained from Applied Biosystems and optimized according to
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Fig. 1. Temporal profile of CBF and ADC shows that CBF in the cortex and hippocampus (A) are significantly decreased at 1 day and return subsequently to the sham-operated level at 28 days after bilateral common carotid artery occlusion. The ADC in the cortex and hippocampus (B) does not show any significant difference at any time point from sham-operated level. Compared with sham-operated group, P < 0.05. CBF, cerebral blood flow; ADC, apparent diffusion coefficient.
the manufacturer’s protocol. The sequences of primer pair were NGF sense, 50 -CCCACCCAGTCTTCCACATG-30 ; antisense, 50 -CAACCCACACACTGACAC- TGT-30 ; probe sequences, 50 -CACACACTGAAAACTC-30 . PCR conditions were 508C for 2 min, 958C for 10 min, followed by 40 cycles at 958C for 15 sec, and 608C for 1 min. 18S rRNA transcript was used as internal control gene and was amplified in a separate tube to normalize for variance in input RNA. A ratio of the mRNA level of ischemic rats at each time point to that of sham-operated rats was determined. The data were calculated with 2[-Delta Delta C(T)].17 Statistical Analysis
The data were analyzed statistically using one-way analysis of variance test followed by Tukey test. All data used Prism 5 software for statistical analysis (GraphPad, San Diego, CA). Values were considered significant if P < 0.05. RESULTS
Data of 40 rats were collected including 30 in BCCAO group and 10 in sham-operated groups. In the MRI study, CBF in the cortex and hippocampus were significantly decreased at 1 day and subsequently returned to the level of sham-operated group at 28 days after BCCAO (Fig. 1). ADC in the cortex and hippocampus at any time point after BCCAO was not significantly different from that of sham-operated group.
Cystometric studies showed intercontraction intervals and voided volumes decreased significantly from 1 day after ischemia and did not return to the level of sham-operated group at 28 days after ischemic insult (Fig. 2). However, the ischemic rats had no significant changes in the peak voiding pressure at any time point. NGF immunoreactivity in both bladder muscle and urothelium increased transiently at 1 day, and declined significantly at 28 days after ischemic insult compared with the shamoperated group (Fig. 3). Likewise, NGF mRNA expressions in both bladder muscle and urothelium increased transiently at 1 day after ischemia, and subsequently decreased at 28 days after ischemic insult compared with the sham-operated group (Fig. 4). DISCUSSION
In recent years, magnetic resonance diffusion- and perfusionweighted imaging has been developed for the measurement of cerebral hemodynamic changes after ischemic injury.15,18 Previous studies have shown the regional CBF in selected brain structures of rats can be reduced immediately after permanent BCCAO.12,19–21 At 2 to 3 days following ischemic insult, the greatest reduction in CBF was recorded 35–45% of the control in the cortical area and 60% of the control in the hippocampus.12,19–21 The CBF can be reduced severely in the early stage after ischemic insult, creating a hypoxic-ischemic condition that may compromise the electrophysiological activity of the
Fig. 2. Cystometric results in the experimental rats (A–C) are presented. Ischemic rats have no significant changes in the peak voiding pressure at any time point (A), but have significant decrease of voided volumes (B), and intercontraction intervals (C) from 1 day to 28 days after bilateral common carotid artery occlusion when compared with sham-operated rats. Compared with sham-operated group, P < 0.05.
Neurourology and Urodynamics DOI 10.1002/nau
Bladder Dysfunction after Cerebral Hypoperfusion
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Fig. 4. The ratio of NGF mRNA signal intensity in ischemic bladder after normalization to that in sham-operated bladder is presented. The expressions of NGF mRNA in ischemic bladder increase transiently at 1 day and decrease subsequently at 28 days after bilateral common carotid artery occlusion compared with sham-operated group. Compared with sham-operated group, P < 0.05. NGF, nerve growth factor.
Fig. 3. The expressions of NGF immunoreactivity in both bladder urothelium (A–D) and smooth muscle (E–H) are transiently increased at 1 day and then decline significantly at 28 days after bilateral common carotid artery occlusion. Bar indicates 50 mm. The optical density ratio is the ratio of immunoreactivity in ischemic bladder to that in sham-operated bladder. Compared with sham-operated group, P < 0.05. NGF, nerve growth factor.
nervous tissue.22 Then, CBF may start to recover gradually at 7 days, but is still lower than the control value at 28 days after BCCAO.12,19–21 Our results are in agreement with the aforementioned studies demonstrating that CBF in the cerebral cortex and hippocampus is significantly decreased at 1 day and returned subsequently to the level of sham-operated group at 28 days after permanent BCCAO. The changes of ADC values in the animal model of permanent BCCAO have been reported rarely. Lee et al.15 found there was Neurourology and Urodynamics DOI 10.1002/nau
cerebral diffusion–perfusion disparity with increased ADC value and reduced CBF in the cortical area after permanent BCCAO in the aged hypertensive rat, which correlated to the infarct size in the brain tissue. Our study demonstrates that in the young non-hypertensive rat, ADC values decreased at 1 day in cortex after ischemic insult and recovered toward normal levels at 28 days, but had no significant difference between BCCAO and sham-operated groups. The most likely explanation for the reduction of ADC in the early stages of ischemia is the migration of extracellular water into intracellular space.23 In an experimental study of transient middle cerebral artery occlusion, Lin et al.18 reported that the ADC value in rats subjected to decreased to 60% of sham-operated level at 1 day after reperfusion, and then slowly reached a value higher than the sham-operated level during the period of 7–14 days after ischemic insult. The role of forebrain in controlling micturition has been studied extensively using rats with cerebral infarction.4–6 Bladder overactivity in rats after middle cerebral artery occlusion was attributed to the interruption of inhibitory neuronal pathways from forebrain to pontine micturition center.4–6 This overactivity has been found to involve receptors such as dopamine, glutamate, and gama-aminobutyric acid in the central nervous system.4,24,25 However, middle cerebral artery occlusion model causes focal brain injury and the mechanism to cause bladder dysfunction is probably different from chronic cerebral hypoperfusion which is associated with cognitive dysfunction and neurodegenerative process. Yotsuyanagi et al.7 used a rat model induced by anastomosis between right external jugular vein and right common carotid artery to evaluate the influence of brain ischemia without cerebral infarction on voiding function.7 They reported that mild forebrain ischemia without infarction may lead to impairment of memory and bladder overactivity.7 Their results also showed that voided volume per micturition in rats with cerebral hypoperfusion was significantly reduced from 14 to 28 days, but became no difference to sham-operated rats at 8 weeks. In our experiments, consistent reduction in voided volume and intercontraction interval was found in the rats with BCCAO from 1 to 28 days after ischemic insult; however, there was no
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significant change in the peak voiding pressure at any time point. In an experimental study of cerebral infarction in rat, peak voiding pressure was not significantly affected after left middle cerebral artery occlusion but bladder capacity had a significant reduction. 26 Accordingly, we speculate that the signs of bladder overactivity maybe result from reduction in bladder capacity in permanent BCCAO rats. NGF is normally present in the bladder muscle cells and urothelium.27 Clinical and experimental studies demonstrated that a direct link between increased NGF levels in the bladder tissue and urine and lower urinary tract dysfunction.28–30 NGF administered intramuscularly to the detrusor muscle can induce rat bladder hyperreflexia and neuronal hypersen sitivity.28 In rats with urethral obstruction, increased NGF production may induce detrusor overactivity via afferent nerve excitability resulting from altered ion channel expression.29 Increased NGF levels in urine have been reported in the patients with detrusor orveractivity.30 Liu et al.31 reported that urinary NGF levels in stroke patients were significantly higher than those in controls and were correlated with the severity of neurological impairment. They inferred that stroke patients with moderate to severe neurological impairment have increased circulating NGF and this causes an increase in urinary NGF level.30 This inference is accordant with the result of previous study that serum NGF levels were significantly associated with stroke severity and lesion volume of brain in the acute phases of stroke, indicating that stroke may modulate peripheral neurotrophin levels.32 In fact, experimental evidence indicates neurotrophins not only may be released from blood cells, but also may be produced in brain tissue after stroke.32 NGF and brain-derived neurotrophic factors are present at the highest level in the hippocampal formation and cerebral cortex.13 Both neurotrophic factors may interact with their high-affinity receptors and have been reported to protect the hippocampal CA1 neurons against ischemic cell damage.13 The reason why the transient change of cerebral hypoperfusion and the elevated (not significantly) NGF levels only lasted for 1 day. It is likely that the change of cerebral perfusion and NGF levels is related to the severity of cerebral ischemia.33 Our previous report in gerbil has shown following non-lethal cerebral ischemia, NGF and brain-derived neurotrophic factors can increase in the hippocampal neurons at the initial several hours, but both neurotrophic factors decreased to a significant level 1 day after lethal ischemia.13 Our present study found that there is significant reduction of CBF at 1 day after BCCAO, which possibly causes a transient increase in circulating NGF levels and then results in a transient increase of bladder NGF immunoreactivity and mRNA. Significant reduction of CBF was related to significant reduction of voided volume and intercontraction interval from 1 day after BCCAO. The increased NGF expression in bladder 1 day after cerebral hypoperfusion may be associated with bladder hyperactivity which can sustain for a certain period even after NGF expression is decreased. This finding suggests that bladder hyperactivity in rats after cerebral hypoperfusion may be due to alterations in brain function rather than a confined dysfunction in bladder. CONCLUSIONS
Our study suggests the cerebral hypoperfusion may cause bladder dysfunction, which could be possibly related to damage to the neural circuitry in the forebrain rather than changes in bladder NGF expression.
Neurourology and Urodynamics DOI 10.1002/nau
ACKNOWLEDGMENTS
This work was supported by the Chang Gung Memorial Hospital under the Medical Research Project (CMRPG371561). We thank the assistance from the Molecular Imaging Center, Chang Gung Memorial Hospital, Linkou Medical center. REFERENCES 1. Skelly J, Flint AJ. Urinary incontinence associated with dementia. J Am Geriatr Soc 1995;43:286–294. 2. Sakakibara R, Fowler CJ, Hattori T. Voiding and MRI analysis of the brain. Int Urogynecol J 1999;10:192–199. 3. Sugiyama T, Hashimoto K, Kiwamoto H, et al. Urinary incontinence in senile dementia of the Alzheimer type. Int J Urol 1994;1:337–340. 4. Yokoyama O, Yoshiyama M, Namiki M, et al. Glutamatergic and dopaminergic contributions to rat bladder hyperactivity after cerebral artery occlusion. Am J Physiol 1999;276:R935–R942. 5. Yokoyama O, Yoshiyama M, Namiki M, et al. Role of the forebrain in bladder overactivity following cerebral infarction in the rat. Exp Neurol 2000; 163:469–476. 6. Yokoyama O, Ootsuka N, Komatsu K, et al. Forebrain muscarinic control of micturition reflex in rats. Neuropharmacology 2001;41:629–638. 7. Yotsuyanagi S, Narimoto K, Namiki M. Mild brain ischemia produces bladder hyperactivity without brain damage in rats. Urol Int 2006;77:57–63. 8. Farkas E, Luiten PG. Cerebral microvascular pathology in aging and Alzheimer’s disease. Prog Neurobiol 2001;64:575–611. 9. Morgan MK, Anderson RE, Spence I, et al. A model of the pathophysiology of cerebral arteriovenous malformations by a carotid-jugular fistula in the rat. Brain Res 1989;496:241–250. 10. Sekhon LH, Morgan MK, Spence I, et al. Chronic cerebral hypoperfusion and impaired neuronal function in rats. Stroke 1994;25:1022–1027. 11. Schultz-Lampel D. Bladder disorders in dementia and Alzheimer’s disease. Rational diagnostic and therapeutic options. Urologe A 2003;42:1579–1587. 12. Farkas E, Luiten PGM, Bari F. Permanent, bilateral common carotid artery occlusion in the rat: A model for chronic cerebral hypoperfusion-related neurodegenerative diseases. Brain Res Rev 2007;54:162–180. 13. Lee TH, Yang JT, Ko YS. et al. Influence of ischemic preconditioning on levels of nerve growth factor, brain-derived neurotrophic factor and their high affinity receptors in hippocampus following forebrain ischemia. Brain Res 2008; 1187:1–11. 14. Ochodnicky P, Cruz CD, Yoshimura N, et al. Nerve growth factor in bladder dysfunction: Contributing factor, biomarker, and therapeutic target. Neurourol Urodyn 2011;30:1227–1241. 15. Lee TH, Liu HL, Yang ST, et al. Effects of aging and hypertension on cerebral ischemic susceptibility: Evidenced by MR diffusion-perfusion study in rat. Exp Neurol 2011;227:314–321. 16. Lin YH, Liu G, Kavran M, et al. Lower urinary tract phenotype of experimental autoimmune cystitis in mouse: A potential animal model for interstitial cystitis. BJU Int 2008;102:1724–1730. 17. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25:402–408. 18. Lin TN, Sun SW, Cheung WM, et al. Dynamic changes in cerebral blood flow and angiogenesis after transient focal cerebral ischemia in rats. Evaluation with serial magnetic resonance imaging. Stroke 2002;33:2985–2991. 19. Tsuchiya M, Sako K, Yura S, et al. Cerebral blood flow and histopathological changes following permanent bilateral carotid artery ligation in Wistar rats. Exp Brain Res 1992;89:87–92. 20. Schmidt-Kastner R, Truettner J, Lin B, et al. Transient changes of brain derived neurotrophic factor mRNA expression in hippocampus during moderate ischemia induced by chronic bilateral common carotid artery occlusion in the rat. Brain Res Mol Brain Res 2001;92:157–166. 21. Otori T, Katsumata T, Muramatsu H, et al. Long-term measurements of cerebral blood flow and metabolism in a rat chronic hypoperfusion model. Clin Exp Pharmacol Physiol 2003;30:266–272. 22. Marosi M, Rakos G, Robotka H, et al. Hippocampal (CA1) activities in Wistar rats from different vendors. Fundamental differences in acute ischemia. J Neurosci Methods 2006;156:231–235. 23. Matsumoto K, Lo EH, Pierce AR, et al. Role of vasogenic edema and tissue cavitation in ischemic evolution on diffusion-weighted imaging: Comparison with multiparameter MR and immunohistochemistry. AJNR Am J Neuroradiaol 1995;16:1107–1115. 24. Yokoyama O, Yoshiyama M, Namiki M, et al. Changes in dopaminergic and glutamatergic excitatory mechanisms of micturition reflex after middle cerebral artery occlusion in conscious rats. Exp Neurol 2002;173:129–135. 25. Kanie S, Yokoyama O, Komatsu K, et al. GABAergic contribution to rat bladder hyperactivity after middle cerebral artery occlusion. Am J Physiol 2000;279: R1230–R1238.
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