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Keywords:

  • afferent;
  • bladder;
  • bladder outlet obstruction;
  • glutamic acid;
  • glycinergic neuron

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Objectives:  We investigated the time course of changes in bladder activity as well as in spinal and serum levels of glutamate and glycine after partial bladder outlet obstruction (BOO) in rats.

Methods:  A total of 36 female rats were divided into six groups: sham operation (control); 3 days, 14 days, and 28 days after BOO; 3 days and 28 days after relief of BOO. Under urethane anesthesia, isovolumetric cystometry was carried out in each group. Then, spinal and serum levels of glutamate and glycine were measured.

Results:  The interval between bladder contractions was shorter in all of the groups compared with the control group. The amplitude and duration of bladder contractions was decreased at 3 days, 14 days, and 28 days after BOO, and at 3 days after relief of BOO. Spinal and serum glutamate levels showed no changes. However, the spinal glycine level was decreased at 14 days and 28 days after BOO, and at 28 days after relief of BOO. Serum glycine level was also decreased at 28 days after BOO and 28 days after relief of BOO.

Conclusions:  Detrusor overactivity during the chronic phase of partial BOO is partly caused by a decrease of glycinergic neuronal activity in the lumbosacral cord. A 3-day period of BOO produces detrusor overactivity, which might be due to an irreversible decrease of spinal glycinergic neuronal activity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Overactive bladder syndrome (OAB) is one of the most common urological problems in elderly patients.1 Bladder outlet obstruction (BOO) caused by benign prostatic hyperplasia (BPH) in elderly men is associated with detrusor overactivity, which is also the most common cause of OAB.2 In the rat, partial BOO leads to bladder hypertrophy and detrusor overactivity,3 and the structural and functional changes noted in rats with BOO are similar to those seen in patients with BPH. It has been reported that an increase of gap junctions in bladder smooth muscle cells,4,5 or the release of acetylcholine or adenosine triphosphate from the urothelium6,7 plays a key role in the genesis of detrusor overactivity in a rat model of BOO and in patients with BPH. Another proposed explanation for these changes of bladder function is plasticity of the micturition reflex pathway that is mediated by an increase of afferent activity after bladder obstruction.8 However, the response of the afferent mechanism to BOO is relatively unexplored.

In the central nervous system, glutamate is a major excitatory neurotransmitter, whereas glycine and gamma-aminobutyric acid are the most abundant inhibitory amino acid neurotransmitters.9,10 Both of these neurotransmitters play an important role in the micturition reflex. When glycine is given intrathecally, intravenously, or orally, it reduces detrusor overactivity and urethral resistance in rats.11–14 In our previous study, rats with chronic spinal cord injury showed frequent bladder contractions on cystometry, and also had lower glycine levels in the lumbosacral cord and serum compared with intact rats.11,13 Therefore, the loss of inhibitory glycinergic neuronal activity is likely to be involved in the genesis of detrusor overactivity after partial BOO. To confirm this hypothesis, we examined the time course of changes in bladder activity, as well as changes of the glutamate and glycine levels in the lumbosacral cord and serum, after partial BOO and after relief of BOO in rats.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals and partial BOO model

A total of 36 female Sprague-Dawley rats weighing 250–300 g were used. The experimental protocol was approved by the University of the Ryukyus. Rats in the BOO group were anesthetized with 2% halothane, and the bladder and proximal urethra were exposed through a lower abdominal incision. A polyethylene catheter (PE-50; outer diameter 0.965 mm) was placed beside the urethra, and a 4-0 silk ligature was tied around both the urethra and the catheter to create partial BOO. After the ligature was secured, the catheter was removed. Then the abdomen was sutured, and the rats were placed in a cage to recover from anesthesia. In the sham-operated (control) group, the surgical procedure was identical to that described above, except that the urethra was not ligated. The rats were divided into the following six groups: a control group (n = 6); groups examined at 3 days, 14 days, and 28 days after BOO (n = 6 each); and groups examined at 3 days and 28 days after the relief of BOO (n = 6 each). In the last two groups, the ligature was cut and removed at 3 days after BOO. The animals were treated with an antibiotic (ampicillin at 100 mg/day intramuscularly for 2 days) after surgery to prevent infection.

Isovolumetric cystometry

Cystometry was carried out under isovolumetric conditions in each group (n = 6 each). The rats were anesthetized by combined intraperitoneal (0.4 g/kg) and subcutaneous (0.8 g/kg) injection of urethane. After micturition was induced by tail pinching, a polyethylene catheter (PE-50) was inserted into the bladder through the urethra and the residual urine volume was determined. Next, an abdominal incision was made and the ureters were transected, with the distal being ligated. The urethra was also ligated to the catheter near the external urethral meatus to produce isovolumetric conditions in the bladder. Bladder activity was monitored via the urethral catheter, which was connected to a pressure transducer and an infusion pump through a three-way stopcock. The bladder was filled with physiological saline (0.05 mL/min) to above the threshold volume that induced rhythmic isovolumetric contractions. After the contractions had been stable for over 60 min, the interval, amplitude, and duration of isovolumetric bladder contractions were evaluated. These parameters were averaged for a 30-min period and then compared between the six groups.

Measurement of bladder weight and glutamate and glycine levels in the lumbosacral cord and serum by capillary electrophoresis

Following cystometry, the bladder was removed in each group (n = 6) and weighed. After the bladder was removed, blood was withdrawn from the inferior vena cava, and then each rat was killed. The blood was centrifuged for 10 min at 1960 g to separate serum, which was then deproteinized. The lumbosacral cord was also removed and was homogenized in cold 0.5 M hydrochloric acid (1.0 mL/0.1 g tissue), after which the homogenate was centrifuged at 17 640 g for 5 min. The supernatant was dechlorinated and was deproteinized by using an Ultrafree C3 THK filter (Millipore, Bedford, MA, USA). Then the amino acid levels in each sample were measured by a capillary electrophoresis system (3DCE, Hewlett-Packard, Waldbronn, Germany).11

Statistical analysis

Results are reported as the mean ± standard error (SE). Student's t-test for unpaired data was used for statistical analysis, and significance was defined as P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Bladder activity after BOO or relief of BOO

At 3 days, 14 days, and 28 days after BOO, the residual urine volume and the intravesical threshold volume that induced isovolumetric bladder contractions were both significantly increased (P < 0.05) compared with those in control rats (Table 1). When isovolumetric bladder contractions were induced, the interval between contractions was significantly shorter (P < 0.01) in all BOO rats (3 days, 14 days, and 28 days) compared with that in control rats (Table 1, Figs 1,2A). In addition, the amplitude and duration were both significantly (P < 0.05) reduced in all BOO rats (3 days, 14 days, and 28 days) compared with those in control rats (Table 1, Figs 1,2B,C).

Table 1.  Changes in cystometric parameters in rats after bladder outlet obstruction (BOO) and after relief of 3-day period of BOO
GroupResidual urine volume (mL)Intravesical threshold volume (mL)Interval (min)Amplitude (cm H2O)Duration (min)
  1. Significant differences from control are indicated by *P < 0.05, and **P < 0.01.

Control0.01 ± 0.010.59 ± 0.082.42 ± 0.20 33.2 ± 2.91.72 ± 0.33
After BOO
 3 days0.12 ± 0.03*1.73 ± 0.11**1.19 ± 0.14**21.4 ± 3.7*0.77 ± 0.17*
 14 days1.68 ± 0.59*2.43 ± 0.28**1.04 ± 0.23**18.3 ± 4.6*0.70 ± 0.15*
 28 days2.58 ± 0.52**3.42 ± 0.38**0.60 ± 0.09**14.0 ± 4.5**0.53 ± 0.13*
After relief of 3-day period of BOO
 3 days0.03 ± 0.011.32 ± 0.18**0.92 ± 0.08**17.5 ± 2.5**0.55 ± 0.02*
 28 days0.02 ± 0.010.68 ± 0.061.84 ± 0.12* 35.5 ± 2.51.15 ± 0.02
image

Figure 1. Isovolumetric cystometry in sham (control) rats, and rats after bladder outlet obstruction (BOO) (3 days to 28 days). At 3 days after BOO, the interval between bladder contractions was irregular and shortened, along with a decrease of amplitude and duration, compared with the findings in control rats. At 14 and 28 days after BOO, the interval between contractions was regular, but was still shortened with a decrease of amplitude and duration, compared with the data for control rats.

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image

Figure 2. Effect of bladder outlet obstruction (BOO) (3 days to 28 days; closed circles) and relief of BOO (3 days and 28 days; open circles) on the interval (a), amplitude (b), and duration (c) of isovolumetric bladder contractions. (a) At 3 days, 14 days, and 28 days after BOO, as well as at 3 days and 28 days after relief of BOO, the interval was significantly shorter compared with that in sham (control) rats. (b) At 3 days, 14 days, and 28 days after BOO, the amplitude was significantly decreased compared with that in control rats. The amplitude at 3 days after relief of BOO was also significantly decreased compared with that in control rats, but it returned to the control level by 28 days after relief of BOO. (c) At 3 days, 14 days, and 28 days after BOO, the duration was significantly shorter compared with that in control rats. The duration at 3 days after relief of BOO was also significantly shorter compared with that in control rats, but it returned to the control level by 28 days after relief of BOO. The number of rats in each group was six. Values are the mean ± standard error (SE). Significant differences from control (Cont) are indicated by: *P < 0.05, and **P < 0.01.

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At 3 days and 28 days after relief of BOO, the residual urine volume did not differ from that in control rats. However, the intravesical threshold volume inducing isovolumetric bladder contractions was significantly increased (P < 0.01) at 3 days after the relief of BOO compared with that in control rats, whereas it returned to the control level by 28 days after relief of BOO (Table 1). When isovolumetric bladder contractions were induced, the interval between contractions was significantly shorter (P < 0.05) at 3 days and 28 days after relief of BOO compared with that in control rats (Table 1, Fig. 2A). The amplitude of bladder contractions was significantly reduced (P < 0.01) at 3 days after relief of BOO compared with that in control rats, but it returned to the control level by 28 days after relief of BOO (Table 1, Fig. 2B). The duration was also significantly shortened (P < 0.05) at 3 days after relief of BOO compared with that in control rats. At 28 days after relief of BOO, this duration still tended to be shorter but was not significantly different from that of control rats (Table 1, Fig. 2C).

Changes of bladder weight after BOO or relief of BOO

At 14 days and 28 days after BOO, the bladder weight was significantly increased (P < 0.05). At 3 days and 28 days after relief of BOO; however, the bladder weight was not different from that in control rats (Table 2).

Table 2.  Changes in bladder weight and glutamate and glycine levels in the lumbosacral cord and serum in rats after bladder outlet obstruction (BOO) and after relief of 3-day period of BOO
GroupBladder weight (g)LSC amino acids (µmol/g tissue)Serum amino acids (mg/L)
GlutamateGlycineGlutamateGlycine
  1. Significant differences from control are indicated by *P < 0.05, and **P < 0.01. LSC, lumbosacral cord.

Control0.10 ± 0.014.26 ± 0.161.26 ± 0.049.32 ± 0.6315.7 ± 1.37
After BOO
 3 days0.13 ± 0.014.12 ± 0.171.01 ± 0.1110.7 ± 0.5317.0 ± 0.69
 14 days0.22 ± 0.04*4.43 ± 0.091.00 ± 0.03**9.40 ± 0.3114.8 ± 1.11
 28 days0.45 ± 0.12*4.15 ± 0.140.80 ± 0.10**8.96 ± 0.4710.1 ± 0.37**
After relief of 3-day period of BOO
 3 days0.13 ± 0.014.01 ± 0.061.02 ± 0.219.69 ± 0.2115.9 ± 0.76
 28 days0.11 ± 0.014.17 ± 0.150.65 ± 0.12**8.91 ± 1.5710.3 ± 0.78*

Glutamate and glycine levels in the lumbosacral cord after BOO or relief of BOO

After BOO or relief of BOO, the glutamate level measured in the lumbosacral cord did not differ from that of control rats. In contrast, the glycine level in the lumbosacral cord was significantly decreased (P < 0.01) at 14 days and 28 days after BOO, as well as at 28 days after relief of BOO, compared with that in control rats (Table 2, Fig. 3A).

image

Figure 3. Changes of the glycine level in the lumbosacral cord (LSC, a) and the serum (b) after bladder outlet obstruction (BOO) (3 days to 28 days; closed circles) and after relief of BOO (3 days and 28 days; open circles). (a) The glycine level in the lumbosacral cord was significantly decreased at 14 days and 28 days after BOO and at 28 days after relief of BOO compared with that in sham (control) rats. The serum glycine level was significantly decreased at 28 days after BOO and at 28 days after relief of BOO compared with that in control rats. The number of rats in each group was six. Values are the mean ± standard error (SE). Significant differences from control (Cont) are indicated by: *P < 0.05, and **P < 0.01.

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Serum glutamate and glycine levels after BOO or relief of BOO

After BOO or relief of BOO, the serum glutamate level did not differ from that in control rats. In contrast, the serum glycine level was significantly decreased (P < 0.01) at 28 days after BOO and at 28 days after relief of BOO compared with that in control rats (Table 2, Fig. 3B).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The results of the present study indicate that: (i) detrusor overactivity during the chronic phase of partial BOO is partly due to a decrease of spinal glycinergic neuronal activity, as evidenced by a decrease of glycine level in the lumbosacral cord at 14 to 28 days after BOO; (ii) the existence of partial BOO for only 3 days can cause detrusor overactivity and an irreversible decrease of spinal glycinergic neuronal activity, as evidenced by the shortened interval between bladder contractions and a decrease of glycine level in the lumbosacral cord even at 28 days after relief of a 3-day period of BOO; and (iii) the serum glycine level reflects the spinal glycine level, as evidenced by a decrease of serum glycine level at 28 days after BOO and after relief of a 3-day period of BOO with a delay of 2 weeks.

The present study showed that the interval between isovolumetric bladder contractions was shortened at 3 days, 14 days, and 28 days after BOO. In addition, the amplitude and duration of isovolumetric bladder contractions were both reduced at these times after BOO, while the residual urine volume and intravesical threshold volume were increased at the same times. Furthermore, the bladder weight was increased at 14 days and 28 days after BOO. These results suggest that the initial response of the bladder to severe experimental obstruction is acute overdistension, followed by a massive increase of bladder muscle mass and a reduced contractile response (as indicated by the decreased amplitude and duration of bladder contractions). The result is an increase of the residual urine volume and intravesical threshold volume. Therefore, detrusor overactivity during the early phase (3 days) of partial BOO might have a myogenic origin and might be dependent on the modulation of smooth muscle cells by mediators released from the urothelium, as reported previously.3,4–7

Bladder activity can be modulated by a variety of afferent inputs from other pelvic organs, such as the rectum, vagina, penis, and urethra.15 Schroder et al.8 have shown that detrusor overactivity develops in obstructed mice without an increase of bladder weight and with only subtle changes of smooth muscle function in vitro. These findings suggest that changes of afferent nerve function may have a greater role than changes of bladder smooth muscle in the consequences of BOO. In our previous study, rats with chronic spinal cord injury showed frequent bladder contractions on cystometry, and had lower glycine levels in the lumbosacral cord and serum compared with intact rats.11,13 In the present study, rats with partial BOO (2–4 weeks) also showed frequent bladder contractions and changes of the amplitude/duration of contractions, as well as having lower glycine levels in the lumbosacral cord and serum compared with control rats. Since changes of the interval between contractions, and the amplitude/duration of contractions are thought to be due to alterations of afferent and efferent activity in the micturition reflex pathway, respectively,16 both the afferent and efferent limbs of the spinobulbospinal micturition reflex pathway may be involved in the development of detrusor overactivity after BOO. However, the amplitude and duration of bladder contractions might have been influenced by acute overdistension in the present study, so the major disturbances caused by BOO may be confined to the afferent arm of the pathway. Glycinergic neurons may mediate the change of the afferent pathway for the micturition reflex during the late phase after BOO. At least two possible explanations can be suggested for the decrease of glycinergic neuronal activity in the late phase after BOO. One possibility is that ischemia around the bladder neck after BOO could induce hyperexcitability of C-fibers in the afferent pathway, which would lead to a decrease of glycinergic neuronal activity. The other possibility is that adaptation may lead to a decrease of spinal glycinergic neuronal activity during the late phase after BOO, in order to allow the expulsion of urine.

After relief of BOO, the interval between isovolumetric bladder contractions was shortened at 3 days and 28 days, while the amplitude and duration of contractions were reduced at 3 days. The shorter interval between contractions and decreased amplitude, duration at 3 days after relief of BOO might have been due to acute overdistension during the 3-day period of BOO, as seen in the rats with BOO. However, our previous study showed that the interval between isovolumetric contractions was shortened and the glycine levels in the lumbosacral cord and serum were decreased without any change of bladder weight even 8 weeks after the relief of a 3-day period of BOO.17 Therefore, these results suggest that the existence of partial BOO for only 3 days can cause detrusor overactivity that persists even 4 weeks after obstruction is relieved, and this change may be due to a an irreversible decrease of glycinergic neuronal activity in the spinal cord. Detrusor overactivity is often a persistent finding after transurethral resection of the prostate,18 and one reason for this persistence may be an irreversible decrease of spinal glycinergic neuronal activity in patients with BPH.

We also found that the serum glycine level showed a decrease at 28 days after BOO that followed the decrease of the spinal glycine level, with a short delay (2 weeks), and serum glycine was also decreased at 28 days after relief of BOO. In our previous study, rats with cerebral infarction showed detrusor overactivity, and had lower glycine levels in the lumbosacral cord compared with intact rats. The serum glycine level was also decreased with a delay of 1–2 weeks.19 Therefore, the serum glycine level may reflect the spinal glycine level, because glycine can cross the blood–brain barrier.20 The serum glycine level was also lower in BPH patients compared with that of healthy controls in our recent study,21 suggesting that it may be possible to use serum glycine as an indicator of glycinergic neuronal activity in the spinal cord.

Clinically, detrusor overactivity is common in patients with BPH and bladder outlet obstruction,2 but its pathogenesis is not well understood. Our present findings suggest that detrusor overactivity during the chronic phase of partial BOO is partly due to a decrease of glycinergic neuronal activity in the lumbosacral cord. Therefore, improving glycinergic neuronal activity in the lumbosacral cord may be a target for the treatment of detrusor overactivity in patients with BPH.

In conclusion, partial BOO led to detrusor overactivity in rats that was associated with decreased glycine levels in the lumbosacral cord and serum during the late phase, suggesting that chronic changes of bladder activity after partial BOO are partly due to a decrease of glycinergic neuronal activity in the lumbosacral cord. A 3-day period of BOO also led to detrusor overactivity, which may have been partly due to an irreversible decrease of spinal glycinergic neuronal activity.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was supported by a Grant-in-Aid for Scientific Research (B-14370516) from the Japan Society for the Promotion of Science and by a 2003 grant from Okinawa Medical Science Research Foundation and a 2005 grant from Daiwa Securities Health Foundation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Abrams P, Cardozo L, Fall M et al. Standardisation Sub-committee of The International Continence Society. The Standardisation of terminology of lower urinary tract function: report from The Standardisation Sub-committee of The International Continence Society. Neurourol. Urodyn. 2002; 21: 16778.
  • 2
    Knutson T, Edlund C, Fall M, Dahlstrand C. BPH with coexisting overactive bladder dysfunction–an everyday urological dilemma. Neurourol. Urodyn. 2001; 20: 23747.
  • 3
    Uvelius B, Persson L, Mattiasson A. Smooth muscle cell hypertrophy and hyperplasia in the rat detrusor after short-time infravesical outflow obstruction. J. Urol. 1984; 131: 1736.
  • 4
    Christ GJ, Day NS, Day M et al. Increased connexin 43-mediated intercellular communication in a rat model of detrusor overactivity in vivo. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2003; 284. R1241–8.
  • 5
    Haferkamp A, Mundhenk J, Bastian PJ et al. Increased expression of connexin 43 in the overactive neurogenic detrusor. Eur. Urol. 2004; 46: 799805.
  • 6
    Banks FC, Knight GE, Calvert RC, Morgan RJ, Burnstock G. Alterations in purinergic and cholinergic components of contractile responses of isolated detrusor contraction in a rat model of partial bladder outlet obstruction. BJU Int. 2006; 97: 3728.
  • 7
    Sun Y, MaLossi J, Jacobs SC, Chai TC. Effect of doxazosin on stretch-activated adenosine triphosphate release in bladder urothelial cells from patients with benign prostatic hyperplasia. Urology 2002; 60: 3516.
  • 8
    Schroder A, Uvelius B, Newgreen D, Andersson KE. Detrusor overactivity in mice after 1 week of outlet obstruction. Mainly afferent dysfunction? J. Urol. 2003; 170: 101721.
  • 9
    Desarmenien M, Santangelo F, Loeffler JP, Feltz P. Comparative study of GABA-mediated depolarizations of lumbar A delta and C primary afferent neurones of the rat. Exp. Brain Res. 1984; 54: 5218.
  • 10
    Shapiro S. Neurotransmission by neurons that use serotonin, noradrenaline, glutamate, glycine, and γ-aminobutyric acid in the normal and injured spinal cord. Neurosurgery 1997; 40: 16876.
  • 11
    Miyazato M, Sugaya K, Nishijima S, Ashitomi K, Hatano T, Ogawa Y. Inhibitory effect of intrathecal glycine on the micturition reflex in normal and spinal cord injury rats. Exp. Neurol. 2003; 183: 23240.
  • 12
    Miyazato M, Sugaya K, Nishijima S, Shimabukuro S, Ashitomi K, Ogawa Y. Intravenous glycine inhibits the micturition reflex in normal and spinal cord injury rats. Biomed. Res. 2003; 24: 2859.
  • 13
    Miyazato M, Sugaya K, Nishijima S, Ashitomi K, Morozumi M, Ogawa Y. Dietary glycine inhibits bladder activity in normal rats and rats with spinal cord injury. J. Urol. 2005; 173: 31417.
  • 14
    Miyazato M, Sugaya K, Nishijima S, Kadekawa K, Ashimine S, Ogawa Y. Intrathecal or dietary glycine inhibits bladder and urethral activity in rats with spinal cord injury. J. Urol. 2005; 174: 2397400.
  • 15
    Kruse MN, Mallory BS, Noto H, Roppolo JR, de Groat WC. Modulation of the spinobulbospinal micturition reflex pathway in cats. Am. J. Physiol. 1992; 262: R478–84.
  • 16
    Sugaya K, Nishijima S, Miyazato M, Ashitomi K, Hatano T, Ogawa Y. Effects of intrathecal injection of tamsulosin and naftopidil, alpha-1A and -1D adrenergic receptor antagonists, on bladder activity in rats. Neurosci. Lett. 2002; 328: 746.
  • 17
    Miyazato M, Sugaya K, Nishijima S, Uchida A, Morozumi M, Ogawa Y. Changes of Connexin43-derived gap junctions in the bladder and glycine in the lumbosacral cord after partial bladder outlet obstruction in rats. J. Urol. 2005; 173 (Suppl): 326 (Abstract 1201).
  • 18
    Gormley EA, Griffiths DJ, McCracken PN, Harrison GM, McPhee MS. Effect of transurethral resection of the prostate on detrusor instability and urge incontinence in elderly males. Neurourol. Urodyn. 1993; 12: 44553.
  • 19
    Nishijima S, Sugaya K, Miyazato M, Morozumi M, Ogawa Y. Relationship between bladder activity and amino acid levels in the central nervous system in rats with cerebral infarction. Biomed. Res. 2003; 24: 17380.
  • 20
    Wright EM. Active transport of glycine across the frog arachnoid membrane. Brain Res. 1974; 16: 3548.
  • 21
    Nishijima S, Sugaya K, Fukuda T, Miyazato M, Ashimine S, Ogawa Y. Serum amino acids as indicators of cerebrospinal neuronal activity in patients with micturition disorders. Int. J. Urol. 2006; 13: 147983.