Nerve growth factor combined with vascular endothelial growth factor enhances regeneration of bladder acellular matrix graft in spinal cord injury-induced neurogenic rat bladder

Authors

  • Nobuyuki Kikuno,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Ken Kawamoto,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Hiroshi Hirata,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Kaveh Vejdani,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Kazumori Kawakami,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Thomas Fandel,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Lora Nunes,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Shinji Urakami,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Hiroaki Shiina,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Mikio Igawa,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Emil Tanagho,

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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  • Rajvir Dahiya

    1. Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA, USA, and Department of Urology, Shimane University School of Medicine, Izumo, Japan
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Rajvir Dahiya, Urology Research Center (112F), Veteran Affairs Medical Center and University of California, San Francisco, 4150 Clement Street, San Francisco, CA 94121, USA.
e-mail: rdahiya@urology.ucsf.edu

Abstract

OBJECTIVE

To determine the combined effects of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) on regeneration of the bladder acellular matrix graft (BAMG) in spinal cord injury (SCI)-mediated neurogenic bladder in rats.

MATERIALS AND METHODS

In all, 40 female Sprague-Dawley rats were used. At 8 weeks after spinalization surgery (neurogenic bladder), they were divided into five groups consisting of untreated controls and those whose bladders were injected with either no growth factor, NGF (2 µg/rat), VEGF (2 µg/rat) or both at partial BAMG replacement surgery. After 8 weeks, bladder function was assessed by urodynamic studies and the bladders were harvested for histological examination. Smooth muscle induction, collagen and nerve fibre regeneration were assessed immunohistochemically using antibodies to smooth muscle actin (α-actin), Masson’s trichrome and protein gene product 9.5, respectively.

RESULTS

Bladder capacity and compliance were significantly increased in all BAMG groups 8 weeks after surgery compared with that before bladder replacement surgery. Bladder capacity and compliance were much higher in the VEGF and NGF combined group than in the control, or NGF and VEGF alone groups. There was no significant difference in the residual volume ratio among all groups.

CONCLUSIONS

This is the first report showing that NGF has a significant synergistic effect on the development, differentiation and functional restoration of the BAMG when administered with VEGF in neurogenic bladder. Our results indicate that NGF may be a useful cytokine for enhancing the regeneration of a functional bladder following acellular matrix grafting in a neurogenic rat model.

Abbreviations
NGF

nerve growth factor

VEGF

vascular endothelial growth factor

BAMG

bladder acellular matrix graft

SCI

spinal cord injury

PGP 9.5

protein gene product 9.5

H&E

haematoxylin and eosin

UIC

uninhibited detrusor contraction.

INTRODUCTION

Neurogenic bladder has recently become a common indication for augmentation surgery in clinical situations. Augmentation enterocystoplasty is suitable for selected patients with overactive or end-stage bladder disorders that have failed to respond to conservative therapies [1–3]. Although this technique can potentially solve the problems associated with bladder capacity, the potential surgical complications such as dysbolism, urolithiasis, and absorptive disadvantage related to the usage of bowel segments, and cancer induction have limited its application [1–3]. Recently, we and other investigators have shown the applicability of bladder replacement using bladder acellular matrix graft (BAMG) as one of the alternatives to augmentation enterocystoplasty in normal or spinal cord injury (SCI)-induced neurogenic bladder rats [4,5]. Nevertheless, these promising results have not yet become clinically relevant [6]. One major problem in these studies is the lack of a clear distinction between native and regenerated bladder in total bladder function after augmentation. Another crucial problem is the absorption and shrinkage of larger grafts, which may result from insufficient vascular supply and smooth muscle regeneration [6]. Several growth factors are thought to play an important role in organogenesis [7,8]. Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that functions as an inducer of vascular permeability and endothelial cell-specific mitogen [9]. In addition to its angiogenic function, VEGF also serves as an inhibitor of apoptosis in smooth muscle [10] and endothelial cells [11], and as a neurotrophic factor involved in nerve regeneration [12]. By contrast, nerve growth factor (NGF) is the first and best-characterized member of the neurotrophin family. NGF supports the survival and maintenance of peripheral sensory and autonomic neurones, during development and adult stages [13,14]. Exogenous NGF administration in developing animals prevents or reduces peripheral neuropathies induced by chemical and surgical insult [15–17]. However, the optimal choice and combination of growth factors to be added, and the specific contribution of each growth factor to tissue regeneration remain unclear. We have previously reported that VEGF could enhance BAMG regeneration at early but not late periods after grafting in normal rats [18].

In the present study, we investigated whether NGF has a synergistic effect with VEGF on the development and differentiation of the BAMG for partial bladder replacement, and whether the effect of NGF continues until the late periods after grafting in a SCI-induced neurogenic rat bladder.

MATERIALS AND METHODS

In all, 40 female Sprague-Dawley rats (Charles River, Montreal, Quebec, Canada) were used. The rats were divided into the following five groups: group 1 received only spinalization surgery, group 2 received BAMG surgery with no growth factors at 8 weeks after the spinalization surgery, groups 3 and 4 received NGF or VEGF, and in group 5, both NGF and VEGF were injected into the host bladder. The rats were maintained and treated according to our institutional guidelines. The local Animal Care Committee approved the experimental protocol.

The bladder from adult healthy rats were harvested and decellularized by sequential 48-h treatments with DNase (400 U/40 mL of 2 m NaCl, Sigma, St. Louis, MO, USA), ammonium hydroxide (0.05 mm), and sodium deoxycholate (2%), followed by extensive washes with distilled water for 5 days. Acellularity was confirmed by light microscopy using haematoxylin and eosin (H&E), and methyl green pyronin staining before grafting.

Rats were anaesthetized by inhalation of 2% isoflurane. A midline dorsal incision was made over lower thoracic vertebra to expose the vertebral spines and paravertebral muscles. A laminectomy of the T10 spinal vertebrae was performed and the spinal cord was completely transected at the lower thoracic level. After surgery, the bladder was manually emptied three times daily until reflex voiding recovered after SCI.

After 8 weeks, the spinalized rats were prepared for bladder replacement surgery. Under anaesthesia, 50% partial cystectomy was performed and 50% of the acellular scaffold was stitched to the bladder using absorbable 7/0 polydioxanone running sutures. The bladder was irrigated with normal saline to test the anastomosis for leakage. Non-absorbable 6/0 nylon marking sutures were placed at four points immediately under the polydioxanone suture line. In the groups that received growth factors, a total of 2 µg of each growth factor was injected at the four points into the bladder submucosa after partial cystectomy.

Open-abdomen cystometrography was done before grafting and repeated 8 weeks after bladder replacement. All rats underwent cystometrography under ketamine anaesthesia (100 mg/kg) [19]. Cystometrography was performed according to previously published methods [18]. After the bladder was emptied, saline was infused into the bladder at 0.2 mL/min. The variables recorded during saline infusion included resting bladder pressure, threshold bladder volume (bladder capacity), threshold voiding pressure, bladder compliance, voided volume, residual urine volume, leak-point pressure, voiding efficacy, maximal amplitude of uninhibited detrusor contractions (UICs). The voided volume was the amount that was leaked from the meatus around the tube and the residual volume was the amount that was drained by the tube after the leakage. A contraction of ≥15 cmH2O was considered as an UIC. Bladder compliance was calculated by dividing the bladder capacity by the threshold voiding pressure. The presence of UICs in this model of neurogenic bladder was categorized as a hyper-reflexic bladder, whereas the absence of UICs indicated an underactive bladder.

After completion of all procedures, the bladder was filled with a 10% formalin solution and removed. The bladder specimens were embedded in paraffin wax. Deparaffinized sections (4 µm) were used for staining with H&E and Masson’s trichrome. Immunohistochemical staining was performed using anti-smooth muscle actin (α-actin; Sigma) to confirm the presence of smooth muscle fibres. Immunostaining of anti-protein gene product 9.5 (PGP 9.5) (Biogenesis, Poole, UK) was used to detect nerve fibres.

The relationship between the urodynamic variables before and after augmentation were analysed using a Wilcoxon signed-rank test. The Mann–Whitney test was used for other statistical analyses; P < 0.05 was considered to indicate statistical significance.

RESULTS

All SCI-control rats with no bladder replacement (group 1) showed similar urodynamic data at 8 weeks and 16 weeks after spinalization. In other words, bladder functions stabilized at 8 weeks after SCI. Therefore, we compared the urodynamic variables at 8 weeks after SCI (as before replacement) and 8 weeks after bladder replacement surgery (as after replacement) in each group. Of the 40 SCI rats, 27 rats (67.5%) developed hyper-reflexic bladders with UICs and eight (20.0%) had underactive bladders with no UICs 8 weeks after spinalization (Fig. 1). Urodynamic data after replacement was obtained from 23 rats. The bladder capacity and compliance were significantly increased in the BAMG groups 8 weeks after surgery compared with those before bladder replacement surgery. As mentioned previously, there was no significant difference in these variables in group 1 between 8 weeks and 16 weeks after spinalization. Furthermore, both capacity and compliance were much higher in the VEGF and NGF combined group (group 5) than in the control (group 2), NGF alone (group 3) or VEGF alone (group 4) (Fig. 2). There was no significant difference in the recorded variables before and after augmentation except for bladder capacity and compliance.

Figure 1.

A, Typical cystometrogram of SCI-induced hyper-reflexic bladder (hyper-reflexia): this spinalized rat showed many large UICs (>15 cm/H2O) during saline infusion. The size of the contractions increased with time. Bladder capacity was ≈3.4 mL, threshold pressure was 43.5 cmH2O, and bladder compliance was 0.078 mL/cmH2O. B, Typical cystometrogram of SCI-induced underactive bladder (areflexia): this spinalized rat showed no UICs during saline infusion. The bladder capacity was ≈4.7 mL, threshold pressure was 28.0 cmH2O, and bladder compliance was 0.168 mL/cmH2O.

Figure 2.

Bladder capacity (A) and compliance (B) at 8 weeks and 16 weeks after spinalization surgery in group 1 (G1) and before and after BAMG replacement surgery in groups 2–5 (G2–5). The (pre) and (post) means before and after BAMG replacement in groups 2–5, respectively. *statistical difference of bladder capacity and compliance between before-BAMG replacement and after-BAMG replacement in each group. ♯statistical difference of bladder capacity and compliance between after-BAMG replacement of G2 and G5.

H&E and Masson’s trichrome stains showed regeneration of the urothelium, blood vessels, smooth muscle, and nerve fibres into the acellular collagen scaffold at 8 weeks in all groups. The luminal surface of the implanted BAMG was completely covered with urothelium 8 weeks after grafting with no significant difference between the groups. There were aggregated bundles of smooth muscle underneath the urothelium and a well-defined detrusor layer was identified in all groups. Smooth muscle content as determined by α-actin staining was significantly greater in group 5 (VEGF and NGF combined group) than in other groups. Specific nerve cell staining using PGP 9.5 showed regeneration of nerve fibres in the groups with growth factors. PGP 9.5 positive nerve fibres were observed most abundantly in group 5 (Fig. 3). There was no harmful effect of growth factors on bladder regeneration.

Figure 3.

G1, Representative H&E, Masson’s trichrome, α-actin, and PGP 9.5 staining of native bladder at 16 weeks after spinalization surgery in group 1 (×200). G2–5, Representative H&E, Masson’s trichrome, α-actin, and PGP 9.5 staining of BAMG at 8 weeks after grafting in groups 2–5 (×200).

DISCUSSION

The present data show that BAMG combined with the administration of NGF and VEGF improves bladder capacity and compliance in SCI-induced neurogenic rat bladder accompanied by aggregated bundles of smooth muscle and regeneration of nerve fibres. These results indicate that NGF has a significant synergistic effect on the development, differentiation and functional restoration of the BAMG when administered with VEGF and that the combination may be useful for enhancing the regeneration of a functional bladder after grafting. Other factors might also contribute to the bladder regeneration.

When the spinal cord above the sacral segment is damaged in rats, the SCI produces an initial period of bladder areflexia that lasts for several days. After this period, rats usually develop hyper-reflexic bladders [4]. On the other hand, it has been reported that detrusor areflexia happens in 30% of human patients with suprasacral SCI or disease [20,21]. Based on these previous reports, the present data where hyper-reflexic bladder was induced in 67.5% of rats and underactive bladder in 20% at 8 weeks after spinalization surgery is close to previously reported values. There were no differences among the groups for complications such as gross haematuria, UTI or vesical stone formation at 8 weeks after spinalization surgery. However, these complications were more prevalent in rats with underactive bladders than in rats with hyper-reflexic bladders at 8 weeks after spinalization surgery.

We and several other investigators have already shown the histological and functional regeneration of healthy and SCI-induced neurogenic bladders augmented with BAMG in a rat model [4,5]. We have also shown a favourable effect of VEGF on bladder regeneration using BAMG at the early period in the healthy rat model [18]. However, the effects of NGF and VEGF on regeneration of neurogenic bladder have not been investigated. We think that bladder regeneration using BAMG could become an effective approach if it was adopted to treat specific pathological conditions such as sphincteric deficiency, muscle hypertrophy and fibrosis, weak detrusor, or impaired innervations of the bladder. In the present study, we investigated whether bladder augmentation surgery using BAMG combined with additional cytokines can effect bladder regeneration and development of tissues at early and late periods to enhance functional restoration of SCI-induced neurogenic bladders. As autonomic neural modulation has been a major target for both neurogenic and non-neurogenic bladder dysfunction, the restoration of neural function may represent a major aspect of functional bladder regeneration [6].

Recent studies provide mounting evidence for a direct involvement of NGF in the angiogenic process, in addition to the well-established ‘classical’ angiogenic growth factors such as VEGF and basic fibroblast growth factor. For example, NGF induces in vitro proliferation of umbilical cord [22] and brain capillaries [23]. The few studies in animal models to date, indicate that NGF up-regulates VEGF production [24,25] and induces reparative neoangiogenesis, arteriogenesis, and wound healing [26–29]. Whether these processes occur cumulatively, directly via NGF-induced angio/arteriogenesis or indirectly via the induction of classical ‘angiogenic factors’ such as VEGF remains to be clarified. However, these previous reports indicate that NGF could enhance the potential effect of VEGF.

The present study showed that both bladder capacity and compliance were significantly improved at 8 weeks after BAMG when either NGF or VEGF alone or in combination were administered before augmentation surgery compared with no growth factor. Furthermore, the grafted BAMG of NGF/VEGF (+/+) rats immunohistochemically had numerous α-actin-positive spindle cells and PGP 9.5-positive nerve cells when compared with the other groups of rats (Fig. 3). These findings support the hypothesis that the combined administration of NGF and VEGF can accelerate the process of angiogenesis and neurogenesis in the grafted BAMG and keep them stable until the late phase, leading to aggregation of smooth muscle bundles and enhancement of bladder function in the BAMG.

Although additional studies are necessary to elucidate the precise mechanism of how NGF functions synergistically with VEGF in BAMG, the present results indicate that NGF may be a useful cytokine for enhancing the regeneration of a functional bladder after grafting.

In conclusion, NGF had a significant synergistic effect on the development, differentiation and functional restoration of the BAMG when administered with VEGF. The present results indicate that NGF may be a useful cytokine for enhancing the regeneration of a functional bladder after grafting.

ACKNOWLEDGEMENTS

We thank Dr Roger Erickson for his support and assistance with the preparation of the manuscript. This study was supported by Department of Defense (DOD), VA Merit Review and Deutsche, Forshungsgemeinschaft FA 479/1-1.

CONFLICT OF INTEREST

None declared.

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