Can Herpes Simplex Virus Vector-Mediated Gene Transfer of Kynurenine Aminotransferase Reduce Urethral Resistance in Spinal Cord Injured Rats?

Research Article

Austin J Urol. 2014;1(3): 5.

Can Herpes Simplex Virus Vector-Mediated Gene Transfer of Kynurenine Aminotransferase Reduce Urethral Resistance in Spinal Cord Injured Rats?

Limin Liao1,2*, Chunsong Jia1,2 and Naoki Yoshimura3

1Department of Urology, China Rehabilitation Research Center, China

2Department of Urology, Capital Medical University, China

3Department of Urology, University of Pittsburgh, USA

*Corresponding author: Limin Liao, Department of Urology, China Rehabilitation Research Center, No 10 Jiaomen Beilu, Beijing 100068, China

Received: August 07, 2014; Accepted: September 15, 2014; Published: September 20, 2014


Aim: To explore if replication-defective herpes simplex virus (HSVrd) vector-mediated gene transfer of kynurenine aminotransferase (KAT) can reduce urethral resistance in spinal cord injured rats.

Methods: The HSVrd-KAT II vector was constructed, Sprague-Dawley rats was spinalized with complete transection of the T10 spinal cord. Viral suspension of HSVrd or HSVrd-KAT II (1×107 plaque-forming units) was injected into 4 sites around the bladder base, cystometry was recorded. The KAT II positive neurons, ratio of KAT II/GADPH proteins and KAT II/β-actin mRNA in L6-S1 dorsal root ganglia (DRG) were examined.

Results: The number and amplitude of non-voiding bladder contraction (NVC), Detrusor Leak Point Pressur (DLPP) and Maximum Cystometric Capacity (MCC) were decreased significantly by 59.6-61.1%, 21.6-24.2%, 30.3- 34.4% and 44.1-46.5% (P<0.01), and the Bladder Emptying Efficiency (BEE) and the time to first NVC were increased significantly by 40.7-47.7% and 30.1- 49.0% (P<0.01), respectively, in the HSVrd-KAT II group compared to sham or HSVrd group. The vectors are transported to L6-S1 dorsal root ganglia and up-regulate the expression of KAT II.

Conclusion: The HSVrd-KAT II vectors injected into the bladder wall can reduce the urethral resistance, improved DSD and BEE in SCI rats, possibly due to suppressing C-fiber bladder afferents and N-Methyl-D-Aspartate Receptors (NMDAR) blockade in the DRG and spinal cord.

Keywords: Herpes simplex virus; Gene therapy; Kynurenine aminotransferase; Urethral resistance; Spinal cord injury; Rat


Detrusor-Sphincter Dyssynergia (DSD) caused by spinal cord injury (SCI) usually lead to an increased urethral resistance, low compliant, elevated pressures in the bladder, hydronephrosis and kidney dysfunction [1,2]. The goal of management for neurogenic bladder is to protect renal function by reducing the bladder pressure during the storage and voiding phase through inhibiting Detrusor Over activity (DO) and reducing urethral resistance.

The glutamatergic system via N-methyl-D-aspartate receptors (NMDARs) activation plays an important role in the control of micturition. MK-801, a potent non-competitive NMDAR antagonist, reportedly increases bladder capacity, reduces bladder contractions [3], inhibits external urethral sphincter contraction and increases the maximum flow rate [4,5] in SCI rats. These results suggest that blocking NMDARs might improve SCI-induced DO and DSD.

Kynurenic acid (KYNA), the only known endogenous antagonist of glutamate receptors, is able to block NMDARs [6,7]. A study showed that KYNA intrathecal application to the sacral cord completely inhibits bladder contractions in cats [8]. In the rat and human brain, over 70% of KYNA is formed from L-kynurenine catalyzed by kynurenine aminotransferase (KAT) II [9,10]. Human KAT II cDNA is about 1.5 kb, encoding a mitochondrial protein of 425 amino residues (NP_057312). Thus, gene delivery of KAT II that increases KYNA, thereby suppressing NMDAR mediated action in micturition, could be a useful approach for the treatment of SCI-induced DO and DSD.

The prosperous researches using replication-defective herpes simplex virus vectors (HSVrd) to treat neuropathies and pain imply the bright future for them in the treatment of bladder dysfunction [11]. HSVrd vectors are non-toxic and neurotropic gene transfer tools so that they could express therapeutic genes after latent infections in dorsal root ganglia (DRG) [12]. These characteristics guarantee the vectors to specifically deliver the KAT II gene to targeted afferent pathways after bladder wall injection with limited side effects in the treatment of SCI-induced neurogenic bladder. Previous reports showed HSVrd-mediated gene transfer of glutamic acid decarboxylase, the enzyme synthesizing GABA, delivered by bladder wall inoculation can reduce DO and DSD in SCI rats [13,14]. In our previous publication [15], we confirmed that HSVrd-mediated gene transfer of KAT II could improve DO in SCI rats. Here we would report if the HSVrd vectors encoding KAT II (HSVrd-KAT II) injection into the bladder wall could reduce urethral resistance.

Materials and Methods

The HSVrd-KAT II vector was constructed by recombining the RG223277 plasmid (Origene Technologies, Beijing, China) human KAT II cDNA into the ICP4, ICP27 and ICP 34.5 deleted HSVrd vector (Sino Geno Max Co., Beijing, China) with 6 His tag, which derived from wild HSV-I virus. Both HSVrd and HSVrd-KAT II express the viral reporter protein RFP under the control of EF1a promoter. The vectors were propagated in the OG01 cell line (Sino Geno Max Co.), which derived from Vero cell (ATCC CCL-81) and supplies ICP4 and ICP27 in trans.

Forty-eight male Sprague-Dawley rats (Academy of military medical sciences, Beijing, China) weighing 240-280 g were spinalized with complete transection of the T 10 spinal cord under 2% Isofluorane anesthesia according to the experimental protocol approved by the ethic committee of China Rehabilitation Research Center. Postoperatively, the rats were treated with ampicillin (100 mg/ kg, i.m.) for three days. The bladder of spinalized rats was expressed manually three times daily until reflex voiding recovered (9-14 days), then once daily afterwards. One week post spinalization, rats were divided into three groups randomly and received bladder wall injection under 2.0% Isofluorane anesthesia: (1) with normal saline (sham; n=16); (2) with HSVrd control vector (HSVrd; n=16) and (3) with HSVrd-KAT II (HSVrd-KAT II; n=16). After exposure of the rat bladder via midline abdominal incision, a total of 40 μl normal saline, viral suspension of HSVrd or HSVrd-KAT II (1×107 plaque-forming units) was injected into 4 sites around the bladder base and 4 sites around the middle of the bladder wall using a 30-gauge syringe (10 μl, Hamilton). After the abdomen was closed, the rats received ampicillin (100 mg/kg, i.m.) treatment for three days.

Three weeks post bladder injection, the rat bladder dome was exposed via a midline abdominal incision and the ureters were ligated at the level of the aortic bifurcation under 2% isoflurane anesthesia. The bladder was emptied and a PE-50 catheter was inserted from the bladder dome to record intravesical pressure. The intravesical catheter was connected to an a PT 100 Pressure Transducer (Chengdu Technology & Market Co., Sichuan, China) for pressure monitoring and to a WZ-50C66T micro infusion pump (Smiths Medical Instrument Co., Zhejiang, China) for bladder infusion through a three-way stopcock. After the abdomen was closed, the rats were allowed to recover from anesthesia for 1-2 h, and then normal saline at room temperature was infused into the bladder at 0.08 ml/min. Once fluid release from urethra occurred, infusion was stopped and residual volume was measured. The data was recorded and analyzed by BL-420F software (Chengdu Technology & Market Co.). Cystometric parameters were recorded after at least two stable micturition cycles. The number and amplitude of all non-voiding bladder contraction NVC, detrusor leak point pressur [(DLPP), cm H2O] and maximum cystometric capacity [(MCC), ml], time to first NVC [(TF), min), leakage volume [(LV), ml] were measured. NVCs were defined as rhythmic intravesical pressure increases greater than 7 cm H2O from baseline pressure without release of fluid from the urethra [13]. MCC was defined as the cystometric volume at DLPP. The bladder emptying efficiency [(BEE), %] was defined as a percentage of leakage volume compared to the MCC.

After cystometry, rats were perfused by 4% paraformaldehyde and L6-S1 DRGs were removed. Cryostat sections (10 μm) of DRG were mounted onto poly-L-lysine-coated slides. Primary antibodies of rabbit anti-human KAT II (H-301) antibody, mouse anti-DsRed (N9) antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and secondary antibodies of goat anti-rabbit IgG conjugated TRITC, Alexa Fluor 488 conjugated rabbit anti- mouse IgG (1: 200; Invitrogen, Carlsbad, CA) were used for the detection of KAT II and RFP. A negative control was prepared without primary antibody. In randomly selected DRG sections (n=3 sections per animal), the intensity of RFP or KAT II immuno reactivity was rated on a four point scale (0-3) and the neurons that exhibited grade 2 or 3 were regarded as positively stained cells. The ratio of positively stained cells/total DRG cells per section was calculated.

L6-S1 DRG were homogenized with RIPA buffer (Sigma- Aldrich, Shanghai, China) containing protease inhibitor cocktail (Sigma-Aldrich) and centrifuged at 10 000 rpm for 10 min at 4°C, and 30 μg protein in the supernatant was separated in a 12% Tris- HCl PAGE-Gel (Bio-Rad laboratories Inc), transferred onto a nitrocellulose membrane (Bio-Rad laboratories Inc), incubated with rabbit anti-human KAT II (H-301) antibody and GAPDH (H- 12) monoclonal antibody (1: 500 and 1: 1 000, respectively) (Santa Cruz Biotechnology) for 2 h at room temperature, followed by HRP-conjugated goat anti-rabbit antibody and HRP-conjugated goat anti-mouse antibody (1:5 000; Santa Cruz Biotechnology) for 1 h at room temperature. The intensity of each band was determined by Versa Doc™ image acquisition and analysis software (Bio-Rad laboratories Inc). The ratio of KAT II/ GAPDH was calculated.

Total RNA was extracted from L6-S1 DRG using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using TIANScript RT Reagent kit (Tiangen Biotech, Beijing, China). KAT II levels were examined with quantitative real-time PCR with TIANScript RT Reagent kit (Tiangen Biotech) and SYBR Premix Ex Taq II (Invitrogen) using a TL988 II System (Tianlong, Xian, China). Primers for KAT II (forward primer: 5’-TAGTAACCAGAAGGATGCAA-3’; reverse primer: 5’-GCTGAAGAGAAGGATGCTC-3’) and β-actin (forward primer: 5’-ACACCCGCCACCAGTTC-3’; reverse primer: 5’-TGACCCATACCCACCATC-3’) were used. Amplification of cDNA was performed as following: 95°C 2 min, then 45 cycles of 94oC 30s, 54oC 30s, and 72oC 30s. The reaction system was as following: SYBR Premix Ex Taq II 1μl, 10×PCR Buffer 2.5μl, dUTP 0.5μl, each primer 0.5μl, cDNA 2μl and Taq DNA polymerase 0.5μl with RNase Free ddH2O added up to 25μl. Each sample was tested three times and melting curve analysis was performed to confirm primer specificity. Standard curves were constructed from serial dilution of cDNA in each tissue and quantification of the samples was achieved from the standard curve. The ratio of KAT II/β-actin mRNA was compared between the two groups.

The experiments were performed on female Sprague-Dawley rats (100-140g; Hilltop Animal Care, Pittsburgh, PA) in accordance with the requirements and recommendations in the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, 1985) approved by the University of Pittsburgh (Pittsburgh, PA) Institutional Animal Care and Use Committee (IACUC). Under 2.0% isoflurane anesthesia, a low dorsal midline incision was performed to remove L6-S1 DRG quickly. DRG were minced, placed in medium consisting of 4.5 ml NEUROBASAL A Medium (Invitrogen, Carlsbad, CA), 5μl 5% B27 supplements (Invitrogen), 45μl 1% Penicillin- Streptomycin-Neomycin solution (Sigma-Aldrich), 0.45ml FBS (Invitrogen), 5mg trypsin (Worthingtown Biochemical; Lakewood, NJ ) and 10mg collagenase type 4 (worthing town Biochemical), and incubated at 37°C for 20–30 min. After trituration using a pipette, the cell suspension was centrifuged for 5 min at 416g. The cell pellet was then re suspended in the medium consisted as above without Trypsin and Collagenase type 4. Neurons were placed on poly-L-lysine-coated 35-mm culture dishes and cultured at 37°C in a 5% CO2 incubator. Vector-untreated neurons were studied after 24–72 h in culture. HSVrd or HSVrd-KAT II-treated neurons (multiplicity of infection: 1:10) were studied after 48-72 h transinfection.

Data are expressed as mean ± SE. One-way ANOVA, Two related samples Wilcoxon tests, Mann-Whitney U tests or Independent t-test was used with P < 0.05 considered statistically significant.


At one week after complete transection of the T 10 spinal cord in male rats, normal saline (sham group; n=16), HSVrd control vector (HSVrd group; n=16) or HSVrd-KAT II (HSVrd-KAT II group; n=16) was injected into the rat bladder wall. At three weeks after injection, the cystometry and the gene expression of KAT II in L6-S1 DRG were evaluated. Representative traces of cystometry in three groups are shown in Figure 1. In sham and HSVrd groups, all rats showed high amplitudes of NVCs before fluid leak from the urethra. There were no significant differences in cystometric parameters between sham and HSVrd groups (Table 1). However, the number and amplitude of NVCs, DLPP and MCC were decreased significantly by 59.6-61.1%, 21.6-24.2%, 30.3-34.4% and 44.1-46.5% (P<0.01), and the BEE and the TF were increased significantly by 40.7-47.7% and 30.1-49.0% (P<0.01), respectively, in the HSVrd-KAT II group compared to sham or HSVrd group (Table 1). The DLPP decreased significantly and the BEE increased significantly suggested that the urethral resistance or bladder outflow resistance reduced in the HSVrd-KAT II group (Figure 2).