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We examined whether replication-defective herpes simplex virus (HSV) vectors encoding the 67 Kd form of the glutamic acid decarboxylase (GAD67) gene product, the gamma-aminobutyric acid (GABA) synthesis enzyme, can suppress detrusor overactivity (DO) in spinal cord injury (SCI) rats. One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (GFP) (HSV-GAD) were injected into the bladder wall. SCI rats without HSV injection (HSV-untreated) and those injected with lacZ-encoding reporter gene HSV vectors (HSV-LacZ) were used as controls. Three weeks after viral injection, continuous cystometry was performed under awake conditions in all three groups. In the HSV-GAD group, the number and amplitude of non-voiding contractions (NVCs) were significantly decreased (40–45% and 38–40%, respectively) along with an increase in voiding efficiency, compared with HSV-untreated and HSV-LacZ groups, but micturition pressure was not different among the three groups. Intrathecal application of bicuculline partly reversed the decreased number and amplitude of NVCs, and decreased voiding efficiency in the HSV-GAD group. In the HSV-GAD group, GAD67 mRNA and protein levels were significantly increased in L6-S1 dorsal root ganglia (DRG) compared with the HSV-LacZ group while 57% of DRG cells were GFP-positive, and these neurons showed increased GAD67-like immunoreactivity compared with the HSV-LacZ group. These results indicate that GAD gene therapy effectively suppresses DO following SCI predominantly via activation of spinal GABAA receptors. Thus, HSV-based GAD gene transfer to bladder afferent pathways may represent a novel approach for the treatment of neurogenic DO.
Micturition depends on the coordinated activity of the urinary bladder and external urethral sphincter.1 However, spinal cord injury (SCI) rostral to the lumbosacral level, which impairs voluntary and supraspinal control of voiding, disrupts this coordination. SCI initially induces areflexic bladder and urinary retention, followed by the emergence of automatic micturition and eventually detrusor overactivity (DO) mediated by spinal reflex mechanisms. Impaired bladder-sphincter coordination termed detrusor-sphincter dyssynergia (DSD) reduces voiding efficiency, leading to urinary retention.2 These lower urinary tract dysfunctions then lead to more serious problems, such as urinary incontinence, recurrent urinary tract infection and upper urinary tract deterioration.
In the central nervous system (CNS), glutamate is a major excitatory neurotransmitter, while glycine and gamma-aminobutyric acid (GABA) are the most abundant inhibitory amino acid neurotransmitters.3–4 GABA is synthesized from glutamate by glutamic acid decarboxylase (GAD),4 and is known to have an important role in the inhibitory regulation of micturition in normal spinal cord intact rats.5 Our recent study revealed that chronic SCI rats had lower GAD67 levels in the lumbosacral spinal cord and in L6-S1 dorsal root ganglia (DRG), where bladder afferent fibers originate. These SCI animals also showed DO on continuous cystometry, which was suppressed by intrathecally administered GABA receptor agonists.6 Therefore, GABA is an important messenger in the inhibitory effect on micturition in SCI rats, and hypofunction of inhibitory GABAergic neuronal activity in the spinal cord is likely to be involved in the genesis of DO after SCI.
Although baclofen, a GABAB receptor agonist, is approved for treatment of DO in SCI patients,7 this agent has not been widely used because the therapeutic window of this drug is modest and the dose is limited by side effects. One potential approach for increasing GABA levels in the spinal cord with limited side effects would be to use viral-mediated gene delivery so that the gene product of interest is delivered specifically to the DRG neurons, or moreover the neighboring non-transduced neurons to which GABA can bind to, that require GABA to reduce DO. Herpes simplex virus (HSV) has several significant advantages over other viral vectors for the treatment of peripheral nervous system (PNS) disorders.8. Replication-defective recombinant vectors, which lack multiple essential gene functions and therefore are non-toxic in vivo8–9 have been constructed to reduce immune clearance of the vectors and increase the overall safety of the vector for clinical therapeutic application. It has been reported that replication-defective HSV mediated gene transfer of GAD67 delivered by subcutaneous inoculation can reduce neuropathic pain in spinal cord hemisected or spinal nerve ligated rats.10–11 Therefore, we hypothesized that DO in SCI rats might be suppressed if GABA levels are increased using replication-defective HSV mediated gene transfer of GAD in bladder afferent pathways.
Thus, in the present study, we investigated the feasibility of HSV vector mediated GAD67 gene therapy for the treatment of DO following SCI. We examined bladder activity and confirmed vector-mediated GAD67 gene delivery by measuring expression of GAD67 mRNA and protein, as well as immunoreactivity of GAD67, in L6-S1 DRG following SCI and HSV vector administration.
Representative traces of continuous cystometry in HSV-LacZ and HSV-GAD groups are shown in Fig. 1. In HSV-untreated and HSV-LacZ groups, all spinalized rats showed many NVCs before large-amplitude voiding bladder contractions occurred. There were no significant differences in cystometric parameters between HSV-untreated and HSV-LacZ groups (Table 1). However, in the HSV-GAD group (3 weeks after viral injection), the number of NVCs was significantly decreased by 37–43% (p<0.01) compared with HSV-untreated or HSV-LacZ groups (Table 1, Fig. 2A). The amplitude of NVCs was also significantly reduced by 39–41% (p<0.01) in the HSV-GAD group compared with HSV-untreated and HSV-LacZ groups (Table 1, Fig. 2B). In addition, the residual volume and voiding efficiency were significantly (p<0.05) decreased and increased, respectively, in the HSV-GAD group compared with HSV-untreated and HSV-LacZ groups. However, MVP, voided volume or bladder capacity was not significantly different among the three groups (Table 1, Fig. 2C).
In the HSV-GAD group (3 weeks after viral injection), continuous cystometry showed a reduction in the number and amplitude of NVCs (Fig. 3A). However, after intrathecal application of bicuculline (0.1 µg), the number and amplitude of NVCs were significantly (p<0.05) increased (Fig. 3B Table 2), and there were no significant differences in the number of NVCs between untreated HSV-LacZ and bicuculline-treated HSV-GAD rats (4.38 ± 0.46 vs. 6.00 ± 2.02, p = 0.46). However, the amplitude of NVC was still lower in the HSV-GAD group after intrathecal application of bicuculline compared with the HSV-LacZ group (13.1 ± 0.8 vs. 28.3 ± 3.4, p < 0.01). After intrathecal application of bicuculline, MVP was not changed while voiding efficiency was reduced in HSV-GAD-treated rats (Table 2). In a separate group of HSV-GAD-treated rats, intrathecal application of saclofen (1 µg) did not affect any cystometric parameters (Table 2).
An RT-PCR study was carried out in order to examine the expression levels of GAD67 mRNA in L6-S1 DRG. In the HSV-GAD group (3 weeks after viral injection), the GAD67 mRNA/β-actin mRNA ratio in L6-S1 DRG was significantly higher (1.29 ± 0.14, p = 0.015) compared with the HSV-LacZ group (0.74 ± 0.08) (Fig. 4A) using primer pairs specific for the GAD67 and β-actin mRNAs.
In the next step, Western blot analysis was carried out in order to evaluate the GAD67 protein expression levels in L6-S1 DRG 3 weeks following treatment with the different vectors. The bands of GAD67 and GAPDH were visualized at 67kDa and 37kDa, respectively, and the intensities of the bands determined as per the Materials and Methods. In the HSV-GAD group (3 weeks after viral injection), the band density of GAD67 protein relative to that of GAPDH was significantly higher (0.44 ± 0.03, p = 0.016) compared with the HSV-LacZ group (0.35 ± 0.02) (Fig. 4B), that correlates with the increase in GAD67-specific message detected by qPCR (Fig. 4A).
To confirm the efficiency of HSV vector transduction into sensory nerves after SCI (3 weeks after viral injection), the expression profile of the marker gene, GFP in L6-S1 DRG was investigated. In 3 DRG sections randomly selected from each of 4 HSV-GAD injected animals, GFP immunoreactivity (i.e., grades 2 or 3 staining intensity) was observed in 57.4 ± 6.4% of DRG cells per section (Fig. 5A).
In the HSV-GAD group, the number of small and medium sized (less than 35 µm in diameter) GAD67-positive cells was increased in L6-S1 DRG neurons when compared with the HSV-LacZ group (Fig. 5B). In 3 DRG sections randomly selected from each of 4 HSV-GAD animals, 42.6 ± 8.4% of DRG cells per section were GAD67 positive (i.e., grades 2 or 3 staining intensity) while GAD67 positive DRG neurons were found in only 13.8 ± 1.4% of DRG cells per section in HSV-LacZ rats.
The present study indicates that; (1) HSV-GAD treatment inhibits DO as evidenced by a reduction in NVCs without affecting voiding in SCI rats, (2) intrathecal administration of the GABAA receptor antagonist bicuculline partly restores DO as evidenced by induction of large NVCs in HSV-GAD-treated SCI rats, (3) 57% of L6-S1 DRG cells where bladder afferents originate express a reporter gene within the vector as evidenced by GFP positive staining after HSV-GAD treatment, (4) expression of GAD67, the GABA synthesizing enzyme, is increased in the L6-S1 DRG after HSV-GAD treatment as evidenced by increased GAD67 mRNA and protein levels. Thus, HSV vectors can be efficiently transported to bladder afferent pathways and inhibit DO predominantly through GABAA receptor activation.
The organization of the micturition reflex undergoes marked changes after SCI. Following SCI, due to the disruption of supraspinal micturition reflex pathways, a spinal micturition reflex is unmasked, resulting in DO. Prior work by our group6,12 and others5 have suggested that the GABA modifier GAD, was reduced following SCI suggesting that a therapeutic approach to deliver a gene product involved in GABA synthesis may prove useful in altering the micturition pattern in SCI. Moreover, our prior work using HSV vectors has shown that vector administered to the rat bladder wall very efficiently transduces the bladder muscle layer and L6-S1 DRG neurons as evidenced by expression of the reporter gene (E. coli lacZ) product, and is capable of expressing a variety of therapeutic gene products.13–15 These studies, along with the use of the same HSV-GAD vector to treat neuropathic pain associated with other forms of SCI provided the underpinning for the current study.
In our recent study, intrathecal application of muscimol and baclofen (GABAA and GABAB receptor agonists, respectively) inhibited NCVs as well as MVP in SCI rats.6 In contrast, the present study showed that GAD gene therapy using replication-defective HSV vectors (HSV-GAD) was effective in reducing the number and amplitude of NVCs (DO) without affecting MVP in SCI rats. Since decreased NVCs (DO) and bladder contractility (i.e., a reduction in MVP) is assumed to reflect the suppression of afferent and efferent activity in the micturition reflex pathway, respectively,2 HSV-GAD treatment is likely to predominantly inhibit the afferent limb of the micturition reflex. Thus, this suggests that GAD gene therapy using HSV vectors can target bladder afferent pathways rather efferent pathways when injected into the bladder. It is well known that two types of afferent fibers, namely Aδ and C-fibers, carry sensory information from the bladder to the spinal cord.16–17 In cats and rats, Aδ-fiber bladder afferents trigger normal micturition via a long latency supraspinal pathway passing through the pons.2,17 In chronic SCI rats, C-fiber afferents appear to initiate DO, although Aδ bladder afferents still trigger the voiding reflex.2,17 Desensitizing C-fiber afferents by systemic capsaicin administration suppressed NVCs without affecting the voiding reflex in chronic SCI rats.18 This capsaicin effect is similar to that seen in the current experiments (i.e., reduced NVCs without affecting voiding) following GAD gene therapy in SCI rats, suggesting that GAD gene therapy preferentially inhibits DO by suppressing C-fiber bladder afferents without affecting Aδ-fiber-dependent voiding contractions. Therefore, GAD gene therapy using non-replicating HSV vectors that can improve urine storage function without affecting voiding function would be more beneficial than systemic or intrathecal drug therapy for the treatment of urinary problems in SCI patients due to the specificity of the afferents involved.
Our therapeutic strategy takes advantage of the natural neurotropism of HSV. HSV has adapted itself to infect neurons of the PNS and remain in a latent or quiescent state within these neurons for the life of the host.19 Replication-defective vectors can be employed to deliver and express genes in the peripheral and central nervous systems. The ability of the virus to establish a latent infection does not depend on viral gene expression. Thus, non-cytotoxic, replication-defective vectors can be employed, which adds a measure of safety to the therapeutic application. We also employed the strong HCMV promoter to drive expression of GAD. Expression from this promoter is transient typically lasting 7–21 days post-infection depending on the animal model and tissue. If longer term expression of GAD is required to achieve the desired effect, HSV already possesses a natural promoter system LAP2 that is active at lower, yet significant long-term levels in the nervous system.20–22
In the present study, we evaluated cystometric parameters 3 weeks after HSV-GAD injection because, in our preliminary data, DO (i.e., NVCs) was not suppressed 2 weeks after HSV-GAD injection in SCI rats (data not shown). However, in our previous study, HSV vectors encoding the preproenkephalin genes (SHPE), which were injected into the bladder wall of normal rats, elevated preproenkephalin gene expression in L6-S1 DRG and reduced bladder hyperactivity induced by capsaicin one week after viral injection.13 Although the reason for the delayed onset of HSV treatment efficacy in SCI animals is not known, the efficiency of gene delivery and transfer to the nerves may be reduced because of sparse distribution of nerves in the bladder due to bladder distension and smooth muscle hypertrophy induced after SCI. This difference in onset of therapeutic effect may also relate to the nature of the transgene and it’s role in synthesizing either met- and leu-enkephalin in the case of SHPE or GABA in the case of the QHGAD67 vector, the release of these peptides and their action. In addition, although we did not measure GABA production in this study, our previous study demonstrated that the amount of GABA released at the dorsal spinal cord from the central terminals of DRG transduced by subcutaneous inoculation into the foot is increased in QHGAD67-treated rats compared with those treated with the control vector.10
The current study also showed that intrathecal bicuculline partly restored NVCs without affecting MVP while intrathecal saclofen did not affect cystometric parameters in HSV-GAD-treated SCI rats. In contrast, in our previous study, intrathecal muscimol and baclofen at the level of L6-S1 spinal cord reduced NVCs in SCI rats, and the effects were antagonized by bicuculline and saclofen, respectively, indicating both GABAA and GABAB receptors are involved in suppression of DO in SCI.6 GABA reportedly inhibits bladder activity by acting through at least four distinct sites in spinalized rats; (1) at the spinal level by reducing afferent inputs from the detrusor through afferent nerves, (2) by inhibiting the neurons of the sacral parasympathetic nucleus, (3) at the pelvic ganglionic level by inhibiting excitatory neurotransmission and (4) at the postganglionic level by reducing neurotransmitter release from neurons innervating the detrusor.23 Thus, it seems reasonable to assume that GABAA receptor activation primarily contributes to suppression of sensory inputs through C-fiber bladder afferent pathways in the spinal cord after HSV-GAD treatment in SCI rats.
A decrease in GABAA receptor-mediated inhibitory postsynaptic potentials, a reduction in GAD65 expression, and apoptosis in the dorsal horn of the spinal cord have been reported in chronic nerve injury models of neuropathic pain.24 We also previously found that the GAD67 mRNA level in the L6-S1 spinal cord and DRG was significantly decreased after SCI.6 Thus, hypofunction of the GABAergic inhibitory system is at least in part responsible for the development of DO after SCI. Therefore, it is likely that GABA supplemental therapy represents a reasonable approach for treating DO after SCI. However, the ubiquitous distribution of GABA receptors in the CNS results in side effects that impose severe restriction on the dose of GABA receptors agonists such as baclofen, even when administered intrathecally as an attempt to control DO after SCI.7 These complications may be avoided by inducing the expression of neurotransmitters in a specific population of neurons. In the present study, after HSV-GAD injection in the bladder wall, GAD expression was increased in L6-S1 DRG that contains bladder afferent neurons, and C-fiber-dependent DO was suppressed even though the Margolis lab has shown that the vector can infect both A-fibers as well as peptidergic small unmyelinated C-fibers that project to lamina I and lamina IIo and non-peptidergic small to medium unmyelinated C-fibers that project to lamina IIiC.25–26 Liu et al. have reported that replication-defective HSV mediated gene transfer of GAD delivered by subcutaneous inoculation can increase the release of GABA in the spinal cord, and can reduce neuropathic pain in spinal cord hemisection rats.10 Therefore, HSV-GAD vector injection into bladder wall could lead to a local increase in GABA synthesis in bladder afferent pathways. We have also reported that nerve growth factor expression in afferent pathways occurs in L6-S1 DRG after HSV-mediated nerve growth factor gene delivery into the bladder wall, but not in adjacent DRGs in rats.14 This suggested that GAD gene therapy using HSV vectors that can target the treated organ and can be efficiently transported to its afferent pathways with limited side effects.
We also found that HSV-mediated GAD gene delivery into the bladder wall decreased the residual volume and increased voiding efficiency, compared with HSV-untreated or HSV-LacZ-treated SCI rats. However, no differences were observed in MVP among HSV-untreated, HSV-LacZ, and HSV-GAD groups. These results suggest that HSV-mediated GAD gene delivery reduces urethral resistance during voiding, resulting in improved residual volume and voiding efficiency. Our previous study indicated that hyperexcitability of C-fiber bladder afferents is involved in DSD (i.e., simultaneous urethral sphincter contraction during bladder contraction) after SCI because C-fiber desensitization by capsaicin pretreatment reduces urethral contraction pressure during bladder contraction in SCI rats.27 We also recently reported that intrathecal application of GABA receptor agonists can suppress DSD in SCI rats.12 Therefore, suppression of C-fiber bladder afferent activity after HSV-mediated GAD gene delivery may also have an inhibitory effect on DSD in SCI although further studies are needed to clarify this point .
In conclusion, the present study provides the first evidence of the efficacy of GAD gene therapy using replication-defective HSV vectors for DO following SCI. GAD gene therapy mainly inhibits C-fiber bladder afferent pathways to exert its effects predominantly through GABAA receptors. Therefore, the novel GAD gene therapy using replication-defective HSV vectors could be effective for the treatment of DO by restoring impaired GABA mechanisms in patients with SCI. The ultimate goal of these studies is to use an HSV vector-based approach in human clinical trials.
A total of 38 adult female Sprague-Dawley rats weighing 236–270 g were used according to the experimental protocol approved by the University of Pittsburgh Institutional Animal Care and Use Committee. To produce SCI, rats were anesthetized with pentobarbital (30 mg/kg i.p., Ovation, Deerfield, IL) and Th9 laminectomy was performed. The dura was opened and the complete transection of the T9–T10 spinal cord was performed with scissors. A sterile Surgifoam sponge (Ferrosan, Soeborg, Denmark) was placed between the cut ends of the spinal cord. The overlying muscle and skin were then sutured to close the wound. Rats were then put on an electric warmer to maintain body temperature and were allowed to recover from anesthesia. SCI animals were postoperatively treated with ampicillin (100 mg/kg, i.m.) for 5 days. The bladder of spinalized rats was emptied by abdominal compression twice a day until reflex voiding recovered, usually 10 to 14 days after spinalization. SCI rats were divided into the following three groups: 1) SCI rats without HSV injection (HSV-untreated, n = 10), 2); SCI rats with replication-defective HSV vector Q0ZHG (control vector) administration (HSV-LacZ, n = 10); and 3) SCI rats with replication-defective, GAD67-expressig HSV vector QHGAD67 administration (HSV-GAD, n = 18).
The QHGAD67 vector (HSV-GAD) was constructed by recombining the UL41-targeting plasmid that contains the human cytomegalovirus immediate early promoter (HCMV IEp) driving expression of the GAD67 cDNA into the vector Q0ZHG as described (Fig. 6).10 The replication-defective vectors QHGAD67 and Q0ZHG were propagated on the 7b cell line 28 that supplies both ICP4 and ICP27 in trans. Both QHGAD67 and Q0ZHG express the green fluorescent protein (GFP), while the Q0ZHG (HSV-LacZ) control vector also expresses lacZ.
In HSV-LacZ and HSV-GAD groups, animals were anesthetized with pentobarbital (30 mg/kg) 1 week after SCI. A lower abdominal incision was made to expose the bladder, and the vectors were injected into the bladder wall using a 30-gauge Hamilton syringe (10 µl, Hamilton, Reno, NV). A total of 40 µl viral suspension (2 × 107 plaque-forming units [PFU] in total) of either Q0ZHG or QHGAD67 was injected at four sites of the bladder wall around the bladder base. After the abdomen was closed, rats were allowed to recover from anesthesia and were housed in an approved Biosafety level-2 animal facility.
In the three groups, 4 weeks after SCI and 3 weeks post vector treatment, the animals were anesthetized with 2% isoflurane, and the abdomen was opened via midline laparotomy in the Experiment #1 and #2 described below. The ureters were transected at the level of the aortic bifurcation, and the distal ends were ligated.27,29 A polyethylene catheter (PE-90, Clay-Adams, Parsippany, NJ) was inserted into the bladder through the bladder dome to record intravesical pressure. After the abdomen was closed, the rats were placed in a restrainer (Braintree Scientific Inc., Braintree, MA) and were allowed to recover from anesthesia for 1 to 2 hours.
For 8 SCI rats of the HSV-GAD group (Experiment #2), a laminectomy was performed at the level of the 3rd lumbar vertebra under isoflurane anesthesia 1–2 days before cystometry. A PE-10 catheter was implanted intrathecally at the level of the L6-S1 spinal cord through a small hole of the dura. The end of the catheter was heat sealed and placed subcutaneously. The implanted intrathecal catheter was exteriorized through the skin incision for drug administration when cystometry was performed.
For intrathecal application in Experiment #2, 1 µl of drug solution was given via the implanted intrathecal catheter and flushed with 10 µl of saline. After the experiments, 2 µl of Evans blue flushed with 10 µl of saline was injected intrathecally, the position of the intrathecal catheter was checked in all animals, and the extent of dye distribution in the subarachnoid space was evaluated. (−)–Bicuculline methobromide and saclofen (Tocris Cookson Inc, Ellisville, MO) were dissolved in distilled water. Intrathecal catheter implantation or vehicle (saline or distilled water) application did not alter bladder activity during cystometry in SCI rats (data not shown).
The intravesical catheter was connected to a pressure transducer and an infusion pump through a three-way stopcock. Physiological saline at room temperature was infused at a rate of 0.08 ml/min to elicit repetitive bladder contractions. Cystometric parameters were recorded after at least two stable micturition cycles were observed.6
The number and amplitude of nonvoiding bladder contractions (NVCs) were measured during a 2–4 min period prior to micturition. NVCs were defined as rhythmic intravesical pressure increases greater than 7 cm H2O from baseline pressure without release of fluid from the urethra. Maximal voiding pressure (MVP) was also measured. Saline volume from the urethral meatus during voiding was collected and measured to determine voided volume. After each voiding, the infusion was stopped and residual volume was measured. Residual saline was withdrawn through the intravesical catheter by gravity and then the bladder was completely emptied by manual compression through the abdominal wall. Bladder capacity was calculated as the sum of voided volume and residual volume. Voiding efficiency was then calculated using the formula: voiding efficiency (%) = [(voided volume / bladder capacity) × 100].
In order to examine the effect of GAD67 delivery, (−)-bicuculline methobromide (GABAA antagonist; 0.1 µg; n = 4) or saclofen (GABAB antagonists; 1 µg; n = 4) were administered through the intrathecal catheter using a Hamilton microsyringe in the HSV-GAD group. Then cystometric parameters were compared before and after administration of GABA receptor antagonists.
After cystometry, L6-S1 DRG were rapidly removed, and total RNA was extracted from the pooled DRG using TRIzol reagent (Invitrogen, Carlsbad, CA). One µg of RNA was reverse-transcribed into cDNA using Superscript II (Invitrogen). Primers for GAD67 (GAD67 forward primer, 5’-GCGGGAGCGGATCCTAATA-3’ and reverse primer, 5’-TGGTGCATCCATGGGCTAC-3’)10 and β-actin (Ambion, Austin, TX) were used. The GAD67 and β-actin mRNA levels were quantified with an MX3000P real-time PCR system (Stratagene, La Jolla, CA) in a 25 µl volume using SYBR Green PCR Master Mix (QIAGEN, Valencia, CA). Amplification of cDNA was performed under the following condition; one cycle of 95 °C for 15 min, followed by 40 cycles of 1 min at 95 °C, 1 min at 55 °C, and 1 min at 72 °C for both GAD67 and β-actin. Specificity of GAD67 in DRG was confirmed by melting curve analysis. Standard curves constructed from serial dilution of cDNA in each tissue were analyzed with an MX3000P real-time PCR system. Quantification of the samples was achieved from the threshold cycle by interpolation from the standard curve to calculate a copy number for GAD67, and ratio of GAD67 to β-actin mRNA was compared.
L6-S1 DRG were sonicated in RIPA buffer containing 1X RIPA Lysis buffer, PMSF, sodium orthovanadate, and protease inhibitor cocktail (0.3 ml/100 mg tissue, Santa Cruz Biotechnology, Santa Cruz, CA). The homogenate was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was collected, and the protein was determined by the Bradford method using a protein assay kit (Pierce Biotechnology, Rockford, IL) with bovine serum albumin as a standard. For each sample, 30 µg of the protein extracts were subjected to a standard 12% Tris-HCl PAGE-Gel (Bio-Rad laboratories Inc., Hercules, CA). After electrophoresis, proteins were transferred to nitrocellulose membrane (Bio-Rad laboratories Inc). The nitrocellulose blots were blocked overnight at 4°C with 5% dry milk (Nestle, Glendale, CA) in PBS with 0.05% Tween-20 and incubated with rabbit anti-GAD67 (1:1000; Chemicon, Temecula, CA) for 2 h at room temperature followed by horseradish peroxidase-conjugated goat anti-rabbit (1:1,000; Vector Labs, Burlingame, CA) for 2 h at room temperature. Western blot was visualized using an ECL detection system (GE Healthcare, Buckinghamshire, UK). The membrane was stripped and reprobed with rabbit anti-GAPDH (Santa Cruz Biotechnology) as a loading control. The intensity of each band was determined by quantitative densitometry using VersaDoc™ image acquisition and analysis software (Bio-Rad laboratories Inc).
Rats randomly selected from the HSV-LacZ and HSV-GAD groups (n = 4 each) were perfused intracardially with 4% paraformaldehyde solution in 0.16% picric acid and 0.1 M phosphate buffer, and L6-S1 DRG were removed. Removed DRG were postfixed in the same solution for about 48h, and then immersed in 0.1 M phosphate buffer containing 25% sucrose for about 48h. Serial sections were cut at 14-µm thickness on a cryostat and thaw mounted onto cold Superfrost microscope slides (Fisher Scientific, Pittsburgh, PA). The sections were air dried, rinsed in 0.01 M phosphate-buffered saline (PBS) and then preincubated for 1 h in 0.1 M PBS containing 2% normal donkey serum and 0.5% Triton X-100 (PBDT). The sections were then incubated overnight with primary antibodies diluted in PBDT in a humidity chamber at 4 °C. A negative control was prepared by using PBDT without primary antibodies. The following primary antibodies were used; rabbit anti-GFP (1:200; Santa Cruz Biotechnology) or rabbit anti-GAD67 (1:1000, Chemicon). After rinsing in 0.01 M PBS, the sections were incubated with biotinylated anti-rabbit immunoglobulin G (IgG) (1:200; Vectors Labs) and anti-rabbit fluorescent Cy3-conjugated fragment IgG (1:600, Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in PBDT for 2 h at room temperature for GFP and GAD67 stainings, respectively. After rinsing in 0.01 M PBS, the sections were incubated with Alexa Fluor 488-conjugated strept-avidin (1:600; Molecular Probes Invitrogen, Carlsbad, CA) diluted in PBDT for 2 h at room temperature for GFP staining. After rinsing in 0.01 M PBS, the sections were coverslipped. The distribution of GFP and GAD67 immunoreactivity in DRG neuronal profiles was analyzed under fluorescence illumination with FITC and Cy3 filters, respectively. Fluorescent images were captured directly off the microscope using a digital camera. The intensity of GFP or GAD67 immunoreactivity was rated on a four point scale, from completely negative (grade 0) to intense staining (grade 3), and the neurons that exhibited grades 2 or 3 were regarded as positively stained cells. We also counted the number of GFP or GAD-positive cells among DRG neurons, in which their nuclei were clearly seen, in order to avoid counting of non-neuronal staining. The number of GFP or GAD67-positive cells as well as the total DRG neurons in each 14-µm thick DRG section were counted, and the percent ratio of positively stained cells against the total DRG cells per section calculated and then averaged.
Data are expressed as the mean ± SE. Statistical comparisons were performed by paired or unpaired t-test as well as Wilcoxon test where applicable with p < 0.05 considered statistically significant.
This work was supported by NIH DK057267, DK068557, HD039768 and DK044935. We thank Vickie L. Erickson, Ryuichi Kato, and Kurumi Sasatomi for their excellent technical assistance and Ali Ozuer and Dave Kopp of the Vector Core for production and purification of the GAD and control vectors.