1. Volume 15, Issue 4, Pages 687-697 (April 2007)
Lentiviral-mediated Targeted NF-κB Blockade in Dorsal Spinal Cord Glia Attenuates Sciatic Nerve Injury–induced Neuropathic Pain in the Rat 
Alice Meunier, Alban Latrémolière, Elisa Dominguez, Annie Mauborgne, Stéphanie Philippe, Michel Hamon, Jacques Mallet, Jean- Jacques Benoliel, Michel Pohl 
Molecular Therapy 
Volume 15, Issue 4, Pages (April 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
LV-srIκBα drives the production of srIκBα in glial cells in vitro. Control (non-infected) or LV-srIκBα-infected (300 of p24/ml) glial cell primary cultures were incubated (or not) in the presence of 10 μM reverse-transcriptase inhibitor AZT. The presence of mRNA encoding sr-IκBα was assessed using semi-quantitative RT-PCR on 0.5 μg total RNA extracted from cell cultures 48 hours after infection. srIκBα specific amplification was compared with amplification from control GPDH mRNA. Production of srIκBα protein was further verified in glial cells infected with LV-srIκBα (300 or 30 ng of p24/ml) using western blot analysis, which showed a viral titer–dependent accumulation of srIκBα.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
LV-srIκBα–mediated overexpression of srIκBα in primary cultures of glial cells blocks intranuclear translocation of NF-κB and luciferase reporter gene activity evoked by LPS. (a) In control (untreated) glial cell cultures, NF-κB–like immunoreactive material (NF-κB-IR, p65 subunit, in red) is distributed throughout the cytoplasm. (b) After treatment with LPS (1 μg/ml, 1 hour) NF-κB-IR is detected mainly in cell nuclei. (c) Forty-eight hours after infection with LV-srIκBα (300 ng of p24/ml), the majority of the cells (bold arrows) showed transgene-derived IκBα-IR (in green). Dashed arrows point to cells without detectable IκBα-IR (presumably non-infected). (d) Double-labeling experiments revealed that LPS treatment evoked NF-κB-IR (in red) translocation and accumulation only in the nucleus of uninfected cells, devoid of IκBα-IR (dashed arrow). In contrast, in cells overproducing IκBα-IR, nuclear translocation of NF-κB-IR was completely abolished and NF-κB-IR was sequestered in the cell cytoplasm (bold arrow). After stimulation with LPS, LV-EGFP pre-treated glial cell cultures (300 ng of p24/ml, 48 hours; EGFP green fluorescence) (e) showed strict nuclear localization of NF-κB-IR (in red) (f). Panels (g) and (h) represent combined IκBα-IR/NF-κB-IR immunofluorescence and EGFP fluorescence/NF-κB immunofluorescence, respectively. Scale bar = 20 μm. (i) In a comparable set of experiments, we analyzed nuclear protein fraction by western blotting. LPS-induced accumulation of NF-κB-IR in nuclear fraction was prevented in LV-srIκBα–infected cells. (j) In cells transfected with 2 X NFB-FLuc reporter gene plasmid and TK-RLuc plasmid, and infected (or not) with control LV-EGFP, LPS treatment (1 hour) induced Luc activity. On the other hand, infection of transfected cells with LV-srIκBα completely prevented the LPS-induced Luc activity. Results represent ratios of luminescence from the NFB-FLuc to TK-RLuc luminescence (means ± SEM from three independent experiments).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Infection of glial cell cultures with LV-srIκBα reduces LPS-induced expression of pro-inflammatory cytokines and iNOS. Semi- quantitative RT-PCR was performed with specific primers on 0.5 μg of total RNA extracted from control, LV-EGFP, and LV-srIκBα–infected cells incubated with LPS (1 μg/ml, 3 hours). Data (n = 3 for each group) are expressed in arbitrary units representing 260 nm optical density of cytokines or iNOS specific RT-PCR products/260 nm optical density of GPDH RT-PCR products. Data represent means ± SEM of three independent experiments.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
In vivo injection of lentiviral-derived vectors into the rat spinal cord allowed dorsal horn–restricted expression of transgene preferentially in glial cells. (a) Micro-injection of LV-EGFP resulted in EGFP fluorescence restricted to the ipsilateral dorsal horn of the spinal cord. (b) Single injections of viral suspension resulted in a rostro-caudal distribution of the vector through approximately 5 mm. Immunofluorescence experiments performed with antibodies raised against specific markers of astrocytes (GFAP), microglia (Ox42) or neurons (NeuN) showed that most EGFP fluorescent cells (c,d,e) contained also GFAP-LM (f) or Ox42-LM (g). Only scarce cells with weak EGFP fluorescence (dashed arrow, e) were colabeled with NeuN antibodies (h). Panels i, j and k represent combined fluorescence for EGFP and GFAP, Ox-42 or NeuN immunofluorescence, respectively. Scale bar = 50 μm or 20 μm in small windows. Estimated from spinal cords of four LV-EGFP–injected rats, neuronal profiles containing EGFP represented approximately 8% of total EGFP expressing cells (graph in k).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
LV-srIκBα delivery into the rat spinal cord resulted in IκBα-IR accumulation in the dorsal horn of the spinal cord. (a) IκBα-IR–positive cellular profiles were observed only in LV-srIκBα–infected right (ipsilateral) dorsal horn 2 weeks after vector injection. Scale bar = 25 μm. (b) Western blot analysis of protein extracts from the right dorsal part of the lumbar spinal cord (L3–L5) of naïve, control LV vector–injected rats or animals (two distinct rats) injected with LV-srIκBα was performed 1 week after injection. The blot was successively incubated with IκBα and α-tubulin antibodies.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Chronic constriction injury (CCI)–induced nuclear accumulation of NF-κB in the dorsal lumbar spinal cord was abolished in rats injected with LV-srIκBα. (a) Western blot analysis of nuclear fraction proteins extracted from the right dorsal part of the lumbar spinal cord (L3–L5) of naïve animals or animals 6 and 48 hours after CCI. (b) In animals injected with LV-srIκBα (CCI-LV-srIκBα, two distinct rats) and subjected 1 week later to CCI, western blot analysis revealed complete inhibition of NF-κB accumulation in nuclear extracts (48 hours after the nerve lesion) from the right dorsal horn of the spinal cord. The blot was successively incubated with NF-κB and α-tubulin antibodies.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Injection of LV-srIκBα into the dorsal spinal cord prevented chronic constriction injury (CCI)–evoked enhanced expression of IL-6 and iNOS but not of IL-1β in the dorsal lumbar spinal cord. Real-time RT-PCR was performed on total RNA extracted from the right dorsal horn of the lumbar (L3–L5) spinal cord of naïve, CCI, or LV-srIκBα–treated CCI rats (n = 3–4 for each group). (a) IL-6 and IL-1β mRNA relative quantities 7 days after CCI. LV-srIκBα treatment prevented the CCI-induced accumulation of IL-6 mRNA. (b) The late increase of iNOS mRNA relative concentrations observed 28 days after CCI was also abolished in LV-srIκBα–treated CCI rats. Each sample, PCR-amplified in triplicate, was normalized with GPDH as reporter gene. Data represent mean ± SEM.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

9. Figure 8
Rats injected with LV-srIκBα into the dorsal horn of the spinal cord exhibited attenuated chronic constriction injury (CCI)–induced thermal hyperalgesia and mechanical and cold allodynia. Behavioral studies were performed in control (representing both naïve and LV-EGFP–injected animals), control-CCI (representing both naïve and LV-EGFP–injected rats with CCI surgery), and LV-srIκBα–infected CCI rats. (a) Nociceptive responses elicited by radiant heating were measured as paw withdrawal latencies (PWLs) in seconds, and for each animal ΔPWL was calculated as the difference between PWL at days 7, 14, and 21 and the baseline PWL (day 0 before CCI). Seven days after LV vector spinal injection (day 0 before CCI), PWLs were comparable between different groups. Starting at day 7 and then throughout the experimental procedure (21 days), ΔPWLs were significantly different between control and control-CCI groups (P < ). Note that single LV-srIκBα injection resulted in constant significant antihyperalgesic effect throughout the 21 days of experiment. (b) Sensitivity to mechanical stimulation was assessed using von Frey filaments. The mean of first reaction (increasing testing) and lowest test result (decreasing testing) was taken as the mechanical paw withdrawal threshold (PWT). These data were log transformed, and the percentage decrease of the withdrawal threshold was then calculated in relation to the withdrawal threshold before surgery (baseline): ΔPWT = (CCI paw − baseline paw)/baseline paw × 100. (c) Reactivity of animals to cold stimulus was measured after application of acetone (100 μl) onto the right footpad. The time that animals spent with paw withdrawn (PWt) was measured during the next 120-second period. Data represent mean ± SEM.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions