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Results: 4

1.
Figure 4

Figure 4. From: Sustained relief of neuropathic pain by AAV-targeted expression of CBD3 peptide in rat dorsal root ganglion.

EGFP-CBD3 expression does not suppress nerve injury-triggered inflammatory reactions. Representative sections from DRG (a) and spinal cord dorsal horn (b) showing Iba1 (top row) and GFAP (bottom row) immunoreactivity. Tissues are from rats 6 weeks after vehicle injection without injury (first column), 6 weeks after vehicle injection and 4 weeks after SNI (second column), 6 weeks after injection of AAV6-EGFP followed by SNI (third column) and 6 weeks after AAV6-EGFP-CBD3 injection followed by SNI (fourth column). Scale bars: 50 μm, and apply to all images in the panel.

G Fischer, et al. Gene Ther. 2014 January;21(1):44-51.
2.
Figure 2

Figure 2. From: Sustained relief of neuropathic pain by AAV-targeted expression of CBD3 peptide in rat dorsal root ganglion.

Electrophysiological properties of neurons expressing EGFP-CBD3. Transduced neurons were identified after dissociation by EGFP epifluorescence (a, bottom image; top image: phase-contrast). N-type current was identified as the difference between current at baseline (BL) and after application of ω-Conotoxin-GVIA (GVIA, 200 nM) in response to depolarizations from −100 mV to VTest of 0 mV (b, sample traces; c, time course for group average) for neurons expressing EGFP (n=10) or EGFP-CBD3 (n=9). Summary data (d) show a significant depression of N-type current in neurons expressing EGFP-CBD3 compared with EGFP (*P<0.05). T-type current traces triggered by depolarizations from VH −100 mV to VTest of −30 mV (e) after incubation (20 min) with R-type current blocker SNX-482 (200 nM) and P/Q-type current blocker ω-Conotoxin MVIIC (200 nM), and addition of L-type current blocker nimodipine (5 μM) and GVIA (200 nM) to the recording bath. Summary data (f) show a significant depression of T-type current in neurons expressing EGFP-CBD3 compared with EGFP (**P<0.01).

G Fischer, et al. Gene Ther. 2014 January;21(1):44-51.
3.
Figure 3

Figure 3. From: Sustained relief of neuropathic pain by AAV-targeted expression of CBD3 peptide in rat dorsal root ganglion.

Therapeutic efficacy and safety of intraganglionic AAV-encoded CBD3 in SNI-induced neuropathic pain. Left panels show the time courses for the group averages of sensitivity to innocuous punctate mechanical stimulation (von Frey, a), hyperalgesia behavior after touch with a pin (Pin, b), and sensitivity to acetone stimulation (Cold, c) before and after DRG injection of either AAV6-EGFP (filled circle, b=7) or AAV6-EGFP-CBD3 (open square, n=7). Injection of AAV vector into the fourth and fifth lumbar DRGs was performed immediately after the baseline (BL) behavioral determinations, and spared nerve injury (SNI) was performed immediately after the week 2 determinations. Right panels show averaged area under the curve calculated for each individual for the time period following SNI for von Frey (d), pin (e) and cold (f). Results are means±s.e.m. *P<0.05, **P<0.01, ***P<0.001 for comparisons to BL (a–c) and for comparisons between groups (df).

G Fischer, et al. Gene Ther. 2014 January;21(1):44-51.
4.
Figure 1

Figure 1. From: Sustained relief of neuropathic pain by AAV-targeted expression of CBD3 peptide in rat dorsal root ganglion.

Expression of fluorescent CBD3 in sensory neurons. DRG sections from rats in which AAV6-EGFP-CBD3 was injected 6 weeks previously and SNI traumatic nerve injury was performed 2 weeks thereafter were immunostained with antibodies to EGFP as well as CGRP (a), IB4 (b) or NF-200 (c). Arrowheads point to examples of co-labeled neurons. Spinal cord sections show EGFP-CBD3 expression (d, highlighted area magnified in (e) with enumerated laminae). No colocalization is observed with NeuN staining of dorsal horn neuronal somata (f, magnified in the inset, showing synaptic varicosities of transduced fibers). Sensory neuron fibers in the dorsal horn show cluster with the synaptic marker synaptophysin (g, magnified in the inset). The EGFP-CBD3 signal was also observed in sciatic nerve (h). Scale bars: 100 μm for all images except 50 μm for inset images. Western analysis (i) of HEK293T cell lysates following plasmid transfection show EGFP immunoreactivity at distinct molecular weights (MWs) for expressed EGFP (lane 2) versus EGFP-CBD3 (lane 3) as positive controls. DRG homogenates show no immunoreactivity in DRGs contralateral from the injection (lane 4), and appropriate MW in homogenates from DRGs injected with AAV6-EGFP-CBD3 (lanes 5 and 6) or AAV6-EGFP (lanes 7 and 8). Lane 1 shows marker protein standards (M; MagicMark, Life Technologies). Arrows point to the expected size bands for EGFP-CBD3 and EGFP (top panel), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, bottom panel) as a loading control.

G Fischer, et al. Gene Ther. 2014 January;21(1):44-51.

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