Gene-based Neuromodulation in the Central Nervous System via an Inwardly Rectifying Potassium Channel Público
Krudy, Christina Ann (2010)
Abstract
Abstract
Gene-based Neuromodulation in the Central Nervous System via an Inwardly Rectifying Potassium Channel
By Christina A. Krudy
Neuromodulation therapies aim to correct imbalanced neural activity, which underlie diseases such as epilepsy, pain, and spasticity. Gene-based neuromodulation employs viral vectors to alter neural activity at a molecular level with greater specificity and reversibility. The inward rectifying potassium channel 2.1 (Kir2.1) has the potential to inhibit excessive neural activation. Kir2.1 stabilizes membrane potential below the activation potential of voltage gated sodium channels. Therefore, we hypothesized that with adenoviral-mediated delivery of Kir2.1 to the spinal cord, motor neurons would become stabilized below the level of action potential threshold and exhibit an inhibitory effect on neuromuscular function. Additionally, the Kir2.1 transgene was placed under an inducible promoter, Rheoswitch®, which becomes activated only in the presence of its orally administered ligand. In vitro analysis confirmed the ability of the Rheoswitch® promoter to effectively regulated Kir2.1 gene expression. Unilateral injections of the viral vector to lumbar spinal cord of rats demonstrated neuromuscular inhibition exclusively in rats that received the ligand. Histological analysis showed evidence of both Kir2.1 gene expression as well as motor neuron loss in rats that received ligand. Rats that did not receive ligand did not express the transgene, had no functional deficits, and contained normal numbers of motor neurons. Expression of Kir2.1 appears to be toxic to motor neurons. To compare vector expression in a different neuronal population, unilateral injections targeting the hippocampus were performed. Hippocampal neurons are known to endogenously express the Kir2.1 channel, whereas it is not normally found in motor neurons. However, the vector was unsuccessful at transducing hippocampal neurons at low volumes. Future experiments are needed to confirm the safety of this vector and whether Kir2.1 toxicity is exclusive to motor neurons.
Table of Contents
Table of Contents
I. Introduction...1-6
II. Materials and Methods
a. Vector Construction...6-7
b. Cell Culture Assay...7-8
c. Intraspinal Cord Delivery...8-9
d. Behavioral Analysis...9-10
e. Histological Analysis of Spinal Cord Tissue...10
f. Statistical Analysis...11
g. Intrahippocampal Delivery...11-12
h. Histological Analysis of Brain Tissue...12
III. Results
a. Transfection of SHSY5Y Cell
Culture...12-13
b. Ad.Rheo.Kir2.1-GFP Causes Neuromuscular Inhibition...13
c. Ad.Rheo.Kir2.1-GFP Results in Cell Loss...13-14
d. Ad.Rheo.Kir2.1-GFP Expression in Hippocampus...15
IV. Discussion...15 -19
V. Conclusion...19
VI. References...20-23
VII. Figures
a. Figure 1...24
b. Figure 2...25
c. Figure 3...26
d. Figure 4...27
e. Figure 5...28
f. Figure 6...29
g. Figure 7...30
h. Figure 8...31
i. Figure 9...32
j. Figure 10...33
k. Figure 11...33
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