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Expression Levels of the Hypothalamic AMPK Gene Determines the Responsiveness of the Rats to Electroacupuncture-Induced Analgesia

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Expression Levels of the Hypothalamic AMPK Gene Determines the Responsiveness of the Rats to Electroacupuncture-Induced Analgesia

Sun Kwang Kim et al. BMC Complement Altern Med.

Abstract

Background: Although electroacupuncture (EA) relieves various types of pain, individual differences in the sensitivity to EA analgesia have been reported, causing experimental and clinical difficulties. Our functional genomic study using cDNA microarray identified that 5'-AMP-activated protein kinase (AMPK), a well-known factor in the regulation of energy homeostasis, is the most highly expressed gene in the hypothalamus of the rats that were sensitive to EA analgesia ("responder"), as compared to the rats that were insensitive to EA analgesia ("non-responder"). In this study, we investigated the causal relationship between the hypothalamic AMPK and the individual variation in EA analgesia.

Methods: Sprague-Dawley (SD) rats were divided into the responder and the non-responder groups, based on EA-induced analgesic effects in the tail flick latency (TFL) test, which measures the latency of the tail flick response elicited by radiant heat applied to the tail. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to quantify the expression levels of AMPK mRNA in the hypothalamus of the responder and non-responder rats. Further, we examined whether viral manipulation of the AMPK expression in the hypothalamus modulates EA analgesia in rats.

Results: The real-time RT-PCR analysis showed that mRNA expression levels of AMPK in the hypothalamus of the responder rats are significantly higher than those of the non-responder rats, validating the previous microarray results. Microinjection of dominant negative (DN) AMPK adenovirus, which inhibits AMPK activity, into the rat hypothalamus significantly attenuates EA analgesia (p < 0.05), whereas wild type (WT) AMPK virus did not affect EA analgesia (p > 0.05).

Conclusions: The present results demonstrated that levels of AMPK gene expression in the rat hypothalamus determine the individual differences in the sensitivity to EA analgesia. Thus, our findings provide a clinically useful evidence for the application of acupuncture or EA for analgesia.

Figures

Figure 1
Figure 1
Verification of the correct injection and transfection of the adenovirus into hypothalamus. (A) Representative photograph (×40) of the Nissle staining showing the injection position (arrowhead). (B) Representative confocal microphotograph of GFP fluorescence in the hypothalamic arcuate nucleus (ARC) from the rat injected with adenovirus. 3v, 3rd ventricle. Scale bar, 100 μm.
Figure 2
Figure 2
Normalized mRNA level of the hypothalamic AMPK in the “responder” and “non-responder” rats. Real-time RT-PCR experiments show the amount of AMPK mRNA expression that normalized by dividing AMPK intensities by that of the house keeping gene, GAPDH. Data are presented as mean ± SEM. **p < 0.01, responder (n = 4) vs. non-responder (n = 4) by the unpaired t-test.
Figure 3
Figure 3
Time course of pre-EA TFL in WT AMPK and DN AMPK virus-injected rats. The TFL was measured before EA stimulation on days -1, 3, 7 and 14 following viral injection. No significant differences in pre-EA TFL were observed between the WT virus-injected and DN virus-injected rats during the whole experimental period. Data are presented as mean ± SEM. N = 8/group.
Figure 4
Figure 4
Comparison of TFL increase ratio after EA between WT AMPK and DN AMPK virus-injected rats. DN: dominant negative form AMPK virus-injected rats (n = 8); WT: wild-type AMPK α subunit virus-injected rats (n = 8). Pre: before the microinjection of virus; Post: after virus microinjection. Data are presented as mean ± SEM. *p < 0.05 and ***p < 0.001, WT vs. DN by the unpaired t-test.

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