Aldehyde dehydrogenase-2 regulates nociception in rodent models of acute inflammatory pain

Abstract

Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase-2 (ALDH2), which metabolizes many aldehydes, may regulate nociception. To test this hypothesis, we generated a knock-in mouse with an inactivating point mutation in ALDH2 (ALDH2*2), which is also present in human ALDH2 of ~540 million East Asians. The ALDH2*1/*2 heterozygotic mice exhibited a larger response to painful stimuli than their wild-type littermates, and this heightened nociception was inhibited by an ALDH2-selective activator (Alda-1). No effect on inflammation per se was observed. Using a rat model, we then showed that nociception tightly correlated with ALDH activity (R(2) = 0.90) and that reduced nociception was associated with less early growth response protein 1 (EGR1) in the spinal cord and less reactive aldehyde accumulation at the insult site (including acetaldehyde and 4-hydroxynonenal). Further, acetaldehyde- and formalin-induced nociceptive behavior was greater in the ALDH2*1/*2 mice than in the wild-type mice. Finally, Alda-1 treatment was even beneficial when given after the inflammatory agent was administered. Our data in rodent models suggest that the mitochondrial enzyme ALDH2 regulates nociception and could serve as a molecular target for pain control, with ALDH2 activators, such as Alda-1, as potential non-narcotic, cardiac-safe analgesics. Furthermore, our results suggest a possible genetic basis for East Asians' apparent lower pain tolerance.

Conflict of interest statement

Competing Interests: DM-R and C-HC are the founders of ALDEA Pharmaceuticals and C-HC is a consultant for the company. However, none of the research in the academic laboratory is supported by or is reported to the company. DM-R, C-HC and VOZ hold a patent on “Methods and Compositions for Treating Pain”. The other authors have no disclosures.

Copyright © 2014, American Association for the Advancement of Science.

Figures

Figure 1. Characterization of heterozygotic ALDH2*1/*2 mice and ALDH2*1/*1 wild type mice

A. Wild type (WT) or heterozygous ALDH2*1/*2 (*1/*2) mice were given 4g/kg ethanol or water of equal volume by gavage, and acetaldehyde blood levels were measured 60 minutes later (n=4 animals/treatment group). B–D. Motor skill tests of wild type ALDH2*1/*1 (blue bars) and heterozygous ALDH2*1/*2 mice (red bars). Mean number of squares crossed in an open field test in (B); number of rearing events in 3 minutes in (C); and latency to fall from a rotating rod (D)(for B and C n=6–7 animals/group, for D n=10 animals/group), tested once per animal. E. Western blot quantitation of ALDH2 protein in wild type and heterozygotic ALDH2*1/*2 knock-in mice. (50μg liver protein homogenate was loaded per lane) (n=3 biological replicates/group). F. ALDH enzyme activity in wild type and ALDH2*1/*2 mice, expressed as μmol NADH per minute per mg of protein (n=3 biological replicates/group). All data are mean±SEM; *P<0.01 compared to wild type given water, ◇P<0.01 compared to all other groups, †P<0.05 compared to wild type, as assessed by t-test (B, C, E, and F), one-way ANOVA (A), and two-way ANOVA (D).

Fig. 2. Experimental protocol and nociceptive behavioral results for ALDH2*1/*2 mice (red bars) and wild type mice (blue bars)

A. Experimental protocol: Nociceptive threshold testing (closed circles) using von Frey hairs performed at baseline and 3 hours after induction of the inflammatory insult. Red arrows indicate treatment with either vehicle or with the ALDH2 activator Alda-1 (2mg/kg, subcutaneously). At time zero (black arrow), carrageenan (100μg) was injected to the right hind paw. B. Nociceptive threshold in mice assessed at baseline and 3 hours after carrageenan-induced inflammatory insult using the up-down technique (n=8 animals/group, tested once per animal). C. A representative Western blot and % change in 4-HNE protein adducts in proteins extracted from paws treated with saline or with carrageenan from wild type and heterozygous ALDH2*1/*2 mice (n=8 animals/group, GAPDH used as loading control). D. In a separate subset of mice, nociceptive threshold was assessed with a scoring scale 3 hours after the inflammatory insult (2=no response, 1= withdrawal from stimulus, 0= immediate withdrawal from stimulus with licking or flinching) (n=6–7 animals/group, repeated 6 times per animal). The observers were blinded to the experimental conditions. Baseline measurements for D. are provided in Fig. S3. E. Nociceptive threshold for wild type and ADLH2*1/*2 mice after administration of Alda-1 (2mg/kg per treatment) as assessed by the scoring technique used in (D) (n=8 animals/group, repeated 6 times per animal). Additional data for other von Frey hairs tested is provided in Fig. S3. All data are expressed as mean±SEM, *P<0.05 compared to wild type, ◇P<0.05 compared to all other groups, as assessed by ANOVA with Tukey’s correction; behavioral analyses were carried out by observers blinded to the experimental conditions.

Figure 3. Experimental protocol and nociceptive behavior in rats

A. Experimental protocol: Nociceptive threshold testing (closed circles) assessed by Randall-Selitto at baseline and 3 hours after inflammatory insult. Red arrows indicate treatment with either vehicle or with the ALDH2 activator, Alda-1 (2mg/kg, subcutaneously). At time zero (black arrow) the inflammatory insults (carrageenan or prostaglandin E2; PGE2) were given to the right hind paw. B. Latency to thermal stimulation for rat hind paw withdrawal with and without treatment with Alda-1 C. Nociceptive threshold in rats subjected to carrageenan or prostaglandin E2 pro-inflammatory insults. All data expressed as mean±SEM, *P<0.01 compared to untreated and Alda-1 alone group, ◇P<0.01 compared to all other groups assessed by ANOVA with Tukey’s correction (B) and linear regression (C), n=6–8 animals/group, tested once per animal; the observers were blinded to the experimental conditions.

Figure 4. Effect of ALDH2 activation by Alda-1 on aldehydic load and hyperalgesia

For A–E, rats were subjected to the protocol described in Fig. 3A, and all analyses were carried out 3 hours after carrageenan-induced insult by an observer blinded to the experimental conditions. A. Paw volume 3 hr after carrageenan injection. B. Mast cell chymase in the paw after carrageenan injection. C. Histological analysis of slices stained for EGR1, a marker of hyperalgesia in the dorsal horn superficial laminae for L4-L6 regions of the spinal cord. (n=6 animals/group, expressed as number of positive nuclei counted per mm2). D. A representative Western blot for 4-HNE protein adducts in the paws of vehicle- (V), carrageenan- (Car) and carrageenan + Alda-1- (Car+ Alda-1) treated groups. (n=5–6 biological replicates/group). E. Malondialdehyde (MDA) levels assessed as percent change relative to naïve rats as assessed by TBARs assay. (n=6 biological replicates/group). All data are expressed as mean±SEM, *P<0.01 versus vehicle, †P<0.05 compared to vehicle assessed by ANOVA with Tukey’s correction.

Figure 5. ALDH2 involvement in acetaldehyde and formaldehyde-induced acute nociception

A. Quantification of acetaldehyde in carrageenan-induced inflamed paw. (n=3 biological replicates/group, repeated 3 times for each replicate). B. Acetaldehyde at the amounts measured in the paw (in A.) was injected into the hind paw, and licking and flinching were measured. C. Nociceptive threshold after administration of 600 ng of acetaldehyde (n=4–7/ animals group, tested once for each animal). D. A single acetaldehyde (100ng) injection was administered to the paw and licking and flinching were measured (n=8/group, tested once per animal). E. Using the up-down technique, the intensity and duration of the acetaldehyde-induced nociceptive behavior was measured in ALDH2*1/*2 mice and wild type ALDH2*1/*1 mice (n=8/group, tested once per animal). F. Phase II cumulative flinching and licking for wild type ALDH2*1/*1 mice and ALDH2*1/*2 mice. (n=8/group, tested once per animal). All data are expressed as mean±SEM, *P<0.001 versus vehicle, †P<0.05 versus all other groups, ◇P<0.001 compared to all other groups, △P<0.001 compared to respective vehicle treated groups, assessed by ANOVA with Tukey’s correction, blue data points and bars= wild type ALDH2*1/*1 mice, red data points and bars= ALDH2*1/*2 mice.

Figure 6. Effect of Alda-1 after the inflammatory insult on nociceptive threshold

A. Nociceptive threshold testing (closed circles) assessed by either Hargreaves or Randall-Selitto at baseline and 3 hours after inflammatory insult. Red arrows, treatment either of vehicle or the ALDH2 activator, Alda-1 (2mg/kg, subcutaneously). Naloxone (Nal, 1mg/kg) or vehicle was given subcutaneously (green arrows) 10 minutes before each Alda-1 treatment. At time zero (black arrow) the inflammatory insult (carrageenan) was given to the right hind paw. B. and C. Alda-1 was given 30 and 150 minutes after carrageenan (n=9/group, tested once per animal *P<0.0001 assessed by ANOVA followed by Tukey’s correction). Error bars represent mean±SEM. The observers were blinded to the experimental conditions.