Identifying genetic determinants of inflammatory pain in mice using a large-scale gene-targeted screen

Abstract

Identifying the genetic determinants of pain is a scientific imperative given the magnitude of the global health burden that pain causes. Here, we report a genetic screen for nociception, performed under the auspices of the International Mouse Phenotyping Consortium. A biased set of 110 single-gene knockout mouse strains was screened for 1 or more nociception and hypersensitivity assays, including chemical nociception (formalin) and mechanical and thermal nociception (von Frey filaments and Hargreaves tests, respectively), with or without an inflammatory agent (complete Freund's adjuvant). We identified 13 single-gene knockout strains with altered nocifensive behavior in 1 or more assays. All these novel mouse models are openly available to the scientific community to study gene function. Two of the 13 genes (Gria1 and Htr3a) have been previously reported with nociception-related phenotypes in genetically engineered mouse strains and represent useful benchmarking standards. One of the 13 genes (Cnrip1) is known from human studies to play a role in pain modulation and the knockout mouse reported herein can be used to explore this function further. The remaining 10 genes (Abhd13, Alg6, BC048562, Cgnl1, Cp, Mmp16, Oxa1l, Tecpr2, Trim14, and Trim2) reveal novel pathways involved in nociception and may provide new knowledge to better understand genetic mechanisms of inflammatory pain and to serve as models for therapeutic target validation and drug development.

Figure 1.

Relative effect size of mutant compared with wild type (WT) mice for chemical nociception. The subacute or late phase response to formalin was measured using the sum of the time spent licking or biting between 10 and 60 minutes after formalin was administered. The percentage effect size of genotype (mutant—WT control) on late phase response to formalin is plotted by gene symbol. The unadjusted P value is inserted after the gene symbol for all significant gene effects and genes of potential interest, reporting the effect of genotype of the null model hypothesis analysis where both sexes are affected or from the sex × genotype interaction where sexual dimorphism is present. A positive effect represents an increase in licking or biting by mutant relative to control (hypersensitive) and a negative effect a decrease in licking or biting (hyposensitive). Only genes with P values less than or equal to 0.1 are plotted, and all other genes with a P value greater than 0.1 are named in the text box. A brown bar indicates that the P value was less than 0.01 but not considered significant, and a green bar indicates that the P value for this test was considered significant with a P value below 0.001. Bars with a single symbol (black circle) represent both sexes. Where sexual dimorphism was detected, the male value is indicated by a grey square and female by a white square.

Figure 2.

Relative effect size of mutant compared with wild type (WT) mice for mechanical nociception. (A) The percentage effect size of genotype for the baseline von Frey measurement plotted by gene symbol. The mixed model analysis tested log10 baseline scores, and effect sizes are calculated on that basis. The x-axis is reversed to indicate that a negative effect represents a measure of lower force and therefore a hypersensitive response by the mutant, and a positive change indicates higher force (hyposensitive). Only genes with P values less than or equal to 0.1 are plotted, and all other genes with P value greater than 0.1 are named in the text box. The unadjusted P value is inserted after the gene symbol for all significant gene effects and genes of potential interest, reporting the effect of genotype of the null model hypothesis analysis where both sexes are affected or from the sex×genotype interaction where sexual dimorphism is present. A brown bar indicates that the P value was less than 0.01 but not considered significant, and a green bar indicates that the P value for this test was considered significant with a P value below 0.001. Bars with a single symbol represent both sexes. Where sexual dimorphism was detected, the male value is indicated by a gray square and female by a white square. The colors and symbols are maintained for part B. (B) The percentage effect size of genotype for the measured peak change from baseline in response to CFA administration of the von Frey assay is plotted by gene symbol. The mixed model analysis tested the difference of log10 scores, and effect sizes are calculated on that basis. A positive effect indicates a greater change from baseline and represents a hypersensitive response to CFA administration for the mutant, and a negative effect indicates a smaller response (hyposensitive). All genes are shown regardless of P value. The unadjusted P value is inserted after the gene symbol for all significant gene effects and genes of potential interest, reporting the effect of genotype of the null model hypothesis analysis where both sexes are affected or from the sex×genotype interaction where sexual dimorphism is present. The data above the break in the y-axis are from TCP and UCD and use 24 hours postadministration as the comparison to baseline; data below the break are from JAX and used 48 hours postadministration as the comparison to baseline (yellow shade). CFA, complete Freund's adjuvant.

Figure 3.

Relative effect size of mutant compared with wild type (WT) mice for thermal nociception. (A) The percentage effect size of genotype for the baseline Hargreaves measurement plotted by gene symbol. The x-axis is reversed to indicate that a negative effect represents a measure of shorter latency to withdraw and therefore a hypersensitive response by the mutant, and a positive change indicates longer latency (hyposensitive). All genes are shown regardless of P value. The unadjusted P value is inserted after the gene symbol for all significant gene effects and genes of potential interest, reporting the effect of genotype of the null model hypothesis analysis where both sexes are affected or from the sex×genotype interaction where sexual dimorphism is present. A brown bar indicates that the P value was less than 0.01 but not considered significant, and a green bar indicates that the P value for this test was considered significant with a P value below 0.001. Bars with a single symbol represent both sexes. Where sexual dimorphism was detected, the male value is indicated by a gray square and female by a white square. The colors and symbols are maintained for part B. (B) The percentage effect size of genotype for the peak change from baseline in response to CFA administration of the Hargreaves assay is plotted by gene symbol. A positive effect indicates a greater change from baseline and represents a hypersensitive response to CFA administration for the mutant, and a negative effect indicates a smaller response (hyposensitive). All genes are shown regardless of P value. The unadjusted P value is inserted after the gene symbol for all significant gene effects and genes of potential interest, reporting the effect of genotype of the null model hypothesis analysis where both sexes are affected or from the sex × genotype interaction where sexual dimorphism is present. For all contributing centers, the peak change from baseline was measured 24 hours after CFA administration. CFA, complete Freund's adjuvant.

Figure 4.

Single-gene knockout strains grouped by nocifensive, locomotor, and anxiety-like or exploratory behavior. One hundred ten single-gene knockout strains were screened for nociception or hypersensitivity. A separate cohort of mice for 103 of these 110 strains was phenotyped using open field. Gene symbols for these knockout strains are grouped by outcome of nociception testing [abnormal (yellow) or normal (blue) nocifensive behavior], the presence or absence of open field data (open field data or no open field data), and the outcome of open field data statistical analyses where abnormalities were detected [abnormal locomotor activity (defined using the MP terms “hyperactive,” and “hypoactive,” green) or anxiety-like or abnormal exploratory behavior (defined using the MP terms “increased thigmotaxis,” “decreased thigmotaxis,” “increased vertical activity,” “decreased vertical activity,” “increased anxiety-related response,” “increased exploration in new environment,” and “abnormal behavior”, pink)].

Figure 5.

Abnormalities in nocifensive behavior after Tecpr2 inactivation. (A and B) Latency to withdraw the paw from a thermal stimulus is shown (quartile boxplots with error bars for 5%-95% percentile) in seconds for each test day in the Hargreaves assay for wild type C57BL/6NJ (WT) mice [8 and 17-18 weeks (n = 96F, n = 100M), gray] and Tecpr2 [17 weeks (n = 12F, n = 10M), orange] homozygous null female (A) and male (B) mice. Baseline withdrawal latency was significantly longer for Tecpr2 mutant mice (mixed model genotype F_(1,196) =15.1, P < 0.001,***) than their WT controls. (C and D) Paw withdrawal threshold is plotted in log10 scale as quartile boxplots (with error bars for 5%-95% percentile) for each test day for wild type C57BL/6NJ (WT) [8 and 17-18 weeks (n = 119F, n = 122M), gray] and Tecpr2 [17 weeks (n = 12F, n = 10M), orange] homozygous null females (C) and males (D) for the von Frey assay. Baseline threshold was significantly lower for Tecpr2 mutant mice (F_(1,256) = 15.2, P < 0.001, ***), and the CFA response was significantly smaller for Tecpr2 mutant mice 48 hours postadministration (F_(1,255) = 12.7, P < 0.001, ***) compared with WT controls. (E and F) The mean (with SEM) of licking or biting behavior duration summed over 10 to 60 minutes is shown for wild type C57BL/6NJ (WT) [aged 12-21 weeks (n = 346F, n = 398M), gray] and Tecpr2 [16-17 weeks (n = 12F, n = 11M), orange] homozygous null mice for females (E) and males (F). No statistically significant difference was detected.

Figure 6.

Abnormalities in nocifensive behavior after Cp inactivation. (A and B) Latency to withdraw the paw from a thermal stimulus is shown (quartile boxplots with error bars for 5%-95% percentile) in seconds for each test day in the Hargreaves assay for wild type C57BL/6NJ (WT) mice [8 and 17-18 weeks (n = 96F, n = 100M), gray] and Cp [16-17 weeks (n = 12F, n = 12M), orange] homozygous null female (A) and male (B) mice. Thermal hyperalgesia resolved more rapidly in Cp mutant mice (null genotype P < 0.001; sex × genotype F_(2,216) = 7.41, P < 0.001,***) than their WT controls. (C and D) Paw withdrawal threshold is plotted in log10 scale as quartile boxplots (with error bars for 5%-95% percentile) for each test day for wild type C57BL/6NJ (WT) [8 and 17-18 weeks (n = 119F, n = 122M), gray] and Cp [16-17 weeks (n = 12F, n = 12M), orange] homozygous null females (C) and males (D) for the von Frey assay. Peak mechanical hyperalgesia 48 hours after CFA administration was significantly reduced for Cp mutant mice (F_(1,257) = 16.3, P < 0.001, ***) compared with WT controls. (E and F) The mean (with SEM) of licking or biting behavior duration summed over 10 to 60 minutes is shown for wild type C57BL/6NJ (WT) [aged 12-21 weeks (n = 346F, n = 398M), gray] and Cp [16-17 weeks (n = 6F, n = 9M), orange] homozygous null mice for females (E) and males (F). No statistically significant difference was detected.