Reduced intraepidermal nerve fibre density, glial activation, and sensory changes in HIV type-1 Tat-expressing female mice: involvement of Tat during early stages of HIV-associated painful sensory neuropathy

Abstract

Introduction: HIV infection is associated with chronic pain states, including sensory neuropathy, which affects greater than 40% of patients.

Objectives and methods: To determine the impact of HIV-Tat induction on nociceptive behaviour in female mice conditionally expressing HIV Tat_1-86 protein through a doxycycline (DOX)-driven glial fibrillary acidic protein promoter, intraepidermal nerve fibre density and immune cell activation in the dorsal root ganglion (DRG) and spinal cord were assessed by immunohistochemistry. Mice were assessed for mechanical and thermal sensitivity for 9 weeks using von-Frey and Hargreaves tests.

Results: Intraepidermal nerve fibre density was significantly reduced after 6 weeks of Tat induction, similar to sensory neuropathy seen in clinical HIV infection. Tat induction through DOX caused a significant reduction in paw withdrawal thresholds in a time-dependent manner starting the 4th week after Tat induction. No changes in paw withdrawal latencies were seen in Tat(-) control mice lacking the tat transgene. Although reductions in paw withdrawal thresholds increased throughout the study, no significant change in spontaneous motor activity was observed. Spinal cord (cervical and lumbar), DRG, and hind paw skin were collected at 8 days and 6 weeks after Tat induction. HIV-Tat mRNA expression was significantly increased in lumbar DRG and skin samples 8 days after DOX treatment. Tat induced a significant increase in the number of Iba-1 positive cells at 6 weeks, but not after 8 days, of exposure. No differences in glial fibrillary acidic protein immunoreactivity were observed.

Conclusion: These results suggest that Tat protein contributes to painful HIV-related sensory neuropathy during the initial stages of the pathogenesis.

Keywords: Digital PCR; HIV-1; Rodent behaviour; Sensory neuropathy; Transactivator of transcription; Viral protein.

Figure 1.

Relative HIV-1 Tat mRNA expression in skin, DRG, and spinal cord of Tat(−) and Tat(+) mice. (A) Relative level of HIV-1 Tat mRNA in the lumbar spinal cord. (B) Relative level of HIV-1 Tat mRNA in lumbar DRG. (C) Relative level of HIV-1 Tat mRNA in skin. Data are shown as box and dot plots. The box represents the interquartile range, with the line representing the median and the square representing the mean. Whiskers show the SD. Diamonds represent individual values for each animal. Data were analysed by a Mann–Whitney test. The P value in the upper left corner represents the overall group difference. DRG, dorsal root ganglion.

Figure 2.

Reduced epidermal nerve fibre density in Tat(+) mice. (A) Intraepidermal nerve fibre density in Tat(−) and Tat(+) mice. Data are shown as box and dot plots with diamonds representing individual animal values. The box represents the interquartile range, with the line representing the median and the square representing the mean. Whiskers show the SD. Data were analysed by a 1-way ANOVA (the Bonferroni post-hoc test). The P value in the upper left corner represents the overall group difference. (B) Immunoreactivity to PGP9.5 in Tat(−) and Tat(+) mice. Arrowheads in B indicate individual fibres. ANOVA, analysis of variance; IENF, intraepidermal nerve fibre.

Figure 3.

No measurable macrophage and satellite cell activation in the DRG of Tat(+) mice. (A) Immunoreactivity to Iba-1 and GFAP in DRG sections of Tat(−) and Tat(+) mice 8 days and 6 weeks after DOX treatment. Arrows in A indicate Iba-1 or GFAP immunoreactive cells and/or their processes. (B) Number of macrophages per mm² in Tat(−) and Tat(+) mice. Data are shown as box and dot plots with diamonds representing single animal values. The box represents the interquartile range, with the line representing the median and the square representing the mean. Whiskers show the SD. Data were analysed by 1-way ANOVA (not significant). The P value in the upper left corner represents the overall group difference. (C) Percent of area with positive immunoreactivity to GFAP in Tat(−) and Tat(+) mice. Data are shown as box and dot plots with diamonds representing single animal values. The box represents the interquartile range, with the line representing the median and the square representing the mean. Whiskers show the SD. Data were analysed by a 1-way ANOVA (NS). The P value in the upper left corner represents the overall group difference. ANOVA, analysis of variance; DOX, doxycycline; DRG, dorsal root ganglion; GFAP, glial fibrillary acidic protein.

Figure 4.

Microglial, but not astroglial, activation in the dorsal spinal cord of Tat(+) mice. (A) Immunoreactivity to Iba-1 and GFAP in spinal cord sections of Tat(−) and Tat(+) mice at 8 days and 6 weeks after DOX treatment. Arrows in A indicate Iba-1 or GFAP immunoreactive cells and/or their processes. (B) Number of microglial profiles per mm² in Tat(−) and Tat(+) mice. Data are shown as box and dot plots with diamonds representing single animal values. The box represents the interquartile range, with the line representing the median and the square representing the mean. Whiskers show the SD. Data were analysed by 1-way ANOVA (the Bonferroni post hoc test). The P value in the upper left corner represents the overall group difference. (C) Percentage of area with positive immunoreactivity to GFAP in Tat(−) and Tat(+) mice. Data are shown as box and dot plots with diamonds representing single animal values. The box represents the interquartile range, with the line representing the median and the square representing the mean. Whiskers show the SD. Data were analysed by 1-way ANOVA (not significant). The P value in the upper left corner represents the overall group difference. ANOVA, analysis of variance; DOX, doxycycline; GFAP, glial fibrillary acidic protein.

Figure 5.

Behavioural outcomes in Tat(−) and Tat(+) mice. (A) Paw withdrawal thresholds (PWTs) over time—von-Frey test. (B) Paw withdrawal latencies (PWLs) over time—Hargreaves test. (C) Body weight over time. (D) Locomotor activity (number of infrared beam interruptions). Data shown as mean ± SEM (n = 10 mice per group). Data were analysed by a 2-way repeated-measures ANOVA (Sidak) for multiple group comparison and by an unpaired Student t test for comparison of 2 groups. *P < 0.05. ANOVA, analysis of variance.