Background & Aims

HIV-associated peripheral neuropathy (HIV-PN) is the most prevalent neurological complication of HIV infection. Previously, we have shown that doxycycline-inducible HIV-1 Tat transgenic (iTat) mice exhibit neuropathy-like behaviors following Tat induction. Bioinformatic pathway analysis based on the NanoString® RNA assessment of a panel of 200+ inflammation-related genes within the lumbar spinal cord of iTat mice revealed altered gene expression clustered in three major Tat-regulated signaling pathways: apoptosis, inflammation mediated by chemokines and cytokines, and Toll-like receptor (TLR) signaling pathways. While all of these pathways are known to be important regulators of inflammation and pain, their interplay in HIV-PN is less clear. To begin this investigation, we used quantitative real-time PCRs (RT-PCRs) to further verify expression changes of the 37 genes that were identified as associated with these above pathways during bioinformatic pathway analysis.

Methods

RNA samples used in the NanoString® analysis were also used in the qRT-PCR measurement. Briefly, adult male and female B6.iTat (2-4 months old, n=3 per sex per group) mice were used. Tat induction was achieved via daily intra-peritoneal (i.p.) injection of doxycycline hyclate (Dox) at 100 mg/kg for 14 days from days 0-13. pH-matched PBS (pH 3.0) was used as control. Lumbar spinal cord tissues were collected at days 0, 3, 7, 14, 21, 28, and 35 for Tat-induced mice and day 14 for the PBS controls. Total RNA was extracted and an aliquot was used for cDNA synthesis. qRT-PCRs were performed for all 37 identified genes with GAPDH as the internal control for data normalization. Two-way ANOVA on the relative gene expression for samples collected from naïve and Dox-treated animals was performed using time and sex as the factors, followed by the Holm Sidak post-hoc test. p<0.05 was considered statistically significant.

Results

Expression of 13 out of 37 genes measured via qRT-PCR showed statistically significant time-dependent changes following Tat induction. Of these, 7 genes showed the same or similar Tat-induced gene expression patterns when measured by qRT-PCR as was observed in the NanoString® (based on copy counts) assessment. Four genes (Cxcr4, CCL5, Alox12, and Shc1) were associated with the inflammation mediated by chemokines and cytokines signaling pathway, two (TRAF2 and MAP3K1) were associated with the apoptosis pathway, and two were associated with the TLR pathways (Tollip and MAP3K1). Except for TRAF2, which showed a continued increase and plateaued at day 14, all genes displayed a transient increase that peaked between days 14-21 post-Tat induction and ranged from a 1.5- to 3-fold change compared with day 0 levels.

Conclusions

Lumbar spinal cord inflammatory responses are regulated by Tat induction and the involvement of selected inflammatory markers in Tat-associated HIV-PN is suggested. Further investigation is needed to elucidate the specific contributions of these selected genes and the interplay between the apoptosis, inflammation mediated by chemokines and cytokines, and TLR signaling pathways. The study also emphasized the importance of verification tests following multi-gene assessment.

References

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Presenting Author

Ling Cao

Poster Authors

Ling Cao

MD, PhD

University of New England

Lead Author

James Withers BS

Lead Author

Karl Mohr

Lead Author

Elizabeth Bean PhD

University of New England

Lead Author

Topics

  • Models: Chronic Pain - Neuropathic