Expression of Human Immunodeficiency Virus Transactivator of Transcription (HIV-Tat1-86) Protein Alters Nociceptive Processing that is Sensitive to Anti-Oxidant and Anti-Inflammatory Interventions

Abstract

Despite the success of combined antiretroviral therapy (cART) in reducing viral load, a substantial portion of Human Immunodeficiency Virus (HIV)+ patients report chronic pain. The exact mechanism underlying this co-morbidity even with undetectable viral load remains unknown, but the transactivator of transcription (HIV-Tat) protein is of particular interest. Functional HIV-Tat protein is observed even in cerebrospinal fluid of patients who have an undetectable viral load. It is hypothesized that Tat protein exposure is sufficient to induce neuropathic pain-like manifestations via both activation of microglia and generation of oxidative stress. iTat mice conditionally expressed Tat_(1-86) protein in the central nervous system upon daily administration of doxycycline (100 mg/kg/d, i.p., up to 14 days). The effect of HIV-Tat protein exposure on the well-being of the animal was assessed using sucrose-evoked grooming and acute nesting behavior for pain-depressed behaviors, and the development of hyperalgesia assessed with warm-water tail-withdrawal and von Frey assays for thermal hyperalgesia and mechanical allodynia, respectively. Tissue harvested at select time points was used to assess ex vivo alterations in oxidative stress, astrocytosis and microgliosis, and blood-brain barrier integrity with assays utilizing fluorescence-based indicators. Tat protein induced mild thermal hyperalgesia but robust mechanical allodynia starting after 4 days of exposure, reaching a nadir after 7 days. Changes in nociceptive processing were associated with reduced sucrose-evoked grooming behavior without altering acute nesting behavior, and in spinal cord dysregulated free radical generation as measured by DCF fluorescence intensity, altered immunohistochemical expression of the gliotic markers, Iba-1 and GFAP, and increased permeability of the blood-brain barrier to the small molecule fluorescent tracer, sodium fluorescein, in a time-dependent manner. Pretreatment with the anti-inflammatory, indomethacin (1 mg/kg/d, i.p.), the antioxidant, methylsulfonylmethane (100 mg/kg/d i.p.), or the immunomodulatory agent, dimethylfumarate (100 mg/kg/d p.o.) thirty minutes prior to daily injections of doxycycline (100 mg/kg/d i.p.) over 7 days significantly attenuated the development of Tat-induced mechanical allodynia. Collectively, the data suggests that even acute exposure to HIV-1 Tat protein at pathologically relevant levels is sufficient to produce select neurophysiological and behavioral manifestations of chronic pain consistent with that reported by HIV-positive patients.

Keywords: Allodynia; HIV; Microglia; Oxidative stress; Pain; Tat.

Figure 1.

Evaluation of HIV-Tat induced hyperalgesia and mechanical allodynia. G-tg and iTat mice were tested before, during and after 14 days of doxycycline treatment (n = 8 per group). Latency of tail-withdrawal after various durations of exposure to the Tat protein was assessed using a 48°C (A) and a 52°C (B) water bath. The duration of paw licking after acetone application (C), as well as mechanical allodynia (D) were assessed as well. Triangles represent mean and SEM. Two-Way Repeated Measures ANOVA with Bonferroni’s Post Hoc analysis was conducted. * = p < 0.05 vs. matching time point of control G-tg mice.

Figure 2.

HIV-Tat protein exposure reduces evoked grooming but not nesting behavior. Total grooming time (A), latency to first groom (B), and nesting behavior (C) were evaluated after 7 days of Tat protein exposure (n = 9 for G-tg and 15 for iTat group). Bars represent mean with SEM, with data points (circles) plotted representing the response of each individual animal. Student’s t-test was used to compare between these two groups for each behavioral outcome. * = p <0.05 vs. response of G-tg mice.

Figure 3.

Evaluation of HIV-Tat induced gliosis in the lumbar spinal cord. G-tg and iTat mice (n = 3 mice per group) were administered doxycycline (100 mg/kg/d i.p) for either 7 or 14 days and their spinal cords were harvested. Sections of the lumbar spinal cord were stained for either Iba-1 (A-D) or GFAP (F-I). Images were collected for 3 sections of the lumbar spinal cord per mouse and analyzed for mean fluorescent intensity (arbitrary fluorescent units) (E, H). Bars represent mean and SEM, with superimposed circles representing data collected from a unique tissue section. Two-way ANOVA with Bonferroni’s Post Hoc analysis was conducted. * = p < 0.05 vs. control G-tg time-point.

Figure 4.

Evaluation of HIV-Tat induced oxidative stress and blood-brain-barrier dysregulation. DCF fluorescence (arbitrary fluorescent units) was used as a direct measure of ROS/RNS levels in supernatant from brain (A) and spinal cord (B) of G-tg (red) and iTat (blue) mice treated with 7 or 14 days of doxycycline (100 mg/kg/d i.p). In a separate cohort of animals treated in the same conditions, the penetration of the small fluorescent tracer, sodium fluorescein, a marker of blood-brain-barrier integrity was measured in the brain (C) and spinal cord (D) of each animal. Bars represent mean and SEM for each treatment group, with superimposed circles representing individual responses (including replicates) from subjects. One-Way ANOVA was utilized with post-hoc comparisons utilizing Bonferroni correction. * = p < 0.05 to respective control condition (doxycycline treated G-tg group).

Figure 5.

Pharmacological interventions targeting oxidative stress/inflammation attenuate Tat-Induced mechanical allodynia. Pretreatment with methylsulfonylmethane (A), indomethacin (B) or dimethyl fumarate (C) were all able to significantly attenuate the development of mechanical allodynia after Tat protein exposure (n = 8 mice per group). Circles indicate mean and SEM. Two-Way Repeated Measures ANOVA was used. Post-hoc comparisons utilized Bonferroni correction. * = p < 0.05 to respective control condition, ╫ = p < 0.05 to respective iTat condition, and ┼ = p < 0.05 to respective baseline on day 0.