Conditional expression of HIV-1 tat in the mouse alters the onset and progression of tonic, inflammatory and neuropathic hypersensitivity in a sex-dependent manner.

BACKGROUND: At least one-third of HIV-1-afflicted individuals experience peripheral neuropathy. Although the underlying mechanisms are not known, they may involve neurotoxic HIV-1 proteins. METHODS: We assessed the influence of the neurotoxic HIV-1 regulatory protein, Tat, on inflammatory and neuropathic nociceptive behaviours using transgenic male and female mice that conditionally expressed (or did not express) HIV-1 Tat1-86 in fibrillary acidic protein-expressing glia in the central and peripheral nervous systems. RESULTS: Tat induction significantly attenuated the time spent paw-licking following formalin injection (2.5%, i.pl.) in both male and female mice. However, significant sex differences were observed in the onset and magnitude of inflammation and sensory sensitivity following complete Freund's adjuvant (CFA) injection (10%, i.pl.) after Tat activation. Unlike female mice, male mice showed a significant attenuation of paw swelling and an absence of mechanical/thermal hypersensitivity in response to CFA after Tat induction. Male Tat(+) mice also showed accelerated recovery from chronic constrictive nerve injury (CCI)-induced neuropathic mechanical and thermal hypersensitivity compared to female Tat(+) mice. Morphine (3.2 mg/kg) fully reversed CCI-induced mechanical hypersensitivity in female Tat(-) mice, but not in Tat(+) females. CONCLUSIONS: The ability of Tat to decrease oedema, paw swelling, and limit allodynia suggests a sequel of events in which Tat-induced functional deficits precede the onset of mechanical hypersensitivity. Moreover, HIV-1 Tat attenuated responses to inflammatory and neuropathic insults in a sex-dependent manner. HIV-1 Tat appears to directly contribute to HIV sensory neuropathy and reveals sex differences in HIV responsiveness and/or the underlying peripheral neuroinflammatory and nociceptive mechanisms.

Animal models of chemotherapy-induced peripheral neuropathy: A machine-assisted systematic review and meta-analysis.

We report a systematic review and meta-analysis of research using animal models of chemotherapy-induced peripheral neuropathy (CIPN). We systematically searched 5 online databases in September 2012 and updated the search in November 2015 using machine learning and text mining to reduce the screening for inclusion workload and improve accuracy. For each comparison, we calculated a standardised mean difference (SMD) effect size, and then combined effects in a random-effects meta-analysis. We assessed the impact of study design factors and reporting of measures to reduce risks of bias. We present power analyses for the most frequently reported behavioural tests; 337 publications were included. Most studies (84%) used male animals only. The most frequently reported outcome measure was evoked limb withdrawal in response to mechanical monofilaments. There was modest reporting of measures to reduce risks of bias. The number of animals required to obtain 80% power with a significance level of 0.05 varied substantially across behavioural tests. In this comprehensive summary of the use of animal models of CIPN, we have identified areas in which the value of preclinical CIPN studies might be increased. Using both sexes of animals in the modelling of CIPN, ensuring that outcome measures align with those most relevant in the clinic, and the animal's pain contextualised ethology will likely improve external validity. Measures to reduce risk of bias should be employed to increase the internal validity of studies. Different outcome measures have different statistical power, and this can refine our approaches in the modelling of CIPN.

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.

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 Tat1-86 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.

Cross-centre replication of suppressed burrowing behaviour as an ethologically relevant pain outcome measure in the rat: a prospective multicentre study.

Burrowing, an ethologically relevant rodent behaviour, has been proposed as a novel outcome measure to assess the global impact of pain in rats. In a prospective multicentre study using male rats (Wistar, Sprague-Dawley), replication of suppressed burrowing behaviour in the complete Freund adjuvant (CFA)-induced model of inflammatory pain (unilateral, 1 mg/mL in 100 µL) was evaluated in 11 studies across 8 centres. Following a standard protocol, data from participating centres were collected centrally and analysed with a restricted maximum likelihood-based mixed model for repeated measures. The total population (TP-all animals allocated to treatment; n = 249) and a selected population (SP-TP animals burrowing over 500 g at baseline; n = 200) were analysed separately, assessing the effect of excluding "poor" burrowers. Mean baseline burrowing across studies was 1113 g (95% confidence interval: 1041-1185 g) for TP and 1329 g (1271-1387 g) for SP. Burrowing was significantly suppressed in the majority of studies 24 hours (7 studies/population) and 48 hours (7 TP, 6 SP) after CFA injections. Across all centres, significantly suppressed burrowing peaked 24 hours after CFA injections, with a burrowing deficit of -374 g (-479 to -269 g) for TP and -498 g (-609 to -386 g) for SP. This unique multicentre approach first provided high-quality evidence evaluating suppressed burrowing as robust and reproducible, supporting its use as tool to infer the global effect of pain on rodents. Second, our approach provided important informative value for the use of multicentre studies in the future.

Cross-centre replication of suppressed burrowing behaviour as an ethologically relevant pain outcome measure in the rat: a prospective multicentre study.

Burrowing, an ethologically relevant rodent behaviour, has been proposed as a novel outcome measure to assess the global impact of pain in rats. In a prospective multicentre study using male rats (Wistar, Sprague-Dawley), replication of suppressed burrowing behaviour in the complete Freund adjuvant (CFA)-induced model of inflammatory pain (unilateral, 1 mg/mL in 100 µL) was evaluated in 11 studies across 8 centres. Following a standard protocol, data from participating centres were collected centrally and analysed with a restricted maximum likelihood-based mixed model for repeated measures. The total population (TP-all animals allocated to treatment; n = 249) and a selected population (SP-TP animals burrowing over 500 g at baseline; n = 200) were analysed separately, assessing the effect of excluding "poor" burrowers. Mean baseline burrowing across studies was 1113 g (95% confidence interval: 1041-1185 g) for TP and 1329 g (1271-1387 g) for SP. Burrowing was significantly suppressed in the majority of studies 24 hours (7 studies/population) and 48 hours (7 TP, 6 SP) after CFA injections. Across all centres, significantly suppressed burrowing peaked 24 hours after CFA injections, with a burrowing deficit of -374 g (-479 to -269 g) for TP and -498 g (-609 to -386 g) for SP. This unique multicentre approach first provided high-quality evidence evaluating suppressed burrowing as robust and reproducible, supporting its use as tool to infer the global effect of pain on rodents. Second, our approach provided important informative value for the use of multicentre studies in the future.

Short-term effect of acute and repeated urinary bladder inflammation on thigmotactic behaviour in the laboratory rat.

Understanding the non-sensory components of the pain experience is crucial to developing effective treatments for pain conditions. Chronic pain is associated with increased incidence of anxio-depressive disorders, and patients often report feelings of vulnerability which can decrease quality of life. In animal models of pain, observation of behaviours such as thigmotaxis can be used to detect such affective disturbances by exploiting the influence of nociceptive stimuli on the innate behavioural conflict between exploration of a novel space and predator avoidance behaviour. This study investigates whether acute and repeated bladder inflammation in adult female Wistar rats increases thigmotactic behaviour in the open field paradigm, and aims to determine whether this correlates with activation in the central amygdala, as measured by c-Fos immunoreactivity. Additionally, up-regulation of inflammatory mediators in the urinary bladder was measured using RT-qPCR array featuring 92 transcripts to examine how local mediators change under experimental conditions. We found acute but not repeated turpentine inflammation of the bladder increased thigmotactic behaviour (decreased frequency of entry to the inner zone) in the open field paradigm, a result that was also observed in the catheter-only instrumentation group. Decreases in locomotor activity were also observed in both models in turpentine and instrumentation groups. No differences were observed in c-Fos activation, although a general increased in activation along the rostro-caudal axis was seen. Inflammatory mediator up-regulation was greatest following acute inflammation, with CCL12, CCL7, and IL-1ß significantly up-regulated in both conditions when compared to naïve tissue. These results suggest that acute catheterisation, with or without turpentine inflammation, induces affective alterations detectable in the open field paradigm accompanied by up-regulation of multiple inflammatory mediators.

One night of total sleep deprivation promotes a state of generalized hyperalgesia: a surrogate pain model to study the relationship of insomnia and pain.

Sleep disturbances are highly prevalent in chronic pain patients. Understanding their relationship has become an important research topic since poor sleep and pain are assumed to closely interact. To date, human experimental studies exploring the impact of sleep disruption/deprivation on pain perception have yielded conflicting results. This inconsistency may be due to the large heterogeneity of study populations and study protocols previously used. In addition, none of the previous studies investigated the entire spectrum of nociceptive modalities. To address these shortcomings, a standardized comprehensive quantitative sensory protocol was used in order to compare the somatosensory profile of 14 healthy subjects (6 female, 8 male, 23.5 ± 4.1 year; mean ± SD) after a night of total sleep deprivation (TSD) and a night of habitual sleep in a cross-over design. One night of TSD significantly increased the level of sleepiness (P<0.001) and resulted in higher scores of the State Anxiety Inventory (P<0.01). In addition to previously reported hyperalgesia to heat (P<0.05) and blunt pressure (P<0.05), study participants developed hyperalgesia to cold (P<0.01) and increased mechanical pain sensitivity to pinprick stimuli (P<0.05) but no changes in temporal summation. Paradoxical heat sensations or dynamic mechanical allodynia were absent. TSD selectively modulated nociception, since detection thresholds of non-nociceptive modalities remained unchanged. Our findings show that a single night of TSD is able to induce generalized hyperalgesia and to increase State Anxiety scores. In the future, TSD may serve as a translational pain model to elucidate the pathomechanisms underlying the hyperalgesic effect of sleep disturbances.

Cathepsin S release from primary cultured microglia is regulated by the P2X7 receptor.

Microglia respond rapidly to injury, increasing their synthesis and release of inflammatory mediators, many of which contribute to the maintenance of persistent pain following CNS or PNS injury. We have recently shown that the lysosomal cysteine protease Cathepsin S (CatS) expressed by spinal microglia is vital for the full expression of neuropathic pain. Here we evaluated the mechanisms by which CatS release occurs from primary microglia in culture. Stimulation of microglia with lipopolysaccharide (LPS) or adenosine tri-phosphate (ATP) alone was insufficient to induce release of enzymatically active CatS in extracellular media. However, following priming with LPS, ATP at 1 mM but not 50 µM resulted in significant release of CatS in the media and maturation of CatS protein in cell extracts. The enzymatic activity measured in media at neutral pH was specific for CatS as it was completely prevented by the CatS inhibitor LHVS. ATP-induced release of CatS required potassium efflux and both extracellular calcium influx and mobilization of intracellular calcium. Pharmacological modulation of ATP-induced release of CatS enzymatic activity revealed that this was dependent on activation of the P2X7 receptor and intracellular phospholipase C and phospholipase A(2). In addition, ATP-induced CatS release involved p38 mitogen activated protein kinase (MAPK) phosphorylation, but not ERK and PI3K signalling pathways. Thus, as high concentration of extracellular ATP promotes release of active CatS from microglia via P2X7 receptor activation, we suggest that the inhibition of CatS release is one of the mechanisms responsible for P2X7 antagonist efficacy in neuropathic pain.

Reduced inflammatory and neuropathic pain and decreased spinal microglial response in fractalkine receptor (CX3CR1) knockout mice.

The chemokine fractalkine (FKN) is a critical mediator of spinal neuronal-microglial communication in chronic pain. Mature FKN is enzymatically cleaved from neuronal membranes and activation of its receptor, CX3CR1, which is expressed by microglia, induces phosphorylation of p38 MAPK. We used CX3CR1 knockout (KO) mice to examine pain behaviour in the absence of FKN signalling. Naive CX3CR1 KO mice had normal responses to acute noxious stimuli. However, KO mice showed deficits in inflammatory and neuropathic nociceptive responses. After intraplantar zymosan, KO mice did not display thermal hyperalgesia, whereas mechanical allodynia developed fully. In the partial sciatic nerve ligation model of neuropathic pain, both mechanical allodynia and thermal hyperalgesia were less severe in KO mice than in wild-types (WT). Dorsal horn Iba1 immunostaining and phosphorylation of p38 MAPK increased after injury in WT controls but not in KO animals. In WT mice, inflammation and nerve injury increased spinal cord CX3CR1 and FKN expression. FKN protein was also increased in KO mice following inflammation but not after neuropathy, suggesting the FKN/CX3CR1 system is differently affected in the two pain models. Loss of FKN/CX3CR1 neuroimmune communication attenuates hyperalgesia and allodynia in a modality-dependent fashion highlighting the complex nature of microglial response in pathological pain models.

Direct activation of transient receptor potential V1 by nickel ions.

TRPV1 is a member of the transient receptor potential (TRP) family of cation channels. It is expressed in sensory neurons of the dorsal root and trigeminal ganglia as well as in a wide range of non-neuronal tissues. The channel proteins serve as polymodal receptors for various potentially harmful stimuli to prevent tissue damage by mediating unpleasant or painful sensations. Using Ca imaging and voltage-clamp recordings, we found that low millimolar doses of Ni2+ (NiSO4) are able to induce non-specific cation currents in a capsaicin-sensitive population of cultured mouse trigeminal ganglion neurons. In addition, we show that NiSO4 elicits intracellular Ca2+ transients and membrane currents in HEK293 and CHO cells heterologously expressing rat TRPV1. The use of voltage ramps from -100 to +100 mV revealed a strong outward rectification of these currents. Application of NiSO4 to the cytoplasmic face of inside-out membrane patches did not induce any currents. However, delivering NiSO4 to the extracellular face during outside-out recordings, we observed a significant increase in open probability paralleled by a decrease in channel conductance. When combined with other TRPV1 agonists, NiSO4 produces a bimodal effect on TRPV1 activity, depending on the strength and concentration of the second stimulus. Outwardly directed currents induced by low doses of capsaicin and nearly neutral pH values ( approximately pH = 7.0-6.5) were augmented by low doses of NiSO4. In contrast, responses to stronger stimuli were reduced by NiSO4. Moreover, we were able to identify amino acids involved in the effect of NiSO4 on TRPV1.

Hydrogen peroxide is a novel mediator of inflammatory hyperalgesia, acting via transient receptor potential vanilloid 1-dependent and independent mechanisms.

Inflammatory diseases associated with pain are often difficult to treat in the clinic due to insufficient understanding of the nociceptive pathways involved. Recently, there has been considerable interest in the role of reactive oxygen species (ROS) in inflammatory disease, but little is known of the role of hydrogen peroxide (H(2)O(2)) in hyperalgesia. In the present study, intraplantar injection of H(2)O(2)-induced a significant dose- and time-dependent mechanical and thermal hyperalgesia in the mouse hind paw, with increased c-fos activity observed in the dorsal horn of the spinal cord. H(2)O(2) also induced significant nociceptive behavior such as increased paw licking and decreased body liftings. H(2)O(2) levels were significantly raised in the carrageenan-induced hind paw inflammation model, showing that this ROS is produced endogenously in a model of inflammation. Moreover, superoxide dismutase and catalase significantly reduced carrageenan-induced mechanical and thermal hyperalgesia, providing evidence of a functionally significant endogenous role. Thermal, but not mechanical, hyperalgesia in response to H(2)O(2) (i.pl.) was longer lasting in TRPV1 wild type mice compared to TRPV1 knockouts. It is unlikely that downstream lipid peroxidation was increased by H(2)O(2). In conclusion, we demonstrate a notable effect of H(2)O(2) in mediating inflammatory hyperalgesia, thus highlighting H(2)O(2) removal as a novel therapeutic target for anti-hyperalgesic drugs in the clinic.

Gabapentin reverses microglial activation in the spinal cord of streptozotocin-induced diabetic rats.

Diabetes mellitus is the leading cause of peripheral neuropathy worldwide. Despite this high level of incidence, underlying mechanisms of the development and maintenance of neuropathic pain are still poorly understood. Evidence supports a prominent role of glial cells in neuropathic pain states. Gabapentin is used clinically and shows some efficacy in the treatment of neuropathic pain. Here we investigate the distribution and activation of spinal microglia and astrocytes in streptozotocin (STZ)-diabetic rats and the effect of the gold standard analgesic, Gabapentin, on these cells. Mechanical allodynia was observed in four week-diabetic rats. Oral administration of Gabapentin significantly attenuated mechanical allodynia. Quantification of cell markers Iba-1 for microglia and GFAP for astrocytes revealed extensive activation of microglia in the dorsal horn of diabetic rats, whereas a reduction in the number of astrocytes could be observed. In addition, an attenuation of microglial activation correlated with reduced allodynia following Gabapentin treatment, while Gabapentin had no effect on the number of astrocytes. Here we show a role of microglia in STZ-induced mechanical allodynia and furthermore, that the anti-allodynic effect of Gabapentin may be linked to a reduction of spinal microglial activation. Astrocytic activation in this model appears to be limited and is unaffected by Gabapentin treatment. Consequently, spinal microglial activation is a key mechanism underlying diabetic neuropathy. Furthermore, we suggest that Gabapentin may exert its anti-allodynic actions partially through alterations of microglial cell function.