Effects of HIV-1 Tat on oligodendrocyte viability are mediated by CaMKIIß-GSK3ß interactions.

Myelin disruptions are frequently reported in human immunodeficiency virus (HIV)-infected individuals and can occur in the CNS very early in the disease process. Immature oligodendrocytes (OLs) are quite sensitive to toxic increases in [Ca2+ ]i caused by exposure to HIV-1 Tat (transactivator of transcription, a protein essential for HIV replication and gene expression), but sensitivity to Tat-induced [Ca2+ ]i is reduced in mature OLs. Tat exposure also increased the activity of Ca2+ /calmodulin-dependent kinase IIß (CaMKIIß), the major isoform of CaMKII expressed by OLs, in both immature and mature OLs. Since CaMKIIß is reported to interact with glycogen synthase kinase 3ß (GSK3ß), and GSK3ß activity is implicated in OL apoptosis as well as HIV neuropathology, we hypothesized that disparate effects of Tat on OL viability with maturity might be because of an altered balance of CaMKIIß-GSK3ß activities. Tat expression in vivo led to increased CaMKIIß and GSK3ß activity in multiple brain regions in transgenic mice. In vitro, immature murine OLs expressed higher levels of GSK3ß, but much lower levels of CaMKIIß, than did mature OLs. Exogenous Tat up-regulated GSK3ß activity in immature, but not mature, OLs. Tat-induced death of immature OLs was rescued by the GSK3ß inhibitors valproic acid or SB415286, supporting involvement of GSK3ß signaling. Pharmacologically inhibiting CaMKIIß increased GSK3ß activity in Tat-treated OLs, and genetically knocking down CaMKIIß promoted death in mature OL cultures treated with Tat. Together, these results suggest that the effects of Tat on OL viability are dependent on CaMKIIß-GSK3ß interactions, and that increasing CaMKIIß activity is a potential approach for limiting OL/myelin injury with HIV infection.

Altered glial glutamate transporter expression in descending circuitry and the emergence of pain chronicity.

BACKGROUND: The glutamate type 1 transporter (GLT1) plays a major role in glutamate homeostasis in the brain. Although alterations of GLT1 activity have been linked to persistent pain, the significance of these changes is poorly understood. Focusing on the rostral ventromedial medulla, a key site in pain modulation, we examined the expression and function of GLT1 and related transcription factor kappa B-motif binding phosphoprotein (KBBP) in rats after adjuvant-induced hind paw inflammation. RESULTS: After inflammation, GLT1 and KBBP showed an early upregulation and gradual transition to downregulation that lasted throughout the eight-week observation period. Nitration of GLT1 was reduced at 30 min and increased at eight weeks after inflammation, suggesting an initial increase and later decrease in transporter activity. Mechanical hyperalgesia and paw edema exhibited an initial developing phase with peak hyperalgesia at 4 to 24 h, a subsequent attenuating phase, followed by a late persistent phase that lasted for months. The downregulation of GLT1 occurred at a time when hyperalgesia transitioned into the persistent phase. In the rostral ventromedial medulla, pharmacological block with dihydrokainic acid and RNAi of GLT1 and KBBP increased nociception and overexpression of GLT1 reversed persistent hyperalgesia. Further, the initial upregulation of GLT1 and KBBP was blocked by local anesthetic block, and pretreatment with dihydrokainic acid facilitated the development of hyperalgesia. CONCLUSIONS: These results suggest that the initial increased GLT1 activity depends on injury input and serves to dampen the development of hyperalgesia. However, later downregulation of GLT1 fosters the net descending facilitation as injury persists, leading to the emergence of persistent pain.

A central role for glial CCR5 in directing the neuropathological interactions of HIV-1 Tat and opiates.

BACKGROUND: The collective cognitive and motor deficits known as HIV-associated neurocognitive disorders (HAND) remain high even among HIV+ individuals whose antiretroviral therapy is optimized. HAND is worsened in the context of opiate abuse. The mechanism of exacerbation remains unclear but likely involves chronic immune activation of glial cells resulting from persistent, low-level exposure to the virus and viral proteins. We tested whether signaling through C-C chemokine receptor type 5 (CCR5) contributes to neurotoxic interactions between HIV-1 transactivator of transcription (Tat) and opiates and explored potential mechanisms. METHODS: Neuronal survival was tracked in neuronal and glial co-cultures over 72 h of treatment with HIV-1 Tat ± morphine using cells from CCR5-deficient and wild-type mice exposed to the CCR5 antagonist maraviroc or exogenously-added BDNF (analyzed by repeated measures ANOVA). Intracellular calcium changes in response to Tat ± morphine ± maraviroc were assessed by ratiometric Fura-2 imaging (analyzed by repeated measures ANOVA). Release of brain-derived neurotrophic factor (BDNF) and its precursor proBDNF from CCR5-deficient and wild-type glia was measured by ELISA (analyzed by two-way ANOVA). Levels of CCR5 and µ-opioid receptor (MOR) were measured by immunoblotting (analyzed by Student's t test). RESULTS: HIV-1 Tat induces neurotoxicity, which is greatly exacerbated by morphine in wild-type cultures expressing CCR5. Loss of CCR5 from glia (but not neurons) eliminated neurotoxicity due to Tat and morphine interactions. Unexpectedly, when CCR5 was lost from glia, morphine appeared to entirely protect neurons from Tat-induced toxicity. Maraviroc pre-treatment similarly eliminated neurotoxicity and attenuated neuronal increases in [Ca2+]i caused by Tat ± morphine. proBDNF/BDNF ratios were increased in conditioned media from Tat ± morphine-treated wild-type glia compared to CCR5-deficient glia. Exogenous BDNF treatments mimicked the pro-survival effect of glial CCR5 deficiency against Tat ± morphine. CONCLUSIONS: Our results suggest a critical role for glial CCR5 in mediating neurotoxic effects of HIV-1 Tat and morphine interactions on neurons. A shift in the proBDNF/BDNF ratio that favors neurotrophic support may occur when glial CCR5 signaling is blocked. Some neuroprotection occurred only in the presence of morphine, suggesting that loss of CCR5 may fundamentally change signaling through the MOR in glia.

Oligodendrocytes Are Targets of HIV-1 Tat: NMDA and AMPA Receptor-Mediated Effects on Survival and Development.

Myelin pallor in HIV(+) individuals can occur very early during the disease process. While myelin damage might partly originate from HIV-induced vascular changes, the timing suggests that myelin and/or oligodendrocytes (OLs) may be directly affected. Histological (Golgi-Kopsch, electron microscopy) and biochemical studies have revealed an increased occurrence of abnormal OL/myelin morphology and dysregulated myelin protein expression in transgenic mice expressing the HIV-1 transactivator of transcription (Tat) protein. This suggests that viral proteins by themselves might cause OL injury. Since Tat interacts with NMDARs, we hypothesized that activation of NMDARs and subsequent disruption of cytoplasmic Ca(2+) ([Ca(2+)]i) homeostasis might be one cause of white matter injury after HIV infection. In culture, HIV-1 Tat caused concentration-dependent death of immature OLs, while more mature OLs remained alive but had reduced myelin-like membranes. Tat also induced [Ca(2+)]i increases and Thr-287 autophosphorylation of Ca(2+)/calmodulin-dependent protein kinase II ß (CaMKIIß) in OLs. Tat-induced [Ca(2+)]i was attenuated by the NMDAR antagonist MK801, and also by the AMPA/kainate receptor antagonist CNQX. Importantly, both MK801 and CNQX blocked Tat-induced death of immature OLs, but only MK801 reversed Tat effects on myelin-like membranes. These results suggest that OLs can be direct targets of HIV proteins released from infected cells. Although viability and membrane production are both affected by glutamatergic receptor-mediated Ca(2+) influx, and possibly the ensuing CaMKIIß activation, the roles of AMPARs and NMDARs appear to be different and dependent on the stage of OL differentiation. SIGNIFICANCE STATEMENT: Over 33 million individuals are currently infected by HIV. Among these individuals, ∼60% develop HIV-associated neurocognitive disorders. Myelin damage and white matter injury have been frequently reported in HIV patients but not extensively studied. Clinical studies using combined antiretroviral therapy (cART) together with adjunctive "anti-inflammatory" drugs show no improvement over cART alone, suggesting existence of injury mechanisms in addition to inflammation. In our studies, oligodendrocytes exhibited rapid increases in intracellular Ca(2+) level upon HIV-1 transactivator of transcription (Tat) exposure. Thus, immature and mature oligodendrocytes can be direct targets of Tat. Since ionotropic glutamate receptor antagonists can partially or fully reverse the detrimental effects of Tat, glutamate receptors could be a potential therapeutic target for white matter damage in HIV patients.

Further observations on the behavioral and neural effects of bone marrow stromal cells in rodent pain models.

BACKGROUND: Bone marrow stromal cells (BMSCs) have shown potential to treat chronic pain, although much still needs to be learned about their efficacy and mechanisms of action under different pain conditions. Here, we provide further convergent evidence on the effects of BMSCs in rodent pain models. RESULTS: In an orofacial pain model involving injury of a tendon of the masseter muscle, BMSCs attenuated behavioral pain conditions assessed by von Frey filaments and a conditioned place avoidance test in female Sprague-Dawley rats. The antihyperalgesia of BMSCs in females lasted for <8 weeks, which is shorter than that seen in males. To relate preclinical findings to human clinical conditions, we used human BMSCs. Human BMSCs (1.5 M cells, i.v.) attenuated mechanical and thermal hyperalgesia induced by spinal nerve ligation and suppressed spinal nerve ligation-induced aversive behavior, and the effect persisted through the 8-week observation period. In a trigeminal slice preparation, BMSC-treated and nerve-injured C57B/L mice showed reduced amplitude and frequency of spontaneous excitatory postsynaptic currents, as well as excitatory synaptic currents evoked by electrical stimulation of the trigeminal nerve root, suggesting inhibition of trigeminal neuronal hyperexcitability and primary afferent input by BMSCs. Finally, we observed that GluN2A (N-methyl-D-aspartate receptor subunit 2A) tyrosine phosphorylation and protein kinase Cgamma (PKCg) immunoreactivity in rostral ventromedial medulla was suppressed at 8 weeks after BMSC in tendon-injured rats. CONCLUSIONS: Collectively, the present work adds convergent evidence supporting the use of BMSCs in pain control. As PKCg activity related to N-methyl-D-aspartate receptor activation is critical in opioid tolerance, these results help to understand the mechanisms of BMSC-produced long-term antihyperalgesia, which requires opioid receptors in rostral ventromedial medulla and apparently lacks the development of tolerance.

5α-reduced progestogens ameliorate mood-related behavioral pathology, neurotoxicity, and microgliosis associated with exposure to HIV-1 Tat.

Human immunodeficiency virus (HIV) is associated with motor and mood disorders, likely influenced by reactive microgliosis and subsequent neural damage. We have recapitulated aspects of this pathology in mice that conditionally express the neurotoxic HIV-1 regulatory protein, trans-activator of transcription (Tat). Progestogens may attenuate Tat-related behavioral impairments and reduce neurotoxicity in vitro, perhaps via progesterone's 5α-reductase-dependent metabolism to the neuroprotective steroid, allopregnanolone. To test this, ovariectomized female mice that conditionally expressed (or did not express) central HIV-1 Tat were administered vehicle or progesterone (4mg/kg), with or without pretreatment of a 5α-reductase inhibitor (finasteride, 50mg/kg). Tat induction significantly increased anxiety-like behavior in an open field, elevated plus maze and a marble burying task concomitant with elevated protein oxidation in striatum. Progesterone administration attenuated anxiety-like effects in the open field and elevated plus maze, but not in conjunction with finasteride pretreatment. Progesterone also attenuated Tat-promoted protein oxidation in striatum, independent of finasteride pretreatment. Concurrent experiments in vitro revealed Tat (50nM)-mediated reductions in neuronal cell survival over 60h, as well as increased neuronal and microglial intracellular calcium, as assessed via fura-2 AM fluorescence. Co-treatment with allopregnanolone (100nM) attenuated neuronal death in time-lapse imaging and blocked the Tat-induced exacerbation of intracellular calcium in neurons and microglia. Lastly, neuronal-glial co-cultures were labeled for Iba-1 to reveal that Tat increased microglial numbers in vitro and co-treatment with allopregnanolone attenuated this effect. Together, these data support the notion that 5α-reduced pregnane steroids exert protection over the neurotoxic effects of HIV-1 Tat.

Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na⁺ influx, mitochondrial instability, and Ca²âº overload.

Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na(+)]i) and calcium ([Ca(2+)]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca(2+)]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the µ-opioid receptor antagonist CTAP and were not observed in neurons cultured from µ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca(2+)]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na(+)]i, mitochondrial instability, excessive Ca(2+) influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via µ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca(2+)]i and by further disrupting [Ca(2+)]i homeostasis. We hypothesize that the spatiotemporal relationship of µ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuroAIDS.

Transition to persistent orofacial pain after nerve injury involves supraspinal serotonin mechanisms.

The orofacial region is a major focus of chronic neuropathic pain conditions characterized by primary hyperalgesia at the site of injury and secondary hyperalgesia outside the injured zone. We have used a rat model of injury to the maxillary branch (V2) of the trigeminal nerve to produce constant and long-lasting primary hyperalgesia in the V2 territory and secondary hyperalgesia in territories innervated by the mandibular branch (V3). Our findings indicate that the induction of primary and secondary hyperalgesia depended on peripheral input from the injured nerve. In contrast, the maintenance of secondary hyperalgesia depended on central mechanisms. The centralization of the secondary hyperalgesia involved descending 5-HT drive from the rostral ventromedial medulla and the contribution of 5-HT3 receptors in the trigeminal nucleus caudalis (Vc), the homolog of the spinal dorsal horn. Electrophysiological studies further indicate that after nerve injury spontaneous responses and enhanced poststimulus discharges in Vc nociresponsive neurons were time-dependent on descending 5-HT drive and peripheral input. The induction phase of secondary hyperalgesia involved central sensitization mechanisms in Vc neurons that were dependent on peripheral input, whereas the maintenance phase of secondary hyperalgesia involved central sensitization in Vc neurons conducted by a delayed descending 5-HT drive and a persistence of peripheral inputs. Our results are the first to show that the maintenance of secondary hyperalgesia and underlying central sensitization associated with persistent pain depend on a transition to supraspinal mechanisms involving the serotonin system in rostral ventromedial medulla-dorsal horn circuits.

Activation of sodium-dependent glutamate transporters regulates the morphological aspects of oligodendrocyte maturation via signaling through calcium/calmodulin-dependent kinase IIß's actin-binding/-stabilizing domain.

Signaling via the major excitatory amino acid glutamate has been implicated in the regulation of various aspects of the biology of oligodendrocytes, the myelinating cells of the central nervous system (CNS). In this respect, cells of the oligodendrocyte lineage have been described to express a variety of glutamate-responsive transmembrane proteins including sodium-dependent glutamate transporters. The latter have been well characterized to mediate glutamate clearance from the extracellular space. However, there is increasing evidence that they also mediate glutamate-induced intracellular signaling events. Our data presented here show that the activation of oligodendrocyte expressed sodium-dependent glutamate transporters, in particular GLT-1 and GLAST, promotes the morphological aspects of oligodendrocyte maturation. This effect was found to be associated with a transient increase in intracellular calcium levels and a transient phosphorylation event at the serine (S)(371) site of the calcium sensor calcium/calmodulin-dependent kinase type IIß (CaMKIIß). The potential regulatory S(371) site is located within CaMKIIß's previously defined actin-binding/-stabilizing domain, and phosphorylation events within this domain were identified in our studies as a requirement for sodium-dependent glutamate transporter-mediated promotion of oligodendrocyte maturation. Furthermore, our data provide good evidence for a role of these phosphorylation events in mediating detachment of CaMKIIß from filamentous (F)-actin, and hence allowing a remodeling of the oligodendrocyte's actin cytoskeleton. Taken together with our recent findings, which demonstrated a crucial role of CaMKIIß in regulating CNS myelination in vivo, our data strongly suggest that a sodium-dependent glutamate transporter-CaMKIIß-actin cytoskeleton axis plays an important role in the regulation of oligodendrocyte maturation and CNS myelination.

Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade.

BACKGROUND: It has been recently recognized that the descending serotonin (5-HT) system from the rostral ventromedial medulla (RVM) in the brainstem and the 5-HT3 receptor subtype in the spinal dorsal horn are involved in enhanced descending pain facilitation after tissue and nerve injury. However, the mechanisms underlying the activation of the 5-HT3 receptor and its contribution to facilitation of pain remain unclear. RESULTS: In the present study, activation of spinal 5-HT3 receptors by intrathecal injection of a selective 5-HT3 receptor agonist SR 57227 induced spinal glial hyperactivity, neuronal hyperexcitability and pain hypersensitivity in rats. We found that there was neuron-to-microglia signaling via the chemokine fractalkine, microglia to astrocyte signaling via cytokine IL-18, astrocyte to neuronal signaling by IL-1ß, and enhanced activation of NMDA receptors in the spinal dorsal horn. Glial hyperactivation in spinal dorsal horn after hindpaw inflammation was also attenuated by molecular depletion of the descending 5-HT system by intra-RVM Tph-2 shRNA interference. CONCLUSIONS: These findings offer new insights into the cellular and molecular mechanisms at the spinal level responsible for descending 5-HT-mediated pain facilitation during the development of persistent pain after tissue and nerve injury. New pain therapies should focus on prime targets of descending facilitation-induced glial involvement, and in particular the blocking of intercellular signaling transduction between neurons and glia.

[Regional CT value in prediction of proximal femoral fracture].

OBJECTIVE: To investigate CT values of cancellous bone in femoral neck in adults over 60 years with proximal femoral fractures. METHODS: From January 2020 to December 2020, a retrospective analysis was performed on 280 subjects aged 60 years or older who underwent bilateral hip CT examination, including 85 males and 195 females, 120 on the left side and 160 on the right side, aged 75 (66, 82) years old. One hundred thirty-six patients with proximal femoral fractures were included in study group and 144 patients without fractures were included in control group. GEOptima CT was used to scan and reconstruct horizontal, coronal and sagittal layers of proximal femur. CT values of cancellous bone in femoral neck were measured and compared between two groups. The relationship between CT values of cancellous bone of femoral neck and proximal femoral fracture was analyzed statistically. RESULTS: In terms of age, fracture group aged 79(73.3, 85.0) years old, non-fracture group aged 69.5 (64.0, 78.8) years old, and had significant difference in age between two groups (P<0.05). In terms of CT value, regional CT value in fracture group was 8.62(-3.62, 27.15) HU, which was lower than that in non-fracture group 34.31(-5.93, 71.74) HU(P<0.05). CT value on coronal view in fracture group was -8.48(-30.96, 17.46) HU, which was lower than that in non-fracture group 40.49(5.55, 80.71) HU (P<0.05). CT value on sagittal view in fracture group was -31.28(-54.91, -5.11) HU, which was lower than that in non-fracture group 7.74(-20.12, 44.54) HU (P<0.05). CT values on horizontal view in fracture group was 0.17(-23.13, 24.60) HU, which was lower than that in non-fracture group 46.40(10.42, 85.18) HU(P<0.05). The mean regional CT values among three planes in the fracture group were lower than those in the non-fracture group. Logistic regression analysis showed coronal CT value was influencing factors of proximal femoral fracture, and it could be written into regression equations that predict probability of fracture. CONCLUSION: In adults aged over 60 years old, CT values of cancellous bone of femoral neck decreased with increasing age. The smaller CT value of cancellous bone of femoral neck, the greater risk of proximal femoral fracture.

Spinal 5-HT(3) receptor activation induces behavioral hypersensitivity via a neuronal-glial-neuronal signaling cascade.

Recent studies indicate that the descending serotonin (5-HT) system from the rostral ventromedial medulla (RVM) in the brainstem and the 5-HT(3) receptor subtype in the spinal dorsal horn are involved in enhanced descending pain facilitation after tissue and nerve injury. However, the mechanisms underlying the activation of the 5-HT(3) receptor and its contribution to facilitation of pain remain unclear. In the present study, activation of spinal 5-HT(3) receptor by intrathecal injection of a selective 5-HT(3) receptor agonist, SR57227, induced spinal glial hyperactivity, neuronal hyperexcitability, and pain hypersensitivity in rats. We found that there was neuron-to-microglia signaling via chemokine fractalkine, microglia to astrocyte signaling via the cytokine IL-18, astrocyte to neuronal signaling by IL-1ß, and enhanced activation of GluN (NMDA) receptors in the spinal dorsal horn. In addition, exogenous brain-derived neurotrophic factor-induced descending pain facilitation was accompanied by upregulation of CD11b and GFAP expression in the spinal dorsal horn after microinjection in the RVM, and these events were significantly prevented by functional blockade of spinal 5-HT(3) receptors. Enhanced expression of spinal CD11b and GFAP after hindpaw inflammation was also attenuated by molecular depletion of the descending 5-HT system by intra-RVM Tph-2 shRNA interference. Thus, these findings offer new insights into the cellular and molecular mechanisms at the spinal level responsible for descending 5-HT-mediated pain facilitation during the development of persistent pain after tissue and nerve injury. New pain therapies should focus on prime targets of descending facilitation-induced glial involvement, and in particular the blocking of intercellular signaling transduction between neuron and glia.

Bone marrow stromal cells produce long-term pain relief in rat models of persistent pain.

Chronic pain conditions are difficult to treat and are major health problems. Bone marrow stromal cells (BMSCs) have generated considerable interest as a candidate for cell-based therapy. BMSCs are readily accessible and are easy to isolate and expand ex vivo. Clinical studies show that direct injection of BMSCs does not produce unwanted side effects and is well tolerated and safe. Here, we show that a single systemic (intravenous) or local injection (into the lesion site) of rat primary BMSCs reversed pain hypersensitivity in rats after injury and that the effect lasted until the conclusion of the study at 22 weeks. The pain hypersensitivity was rekindled by naloxone hydrochloride, an opioid receptor antagonist that acts peripherally and centrally, when tested at 1-5 weeks after BMSC infusion. In contrast, naloxone methiodide, a peripherally acting opioid receptor antagonist, only rekindled hyperalgesia in the first 3 weeks of BMSC treatment. Focal downregulation of brainstem mu opioid receptors by RNA interference (RNAi) reversed the effect of BMSCs, when RNAi was introduced at 5- but not 1-week after BMSC transplantation. Thus, BMSCs produced long-term relief of pain and this effect involved activation of peripheral and central opioid receptors in distinct time domains. The findings prompt studies to elucidate the cellular mechanisms of the BMSC-induced pain relieving effect and translate these observations into clinical settings.

Morphine potentiates neurodegenerative effects of HIV-1 Tat through actions at µ-opioid receptor-expressing glia.

Individuals infected with human immunodeficiency virus-1 who abuse opiates can have a higher incidence of virus-associated neuropathology. Human immunodeficiency virus does not infect neurons, but viral proteins such as transactivator of transcription and glycoprotein 120, originating from infected glia, are neurotoxic. Moreover, functional changes in glial cells that enhance inflammation and reduce trophic support are increasingly implicated in human immunodeficiency virus neuropathology. In previous studies, co-exposure with morphine enhanced transactivator of transcription neurotoxicity towards cultured striatal neurons. Since those cultures contained µ-opioid receptor-expressing astroglia and microglia, and since glia are the principal site of infection in the central nervous system, we hypothesized that morphine synergy might be glially mediated. A 60 hour, repeated measures paradigm and multiple co-culture models were used to investigate the cellular basis for opiate-enhanced human immunodeficiency virus neurotoxicity. Morphine co-exposure significantly enhanced transactivator of transcription-induced neuron death when glia were present. Synergistic effects of morphine on transactivator of transcription neurotoxicity were greatest with neuron-glia contact, but also occurred to a lesser extent with glial conditioned medium. Importantly, synergy was lost if glia, but not neurons, lacked µ-opioid receptors, indicating that opiate interactions with human immunodeficiency virus converge at the level of µ-opioid receptor-expressing glia. Morphine enhanced transactivator of transcription-induced inflammatory effectors released by glia, elevating reactive oxygen species, increasing 3-nitrotyrosine production by microglia, and reducing the ability of glia to buffer glutamate. But neuron survival was reduced even more with glial contact than with exposure to conditioned medium, suggesting that noxious elements associated with cell contact augment the toxicity due to soluble factors. Similar morphine-transactivator of transcription synergy was also observed in studies with the clade C sequence of HIV-1 transactivator of transcription, which did not cause neuron death unless morphine was present. Several paradoxical observations related to opiate effects were noted when µ-opioid receptors were specifically ablated from either glia or neurons. This suggests that µ-opioid receptor loss in isolated cell types can fundamentally distort cell-to-cell signalling, revealing opponent processes that may exist in individual cell types. Our findings show the critical role of glia in orchestrating neurotoxic interactions of morphine and transactivator of transcription, and support the emerging concept that combined exposure to opiates and human immunodeficiency virus drives enhanced pathology within the central nervous system.

[Application of porous tantalum Jumbo cup in revision of hip arthroplasty].

OBJECTIVE: To investigate the clinical effect of porous tantalum Jumbo cup on acetabular reconstruction in revision of total hip arthroplasty. METHODS: From September 2014 to December 2017, 18 patients(18 hips) with acetabular defect were reconstructed by porous tantalum Jumbo cup technology, including 6 males and 12 females;the age ranged from 54 to 76 years old with an average of(63.8±15.3) years. There were 6 cases of paprosky typeⅡA, 8 cases of typeⅡB, 2 cases of typeⅡC and 2 cases of type Ⅲ a. Harris score and visual analogue scale (VAS) were performed before and after operation. Imaging examination was performed to evaluate the position of hip rotation center and prosthesis, and to judge whether acetabular loosening, displacement and complications existed. RESULTS: All cases were followed up for 13 to 49 months, with an average of 20.6 months. Harris score increased from 54.6±4.7 to 86.5±3.2 one year after operation(P<0.01), and VAS score decreased from 6.8±0.7 to 0.8±0.6 one year after operation (P<0.01). The transverse coordinate of hip rotation center was (3.52±0.72) cm before operation and (3.47±0.54) cm after operation (P>0.05). The longitudinal coordinate of hip rotation center was improved from (3.02±0.84) cm before operation to (2.35±0.53) cm after operation (P<0.01). During the follow-up period, the Jumbo cup was well fixed without loosening and displacement, the acetabular cup had bone ingrowth in varying degrees, and no light transmission line and osteolysis around the acetabular cup were found. No complications such as infection and nerve injury occurred. CONCLUSION: The method of reconstructing acetabular bone defect with porous tantalum Jumbo cup is simple and easy, the early stability of acetabulum is good, and the short-term follow-up effect is good.

Protective effects of thiamine on Wickerhamomyces anomalus against ethanol stress.

Wickerhamomyces anomalus (W. anomalus) is widely reported in the brewing industry and has positive effects on the aromatic profiles of wines because of its unique physiological characteristics and metabolic features. However, the accumulation of ethanol during fermentation inhibits the growth of W. anomalus. Thiamine is involved in the response against various abiotic stresses in microorganisms. Therefore, we used transcriptomic and metabolomic analyses to study the effect of thiamine on ethanol-stressed W. anomalus. The results indicate that thiamine could alleviate the inhibitory effect of ethanol stress on the survival of W. anomalus. Differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) caused by the thiamine intervention were identified as oxidative phosphorylation through integrated transcriptomic and metabolomic analyses. In addition, ethanol treatment decreased the content of intracellular adenosine triphosphate (ATP), while thiamine partially alleviated this phenomenon. The present comprehensive transcriptional overview and metabolomic analysis provide insights about the mechanisms of thiamine protection on W. anomalus under ethanol stress and promote the potential applications of W. anomalus in the fermentation industry.

Molecular depletion of descending serotonin unmasks its novel facilitatory role in the development of persistent pain.

Recent studies indicate that persistent pain after tissue or nerve injury is accompanied by an enhanced net descending facilitatory drive that contributes to an amplification and spread of pain. Although 5-HT-containing neurons in the rostral ventromedial medulla (RVM) provide the major descending serotonergic projection to the spinal cord, it is not clear whether the neurotransmitter 5-HT itself released from RVM-spinal neurons contributes to descending pain modulation. In the present study, we determined the role of the descending 5-HT in rat nocifensive behaviors after persistent pain by selectively depleting functional phenotypes of 5-HT in RVM neurons with regional shRNA interference (RNAi) of tryptophan hydroxylase-2 (Tph-2), the rate-limiting enzyme in the synthesis of neuronal 5-HT. Compared to negative control shRNA, Tph-2 shRNA induced significantly prolonged downregulation of Tph-2 in the RVM and 5-HT in spinal dorsal horn. The 5-HT-depleted rats showed normal pain sensitivity in responses to acute noxious stimulation. However, the same RNAi treatment attenuated formalin-induced spontaneous nocifensive responses and tissue or nerve injury-induced allodynia and hyperalgesia. Furthermore, in control shRNA-treated animals, intra-RVM microinjection of brain-derived neurotrophic factor produced a reversible hyperalgesia, which was completely prevented by Tph-2 RNAi pretreatment. Descending inhibition induced by intra-RVM electrical stimulation, but not microinjection of the mu- or kappa-opioid receptor agonists in control shRNA-treated animals was eliminated in 5-HT-depleted rats. These results indicate that the descending 5-HT from the RVM is an important contributor to pain facilitation during the development of persistent pain, and may not mediate opioid-induced descending inhibition in acute pain.

PTEN gene silencing prevents HIV-1 gp120(IIIB)-induced degeneration of striatal neurons.

To assess the role of the phosphatase and tensin homologue on chromosome 10 (PTEN) in mediating envelope glycoprotein 120 (gp120)-induced neurotoxicity in the striatum, PTEN was silenced using short interfering RNA (siRNA) vectors. PTEN activity directs multiple downstream pathways implicated in gp120-induced neuronal injury and death. PTEN is a negative regulator of Akt (protein kinase B) phosphorylation, but has also been shown to directly activate extrasynaptic NMDA receptors and dephosphorylate focal adhesion kinase. Rodent striatal neurons were nucleofected with green fluorescent protein (GFP)-expressing siRNA constructs to silence PTEN (PTENsi-GFP) or with negative-control (NCsi-GFP) vectors, and exposed to HIV-1 gp120(IIIB) using rigorously controlled, cell culture conditions including computerized time-lapse microscopy to track the fate of individual neurons following gp120 exposure. Immunofluorescence labeling showed that subpopulations of striatal neurons possess CXCR4 and CCR5 co-receptor immunoreactivity and that gp120(IIIB) was intrinsically neurotoxic to isolated striatal neurons. Importantly, PTENsi-GFP, but not control NCsi-GFP, constructs markedly decreased PTEN mRNA and protein levels and significantly attenuated gp120-induced death. These findings implicate PTEN as a critical factor in mediating the direct neurotoxic effects of HIV-1 gp120, and suggest that effectors downstream of PTEN such as Akt or other targets are potentially affected. The selective abatement of PTEN activity in neurons may represent a potential therapeutic strategy for the CNS complications of HIV-1.

Regional heterogeneity and diversity in cytokine and chemokine production by astroglia: differential responses to HIV-1 Tat, gp120, and morphine revealed by multiplex analysis.

HIV-infected individuals who abuse opiates show a faster progression to AIDS and higher incidence of encephalitis. The HIV-1 proteins Tat and gp120 have been shown to cause neurodegenerative changes either in vitro or when injected or expressed in the CNS, and we have shown that opiate drugs can exacerbate neurotoxic effects in the striatum through direct actions on pharmacologically discrete subpopulations of mu-opioid receptor-expressing astroglia. Opiate coexposure also significantly enhances release of specific inflammatory mediators by astroglia from the striatum, and we theorize that astroglial reactivity may underlie aspects of HIV neuropathology. To determine whether astroglia from different regions of the central nervous system have distinct, intrinsic responses to HIV-1 proteins and opiates, we used multiplex suspension array analyses to define and compare the inflammatory signature of cytokines released by murine astrocytes grown from cerebral cortex, cerebellum, and spinal cord. Results demonstrate significant regional differences in baseline secretion patterns, and in responses to viral proteins. Of importance for the disease process, astrocytes from all regions have very limited inflammatory response to gp120 protein, as compared to Tat protein, either in the presence or absence of morphine. Overall, the chemokine/cytokine release is higher from spinal cord and cortical astroglia than from cerebellar astroglia, paralleling the relatively low incidence of HIV-related neuropathology in the cerebellum.

Inhibition of class II histone deacetylases in the spinal cord attenuates inflammatory hyperalgesia.

BACKGROUND: Several classes of histone deacetylases (HDACs) are expressed in the spinal cord that is a critical structure of the nociceptive pathway. HDAC-regulated histone acetylation is an important component of chromatin remodeling leading to epigenetic regulation of gene transcription. To understand the role of histone acetylation in epigenetic regulation of pathological pain, we have studied the impact of different classes of HDACs in the spinal cord on inflammatory hyperalgesia induced by complete Freund's adjuvant (CFA). RESULTS: We intrathecally applied inhibitors specific to different classes of HDACs and evaluated their impact on inflammatory hyperalgesia. Pre-injected inhibitors targeting class I as well as II (SAHA, TSA, LAQ824) or IIa (VPA, 4-PB) HDACs significantly delayed the thermal hyperalgesia induced by unilateral CFA injection in the hindpaw. Existing hyperalgesia induced by CFA was also attenuated by the HDAC inhibitors (HDACIs). In contrast, these inhibitors did not interfere with the thermal response either in naïve animals, or on the contralateral side of inflamed animals. Interestingly, MS-275 that specifically inhibits class I HDACs failed to alter the hyperalgesia although it increased histone 3 acetylation in the spinal cord as SAHA did. Using immunoblot analysis, we further found that the levels of class IIa HDAC members (HDAC4, 5, 7, 9) in the spinal dorsal horn were upregulated following CFA injection while those of class I HDAC members (HDAC1, 2, 3) remained stable or were slightly reduced. CONCLUSIONS: Our data suggest that activity of class II HDACs in the spinal cord is critical to the induction and maintenance of inflammatory hyperalgesia induced by CFA, while activity of class I HDACs may be unnecessary. Comparison of the effects of HDACIs specific to class II and IIa as well as the expression pattern of different HDACs in the spinal cord in response to CFA suggests that the members of class IIa HDACs may be potential targets for attenuating persistent inflammatory pain.