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1.  Monoarticular antigen-induced arthritis leads to pronounced bilateral upregulation of the expression of neurokinin 1 and bradykinin 2 receptors in dorsal root ganglion neurons of rats 
Arthritis Research  2000;2(5):424-427.
This study describes the upregulation of neurokinin 1 and bradykinin 2 receptors in dorsal root ganglion (DRG) neurons in the course of antigen-induced arthritis (AIA) in the rat knee. In the acute phase of AIA, which was characterized by pronounced hyperalgesia, there was a substantial bilateral increase in the proportion of lumbar DRG neurons that express neurokinin 1 receptors (activated by substance P) and bradykinin 2 receptors. In the chronic phase the upregulation of bradykinin 2 receptors persisted on the side of inflammation. The increase in the receptor expression is relevant for the generation of acute and chronic inflammatory pain.
Ongoing pain and hyperalgesia (enhanced pain response to stimulation of the tissue) are major symptoms of arthritis. Arthritic pain results from the activation and sensitization of primary afferent nociceptive nerve fibres ('pain fibres') supplying the tissue (peripheral sensitization) and from the activation and sensitization of nociceptive neurons in the central nervous system (central sensitization). After sensitization, nociceptive neurons respond more strongly to mechanical and thermal stimulation of the tissue, and their activation threshold is lowered. The activation and sensitization of primary afferent fibres results from the action of inflammatory mediators such as bradykinin (BK), prostaglandins and others on membrane receptors located on these neurons. BK is a potent pain-producing substance that is contained in inflammatory exudates. Up to 50% of the primary afferent nerve fibres have receptors for BK. When primary afferent nerve fibres are activated they can release neuropeptides such as substance P (SP) and calcitonin gene-related peptide from their sensory endings in the tissue. SP contributes to the inflammatory changes in the innervated tissue (neurogenic inflammation), and it might also support the sensitization of nociceptive nerve fibres by binding to neurokinin 1 (NK1) receptors. NK1 receptors are normally expressed on a small proportion of the primary afferent nerve fibres.
Because the expression of receptors on the primary afferent neurons is essential for the pain-producing action of inflammatory mediators and neuropeptides, we investigated in the present study whether the expression of BK and NK1 receptors on primary afferent neurons is altered during the acute and chronic phases of an antigen-induced arthritis (AIA). AIA resembles in many aspects the inflammatory process of human rheumatoid arthritis. Because peptide receptors are expressed not only in the terminals of the primary afferent units but also in the cell bodies, we removed dorsal root ganglia (DRGs) of both sides from control rats and from rats with the acute or chronic phase of AIA and determined, after short-term culture of the neurons, the proportion of DRG neurons that expressed the receptors in the different phases of AIA. We also characterized the inflammatory process and the nociceptive behaviour of the rats in the course of AIA.
Materials and methods:
In 33 female Lewis rats 10 weeks old, AIA was induced in the right knee joint. First the rats were immunized in two steps with methylated bovine serum albumin (m-BSA) emulsified with Freund's complete adjuvant, and heat-inactivated Bordetella pertussis. After immunization, m-BSA was injected into the right knee joint cavity to induce arthritis. The joint swelling was measured at regular intervals. Nociceptive (pain) responses to mechanical stimulation of the injected and the contralateral knee were monitored in the course of AIA. Groups of rats were killed at different time points after the induction of AIA, and inflammation and destruction in the knee joint were graded by histological examination. The DRGs of both sides were dissected from segments L1–L5 and C1–C7 from arthritic rats, from eight immunized rats without arthritis and from ten normal control rats. Excised DRGs were dissociated into single cells which were cultured for 18 h.
The expression of the receptors was determined by assessment of the binding of SP-gold or BK-gold to the cultured neurons. For this purpose the cells were slightly fixed. Binding of SP-gold or BK-gold was detected by using enhancement with silver and subsequent densitometric analysis of the relative grey values of the neurons. Displacement controls were performed with SP, the specific NK1 receptor agonist [Sar9, Met(O2)11]-SP, BK, the specific BK 1 (B1) receptor agonist D-Arg (Hyp3-Thi5,8-D-Phe7)-BK and the specific BK 2 (B2) receptor agonist (Des-Arg10)-Lys-BK.
The inflammatory process in the injected right knee joint started on the first day after induction of AIA and persisted throughout the observation period of 84 days (Fig. 1). The initial phase of AIA was characterized by strong joint swelling and a predominantly granulocytic infiltration of the synovial membrane and the joint cavity (acute inflammatory changes). In the later phases of AIA (10–84 days after induction of AIA) the joint showed persistent swelling, and signs of chronic arthritic alterations such as infiltration of mononuclear leucocytes, hyperplasia of synovial lining layer (pannus formation) and erosions of cartilage and bone were predominant. The contralateral knee joints appeared normal at all time points. Destruction was observed only in the injected knee but some proteoglycan loss was also noted in the non-injected, contralateral knee. In the acute and initial chronic phases of AIA (1–29 days) the rats showed mechanical hyperalgesia in the inflamed knee (limping, withdrawal response to gentle pressure onto the knee). In the acute phase (up to 9 days) a pain response was also seen when gentle pressure was applied to the contralateral knee.
Figure 2 displays the changes in the receptor expression in the DRG neurons during AIA. The expression of SP–gold-binding sites in lumbar DRG neurons (Fig. 2a) was substantially increased in the acute phase of arthritis. In untreated control rats (n = 5), 7.7 ± 3.8% of the DRG neurons from the right side and 10.0 ± 1.7% of the DRG neurons from the left side showed labelling with SP–gold. The proportion of SP–gold-labelled neurons in immunized animals without knee injection (n = 3) was similar. By contrast, at days 1 (n = 2 rats) and 3 (n = 5 rats) of AIA in the right knee, approximately 50% of the DRG neurons exhibited labelling with SP–gold, and this was seen both on the side of the injected knee and on the opposite side. At day 10 of AIA (n = 3 rats), 26.3 ± 6.1% of the ipsilateral DRG neurons but only 15.7 ± 0.6% of the contralateral neurons exhibited binding of SP–gold. At days 21 (n = 5 rats), 42 (n = 3 rats) and 84 (n = 5 rats) of AIA, the proportion of SP–gold-positive neurons had returned to the control values, although the arthritis, now with signs of chronic inflammation, was still present. Compared with the DRG neurons of the untreated control rats, the increase in the proportion of labelled neurons was significant on both sides in the acute phase (days 1 and 3) and the intermediate phase (day 10) of AIA (Mann–Whitney U-test). The size distribution of the neurons was similar in the DRG neurons of all experimental groups. Under all conditions and at all time points, SP–gold binding was found mainly in small and medium-sized (less than 700 μm2) neurons. In the cervical DRGs the expression of NK1 receptors did not change in the course of AIA. The binding of SP–gold to the neurons was suppressed by the coadministration of the specific NK1 receptor agonist [Sar9, Met(O2)11]–SP in three experiments, showing that SP–gold was bound to NK1 receptors.
The expression of BK–gold-binding sites in the lumbar DRG neurons showed also changes in the course of AIA, but the pattern was different (Fig. 2b). In untreated control rats (n = 5), 42.3 ± 3.1% of the DRG neurons of the right side and 39.6 ± 2.6% of the DRG neurons of the left side showed binding of BK–gold. At days 1 (n = 2 rats) and 3 (n = 5 rats) of AIA, approximately 80% of the DRG neurons on the side of the knee injection (ipsilateral) and approximately 70% on the opposite side were labelled. In comparison with the untreated control group, the increase in the proportion of labelled neurons was significant on both sides. The proportion of labelled neurons in the ipsilateral DRGs remained significantly increased in both the intermediate phase (day 10, n = 3 rats) and chronic phase (days 21, n = 5 rats, and 42, n = 3 rats) of inflammation. At 84 days after the induction of AIA (n = 5 rats), 51.0 ± 12.7% of the neurons showed an expression of BK–gold-binding sites and this was close to the prearthritic values. However, in the contralateral DRG of the same animals the proportion of BK–gold-labelled neurons declined in the intermediate phase (day 10) and chronic phase (days 21–84) of AIA and was not significantly different from the control value. Thus the increase in BK–gold-labelled neurons was persistent on the side where the inflammation had been induced, and transient on the opposite side. The size distribution of the DRG neurons of the different experimental groups was similar. In the cervical DRGs the expression of BK receptors did not change in the course of AIA. In another series of experiments, we determined the subtype(s) of BK receptor(s) that were expressed in DRGs L1–L5 in different experimental groups. In neither untreated control animals (n = 5) nor immunized rats without knee injection (n = 5) nor in rats at 3 days (n = 5) and 42 days (n = 5) of AIA was the binding of BK–gold decreased by the coadministration of BK–gold and the B1 agonist. By contrast, in these experimental groups the binding of BK–gold was suppressed by the coadministration of the B2 agonist. These results show that B2 receptors, but not B1 receptors, were expressed in both normal animals and in animals with AIA.
These results show that in AIA in the rat the expression of SP-binding and BK-binding sites in the perikarya of DRGs L1–L5 is markedly upregulated in the course of knee inflammation. Although the inflammation was induced on one side only, the initial changes in the binding sites were found in the lumbar DRGs of both sides. No upregulation of SP-binding or BK-binding sites was observed in the cervical DRGs. The expression of SP-binding sites was upregulated only in the first days of AIA, that is, in the acute phase, in which the pain responses to mechanical stimulation were most pronounced. By contrast, the upregulation of BK-binding sites on the side of AIA persisted for up to 42 days, that is, in the acute and chronic phase of AIA. Only the B2 receptor, not the B1 receptor, was upregulated. The coincidence of the enhanced expression of NK1 and BK receptors on sensory neurons and the pain behaviour suggests that the upregulation of these receptors is relevant for the generation and maintenance of arthritic pain.
In the acute phase of AIA, approximately 50% of the lumbar DRG neurons showed an expression of SP-binding sites. Because peptide receptors are transported to the periphery, the marked upregulation of SP-binding receptors probably leads to an enhanced density of receptors in the sensory endings of the primary afferent units. This will permit SP to sensitize more neurons under inflammatory conditions than under normal conditions. However, the expression of NK1 receptors was upregulated only in the acute phase of inflammation, suggesting that SP and NK1 receptors are less important for the generation of hyperalgesia in the chronic phase of AIA.
Because BK is one of the most potent algesic compounds, the functional consequence of the upregulation of BK receptors is likely to be of immediate importance for the generation and maintenance of inflammatory pain. The persistence of the upregulation of BK receptors on the side of inflammation suggests that BK receptors should be an interesting target for pain treatment in the acute and chronic phases. Only B2 receptors were identified in normal animals and in rats with AIA. This is surprising because previous pharmacological studies have provided evidence that, during inflammation, B1 receptors can be newly expressed.
Receptor upregulation in the acute phase of AIA was bilateral and almost symmetrical. However, hyperalgesia was much more pronounced on the inflamed side. It is most likely that receptors on the contralateral side were not readily activated because in the absence of gross inflammation the local concentration of the ligands BK and SP was probably quite low. We hypothesize that the bilateral changes in receptor expression are generated at least in part by mechanisms involving the nervous system. Symmetrical segmental changes can be produced only by the symmetrical innervation, involving either the sympathetic nervous system or the primary afferent fibres. Under inflammatory conditions, primary afferent fibres can be antidromically activated bilaterally in the entry zone of afferent fibres in the spinal cord, and it was proposed that this antidromic activation might release neuropeptides and thus contribute to neurogenic inflammation. Because both sympathetic efferent fibres and primary afferent nerve fibres can aggravate inflammatory symptoms, it is also conceivable that they are involved in the regulation of receptor expression in primary afferent neurons. A neurogenic mechanism might also have been responsible for the bilateral degradation of articular cartilage in the present study.
PMCID: PMC17819  PMID: 11056677
antigen-induced arthritis; bradykinin receptor; dorsal root ganglion neurons; neurokinin 1 receptor; pain
2.  Nerve growth factor acts through the TrkA receptor to protect sensory neurons from the damaging effects of the HIV-1 viral protein, Vpr 
Neuroscience  2013;252:10.1016/j.neuroscience.2013.07.046.
Distal sensory polyneuropathy (DSP) with associated neuropathic pain is the most common neurological disorder affecting patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Viral protein R (Vpr) is a neurotoxic protein encoded by HIV-1 and secreted by infected macrophages. Vpr reduces neuronal viability, increases cytosolic calcium and membrane excitability of cultured dorsal root ganglion (DRG) sensory neurons, and is associated with mechanical allodynia in vivo. A clinical trial with HIV/AIDS patients demonstrated that nerve growth factor (NGF) reduced the severity of DSP-associated neuropathic pain, a problem linked to damage to small diameter, potentially NGF responsive fibers. Herein, the actions of NGF were investigated in our Vpr model of DSP and we demonstrated that NGF significantly protected sensory neurons from the effects of Vpr. Footpads of immunodeficient Vpr transgenic (vpr/RAG1−/−) mice displayed allodynia (p<0.05), diminished epidermal innervation (p<0.01) and reduced NGF mRNA expression (p<0.001) compared to immunodeficient (wildtype/RAG1−/−) littermate control mice. Compartmented cultures confirmed recombinant Vpr exposure to the DRG neuronal perikarya decreased distal neurite extension (p<0.01), whereas NGF exposure at these distal axons protected the DRG neurons from the Vpr-induced effect on their cell bodies. NGF prevented Vpr-induced attenuation of the phosphorylated glycogen synthase-3 axon extension pathway and tropomyosin related kinase A (TrkA) receptor expression in DRG neurons (p<0.05) and it directly counteracted the cytosolic calcium burst caused by Vpr exposure to DRG neurons (p<0.01). TrkA receptor antagonists indicated that NGF acted through the TrkA receptor to block the Vpr-mediated decrease in axon outgrowth in neonatal and adult rat and fetal human DRG neurons (p<0.05). Similarly, inhibiting the lower affinity NGF receptor, p75, blocked Vpr’s effect on DRG neurons. Overall, NGF/TrkA signalling or p75 receptor inhibition protects somatic sensory neurons exposed to Vpr, thus laying the groundwork for potential therapeutic options for HIV/AIDS patients suffering from DSP.
PMCID: PMC3829629  PMID: 23912036
3.  Peripheral expression and biological activities of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons 
The Journal of Cell Biology  1995;130(1):137-148.
Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic polypeptide, distantly related to transforming growth factor-beta (TGF- beta), originally isolated by virtue of its ability to induce dopamine uptake and cell survival in cultures of embryonic ventral midbrain dopaminergic neurons, and more recently shown to be a potent neurotrophic factor for motorneurons. The biological activities and distribution of this molecule outside the central nervous system are presently unknown. We report here on the mRNA expression, biological activities and initial receptor binding characterization of GDNF and a shorter spliced variant termed GDNF beta in different organs and peripheral neurons of the developing rat. Both GDNF mRNA forms were found to be most highly expressed in developing skin, whisker pad, kidney, stomach and testis. Lower expression was also detected in developing skeletal muscle, ovary, lung, and adrenal gland. Developing spinal cord, superior cervical ganglion (SCG) and dorsal root ganglion (DRG) also expressed low levels of GDNF mRNA. Two days after nerve transection, GDNF mRNA levels increased dramatically in the sciatic nerve. Overall, GDNF mRNA expression was significantly higher in peripheral organs than in neuronal tissues. Expression of either GDNF mRNA isoform in insect cells resulted in the production of indistinguishable mature GDNF polypeptides. Purified recombinant GDNF promoted neurite outgrowth and survival of embryonic chick sympathetic neurons. GDNF produced robust bundle-like, fasciculated outgrowth from chick sympathetic ganglion explants. Although GDNF displayed only low activity on survival of newborn rat SCG neurons, this protein was found to increase the expression of vasoactive intestinal peptide and preprotachykinin-A mRNAs in cultured SCG neurons. GDNF also promoted survival of about half of the neurons in embryonic chick nodose ganglion and a small subpopulation of embryonic sensory neurons in chick dorsal root and rat trigeminal ganglia. Embryonic chick sympathetic neurons expressed receptors for GDNF with Kd 1-5 x 10(-9) M, as measured by saturation and displacement binding assays. Our findings indicate GDNF is a new neurotrophic factor for developing peripheral neurons and suggest possible non-neuronal roles for GDNF in the developing reproductive system.
PMCID: PMC2120511  PMID: 7790368
4.  The Molecular and Pharmacological Mechanisms of HIV-Related Neuropathic Pain 
Current Neuropharmacology  2013;11(5):499-512.
Infection of the nervous system with the human immunodeficiency virus (HIV-1) can lead to cognitive, motor and sensory disorders. HIV-related sensory neuropathy (HIV-SN) mainly contains the HIV infection-related distal sensory polyneuropathy (DSP) and antiretroviral toxic neuropathies (ATN). The main pathological features that characterize DSP and ATN include retrograde (“dying back”) axonal degeneration of long axons in distal regions of legs or arms, loss of unmyelinated fibers, and variable degree of macrophage infiltration in peripheral nerves and dorsal root ganglia (DRG). One of the most common complaints of HIV-DSP is pain. Unfortunately, many conventional agents utilized as pharmacologic therapy for neuropathic pain are not effective for providing satisfactory analgesia in painful HIV-related distal sensory polyneuropathy, because the molecular mechanisms of the painful HIV-SDP are not clear in detail. The HIV envelope glycoprotein, gp120, appears to contribute to this painful neuropathy. Recently, preclinical studies have shown that glia activation in the spinal cord and DRG has become an attractive target for attenuating chronic pain. Cytokines/chemokines have been implicated in a variety of painful neurological diseases and in animal models of HIV-related neuropathic pain. Mitochondria injured by ATN and/or gp120 may be also involved in the development of HIV-neuropathic pain. This review discusses the neurochemical and pharmacological mechanisms of HIV-related neuropathic pain based on the recent advance in the preclinical studies, providing insights into novel pharmacological targets for future therapy.
PMCID: PMC3763758  PMID: 24403874
HIV; neuropathic pain; gp120; NRTI; spinal cord; DRG.
5.  Differential effects of HIV infected macrophages on dorsal root ganglia neurons and axons 
Experimental neurology  2007;210(1):30-40.
Human immunodeficiency virus-associated distal-symmetric neuropathy (HIV-DSP) is the most common neurological complication of HIV infection. The pathophysiology of HIV-DSP is poorly understood and no treatment is available for this entity. The dorsal root ganglia (DRG) are the principal sites of neuronal damage and are associated with reactive mononuclear phagocytes as well as HIV-infected macrophages. To determine the role of HIV-infected macrophages in the pathogenesis of HIV-DSP, we developed a technique for culturing human DRG’s. When the dissociated DRG neurons were exposed to supernatants from macrophages infected with CXCR4 or CCR5 tropic HIV-1 strains axonal retraction was observed without neuronal cell death but there was mitochondrial dysfunction in the neuronal cell body. Even though CXCR4 and CCR5 were expressed on the DRG neurons, the effects were independent of these receptors. Antioxidants rescued the neuronal cell body but not the axon from the toxic effects of the culture supernatants. Further, peripheral nerves of HIV-infected patients obtained at autopsy did not show evidence of increased oxidative stress. These observations suggest a differential effect on the axon and cell body. Different mechanisms of injury may be operative in these two structures.
PMCID: PMC2270478  PMID: 18177640
neuropathy; dorsal root ganglia; macrophages; neurotoxicity; axonal retraction; mitochondria; chemokine; AIDS
6.  Modulation of Feline Immunodeficiency Virus Infection by Stromal Cell-Derived Factor 
Journal of Virology  1998;72(3):2097-2104.
The α-chemokine receptor CXCR4 has recently been shown to support syncytium formation mediated by strains of feline immunodeficiency virus (FIV) that have been selected for growth in the Crandell feline kidney cell line (CrFK-tropic virus). Given that both human and feline CXCR4 support syncytium formation mediated by FIV, we investigated whether human stromal cell-derived factor (SDF-1) would inhibit infection with FIV. Human SDF-1α and SDF-1β bound with a high affinity (KDs of 12.0 and 10.4 nM, respectively) to human cells stably expressing feline CXCR4, and treatment of CrFK cells with human SDF-1α resulted in a dose-dependent inhibition of infection by FIVPET. No inhibitory activity was detected when the interleukin-2 (IL-2)-dependent feline T-cell line Mya-1 was used in place of CrFK cells, suggesting the existence of a CXCR4-independent mechanism of infection. Furthermore, neither the human β-chemokines RANTES, MIP-1α, MIP-1β, and MCP-1 nor the α-chemokine IL-8 had an effect on infection of either CrFK or Mya-1 cells with CrFK-tropic virus. Envelope glycoprotein purified from CrFK-tropic virus competed specifically for binding of SDF-1α to feline CXCR4 and CXCR4 expression was reduced in FIV-infected cells, suggesting that the inhibitory activity of SDF-1α in CrFK cells may be the result of steric hindrance of the virus-receptor interaction following the interaction between SDF and CXCR4. Prolonged incubation of CrFK cells with SDF-1α led to an enhancement rather than an inhibition of infection. Flow cytometric analysis revealed that this effect may be due largely to up-regulation of CXCR4 expression by SDF-1α on CrFK cells, an effect mimicked by treatment of the cells with phorbol myristate acetate. The data suggest that infection of feline cells with FIV can be mediated by CXCR4 and that, depending on the assay conditions, infection can be either inhibited or enhanced by SDF-1α. Infection with FIV may therefore prove a valuable model in which to study the development of novel therapeutic interventions for the treatment of AIDS.
PMCID: PMC109504  PMID: 9499065
7.  Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy 
BMC Neurology  2007;7:11.
Transient receptor potential (TRP) receptors expressed by primary sensory neurons mediate thermosensitivity, and may play a role in sensory pathophysiology. We previously reported that human dorsal root ganglion (DRG) sensory neurons co-expressed TRPV1 and TRPV3, and that these were increased in injured human DRG. Related receptors TRPV4, activated by warmth and eicosanoids, and TRPM8, activated by cool and menthol, have been characterised in pre-clinical models. However, the role of TRPs in common clinical sensory neuropathies needs to be established.
We have studied TRPV1, TRPV3, TRPV4, and TRPM8 in nerves (n = 14) and skin from patients with nerve injury, avulsed dorsal root ganglia (DRG) (n = 11), injured spinal nerve roots (n = 9), diabetic neuropathy skin (n = 8), non-diabetic neuropathic nerve biopsies (n = 6), their respective control tissues, and human post mortem spinal cord, using immunohistological methods.
TRPV1 and TRPV3 were significantly increased in injured brachial plexus nerves, and TRPV1 in hypersensitive skin after nerve repair, whilst TRPV4 was unchanged. TRPM8 was detected in a few medium diameter DRG neurons, and was unchanged in DRG after avulsion injury, but was reduced in axons and myelin in injured nerves. In diabetic neuropathy skin, TRPV1 expressing sub- and intra-epidermal fibres were decreased, as was expression in surviving fibres. TRPV1 was also decreased in non-diabetic neuropathic nerves. Immunoreactivity for TRPV3 was detected in basal keratinocytes, with a significant decrease of TRPV3 in diabetic skin. TRPV1-immunoreactive nerves were present in injured dorsal spinal roots and dorsal horn of control spinal cord, but not in ventral roots, while TRPV3 and TRPV4 were detected in spinal cord motor neurons.
The accumulation of TRPV1 and TRPV3 in peripheral nerves after injury, in spared axons, matches our previously reported changes in avulsed DRG. Reduction of TRPV1 levels in nerve fibres in diabetic neuropathy skin may result from the known decrease of nerve growth factor (NGF) levels. The role of TRPs in keratinocytes is unknown, but a relationship to changes in NGF levels, which is produced by keratinocytes, deserves investigation. TRPV1 represents a more selective therapeutic target than other TRPs for pain and hypersensitivity, particularly in post-traumatic neuropathy.
PMCID: PMC1892784  PMID: 17521436
8.  Increased chemokine signaling in a model of HIV1-associated peripheral neuropathy 
Molecular Pain  2009;5:48.
Painful distal sensory polyneuropathy (DSP) is the most common neurological complication of HIV1 infection. Although infection with the virus itself is associated with an incidence of DSP, patients are more likely to become symptomatic following initiation of nucleoside reverse transcriptase inhibitor (NRTI) treatment. The chemokines monocyte chemoattractant protein-1 (MCP1/CCL2) and stromal derived factor-1 (SDF1/CXCL12) and their respective receptors, CCR2 and CXCR4, have been implicated in HIV1 related neuropathic pain mechanisms including NRTI treatment in rodents. Utilizing a rodent model that incorporates the viral coat protein, gp120, and the NRTI, 2'3'-dideoxycytidine (ddC), we examined the degree to which chemokine receptor signaling via CCR2 and CXCR4 potentially influences the resultant chronic hypernociceptive behavior. We observed that following unilateral gp120 sciatic nerve administration, rats developed profound tactile hypernociception in the hindpaw ipsilateral to gp120 treatment. Behavioral changes were also present in the hindpaw contralateral to the injury, albeit delayed and less robust. Using immunohistochemical studies, we demonstrated that MCP1 and CCR2 were upregulated by primary sensory neurons in lumbar ganglia by post-operative day (POD) 14. The functional nature of these observations was confirmed using calcium imaging in acutely dissociated lumbar dorsal root ganglion (DRG) derived from gp120 injured rats at POD 14. Tactile hypernociception in gp120 treated animals was reversed following treatment with a CCR2 receptor antagonist at POD 14. Some groups of animals were subjected to gp120 sciatic nerve injury in combination with an injection of ddC at POD 14. This injury paradigm produced pronounced bilateral tactile hypernociception from POD 14–48. More importantly, functional MCP1/CCR2 and SDF1/CXCR4 signaling was present in sensory neurons. In contrast to gp120 treatment alone, the hypernociceptive behavior associated with the injury plus drug combination was only effectively reversed using the CXCR4 antagonist AMD3100. These studies indicate that the functional upregulation of CCR2 and CXCR4 signaling systems following a combination of gp120 and an NRTI are likely to be of central importance to associated DSP and may serve as potential therapeutic targets for treatment of this syndrome.
PMCID: PMC2734548  PMID: 19674450
9.  Rapid inflammasome activation in microglia contributes to brain disease in HIV/AIDS 
Retrovirology  2014;11:35.
Human immunodeficiency virus type 1(HIV-1) infects and activates innate immune cells in the brain resulting in inflammation and neuronal death with accompanying neurological deficits. Induction of inflammasomes causes cleavage and release of IL-1β and IL-18, representing pathogenic processes that underlie inflammatory diseases although their contribution HIV-associated brain disease is unknown.
Investigation of inflammasome-associated genes revealed that IL-1β, IL-18 and caspase-1 were induced in brains of HIV-infected persons and detected in brain microglial cells. HIV-1 infection induced pro-IL-1β in human microglia at 4 hr post-infection with peak IL-1β release at 24 hr, which was accompanied by intracellular ASC translocation and caspase-1 activation. HIV-dependent release of IL-1β from a human macrophage cell line, THP-1, was inhibited by NLRP3 deficiency and high extracellular [K+]. Exposure of microglia to HIV-1 gp120 caused IL-1β production and similarly, HIV-1 envelope pseudotyped viral particles induced IL-1β release, unlike VSV-G pseudotyped particles. Infection of cultured feline macrophages by the related lentivirus, feline immunodeficiency virus (FIV), also resulted in the prompt induction of IL-1β. In vivo FIV infection activated multiple inflammasome-associated genes in microglia, which was accompanied by neuronal loss in cerebral cortex and neurological deficits. Multivariate analyses of data from FIV-infected and uninfected animals disclosed that IL-1β, NLRP3 and caspase-1 expression in cerebral cortex represented key molecular determinants of neurological deficits.
NLRP3 inflammasome activation was an early and integral aspect of lentivirus infection of microglia, which was associated with lentivirus-induced brain disease. Inflammasome activation in the brain might represent a potential target for therapeutic interventions in HIV/AIDS.
PMCID: PMC4038111  PMID: 24886384
Inflammmasome; NLRP3; IL-1beta; HIV-1; FIV; Caspase-1; ASC microglia; Nervous system
10.  Feline Tetherin Efficiently Restricts Release of Feline Immunodeficiency Virus but Not Spreading of Infection▿ 
Journal of Virology  2011;85(12):5840-5852.
Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and “OrfA” proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.
PMCID: PMC3126296  PMID: 21490095
11.  Temporomandibular joint inflammation activates glial and immune cells in both the trigeminal ganglia and in the spinal trigeminal nucleus 
Molecular Pain  2010;6:89.
Glial cells have been shown to directly participate to the genesis and maintenance of chronic pain in both the sensory ganglia and the central nervous system (CNS). Indeed, glial cell activation has been reported in both the dorsal root ganglia and the spinal cord following injury or inflammation of the sciatic nerve, but no data are currently available in animal models of trigeminal sensitization. Therefore, in the present study, we evaluated glial cell activation in the trigeminal-spinal system following injection of the Complete Freund's Adjuvant (CFA) into the temporomandibular joint, which generates inflammatory pain and trigeminal hypersensitivity.
CFA-injected animals showed ipsilateral mechanical allodynia and temporomandibular joint edema, accompanied in the trigeminal ganglion by a strong increase in the number of GFAP-positive satellite glial cells encircling neurons and by the activation of resident macrophages. Seventy-two hours after CFA injection, activated microglial cells were observed in the ipsilateral trigeminal subnucleus caudalis and in the cervical dorsal horn, with a significant up-regulation of Iba1 immunoreactivity, but no signs of reactive astrogliosis were detected in the same areas. Since the purinergic system has been implicated in the activation of microglial cells during neuropathic pain, we have also evaluated the expression of the microglial-specific P2Y12 receptor subtype. No upregulation of this receptor was detected following induction of TMJ inflammation, suggesting that any possible role of P2Y12 in this paradigm of inflammatory pain does not involve changes in receptor expression.
Our data indicate that specific glial cell populations become activated in both the trigeminal ganglia and the CNS following induction of temporomandibular joint inflammation, and suggest that they might represent innovative targets for controlling pain during trigeminal nerve sensitization.
PMCID: PMC3017032  PMID: 21143950
12.  Expression of estrogen receptor GPR30 in the rat spinal cord, autonomic and sensory ganglia 
Journal of neuroscience research  2009;87(7):1610-1619.
The G protein-coupled receptor GPR30 has recently been identified as a non nuclear estrogen receptor. RT-PCR revealed expression of GPR30 mRNA in varying quantities in the rat spinal cord, dorsal root ganglia, nodose ganglia, trigeminal ganglia, hippocampus, brain stem and hypothalamus. Immunohistochemical studies using a rabbit polyclonal antiserum against the human GPR30 C-terminus revealed a fine network of GPR30-immunoreactive (irGPR30) cell processes in the superficial layers of the spinal cord; some of which extended into deeper laminae. A population of neurons in the dorsal horn and ventral horn were irGPR30. Dorsal root, nodose and trigeminal ganglionic neurons displayed varying intensities of irGPR30. Positively labeled neurons were detected in the major pelvic ganglion, but not in the superior cervical ganglion. A population of chromaffin cells in the adrenal medulla was irGPR30, so were cells of the zona glomerulosa. Double-labeling the adrenal medulla with GPR30 antiserum and tyrosine hydroxylase (TH) antibody or phenylethanolamine-N-methyltransferase (PNMT) antiserum revealed that irGPR30 is expressed in the majority of TH-positive chromaffin cells. Lastly, some of the myenteric ganglion cells were irGPR30. Tissues processed with pre-immune serum resulted in no staining. Voltage-sensitive dye imaging studies showed that the selective GPR30 agonist G-1 (1, 10 and 100 nM) depolarized cultured spinal neurons in a concentration dependent manner. Collectively, our result provides the first evidence that GPR30 is expressed in neurons of the dorsal and ventral horn as well as in sensory and autonomic neurons, and activation of GPR30 by the selective agonist G-1 depolarizes cultured spinal neurons.
PMCID: PMC2692324  PMID: 19125412
adrenal gland; myenteric plexus; nodose ganglia; pelvic ganglia; trigeminal ganglia
13.  Suppression of immunodeficiency virus-associated neural damage by the p75 neurotrophin receptor ligand, LM11A-31, in an in vitro feline model 
Feline immunodeficiency virus (FIV) infection like human immunodeficiency virus (HIV), produces systemic and central nervous system disease in its natural host, the domestic cat, that parallels the pathogenesis seen in HIV-infected humans. The ability to culture feline nervous system tissue affords the unique opportunity to directly examine interactions of infectious virus with CNS cells for the development of models and treatments that can then be translated to a natural infectious model. To explore the therapeutic potential of a new p75 neurotrophin receptor ligand, LM11A-31, we evaluated neuronal survival, neuronal damage and calcium homeostasis in cultured feline neurons following inoculation with FIV. FIV resulted in the gradual appearance of dendritic beading, pruning of processes and shrinkage of neuronal perikarya in the neurons. Astrocytes developed a more activated appearance and there was an enhanced accumulation of microglia, particularly at longer times post-inoculation. Addition of 10 nM LM11A-31, to the cultures greatly reduced or eliminated the neuronal pathology as well as the FIV effects on astrocytes and microglia. LM11A-31 also, prevented the development of delayed calcium deregulation in feline neurons exposed to conditioned medium from FIV treated macrophages. The suppression of calcium accumulation prevented the development of foci of calcium accumulation and beading in the dendrites. FIV replication was unaffected by LM11A-31. The strong neuroprotection afforded by LM11A-31 in an infectious in vitro model indicates that LM11A-31 may have excellent potential for the treatment of HIV-associated neurodegeneration.
PMCID: PMC3746485  PMID: 22161560
14.  Neuroprotective effect of herpes simplex virus-mediated gene transfer of erythropoietin in hyperglycemic dorsal root ganglion neurons 
Brain  2009;132(4):879-888.
We examined the efficacy of herpes simplex virus vector-mediated gene transfer of erythropoietin in preventing neuropathy in mouse model of streptozotocin-diabetes. A replication-incompetent herpes simplex virus vector with erythropoietin under the control of the human cytomegalovirus promoter (vector DHEPO) was constructed. DHEPO expressed and released erythropoietin from primary dorsal root ganglion neurons in vitro, and following subcutaneous inoculation in the foot, expressed erythropoietin in dorsal root ganglion neurons in vivo. At 2 weeks after induction of diabetes, subcutaneous inoculation of erythropoietin prevented the reduction in sensory nerve amplitude characteristic of diabetic neuropathy measured 4 weeks later, preserved autonomic function measured by pilocarpine-induced sweating, and prevented the loss of nerve fibres in the skin and reduction of neuropeptide calcitonin gene-related peptide in the dorsal horn of spinal cord of the diabetic mice. We further investigated whether vector-mediated local expression of erythropoietin in dorsal root ganglion neurons can protect in vivo as well as in vitro hyperglycemia-induced axonal degeneration. Our findings show that the AKT/GSK-3β dependent pathway plays an important role in mediating the protection of erythropoietin against diabetic neuropathy. Herpes simplex virus-mediated transfer of erythropoietin to dorsal root ganglia may prove useful in treatment of diabetic neuropathy.
PMCID: PMC2724909  PMID: 19244253
diabetes; gene therapy; herpes simplex virus; erythropoietin
15.  Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses. 
Journal of Virology  1997;71(9):6407-6415.
Feline immunodeficiency virus (FIV) induces a disease state in the domestic cat that is similar to AIDS in human immunodeficiency virus (HIV)-infected individuals. As with HIV, FIV can be divided into primary and cell culture-adapted isolates. Adaptation of FIV to replicate and form syncytia in the Crandell feline kidney (CrFK) cell line is accompanied by an increase in the net charge of the V3 loop of the envelope glycoprotein, mirroring the changes observed in the V3 loop of HIV gp120 with the switch from a non-syncytium-inducing phenotype to a syncytium-inducing phenotype. These data suggest a common mechanism of infection with FIV and HIV. In this study, we demonstrate that cell culture-adapted strains of FIV are able to use the alpha-chemokine receptor CXCR4 for cell fusion. Following ectopic expression of human CXCR4 on nonpermissive human cells, the cells are able to fuse with FIV-infected feline cells. Moreover, fusion between FIV-infected feline cells and CXCR4-transfected human cells is inhibited by both anti-CXCR4 and anti-FIV antibodies. cDNAs encoding the feline CXCR4 homolog were cloned from both T-lymphoblastoid and kidney cell lines. Feline CXCR4 displayed 94.9% amino acid sequence identity with human CXCR4 and was found to be expressed widely on cell lines susceptible to infection with cell culture-adapted strains FIV. Ectopic expression of feline CXCR4 on human cells rendered the cells susceptible to FIV-dependent fusion. Moreover, feline CXCR4 was found to be as efficient as human CXCR4 in supporting cell fusion between CD4-expressing murine fibroblast cells and either HIV type 1 (HIV-1) or HIV-2 Env-expressing human cells. Previous studies have demonstrated that feline cells expressing human CD4 are not susceptible to infection with HIV-1; therefore, further restrictions to HIV-1 Env-dependent fusion may exist in feline cells. As feline and human CXCR4 support both FIV- and HIV-dependent cell fusion, these results suggest a close evolutionary link between FIV and HIV and a common mechanism of infection involving an interaction between the virus and a member of the seven-transmembrane domain chemokine receptor family of molecules.
PMCID: PMC191914  PMID: 9261358
16.  Productive Replication of vif-Chimeric HIV-1 in Feline Cells▿  
Journal of Virology  2010;84(14):7378-7395.
Nonprimate animal models of HIV-1 infection are prevented by missing cellular cofactors and by antiviral actions of species-specific host defense factors. These blocks are profound in rodents but may be less abundant in certain Carnivora. Here, we enabled productive, spreading replication and passage of HIV-1 in feline cells. Feline fibroblasts, T-cell lines, and primary peripheral blood mononuclear cells supported early and late HIV-1 life cycle phases in a manner equivalent to that of human cells, except that produced virions had low infectivity. Stable expression of feline immunodeficiency virus (FIV) Vif-green fluorescent protein (GFP) in HIV-1 entry receptor-complemented feline (CrFK) cells enabled robust spreading HIV-1 replication. FIV Vif colocalized with feline APOBEC3 (fA3) proteins, targeted them for degradation, and prevented G→A hypermutation of the HIV-1 cDNA by fA3CH and fA3H. HIV-1 Vif was inactive against fA3s as expected and even paradoxically augmented restriction in some assays. In an interesting contrast, simian immunodeficiency virus SIVmac Vif had substantial anti-fA3 activities, which were complete against fA3CH and partial against fA3H. Moreover, both primate lentiviral Vifs colocalized with fA3s and could be pulled down from cell lysates by fA3CH. HIV-1 molecular clones that encode FIV Vif or SIVmac Vif (HIV-1VF and HIV-1VS) were then constructed. These viruses replicated productively in HIV-1 receptor-expressing CrFK cells and could be passaged serially to uninfected cells. Thus, with the exception of entry receptors, the cat genome can supply the dependency factors needed by HIV-1, and a main restriction can be countered by vif chimerism. The results raise the possibility that the domestic cat could yield an animal model of HIV-1 infection.
PMCID: PMC2898257  PMID: 20463079
17.  In vivo lymphocyte tropism of feline immunodeficiency virus. 
Journal of Virology  1993;67(9):5175-5186.
Feline immunodeficiency virus (FIV) infection in the cat is similar to human immunodeficiency virus type 1 infection in causing a selective reduction in CD4+ cell numbers, leading to inversion of the CD4+/CD8+ ratio. To determine whether FIV, similar to human immunodeficiency virus type 1, has a tropism for CD4+ cells, we examined the in vitro and in vivo susceptibilities of feline lymphocyte subpopulations to FIV infection. Infection of interleukin-2-dependent CD4+ or CD8+ lymphocyte cultures with the NCSU1 isolate of FIV (FIV-NCSU1) resulted in syncytium formation, cell death, and Mg(2+)-dependent reverse transcriptase (RT) activity in both cases. Monoclonal antibodies to feline lymphocyte subsets were used to sort peripheral blood mononuclear cells from FIV-infected cats into highly (> 95%) purified CD4+ cell, CD8+ cell, immunoglobulin-positive (Ig+) cell, and monocyte subpopulations. The mononuclear cell subpopulations were analyzed for FIV provirus by polymerase chain reaction and Southern blot analysis and for virus expression by RT activity. All 16 cats infected with FIV-NCSU1 demonstrated FIV provirus in CD4+ cell-, CD8+ cell-, and Ig+ cell-enriched lymphocyte populations. Southern blot detection of amplified gag gene sequences and limiting-cell-dilution polymerase chain reaction analysis indicated that Ig+ cells carried a higher FIV provirus burden in chronically (> or = 1-year) infected cats than either CD4+ or CD8+ cells. In contrast, CD4+ cells carried the greatest provirus burden in acutely (2- to 4-week) infected cats. FIV provirus was detected in monocytes from only 1 of 10 cats with asymptomatic infection. Addition of culture supernatants from enriched CD4+, CD8+, and Ig+ cells from FIV-infected cats to an FIV-susceptible CD4+ lymphocyte culture resulted in syncytium formation, cell death, and RT activity. Infection of Ig+ cells is not unique to FIV-NCSU1, as lymphocyte subpopulations from other cats with natural infections and cats infected with the Petaluma or Mount Airy isolate of FIV demonstrated a similar distribution of FIV provirus and RT activity. These data suggest that FIV possesses a broad tropism for peripheral blood mononuclear cells and that an Ig+ cell may serve as a major reservoir for the virus in chronically infected cats.
PMCID: PMC237915  PMID: 7688819
18.  Upregulation of Surface Feline CXCR4 Expression following Ectopic Expression of CCR5: Implications for Studies of the Cell Tropism of Feline Immunodeficiency Virus 
Journal of Virology  2002;76(18):9242-9252.
Feline CXCR4 and CCR5 were expressed in feline cells as fusion proteins with enhanced green fluorescent protein (EGFP). Expression of the EGFP fusion proteins was localized to the cell membrane, and surface expression of CXCR4 was confirmed by using a cross-species-reactive anti-CXCR4 monoclonal antibody. Ectopic expression of feline CCR5 enhanced expression of either endogenous feline CXCR4 or exogenous feline or human CXCR4 expressed from a retrovirus vector, indicating that experiments investigating the effect of CCR5 expression on feline immunodeficiency virus (FIV) infection must be interpreted with caution. Susceptibility to infection with cell culture-adapted strains of FIV or to syncytium formation following transfection with a eukaryotic vector expressing an env gene from a cell culture-adapted strain of virus correlated with expression of either human or feline CXCR4, whereas feline CCR5 had no effect. In contrast, neither CXCR4 nor CCR5 rendered cells permissive to either productive infection with primary strains of FIV or syncytium formation following transfection with primary env gene expression vectors. Screening a panel of Ghost cell lines expressing diverse human chemokine receptors confirmed that CXCR4 alone supported fusion mediated by the FIV Env from cell culture-adapted viruses. CXCR4 expression was upregulated in Ghost cells coexpressing CXCR4 and CCR5 or CXCR4, CCR5, and CCR3, and susceptibility to FIV infection could be correlated with the level of CXCR4 expression. The data suggest that β-chemokine receptors may influence FIV infection by modulating the expression of CXCR4.
PMCID: PMC136470  PMID: 12186908
19.  Increased levels of SV2A botulinum neurotoxin receptor in clinical sensory disorders and functional effects of botulinum toxins A and E in cultured human sensory neurons 
Journal of Pain Research  2011;4:347-355.
There is increasing evidence that botulinum neurotoxin A may affect sensory nociceptor fibers, but the expression of its receptors in clinical pain states, and its effects in human sensory neurons, are largely unknown.
We studied synaptic vesicle protein subtype SV2A, a receptor for botulinum neurotoxin A, by immunostaining in a range of clinical tissues, including human dorsal root ganglion sensory neurons, peripheral nerves, the urinary bladder, and the colon. We also determined the effects of botulinum neurotoxins A and E on localization of the capsaicin receptor, TRPV1, and functional sensitivity to capsaicin stimuli in cultured human dorsal root ganglion neurons.
Image analysis showed that SV2A immunoreactive nerve fibers were increased in injured nerves proximal to the injury (P = 0.002), and in painful neuromas (P = 0.0027); the ratio of percentage area SV2A to neurofilaments (a structural marker) was increased proximal to injury (P = 0.0022) and in neuromas (P = 0.0001), indicating increased SV2A levels in injured nerve fibers. In the urinary bladder, SV2A nerve fibers were found in detrusor muscle and associated with blood vessels, with a significant increase in idiopathic detrusor over-activity (P = 0.002) and painful bladder syndrome (P = 0.0087). Colon biopsies showed numerous SV2A-positive nerve fibers, which were increased in quiescent inflammatory bowel disease with abdominal pain (P = 0.023), but not in inflammatory bowel disease without abdominal pain (P = 0.77) or in irritable bowel syndrome (P = 0.13). In vitro studies of botulinum neurotoxin A-treated and botulinum neurotoxin E-treated cultured human sensory neurons showed accumulation of cytoplasmic vesicles, neurite loss, and reduced immunofluorescence for the heat and capsaicin receptor, TRPV1. Functional effects included dose-related inhibition of capsaicin responses on calcium imaging after acute treatment with botulinum neurotoxins A and E.
Differential levels of SV2A protein expression in clinical disorders may identify potential new targets for botulinum neurotoxin therapy. In vitro studies indicate that treatment with botulinum neurotoxins A and E may affect receptor expression and nociceptor function in sensory neurons.
PMCID: PMC3215514  PMID: 22090803
SV2A; human; pain; botulinum neurotoxin; neurons
20.  Strain-Specific Viral Distribution and Neuropathology of Feline Immunodeficiency Virus 
Feline immunodeficiency virus (FIV) is a naturally-occurring lentivirus of domestic cats, and is the causative agent of feline AIDS. Similar to human immunodeficiency virus (HIV), the pathogenesis of FIV involves infection of lymphocytes and macrophages, and results in chronic progressive immune system collapse and death. Neuropathologic correlates of FIV infection have not yet been elucidated, and may be relevant to understanding HIV-associated neurologic disease (neuroAIDS). As in HIV, FIV strains have been shown to express differential tendencies towards development of clinical neuroAIDS. To interrogate viral genetic determinants that might contribute to neuropathogenicity, cats were exposed to two well-characterized FIV strains with divergent clinical phenotypes and a chimeric strain as follows: FIVPPR (PPR, relatively apathogenic but associated with neurologic manifestations), FIVC36 (C36, immunopathogenic but without associated neurologic disease), and Pcenv (a chimeric virus consisting of a PPR backbone with substituted C36 env region). A sham inoculum control group was also included. Peripheral nerve conduction velocity, CNS imaging studies, viral loads and hematologic analysis were performed over a 12 month period. At termination of the study (350 days post-inoculation), brain sections were obtained from four anatomic locations known to be involved in human and primate lentiviral neuroAIDS. Histological and immunohistochemical evaluation with seven markers of inflammation revealed that Pcenv infection resulted in mild inflammation of the CNS, microglial activation, neuronal degeneration and apoptosis, while C36 and PPR strains induced minimal neuropathologic changes. Conduction velocity aberrations were noted peripherally in all three groups at 63 weeks post-infection. Pcenv viral load in this study was intermediate to the parental strains (C36 demonstrating the highest viral load and PPR the lowest). These results collectively suggest that (i) 3′ C36 genomic elements contribute to viral replication characteristics, and (ii) 5′ PPR genomic elements contribute to CNS manifestations. This study illustrates the potential for FIV to provide valuable information about neuroAIDS pathogenesis related to genotype and viral kinetics, as well as to identify strains useful to evaluation of therapeutic intervention.
PMCID: PMC3167954  PMID: 21715019
Lentiviral neuropathology; neuroAIDS; feline immunodeficiency virus
21.  Bortezomib-Induced Painful Peripheral Neuropathy: An Electrophysiological, Behavioral, Morphological and Mechanistic Study in the Mouse 
PLoS ONE  2013;8(9):e72995.
Bortezomib is the first proteasome inhibitor with significant antineoplastic activity for the treatment of relapsed/refractory multiple myeloma as well as other hematological and solid neoplasms. Peripheral neurological complications manifesting with paresthesias, burning sensations, dysesthesias, numbness, sensory loss, reduced proprioception and vibratory sensitivity are among the major limiting side effects associated with bortezomib therapy. Although bortezomib-induced painful peripheral neuropathy is clinically easy to diagnose and reliable models are available, its pathophysiology remains partly unclear.
In this study we used well-characterized immune-competent and immune-compromised mouse models of bortezomib-induced painful peripheral neuropathy. To characterize the drug-induced pathological changes in the peripheral nervous system, we examined the involvement of spinal cord neuronal function in the development of neuropathic pain and investigated the relevance of the immune response in painful peripheral neuropathy induced by bortezomib.
We found that bortezomib treatment induced morphological changes in the spinal cord, dorsal roots, dorsal root ganglia (DRG) and peripheral nerves. Neurophysiological abnormalities and specific functional alterations in Aδ and C fibers were also observed in peripheral nerve fibers. Mice developed mechanical allodynia and functional abnormalities of wide dynamic range neurons in the dorsal horn of spinal cord. Bortezomib induced increased expression of the neuronal stress marker activating transcription factor-3 in most DRG. Moreover, the immunodeficient animals treated with bortezomib developed a painful peripheral neuropathy with the same features observed in the immunocompetent mice.
In conclusion, this study extends the knowledge of the sites of damage induced in the nervous system by bortezomib administration. Moreover, a selective functional vulnerability of peripheral nerve fiber subpopulations was found as well as a change in the electrical activity of wide dynamic range neurons of dorsal horn of spinal cord. Finally, the immune response is not a key factor in the development of morphological and functional damage induced by bortezomib in the peripheral nervous system.
PMCID: PMC3772181  PMID: 24069168
22.  Prostanoid receptor EP1 and Cox-2 in injured human nerves and a rat model of nerve injury: a time-course study 
BMC Neurology  2006;6:1.
Recent studies show that inflammatory processes may contribute to neuropathic pain. Cyclooxygenase-2 (Cox-2) is an inducible enzyme responsible for production of prostanoids, which may sensitise sensory neurones via the EP1 receptor. We have recently reported that while macrophages infiltrate injured nerves within days of injury, they express increased Cox-2-immunoreactivity (Cox-2-IR) from 2 to 3 weeks after injury. We have now investigated the time course of EP1 and Cox-2 changes in injured human nerves and dorsal root ganglia (DRG), and the chronic constriction nerve injury (CCI) model in the rat.
Tissue sections were immunostained with specific antibodies to EP1, Cox-2, CD68 (human macrophage marker) or OX42 (rat microglial marker), and neurofilaments (NF), prior to image analysis, from the following: human brachial plexus nerves (21 to 196 days post-injury), painful neuromas (9 days to 12 years post-injury), avulsion injured DRG, control nerves and DRG, and rat CCI model tissues. EP1 and NF-immunoreactive nerve fibres were quantified by image analysis.
EP1:NF ratio was significantly increased in human brachial plexus nerve fibres, both proximal and distal to injury, in comparison with uninjured nerves. Sensory neurones in injured human DRG showed a significant acute increase of EP1-IR intensity. While there was a rapid increase in EP1-fibres and CD-68 positive macrophages, Cox-2 increase was apparent later, but was persistent in human painful neuromas for years. A similar time-course of changes was found in the rat CCI model with the above markers, both in the injured nerves and ipsilateral dorsal spinal cord.
Different stages of infiltration and activation of macrophages may be observed in the peripheral and central nervous system following peripheral nerve injury. EP1 receptor level increase in sensory neurones, and macrophage infiltration, appears to precede increased Cox-2 expression by macrophages. However, other methods for detecting Cox-2 levels and activity are required. EP1 antagonists may show therapeutic effects in acute and chronic neuropathic pain, in addition to inflammatory pain.
PMCID: PMC1361784  PMID: 16393343
23.  Varicella-Zoster Virus Glycoprotein E Is a Critical Determinant of Virulence in the SCID Mouse-Human Model of Neuropathogenesis ▿  
Journal of Virology  2010;85(1):98-111.
Varicella-zoster virus (VZV) is a neurotropic alphaherpesvirus. VZV infection of human dorsal root ganglion (DRG) xenografts in immunodeficient mice models the infection of sensory ganglia. We examined DRG infection with recombinant VZV (recombinant Oka [rOka]) and the following gE mutants: gEΔ27-90, gEΔCys, gE-AYRV, and gE-SSTT. gEΔ27-90, which lacks the gE domain that interacts with a putative receptor insulin-degrading enzyme (IDE), replicated as extensively as rOka, producing infectious virions and significant cytopathic effects within 14 days of inoculation. Since neural cells express IDE, the gE/IDE interaction was dispensable for VZV neurotropism. In contrast, gEΔCys, which lacks gE/gI heterodimer formation, was significantly impaired at early times postinfection; viral genome copy numbers increased slowly, and infectious virus production was not detected until day 28. Delayed replication was associated with impaired cell-cell spread in ganglia, similar to the phenotype of a gI deletion mutant (rOkaΔgI). However, at later time points, infection of satellite cells and other supportive nonneuronal cells resulted in extensive DRG tissue damage and cell loss such that cytopathic changes observed at day 70 were more severe than those for rOka-infected DRG. The replication of gE-AYRV, which is impaired for trans-Golgi network (TGN) localization, and the replication of gE-SSTT, which contains mutations in an acidic cluster, were equivalent to that of rOka, causing significant cytopathic effects and infectious virus production by day 14; genome copy numbers were equivalent to those of rOka. These experiments suggest that the gE interaction with cellular IDE, gE targeting to TGN sites of virion envelopment, and phosphorylation at SSTT are dispensable for VZV DRG infection, whereas the gE/gI interaction is critical for VZV neurovirulence.
PMCID: PMC3014186  PMID: 20962081
24.  Mitochondrial dysfunction in distal axons contribute to HIV sensory neuropathy 
Annals of neurology  2010;69(1):100-110.
Accumulation of mitochondrial DNA (mtDNA) damage has been associated with aging and abnormal oxidative metabolism. We hypothesized that in human immunodeficiency virus associated sensory neuropathy (HIV-SN), damaged mtDNA accumulates in distal nerve segments and that a spatial pattern of mitochondrial dysfunction contribute to the distal degeneration of sensory nerve fibers.
We measured levels of common deletion mutations in mtDNA and expression levels of mitochondrial respiratory chain complexes of matched proximal and distal nerve specimens from patients with and without HIV-SN. In mitochondria isolated from peripheral nerves of simian immunodeficiency virus (SIV) infected macaques, a model of HIV-SN, we measured mitochondrial function and generation of reactive oxygen species.
We identified increased levels of mtDNA common deletion mutation in post-mortem sural nerves of patients with HIV-SN as compared to uninfected patients or HIV patients without sensory neuropathy. Furthermore, we found that common deletion mutation in mtDNA was more prevalent in distal sural nerves compared to dorsal root ganglia. In a primate model of HIV-SN, freshly isolated mitochondria from sural nerves of macaques infected with a neurovirulent strain of SIV showed impaired mitochondrial function compared to mitochondria from proximal nerve segments.
Our findings suggest that mtDNA damage accumulates in distal mitochondria of long axons, especially in patients with HIV-SN, and that this may lead to reduced mitochondrial function in distal nerves relative to proximal segments. Although our findings are based on HIV-SN, if confirmed in other neuropathies, these observations could explain the length-dependent nature of most axonal peripheral neuropathies.
PMCID: PMC3051401  PMID: 21280080
HIV; peripheral neuropathy; mitochondria
25.  Differential expression of microRNAs in dorsal root ganglia after sciatic nerve injury 
Neural Regeneration Research  2014;9(10):1031-1040.
This study investigated the possible involvement of microRNAs in the regulation of genes that participate in peripheral neural regeneration. A microRNA microarray analysis was conducted and 23 microRNAs were identified whose expression was significantly changed in rat dorsal root ganglia after sciatic nerve transection. The expression of one of the downregulated microRNAs, microRNA-214, was validated using quantitative reverse transcriptase-PCR. MicroRNA-214 was predicted to target the 3′-untranslated region of Slit-Robo GTPase-activating protein 3. In situ hybridization verified that microRNA-214 was located in the cytoplasm of dorsal root ganglia primary neurons and was downregulated following sciatic nerve transection. Moreover, a combination of in situ hybridization and immunohistochemistry revealed that microRNA-214 and Slit-Robo GTPase-activating protein 3 were co-localized in dorsal root ganglion primary neurons. Western blot analysis suggested that Slit-Robo GTPase-activating protein 3 was upregulated in dorsal root ganglion neurons after sciatic nerve transection. These data demonstrate that microRNA-214 is located and differentially expressed in dorsal root ganglion primary neurons and may participate in regulating the gene expression of Slit-Robo GTPase-activating protein 3 after sciatic nerve transection.
PMCID: PMC4146302  PMID: 25206756
nerve regeneration; peripheral nerve injury; sciatic nerve injury; Slit-Robo GTPaseactivating protein 3; microRNA-214; dorsal root ganglia; gene expression; microarray; bioinformatics; NSFC grant; neural regeneration

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