Julie A. Gurwell

Synergistic increases in intracellular Ca2+, and the release of MCP-1, RANTES, and IL-6 by astrocytes treated with opiates and HIV-1 Tat

Recent evidence suggests that injection drug users who abuse heroin are at increased risk of CNS complications from human immunodeficiency virus (HIV) infection. Opiate drugs may intrinsically alter the pathogenesis of HIV by directly modulating immune function and by directly modifying the CNS response to HIV. Despite this, the mechanisms by which opiates increase the neuropathogenesis of HIV are uncertain. In the present study, we describe the effect of morphine and the HIV‐1 protein toxin Tat1‐72 on astroglial function in cultures derived from ICR mice. Astroglia maintain the blood‐brain barrier and influence inflammatory signaling in the CNS. Astrocytes can express μ‐opioid receptors, and are likely targets for abused opiates, which preferentially activate μ‐opioid receptors. While Tat alone disrupts astrocyte function, when combined with morphine, Tat causes synergistic increases in [Ca2+]i. Moreover, astrocyte cultures treated with morphine and Tat showed exaggerated increases in chemokine release, including monocyte chemoattractant protein‐1 (MCP‐1) and regulated on activation, normal T cell expressed and secreted (RANTES), as well as interleukin‐6 (IL‐6). Morphine‐Tat interactions were prevented by the μ‐opioid receptor antagonist β‐funaltrexamine, or by immunoneutralizing Tat1‐72 or substituting a nontoxic, deletion mutant (TatΔ31‐61). Our findings suggest that opiates may increase the vulnerability of the CNS to viral entry (via recruitment of monocytes/macrophages) and ensuing HIV encephalitis by synergistically increasing MCP‐1 and RANTES release by astrocytes. The results further suggest that astrocytes are key intermediaries in opiate‐HIV interactions and disruptions in astroglial function and inflammatory signaling may contribute to an accelerated neuropathogenesis in HIV‐infected individuals who abuse opiates.

Synergistic neurotoxicity of opioids and human immunodeficiency virus-1 Tat protein in striatal neurons in vitro

Human immunodeficiency virus (HIV) infection selectively targets the striatum, a region rich in opioid receptor-expressing neural cells, resulting in gliosis and neuronal losses. Opioids can be neuroprotective or can promote neurodegeneration. To determine whether opioids modify the response of neurons to human immunodeficiency virus type 1 (HIV-1) Tat protein-induced neurotoxicity, neural cell cultures from mouse striatum were initially characterized for mu and/or kappa opioid receptor immunoreactivity. These cultures were continuously treated with morphine, the opioid antagonist naloxone, and/or HIV-1 Tat (1-72) protein, a non-neurotoxic HIV-1 Tat deletion mutant (TatDelta31-61) protein, or immunoneutralized HIV-1 Tat (1-72) protein. Neuronal and astrocyte viability was examined by ethidium monoazide exclusion, and by apoptotic changes in nuclear heterochromatin using Hoechst 33342. Morphine (10nM, 100nM or 1microM) significantly increased Tat-induced (100 or 200nM) neuronal losses by about two-fold at 24h following exposure. The synergistic effects of morphine and Tat were prevented by naloxone (3microM), indicating the involvement of opioid receptors. Furthermore, morphine was not toxic when combined with mutant Tat or immunoneutralized Tat. Neuronal losses were accompanied by chromatin condensation and pyknosis. Astrocyte viability was unaffected. These findings demonstrate that acute opioid exposure can exacerbate the neurodegenerative effect of HIV-1 Tat protein in striatal neurons, and infer a means by which opioids may hasten the progression of HIV-associated dementia.

Opioid system diversity in developing neurons, astroglia, and oligodendroglia in the subventricular zone and striatum: Impact on gliogenesis in vivo

Accumulating evidence, obtained largely in vitro, indicates that opioids regulate the genesis of neurons and glia and their precursors in the nervous system. Despite this evidence, few studies have assessed opioid receptor expression in identified cells within germinal zones or examined opioid effects on gliogenesis in vivo. To address this question, the role of opioids was explored in the subventricular zone (SVZ) and/or striatum of 2–5‐day‐old and/or adult ICR mice. The results showed that subpopulations of neurons, astrocytes, and oligodendrocytes in the SVZ and striatum differentially express μ‐, δ‐, and/or κ‐receptor immunoreactivity in a cell type‐specific and developmentally regulated manner. In addition, DNA synthesis was assessed by examining 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation into glial and nonglial precursors. Morphine (a preferential μ‐agonist) significantly decreased the number of BrdU‐labeled GFAP+ cells compared with controls or mice co‐treated with naltrexone plus morphine. Alternatively, in S100β+ cells, morphine did not significantly decrease BrdU incorporation; however, significant differences were noted between mice treated with morphine and those treated with morphine plus naltrexone. Most cells were GFAP−/S100β−. When BrdU incorporation was assessed within the total population (glia and nonglia), morphine had no net effect, but naltrexone alone markedly increased BrdU incorporation. This finding suggests that DNA synthesis in GFAP−/S100β− cells is tonically suppressed by endogenous opioids. Assuming that S100β and GFAP, respectively, distinguish among younger and older astroglia, this implies that astroglial replication becomes increasingly sensitive to morphine during maturation, and suggests that opioids differentially regulate the development of distinct subpopulations of glia and glial precursors. GLIA 36:78–88, 2001.

Morphine alters astrocyte growth in primary cultures of mouse glial cells: evidence for a direct effect of opiates on neural maturation

To determine whether exogenous opiate drugs with abuse liability directly modify neural growth, the present study investigated the effects of morphine on astrocyte proliferation and differentiation in primary cultures of murine glial cells. The results indicate that morphine decreases glial cell production in a dose-dependent, naloxone-reversible manner. Most notably, gliogenesis virtually ceased in the presence of 10(-6) M morphine during the first week in culture, whereas 10(-8) M or 10(-10) M morphine caused an intermediate suppression of growth compared to control or 10(-6) M morphine treated cultures. Moreover, morphine treatment inhibited [3H]thymidine incorporation by glial fibrillary acidic protein (GFAP) immunoreactive, flat (type 1) astrocytes, suggesting that the decrease in glial cell production was due in part to an inhibition of astrocyte proliferation. Morphine also caused significant increases in both cytoplasmic area and process elaboration in flat (type 1) astrocytes indicating greater morphologic differentiation. In the above experiments, morphine-dependent alterations in astrocyte growth were antagonized by naloxone, indicating that morphine action was mediated by specific opioid receptors. These observations suggest that opiate drugs can directly modify neural growth by influencing two critical developmental events in astrocytes, i.e., inhibiting proliferation and inducing morphologic differentiation.

JC Virus Antibody Status Underestimates Infection Rates

JC virus (JCV) seropositivity is a risk factor for progressive multifocal leukoencephalopathy (PML) in patients on natalizumab. Accordingly, the JCV serological antibody test is of paramount importance in determining disease risk.

κ-Opioid receptor expression defines a phenotypically distinct subpopulation of astroglia: relationship to Ca2+ mobilization, development, and the antiproliferative effect of opioids

To assess the role of kappa-opioid receptors in astrocyte development, the effect of kappa-agonists on the growth of astroglia derived from 1-2-day-old mouse cerebra was examined in vitro. kappa-Opioid receptor expression was assessed immunocytochemically (using KA8 and KOR1 antibodies), as well as functionally by examining the effect of kappa-receptor activation on intracellular calcium ([Ca2+]i) homeostasis and DNA synthesis. On days 6-7, as many as 50% of the astrocytes displayed kappa-receptor (KA8) immunoreactivity or exhibited increases in [Ca2+]i in response to kappa-agonist treatment (U69,593 or U50,488H). Exposure to U69,593 (100 nM) for 72 h caused a significant reduction in number and proportion of glial fibrillary acidic protein-immunoreactive astrocytes incorporating bromodeoxyuridine (BrdU) that could be prevented by co-administering the kappa-antagonist, nor-binaltorphimine (300 nM). In contrast, on day 14, only 5 or 14%, respectively, of the astrocytes were kappa-opioid receptor (KA8) immunoreactive or displayed functional increases in [Ca2+]i. Furthermore, U69,593 (100 nM) treatment failed to inhibit BrdU incorporation at 9 days in vitro. Experimental manipulations showed that kappa-receptor activation increases astroglial [Ca2+]i both through influx via L-type channels and through mobilization of intracellular stores (which is an important Ca2+ signaling pathway in cell division). Collectively, these results indicate that a subpopulation of developing astrocytes express kappa-opioid receptors in vitro, and suggest that the activation of kappa-receptors mobilizes [Ca2+]i and inhibits cell proliferation. Moreover, the proportion of astrocytes expressing kappa-receptors was greatest during a period of rapid cell growth suggesting that they are preferentially expressed by proliferating astrocytes.

Morphine Inhibits Purkinje Cell Survival and Dendritic Differentiation in Organotypic Cultures of the Mouse Cerebellum

The effects of morphine on the morphogenesis and survival of calbindin-D28k-immunoreactive Purkinje cells were studied in organotypic explant cultures isolated from 1- or 7-day-old mouse cerebella. To reduce experimental variability, bilaterally matched pairs of organotypic cultures were used to compare the effects of opiate drug treatment. One explant within each pair was untreated, while the remaining explant was continuously treated for 7 to 10 days with morphine, morphine plus naloxone, or naloxone alone. In explants derived from 1-day-old mice, morphine treatment significantly reduced Purkinje cell dendritic length compared to symmetrically matched untreated control explants. The concentration of morphine estimated to cause a half-maximal reduction (EC50) in dendritic length was 4.9 x 10(-8) M. At higher concentrations (EC50 = 3.6 x 10(-6) M), morphine also significantly decreased the number of Purkinje cells in explants from 1-day-old mice compared to untreated explants. Electron microscopy identified increased numbers of degenerating Purkinje cells in explants derived from 1-day-old mice. This showed that high concentrations (10(-5) M) of morphine reduced Purkinje cell numbers by decreasing their rate of survival. In explants derived from 7-day-old mice, morphine (10(-5) M) neither affected Purkinje cell dendritic length nor cell numbers compared to symmetrically matched untreated (control) explants. Collectively, these findings suggest that morphine per se, through a direct action on the cerebellum, can affect Purkinje cell differentiation and survival. The results additionally suggest that there is a critical period during development when Purkinje cells are especially vulnerable to the effects of morphine.

Morphine does not affect astrocyte survival in developing primary mixed-glial cultures.

In mixed-glial cultures, high concentrations of morphine (1 microM) have previously been shown to completely inhibit any increase in glial numbers, although DNA synthesis continues in flat, polyhedral astrocytes (type 1 astrocytes). This suggests that high concentrations of morphine are toxic to glia. Morphine toxicity was assessed in mixed-glial cultures using calcein-AM and ethidium homodimer dyes as viability markers to identify live and dead cells, respectively. At 3, 5, and 7 days in vitro there was no significant difference in the number of dead cells between untreated and opiate-treated groups. Comparable numbers of ethidium homodimer-labeled cells were present in all groups. The greatest amount of cell death (16-19%) occurred at 3 days in vitro, while fewer cells (8-12%) were dying at 7 days in vitro. To further characterize the dying glia, glial fibrillary acidic protein (GFAP) and A2B5 immunocytochemistry were combined with viability markers. Only GFAP immunoreactive process-bearing cells and A2B5 immunoreactive cells (process-bearing cells and possibly some neurons) were dying in culture, whereas the death of flat, polyhedral GFAP-positive cells was not observed. Cell survival was not affected by morphine, but may be affected by culture conditions. Thus, morphine-induced reductions in glial numbers did not result from an increased rate of cell death. Collectively, the present and previous findings suggest that morphine inhibits the production of flat, polyhedral astrocytes solely by decreasing their rate of proliferation.

Implantation of autologous peripheral nerve grafts into the substantia nigra of subjects with idiopathic Parkinson's disease treated with bilateral STN DBS: a report of safety and feasibility

OBJECTIVE One avenue of intense efforts to treat Parkinsons disease (PD) involves the delivery of neurotrophic factors to restore dopaminergic cell function. A source of neurotrophic factors that could be used is the Schwann cell from the peripheral nervous system. The authors have begun an open-label safety study to examine the safety and feasibility of implanting an autologous peripheral nerve graft into the substantia nigra of PD patients undergoing deep brain stimulation (DBS) surgery. METHODS Multistage DBS surgery targeting the subthalamic nucleus was performed using standard procedures in 8 study participants. After the DBS leads were implanted, a section of sural nerve containing Schwann cells was excised and unilaterally delivered into the area of the substantia nigra. Adverse events were continuously monitored. RESULTS Eight of 8 participants were implanted with DBS systems and grafts. Adverse event profiles were comparable to those of standard DBS surgery. Postoperative MR images did not reveal edema, hemorrhage, or significant signal changes in the graft target region. Three participants reported a patch of numbness on the outside of the foot below the sural nerve harvest site. CONCLUSIONS Based on the safety outcome of the procedure, targeted peripheral nerve graft delivery to the substantia nigra at the time of DBS surgery is feasible and may provide a means to deliver neurorestorative therapy. Clinical trial registration no.: NCT01833364 ( clinicaltrials.gov ).

Peripheral nerve grafts implanted into the substantia nigra in patients with Parkinson’s disease during deep brain stimulation surgery: 1-year follow-up study of safety, feasibility, and clinical outcome

OBJECTIVECurrently, there is no treatment that slows or halts the progression of Parkinsons disease. Delivery of various neurotrophic factors to restore dopaminergic function has become a focus of study in an effort to fill this unmet need for patients with Parkinsons disease. Schwann cells provide a readily available source of such factors. This study presents a 12-month evaluation of safety and feasibility, as well as the clinical response, of implanting autologous peripheral nerve grafts into the substantia nigra of patients with Parkinsons disease at the time of deep brain stimulation (DBS) surgery.METHODSStandard DBS surgery targeting the subthalamic nucleus was performed in 8 study participants. After DBS lead implantation, a section of the sural nerve containing Schwann cells was harvested and unilaterally grafted to the substantia nigra. Adverse events were continually monitored. Baseline clinical data were obtained during standard preoperative evaluations. Clinical outcome data were obtained with postoperative clinical evaluations, neuropsychological testing, and MRI at 1 year after surgery.RESULTSAll 8 participants were implanted with DBS systems and grafts. Adverse event profiles were comparable to those of standard DBS surgery with the exception of 1 superficial infection at the sural nerve harvest site. Three participants also reported numbness in the distribution of the sural nerve distal to the harvest site. Motor scores on Unified Parkinsons Disease Rating Scale (UPDRS) part III while the participant was off therapy at 12 months improved from baseline (mean ± SD 25.1 ± 15.9 points at 12 months vs 32.5 ± 9.7 points at baseline). An analysis of the lateralized UPDRS scores also showed a greater overall reduction in scores on the side contralateral to the graft.CONCLUSIONSPeripheral nerve graft delivery to the substantia nigra at the time of DBS surgery is feasible and safe based on the results of this initial pilot study. Clinical outcome data from this phase I trial suggests that grafting may have some clinical benefit and certainly warrants further study to determine if this is an efficacious and neurorestorative therapy.Clinical trial registration no.: NCT01833364 (clinicaltrials.gov).