J Antimicrob Chemother 2016; 71: 685 – 691 doi:10.1093/jac/dkv386 Advance Access publication 26 November 2015
Toxicity to sensory neurons and Schwann cells in experimental linezolid-induced peripheral neuropathy Ilja Bobylev1,2, Helina Maru1,2, Abhijeet R. Joshi1,2 and Helmar C. Lehmann1,2* 1
Department of Neurology, University Hospital of Cologne, Cologne, Germany; 2Centre for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany *Corresponding author. Department of Neurology, University Hospital of Cologne, Kerpener Str. 62, 50937 Cologne, Germany. Tel: +49-221-478-87091; Fax: +49-221-478-87309; E-mail:
[email protected]
Received 14 August 2015; returned 24 August 2015; revised 30 September 2015; accepted 17 October 2015 Objectives: Peripheral neuropathy is a common side effect of prolonged treatment with linezolid. This study aimed to explore injurious effects of linezolid on cells of the peripheral nervous system and to establish in vivo and in vitro models of linezolid-induced peripheral neuropathy. Methods: C57BL/6 mice were treated with linezolid or vehicle over a total period of 4 weeks. Animals were monitored by weight, nerve conduction studies and behavioural tests. Neuropathic changes were assessed by morphometry on sciatic nerves and epidermal nerve fibre density in skin sections. Rodent sensory neuron and Schwann cell cultures were exposed to linezolid in vitro and assessed for mitochondrial dysfunction. Results: Prolonged treatment with linezolid induced a mild, predominantly small sensory fibre neuropathy in vivo. Exposure of Schwann cells and sensory neurons to linezolid in vitro caused mitochondrial dysfunction primarily in neurons (and less prominently in Schwann cells). Sensory axonopathy could be partially prevented by coadministration of the Na+/Ca2+ exchanger blocker KB-R7943. Conclusions: Clinical and pathological features of linezolid-induced peripheral neuropathy can be replicated in in vivo and in vitro models. Mitochondrial dysfunction may contribute to the axonal damage to sensory neurons that occurs after linezolid exposure.
Introduction Linezolid (Zyvoxidw) is an oxazolidinone antibiotic that is one of the mainstays in the treatments for infections of vancomycinresistant Gram-positive bacteria.1,2 In addition, linezolid is also frequently used for the treatment of drug-resistant strains of Mycobacterium tuberculosis.3 – 6 Short-term treatment with linezolid has revealed an overall favourable safety profile in clinical trials and in post-marketing surveillance. However, use of linezolid for more than 28 days is associated with more severe side effects that include myelosuppression as well as optic and/or peripheral neuropathy.7 – 13 Many infections that are caused by multiresistant staphylococci (for instance osteomyelitis) or Mycobacterium tuberculosis require continued treatment over weeks and months. Therefore, neurotoxicity represents the major dose-limiting side effect that restricts the therapeutic efficacy of an otherwise highly effective drug. Because linezolid may also interfere with mitochondrial protein synthesis, it has been assumed that neuronal damage is caused by toxicity to neuronal mitochondria, but the exact underlying pathomechanism is poorly understood.14 To address this
issue, we investigated the effects of long-term linezolid treatment in an animal model and in vitro by use of primary dorsal root ganglia neurons and Schwann cell cultures.
Materials and methods Animals Male (8 – 9 weeks old) WT mice (C57BL/6) were used for the study. Mice were housed under standard conditions at 21+18C under a reversed 12 h/12 h light/dark cycle with food and water ad libitum. All animal procedures were approved by the governmental animal welfare committee of Nordrhein-Westfalen.
Drug administration Doses of 0.6 or 3.0 mg/kg/day linezolid (Pfizer) or vehicle (saline) were injected intraperitoneally (ip) for 4 weeks (n ¼ 5 in each linezolid and each control group). General clinical condition was monitored daily. Body weight was measured at the baseline (before the treatment) and every week afterwards for each mouse. At the end of the experiment, mice were deeply anaesthetized and sacrificed by decapitation.
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Behavioural/cold stimulation testing Cold stimulation was measured by placing a drop of acetone (50 mL) onto the hind paw of the linezolid-treated and control mice. Subsequently, the mice were then placed in an empty plastic cage with planar surface. Time was measured until the mice tried to remove the acetone. The measurements were performed up to 60 s and repeated three times in 10–15 min intervals as previously described.15
Electrophysiological studies Electrophysiological studies were recorded with a PowerLab signal acquisition set-up (ADInstruments). The mice were anaesthetized with isoflurane and their body temperature was maintained at 378C using an external heating device. Compound muscle action potential (CMAP) amplitudes were measured bilaterally. Briefly, recording needle electrodes were inserted in the sole of the foot and the sciatic nerve was stimulated with needle electrodes at the sciatic notch. The sensory nerve action potential (SNAP) latencies were recorded as described previously.16
Histological evaluation For analysis of epidermal nerve fibre density (ENFD) in the hind paw skin, 30 mm transverse sections were prepared and immunostained with antibodies against Protein Gene Product 9.5 (1:1.000, ab8189, Abcam) in combination with biotinylated anti-mouse IgG and diaminobenzidine (DAB; Merck Millipore), following standard protocols. The ENFD was quantified by the method of Ko et al.17 Briefly, nerve fibres with branching points inside the epidermis were counted as one, and in the dermis each branching nerve fibre was counted separately. The density was determined as the number of nerve fibres per epidermal length.
Morphometry Sciatic nerves were immersion-fixed in 3% glutaraldehyde overnight, osmicated (1% OsO4), dehydrated and embedded in epoxide resin (Fluka). For morphology studies, 1 mm cross-sections were stained with toluidine blue. All myelinated axons in a single whole cross-section of the nerve were examined with a BZ-9000 microscope (Keyence) equipped with a ×40/0.95 numerical aperture objective lens (Nikon), as previously described.15
Cell culture and pharmacology Sensory cultures Dissociated sensory cultures were prepared from E15 rat embryos, as described previously.15 Briefly, the entire spinal column was dissected out to reveal the spinal cord with attached dorsal root ganglia (DRGs). The DRGs were removed from each column and digested with trypsin for 1 h at 378C. After incubation, DRGs were washed with FBS. The tissue was triturated with a glass pipette to obtain the cell suspension. Neurons were plated in low density on coverslips coated with collagen and incubated in neurobasal medium (Gibco, Life Technology) containing 1% FBS (Gibco, Life Technology), 0.2 M L-glutamine, N2 supplement (Gibco, Life Technology) and 10 ng/mL rat nerve growth factor.
Schwann cell cultures Schwann cells were prepared as described previously from the sciatic nerve of an adult Wistar rat.18 For experimental procedures, Schwann cells were maintained in DMEM—F12 medium.
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Cells were treated either with medium/linezolid (5, 10 or 15 mg/mL) or medium for 24, 48 or 72 h. For the rescue experiment, cells were treated with the Na+/Ca2+ exchange-blocker KB-R7943 (0.5 mM).
Cell viability assay In order to label dead cells, 100 mL of propidium iodide was added to cells and incubated for 15 min at room temperature. Subsequently, the staining solution was discarded, followed by a washing step with 1× PBS. For the additional staining of the nuclei, each well was incubated with Hoechst 33342 (Thermo Scientific). Eight images of the fluorescent cells were randomly taken for quantification in each group with a BZ-9000 microscope (Keyence) equipped with a ×10/0.45 numerical aperture objective lens (Nikon). Living and dead cells were counted with the cell counter plugin for the ImageJ software and evaluated with the GraphPad Prism 5.0 software.
Neurite outgrowth assay Cells were then stained with the primary anti-b-III tubulin antibody (1:1.000 G712A, Promega) and detected with a 1:200 anti-mouse fluorescein secondary antibody (Vector). In addition, those cells were co-stained with Hoechst 33342 (Thermo Scientific) and mounted with Fluoromount-GTM (SouthernBiotech). The analysis of the axon length was performed with ImageJ software.
Ca21 imaging Neuronal and Schwann cell cultures were stained with 3 mM Fluo-4 (F-14201, Life Technologies) dissolved in PBS for 30 min in an incubator (378C, 5% CO2). After 30 min of incubation, Fluo-4 was bound to Ca2+. The unbound Fluo-4 was removed by an additional washing step with PBS for 30 min, after which the wells were filled again with medium. Following an additional waiting period of 30 min, the fluorescence was measured with a plate reader (ex. 485 nm, em. 520 nm, BMG Labtech FLUOstar Omega). After each time point (1 – 3 h), cell cultures were washed with PBS and measured again.
MitoTracker assay Sensory cultures and Schwann cell cultures were incubated with 0.05 mM MitoTracker (M-7512, Life Technologies) for 30 min at 378C. During the incubation, MitoTracker was bound to mitochondria depending on the intact membrane potential in live cells. After the incubation, the wells were rinsed in PBS and subsequently refilled with medium. The fluorescence was measured with a plate reader (excitation 570 nm, emission 600 nm, BMG Labtech FLUOstar Omega). After each time point (24, 48 and 72 h), cell cultures were washed with PBS and measured again.
Statistical analysis Data were collected randomly and assessed blindly. The data distribution was normal, and it was analysed using GraphPad Prism 5.0 (GraphPad Software) and presented as the mean of n+SEM. Statistical analysis was performed by the unpaired two-tailed Student’s t-test for comparisons between two groups and ANOVA for multiple groups. P, 0.05 was considered statistically significant.
Results Clinical signs and body weight The administration of linezolid or vehicle was well tolerated by the mice, without any cases of mortality. Mice that received
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Pilot experiments (data not shown) of the used viability assay in Schwann cell cultures showed that the survival did not change at lower concentrations of linezolid (,5 mg/mL) at selected time points of the treatment (24 h, 48 h, 72 h). To assess Schwann cell viability, cell cultures were exposed to three different concentrations of linezolid: 5, 10 and 15 mg/mL. In order to analyse the viability, ratios from cells stained with propidium iodide and Hoechst dye were compared between the groups (Figure 3a). A significant increase in dead Schwann cells could be observed 24 h after treatment with 10 mg/mL linezolid (Figure 3b). There was also a nonsignificant increase in dead cells in the 15 mg/mL treatment group after 24 h. After 48 h of treatment with linezolid, a significant increase in dead Schwann cells could be observed in the 15 mg/mL linezolid-treated group. The results of the third time point (72 h) revealed that linezolid led to an increased amount of dead cells in the 5 and 15 mg/mL linezolid-treated groups.
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Figure 1. Behaviour and neurophysiological analysis in vivo. (a) Cold sensation test of linezolid-treated and control mice (n ¼ 5). (b) CMAP amplitudes of linezolid-treated and control mice (n¼5). (c) SNAP latencies of linezolid-treated and control mice (n ¼ 5). (d) SNAP amplitudes of linezolid-treated and control mice (n¼5). d, days.
the lower concentration of linezolid showed a slight, but not significant, reduction in body weight compared with that of the controls.
Behaviour and neurophysiological analysis in vivo The two groups of mice that were treated with lower and higher doses of linezolid, showed similar results during the behaviour and neurophysiological analysis (Figure 1). Compared with controls, linezolid-treated mice showed increased reaction time during the cold stimulation testing, indicating dysfunction of sensory neurons in the skin (Figure 1a). Nerve conduction studies revealed no change in measures of motor neuron function (CMAP; Figure 1c); however, sensory nerve latencies were increased (Figure 1d) and amplitudes (SNAP) were decreased (Figure 1e), indicating a predominating sensory neuropathy.
To analyse toxic effects to neurons after linezolid treatment, sensory neuron cells were incubated with 5, 10 and 15 mg/mL linezolid for 24 h. The cultures were then stained with anti-b-III tubulin antibody, and mean cell number and neurite length were compared between linezolid-treated cultures and controls (Figure 3c). Although the total number of neurons was similar between control and linezolid-exposed cell cultures, a significantly lower neurite length was observed after linezolid exposure, indicating axonopathy induced by linezolid (Figure 3d).
Ca21 and mitochondrial membrane potential in sensory neuron and Schwann cell cultures Ca2+ examination of linezolid-treated sensory neurons showed a significant increase in cytosolic Ca2+ in cells treated with 10 mg/mL linezolid for 24 h (Figure 4a). Further examination of Ca 2+ in Schwann cells after 72 h of treatment showed a significant increase in cytosolic Ca2+ in all conditions (Figure 4b). In order to analyse the mitochondrial membrane potential during the increase in cytosolic Ca 2+, sensory neuron and Schwann cell cultures were tested with the MitoTracker assay. Treatment with 10 and 15 mg/mL linezolid resulted in a significant reduction of the mitochondrial membrane potential in sensory neurons (Figure 4c). In Schwann cells, a decrease in the mitochondrial membrane potential could be observed 72 h after treatment with 10 mg/mL linezolid (Figure 4d).
Neurite outgrowth and Ca21 assay in sensory neuron and Schwann cell cultures after treatment with KB-R7943 Previous studies have shown that Na+/Ca2+ exchanger blockers can improve axonal growth by inhibition of Ca2+ release.19 In order
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Figure 2. Histological analysis of peripheral nerves. (a) Representative immunohistochemistry staining for PGP9.5 in hind paw skin from linezolid-treated and control mice (n¼5). The broken line indicates the border between the epidermis and the dermis; scale bar¼50 mm. The density of sensory nerve fibres (nerve fibres/cm skin length) was analysed with ImageJ and compared between the groups. (b) Representative semi-thin sections of tibial nerves, prepared from linezolid-treated and control mice (n¼4); scale bar¼20 mm. The mean number of myelinated axons per tibial nerve cross-profile was analysed with ImageJ and compared between the groups. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
to prevent linezolid-induced axonopathy, sensory neuron cultures were incubated with 15 mg/mL linezolid and the Na+/Ca2+ exchanger blocker KB-R7943 (Figure 4e). The presence of KB-R7943 resulted in a significant increase in neurite length after linezolid exposure compared with that of cells that were treated with linezolid only (Figure 4f). However, the axonal length did not reach the length of the controls, thus indicating only a partial rescue. In order to prevent the release of Ca2+ in Schwann cells, Schwann cells were incubated with 15 mg/mL linezolid for 72 h with or without KB-R7943 (Figure 4g). KB-R7943 together with linezolid decreased the release of Ca2+ compared with that in cells that were treated with linezolid only. Schwann cells that were treated with KB-R7943 only showed a slight increase in cytosolic Ca2+, which indicates that KB-R7943 might be toxic to Schwann cells after prolonged treatment.
Discussion The neurotoxic side effects of linezolid are a frequent reason for treatment discontinuation. So far, no in vivo or in vitro models have been established in order to replicate the main clinical features of linezolid-induced peripheral neuropathy. To provide a base for future prevention approaches, we characterized the neurotoxic effects of prolonged use of linezolid in a rodent model and in cell cultures. The doses for the in vivo (0.6 and
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3.0 mg/kg) and in vitro (5, 10 and 15 mg/mL) models were chosen to be in the range of antibacterial activity and are similar to those used in humans.20,21 Our data demonstrate that daily administration of linezolid during a 4 week period causes a mild peripheral neuropathy in C57BL/6 mice. The behavioural and neurophysiological analysis revealed that both doses tested in vivo caused a significant increase in reaction time, indicating a cold allodynia. Simultaneously, linezolid treatment increased the latency time and decreased the amplitudes in SNAP compared with those of controls, whereas CMAP amplitudes of motor neurons remained unchanged. The behavioural and neurophysiological alterations were confirmed by additional analysis of ENFD, which was reduced in linezolid-treated mice. In contrast, no pathological changes were evident after the morphometry analysis of the sciatic nerves of mice treated with linezolid. These results indicate that both doses of linezolid induce a mild, predominantly small sensory neuropathy in vivo, as previously described in patients.8,10,22 Our in vitro studies indicate that linezolid is primarily toxic to sensory axons but also to Schwann cells. They thereby point to a non-cell-specific toxic mode of action, which may include mitochondrial dysfunction. We observed that the viability of Schwann cells did not decrease before 72 h of linezolid treatment. This observation can be explained by the excessive influx of Ca2+ after 72 h,
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Figure 3. Viability assay and neurite outgrowth assay in vitro. (a) Representative staining of dead Schwann cells after treatment with linezolid or vehicle (control); scale bar¼10 mm. Dead cells are detected by co-localization of Hoechst (blue) and increased propidium iodide (red) staining (white arrow). (b) Mean number of dead Schwann cells (SC) after 24, 48 and 72 h of linezolid treatment compared with those of controls (n≥ 16). (c) Representative images of sensory neurons stained for b-III-tubulin 24 h after treatment with linezolid (15 mg/mL); scale bar ¼20 mm. (d) Mean neurite length of sensory neurons (SN) 24 h after 24, 48 and 72 h of linezolid treatment compared with those of controls (n¼680). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
which could induce apoptosis in Schwann cells.23 The influx of Ca2+ was partially prevented by KB-R7943, a Na+/Ca2+ exchanger 1 (NCX1) blocker. This blocker binds only to NCX1, which leaves out other members of the NCX family (NCX2-3).24,25 Those family members are exchangers, which potentially allow further Ca2+ influx in the cytosol. Additional analysis of mitochondrial membrane potential showed only a moderate decrease in Schwann cell cultures, compared with that in neurons, indicating that linezolid has a less toxic effect on mitochondria in Schwann cells compared with those in neurons. To test whether linezolid has an injurious effect on axons, sensory neuron cultures were treated with linezolid. The results of these experiments showed a significant reduction of the axon length in linezolid-treated cultures compared with that of controls. This observation can be explained by axonal damage induced by excessive influx of Ca2+ and decreased mitochondrial membrane potential. As the cytosolic Ca2+ concentration rises, cytoskeletal break-down occurs via Ca2+-dependent activation of the serine-threonine protease calpain.26 – 28 This activation
leads to the degradation of axonal neurofilaments and microtubule-associated components.28 Moreover, decreased mitochondrial membrane potential reflects the loss of mitochondrial function, which is necessary for ATP production and therefore maintenance of axonal structure and physiological performance.29 – 31 The reduction of the axon length was partially prevented by KB-R7943, an Na+/Ca2+ exchanger 1 (NCX1) blocker. Similar to the case with Schwann cells, we expect that KB-R7943 blocks only NCX1, allowing further influx of Ca2+ from other members of the NCX family.24,25 In summary, our study demonstrates that linezolid-induced neurotoxicity can be partially replicated in vivo and in vitro. Our data also indicate that toxic effects to mitochondria in sensory axons and Schwann cells contribute to linezolid-induced peripheral neuropathy. The models described here may be suitable for preclinical evaluation of neuroprotective strategies. Furthermore, a more detailed examination of the cellular changes in these models may allow a better understanding of the pathogenesis of linezolid-induced neuropathy.
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Figure 4. Ca2+ release and mitochondrial membrane potential after treatment with linezolid and KB-R7943 in vitro. Mean levels of cytosolic Ca2+ in (a) sensory neurons (SNs) after 24 h and (b) Schwann cells (SCs) after 72 h of linezolid treatment (n≥ 16). Mean levels of MitoTracker fluorescence in (c) sensory neurons after 24 h and (d) SCs after 72 h of linezolid treatment (n ≥32). (e) Representative images of neuronal cell cultures after 24 h of treatment with linezolid (15 mg/mL), KB-R7943, the combination of linezolid (15 mg/mL) and KB-R7943, or vehicle; scale bar ¼ 50 mm. (f) Mean axonal length of sensory neurons after 24 h of treatment with linezolid (15 mg/mL), KB-R7943, the combination of linezolid (15 mg/mL) and KB-R7943, or vehicle (n¼386). (g) Mean levels of cytosolic Ca2+ in SCs after 72 h of treatment with linezolid (15 mg/mL), KB-R7943, the combination of linezolid (15 mg/mL) and KB-R7943, or vehicle (n≥ 48). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
Funding
References
This study was supported by internal funding.
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