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Bortezomib is part of a newer class of chemotherapeutic agents whose mechanism of action is inhibition of the proteasome-ubiquitination system. Primarily used in multiple myeloma, bortezomib causes a sensory-predominant axonal peripheral neuropathy in approximately 30% of patients. There are no established useful preventative agents for bortezomib-induced peripheral neuropathy (BIPN), and the molecular mechanisms of BIPN are unknown. We have developed an in vitro model of BIPN using rat dorsal root ganglia neuronal cultures. At clinically–relevant dosages, bortezomib produces a sensory axonopathy as evidenced by whole explant outgrowth and cell survival assays. This sensory axonopathy is associated with alterations in tubulin and results in accumulation of somatic tubulin without changes in microtubule ultrastructure. Furthermore, we observed an increased proportion of polymerized tubulin, but not total or acetylated tubulin, in bortezomib-treated DRG neurons. Similar findings are observed with lactacystin, an unrelated proteasome-inhibitor, which argues for a class effect of proteasome inhibition on dorsal root ganglion neurons. Finally, there is a change in axonal transport of mitochondria induced by bortezomib in a time-dependent fashion. In summary, we have developed an in vitro model of BIPN that recapitulates the clinical sensory axonopathy; this model demonstrates that bortezomib induces an alteration in microtubules and axonal transport. This robust model will be used in future mechanistic studies of BIPN and its prevention.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major dose limiting side effect that can lead to long-term morbidity. Approximately one-third of patients receiving chemotherapy with taxanes, vinca alkaloids, platinum compounds or proteasome inhibitors develop this toxic side effect. It is not possible to predict who will get CIPN, however, genetic susceptibility may play a role. We explored this hypothesis using an established in vitro dorsal root ganglia neurite outgrowth (DRG-NOG) assay to assess possible genetic influences for cisplatin- and bortezomib-induced neurotoxicity. Almost all previous in vitro studies have used rats or mice. We compared DRG-NOG between four genetically defined, inbred mouse strains (C57BL/6J, DBA/2J, BALB/cJ, and C3H/HeJ) and one rat strain (Sprague Dawley). Our studies found differences in cisplatin and bortezomib-induced neurotoxicity between mouse and rat strains and between the different mouse strains. C57BL/6J and Balb/cJ DRG-NOG was more sensitive to cisplatin than DBA/2J and C3H/HeJ DRG-NOG, and all mouse strains were more sensitive to cisplatin than rat. Bortezomib induced a biphasic dose response in DBA/2J and C3H/H3J mice. C57BL/6J DRG-NOG was most sensitive and Balb/cJ DRG-NOG was least sensitive to bortezomib. Our animal data supports the hypothesis that genetic background may play a role in CIPN and care must be taken when rodent models are used to better understand the contribution of genetics in patient susceptibility to CIPN.

Using dorsal root ganglion neurons (DRG), in vitro, we studied the effects of nerve growth factor (NGF) on a toxin extracted from ethylene oxide (EO) sterilized hemodialyzers. Tissue culture medium passed through dialyzers produced beading of DRG axons that was inhibited by increasing the concentration of NGF from 3.5 to 10 ng/ml. The antioxidant enzymes, catalase and glutathione peroxidase (GPx), prevented neurite beading while superoxide dismutase (SOD) alone did not. 3-Amino-1,2,4-triazole (Az), an inhibitor of catalase blocked the protective effects of catalase and NGF. 1,3 bis[Chloromethyl]-1-nitrosourea (BCNU) inhibits glutathione reductase, and reduces intracellular glutathione levels; it blocked the protective effects of NGF. Dialyzer treated medium was found to have increased peroxide content. In parallel experiments, NGF protected DRG neurons from hydrogen peroxide (H 2 O 2 ) toxicity that was inhibited by Az and BCNU. NGF was also shown to upregulate glutathione in DRG neurons. We propose that EO gas used in the sterilization of hemodialyzers is responsible for the neurotoxicity and is most likely due to oxidative damage in DRG neurons. NGF protects DRG from this toxin by upregulating antioxidants such as catalase, GPx and GSH.

Nerve growth factor (NGF) rescues dorsal root ganglion neurons and PC12 cells from cisplatin-induced cell death. Two model systems were used to demonstrate that rescue is mediated through the high affinity NGF receptor. In dorsal root ganglion (DRG) neurons isolated from p75 −/− and control mice, 20 ng/ml NGF completely prevented cisplatin-induced death. In PC12 cells, we overexpressed receptor chimeras between the tumor necrosis factor and NGF receptors. We demonstrated that activation of the intracellular domain of Trk A is responsible for the NGF rescue effect.

Assembly of the extracellular matrix (ECM) has been tightly linked to compact myelin formation in the peripheral nervous system. We recently demonstrated that myelination of dorsal root ganglion (DRG) axons by Schwann cells may occur in the absence of basal lamina. We have now determined whether laminin deposition occurs around myelinating SC, even though basal lamina has not been assembled. DRG/SC co-cultures were prepared from E15 rat embryos and incubated in fully defined medium (B27) with and without ascorbic acid for 21-24 days. Cultures were stained with a rabbit anti-laminin antibody and examined by laser confocal fluorescence microscopy. Myelination occurred in both groups. In the presence of ascorbic acid, there was dense even laminin staining around myelinating SC. In the absence of ascorbic acid, laminin staining was also present but was irregular and less dense. DRG and SC were co-cultured without ascorbic acid in the presence or absence of a function blocking anti-beta(1) integrin receptor antibody. The antibody completely inhibited myelination. Finally, DRG/SC co-cultures were prepared both with and without ascorbic acid and incubated under control conditions or in the presence of continual, gentle motion. Movement in the absence of ECM significantly inhibited myelination. This demonstrates that laminin deposition on the surface of SC but not ECM assembly is required for formation of compact myelin. ECM is required to provide mechanical stability during the process of myelination.

Cisplatin has been in use for 40 years, primarily for treatment of ovarian and testicular cancer. Oxaliplatin is the only effective treatment for metastatic colorectal cancer. Neurotoxicity occurs in up to 30% of patients and is dose-limiting for both drugs. The neuropathy is characterized by selective sensory loss in the extremities. Cisplatin treatment is associated with high levels of Pt-DNA binding and apoptosis of dorsal root ganglion (DRG) neurons. In this study, we directly compared the effects of oxaliplatin on DRG in vitro. Compared with cisplatin, oxaliplatin formed fewer Pt-DNA adducts following 6, 12, 24, and 48h (0.007ng Pt/mug DNA, 0.012ng/microg, 0.011ng/microg, 0.011ng/microg versus 0.014ng/microg, 0.022ng/microg, 0.041ng/microg, 0.030ng/microg), respectively. These findings closely correlated with data on cell survival where equimolar concentrations of oxaliplatin induced less cell death than cisplatin. Oxaliplatin-induced DRG death was associated with the morphological characteristics of apoptosis defined by 4'-6-diamidino-2-phenylindole and annexin/propidium iodide staining. Death was completely inhibited by the caspase inhibitor z-VAD-fmk. Our results demonstrate that both compounds cause apoptosis of DRG neurons but compared to cisplatin, oxaliplatin forms fewer Pt-DNA adducts and is less neurotoxic to DRG neurons in vitro.

We have demonstrated that myelination of dorsal root ganglion (DRG) axons occurs in a fully defined, serum-free medium (B27). This implies that there may be components in B27 medium that support myelination. To determine which of the components in B27 were essential for myelination, we systematically removed components from B27 until myelination was lost. We added these components to a fully defined minimal medium (N2) that supports neuron survival but not myelination. When antioxidants were removed from B27, myelination was lost. However, the individual antioxidants did not induce myelination when added to N2 medium. Addition of ascorbic acid along with the B27 antioxidants was sufficient to induce myelination in N2 medium, which was enhanced by retinyl acetate. Removal of vitamin E from B27 caused a partial loss of myelination, and addition of vitamin E to N2 medium containing ascorbic acid induced partial myelination. Addition of serum to the B27 myelinating medium inhibited myelination completely. These results indicate that antioxidants are important for myelination, in vitro. Vitamin E may play an important role. Use of a serum-free medium may be beneficial for in vitro myelination studies because serum has unknown inhibitory effects.

Objective: Our aims were to better understand the mechanisms underlying peripheral neuropathy with diabetes mellitus and to test the hypothesis that acute lowering of glucose levels induces apoptosis in hypoxic neurons. Methods: We used rat dissociated dorsal root ganglion (DRG) neurons incubated in a medium high in glucose concentration (700 mg%) and room air (PO2 150 torr). After 5 days, DRG neurons were placed in hypoxic conditions (PO2 7.6 torr) with a normal-glucose (100 mg%) or high-glucose (700 mg%) medium containing 3 or 100 ng/mL of nerve growth factor. Acute lowering of glucose levels under hypoxic conditions led to apoptosis of DRG neurons. Apoptosis was demonstrated by bis-benzimide staining for nuclear fragmentation, electron microscopy, DNA laddering, and TUNEL staining. Caspase 3 immunocytochemistry and inhibition of neuronal death by the caspase inhibitor z-VAD-fmk (100 μM) confirmed that death was apoptotic. Results: Hypoxia-induced death was decreased when DRG neurons were maintained in high-glucose medium, suggesting that high levels of substrate protected against hypoxia. Apoptosis was completely prevented by increasing the concentration of nerve growth factor from 3 to 100 ng/mL and was partially prevented by the addition of the antioxidant α-lipoic acid (500 μM). Conclusions: This model provides a novel means for studying the pathogenesis and treatment of early stages of diabetic neuropathy.

The PC12 cell line may be used as a model of NGF-induced neuronal differentiation. Exposure to NGF is accompanied by extension of neurites, cessation of growth and differentiation into cells resembling sympathetic neurons. In this study neurite outgrowth from PC12 cells was induced in serum-free, NGF-free medium conditions. Neurite outgrowth in serum-free conditions was abolished by exposure to anti-NGF antisera. Reverse transcription combined with polymerase chain reaction (RT-PCR) and in situ hybridization of PC12 cells in serum-free medium conditions revealed NGF transcripts. Western blot analysis of these cells revealed tyrosine phosphorylation of the high affinity NGF receptor (TrkA/gp140) and activation of a downstream signal cascade element, ERK-1/MAP kinase. NGF was also detected by a specific enzyme-linked immunoabsorbant assay (ELISA) revealing picogram levels of protein in conditioned medium and cell lysates. Survival of embryonic rat dorsal root ganglion neurons was maintained in cultures grown in this serum-free conditioned medium. This demonstrated that NGF may act as an autocrine or paracrine growth factor for PC12 cell differentiation.

Recently we demonstrated that oxidized cellulose (OC), a surgical topical hemostatic agent, induces subjacent nerve fiber degeneration by a diffusible chemical mechanism. Since OC is highly acidic, we examined the role of acidity in the development of neuropathy by OC in this study. Fifteen minutes' exposure to culture media containing OC (2 mg/ml, pH 3.47 or 10 mg/ml, pH 2.57) suppressed the subsequent neurite outgrowth of precultured rat DRG neurons in vitro. However, the neurotoxicity of OC disappeared when the pH of the media was restored to 7.42. Topical application of 20 mg OC lowered the pH in the subperineurium of the adjacent rat sciatic nerve to around 3, and kept it below 4 for 2 h in vivo. Application of 0.1 ml neutralized physiological saline containing 40 mg OC did not produce pathological changes in the adjacent rat sciatic nerve in vivo, in contrast to the marked subperineurial nerve damage by direct application of 20 or 40 mg OC observed in our previous study. These results strongly indicate that local neurotoxicity of OC is due to its high acidity. Further care is needed to avoid direct application of large amounts of OC to peripheral nerve.

We determined whether suramin neurotoxicity can be prevented by nerve growth factor (NGF) and if this interaction occurs at the level of the NGF receptor. Neurite outgrowth from rat dorsal root ganglia in vitro was measured serially in the presence of suramin (100-600 microM) alone or with beta-NGF (50-1,000 ng/ml). Competitive NGF receptor-binding studies were done with 125I-labeled NGF in the presence or absence of suramin. Neurite growth was inhibited in a dose-dependent manner, but at usual neurotoxic levels this inhibition could be overcome completely by increasing the concentration of NGF. Receptor-binding assays showed similar dose-dependent inhibition of 125I-labeled NGF binding. In the presence of suramin, the dissociation constant for high-affinity binding was decreased from 1.2 x 10(-11) to 3.9 x 10(-10) and low-affinity binding from 2.7 x 10(-9) to 1.2 x 10(-8). Increasing doses of suramin inhibited 125I-labeled NGF specific binding in a dose-dependent fashion, and doses of suraminor = 1,000 microM were able to completely inhibit 125I-labeled NGF specific binding. Suramin-induced dorsal root ganglia damage can be ameliorated by high-dose NGF. This effect is most likely due to competition between suramin and NGF at the high-affinity NGF receptor.