A calpain inhibitor enhances the survival of Schwann cells in vitro and after transplantation into the injured spinal cord

Abstract

Despite the diversity of cells available for transplantation into sites of spinal cord injury (SCI), and the known ability of transplanted cells to integrate into host tissue, functional improvement associated with cellular transplantation has been limited. One factor potentially limiting the efficacy of transplanted cells is poor cell survival. Recently we demonstrated rapid and early death of Schwann cells (SCs) within the first 24 h after transplantation, by both necrosis and apoptosis, which results in fewer than 20% of the cells surviving beyond 1 week. To enhance SC transplant survival, in vitro and in vivo models to rapidly screen compounds for their ability to promote SC survival are needed. The current study utilized in vitro models of apoptosis and necrosis, and based on withdrawal of serum and mitogens and the application of hydrogen peroxide, we screened several inhibitors of apoptosis and necrosis. Of the compounds tested, the calpain inhibitor MDL28170 enhanced SC survival both in vitro in response to oxidative stress induced by application of H2O2, and in vivo following delayed transplantation into the moderately contused spinal cord. The results support the use of calpain inhibitors as a promising new treatment for promoting the survival of transplanted cells. They also suggest that in vitro assays for cell survival may be useful for establishing new compounds that can then be tested in vivo for their ability to promote transplanted SC survival.

FIG. 1.

Serum and mitogen withdrawal induces apoptosis of adult Schwann cells (SCs) in culture, which is prevented by caspase inhibitors. Serum and mitogen withdrawal was sufficient to induce significant apoptosis in adult SCs after 3 h [ANOVA: F(2,5) = 23.34] (A). Three hours of exposure to DMEM was used to test caspase inhibitors. Preincubation of SCs with the caspase inhibitors ZVAD or YVAD for 1 h prior to apoptosis induction significantly decreased caspase 3/7 activity 3 h after serum and mitogen withdrawal [ANOVA: F(7,120) = 98.99] (B). Images of cells in phase (C, E, G, and I), and nuclear labeling (D, F, H, and J), in D10 + 3M, DMEM, DMEM + ZVAD, and DMEM + YVAD, show condensed cells and nuclei (arrowheads). A reduction in cell number is seen following serum and mitogen withdrawal and partial return following caspase inhibition, as shown by changes in the numbers of healthy nuclei (arrow). Cleavage of a luciferase substrate, measured in relative luminescent units (RLU), was used to detect caspase 3/7 activity, a measure of apoptosis. Luciferase levels in D10 + 3M were used to normalize data across the experiments in A. To assess the effects of inhibitors, luciferase levels following serum and mitogen withdrawal were set to 100% (**p ≤ 0.005; scale bar = 10 μm; DMEM, Dulbecco modified Eagle medium; DMSO, dimethyl sulfoxide; ANOVA, analysis of variance).

FIG. 2.

H₂O₂-induced death of adult cultured Schwann cells (SCs) is concentration-dependent and is reduced following inhibition of lipid peroxidation and calpain activity. H₂O₂ application in vitro induces SC death, as measured by LDH release (A), and reduces the number of surviving adult SCs, as measured by GFP fluorescence (C), in a concentration-dependent manner when assessed after 3 h of exposure. To determine the LD₅₀ of H₂O₂, data were normalized to 500 μM H₂O₂ for cell death (A), and 0 μM H₂O₂ (D10 + 3M) for all live cells (C), and is presented as percent survival. The LD₅₀ of H₂O₂, determined to be ∼ 50 μM H₂O₂, was subsequently used to test inhibitors. Data are normalized to 100% cell death following treatment with the cell lysis solution included in the CytoTox-One kit for maximal LDH release. Application of the lipid peroxidase inhibitor U83836E significantly reduced the percentage of dead cells (B), when cultures were treated with it either at the time of H₂O₂ application, or 1 h before. [ANOVA: F(21,238) = 37.9]. Pretreatment of cultures with the lipid peroxidase inhibitor U83836E, or the calpain inhibitor MDL28170, for 1 h prior to application of H₂O₂, or application of the lipid peroxidase inhibitor U83836E at the time of H₂O₂ application, significantly enhanced GFP fluorescence after 3 h over H₂O₂ application alone [D; ANOVA: F(21,242) = 23.2]. Inhibition of iNOS did not prevent H₂O₂-induced cell death. Data were normalized so that D10-3M is 100% live cells (*p ≤ 0.05, **p ≤ 0.005; GFP, green fluorescence protein; ANOVA, analysis of variance; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; LD₅₀, lethal dose of ∼ 50% of cells).

FIG. 3.

More GFP-positive Schwann cells (SCs) survive H₂O₂-induced injury in vitro following application of inhibitors of lipid peroxidation and calpain activity. Representative cultures from each condition are shown following application of inhibitors before (A–D), or at the time (E–H) of H₂O₂-induced injury. SCs were bipolar and covered the dishes when maintained in D10 + 3M (D). Addition of H₂O₂ decreased the number of GFP-positive SCs after 3 h (H). Treatment of SCs before injury with the lipid peroxidase inhibitor (A), and the calpain inhibitor (C), or at the time of injury with the lipid peroxidase inhibitor (E), resulted in improved SC survival compared to H₂O₂-treated cells (H; scale bar = 100 μm; GFP, green fluorescence protein; iNOS, inducible nitric oxide synthase).

FIG. 4.

Pretreatment of Schwann cells (SCs) with a calpain inhibitor increases SC survival 7 days after transplantation. Grafts of each of the treatment conditions (A–H), with enlargements of individual cells within the grafts (I–P), are shown here. The insets in A–H outline the injury and transplant sites for each condition. Surviving GFP-positive SCs were quantified sterologically (Q), or by real-time PCR for GFP DNA (R). Pretreatment of SCs with the calpain inhibitor resulted in a significant increase in the percentage of surviving SCs [ANOVA: F(7,31) = 3.422; Q). Inhibiting caspase activity with YVAD showed a trend toward increased cell survival. No significant differences were observed in transplant volume (S). Dots on the grafts in Q, R, and S represent values of individual animals within each group (*p ≤ 0.05; scale bar = 10 μm; ANOVA, analysis of variance; GFP, green fluorescence protein; PCR, polymerase chain reaction).