Inhibiting epidermal growth factor receptor improves structural, locomotor, sensory, and bladder recovery from experimental spinal cord injury

Abstract

Lack of axon regeneration in the adult CNS has been attributed partly to myelin inhibitors and the properties of astrocytes. After spinal cord injury, proliferating astrocytes not only represent a physical barrier to regenerating axons but also express and secrete molecules that inhibit nerve growth, including chondroitin sulfate proteoglycans (CSPGs). Epidermal growth factor receptor (EGFR) activation triggers astrocytes into becoming reactive astrocytes, and EGFR ligands stimulate the secretion of CSPGs as well as the formation of cribriform astrocyte arrangements that contribute to the formation of glial scars. Recently, it was shown that EGFR inhibitors promote nerve regeneration in vitro and in vivo. Blocking a novel Nogo receptor interacting mechanism and/or effects of EGFR inhibition on astrocytes may underlie these effects. Here we show that rats subjected to weight-drop spinal cord injury can be effectively treated by direct delivery of a potent EGFR inhibitor to the injured area, leading to significantly better functional and structural outcome. Motor and sensory functions are improved and bladder function is restored. The robust effects and the fact that other EGFR inhibitors are in clinical use in cancer treatments make these drugs particularly attractive candidates for clinical trials in spinal cord injury.

Figure 1.

BBB scores and subscores, residual urine, and weight gain. A, PD168393 causes significantly better recovery from SCI. The first BBB score was obtained 4 d after injury, when treated animals (n = 13) already showed a higher degree of joint movements. Control animals (n = 14) reach a plateau after 3 weeks at a BBB score of ≈10, which corresponds to occasional stepping. Treated animals continue to improve until 7 weeks after injury and reach a score of >14, corresponding to constant stepping with frequent forelimb–hindlimb coordination. Four of the 13 treated animals showed consistent limb coordination. Repeated-measurement ANOVA showed the difference between control and treated animals to be significant (p = 0.009). B, The BBB subscore reveals improvement of the treated group during the first 9 weeks, whereas the control group did not improve. C, Residual urine volumes differed dramatically between control and treated groups. As soon as 4 d after injury, this difference became obvious. Residual urine normalized in treated animals after 12 d. Control animals needed longer bladder assistance and did not reach normal residual urine volumes within the 21 d observation period. D, Treated animals (n = 6) lost less weight after injury, recovered faster, and gained more weight after injury compared with control animals (n = 6).

Figure 2.

Myelin sparing and tissue analysis. A, A′, After SCI, most dorsal tracts were destroyed and replaced by scar tissue, whereas ventral tracts were spared, visualized here by luxol fast blue to stain myelin. B, Tissue analysis reveals that control spinal cords (n = 6) contain half as much myelin in the injury zone compared with treated spinal cords (n = 6). Treated cords also show significantly more spared tissue. C, Proportions of different tissue types in the spinal cord. Spinal cords of control animals consisted of a significantly higher degree of scar tissue (63%) compared with control animals (46%). Scale bar, 200 μm.

Figure 3.

5-HT- and TH-IR fibers in gray and white matter. A, A′, In treated animals (n = 6), significantly larger amounts of 5-HT-IR fibers were found in gray matter compared with control animals (n = 6). B, In ventral white matter, fibers run longitudinally. C, Numbers of 5-HT- and TH-IR axons were higher in spinal cords from rats treated with the EGFR inhibitor. Scale bars, 100 μm.

Figure 4.

EGFR immunoreactivity and pEGFR expression in the SC. A, EGFR immunoreactivity in the injured spinal cord was mainly located in astrocytes and gray matter. B, Double labeling for GFAP (green) and EGFR (red). C, Peripheral astrocytes are double labeled and appear yellow. pEGFR immunoreactivity in the intact spinal cord is restricted to glia limitans and adjacent astrocytes. D, After injury, a massive upregulation of pEGFR was observed, confined to white matter. E, In the injury zone, a dense network of astrocytes surrounding the lesion is observed. F, Some round-shaped OX-42-positive cells, presumably macrophages, in the lesion center show EGFR-IR colocalization (arrowheads). Scale bars: A, B, F, 100 μm; E, 200 μm.