Crocetin Potentiates Neurite Growth in Hippocampal Neurons and Facilitates Functional Recovery in Rats with Spinal Cord Injury

Abstract

Crocetin is an ingredient of traditional Chinese medicine and has therapeutic potential in various diseases due to its pharmacological properties, such as neuroprotection, anti-oxidative stress, and anti-inflammation. These properties might benefit the treatment of spinal cord injury. In the present study, we tested the effect of crocetin on neurite growth and sensorimotor dysfunction in a rat model of spinal cord injury. We evaluated the viability of cultured hippocampal neurons with tetrazolium dye and lactate dehydrogenase assays, visualized neurites and axons with antibody staining, and monitored motor and sensorimotor functions in rats with spinal cord injury using the Basso, Beattie, and Bresnahan assay and the contact plantar placement test, respectively, and measured cytokine expression using enzyme-linked immuno-absorbent assays. We found that crocetin (1) did not alter the viability of cultured hippocampal neurons; (2) accelerated neurite growth with preference for the longest process in individual hippocampal neurons; (3) reversed the inhibition of neurite growth by chondroitin sulfate proteoglycan and NogoA; (4) facilitated the recovery of motor and sensorimotor functions after spinal cord injury; and (5) did not inhibit pro-inflammatory responses, but restored the innervation of the descending 5-HT system in injured spinal cord. Crocetin promotes neurite growth and facilitates the recovery of motor and sensorimotor functions after spinal cord injury, likely through repairing neuronal connections.

Keywords: Crocetin; Hippocampal neurons; Inflammation; Spinal cord injury.

Fig. 1

Effects of crocetin on the viability of primary neurons in vitro. Cell viability was measured using the MTT assay and cell death was assessed by the LDH test. Primary neurons were treated with 5 μmol/L, 10 μmol/L, or 20 μmol/L crocetin for 24 h. A Effects of crocetin on the viability of primary cultures of hippocampal neurons. B Effects of crocetin on the death of hippocampal neurons in culture. Data are presented as mean ± SD.

Fig. 2

Crocetin increases neurite growth in cultured hippocampal neurons. A, B Representative images of cultured hippocampal neurons two days after plating without (Control) and with 10 μmol/L crocetin. Cells were immunostained with β-tubulin antibody. Crocetin (5 μmol/L–20 μmol/L) promoted growth of the longest neurite (C) in individual neurons. At least 100 neurites were analyzed in 20 fields from each group. Data are presented as mean ± SD; *P < 0.05, **P < 0.01 versus control.

Fig. 3

Crocetin reverses the inhibition of neurite growth in cultured hippocampal neurons by CSPG and NogoA. A Typical image showing that CSPG inhibited the neurite growth in cultured hippocampal neurons. B Typical image showing that crocetin treatment reversed the inhibition of neurite growth by CSPG. C Summary of the length of the longest neurite in cultured hippocampal neurons in control (Control) and in the presence of CSPG (1 μg/mL) and CSPG + crocetin (10 µmol/L). D Summary of the length of the longest neurite in cultured hippocampal neurons in the presence of NogoA (2 μg/mL) and NogoA + crocetin. At least 100 neurites from 20 fields in each group were analyzed. Data are mean ± SD; **P < 0.01 compared with Control; ^## P < 0.01 compared with CSPG or NogoA.

Fig. 4

Crocetin facilitates the recovery of motor and sensorimotor functions in a rat model of spinal cord injury. A Time-course of BBB scores showing the recovery of motor function after spinal cord injury with and without crocetin. B Time-course of contact plantar placement (CPP) scores showing the recovery of sensorimotor function after spinal cord injury with and without crocetin (40 mg/kg). Data are presented as mean ± SD; n = 6/group; *P < 0.05, **P < 0.01.

Fig. 5

Effects of crocetin on pro-inflammatory cytokines and 5-HT boutons in rats with spinal cord injury (SCI). The levels of TNF-α (A), IL-1β (B), IL-6 (C), and IL-8 (D) were monitored within 5 days post-injury in the SCI rat model. The SCI rats were treated with i.p. injection of 40 mg/kg crocetin (Crocetin), vehicle (SCI), or sham surgery. 5-HT antibody staining was used to visualize 5-HT bouton and fibers in sections from T5-L1 spinal segments of sham (E), SCI (F), and SCI + crocetin (G) rats. Mean ± SD, n = 6/group; **P < 0.01, ***P < 0.001.