Early treatment with dapsone after spinal cord injury in rats decreases the inflammatory response and promotes long-term functional recovery

Abstract

Failure of therapeutic strategies for the management and recovery from traumatic spinal cord injury (SCI) is a serious concern. Dapsone (DDS) has been reported as a neuroprotective drug after SCI, although the phase after SC damage (acute or chronic) of its major impact on functional recovery has yet to be defined. Here, we evaluated DDS acute-phase anti-inflammatory effects and their impact on early functional recovery, one week after moderate SCI, and late functional recovery, 7 weeks thereafter. Female Wistar rats were randomly assigned to each of five experimental groups: sham group; four groups of rats with SCI, treated with DDS (0, 12.5, 25.0, and 37.5 mg/kg ip), starting 3 h after injury. Plasma levels of GRO/KC, and the number of neutrophils and macrophages in cell suspensions from tissue taken at the site of injury were measured as inflammation biomarkers. Hindlimb motor function of injured rats given DDS 12.5 and 25.0 mg/kg daily for 8 weeks was evaluated on the BBB open-field ordinal scale. Six hours after injury all DDS doses decreased GRO/KC plasma levels; 24 h after injury, neutrophil numbers decreased with DDS doses of 25.0 and 37.5 mg/kg; macrophage numbers decreased only at the 37.5 mg/kg dose. In the acute phase, functional recovery was dose-dependent. Final recovery scores were 57.5 and 106.2% above the DDS-vehicle treated control group, respectively. In conclusion, the acute phase dose-dependent anti-inflammatory effects of DDS impacted early motor function recovery affecting final recovery at the end of the study.

Keywords: Dapsone; Inflammatory response; Interleukin-8; Motor function recovery; Spinal cord injury.

Fig. 1

Percentage IL-8 (rat GRO/KC) plasma levels 6 h after SCI. Results are expressed as percentage vs control group ± SEM (n = 4 to 5) rats per experimental group. Sham (laminectomy only); SCI, moderately contused rats treated with the vehicle (Veh, 4% PEG in a saline solution) or with a single dose of increasing DDS dosages (12.5, 25.0, and 37.5 mg/kg), all administered 3 h after injury. Statistical analyses were performed using parametric one-way ANOVA test, followed by the multiple comparisons Dunnett’s test vs SCI/Veh. *F_4,18 = 3.111, p = 0.0411.

Fig. 2

Set of representative two-dimensional dot plots obtained by flow cytometry of RP-1-PE/MPO-FITC double-labeled neutrophils (plots 2a-e) in single-cell suspensions of spinal cord tissue approximately 5 mm in length, taken 24 h after SCI from the site of injury of one rat from each experimental group. Images are from: 2a and 2b, a sham and a moderately contused rat treated with the vehicle (4% PEG in a saline solution), respectively; 2c, 2d, and 2e are from a moderately contused rat given a single dose of DDS of increasing dosage (mg/kg): 12.5, 25.0, and 37.5, respectively. The histogram in Fig. 2f illustrates mean ± SEM neutrophil numbers present in cell suspensions from spinal cord tissue specimens taken at the site of injury from all animals of each experimental group (n = 6). Statistical analyses were performed using parametric one-way ANOVA test, followed by the multiple comparisons Dunnett’s test vs SCI/Veh. *F_4,25 = 58.624, p = 0.0001.

Fig. 3

Set of representative two-dimensional dot plots obtained by flow cytometry of CD-68-FITC/MPO-FITC double-labeled macrophages (plots 3a-e) in single-cell suspensions of spinal cord tissue approximately 5 mm in length, taken 24 h after SCI from the site of injury of one rat from each experimental group. Images are from: A and B, a sham and a moderately contused rat treated with the vehicle (4% PEG in a saline solution), respectively; 3c, 3d, and 3e are from a moderately contused rat given a single dose of DDS of increasing dosage (mg/kg): 12.5, 25.0, and 37.5, respectively. The histogram in Fig. 3f illustrates mean ± SEM macrophage numbers present in cell suspensions from spinal cord tissue specimens taken at the site of injury from all animals of each experimental group (n = 6). Statistical analyses were performed using parametric one-way ANOVA test, followed by the multiple comparisons Dunnett’s test vs SCI/Veh. *F_4,25 = 6.494, p = 0.001.

Fig. 4

Shown are representative micrographs (100X) of H/E staining panels, 4a-d of longitudinal sections from damaged spinal cord rat tissue from each contused experimental group obtained at the site of rod impact 24 h after SCI. Contused rats (n = 3/group, for all four experimental groups) were treated 3 h after surgery with: (4a) the vehicle (4% PEG in a saline solution) or single doses of DDS of increasing dosage (mg/kg): (4b) 12.5, (4c) 25.0, and (4d) 37.5. In 4a and 4b images, from panels show significant numbers of PMN leukocytes (white arrows), while the number of inflammatory cells in micrographs 4c and 4d (white arrows) are noticeably decreased.

Fig. 5

Shown are representative micrographs (100X) of neutrophil MPO immunohistochemistry panels 5a-d of PMN leukocytes of longitudinal sections from damaged spinal cord rat tissue from each contused experimental group obtained at the site of rod impact 24 h after SCI. Contused rats (n = 3/group, for all four experimental groups) were treated 3 h after surgery with: (5a) the vehicle (4% PEG in a saline solution) or single doses of DDS of increasing dosage (mg/kg): (5-b) 12.5, (5c) 25.0, and (5d) 37.5. Both 5a and 5b images, from left and right panels show significant numbers of PMN leukocytes (white arrows), while the number of inflammatory cells in micrographs 5c and 5d (white arrows) are noticeably decreased.

Fig. 6

Hind-limb motor function of rats after moderate spinal cord contusion and long-term DDS treatment. Depicted are the time courses of hindlimb motor function recovery on the BBB open-field scale of contused rats given DDS daily (12.5 or 25.0 mg/kg) or vehicle only, starting 3 h after SCI and then every 24 h for 2 months. Rats were evaluated 24 h after injury and then weekly for 8 weeks. Results are expressed as the mean ± SEM of 3–4 rats/group. Statistical analyses were performed using the repeated-measures ANOVA, followed by Dunnett’s test. *Different from the SCI/Veh group (F_2,8 = 12.774, p = 0.003).