A selective phosphodiesterase-4 inhibitor reduces leukocyte infiltration, oxidative processes, and tissue damage after spinal cord injury

Abstract

We tested the hypothesis that a selective phosphodiesterase type 4 inhibitor (PDE4-I; IC486051) would attenuate early inflammatory and oxidative processes following spinal cord injury (SCI) when delivered during the first 3 days after injury. Rats receiving a moderately severe thoracic-clip-compression SCI were treated with the PDE4-I (0.5, 1.0, and 3.0 mg/kg IV) in bolus doses from 2-60 h post-injury. Doses at 0.5 mg/kg and 1.0 mg/kg significantly decreased myeloperoxidase (MPO) enzymatic activity (neutrophils), expression of a neutrophil-associated protein and of ED-1 (macrophages), and estimates of lipid peroxidation in cord lesion homogenates at 24 h and 72 h post-injury by 25-40%. The 3.0 mg/kg dose had small or no effects on these measures. The PDE4-I treatment (0.5 or 1.0 mg/kg) reduced expression of the oxidative enzymes gp91(phox), inducible nitric oxide synthase, and cyclooxygenase-2, and diminished free radical generation by up to 40%. Treatment with 0.5 mg/kg PDE4-I improved motor function (as assessed by the Basso-Beattie-Bresnahan scale) significantly from 4-8 weeks after SCI (average difference 1.3 points). Mechanical allodynia elicited from the hindpaw decreased by up to 25%. The PDE4-I treatment also increased white matter volume near the lesion at 8 weeks after SCI. In conclusion, the PDE4-I reduced key markers of oxidative stress and leukocyte infiltration, producing cellular protection, locomotor improvements, and a reduction in neuropathic pain. Early inhibition of PDE4 is neuroprotective after SCI when given acutely and briefly at sufficient doses.

Figures

FIG. 1.

Phosphodiesterase type 4 inhibitor (PDE4-I) treatment decreases intraspinal neutrophil and monocyte/macrophage influx at 24 h and 72 h after SCI. (A) Myeloperoxidase (MPO) activity in cord homogenates increased after injury in control rats (n=4/5 per group to match treated rat numbers), and in the PDE4-I-treated rats compared to uninjured rats (n=7). These increases were significantly smaller in the PDE4-I-treated rats than in controls at the doses of 0.5 mg/kg or 1.0 mg/kg at 24 h and 72 h post-injury (n=4–5/group; see Table 1), and 3.0 mg/kg at 24 h (n=4/group), but not 72 h (n=4/group) after SCI (analysis of variance [ANOVA] F12,50=19.08, p<0.001; Student-Neuman-Keuls [SNK] test, p<0.05). Values are means±standard error (SE; *significantly different from control injured). All groups were significantly different from uninjured (SCI, SCI control injured; SCI+PDE4-I, treated injured). (B) Neutrophil protein, as identified by Western blotting, in cord homogenates and expressed in arbitrary units (A.U.), increased after injury in control rats and in the PDE4-I-treated rats compared to uninjured rats (n=4 for all groups). Representative autoradiograms of Western blots are shown in the bottom panel (U, uninjured; S, SCI, injured; S+I, injured+treated). These increases were significantly smaller in the PDE4-I-treated rats than in controls at the doses of 0.5 mg/kg or 1.0 mg/kg at 24 h post-injury, and 0.5 mg/kg at 72 h after SCI (ANOVA, F12,39=8.5, p<0.001; by SNK test p<0.05). The MPO activity at 72 h post-injury in the 0.5-mg/kg treatment group tended to be smaller that in the 3.0-mg/kg treatment group (p=0.09). Values are means±SE (*significantly different from control injured; #tended to differ from 3.0-mg/kg PDE4-I treated at 72 h). All groups were significantly different from uninjured animals. Abbreviations as above. (C) Photomicrographs of longitudinal sections of spinal cord immunostained by an anti-neutrophil antibody. The sections from control injured (panels C1 and C3), and 0.5-mg/kg PDE4-I-treated rats (panels C2 and C4) at 72 h following T4 clip compression injury. Panels C3 and C4 show detail of area indicated by the arrows in panels C1 and C2, respectively (scale bar=500 μm in C1 and C2 and 100 μm in C3 and C4). The section from the PDE4-I-treated rat appears to have fewer neutrophils.

FIG. 2.

Phosphodiesterase type 4 inhibitor (PDE4-I) treatment decreases intraspinal monocyte/macrophage influx at 24 h and 72 h after SCI. (A) Western blotting with ED-1, expressed in arbitrary units (A.U.), in cord homogenates (n=4–6/group; see Table 1) showed an increase at 24 and 72 h after SCI compared to uninjured cords; these increases were reduced by PDE4-I administration (analysis of variance [ANOVA] F12,39=11.26, p<0.001; Student-Neuman-Keuls [SNK] test p<0.05). A representative autoradiogram of a Western blot is shown in the bottom panel Values are means±standard error (SE; *significantly different from control injured; #significantly different from 3-mg/kg PDE4-I treated at 72 h; all groups were significantly different from uninjured animals; U, uninjured; S, SCI control injured; S+I, PDE4-I-treated injured; SCI+PDE4-I, treated injured). (B) Photomicrographs of longitudinal sections of spinal cord immunostained by an anti-ED1 antibody. The sections from control injured (A and C), and PDE4-I-treated rats (0.5 mg/kg, B and D) at 72 h following T4 clip-compression injury. C and D show detail of area indicated by the arrows in A and B, respectively (scale bars=500 μm in A and B, and 100 μm in C and D). The section from the PDE4-I-treated rat appears to have fewer monocytes/macrophages.

FIG. 3.

Phosphodiesterase type 4 inhibitor (PDE4-I) treatment decreased lipid peroxidation in spinal cord homogenates at 24 and 72 h after spinal cord injury (SCI). Lipid peroxidation was assessed using the thiobarbituric acid reactive substances (TBARS) assay for malondialdehyde. (A) TBARS concentration increased significantly in controls (n=4–5 to match numbers in the treated groups), and the PDE4-I-treated rats (n=4–5; see Table 1), when compared to that in the cords of uninjured rats (n=6). In rats treated with PDE4-I at the 0.5-mg/kg and 1.0-mg/kg doses, the increases were significantly smaller than those of controls at 24 and 72 h after injury (analysis of variance [ANOVA] F12,49=13.69, p<0.001; Student-Neuman-Keuls [SNK] test p<0.05). After the 3.0-mg/kg dose (n=4) the TBARS concentration was not significantly changed. The TBARS concentration at 24 h after injury in the 1.0-mg/kg treatment group tended to be smaller than that in the 3.0-mg/kg group (p=0.08). Lipid peroxidation was also assayed by Western blotting for 4-hydroxynonenol (HNE, B, n=4–6/group; see Table 1), and the PDE4-I treatment reduced HNE protein damage significantly (ANOVA F8,27=44.56, p<0.001; SNK test p<0.05). Western blots illustrate HNE-bound protein expression compared to loading controls (β-actin). Values are means±standard error (SE). All groups were significantly different from uninjured animals (*significantly different from SCI controls; tended to differ from 3-mg/kg PDE4-I-treated at 24 h; U, uninjured; S, SCI control injured; S+I, PDE4-I-treated injured; SCI+PDE4-I, treated injured).

FIG. 4.

Phosphodiesterase type 4 inhibitor (PDE4-I) treatment decreased 2′-7′-dichlorofluorescein (DCF) fluorescence, a marker of reactive oxygen species, in cord homogenates at 24 and 72 h after spinal cord injury (SCI). DCF levels increased in the injured controls (n=5–6/group; see Table 1), and in the PDE4-I-treated rats (n=5/group) compared to uninjured rats (n=6). DCF levels, however, were significantly lower in the PDE4-I-treated rats than in controls, both at 0.5-mg/kg and 1.0-mg/kg doses (analysis of variance [ANOVA] F8,35=21.18, p<0.001; Student-Neuman-Keuls [SNK] test p<0.05). Values are means±standard error (*significantly different from controls). All groups were significantly different compared to uninjured animals.

FIG. 5.

Phosphodiesterase type 4 inhibitor (PDE4-I) treatment decreases the production of oxidative enzymes in cord homogenates at 24 and 72 h after spinal cord injury (SCI). When assayed by Western blotting, expression of (A) gp91phox, a subunit of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (n=4–6/group; see Table 1), (B) inducible nitric oxide synthase (iNOS; n=4–6/group), and (C) cyclooxygenase-2 (COX-2; n=4–6/group) increased after SCI (both controls and PDE4-I-treated animals) compared to uninjured rats. See Table 1 for details of animal numbers. These increases were significantly smaller in the PDE4-I-treated rats than in controls with the 0.5-mg/kg and 1.0-mg/kg doses at 24 and 72 h after SCI (analysis of variance [ANOVA]: gp91phox F8,27=30.1, p<0.001; iNOS F8,27=62.2, p<0.001; COX-2 F4,15=57.7, p<0.001). All comparisons of means by Student-Neuman-Keuls test (p<0.05). Representative Western blots illustrate gp91phox, iNOS, and COX-2 expression compared to loading controls (β-actin). Values are means±standard error (all groups were significantly different from uninjured animals; *significantly different from controls; U, uninjured; S, SCI control injured; S+I, PDE4-I-treated injured; SCI+PDE4-I, treated injured).

FIG. 6.

Phosphodiesterase type 4 inhibitor (PDE4-I) treatment improves locomotor function and decreases mechanical allodynia after spinal cord injury (SCI). Basso-Beattie-Bresnahan (BBB) locomotor scores (A) were obtained from the PDE4-I-treated injured rats and SCI controls twice weekly for 8 weeks. The left and right leg scores were averaged after each test, and the two scores per week were averaged. Rats receiving the 0.5-mg/kg dose (n=6) had BBB scores significantly greater than those of SCI controls (n=11; see Table 1) from 4–8 weeks after SCI (analysis of variance [ANOVA] F11,90=73.5, p<0.001; Student-Neuman-Keuls [SNK] test p<0.05). BBB scores for the PDE4-I-treated rats at 1.0 mg/kg (n=6) were not different from those of SCI controls (ANOVA F11,90=78.0, p<0.001; SNK test p<0.05). (B) Mechanical allodynia was detected by avoidance responses to hindpaw stimulation in SCI control rats (n=11), and in PDE4-I-treated injured rats (n=6). The rats were tested once per week, commencing at 2 weeks after SCI; scores of the left and right hindpaws were averaged. The PDE4-I treatment caused significant decreases in avoidance responses at 1 week (1.0-mg/kg group only) after SCI, and at 4–6 weeks (both doses) after SCI (0.5 mg/kg ANOVA F9,75=45.3, p<0.001; SNK test p<0.05; 1.0 mg/kg ANOVA F9,75=54.0, p<0.001; SNK test p<0.05). Values are means±standard error (*significantly different between control and PDE4-I-treated rats; *0.05 mg/kg PDE4-I-treated rats tended to differ from controls, p=0.07).

FIG. 7.

Phosphodiesterase type 4 inhibitor (PDE4-I) treatment at 0.5 mg/kg preserves tissue at 8 weeks after SCI. The top panel shows photomicrographs of Luxol fast blue- and hematoxylin and eosin (H&E)-stained cross-sections of spinal cord from control (A) and PDE4-I-treated rats (B). Normal-appearing white matter used for quantification is outlined with a green line. Note the greater overall tissue preservation in the spinal cord of the PDE4-I-treated rat (scale bar=500 μm). The volumes of gray matter, white matter, and lesion size and neuron number within the T3–T5 spinal cord of controls (n=5) and PDE4-I-treated rats (n=5) are plotted in the lower panel. PDE4-I treatment significantly increased the white matter volume (p=0.004 by Student's t-test), but did not significantly change gray matter, lesion volume, or neuron number. The coefficient of error for the counts of neurons ranged from 0.024–0.048 among the control and PDE4-I-treated rats, and the average value was 0.032 (SCI, SCI control injured; SCI+PDE4-I, treated injured).