thalidomide treatment improves tissue sparing and locomotion after experimental spinal cord injury

Experimental Neurology 216 (2009) 490–498

Contents lists available at ScienceDirect

Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r

Acute rolipram/thalidomide treatment improves tissue sparing and locomotion after experimental spinal cord injury Guido C. Koopmans a,⁎,1,2, Ronald Deumens a, Armin Buss b, Liam Geoghegan c, Aye Mu Myint c,3, Wiel H.H. Honig a, Nadine Kern b, Elbert A. Joosten a, Johannes Noth b, Gary A. Brook b,d,1 a

Department of Anesthesiology, Academic Hospital Maastricht, Maastricht, 6200 AZ, The Netherlands Department of Neurology, Aachen University Hospital, RWTH Aachen, D-52074, Germany Department of Psychiatry and Neuropsychology, Division of Cellular Neuroscience, Academic Hospital Maastricht, Maastricht, 6200 MD, The Netherlands d Department of Neuropathology, Aachen University Hospital, RWTH Aachen, D-52074 Germany b c

a r t i c l e

i n f o

Article history: Received 15 August 2008 Revised 22 December 2008 Accepted 5 January 2009 Available online 15 January 2009 Keywords: Spinal cord injury Immunomodulation Rolipram Thalidomide TNF-α IL-1β Cytokines Functional recovery Neuroprotection

a b s t r a c t Traumatic spinal cord injury (SCI) causes severe and permanent functional deficits due to the primary mechanical insult followed by secondary tissue degeneration. The cascade of secondary degenerative events constitutes a range of therapeutic targets which, if successfully treated, could significantly ameliorate functional loss after traumatic SCI. During the early hours after injury, potent pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) are synthesized and released, playing key roles in secondary tissue degeneration. In the present investigation, the ability of rolipram and thalidomide (FDA approved drugs) to reduce secondary tissue degeneration and improve motor function was assessed in an experimental model of spinal cord contusion injury. The combined acute single intraperitoneal administration of both drugs attenuated TNF-α and IL-1β production and improved white matter sparing at the lesion epicenter. This was accompanied by a significant (2.6 point) improvement in the BBB locomotor score by 6 weeks. There is, at present, no widely accepted intervention strategy that is appropriate for the early treatment of human SCI. The present data suggest that clinical trials for the acute combined application of rolipram and thalidomide may be warranted. The use of such “established drugs” could facilitate the early initiation of trials. © 2009 Elsevier Inc. All rights reserved.

Introduction Spinal cord injury (SCI) causes neuronal and axonal destruction as well as demyelination at or close to the primary site of injury (Bunge et al., 1993). The initial, direct tissue damage is followed by a second phase of tissue degeneration which may take place over a period of weeks or even months (Tator and Fehlings, 1991). A key mediator of this process is an acute and robust inflammatory response which involves the synthesis and release of chemo- and cytokines and a coordinated recruitment of circulating leucocytes as well as microglia from the CNS parenchyma (Schwab and Bartholdi, 1996; Brook et al., 1998; Fitch et al., 1999; Donnnelly and Popovich, 2008; Ankeny and Popovich, in press). During the first 24 h post-injury, the synthesis and release of the potent pro-inflammatory mediators, tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) are markedly ⁎ Corresponding author. Spinal Cord Therapeutics GmbH, Max-Planck-Str. 15a, 40699 Erkrath, Germany. Fax: +49 211 617 851 50. E-mail address: [email protected] (G.C. Koopmans). 1 Contributed equally to this manuscript. 2 Current address: Spinal Cord Therapeutics GmbH, Erkrath, Germany. 3 Current address: Laboratory for PNI and TDM, Psychiatric Hospital Ludwig Maximilians University, Munich, Germany. 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.01.005

elevated at the lesion site where they play a major role in the development of secondary tissue degeneration after experimental SCI in experimental animals and in humans (Bethea et al., 1999; Leskovar et al., 2000; Hausmann, 2003; Yang et al., 2004). This presents an early therapeutic window of opportunity, during which intervention strategies directed at reducing the influence of these cytokines might deliver significant beneficial effects. Acute treatment with anti-inflammatory agents such as pregnenolone or methylprednisolone (Guth et al., 1994; Oudega et al., 1999), or interleukin10 (Bethea et al., 1999; Takami et al., 2002) limits injury-induced tissue damage. However, despite application of neuroprotective treatments, severe functional deficits still remain and the prognosis for human SCI still remains poor. The rationale for targeting pro-inflammatory cytokines has been vindicated in a number of experimental lesion models (Klusman and Schwab, 1997; Penkowa et al., 1999; Yan et al., 2001). A substantial neuroprotective effect of IL-1 receptor antagonist (IL-RA) has been demonstrated following focal or global ischemia, excitotoxicity or traumatic brain injury in rodents (Mulcahy et al., 2003; Lucas et al., 2006). Furthermore, the reduction of TNF-α by systemic IL-10 infusion has been reported to improve functional recovery following experimental spinal cord injury (Bethea et al., 1999). More recently, the acute

G.C. Koopmans et al. / Experimental Neurology 216 (2009) 490–498

administration of etanercept, a TNF-α antagonist, alone or in combination with dexamethasone has been shown to reduce the development of inflammation and secondary injury damage as well as to ameliorate locomotor function in a mouse model of SCI (Genovese et al., 2006, 2007). Alternative candidates for clinically relevant pharmacological strategies, aimed at reducing secondary tissue degeneration by modulating the immune response are thalidomide and rolipram. Both drugs have potent anti-inflammatory effects and have already been FDA approved and assessed clinically for the treatment of human ailments other than traumatic spinal cord injury. Thus, the goal of the present investigation was to assess the potential usefulness of such, already available and clinically tested drugs, in a clinically relevant model of experimental SCI. Thalidomide is a psychoactive drug, which readily crosses the blood brain barrier. It induces a number of well documented anti-inflammatory effects, including the reduction of TNF-α release from LPS-stimulated macrophages as well as promoting the expression of the antiinflammatory cytokine IL-10 (George et al., 2000; Meierhofer and Wiedermann, 2003). Its potent anti-inflammatory effects have resulted in its use in the treatment of erythema nodosum leprosa (Haslett et al., 2005; Villahermosa et al., 2005). Phosphodiesterase 4 (PDE4)-specific inhibitors, such as rolipram, have antidepressant activity (Fleischhacker et al., 1992; Scott et al., 1991), however their therapeutic potential is limited due to side effects such as nausea, vomiting and sedation (O'Donnell and Zhang, 2004). Rolipram also has both axon regenerative (Nikulina et al., 2004; Pearse et al., 2004) and anti-inflammatory effects mediated via elevated intracellular cAMP levels (Zhu et al., 2001). In its anti-inflammatory role, rolipram has been demonstrated to suppress the expression and release of TNF-α and IL-1 from LPS-stimulated macrophages in vitro (Buttini et al., 1997; Witkamp and Monshouwer, 2000; Yoshikawa et al., 1999). Furthermore, when applied via an osmotic pump over a 14 day period following experimental SCI, rolipram has also been reported to reduce TNF-α protein levels and to increase the number of oligodendrocytes (Whitaker et al., 2008) and oligodendrocytemyelinated axons (Pearse et al., 2004). In the present investigation, the effect of a combined, single i.p. injection of thalidomide and rolipram on tissue sparing and functional recovery following a moderate experimental spinal contusion injury has been investigated. This combined pharmacological approach attenuated both TNF-α and IL-1β production, significantly enhanced white matter sparing and improved motor function. Methods and materials Animal housing and care Procedures complied with the ethical committee of the University Maastricht and met governmental guidelines. Twelve week-old male Lewis rats were housed under a 12:12 h dark/light regime and allowed free access to water and a restricted daily diet of 10–12 g standard laboratory chow. Seventy four animals were divided into 6 groups. Group 1: contusion injury followed by immediate i.p. vehicle injection (1% methylcellulose, 0.1% Tween 80 in sterile saline, n = 20), Group 2: contusion injury followed by immediate i.p. rolipram injection (3 mg/ kg, n = 12), Group 3: contusion injury followed by immediate i.p. thalidomide injection (100 mg/kg, n = 12), Group 4: contusion injury followed by immediate i.p. thalidomide/rolipram injection (100 mg/ kg and 3 mg/kg respectively, n = 20), Group 5: sham operation animals (n = 6), Group 6: control, unlesioned animals (n = 4). The i.p. doses of 3 mg/kg (rolipram) and 100 mg/kg (thalidomide) were chosen for the present investigation since earlier reports had demonstrated the efficacy of similar doses in modulating pro-inflammatory cytokine production in vivo (Buttini et al., 1997; Ribeiro et al., 2000).

491

From the seventy four animals included in the experiment seven animals died, presumably due to infection of the bladder and urine tract. Most animals (n = 4) died in the vehicle control group. In the rolipram-, thalidomide- and combi treated group only one animal died in each group. Contusion injury Animals were anesthetized with a mixture of isoflurane and air (induction: 4% isoflurane, maintenance: 1.8% isoflurane). A Th10 laminectomy was performed without rupturing the dura and a 12.5 gcm MASCIS impactor contusion injury (Gruner, 1992) was induced. After suturing muscle and skin, a subcutaneous (s.c.) injection of 5 ml of saline was given. Bladders were emptied manually 2 times a day until spontaneous voiding returned (usually within 1 week). Ten minutes prior to surgery, a s.c. injection of Buprenorfine (Temgesic 0.1 mg/kg) was given as pre-operative pain treatment. A second s.c. injection (same dose) was given 6 h after surgery as postoperation pain treatment. Immediately after injury the lesion severity was verified by the impact velocity of the impactor rod. Animals with an impact velocity error N5% were excluded from further analysis. In total 2 animals were excluded. Functional analysis The BBB open field locomotor rating scale was used to assess general locomotor performance (Basso et al., 1995). The score was assessed before injury and at 1, 3, 5, 7, 14, 21, 28, 35, and 42 days post-operation (dpo) by 2 blinded observers. The BBB sub-score, which grades fine locomotor behavior independently, has been developed to improve the sensitivity of the BBB score by scoring some of the behavioral attributes independently (Lankhorst et al., 1999). The application of sub-scoring is necessary when a certain treatment will affect some but not all aspects of locomotion (Basso, 2004). The BBB sub-score has been applied successfully to show significant differences in treatment effect across groups (Popovich et al., 1999; Lankhorst et al., 2001). The CatWalk automated gait analysis was used to objectively quantify static and dynamic gait parameters (Hamers et al., 2001, 2006; Koopmans et al., 2005, 2006). CatWalk measurements were made from 3 uninterrupted runs before, as well as at 21 and 42 dpo. The regularity index (RI), as determined by the CatWalk, is a measure of coordination which can be implemented into the BBB score. The RI is defined as: RI = NSSP
4 PP
100k. (where NSSP: the number of normal step sequence patterns, PP: total number of paw placements (Hamers et al., 2001, 2006)). Traversing the walk-way with a RI of 100% is considered as coordinated. The CatWalk based BBB score implements this CatWalk based coordination into the normal BBB score. Thus, if three-, two-, one-, or zero of the walk-way crossings are coordinated this was classified as consistent-, frequent-, occasional-, or no coordination for subsequent implementation into the BBB score (Koopmans et al., 2005). Perfusion and tissue processing In all SCI groups, a minimum of 9 animals were terminally anaesthetized and transcardially perfused 6 weeks after injury with 4% paraformaldehyde (pH 7.4). The spinal cords were removed, post-fixed and processed for serial transverse cryosectioning (30 μm), sections being collected on 10 adjacent slides such that the effective interval between neighboring sections on the same slide was 300 μm. A sample series, composed of each 5th slide was stained with thionin (a form of Nissl stain), and another series was processed for 200 kDa neurofilament (NF, Clone 52, Sigma, diluted 1: 1000) and myelin basic protein (MBP, rabbit polyclonal antibody, Chemicon, diluted 1:300) double immunofluorescence according to

492

G.C. Koopmans et al. / Experimental Neurology 216 (2009) 490–498

a protocol described earlier (Buss and Schwab, 2003). The thionin stained sections were then employed for computer-assisted morphometric analysis. In total fifteen thionin stained sections per animal, with a spatial sampling interval of 300 μm, were used to delineate and measure the total spinal cord area and the lesion area, allowing the calculation of the percentage (%) tissue sparing. The section with the lowest amount of tissue sparing was considered to be the epicenter of the lesion. The sections of the lesion epicenter were then further analyzed for white matter sparing. Finally, the spared white matter (SWM) was calculated by subtracting the lesion area, the cavitation areas and incidental gray matter remnants from the total spinal cord area. Immediate adjacent sections processed for NF/MBP double immunofluorescence were used to demonstrate the preservation of NF-positive axons as well as their associated MBPpositive myelin in the ventral white matter. ELISA measurement of TNF-α and IL-1β For the measurement of cytokines levels, 6 sham operated, 6 lesioned-vehicle injected, 6 lesioned-drug combination drug injected, as well as 4 unoperated, control animals were sacrificed 1 h after injury. A 1 cm sample containing the lesion site (or comparable region of sham operated animals) was rapidly dissected and homogenized in 1 ml PBS containing protease inhibitors (Complete protease inhibitor tablets, Roche). Homogenate protein concentrations were determined using the Lowry method, with BSA as a standard. TNF-α and IL-1β levels were assayed using DuoSet ELISA Development System (R&D Systems). All assays were carried out in duplicate using recommended buffers, diluents and substrates. Costar high affinity EIA 96-well plates (Corning) were used and absorbency determined using a microplate reader at 450 nm. The intra-assay coefficient of variations for both assays were less than 10%. The concentration of the cytokines in the tissue was mentioned as pg/μg protein. Statistics All data were analyzed using SPSS statistical software. The ELISA data were analyzed using Kruskal–Wallis H and Mann–Whitney U non-parametric tests. BBB and the BBB sub-score data were analyzed using analysis of variance with repeated measurements over “time” (ANOVAR). In addition, one-way analysis of variance (ANOVA) was used to assess RI, SWM and CatWalk-based BBB data at single timepoints. Bonferroni post hoc test was used in all parametric analyses to determine group differences. Interdependency between locomotor performance and SWM was analyzed using Pearson's correlation coefficient. Results Acute combined drug treatment improves locomotor performance Locomotor performance of all experimental groups demonstrated progressive improvement over the first 2–3 weeks after injury. However, the extent of recovery differed between treatment groups [treatment: F3,39 = 4.6, p b 0.01; Fig. 1A]; post-hoc comparison revealed that the animals treated with a combination of rolipram and thalidomide recovered significantly better than both the rolipram and vehicle control treated animals. At 7 days after injury, the combined treatment animals (BBB: 8.8 ± 0.6) were shown to perform better than all other groups: thalidomide treated (BBB: 6.9 ± 0.3), rolipram treated (BBB: 7.0 ± 0.33) and vehicle treated animals (BBB: 7.0 ± 0.1) [dpo7: F3,39 = 5.8, p b 0.01; Fig. 1A]. At the end of the experiment (6 weeks post-injury) the combination treatment group (BBB: 11.8 ± 0.23) performed significantly better than both the rolipram (BBB: 11.0 ± 0.00) and vehicle treated groups (BBB: 10.7 ± 0.30) [dpo 42: F3,39 = 5.3, p b 0.01; Fig. 1A]. The thalidomide treated group (BBB:

Fig. 1. Behavioural analyses of rats receiving a moderate contusion injury. (A) BBB score for recovery of locomotor performance revealed a significant improvement of functional recovery in combined rolipram/thalidomide treated animals when compared to the other groups (⁎⁎p b 0.01). (B) The BBB sub-score (Lankhorst et al., 1999), revealed a significant combined drug treatment effect at 14, 21 and 42 dpo (⁎⁎p b 0.01). (C) The recovery of full weight bearing plantar stepping (BBB 11) was achieved earliest in combined drug treatment group. Values in A–C are expressed as mean + SEM. ⁎p b 0.05.

G.C. Koopmans et al. / Experimental Neurology 216 (2009) 490–498

493

11.2 ± 0.13) demonstrated an intermediate BBB score that was not significantly different from the other three groups. The use of the BBB sub-score (Lankhorst et al., 1999) clearly demonstrated recovery of fine locomotor skills over the postoperative period for all treatment groups. However, the extent of recovery was substantially affected by the treatment strategies [treatment: F3,39 = 6.9, p b 0.001; Fig. 1B]. The vehicle- and rolipram treated groups demonstrated minimal recovery over 6 weeks, while combined treatment induced a striking and statistically significant improvement. The thalidomide treated group, again, adopted an intermediate level of recovery. Comparison of the treatment groups at individual survival times of 14, 21 and 42 dpo all revealed significant improvements following combined rolipram/thalidomide treatment [F's N 5.8, p b 0.01; Fig. 1B]. An important stage in functional recovery is the return of consistent plantar stepping. For this reason, particular emphasis was focused on determining the return of plantar stepping (Fig. 1C). By 7 days, none of animals in the rolipram-, thalidomide- or vehicle treated groups could generate plantar stepping. In contrast, 40% of the animals in the combined treatment group demonstrated consistent plantar stepping. By 14 days, 90% of the combined treatment animals demonstrated consistent plantar stepping, and by 21 days there was complete recovery of plantar stepping in the combined treatment animals. The complete recovery of plantar stepping in rolipram-,

Fig. 3. Pro-inflammatory cytokine production following moderate contusion injury. Il-β and TNF-α protein levels 1 h after injury at the injury site. (A) Il-β protein levels increased significantly after injury and sham operation when compared to control animals. Acute administration of rolipram/thalidomide (SCI-combi) after injury significantly reduced IL-1β levels by 28%. (B) The combined treatment also caused a reduction of TNF-α protein levels by 21%. Both sham operated animals and SCI-vehicle treated animals showed to be significantly increased after operation whereas the SCIcombi treated animals were not. All values are expressed as mean + SEM, ⁎p b 0.05.

Fig. 2. CatWalk-assisted BBB score. (A) The CatWalk regularity index (RI) revealed complete recovery of inter-limb coordination in combined drug treated group by 42 dpo. Values are expressed as mean + SEM. ⁎p b 0.05. (B) The Catwalk-based BBB score showed a significant improvement in the recovery of locomotor performance by the combined drug treatment group by 42 dpo. Values are individual scores and means (black bars). ⁎⁎p b 0.01.

thalidomide treated animals was much slower, being apparent by 35 days post-injury (2 weeks later than the combined treatment group, Fig. 1C). Consistent plantar stepping did not return completely in the vehicle-treated group. The next phase of functional recovery is the return of inter-limb coordination, which has been suggested to be a milestone in the recovery process (Basso, 2004). The computer assisted CatWalk regularity index (RI) was assessed at 28, 35 and 42 days post-injury and demonstrated a complete return of coordination in the combined treatment group by 42 days (98% ± 1%, Fig. 2A). By 4 weeks, the mean RI was significantly reduced (p b 0.05) in all groups (vehicle treated group: 81% ± 7%; thalidomide treated group: 90% ± 3%, rolipram treated group: 85% ± 3%, combined treatment group: 92% ± 2%). Although, some degree of recovery was also observed in all other groups, the values never achieved pre-operative values (vehicle treated group: 91% ± 2%, thalidomide treated group: 93% ± 1%, rolipram treated group: 93% ± 2%, p b 0.05, Fig. 2A). Implementation of the RI into the BBB score (see Methods and materials section, CatWalk-based BBB score) at 42 days was able to demonstrate a highly significant improvement of locomotor function [F3,38 = 6.6, p = 0.001; Fig. 2B]. Bonferroni post hoc analysis revealed that the recovery of the combined treatment group (CatWalk-based BBB: 13.6 ± 0.5) was substantially better that of the thalidomide-

494

G.C. Koopmans et al. / Experimental Neurology 216 (2009) 490–498

(CatWalk based BBB: 12.1 ± 0.3), rolipram- (CatWalk based BBB: 12.1 ± 0.3) and vehicle treated groups (CatWalk based BBB: 11.3 ± 0.2, Fig. 2B).

Acute combined drug treatment retards the injury-induced increase of IL-1β and TNF-α The production of the pro-inflammatory cytokines IL-1β and TNFα were measured by ELISA at the lesion site 1 h after SCI. Levels of IL1β increased significantly (60.4 ± 5.4 pg/μg protein) at 1 h after injury as compared to control animals (27.3 ± 2.4 pg/μg protein). The acute i.p. administration of combined rolipram/thalidomide after injury significantly reduced IL-1β levels by 28% (p b 0.05, Fig. 3). Levels of TNF-α increased significantly (37.6 ± 7.3 pg/μg protein) at 1 h after injury as compared to control animals (14.4 ± 4.4 pg/μg protein). The combined treatment also caused a reduction of TNF-α protein levels by 21%, but this did not reach the level of statistical significance (Fig. 3). Interestingly, sham operated animals also demonstrated a significant increase of both pro-inflammatory cytokines at 1 h after injury (IL-1β: 52.6 ± 5.8 and TNF-α: 44.5 ± 5.7 pg/μg protein). White matter sparing Morphometric analysis of thionin stained sections throughout the lesion site was used to determine the lesion epicenter (Fig. 4A). The sections containing the lowest amount of tissue sparing were defined as being at the lesion epicenter and were used for subsequent analyses. The moderate contusion injury used in the present investigation caused total destruction of the dorsal funiculus and spinal cord grey matter, with little- or no residual central canal could be detected at the lesion epicenter (Fig. 5C). By 42 days, the lesion site was largely composed of dense populations of rounded, phagocytic macrophages located within- and around numerous cavities or cysts (Fig. 5C). The use of high power objectives during the morphometric quantification allowed a clear delineation of these cavities within the white matter as well as those areas that had degenerated and been infiltrated by phagocytic macrophages. Tissue sparing appeared to be most prominent in the dorso-lateral, lateral, ventro-lateral and ventral white matter (Figs. 5A–D). Quantification of total white matter sparing of thionin stained sections at the lesion epicenter clearly revealed significant differences between the treatment groups [F3,35 = 7.1, p b 0.001; Fig. 4B]. Post-hoc comparison revealed that the greatest degree of white matter sparing was caused by combined rolipram/ thalidomide treatment (Fig. 4B). This was supported qualitatively by the NF/MBP double-immunofluorescence which demonstrated the preservation of more NF/MBP-positive axons/myelin sheaths in the ventral white matter following combined rolipram/thalidomide treatment than following the injection of vehicle (large white arrows, Figs. 6C, D). Some of the NF-positive axons in the vehicle treated groups appeared to be devoid of any MBP-positive myelin (e.g. short arrow, Fig. 6D). Furthermore, substantially more myelin debris could be observed throughout the white matter regions, appearing as rounded, amorphous accumulations of MBP-immunoreactivity, devoid of any associated NF-positive axons (white asterisks, Fig. 6D) The Pearson's correlation coefficient demonstrated a significant positive correlation (r = 0.414) between the extent of white matter sparing and functional recovery (Fig. 4C). Discussion

Fig. 4. Morphometric analysis of tissue sparing based on thionin stained sections. (A) The effect of the different treatment strategies can be seen throughout most of the rosto-caudal extent of the lesion site. The lesion epicenter was defined as the area of least tissue sparing. (B) Total white matter sparing at the lesion epicenter was significantly larger in the combined drug treated group. (C) Locomotor recovery was shown to be positively correlated with the extent of white matter sparing. All individual values are expressed as well as the linear regression. The values in A are expressed as mean + SEM, the values in B are individual scores and means (black bars) ⁎p b 0.05, ⁎⁎p b 0.01.

The present investigation has demonstrated the value of assessing drugs that are already available for clinical application in experimental models of SCI. This “off-the-shelf” strategy has focused on determining the potential therapeutic benefit of 2 drugs, rolipram and thalidomide, in a clinically relevant model of spinal cord contusion injury. The combined acute administration of both drugs was clearly capable of attenuating the production of the proinflammatory cytokine IL-1β, and of reducing secondary tissue degeneration at the lesion epicenter leading to an improved functional outcome.

G.C. Koopmans et al. / Experimental Neurology 216 (2009) 490–498

495

Fig. 5. Thionin stained sections and corresponding camera lucida of lesion epicenter. (A) Thionin stained section of combined drug treatment animal and (C) vehicle treated animal. The respective camera lucida demonstrated enhanced tissue sparing of the lateral and ventral white matter of the combined treated animals (B) in comparison to the vehicle treated controls (D).

The severe and often permanent functional deficits that occur following SCI are due to a combination of primary and secondary degenerative events that are initiated by the initial insult and can extend over a number of weeks (Schwab and Bartholdi, 1996; Green and Wagner, 1973; Osterholm, 1974; Senter and Venes, 1978; Young, 1993; Donnnelly and Popovich, 2008; Ankeny and Popovich, in press).

This presents a therapeutic window of opportunity in which the early application of pharmacological agents could improve tissue sparing, the function of which would contribute to an improved behavioural outcome. Numerous experimental intervention strategies have thus been developed which aim to either reduce secondary tissue degeneration or promote axonal regeneration or enhanced

Fig. 6. Double immunofluorescence demonstration of white matter sparing. Axonal neurofilament content (red signal) and myelin basic protein (green signal) in combined drug treated (A, C) and vehicle treated (B, D) animals. Areas boxed in A and B are shown at higher magnification in C and D respectively. (C, D) More spared myelinated axons (NF/MBPpositive, large white arrows) could be observed in the ventral and ventro-lateral white matter following combined drug treatment than after vehicle injection. NF-positive axons that were devoid of myelin could also be observed more frequently in vehicle injected animals (small white arrow in D). Furthermore, myelin debris, identified as amorphous balls of MPB immunoreactivity that had no apparent association with any NF-positive profiles, was more apparent in the vehicle treated group (e.g. white asterisks in D).

496

G.C. Koopmans et al. / Experimental Neurology 216 (2009) 490–498

compensatory sprouting, some of which have entered clinical trials (Schwab and Bartholdi, 1996; Baptiste and Fehlings, 2006; David and Lacroix, 2003; Schwab et al., 2006; Spencer and Bazarian, 2003; Thuret et al., 2006; Deumens et al., 2005). The evolution of the moderate contusion injury was similar to that already described by others (Bethea et al., 1999), with the early loss of Nissl substances from neurons within the lesion as well as the formation of numerous petechial hemorrhages. The cardinal features of early inflammation are infiltration by inflammatory cells such as polymorphonuclear neutrophils (PMNs), macrophages and lymphocytes (Donnnelly and Popovich, 2008). This takes place in parallel to endothelial cell activation and injury, leading to increased disruption of vascular and blood–spinal cord barrier permeability, oedema and the production of inflammatory mediators (Lucas et al., 2006; Arvin et al., 1996). The rapid, lesion-induced increase of the proinflammatory cytokines, TNF-α and IL-1β, observed in the present investigation supports previous studies which reported increased expression in a range of neuronal and non-neuronal cell types in both experimental animal models and in human SCI (Schwab and Bartholdi, 1996; Leskovar et al., 2000; Yang et al., 2004; Pearse et al., 2004; Wang et al., 1996; Xu et al., 1998; Yakovlev and Faden, 1994; Yan et al., 2001). TNF-α and IL-1β are involved in a wide range of cellular events including transcriptional regulation of genes, cytotoxicity and immuno-regulation (Arvin et al., 1996; Yan et al., 2001; Bonmann et al., 1997; Tartaglia et al., 1993). The acute combined administration of thalidomide and rolipram reduced levels of TNF-α and IL-1β by 21% and 28% respectively (changes of IL-1β being statistically significance while those of TNF-α could only be described as a trend). The therapeutic value of interfering with either TNF-α or IL-1β production or signalling has been demonstrated in other models of CNS diseases or disorders including ischemia, traumatic brain injury and SCI, all of which demonstrated reduced secondary tissue degeneration and functional improvement following modulation of the cytokines (Bethea et al., 1999; Lucas et al., 2006; Pearse et al., 2004; Demjen et al., 2004; Knoblach and Faden, 1998). In parallel to a reduction of pro-inflammatory cytokines at 1 h after injury, a significant reduction of lesion size at its epicenter, spared white matter being particularly evident in the lateral and ventral funiculi was detected at 6 weeks after moderate spinal cord contusion injury and combined rolipram/thalidomide treatment. There is substantial evidence that the sparing of as little as 10% of spinal cord axons can have significant benefits on functional outcome (Blight, 1983; Blight and Decrescito, 1986; Eidelberg et al., 1977). The lateral and ventral white matter contain nerve fibers of the descending reticular system — a population of axons important for activating the central pattern generator and supporting coordinated stepping (Schucht et al., 2002). The combined treatment strategy used in the present investigation clearly demonstrated a rapid return of consistent plantar stepping. The increase white matter sparing induced by the combined rolipram/thalidomide treatment was paralleled by a complete recovery of inter-limb coordination and a statistically significant, 2.5 point increment of the CatWalk-based BBB score, the improved locomotor performance being positively correlated with the amount of spared white matter at the lesion epicenter. Sparing and/or regeneration of reticular and raphe spinal axons has been suggested to contribute to the improved motor function following combined treatment with cAMP, rolipram infusion and Schwann cell transplantation (Pearse et al., 2004). Moreover, prolonged treatment with rolipram alone had beneficial effects on these measures. This latter aspect strongly suggests that the period of drug delivery may determine its beneficial effects on motor outcome and tissue preservation after spinal cord injury. The notion that white matter sparing in the ventral and ventro-lateral areas (containing retuclospinal and propriospinal axons) is important for the maintenance or recovery of hind-limb function and coordination has been

supported by a number of observations (Schucht et al., 2002; Jordan, 1998; Cao et al., 2005; Loy et al., 2002). The early use of high dose methylprednisolone has been widely used in some countries to reduce neutrophil infiltration and lipid peroxidation at the lesion site and promote tissue sparing during the acute phase of experimental and human SCI. Despite promising experimental data, controversies surrounding the interpretation of the NASCIS II/III clinical trials has resulted in a variable application of the approach world-wide (Hugenholtz et al., 2002). Other clinically relevant neuroprotective pharmacotherapeutic approaches have included tests for monosialotetrahexosylganglioside (GM-1) gangliosides, tirilazad mesylate, naloxone, nimodipine, recombinant human erythropoietin, and enterocept, the beneficial actions of the latter 2 being due to associated with reduced inflammatory cell infiltration and attenuated TNF-α production Gorio et al., 2002; King et al., 2007; Sayer et al., 2006). It was clear that the single dose injection of either rolipram or thalidomide alone was not capable of inducing any significant improvement in the CatWalk-assisted BBB. It is probable that further studies to optimize the dose for each component of this combination strategy would yield improvements in the efficacy of the treatment. The combination strategy, using 2 agents which have anti-inflammatory effects via different mechanisms, was essential to reduce both cytokines, leading to enhanced sparing of white matter and improved motor function. It is of particular significance that both rolipram and thalidomide have already been assessed for their clinical efficacy in the treatment of a range of disorders including depression, erythoma lupus, rheumatoid arthritis and Crohn's disease (Thome et al., 2002; Wachtel, 1983). Rolipram and thalidomide exert their anti-inflammatory effects via different mechanisms; rolipram acting via the elevation of intracellular cAMP levels and thalidomide enhancing the rate of TNF-α mRNA degradation (Kim et al., 2004). However, in addition to the above anti-inflammatory mechanisms, thalidomide may have also promoted the expression of the anti-inflammatory cytokine IL-10 (George et al., 2000). The potential advantages of using a combination of drugs, each of which having a number of supplementary beneficial effects, are considerable. Thalidomide has been reported to suppress TNF-α mediated NF-kappa B activation (Majumdar et al., 2002) and to also act as an immunomodulator by interfering with the recruitment of leukocytes by down-regulating endothelial cell expression of cell adhesion molecules i.e. ICAM-1 and LFA-1; (Settles et al., 2001). Rolipram has also been reported to have additional beneficial effects in CNS tissue injury by inducing the expression of mRNA for the neurotrophin, brain derived neurotrophic factor Fujimaki et al., 2000). All of these additional effects of rolipram and thalidomide would, to a greater or lesser degree, contribute to improved functional outcome following experimental SCI and thereby compliment the original choice of rolipram and thalidomide as mediators of reduced secondary tissue damage via reduced pro-inflammatory cytokine production. Thalidomide has suffered notoriety since its withdrawal from the market as a sedative to combat morning sickness during the early stages of pregnancy. The drug tragically provoked teratogenic sideeffects in several thousand children born in the early 1969's. However, more recently thalidomide or its analogues have undergone a number of clinical trials and are currently being used in the treatment of a range diseases and disorders including cancerinduced pain, erythema nodosum leprosum, and inflammatory bowel disease that is refractory to routine anti-inflammatory agents (e.g. Bariol et al., 2002; Laffitte, 2004; Yennurajalingam et al., 2004; Wu et al., 2005). The addition of spinal cord injury to the growing list of potential indications for thalidomide, or its analogues (in combination with other drugs) highlights the potential therapeutic benefits of applying already established drugs to new medical applications.

G.C. Koopmans et al. / Experimental Neurology 216 (2009) 490–498

Conclusion There is a growing awareness that the costs of developing pharmacological agents that will prove successful in clinical is trials is becoming prohibitively expensive. The potential benefits of assessing drugs that have already passed clinical trials for their effectiveness in the treatment of alternative maladies are becoming increasingly clear. The present demonstration of the reduced secondary tissue degeneration and improved motor function brought about by combined rolipram and thalidomide treatment strongly suggests that effective pharmacological agents, that may be applied during the acute phase after injury, are already available in the clinic. Suitable candidate drugs should be “fast-tracked” to early clinical trial in selected cases of human SCI. Acknowledgments This work was supported by a grant from the Institutional Institute for Research in Paraplegia (P80/04) and forms part of the doctoral thesis of Nadine Kern (D82, Diss. RWTH Aachen). The authors have no conflicting financial interests. References Arvin, B., Neville, L.F., Barone, F.C., Feuerstein, G.Z., 1996. The role of inflammation and cytokines in brain injury. Neurosci. Biobehav. Rev. 20, 445–452. Ankeny, D.P., Popovich, P.G., in press. Mechanisms and implications of adaptive immune responses after traumatic spinal cord injury. Neuroscience. doi:10.1016/j. neuroscience.2008.07.001 Baptiste, D.C., Fehlings, M.G., 2006. Pharmacological approaches to repair the injured spinal cord. J. Neurotrauma 23, 318–334. Bariol, C., Maegher, A.P., Vickers, C.R., Byrnes, D.J., Edwards, P.D., Hing, M., Wettstein, A.R., Field, A., 2002. Early studies on the safety and efficacy of thalidomide for symptomatic inflammatory bowel disease. J. Gastroenterol. Hepatol.17, 233–235. Basso, D.M., 2004. Behavioral testing after spinal cord injury: congruities, complexities, and controversies. J. Neurotrauma 21, 395–404. Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 12, 1–21. Bethea, J.R., Nagashima, H., Acosta, M.C., Briceno, C., Gomez, F., Marcillo, A.E., Loor, K., Green, J., Dietrich, W.D., 1999. Systemically administered interleukin-10 reduces tumor necrosis factor-alpha production and significantly improves functional recovery following traumatic spinal cord injury in rats. J. Neurotrauma 16, 851–863. Blight, A.R., 1983. Cellular morphology of chronic spinal cord injury in the cat: analysis of myelinated axons by line-sampling. Neuroscience 10, 521–543. Blight, A.R., Decrescito, V., 1986. Morphometric analysis of experimental spinal cord injury in the cat: the relation of injury intensity to survival of myelinated axons. Neuroscience 19, 321–341. Bonmann, E., Suschek, C., Spranger, M., Kolb-Bachofen, V., 1997. The dominant role of exogenous or endogenous interleukin-1 beta on expression and activity of inducible nitric oxide synthase in rat microvascular brain endothelial cells. Neurosci. Lett. 230, 109–112. Brook, G.A., Plate, D., Franzen, R., Martin, D., Moonen, G., Schoenen, J., Schmitt, A.B., Noth, J., Nacimiento, W., 1998. Spontaneous longitudinally orientated axonal regeneration is associated with the Schwann cell framework within the lesion site following spinal cord compression injury of the rat. J. Neurosci. Res. 53, 51–65. Bunge, R.P., Puckett, W.R., Becerra, J.L., Marcillo, A., Quencer, R.M., 1993. Observations on the pathology of human spinal cord injury. A review and classification of 22 new cases with details from a case of chronic cord compression with extensive focal demyelination. Adv. Neurol. 59, 75–89. Buss, A., Schwab, M.E., 2003. Sequential loss of myelin proteins during Wallerian degeneration in the rat spinal cord. Glia 42, 424–432. Buttini, M., Mir, A., Appel, K., Wiederhold, K.H., Limonta, S., Gebicke-Haerter, P.J., Boddeke, H.W., 1997. Lipopolysaccharide induces expression of tumour necrosis factor alpha in rat brain: inhibition by methylprednisolone and by rolipram. Br. J. Pharmacol. 122, 1483–1489. Cao, Q., Zhang, Y.P., Iannotti, C., DeVries, W.H., Xu, X.M., Shields, C.B., Whittemore, S.R., 2005. Functional and electrophysiological changes after graded traumatic spinal cord injury in adult rat. Exp. Neurol. 191, S3–16. David, S., Lacroix, S., 2003. Molecular approaches to spinal cord repair. Annu. Rev. Neurosci. 26, 411–440. Demjen, D., Klussmann, S., Kleber, S., Zuliani, C., Stieltjes, B., Metzger, C., Hirt, U.A., Walczak, H., Falk, W., Essig, M., Edler, L., Krammer, P.H., Martin-Villalba, A., 2004. Neutralization of CD95 ligand promotes regeneration and functional recovery after spinal cord injury. Nat. Med. 10, 389–395. Deumens, R., Koopmans, G.C., Joosten, E.A.J., 2005. Regeneration of descending axon tract after spinal cord injury. Prog. Neurobiol. 77, 57–89. Donnnelly, P.G., Popovich, P.G., 2008. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Exp. Neurol. 209, 378–388.

497

Eidelberg, E., Straehley, D., Erspamer, R., Watkins, C.J., 1977. Relationship between residual hindlimb-assisted locomotion and surviving axons after incomplete spinal cord injuries. Exp. Neurol. 56, 312–322. Fitch, M.T., Doller, C., Combs, C.K., Landreth, G.E., Silver, J., 1999. Cellular and molecular mechanisms of glial scarring and progressive cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma. J. Neurosci. 19, 8182–8198. Fleischhacker, W.W., Hinterhuber, H., Bauer, H., Pflug, B., Berner, P., Simhandl, C., Wolf, R., Gerlach, W., Jaklitsch, H., Sastre-y-Hernandez, M., 1992. A multicenter doubleblind study of three different doses of the new cAMP-phosphodiesterase inhibitor rolipram in patients with major depressive disorder. Neuropsychobiology 26, 59–64. Fujimaki, K., Morinobu, S., Duman, R.S., 2000. Administration of a cAMP phosphodiesterase 4 inhibitor enhances antidepressant-induction of BDNF mRNA in rat hippocampus. Neuropsychopharmacology 22, 42–51. Guth, L., Zhang, Z., Roberts, E., 1994. Key role for pregnenolone in combination therapy that promotes recovery after spinal cord injury. Proc. Natl. Acad. Sci. 91, 12308–12312. Genovese, T., Mazzon, E., Crisafulli, C., Di Paola, R., Muia, C., Bramanti, P., Cuzzocrea, S., 2006. Immunomodulatory effects of etanercept in an experimental model of spinal cord injury. J. Pharmacol. Exp. Ther. 316, 1006–1016. Genovese, T., Mazzona, E., Crisafulli, C., Esposito, E., Di Paola, R., Muira, C., Di Bella, P., Meli, R., Bramanti, P., Cuzzocrea, S., 2007. Combination of dexamethasone and enteracept reduces secondary damage in experimental spinalcord trauma. J. Neurosci. 150, 168–181. George, A., Marziniak, M., Schafers, M., Toyka, K.V., Sommer, C., 2000. Thalidomide treatment in chronic constrictive neuropathy decreases endoneurial tumor necrosis factor-alpha, increases interleukin-10 and has long-term effects on spinal cord dorsal horn met-enkephalin. Pain 88, 267–275. Gorio, A., Gokmen, N., Erbayraktar, S., Yilmaz, O., Madaschi, L., Cichetti, C., Di Giulio, A.M., Vardar, E., Cerami, A., Brines, M., 2002. Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc. Natl. Acad. Sci. 99, 9450–9455. Green, B.A., Wagner Jr., F.C., 1973. Evolution of edema in the acutely injured spinal cord: a fluorescence microscopic study. Surg. Neurol. 1, 98–101. Gruner, J.A., 1992. A monitored contusion model of spinal cord injury in the rat. J. Neurotrauma 9, 123–128. Hamers, F.P., Lankhorst, A.J., van Laar, T.J., Veldhuis, W.B., Gispen, W.H., 2001. Automated quantitative gait analysis during overground locomotion in the rat: its application to spinal cord contusion and transection injuries. J. Neurotrauma 18, 187–201. Hamers, F.P., Koopmans, G.C., Joosten, E.A., 2006. CatWalk-assisted gait analysis in the assessment of spinal cord injury. J. Neurotrauma 23, 537–548. Haslett, P.A., Roche, P., Butlin, C.R., Macdonald, M., Shrestha, N., Manandhar, R., Lemaster, J., Hawksworth, R., Shah, M., Lubinsky, A.S., Albert, M., Worley, J., Kaplan, G., 2005. Effective treatment of erythema nodosum leprosum with thalidomide is associated with immune stimulation. J. Infect. Dis. 192, 2045–2053. Hausmann, O.N., 2003. Post-traumatic inflammation following spinal cord injury. Spinal Cord 41, 369–378. Hugenholtz, H., Cass, D.E., Dvorak, M.F., Fewer, D.H., Fox, R.J., Izukawa, D.M., Lexchin, J., Tuli, S., Bharatwal, N., Short, C., 2002. High-dose methylprednisolone for acute closed spinal cord injury—only a treatment option. Can. J. Neurol. Sci. 29, 227–235. Jordan, L.M., 1998. Initiation of locomotion in mammals. Ann. N.Y. Acad. Sci. 860, 83–93. Kim, Y.S., Kim, J.S., Jung, H.C., Song, I.S., 2004. The effects of thalidomide on the stimulation of NF-kappaB activity and TNF-alpha production by lipopolysaccharide in a human colonic epithelial cell line. Mol. Cells 17, 210–216. King, V.R., Averill, S.A., Hewazy, D., Priestley, J.V., Torup, L., Michael-Titus, A.T., 2007. Erythropoietin and carbamylated erythropoietin are neuroprotective following spinal cord hemisection in the rat. Eur. J. Neurosci. 26, 90–100. Klusman, I., Schwab, M.E., 1997. Effects of pro-inflammatory cytokines in experimental spinal cord injury. Brain Res. 762, 173–184. Koopmans, G.C., Deumens, R., Honig, W.M., Hamers, F.P., Steinbusch, H.W., Joosten, E.A., 2005. The assessment of locomotor function in spinal cord injured rats: the importance of objective analysis of coordination. J. Neurotrauma 22, 214–225. Koopmans, G.C., Brans, M., Gomez-Pinilla, F., Duis, S., Gispen, W.H., Torres-Aleman, I., Joosten, E.A., Hamers, F.P., 2006. Circulating insulin-like growth factor I and functional recovery from spinal cord injury under enriched housing conditions. Eur. J. Neurosci. 23, 1035–1046. Knoblach, S.M., Faden, A.I., 1998. Interleukin-10 improves outcome and alters proinflammatory cytokine expression after experimental traumatic brain injury. Exp. Neurol. 153, 143–151. Laffitte, E., 2004. Thalidomide: an old drug with new clinical implications. Expert Opin. Drug Safety 3, 47–56. Lankhorst, A.J., Duis, S.E., ter Laak, M.P., Joosten, E.A., Hamers, F.P., Gispen, W.H., 1999. Functional recovery after central infusion of alpha-melanocyte-stimulating hormone in rats with spinal cord contusion injury. J. Neurotrauma 16, 323–331. Lankhorst, A.J., ter Laak, M.P., van Laar, T.J., van Meeteren, N.L., de Groot, J.C., Schrama, L.H., Hamers, F.P., Gispen, W.H., 2001. Effects of enriched housing on functional recovery after spinal cord contusive injury in the adult rat. J. Neurotrauma 18, 203–215. Leskovar, A., Moriarty, L.J., Turek, J.J., Schoenlein, I.A., Borgens, R.B., 2000. The macrophage in acute neural injury: changes in cell numbers over time and levels of cytokine production in mammalian central and peripheral nervous systems. J. Exp. Biol. 203, 1783–1795. Loy, D.N., Talbott, J.F., Onifer, S.M., Mills, M.D., Burke, D.A., Dennison, J.B., Fajardo, L.C., Magnuson, D.S., Whittemore, S.R., 2002. Both dorsal and ventral spinal cord

498

G.C. Koopmans et al. / Experimental Neurology 216 (2009) 490–498

pathways contribute to overground locomotion in the adult rat. Exp. Neurol. 177, 575–580. Lucas, S.M., Rothwell, N.J., Gibson, R.M., 2006. The role of inflammation in CNS injury and disease. Br. J. Pharmacol. 147, S232–240. Majumdar, S., Lamothe, B., Aggarwal, B.B., 2002. Thalidomide suppresses NF-kappa B activation induced by TNF and H2O2, but not that activated by ceramide, lipopolysaccharides, or phorbol ester. J. Immunol. 168, 2644–2651. Mulcahy, N.J., Ross, J., Rothwell, N.J., Loddick, S.A., 2003. Delayed administration of interleukin-1 receptor antagonist protects against transient cerebral ischaemia in the rat. Br. J. Pharmacol. 140, 471–476. Meierhofer, C., Wiedermann, C.J., 2003. New insights into the pharmacological and toxicological effects of thalidomide. Curr. Opin. Drug Discov. Dev. 6, 92–99. Nikulina, E., Tidwell, J.L., Dai, H.N., Bregman, B.S., Filbin, M.T., 2004. The phosphodiesterase inhibitor rolipram delivered after a spinal cord lesion promotes axonal regeneration and functional recovery. Proc. Natl. Acad. Sci. 101, 8786–8790. O'Donnell, J.M., Zhang, H.T., 2004. Antidepressant effects of inhibitors of cAMP phosphodiesterase (PDE4). Trends Pharmacol. Sci. 25, 158–163. Osterholm, J.L., 1974. The pathophysiological response to spinal cord injury. The current status of related research. J. Neurosurg. 40, 5–33. Oudega, M., Vargas, C.G., Weber, A.B., Kleitman, N., Bunge, M.B., 1999. Long-term effects of methylprednisolone following transection of adult rat spinal cord. Eur. J. Neurosci. 11, 2453–2464. Pearse, D.D., Pereira, F.C., Marcillo, A.E., Bates, M.L., Berrocal, Y.A., Filbin, M.T., Bunge, M.B., 2004. cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Nat. Med. 10, 610–616. Penkowa, M., Moos, T., Carrasco, J., Hadberg, H., Molinero, A., Bluethmann, H., Hidalgo, J., 1999. Strongly compromised inflammatory response to brain injury in interleukin6-deficient mice. Glia 25, 343–357. Popovich, P.G., Guan, Z., Wei, P., Huitinga, I., van Rooijen, N., Stokes, B.T., 1999. Depletion of hematogenous macrophages promotes partial hindlimb recovery and neuroanatomical repair after experimental spinal cord injury. Exp. Neurol. 158, 351–365. Ribeiro, R.A., Vale, M.L., Ferreira, S.H., Cunha, F.Q., 2000. Analgesic effect of thalidomide on inflammatory pain. Eur. J. Pharmacol. 391, 97–103. Sayer, F.T., Kronvall, E., Nilsson, O.G., 2006. Methylprednisolone treatment in acute spinal cord injury: the myth challenged through a structured analysis of published literature. Spine J. 6, 335–343. Schucht, P., Raineteau, O., Schwab, M.E., Fouad, K., 2002. Anatomical correlates of locomotor recovery following dorsal and ventral lesions of the rat spinal cord. Exp. Neurol. 176, 143–153. Schwab, M.E., Bartholdi, D., 1996. Degeneration and regeneration of axons in the lesioned spinal cord. Physiol. Rev. 76, 319–370. Schwab, J.M., Brechtel, K., Mueller, C.A., Failli, V., Kaps, H.P., Tuli, S.K., Schluesener, H.J., 2006. Experimental strategies to promote spinal cord regeneration—an integrative perspective. Prog. Neurobiol. 78, 91–116. Scott, A.I., Perini, A.F., Shering, P.A., Whalley, L.J., 1991. In-patient major depression: is rolipram as effective as amitriptyline? Eur. J. Clin. Pharmacol. 40, 127–129. Senter, H.J., Venes, J.L., 1978. Altered blood flow and secondary injury in experimental spinal cord trauma. J. Neurosurg. 49, 569–578. Settles, B., Stevenson, A., Wilson, K., Mack, C., Ezell, T., Davis, M.F., Taylor, L.D., 2001. Down-regulation of cell adhesion molecules LFA-1 and ICAM-1 after in vitro treatment with the anti-TNF-alpha agent thalidomide. Cell. Mol. Biol. 47, 1105–1114. Spencer, M.T., Bazarian, J.J., 2003. Evidence-based emergency medicine/systematic review abstract. Are corticosteroids effective in traumatic spinal cord injury? Ann. Emerg. Med. 41, 410–413.

Takami, T., Oudega, M., Bethea, J.R., Wood, P.M., Kleitman, N., Bunge, M.B., 2002. Methylprednisolone and interleukin-10 reduce gray matter damage in the contused Fischer rat thoracic spinal cord but do not improve functional outcome. J. Neurotrauma 19, 653–666. Tartaglia, L.A., Rothe, M., Hu, Y.F., Goeddel, D.V., 1993. Tumor necrosis factor's cytotoxic activity is signaled by the p55 TNF receptor. Cell 73, 213–216. Tator, C.H., Fehlings, M.G., 1991. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J. Neurosurg. 75, 15–26. Thome, J., Duman, R.S., Henn, F.A., 2002. Molecular aspects of antidepressive therapy. Transsynaptic effects on signal transduction, gene expression and neuronal plasticity. Nervenarzt 73, 595–599. Thuret, S., Moon, L.D., Gage, F.H., 2006. Therapeutic interventions after spinal cord injury. Nat. Rev. Neurosci. 7, 628–643. Villahermosa, L.G., Fajardo Jr., T.T., Abalos, R.M., Balagon, M.V., Tan, E.V., Cellona, R.V., Palmer, J.P., Wittes, J., Thomas, S.D., Kook, K.A., Walsh, G.P., Walsh, D.S., 2005. A randomized, double-blind, double-dummy, controlled dose comparison of thalidomide for treatment of erythema nodosum leprosum. Am. J. Trop. Med. Hyg. 72, 518–526. Wachtel, H., 1983. Potential antidepressant activity of rolipram and other selective cyclic adenosine 3′,5′-monophosphate phosphodiesterase inhibitors. Neuropharmacology 22, 267–272. Wang, C.X., Nuttin, B., Heremans, H., Dom, R., Gybels, J., 1996. Production of tumor necrosis factor in spinal cord following traumatic injury in rats. J. Neuroimmunol. 69, 151–156. Witkamp, R., Monshouwer, M., 2000. Signal transduction in inflammatory processes, current and future therapeutic targets: a mini review. Vet. Q. 22, 11–16. Whitaker, C.M., Beaumont, E., Wells, M.J., Magnuson, D.S., Hetman, M., Onifer, S.M.m., 2008. Rolipram attenuates acute oligodendrocyte death in the adult rat ventrolateral funiculus following contusive cervical spinal cord injury. Neurosci. Lett. 438, 200–204. Wu, J.J., Huang, D.B., Pang, K.R., Hsu, S., Tyring, S.K., 2005. Thalidomide: dermatological indications, mechanisms of action and side effects. Br. J. Dermatol. 153, 223–254. Xu, J., Fan, G., Chen, S., Wu, Y., Xu, X.M., Hsu, C.Y., 1998. Methylprednisolone inhibition of TNF-alpha expression and NF-kB activation after spinal cord injury in rats. Brain Res. Mol. Brain Res. 59, 135–142. Yakovlev, A.G., Faden, A.I., 1994. Sequential expression of c-fos protooncogene, TNFalpha, and dynorphin genes in spinal cord following experimental traumatic injury. Mol. Chem. Neuropathol. 23, 179–190. Yan, P., Li, Q., Kim, G.M., Xu, J., Hsu, C.Y., Xu, X.M., 2001. Cellular localization of tumor necrosis factor-alpha following acute spinal cord injury in adult rats. J. Neurotrauma 18, 563–568. Yang, L., Blumbergs, P.C., Jones, N.R., Manavis, J., Sarvestani, G.T., Ghabriel, M.N., 2004. Early expression and cellular localization of proinflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in human traumatic spinal cord injury. Spine 29, 966–971. Yennurajalingam, S., Peuckmann, V., Bruera, E., 2004. Recent developments in cancer pain assessment and management. Support. Cancer Ther. 1, 97–110. Yoshikawa, M., Suzumura, A., Tamaru, T., Takayanagi, T., Sawada, M., 1999. Effects of phosphodiesterase inhibitors on cytokine production by microglia. Mult. Scler. 5, 126–133. Young, W., 1993. Secondary injury mechanisms in acute spinal cord injury. J. Emerg. Med. 11, S13–22. Zhu, J., Mix, E., Winblad, B., 2001. The antidepressant and antiinflammatory effects of rolipram in the central nervous system. CNS Drug Rev. 7, 387–398.