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NEUROPHARMACOLOGY
Agonists Prevents Neuronal Damage, Motor Dysfunction, Myelin Loss, Neuropathic Pain, and Inflammation after Spinal Cord Injury in Adult Rats
Department of Neurological Surgery (S.-W.P., J.-H.Y., G.M., I.S., K.B., D.K.R., R.V.), Neuroscience Training Program (R.V.), Cardiovascular Research Center (R.V.), and Regenerative Medicine Program (R.V.), University of Wisconsin, Madison, Wisconsin; and Department of Neurological Surgery, Chung-Ang University, Seoul, Korea (S.-W.P.)
Received September 6, 2006; accepted December 11, 2006.
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Abstract |
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(PPAR). TZDs were shown to induce neuroprotection after cerebral ischemia by blocking inflammation. As spinal cord injury (SCI) induces massive inflammation that precipitates secondary neuronal death, we currently analyzed the therapeutic efficacy of TZDs pioglitazone and rosiglitazone after SCI in adult rats. Both pioglitazone and rosiglitazone (1.5 mg/kg i.p.; four doses at 5 min and 12, 24, and 48 h) significantly decreased the lesion size (by 57 to 68%, p < 0.05), motor neuron loss (by 3- to 10-fold, p < 0.05), myelin loss (by 66 to 75%, p < 0.05), astrogliosis (by 46 to 61%, p < 0.05), and microglial activation (by 59 to 78%, p < 0.05) after SCI. TZDs significantly enhanced the motor function recovery (at 7 days after SCI, the motor scores were 37 to 45% higher in the TZD groups over the vehicle group; p < 0.05), but the treatment was effective only when the first injection was given by 2 h after SCI. At 28 days after SCI, chronic thermal hyperalgesia was decreased significantly (by 31 to 39%; p < 0.05) in the pioglitazone group compared with the vehicle group. At 6 h after SCI, the pioglitazone group showed significantly less induction of inflammatory genes [interleukin (IL)-6 by 83%, IL-1
by 87%, monocyte chemoattractant protein-1 by 75%, intracellular adhesion molecule-1 by 84%, and early growth response-1 by 67%] compared with the vehicle group (p < 0.05 in all cases). Pioglitazone also significantly enhanced the post-SCI induction of neuroprotective heat shock proteins and antioxidant enzymes. Pretreatment with a PPAR antagonist, 2-chloro-5-nitro-N-phenyl-benzamide (GW9662), prevented the neuroprotection induced by pioglitazone.
). PPAR exists as three isoforms (
, , and 
/) that control many cellular functions including lipid metabolism, glucose absorption, and cell growth and differentiation (Escher and Wahli, 2000). 15-Deoxy-
-12,14-prostaglandin J2 (15-d-PGJ2) is the natural agonist and thiazolidinediones (TZDs) (troglitazone, ciglitazone, rosiglitazone, and pioglitazone) are potent synthetic agonists of PPAR. Of these, troglitazone was removed from the market because of hepatotoxicity, whereas rosiglitazone and pioglitazone are currently approved by the United States Food and Drug Administration for type 2 diabetes treatment (Tolman and Chandramouli, 2003). PPAR agonists were also shown to prevent inflammation associated with gut, myocardial, and lung ischemia (Michalik and Wahli, 2006). More recently, PPAR agonists were shown to prevent inflammation and neuronal death after focal cerebral ischemia in rodents (Shimazu et al., 2005; Sundararajan et al., 2005; Zhao et al., 2006; Tureyen et al., 2007).
Every year, thousands of individuals have a spinal cord injury (SCI), and many die within days because of the severity of their injury. Most of the survivors are paralyzed and require use of a wheelchair. Inflammation that starts within minutes and continues for days after the injury is known to significantly contribute to the secondary neuronal damage, which is a major cause of motor dysfunction after SCI in rodents (Jones et al., 2005). Accordingly, anti-inflammatory therapies were shown to be neuroprotective after SCI in rodents (Stirling et al., 2004; Isaksson et al., 2005; Sribnick et al., 2005). In addition, SCI-induced neuronal damage was observed to be less severe in knockout mice that lack intracellular adhesion molecule-1 (ICAM-1), P-selectin, tumor necrosis factor receptor, and nuclear factor-
B (NF-B) (Farooque et al., 1999; Kim et al., 2001; Brambilla et al., 2005).
Controlling cerebral inflammation at the level of transcription is an effective strategy for preventing secondary neuronal damage after CNS injury. Thus, we tested the efficacy of rosiglitazone and pioglitazone to prevent inflammation, neuronal death, motor dysfunction, and neuropathic pain after SCI in adult rats.
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Materials and Methods |
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Spinal Cord Injury. A moderate-grade SCI was induced in adult Sprague-Dawley rats (300 
325 g) with the Multicenter Animal Spinal Cord Injury Study impactor as described earlier (Song et al., 2001). In brief, under halothane anesthesia [induction, 4%; maintenance, 2% in an oxygen and nitrous oxide (50:50) mixture], a T9 laminectomy was performed, and the spinal cord was injured by dropping a 10-g weight from a height of 12.5 mm. Sham-operated controls were laminectomized but not contused. The incisions were closed, and the rats were returned to their cages after recovering from anesthesia. Throughout the procedure, body temperature was maintained at 37°C with a heating pad. Injured rats underwent manual bladder expression until reflexive bladder emptying was established.
Drug Administration. Potassium salts of pioglitazone and rosiglitazone (Cayman Chemical, Ann Arbor, MI) were dissolved in phosphate-buffered saline (PBS) (pH 7.2) and administered i.p. at a dose of 0.5, 1.5, or 3 mg/kg at various time points. In some experiments, the rats were pretreated with the PPAR antagonist GW9662 (2 mg/kg i.p.) dissolved in 3% dimethyl sulfoxide. Control groups were injected with either PBS, 3% dimethyl sulfoxide, or both, based on the experiment. We used several groups of rats and different drug administration schedules and sacrifice times to measure various outcome parameters. The details are given for each set of experiment under Results. In all the experiments, drug administrations were randomized and blinded.
Motor Function Assessment. Motor function recovery after SCI was studied with the Basso-Beattie-Bresnahan (BBB) scoring system that uses a 21-point open-field locomotor scale as described previously (Basso et al., 1996). On days 1, 3, 7, 14, 21, 28, 35, and 42 (or up to the day the animal was killed), each rat was placed in a 3-foot diameter circular plastic molded tub (rats were acclimatized to the observation tub for 2 days before injury), and the movements were scored for 4 min by two evaluators blinded to the study groups. The score for each animal was computed as the average of the scores of the two evaluators.
Histopathology. Rats were deeply anesthetized with halothane and subjected to transcardiac perfusion with 0.1 M PBS followed by 4% paraformaldehyde in PBS (pH 7.4). The spinal cord was dissected from the spinal canal, and the segment covering the injury epicenter and rostral and caudal areas was harvested. The spinal cords were postfixed, cryoprotected, and serially sectioned (each section was 35 µm thick). From each rat, 40 to 46 serial sections (360 µm apart) covering the injury were stained with thionine (Nissl). Parallel sets of sections from each rat were stained to observe astrogliosis [polyclonal rabbit anti-cow glial fibrillary acidic protein (GFAP) antibody; Dako North America, Inc., Carpentaria, CA], reactive microglia (Campbell-Switzer staining method) (Campbell et al., 1987), and myelin (Luxol fast blue staining) using standard procedures. For each of these stains, 15 to 20 sections from each rat were used.
Lesion Length, Area, and Volume Assessment. The thionine-stained serial sections were microscopically analyzed with a Zeiss Axioplan2 stereomicroscope fitted with a charge-coupled device camera. Using the design-based, unbiased stereology software Stereo Investigator 6.5 (MicroBrightField, Inc., Williston, VT) we calculated the length, area, and volume of the lesion with the Cavelieri estimator, considering the section thickness and sectional interval. The lesion volume was delineated by its bounding surface, which is defined by a series of closed contours in the serial sections. The software reconstructed the injured spinal cords showing three-dimensional (3-D) structures from the two-dimensional images of the sequences of sections (see supplemental Figs. 1 and 2). The contours and structures in each spinal cord section (photographed with the microscope) were traced to reconstruct the stacked image for volumetric analysis. The sections were stacked by visually matching the outlines of adjacent sections. The central canal of the spinal cord was used as the fiducial point to align the sections with one another. For each rat, the lesion area was computed using the sections from the epicenter, 1, 2, 3, and 4 mm rostral and 1, 2, 3, and 4 mm caudal to epicenter. The epicenter of injury was characterized by a peripheral and in some cases incomplete rim of residual, hypomyelinated white matter. The residual rim surrounded a central lesion that consisted of cavities and a loose network of non-neuronal cells. Using Stereo Investigator 6.5, we estimated the volume of myelin loss with the Cavelieri estimator, considering the section thickness and sectional interval. The Luxol fast blue-stained serial sections of the three groups (vehicle-, rosiglitazone-, and pioglitazone-treated) photographed microscopically were used for these estimations.
Cell Counting. The numbers of surviving ventral horn motor neurons were counted in three 300x fields using the thionine-stained sections from epicenter, 1, 2, and 3 mm rostral, and 1, 2, and 3 mm caudal areas of each rat in four sequential sections in each case using Stereo Investigator 6.5. The GFAP-positive astrocytes and the Campbell-Switzer stain-positive reactive microglia were counted in the ventral horn of the three groups in the epicenter, 3 mm rostral, and 3 mm caudal segments in three 300x fields in each case using Stereo Investigator 6.5.
Assessment of Thermal Sensitivity. Withdrawal of a hind paw from a movable radiant heat source was shown previously to be a sensitive and reproducible test for neuropathic pain (Mitsui et al., 2005). The latency of hind paw withdrawal to thermal noxious stimulus was measured in rats subjected to SCI as described previously (DomBourian et al., 2006). In brief, an animal was placed inside the plastic cabinet of the Plantar Test Apparatus (UGO Basile Biological Research Apparatus, Comerio, Italy) with a movable focused beam of radiant heat positioned under the animal's hind paw. A photocell automatically turned the heat off as soon as the animal moved its paw and the latency time for the animal to withdraw its paw was recorded. Strength of stimulation was adjusted to produce a baseline latency of 8 to 9 s (typically 5258°C). A safety cutoff (
20 s) was used to prevent prolonged exposure to the noxious heat. Hind limb thermal hyperalgesia testing was performed 1 day before the injury to establish the animal's baseline and on postinjury day 28. To avoid a training effect, different groups of animals were tested randomly. A decrease in withdrawal latency of 
3 s was considered as the presence of neuropathic pain.
Real-Time PCR Analysis. The mRNA levels of the following transcripts were estimated using real-time PCR as described earlier (Vemuganti et al., 2006): interleukin (IL)-1, IL-6, ICAM-1, monocyte chemoattractant protein-1 (MCP-1), interferon regulatory factor-1 (IRF-1), NF-B, early growth response-1 (Egr-1), catalase, glutathione peroxidase (GPx), heat shock protein (HSP) 70, HSP27, and heme oxygenase-1 (HO-1)/HSP32. Cohorts of rats subjected to SCI and treated with either vehicle or pioglitazone (1.5 mg at 5 min, 6 h, and 12 h; n = 4/group) were sacrificed at 24 h. A separate cohort of rats subjected to SCI and treated with vehicle or pioglitazone (1.5 mg at 5 min and 3 h; n = 4/group) were sacrificed at 6 h. From each rat, total RNA was extracted from the segment 1 mm rostral to the injury epicenter using the TRIzol reagent (Invitrogen, Carlsbad, CA). RNA (1 µg) from each sample was reverse-transcribed with oligo(dT)15 and random hexamer primers using Maloney murine leukemia virus reverse transcriptase (Invitrogen, Rockville, MD). Ten nanograms of cDNA and gene-specific primers were added to SYBR Green PCR Master Mix (SYBR Green I Dye, AmpliTaq DNA polymerase, dNTPs with dUTP, and optimal buffer components; Applied Biosystems, Foster City, CA) and subjected to PCR amplification in a Perkin-Elmer TaqMan 5700 Sequence Detection System (1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, and 40 cycles at 95°C for 15 s and 60°C for 1 min). PCR reactions were conducted in duplicate. The amplified transcripts were quantified with the comparative CT method using 18S rRNA as the internal control as described earlier (Vemuganti et al., 2006). The real-time PCR primers were designed using Primer Express software (Applied Biosystems) on the basis of the GenBank accession numbers given in Table 1. The primer sequences were same as those used previously by our laboratory (Song et al., 2001; Vemuganti et al., 2006).
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Statistical Analysis. The data are expressed as means ± S.D. Comparisons among groups were performed by Student's t test or a one-way analysis of variance (ANOVA) followed by a Tukey-Kramer multiple comparisons post-test.
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16%) in blood glucose levels (vehicle group: 91 ± 7 mg/dl, rosiglitazone group: 85 ± 8 mg/dl, and pioglitazone group: 87 ± 7 mg/dl; n = 9/group), although there were no statistically significant difference between the three groups. At 3 days after SCI, the blood glucose levels returned to the preinjury range in all three groups. The decrease in blood glucose at 24 h is understandable as rats consume less chow in the 1st day after SCI.
TZD Treatment Increased the Neuronal Survival after SCI. The numbers of surviving ventral horn motor neurons at 1 week after SCI were counted microscopically in the sections from epicenter, 1, 2, and 3 mm rostral, and 1, 2, and 3 mm caudal to the injury (n = 9 rats/group). Four sections at each level were used for each animal. The mean numbers of surviving neurons were significantly higher in the rosiglitazone/SCI and pioglitazone/SCI groups than in the vehicle/SCI group at 1 mm rostral (by 10-fold; p < 0.05), at 2 mm rostral (by 6-fold; p < 0.05), at 3 mm rostral (by 3.5-fold, p < 0.05), and 3 mm caudal (by 2.5-fold; p < 0.05) levels (Fig. 2). The pioglitazone/SCI group also showed a significantly higher neuronal number at the 2 mm caudal level compared with vehicle/SCI group (by 1.8-fold; p < 0.05) (Fig. 2).
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PPAR Agonists Decreased Astrogliosis, Microglial Activation, and Myelin Loss. The vehicle/SCI group showed several GFAP-positive swollen astrocytes and reactive microglia (stained with Campbell-Switzer stain) in the ventral horn 1 to 3 mm rostral and caudal to the injury epicenter at 7 days after the injury (Fig. 4). The rosiglitazone/SCI and pioglitazone/SCI groups showed significantly fewer numbers of GFAP-positive astrocytes (by 46 to 61%, p < 0.05) and activated microglia (by 59 to 78%; p < 0.05) compared with vehicle/SCI group (n = 9/group) (Fig. 4). The 1.5 mm rostral to injury area of the vehicle/SCI group also showed several infiltrated macrophages (ED-1-positive and Iba-1-positive) in the spinal cord parenchyma, and the pioglitazone/SCI group showed significantly decreased numbers of both ED-1-positive (by 74 to 86%; p < 0.05; n = 6/group) and Iba-1-positive (by 69 to 81%; p < 0.05; n = 6/group) cells (Fig. 5). The rosiglitazone/SCI and pioglitazone/SCI groups also showed more myelin preservation compared with the vehicle group in the areas 1 to 3 mm rostral and caudal to the injury epicenter at 7 days after injury (Fig. 6). The volume of the myelin loss computed by Stereo Investigator 6.5 using the Luxol fast blue-stained serial sections was observed to be 13.98 ± 3.84 mm3 in the vehicle/SCI group (n = 9). The myelin loss was significantly lower (by 66 to 75%, p < 0.05) in the pioglitazone/SCI (3.49 ± 0.81 mm3) and rosiglitazone/SCI (4.83 ± 0.97 mm3) groups compared with the vehicle/SCI group (n = 9/group).
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Long-Term Motor Function Recovery by Pioglitazone. In the experiments described above, the BBB scores were estimated only up to 1 week after SCI (as rats were sacrificed at that time for histopathological analysis). To determine whether PPAR agonist treatment induces a sustained improvement in motor function, we measured BBB scores for 6 weeks in a cohort of SCI rats injected with vehicle or pioglitazone (1.5 mg/kg at 5 min, 12 h, 24 h, and 48 h; n = 6/group). Similar to the first study, the pioglitazone/SCI group showed significantly higher BBB scores on days 3 and 7 compared with the vehicle/SCI group (Fig. 7A). In addition, the BBB score was significantly higher at all weeks in the pioglitazone group than that in the vehicle group (Fig. 7A). This result shows that the benefit of PPAR activation is not transient and indicates that preventing inflammation (assuming PPAR is acting by controlling inflammation) in the acute phase after SCI leads to long-term motor benefits. A third group of rats subjected to SCI (n = 6) was treated with the PPAR antagonist GW9662 (2 mg/kg) (four doses given 1 h before each pioglitazone injection) followed by pioglitazone (same schedule as above). In the GW9662 plus pioglitazone group, the motor function recovery was not significantly different from that in the vehicle group at any time point studied (3 42 days) (Fig. 7A), indicating that the effects of pioglitazone after SCI were mediated by direct activation of PPAR. A fourth group of rats injected with GW9662 alone (four injections of 2 mg/kg; n = 6) showed no significant difference in the BBB scores compared with the vehicle control group (Fig. 7A). In these cohorts of rats sacrificed at 6 weeks after SCI, the lesion shrunk in size to 
of that observed at 1 week, but the lesion volume was significantly smaller (by 53%, p < 0.05) in the pioglitazone group compared with the vehicle group, whereas the GW9662 plus pioglitazone group showed a lesion size similar to that in vehicle group (data not shown).
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). We tested the effect of PPAR stimulation on neuropathic pain using the three groups of rats (vehicle, pioglitazone, and GW9662 plus pioglitazone; n = 6/group) on day 28 after SCI. These are the same groups of rats used for the long-term motor function recovery experiments described above. In plantar thermal hyperalgesia testing, rats withdrew the palm of the hind leg from the heated surface (55 ± 3°C) in 8.37 ± 1.41 s (preinjury baseline; all three groups; n = 21). At 28 days after SCI, the latency to withdraw the palm decreased to 5.62 ± 0.69 s in the vehicle/SCI group (p < 0.05 versus baseline) (Fig. 7B), whereas the pioglitazone/SCI group showed no statistically significant thermal latency compared with the baseline (Fig. 7B). The GW9662 plus pioglitazone/SCI group showed a thermal latency of 5.41 ± 0.64 s (p < 0.05 versus baseline), which is similar to that in the vehicle/SCI group (Fig. 7B). In the group of rats treated with GW9662 alone, there was no significant difference in the post-SCI pain perception compared with that in the vehicle group (Fig. 7B).
Therapeutic Window of Pioglitazone Neuroprotection after SCI. To identify the effective time of TZD action, a cohort of rats subjected to SCI were divided into four groups (n = 6 rats/group) and groups 1 to 3 were injected with pioglitazone. The first dose was given at 5 min to group 1, at 2 h to group 2, and at 4 h to group 3. The remaining three doses were given at 12, 24, and 48 h to all three groups. Group 4 rats were injected with vehicle at 5 min and 12, 24, and 48 h. As in the previous experiments, the motor function recovery started earlier in the three pioglitazone groups than in the vehicle group (Fig. 8A). The pioglitazone groups showed higher BBB scores than the vehicle group at all time points studied (days 1, 3, 7, 14, 21, and 28 after SCI) (Fig. 8A). However, there was a gradual loss of neuroprotection when the first dose of pioglitazone was delayed (Fig. 8A). At all the time points, the BBB scores showed a significant improvement when the first dose of pioglitazone was administered either at 5 min or at 2 h after SCI compared with the vehicle group (Fig. 8A). The lesion volume was also significantly smaller in these two groups (by 63 ± 11% in the 5-min group and by 49 ± 7% in the 2-h group; both p < 0.05) compared with the vehicle group (Fig. 8B). When the first dose of pioglitazone was given at 4 h after SCI, neither the BBB score nor the lesion volume was significantly different from that for the vehicle group (Fig. 8, A and B).
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Pioglitazone Prevented Induction of Inflammatory Genes. Many inflammatory genes are known to be up-regulated in the acute phase after SCI. Real-time PCR analysis showed increased mRNA levels of the proinflammatory cytokines IL-6 and IL-1 (by 83.9- and 38.2-fold, respectively; p < 0.05), the chemokine MCP-1 (by 38.6 fold; p < 0.05), and the adhesion molecule ICAM-1 (by 17.1-fold; p < 0.05) at 24 h after SCI in the 1-mm rostral segment compared with sham-operated controls (Table 1). Pioglitazone treatment (1.5 mg/kg at 5 min and 12 h) significantly curtailed the induction of IL-6 (by 71%; p < 0.05), IL-1 (by 61%; p < 0.05), MCP-1 (by 46%; p < 0.05), and ICAM-1 (by 80%; p < 0.05) compared with vehicle treatment (Table 1). The vehicle/SCI group also showed significantly increased expression of the proinflammatory transcription factors IRF-1 (by 3.1-fold; p < 0.05), NF-B (by 7.8-fold; p < 0.05), and Egr-1 (by 21-fold; p < 0.05) (Table 1). The pioglitazone/SCI group showed significantly curtailed postinjury induction of IRF-1 (by 57%; p < 0.05), NF-B (by 50%; p < 0.05), and Egr-1 (by 43%; p < 0.05) compared with the vehicle/SCI group (Table 1). Previous studies from our laboratory showed that some of the inflammatory genes will be up-regulated as early as 3 to 6 h after SCI (Song et al., 2001). Hence, to understand whether prevention of inflammatory gene expression by pioglitazone treatment precedes the secondary neuronal death after SCI, expression of IL-1, IL-6, MCP-1, ICAM-1, and Egr-1 was analyzed in a cohort of rats sacrificed at 6 h after SCI (n = 8). Four of these rats were treated with pioglitazone (1.5 mg/kg at 5 min and 3 h) and the other four with vehicle. The pioglitazone/SCI group showed a significant curtailment of the expression of IL-1 (by 88%; p < 0.05), IL-6 (by 83%; p < 0.05), MCP-1 (by 75%; p < 0.05), ICAM-1 (by 84%; p < 0.05), and Egr-1 (by 67%; p < 0.05) compared with the vehicle/SCI group (Table 1). In a separate cohort of rats sacrificed at 1 day after SCI, pioglitazone treatment (1.5 mg/kg at 5 min and 12 h; n = 4) significantly decreased IL-1-immunopositive cells (by 82 to 88%; p < 0.05), ICAM-1-immunopositive capillaries (by 76 to 84%; p < 0.05), and MCP-1-immunopositive macrophage-like cells (by 83 to 91%; p < 0.05) compared with vehicle treatment (n = 4) (Fig. 5).
Pioglitazone Increased the Expression of Neuroprotective Genes. In the vehicle/SCI group, there was a significant induction of HSP27 (by 16.9-fold; p < 0.05), HSP70 (by 4.2-fold; p < 0.05), and HSP32/HO-1 (by 13.4-fold; p < 0.05) over that in sham-operated controls (Table 1). The pioglitazone/SCI group showed an enhanced expression of these three neuroprotective HSPs (HSP27 by 47%, HSP70 by 140%, and HO-1 by 67%; all p < 0.05) over that in the vehicle/SCI group. The antioxidant enzymes GPx and catalase also showed a significant induction by 2.2- and 2.9-fold, respectively, in the vehicle/SCI group over that in sham-operated controls (Table 1). In the pioglitazone/SCI group, there was a significantly higher induction of both GPx (by 92%, p < 0.05) and catalase (by 152%, p < 0.05) mRNA compared with that in the vehicle/SCI group (Table 1).
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Discussion |
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agonists (rosiglitazone and pioglitazone) decreases secondary neuronal damage, astrogliosis, microglial activation, myelin loss, and neuropathic pain and improves motor function recovery after SCI. Four injections of 1.5 mg/kg of either rosiglitazone or pioglitazone during the first 24 h after the injury induced good neuroprotection, but the treatment was effective only when the first injection was given by 2 h after SCI. Although a higher dose of 3.0 mg/kg pioglitazone induced neuroprotection similar to that with the 1.5 mg/kg dose, a lower dose of 0.5 mg/kg was relatively ineffective. Mechanistically, TZD treatment prevented the mRNA expression of the proinflammatory transcription factors, cytokines, chemokines, and adhesion molecules and promoted the mRNA expression of the neuroprotective HSPs and antioxidant enzymes after SCI.
Rosiglitazone and pioglitazone are United States Food and Drug Administration-approved to control blood glucose levels in millions of type 2 diabetics. TZDs were also reported to be beneficial in several neuroinflammatory conditions. In patients with early Alzheimer's disease, rosiglitazone promotes cognitive preservation (Watson et al., 2005). Rosiglitazone was also shown to attenuate learning and memory deficits in transgenic Alzheimer's mice (Pedersen et al., 2006). In animal models of amyotrophic lateral sclerosis (ALS) and Parkinson's disease, pioglitazone was shown to relieve the disease symptoms (Breidert et al., 2002; Schutz et al., 2005). Both pioglitazone and rosiglitazone were shown to decrease the infarct volume in rodent focal ischemia models (Shimazu et al., 2005; Sundararajan et al., 2005; Tureyen et al., 2007). The PPAR natural agonist 15-d-PGJ2 was shown to decrease neurological deficits after experimental intracerebral hemorrhage (Zhao et al., 2006) and disease severity after experimental autoimmune encephalopathy (Diab et al., 2004), and its plasma levels were shown to correlate to the neurological function in stroke patients (Blanco et al., 2005). These studies indicate that TZDs have significant therapeutic potential in neuroinflammatory disorders in addition to diabetes.
Regarding the window of opportunity for TZD-induced neuroprotection after SCI, starting treatment as early as possible after the injury is beneficial. This is understandable as the putative mechanism of PPAR-induced neuroprotection is minimizing the inflammation, which starts within minutes after SCI. After SCI, inflammation continues to increase in the 1st day and accordingly we observed that at least three injections of TZDs in the 1st day are essential for optimal neuroprotection. Both 1.5 and 3 mg/kg doses of pioglitazone induced similar neuroprotection, indicating that higher doses might not be necessary.
The elimination half-life of rosiglitazone and pioglitazone was known to be 3 to 7 h and was independent of dose (see http://www.fda.gov/medwatch/Safety/2003/03Jan_labels/Actos_PI.pdf and http://www.fda.gov/Medwatch/safety/2002/Avandia_hilite.pdf). Although the potential to cross the blood-brain barrier is a concern for a neuroprotective compound, most drugs can enter the CNS after insults like SCI and focal ischemia that are known to cause blood-brain barrier breakdown. Several previous studies also demonstrated the neuroprotective efficacy of TZDs in animal models of acute as well as chronic CNS insults including focal ischemia, Parkinson's disease, and ALS (Breidert et al., 2002; Schutz et al., 2005; Shimazu et al., 2005; Sundararajan et al., 2005; Zhao et al., 2006; Tureyen et al., 2007). A potential concern is the possible toxicity of rosiglitazone and pioglitazone at doses higher than those used presently. Although most of the experiments in the present study used a dose of 1.5 mg/kg of rosiglitazone or pioglitazone, a dose of 3 mg/kg that was tested in some experiments, and this dose resulted in no significant mortality. Furthermore, we recently tested both rosiglitazone and pioglitazone at a dose of 6 mg/kg (four times the neuroprotective dose used in the present study) in adult rats and observed no mortality or overt symptoms such as altered grooming or feeding behavior (Tureyen et al., 2007).
A previous in vitro study that used cultured microglial cells indicated that TZDs can act directly without activating PPAR under certain conditions (Park et al., 2003). However, we currently observed that pretreating animals with a PPAR antagonist GW9622 prevents pioglitazone-induced neuroprotection after SCI. Furthermore, recent studies showed that the in vivo neuroprotective actions of rosiglitazone after focal ischemia can be prevented by the GW9662 (Tureyen et al., 2007), and 15-d-PGJ2 causes a rapid activation of PPAR, which correlates with prevention of NF-B activation after intracerebral hemorrhage (Zhao et al., 2006). Thus, in vivo TZD neuroprotection after acute CNS insults might require activation of PPAR. On the basis of the studies using animal models of stroke, ALS, and Parkinson's disease, prevention of inflammation was thought to be the major mechanism underlying TZD neuroprotection (Hirsch et al., 2003; Sundararajan et al., 2005; Zhao et al., 2006; Tureyen et al., 2007). In support of this concept, we presently observed significant reduction in the post-SCI induction of several proinflammatory genes including chemokines, cytokines, and adhesion molecules in the pioglitazone-treated rats. We further show that TZDs act at the level of transcription by preventing post-SCI expression of proinflammatory transcription factors such as NF-B, Egr-1, and IRF-1, leading to effective curtailment of downstream inflammatory gene expression. We also observed that pioglitazone treatment curtailed the inflammatory gene expression even at an early stage of 6 h after SCI, indicating that prevention of inflammation is a contributing factor to the neuroprotection induced by TZDs.
Oxygen free radicals formed in excess during the acute phase after CNS injury mediate the secondary neuronal damage, and antioxidant enzymes such as catalase, GPx, and superoxide dismutase are the major defense mechanism by which free radicals will be metabolized to minimize oxidative neuronal damage. In a cell, the antioxidant enzymes are localized primarily within the peroxisomes, and hence PPAR agonists could efficiently induce their expression. Rosiglitazone and 15-d-PGJ2 were shown to induce the expression of catalase and Cu/Zn-superoxide dismutase in rodent brain (Shimazu et al., 2005; Zhao et al., 2006; Tureyen et al., 2007). We presently observed that pioglitazone treatment increases the post-SCI mRNA levels of catalase and GPx in the spinal cord area rostral to the injury, which showed better neuronal survival compared with the vehicle control. Thus, promotion of antioxidant mechanisms might be one of the underlying mechanisms of TZD-induced neuroprotection.
Previous GeneChip studies showed that SCI leads to significant up-regulation of several neuroprotective HSPs (Song et al., 2001). We presently observed that pioglitazone significantly enhanced the post-SCI induction of HSP70, HSP27, and HSP32/HO-1. By acting as chaperones, HSPs prevent aggregation and denaturation of many proteins. HSP70 is known to attenuate the increased glutamate release to minimize excitotoxic neuronal damage after CNS injury (Kelty et al., 2002). HSP27 is known to reduce reactive oxygen species levels, prevent apoptosis, and stabilize actin filaments in CNS (Mehlen et al., 1996; Guay et al., 1997). The stress-inducible astroglial isoform of heme oxygenase HSP32/HO-1 is known to prevent cell death by regulating cellular iron levels and to mediate the anti-inflammatory effects of interleukin-10 via a p38 mitogen-activated protein kinase pathway (Lee and Chau, 2002). As these HSPs (HSP70, HSP27, and HSP32/HO-1) are known to be neuroprotective, the beneficial effects of PPAR stimulation after SCI extend beyond preventing inflammation.
After SCI, both humans and animals develop chronic neuropathic pain (DomBourian et al., 2006; Waxman and Hains, 2006). Currently there are few therapeutic options to prevent the chronic pain after SCI. The present studies showed that pioglitazone significantly curtailed the development of thermal hyperalgesia, which was prevented by the PPAR antagonist GW9662. This indicates that in addition to inducing better motor function recovery and reduced inflammation, PPAR stimulation also helps in pain management after SCI. The molecular mechanisms underlying this response need to be elucidated.
We observed that the effective neuroprotective doses of rosiglitazone and pioglitazone after SCI are similar. This is puzzling as the Kd values for rosiglitazone and pioglitazone to PPAR are 45 and 500 nM, respectively. Although pioglitazone binds to PPAR with 10-fold lower affinity than rosiglitazone, pioglitazone has been shown to cross the blood-brain barrier and accumulate in brain more efficiently (Maeshiba et al., 1997). Hence, higher doses of rosiglitazone may be needed to achieve sufficient levels in CNS to provide neuroprotection. Furthermore, TZDs are known to exert some PPAR-independent effects, and in most cases pioglitazone was shown to be equally as or more effective than rosiglitazone (Feinstein et al., 2005). For example, TZDs can increase astrocyte glucose uptake and lactate production in a PPAR-independent manner (Dello Russo et al., 2003), effects that could contribute to the observed neuroprotective actions.
In conclusion, our studies show the neuroprotective potential of TZDs after SCI. We demonstrated that TZDs induce significantly better motor function recovery and decreased neuropathic pain after SCI. We also indicated that curtailing inflammation and inducing antioxidant enzymes and HSPs are potential mechanisms of TZD-induced neuroprotection. We also showed that after SCI, TZDs act directly by stimulating PPAR. A recent study showed that PPAR-knockout mice show less neutrophil infiltration and neuronal damage after SCI (Genovese et al., 2005). Currently several dual agonists that can activate both PPAR and PPAR are under development, and in the future these compounds might help prevent the detrimental effects of SCI by curtailing inflammation at the level of transcription.
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This work was presented in part at the 37th Annual Meeting of the American Society for Neurochemistry; 2006 Mar 1116; Portland, OR. American Society for Neurochemistry, Windemere, FL; Paris Anti-Inflammatory Drugs; 2005 Oct 6 7; Paris, France; and The Society for Neuroscience 35th Annual Meeting; 2005 Nov 1216; Washington, DC. Society for Neuroscience, Washington, DC.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; 15-d-PGJ2, 15-deoxy--12,14-prostaglandin J2; TZD, thiazolidinedione; SCI, spinal cord injury; ICAM-1, intracellular adhesion molecule-1; NF-B, nuclear factor-B; CNS, central nervous system; PBS, phosphate-buffered saline; GW9662, 2-chloro-5-nitro-N-phenyl-benzamide; BBB, Basso-Beattie-Bresnahan; GFAP, glial fibrillary acidic protein; 3-D, three-dimensional; PCR, polymerase chain reaction; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; IRF-1, interferon regulatory factor-1; Egr-1, early growth response-1; GPx, glutathione peroxidase; HSP, heat shock protein; HO-1, heme oxygenase-1; ANOVA, analysis of variance; Iba-1, ionized calcium-binding adapter molecule 1; ALS, amyotrophic lateral sclerosis.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Raghu Vemuganti, Department of Neurological Surgery, University of Wisconsin, K4/8 (Mail stop code CSC-8660), 600 Highland Ave., Madison WI 53792. E-mail: vemugant{at}neurosurg.wisc.edu
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