Annexin A1 Reduces Inflammatory Reaction and Tissue Damage Through Inhibition of Phospholipase A^Sub 2^ Activation in Adult Rats Following Spinal Cord Injury
By Liu, Nai-Kui Zhang, Yi Ping; Han, Shu; Pei, Jiong; Xu, Lisa Y; Lu, Pei-Hua; Shields, Christopher B; Xu, Xiao-Ming
Abstract Annexin A1 (ANXA1) has been suggested to be a mediator of the anti-inflammatory actions of glucocorticoids and more recently an endogenous neuroprotective agent. In the present study, we investigated the anti-inflammatory and neuroprotective effects of ANXA1 in a model of contusive spinal cord injury (SCI). Here we report that injections of ANXA1 (Ac 2-26) into the acutely injured spinal cord at 2 concentrations (5 and 20 [mu]g) inhibited SCI- induced increases in phospholipase A^sub 2^ and myeloperoxidase activities. In addition, ANXA1 administration reduced the expression of interleukin-1beta and activated caspase-3 at 24 hours, and glial fibrillary acidic protein at 4 weeks postinjury. Furthermore, ANXA1 administration significantly reversed phospholipase A^sub 2^- induced spinal cord neuronal death in vitro and reduced tissue damage and increased white matter sparing in vivo, compared to the vehicle-treated controls. Fluorogold retrograde tracing showed that ANXA1 administration protected axons of long descending pathways at 6 weeks post-SCI. ANXA1 administration also significantly increased the number of animals that responded to transcranial magnetic motor- evoked potentials. However, no measurable behavioral improvement was found after these treatments. These results, particularly the improvements obtained in tissue sparing and electrophysiologic measures, suggest a neuroprotective effect of ANXA1.
Key Words: Annexin A1, Inflammation, Neuroprotection, Phospholipase A^sub 2^, Spinal cord injury.
INTRODUCTION
There are 2 mechanisms of damage to the spinal cord after acute spinal cord injury (SCI): a primary mechanical injury and a secondary injury due to multiple damaging processes initiated by the primary injury. Mounting evidence suggests that post-traumatic inflammation plays a key role in the pathogenesis of secondary SCI (1-3). Clinically, methylprednisolone, a glucocorticoid and anti- inflammatory agent, is the recommended drug routinely administered after SCI in the United States. Annexin A1 (ANXA1, also named annexin I and lipocortin 1) is a member of the annexin family of calcium- and phospholipid-binding proteins (4, 5) that is suggested to be one of the “second messengers” mediating the anti- inflammatory actions of the glucocorticoids. This protein also plays a regulatory role in conditions such as cell growth regulation and differentiation, neutrophil migration, CNS response to cytokines, neuroendocrine secretion, and neurodegeneration (4-6).
ANXA1 may play a neuroprotective role in the CNS in response to injury. For example, central injection of ANXA1^sub 1-188^ (1 [mu]g) markedly inhibited neuronal death (by up to 70%) and edema induced by focal cerebral ischemia in rats, whereas injection of a neutralizing ANXA1 antiserum significantly exacerbated the damage (7). Neurodegeneration caused by pharmacologic activation of N- methyl-D-aspartate receptors (striatal infusions of an N-methyl-D- aspartate receptor agonist) also was significantly inhibited by ANXA1 fragment administration and exacerbated by injection of anti- ANXA1 antiserum (8). ANXA1 (Ac^sub 2-26^) inhibited lipopolysaccharide-induced expression of microglial cyclooxygenase- 2 and inducible nitric oxide synthase, the major isoforms expressed in activated inflammatory cells, including the microglia (9). The neuroprotective and anti-inflammatory effects of ANXA1 were demonstrated also in experimental autoimmune encephalomyelitis in rats (10) and in a gene knockout model in mice (11, 12), respectively.
ANXA1 is an endogenous inhibitor of phospholipase A^sub 2^ (PLA^sub 2^), a key enzyme responsible for inflammation and cytotoxicity. ANXA1 has been suggested to exert its potent anti- inflammatory effect through inhibiting the activity of PLA^sub 2^ (4, 5, 13, 14). Recently, we demonstrated that SCI resulted in a significant increase in PLA^sub 2^ activity (15). Moreover, administration of PLA^sub 2^ in vitro induced a dose-dependent spinal cord neuronal death and in vivo tissue damage in the spinal cord. Importantly, both effects could be substantially reversed by mepacrine, a PLA^sub 2^ inhibitor (15). These findings suggest that PLA^sub 2^ may be a novel therapeutic target for SCI. In the present study, we sought to determine whether ANXA1 played a neuroprotective role after SCI and, if so, whether its effect was mediated by the inhibition of PLA^sub 2^ activation.
MATERIALS AND METHODS
All of the chemicals used in this study were from Sigma Chemical (St. Louis, MO) except for those specifically indicated. Polyvinylidene diflouride membrane was purchased from Osmonics (Westborough, MA).
Animals
A total of 112 adult female Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing 210 to 230 g, were used in this study. Among them, 72 were randomly assigned into 4 groups (6 rats/group): 1) sham + vehicle; 2) SCI + vehicle; 3) SCI + 5 [mu]g of ANXA1 (Ac^sub 2-26^; Phoenix Pharmaceuticals, Inc., Belmont, CA); 4) SCI + 20 [mu]g of ANXA1 (Ac^sub 2-26^) for Western blot analysis of interleukin (IL)-1beta and active caspase-3 expressions and myeloperoxidase (MPO) and PLA^sub 2^ activities. Forty rats were used for behavioral (Basso, Beattie, Bresnahan [BBB] locomotor rating scale and grid walking), electrophysiologic, and histologic analyses.
Spinal Cord Injury
Spinal cord contusion injury was produced using an IH Impactor (Precision Systems and Instrumentation, Lexington, KY) according to a previously published method (16). Briefly, rats were anesthetized with pentobarbital (50 mg/kg i.p.) and a laminectomy was performed at the T9-T10 level. After the exposed vertebral column was stabilized by clamping the rostral T8 and caudal T10 vertebral bodies with forceps, the exposed dorsal surface of the cord was subjected to an impact of 150 kDyne. This injury severity is severe enough to eliminate trans-cranial magnetic motor-evoked potentials (tcMMEPs) but spare a rim of tissue at the injury epicenter (17). For the sham-operated controls, the animals underwent a T9 laminectomy without the impact. The rats were killed at 24 hours or at 6 weeks post-SCI, and a 10-mm spinal cord segment containing the injury epicenter was removed quickly and prepared for Western blot, MPO activity, PLA^sub 2^ activity, or histology analysis. All surgical interventions and postoperative animal care were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and the Guidelines of the University of Louisville Institutional Animal Care and Use Committee.
Intraspinal Injection of Annexin A1
After contusion injury, each rat received 4 injections around the injury epicenter within 5 minutes post-SCI. The bilateral microinjections (2 injections/side, 1 [mu]L/injection; total = 4 [mu]L) of vehicle or ANXA1 Ac^sub 2-26^ (5 or 20 [mu]g/rat) from Phoenix Pharmaceuticals, Inc. were made into the spinal cord at 0.6 mm from the midline and at a depth of 1.5 mm from the dorsal cord surface on both sides using a glass micropipette attached to a pneumatic picopump (World Precision Instruments, Inc., Sarasota, FL). There was a 2 mm distance between the 2 injections on each side. Once the micropipettes were in place a total of 1 [mu]L of solution was injected over a period of 3 minutes. When the injections were finished, the fluid was allowed to disperse over a 10-minute period, and the micropipette was slowly withdrawn. This dose of ANXA1 Ac^sub 2-26^ (amino acid sequence 2-26 of the NH^sub 2^-terminal of human ANXA1) was chosen, on the basis of a previous report in which 5 to 20 [mu]g/paw of ANXA1 Ac^sub 2-26^ significantly inhibited inflammatory paw edema induced by carrageenin and PLA^sub 2^ (18).
Myeloperoxidase Activity Assay
MPO activity was measured according to previously reported methods (19, 20) with minor modifications. Briefly, the injured spinal cord segment (10 mm) was removed and homogenized. The supernatant, after centrifugation at 14,000 x g for 25 minutes, was assayed for MPO activity. This was determined in duplicate by mixing 0.1 mL of the supernatant with 2.9 mL of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine and 0.0005% H^sub 2^O^sub 2^. Absorbance at 460 nm was recorded with a Biophotometer (Eppendorf AG, Hamburg, Germany).
Western Blotting
Western blotting followed the previously described procedure (21). Briefly, a 10-mm spinal cord segment containing the injury epicenter was dissected after intracardial perfusion of the rat with 200 mL of saline under anesthesia. The cord segment was homogenized in 0.4 mL of RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM Na^sub 3^VO, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 100 [mu]g/mL phenylmethylsulfonyl fluoride, 30 [mu]L/mL aprotinin, and 4 [mu]g/mL leupeptin, pH 7.5) and centrifuged at 10,000 x g for 10 minutes at 4[degrees]C. The supernatant was removed and centrifuged again. Forty micrograms of proteins from the supernatant of each sample was loaded onto a 12% polyacrylamide gel, separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane by electrophoresis. The membrane was blocked in TBST buffer (20 mM Tris-HCl, 5% nonfat milk, 500 mM NaCl, and 0.1% Tween 20, pH 7.5) for 1 hour at room temperature. The primary rabbit polyclonal anti-IL-1beta (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-caspase 3 (1:500; Cayman Chemical, Ann Arbor, MI), or mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:3000; Sigma Chemical) was added to the membrane, and the mixture was incubated for 1 hour at room temperature. The membrane was washed once for 15 minutes and twice for 5 minutes with TBST at room temperature, incubated with a secondary horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody or horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (1:5000; Amersham Pharmacia Biotech Inc., Piscataway, NJ) for 1 hour and then washed once for 15 minutes and twice for 5 minutes with TBST. The Western blot was visualized using the ECL Plus detection system as described in the technical manual provided by Amersham Pharmacia Biotech, Inc. The blots were then re-probed with a monoclonal anti-beta-tubulin antibody (1:1000; Sigma Chemical) after stripping of bound antibodies, and each single band was normalized by beta-tubulin. For the negative control the primary antibody was omitted. Phospholipase A^sub 2^ Activity Assay
Rats were killed with or without ANXA1 at 24 hours post-SCI. A 10- mm spinal cord segment containing the injury epicenter was dissected after intracardial perfusion of the rat with 200 mL of saline under anesthesia. The cord segment was homogenized in 0.4 mL of 50 mM Hepes, pH 7.4, containing 1 mM EDTA and centrifuged at 10,000 x g for 15 minutes at 4[degrees]C. The supernatant was removed for PLA^sub 2^ activity analysis. PLA^sub 2^ activity was measured according to the protocol in the PLA^sub 2^ Assay Kits (Cayman Chemical Co., Ann Arbor, MI).
Spinal Cord Neuronal Culture, Cell Treatment, and Viability Assessment
Cells obtained from embryonic (E) day 14 rat spinal cords were dissociated by incubation in 0.05% trypsin/EDTA followed by gentle trituration. The cell suspension was plated onto poly-L-lysine- coated 96-well cell culture plates at a density of 0.5 x 10^sup 5^ cells/well (for lactate dehydrogenase [LDH] release assays) and 1 x 10^sup 5^ cells/well to a 24-well chamber slide (for immunocytochemistry). The cells were grown in serum-free neurobasal medium supplemented with 2% B27 and 2 mM glutamine (all from Invitrogen, Carlsbad CA). Three days later, 5 [mu]M cytosine-beta-D- arabinofuranoside was added to the medium for 24 hours to inhibit non-neuronal cell division. Under this culture condition, a purity of >85% spinal cord neuronal population was obtained at the 7th day in vitro. The cultures were then treated with PLA^sub 2^ (1 nM; Type III; Cayman Chemical Co.) or melittin (an activator of endogenous PLA^sub 2^, 500 nM; Sigma Chemical) with or without ANXA1 Ac^sub 2- 26^ (0.4 [mu]M), respectively. The cultures were maintained for an additional 24 hours, and the culture medium of each well was removed for a LDH release assay using a LDH cytotoxicity assay kit (Biovision, Inc., Mountain View, CA) according to the manufacturer’s protocol, the relative absorbance of all samples were measured at 490 nm with a ELX 800 UV microtiter plate reader (Bio-Tek Instruments, Winooski, VT).
Behavioral Assessments
The BBB locomotor test was performed weekly up to 6 weeks post- SCI by 2 observers lacking knowledge of the experimental groups according to a method published previously (22). Grid walking was used also to assess hindlimb locomotor deficits (23). Footfalls were evaluated at 3 and 6 weeks post-SCI. During the test the rats were allowed to walk on a plastic mesh (3 x 3 square foot) containing 4.5 x 5 cm diamond holes. Total hindlimb footfalls were counted by 2 observers unaware of the experimental groups during each trial. For testing, each animal was placed on the grid and allowed to perform active grid walking task for a period of 3 minutes. During this time period, the number of footfalls (fall of the hindlimb, including at least the ankle joint, through the grid surface) was determined individually for each hindlimb.
tcMMEP Responses
As an in vivo electrophysiologic measure of motor pathway function, The recordings of tcMMEP were performed at the sixth week after contusive SCI, using methods described previously (15, 17, 24). The tcMMEP relies on the activation of subcortical structures with an electromagnetic coil placed over the cranium. Action potentials descend in the ventral spinal cord and synapse onto motoneuron pools in which output signals can be recorded from both of the gastrocnemius muscles. Changes in the latency of onset and peak-to-peak amplitude among the experimental groups were recorded and statistically compared.
Histologic and Stereologic Assessment of Lesion Volume and White Matter Sparing
After 6 weeks of behavioral evaluation, all of the sham-operated rats (n = 4) and half of SCI rats (n = 6/group) that had received different treatments were perfused for histologic analysis. These rats received anesthesia with pentobarbital (80 mg/kg i.p.) and transcardial perfusion of 0.01 M PBS followed by 4% paraformaldehyde (400 mL/rat). After perfusion, a 10-mm-long piece of the spinal cord was dissected out from each rat, left in the same fixative for 4 hours, and then transferred to 30% sucrose in 0.01 M PBS (pH 7.4). Spinal cord segments containing the epicenter were isolated from each animal, embedded, and cut into 25-[mu]m-thick serial sections (250 [mu]m apart and spanning the entire rostrocaudal extent of the lesion) in 2 identical sets. One set of the sections was stained for myelin with Luxol fast blue and the other was counterstained with cresyl violeteosin. The lesion and spared white matter area of the injured cord were visualized, outlined, and quantified using an Olympus BX60 microscope equipped with a Neurolucida system (MicroBrightField, Colchester, VT). An unbiased estimation of the percentage of spared tissue was calculated using the Cavalieri method (25). The total volume of spared white and gray matter was calculated by summing their individual subvolumes (26). Individual subvolumes of spared tissue were calculated by multiplying the cross- sectional area A x d, where d represents the distance between sections. Septae or fibrous bands of tissue observed within and/or spanning areas of cystic cavitation were not considered to represent spared tissue. The percent total volume of spared white and gray matter was calculated by dividing the total volume of spared white and gray matter by the total tissue volume of the corresponding region ( x 100), respectively. Estimation of total and percent total lesion volume (which included areas of cavitation and fibrosis) was determined using identical procedures.
Fluorogold Retrograde Tracing
After 6 weeks of behavioral evaluation, the other half of the rats (n = 6/group) from SCI and treatment groups were used for Fluorogold (FG) (Fluorochrome, Inc, Denver, CO) retrograde tracing, which was used to determine the extent to which spared descending axons reached the rostral lumbar enlargement. The retrograde tracing method with FG followed a previously described method (16). Briefly, 4% FG was injected into the lumbar enlargement at 0.6 and 1 mm from the midline and a depth of 0.7 and 1.5 mm from the dorsal cord surface on both sides (4 injections/side, 0.5 [mu]L/injection) using a glass micropipette attached to a pneumatic picopump, approximately 12 mm distal to the lesion epicenter. At 7 weeks postinjury, rats were killed and tissues fixed by transcardial perfusion of 4% paraformaldehyde as described above. Transverse sections (40 [mu]m) from the C6 and T5 segments were chosen as representative cervical and thoracic segments because axons from these regions form propriospinal projections that descend to the spinal cord caudal to the injury. Counting the number of FG-labeling neurons in selected propriospinal and supraspinal regions whose axons traversing through the SCI site and retrogradely transporting FG allowed us to compare axonal sparing between different treatment groups. To avoid counting a single neuron more than once in adjacent sections, only neurons containing nuclei were counted. Transverse sections (40 [mu]m, every fourth section) from the entire brainstem and sensorimotor cortex also were cut in an identical manner. The total number of FG- labeled neurons in the raphe nuclei, the lateral vestibular nucleus, the locus coeruleus, the subcoeruleus, alpha, the caudal pontine reticular nucleus, the red nucleus, and the hindlimb area of motor cortex was counted bilaterally according to previously reported methods (16, 27).
Statistical Analysis
Except the rate of tcMMEP responses, all of data are presented as mean +- SEM values. One-way analysis of variance with Tukey honestly significant difference post hoc t-tests were used to determine levels of statistical significance. p < 0.05 was considered statistically significant. The nonparametric analysis, chi^sup 2^, was used for comparisons of the rate of tcMMEP responses. Differences were accepted to be statistically significant at p < 0.05.
RESULTS
ANXA1 Inhibited Inflammatory Reaction and Apoptosis After SCI
To assess whether ANXA1 inhibited SCI-induced inflammation, we examined MPO activity (a marker for neutrophil infiltration) and expression of a cytokine, IL-beta1 (IL-beta1), in the rat spinal cord at 24 hours after SCI and injections of either vehicle or ANXA1 Ac^sub 2-26^ (5 or 20 [mu]g/rat). ANXA1 injection at a dose of 5 or 20 [mu]g resulted in a significant decreases of MPO activity by 48.5% (p < 0.05) and 65.3% (p < 0.01), respectively (Fig. 1A). However, only the high dose of ANXA1 (20 [mu]g) resulted in a significant decrease of IL-1beta expression by 71% (p < 0.01) at 24 hours post-SCI (Fig. 1B).
FIGURE 1. Effects of annexin A1 (ANXA1) on myeloperoxidase (MPO) activity and interleukin (IL)-1beta expression after spinal cord injury (SCI). (A) Both low (5 [mu]g) and high (20 [mu]g) doses of ANXA1 Ac^sub 2-26^ resulted in a decrease of MPO activity at 24 hours post-SCI. (B) Only the high dose of ANXA1 Ac^sub 2-26^ resulted in a decrease of IL-1beta expression. Upper panel, representative photograph of IL-1beta expression. Lower panel, compiled results in a bar graph. n = 6 rats/group. ##, p < 0.01 versus sham group; *, p < 0.05; **, p < 0.01 versus SCI group. To test whether the anti-inflammatory effect of ANXA1 resulted in inhibition of SCI-induced cell death, particularly apoptosis, we examined the expression of active caspase-3, an enzyme that is known to be expressed after SCI and that is critically involved in the execution of the mammalian apoptotic cell death program (28). Western blot analysis revealed that SCI induced a significant increase of active caspase-3 expression (p < 0.01), which was significantly reversed by 48.8% (p < 0.01) and 49.7% (p < 0.01) after the 5- and 20-[mu]g doses of ANXA1 treatments, respectively (Fig. 2). No significant difference was found in active caspase-3 expression between the 2 ANXA1-treated groups.
FIGURE 2. Effect of annexin A1 (ANXA1) on active caspase-3 expression after spinal cord injury (SCI). Both 5- and 20-[mu]g doses of ANXA1 Ac^sub 2-26^ resulted in a decrease in active caspase- 3 expression at 24 hours post-SCI. Upper panel, representative photograph of active caspase-3 expression. Lower panel, compilation of results in a bar graph. n = 6 rats/group; ##, p < 0.01 versus sham group; **, p < 0.01 versus SCI group.
ANXA1 Inhibited SCI-Induced Phospholipase A^sub 2^ Activity and Phospholipase A^sub 2^-Induced Neuronal Death
Because we demonstrated that ANXA1 inhibited inflammation and apoptosis after SCI, our next step was to determine whether such an inhibition was mediated through the inhibition of PLA^sub 2^. We examined the effect of ANXA1 on PLA^sub 2^ activity both in the injured spinal cord and in spinal cord neuronal cultures when PLA^sub 2^ was administrated. In the first experiment, we found that SCI induced a significant increase in PLA^sub 2^ activity (p < 0.01) at 24 hours postinjury Fig. 3). Such an increase, however, was significantly reversed by 28.4% (p < 0.05) and 43.6% (p < 0.01), respectively, after the 5 and 20 [mu]g ANXA1 treatments, indicating that PLA^sub 2^ activity, induced by SCI, can be effectively inhibited by locally administrated ANXA1. No significant difference was found in PLA^sub 2^ activity between the 2 ANXA1-treated groups.
FIGURE 3. Effect of annexin A1 (ANXA1) on phospholipase A^sub 2^ (PLA^sub 2^) activity after spinal cord injury (SCI). ANXA1 Ac^sub 2- 26^ at both 5- and 20-[mu]g doses inhibited an increase of PLA^sub 2^ activity induced by SCI. n = 6 rats/group; ##, p < 0.01 versus sham group; *, p < 0.05; **, p < 0.01 versus the SCI group.
FIGURE 4. Effect of annexin A1 (ANXA1) on phospholipase^sub 2^ (PLA^sub 2^)-induced spinal cord neuronal death in vitro. Both PLA^sub 2^ (A) and melittin (B), a potent activator of endogenous PLA^sub 2^, induced spinal cord neuronal death that was significantly reversed by ANXA1 Ac^sub 2-26^. n = 6/group; ##, p < 0.01 versus sham group; **, p < 0.01 versus SCI group. LDH, lactate dehydrogenase.
We next determined whether ANXA1 could reverse PLA^sub 2^- induced cell death of spinal cord neurons. Our previous work showed that PLA^sub 2^ or melittin, a potent activator of endogenous PLA^sub 2^ activity, induced cultured embryonic spinal cord neuronal death in a dose-dependent manner (15). The PLA^sub 2^-induced cell death was determined by measuring the release of LDH from degenerating spinal neurons. As shown in Figure 4, both PLA^sub 2^ (1 nM; p < 0.01) and melittin (0.5 [mu]M; p < 0.01) significantly increased cultured spinal cord neuronal death, which was significantly reversed by ANXA1 (0.4 [mu]M; p < 0.01). These results collectively suggest that SCI-induced PLA^sub 2^ activation can be effectively suppressed by ANXA1 and that such a suppression leads to neuroprotection of spinal cord neurons from PLA^sub 2^-induced cell death.
ANXA1 Reduced Tissue Damage, Demyelination, and Reactive Gliosis After SCI
We showed that ANXA1 treatment reduced PLA^sub 2^ activation in vivo and PLA^sub 2^-induced spinal cord neuronal death in vitro; therefore, we next examined whether such a treatment would also result in tissue protection in vivo. We examined percent lesion area, percent white matter sparing area at the injury epicenter, and percent total lesion volume (relative to the corresponding total spinal cord volume) in rats killed at the sixth week after a moderate contusive SCI. To ensure that the entire rostrocaudal expansion of the lesion was examined, a 1.5-cm-long cord segment was serially sectioned. Measurements of percent total lesion volume, lesion area, and white matter sparing area were made from cresyl violet and eosin-stained transverse sections spanning the entire lesion (Fig. 5). Comparison of the lesion area at the injury epicenter demonstrated that ANXA1 treatments at doses of 5 and 20 [mu]g resulted in a reductions of lesion area by 19.2% (p < 0.05) and 30.8% (p < 0.01), respectively, compared with the vehicle- treated group (Fig. 5A-C, J). Such reductions in lesion area were accompanied also by a corresponding increase in the area of white matter sparing by 17.7% (p < 0.05) and 28.5% (p < 0.01) at 6 weeks post-SCI (Fig. 5K). No statistically significant differences were found in average lesion area and white matter sparing between the 2 ANXA1-treated groups (Fig. 5J, K). Stereologic assessments of lesion volume indicated that microinjections of 2 concentrations of ANXA1 resulted in a significant reduction in the percent total lesion volume by 26.6% (Fig. 5H, M; p < 0.05) and 33.8% (Fig. 5I, M; p < 0.01), respectively, compared with vehicle-treated controls (Fig. 5G, M). In addition, Luxol fast blue staining showed that ANXA1 treatments at doses of 5 and 20 [mu]g resulted in a corresponding increase in myelin sparing by 35.2% (Fig. 5E, L; p < 0.05) and 51.3% (Fig. 5F, L; p < 0.01) at 6 weeks post-SCI, compared with the vehicle-treated controls (Fig. 5D, L). However, no statistically significant differences were found in the lesion volume and the spared white matter/myelin between the 2 ANXA1-treated groups.
FIGURE 5. Histologic and stereologic analysis of protective effects of annexin A1 (ANXA1). (A-C) Representative sections show the lesion epicenter stained with cresyl violet and eosin. After spinal cord injury (SCI), a large centrally located cystic cavity was present with a thin rim of spared tissue surrounding the cavity (A). A low (B) or high (C) dose of ANXA1 Ac^sub 2-26^ enhanced tissue sparing and reduced lesion size at the injury epicenter. (D- F) Representative sections show the lesion epicenter stained with Luxol fast blue. Note that both 5 and 20 [mu]g of ANXA1 Ac-^sub 2- 26^ enhanced spared myelination after SCI. (G-I) Three-dimensional reconstruction of a spinal cord segment from each group illustrates rostrocaudal extension of the lesion (blue). Bar graphs show quantitative data of lesion area (J), spared white matter area (K), myelination area (L), and lesion volume (M). n = 6 rats/group; *, p < 0.05; **, p < 0.01 versus vehicle group. Scale bar = (A-F) 500 [mu]m.
FIGURE 6. Effect of annexin A1 (ANXA1) on glial fibrillary acidic protein (GFAP) expression after spinal cord injury (SCI). The upper panel shows a representative photograph of GFAP expression. The 50- kDa band indicates intact GFAP and the bands with molecular mass lower than 37 kDa indicates degraded GFAP. Lower panel, compilation of results in a bar graph. = 6 rats/group; ##, p < 0.01 versus sham group; **, p < 0.01 versus SCI group.
FIGURE 7. Effect of annexin A1 (ANXA1) on propriospinal axon sparing measured by counting the number of Fluorogold (FG)-labeled neurons in the T5 and C6 spinal segments. FG was injected into the lumbar enlargement, approximately 12 mm caudal to the injury. In all cases, FG-labeled neurons were predominantly seen within lamina VII and the medial portion of lamina VIII that form propriospinal projections. An increase in the number of FG-labeled neurons was found after treatment with the low (C, D) or high (E, F) dose of ANXA1 Ac^sub 2-26^, compared with the vehicle-infused group (A, B). Scale bar = 100 [mu]m. (G) Quantitative analysis of FG-labeled propriospinal neurons shows that both low and high doses of ANXA1 Ac^sub 2-26^ resulted in a significant increase in the number of FG- labeled neurons located at both T5 and C6 spinal levels, n = 6 rats/ group. **, p < 0.01 versus vehicle group.
To assess whether ANXA1 inhibited SCI-induced reactive gliosis, we examined expression of GFAP, a marker for astrocyte, in the rat spinal cord at 4 weeks after SCI and injections of either vehicle or ANXA1 Ac^sub 2-26^ (5 or 20 [mu]g/rat). Western blot analysis of GFAP protein expression levels revealed that SCI not only induced the expression of intact GFAP with a molecular mass of 50 kDa, but also degraded GFAP with molecular masses smaller than 35 kDa (Fig. 6). ANXA1 injection at a dose of 5 or 20 [mu]g resulted in a marked decrease of GFAP expression by 16.7% and 28.2% (Fig. 6; p < 0.01), respectively. These results indicate that ANXA1 has an inhibitory effect on the development of astrogliosis after SCI.
ANXA1 Increased Axonal Sparing of Proprio- and Supraspinal Origins
If more white matter tissues are spared at the lesion epicenter, one anticipates that more axons may have been spared at the site of injury. We thus examined this possibility by using the FG retrograde tracing approach. At the sixth week post-SCI, a retrograde tracer FG was injected into the lumbar enlargement to assess the extent of spared descending axons at the site of injection in both vehicle and ANXA1 -treated rats. ANXA1 treatments at doses of 5 and 20 [mu]g resulted in a significant increase (p < 0.01) of FG-labeled neurons located at both T5 and C6 spinal levels compared with the vehicle- infused group (Fig. 7A-G). However, no statistically significant difference was found in the number of FG-labeled neurons between the 2 ANXA1-treated groups at both cord levels. FIGURE 8. Effects of annexin A1 (ANXA1) on supraspinal axonal sparing measured by counting the number of Fluorogold (FG)-labeled neurons in selective brainstem nuclei at 7 weeks post-spinal cord injury (SCI). (A) Representative photographs of the red nucleus show an increase in the number of FG-labeled neurons after 5 and 20 [mu]g of ANXA1 Ac^sub 2-26^ treatments compared with the vehicle-treated control. Scale bar = 200 [mu]m. (B) A bar graph shows the number of FG- labeled neurons counted in 7 selective supraspinal regions at 7 weeks post-SCI. Ra, raphe nuclei; LVe, lateral vestibular nuclei; LC, locus coeruleus; SubCA, subcoeruleus, alpha; PnC, caudal pontine reticular nucleus; RN, red nucleus; Ctx-HL, hindlimb area of motor cortex. *, p < 0.05; **, p < 0.01 versus vehicle group.
FG-labeled neurons in selected supraspinal nuclei also were counted to determine whether ANXA1-induced reduction in lesion volume resulted in an increase in axon sparing of neurons in these areas (Fig. 8A, B). Figure 8A shows representative examples of FG- labeled neurons in the red nucleus of vehicle- and 5- and 20 [mu]g ANXA1-treated groups (Fig. 8A). Counts of neurons retrogradely labeled by FG are summarized in Figure 8B. Statistically significant differences in the number of FG-labeled neurons between the vehicle- and ANXA1 -treated groups were observed in 4 of 7 nuclei that were examined (p < 0.05-0.01) (Fig. 9B). They were raphe nuclei, lateral vestibular nuclei, locus coeruleus, and red nucleus. No statistically significant differences were found in the number of FG- labeled neurons in the subcoeruleus, alpha, the caudal pontine reticular nucleus, and the hindlimb area of motor cortex between the vehicle- and ANXA1-treated groups (Fig. 8B). In addition, no statistically significant difference was found in the number of FG- labeled neurons between microinjections of 2 different ANXA1 concentrations in the above-indicated nuclei. There was a trend for increased neuronal counts in the hindlimb area of motor cortex with ANXA1 treatment compared with the vehicle-treated group, but no statistically significant difference was found between these groups. These data indicate that microinjection of ANXA1 into the contused adult rat spinal cord resulted in increased axonal sparing of some but not all of the descending pathways that were examined.
ANXA1 Improved tcMMEP Responses
To determine whether anatomical sparing of tissues and axons after AXA1 treatments increased axonal integrity of descending motor pathways in the ventrolateral funiculus, tcMMEP responses were recorded from the left and right gastrocnemius muscles at the sixth week post-SCI. The tcMMEP responses were classified into either an early response (onset latency: 4-8 ms), a late response (onset latency: >16 ms) or no response (absent) (Fig. 9A). In sham- operated animals, all had tcMMEP responses in the category of early response with an onset latency of 5.70 +- 0.19 ms and amplitude of 28.43 +- 1.23 mV (Table). In the SCI group, hindlimb recordings from 67% of animals had no tcMMEP responses. Among the remaining 33% of recordings that showed tcMMEP responses, 13% were early responses with an onset latency of less than 6 ms and 20% were late responses with an onset latency of <18 ms (Fig. 9A; Table). After ANXA1 treatment at a dose of 20 [mu]g, the early responses of tcMMEPs were increased from 13% to 29% to 42% (p < 0.05) and total responses were increased from 33% to 54% to 71% (p < 0.05) (Fig. 9B; Table). However, no significant differences in peak-to-peak amplitude was found among different groups.
FIGURE 9. Effect of annexin A1 (ANXA1) on tcMMEP responses at the sixth week after spinal cord injury (SCI). (A) Representative tcMMEP recordings were classified into 3 categories: early response (ER, 5- 7 ms), late response (LR, 16-20 months), and no response (absent). (B) Comparison of total responses of tcMMEPs between ANXA1- and vehicle-treated groups is shown. n = 24/group; p < 0.05.
TABLE. Effect of ANXA1 on tcMMEP Responses
FIGURE 10. Effect of annexin A1 (ANXA1) on behavioral outcomes after spinal cord injury (SCI). No statistically significant difference was found in Basso, Beattie, Bresnahan (BBB) locomotor rating scores nor in footfalls between ANXA1- and vehicle-treated groups (sham: n = 4; ANXA1-treated groups: n = 12/group).
ANXA1 Did Not Improve Behavioral Function After SCI
To determine whether AXA1 treatments also improve functional outcomes, we evaluated the ANXA1 effect on hindlimb functional recovery using the BBB locomotor rating scale and grid-walking test by 2 observers under experimentally blinded conditions. Testing was performed once a week for 6 weeks starting 1 week post-injury. In the BBB locomotor results, progressive functional recovery of the hindlimbs involving frequent to consistent weight supported plantar stepping and occasional forelimb-hindlimb coordination was found in both treated and untreated rats. However, no statistically significant difference was found between them at all time points that were studied (Fig. 10A). In the grid-walking test, a trend toward a decrease in the number of footfalls was found after the high dose (20 [mu]g) ANXA1 treatment. However, no statistically significant difference was found in the number of footfalls between this and the vehicle-treated group (Fig. 10B).
DISCUSSION
To our knowledge, this is the first study demonstrating a neuroprotective effect of ANXA1 on spinal cord tissues and axons of long descending pathways in adult rats after SCI. Administration of ANXA1 Ac^sub 2-26^ shortly after a moderate contusion injury of the adult rat thoracic spinal cord significantly inhibited PLA^sub 2^ activity, inflammatory reaction, reactive astrogliosis, neuronal death, and tissue damage. In addition, ANXA1 significantly increased spared white matter and myelin at the injured epicenter. Although no statistically significant improvements were found in BBB locomotor and grid walking analyses in ANXA1-treated groups at 6 weeks post- injury, ANXA1 was shown to protect spinal tissue and increase the number of animals that responded to tcMMEPs. These results collectively suggest that ANXA1 is a potent neuroprotective agent and may be of therapeutic benefit for the treatment of SCI.
In the present study, we found that administration of ANXA1 Ac^sub 2-26^ inhibited SCI-induced MPO activity at both doses. This suggests that ANXA1 suppressed SCI-induced neutrophil infiltration, which has been determined to be one of its important anti- inflammatory mechanisms (29, 30). Our observation is consistent with previous studies in which ANXA1 Ac^sub 2-26^ inhibited MPO activation and infiltrations of neutrophils and monocytes in other models of inflammation and ischemia-reperfusion injury (31-37).
The anti-inflammatory effect of ANXA1 also was demonstrated by its inhibition on IL-1beta overexpression after SCI. Our study agrees with previous findings in which ANXA1 inhibited expression and release of inflammatory cytokines such as IL-1beta and TNF- alpha. For example, ANXA1 Ac^sub 2-26^ was shown to suppress increased levels of IL-1beta induced by myocardial ischemia- reperfusion injury (36). In endotoxemic IL-6 knockout mice, human recombinant ANXAl inhibited TNF-alpha release and reduced circulating TNF-alpha and IL-1beta levels (38). The inhibitory role of ANXA1 on IL-1beta expression was shown in an ANXA1 gene knockout mouse model (12).
It has been suggested that ANXA1 exerts its antiinflammatory effect by inhibiting PLA^sub 2^ activity (4, 5). PLA^sub 2^ is a diverse family of enzymes that hydrolyze the acyl bond at the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. These products are precursors of bioactive eicosanoids and platelet-activating factor that lead to the concomitant production of reactive oxygen intermediates. The eicosanoids, platelet-activating factor, and reactive oxygen intermediates are well-known mediators of tissue injury and inflammatory processes and have been implicated in pathologic states of the CNS including SCI (39-41). Recently, we demonstrated that PLA2 mediated the secondary injury after SCI (15). The present study further showed that the SCI-induced PLA^sub 2^ activation could be effectively suppressed by ANXA1 in vivo. These findings suggest that inhibition of PLA^sub 2^ by ANXA1 may represent an important mechanism of neuroprotection.
Apoptosis has been demonstrated as a mechanism of cell death after SCI (42, 43). After SCI, apoptotic cells were mainly neurons and oligodendrocytes as we and others have demonstrated previously (42-44). Caspase-3 is one of the key executors of apoptosis and is responsible for the cleavage of proteins such as the nuclear enzyme poly(ADP-ribose) polymerase. Increased expression of caspase-3 has been shown after SCI (28). Our results showing that ANXA1 inhibited the expression of active caspase-3 after SCI suggested an antiapoptotic role of ANXA1. This finding is in contrast to the studies in which ANXA1 was shown to be an inducer of apoptosis of inflammatory cells including neutrophils and T lymphocytes (30, 45, 46). Although the reason for such differences remains unclear, it is possible that different cells may respond to ANXA1 differently. However, ANXA1-induced apoptosis of inflammatory cells may add to the overall anti-inflammatory actions of this molecule, which in turn suppresses neuronal and glial apoptosis after SCI.
Histologic analysis provides important evidence that administration of ANXA1 elicited neuroprotection on the contused adult rat spinal cord. Stereologic analysis showed that ANXA1 at both 5 and 20 [mu]g doses promoted reductions of 26.6% and 33.8% in total lesion volume, respectively, compared with the vehicle control group. The reduction in lesion size was well correlated with an increase in white matter sparing surrounding the lesion epicenter. Luxol fast blue staining demonstrated that injury-induced demyelination was significantly decreased after microinjections of ANXA1 Ac^sub 2-26^. Importantly, the increase in white matter sparing was accompanied by an increase in the sparing of some descending proprio- and supraspinal axons, as demonstrated by FG- retrograde tracing. This includes a 2.01- to 2.34-fold increase in the number of FG-labeled propriospinal neurons in the C6 and T5 cord segments, respectively, and an increase in the number of labeled neurons in several brainstem nuclei including Ra, LVe, LC, and RN. No statistically significant differences were found in the number of FG-labeled neurons in the SubCA, PnC, and Ctx-HL between ANXA1- and vehicle-treated groups. Thus, ANXA1 not only protected spinal cord tissues from secondary damage but also enhanced the sparing of selective proprio- and supraspinal axons that traverse the lesion site. It should be noted that although both high and low doses of ANXA1 showed tissue-sparing effects in the adult spinal cord after injury, no statistically significant differences were found in the total lesion volume, percent lesion volume, and the number of FG- labeled proprio- and supraspinal neurons between the 2 ANXA1 Ac^sub 2-26^ concentrations. This indicates that the high dose that we chose might not be high enough to achieve a maximal biologic effect of ANXA1 and that an increase of ANXA1 levels may promote further tissue sparing. Reactive gliosis has been considered as a major impediment for SCI repair. Our observation that ANXA1 inhibited GFAP overexpression indicates that the protective effects of ANXA1 may be mediated, at least in part, by its inhibition on reactive astrogliosis after SCI. Interestingly, both the intact and degraded GFAP isoforms were increased after SCI, similar to those reported previously (47). Such an effect may be a result of the activation of Ca^sup 2+^-dependent enzymes, such as calpain (47). Importantly, ANXA1 inhibited the expression of both intact and degraded GFAP isoforms.
In the present study, we used tcMMEP assessment to determine the degree of axonal sparing of functional descending pathways across an injury site and to predict functional outcomes after SCI. Although there was no significant difference in the peak-to-peak amplitude of tcMMEPs between ANXA1-treated and untreated groups, microinjection of ANXA1 Ac^sub 2-26^ resulted in a significant increase in the rate of tcMMEP responses including the percentage of early and total responses at 6 weeks postinjury, suggesting that ANXA1 has a neuroprotective effect on axons after SCI. This finding was consistent with the histologic results in which ANXA1 significantly reduced lesion volume and increased white matter sparing after SCI.
Despite the fact that ANXA1 significantly reduced lesion volume and increased white matter sparing after SCI, no statistically significant differences in the BBB score and number of footfalls were found between ANXA1- and vehicle-infused groups. This suggests that the spared cord tissue protected by ANXA1 was not sufficient to improve behavioral function assessed by these measures, which indicates that higher doses or longer administration (rather than a bolus injection) schedules of ANXA1 are needed to achieve maximal biologic and functional effects. The fact that no statistically significant differences were found in tissue and axonal sparing between the groups administered the 2 doses of ANXA1 indicates that the high dose we chose might not be sufficient for a stronger neuroprotective effect of ANXA1. Further studies with the aim of optimizing ANXA1 doses and/or delivery means may show greater improvement in the histologie and electrophysiologic outcomes and eventually behavioral recovery after contusive SCI. Experiments along these lines are currently in progress in our laboratory.
ACKNOWLEDGMENTS
We are thankful to Christine Nunn and Aaron Puckett for care of animals, and Darlene Burke and Kim Fentress for behavioral and electrophysiologic assessments.
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Nai-Kui Liu, MD, PhD, Yi Ping Zhang, MD, Shu Han, PhD, Jiong Pei, MD, Lisa Y. Xu, MD, Pei-Hua Lu, MD, Christopher B. Shields, MD, and Xiao-Ming Xu, MD, PhD
From the Department of Neurological Surgery, Kentucky Spinal Cord Injury Research Center (N-KL, YPZ, JP, LYX, CBS, X-MX), University of Louisville School of Medicine, Louisville, Kentucky; and Department of Neurobiology (SH, P-HL, X-MX), Shanghai Jiaotong University School of Medicine, Shanghai, People’s Republic of China.
Send correspondence and reprint requests to: Xiao-Ming Xu, MD, PhD, James R. Petersdorf Professor, Department of Neurological Surgery, University of Louisville School of Medicine, 511 S. Floyd Street, MDR 616, Louisville, KY 40292; E-mail: xmxu0001@louisville.edu
This work was supported by National Institutes of Health (NIH) Grants NS36350 and NS52290, the Kentucky Spinal Cord and Head Injury Research Trust #4-16, the Daniel Heumann Fund for Spinal Cord Research (XMX), and the Paralysis Project of America (NKL). We are also thankful to Norton Healthcare, the Kentucky Spinal Cord and Head Injury Research Trust Board, The Commonwealth of Kentucky Bucks for Brains Program, and the University of Louisville through the James R. Petersdorf Endowment and appreciate the use of the Kentucky Spinal Cord Injury Research Center’s Core facility supported by NIH COBRE RR15576.
Copyright Lippincott Williams & Wilkins Oct 2007
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