==== Front BMC NeurosciBMC NeurosciBMC Neuroscience1471-2202BioMed Central 1471-2504-29-4515423855710.1186/1471-2202-14-146Research ArticleThe temporal and spatial profiles of cell loss following experimental spinal cord injury: effect of antioxidant therapy on cell death and functional recovery Ling Xiang amberalvarez@example.com Feng amberalvarez@example.com Hao amberalvarez@example.com Danxia amberalvarez@example.com Department of Neurology, University of Texas Medical Branch, 301 University Blvd., Rt. 0881, Galveston, TX 77555-0881, USA2 Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Rt. 0881, Galveston, TX 77555-0881, USA2013 18 11 2013 14 146 146 23 5 2013 12 11 2013 Copyright © 2013 Ling et al.; licensee BioMed Central Ltd.2013Ling et al.; licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Traumatic spinal cord injury (SCI)-induced overproduction of endogenous deleterious substances triggers secondary cell death to spread damage beyond the initial injury site. Substantial experimental evidence supports reactive species (RS) as important mediators of secondary cell death after SCI. This study established quantitative temporal and spatial profiles of cell loss, characterized apoptosis, and evaluated the effectiveness of a broad spectrum RS scavenger - Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) and a combination of MnTBAP plus nitro-L-arginine to prevent cell loss and neurological dysfunction following contusion SCI to the rat spinal cord. Results By counting the number of surviving cells in spinal cord sections removed at 1, 6, 12, 24, 48, 72 h and 1 week post-SCI and at 0 – 4 mm from the epicenter, the temporal and spatial profiles of motoneuron and glia loss were established. Motoneurons continued to disappear over a week and the losses decreased with increasing distance from the epicenter. Significant glia loss peaked at 24 to 48 h post-SCI, but only at sections 0–1.5 mm from the epicenter. Apoptosis of neurons, motoneurons and astrocytes was characterized morphologically by double immuno-staining with cell-specific markers and apoptosis indicators and confirmed by transmission electron microscopy. DNA laddering, ELISA quantitation and caspase-3 activation in the spinal cord tissue indicated more intense DNA fragments and greater caspase-3 activation in the epicenter than at 1 and 2 cm away from the epicenter or the sham-operated sections. Intraperitoneal treatment with MnTBAP + nitro-L-arginine significantly reduced motoneuron and cell loss and apoptosis in the gray and white matter compared with the vehicle-treated group. MnTBAP alone significantly reduced the number of apoptotic cells and improved functional recovery as evaluated by three behavioral tests. Conclusions Our temporal and spatial profiles of cell loss provide data bases for determining the time and location for pharmacological intervention. Our demonstration that apoptosis follows SCI and that MnTBAP alone or MnTBAP + nitro-L-arginine significantly reduces apoptosis correlates SCI-induced apoptosis with RS overproduction. MnTBAP significantly improved functional recovery, which strongly supports the important role of antioxidant therapy in treating SCI and the candidacy of MnTBAP for such treatment. Antioxidant therapyApoptotic cell deathBehavioral testMn (III) tetrakis (4-benzoic acid) porphyrinNitro-L-arginineSecondary spinal cord injury ==== Body Background Spinal cord injury (SCI) develops in two phases: primary injury and secondary injury. The initial mechanical injury not only directly kills cells and destroys various elements of the tissue, but also induces overproduction of endogenous deleterious substances, which trigger secondary damage processes that lead to secondary cell death, thereby spreading damage beyond the initial injury site [1,2]. Apoptosis is an important contributor to secondary cell death after SCI. Numerous studies have shown that neuronal and glial apoptosis follows SCI [3-6]. Cells die by necrosis or apoptosis depending on the types and concentrations of the triggering death signals. At the injury epicenter, the concentration of endogenous deleterious substances produced upon the initial trauma reaches a maximum, and then gradually declines with distance from the epicenter; in like manner, the type of cell death may also depend on the distance from the injury epicenter. Therefore, it is critical to explore the temporal and spatial profiles of cell death by necrosis and apoptosis for each type of cell to precisely define the time window and the damage area for therapeutic intervention. Reactive species (RS), including free radicals and non-radical oxidants, are believed to contribute to secondary destruction after central nervous system (CNS) injury by oxidatively damaging the major cellular components proteins: DNA, and phospholipids [7-9]. Substantial experimental evidence supports RS as important mediators of secondary damage after SCI [10-13]. We and others have demonstrated that the levels of superoxide anion [6,14], hydrogen peroxide [15], hydroxyl radical [16], catalytic iron [17], nitric oxide (•NO) [18,19] and peroxynitrite [18,20], as well as the products of oxidation and nitration of proteins [6,18,21-23] and membrane lipid peroxidation (MLP) [24-26], all significantly increase following SCI. By administering the increased levels of RS into an un-injured rat spinal cord, we demonstrated that they caused oxidative damage to major cellular components [27,28], neuronal death by necrosis and apoptosis [29-31] and neurological dysfunction [29]. Our in vivo demonstration unequivocally links SCI-induced RS elevation to necrotic and apoptotic cell death and neurological dysfunction in SCI. Therefore, a broad spectrum scavenger of RS should more effectively reduce secondary cell death and the resulting neurological dysfunction than would agents with a single target. Metalloporphyrins, a novel class of catalytic antioxidants, not only scavenge a wide range of RS such as superoxide anion, hydrogen peroxide, peroxynitrite and lipid peroxyl radicals [32], but also modulate RS-based redox signaling pathways [33]. The metalloporphyrin Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) possesses both superoxide dismutase and catalase-like activity [34] and scavenges peroxynitrite [35]. It is also a potent inhibitor of MLP [36]. In the CNS, cerebroventricular injection of MnTBAP inhibited kainate-induced mitochondrial superoxide production, DNA oxidation and neuronal loss in the hippocampus of rat [37]. We demonstrated in vivo that MnTBAP reduced peroxynitrite-induced oxidation and nitration of proteins [27] and MLP [28] in the rat spinal cord. It prevented hydroxyl radical-induced necrotic and apoptotic cell death [31]. We recently demonstrated that intrathecal administration of MnTBAP reduced superoxide and hydrogen peroxide production, reduced oxidation and nitration of proteins and increased the number of surviving neurons, motoneurons, astrocytes and oligodendrocytes after SCI [38-40]. These results suggest that the catalytic antioxidant MnTBAP may be a potential agent for antioxidant therapy, owing to its cell permeability, low toxicity and broad scavenging of RS. However, MnTBAP was reported poorly able to penetrate the blood–brain barrier [41], so it does not seem to be a favorable candidate for antioxidant therapy for CNS injury and degenerative disorders. We compared the penetrating ability of MnTBAP to methylprednisolone (MP), the only drug used clinically to treat SCI. We found that, despite the much lower penetration of the blood-spinal cord barrier (BSB) by MnTBAP compared with MP, its higher stability allows a lower dose of MnTBAP to produce a higher concentration in the CSF than does higher doses of MP [42]. Moreover, we demonstrated that MnTBAP (10 mg/kg) given intraperitoneally (i.p.) increased the number of neurons and attenuated the number of apoptotic neurons after SCI [43]. Treatment with this dose of MnTBAP (i.p.) more effectively improved the functional recovery after SCI than did the standard MP regimen [40]. These results suggest that MnTBAP indeed crossed the BSB and reached the appropriate targets, perhaps in part, because the injury disrupted the BSB to allow MnTBAP to pass through. Therefore, MnTBAP warrants further examination of its antioxidative efficacy. In our previous study, we established the temporal and spatial profile of neuron death, the temporal and spatial profiles of the appearance of apoptotic cells in the gray and white matter and the temporal and spatial profiles of neuronal apoptosis after SCI [43]. The goals of the present study were the following. 1) To established the temporal and spatial profiles of glial cell loss in the white matter and motoneuron loss in the gray matter for up to one week following severe injury to the rat lumbar enlargement. 2) To characterize SCI-induced apoptosis of neurons, motoneurons and glial cells morphologically and biochemically. 3) To evaluate the efficacy of cell protection by MnTBAP and a combination of MnTBAP plus nitro-L-arginine (L-NA) ― a non-selective nitric oxide synthase (NOS) inhibitor. It was reported that MnTBAP is not a scavenger of •NO [44]. However, the toxicity of •NO occurs when it reacts with overproduced superoxide to form peroxynitrite - a strong oxidant, whereas MnTBAP scavenges peroxynitrite. Therefore, both MnTBAP alone and the combination of MnTBAP + L-NA will be tested for their protection against cell loss following SCI. 4) To test the ability of MnTBAP to improve functional recovery after SCI by behavioral tests. Accomplishment of these goals will provide a foundation for determining the optimal time and location to test the therapeutic potential of pharmaceutical agents to prevent cell death and apoptosis and add new information for exploring the therapeutic potential of the catalyst antioxidant MnTBAP in SCI treatment. Methods All animal experimental procedures were approved by the University of Texas Medical Branch Animal Care and Use Committee and were in accord with the NIH Guide for the Care and Use of Laboratory Animals. Establishing cell loss profiles and characterizing cellular apoptosis morphologically following SCI Animal preparation, spinal cord injury and drug administration To obtain the temporal and spatial profiles of cell loss after SCI, male Sprague–Dawley rats (250–300 g) were randomly divided into two groups: injured and sham operated. Animals were anesthetized with pentobarbital (50 mg/kg, i.p.). When the rat was fully anesthetized, a laminectomy was performed on vertebra T13 and the dura was kept intact. Care was taken not to injure the cord. Then the animal was clamped in a frame by attachments to its dorsal vertebral processes. The cord was injured severely by dropping a weight (10 g) through a guide tube 5 cm onto the cord (50 g.cm) according to the impact method originally devised by Allen [45] and modified by us. The weight was fitted with a Teflon tip with an area of 10 mm2. After injury, the incision was surgically repaired and the rats were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle. Animals had free access to food and water. The procedures for anesthesia, surgery and impact injury are described in detail in our previous publications [14-18,21]. The only difference in animal preparation between the sham-operated control and the injured group was that no impact injury was performed in the sham rats. To test the protection against cell death by the combination of MnTBAP (Calbiochem, San Diego, CA, USA) and L-NA, the animal was injured as above, then MnTBAP (10 mg/kg) plus L-NA (1 mg/kg) dissolved in saline was given i.p. at 15 min pre- and 6 h post-SCI. To obtain the spatial profiles of MnTBAP protection against apoptotic cell death, MnTBAP alone (10 mg/kg in saline) was administered by i.p. injection as described above. In the vehicle control group, animals were injured and saline was administered as in the treated group. Spinal cord tissue processing for morphological examination To establish the temporal and spatial profiles of motoneuron and glial cell loss, the injured cords were harvested at 1, 6, 12, 24, 48, 72 h and 1 week post-SCI as the same time points as for establishing the temporal and spatial profile of neuron loss [43], so the profiles of different cell types can be compared. At the time of cord removal, the animals were re-anesthetized with pentobarbital (50 mg/kg), perfused intracardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.2-7.4) while the animal was still deeply anesthetized. A second laminectomy was performed on vertebrae T11-L3 to extend the exposed cord, so that segments approximately 1.5 cm long centered at the injury site were removed and fixed in the same fixative overnight and embedded in paraffin. Rats that served as sham-operated controls were similarly perfused at 12 h post-operation. Serial sections (10 μm thick) were cut and mounted on glass slides coated with poly-L-lysine. The amberalvarez@example.com mm (10 sections) intervals from every cord were deparaffinized and stained histochemically with cresyl violet (CV) as describe in our previous publications [31]. The CV-stained sections were observed under a light microscope to locate the epicenter (with the lowest cell density). After the epicenter was determined, the spinal sections at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mm caudal from the epicenter at each time point post-SCI were CV-stained to obtain the temporal and spatial profiles of cell death. From our temporal and spatial profiles, we found that severe neuron [43] and motoneuron loss near the epicenter starting at 12 and 24 h post-SCI and gradually spread to longer distance over time. Our temporal and spatial profiles of apoptosis published previously [43], indicated that the peak time of TUNEL-positive cell appearances are from 12 to 48 h post-SCI. Therefore, 12 to 48 h post-SCI is a reasonable time frame in which to evaluate the efficiency of treatment to reduce cell death and apoptosis. The rostral sections removed at 12 h post-SCI at 0–4.0 mm from the epicenter were also CV-stained to be used as vehicle controls to establish the spatial profile of MnTBAP + L-NA protection against cell death and for terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-(dUTP)-biotin nick end labeling (TUNEL) staining to evaluate the protection of the combined treatment against apoptosis. The rostral sections obtained at 24 h post-SCI at 0, 1.0, 2.0 and 4.0 mm from the epicenter were used for TUNEL staining as vehicle-treated group to evaluate MnTBAP protection against apoptotic cell death and for characterization of apoptosis. To match with the vehicle-treated groups, the injured spinal cord in the MnTBAP + L-NA-treated rats was removed at 12 h post-SCI and the injured cord in the MnTBAP-treated rats was removed at 24 h post-SCI. TUNEL staining and TUNEL plus immunohistochemical or immuno-fluorescence double staining to characterize apoptosis in different types of cells in injured spinal sections DNA fragmentation in the nuclei is an indicator of apoptosis. Fragmented DNA in the nuclei of cells was characterized by TUNEL staining using an ApopoTag peroxidase in situ apoptosis detection kit according to the manufacturer's instructions. The TUNEL staining and visualization of spinal cord sections were performed according to procedures we previously reported [30,31,43]. The only difference was that, in the present study, the TUNEL detecting kit was purchased from Millipore (Billerica, MA, USA). Nonspecific labeling was investigated by using water or equilibration buffer to substitute for the volume of TdT in the TUNEL reaction mixture. To characterize apoptosis in different types of cells, TUNEL plus immunohistochemical double staining was performed. The spinal cord sections were first stained with TUNEL, then stained with antibodies specific to different types of cells. Following TUNEL staining, the sections were washed in three changes of PBS for 5 min each, blocked with 5% goat serum and incubated with: a mouse monoclonal antibody directed against neuron specific enolase (NSE, 1:100, DAKO, Carpinteria, CA, USA) to stain neurons; a rabbit polyclonal antibody directed against choline acetyltransferase (ChAT, 1:500, Abcam, Cambridge, UK) to stain motoneurons; or a monoclonal antibody against glial fibrillary acidic protein (GFAP, 1:200, Sigma, St. Louis, MO, USA) to stain astrocytes. After washing in PBS, the sections were incubated with biotinylated goat anti-rabbit-IgG (1:200, Sigma, St. Louis, MO, USA) or goat anti-mouse-IgG (1:200, Sigma, St. Louis, MO, USA) followed by a solution of an avidin-biotin-horseradish peroxidase complex (1:200, Vector Laboratories, Inc., Burlingame, CA, USA). The sections were then visualized with SG Substrate Kit (Vector Laboratories, Inc., Burlingame, CA, USA), washed in water, dehydrated, cleaned and coverslipped with Permount as described in our previous report [30,31,43]. Negative controls were similarly stained without the primary antibody. Motoneurons were also characterized with TUNEL and immuno-fluorescence double staining. The TUNEL staining was performed similarly as above, but using the ApopTag plus fluorescein In Situ apoptosis detection kit (Chemicon Intl. Temecula, CA, USA) following the manufacturer's instruction. After TUNEL staining, the sections were immuno-stained as in the above procedures with the primary antibody, mouse anti-ChAT (1:100, ABcam, Cambridge, MA, USA). After washing in PBS for 3 times, a secondary antibody goat anti-mouse Alexa Flour 594 (Invitrogen, Carlsbad, CA, USA) was added to the sections and the sections were incubated for 1 h at room temperature. The sections were washed in PBS and coversliped with anti-fade solution (Invitrogen) and kept in the dark. Negative controls were similarly stained without the primary antibody. Active caspase-3 and cellular marker immuno-fluorescence double staining Caspase-3 activation is another indicator of apoptosis. To characterize apoptosis in different types of cells, double immuno-fluorescence staining with antibodies against active caspase-3 and specific cellular markers was performed. The procedures for immuno-fluorescence staining were similar to those described above. The deparaffinized spinal cord sections were rinsed in PBS and placed in 5% goat serum in PBS for 30 min, then incubated with the primary antibody: a monoclonal antibody anti-NSE (1:100, DAKO, Carpinteria, CA, USA) or a monoclonal antibody anti-GFAP (1:200, Sigma, St. Louis, MO, USA) to stain neurons or astrocytes, respectively. For immunohistochemical staining of active caspase-3, the immuno-stained sections were incubated with the secondary antibody, a polyclonal antibody anti-p20 active fragment of caspase-3 (1:200, R&D systems, Inc. Minneapolis, MN) in PBS with 0.2% Triton X-100 and 0.1% bovine serum albumin overnight at 4°C. For visualization, after washing in PBS, the sections were incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (1:100, Sigma, St. Louis, MO, USA) and crystalline tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit IgG (1:100, Sigma, St. Louis, MO, USA) in PBS with 0.2% Triton X-100 and 0.1% BSA for 1 hour in the dark to visualize cells and active caspase-3, respectively. The slides were coverslipped with PBS/glycerol and stored at 4°C after washing with PBS in the dark. The FITC-labeled neurons or astrocytes display green and the TRITC-labeled activated caspase-3 displays red fluorescence. Negative controls were similarly stained without the primary antibody. Confirmation of apoptosis by transmission electron microscopy (TEM) For TEM examination of neuronal and glial apoptosis, the animal preparation and spinal cord injury were done as described above. At 24 h post-SCI, the rat was perfused intracardially with 0.9% saline, followed by 4% paraformaldehyde with 2.0% glutaraldehyde in 0.01 M PBS (pH 7.4). After perfusion, the spinal cord was further exposed and a 1.5 cm segment centered at the injury site was removed as described above and fixed in the same fixative overnight. Then, the segment was fixed with 4% osmium tetroxide in 0.01 M PBS for 2 h, dehydrated and embedded in epoxy resin. Semi-thin (1 μm) plastic amberalvarez@example.com mm from the epicenter were cut and stained with 1% toluidine blue to screen for apoptotic neurons under the light microscope. Ultrathin sections (in the Rexed lamina VIII, gold interference color) were cut with an ultramicrotome, stained with uranyl acetate and lead citrate, then examined using a Hitachi H600 electron microscope (Tokyo, Japan). Quantitation of cell loss and apoptosis by counting surviving or TUNEL-positive cells in spinal cord sections Surviving cells in the CV-stained sections or TUNEL-positive cells in the TUNEL-stained sections were counted in an area of the bottom left and bottom right quarter of the gray matter of the cord. Quarters were defined by horizontal and vertical crossed lines drawn through the central canal in the center of the sections [18,38-40,43]. This area of gray matter includes motoneurons, middle- and small-sized neurons, and glial cells. Neurons in the CV-stained sections were identified by the presence of bright nuclei with nucleolus and enrichment in Nissl bodies. Neurons with soma diameters greater than 25 μm were identified and counted as motor neurons [46]. Glial cells in the CV-stained sections were counted in 250 × 250 μm2 areas of the left and right ventromedial white matter. TUNEL-positive cells were identified by dark nuclei and were counted in TUNEL-stained sections. The cells in the left or right quarters of each section were counted 3 times and the counts averaged. Then, the average counts of the left and right quarters in each section were averaged to obtain the final counts for that section. Three adjacent sections were counted and the counts averaged to obtain the final counts for that distance. To avoid bias, the person processing the cord into sections and the person counting the cells in the sections were blinded. This counting method has been validated by our previous publications [18,38-40,43] and by those of many others. Biochemical characterization and quantification of apoptosis in spinal cord tissues following SCI Animal preparation and tissue processing The animal preparation and impact injury were the same as described above. At 48 h post-SCI (or post-laminectomy for sham controls), the animal was re-anesthetized and the cord was further exposed from the vertebrae T10 to L5 by a second laminectomy. A cavity was formed by pulling up the skin around the exposed cord and the spinal cord was frozen in situ by pouring liquid nitrogen (−196°C) into the cavity as described previously [21]. The frozen tissue was cut into three sections of equal length (1 cm each) with section 1 centered at the epicenter and sections 2 and 3 centered at 1 and 2 cm caudal to the epicenter. The sections were immediately stored in a −80°C freezer for characterization of specific laddering of DNA fragments by agarose gel electrophoresis and for quantitation by enzyme-linked immuno-sorbent assay (ELISA). Since caspase-3 activates as early as 4 h post-SCI [6], to analyze caspase-3 activity, the frozen tissue was removed at 4 h post-SCI. The cord was cut into 2 sections (1 cm each) with section 1 centered at the epicenter and section 2 centered 1 cm from the epicenter. DNA laddering by agarose gel electrophoresis DNA fragmentation was characterized by radio-end labeling using [32P]dCTP and agarose gel electrophoresis following procedures reported [47]. Briefly, the spinal cord sections were removed and divided at 48 h post-SCI (5–10 mg wet weight), then gently disrupted with a hand-operated glass/glass Dounce homogenizer in lysis buffer (5 mM Tris–HCl, pH 7.4, containing 0.5% Triton X-100, 1:15 w:v), left on ice for 20 min, centrifuged for 30 min at 4°C at 15,000 g to remove the nuclei and cellular debris, then extracted with phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation, DNA in the aqueous phase was precipitated using ethanol. The DNA pellet was resuspended in 20 μl of Tris-EDTA buffer (pH 8.0) and treated with 20 μg/ml RNase A for 1 h at 37°C to digest the RNA. After quantitation, 2 μg of DNA from each sample was labeled with [32P]dCTP (50 μCi) by the standard method involving the Klenow fragment of DNA polymerase I. Samples were then re-extracted as above to remove unincorporated nucleotides, resuspended in DNA gel loading buffer and loaded onto a 2% agarose gel. After electrophoresis, the gel was dried under vacuum and exposed for 24 h to autoradiographic film. Quantitation of DNA fragmentation by ELISA Frozen spinal cord removed at 48 h post-injury was homogenized in lysis buffer containing 8 M urea, 0.24 M sodium phosphate, 1% sodium dodecyl sulfate and 10 mM EDTA, pH 6.8 using a 20:1 solution to tissue ratio (v:w). The homogenates were extracted with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) saturated with the homogenization buffer. The resulting emulsion was separated into two phases by centrifugation at 4,500 g for 3 min in an Eppendorf centrifuge. The aqueous phase above the organic phase was removed for DNA analysis as reported originally by Beland and coworkers [48] and slightly modified by us [21]. Aliquots (10 μl) of each sample were used to quantitate the nucleosome-associated apoptotic DNA fragments by ELISA using a commercial cell death detection kit (Boehringer Mannheim, Indianapolis, IN, USA) which detects histone-associated DNA fragments enriched in the cytoplasm of a dying cell. The analysis was performed in duplicate according to the manufacturer's instructions. Analysis of caspase-3-like protease activity Caspase-3-like protease activity was measured according to the reported procedure [49,50]. Briefly, approximately 40 mg of spinal cord tissue was homogenized in a 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.5) containing 1 mM ethylene glycol-bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid, 5 mM MgCl2, 1 mM phenylmethyl-sulphonyl fluoride and 1 μg/ml leupetin and aprotinin, then centrifuged at 12,000 g for 15 min. The protein concentration in the supernatant was detected using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Then, 75 μg protein was incubated with synthetic fluorescent substrates benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC, Enzyme Systems Products, Livermore, CA, USA) to assay the caspase-3-like protease activity at a concentration of 50 μM in 300 μl of 0.1 M HEPES buffer (pH 7.4) at 37°C for 1 h. The HEPES buffer contains 2 mM dithiothreitol, 0.1% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate) and 10% sucrose. DEVD-AFC cleavage was measured using a spectrafluorimetric detector (Shimadzu RF-10A, Shimadzu Europa GmbH, Duisburg, Germany) at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. The fluorescence intensity was calibrated with a standard concentration of AFC and expressed in pmol per minute per mg of protein. Behavioral assessments The protective effect of MnTBAP on the functional recovery after SCI was assessed by the Basso-Beattie-Bresnahan locomotor rating scale (BBB test), the inclined plane test, and the beam walk test for 9 weeks. SCI causes hind limb paralysis and inability of the rat to support its body weight, so the scrotum of male rats touched and dragged around the bottom of cage, increasing the incidence of infection. Therefore, most publications using behavioral tests to study SCI use female rats. This study examined the efficacy of MnTBAP to attenuate neurological deficits in three groups of female rats (200–225 g): injured groups treated with saline or MnTBAP (n = 8 for injured groups) and sham-operated controls (n = 4). The injuries for behavioral tests to evaluate functional recovery after SCI are performed on T9 or T10 by using the standard New York University (NYU) weight drop device [40,51,52] or the Infinite Horizons (IH, Lexington, KY) device [53-55] in many publications. To explore the therapeutic potential of MnTBAP in treating SCI by behavioral tests, in the present study, the cord was injured on vertebra T10 using the NYU device, so that the results can be compared with other publications using behavioral tests. Animal preparation, impact injury and drug administration The rats were given 0.1 mg/kg buprenorphine subcutaneously (s.c.) 15 min prior to anesthesia, then anesthetized with pentobarbital (50 mg/kg, i.p.). After a laminectomy, the exposed cords were injured on vertebra T10 by a 10 g rod dropping 1.25 cm onto the exposed cord (12.5 g.cm force) using NYU device. Then, the muscle and skin were sutured in layers and 10 ml saline was injected (i.p.) to prevent dehydration. The body temperature of the rat was maintained using a feedback-controlled heating pad during surgery and until the animal awoke from surgery. MnTBAP (10 mg/kg) or saline was given by i.p. injection 15 min pre- and 4 h post-SCI for behavioral tests. In the sham-operated control group, animals underwent the same surgical procedures but no weight drop injury was performed. These rats were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle. In the first week following SCI, the bladder was emptied manually until the voiding reflex was recovered; buprenorphrine (0.5 mg/kg, s.c.) was given post-injury to minimize the impairment or discomfort of injured rats. The body weight of the animal was monitored during behavioral tests. If the loss of body weight was more than 20% pre-injury weight, the animal was terminated. Since only the hindlimbs were paralyzed, the rat could still reach food and water. All experiments were triple blinded: 2 blinded observers gave scores and the third one administered MnTBAP or vehicle, blinded to the 2 observers. BBB test The motor function of rats was assessed using the BBB locomotor rating scale as reported [56,57]. The test is designed to assess progressive locomotor recovery after SCI. The BBB score was assessed by observing each animal walking in an open field for 4 min. The open field was simply a molded plastic pool with a dimpled floor (100 cm diameter, 21 cm height). To observe animal locomotion requires pretest training. Before surgery, the rats were exposed to the testing environment twice a day for 5 days. Each rat was handled several times during each 30–60 min session so that they no longer showed signs of fear and became accustomed to being picked up and walking continuously in the open field environment. Then the rats were individually placed in the open field for 4 min to assure that all consistently obtained a maximum score of 21. After SCI, the animals were evaluated for hind limb motor function either alone for 4 min or in pairs for 5 min every day for 3 days, then on day 5 and weekly from 1 – 9 weeks by two blinded observers. BBB scores (0 – 21 points) categorize combinations of rat hindlimb movement, trunk position and stability, stepping, coordination, paw placement, toe clearance and tail position, representing sequential recovery stages of the rat's hind limb motor function after SCI. A score of 0 indicates a paralyzed rat. A score of 21 indicates normal locomotion. Scores from 0 to 8 focus on limb movement of three joints (hip, knee and ankle), representing the early phase of recovery. Scores from 9 to 14 describe the intermediate recovery phase with return of standing and paw placement, stepping with varying degrees of forelimb-hindlimb coordination. Scores from 14 to 20 indicate the rate with return of toe clearance during the step phase, predominant paw position, trunk stability and tail position, reflecting the late and final phase of recovery. Two examiners were positioned across from each other to observe both sides of the rat. Inclined plane test The inclined plane test measures the animal's ability to maintain its body position on an inclined board. Each rat was placed on an inclined plane covered by a rubber mat (with 0.6 mm deep grooves) constructed as reported [58,59]. The plane can be adjusted to different slopes to measure the animal's ability to maintain its body position for 5 sec without falling. The animal was placed on an inclined plane with the head to the right and the left. The average score of the two positions was used. The angle of the board was increased from 0° until it reached an angle at which the rat could maintain its position only for 5 seconds. Final readings to the nearest 5° were taken for stationary positions. All animals were assessed every week for 9 weeks after SCI. The beam walk test The beam walk was performed as described [60] using seven wooden beams of different widths (1.7, 2.7, 3.7, 4.7, 5.7, 6.7 and 7.7 cm). Before surgery, the rats were trained for 5 days on the plank from the widest to the narrowest for several times each day, so that all of the rats could walk across the 1.7 cm plank. The beam walk performance test was conducted once a week for 9 weeks post-SCI. The animals were allowed to walk on the planks and the narrowest plank on which they could walk without foot slip in two trials was recorded. Rats that could walk only on the widest plank (7.7 cm) were scored 1, and those who could walk on the narrowest plank (1.7 cm) were scored 7. Statistical analysis To obtain the temporal and spatial profiles of cell loss, the counts at each distance were compared with those obtained in the sections at the same distance in the sham control group over time and the counts at each time were also compared over distance by using one-way repeated measures analysis of variance (RMANOVA) followed by the post-hoc Tukey test. Two-way RMANOVA followed by all pairwise multiple comparison using the Bonferroni correction were used to evaluate the spatial protection against different types of cell loss by MnTBAP + L-NA and by MnTBAP alone against apoptosis, and to assess the effect of MnTBAP treatment on the hind limb motor function after SCI by behavioral tests. This statistical procedure was also used to compare DNA fragmentation between the SCI and sham groups at different distances from the epicenter obtained by ELISA. Caspase-3 activation in sham controls and by distance from the epicenter was analyzed by analysis of variance (ANOVA) followed by Dunnett's test. The protection of MnTBAP + L-NA against apoptotic cell death in injured and treated groups was compared by the unpaired t-test. P < 0.05 was considered a statistically significant difference. Results The quantitative temporal and spatial profiles of motoneuron and glia loss after SCI To obtain quantitative temporal and spatial profiles of motoneuron and glia cell loss, the spinal cord in injured animals were removed at 1, 6, 12, 24, 48, 72 h and 1 week post-SCI (50 g.cm). The cords in sham-operated controls were removed at 12 h post-laminectomy and served as the control for all time points. The sham control animals underwent the same surgical procedures without weight drop injury. The spinal cords were processed for CV-staining. After staining, the number of cells in the CV-stained sections was counted over time from 1 h to 1 week post-SCI and over distance from 0 to 4 mm caudal from the injury amberalvarez@example.com mm intervals as described in the Methods. The counts were then compared between sham controls and the post-SCI groups for all time points at each distance and by distance at each time point using one way RMANOVA followed by the post-hoc Tukey test. Figure 1 illustrates photomicrographs of CV-stained sections at different times post-SCI (upper panel) and at different distances from the epicenter (lower panel). The sham-operated section (A, D) had many cells, including large motoneurons in the ventral gray matter which showed bright nuclei with nucleoli and enrichment in the Nissl bodies. In the upper panel, at 1 h post-SCI, many cells had been lost in the gray matter (B, E) and some neurons lost their normal morphological features. At 24 h post-SCI (C, F), almost no neurons could be observed in the ventral gray matter. In the lower panel, at the epicenter (0 mm), few cells and almost no neurons were observed (B, E). At 2 mm from the epicenter (C, F), some surviving cells including neurons were observed in the ventral gray matter. Figure 1 Photomicrographs of CV-stained spinal cord sections at different times post-SCI and different distances from the epicenter. Following a laminectomy, the rat spinal cords were injured (50 g.cm), the injured cords were removed at different times post-SCI and processed for CV staining. The upper panel shows surviving cells in CV-stained sections at 1 mm caudal from the epicenter removed at 1 and 24 h post-SCI. The lower panel shows surviving cells in CV-stained sections at 0 and 2 mm caudal from the epicenter removed at 24 h post-SCI. A-C: Lower magnification, D-F: higher magnification of A-C. Scale bar: A-C, 100 μm, D-F, 50 μm. Figure 2 shows the temporal and spatial profiles of cell loss. Figure 2A shows the motoneuron loss in the ventral gray matter following SCI. Neurons larger than 25 μm in diameter in the CV-stained sections were counted as motoneurons. The average counts presented as mean ± standard deviation (SD) of motoneurons at each time and each distance from the epicenter are presented in Table 1. The counts were compared between sham control sections and all post-SCI sections over time at 1, 6, 12, 24, 48 and 72 h, and at 1 week post-SCI for each distance (n = 3 for all). The count at each time point was also compared to that in the sham controls at the same distance. We found significant differences over time at all distances examined (p = 0.02 - <0.001). Motoneurons were significantly fewer at 0 to 1.5 mm from 1 h to 1 week (p = 0.003 - < 0.001); at 2 mm from 12 h to 1 week (p = 0.002 - 0.01); amberalvarez@example.com - 4.0 mm from 48 h to 1 week post-SCI (p = 0.002 - 0.046). Comparisons by distance (0 - 4 mm) from the epicenter at each time point revealed no significant differences in the sham control (p = 0.9) or in the post-SCI groups at 1 h (p = 0.7), 48 h (p = 0.2), 72 h (p = 0.1) and 1 week (p = 0.9). Figure 2 The temporal and spatial profiles of cell death after SCI. Cells in the CV-stained sections caudal from the epicenter as shown in Figure 1 in injured or sham control sections were counted as described in the Methods and counts were then statistically compared over time and by distance from the epicenter. The counts were presented as mean ± SD. A: The temporal and spatial profile of motoneuron loss in the ventral gray matter of the cord after SCI. The number of surviving motoneurons increased with the distance from the epicenter at each time point, and decreased over time at each distance after SCI. All significant differences were indicated under the dotted lines. B: The temporal and spatial profiles of glial cell loss in the ventromedial white matter following SCI. Significant glial loss in the white matter was found only near the epicenter; such loss was not significant at 2 mm from the epicenter at any time post-SCI. * indicates significant difference between sham control and SCI (p < 0.05). Table 1 The average number of motoneurons in the ventral gray matter of rat spinal cords Time post-SCI (h) Distances caudal from the epicenter (mm) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Sham 9.3 ± 1.0 10.2 ± 1.3 9.4 ± 0.3 9.8 ± 0.8 9.8 ± .8 8.4 ± 1.5 8.9 ± 1.0 9.2 ± 0.7 8.6 ± 0.3 1 2.6 ± 2.2 3.1 ± 2.0 3.3 ± 0.3 3.1 ± 2.1 3.7 ± 2.0 3.9 ± 1.3 5.4 ± 1.3 4.3 ± 1.3 4.9 ± 0.4 6 0.8 ± 1.3 0.4 ± 0.7 1.4 ± 1.3 2.3 ± 2.0 4.0 ± 1.2 4.7 ± 0.8 5.2 ± 1.6 5.6 ± 2.1 6.1 ± 3.5 12 0.2 ± 0.2 0.1 ± 0.3 0.8 ± 1.2 1.7 ± 1.2 2.5 ± 2.7 3.7 ± 2.8 4.9 ± 2.8 5.4 ± 2.4 6.6 ± 1.6 24 0 0 0 1.04 ± 1.9 1.9 ± 2.5 3.2 ± 2.5 4.3 ± 2.2 4.8 ± 2.6 5.4 ± 1.0 48 0.3 ± 0.3 0.2 ± 0.3 0.1 ± 0.1 0.22 ± 0.3 1.4 ± 1.2 1.5 ± 1.2 0.8 ± 0.6 1.7 ± 1.1 1.9 ± 0.8 72 0 0 0.2 ± 0.4 0.1 ± 0.1 0 0.7 ± 0.9 0.8 ± 1.1 1.4 ± 0.3 1.4 ± 1.1 168 0 0 0 1.6 ± 2.7 0.8 ± 1.3 0.9 ± 3.1 2.5 ± 3.7 0.8 ± 0.6 2.1 ± 0.8 The number of motoneurons at different times post-SCI and different distances from the epicenter was presented as mean ± SD (n = 3 for all). The absence of significant differences over distance and the small number of surviving motoneurons at times from 48 h to 1 week show that most motoneurons had been lost by 48 h to at least 4.0 mm from the epicenter. Significant differences were found at 6, 12 and 24 h (p = 0.03 - <0.001) post-SCI. At 6 h, the difference was significant over distance (p = 0.03), but no difference was found among sections; at 12 h, the difference was significant over distance (p < 0.001) with significantly more motoneurons at 3 mm than at 0 and 0.5 mm (p = 0.03 for both), at 3.5 mm amberalvarez@example.com - 1 mm (p = 0.01 - 0.04) and at 4 mm than at 0–1.5 mm (p = 0.001- 0.03); at 24 h post-SCI, a significant different was found only between 4 mm and 0–1 mm. By 48 h to 1 week, no significant differences were found over distance (p = 0.1 - 0.9), indicating that most motoneurons had already been lost at all distance examined. Figure 2B shows the temporal and spatial profile of glial cell loss in the ventral white matter following SCI. Glial cells in the ventral white matter of CV-stained sections were counted as described in the Methods. The average counts (mean ± SD) at each time and each distance from the epicenter are presented in Table 2. Significant glial cell loss appeared at 24–72 h post-SCI in 0 mm sections (p = 0.03 - 0.01) and at 24 and 48 h post-SCI in 0.5 - 1.5 mm sections compared to sections from sham-operated controls (p = 0.047 - 0.003). At 72 h post-SCI, glial cells began proliferating and significantly more glial cells were found at 72 h than at 24 and 48 h post-SCI, but only in 1.5 mm sections (p = 0.02-0.03). By 1 week, the number of glial amberalvarez@example.com mm was significantly higher than at 24 and 48 h (p = 0.006 - 0.03); higher at 1 mm than at 6–48 h; and amberalvarez@example.com mm than at 24 and 48 h (p = 0.047 - <0.001) post-SCI. Glial loss in the ventral white matter was much slighter than motoneuron loss in the ventral gray matter, and the loss was quickly offset by proliferation (n = 3 for all time points and all distances). Table 2 The average number of glial cells in the ventral white matter of rat spinal cords Time post-SCI (h) Distances caudal from the epicenter (mm) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Sham 41.4 ± 0.7 41.3 ± 1.8 41.6 ± 2.5 40.0 ± 1.3 40.5 ± 0.9 42.3 ± 1.5 41.1 ± 2.7 39.3 ± 2.4 40.0 ± 3.1 1 35.7 ± 4.9 34.2 ± 0.4 34.8 ± 3.1 37.2 ± 1.2 33.2 ± 0.7 37.9 ± 0.4 39.4 ± 0.4 39.3 ± 0.5 39.2 ± 1.6 6 28.9 ± 4.0 32.0 ± 3.8 30.1 ± 8.3 33.8 ± 4.2 33.5 ± 2.2 34.6 ± 3.2 36.7 ± 0.7 34.8 ± 2.4 35.6 ± 0.3 12 26.1 ± 8.1 27.0 ± 5.4 28.3 ± 6.7 33.5 ± 4.0 37.2 ± 4.9 34.1 ± 3.1 34.2 ± 3.3 35.7 ± 2.8 35.7 ± 0.3 24 13.7 ± 6.6 18.4 ± 1.1 23.0 ± 3.9 22.2 ± 4.2 34.1 ± 5.5 36.8 ± 4.8 35.1 ± 2.2 35.7 ± 4.0 36.2 ± 0.3 48 10.9 ± 5.3 12.7 ± 6.1 17.2 ± 8.0 21.8 ± 9.7 36.8 ± 3.4 34.4 ± 5.3 38.1 ± 0.8 32.3 ± 0.7 38.5 ± 0.8 72 14.0 ± 1.5 28.3 ± 11.5 32.1 ± 9.0 38.1 ± 6.2 34.8 ± 4.5 39.3 ± 4.3 41.7 ± 2.07 37.3 ± 0.7 40.3 ± 4.4 168 28.7 ± 21 42.8 ± 17.3 47.6 ± 3.4 41.7 ± 5.4 42.2 ± 7.0 39.4 ± 4.3 40.2 ± 2.8 34 ± 8.1 34 ± 7.2 The number of glial cells at different times post-SCI and different distances from the epicenter was presented as mean ± SD (n = 3 for all). Morphological characterization of SCI-induced neuronal and glial apoptosis By counting TUNEL-positive cells in the sections 0, 1, 2 and 4 mm from the epicenter in the cord removed at 1 h to 1 week post-SCI, we found that the number of TUNEL-positive cells in the ventral gray matter peaked at 12 to 48 h post-SCI and maximized at the distances of 1 and 2 mm from the epicenter [43]. Therefore, in this study, neuronal and glial apoptosis was further characterized morphologically as described in the Methods at 12 or 24 h post-SCI and at 2 mm rostral from the epicenter by the following staining: 1) TUNEL and immunohistochemical double staining with antibodies against specific cell markers of different types of cells (anti-NSE for neurons, anti-ChAT for motoneurons and anti-GFAP for astrocytes); 2) TUNEL and anti-ChAT immuno-histochemical and immuno-fluorescence double staining of apoptotic motoneurons; and 3) active caspase-3 and specific cell marker double immuno-fluorescence staining of apoptotic neurons and astrocytes. Neuronal and glial apoptosis was confirmed by transmission electron microscopy. Figure 3 illustrates the photomicrographs of the morphological characterization of apoptosis in neurons and astrocytes. DNA fragmentation is an event of apoptosis and TUNEL stains the fragmented DNA in the nuclei. Anti-NSE and anti-GFAP immunohistochemical staining identifies neurons and reactive astrocytes, respectively. A to D in Figure 3 show the photomicrographs of TUNEL and NSE or GFAP immunohistochemically double-stained sections in the ventral horn of the gray matter in an injured spinal cord sections 2 mm rostral from the epicenter removed at 24 h post-SCI. The arrowheads in B indicate TUNEL-positive nuclei in NSE-positive neurons. The arrows in D indicate TUNEL-positive nuclei in GFAP-positive astrocytes. These double-stained spinal cord sections localize the apoptotic DNA fragmentation in the nuclei of neurons (A and B) and astrocytes (C and D). This is the morphological indication of neuronal and glial apoptosis. Since caspase-3 activation occurs prior to DNA fragmentation, the injured spinal cord was removed at 12 h post-SCI in this study for active caspase-3 staining. E to J in Figure 3 show the photomicrographs of active caspase-3 and NSE or GFAP immuno-fluorescence double-stained apoptotic astrocytes (E - G) and neurons (H - J), respectively, in the spinal cord sections 2.05 mm rostral from the epicenter removed at 12 h post-SCI. E to G show double immuno-fluorescence-stained sections with antibodies against an active fragment p20 of caspase-3 and anti-GFAP. E shows astrocytes (GFAP-positive, arrows) and F shows the same astrocytes stained for p20 (arrows). G is the colocalization of E and F, showing caspase-3-positive astrocytes (arrows). H to J show double immuno-fluorescence-stained neurons. H shows NSE-positive neurons (arrow head). I shows that the same neurons are also p20-positive (arrow head). J is colocalization of H and I, showing active caspase-3-positive neurons (arrow head). These double immuno-fluorescence staining co-localized caspase-3 activation in the neurons and astrocytes and provided another morphological indication of neuronal and glial apoptosis. Figure 3 Photomicrographs of the morphological characterization of apoptotic neurons and astrocytes. A-D: photomicrographs of TUNEL and immunohistochemical double-stained sections 2 mm rostral from the epicenter in the ventral horn of injured spinal cords removed at 24 h post-SCI. A and B: TUNEL and NSE double-stained TUNEL-positive neurons at lower (A) and higher (B) magnifications. The arrowheads indicate TUNEL-positive nuclei in NSE-positive neurons. C and D: TUNEL and GFAP double-stained TUNEL-positive astrocyte at lower (C) and higher (D) magnifications. The arrows indicate TUNEL-positive nuclei in GFAP-positive astrocytes. E-J: photomicrographs of active caspase-3 and immuno-fluorescence double-stained sections 2.05 mm rostral from the epicenter in the ventral gray matter of injured spinal cords removed at 12 h post-SCI. E-G: p20 fragment and GFAP double-stained active caspase-3 with active astrocytes. E: active astrocytes (GFAP-positive, arrows); F: the same astrocytes stained for p20 (arrows). G: immuno-colocalization of E and F, showing caspase-3-positive astrocytes (arrows, yellow). H-J: p20 fragment and NSE double-stained neurons. H: NSE-positive neurons (arrowhead); I: the same neurons were also p20-positive (arrowhead). J: immuno-colocalization of H and I showing caspase-3-positive neurons (arrowhead, yellow). Scale bar = 100 μm. In the spinal cord, most ChAT immuno-reactive neurons are motoneurons. Using ChAT and TUNEL double-staining, we found that a number of ChAT-positive neurons were also TUNEL-positive in the ventral horn of spinal cord sections 2 mm rostral from the epicenter removed at 24 h post-SCI, indicating motoneuron apoptosis (Figure 4A-C). Motoneuron apoptosis was further shown by the ChAT and TUNEL double immuno-fluorescence colocalization (Figure 4D-F). ChAT-immuno-staining showed red cell bodies of motoneurons (D) and TUNEL staining showed blue nuclei (E) in the ventral horn of the gray matter of the cord from a section 2.05 mm rostral from the epicenter removed at 24 h post-SCI. The blue TUNEL-stained nuclei were localized clearly in the red ChAT-positive cell bodies of motoneurons (F. arrows). Figure 4 Photomicrographs of the morphological characterization of apoptotic motoneurons. A-C: Photomicrographs of TUNEL and ChAT double immunohistochemical stained section at 2 mm rostral from the epicenter removed at 24 h post-SCI. A: lower magnification of the ventral gray matter; B: higher magnification corresponding to the frame in A; C: high magnification corresponding to the frame in B. Two enlarged TUNEL-positive motoneurons were shown in the frames of C. Scale bar, 100 μm. D-F: Photomicrographs of TUNEL and ChAT double immuno-fluorescence stained amberalvarez@example.com mm rostral to the epicenter removed at 24 h post-SCI. The section was double immuno-stained with anti-ChAT primary antibody (D, red) and TUNEL (E, blue), followed by anti-mouse Alexa Fluor 594 secondary antibody. F: immuno-colocalization of D and E showing apoptotic motoneurons (bluish red) in the ventral gray matter of the cord from a vehicle control rat. Photomicrographs are taken at high magnification (scale 100 μm). SCI-induced apoptosis was confirmed by TEM identification based on the specific morphology of apoptosis. Figure 5 illustrates the ultra-fine structural changes in apoptotic cells in layer VIII of the ventral horn in the gray matter of the spinal cord amberalvarez@example.com mm rostral from the epicenter removed at 24 h post-SCI (50 g.cm). Figure 5A, a sham control section 2 mm rostral from the epicenter, shows a normal neuron with a large, euchromatic (pale-staining) nucleus (N), abundant rough endoplasmic reticulum (R), mitochondria (M), and Golgi complexes (G) in the cytoplasm. Figure 5B-F show different stages of apoptosis and degeneration from injured animals. B shows
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