Publish in this journal
Journal Information
Vol. 24. Num. 1.January - February 2013Pages 1-50
Share
Share
Download PDF
More article options
Visits
2219
Vol. 24. Num. 1.January - February 2013Pages 1-50
Clinical Research
DOI: 10.1016/j.neucir.2011.12.006
AR-A014418 as a glycogen synthase kinase-3 inhibitor: Anti-apoptotic and therapeutic potential in experimental spinal cord injury
AR-A014418 como inhibidor de la glucógeno sintasa quinasa 3: potencial anti-apoptótico y terapéutico en lesión medular
Visits
2219
Matem Tunçdemira, Aziz Yıldırımb, Alper Karaoğlanc,??
Corresponding author
drkaraoglan@yahoo.com

Corresponding author.
, Osman Akdemird, Melek Öztürka
a Department of Medical Biology, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey
b Department of Neurosurgery, Bingöl State Hospital, Bingöl, Turkey
c Department of Neurosurgery, Maltepe University, School of Medicine, Istanbul 34857, Turkey
d Department of Neurosurgery, Taksim Education and Research Hospital, Istanbul, Turkey
Visits
2219
Article information
Abstract
Full Text
Bibliography
Download PDF
Statistics
Figures (6)
Show moreShow less
Abstract
Objectives

We aimed to investigate the effects of AR-A014418, a strong inhibitor specific to GSK-3beta, on neuronal apoptosis and neuroprotection in the traumatic SCI model.

Materials and methods

In this study, three groups were generated from 36 Wistar rats; (1) control, (2) spinal cord trauma group created by clip compression technique after laminectomy, and (3) AR-A014418 (4mg/kg, i.p., DMSO) treatment group after laminectomy and spinal cord trauma. The TUNEL assay for apoptosis detection, immunohistochemical staining for bax and TGF-beta were applied in spinal cord tissues. For light microscopic examination, necrotic, and apoptotic cells were counted, and PMNL counting was applied to detect inflammation. Functional recovery was tested by field locomotor test in the 3rd and 7th days following surgery.

Results

In the trauma group, diffuse hemorrhage, cavitation, necrosis and edematous regions, degeneration in motor neurons and leukocyte infiltration were observed in gray matter. In the AR-A014418-treated groups, healthy cells were observed in more places compared to the trauma groups, however, cavitation, hemorrhagic, and edematous areas were seen in gray matter. In the AR-A014418-treatment groups, the number of apoptotic cells in the 3rd and 7th days (respectively; p<0.05, p<0.01), were significantly decreased compared to the trauma groups, as were the levels of bax (p<0.01) and TGF-beta 1 immunoreactivity. Results of the locomotor test were significantly increased in the treatment group (p<0.001) as compared to the trauma group.

Conclusions

In this experimental spinal cord trauma model study neural apoptosis was significantly triggered in secondary damage developed after trauma, however, neurological healing was expedited by preventing mitochondrial apoptosis and reducing the inflammation by the potent inhibitor AR-A014418, which is GSK-3beta selective.

Keywords:
Spinal cord injury
GSK-3beta
AR-A014418
Apoptosis
TGF-beta
Abbreviations:
SCI
GSK-3
DMSO
PMNL
H+E
CV
TUNEL
Resumen
Objetivo

Este trabajo tuvo como objetivo investigar los efectos del AR-A014418, un potente inhibidor específico de la GSQ-3beta, en la apoptosis y neuroprotección neuronales en el modelo de lesión medular traumática.

Materiales y métodos

Se generaron tres grupos a partir de 36 ratas Wistar: (1) control, (2) grupo de traumatismo medular obtenido mediante técnica de pinzamiento post-laminectomía, y (3) grupo de tratamiento mediante AR-A014418 (4mg/kg, i.v., DMSO) post-laminectomía y traumatismo medular. Se aplicaron el test TUNEL para la detección de la apoptosis, tinción inmunohistoquímica para bax y TGF-beta en los tejidos medulares. Se llevo a cabo un examen microscópico y recuento de células necróticas y apoptóticas, así como recuento de LPMN para detectar inflamación. La recuperación funcional fue verificada mediante la prueba de campo del aparato locomotor en los días 3 y 7 después de la cirugía.

Resultados

Se observo hemorragia difusa, cavitación, necrosis y regiones edematosas, degeneración de las neuronas motoras e infiltración leucocítica en la materia gris en los grupos traumáticos. En los grupos con tratamiento AR-A014418 se observaron células sanas con mayor abundancia en comparación con los grupos traumáticos. Sin embargo, se observaron áreas de cavitación, hemorragia y edema en la materia gris. En los grupos con tratamiento AR-A014418, el número de células apoptóticas en los días 3 y 7 (p<0,05 y p<0,01, respectivamente), se redujo significativamente en comparación con los grupos traumáticos, así como los niveles de bax (p<0,01) y las células inmunopositivas para TGF-beta 1. Los resultados de la prueba del aparato locomotor se incrementaron significativamente en el grupo de tratamiento (p<0,001) en comparación con el grupo traumático.

Conclusiones

Mediante un estudio experimental de un modelo de traumatismo medular se observó como la apoptosis neuronal era desencadenada de manera significativa por daños secundarios postraumáticos. Sin embargo, la mejora neurológica se vio acelerada por la prevención de la apoptosis mitocondrial y reducción de la inflamación causadas por el inhibidor de GSQ-3beta, AR-A014418.

Palabras clave:
Lesión medular
GSQ-3beta
AR-A014418
Apoptosis
TGF-beta
Full Text
Introduction

Spinal cord injury (SCI) can be caused by a number of injuries to the spine that result in a loss of function such as mobility or feeling. Frequent causes of damage are trauma such as car accidents, falls or gunshots, or diseases such as polio or spina bifida. Following mechanical injury to the spinal cord, primary and secondary pathological changes occur. Primary damage leads to irreversible tissue injury that is almost impossible to treat. Secondary damage amplifies the extent of initial damage and apoptosis is critical for triggering collateral damage following primary injury to the spinal cord.1–7 Depending on the trauma grade, a variety of sensory and motor neurologic deficits are seen in SCI.8 Total spinal cord injury is seen neurologically in almost half of the patients, however, on the other hand, 54% of patients are quadriplegic and 46% of them suffer from paraplegia.9 In 1911, Allen defined secondary spinal cord damage, which resulted in a change to the classical approach.10 Experimental and clinical studies show that neurological deficits after SCI are the result of the secondary damage due to apoptotic cascade activation after primary damage.8 Primary damage was shown to appear as a result of both tissue injury during trauma and neuronal necrotic cell death. On the other hand, secondary damage occurs after a chain of events as a result of activation of a variety of cell death pathways.11 The aim of the clinical and experimental research on neuron trauma is to prevent or to reduce the secondary damage. Although a lot of research has been done on SCI, providing a full recovery treatment on spinal cord damage has not been found yet.1–4,12,13

Apoptosis is carried out by death activators binding to cell surface receptors and triggering the mitochondria pathway. Cytochrome-c is released into the cytoplasm by the effect of pro-apoptotic members on the mitochondria and through the formation of a complex by binding the latent cytosolic Apaf-1 protein and activation of the inactive precursor of caspase-9. The apoptotic process is carried on by the activation of caspase-9 and caspase effectors (caspase-3).7,12,14 Apoptosis plays an active part in secondary damage, and is seen as a result of central nervous system ischemia, degenerations, inflammations, and traumatic injuries.15 In the recent years, the existence of neuronal apoptosis after traumatic human SCI was reported. Apoptosis can be triggered by a variety of insults including free radical damage, releasing of cytokines, inflammatory injury, and excitotoxicity.16 The apoptosis seen on microglia deteriorates the inflammatory secondary damage.17 In experimental studies, apoptosis in oligodendrocytes is shown to aggravate the occurrence of demyelination after the first few weeks of spinal cord damage, and serious consequences are caused by increasing the loss of neural cells.6,18 In studies that looked at apoptotic cell death after spinal cord trauma, TNF19 and Fas20 were found to stimulate the receptor pathway and activate caspase-3 and -8.1,21 Release of cytochrome-c also causes caspase activation via the mitochondrial pathway by the increase of neuronal Ca2+ concentration.22

TGF-beta is a regulatory cytokine and plays a part in brain damage, angiogenesis, inflammation, anti-inflammation, neuroprotection, and scar accumulation.23 TGF-beta is involved in wound healing after SCI and reduces the probability of secondary damage by reducing the mononuclear phagocyte accumulation around the wound.24

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase. It plays an important role in the pathophysiology of a lot of diseases. In addition, it is involved in the mechanism of neural cell death related with the disease.25,26 GSK-3 has two major isoforms; alpha and beta. GSK-3beta (tau phosphorylation kinase-1) is a serine-threonine kinase that is primarily localized at neurons, and found in brain at very high levels. Throughout growth it is present at very high levels, but its expression is reduced in adults. Active GSK-3beta cell death stimulates the signal transduction involving in neuron loss and loss of synaptic plasticity.25,26 An abnormal increase in GSK-3beta expression and activity in the adult brain is directly related with the key neuropathologic mechanism of Alzheimer's disease.26 GSK-3 induces cell death by the intrinsic apoptotic mitochondrial pathway but inhibits the death receptor-mediated extrinsic apoptotic signaling pathways. There may be differences in the effects of each GSK-3 isoform in different types of cells, so the balance between GSK-3 isoforms may be important in regulating the extrinsic apoptosis signaling pathway. Intrinsic apoptotic signals that are activated by cell damage are induced with GSK-3. These signals cause abnormal control mechanisms of expression in anti-apoptotic or pro-apoptotic proteins during the mitochondrial pathway.27 GSK-3 is known to regulate the activity of a large number of transcription factors that control gene expression of proteins participating in the mitochondrial pathway of the apoptotic cascade. Therefore, mitochondrial function is also influenced by GSK-3 mediated regulation during intrinsic apoptotic signaling. Some of these proteins are regulated by nuclear GSK-3 where a small, but dynamic, portion of cellular GSK-3beta is located. Nuclear GSK-3beta is in a relatively greater activation state than cytosolic GSK-3beta, and apoptotic conditions can increase the level and/or activity of GSK-3beta in the nucleus.28,29 GSK-3beta inhibitors are used as key regulators in apoptosis.27 Disorders of neuro-behavioral functions are reported to be overcome by the neuroprotective effects of GSK-3beta inhibition.30

AR-A014418 is a new member of a family of particularly effective and selective inhibitors of GSK-3beta enzyme activity. It has an ability to reduce tau phosphorylation and cell death. AR-A014418 can pass the blood brain barrier and has a powerful effect.28,31 It has been shown that treatment with TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), a potent and selective GSK-3beta inhibitor, is able to reduce tissue damage, inflammation, and apoptosis in the spinal cord injury, thereby significantly ameliorating the recovery of limb function.25 AR-A014418 is much more effective than other GSK-3beta inhibitors in the cerebral ischemia model.28,32

In the literature, AR-A014418 has not yet been used in spinal cord injury, either clinically or experimentally. In this study, we aimed to investigate the effects of AR-A014418 on neuronal apoptosis and evaluate its neuroprotective effects in the experimental spinal cord trauma model.

Materials and methods

All experiments were approved by the Ethics Committee for Animal Experiments of Istanbul University and followed the NIH Guide for the Care and Use of Laboratory Animals.

Anesthesia

All the rats used in the study were starved a day before the operation. Anesthesia was induced by the intramuscular application of 60mg/kg ketamine (Ketalar, Parke-Davis. Eczacıbası, Istanbul, Turkey) and 9mg/kg Xylazine (Rompun, Bayer, Istanbul, Turkey). In case of need 20% of the initial dose was repeated intermittently.

Surgical procedure

The backs of the animals were shaved after anesthesia and they were placed on the operation table in the prone position. Body temperature was, as far as possible, maintained at 37°C with heating pads throughout the operation until the end of the anesthesia. After local antisepsis was applied by povidone iodine (Poviiodeks, KIM-PA, Istanbul, Turkey), a skin incision was made using interscapular distance as a reference point. Subdermal tissues and the paravertebral muscle fascia were advanced. Paravertebral muscles were swabbed by blunt dissection. The thoracic 6th, 7th and 8th vertebral laminae were set forth and a total laminectomy was done. Through this procedure the dura mater was left intact. Different procedures were applied to each group through further steps of operation.

Spinal cord injury

Spinal cord trauma was performed by the vascular clip compression technique developed by Rivlin and Tator.33 This method can be applicable to small animals and has been reported as much more reliable than balloon compression and weight reducing methods.34,35 In this study, the thoracic 6, 7 and 8 total laminectomy was applied to set front the durameter. Afterwards, FE 781 K coded Yasargil aneurysm clips with 70g closing power were used to constitute spinal cord trauma with 60s application.

Experimental groups

Animals (Wistar type albino male rats) were separately divided into 3 randomized groups such that each group had 12 animals. Animals whose weights lay between 240 and 320g were used in this study.

Group I (n=12, sham-operated control group)

After skin incision and dissection of paravertebral muscles; the laminae were set front and total laminectomy was applied at thoracic 6–8 levels. A 3.0 silk suture was used to sew the muscles and the skin. In 6 of the rats, intercardiac perfusion was used at the third day with 10% formaldehyde. In the remaining 6 rats the same procedure was conducted on the seventh day. After the intracardiac perfusion with 10% formaldehyde was complete, the surgical zone was opened again and approximately 2cm spinal cord was removed under microscope.

Group II (n=12, trauma control group)

The laminectomy was applied to the animals as described in first (I) group and then the clip compression technique was used to constitute spinal cord damage. Dimethylsulfoxide (DMSO) in an equivalent dose to that of AR-A014418 was administered intra-peritoneally (i.p.) just after the trauma. As described in the first group, spinal cord samples were taken at the third and the seventh days.

Group III (n=12, AR-A014418-treated group)

The same surgical and trauma procedures were applied to each animal in the second (II) group. A single dose of 4mg/kg AR-A014418 (Sigma, A3230) dissolved in DMSO solution was given i.p. just after the trauma. Spinal cord samples were taken at third and seventh days as described for the first (I) group.

Neurological evaluation of functional healing of all the rats in each group at the third and the seventh days after surgical operation was done before sacrificing the animals.

Light microscopy—histopathology

Spinal cord tissues were defined by neutral formalin solution and embedded in paraffin by following the conventional light microscopy techniques. 5μm paraffin cross-sections were taken and put on microscope slides covered with poly-l-lysine (PLL, Sigma P-8920, St. Louis, MO). The cross-sections of spinal cord tissue were stained with hematoxylin and eosin (H+E), and cresyl violet (CV) dyes for histopathological evaluation.

In order to make the statistical evaluation, polymorphonuclear leukocytes (PMNL) of 10 different areas were counted on the H+E stained tissue cross-section of each animal in every group. The average value of the PMNL number was calculated by an independent researcher to evaluate the inflammatory response in trauma groups.

Immunohistochemistry

Histostain-Plus Bulk Kit (Zymed LAB-SA DetEction System, 85-9043), rabbit polyclonal Bax (Delta Biolabs, DB005, 1:50 dilution), and rabbit polyclonal TGF-beta 1 (Santa Cruz Biotech., sc-146, 1:100 dilution) antibodies were used with the Streptavidin-Biotin-Peroxidase method in order to stain the paraffin cross-sections by the immunohistochemistry stain procedure offered in the kit.

To determine the specificity of immunostaining, sections were incubated following the above procedure, except for omission of incubation with the primary antibody. Instead of primary antibody, control serum was also used as control.

Apoptosis assay (TUNEL method)

The in situ DNA end marking method was used to assess apoptosis and was conducted according to the protocol provided in the kit (ApopTag Plus, In Situ Apoptosis Detection Peroxidase kit, S7101-KIT, Chemicon). For staining specificity controls the thymus tissue of dexametazone-treated rat was used as positive control. Distilled water was used instead of Tdt enzyme as a negative control.

Apoptotic cell count

Marked apoptotic cell counting (TUNEL) for all groups of spinal cord tissue was performed under high-power fields (original magnification 40×) with a light microscope. Apoptotic cells in 12 different unit areas were counted on the cross-sections by two independent researchers. These 12 fields were randomly selected from slides without discrimination between white and gray matter. Average cell per unit area number was found by taking the average of the results.

Evaluation of the immunohistochemical stain signals

TGF-beta 1 immunoperoxidase staining; under a microscope at 40× magnification, 15 areas were selected randomly and immunostained cells were evaluated semi-quantitatively. The results were scored as −: no staining, +: weak staining, ++: moderate staining, +++: strong staining.

Bax immunoperoxidase staining; 15 randomized areas were chosen from all gray and white substances of the spinal cord tissue, and were evaluated under a microscope at 40× magnification by counting of the positive staining cells. Statistical comparison was conducted for each.

Locomotor evaluation

The locomotor test was applied to the animals according to the open field BBB scoring.36 The animals were observed for scoring in a 75cm×120cm field at the third and the seventh days before euthanasia was carried out by two researchers.

Statistical evaluation

In order to evaluate the collected data statistically, the nonparametric Kruskal–Wallis test was used for comparison among the groups. If the results of Kruskal–Wallis test were significant with p<0.05, “post hoc” analysis between two groups was performed by the Dunn test. Data are expressed as means±SD where applicable.

ResultsHistopathologic findings

In the spinal cord tissue sections stained with HE and CV, edema, hemorrhage, leukocyte infiltration, and necrosis were evaluated through histological examination. The neural and vascular structure of the spinal cord was obtained normally in control groups (Figs. 1A and 2A). Cross-sections from the third and the seventh days of trauma groups showed hemorrhage, cavitation, necrotic, and edematous areas in gray matter and degeneration in motor neurons and leukocyte infiltration (Fig. 1B and C). The third day trauma group exhibited areas containing abundant necrotic (??) and apoptotic cells () (Fig. 2B). The seventh day trauma group showed areas containing necrotic cells (Fig. 2C). On the third day AR-A014418-treated trauma group showed moderate cavitation areas, hemorrhagic and edematous regions, although the healthy cell populations occupied more space (Fig. 1D). In the seventh day of the AR-A014418-treated group, there were fewer hemorrhagic areas and decreased cavitation degeneration, accompanied by an increased number of healthy cells, as compared to the trauma groups (Figs. 1E and 2E).

Fig. 1.
(0.61MB).

Hematoxylin and eosin staining of spinal cord tissues. Control (A); trauma 3rd day (B), trauma 7th day (C), AR-A014418-treated 3rd day (D), AR-A014418-treated 7th day (E) groups and graph showing the mean numbers of PMNL in trauma groups (F); ap<0.001 vs. all groups, bp<0.001 vs. AR-A014418 3rd and 7th days, cp<0.001 vs. AR-A014418 3rd days.

Fig. 2.
(0.43MB).

Cresyl violet staining of spinal cord tissues. Control (A); trauma 3rd day (B), trauma 7th day (C), AR-A014418-treated 3rd day (D), AR-A014418-treated 7th day (E) groups.

Evaluation of inflammation (PMNL count)

In the third day trauma groups PMNL counts 48.86±3.07 in both gray and white matter were statistically significantly higher than in other groups (p<0.001). In the trauma seventh day group, the PMNL count was found to be 28.74±2.20. In AR-A014418 third day and in AR-A014418 seventh day the PMNL count was found to be 15.5±2.36 and 5.98±0.58, respectively, and average PMNL count was shown to be decreased compared to that of the trauma groups (p<0.001) (Fig. 1F).

TGF-beta 1 immunohistochemical staining

TGF-beta 1 immunopositivity was evaluated in all tissue cross-sections at all gray and white matter. Very weak (−) TGF-beta 1 immunopositivity was observed in the control group (Fig. 3A). Significant TGF-beta 1 immunopositivity (++) was observed in the trauma third day group (Fig. 3 B). In the trauma seventh day group, significantly more TGF-beta 1 immunopositivity (+++) was seen compared to all other groups (Fig. 3C). In the third day and seventh day of the AR-A014418-treated trauma groups, decreased TGF-beta 1 immunopositivity was observed compared to the trauma groups (Fig. 3D and E).

Fig. 3.
(0.66MB).

TGF-beta 1 immunoreaction of spinal cord tissue samples. Control (A), increased TGF-beta 1 immunoreactivity in the trauma 3rd day (B) and trauma 7th day (C), groups, decreased TGF-beta 1 immunoreactivity in AR-A014418-treated 3rd day (D), AR-A014418-treated 7th day (E) groups.

Apoptotic cell count

In the TUNEL staining method, cell nuclei were evaluated as brown TUNEL (+). Positive apoptotic cell number marked with TUNEL per unit area was found to be 0.11±0.04 in control groups (Fig. 4A and F). In trauma groups, apoptosis was commonly observed among oligodendrocytes and other glial cells at the lesion surrounding tissues (Fig. 4B and C). In the trauma third day group apoptotic cells per unit area was found to be 2.15±0.45. In the trauma seventh day group, positive cell number marked with TUNEL per unit area was found to be 3.86±0.58. Apoptotic cell number was found to be 1.21±0.22 in the third day group of AR-A014418 and 0.81±0.07 in the seventh day group that were prominently decreased compared to the trauma groups (p<0.05, p<0.001, respectively) (Fig. 4D–F).

Fig. 4.
(0.47MB).

TUNEL method. Control (A); in the trauma 3rd day (B) and trauma 7th day (C), a marked appearance of dark brown apoptotic cells (arrows) was demonstrated. AR-A014418-treated 3rd day (D), AR-A014418-treated 7th day (E) groups. Graphs showing the mean values of apoptotic cell numbers in the trauma groups (F); ap<0.05 vs. trauma 7th days, bp<0.05 vs. trauma 3rd days, cp<0.01 vs. trauma 7th days.

Bax immunohistochemical staining

In all tissue cross-sections, Bax immunopositivity was counted by scanning of the gray and white matter. Weak Bax immunopositivity was observed in the control group, but in a very small proportion of cells (Fig. 5A). In the trauma third day group, a large number of Bax immunopositive cells were seen (94.6±5.03) (Fig. 5 F). An increase in Bax immunopositive cell number was observed in trauma seventh day compared to the trauma third day (106.6±7.47) (Fig. 5B and C). In the third day of the AR-A014418-treated trauma group, decreased Bax immunopositivity was observed (48.4±7.76) (Fig. 5D). In the seventh day of the AR-A014418 application trauma group (31.4±9.01), a significant decrease in Bax immunopositive cell number was observed compared to the trauma groups and AR-A014418-treated third day groups (p<0.01, p<0.05, respectively) (Fig. 5E and F).

Fig. 5.
(0.69MB).

Immunoreactivity of bax. Control (A); Bax immunoreactivity is stronger in the trauma 3rd day (B), trauma 7th day (C) groups compared to control group. In the AR-A014418-treated 3rd day (D) and AR-A014418-treated 7th day (E) groups immunoreactivity of bax is seen weaker compared to untreated trauma groups. Evaluation of Bax immunopositive cell numbers in the trauma groups (F); ap<0.01 vs. Trauma 3rd and 7th days, bp<0.05 vs. AR-A014418 3rd days.

Results of functional neurological improvement

In neurological evaluation of the subjects, average BBB scores were found to be 0.83±0.75 and 0.83±0.75 for the trauma third day group and trauma seventh day group, respectively. The AR-A014418-treated third day and seventh day trauma groups were found to be 1.33±0.81 and 4.50±1.22, respectively. A significant increase was observed in the BBB values of the AR-A014418-treated seventh day group compared to the other trauma groups (p<0.001). An increase in locomotor abilities of both AR-A014418 groups was observed compared to the control group (Fig. 6).

Fig. 6.
(0.14MB).

Changes in the mean BBB scores of trauma groups (mean±SD). *p<0.001 vs. all groups.

Discussion

There are currently no available treatment options for the recovery of primary injury after SCI. Studies of acute SCI damage treatment have largely focused on preventing secondary neural damage by pharmacological treatment protocols aimed to minimize the neurological sequelae.22

An increase or decrease in cell death by apoptosis is reported to play a part in the pathogenesis of a lot of diseases like cancer, autoimmune disorders, viral infections, and neurodegenerative diseases.37,38 Emery et al. was able to detect the existence of apoptosis in traumatic SCI in humans. In 14 patients out of a total of 15 who died between 3h and 2 months after spinal cord damage, apoptotic cells were shown near viable cells that were located around the lesion center and within the adjacent white matter.39 When clip compression7 and weight reducing1,4 methods were used in animal models, neuronal and glial apoptosis were developed in both. Yong et al. reported apoptosis in neurons, astrocytes, oligodendroglias, and microglias of rats after SCI.40 Liu et al. determined the apoptosis in the neurons and glia within 4h in the lesion region after the trauma. Neuronal apoptosis and glial apoptosis were significantly increased in the 8th and 24th hours, respectively. On the other hand, on the seventh day more cell death was observed with progressive enlargement of the initial lesion and its cavitation. A second wave of apoptosis was found to have occurred in white matter at the seventh day, related to axon degeneration in oligodendrocytes.5,7 In the trauma group of the SCI model, common hemorrhage, cavitation, necrosis and edema regions, degeneration in motor neurons and leukocyte infiltration were observed in the gray matter. Some regions of necrotic and apoptotic cells were found among those. Apoptotic cells were commonly observed in cells which were located near tissues around the lesion. Apoptotic cells marked positively with TUNEL were found to be significantly increased in trauma group compared to the control group. This finding showed that apoptotic cell death plays an important part in the progress of secondary trauma.

Some of the studies have used small molecule inhibitors of GSK-3 such as arylindolemaleimide SB-216763, anilinomaleimide SB-41528641 and anilino-4-arylmaleimide glycogen synthase kinase-3.42 However, almost all of these inhibitors are not selective for GSK-3 and affect cdk-2 and cdk-5, which share 33% amino acid homology with GSK-3.26 In this study, AR-A014418 {N-(4 methoxy benzyl)-N1-(5-nitro 1,3 thiazol-2yl) urea} was used after spinal cord trauma for therapeutic reasons and it is a selective and potent thiazol inhibitor to GSK-3beta. Additionally, AR-A014418 is the first member of a specific inhibitor family of GSK-3beta which does not cause inhibition of cdk-2 and cdk-5. It has been reported that the AR-A014418 has a protective effect on the N2A neuroblastom cells against the cell death via phosphatidylinositol 3 kinase and protein kinase beta.26 In order to find out the most effective dose of AR-A014418, comparative studies were carried out. From these studies, it was determined that a dose of 4mg/kg provided optimum inhibition. The optimal dose of GSK-3 was shown to prevent apoptosis.28

In this study, the 4mg/kg dose was used for AR-A014418 according to the cerebral ischemia studies that were carried out before. Inhibition of GSK-3beta significantly reduced spinal cord inflammation, and tissue damage, neutrophil infiltration, NO syntheses, nitrotyrosine and COX-2 expression, and additionally it was shown to improve motor healing scores.25 In this study, PMNL number was found to be significantly increased in the lesion region at the third day after trauma. After the use of the GSK-3beta inhibitor AR-A014418 in the trauma group, it was determined that PMNL number was significantly reduced in both trauma groups and facilitated the neurological healing.

GSK-3 can control the expression of anti-apoptotic and pro-apoptotic proteins at the intrinsic apoptotic signaling pathway. In general, it was reported that GSK-3 prevents the expression of anti-apoptotic proteins and increases the expression of pro-apoptotic proteins.27 The Bax as the proapoptotic protein plays an important role in the cell death and involves to neurodegeneration in central nervous system. Dong et al. studied the lateral cord cross-section model of spinal cord damage and reported that apoptotic death of the oligodendrocytes can be dependent on Bax activation and axonal degeneration.43 Cuzzocrea et al. reported that the apoptotic cell death was reduced with GSK-3beta inhibitor (TDZD-8) treatment after SCI. They showed that Bax expression diminished after application of the GSK-3beta inhibitor (TDZD-8) in the trauma group; however, Bcl-2 expression was shown to be increased.25 In tissue cross-sections of the AR-A014418-treated groups, healthy cells were found to be in greater number, but significantly there were areas displaying edema, hemorrhage, and cavitation. In the AR-A014418-treated third and seventh day groups, the numbers of apoptotic cells per unit area were found to be reduced compared to the trauma groups. This finding indicated that apoptotic cell death was significantly prevented by the GSK-3beta inhibitor. In this study, Bax immunopositive cells were significantly increased in the third day trauma group compared to the control group. Bax immunopositive cell numbers were increased in trauma seventh day group compared to those of the third day group. In the AR-A014418-treated seventh day trauma group, Bax immunopositive cell numbers were found to be significantly reduced compared to the third day of the AR-A014418-treated and the trauma groups. These findings support the hypothesis that use of AR-A014418 as a GSK-3beta inhibitor is able to prevent the expression of pro-apoptotic proteins, which could develop in the early stages of the mitochondrial apoptosis pathway.

TGF-beta is a powerful regulatory cytokine that is involved in a number of biological processes.23 TGF-beta is very important for inflammatory gene synthesis, which plays a role in healing after SCI, and mediates the eventual termination of inflammation. A reduction in secondary damage progress was reported by a reduction in PMNL accumulation around the lesion.24 In the SCI treated rats, the increased TGF-beta accumulation has been observed in neurons after just 24h and moreover, this accumulation persisted even in 7th day of the trauma despite a gradual reduction. In the erythropoietin treated animals with SCI, this accumulation was determined to be significantly reduced after seven days.44 Tyor et al. determined that there was a 50% reduction in lesion size after 48h of spinal cord trauma compared to the untreated group as a result of TGF-beta treatment. Additionally, they reported a relative reduction in neurotoxin production with significant reduction in mononuclear phagocyte number.24 In this study, an increase in TGF-beta 1 immunopositivity was observed in the trauma third day group. It was more intensive in the seventh day of trauma compared to the all other groups. TGF-beta 1 expression in the AR-A014418-treated third and seventh day trauma groups was determined to be reduced compared to the trauma groups. In this SCI model, our findings showed that treatment with AR-A014418 controls tissue damage, reduces the pathological effects of decreasing of TGF-beta 1 expression, and contributes to the healing process in spinal cord tissues.

In this study, after the evaluation of subject neurological functions, the average BBB values obtained showed a significant increase in seventh day results of the AR-A014418 group compared to all the trauma groups. The locomotor abilities of the subjects of the AR-A014418-treated trauma group were found to be increased compared to the subjects of the trauma groups. This finding showed that administration of AR-A014418 to trauma groups was able to contribute to the healing of neurological functions after spinal cord injury.

In this experimental spinal cord trauma model, neural apoptosis was significantly triggered in secondary damage developed after trauma. However, neurological healing was expedited by treatment with AR-A014418, a GSK-3beta selective inhibitor, which acted to prevent apoptosis via the mitochondrial pathway and reduce inflammation. Our results showed that the GSK-3beta inhibitor AR-A014418 can be used to prevent the progress of trauma and reduce inflammation in the treatment of secondary spinal cord damage.

Author's contribution

Matem Tunçdemir contributed in laboratory methods, microscopic examination, data collection, and article writing. Aziz Yıldırım, Alper Karaoğlan and Osman Akdemir contributed in surgical methods, data collection, and article writing. Melek Öztürk contributed in laboratory methods, microscopic examination, data collection, and article writing.

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgement

This work was supported by Scientific Research Projects Coordination Unit of Istanbul University, project number UDP-17368.

References
[1]
S. Barut,Y.A. Unlü,A. Karaoğlan,M. Tunçdemir,F.K. Dağistanli,M. Oztürk
The neuroprotective effects of z-DEVD.fmk, a caspase-3 inhibitor, on traumatic spinal cord injury in rats
Surg Neurol, 64 (2005), pp. 213-220 http://dx.doi.org/10.1016/j.surneu.2005.03.042
[2]
A. Colak,V. Antar,A. Karaoğlan,O. Akdemir,E. Sahan,O. Celik
Q-VD-OPh, a pancaspase inhibitor, reduces trauma-induced apoptosis and improves the recovery of hind-limb function in rats after spinal cord injury
Neurocirugia (Astur), 20 (2009), pp. 533-540
[3]
A. Colak,M. Kaya,A. Karaoğlan,A. Sağmanligil,O. Akdemir,E. Sahan
Calpain inhibitor AK 295 inhibits calpain-induced apoptosis and improves neurologic function after traumatic spinal cord injury in rats
Neurocirugia (Astur), 20 (2009), pp. 245-254
[4]
A. Karaoğlan,E. Kaya,O. Akdemir,A. Sağmanligil,K. Bilguvar,B. Cirakoğlu
Neuroprotective effects of Ac.YVAD.cmk on experimental spinal cord injury in rats
Surg Neurol, 69 (2008), pp. 561-567 http://dx.doi.org/10.1016/j.surneu.2007.03.041
[5]
X.Z. Liu,X.M. Xu,R. Hu,C. Du,S.X. Zhang,J.W. McDonald
Neuronal and glial apoptosis after traumatic spinal cord injury
J Neurosci, 17 (1997), pp. 5395-5406
[6]
J. Lou,L.G. Lenke,F.J. Ludwig,M.F. O’Brien
Apoptosis as a mechanism of neuronal cell death following acute experimental spinal cord injury
Spinal Cord, 36 (1998), pp. 683-690
[7]
J. Lu,K. Ashwell,W.S. Ken,P. Waite
Advances in secondary spinal cord injury: role of apoptosis
Spine, 25 (2000), pp. 1859-1866
[8]
A.P. Amar,M.L. Levy
Pathogenesis and pharmacological strategies for mitigating secondary damage in acute spinal cord injury
Neurosurgery, 44 (1999), pp. 1027-1039
[9]
V.K.H. Sonntag
History of degenerative and traumatic disease of the spine
A history neurosurgery: in its scientific and professional contexts,
[10]
A.R. Allen
Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column. Preliminatory report
J Am Med Assoc, 57 (1911), pp. 877-880
[11]
M.J. Crowe,J.C. Bresnahan,S.L. Shuman,J.N. Masters,M.S. Beattie
Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys
Nat Med, 3 (1997), pp. 73-76
[12]
R.W. Keane,S. Kraydieh,G. Lotocki,J.R. Bethea,S. Krajewski,J.C. Reed
Apoptotic and anti-apoptotic mechanisms following spinal cord injury
J Neuropathol Exp Neurol, 60 (2001), pp. 422-429
[13]
C.H. Tator
Biology of neurological recovery and functional restoration after spinal cord injury
Neurosurgery, 42 (1998), pp. 696-708
[14]
F. Bao,D. Liu
Peroxynitrite generated in the rat spinal cord induces apoptosis cell death and activates caspase-3
Neuroscience, 116 (2003), pp. 59-70
[15]
D.H. Kim,A.R. Vaccaro,F.C. Henderson,E.C. Benzel
Molecular biology of cervical myelopathy and spinal cord injury: role of oligodendrocyte apoptosis
Spine J, 3 (2003), pp. 510-519
[16]
R.J. Dumont,D.O. Okonkwo,S. Verma,J. Hurlbert
Acute spinal cord injury. Part I: Pathophysiologic mechanisms
Clin Neuropharmacol, 24 (2001), pp. 254-264
[17]
S.L. Shuman,J.C. Bresnahan,M.S. Battie
Apoptosis of microglia and oligodendrocytes after experimental spinal cord contusion in rats
[18]
G.L. Li,M. Faroque,A. Holtz,Y. Olsson
Apoptosis of oligodendrocytes occurs for long distances away from the primary injury after compression trauma to rat spinal cord
Acta Neuropathol (Berl), 98 (1999), pp. 473-480
[19]
T.Y. Yune,M.J. Chang,S.J. Kim,Y.B. Lee,S.W. Shin,H. Rhim
Increased production of tumor necrosis factor-alpha induces apoptosis after traumatic spinal cord injury in rats
J Neurotrauma, 20 (2003), pp. 207-219 http://dx.doi.org/10.1089/08977150360547116
[20]
A.R. Davis,G. Lotocki,A.E. Marcillo,W.D. Dietrich,R.W. Keane
FasL Fas, and death-inducing signaling complex (DISC) proteins are recruited to membrane rafts after spinal cord injury
J Neurotrauma, 24 (2007), pp. 823-834 http://dx.doi.org/10.1089/neu.2006.0227
[21]
V.R. Dasari,D.G. Spomar,C. Cady,M. Gujrati,J.S. Rao,D.H. Dinh
Mesenchymal stem cells from rat bone marrow downregulate caspase-3-mediated apoptotic pathway after spinal cord injury in rats
Neurochem Res, 32 (2007), pp. 2080-2093 http://dx.doi.org/10.1007/s11064-007-9368-z
[22]
R.J. Dumont,S. Verma,D.O. Okonkwo,R.J. Hurlbert
Acute spinal cord injury. Part II: Contemporary pharmacotherapy
Clin Neuropharmacol, 24 (2001), pp. 265-279
[23]
L. Aigner,U. Bogdahn
TGF-beta in neural stem cells and in tumors of the central nervous system
Cell Tissue Res, 331 (2008), pp. 225-241 http://dx.doi.org/10.1007/s00441-007-0466-7
[24]
W.R. Tyor,N. Avgeropoulos,G. Ohlandt,E.L. Hogan
Treatment of spinal cord impact injury in the rat with transforming growth factor-beta
J Neurol Sci, 200 (2002), pp. 33-41
[25]
S. Cuzzocrea,T. Genovese,E. Mazzon,C. Crisafulli,R. Di Paola,C. Muià
Glycogen synthase kinase-3 beta inhibition reduces secondary damage in experimental spinal cord trauma
J Pharmacol Exp Ther, 318 (2006), pp. 79-89 http://dx.doi.org/10.1124/jpet.106.102863
[26]
R. Bhat,Y. Xue,S. Berg,S. Hellberg,M. Ormö,Y. Nilsson
Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418
J Biol Chem, 278 (2003), pp. 45937-45945 http://dx.doi.org/10.1074/jbc.M306268200
[27]
E. Beurel,R.S. Jope
The paradoxical pro- and anti-apoptotic actions of GSK3 in the intrinsic and extrinsic apoptosis signaling pathways
Prog Neurobiol, 79 (2006), pp. 173-189 http://dx.doi.org/10.1016/j.pneurobio.2006.07.006
[28]
G.N. Bijur,R.S. Jope
Glycogen synthase kinase-3β is highly activated in nuclei and mitochondria
[29]
W. Elyaman,F. Terro,N.S. Wong,J. Hugon
In vivo activation and nuclear translocation of phosphorylated glycogen synthase kinase-3β in neuronal apoptosis: links to tau phosphorylation
Eur J Neurosci, 15 (2002), pp. 651-660
[30]
S.H. Koh,A.R. Yoo,D.I. Chang,S.J. Hwang,S.H. Kim
Inhibition of GSK-3 reduces infarct volume and improves neurobehavioral functions
Biochem Biophys Res Commun, 371 (2008), pp. 894-899 http://dx.doi.org/10.1016/j.bbrc.2008.05.006
[31]
T.D. Gould,A.M. Picchini,H. Einat,H.K. Manji
Targeting glycogen synthase kinase-3 in the CNS: implications for the development of new treatments for mood disorders
Curr Drug Targets, 7 (2006), pp. 1399-1409
[32]
A.O. Rosa,J. Egea,A. Martínez,A.G. García,M.G. López
Neuroprotective effect of the new thiadiazolidinone NP00111 against oxygen-glucose deprivation in rat hippocampal slices: implication of ERK1/2 and PPARgamma receptors
[33]
A.S. Rivlin,C.H. Tator
Regional spinal cord blood flow in rats after severe cord trauma
J Neurosurg, 49 (1978), pp. 844-853 http://dx.doi.org/10.3171/jns.1978.49.6.0844
[34]
M. Kahn,R. Griebel
Acute spinal cord injury in the rat. Comparison of three experimental techniques
Can J Neurosci, 10 (1983), pp. 161-164
[35]
C.H. Tator
Review of experimental spinal cord injury with emphasis on the local and systemic circulatory effects
Neurochirurgie, 37 (1991), pp. 291-302
[36]
D.M. Basso,M.S. Beattie,J.C. Bresnahan
A sensitive and reliable locomotor rating scale for open field testing in rats
J Neurotrauma, 12 (1995), pp. 1-21 http://dx.doi.org/10.1089/neu.1995.12.1
[37]
S.W. Hetts
To die or not to die: an overview of apoptosis and its role in disease
JAMA, 278 (1998), pp. 300-307
[38]
C.B. Thompson
Apoptosis in the pathogenesis and treatment of disease
Science, 267 (1995), pp. 1456-1462
[39]
E. Emery,P. Aldana,M.B. Bunge
Apoptosis after traumatic human spinal cord injury
J Neurosurg, 89 (1998), pp. 911-920 http://dx.doi.org/10.3171/jns.1998.89.6.0911
[40]
C. Yong,P.M. Arnold,M.N. Zoubine,B.A. Citron,Watanabe l,N.E. Berman
Apoptosis in cellular compartments of rat spinal cord after severe contusion injury
J Neurotrauma, 15 (1998), pp. 459-472 http://dx.doi.org/10.1089/neu.1998.15.459
[41]
M.P. Coghlan,A.A. Culbert,D.A. Cross,S.L. Corcoran,J.W. Yates,N.J. Pearce
Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription
Chem Biol, 7 (2000), pp. 793-803
[42]
D.G. Smith,M. Buffet,A.E. Fenwick,D. Haigh,R.J. Ife,M. Saunders
3-Anilino-4-arylmaleimides: potent and selective inhibitors of glycogen synthase kinase-3 (GSK-3)
Bioorg Med Chem Lett, 11 (2001), pp. 635-639
[43]
H. Dong,A. Fazzaro,C. Xiang,S.J. Korsmeyer,M.F. Jacquin,J.W. McDonald
Enhanced oligodendrocyte survival after spinal cord injury in Bax-deficient mice and mice with delayed Wallerian degeneration
J Neurosci, 23 (2003), pp. 8682-8691
[44]
X.Q. Fang,M. Fang,S.W. Fan,C.L. Gu
Protection of erythropoietin on experimental spinal cord injury by reducing the expression of thrombospondin-1 and transforming growth factor-beta
Chin Med J (Engl), 122 (2009), pp. 1631-1635
Copyright © 2011. Sociedad Española de Neurocirugía
Neurocirugía (English edition)

Subscribe to our Newsletter

Article options
Tools
Cookies policy
To improve our services and products, we use cookies (own or third parties authorized) to show advertising related to client preferences through the analyses of navigation customer behavior. Continuing navigation will be considered as acceptance of this use. You can change the settings or obtain more information by clicking here.