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Vol. 20. Issue 3.
Pages 245-254 (January 2009)
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Vol. 20. Issue 3.
Pages 245-254 (January 2009)
DOI: 10.1016/S1130-1473(09)70163-0
Calpain inhibitor AK 295 inhibits calpain-induced apoptosis and improves neurologic function after traumatic spinal cord injury in rats
El inhibidor de la calpaina AK 295 inhibe la apoptosis inducida por calpaina y mejora la función neurológica tras traumatismo medular en ratas
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A. Çolak??, A. Karaoğlan
Department of Neurosurgery, Maltepe University, School of Medicine, Istanbul, Turkey
M. Kaya*, A. Sağmanligil*, O. Akdemir*, E. Şahan**, Ö. Çelik***
* Departments of Neurosurgery, Taksim Education and Research Hospital, Taksim, Istanbul, Turkey
** Departments of Pathology, Taksim Education and Research Hospital, Taksim, Istanbul, Turkey
*** Department of Molecular Biology and Genetics, Istanbul Kültür University Faculty of Science and Letters, Istanbul, Turkey
Article information
Summary
Background

An increase in the level of intracellular calcium activates the calcium-dependent neutral protease calpain, which in turn leads to cellular dysfunction and cell death after an insult to the central nervous system. In this study, we evaluated the effect of a calpain inhibitor, AK 295, on spinal cord structure, neurologic function, and apoptosis after spinal cord injury (SCI) in a murine model.

Methods

Thirty albino Wistar rats were divided into 3 groups of 10 each: the sham-operated control group (group 1), the spinal cord trauma group (group 2), and the spinal cord trauma plus AK 295 treatment group (group 3). After having received a combination of ketamine 60mg/kg and xylazine 9mg/kg to induce anesthesia, the rats in groups 2 and 3 were subjected to thoracic trauma by the weight drop technique (40g-cm). One hour after having been subjected to that trauma, the rats in groups 2 and 3 were treated with an intraperitoneal injection of either dimethyl sulfoxide 2mg/kg or AK 295 2mg/kg. The effects of the injury and the efficacy of AK 295 were determined by an assessment of the TUNEL technique and the results of examination with a light microscope. The neurologic performance of 5 rats from group 2 and 5 from group 3 was assessed by means of the inclined plane technique and the modified Tarlov's motor grading scale 1, 3, and 5 days after spinal cord trauma.

Findings

Light-microscopic examination of spinal cord specimens from group 2 revealed hemorrhage, edema, necrosis, and vascular thrombi 24 hours after trauma. Similar (but less prominent) features were seen in specimens obtained from group 3 rats. Twenty-four hours after injury, the mean apoptotic cell numbers in groups 1 and 2 were zero and 4.57±0.37 cells, respectively. In group 3, the mean apoptotic cell number was 2.30±0.34 cells, a value significantly lower than that in group 2 (P<.05). Five days after trauma, the injured rats in group 2 demonstrated significant motor dysfunction (P<.05). In comparison, the motor scores exhibited by group 3 rats were markedly better (P<.05).

Conclusions

AK 295 inhibited apoptosis via calpaindependent pathways and provided neuroprotection and improved neurologic function in a rat model of SCI. To our knowledge, this is the first study to evaluate the use of AK 295, a calpain inhibitor, after SCI. Our data suggest that AK 295 might be a novel therapeutic compound for the neuroprotection of tissue and the recovery of function in patients with a SCI.

Key words:
AK 295
Apoptosis
Calpain inhibitor
Secondary damage
Spinal cord trauma
Resumen
Introducción

Una lesión en el sistema nervioso central origina un incremento en los niveles de calcio intracelular que activa la proteasa neutral calciodependiente calpaina, que a su vez conduce a la producción de disfunción y muerte celular. En este estudio evaluamos el efecto de un inhibidor de la calpaina, AK 295, sobre la estructura de la médula espinal, la función neurológica y apoptosis tras lesión medular en un modelo murino.

Métodos

Treinta ratas Wistar se dividieron en tres grupos de 10 ratas cada uno: Un grupo control (grupo 1), un grupo sometido a trauma espinal (grupo 2) y un grupo de ratas a las que se sometió a trauma medular y tratamiento con AK 295 (grupo 3). Después de recibir una combinación de ketamina 60mg/kg y xylazina 8mg/kg para la inducción anestésica, las ratas del grupo 2 y 3 fueron sometidas a trauma medular torácico mediante la técnica de caída de peso (40g-cm). Una hora después de haber sufrido el traumatismo, las ratas del grupo 2 y 3 fueron tratadas mediante una inyección intraperitoneal bien de dimetil-sulfóxido 2mg/kg o de AK 295 2mg/kg. Los efectos del traumatismo y la eficacia de AK 295 fueron determinados mediante la estimación de la técnica TUNEL y los resultados del examen del tejido mediante microscopía óptica. La función neurológica de 5 ratas del grupo 2 y 5 del grupo 3 fue estimada mediante la técnica del plano inclinado y la escala motora de Tarlov modificada a 1, 3 y 5 días desde el traumatismo medular.

Resultados

El estudio mediante microscopía óptica de las preparaciones de médula espinal del grupo 2 demostró la existencia de hemorragia, edema, necrosis y trombosis vascular 24 horas tras el traumatismo. Hallazgos similares pero menos importantes se encontraron en las preparaciones procedentes del grupo 3. Veinticuatro horas tras el trauma, el número medio de células apoptóticas en los grupos 1 y 2 fueron cero y 4.57±0.37 células respectivamente. En el grupo 3, el número medio de células apoptóticas fue de 2.30±0.34 células, un valor significativamente menor que en el grupo 2 (p < 0.05). Cinco días tras el traumatismo, las ratas lesionadas en el grupo 2 demostraron una significativamente mayor disfunción neurológica (p<0.05). En comparación, la puntuación motora que exhibieron las ratas del grupo 3 fue marcadamente mejor (p<0.05).

Conclusión

AK 295 inhibe la apoptosis a través de vías calpain-dependientes y provee neuroprotección y consigue una mejor función neurológica en el modelo de lesión medular traumática en la rata. En nuestro conocimiento, este es el primer estudio en evaluar el uso de AK 295, un inhibidor de la calpaina, tras lesión medular traumática. Nuestros datos sugieren que AK 295 podría ser un nuevo compuesto terapéutico capaz de ofrecer neuroprotección tisular y recuperación funcional en pacientes con lesión medular traumática.

Palabras clave:
AK 295
Apoptosis
Inhibidor de la calpaina
Lesión secundaria
Lesión medular traumática

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