Spinal cord injury experimental model and motion evaluation protocol in wistar rats

Abstracts

OBJETIVOS: Determinar modelo de contusão medular padronizado e método de avaliação motora em ratos com quadro de paraplegia. MÉTODOS: Utilizados 20 ratos Wistar divididos em quatro grupos, diferenciado o nível de gravidade da lesão raquimedular; realizadas lesões intermediárias padronizadas, através do sistema "MASCIS IMPACTOR" (Multicenter Animal Spinal Cord Injury Study): grupo 1 - 12,5 mm (lesão leve); grupo 2 - 25 mm (lesão moderada); grupo 3 - 50 mm (lesão grave); grupo 4 - animais que não sofreram lesão (grupo controle). Após 48 horas foi avaliada função motora, segundo escala proposta por Basso, Beattie e Bresnahan. RESULTADOS: Utilizando o modelo, notamos que as contusões leves (12,5mm de altura) foram efetivas, e os animais apresentaram infecção urinária na fase aguda uma semana após a contusão. As contusões intermediárias (25 mm de altura) foram efetivas, os animais apresentaram infecção urinária até duas semanas após a contusão. As contusões graves (50mm de altura) foram efetivas, os animais apresentaram infecção urinária durante três a quatro semanas e autofagia. CONCLUSÃO: O modelo de lesão medular utilizando o sistema MASCIS IMPACTOR e a avaliação funcional proposta por Basso, Beattie e Bresnahan é reprodutível, podendo ser utilizada, propiciando troca de informações entre os diferentes pesquisadores.

Traumatismo da medula espinal; Paraplegia; Contusões

ORIGINAL ARTICLE

LIM 41 - Laboratory of Medical Investigation of the Musculoskeletal System of the Orthopedics and TraumatologyDepartment

Mailing address

ABSTRACT

OBJECTIVES: To determine a standardized spinal cord contusion model and a method for motor assessment in rats with paraplegia.

METHODS: This study used 20 Wistar rats divided into 4 groups according to level of severity of spinal cord injury; standardized intermediate lesions were made through system MASCIS IMPACTOR (Multicenter Animal Spinal Cord Injury Study): group 1, 12.5mm (mild injury); group 2,25mm (moderate injury); group 3,50mm (severe injury; in the group 4 the animals suffered no injury (control group). Motor function was assessed after 48 hours, using the scale proposed by Basso, Beattie and Bresnahan.

RESULTS: Using the model, we observed that the mild contusions (12.5mm height) were effective, and the animals presented acute urinary tract infection one week after the injury. Moderate contusions (25mm height) were effective, and the animals presented urinary infection until 2 weeks after injury. The severe contusions (50mm height) were effective, and the animals presented urinary infection for 3 to 4 weeks and autophagy.

CONCLUSION: The model of spinal cord injury using the system MASCIS IMPACTOR and the functional assessment proposed by Basso, Beattie and Bresnahan is reproducible and can be used, enabling information exchange among different researchers.

Keywords: Spinal cord injuries. Paraplegia. Contusions.

INTRODUCTION

Experimental models have been used to accumulate experiences and consequently to improve and refine knowledge about spinal cord injury physiopathology.^1-3

This knowledge applied to clinical and surgical treatments is used to reduce morbidity and mortality in patients with spinal cord injury.⁴

It is important to have a standardized assessment applied in experimental studies to determine the effects of each therapeutic intervention that one wishes to study. Some assessment criteria have been presented in literature, and the scale proposed by Basso, Beattie and Bresnahan, BBB, is that used in this study.^1,2,4

Defined as a scale of 21 points that observes recovery of locomotion in the hind limb after thoracic spinal cord injury in rats, this scale represents a detailed and organized categorization of the recovery of movements in the lower limb following the spinal cord lesion. Each index represents a single and sequential stage of behavioral recovery.⁵

The objective of this study is to evaluate, using the BBB scale, the severity of lesions at different levels of height made using a standardized experimental spinal cord injury model known as a the "Mascis Impactor".⁴

The study subjects were 20 young male adult Wistar rats, with mean age 20 of weeks, weighing around 350g, from the Centro de Bioterismo da Faculdade de Medicinada USP, divided into four groups of five animals.

The group divisions were performed as follows:

Control group: we performed only approach and laminectomy at the level of T9

Group 2: 12.5 mm representing mild injury

Group 3: 15 mm representing moderate injury

Group 4: 50 mm representing severe injury.

The procedures and the accommodation of the animals were performed at the Laboratório de Estudos do Traumatismo Raquimedular e Nervos do Instituto de Ortopedia e Traumatologia da USP. The animals were anesthetized with intraperitoneal pentobarbital, in the concentration of 45mg/kg, in the lower third of the abdomen, then submitted to dorsal trichotomy, antisepsis with chlorhexidine, and longitudinal incision in the topography of T7 to T12 (Figure 1), dissection by planes and exposure of the posterior components of the dorsolumbar spine. (Figure 2)

The laminae from T9 and T10 were resected in the caudocranial direction. (Figure 3)

SPINAL CORD CONTUSION

The lesions were produced in compliance with the international protocol MASCIS (Multicenter Animal Spinal Cord Injury Study), a model that produces spinal cord contusions at different levels, which determines the degree of severity. (Figures 4, 5, 6, 7 and 8)

The injury levels range from 6 mm of height, which is considered a mild injury, to 70 mm of height, which simulates an extremely severe lesion. Intermediate lesions were produced with 12.5 mm representing mild injury, 25 mm moderate injury and 50 mm of height representing severe injury. The lesion procedure was followed by the observation of standardization parameters such as clinical aspects of the injury site and graphs. (Figures 9 and 10)

The injury site was irrigated with saline solution at room temperature, followed by the inspection, cauterization, myorrhaphy and skin suture. (Figures 11 and 12) A dose of 25mg/kg of cephalothin was administered subcutaneously.⁴

The animals were kept in cages (60x40cm) in groups of 3 or 4, and provided with feed and water ad libitum. The massages for vesicle emptying were performed daily, in addition to the evaluation of possible complications, such as urinary infection.

Motor assessments were conducted after 48 hours following the motor assessment protocol proposed by Basso, Beattie and Bresnahan, "BBB", which corresponds to a scale of 21 points, according to the motility of the pelvic limbs and tail of the animal, in a standardized place with dimensions of 90 cm of diameter and 7 cm of height without the presence of noise. (Charts 1 and 2).

RESULTS

The data obtained show statistical differences among the groups and also that the contusions are effective and performed in a standardized manner, since no statistical differences were noticed in the same group, according to Chart 3 and Figure 13.

DISCUSSION

Several experimental models have been described in literature for the study of standardized spinal cord injuries in animals.^1-6 In our protocol we opted for the "MASCIS IMPACTOR" model, which uses weight drop from a standardized height causing spinal cord contusion in rats, after which they are assessed in their locomotor function.^1,2,7 In this experimental study we used Wistar rats due to the low cost and ease in their obtainment.

The functional assessment methods are not yet totally standardized, with several descriptions in literature.^3,8,9

Within the standardized evaluation of spinal cord injury, to provide a safe exchange of data among the different research groups, anatomopathological, biochemical and imaging diagnosis alterations, among others, are being analyzed.^10-11

In our study we opted for the functional assessment model described by Basso, Beattie and Bresnahan (BBB), which uses a standardized locomotor assessment scale. We opted for the methodology adopted in the Multicenter animal spinal cord injury (MASCIS), which uses the BBB scale after spinal cord injury at standardized levels of height (12.5, 25, 50 mm).

The results presented by the groups exhibited differences in the functional assessment of the injury at each standardized height, which confirms the objectivity and reliability of the data from the BBB scale.

It is important to emphasize that this assessment method presents reliability in the exams carried out by different observers as demonstrated by Basso et al.²

CONCLUSION

It is concluded that the spinal cord injury model using the MASCIS IMPACTOR system and the functional assessment model proposed by Basso, Beattie and Bresnahan "BBB" is reproducible and can be used by observers with previous training in the evaluation of animals with spinal cord injury, favoring the exchange of information among the different researchers that use the same scale.

REFERENCES

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• 8. Faconer JC, Naroyama PA, Bhattachojee M, Liv SJ. Caracterization of a experimental spinal cord injury model using waveform and morfhonetic analysis. Spine. 1996;21:104-12.
• 9. Black P, Markowitz RS, Cooper V, Mechanic A, Kushner H, Damjanov I et al. Models of spinal cord injury: Part 1. Static load technique. Neurosurgery.1986;19:752-62.
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Spinal cord injury experimental model and motion evaluation protocol in wistar rats

Gustavo Bispo dos Santos; Alexandre Fogaça Cristante; Raphael Martus Marcon; Fabiano Inácio de Souza; Tarcísio Eloy Pessoa de Barros Filho; Marcelo Loquette Damasceno

Publication Dates

• Publication in this collection
  15 June 2011
• Date of issue
  Apr 2011

History

• Received
  14 Apr 2010
• Accepted
  26 Apr 2010