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Acta Ortopédica Brasileira

Print version ISSN 1413-7852On-line version ISSN 1809-4406

Acta ortop. bras. vol.11 no.2 São Paulo Apr./june 2003

http://dx.doi.org/10.1590/S1413-78522003000200003 

ORIGINAL ARTICLE

 

Effect of methylprednisolone in medullar injury in rats: a functional and histological analysis

 

Efeito da metilprednisolona na lesão medular em ratos: análise funcional e histológica

 

 

Marcos Antônio TebetI; Tarcísio Eloy Pessoa Barros FilhoII; Itibagi Rocha MachadoIII; Márcio Oliveira Penna de CarvalhoIV; Fábio Richieri HananiaV; Kuang DacíVI

IAdjunct Professor, Faculty of Medicine of Jundiai, SP, Brazil
IIAssociate Professor, Department of Orthopedics and Traumatology, FMUSP - Scientific Director of IOT - HC/FMUSP
IIIAdjunct Professor, Faculty of Medicine of Jundiai, SP, Brazil
IVPermanent Member of the Brazilian Society for Orthopedics and Traumatology
V5th year student, FMUSP
VI5th year student, FMUSP

Correspondence

 

 


SUMMARY

The pharmacological effects of the methylprednisolone in the treatment of the spinal cord lesion, have been researched. An experimental study was performed with twelve wistar rats subjected to a contusion lesion of the spinal cord, by the NYU system. The rats were divided in two groups; the MP group which received the methylprednisolone after the contusion lesion and the control group, which received a 0,9% physiological serum. An analysis of the recuperation of the functional movement of the rats was accomplished on the 2nd, 7th and 14th day after the contusion, utilizing the BBB test. On the fourteenth day the rats were sacrificed and the histological findings of the spinal cord lesion were analyzed. It was noted that the rats from the MP group exhibited an improvement in the recuperation of their functional movement compared to that of the control group, and the histological findings of the spinal cord lesion couldn't be correlated with the recuperation of their functional movement.

Key words: Methylprednisolone; Spinal cord; Rats; Traumatism of spinal cord; Motor activity.


RESUMO

Os efeitos farmacológicos da metilprednisolona têm sido investigados no tratamento da lesão medular. Foi realizado estudo experimental com doze ratos wistar submetidos à lesão contusa da medula espinhal pelo sistema NYU. Os animais foram divididos em dois grupos: grupo MP, que recebeu metilprednisolona após a lesão contusa e o grupo controle, que recebeu soro fisiológico a 0,9%. Foi realizada análise da recuperação funcional locomotora utilizando-se o teste de BBB no 2º, 7º e 14º dias após a contusão. No décimo quarto dia, os ratos foram sacrificados e analisados os achados histológicos da medula lesada. Observou-se que os ratos do grupo MP apresentaram melhora na recuperação da função locomotora em comparação com o grupo controle e que os achados histológicos da medula lesada não puderam ser correlacionados com a recuperação da função locomotora.

Descritores: Metilprednisolona; Medula espinhal; Ratos; Traumatismos da medula espinhal; Atividade motora.


 

 

INTRODUCTION

Medullar injury is a devastating problem affecting mainly young males in the productive age. It will affect 20 to 40 people per million each year and generate an annual economic impact of over four million American dollars in the USA, where it is mostly caused by motor vehicle accidents(9,24,25).

Studies have shown that medullar injury occurs in two distinct phases: the primary (or mechanical) phase and the secondary (or ischemic) phase (22,24). The basic biological change of secondary, or ischemic, injury is the lipid peroxidation that triggers a cascade of reactions and promotes the reduction of medullar flow, ischemia and tissular necrosis (2,13,15,22).

In an effort to prevent secondary post-traumatic medullar ischemia and improve the potential for functional recovery of medullar injuries, drugs able to modulate endogenous responses are being progressively investigated and gradually introduced (9,15).

High doses of methylprednisolone have been used in trials performed in rats and cats, with improvements in microvascular perfusion, medullar metabolism and histomorphometric measurements (1,8). It is suggested that its mechanisms of action consist of lipid peroxidation inhibition, reduction of axon degeneration, prevention of ischemia and reversion of intracellular calcium accumulation(1,6,8,14,25).

Multicenter, controlled studies such as the National Acute Spinal Cord Injury Study (NASCIS) I, II and III, started in 1979, advocate the use of methylprednisolone within the first eight hours after the traumatic medullar injury in the clinical practice(9). Although over 85% of the trials showed some beneficial effect with steroids, the mechanisms of action of methylprednisolone in the injured medulla are not yet totally understood(9).

The BBB locomotor scale was developed in 1995 by Basso, Beattie and Bresnahan to evaluate the pattern of locomotor recovery in trials performed in rats submitted to medullar injury by the NYU system (4).

The purpose of this study is to evaluate the locomotor function recovery in rats with medullar injury produced by the NYU system and treated with methylprednisolone, and correlate it with the findings from the histological study on injured medulla.

 

MATERIAL AND METHODS

Twelve young adult male Wistar rats were studied whose average weight was 327 g (minimum 302 g, maximum 346 g, SD = 13.5). The animals were kept in the Laboratory of Microsurgery, IOT-FMUSP, inside individual cages in acclimatized chambers at a temperature of 25°C, with a 12-hour light/dark cycle and ad libitum feeding.

The animals were randomized into two groups: the MP group (n=6) received 30 mg/kg methylprednisolone intraperitoneally five minutes and then three hours following medullar contusion, and the control group (n=6) received placebo as one application of 1 ml 0.9% saline solution.

For the anesthetic procedure a 65-mg/kg dose of pentobarbital was administered intraperitoneally. One skin incision of about 5 cm in length was made in the dorsal median line. The paravertebral musculature was separated from the spinal processes to allow the exposure of spinal cord vertebrae T8 to T12; then, with the aid of a proper punching device, laminectomies of the distal half of vertebrae T9 and T10 were performed without causing any injury to the dural sac, allowing space enough to accommodate the impactor head.

The medullar injury was performed according to the NYU system developed by Gruner (12) in 1992 (Figure 1). A contuse injury equivalent to the fall of a 10-g weight from a 25-mm height was produced.

 

 

After the production of medullar injury, the rat was transferred to a temperature-controlled chamber; the rat's rectal temperature was taken after 15 minutes and its bladder emptied by hand compression after 6 hours, 24 hours and then once daily, until the killing.

The locomotor function was evaluated 2, 7 and 14 days after the medullar contusion, with the aid of the BBB scale. Based on this scale, we observed the movements of the hip, knee and ankle articulations, as well as the positions of the trunk, tail and hind legs. To quantify these observations a score ranging from zero to 21 was used where zero corresponded to complete absence of movements and 21 to the presence of normal movements. Four physicians were trained as observers and formed the study group in charge of the evaluation of the animals with the BBB scale (4).

Fourteen days after the injury the animals were killed by one dosis of 140 mg/kg pentobarbital administered intraperitoneally in accordance with the rules and regulations of the Ethics Committee for Analysis of Research Projects, HC - FMUSP.

The T1 - L5 segment containing the whole osseous enchasement and the paravertebral muscles was removed and laminectomies (vertebrae T5 - L5) were performed in order to remove the medulla. The medullar region with macroscopic evidence of contusion was named "Zone B", while the cephalic end was named "Zone A" and the caudal area, "Zone C", measured within 1 cm from the epicenter of the contusion, 5 mm above and 5 mm below it. The whole set was stored in jars with 10% formol solution.

The medullar segment was submitted to histological cuts in the axial plan with 2-mm intervals. The material was processed and dehydrated using a series of alcohols, and then dehydrated in xylol and embedded in paraffin. After the 5 µ histological cuts were made with the aid of a microtome caliper 5 mm above and 5 mm below the injury, the material was fixed in glass staining dishes and stained with the hematoxylin-eosin technique.

The histological alterations were evaluated for the presence of edema, congestion, cell infiltrate, cavitations and necrosis. The findings regarding the medullar cuts investigated were scored from 0 to 3.

Statistical analysis

In the descriptive analysis the quantitative variables (weight, scores on Days 2, 7 and 14 following the medullar injury) were evaluated through the observation of minimum and maximum values and the calculation of the mean values, standard deviations and average values. Qualitative variables such as edema, congestion, necrosis, cell infiltration and cavitation were also evaluated for each region, and the calculation of both absolute and percentage frequencies was performed.

For the comparative analysis the Fisher's exact test was used to evaluate the homogeneity of the proportions of the groups as regards the qualitative variables. The ANOVA non-parametric analysis compared both groups as regards the variables that were measured for independent samples while the non-parametric Friedman test compared the time points in the same group for the variables measured for dependent samples. All tests were performed based on a 5% significance level (p < 0.05).

 

RESULTS

Motor functional analysis according to the BBB scale

Table 1 shows the result of the functional evaluation according to the BBB locomotor scale of the groups on Days 2, 7 and 14 following the medullar injury, and Graph 1 shows the evolution of the average values of the functional evaluation. The average values found at those three time points are represented in Table 2.

 

 

 

 

 

 

A statistical study of the comparative results obtained with the BBB scale was performed at each time point. The groups showed significant alterations at the time points evaluated (p < 0.01). By paired comparison, on Day 2 following the medullar injury, the results of the evaluation were significantly different from the results found on Day 14, in both groups (p < 0.05). There were no statistical differences on Day 7 after the medullar injury as compared to the other time points (Table 2 and Graph 1).

It was seen that the MP group presented a significantly higher mean value (p < 0.05) than the control group at the third time point (Day 14 after the medullar injury).

Histological analysis

A histological analysis evaluated the variables for the presence of edema, congestion, necrosis, cell infiltrate and cavitation at the center of the injury (zone B), above the injury (zone A) and below the injury (zone C). Each variable was scored from 0 to 3 (see Tables 3, 4 and 5).

 

 

 

 

 

 

Necrosis and edema were the only findings seen in all rats of all groups in the injury zone (Zone B). However, necrosis was present in 11 (91.6%) rats and edema was present in 10 (83.3%) rats in the zone below the injury (zone C). In the zone above the injury (zone A) necrosis and edema were found in 7 (58.3%) and 9 (75.0%) rats, respectively.

Another significant finding was the necrosis ratio in the injury zone (Zone B), with a presence that was significantly higher as compared with the other zones (p < 0.05).

Zone B had a significantly higher histological score as compared with the other zones (p < 0.05). However, when the control group was compared with the MP group in each zone, this difference was not observed (p > 0.05) (Graph 2).

 

 

Photomicrographies of the medulla are presented in Figures 1 and 2 that show the histological findings regarding cavitation, edema, congestion, necrosis and cell infiltration of the hematoxylin-eosin-stained medullar cuts.

 

 

DISCUSSION

Several experimental models were developed to simulate medullar injury and reproduce it in homogeneous form, in order to analyze the locomotor function response in animals submitted to medullar injury(1,5,8, 13,14,17,25).

Qualitative locomotor tests should be easy to use, sensible and able to quickly detect any behavioral changes(4,10). The Tarlov and Klinger test developed in 1954 was the first to approach this ideal; later, other authors(4,10,19) modified this test until 1995, when the so-called BBB test was developed by Basso et al(4).

The BBB locomotor test is a 21-point system based on behavioral findings defined as movements of the hind legs, position of the trunk, position of the abdomen, accommodation of legs, march, coordination, ability to clean the paws, predominant position on the hind legs, instability of the trunk and position of the tail that follow recovery, from complete paralysis (score 0) to normal locomotion (score 21), evaluated in open field for a 4-minute period.

The BBB test was designed to be more specific, trustworthy and comprehensive than the previous tests(4) that might lead to a large variation in the interpretation both by inter-observers and intra-observers(23). However, we noticed that once the observed were trained, the test could be applied in a sensitive, proper form, as noticed also by Basso et al(3,4).

Recent studies indicate that contuse injuries in the spinal cord may be minimized by pharmacological therapies, provided they are administered shortly after the trauma(11). Although their mechanisms of action are not well understood nor universally accepted, corticosteroids such as methylprednisolone have been widely employed in the treatment of experimental medullar injury(13,25). Methylprednisolone was considered a standard therapy following acute medullar injury in humans by some authors(20), although there is no general consensus(16).

Methylprednisolone was used initially, in the decade of 1960, in patients with acute medullar injury due, to its beneficial effect in the treatment of other neurological disorders such as CNS tumors(9).

From then on several trials were performed with the purpose of showing the beneficial effects of methylprednisolone in acute medullar traumatism(9,13,15,25). However, the NASCIS II study, completed in 1990, was the first randomized, double-blind, placebo-controlled study to show unmistakably that methylprednisolone may change beneficially the course of events following acute medullar injury(9,15).

In the present study we observed that the group of rats receiving methylprednisolone showed an improvement in the locomotor scale that was significantly higher (average: 6.1) as compared with the control group (average: 1.7), which agrees with the results found by Constantini and Young (8). We believe that this functional improvement is due to the beneficial effects of methylprednisolone during the ischemic phase of medullar injury.

The presence of necrotic areas mainly in the medullar dorsal zone and in zone B (injury zone) shows that necrosis is a result not only of the initial mechanical injury, but also and mainly of the secondary ischemic injury, as quoted also Balentine(2).

We did not observe the presence of hemorrhagic zones in the medullar segments evaluated, in disagreement with the findings of Constantini and Young(8). However, these authors performed a histological study 24 hours after the medullar injury and, according to Balentine (2), hemorrhage is one of the initial findings, predominating during the first three days and being replaced later by cavitation areas (Figures 2 and 3). In our study the histological evaluation was performed on Day 14, when the hemorrhagic area had already been replaced.

 

 

We interpreted the cavitations surrounded by non-neuronal cells and the presence of cell infiltrate (neutrophils) as local inflammatory response. Peripherally to these cavitation areas, we observed the presence of feasible neuronal tissue, which proves the occurrence of incomplete medullar injury.

As we analyzed the evolution of the locomotor functional results and histological findings, we realized that we could not correlate them, meaning that the functional recovery was not inversely proportional to the evidence of tissular necrosis, in disagreement with the studies by Basso et al (3), but in agreement with the observations of Means et al(18).

In 1996 Basso et al(3) performed an histomorphometric analysis and staining specific for nervous tissues, in order to analyze and quantify the remaining nervous tissue and make a comparison with the functional findings. Differently from these authors, we used hematoxylin-eosin staining with the purpose of quantifying the indirect inflammatory findings that represented the secondary medullar injury and necrosis.

 

CONCLUSIONS

1) Methylprednisolone had a beneficial effect in the functional recovery of rats with medullar injury.

2) The histological findings could not be correlated with the results of the locomotor function.

 

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Correspondence to
Rua Euclides da Cunha, 180
CEP 13200-390 Jundiaí - SP
E-mail: matebet@uol.com.br

Work performed at Laboratory of Rachiomedular Traumatism and Peripheral Nerves of Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo
Trabalho recebido em 21/02/2003
Aprovado em 20/03/2003

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