Indian Journal of Experimental Biology Vol. 42, May 2004, pp. 476-480
Comparison of methylprednisolone with dexamethasone in treatment of acute spinal injury in rats
Alok Sharma*, Rajan Tiwari, Prerna Badhe & Garima Sharma
Department of Neurosurgery, Lokmanya Tilak Municipal Medical College and General Hospital, Sion, Mumbai 400022, India Received 22 April 2003; revised 5 February 2004
Effect of methylprednisolone sodium succinate (MPSS) and its comparison with dexamethasone in experimentally induced acute spinal cord compression in adult rats was studied. The rats were divided into group A (control) and group B, which was subdivided into BI, B2, B3 where MPSS was given after I, 8 and 24 hr and B4 where dexamethasone was given after I hr of cord injury respectively. Proper neurological evaluation was done with mobility, running and climbing score.
Recovery index was evaluated for 7 days. After sacrificing the rats, spinal cord was observed histopathologically. Mean recovery index and microscopic findings based on hemorrhage in gray and white matter, neuronal degeneration, hematomyelia and edema in white matter were recorded. The results suggested that MPSS was effective in promoting post- traumatic clinical and histological recovery and to a greater extent, when given I hr after trauma. MPSS is more effective than dexamethasone in reducing edema when both are given after interval of Ihr.
Kewwords: Dexamethasone, Methylprednisolone, Rat, Spinal injury IPC Code: Int. cf A6I K
Acute spinal cord compression is seen in' patients suffering from vertebral fractures, herniated interver- tebral discs, neoplasms or hemorrhage following which a series of events take place leading to auto destruction. The main being prolonged oligaemia, which produces marked depletion of high-energy phosphate reserves, lactic acidosis and tissue edema in addition to posttraumatic inflammatory responsel. In experimental models of spinal cord injury, methyl- prednisolone has prevented posttraumatic spinal cord ischaemia especially when administered early since pathophysiological changes such as axonal and neuronal degeneration, edema and ischemia begin to occur within first 6 hr of the insu!t2-5. The present study was conducted to compare the efficacy of MPSS with dexamethasone in promoting clinical recovery, decreasing edema and lissue necrosis of the spinal cord following acute compression injury in rats.
Materials and Methods
Immunized and conditioned, 50 Wi star albino rats (250 to 350 g) of either sex were divided in two broad
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groups. Group A (control), undergoing spinal cord compression only and Group B undergoing spinal cord compression and treated with MPSS and dexamethasone. The latter was subdivided into Bl, B2, and B3 where MPSS was administered after 1, 8, and 24 hr of compression respectively, and B4 where dexamethasone was administered after 1 hr of injury.
Each group comprised 10 rats. Fifty rats were anaesthetized after shaving, painting and draping the lower dorsal region and three level laminectomy was performed. The dura was exposed with the intention of producing reversible injury and compressed by placing an object of 25 g weight for . 2 min.
Subsequently, the wound was closed meticulously in layers and sterile dressing done and the rats were placed in their respective labeled cages. One hr after injury, in 10 rats (B 1) MPSS was administered at the dose of 30 mg/kg/day for the first 3 days, 15 mglkg/day for the next 2 days, 7.5 mglkg/day for final 2 days. In 10 rats (B4), dexamethasone was administered as 6 mg/kg/day for 3 days, 3 mg/kglday for next 2 days, 1.5 mg/kg/day for final 2 days. MPSS was administered at the same dose and scheduled as Bl to B2 ar,d B4 after 8 and 24 hr of injury
t esptxti vcly (IO rais each). These fifty rats ~ere
neurologically evaluated on a daily basis for 7-days
SHARMA et.a(: COMPARISON OF METHYLPREDNISOLONE WITH DEXAMETHASONE IN SPINAL INJURY 477
(recovery period). Eugene D Means & Douglas K Anderson 6 methods was taken for the locomotor score (Table 1). The animals were sacrificed by intraperitoneal (ip) sodium pentobarbital on day 8.
The spinal cord was removed after 4 hr. The injured segment of the spinal cord was identified and approximately 2 cm section was cut and fixed by immersing in cold 10% formalin. The dura was carefully · removed and the spinal cord divided transversely into two pieces. Each piece was processed in routine fashion and embedded in paraffin. The tissue was serially sectioned (10 /lm thick) and stained with hematozylin and eosin. Every 10lh section of the tissue was photographed on a Nikon photo microscope using AX I objecti ve.
Microscopically areas of hemorrhage in gray and white matter, neuronal degeneration, necrosis, edema and hematomyelia were observed7.8
. Mean recovery index and microscopic findings were compared between groups A (control) and B and between the group Bland B4. Statistical evaluation was done.
Mean values were computed as arithmetic means ±SD and compared with Chi Square test. Level of signi- ficance was set at p<O.OS. For group A it was S.1±1.37, group B 1 was *8.2±1.23 (*p<O.OS, ~ignifi
cant), B2 @ 6.2±2.04, B3@ S.8±2.04 (@ p>0.05 not
significant), and for B4 it was *6.6± 1.07 (p<O.OS, significant).
Results
On calculation of the mean recovery index, it was observed that MPSS produced better clinical recovery as compared to dexamethasone, given after 1 hr following cord trauma and these results were statistically significant. It was observed in the present study that in 80% of rats (control group) the white matter edema was of moderate to severe degree, while among the ster-oid treated group (B 1, B2, B3 and B4) it was 20, 40, 40 and 60% respectively (Table 2). The histopathological evaluation also showed moderate to severe degree of gray matter hemorrhages in 70% rats of the control group, while among the steroid treated groups (Bl, B2, B4, B4) it was 40, 60, 60, 60%
retrospectively (Fig. 1) and white matter hemorrhages in 60% rats of the control group, while B 1, B2, 83, 84 it was 40, 40, 60, 40% respectively (Table 3).
Neuronal degeneration and necrosis as a result of ischemia was seen in 60% rats of the control group, while it was not there in B 1, 40% in B2, 20% in B3 and nil in 84. But the results were statistically insignificant as P>O.OS in above cases with hemorrhage and degeneration (Fig. 2).
Table I-Eugene D Means and Douglas K Anderson's motility score, 1981 (Ref. 6) Mobility Score
No movements of hind limbs.
Random movements of hind limbs Purposeful movements of hind limbs Can stand, cannot walk
Walks very unstable.
Walks uncoordinated gait
Walks coordinated gait, some deficit Normal
Running Score
Falls frequently when running Runs, some deficit.
Normal
Climbing Score Uses only fore limbs
Places legs on step cannot push to next step with hind limbs Can negotiate several steps Negotiates steps in jumping manner, some deficit Normal
Table 2-Profile of edema in white matter in all groups on microscopy
Severity Group A Group BI Group B2 Group B3 Group B4
No. (%) No. (%) No. (%) No. (%) No. (%)
Nil
Mild 2 (20) *8(80) 6(60) 6(60) 4(40)
Moderate . 8(80) 2(20) 4(40) 4(40) . 6(60)
Total 10(100) 10(100) 10(100) 10(100) 10(100)
*Significant at P> 0.05
478 INDIAN J EXP BIOL, MAY 2004
Discussion
The most dramatic response to spinal cord injury is the reduction in the spinal cord blood flow [SCBF].
At the level of cord injury the gray matter showed a progressive reduction in blood flow within 1-2 hr, while the changes in mean white matter blood flow are less dramatic9. Within 2 hr, the central petechial hemorrhages enlarge while polymorphonuclear and microglial reactions, ghost cells, eosinophilic nerve cells with indistinct nuclei, smudged cytoplasm and loss of Nissle's bodies become evident. At 4 hr, the process advances to coagulation necrosis up to 40%
of the central gray and subjacent white matter 10. By 6 hr, neuronal and axonal degeneration occurs with accompanying edema, ischemia and advanced structural degeneration. Biochemical evidence of significant lipid peroxidation within the spinal cord
has been demonstrated to occur within 1 hr after severe spinal cord contusion II. Thereby,. one can ascertain that ischaemic hypoxia and associated oxygen derived free radical generation that lead to lipid peroxidation probably begin in the central gray matter. This would irreversibly damage myelin and
12-14 Ed - l I b
axons . ema can mvo ve gray matter a so, ut mainly manifests by enlargement of periaxonal spaces, swelling ofaxons and appearance of vacuolation in the white matter of the traumatized cordiS. As seen in our finding, microscopic evaluation of white matter edema as manifested by enlargement of periaxonal spaces, swelling ofaxons vacuolation was of moderate to severe degree in 80% of control group while among the steroid treated group it was 20% in B 1, 40% in B2, 40% in B3 and 60% in B4 respectively. At ultra structural level thickening of
Fig. I--Gross transverse section of the spinal cord (A}--Control group A showing moderate degree of gray matter hemorrhage and white matter edema; and (B}--Treated group showing mild degree of gray matter hemorrhage and white matter edema (Group B; magnification
at 25X) . . ,
Table 3-Profile of hemorrhage in white and gray matter
Severity Group A Group Bl Group B2 Group B3 Group B4
No. (%) No. (%) No. (%) No. (%) No. (%)
Nil in gray matter
Mild in gray matter 3 (30) 6 (60) 4 (40) 4 (40) 4 (40)
Moderate in gray matter 7 (70) 4 (40) 6 (60) 6 (60) 6 (60)
Total in gray matter 10 (100) 10 (100) 10 (100) 10 (100) 10 (100)
Nil in white matter
Mild in white matter 4 (40) 6 (60) 6 (60) 4 (40) 6 (60)
Moderate in white matter 6 (60) 4 (40) 4 (40) 6 (60) 4 (40)
Total in white matter 10 (100) 10 (100) 10(100) 10 (100) 10 (100)
Value of p > 0.05 Not significant.
SHARMA et.at COMPARISON OF METHYLPREDNISOLONE WITH DEXAMETHASONE IN SPINAL INJURY 479
Fig. 2-Transverse section of the spinal cord shows severe gray matter hemorrhage and white matter edema (indicated by arrow;
magnification at 35X)
capillary wall swelling of astrocyte perivascular feet and enlargement of the extra cellular and peri axonal spaces in the white matter is seen. The white matter undergoes graded changes beginning with the enlargement of the peri axonal spaces after 30 min.
and attenuated myelin sheath and splaying of the myelin lamellae by 1 hr after 4 hr, 25% of the fibers manifest myelin breakdown and consequent axonal changes16.18
. These changes become maximal between 2 to 3 days after the injury and resolves within about 7 days. Thus, we can infer that to achieve significant reduction in white matter edema early institution of MPSS therapy is mandatory. This will hasten the neurological recovery by checking the pathological sequele of cord edema. A number of factors contribute to the vulnerability of the center of the cord. In contrast to the tightly packed fiber tracts of white matter, the neurophil of gray matter is easily separated by fluid or blood increase, if any, in the intramedullary pressure is concentrated centrally because of the inelastic pial membrane. Normally the gray matter: white matter blood flow ratio is 5: l. In the injured gray matter, metabolic needs may exceed the available blood flow. At the same time dorsal white matter blood flow is more severely compressed than ventral white matter blood flow. This may reflect the inherent resistance of the ventral spinal artery to compression compared with the smaller dorsal spinal arteries and pial vessels. Experimental studies indicate that the degree of involvement of any cord system is
mainly a function of its distance from the center of the cord. Accordingly peripherally located fibers tend to be spared 19,20. MPSS, given early, may stop this sequence of events by a direct antioxidant effect, inhibiting arachidonic acid release and thromboxane formation, increasing vascular responsiveness of to vasoactive neurotransmitters and direct vasodilata- tion21-23. Thus, we infer that MPSS is potent in promoting recovery post trauma especially if given in right dose and right time i.e. earliest after trauma. On comparing the neurological and histopathological observations on various groups treated with MPSS and dexamethasone, it can be inferred that MPSS is more effective than dexamethasone in promoting clinical and histological recovery and reducing edema when given earliest after trauma and these results are statistically significant.
Acknowledgement
The authors appreciate the contribution of the Department of Pharmacology of Lokmanya Tilak Municipal Medical College and Hospital for providing us with the laboratory facility, drugs and animals.
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