Progesterone is Neuroprotective After Acute Experimental Spinal Cord Trauma in Rats

SPINE Volume 24, Number 20, pp 2134 –2138 ©1999, Lippincott Williams & Wilkins, Inc.

Progesterone is Neuroprotective After Acute Experimental Spinal Cord Trauma in Rats Ajith J. Thomas, MD,* Russ P. Nockels, MD,* Hiu Q. Pan, MD,* Christopher I. Shaffrey, MD,* and Michael Chopp, PhD†

Study Design. A standardized rat contusion model was used to test the hypothesis that progesterone significantly improves neurologic recovery after a spinal cord injury that results in incomplete paraplegia. Objectives. To compare the effect of progesterone versus a variety of control agents to determine its effectiveness in promoting neurologic recovery after an incomplete rat spinal cord injury. Summary of Background Data. Progesterone is a neurosteroid, possessing a variety of functions in the central nervous system. Exogenous progesterone has been shown to improve neurologic function after focal cerebral ischemia and facilitates cognitive recovery after cortical contusion in rats. Methods. A standardized contusion model of spinal cord injury using the New York University impactor that resulted in rats with incomplete paraplegia was used. Forty mature male Sprague–Dawley rats were randomly assigned to four groups: laminectomy with sham contusion, laminectomy with contusion without pharmacologic treatment, laminectomy with contusion treated with dimethylsulfoxide and dissolved progesterone, and laminectomy with contusion treated with dimethylsulfoxide. Functional status was assessed weekly using the Basso– Beattie–Bresnehan (BBB) locomotor rating scale for 6 weeks, after which the animals were killed for histologic studies. Results. Rats treated with progesterone had better outcomes (P 5 0.0017; P 5 0.0172) with a BBB score of 15.5, compared with 10.0 in the dimethylsulfoxide control group and 12.0 in the spinal cord contusion without pharmacologic intervention group. This was corroborated in histologic analysis by relative sparing of white matter tissue at the epicenter of the injury in the progesteronetreated group (P , 0.05). Conclusions. Rats treated with progesterone had a better clinical and histologic outcome compared with the various control groups. These results indicate potential therapeutic properties of progesterone in the management of acute spinal cord injury. [Key words: Basso–Beattie–Bresnehan score, progesterone, spinal cord contusion, spinal cord injury, white matter] Spine 1999;24: 2134 –2138

The optimal management of acute spinal cord injury continues to evolve. Current pharmacologic treatment involves the use of intravenous methylprednisolone within 8 hours of the injury. Results of several randomized clinical trials indicate that methylprednisolone has a From the Departments of *Neurosurgery and †Neurology, Henry Ford Health Sciences Center, Detroit, Michigan. Acknowledgment date: December 2, 1998. Acceptance date: March 24, 1999. Device status category: 1.

2134

real but limited role in providing clinical improvement.4,5 The preclinical search for better pharmacologic treatments has centered on the inhibition of detrimental posttraumatic pathochemical events, such as generation of free radicals, mediators of the inflammatory response, and local neurotransmitter toxicity.18,33 Increasing attention has been focused on the actions of progesterone on the central nervous system. Progesterone receptors are widely distributed in the central nervous system, including the hypothalamus, preoptic area, midbrain, cortex, amygdala, hippocampus, caudate, putamen, and cerebellum.17 Progesterone is considered a neurosteroid because of de novo synthesis and accumulation within the nervous system that is independent of steroidogenic gland secretion rates.24 Classically, the actions of progesterone in nerve cells have been thought to occur through cytosolic–nuclear receptors specific to the steroid.22 More recently, progesterone has been shown to modify the function of traditional neurotransmitter systems in the central nervous system such as the inhibitory g-aminobutyric acid15,16 and excitatory amino acids.30 The presence of receptors and sources of progesterone within the nervous system as well as its modulation of inhibitory and excitatory amino acids indicate a possible broader role for progesterone than simply as a gestational hormone. It has been shown that the administration of progesterone is neuroprotective in a transient focal ischemia model in the rat, either when administered before the onset of ischemia or 2 hours after the onset of ischemia.12 Progesterone also facilitates cognitive recovery and reduces secondary neuron loss caused by cortical contusion injury in an animal model.25,28 Methods Mature male Sprague–Dawley rats (weight range, 295–305 g) were used for the study. Male rats were preferred because their endogenous progesterone levels do not fluctuate. Rats were anesthetized with 50 mg/kg intraperitoneal pentobarbital. Cefazolin (50 mg/kg) was injected subcutaneously before surgery. The surgical site was shaved and swabbed with alcohol and then with povidone alcohol. Rats were placed on a heated surgical table. Rectal temperature was maintained at 37 C throughout the surgical procedure using a feedback-regulated water heating system. A midline skin incision was made to expose the T6 –T10 spinal column. The muscles attached to the spinous process of T6 –T10 were cut and cleared away from the column. The T8 spinous process and the caudal half of the T7 spinous process and lamina were removed with microrongeurs, starting at the caudal edge of the T8 lamina. An opening was

Progesterone in Spinal Cord Injury • Thomas et al 2135 made that was large enough to accommodate the impactor head (2.5 mm). The dura mater is left intact for the procedure. The contusion was produced with the impactor (spinal cord contusion system, with impactor software ver. 7.0; New York University, New York, NY) to monitor and record the contusion (New York University Medical Center, New York, NY). The rat was mounted on the impactor with the rostral clamp attached to the T6 spinous process and the caudal clamp to the T10 spinous process. With the clamps, the rat was slightly raised off the sponge. The 10-g impactor head was raised to a height of 25 mm to produce a spinal cord injury of moderate severity. The injury was deemed successful if it fell within the parameters of the impactor’s software, which monitors the degree and rate of vertebral and spinal cord displacement, as described elsewhere.2 The impactor was immediately lifted from the cord after the impact. The rat was returned to the heating table, paravertebral muscles were sutured, and the skin was closed with stainless-steel clips. After surgery, the rats were warmed using a desk lamp until they recovered from anesthesia. Fifteen to 25 ml of lactated Ringer’s solution was administered subcutaneously immediately after surgery. Buprenorphine was administered subcutaneously twice daily for 2 days. Rats were monitored daily for urinary retention, weight loss, and dehydration. Bladders were manually expressed twice daily until function was regained. Cefazolin was administered subcutaneously twice daily for 3 days after surgery as a prophylaxis for urinary tract infections. The Basso–Beattie–Bresnehan (BBB) scores were evaluated at 6 hours, 24 hours, and 3 days and then once weekly for 6 weeks (Table 1). For evaluation of BBB scores, rats were placed in a shallow plastic tub with a rough surface (1.5 m 3 1.5 m). Two observers independently evaluated the scores for each hind limb and in the event of discrepancy, the average score was used.

Experimental Design. There were four groups with 10 rats in each group. Group A contained sham-procedure rats with laminectomy and exposure of the dura mater, but without any injury to the spinal cord. In rats in Group B, after the laminectomy and exposure of the dura mater the impactor rod was raised to a height of 25 mm and a moderate to severe spinal cord contusion was applied causing incomplete paraplegia in all animals. Group C animals had the same procedure as Group B. Thirty minutes after laminectomy and spinal cord contusion, Group C rats received an intraperitoneal administration of 4.0 mg/kg progesterone (4-pregnene-3,20-dione) dissolved in 8.0 mg/mL dimethylsulfoxide (DMSO). This dose was repeated at 6-hour, 24-hour, 48-hour, 72-hour, 96-hour, and 120-hour intervals. Rats in Group D underwent the same procedure as those in Group B, followed by administration of the vehicle DMSO alone at the 30-minute, 6-hour, 24-hour, 48-hour, 72hour, 96-hour, and 120-hour intervals.

Pathologic Analysis. Rats were killed 42 days after spinal cord contusion. Under pentobarbital anesthesia, the chest was opened and transcardiac perfusion performed with heparinized phosphate-buffered saline followed by 10% formaldehyde. The entire spinal column was removed after which the spinal cord was dissected. Segments 2 cm rostral and caudal to the site of injury were paraffin embedded, and representative sections were taken at 2-mm intervals and stained with hematoxylin and eosin and Klu¨ver–Barrera Luxol stain. The myelinated white matter tracts were calculated as a percentage area of the

Table 1. Basso, Beattie, and Bresnehan Locomotor Rating Scale 0 1 2

3 4 5 6 7 8 9

10 11 12 13 14

15 16 17 18 19 20 21

No observable hindlimb (HL) movement Slight movement of one or two joints, usually the hip and/or knee Extensive movement of one joint OR extensive movement of one joint and slight movement of one other joint Extensive movement of two joints Slight movement of all three joints of the HL Slight movement of two joints and extensive movement of the third Extensive movement of two joints and slight movement of the third Extensive movement of all three joints of the HL Sweeping with no weight support of plantar placement of the paw with no weight support Plantar placement of the paw with weight support in stance only (i.e., when stationary) OR occasional, frequent, or consistent weight supported dorsal stepping and no plantar stepping Occasional weight supported plantar steps and no forelimb (FL)HL coordination Frequent to consistent weight supported plantar steps, no FL-HL coordination Frequent to consistent weight supported plantar steps and occasional FL-HL coordination Frequent to consistent weight supported plantar steps and frequent FL-HL coordination Consistent weight supported plantar steps, consistent FL-HL coordination; and predominant paw position during locomotion is rotated (internally or externally) when it makes initial contact with the surface as well as just before it is lifted off at the end of stance or frequent plantar stepping, consistent FL-HL coordination, and occasional dorsal stepping Consistent FL-HL coordination and toes frequently to consistently dragged across the walking surface; predominant paw position parallel to body at initial contact Consistent FL-HL coordination during gait and toes occasionally dragged; predominant paw position parallel at initial contact and rotated at liftoff Consistent FL-HL coordination during gait and toes occasionally dragged; predominant paw position parallel at initial contact and at liftoff Consistent FL-HL coordination during gait and toes no longer dragged; predominant paw position parallel at initial contact and liftoff Consistent FL-HL coordination during gait and toes no longer dragged; predominant paw position parallel at initial contact and liftoff; tail down part or all the time Consistent coordinated gait: no toe drags: predominant paw position parallel at initial contact and at liftoff; trunk instability present: and tail consistently up Coordinated gait, consistent toe clearance, predominant paw position parallel throughout stance, consistent trunk stability, tail consistently up

cross section of the spinal cord on the Luxol-stained sections using commercial image analysis software (Global Lab Image Analysis; Data Translation, Marlborough, MA). With the help of a charge-coupled device, video microscopic images were collected, highlighted, and measured. The spinal tissue in the axial tissue section was outlined, and the central cavitary area with fibrosis was subtracted, providing the surface area of the intact spinal tissue.

Statistical Analysis. Student’s t test was used to determine the statistical significance of BBB scores at the end of 6 weeks in the different groups.

2136

Spine • Volume 24 • Number 20 • 1999

Table 2. BBB Scores 6 Weeks After Spinal Cord Injury Group

Table 3. Percentage of White Matter Left Intact at 6 Weeks After Injury

BBB Score Group

Group Group Group Group

A: sham B: control C: progesterone 1 DMSO D: control 1 DMSO

21 (0) 12 (1.65) 15.5 (2.73) 10.3 (2.9)

A B C D

% of Intact White Matter 100 (0) 24.32 (8.5) 53.42 (22.9) 31.01 (6.3)

Results

Functional Outcome The BBB locomotor rating scores in the four groups at 6 weeks are shown in Table 2. The maximum score is 21. All Group A (sham-procedure) rats had a score of 21. Rats in Groups B, C, and D all had scores of zero 6 hours after spinal cord injury. Group B (contusion without pharmacologic treatment) and Group D (contusion treated with DMSO alone) scores improved to 12.0 and 10.0, respectively, at final follow-up. The difference between these groups was not statistically significant. Group C (contusion treated with progesterone) had a significantly improved score of 15.5 at final follow-up, compared with the control groups (P 5 0.0017; P 5 0.0172). In real terms, this means that rats in both contusion control groups (Groups B and D) could walk with weight support, whereas those in the progesteronetreated group could walk with consistent weightsupported plantar steps with forelimb and hindlimb coordination. The BBB scale is illustrated in Table 1. The temporal profile of the recovery is outlined in Figure 1. Both control groups (Groups B and D) had an early improvement in neurologic function, which plateaued by the third week. The progesterone-treated group (Group C) exhibited a steady recovery that had

Figure 1. Temporal profile of Basso–Beattie–Bresnehan scores in rats recovering from spinal cord injury.

not plateaued by the sixth week, which was the end point of the experiment.

Histologic Analysis Histology of the epicenter was assessed using hematoxylin and eosin stain and Klu¨ver–Barrera Luxol stain, which is a myelin stain. Results are shown in Table 3. There was no injury apparent in Group A specimens, evidenced by complete sparing of white matter. There was significant sparing of the white matter at the epicenter of injury in Group C (progesterone-treated group) which was more apparent on the myelin stain (P , 0.05). The progesterone-treated rats had 53.42 6 22.9% of myelinated tracts intact as a percentage of the total cross section of the cord Group B (contusion without pharmacologic treatment) control rats had 24.32 6 8.5% left intact, whereas Group D (contusion with DMSO alone) rats had 31.01 6 6.3% of the cord remaining. Representative sections obtained from the epicenter of injury in Group C and Group D are illustrated in Figure 2, A and B. Discussion The pathophysiology of spinal cord injury involves a primary mechanical injury and delayed secondary injury resulting from a number of proposed mechanisms including ischemia, abnormal intracellular shifts of ions including sodium and calcium, free radical–associated lipid peroxidation of cell membranes, ischemia, edema, leukocytic infiltration, and excitotoxic cell death.32 Vascular injury plays an important role in the primary and secondary injury mechanisms that cause damage to the acutely traumatized spinal cord.31 Non-N-methyl-Daspartate glutamate receptors and abnormal sodium influx may mediate this injury.1,8 As a result of these insults, progressive tissue necrosis, which is unique to spinal cord trauma, occurs. The site of injury is gradually transformed into a large, cavity-filled lesion. Substantial residual neurologic function could persist with survival of 5–10% of the original number of axons.9 Once the acute and subacute phases are over, failure of regeneration and formation of a glial scar inhibit neurologic recovery. Clinically, a reduction of secondary injury after spinal cord trauma may optimize the chances for improved neurologic function. Saving 5% of the white matter tracts could translate into significant preservation of function. We have demonstrated that progesterone is neuroprotective when used over a period of 5 days after rat spinal

Progesterone in Spinal Cord Injury • Thomas et al 2137

Figure 2. A, Histologic examination of the spinal cord at the epicenter of injury in Group C (progesterone treated). Note the relative sparing of the myelinated tracts (stained blue) and limited central cavitation. B, Histologic examination of the spinal cord at the epicenter of injury in Group D (dimethylsulfoxide [DMSO] control). Significant central cavitation with a small rim of myelinated tract remains (A, B: Klu¨vers–Barrera Luxol; original magnification, 350).17

cord injury with treatment being initiated 30 minutes after injury. Significant reduction was observed in the central cavitary process that follows spinal cord injury, and the reduction was accompanied by an improvement in the locomotor rating. This is consistent with reports that progesterone offers neuroprotection in cerebral ischemia and cortical contusion.12,25 In cortical contusion injury there is reduction of secondary neuronal loss and attenuation of brain edema.26 The drug was administered for 5 days for two reasons: first, because in a contusion injury model in which progesterone facilitated cognitive recovery, the drug was administered for 5 days, and second, because the cavitary process and the consequent neuronal loss in the spinal cord evolves with stepwise sequential changes and continues for several days after injury.7 Any intervention to protect the neural elements should therefore be continued after the initial insult. The mechanisms behind the neuroprotective effects of progesterone are not known. It may act through several different mechanisms. Excitotoxic cell death is prevented by potentiating g-aminobutyric acid receptors11 and/or

inhibiting excitatory amino acid receptors, especially the N-methyl- D -aspartate subtype of glutamate receptor.19,20 Progesterone has been shown to protect cultured spinal cord neurons from glutamate toxicity19 and to alter the excitability of neurons partly by potentiating g-aminobutyric acid– evoked chloride currents.21 It also potentiates the uptake of adenosine by rat cerebral cortical synaptosomes and its depressant action on the spontaneous firing of these neurons.23 This cortical depressant activity also confers anticonvulsant effects on progesterone.14 Progesterone attenuates the cerebellar Purkinje cell responses by depressing both the kainate and the N-methyl-D-aspartate responses of cerebellar neurons.30 In the acute phase of injury, progesterone also attenuates the severity of postinjury edema,26 probably by reducing the permeability of the blood– brain barrier.3 This effect may be mediated by its antioxidant effects and inhibition of lipid peroxidation. Lipid peroxidation is a major contributor to BBB breakdown, and progesterone limits this free radical–induced damage.28 After traumatic brain damage, edema is almost absent in pseudopregnant female rats,27 a condition in which progesterone levels are high relative to estrogen. Progesterone facilitates cognitive recovery after contusion of the medial frontal cortex in rats and was associated with less neuronal degeneration in the areas in the thalamus that had reciprocal connection with the contused area.25 Some data suggest that progesterone and its metabolites are involved in the development and regeneration of the nervous system. 24 Progesterone, synthesized by Schwann cells, promotes the formation of new myelin sheaths after lesion of the mouse sciatic nerve.13 Progesterone also inhibits the proliferation of the glial cells of the central nervous system.24 It has been suggested that oligodendrocytes are a negative influence on the regenerating axons in the injured spinal cord.32 Thus, inhibition of oligodendrocytes may promote neuronal regeneration. The inefficacy of DMSO may seem surprising. However, previous studies of DMSO29 were performed before the development of the New York University impactor and the BBB locomotor scoring system.2 Both these have helped considerably in the standardization of spinal cord injury models and interpretation of functional outcome. We used DMSO as a solvent because progesterone dissolved in water has very poor penetration of the blood– brain barrier. Advantages of DMSO over vegetable oil include a higher available saturant concentration of progesterone and more rapid absorption. Although DMSO has been proposed as a neuroprotective agent by virtue of its free radical scavenger properties,29 no benefit was found in the present study at a dose of 1 mL/kg when compared with the response of the group with contusion without pharmacologic treatment. Progesterone has many attractive properties that theoretically should limit secondary injury and promote regeneration after spinal cord injury. Further investigation

2138

Spine • Volume 24 • Number 20 • 1999

of progesterone with respect to dosage, duration of treatment, and comparison with other currently used pharmacologic agents is needed before it can be tested clinically as a treatment for use in spinal cord injury. At the completion of the current experiment, improvement in neurologic function was continuing to occur in the progesterone-treated group. The maximum improvement associated with the therapeutic effect of progesterone will be investigated. The dose of progesterone used in these studies was 30 – 40 times the dose used in humans for the treatment of uterine problems.6 Adverse reactions to progesterone include central nervous system reactions such as insomnia and depression, skin rashes, weight gain, and uterine bleeding. Because progesterone is a widely and clinically used compound, further investigation into its therapeutic benefit for treatment of spinal cord injury is warranted. References 1. Agrawal SK, Fehlings MG. Role of NMDA and non-NMDA ionotropic glutamate receptors in traumatic spinal cord axonal injury. J Neurosci 1997;17: 1055– 63. 2. Basso DM, Beattie MS, Bresnehan JC, et al. MASCIS evaluation of open field locomotor scores: Effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study. J Neurotrauma 1996;13:343–59. 3. Betz AL, Coester HC. Effect of steroid on edema and sodium uptake of the brain during focal ischemia in rats. Stroke 1990;21:1199 –204. 4. Bracken MB, Shepard MJ, Holford TR, et al. Administration of methylprednisone for 24 hours or 48 hours or trilazad mesylate for 48 hours in the treatment of acute spinal cord injury. Results of the Third National Acute Spinal Cord Injury Study. JAMA 1997;277:1597– 604. 5. Bracken MB, Shepard MJ, Holford TR, et al. A randomized controlled study of methylprednisone or naloxone in the treatment of acute spinal cord injury. Results of the Second National Acute Spinal Cord Injury Study. N Engl J Med 1990;322:1405–11. 6. Cada DJ, ed. Drug: Facts and Comparisons. St. Louis: Wolters Kluwer, 1999:451–7. 7. Ducker TB, Kindt GW, Kemp LG. Pathologic findings in acute experimental spinal cord trauma. J Neurosurg 1971;35:700 – 8. 8. Fehlings MG, Agrawal S. Role of sodium in the pathophysiology of secondary spinal cord injury. Spine 1995;20:2187–91. 9. Fehlings MG, Tator CH. The relationships among the severity of spinal cord injury, residual neurological function, axon counts, and counts of retrogradely labeled neurons after experimental spinal cord injury. Exp Neurol 1995;132: 220 – 8. 10. Garcia–Estrada J, Del Rio JA, Luquin S, et al. Gonadal hormones downregulate reactive gliosis and astrocyte proliferation after a penetrating brain injury. Brain Res 1993;628:271– 8. 11. Green AR, Cross AJ. The neuroprotective actions of chlormethiazole. Prog Neurobiol 1994;44:463– 84. 12. Jiang N, Chopp M, Stein D, Feit H. Progesterone is neuroprotective after transient middle cerebral artery occlusion in male rats. Brain Res 1996;735: 101–7. 13. Koenig HL, Schumacher M, Ferzaz B, et al. Progesterone synthesis and myelin formation by Schwann cell. Science 1995;268:1500 – 4.

14. Kokate TG, Svensson BE, Togawski MA. Anticonvulsant activity of neurosteroid: Correlation with gammaaminobutyric acid evoked chloride potentiation. J Pharmacol Exp Ther 1994;270:1223–9. 15. Lambert JJ, Peters JA, Sturgess NC, Hales TG. Steroid modulation of GABA receptor complex: Electrophysiological studies. In: Chadwick D, Widdows K, eds. Steroids and Neuronal activity. Chichester, UK: Wiley, 1990:56 –71. 16. Limroth V, Lee WS, Moskowitz MA. GABAA - receptor mediated effects of progesterone, its ring-A-reduced metabolites and synthetic neuroactive steroids on neurogenic edema in the rat meninges. Br J Pharmacol 1996;117:99 –104. 17. MacLusky NJ, McEwen BS. Oestrogen modulates progestin receptor concentration in some rat brain regions but not in others. Nature 1978;274:276 – 8. 18. Nockels R, Young W. Pharmacologic strategies in the treatment of experimental spinal cord injury. J Neurotrauma 1992;9(Suppl):S211–7. 19. Ogata T, Nakamura Y, Tsuji K, et al. Steroid hormones protect spinal cord neurons from glutamate toxicity. Neuroscience 1993;55:445–9. 20. Park CK, McCulloch J, Kang JK, Chio CR. Pretreatment with a competitive NMDA antagonist DCPPene attenuates focal cerebral infarction and brain swelling in awake rats. Acta Neurochir (Wien) 1994:127:220 – 6. 21. Paul SM, Purdy RH. Neuroactive steroids. FASEB J 1992;6:2311–22. 22. Pfaff DW, McEwen BS. Actions of estrogens and progestins on nerve cells. Science 1983;219:808 –14. 23. Phillis JW, Bender AS, Marszalec W. Estradiol and progesterone potentiate adenosine’s depressant action on rat cerebral cortical neurons. Gen Pharmacol 1985;16:609 –12. 24. Robel P, Baulie EE. Neurosteroids: Biosynthesis and function. Trends Endocrinol Metab 1994;5:1– 8. 25. Roof RL, Duvdevani R, Braswell L, Stein DG. Progesterone facilitates cognitive recovery and reduces secondary neuronal loss caused by cortical contusion injury in male rats. Exp Neurol 1994;129:64 –9. 26. Roof RL, Duvdevani R, Braswell L, Stein DG. Progesterone treatment attenuated brain edema following contusion injury in male and female rats. Restorative Neurology Neuroscience 1992;4:425–7. 27. Roof RL, Hofman SW, Stein DG. Gender influences outcome of brain injury: Progesterone plays protective role. Brain Res 1993;607:333– 6. 28. Roof RL, Hofman SW, Stein DG. Progesterone protects against lipid peroxidation following traumatic brain injury in rats. Mol Chem Neuropathol 1997; 31:1–11. 29. Rucker NC, Lumb WV, Scott RJ. Combined pharmacologic and surgical treatments for acute spinal cord trauma. Am J Vet Res 1981;42:1138 – 42. 30. Smith SS. Progesterone administration attenuates excitatory amino acid responses of cerebellar Purkinje cells. Neuroscience 1991;42:309 –20. 31. Tator CH, Koyanagi I. Vascular mechanisms in the pathophysiology of human spinal cord injury. J Neurosurg 1997;86:483–92. 32. Tatagiba M, Brosame C, Schwab ME. Regeneration of injured axons in the adult mammalian central nervous system. Neurosurgery 1196;40:541– 46. 33. Zhang Z, Krebs CJ, Guth L. Experimental analysis of progressive necrosis after spinal cord trauma in the rat: Etiological role of the inflammatory response. Exp Neurol 1997;143:145–52.

Address reprint requests to Russ P. Nockels, MD Department of Neurosurgery Henry Ford Health Sciences Center 2799 West Grand Boulevard Detroit, MI 48202