Neurological Research A Journal of Progress in Neurosurgery, Neurology and Neurosciences
ISSN: 0161-6412 (Print) 1743-1328 (Online) Journal homepage: http://www.tandfonline.com/loi/yner20
Efficacy of the 21-aminosteroid U74006F in improving neurological recovery after spinal cord injury in rats Anders Holtz & Bengt Gerdin To cite this article: Anders Holtz & Bengt Gerdin (1992) Efficacy of the 21-aminosteroid U74006F in improving neurological recovery after spinal cord injury in rats, Neurological Research, 14:1, 49-52, DOI: 10.1080/01616412.1992.11740010 To link to this article: https://doi.org/10.1080/01616412.1992.11740010
Published online: 20 Jul 2016.
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Efficacy of the 21-aminosteroid U74006F in improving neurological recovery after spinal cord injury in rats Anders Holtz1 and Bengt Gerdin 2 Departments of Neurosurgery1 and Surgeif, University Hospital, Uppsala, Sweden
The efficacy of three different regimens of the 21-aminosteroid U74006F in counteracting the neurological damage after spinal cord compression causing paraparesis in rats was investigated. Three groups of ten animals each were given totally 6 mg/ kg of U74006F in different regimens beginning one hour after injury (A: bolus doses of 1.5 mg/ kg at 1, 4, 7 and 10 hours; 8: bolus of 1.5 mg/kg at 1 hour and 4.5 mg / kg as an infusion over the next 9 hours; and C: infusion alone, 6 mg/ kg, given between 1 and 10 hours after trauma ). Two groups of ten animals each received vehicle alone in administration modes comparable to those of the U74006F treated animals. The motor function was assessed daily on the inclined plane. On day one, the capacity angle had decreased from about 62 o preoperatively to 28-30 o in the two vehicle-treated groups and in group C. In these groups there was a similar improvement in neurological function and on day 9 the capacity angles were 49-55 °. In groups A and B, both of which received a bolus dose of U74006F at 1 hour, the neurological outcome improved on day one with capacity angles of 38-40 °. The difference in neurological function between the animals given U74006F as bolus doses and those given vehicle alone persisted over the entire observation span until day 9. The data suggest that early treatment with a bolus dose seems to be required in order to obtain an effect of U74006F on neurological recovery. Keywords: Neurological function; spinal cord injury; U74006F; 21-aminosteroids; rats
INTRODUCTION In recent studies it has been found that the structural damage that follows trauma to the spinal cord is partly caused by secondary ischaemic and biochemical factors 1 . Among these secondary events, lipid peroxidation has been identified as a major culprit 2- 4 . Lipid peroxidation also acts as a potent autodestructive factor in various types of nontraumatic disorders of the central nervous system, such as ischaemic and haemorrhagic stroke5 . Compounds that interfere with lipid peroxidation, such as the iron chelating agent deferoxamine6 , and 9 , have thus antioxidative substances such as vitamin been effective in decreasing the extent of tissue damage in the central nervous system in experimental injury models. In 1985 Braughler and Hall proposed that the ability of high doses of steroids to protect against damage to the central nervous system is related to an antioxidant effect, independent of activation of glucocorticoid receptors 10. On the basis of their conclusion, several compounds of a new group of steroids, the 21-aminosteroids or lazaroids, were synthesized. The aim was to develop a steroid which would exert its action at the cell membrane and would reduce lipid
e-
Address correspondence to : Dr Anders Holtz, MD, PhD, Department of Neurosurgery, University Hospital, 5-75185 Uppsala, Sweden. Accepted for publication October 1991. The study was approved by the Committee for the Care of Laborat ory Animals, Uppsala University.
© 1992 Forefront Publishing Group 0161- 6412 / 92/ 010049-04
peroxidation by virtue of its antioxidant properties. The compounds were found to be more potent inhibitors of lipid peroxidation than methylprednisolone, but as they did not bind to glucocorticoid receptors, they had no gluco- and mineralocorticoid side effects11 . One of the 21-aminosteroids, U74006F, has been used extensively in animal experiments, and has been reported t o reduce vasospasm after experimental subarachnoid haemorrhage12 to diminish infarction ionic shifts and oedema after stroke13•14, and to decrease neuronal necrosis and reduce postischaemic changes in cortical blood flow after brain ischaemia15•16 . Also, it has been found to improve the neurological recovery after head injury17•18 and to reduce posttraumatic ischaemia and improve the neurological recovery after spinal cord injury 19- 22 . In a previous study we demonstrated that U74006F had a beneficial effect on the functional recovery after compression injury to the mid-thoracic level of the spinal cord in the rat when given as an intravenous bolus dose of 3 mg/ kg 60 min after injury23 . Although this finding, and the above-mentioned observations indicate that U74006F is a promising compound for use in patients with trauma to the spinal cord, they provide no information on how to tailor optimal treatment. In the present study we have attempted to gain information on the way in which a defined dose of U74006F should be administered for it to have an optimal effect on the neurological recovery after spinal cord compression in rats. We chose to administer a total dose of 6 mg/ kg and to divide the dose in various fashions.
Neurological Research, 1992, Volume 14, March
49
U74006F in spinal cord trauma: A. Holtz et al.
MATERIALS AND METHODS Animals and operative procedures Fifty male Sprague-Dawley (Aiab, Sollentuna, Sweden) rats weighing between 320-390 g were used. They were given food and water until experimentation. Each animal was anaesthetized with a solution of fluanisone 2.5 mg/ ml and midazolam 1.25 mg/ ml, total 1.52 ml / kg subcutaneously. A PE 50 catheter was inserted into an external jugular vein for infusion of vehicle or U74006F. A similar catheter was inserted into one carotid artery for monitoring the mean arterial blood pressure (MABP) and sampling for arterial blood gas analysis. The rats were placed prone on a heating pad and the body temperature was kept close to 37 with the aid of a rectal thermistor. The technique of exposing and injuring the spinal cord has been described previousll4 . Briefly, a dorsal laminectomy of Th7 and Th8 was performed under microscopic guidance, leaving the dura intact. The spinal cord was injured by a compression device whereby a slightly curved rectangular plate (2.2 x 5.5 mm) loaded with 35 g was applied on the exposed dura for 5 min. After removal of the plate the muscle and skin were closed. The animals were then placed under a heating lamp and allowed to wake up.
oc
Experimental groups The animals were randomly divided into five groups with ten in each and were given vehicle and / or U74006F (total 6 mg/ kg; Upjohn Co, Kalamazoo, USA) in different regimens starting one hour after injury.
Croups A and D thus received a total volume of 4 ml / kg, while groups B, C and E received 5 ml / kg. Neurological examination The motor function was assessed dai~ by the inclined plane technique as described previously 5 . The maximum inclination at which the rat could maintain itself transversely for 5 s was recorded as the 'capacity angle' 26 . Statistical methods A multifactorial analysis of variance with multiple range testing according to the least significant differences was used for comparison between groups. A difference at the 5% level was considered significant. Values are given as mean ± SEM. RESULTS Physiological variables Table 1 shows the data for physiological variables recorded after laminectomy but prior to injury, 60 min after injury and 5 ·min after the start of the infusion or after the bolus dose. There were no significant differences in any of the measured variables. Nor were there any differences in MABP immediately after infusion of vehicle or U74006F. The five groups did not differ regarding the duration of operation, body temperature or body weight (data not shown ). Neurological dysfunction Before the injury was produced, the motor performance was similar in all rats, with very little difference in the capacity angle (mean value 62 °; Figure 1). One day after injury similar, severe impairment of motorfunction was observed in the three groups that received vehicle as a bolus dose (groups C, D and E), with capacity angles of 28-31 °. There was thus no improvement in the group that received U74006F solely as an infusion (group C) compared with the vehicle-infused control (group E). During the following eight days these three groups displayed an equal and gradual recovery, with capacity angles of 35-41 o and 49-55 ° on days 4 and 9, respectively. The two groups treated with a bolus dose of 1.5 mg/ kg of U74006F subsequently followed either by
Croup A received four i.v. bolus doses of U74006F, 1.5 mg( = 1 ml) / kg, 1, 4, 7 and 10 hours after injury. Croup B received an i.v. bolus dose of U74006F, 1.5 mg( = 1 ml) / kg, 1 hour after injury and thereafter an infusion of a mixture of U74006F, 4.5 mg( = 3 mi) / kg, and vehicle, 1 ml / kg, during the next 9 hours. · Croup C received an i.v. bolus of vehicle, 1 ml / kg, 1 hour after injury followed by an infusion of U74006F, 6 mg( =4 mil/ kg, during the following nine hours. Croup 0 received four i.v. bolus doses of vehicle, 1 ml / kg, 1, 4, 7 and 10 hours after injury. Croup E received an i.v. bolus of vehicle, 1 ml / kg, 1 hour after injury followed by an infusion of vehicle, 4 ml / kg, during the next 9 hours.
Table 1: Physiological variables after laminectomy but before spinal cord injury (a) and at the start of infusion of vehicle and / or U74006F, i.e. 60 min after injury (b), and 5 min after the start of infusion (c)
PC0 2 (kPa)
pH Time period Croup A Croup B Croup C Croup D Croup E
50
P0 2 (kPa )
Hct (%)
Glucose (mM)
MABP (mmHg)
a
b
a
b
a
b
b
b
a
b
c
7.44 ±0.01 7.43 ±0.01 7.44 ±0.01 7.42 ±0.01. 7.43 ±0.02
7.46 ± 0.01 7.43 ±0.02 7.44 ±0.02 7.42 ±0.02 7.47 ±0.03
4.7 ± 0.1 5.0 ±0.2 4.9 ±0.3 4.9 ±0.2 4.6 ±0.1
4.7 ±0.1 4.8 ±0.2 4.8 ±0.2 4.9 ±0.2 4.5 ±0.3
12.1 ± 0.5 10.8 ±0.4 12.5 ±0.8 12.2 ±0.5 12.5 ±0.8
10.5 ±0.6 11.1 ±0.8 10.6 ±0.8 11.6 ±1 .0 12.0 ±0.9
43 ± 1 44 ±1 44 ±1 46 ±2 43 ±1
7.4 ±0.5 8.4 ± 0.7 7.9 ±0.9 7.9 ± 0.6 8.4 ±1.2
102 ±3 103 ±3 101 ±3 99 ±3 105 ±5
107 ±3 103 ±4 105 ±2 104 ±3 105 ±2
108 ±3 102 ±3 100 ±3 100 ±3 108 ±4
Neurological Research, 1992, Volume 14, March
U74006F in spinal cord trauma : A. Holtz et al. 70 oo•
60
00
~50 !!! Cl CD
"'-40 CD a,
c: ;30 '13 c. J 20
"'
10 0
2 Preinjury
3
5 4 6 Days after trauma
7
8
9
Figure 1: The motor function (mean ± SEM), expressed as the capacity angle, in the different experimental groups before and at various times after spinal cord injury. Group A: • - - - • ; group group C: 0---0 ; group D: D---0; group E: B:
e---e;
/::o.---/::o.
repeated bolus doses (group A ) or by infusion of the compound (group B) showed significantly better motor performance during the nine days of observation than the animals given vehicle alone (groups D and E). On day one the capacity angle was 40 ° ± 2 in group A and 38 o ± 2 in group B. There was a gradual recovery over time and on day 4 the capacity angle was 2 in these two groups 2 and 50 o improved to 53 o had increased to they nine day on while respectively, 59 o ± 2 and 60 o ± 2.
±
±
DISCUSSION The results of this study show that the neurological dysfunction that occurs after a standardized compressioninduced spinal cord injury in the rat is mitigated by administration of U74006F as a bolus one hour after the injury. There was no difference in efficacy between animals in which the initial bolus dose was followed by a continuous infusion and those in which it was followed by three repeated bolus doses every 3 hours. A 9-hour infusion alone was not sufficient however, to protect against the damage. These results indicate that it is essential to give a sufficient amount of U74006F early after the injury. They do not permit any conclusion, however, as to how to pursue the treatment further. The doses chosen in this study were the current maximal doses allowed in clinical studies in man. The aim was therefore not primarily to identify the best possible effect of the highest possible dose, but rather to determine how a dosing regimen should be tailored to provide an optimal effect at a submaximal dose. Previous studies in the rat have shown that in traumatic shock the efficacy is dosedependent in the dose range 7.5 to 22.5 mg/ kg27, indicating that higher doses than the total dose of 6 mg/ kg over 9 hours given in our study might result in better protective effects. In previous studies on spinal cord compression at the L2 level in cats, however, the beneficial effect did not show a clear dose dependency19 . In a previous investigation 23 with an identical experimental set-up, U74006F given as a single dose of 3 mg/ kg one hour after trauma had a beneficial effect similar t o that observed after the repeated lower doses
given in the present study. The fact that an infusion of U74006F alone, starting at one hour, was not sufficient to protect against damage suggests that a favourable effect is dependent both on the dose and on the time of administration. Regarding the time of administration, it seems that the earlier the dose is given, the better is the effect. In previous studies on an experimental head injuri 7 , an effect was observed when treatment was commenced 5 min after the injury. Administration of U74006F 30 min after spinal cord injury lead both to improvement of neurological recovery and to an increased spinal cord blood flow 19' 21 . In the present study a beneficial effect was only observed when treatment instituted after one hour was commenced with a bolus dose. Current pharmacokinetic studies in healty rats and monkeys (data from Up john ) suggest that only a minor fraction of U74006F crosses the blood-brain barrier. It is therefore reasonable to ask whether the mechanism of the beneficial effect observed in this and other studies on damage to the CNS actually requires that the drug has crossed the blood-brain barrier. Assuming that the beneficial effect is mediated by U74006F located in the CNS, two alternatives have to be considered. Either the minor fraction crossing the blood-brain barrier is sufficient, or the CNS damage involves an alteration in the blood-brain barrier function, including an increase in the permeability to compounds such as U74006F. Another conceivable alternative is that the beneficial effect is mediated on the endothelium itself, as has previously been discussed concerning the effect of methylprednisolone28 . In summary, U74006F given as a bolus dose 60 min after a spinal cord compression injury has a beneficial effect on the impaired neurological function. Additional studies to evaluate the mechanism(s) of action and further dose-response qualities of the drug are required before clinical trials are undertaken.
ACKNOWLEDGEMENTS The study was supported by Trygg-Hansa Insurance Company and the Swedish Medical Research Council. REFERENCES Young W. Blood flow, metabolic and neurophysiological mechanisms in spinal cord injury. In: Decker DP, Povlishock JT, eds. Central Nervous System Trauma Status Report. National Institute of Neurological and Communicative Disorders and Stroke, National Institute of Health : Bethesda, 1985; pp. 2
3
4
5
6
463-473 Hall ED, Braughler JM. Central nervous system t rauma and stroke. 2. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. j Free Radic Bioi Med 1989; 6 : 303- 313 Hall ED, Braughler JM. Role of lipid peroxidation in posttraumatic spinal cord degeneration: A review. CNS Trauma 1986; 3: 281-294 Kinuta Y, Kikuchi H, Ishikawa M, Kimura M, ltokawa Y. Lipid peroxidation in focal cerebral ischemia. J Neurosurg 1989 ; 71 : 421-429 Braughler JM, Hall ED. Central nervous system trauma and stroke. Biochemical considerations for oxygen free radical formation and lipid peroxidation. J Free Radic Bioi Med 1989; 6: 289- 301 Ikeda Y, Ikeda K, Long DM. Protective effect of the iron chelator deferoxamine on cold-induced brain edema. J Neurosurg 1989 ; 71 : 233- 238
Neurological Research, 1992, Volume 14, March
51
U74006F in spinal cord trauma: A. Holtz et al.
7
Anderson OK, Saunders RD, Demediuk P, Dugan LL, Braughler JM, Hall ED, Means ED, Horrocks LA. Lipid hydrolysis and peroxidation in injured spinal cord: Partial protection with methylprednisolone or vitamin E and selenium. CNS Trauma 1985; 2: 257-267 8 Fujimoto S, Mizoi K, Yoshimoto T, Suzuki ]. The protective effect of vitamin E on cerebral ischemia. Surg Neural 1984; 22: 449-454 9 Hara H, Kato H, 1\ogure K. Protective effect of alpha-tocopherol on ischemic neuronal damage in the gerbil hippocampus. Brain Res 1990; 510: 335-338 10 Braughler JM, Hall ED. Current application of "high-dose" steroid therapy for CNS injury: A pharmacological perspective. J Neurosurg 1985; 62: 806-810 11 McCall JM, Hall ED, Braughler JM. A new class of 21aminosteroids which are useful for stroke and trauma. In: R. Capildeo, ed. Steroids in diseases of the central nervous system. Chichester: J Wiley, 1989; pp. 69-80 12 Kanamaru K, Weir BKA, Findlay ]M, Grace M, Macdonald RL. A dosage study of the effect of the 21-aminosteroid U74006F on chronic cerebral vasospasm in a primate model. Neurosurgery 1990; 27: 29-38 13 Dimlich RV, Tornheim PA, Kindel RM, Hall ED, Braughler JM, McCall ]M. Effects of a 21-aminosteroid (U74006F) on cerebral metabolites and edema after severe experimental head trauma. Adv Neurol1990; 52: 365-375 14 Silvia RC, Piercey MF, Hoffmann WE, Chase RL, Braughler ]M, Tang AH. U74006F, an inhibitor of lipid peroxidation protects against lesion development following experimental stroke in the cat: Histological and metabolic analysis. Neurosci Abst 1987; 13: 1495 15 Hall ED, Yonkers PA. Attenuation of post-ischemic cerebral hypoperfusion by the 21-aminosteroid U74006F. Stroke 1988; 19: 340-344 16 Hall ED, Pazara KE, Braughler ]M. 21-aminosteroid lipid peroxidation inhibitor U74006F protects against cerebral ischemia in gerbils. Stroke 1988; 19: 997-1002 17 Hall ED, Yonkers PA, McCall JM, Braughler ]M. Effect of the 21 -aminosteroid U74006F on experimental head injury in mice.
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Neurological Research, 1992, Volume 14, March
J Neurosurg 1988; 68 : 456-461 jacobsen E], McCall JM, Ayer DE, Van-Doornik Fj, Palmer ]R, Braughler ]M, Hall ED, Houser DJ, Krook MA. Novel 21aminosteroids that inhibit iron-dependent lipid peroxidation and protect against central nervous system trauma. j Med Chem 1990; 33: 1145-51 19 Anderson OK, Braughler ]M, Hall ED, Waters TR, McCall JM, Means ED. Effects of treatment with U47006F on neurological outcome following experimental spinal cord injury. J Neurosurg 1988; 69: 562-567 20 Fowl RJ, Patterson RB, Gewirtz Rj, Anderson OK. Protection against postischemic spinal cord injury using a new 21aminosteroid. J Surg Res 1990; 48: 597-600 21 Hall ED. Effects of the 21-aminosteroid U47006F on posttraumatic spinal cord ischemia in cats. J Neurosurg 1988; 68: 462-465 22 Hall ED, Yonkers PA, Horan KL, Braughler ]M. Correlation between attenuation of posttraumatic spinal cord ischemia and preservation of tissue vitamin E by the 21-aminosteroid U47006F: Evidence for an in vivo antioxidant mechanism. J Neurotrauma 1989; 6: 169-176 23 Holtz A, Gerdin B. Blocking weight-induced spinal cord injury in rats ; Therapeutic effect of the 21-aminosteroid U74006F. j Neurotrauma 1992, in press 24 Holtz A, Nystrom B, Gerdin B. Spinal cord blood flow measured by 14C-iodoantipyrine autoradiography during and after graded spinal cord compression in rats. Surg Neurol1989; 31 : 350-360 25 Rivlin AS, Tator CH. Objective clinical assessment of motor function after experimental spinal cord injury in the rat. j Neurosurg 1977; 47 : 577-581 26 Nystrom B, Berglund J-E, Bergqvist E. Methodological analysis of an experimental spinal cord compression model in the rat. Acta Neural Scand 1988; 78: 460-466 27 Aoki N, Lefer AM. Protective effects of a·novel nonglucocorticoid 21-aminosteroid (U47006F) during traumatic shock in rats. J Cardiovasc Pharmacol1990; 15: 205-210 28 Holtz A, Nystrom B, Gerdin B. Effect of methylprednisolone on motor function and spinal cord blood flow after spinal cord compression in rats. Acta Neurol Scand 1990; 82 : 68-73 18
ISSN: 0161-6412 (Print) 1743-1328 (Online) Journal homepage: http://www.tandfonline.com/loi/yner20
Efficacy of the 21-aminosteroid U74006F in improving neurological recovery after spinal cord injury in rats Anders Holtz & Bengt Gerdin To cite this article: Anders Holtz & Bengt Gerdin (1992) Efficacy of the 21-aminosteroid U74006F in improving neurological recovery after spinal cord injury in rats, Neurological Research, 14:1, 49-52, DOI: 10.1080/01616412.1992.11740010 To link to this article: https://doi.org/10.1080/01616412.1992.11740010
Published online: 20 Jul 2016.
Submit your article to this journal
View related articles
Citing articles: 13 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yner20
Efficacy of the 21-aminosteroid U74006F in improving neurological recovery after spinal cord injury in rats Anders Holtz1 and Bengt Gerdin 2 Departments of Neurosurgery1 and Surgeif, University Hospital, Uppsala, Sweden
The efficacy of three different regimens of the 21-aminosteroid U74006F in counteracting the neurological damage after spinal cord compression causing paraparesis in rats was investigated. Three groups of ten animals each were given totally 6 mg/ kg of U74006F in different regimens beginning one hour after injury (A: bolus doses of 1.5 mg/ kg at 1, 4, 7 and 10 hours; 8: bolus of 1.5 mg/kg at 1 hour and 4.5 mg / kg as an infusion over the next 9 hours; and C: infusion alone, 6 mg/ kg, given between 1 and 10 hours after trauma ). Two groups of ten animals each received vehicle alone in administration modes comparable to those of the U74006F treated animals. The motor function was assessed daily on the inclined plane. On day one, the capacity angle had decreased from about 62 o preoperatively to 28-30 o in the two vehicle-treated groups and in group C. In these groups there was a similar improvement in neurological function and on day 9 the capacity angles were 49-55 °. In groups A and B, both of which received a bolus dose of U74006F at 1 hour, the neurological outcome improved on day one with capacity angles of 38-40 °. The difference in neurological function between the animals given U74006F as bolus doses and those given vehicle alone persisted over the entire observation span until day 9. The data suggest that early treatment with a bolus dose seems to be required in order to obtain an effect of U74006F on neurological recovery. Keywords: Neurological function; spinal cord injury; U74006F; 21-aminosteroids; rats
INTRODUCTION In recent studies it has been found that the structural damage that follows trauma to the spinal cord is partly caused by secondary ischaemic and biochemical factors 1 . Among these secondary events, lipid peroxidation has been identified as a major culprit 2- 4 . Lipid peroxidation also acts as a potent autodestructive factor in various types of nontraumatic disorders of the central nervous system, such as ischaemic and haemorrhagic stroke5 . Compounds that interfere with lipid peroxidation, such as the iron chelating agent deferoxamine6 , and 9 , have thus antioxidative substances such as vitamin been effective in decreasing the extent of tissue damage in the central nervous system in experimental injury models. In 1985 Braughler and Hall proposed that the ability of high doses of steroids to protect against damage to the central nervous system is related to an antioxidant effect, independent of activation of glucocorticoid receptors 10. On the basis of their conclusion, several compounds of a new group of steroids, the 21-aminosteroids or lazaroids, were synthesized. The aim was to develop a steroid which would exert its action at the cell membrane and would reduce lipid
e-
Address correspondence to : Dr Anders Holtz, MD, PhD, Department of Neurosurgery, University Hospital, 5-75185 Uppsala, Sweden. Accepted for publication October 1991. The study was approved by the Committee for the Care of Laborat ory Animals, Uppsala University.
© 1992 Forefront Publishing Group 0161- 6412 / 92/ 010049-04
peroxidation by virtue of its antioxidant properties. The compounds were found to be more potent inhibitors of lipid peroxidation than methylprednisolone, but as they did not bind to glucocorticoid receptors, they had no gluco- and mineralocorticoid side effects11 . One of the 21-aminosteroids, U74006F, has been used extensively in animal experiments, and has been reported t o reduce vasospasm after experimental subarachnoid haemorrhage12 to diminish infarction ionic shifts and oedema after stroke13•14, and to decrease neuronal necrosis and reduce postischaemic changes in cortical blood flow after brain ischaemia15•16 . Also, it has been found to improve the neurological recovery after head injury17•18 and to reduce posttraumatic ischaemia and improve the neurological recovery after spinal cord injury 19- 22 . In a previous study we demonstrated that U74006F had a beneficial effect on the functional recovery after compression injury to the mid-thoracic level of the spinal cord in the rat when given as an intravenous bolus dose of 3 mg/ kg 60 min after injury23 . Although this finding, and the above-mentioned observations indicate that U74006F is a promising compound for use in patients with trauma to the spinal cord, they provide no information on how to tailor optimal treatment. In the present study we have attempted to gain information on the way in which a defined dose of U74006F should be administered for it to have an optimal effect on the neurological recovery after spinal cord compression in rats. We chose to administer a total dose of 6 mg/ kg and to divide the dose in various fashions.
Neurological Research, 1992, Volume 14, March
49
U74006F in spinal cord trauma: A. Holtz et al.
MATERIALS AND METHODS Animals and operative procedures Fifty male Sprague-Dawley (Aiab, Sollentuna, Sweden) rats weighing between 320-390 g were used. They were given food and water until experimentation. Each animal was anaesthetized with a solution of fluanisone 2.5 mg/ ml and midazolam 1.25 mg/ ml, total 1.52 ml / kg subcutaneously. A PE 50 catheter was inserted into an external jugular vein for infusion of vehicle or U74006F. A similar catheter was inserted into one carotid artery for monitoring the mean arterial blood pressure (MABP) and sampling for arterial blood gas analysis. The rats were placed prone on a heating pad and the body temperature was kept close to 37 with the aid of a rectal thermistor. The technique of exposing and injuring the spinal cord has been described previousll4 . Briefly, a dorsal laminectomy of Th7 and Th8 was performed under microscopic guidance, leaving the dura intact. The spinal cord was injured by a compression device whereby a slightly curved rectangular plate (2.2 x 5.5 mm) loaded with 35 g was applied on the exposed dura for 5 min. After removal of the plate the muscle and skin were closed. The animals were then placed under a heating lamp and allowed to wake up.
oc
Experimental groups The animals were randomly divided into five groups with ten in each and were given vehicle and / or U74006F (total 6 mg/ kg; Upjohn Co, Kalamazoo, USA) in different regimens starting one hour after injury.
Croups A and D thus received a total volume of 4 ml / kg, while groups B, C and E received 5 ml / kg. Neurological examination The motor function was assessed dai~ by the inclined plane technique as described previously 5 . The maximum inclination at which the rat could maintain itself transversely for 5 s was recorded as the 'capacity angle' 26 . Statistical methods A multifactorial analysis of variance with multiple range testing according to the least significant differences was used for comparison between groups. A difference at the 5% level was considered significant. Values are given as mean ± SEM. RESULTS Physiological variables Table 1 shows the data for physiological variables recorded after laminectomy but prior to injury, 60 min after injury and 5 ·min after the start of the infusion or after the bolus dose. There were no significant differences in any of the measured variables. Nor were there any differences in MABP immediately after infusion of vehicle or U74006F. The five groups did not differ regarding the duration of operation, body temperature or body weight (data not shown ). Neurological dysfunction Before the injury was produced, the motor performance was similar in all rats, with very little difference in the capacity angle (mean value 62 °; Figure 1). One day after injury similar, severe impairment of motorfunction was observed in the three groups that received vehicle as a bolus dose (groups C, D and E), with capacity angles of 28-31 °. There was thus no improvement in the group that received U74006F solely as an infusion (group C) compared with the vehicle-infused control (group E). During the following eight days these three groups displayed an equal and gradual recovery, with capacity angles of 35-41 o and 49-55 ° on days 4 and 9, respectively. The two groups treated with a bolus dose of 1.5 mg/ kg of U74006F subsequently followed either by
Croup A received four i.v. bolus doses of U74006F, 1.5 mg( = 1 ml) / kg, 1, 4, 7 and 10 hours after injury. Croup B received an i.v. bolus dose of U74006F, 1.5 mg( = 1 ml) / kg, 1 hour after injury and thereafter an infusion of a mixture of U74006F, 4.5 mg( = 3 mi) / kg, and vehicle, 1 ml / kg, during the next 9 hours. · Croup C received an i.v. bolus of vehicle, 1 ml / kg, 1 hour after injury followed by an infusion of U74006F, 6 mg( =4 mil/ kg, during the following nine hours. Croup 0 received four i.v. bolus doses of vehicle, 1 ml / kg, 1, 4, 7 and 10 hours after injury. Croup E received an i.v. bolus of vehicle, 1 ml / kg, 1 hour after injury followed by an infusion of vehicle, 4 ml / kg, during the next 9 hours.
Table 1: Physiological variables after laminectomy but before spinal cord injury (a) and at the start of infusion of vehicle and / or U74006F, i.e. 60 min after injury (b), and 5 min after the start of infusion (c)
PC0 2 (kPa)
pH Time period Croup A Croup B Croup C Croup D Croup E
50
P0 2 (kPa )
Hct (%)
Glucose (mM)
MABP (mmHg)
a
b
a
b
a
b
b
b
a
b
c
7.44 ±0.01 7.43 ±0.01 7.44 ±0.01 7.42 ±0.01. 7.43 ±0.02
7.46 ± 0.01 7.43 ±0.02 7.44 ±0.02 7.42 ±0.02 7.47 ±0.03
4.7 ± 0.1 5.0 ±0.2 4.9 ±0.3 4.9 ±0.2 4.6 ±0.1
4.7 ±0.1 4.8 ±0.2 4.8 ±0.2 4.9 ±0.2 4.5 ±0.3
12.1 ± 0.5 10.8 ±0.4 12.5 ±0.8 12.2 ±0.5 12.5 ±0.8
10.5 ±0.6 11.1 ±0.8 10.6 ±0.8 11.6 ±1 .0 12.0 ±0.9
43 ± 1 44 ±1 44 ±1 46 ±2 43 ±1
7.4 ±0.5 8.4 ± 0.7 7.9 ±0.9 7.9 ± 0.6 8.4 ±1.2
102 ±3 103 ±3 101 ±3 99 ±3 105 ±5
107 ±3 103 ±4 105 ±2 104 ±3 105 ±2
108 ±3 102 ±3 100 ±3 100 ±3 108 ±4
Neurological Research, 1992, Volume 14, March
U74006F in spinal cord trauma : A. Holtz et al. 70 oo•
60
00
~50 !!! Cl CD
"'-40 CD a,
c: ;30 '13 c. J 20
"'
10 0
2 Preinjury
3
5 4 6 Days after trauma
7
8
9
Figure 1: The motor function (mean ± SEM), expressed as the capacity angle, in the different experimental groups before and at various times after spinal cord injury. Group A: • - - - • ; group group C: 0---0 ; group D: D---0; group E: B:
e---e;
/::o.---/::o.
repeated bolus doses (group A ) or by infusion of the compound (group B) showed significantly better motor performance during the nine days of observation than the animals given vehicle alone (groups D and E). On day one the capacity angle was 40 ° ± 2 in group A and 38 o ± 2 in group B. There was a gradual recovery over time and on day 4 the capacity angle was 2 in these two groups 2 and 50 o improved to 53 o had increased to they nine day on while respectively, 59 o ± 2 and 60 o ± 2.
±
±
DISCUSSION The results of this study show that the neurological dysfunction that occurs after a standardized compressioninduced spinal cord injury in the rat is mitigated by administration of U74006F as a bolus one hour after the injury. There was no difference in efficacy between animals in which the initial bolus dose was followed by a continuous infusion and those in which it was followed by three repeated bolus doses every 3 hours. A 9-hour infusion alone was not sufficient however, to protect against the damage. These results indicate that it is essential to give a sufficient amount of U74006F early after the injury. They do not permit any conclusion, however, as to how to pursue the treatment further. The doses chosen in this study were the current maximal doses allowed in clinical studies in man. The aim was therefore not primarily to identify the best possible effect of the highest possible dose, but rather to determine how a dosing regimen should be tailored to provide an optimal effect at a submaximal dose. Previous studies in the rat have shown that in traumatic shock the efficacy is dosedependent in the dose range 7.5 to 22.5 mg/ kg27, indicating that higher doses than the total dose of 6 mg/ kg over 9 hours given in our study might result in better protective effects. In previous studies on spinal cord compression at the L2 level in cats, however, the beneficial effect did not show a clear dose dependency19 . In a previous investigation 23 with an identical experimental set-up, U74006F given as a single dose of 3 mg/ kg one hour after trauma had a beneficial effect similar t o that observed after the repeated lower doses
given in the present study. The fact that an infusion of U74006F alone, starting at one hour, was not sufficient to protect against damage suggests that a favourable effect is dependent both on the dose and on the time of administration. Regarding the time of administration, it seems that the earlier the dose is given, the better is the effect. In previous studies on an experimental head injuri 7 , an effect was observed when treatment was commenced 5 min after the injury. Administration of U74006F 30 min after spinal cord injury lead both to improvement of neurological recovery and to an increased spinal cord blood flow 19' 21 . In the present study a beneficial effect was only observed when treatment instituted after one hour was commenced with a bolus dose. Current pharmacokinetic studies in healty rats and monkeys (data from Up john ) suggest that only a minor fraction of U74006F crosses the blood-brain barrier. It is therefore reasonable to ask whether the mechanism of the beneficial effect observed in this and other studies on damage to the CNS actually requires that the drug has crossed the blood-brain barrier. Assuming that the beneficial effect is mediated by U74006F located in the CNS, two alternatives have to be considered. Either the minor fraction crossing the blood-brain barrier is sufficient, or the CNS damage involves an alteration in the blood-brain barrier function, including an increase in the permeability to compounds such as U74006F. Another conceivable alternative is that the beneficial effect is mediated on the endothelium itself, as has previously been discussed concerning the effect of methylprednisolone28 . In summary, U74006F given as a bolus dose 60 min after a spinal cord compression injury has a beneficial effect on the impaired neurological function. Additional studies to evaluate the mechanism(s) of action and further dose-response qualities of the drug are required before clinical trials are undertaken.
ACKNOWLEDGEMENTS The study was supported by Trygg-Hansa Insurance Company and the Swedish Medical Research Council. REFERENCES Young W. Blood flow, metabolic and neurophysiological mechanisms in spinal cord injury. In: Decker DP, Povlishock JT, eds. Central Nervous System Trauma Status Report. National Institute of Neurological and Communicative Disorders and Stroke, National Institute of Health : Bethesda, 1985; pp. 2
3
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463-473 Hall ED, Braughler JM. Central nervous system t rauma and stroke. 2. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. j Free Radic Bioi Med 1989; 6 : 303- 313 Hall ED, Braughler JM. Role of lipid peroxidation in posttraumatic spinal cord degeneration: A review. CNS Trauma 1986; 3: 281-294 Kinuta Y, Kikuchi H, Ishikawa M, Kimura M, ltokawa Y. Lipid peroxidation in focal cerebral ischemia. J Neurosurg 1989 ; 71 : 421-429 Braughler JM, Hall ED. Central nervous system trauma and stroke. Biochemical considerations for oxygen free radical formation and lipid peroxidation. J Free Radic Bioi Med 1989; 6: 289- 301 Ikeda Y, Ikeda K, Long DM. Protective effect of the iron chelator deferoxamine on cold-induced brain edema. J Neurosurg 1989 ; 71 : 233- 238
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Anderson OK, Saunders RD, Demediuk P, Dugan LL, Braughler JM, Hall ED, Means ED, Horrocks LA. Lipid hydrolysis and peroxidation in injured spinal cord: Partial protection with methylprednisolone or vitamin E and selenium. CNS Trauma 1985; 2: 257-267 8 Fujimoto S, Mizoi K, Yoshimoto T, Suzuki ]. The protective effect of vitamin E on cerebral ischemia. Surg Neural 1984; 22: 449-454 9 Hara H, Kato H, 1\ogure K. Protective effect of alpha-tocopherol on ischemic neuronal damage in the gerbil hippocampus. Brain Res 1990; 510: 335-338 10 Braughler JM, Hall ED. Current application of "high-dose" steroid therapy for CNS injury: A pharmacological perspective. J Neurosurg 1985; 62: 806-810 11 McCall JM, Hall ED, Braughler JM. A new class of 21aminosteroids which are useful for stroke and trauma. In: R. Capildeo, ed. Steroids in diseases of the central nervous system. Chichester: J Wiley, 1989; pp. 69-80 12 Kanamaru K, Weir BKA, Findlay ]M, Grace M, Macdonald RL. A dosage study of the effect of the 21-aminosteroid U74006F on chronic cerebral vasospasm in a primate model. Neurosurgery 1990; 27: 29-38 13 Dimlich RV, Tornheim PA, Kindel RM, Hall ED, Braughler JM, McCall ]M. Effects of a 21-aminosteroid (U74006F) on cerebral metabolites and edema after severe experimental head trauma. Adv Neurol1990; 52: 365-375 14 Silvia RC, Piercey MF, Hoffmann WE, Chase RL, Braughler ]M, Tang AH. U74006F, an inhibitor of lipid peroxidation protects against lesion development following experimental stroke in the cat: Histological and metabolic analysis. Neurosci Abst 1987; 13: 1495 15 Hall ED, Yonkers PA. Attenuation of post-ischemic cerebral hypoperfusion by the 21-aminosteroid U74006F. Stroke 1988; 19: 340-344 16 Hall ED, Pazara KE, Braughler ]M. 21-aminosteroid lipid peroxidation inhibitor U74006F protects against cerebral ischemia in gerbils. Stroke 1988; 19: 997-1002 17 Hall ED, Yonkers PA, McCall JM, Braughler ]M. Effect of the 21 -aminosteroid U74006F on experimental head injury in mice.
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J Neurosurg 1988; 68 : 456-461 jacobsen E], McCall JM, Ayer DE, Van-Doornik Fj, Palmer ]R, Braughler ]M, Hall ED, Houser DJ, Krook MA. Novel 21aminosteroids that inhibit iron-dependent lipid peroxidation and protect against central nervous system trauma. j Med Chem 1990; 33: 1145-51 19 Anderson OK, Braughler ]M, Hall ED, Waters TR, McCall JM, Means ED. Effects of treatment with U47006F on neurological outcome following experimental spinal cord injury. J Neurosurg 1988; 69: 562-567 20 Fowl RJ, Patterson RB, Gewirtz Rj, Anderson OK. Protection against postischemic spinal cord injury using a new 21aminosteroid. J Surg Res 1990; 48: 597-600 21 Hall ED. Effects of the 21-aminosteroid U47006F on posttraumatic spinal cord ischemia in cats. J Neurosurg 1988; 68: 462-465 22 Hall ED, Yonkers PA, Horan KL, Braughler ]M. Correlation between attenuation of posttraumatic spinal cord ischemia and preservation of tissue vitamin E by the 21-aminosteroid U47006F: Evidence for an in vivo antioxidant mechanism. J Neurotrauma 1989; 6: 169-176 23 Holtz A, Gerdin B. Blocking weight-induced spinal cord injury in rats ; Therapeutic effect of the 21-aminosteroid U74006F. j Neurotrauma 1992, in press 24 Holtz A, Nystrom B, Gerdin B. Spinal cord blood flow measured by 14C-iodoantipyrine autoradiography during and after graded spinal cord compression in rats. Surg Neurol1989; 31 : 350-360 25 Rivlin AS, Tator CH. Objective clinical assessment of motor function after experimental spinal cord injury in the rat. j Neurosurg 1977; 47 : 577-581 26 Nystrom B, Berglund J-E, Bergqvist E. Methodological analysis of an experimental spinal cord compression model in the rat. Acta Neural Scand 1988; 78: 460-466 27 Aoki N, Lefer AM. Protective effects of a·novel nonglucocorticoid 21-aminosteroid (U47006F) during traumatic shock in rats. J Cardiovasc Pharmacol1990; 15: 205-210 28 Holtz A, Nystrom B, Gerdin B. Effect of methylprednisolone on motor function and spinal cord blood flow after spinal cord compression in rats. Acta Neurol Scand 1990; 82 : 68-73 18
