- Email: [email protected]
Increased thromboxane level in experimental spinal cord injury
Journal of the Neurological Sciences, 1986, 74:289-296 Elsevier
289
JNS 2678
Increased Thromboxane Level in Experimental Spinal Cord Injury C.Y. Hsu, P.V. Halushka, E.L. Hogan and R.D. Cox Departments of Neurology and Pharmacology, Medical University of South Carolina, Charleston, SC (U.S.A.) (Received 4 December, 1985) (Revised, received 17 February, 1986) (Accepted 17 February, 1986)
SUMMARY
An increased accumulation of tissue thromboxane A 2 occurred shortly after spinal cord injury. Prostacyclin formation was not affected. The magnitude of the increase in thromboxane and the extent of post-traumatic vascular damage as determined by extravasation of 125I-labeled human serum albumin were both dependent on the degree of injury. These f'mdings raise the possibility that activation of arachidonic acid metabolism with a preponderance in thromboxane formation may contribute to microvascular injury in experimental spinal cord contusion.
K e y w o r d s : Arachidonic a c i d - E d e m a - Prostacyclin - Spinal cord injury - Thromboxane
- Vascular injury
INTRODUCTION
Post-traumatic necrosis in spinal cord injury is a consequence of the initial physical insult and a series of molecular events fostering progressive damage (Ducker 1975; Balentine 1985; Hogan and Banik 1985; Young 1985). The secondary injury hypothesis has stimulated extensive investigation of potential therapeutic interventions
This work was supported by grants from the National Institutes of Health NS-11066, GM-27673 and HL-29506. Dr. Hsu is a recipient of a Teacher-Investigator Development Award from NINCDS (NS-00792). Dr. Halushka is a Burroughs Welcome Scholar in Clinical Pharmacology. Address correspondence to: C.Y. Hsu, M. D., Ph. D., Department of Neurology, Medical University of South Carolina, Charleston, SC 29425, U.S.A. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
290 seeking to reduce the permanent damage following insult to the spinal cord (Balentine et al. 1985; Faden 1985). Although the detailed molecular pathophysiology of the progressive necrosis after impact injury is not known, several lines suggest that vascular damage plays a major role. After experimental injury using a standard technique, the progressive lesion is preceded by endothelial damage with platelet adherence, intravascular platelet aggregation, microvascular occlusion, dissemination of emboli and vasogenic edema (Dohrman et al. 1971; Griffiths and Miller 1974; Balentine 1978; Demopoulos et al. 1978; Hsu et al. 1985b). Progressive vascular stasis has been observed with an operating microscope (Assenmacher and Ducker 1971). Studies employing fluorescent tracers (Wagner et al. 1969; Dohrman and Wick 1975), microangiography (Fried and Goodkin 1971) and blood flow measurements (Ducker et al. 1978) all implicate evolution of ischemia after impact injury. The prominent vascular changes have prompted proposals of therapeutic trials of antiplatelet or spasmolytic agents (Nelson et al. 1977; Don-Edwards et al. 1980; HaUenbeck et al. 1983). Liberation of arachidonic acid from membrane phospholipids has been noted in the CNS following a variety of perturbations (Bazan 1970; Yoshida et al. 1980). The free arachidonic acid is rapidly metabolized by cyclooxygenase to fatty acid peroxides and prostanoids or by lipoxygenase to hydroxy fatty acids and leukotrienes (Wolfe 1982). An increased production of prostanoids has been reported in both physical and ischemic injury ofCNS (Jonsson and Daniel 1976; Gandet et al. 1980; Ellis et al. 1981 ; Chen et al. 1986). Among the vasoactive prostanoids, thromboxane (TxA2) and prostacyclin (PGI2) are two of the most potent and appear to affect hemostasis and vascular integrity in different ways (Moncada and Vane 1979). TxA 2 stimulates platelet aggregation and vasoconstriction while PGI 2 inhibits platelet aggregation and causes vasodilation (Monacada and Vane 1979; Chen et al. 1986). The prominent vascular changes observed in spinal cord injury and the potent vascular actions of TxA 2 and PGI 2 prompted us to study the formation of these arachidonate metabolites in two experimental spinal cord injury models. We found activation of arachidonic acid metabolism with prepondorence in thromboxane production (Hsu et al. 1985a). How a TxA2-PGI 2 imbalance is related to the pathophysiological consequences of spinal cord injury remains to be determined. The present study was undertaken to investigate the magnitude of the TxA2-PGI 2 imbalance in relation to the extent of the vascular injury in two degrees of rat spinal cord injury. MATERIALS AND METHODS
Spinal cord injury The method for producing spinal cord injury in rats (Sprague-Dawley, 200-300 g) has been previously described (Daniel et al. 1975; Balentine 1978; Hsu et al. 1985b). Briefly, rats were anesthetized with Ketamine (100 mg/kg) and a one-segment laminectomy was made at T12. Spinal cord injury was inflicted by dropping a 5-g cylindrical brass weight from a height of 5 cm (moderate injury) or 20 cm (severe injury) onto a cast plastic impounder with a 0.3 cm diameter which had been carefully placed
291 on the dura overlying the dorsal surface of the spinal cord. In preliminary experiments, laminectomy alone caused an immediate but transient elevation of tissue thromboxane B2 (TxB2) levels, which returned to baseline in 1 h. Impact injury was therefore inflicted in all animals 1 h after laminectomy in order to eliminate the potential artifact attributed to laminectomy. Determination of vascular injury and tissue edema The extent of vascular injury was measured by the degree of extravasation of 125I-labelled human serum albumin ([ 125I]HSA) (Mallinckrodt, St. Louis, MO.) (Hsu etal. 1985b). Rats were labelled with 5 #Ci [t25I]HSA by i.v. infusion 1 h after laminectomy and then assigned to control, moderate and severe injury groups as described earlier. Twenty-four hours after injury, or in controls 25 h after laminectomy, the neurological deficit was scored (see below). The animals were killed and blood samples collected during exsanguination for determination of plasma [125I]HSA content. The T12 cord segment was then removed in a humidity chamber and wet weight determined immediately. The cord segment was dried at 100 °C to constant weight and the dry weight determined. [ 125I]HSA in the cord segment was determined by counting radioactivity simultaneously with the plasma sample from the same animal. The extent of vascular injury was estimated by a vascular injury index (VII) derived from the following formula: VII =
Radioactivity (cpm)/dry weight (g) Radioactivity (cpm) in 200/~1 plasma from the same animal
Tissue edema was determined by calculating tissue water content where ~o tissue water content = (1-dry weight/wet weight) x 100%. Neurological deficit score The neurological deficit scores in rat 25 h after laminectomy or 24 h after spinal cord injury were determined using a modified Tarlov Scale in which 0 = no movement, 1 = barely perceptible movement, 2 = good movement, 3 = support body weight, 4 = walk with mild deficit, and 5 = normal walking/running. An average score lbr the hindlimbs was obtained for each animal. Determination of tissue thromboxane Be and 6-Keto-PGFI~ (6KF) A separate group of animals was used for determination of tissue prostanoid levels (Hsu et al. 1985a). Preliminary studies established that the post-traumatic elevation of TxB 2 peaked between 15 and 30 min. Sampling of spinal cord for determination ofTxB 2 and 6KF levels were therefore conducted 30 min after injury. The T12 cord segment was rapidly removed and homogenized in Tris-HC1 buffer (50 mM, pH 7.8) containing 155 mM NaC1 and 0.3 mM indomethacin. The time interval from dissecting the cord to homogenization was less than 15 s. Control cord segments were removed 90 min after laminectomy. Tissue preparation and measurement of immunoreactive TxB 2 (iTxB2) and 6KF (i6KF) by radioimmunoassay have been described previously (Burch et al. 1979; Wise et al. 1980; Hsu et al. 1985a). All samples were
292 assayed in triplicate with 3 different aliquot sizes. The interassay variability was 8~o (n - 5) for iTxB 2 a n d 10~o (n = 5) for i 6 K F .
Determination of tissue protein content Protein content in tissue h o m o g e n a t e was determined by the m e t h o d o f Lowry et al. (1951) after dissolving the h o m o g e n a t e in 0.5 N N a O H containing 5 ~ sodium lauryl sulfate.
Statistical analysis D a t a is expressed as mean + S E M . T h e difference between control and injured or between two different degrees o f injury was determined for statistical significance by Student's t-test. RESULTS The effects o f m o d e r a t e and severe injury on the extent o f vascular injury reflected by extravasation o f [~25I]HSA, c o r d e d e m a reflected by tissue water content and the neurological deficit scores are in Table 1. A significant difference in all three p a r a m e t e r s was noted between control a n d m o d e r a t e injury groups as well as between m o d e r a t e and severe injury groups. The post-traumatic increase in tissue iTxB 2 levels was d e p e n d e n t on the magnitude o f injury with greatest increase n o t e d in animals with severe injury (Fig. 1). The c o r d i 6 K F levels r e m a i n e d u n c h a n g e d irrespective o f the magnitude o f injury (Fig. 1). Thus a m a r k e d shift o f a T x B 2 - 6 K F ratio which reflects the i m b a l a n c e d p r o d u c t i o n o f T x A 2 and P G I 2 in favor o f platelet aggregation and vasoconstriction was n o t e d with severe injury (5.70 + 0.98; n = 7 in the severe injury vs 1.17 + 0.05; n = 7 in the control, P < 0.001). The T x B 2 - 6 K F ratio is also significant-
TABLE 1 NEUROLOGICAL DEFICIT SCORE, EDEMA, AND VASCULAR INJURY IN TWO DEGREES OF IMPACT INJURY Neurological deficit score was determined by a modified Tarlov scale; cord edema was calculated by % water content; and vascular injury index was estimated by extravasation of 125I-labelledserum albumin (see MATERIALSAND METHODS). Each group consisted of 7 rats.
Control Moderate injury Severe injury
Neurological deficit score
Cord edema (%H20)
Vascular injury index
5.0 + 0 2.83 + 0.31" 0.25 + 0.25*'÷
69.65 + 0.26 71.88 + 0.77* 73.98 _+0.27*'÷
1.17 + 0.05 3.20 + 0.56* 5.70 + 0.98*-÷
* Denotes difference between control and moderate injury or between control and severe injury is significant; ÷ denotes difference between moderate and severe injury is significant by Student's t-test (P < 0.05).
293 t l
¢,
600
-600
500
-500
400
-400
~-5
aoo
"300
O_
200
200
¢.D
100-
-100
0 Q"
0
E Q.
"~
E
cff X b-
C'i'L
MOD
SEV
INJURY Fig. 1. Effects of two degrees of trauma on cord content of thromboxane B 2 (TxB2) and 6-keto-PGF~= (6KF). Control (CTL) group received laminectomy. Moderate (MOD) and severe (SEV) injury groups received impact injury of 5 g from a height of 5 cm and 20 cm, respectively. Mean + SEM from 7 rats in each group is shown. * Denotes difference from CTL is significant; + denotes difference from MOD is significant.
ly elevated with moderate injury (3.20 + 0.56; n = 7, P < 0.05 compared to the control) but less than that with severe injury (P < 0.05 compared to the severe injury). A correlation of TxB2-6KF ratio and extent of vascular injury is shown in Fig. 2. DISCUSSION
The contribution of increased prostanoid formation to the vascular injury in experimental spinal cord contusion remains to be determined. In the present study we found that increased accumulation of TxB2, a stable metabolite of TxA2, depended on the degree of injury, whereas the formation of 6KF, a stable metabolite of PGI2, was not altered irrespective of the magnitude of injury. The selective increase in TxB2, but not 6KF, can be attributed to post-traumatic lipid peroxidation (Demopoulos et al. 1980) for lipid peroxides selectively inhibit PGI 2 production (Warso and Lando 1983). The TxA2-PGI 2 imbalance observed favors platelet aggregation and vasoconstriction, The extent to which this contributes to the vascular damage of experimental spinal cord injury is not clear. Therapy with indomethacin, PGI 2 and heparin, which should affect the TxA2-PGI 2 ratio to favor reduced mierovascular thromboembolism, has been reported to improve the post-traumatic neurologic deficit in cat spinal cord injury (Hallenbeck et al. 1983). This is consistent with the potential contribution of an altered TxA2-PGI 2 balance in the posttraumatic vascular damage in experimental spinal cord
294
~
4-
I,J_ ,¢. 37 X
I-
EV
MOD
2'
CTL o
o
~
'
~
VASCULAR INJURY INDEX
Fig. 2. Correlation of thromboxane B2-6-keto-PGIl~ratio (TxB2/6KF) and vascular injury index among control (CTL), moderate (MOD) and severe (SEV) injury groups: TxB2/6KF was derived from the ratio of the tissue content of TxB2 (in pg/mg protein) and 6KF (in pg/mg protein). Vascular injury index was estimated by extravasation of ~25I-humanserum albumin as described in MATERIALSAND METHODS. Mean + SEM from 7 rats in each group is shown.
injury. We found the extent of vascular injury, reflected by extravasation of [ 125I]HSA, to be closely related to the magnitude of the T x A z - P G I z imbalance as estimated by the T x B 2 - 6 K F ratio (Fig. 2). This is consistent with the contention that T x A z - P G I 2 imbalance may contribute to vascular damage in experimental spinal cord injury. Admittedly our observation o f T x A 2 - P G I 2 imbalance and vascular injury is correlative only and more studies are needed to explore the causal relationship between r x A z - P G I 2 imbalance and vascular injury. The degree of post-traumatic edema also depended on the magnitude of impact injury. Post-traumatic edema is vasogenic in nature (Griffiths and Miller 1974; Hsu ~t al. 1985b) and may develop as a function of vascular damage (Hsu et al. 1985b). Arachidonic acid induces cerebral edema (Chan and Fishman 1978) probably as a consequence of vascular injury (Chan et al. 1983). Therefore release of arachidonic acid and subsequent formation of its metabolites including leukotrienes (Chen et al. 1986) may contribute to the development of posttraumatic edema though the precise role of arachidonic acid and its metabolite in this regard remains to be determined. In conclusion, our results confirm the earlier observation in two different animal models of the existence of an imbalance of TxA 2 and P G I 2 formation with bias to an increased thrombotic activity (Hsu et al. 1985a). The T x A 2 - P G I 2 imbalance also zorrelates with the extent of vascular damage determined by extravasation of[ I25I]HSA and suggests that a selective increase in TxA 2 may contribute to the microvascular damage in experimental spinal cord injury.
295 REFERENCES Assenmacher, D.R. and T. B. Ducker (1971) The vascular and pathological changes seen in reversible spinal cord lesions, J. Bone Joint Surg. [Am.], 53A: 671-680. Balentine, J.D. (1978) Pathology of experimental spinal cord trauma, Part 1 (The necrotic lesions as a function of vascular injury), Lab. Invest., 39: 236-253. Balentine, J.D. (1985) Hypotheses in spinal cord trauma research. In: D.P. Becker and J.T. Povlishock (Eds.), Central Nervous System Trauma Status Report, NINCDS, Bethesda, pp. 455-461. Balentine, J. D., E. L. Hogan, N. L. Banik and P. L. Perot, Jr. (1985) Calcium and the pathogenesis of spinal cord injury. In: H.R. Winn, R. Rimel and J. A. Jane (Eds.), Recent Advances in Neural Trauma, Raven Press, New York, pp. 285-295. Bazan, N.G. (1970) Effects of ischemia and electroconvulsive Shock on free fatty acid pool in the brain, Biochim. Biophys. Acta, 218: 1-10. Burch, R. M., D. P. Knapp, P.V. Halushka (1979) Vasopressin stimulates thromboxane synthesis in the toad urinary bladder - - Effects of imidazole, J. Pharmacol. Exp. Ther., 210: 344-348. Chan, P.H. and R.A. Fishman (1978) Brain edema - - Induction in cortical slices by polyunsaturated fatty acid, Science, 201: 358-360. Chan, P.H., R.A. Fishman, J. Caronna, J.W. Schmidley, G. Prioleau and J. Lee (1983) Induction of brain edema following intracerebral injection of arachidonic acid, Ann. Neurol., 13: 625-632. Chen, S.T., C.Y. Hsu, E.L. Hogan, P.V. Halushka, O.I. Linet, F.M. Yatsu (1986) Thromboxane, prostacyclin and leukotrienes in cerebral ischemia, Neurology, 36: 466-470. Daniel, H.B., W. Francis, W.A. Lee and T.B. Ducker (1975) A method of quantitating injury inflicted in acute spinal cord studies, Paraplegia, 13: 137-142. Demopoulos, H. B., M. Yoder, E. G. Gutman, M. L. Seligman, E. S. Flamm and J. Ransohoff (1978) The fine structure of endothelial surfaces in the microcirculation of experimentally injured feline spinal cords, Scan. Electron Microsc., 2: 677. Demopoulos, H.B., E. S. Flamm, D.D. Pietronigro and M.L. Seligman (1980) The free radical pathology and the microcirculation in the major central nervous system disorders, Acta Physiol. Scand., Suppl. 492:91-119. Dohrman, G.J. and K.M. Wick (1975) Intramedullary blood flow patterns in the transitory traumatic paraplegia, Surg. Neurol., 1: 209-215. Dohrman, G. J., F. C. Wagner and P. C. Bucy (197 I) The microvasculature in transitory traumatic paraplegia - - An electron microscopic study in the monkey, J. Neurosurg., 35: 263-271. Don-Edwards, D., V. Decrescito, J. Tomasula and E.S. Flamm (1980) Effect of aminophylline and isoproterenol on spinal cord blood flow after impact injury, J. Neurosurg., 53: 385-390. Ducker, T.B. (1975) Experimental injury of the spinal cord. In: P.J. Vinken and G.W. Bruyn (Eds.), Handbook of Clinical Neurology, Vol. 25 (Injuries of the Spine and Spinal Cord), North-Holland, Amsterdam, pp. 9-26. Ducker, T.B., M. Salcman, P.L. Perot, Jr. and J.D. Balentine (1978) Experimental spinal cord trauma, Part 1 (Correlation of blood flow, tissue oxygen and neurological status in the dog), Surg. Neurol., 10" 60-70. Ellis, E.F., K.F., Wright, E.P. Wei, H.A. Kontos (1981) Cyclooxygenase products of arachidonic aid metabolism in cerebral cortex after experimental concussive brain injury, J. Neurochem., 37: 892-896. Faden, A. I. (1985) Pharmacologic therapy in acute spinal cord injury - - Experimental strategies and future decisions. In: D.P. Becker and J.T. Povlishock (Eds.), Central Nervous System Trauma Status Report, NINCDS, Bethesda, pp. 481-485. Fried, L. C. and R. Goodkin (1971) Microangiographic observations of the experimental traumatized spinal cord, J. Neurosurg., 35: 709-714. Gaudet, F.J., I. Alam and L. Levine (1980) Accumulation of cyclooxygenase products of arachidonic acid metabolism in gerbil brain during reperfusion after bilateral common carotid occlusion, J. Neurochem., 35: 653-658. Goodman, J.G., W. G. Bingham, W. E. Hunt (1979) Platelet aggregation in experimental spinal cord injury, Arch. Neurol. (Chic.), 30: 197-201. Griffiths, I.R. and R. Miller (1974) Vascular permeability to protein and vasogenic edema in experimental concussive injuries to the canine spinal cord, J. Neurol. Sci., 22: 291-304. Hallenbeck, J.M., T.P. Jacobs, A.I. Faden (1983) Combined PGI2, indomethacin and heparin improves neurological recovery after spinal trauma in cats, J. Neurosurg., 58: 749-754. Hogan, E . L and N.L. Banik (1985) Biochemistry of the spinal cord. In: A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. I0, Plenum, New York, pp. 285-377.
296 Hsu, C.Y., P.V. Halushka, E.L. Hogan, N.L. Banik, W.A. Lee and P.L. Perot, Jr. (1985a) Altered thromboxane and prostacyclin levels in experimental spinal cord injury, Neurology, 35: 1003-1009. Hsu, C.Y., E.L. Hogan, R.H. Gasden, Sr., K.M. Spicer, M.P. Shi and R.D. Cox (1985b) Vascular permeability in experimental spinal cord injury, J. Neurol. Sci., 70: 275-282. Jonsson, H.T. and H.B. Daniel (1976) Altered levels of PGF in cat spinal cord tissue following traumatic injury, Prostaglandins, 11: 51-61. Lowry O.H., N.J. Rosenbrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin reagent. J. Biol. Chem., 193: 265-275. Moncada, S. and J.R. Vane (1979) Arachidonic acid metabolites and their interactions between platelets and blood vessel walls, N. Engl. J. Med., 300:1142-1147. Nelson, E., S.C. Gertz, M.L. Rennels, T.B. Ducker and O.R. Blaumanis (1977) Spinal cord injury - - The role of vascular damage in the pathogenesis of central hemorrhagic necrosis, Arch. Neurol. (Chic.), 34: 332-333. Wagner, R., N. Taslitz, R. F. White, et al, (1969) Vascular phenomena in the normal and traumatized spinal cord, Anat. Rec., 163: 281. Warso, M.A. and W. E. M. Lando (1983) Lipid peroxidation in relation to prostacyclin and thromboxane physiology and pathophysiology, Brit. Med. Bull., 39: 277-280. Wise, W.C., J.A. Cook, T. Eller and P.V. Halushka (1980) Ibuprofen improves survival from endotoxic shock in the rat,,/. PharmacoL Exp. Ther., 215: 160-164. Wolfe, L.S. (1982) Eicosanoids - - Prostaglandins, thromboxanes, leukotrienes and other derivatives of carbon-20 unsaturated fatty acids, J. Neurochem., 38: 1-14. Wolfe, L.S. and H.M. Pappius (1983) Involvement of arachidonic acid metabolites in functional disturbances following brain injury, Adv. Prostaglandins Thromboxane Leukotriene Res., 12: 345-349. Yoshida, S., S. Inoh, T. Asano, et al. (1980) Effect of transient ischemia on free fatty acids and phospholipids in the gerbil brain, J. Neurosurg., 53: 323-331. Young, W. (1985) Blood flow, metabolic and neurophysiological mechanism in spinal cord injury. In: D.P. Beeker and J.T. Povlishock (Eds.), Central Nervous System Trauma Status Report, NINCDS, Bethesda, pp. 463-473.
289
JNS 2678
Increased Thromboxane Level in Experimental Spinal Cord Injury C.Y. Hsu, P.V. Halushka, E.L. Hogan and R.D. Cox Departments of Neurology and Pharmacology, Medical University of South Carolina, Charleston, SC (U.S.A.) (Received 4 December, 1985) (Revised, received 17 February, 1986) (Accepted 17 February, 1986)
SUMMARY
An increased accumulation of tissue thromboxane A 2 occurred shortly after spinal cord injury. Prostacyclin formation was not affected. The magnitude of the increase in thromboxane and the extent of post-traumatic vascular damage as determined by extravasation of 125I-labeled human serum albumin were both dependent on the degree of injury. These f'mdings raise the possibility that activation of arachidonic acid metabolism with a preponderance in thromboxane formation may contribute to microvascular injury in experimental spinal cord contusion.
K e y w o r d s : Arachidonic a c i d - E d e m a - Prostacyclin - Spinal cord injury - Thromboxane
- Vascular injury
INTRODUCTION
Post-traumatic necrosis in spinal cord injury is a consequence of the initial physical insult and a series of molecular events fostering progressive damage (Ducker 1975; Balentine 1985; Hogan and Banik 1985; Young 1985). The secondary injury hypothesis has stimulated extensive investigation of potential therapeutic interventions
This work was supported by grants from the National Institutes of Health NS-11066, GM-27673 and HL-29506. Dr. Hsu is a recipient of a Teacher-Investigator Development Award from NINCDS (NS-00792). Dr. Halushka is a Burroughs Welcome Scholar in Clinical Pharmacology. Address correspondence to: C.Y. Hsu, M. D., Ph. D., Department of Neurology, Medical University of South Carolina, Charleston, SC 29425, U.S.A. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
290 seeking to reduce the permanent damage following insult to the spinal cord (Balentine et al. 1985; Faden 1985). Although the detailed molecular pathophysiology of the progressive necrosis after impact injury is not known, several lines suggest that vascular damage plays a major role. After experimental injury using a standard technique, the progressive lesion is preceded by endothelial damage with platelet adherence, intravascular platelet aggregation, microvascular occlusion, dissemination of emboli and vasogenic edema (Dohrman et al. 1971; Griffiths and Miller 1974; Balentine 1978; Demopoulos et al. 1978; Hsu et al. 1985b). Progressive vascular stasis has been observed with an operating microscope (Assenmacher and Ducker 1971). Studies employing fluorescent tracers (Wagner et al. 1969; Dohrman and Wick 1975), microangiography (Fried and Goodkin 1971) and blood flow measurements (Ducker et al. 1978) all implicate evolution of ischemia after impact injury. The prominent vascular changes have prompted proposals of therapeutic trials of antiplatelet or spasmolytic agents (Nelson et al. 1977; Don-Edwards et al. 1980; HaUenbeck et al. 1983). Liberation of arachidonic acid from membrane phospholipids has been noted in the CNS following a variety of perturbations (Bazan 1970; Yoshida et al. 1980). The free arachidonic acid is rapidly metabolized by cyclooxygenase to fatty acid peroxides and prostanoids or by lipoxygenase to hydroxy fatty acids and leukotrienes (Wolfe 1982). An increased production of prostanoids has been reported in both physical and ischemic injury ofCNS (Jonsson and Daniel 1976; Gandet et al. 1980; Ellis et al. 1981 ; Chen et al. 1986). Among the vasoactive prostanoids, thromboxane (TxA2) and prostacyclin (PGI2) are two of the most potent and appear to affect hemostasis and vascular integrity in different ways (Moncada and Vane 1979). TxA 2 stimulates platelet aggregation and vasoconstriction while PGI 2 inhibits platelet aggregation and causes vasodilation (Monacada and Vane 1979; Chen et al. 1986). The prominent vascular changes observed in spinal cord injury and the potent vascular actions of TxA 2 and PGI 2 prompted us to study the formation of these arachidonate metabolites in two experimental spinal cord injury models. We found activation of arachidonic acid metabolism with prepondorence in thromboxane production (Hsu et al. 1985a). How a TxA2-PGI 2 imbalance is related to the pathophysiological consequences of spinal cord injury remains to be determined. The present study was undertaken to investigate the magnitude of the TxA2-PGI 2 imbalance in relation to the extent of the vascular injury in two degrees of rat spinal cord injury. MATERIALS AND METHODS
Spinal cord injury The method for producing spinal cord injury in rats (Sprague-Dawley, 200-300 g) has been previously described (Daniel et al. 1975; Balentine 1978; Hsu et al. 1985b). Briefly, rats were anesthetized with Ketamine (100 mg/kg) and a one-segment laminectomy was made at T12. Spinal cord injury was inflicted by dropping a 5-g cylindrical brass weight from a height of 5 cm (moderate injury) or 20 cm (severe injury) onto a cast plastic impounder with a 0.3 cm diameter which had been carefully placed
291 on the dura overlying the dorsal surface of the spinal cord. In preliminary experiments, laminectomy alone caused an immediate but transient elevation of tissue thromboxane B2 (TxB2) levels, which returned to baseline in 1 h. Impact injury was therefore inflicted in all animals 1 h after laminectomy in order to eliminate the potential artifact attributed to laminectomy. Determination of vascular injury and tissue edema The extent of vascular injury was measured by the degree of extravasation of 125I-labelled human serum albumin ([ 125I]HSA) (Mallinckrodt, St. Louis, MO.) (Hsu etal. 1985b). Rats were labelled with 5 #Ci [t25I]HSA by i.v. infusion 1 h after laminectomy and then assigned to control, moderate and severe injury groups as described earlier. Twenty-four hours after injury, or in controls 25 h after laminectomy, the neurological deficit was scored (see below). The animals were killed and blood samples collected during exsanguination for determination of plasma [125I]HSA content. The T12 cord segment was then removed in a humidity chamber and wet weight determined immediately. The cord segment was dried at 100 °C to constant weight and the dry weight determined. [ 125I]HSA in the cord segment was determined by counting radioactivity simultaneously with the plasma sample from the same animal. The extent of vascular injury was estimated by a vascular injury index (VII) derived from the following formula: VII =
Radioactivity (cpm)/dry weight (g) Radioactivity (cpm) in 200/~1 plasma from the same animal
Tissue edema was determined by calculating tissue water content where ~o tissue water content = (1-dry weight/wet weight) x 100%. Neurological deficit score The neurological deficit scores in rat 25 h after laminectomy or 24 h after spinal cord injury were determined using a modified Tarlov Scale in which 0 = no movement, 1 = barely perceptible movement, 2 = good movement, 3 = support body weight, 4 = walk with mild deficit, and 5 = normal walking/running. An average score lbr the hindlimbs was obtained for each animal. Determination of tissue thromboxane Be and 6-Keto-PGFI~ (6KF) A separate group of animals was used for determination of tissue prostanoid levels (Hsu et al. 1985a). Preliminary studies established that the post-traumatic elevation of TxB 2 peaked between 15 and 30 min. Sampling of spinal cord for determination ofTxB 2 and 6KF levels were therefore conducted 30 min after injury. The T12 cord segment was rapidly removed and homogenized in Tris-HC1 buffer (50 mM, pH 7.8) containing 155 mM NaC1 and 0.3 mM indomethacin. The time interval from dissecting the cord to homogenization was less than 15 s. Control cord segments were removed 90 min after laminectomy. Tissue preparation and measurement of immunoreactive TxB 2 (iTxB2) and 6KF (i6KF) by radioimmunoassay have been described previously (Burch et al. 1979; Wise et al. 1980; Hsu et al. 1985a). All samples were
292 assayed in triplicate with 3 different aliquot sizes. The interassay variability was 8~o (n - 5) for iTxB 2 a n d 10~o (n = 5) for i 6 K F .
Determination of tissue protein content Protein content in tissue h o m o g e n a t e was determined by the m e t h o d o f Lowry et al. (1951) after dissolving the h o m o g e n a t e in 0.5 N N a O H containing 5 ~ sodium lauryl sulfate.
Statistical analysis D a t a is expressed as mean + S E M . T h e difference between control and injured or between two different degrees o f injury was determined for statistical significance by Student's t-test. RESULTS The effects o f m o d e r a t e and severe injury on the extent o f vascular injury reflected by extravasation o f [~25I]HSA, c o r d e d e m a reflected by tissue water content and the neurological deficit scores are in Table 1. A significant difference in all three p a r a m e t e r s was noted between control a n d m o d e r a t e injury groups as well as between m o d e r a t e and severe injury groups. The post-traumatic increase in tissue iTxB 2 levels was d e p e n d e n t on the magnitude o f injury with greatest increase n o t e d in animals with severe injury (Fig. 1). The c o r d i 6 K F levels r e m a i n e d u n c h a n g e d irrespective o f the magnitude o f injury (Fig. 1). Thus a m a r k e d shift o f a T x B 2 - 6 K F ratio which reflects the i m b a l a n c e d p r o d u c t i o n o f T x A 2 and P G I 2 in favor o f platelet aggregation and vasoconstriction was n o t e d with severe injury (5.70 + 0.98; n = 7 in the severe injury vs 1.17 + 0.05; n = 7 in the control, P < 0.001). The T x B 2 - 6 K F ratio is also significant-
TABLE 1 NEUROLOGICAL DEFICIT SCORE, EDEMA, AND VASCULAR INJURY IN TWO DEGREES OF IMPACT INJURY Neurological deficit score was determined by a modified Tarlov scale; cord edema was calculated by % water content; and vascular injury index was estimated by extravasation of 125I-labelledserum albumin (see MATERIALSAND METHODS). Each group consisted of 7 rats.
Control Moderate injury Severe injury
Neurological deficit score
Cord edema (%H20)
Vascular injury index
5.0 + 0 2.83 + 0.31" 0.25 + 0.25*'÷
69.65 + 0.26 71.88 + 0.77* 73.98 _+0.27*'÷
1.17 + 0.05 3.20 + 0.56* 5.70 + 0.98*-÷
* Denotes difference between control and moderate injury or between control and severe injury is significant; ÷ denotes difference between moderate and severe injury is significant by Student's t-test (P < 0.05).
293 t l
¢,
600
-600
500
-500
400
-400
~-5
aoo
"300
O_
200
200
¢.D
100-
-100
0 Q"
0
E Q.
"~
E
cff X b-
C'i'L
MOD
SEV
INJURY Fig. 1. Effects of two degrees of trauma on cord content of thromboxane B 2 (TxB2) and 6-keto-PGF~= (6KF). Control (CTL) group received laminectomy. Moderate (MOD) and severe (SEV) injury groups received impact injury of 5 g from a height of 5 cm and 20 cm, respectively. Mean + SEM from 7 rats in each group is shown. * Denotes difference from CTL is significant; + denotes difference from MOD is significant.
ly elevated with moderate injury (3.20 + 0.56; n = 7, P < 0.05 compared to the control) but less than that with severe injury (P < 0.05 compared to the severe injury). A correlation of TxB2-6KF ratio and extent of vascular injury is shown in Fig. 2. DISCUSSION
The contribution of increased prostanoid formation to the vascular injury in experimental spinal cord contusion remains to be determined. In the present study we found that increased accumulation of TxB2, a stable metabolite of TxA2, depended on the degree of injury, whereas the formation of 6KF, a stable metabolite of PGI2, was not altered irrespective of the magnitude of injury. The selective increase in TxB2, but not 6KF, can be attributed to post-traumatic lipid peroxidation (Demopoulos et al. 1980) for lipid peroxides selectively inhibit PGI 2 production (Warso and Lando 1983). The TxA2-PGI 2 imbalance observed favors platelet aggregation and vasoconstriction, The extent to which this contributes to the vascular damage of experimental spinal cord injury is not clear. Therapy with indomethacin, PGI 2 and heparin, which should affect the TxA2-PGI 2 ratio to favor reduced mierovascular thromboembolism, has been reported to improve the post-traumatic neurologic deficit in cat spinal cord injury (Hallenbeck et al. 1983). This is consistent with the potential contribution of an altered TxA2-PGI 2 balance in the posttraumatic vascular damage in experimental spinal cord
294
~
4-
I,J_ ,¢. 37 X
I-
EV
MOD
2'
CTL o
o
~
'
~
VASCULAR INJURY INDEX
Fig. 2. Correlation of thromboxane B2-6-keto-PGIl~ratio (TxB2/6KF) and vascular injury index among control (CTL), moderate (MOD) and severe (SEV) injury groups: TxB2/6KF was derived from the ratio of the tissue content of TxB2 (in pg/mg protein) and 6KF (in pg/mg protein). Vascular injury index was estimated by extravasation of ~25I-humanserum albumin as described in MATERIALSAND METHODS. Mean + SEM from 7 rats in each group is shown.
injury. We found the extent of vascular injury, reflected by extravasation of [ 125I]HSA, to be closely related to the magnitude of the T x A z - P G I z imbalance as estimated by the T x B 2 - 6 K F ratio (Fig. 2). This is consistent with the contention that T x A z - P G I 2 imbalance may contribute to vascular damage in experimental spinal cord injury. Admittedly our observation o f T x A 2 - P G I 2 imbalance and vascular injury is correlative only and more studies are needed to explore the causal relationship between r x A z - P G I 2 imbalance and vascular injury. The degree of post-traumatic edema also depended on the magnitude of impact injury. Post-traumatic edema is vasogenic in nature (Griffiths and Miller 1974; Hsu ~t al. 1985b) and may develop as a function of vascular damage (Hsu et al. 1985b). Arachidonic acid induces cerebral edema (Chan and Fishman 1978) probably as a consequence of vascular injury (Chan et al. 1983). Therefore release of arachidonic acid and subsequent formation of its metabolites including leukotrienes (Chen et al. 1986) may contribute to the development of posttraumatic edema though the precise role of arachidonic acid and its metabolite in this regard remains to be determined. In conclusion, our results confirm the earlier observation in two different animal models of the existence of an imbalance of TxA 2 and P G I 2 formation with bias to an increased thrombotic activity (Hsu et al. 1985a). The T x A 2 - P G I 2 imbalance also zorrelates with the extent of vascular damage determined by extravasation of[ I25I]HSA and suggests that a selective increase in TxA 2 may contribute to the microvascular damage in experimental spinal cord injury.
295 REFERENCES Assenmacher, D.R. and T. B. Ducker (1971) The vascular and pathological changes seen in reversible spinal cord lesions, J. Bone Joint Surg. [Am.], 53A: 671-680. Balentine, J.D. (1978) Pathology of experimental spinal cord trauma, Part 1 (The necrotic lesions as a function of vascular injury), Lab. Invest., 39: 236-253. Balentine, J.D. (1985) Hypotheses in spinal cord trauma research. In: D.P. Becker and J.T. Povlishock (Eds.), Central Nervous System Trauma Status Report, NINCDS, Bethesda, pp. 455-461. Balentine, J. D., E. L. Hogan, N. L. Banik and P. L. Perot, Jr. (1985) Calcium and the pathogenesis of spinal cord injury. In: H.R. Winn, R. Rimel and J. A. Jane (Eds.), Recent Advances in Neural Trauma, Raven Press, New York, pp. 285-295. Bazan, N.G. (1970) Effects of ischemia and electroconvulsive Shock on free fatty acid pool in the brain, Biochim. Biophys. Acta, 218: 1-10. Burch, R. M., D. P. Knapp, P.V. Halushka (1979) Vasopressin stimulates thromboxane synthesis in the toad urinary bladder - - Effects of imidazole, J. Pharmacol. Exp. Ther., 210: 344-348. Chan, P.H. and R.A. Fishman (1978) Brain edema - - Induction in cortical slices by polyunsaturated fatty acid, Science, 201: 358-360. Chan, P.H., R.A. Fishman, J. Caronna, J.W. Schmidley, G. Prioleau and J. Lee (1983) Induction of brain edema following intracerebral injection of arachidonic acid, Ann. Neurol., 13: 625-632. Chen, S.T., C.Y. Hsu, E.L. Hogan, P.V. Halushka, O.I. Linet, F.M. Yatsu (1986) Thromboxane, prostacyclin and leukotrienes in cerebral ischemia, Neurology, 36: 466-470. Daniel, H.B., W. Francis, W.A. Lee and T.B. Ducker (1975) A method of quantitating injury inflicted in acute spinal cord studies, Paraplegia, 13: 137-142. Demopoulos, H. B., M. Yoder, E. G. Gutman, M. L. Seligman, E. S. Flamm and J. Ransohoff (1978) The fine structure of endothelial surfaces in the microcirculation of experimentally injured feline spinal cords, Scan. Electron Microsc., 2: 677. Demopoulos, H.B., E. S. Flamm, D.D. Pietronigro and M.L. Seligman (1980) The free radical pathology and the microcirculation in the major central nervous system disorders, Acta Physiol. Scand., Suppl. 492:91-119. Dohrman, G.J. and K.M. Wick (1975) Intramedullary blood flow patterns in the transitory traumatic paraplegia, Surg. Neurol., 1: 209-215. Dohrman, G. J., F. C. Wagner and P. C. Bucy (197 I) The microvasculature in transitory traumatic paraplegia - - An electron microscopic study in the monkey, J. Neurosurg., 35: 263-271. Don-Edwards, D., V. Decrescito, J. Tomasula and E.S. Flamm (1980) Effect of aminophylline and isoproterenol on spinal cord blood flow after impact injury, J. Neurosurg., 53: 385-390. Ducker, T.B. (1975) Experimental injury of the spinal cord. In: P.J. Vinken and G.W. Bruyn (Eds.), Handbook of Clinical Neurology, Vol. 25 (Injuries of the Spine and Spinal Cord), North-Holland, Amsterdam, pp. 9-26. Ducker, T.B., M. Salcman, P.L. Perot, Jr. and J.D. Balentine (1978) Experimental spinal cord trauma, Part 1 (Correlation of blood flow, tissue oxygen and neurological status in the dog), Surg. Neurol., 10" 60-70. Ellis, E.F., K.F., Wright, E.P. Wei, H.A. Kontos (1981) Cyclooxygenase products of arachidonic aid metabolism in cerebral cortex after experimental concussive brain injury, J. Neurochem., 37: 892-896. Faden, A. I. (1985) Pharmacologic therapy in acute spinal cord injury - - Experimental strategies and future decisions. In: D.P. Becker and J.T. Povlishock (Eds.), Central Nervous System Trauma Status Report, NINCDS, Bethesda, pp. 481-485. Fried, L. C. and R. Goodkin (1971) Microangiographic observations of the experimental traumatized spinal cord, J. Neurosurg., 35: 709-714. Gaudet, F.J., I. Alam and L. Levine (1980) Accumulation of cyclooxygenase products of arachidonic acid metabolism in gerbil brain during reperfusion after bilateral common carotid occlusion, J. Neurochem., 35: 653-658. Goodman, J.G., W. G. Bingham, W. E. Hunt (1979) Platelet aggregation in experimental spinal cord injury, Arch. Neurol. (Chic.), 30: 197-201. Griffiths, I.R. and R. Miller (1974) Vascular permeability to protein and vasogenic edema in experimental concussive injuries to the canine spinal cord, J. Neurol. Sci., 22: 291-304. Hallenbeck, J.M., T.P. Jacobs, A.I. Faden (1983) Combined PGI2, indomethacin and heparin improves neurological recovery after spinal trauma in cats, J. Neurosurg., 58: 749-754. Hogan, E . L and N.L. Banik (1985) Biochemistry of the spinal cord. In: A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. I0, Plenum, New York, pp. 285-377.
296 Hsu, C.Y., P.V. Halushka, E.L. Hogan, N.L. Banik, W.A. Lee and P.L. Perot, Jr. (1985a) Altered thromboxane and prostacyclin levels in experimental spinal cord injury, Neurology, 35: 1003-1009. Hsu, C.Y., E.L. Hogan, R.H. Gasden, Sr., K.M. Spicer, M.P. Shi and R.D. Cox (1985b) Vascular permeability in experimental spinal cord injury, J. Neurol. Sci., 70: 275-282. Jonsson, H.T. and H.B. Daniel (1976) Altered levels of PGF in cat spinal cord tissue following traumatic injury, Prostaglandins, 11: 51-61. Lowry O.H., N.J. Rosenbrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin reagent. J. Biol. Chem., 193: 265-275. Moncada, S. and J.R. Vane (1979) Arachidonic acid metabolites and their interactions between platelets and blood vessel walls, N. Engl. J. Med., 300:1142-1147. Nelson, E., S.C. Gertz, M.L. Rennels, T.B. Ducker and O.R. Blaumanis (1977) Spinal cord injury - - The role of vascular damage in the pathogenesis of central hemorrhagic necrosis, Arch. Neurol. (Chic.), 34: 332-333. Wagner, R., N. Taslitz, R. F. White, et al, (1969) Vascular phenomena in the normal and traumatized spinal cord, Anat. Rec., 163: 281. Warso, M.A. and W. E. M. Lando (1983) Lipid peroxidation in relation to prostacyclin and thromboxane physiology and pathophysiology, Brit. Med. Bull., 39: 277-280. Wise, W.C., J.A. Cook, T. Eller and P.V. Halushka (1980) Ibuprofen improves survival from endotoxic shock in the rat,,/. PharmacoL Exp. Ther., 215: 160-164. Wolfe, L.S. (1982) Eicosanoids - - Prostaglandins, thromboxanes, leukotrienes and other derivatives of carbon-20 unsaturated fatty acids, J. Neurochem., 38: 1-14. Wolfe, L.S. and H.M. Pappius (1983) Involvement of arachidonic acid metabolites in functional disturbances following brain injury, Adv. Prostaglandins Thromboxane Leukotriene Res., 12: 345-349. Yoshida, S., S. Inoh, T. Asano, et al. (1980) Effect of transient ischemia on free fatty acids and phospholipids in the gerbil brain, J. Neurosurg., 53: 323-331. Young, W. (1985) Blood flow, metabolic and neurophysiological mechanism in spinal cord injury. In: D.P. Beeker and J.T. Povlishock (Eds.), Central Nervous System Trauma Status Report, NINCDS, Bethesda, pp. 463-473.