- Email: [email protected]
Increased dynorphin immunoreactivity in spinal cord after traumatic injury
Regulatory Peptides, 11 (1985) 35--41 Elsevier
35
RPT 00362
Increased dynorphin immunoreactivity in spinal cord after traumatic injury A.I. F a d e n ' , C.J. Molineaux b, J.G. Rosenberger b, T.P. Jaeobs c and B.M. Cox b "Center for Neural Injury, San Francisco Veterans Administration Medical Center University of California, San Francisco, CA 94121, bDepartment of Pharmacology and *Neurobiology Research Unit, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, U.S.A. (Received 10 December 1984; revised manuscript received 19 February 1985; accepted for publication 21 February 1985)
Summary Opiate antagonists, at high doses, have been shown to improve physiological variables and outcome after experimental spinal injury. Dynorphin appears to be unique amongst opioids in producing hindlimb paralysis after intrathecal injection. Taken together, these findings suggest a possible pathophysiological role for endogenous opioids, particularly dynorphin, in spinal injury. In the present studies we examined the relationship between changes in dynorphin immunoreactivity (Dyn-ir) in rat spinal cord after traumatic injury and the subsequent motor dysfunction. Trauma was associated with significantly increased Dyn-ir at the injury site, but not distant from the lesion. Dyn-ir was found elevated as early as 2 h and as late as 2 weeks after trauma, and was significantly correlated with the degree of injury. These data are consistent with the hypothesis that dynorphin systems may be involved in the secondary injury that follows spinal trauma. endogenous opioids; dynorphin; trauma; spinal injury; neurological function
Introduction Traumatic injury to the spinal cord causes neurological dysfunction, in part, through delayed secondary mechanisms which appear to involve endogenous factors [1]. The opiate antagonist naloxone, administered even hours after injury, improves Address correspondence to: Dr. Alan I. Faden, Neurology Serviee, San Francisco Veterans Administration Medical Center, 4150 Clement Street, San Francisco, CA 94121, U.S.A. Telephone: 415-750-2011. 0167-0115/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
36
physiological variables and neurological recovery after experimental spinal injury, implicatingendogenous opioid systems and opiate receptorsin thissecondary injury process [2-5].Naloxone's beneficialactions in these models are dose-dependent and optimally effectivein the mg/kg range, suggesting possible activityat non-# opiate receptors [6].Opiate antagonists showing increased activityat K sites[7] but not 6 sites[8] arc far more active than naloxone in spinal injury,implicating the it-opiate receptor in this process. Immunoreactivc dynorphin (Dyn-ir) is found in spinal cord [9-II]. Since dynorphin has selectivityfor the z-receptor and may bc an endogenous ligand for this receptor, the above findingsfurtherraisethe possibilitythat dynorphin systems may contribute to secondary spinal cord injury aftertrauma. Additional support for this hypothesis comes from the observation that prodynorphin-derivcd peptides are unique amongst opioids in producing hindlimb paralysisafter intrathecalinjection in rats [12-14]; such paralysisis dose-relatedand irreversibleat high doses [12].In the present studies wc examined the changes in Dyn-ir in spinal cord after graded traumatic spinal injury in the rat.
Materials and Methods
Dyn-ir was assessed in the spinal cord of rats subjected to traumatic spinal injury. Male Sprague-Dawley rats (300 g) were anesthetized with ketamine hydrochloride (50 mg/kg, i.m.) and sodium pentobarbital (30 mg/kg, i.m.). Laminectomy was performed to expose the T-10 spinal segment. The spinal cord was injured using the Allen method [2],in which a fixed weight (I0 g) was dropped a fixed distance (I, 5, 7.5 or 10 cm) onto a plasticimpounder resting on the exposed dura mater. These parameters yielded increasinggraded injury,referredto as low (I0 g/cm), moderate (50 g/cm) or high (75-I00 g/cm) degrees of injury. Following trauma, the incision siteswere surgicallyrepaired and the animals removed to home cages. Injured animals were maintained for 2 h, 24 h or 2 weeks following trauma, and subsequently killedwith sodium pentobarbital in order to assessdynorphin levels.Control animals were subjected to the same surgicalprocedure, including leminectomy, but received no trauma; separate groups of surgical controls were maintained for 2 h, 24 h and 2 weeks to parallelthe injured animals. In addition,a group of normal animals served as non-surgical controls. Neurological examination was made on all animals maintained 24 h or 2 weeks afterinjury.Grading was performed utilizingordinal scalesbased on motor function as follows: 0 = paraplegia; I = spontaneous movement, unable to walk; 2 = able to walk with spasticity;3 -- normal motor function. Dyn-ir was determined using the 'Lucia' antiserum as previously described [15].This antiserum recognizes Dyn A-(I-17), (I-13) and (I-12), as well as larger precursors of Dyn A. The smaller fragments of Dyn A, including (I-9),(I-I0) and (I-I I),show measurable but smaller cross-reactivity(< 10%), whereas Dyn A-(I-8) and shorter fragments, as well as cnkephalins and/1-endorphin, have no significantcross-reactivitywith thisantiserum.
37
Results
At the injury site, Dyn-ir was significantly correlated with the degree of injury (regression ANOVA, F = 8.37, P < 0.01): low, medium and high injury produced progressively larger increases in Dyn-ir which were evident at 24 h (Fig. 1). Smaller but significant increases were also found just below the injury site in the lumbar region, but not at more distant cervical sites away from the lesion. This graded injury was highly correlated with neurological dysfunction at both 24 h and 2 weeks after injury (regression ANOVA, F = 68.88, P < 0.01; F = 38.7, P < 0.01, respectively). Moreover, concentrations of Dyn-ir at the injury site were significantly correlated with the degree of neurological deficit (Spearman's rank correlation test, P < 0.05). This relationship is clearly seen in Fig. 2, which compares neurological function after injury with the concentration of Dyn-ir at the injury site; significantly higher levels of Dyn-ir were found in non-walkers (neurological score = 0 or 1) as compared to walkers (neurological score = 2 or 3) at both 24 h and 2 weeks (t-tests, each P < 0.05). There was a trend toward increased Dyn-ir levels at the injury site by 2 h post-trauma, with significant increases at 24 h (t-test, P < 0.05) and at 2 weeks (ttest, P < 0.05) (Fig. 3). Laminectomy control animals showed consistent levels of Dyn-ir at 2 h, 24 h and 2 weeks (ANOVA, F -- 2.39, P > 0.05) (Fig. 3); these levels did not differ from those of normal control animals not subjected to surgery (ANOVA, F = 1.86, P > 0.05). Dyn-ir levels in thoracic cord were significantly higher than those in cervical cord for both normal controls and laminectomy controls (ttests, each P < 0.01). 0.3
Z
o it. O
~
0.2
0.1
I:1.
0.0
C
L M H CERVICAL
C
L M H THORACIC
C
L M H LUMBAR
Fig. 1. Levels ofdynorphin A immunoreactivity (Dyn-ir) in the rat spinal cord 24 h after traumatic injury. At the injury site (thoracic region), Dyn-ir was significantly and progressively elevated with increasingly severe injury (regression ANOVA; P < 0.01). Smaller but significant increases were also noted in the adjacent lumbar region, but not at more distant cervical sites. C = laminectomy controls; L = low injury; M = medium injury; H ffi high injury (see text for details).
38
A. 24 HOURS = 0'4f i 0.3 F g. 0.2
-'x.
4~ n 0
l= 0"
0.1
B, 2 WEEKS 0.4
ii' g"
0.3 0.2
m 0
E 0.1
WALKERSNON-WALKIBIS Fig. 2. Levels of Dyn-ir associated with neurological function at 24 h and 2 weeks after traumatic cord injury in the rat. Animals that were unable to walk (neurological score = 0 or 1) had ~a,nlflcantly higher levels of" Dyn-ir than animals that could walk (neurological score -- 2 or 3) (unpaired t-tests, each
P < 0.05).
0.4
_z 0.3 0 no. 0 X nu.I a.
~
0.2
0.1
0.0
Fig. 3. Levels of Dyn-b" at the injury site (thoracic region) at different times after a high degree of injury. Open bars represent l ~ d n ~ t o m y control animals, and ~aaded bars r e p o t injured amrmds. A t~dency for increased levels of Dyn-ir is present at 2 h with significant increases at 24 h and 2 weeks (t-tests; each
P < 0.05).
39 Discussion
A number of groups have now reported beneficial effects of the opiate receptor antagonist naloxone following central nervous system (CNS) injury, including traumatic [2-5] and ischemic [6-8] spinal cord injury, traumatic brain injury [16] and ischemic brain injury [17]. The doses of naloxone required for these therapeutic actions have been quite high (between 2 and 10 mg/kg) suggesting actions at non-# opiate receptors such as the ~ or r receptor [17]. We have found that WIN44,441-3, an opiate antagonist that is more active than naloxone at r sites [18], stereospecifically improved neurological recovery following ischemic or traumatic spinal injury [7]; in the former model, this antagonist proved to be approximately 50 times more potent than naloxone, whereas the selective &receptor antagonist ICI 154,129 was totally ineffective [8]. Since the J¢ receptor appears to be the predominant opioid receptor in the spinal cord of a variety of species [19-21], the findings to date support the view that the ~¢receptor may play a role in the pathophysiology of traumatic and ischemic CNS injury. Dyn A has selectivity for the r receptor and may be an endogenous ligand for that receptor [22,23], whereas smaller fragments such as Dyn (1-8) also have activity at # sites [24]. Given intrathecally, Dyn A produces dose-related hindlimb paralysis in the rat [12-14]; this effect is not produced by #- or &selective enkephalins, or by flendorphin. Smaller dynorphin fragments also have this paralytic action, but with considerably reduced potency [14]. Whether the ability of dynorphin-related opioids to produce motor dysfunction represents an opiate receptor-mediated effect has been questioned [14]; however, one group reported that paralysis produced by low doses of Dyn A-(l-13) is blocked by high (10 mg/kg) but not by lower (1 mg/kg) doses of naloxone [13]. The present studies demonstrate a striking correlation between degree of traumatic spinal injury, neurological dysfunction and concentration of Dyn-ir at the injury site. If the increase in Dyn-ir is causally related to the paralysis observed after injury through actions at r-receptor sites, then changes in Dyn-ir over time should be evaluated within the context of opiate receptor binding after injury. Preliminary data from our laboratory indicates that r-receptor binding, but not that of # or ~ receptors, is substantially and significantly increased at 24 h after traumatic spinal injury in the rat; these changes occur at the injury site and return to normal by 1 week. Such data may explain the observation that naloxone treatment is effective relatively early after injury, whereas Dyn-ir remains elevated for a more prolonged period. Levels of Dyn-ir in spinal cord are higher than in most brain regions except hypothalamus, and dynorphin-immunoreactive cell bodies and fibers have been observed in the spinal cord [9]. The largest amounts of immunoreactive dynorphin are found in dorsal spinal cord; relative concentrations appear to vary among species, with approximately twofold higher concentrations in dorsal over ventral cord in the rat [9]. The majority of cell bodies containing Dyn-ir are found in dorsal horn, primarily lamina I and II [25]; however, little information is available regarding dynorphin terminals in spinal cord. Levels of Dyn-ir are found in the spinal cord of control animals in the present studies are similar to levels found by other investigators in this
40
species [9]. However, in contrast to an earlier study [9], significant differences were observed in Dyn-ir levels between thoracic and cervical spinal segments. The mechanism by which dynorphin may affect motor function after injury remains speculative. No information is available regarding the effects of dynorphin on spinal cord blood flow or metabolism. Nor is there any published data on the electrophysiological actions of dynorphin on spinal neurons or reflexes in vivo. However, dynorphin reduces calcium-dependent action potential duration in mouse spinal cord-dorsal root ganglion co-cultures; this action appears to be at opiate receptors separate from # or 6 (presumably r), and is antagonized by naloxone [26]. Increases in Dyn-ir after spinal trauma contrast with changes in levels of other neuropeptides in the spinal cord after similar injury: preliminary studies indicate that substance P, somatostatin, Met-enkephalin and Leu-enkephalin show substantial decreases in immunoreactivity at the trauma site (unpublished observations). Changes in Dyn-ir do not appear to reflect inhibition of axonal transport produced by injury, since Dyn-ir in the synaptosomal fraction of tissue homogenates of spinal cord is unchanged by trauma [27]. Since Leu-enkephalin may be derived from prodynorphin precursors [28], the increased Dyn-ir combined with reduced Leu-enkephalin-ir may suggest that spinal injury alters dynorphin processing. Independent of the mechanism by which Dyn-ir is increased, the present findings show a remarkable correlation between change in spinal cord Dyn-ir and degree of neurological injury after spinal trauma, and are consistent with the hypothesis that this particular opioid system may be involved in the pathophysiology of spinal cord injury.
Acknowledgements This work was supported in part by the Office of Naval Research (Contract #N0001482WR20257) and Uniformed Services University of the Health Sciences protocol # R07542. Christopher J. Molineaux was supported by United States Public Health Service post-doctoral fellowship # HL06916. The opinions or assertions conmined herein are the private ones of the authors and are not to be construed as official or reflecting the view of the Department of Defense or the Uniformed Services University of the Health Sciences. The experiments reported herein were conducted according to the principles set forth in the "Guide for the Care and Use of Laboratory Animals", Institute of Laboratory Animal Resources, National Research Council (DHEW Publication #[NIH] 78-23, 1978). The authors wish to thank Susan Knoblach and Edward Burgard for their technical assistance, and Eleanor M. Bell for preparation of this manuscript.
References 1 Faden, A.I., Recent pharmacological advances in experimental spinal injury: Theoretical and methodolo#cal considerations, Tt~-nds Nenro~., 6 (1983) 375-377. 2 Faden, A.I., Jacobs, T.P. and Holaday, J.W., Opiate antagonist improves neurolo$ic recovery after spinal injury, Science, 211 (1981) 493-494.
41 3 Faden, A.I., Jacobs, T.P. and Holaday, J.W., Endorphins in experimental spinal injury: therapeutic effect of naloxune, Ann. Neurol., 10 (1981) 326-332. 4 Faden, A.I., Jacobs, T.P. and Holaday, J.W., Comparison of early and late naloxone treatment in experimental spinal injury, Neurology, 32 (1982) 677-681. 5 Flamm, E.S., Young, W., Demopoulos, H.B., DeCrescito, V. and Tomasula, J.J., Experimental spinal cord injury: treatment with naloxone, Neurosurgery, 10 (1982) 227-231. 6 Faden, A.I., Jacobs, T.P., Smith, M.T. and Zivin, J.A., Naloxone in experimental spinal cord ischemia: dose-response studies, Eur. J. PharmacoL, 103 (1984) 115-120. 7 Faden, A.I., Knobiach, S., Mays, C. and Jacobs, T.P., Motor dysfunction after spinal cord injury is mediated by opiate receptors, Peptides (1985) in press. 8 Faden, A.I., Jacobs, T.P. and Zivin, J.A., Comparison of naloxone and a delta-selective antagonist in experimental spinal 'stroke', Life Sci., 33 (Suppl. I) (1983) 707-710. 9 Botticelli, L.J., Cox, B.M. and Goldstein, A., Immunoreactive dynorphin in mammalian spinal cord and dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 7783-7786. 10 Millan, M.J., Tsang, Y.F., Przewlocki, R., Hollt, V. and Herz, A., The influence of foot shock stress upon brain, pituitary and spinal cord pools of immunoreactive dynorphin in rats, Neurosci. Lett., 24 (1981) 75-79. I 1 Watson, S.J., Khachaturian, H., Akil, H., Coy, D.H. and Guldstein, A., Comparison of the distribution of dynorphin systems and enkephalin systems in brain, Science, 218 (1982) 1134-1136. 12 Faden, A.I. and Jacobs, T.P., Dynorphin induces partially reversible paraplegia in the rat, Eur. J. PharmacoL, 91 (2/3) (1983) 321-324. 13 Przewlocki, R., Shearman, G.T. and Herz, A., Mixed opioid/non-opioid effects of dynorphin and dynorphin-related peptides after their intrathecal injection in rats, Neuropeptides, 3 (1983) 233-240. 14 Faden, A.I. and Jacobs, T.P., Dynorphin-related peptides cause motor dysfunction in the rat through a non-opiate action, Br. J. Pharmacol., 81 (1984) 271-276. 15 Molineaux, C.J., Feuerstein, G., Faden, A.I. and Cox, B.M., Distribution of immunoreactive dynorphin in discrete brain nuclei; comparison with vasopressin, Neurosci. Lett., 33 (1982) 179-184. 16 Hayes, R.L., Galinet, B.J., Kulkarne, P. and Becker, D.P., Effects of naloxone on systemic and cerebral responses to experimental concussive brain injury in eats, J. Neurosurg., 58 (1983) 720-728. 17 Faden, A.I., Opiate antagonists in the treatment of stroke, CurT. Concepts Cerebrovasc. Dis., 18 (1983) 27-31. 18 Michne, W.F., Lewis, T.R., Michalec, S.J., Pierson, A.K., Gillan, M.C., Paterson, S.J, Robson, L.E. and Kosterlitz, H.W., Novel developments of N-methylbenzomorphan narcotic antagonists. In J.M. Van Ree and L. Terenius (Eds.), Characteristics and Functions of Opioids, Elsevier/North-Holland Biomedical Press, 1978, pp. 197-206. 19 Traynor, J.R., Kelley, P.D. and Rance, M.J., Multiple opiate binding sites in rat spinal cord, Life Sci., 31 (1982) 1377-1380. 20 Guarderes, C., Attali, B., Audigier, Y. and Cros, J., In Advances in Endogenous and Exogenous Opiates, Kodansha Ltd., Tokyo, 1981, pp. 18-20. 21 Czlonkowski, A., Costa, T., Przewlocki, R., Pasi, A. and Herz, A., Opiate receptor binding sites in human spinal cord, Brain Res., 267 (1983) 392-396. 22 Chavkin, C., James, I.F. and Goldstein, A., Dynorphin is a specific endogenous ligand of the K opiate receptor, Science, 215 (1982) 413-415. 23 Huidobro-Toro, J.P., Yoshimura, K., Lee, N.M., Loh, H.H. and Way, E.L., Dynorphin interaction at the ~c-opiatesite, Eur. J. Pharmacol., 72 (1981) 265-266. 24 Cox, B.M., Endogenous opioid peptides: a guide to structures and terminology, Life Sci., 31 (1982) 1645-1658. 25 Basbaum, A., personal communication. 26 Werz, M.A. and Macdonald, R.L., Dynorphin reduces calcium-dependent action - potential duration by decreasing voltage-dependent calcium conductance, Neurosci. Lett., 46 (1984) 185-190. 27 Cox, B.M., Molineaux, C.J., Jacobs, T.P., Rosenberger, J.G. and Faden, A.I., Effects of traumatic injury on dynorphin imunoreactivity in spinal cord, Neuropeptides, (1985) in press. 28 Zamir, N., Palkovits, M., Weber, E., Mezey, E. and Brownstcin, M.J., A dynorphinergic pathway of Leu-enkephalin production in rat substantia nigra, Nature, 307 (1984) 643--645.
35
RPT 00362
Increased dynorphin immunoreactivity in spinal cord after traumatic injury A.I. F a d e n ' , C.J. Molineaux b, J.G. Rosenberger b, T.P. Jaeobs c and B.M. Cox b "Center for Neural Injury, San Francisco Veterans Administration Medical Center University of California, San Francisco, CA 94121, bDepartment of Pharmacology and *Neurobiology Research Unit, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, U.S.A. (Received 10 December 1984; revised manuscript received 19 February 1985; accepted for publication 21 February 1985)
Summary Opiate antagonists, at high doses, have been shown to improve physiological variables and outcome after experimental spinal injury. Dynorphin appears to be unique amongst opioids in producing hindlimb paralysis after intrathecal injection. Taken together, these findings suggest a possible pathophysiological role for endogenous opioids, particularly dynorphin, in spinal injury. In the present studies we examined the relationship between changes in dynorphin immunoreactivity (Dyn-ir) in rat spinal cord after traumatic injury and the subsequent motor dysfunction. Trauma was associated with significantly increased Dyn-ir at the injury site, but not distant from the lesion. Dyn-ir was found elevated as early as 2 h and as late as 2 weeks after trauma, and was significantly correlated with the degree of injury. These data are consistent with the hypothesis that dynorphin systems may be involved in the secondary injury that follows spinal trauma. endogenous opioids; dynorphin; trauma; spinal injury; neurological function
Introduction Traumatic injury to the spinal cord causes neurological dysfunction, in part, through delayed secondary mechanisms which appear to involve endogenous factors [1]. The opiate antagonist naloxone, administered even hours after injury, improves Address correspondence to: Dr. Alan I. Faden, Neurology Serviee, San Francisco Veterans Administration Medical Center, 4150 Clement Street, San Francisco, CA 94121, U.S.A. Telephone: 415-750-2011. 0167-0115/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
36
physiological variables and neurological recovery after experimental spinal injury, implicatingendogenous opioid systems and opiate receptorsin thissecondary injury process [2-5].Naloxone's beneficialactions in these models are dose-dependent and optimally effectivein the mg/kg range, suggesting possible activityat non-# opiate receptors [6].Opiate antagonists showing increased activityat K sites[7] but not 6 sites[8] arc far more active than naloxone in spinal injury,implicating the it-opiate receptor in this process. Immunoreactivc dynorphin (Dyn-ir) is found in spinal cord [9-II]. Since dynorphin has selectivityfor the z-receptor and may bc an endogenous ligand for this receptor, the above findingsfurtherraisethe possibilitythat dynorphin systems may contribute to secondary spinal cord injury aftertrauma. Additional support for this hypothesis comes from the observation that prodynorphin-derivcd peptides are unique amongst opioids in producing hindlimb paralysisafter intrathecalinjection in rats [12-14]; such paralysisis dose-relatedand irreversibleat high doses [12].In the present studies wc examined the changes in Dyn-ir in spinal cord after graded traumatic spinal injury in the rat.
Materials and Methods
Dyn-ir was assessed in the spinal cord of rats subjected to traumatic spinal injury. Male Sprague-Dawley rats (300 g) were anesthetized with ketamine hydrochloride (50 mg/kg, i.m.) and sodium pentobarbital (30 mg/kg, i.m.). Laminectomy was performed to expose the T-10 spinal segment. The spinal cord was injured using the Allen method [2],in which a fixed weight (I0 g) was dropped a fixed distance (I, 5, 7.5 or 10 cm) onto a plasticimpounder resting on the exposed dura mater. These parameters yielded increasinggraded injury,referredto as low (I0 g/cm), moderate (50 g/cm) or high (75-I00 g/cm) degrees of injury. Following trauma, the incision siteswere surgicallyrepaired and the animals removed to home cages. Injured animals were maintained for 2 h, 24 h or 2 weeks following trauma, and subsequently killedwith sodium pentobarbital in order to assessdynorphin levels.Control animals were subjected to the same surgicalprocedure, including leminectomy, but received no trauma; separate groups of surgical controls were maintained for 2 h, 24 h and 2 weeks to parallelthe injured animals. In addition,a group of normal animals served as non-surgical controls. Neurological examination was made on all animals maintained 24 h or 2 weeks afterinjury.Grading was performed utilizingordinal scalesbased on motor function as follows: 0 = paraplegia; I = spontaneous movement, unable to walk; 2 = able to walk with spasticity;3 -- normal motor function. Dyn-ir was determined using the 'Lucia' antiserum as previously described [15].This antiserum recognizes Dyn A-(I-17), (I-13) and (I-12), as well as larger precursors of Dyn A. The smaller fragments of Dyn A, including (I-9),(I-I0) and (I-I I),show measurable but smaller cross-reactivity(< 10%), whereas Dyn A-(I-8) and shorter fragments, as well as cnkephalins and/1-endorphin, have no significantcross-reactivitywith thisantiserum.
37
Results
At the injury site, Dyn-ir was significantly correlated with the degree of injury (regression ANOVA, F = 8.37, P < 0.01): low, medium and high injury produced progressively larger increases in Dyn-ir which were evident at 24 h (Fig. 1). Smaller but significant increases were also found just below the injury site in the lumbar region, but not at more distant cervical sites away from the lesion. This graded injury was highly correlated with neurological dysfunction at both 24 h and 2 weeks after injury (regression ANOVA, F = 68.88, P < 0.01; F = 38.7, P < 0.01, respectively). Moreover, concentrations of Dyn-ir at the injury site were significantly correlated with the degree of neurological deficit (Spearman's rank correlation test, P < 0.05). This relationship is clearly seen in Fig. 2, which compares neurological function after injury with the concentration of Dyn-ir at the injury site; significantly higher levels of Dyn-ir were found in non-walkers (neurological score = 0 or 1) as compared to walkers (neurological score = 2 or 3) at both 24 h and 2 weeks (t-tests, each P < 0.05). There was a trend toward increased Dyn-ir levels at the injury site by 2 h post-trauma, with significant increases at 24 h (t-test, P < 0.05) and at 2 weeks (ttest, P < 0.05) (Fig. 3). Laminectomy control animals showed consistent levels of Dyn-ir at 2 h, 24 h and 2 weeks (ANOVA, F -- 2.39, P > 0.05) (Fig. 3); these levels did not differ from those of normal control animals not subjected to surgery (ANOVA, F = 1.86, P > 0.05). Dyn-ir levels in thoracic cord were significantly higher than those in cervical cord for both normal controls and laminectomy controls (ttests, each P < 0.01). 0.3
Z
o it. O
~
0.2
0.1
I:1.
0.0
C
L M H CERVICAL
C
L M H THORACIC
C
L M H LUMBAR
Fig. 1. Levels ofdynorphin A immunoreactivity (Dyn-ir) in the rat spinal cord 24 h after traumatic injury. At the injury site (thoracic region), Dyn-ir was significantly and progressively elevated with increasingly severe injury (regression ANOVA; P < 0.01). Smaller but significant increases were also noted in the adjacent lumbar region, but not at more distant cervical sites. C = laminectomy controls; L = low injury; M = medium injury; H ffi high injury (see text for details).
38
A. 24 HOURS = 0'4f i 0.3 F g. 0.2
-'x.
4~ n 0
l= 0"
0.1
B, 2 WEEKS 0.4
ii' g"
0.3 0.2
m 0
E 0.1
WALKERSNON-WALKIBIS Fig. 2. Levels of Dyn-ir associated with neurological function at 24 h and 2 weeks after traumatic cord injury in the rat. Animals that were unable to walk (neurological score = 0 or 1) had ~a,nlflcantly higher levels of" Dyn-ir than animals that could walk (neurological score -- 2 or 3) (unpaired t-tests, each
P < 0.05).
0.4
_z 0.3 0 no. 0 X nu.I a.
~
0.2
0.1
0.0
Fig. 3. Levels of Dyn-b" at the injury site (thoracic region) at different times after a high degree of injury. Open bars represent l ~ d n ~ t o m y control animals, and ~aaded bars r e p o t injured amrmds. A t~dency for increased levels of Dyn-ir is present at 2 h with significant increases at 24 h and 2 weeks (t-tests; each
P < 0.05).
39 Discussion
A number of groups have now reported beneficial effects of the opiate receptor antagonist naloxone following central nervous system (CNS) injury, including traumatic [2-5] and ischemic [6-8] spinal cord injury, traumatic brain injury [16] and ischemic brain injury [17]. The doses of naloxone required for these therapeutic actions have been quite high (between 2 and 10 mg/kg) suggesting actions at non-# opiate receptors such as the ~ or r receptor [17]. We have found that WIN44,441-3, an opiate antagonist that is more active than naloxone at r sites [18], stereospecifically improved neurological recovery following ischemic or traumatic spinal injury [7]; in the former model, this antagonist proved to be approximately 50 times more potent than naloxone, whereas the selective &receptor antagonist ICI 154,129 was totally ineffective [8]. Since the J¢ receptor appears to be the predominant opioid receptor in the spinal cord of a variety of species [19-21], the findings to date support the view that the ~¢receptor may play a role in the pathophysiology of traumatic and ischemic CNS injury. Dyn A has selectivity for the r receptor and may be an endogenous ligand for that receptor [22,23], whereas smaller fragments such as Dyn (1-8) also have activity at # sites [24]. Given intrathecally, Dyn A produces dose-related hindlimb paralysis in the rat [12-14]; this effect is not produced by #- or &selective enkephalins, or by flendorphin. Smaller dynorphin fragments also have this paralytic action, but with considerably reduced potency [14]. Whether the ability of dynorphin-related opioids to produce motor dysfunction represents an opiate receptor-mediated effect has been questioned [14]; however, one group reported that paralysis produced by low doses of Dyn A-(l-13) is blocked by high (10 mg/kg) but not by lower (1 mg/kg) doses of naloxone [13]. The present studies demonstrate a striking correlation between degree of traumatic spinal injury, neurological dysfunction and concentration of Dyn-ir at the injury site. If the increase in Dyn-ir is causally related to the paralysis observed after injury through actions at r-receptor sites, then changes in Dyn-ir over time should be evaluated within the context of opiate receptor binding after injury. Preliminary data from our laboratory indicates that r-receptor binding, but not that of # or ~ receptors, is substantially and significantly increased at 24 h after traumatic spinal injury in the rat; these changes occur at the injury site and return to normal by 1 week. Such data may explain the observation that naloxone treatment is effective relatively early after injury, whereas Dyn-ir remains elevated for a more prolonged period. Levels of Dyn-ir in spinal cord are higher than in most brain regions except hypothalamus, and dynorphin-immunoreactive cell bodies and fibers have been observed in the spinal cord [9]. The largest amounts of immunoreactive dynorphin are found in dorsal spinal cord; relative concentrations appear to vary among species, with approximately twofold higher concentrations in dorsal over ventral cord in the rat [9]. The majority of cell bodies containing Dyn-ir are found in dorsal horn, primarily lamina I and II [25]; however, little information is available regarding dynorphin terminals in spinal cord. Levels of Dyn-ir are found in the spinal cord of control animals in the present studies are similar to levels found by other investigators in this
40
species [9]. However, in contrast to an earlier study [9], significant differences were observed in Dyn-ir levels between thoracic and cervical spinal segments. The mechanism by which dynorphin may affect motor function after injury remains speculative. No information is available regarding the effects of dynorphin on spinal cord blood flow or metabolism. Nor is there any published data on the electrophysiological actions of dynorphin on spinal neurons or reflexes in vivo. However, dynorphin reduces calcium-dependent action potential duration in mouse spinal cord-dorsal root ganglion co-cultures; this action appears to be at opiate receptors separate from # or 6 (presumably r), and is antagonized by naloxone [26]. Increases in Dyn-ir after spinal trauma contrast with changes in levels of other neuropeptides in the spinal cord after similar injury: preliminary studies indicate that substance P, somatostatin, Met-enkephalin and Leu-enkephalin show substantial decreases in immunoreactivity at the trauma site (unpublished observations). Changes in Dyn-ir do not appear to reflect inhibition of axonal transport produced by injury, since Dyn-ir in the synaptosomal fraction of tissue homogenates of spinal cord is unchanged by trauma [27]. Since Leu-enkephalin may be derived from prodynorphin precursors [28], the increased Dyn-ir combined with reduced Leu-enkephalin-ir may suggest that spinal injury alters dynorphin processing. Independent of the mechanism by which Dyn-ir is increased, the present findings show a remarkable correlation between change in spinal cord Dyn-ir and degree of neurological injury after spinal trauma, and are consistent with the hypothesis that this particular opioid system may be involved in the pathophysiology of spinal cord injury.
Acknowledgements This work was supported in part by the Office of Naval Research (Contract #N0001482WR20257) and Uniformed Services University of the Health Sciences protocol # R07542. Christopher J. Molineaux was supported by United States Public Health Service post-doctoral fellowship # HL06916. The opinions or assertions conmined herein are the private ones of the authors and are not to be construed as official or reflecting the view of the Department of Defense or the Uniformed Services University of the Health Sciences. The experiments reported herein were conducted according to the principles set forth in the "Guide for the Care and Use of Laboratory Animals", Institute of Laboratory Animal Resources, National Research Council (DHEW Publication #[NIH] 78-23, 1978). The authors wish to thank Susan Knoblach and Edward Burgard for their technical assistance, and Eleanor M. Bell for preparation of this manuscript.
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