J,~urniil,I/ N ~ . a r n ~ k r , n i \ l nVol. . 33. pp. 397 to 401 I’crgamon P r e s Ltd 1079. Prinlcd In Great Britain Q , 1ntern.itional Society for Ncurochemisrry Ltd
SHORT COMMUNICATION
Regional canine spinal cord energy state after experimental trauma (Receiaed 16 October 1978 Accepted 8 Februury 1979)
el ul., WE HAVE reported (WALKERet a/., 1977b) that a marked nary report of these results has appeared (WALKER decrease in the concentrations of the energy metabolites 1978). ATP and phosphocreatine and an increase in the concenEXPERIMENTAL PROCEDURES trations of lactic acid and AMP occur at the trauma site Adult mongrel dogs weighing 12-14 kg were anestheof total canine spinal cord within 15 min after a midthoracic injury which is sufficient to cause permanent paralysis. tized with pentobarbital (Nembutal, Abbott, 28 mg/kg) The earliest demonstrable pathology after spinal injury with additional dosage as required during the experiment. 1969; A dorsal laminectomy was performed at the T2-T6 level occurs in the vasculature (DUCKER& ASSENMACHER, ct d., 1971; WAGNERet ol., 1971; GOODMANof the spinal cord. Physiological parameters were moniDOHRMANN r t a/., 1974, 1976; GRIFFITHS et al., 1978) of the central tored and the surgical and experimental procedures were grey matter which then becomes the focus for the spread- carried out as described previously (WALKER et a/., ing hemorrhagic necrosis which occurs at the trauma site 1977a, b). At various times from 5 min to 4 h after laminec(ALLEN,1914; GOODKIN & CAMPBELL, 1969; DUCKERe t tomy or after a 400 g cm injury produced by the method d., 1971; WAGNERet a/., 1971; OSTERHOLM, 1974). Deter- of ALBINet a/. (1967, 1968) the spinal cord was frozen in minations of blood flow to the traumatized segment of sim with the dura intact as described previously ( W A L K ~ K the spinal cord (DUCKER & PEROT, 1971; FAIRHOLM & et a/., 1977~).A 1-li inch piece of intact, total spinal cord was removed from the experimental animal and dissected TURNBULL, 1971: FRIED & GOODKIN,1971; DOHRMANN er ul.. 1973; BINCHAMet ul., 1975; DOHRMANN & ALLEN, in a cryostat without warming beyond -2O’C. The 1975; KOBRINEet ul., 1975; GRIFFITHS,1976; SANDLER& 8-10 mm length of tissue lying directly below the impact TAYLOR, 1976; SMITHef ul., 1978) revealed a markedly de- site was cut into rostral and caudal sections. A core was creased or permanently compromised flow to the central bored from the center of each piece with a squared off grey matter. In the peripheral white matter there was either 2 m m diameter biopsy needle to separate the tissue into a hyperemic response or the blood flow was decreased a central grey core and a ring of surrounding white matter to a lesser extent after an equivalent injury. GRIFFITHSwhich contained a small amount of undissected grey (1976), however, has reported a greater decrease in white matter. The tissue was processed as described previously et al., 1977u, b ) with the exception that the frozen matter blood flow after a 500 g cm injury to canine spinal (WALKER tissues were transferred directly into homogenizer tubes cord Our previous study of the effect of trauma on the energy containing frozen 3 N-HCIO., which were immersed in an metabolites in total canine spinal cord (WALKERet a[,, ethanol-dry ice bath (-20 to -40°C). The frozen tissues 19776) provides data for the average value for the concen- were powdered with a glass rod and then thoroughly tration of the various energy metabolites present in all mixed with the perchloric acid during thawing. This proregions of the spinal cord. Because of the regional differ- cedure greatly facilitated the handling of the small tissue ences in tissue perfusion and vascular response which samples and avoided the delays involved in powdering the occur after spinal trauma, it appeared important to deter- frozen tissue within tygon tubing (SIMON,1968). In addimine the effect of injury on the levels of the labile energy tion, the concentration of EDTA in the neutralizing solumetabolites present in the tissue regionally. Such studies tion was decreased from 15 to 5mM. The concentrations would determine whether differential energy deficits occur of ATP, ADP, AMP, phosphocreatine and lactic acid which might be related to the eventual loss of function. present in the tissue extracts were analyzed fluorometriThese studies would also permit comparison between the cally by the enzymatic assay techniques of LOWRY& PASregional changes in tissue metabolites and the regional SONNEAU (1972). The value for the level of each metabolite changes in blood flow after trauma which have been present in the central grey and peripheral white matter reported by others. In the present study, therefore, we have was obtained by determining the mean value for the conmeasured the tissue levels of the labile energy metabolites centration of metabolite present in these respective regions in the separate grey and white regions of the canine spinal of the rostral and caudal tissue samples. The mean value cord as a function of time after trauma for the first 4 h for the concentration of each metabolite found after experiafter injury. For comparison the regional levels of the mental trauma was compared by the Student t-test with energy metabolites present in the spinal cords of uninjured the mean value for the concentration of the same metabosurgical control dogs have also been determined during lite present in the corresponding region of surgical control the 4 h interval following laminectomy alone. A prelimi- tissue sampled at the same experimental time.
398
Short communication
Phosphocreatine
ATP
Lactate
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FIG. 1. Major metabolites (mmol/kg fresh wt) present in the central grey and the peripheral white matter at the T2-T6 level of the canine spinal cord at various times after laminectomy or after a 400g cm injury delivered as described by ALBIN et al. (1967, 1968). The spinal cord was sampled et al., 19770) and the metabolites were assayed fluorometrically by after in situ freezing (WALKER (1972). Probabilities d0.05 as determined the enzymatic assay techniques of LOWRY& PASSONNEAU by the Student t-test are indicated by an asterisk. The number of animals studied at the consecutive sampling times after laminectomy were 4, 3, 3, 3, 3, 4 and 3 for both the central grey and peripheral white matter. After experimental trauma the numbers of animals studied were 3, 4, 4, 4, 3, 4 and 5 for central grey matter and 4, 4, 5, 5, 3, 4 and 6 for peripheral white matter for the consecutive post trauma sampling times. RESULTS The mean value f S.E.M.for the concentrations of ATP, phosphocreatine and lactic acid are presented in Fig. 1. Figure 1 represents the change in the major metabolites found in the central grey and peripheral white matter of surgical control tissue or of the spinal cord trauma site during the first 4 h after laminectomy or experimental trauma. The mean values +_ S.E.M.for the regional concentrations of ADP and AMP and for the sum of the adenine nucleotides at the trauma site as a function of time after injury are presented in Table 1 . In surgical control dogs laminectomy had little effect on the concentrations of the energy metabolites present in spinal cord white matter. There was a rise in the concentration of phosphocreatine in the central grey matter at 15 min after laminectomy and smaller increases at 30 min and 3 h after surgery. Lactic acid in grey matter was also slightly elevated at 4 h after laminectomy. There was no
effect on the tissue concentrations of ADP or AMP or on the total adenine nucleotides in either region of the surgical control spinal cord with increasing time after laminectomy. After a 400g cm injury to the canine spinal cord, a precipitous decline in the tissue levels of the labile high energy metabolites ATP and phosphocreatine and a rise in the tissue concentrations of lactic acid occurred in both the grey and white regions of the trauma site. These changes reached their maximum within the first 30 min after injury. By 30min post trauma, the time of its greatest depletion, ATP was reduced in the central grey and peripheral white regions of the trauma site to 21 and 57% of the levels present in the respective surgical control tissue sampled at the same experimental time. The phosphocreatine concentrations in the traumatized tissue at 30 min post trauma were 12% in grey matter and 40”/, in white matter compared to the levels present in the corresponding surgical control tissue. During this same time interval there was a greater than 2-fold rise in lactic acid in grey matter and a greater than 10-fold increase in lactic acid in white matter compared to the levels in the corresponding surgical control tissue. The concentration of AMP was also increased at least 2-fold in both regions of the trauma site during the first 30min after injury. The ADP concentrations in both regions of the traumatized tissue were unchanged, but the tissue total adenine nucleotides declined
Short communication TABLE1. EFFECTOF
399
EXPERIMENTAL TRAUMA ON REGIONAL CANINE SPINAL CORD CONCENTRATIONS OF TOTAL ADENINE NUCLEOTIDES
Surgical control 5 min 15 min
30 min lh 2h 3h 4h
Z Ad.
ADP
AMP
nucl.
0.16 _+ 0.05 (4) 0.25 0.03 (3) 0.26 +_ 0.09 (4) 0.21 0.07 (4) 0.16 0.06 (4) 0.14 It 0.05 (3) 0.12 _+ 0.02 (4) 0.16 _+ 0.03 (5)
0.020 k 0.009 (4) 0.008 0.00 (3) 0.13 f 0.06 (4) 0.07 f 0.02 (4) 0.02 k 0.01 (4) 0.04 0.00 (3) 0.05 k 0.01 (4) 0.03 0.01 (5)
1.8 f 0.1 (4) 1.3 & 0.2 (3) 1.0 f 0.3 (4) 0.6 f 0.1 (4) 0.8 1 0 . 2 (4) 1.0 f 0.3 (3) 0.59 rl: 0.04 (4) 1.1 k 0.2 (5)
+
AND
Peripheral white matter
Central grey matter Time post-trauma of tissue sampling
ADP, AMP
Z Ad.
ADP 0.14
0.01
AMP 0.02
(4)
0.14 k 0.04 (4) 0.24 k 0.07 (4) 0.18 It 0.04 (5) 0.13 k 0.03 (5) 0.14 f 0.01 (3) 0.11 & 0.01 (4) 0.14 k 0.03 (6)
k 0.03 (4)
0.020
+ 0.003
(4)
k 0.05 (4) 0.12 & 0.05 (5) 0.08 0.07 (5) 0.02 +_ 0.02 (3) 0.02 k 0.00 (4) 0.007 0.004 (6) 0.13
nucl. 1.60 f 0.07 (4) 1.3 k 0.1 (4) 1.2 k 0.1 (4) 1.0 & 0.1 (5) 1.18 f 0.08 (5) 1.2 f 0.1 (3) 0.89 0.09 (4) 1.2 t0.1 (6)
Data are means & S.E.M.for the concentration expressed in mmol/kg fresh wt of biochemical intermediate present in the central grey or the peripheral white matter of canine thoracic (T2-T6) spinal cord sampled after in situ freezing (WALKERet al., 1977~)at various times after a 400g cm injury produced as described by ALBIN et al. (1967, 1968). Intermediates were assayed fluorometrically by the enzymatic assay technique of LOWRY& PASSONNEAU (1972). Numbers in brackets are the number of experimental animals studied at each sampling time. in both regions of the trauma site after experimental injury. At times greater than 30min after injury the levels of all metabolites measured began to normalize. By 2 h post trauma their concentrations were returning toward the concentrations found in the surgical control tissue sampled immediately after laminectomy . A secondary smaller depletion of ATP, phosphocreatine and total adenine nucleotides occurred at 3 h after injury and was followed by a secondary partial normalization of these metabolites at 4 h post trauma in both regions of the spinal cord. At the time of tissue sampling the physiological data for both the surgical control and the traumatized animals were well within the normal range.
DISCUSSION After an experimental injury to the spinal cord which is severe enough to cause permanent paralysis, the response pattern for the levels of the major energy metabolites is the same in both regions of the trauma site during the 4 h post trauma recovery period which was studied. The effect of injury on the concentration of the labile energy metabolites is considerably greater in the central grey matter than it is in the peripheral white regions of the spinal cord trauma site. During the first 0.5 h after injury, however, the rise in lactic acid levels after trauma is at least 5-fold greater in peripheral white matter than it is in the central grey matter. The coring technique which was used to obtain the central grey matter samples for this study included a small percentage of white matter. If the grey and white regions had been separated in their entirety the differences in the energy metabolite levels in these regions of the trauma site would have been even greater. The regional changes in energy metabolite concentrations in the canine spinal cord are qualitatively similar to those occurring at the trauma site in total canine spinal cord, which we have reported previously WALKERet a/., 1977b).The increased values which we have found for ATP
and phosphocreatine in surgical control tissue sampled at the initial time compared to those reported previously et ul., 1977a) we believe to be due to the modifi(WALKER cation in the method of powdering the tissue used in the present study. Initially it was not clear whether the laminectomy or maintaining the animals for as long as 4 h after surgery would influence the levels of the spinal cord energq metabolites in the absence of the experimental injury. Laminectomy alone has been reported to decrease blood flow to the feline spinal cord (ANDERSON et a/., 1978) and to cause minimal injury to the spinal roots in the monkey (HRESNAHAN et al., 1976). The present results indicate that l-minectomy alone had little effect on the energy metabolite levels in the peripheral white matter of the canine spinal cord. The central grey matter was affected to a somewhat greater extent by the surgical procedure; however, the effects were not large compared to the limits of error that were noted. These results indicate that the regional effects of experimental trauma on spinal cord energy metabolites are easily visible in the presence of a laminectomy. The nature of the present experiments requires the use of sufficient anesthesia to carry out the necessary surgical procedures. It is not possible to duplicate experimentally the unanesthetized state which is present in cases of human spinal cord injury. We have previously utilized barbiturate anesthesia or nitrous oxide-oxygen analgesia combined with metubine paralysis to determine the concentration of energy metabolites present in total uninjured canine spinal ei d,1977a). I t is now cord after in situ freezing (WALKER well documented (YATSUei a/., 1972; SMITHet a/., 1974; et a/., 1976; CORKHILL HOFFet al., 1975; MICHENFELDER et al., 1978) that barbiturate anesthesia significantly reduces tissue infarcation and the eventual neurological deficit of animals subjected to experimental cerebral ischemia. Barbiturate anesthesia also protects functional cerebral tissue by mitigating the effects of severe hypotension and hypoxia on the tissue energy metabolites and prolongs
400
Short communication
cerebral electrical activity during experimental anoxia (MICHENFELDER & THEYE,1973). We have found that it is easier to maintain normal physiological parameters over the experimental time course in barbiturate anesthetized animals than in animals maintained with the more superficial nitrous oxide-oxygen with metubine paralysis anesthetic regimen. The use of barbiturate anesthesia for studies of spinal trauma during which tissue ischemia and hypoxia occur may however have protective effects on the injured tissue. It is possible that the effects of spinal trauma on tissue energy metabolites in the unanesthetized animal may be more severe than the results reported here. The present data for the change in the concentration of the various energy metabolites as a function of time after trauma or after laminectomy permit the calculation of the ratio of the concentration of metabolite in injured tissue to the level of the same metabolite in the corresponding surgical control tissue at the same sampling time. This ratio expresses the effect of trauma alone o n the regional concentration of each energy metabolite. We have compared the regional ratios for ATP obtained in the present study with the blood flow ratios, i.e. the rate of blood flow in injured tissue/rate of blood flow in control tissue calculated from the studies of total (DUCKER& PEROT,1971) and regional (GRIFFITHS, 1976) spinal cord blood flow after trauma in the canine. There is good quantitative agreement between the ratios for ATP and for blood flow after trauma in the canine. Maintenance of normal levels of the labile energy metabolites in any tissue depends upon an adequate blood supply to provide substrate and oxygen for their synthesis. Little is known about the rate of change of either the substrate or the oxygen supply to the injured spinal cord. The data of KELLYef ul. (1970) o n tissue oxygen after spinal trauma are not consistent with the increased levels of ATP which we have found at 4 h after injury. The tissue oxygen data of DUCKER & PEROT (1971) are in much better agreement with the present metabolite data at 4 h after injury; however, DUCKER & PEROT(1971) do not present tissue oxygen data which would account for the striking initial rise in lactic acid which we have found. If the fall in the tissue oxygen concentration is very great compared to the decline in the substrate pool, the large initial rise in lactic acid which we have found is not surprising. In view of the known vasodilatory effects of lactic acid in cerebral tissue (LASSEN,1974), the large initial increase in lactic acid in white matter which we have found may be related to the increased blood flow in white matter reported by BINGHAMet nl. (1975) and KOBRINEet ul. (1975) after spinal trauma in the monkey. Our findings of recovery of ATP levels to 56% in the central grey and 70% in the peripheral white matter compared to levels present in zero time surgical control tissue by as late as 4 h after a permanently paralyzing injury indicate that considerable metabolic recovery has occurred in both regions of the spinal cord in the untreated animal. These results suggest the potential for additional restoration of the tissue energy metabolites which are required for function provided early treatment with a suitable therapeutic regimen were available.
Acknowledgements-This research was supported by the Paralyzed Veterans of America and Grant No. NS 10165 from NIH. We are grateful for the excellent surgical assistance of Mr. WARRENWILSONand the continued interest of Dr. JOHNJ. O’NEILL.
Depurtment of Surgery, Neurosurgery Diiision, Ohio State University College of Medicine. Columbus, OH 43210, U.S.A.
JOANNE C. W A L K ~ R RONALDR. YATES DAVIDYASHON
REFERENCES ALBIN M. S., WHITE R. J.. LOCKE G. S., MASSOP~JST H. E. (1967) Localized spinal L. C., JR. & KRETCHMER cord hypothermia-anesthetic effects and application to spinal cord injury. Anesth. Anulg. curr. Res. 46, 8-15, ALBINM. S., WHITER. J., ACOUSTA-RAU G . & YASHON D. (1968) Study of functional recovery produced by delayed localized cooling after spinal cord injury in primates. J . Neurosury. 29, 113-120. ALLENA. R. (1914) Remarks on the histopathological changes in the spinal cord due to impact, an experimental study. J . nerij. meni. Dis. 41, 141-147. ANDERSOND. K., NICOLOSI G. R., MEANSE. D. & HARTLEY L. E. (1978) Effects of laminectomy on spinal cord blood flow. J . Neurosurg. 48, 131-138. H., FRIEDMAN S. J., MURPHY BINGHAM W. G., GOLDMAN S., YASHOND. & HUNTW. E. (1975) Blood Row in normal and injured monkey spinal cord. J. Neurosurg. 43, 162- 171. BRESNAHAN J. C., KIN(; J. S., MARTING. F. & YASHON D. (1976) A neuroanatomical analysis of spinal cord injury in the Rhesus monkey ( M u c u c ~mulottu). J . Neurol. Sci. 28, 521-542. CORKHILL G., SIVALINGHAM S., REITANJ. A., GII.ROYB. A. & HELPHREY M. G. (1978) Dose dependency of the postinsult protective effect of pentobarbital in the canine experimental stroke model. Stroke 9, 10-12. DOHRMANN G. J. & ALLEN W. E. (1975) Microcirculation of traumatized spinal cord, a correlation of microangiography and blood flow patterns in transitory and permanent paraplegia. J . Truiiniu 15, 1003-1013. G. J.. WAGNERF. C. & Bucu P. C. (1971) DOHRMANN The microvasculature in transitory traumatic paraplegia, an electron microscopic study in the monkey. J . Neurosury. 35, 263-271. DOHRMANN G. J., WICKK. M. & BUW P. C. (1973) Spinal cord blood flow patterns in experimental traumatic paraplegia. J . Neurosurg. 38, 52-58. T. B. & ASSENMACHERD. R. (1969) Microvascular DUCKER response to experimental spinal cord trauma. Sury. Forum 20, 428-430. DUCKERT. B. & PEROTP. L. (1971) Spinal cord oxygen and blood flow in trauma. Surg. Forum. 22, 413-415. DUCKERT. B., KINDTG. W. & KEMPEL. G. (1971) Pathological findings in acute experimental spinal cord trauma. J . Neurosurg. 35, 700-708. 1. M. (1971) MicroangioFAIRHOLMD. J. & TURNBULL graphic study of experimental spinal cord injuries, .I. Neurosurg. 35, 277-286. FRIED L. C. & GOODKINR. (1971) Microangiographic observations of the cxperimentally traumatized spinal cord. J . Neurosurg. 35, 709-714. GOODKIN R. & CAMPIlFI.1. J. B. (1969) Sequential pathologic changes in spinal cord injury: a preliminary report. Sury. Forum 20, 430 432. GOODMAN J. H., BINGHAM W. G. & HUNTW. E. (1974) Edema formation and central hemorrhagic necrosis following impact injury to primate spinal cord. Sury. Forum 25, 440442. I
Short communication GOODMAN J . H., BINGHAM W. G. & H w r W. E. (1976) Untrastructural blood-brain barrier alterations and edema formation in acute spinal trauma. J . Neurosurg. 44, 418-424. GRIPFITHS 1. R . (1976) Spinal cord blood flow after acute experimental cord injury in dogs. J . Neurol. Sci. 27, 147-259. GRIFFITHS I. R.. BURNSN. & CRAWFORD A. R. (1978) Early vascular changes in the spinal grey matter following impact injury. Acra Neuropath.. Berl. 41, 33-39. HUFFJ . T., SMITHA. L.. HANKINSON H. L. & NIELSEN S. L. (1975) Barbiturate protection from cerebral infarction in primates. Stroke 6, 28-33. KELLYD. L., LASSITER K. R. L., CALOCERO J . A. & ALEXANDER E. (1970) Effects of local hypothermia and tissue oxygen studies in experimental paraplegia. J . Neurosurg. 33, 55&563. KOBRINE A. I., DOYLET. F. & MARTINS A. N. (1975) Local spinal cord blood flow in experimental traumatic myelopathy. J. Neurosurg. 42, 144.~149. LASSESN. A. (1974) Control of cerebral circulation in health and disease. Circ. Res. 34, 749--760. LOWRY0. H. & PASSONNEAUJ. V. (1972) A Flexible System of Enzymatic Analysis. Academic Press, New York. MICHENFELDER J. D. & THEYE R. A. (1973) Cerebral protection by thiopental during hypoxia. Anesrhesioloyy 39, 51S-517. MICHENFELDER J. D.. MILLIE J. H. & SUNUTT. M. (1976) Cerebral protection by barbiturate anesthesia. Archs Neural. 33, 345-350.
40 1
OSTPRHOLM J . L. (1974) The pathophysiological response to spinal cord injury, the current status of related research. J . Neurosurg. 40, 5-33. SANDLER A. N. & TATORC. H. (1976) Effect of acute spinal cord compression injury on regional spinal cord blood flow in primates. J . Neurosurg. 45, 66G676. SIMONS. (1968) Elucidation of pyruvate metabolism in cerebral cortex. M.S. thesis, University of Maryland. S. L. & LARSONC. P. SMITHA. L., HOFF J. T., NIELSEN (1974) Barbiturate protection in acute focal cerebral ischemia. Stroke 5, 1-7. SMITHA. J. K., MCCREERY D. B.. BLOEDEL J. R. & C H O ~ J S. N. (1978) Hyperemia, C 0 2 responsiveness, and autoregulation in the white matter following experimental spinal cord injury. J . Neurosurg. 48, 239--251. WAGNER F. c., DOHRMANN G. J. & BUCY P. c'. (1971) Histopathology of transitory traumatic paraplegia in thc monkey. J . Neurosurg. 35, 272-276. WALKERJ. G.. YATESR. R. & YASHON D. (19770) Canine spinal cord energy state after in situ freezing. J . NeuroC ~ C J V29, I . 171 - 173. WALKERJ. G., YATES R. R., O"EII.I. J. J. & YASHOND. (1977b3 Canine spinal cord energy state after experimental trauma. J . Nrurochem. 29, 929-~932. WALKER J. G., YATESR. R. & YASHON D. (1978) Regional spinal cord energy state after experimental trauma. Fcdn Prac. F'edn Am. SUCSp x p Biol. 37, 277 (Abstr.). YATW F. M., DIAMOND I., GRAZIANO C. & LINDQUIST P. (1972) Experimental brain ischemia: protection from irreversible damage with a rapid-acting barbiturate (methohexital). Struke 3, 726-732.
SHORT COMMUNICATION
Regional canine spinal cord energy state after experimental trauma (Receiaed 16 October 1978 Accepted 8 Februury 1979)
el ul., WE HAVE reported (WALKERet a/., 1977b) that a marked nary report of these results has appeared (WALKER decrease in the concentrations of the energy metabolites 1978). ATP and phosphocreatine and an increase in the concenEXPERIMENTAL PROCEDURES trations of lactic acid and AMP occur at the trauma site Adult mongrel dogs weighing 12-14 kg were anestheof total canine spinal cord within 15 min after a midthoracic injury which is sufficient to cause permanent paralysis. tized with pentobarbital (Nembutal, Abbott, 28 mg/kg) The earliest demonstrable pathology after spinal injury with additional dosage as required during the experiment. 1969; A dorsal laminectomy was performed at the T2-T6 level occurs in the vasculature (DUCKER& ASSENMACHER, ct d., 1971; WAGNERet ol., 1971; GOODMANof the spinal cord. Physiological parameters were moniDOHRMANN r t a/., 1974, 1976; GRIFFITHS et al., 1978) of the central tored and the surgical and experimental procedures were grey matter which then becomes the focus for the spread- carried out as described previously (WALKER et a/., ing hemorrhagic necrosis which occurs at the trauma site 1977a, b). At various times from 5 min to 4 h after laminec(ALLEN,1914; GOODKIN & CAMPBELL, 1969; DUCKERe t tomy or after a 400 g cm injury produced by the method d., 1971; WAGNERet a/., 1971; OSTERHOLM, 1974). Deter- of ALBINet a/. (1967, 1968) the spinal cord was frozen in minations of blood flow to the traumatized segment of sim with the dura intact as described previously ( W A L K ~ K the spinal cord (DUCKER & PEROT, 1971; FAIRHOLM & et a/., 1977~).A 1-li inch piece of intact, total spinal cord was removed from the experimental animal and dissected TURNBULL, 1971: FRIED & GOODKIN,1971; DOHRMANN er ul.. 1973; BINCHAMet ul., 1975; DOHRMANN & ALLEN, in a cryostat without warming beyond -2O’C. The 1975; KOBRINEet ul., 1975; GRIFFITHS,1976; SANDLER& 8-10 mm length of tissue lying directly below the impact TAYLOR, 1976; SMITHef ul., 1978) revealed a markedly de- site was cut into rostral and caudal sections. A core was creased or permanently compromised flow to the central bored from the center of each piece with a squared off grey matter. In the peripheral white matter there was either 2 m m diameter biopsy needle to separate the tissue into a hyperemic response or the blood flow was decreased a central grey core and a ring of surrounding white matter to a lesser extent after an equivalent injury. GRIFFITHSwhich contained a small amount of undissected grey (1976), however, has reported a greater decrease in white matter. The tissue was processed as described previously et al., 1977u, b ) with the exception that the frozen matter blood flow after a 500 g cm injury to canine spinal (WALKER tissues were transferred directly into homogenizer tubes cord Our previous study of the effect of trauma on the energy containing frozen 3 N-HCIO., which were immersed in an metabolites in total canine spinal cord (WALKERet a[,, ethanol-dry ice bath (-20 to -40°C). The frozen tissues 19776) provides data for the average value for the concen- were powdered with a glass rod and then thoroughly tration of the various energy metabolites present in all mixed with the perchloric acid during thawing. This proregions of the spinal cord. Because of the regional differ- cedure greatly facilitated the handling of the small tissue ences in tissue perfusion and vascular response which samples and avoided the delays involved in powdering the occur after spinal trauma, it appeared important to deter- frozen tissue within tygon tubing (SIMON,1968). In addimine the effect of injury on the levels of the labile energy tion, the concentration of EDTA in the neutralizing solumetabolites present in the tissue regionally. Such studies tion was decreased from 15 to 5mM. The concentrations would determine whether differential energy deficits occur of ATP, ADP, AMP, phosphocreatine and lactic acid which might be related to the eventual loss of function. present in the tissue extracts were analyzed fluorometriThese studies would also permit comparison between the cally by the enzymatic assay techniques of LOWRY& PASregional changes in tissue metabolites and the regional SONNEAU (1972). The value for the level of each metabolite changes in blood flow after trauma which have been present in the central grey and peripheral white matter reported by others. In the present study, therefore, we have was obtained by determining the mean value for the conmeasured the tissue levels of the labile energy metabolites centration of metabolite present in these respective regions in the separate grey and white regions of the canine spinal of the rostral and caudal tissue samples. The mean value cord as a function of time after trauma for the first 4 h for the concentration of each metabolite found after experiafter injury. For comparison the regional levels of the mental trauma was compared by the Student t-test with energy metabolites present in the spinal cords of uninjured the mean value for the concentration of the same metabosurgical control dogs have also been determined during lite present in the corresponding region of surgical control the 4 h interval following laminectomy alone. A prelimi- tissue sampled at the same experimental time.
398
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Phosphocreatine
ATP
Lactate
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2 3 4 Hours after laminectomy (0)or trauma 0
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2
(0)
FIG. 1. Major metabolites (mmol/kg fresh wt) present in the central grey and the peripheral white matter at the T2-T6 level of the canine spinal cord at various times after laminectomy or after a 400g cm injury delivered as described by ALBIN et al. (1967, 1968). The spinal cord was sampled et al., 19770) and the metabolites were assayed fluorometrically by after in situ freezing (WALKER (1972). Probabilities d0.05 as determined the enzymatic assay techniques of LOWRY& PASSONNEAU by the Student t-test are indicated by an asterisk. The number of animals studied at the consecutive sampling times after laminectomy were 4, 3, 3, 3, 3, 4 and 3 for both the central grey and peripheral white matter. After experimental trauma the numbers of animals studied were 3, 4, 4, 4, 3, 4 and 5 for central grey matter and 4, 4, 5, 5, 3, 4 and 6 for peripheral white matter for the consecutive post trauma sampling times. RESULTS The mean value f S.E.M.for the concentrations of ATP, phosphocreatine and lactic acid are presented in Fig. 1. Figure 1 represents the change in the major metabolites found in the central grey and peripheral white matter of surgical control tissue or of the spinal cord trauma site during the first 4 h after laminectomy or experimental trauma. The mean values +_ S.E.M.for the regional concentrations of ADP and AMP and for the sum of the adenine nucleotides at the trauma site as a function of time after injury are presented in Table 1 . In surgical control dogs laminectomy had little effect on the concentrations of the energy metabolites present in spinal cord white matter. There was a rise in the concentration of phosphocreatine in the central grey matter at 15 min after laminectomy and smaller increases at 30 min and 3 h after surgery. Lactic acid in grey matter was also slightly elevated at 4 h after laminectomy. There was no
effect on the tissue concentrations of ADP or AMP or on the total adenine nucleotides in either region of the surgical control spinal cord with increasing time after laminectomy. After a 400g cm injury to the canine spinal cord, a precipitous decline in the tissue levels of the labile high energy metabolites ATP and phosphocreatine and a rise in the tissue concentrations of lactic acid occurred in both the grey and white regions of the trauma site. These changes reached their maximum within the first 30 min after injury. By 30min post trauma, the time of its greatest depletion, ATP was reduced in the central grey and peripheral white regions of the trauma site to 21 and 57% of the levels present in the respective surgical control tissue sampled at the same experimental time. The phosphocreatine concentrations in the traumatized tissue at 30 min post trauma were 12% in grey matter and 40”/, in white matter compared to the levels present in the corresponding surgical control tissue. During this same time interval there was a greater than 2-fold rise in lactic acid in grey matter and a greater than 10-fold increase in lactic acid in white matter compared to the levels in the corresponding surgical control tissue. The concentration of AMP was also increased at least 2-fold in both regions of the trauma site during the first 30min after injury. The ADP concentrations in both regions of the traumatized tissue were unchanged, but the tissue total adenine nucleotides declined
Short communication TABLE1. EFFECTOF
399
EXPERIMENTAL TRAUMA ON REGIONAL CANINE SPINAL CORD CONCENTRATIONS OF TOTAL ADENINE NUCLEOTIDES
Surgical control 5 min 15 min
30 min lh 2h 3h 4h
Z Ad.
ADP
AMP
nucl.
0.16 _+ 0.05 (4) 0.25 0.03 (3) 0.26 +_ 0.09 (4) 0.21 0.07 (4) 0.16 0.06 (4) 0.14 It 0.05 (3) 0.12 _+ 0.02 (4) 0.16 _+ 0.03 (5)
0.020 k 0.009 (4) 0.008 0.00 (3) 0.13 f 0.06 (4) 0.07 f 0.02 (4) 0.02 k 0.01 (4) 0.04 0.00 (3) 0.05 k 0.01 (4) 0.03 0.01 (5)
1.8 f 0.1 (4) 1.3 & 0.2 (3) 1.0 f 0.3 (4) 0.6 f 0.1 (4) 0.8 1 0 . 2 (4) 1.0 f 0.3 (3) 0.59 rl: 0.04 (4) 1.1 k 0.2 (5)
+
AND
Peripheral white matter
Central grey matter Time post-trauma of tissue sampling
ADP, AMP
Z Ad.
ADP 0.14
0.01
AMP 0.02
(4)
0.14 k 0.04 (4) 0.24 k 0.07 (4) 0.18 It 0.04 (5) 0.13 k 0.03 (5) 0.14 f 0.01 (3) 0.11 & 0.01 (4) 0.14 k 0.03 (6)
k 0.03 (4)
0.020
+ 0.003
(4)
k 0.05 (4) 0.12 & 0.05 (5) 0.08 0.07 (5) 0.02 +_ 0.02 (3) 0.02 k 0.00 (4) 0.007 0.004 (6) 0.13
nucl. 1.60 f 0.07 (4) 1.3 k 0.1 (4) 1.2 k 0.1 (4) 1.0 & 0.1 (5) 1.18 f 0.08 (5) 1.2 f 0.1 (3) 0.89 0.09 (4) 1.2 t0.1 (6)
Data are means & S.E.M.for the concentration expressed in mmol/kg fresh wt of biochemical intermediate present in the central grey or the peripheral white matter of canine thoracic (T2-T6) spinal cord sampled after in situ freezing (WALKERet al., 1977~)at various times after a 400g cm injury produced as described by ALBIN et al. (1967, 1968). Intermediates were assayed fluorometrically by the enzymatic assay technique of LOWRY& PASSONNEAU (1972). Numbers in brackets are the number of experimental animals studied at each sampling time. in both regions of the trauma site after experimental injury. At times greater than 30min after injury the levels of all metabolites measured began to normalize. By 2 h post trauma their concentrations were returning toward the concentrations found in the surgical control tissue sampled immediately after laminectomy . A secondary smaller depletion of ATP, phosphocreatine and total adenine nucleotides occurred at 3 h after injury and was followed by a secondary partial normalization of these metabolites at 4 h post trauma in both regions of the spinal cord. At the time of tissue sampling the physiological data for both the surgical control and the traumatized animals were well within the normal range.
DISCUSSION After an experimental injury to the spinal cord which is severe enough to cause permanent paralysis, the response pattern for the levels of the major energy metabolites is the same in both regions of the trauma site during the 4 h post trauma recovery period which was studied. The effect of injury on the concentration of the labile energy metabolites is considerably greater in the central grey matter than it is in the peripheral white regions of the spinal cord trauma site. During the first 0.5 h after injury, however, the rise in lactic acid levels after trauma is at least 5-fold greater in peripheral white matter than it is in the central grey matter. The coring technique which was used to obtain the central grey matter samples for this study included a small percentage of white matter. If the grey and white regions had been separated in their entirety the differences in the energy metabolite levels in these regions of the trauma site would have been even greater. The regional changes in energy metabolite concentrations in the canine spinal cord are qualitatively similar to those occurring at the trauma site in total canine spinal cord, which we have reported previously WALKERet a/., 1977b).The increased values which we have found for ATP
and phosphocreatine in surgical control tissue sampled at the initial time compared to those reported previously et ul., 1977a) we believe to be due to the modifi(WALKER cation in the method of powdering the tissue used in the present study. Initially it was not clear whether the laminectomy or maintaining the animals for as long as 4 h after surgery would influence the levels of the spinal cord energq metabolites in the absence of the experimental injury. Laminectomy alone has been reported to decrease blood flow to the feline spinal cord (ANDERSON et a/., 1978) and to cause minimal injury to the spinal roots in the monkey (HRESNAHAN et al., 1976). The present results indicate that l-minectomy alone had little effect on the energy metabolite levels in the peripheral white matter of the canine spinal cord. The central grey matter was affected to a somewhat greater extent by the surgical procedure; however, the effects were not large compared to the limits of error that were noted. These results indicate that the regional effects of experimental trauma on spinal cord energy metabolites are easily visible in the presence of a laminectomy. The nature of the present experiments requires the use of sufficient anesthesia to carry out the necessary surgical procedures. It is not possible to duplicate experimentally the unanesthetized state which is present in cases of human spinal cord injury. We have previously utilized barbiturate anesthesia or nitrous oxide-oxygen analgesia combined with metubine paralysis to determine the concentration of energy metabolites present in total uninjured canine spinal ei d,1977a). I t is now cord after in situ freezing (WALKER well documented (YATSUei a/., 1972; SMITHet a/., 1974; et a/., 1976; CORKHILL HOFFet al., 1975; MICHENFELDER et al., 1978) that barbiturate anesthesia significantly reduces tissue infarcation and the eventual neurological deficit of animals subjected to experimental cerebral ischemia. Barbiturate anesthesia also protects functional cerebral tissue by mitigating the effects of severe hypotension and hypoxia on the tissue energy metabolites and prolongs
400
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cerebral electrical activity during experimental anoxia (MICHENFELDER & THEYE,1973). We have found that it is easier to maintain normal physiological parameters over the experimental time course in barbiturate anesthetized animals than in animals maintained with the more superficial nitrous oxide-oxygen with metubine paralysis anesthetic regimen. The use of barbiturate anesthesia for studies of spinal trauma during which tissue ischemia and hypoxia occur may however have protective effects on the injured tissue. It is possible that the effects of spinal trauma on tissue energy metabolites in the unanesthetized animal may be more severe than the results reported here. The present data for the change in the concentration of the various energy metabolites as a function of time after trauma or after laminectomy permit the calculation of the ratio of the concentration of metabolite in injured tissue to the level of the same metabolite in the corresponding surgical control tissue at the same sampling time. This ratio expresses the effect of trauma alone o n the regional concentration of each energy metabolite. We have compared the regional ratios for ATP obtained in the present study with the blood flow ratios, i.e. the rate of blood flow in injured tissue/rate of blood flow in control tissue calculated from the studies of total (DUCKER& PEROT,1971) and regional (GRIFFITHS, 1976) spinal cord blood flow after trauma in the canine. There is good quantitative agreement between the ratios for ATP and for blood flow after trauma in the canine. Maintenance of normal levels of the labile energy metabolites in any tissue depends upon an adequate blood supply to provide substrate and oxygen for their synthesis. Little is known about the rate of change of either the substrate or the oxygen supply to the injured spinal cord. The data of KELLYef ul. (1970) o n tissue oxygen after spinal trauma are not consistent with the increased levels of ATP which we have found at 4 h after injury. The tissue oxygen data of DUCKER & PEROT (1971) are in much better agreement with the present metabolite data at 4 h after injury; however, DUCKER & PEROT(1971) do not present tissue oxygen data which would account for the striking initial rise in lactic acid which we have found. If the fall in the tissue oxygen concentration is very great compared to the decline in the substrate pool, the large initial rise in lactic acid which we have found is not surprising. In view of the known vasodilatory effects of lactic acid in cerebral tissue (LASSEN,1974), the large initial increase in lactic acid in white matter which we have found may be related to the increased blood flow in white matter reported by BINGHAMet nl. (1975) and KOBRINEet ul. (1975) after spinal trauma in the monkey. Our findings of recovery of ATP levels to 56% in the central grey and 70% in the peripheral white matter compared to levels present in zero time surgical control tissue by as late as 4 h after a permanently paralyzing injury indicate that considerable metabolic recovery has occurred in both regions of the spinal cord in the untreated animal. These results suggest the potential for additional restoration of the tissue energy metabolites which are required for function provided early treatment with a suitable therapeutic regimen were available.
Acknowledgements-This research was supported by the Paralyzed Veterans of America and Grant No. NS 10165 from NIH. We are grateful for the excellent surgical assistance of Mr. WARRENWILSONand the continued interest of Dr. JOHNJ. O’NEILL.
Depurtment of Surgery, Neurosurgery Diiision, Ohio State University College of Medicine. Columbus, OH 43210, U.S.A.
JOANNE C. W A L K ~ R RONALDR. YATES DAVIDYASHON
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