JOURNAL OF NEUROTRAUMA Volume 7, Number 1, 1990 Mary Ann Liebert, Inc., Publishers
Mitochondrial Metabolism Following Traumatic Brain Injury in Rats ROBERT VINK,1 VALERIE A. HEAD,2 PETER J. ROGERS,3 TRACY K. McINTOSH,4 and ALAN I. FADEN2
ABSTRACT
Although a number of studies of traumatic brain injury have implicated mitochondrial dysfunction as a cause of altered posttraumatic energy metabolism, no studies to date have isolated mitochondria and measured their respiratory capacity following trauma. The present study sought to determine whether mitochondrial capacity for oxidative phosphorylation is adversely affected by fluid-percussion-induced traumatic brain injury in rats. Prior to brain injury, the mitochondrial respiratory control ratio was 4.3 ± 0.2 and the ratio of nmoles of ADP phosphorylated per natom oxygen consumed (ADP/O ratio) was 2.66 ± 0.09. After injury (2.8 atm; t- 4 h), there were slight but not significant alterations in ADP/O ratio (2.41 ± 0.07) and state 3 respiratory rate (ADP stimulated); however, there were no changes in the respiratory control ratio. These data suggest that traumatic brain injury, unlike ischemia, does not cause uncoupling of ATP synthesis from respiration, and that brain mitochondria are quite resistant to trauma-induced injury.
INTRODUCTION
following neurotrauma involves a secondary injury of biochemical changes including release of endogenous response that is made up of a series autodestructive factors such as opioid peptides, excitotoxins, oxygen free radicals, and membrane breakdown products (Siesjo, 1981 ; Siesjo and Weiloch, 1985; Faden, 1986; Kontos and Povlishock, 1986; Faden et al., 1989). Alterations in ionic fluxes, especially those of calcium and magnesium, have also been observed (Young, 1987; Vink et al., 1988a). Mitochondrial dysfunction has also been implicated as a possible factor in injury (Yang et al., 1985; Vink et al., 1988b), causing a decreased bioenergetic capacity of tissue and thereby limiting certain protective mechanisms. Despite the implications, no studies have actually isolated mitochondria following neurotrauma and determined their respiratory capacity.
Development
of irreversible tissue damage
'Department of Chemistry and Biochemistry, James Cook University, Townsville, Australia, department of Neurology, University of California, San Francisco, and Center for Neural Injury,
Administration Medical Center, San Francisco, California. 3School of Science, Griffith University, Nathan, Brisbane, Australia. 4Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut.
21
Veterans
VINK ET AL. A number of studies have, however, examined mitochondrial function following cerebral ischemia (Ozawa etal., 1967;Rehncronaetal., 1979; Hilleredet al, 1984; Sims and Pulsinelli, 1987). They have shown that ischemia causes uncoupling of ATP synthesis from respiration, leading to a reduced capacity for oxidative phosphorylation. Furthermore, significant declines in state 3 respiration (ADP stimulated) have been noted (Ginsburg et al., 1977), with the degree of depression tending to be associated with eventual outcome. Focal reductions in blood flow are also a feature of fluid-percussion-induced traumatic brain injury (Yuan et al., 1986; Yamakami and Mclntosh, 1989). Together with the well-characterized decline in bioenergetic parameters of brain following traumatic injury (Yang et al., 1985; Vink et al., 1988b,c), it becomes important to establish whether alterations in mitochondrial metabolism as observed in ischemia, occur similarly following brain trauma. In the present study we demonstrate in isolated mitochondria that there is no mitochondrial uncoupling or depression of state 3 respiration following traumatic brain injury in rats.
MATERIALS AND METHODS Induction
of Traumatic Brain Injury
Male Sprague-Dawley rats (350-450 g) were prepared and injured by lateral fluid percussion as described in detail elsewhere (Vink et al., 1988b; Mclntosh et al., 1989). Animals (n 21) were anesthetized with 60 mg/kg sodium pentobarbital, cannulated for monitoring of arterial blood pressure and gases, and a craniectomy was performed over the left parietal cortex. After surgery and throughout the posttraumatic experimental procedures, the animals were maintained on a constant infusion of sodium pentobarbital (15 mg/kg/h). Fluid-percussion injury was induced at 2.8 atm (« = 5). This form of injury consists of rapidly injecting a saline pressure pulse into the cranial cavity to produce a transient deformation of the brain, resulting in irreversible injury and associated neurologic deficit. In addition to the 5 injured animals, 5 animals served as noninjured, anesthesia controls, while another 11 animals served as "normal" controls (anesthetized and immediately decapitated). =
Isolation
of Mitochondria
Mitochondria were isolated at 4 h posttrauma using a modified version of the method described by Lai and Clark (1979). After they were decapitated, the brain was rapidly removed (< 30 s), dissected, and the left hemisphere placed in ice-cold isolation medium (0.225 M mannitol, 0.075 M sucrose, 5 mM Tris, 120 mM KC1,1 mM EGTA, 0.1 % BSA, pH 7.4) where it was finely chopped. After being washed twice, the tissue was resuspended in isolation buffer containing 0.5% Nagarse and 0.5% BSA and homogenized using a handheld teflon/glass homogenizer. The homogenate was then centrifuged twice (2000 g x 10 min; 5°C) before the supernatant was recentrifuged at 12,500 g for 10 min. After the pellet was resuspended in 3% Ficoll in respiratory buffer (20 mM potassium phosphate, 160 mM KC1,0.1 mM EGTA, pH 7.1), the suspension was layered onto a 6% Ficoll respiratory solution and centrifuged for 30 min (11,000 g). The resultant pellet was washed once before being finally resuspended in 50 jxl of respiratory buffer.
Protein Determinations All mitochondrial preparations were subject to protein determinations using Lowry assays (Lowry et al., 1951). Previous studies have shown that the use of Nagarse in isolating brain mitochondria may degrade a significant amount of mitochondrial protein; however, it does not have a detrimental effect on respiratory parameters (Wilson, 1987).
Respiratory Assays Mitochondria were assayed for rate of oxygen consumption using a Clarke-type oxygen electrode (Yellow Springs Instrument Co.) as described by Estabrook (1967). Mitochondrial protein (0.5-1.0 mg) was added to 600 pi respiratory buffer in the assay chamber (25°C). Substrate (glutamate/malate) was added to 5 mM final 22
MITOCHONDRIAL METABOLISM AFTER BRAIN INJURY state 3 respiration was induced by the addition of 100 or 150 nmoles of ADP. Each mitochondrial suspension isolated from individual animals was assayed at least four times. Respiratory control ratio (RCR) was calculated by dividing rate of oxygen consumption in state 3 (ADP stimulated) by that in state 4 (ADP depleted). ADP/O ratio was determined by dividing the number of nmoles of ADP phosphorylated during state 3 respiration by the number of natoms of oxygen consumed.
concentration, and
Data
Analysis
Data were analyzed for statistical significance using analysis of variance (ANOVA) followed by individual Student Newman-Keuls tests. A value for p of < 0.05 was considered significant. All data are expressed as mean ± standard error.
RESULTS Total mitochondrial protein yield from the left hemisphere of normal uninjured controls was 2.5 ± 0.2 mg. Results reported by others for brain mitochondrial isolations (Rehnchrona et al., 1979; Fullered and Ernster, 1983) show mean rat whole brain yields of 7.6-7.9 mg with and without Nagarse treatment, respectively. If we consider that in the present studies the cerebellum and part of the brainstem were removed, and only the left hemisphere was used for mitochondrial isolation, the present yield is comparable to the protein yields reported previously. Traumatic brain injury did not cause any significant changes in total mitochondrial protein yield at 4 h posttrauma (2.2 ± 0.1 mg), thus indicating no selective loss of damaged mitochondria following trauma.
Figure 1 shows a typical oxygraph trace obtained from mitochondria isolated from normal controls. Prior to 0.5 mg mitochondrial
protein
Glutamate/malate (5mM) 100 nmoles ADP
150 nmoles ADP
20 natoms O min
FIG. 1. Typical oxygen electrode recording of respiring brain mitochondria isolated from normal controls. Concentration of additions is final concentration in the sample chamber. All experiments were performed at 25°C.
23
VINK ET AL. Table 1. Respiratory Control Ratios (RCR), ADP/O Ratios, and Respiratory Rates in Brain Mitochondria Isolated from Normal Control Animals, Anesthetized Control Animals, and Brain-Injured Animals
State 3 Normal controls (t = 0 b.) Anesthesia controls (t 4 h) Injured animals (t 4 h) =
=
RCR
A DPIO
4.3 ± 0.2 4.1+0.2 4.2 + 0.2
2.66 ± 0.09 2.53 + 0.14 2.41+0.07
State 4
(natoms OImini mg protein) 105 + 5 103 + 5 120 + 7
26 ± 3 26 + 4 29 + 3
the addition of ADP, oxygen consumption was 26 ± 3 natoms (O/min/mg mitochondrial protein). With the addition of ADP, oxygen consumption increased to 105 ± 5 natoms (O/min/mg mitochondrial protein) before declining to approximate pre-ADP levels as ADP became depleted. The calculated RCR from normal controls was 4.3 ± 0.2, while the ADP/O ratio was determined to be 2.66 ± 0.09. These values are in excellent agreement with previously published values for isolated brain mitochondria (Lai and Clark, 1979; Rehncrona
etal., 1979).
The effects of pentobarbital anesthesia on brain mitochondrial respiratory parameters are summarized in Table 1. There were no changes in RCR, ADP/O ratio, or state 3 respiration rate induced by 4 h of pentobarbital anesthesia. Animals subjected to fluid-percussion-induced brain injury demonstrated similar pre-ADP basal rates of oxygen consumption (29 ± 3 natoms/min/mg protein) as controls. However, with the initiation of state 3 respiration, oxygen consumption rate showed a trend towards an increase that did not reach significance (120 ± 7 natoms [O/min/mg protein]; p 0.08). In a similar fashion, while indicating a trend towards a decline, the calculated ADP/O ratio of 2.41 ±0.07 was not significantly lower than that observed for the normal controls (p 0.08). There was no change in the calculated RCR value posttrauma (4.2 ± 0.2) as compared to normal controls (Table 1). =
=
DISCUSSION
Despite the suggestion that traumatic injury to the CNS causes mitochondrial dysfunction (Yang et al.,
1985; Vink et al., 1988b), no studies to date have isolated mitochondria from traumatized tissue and assayed them directly for respiratory capacity. The present study has examined the effects of traumatic brain injury in rats on cerebral mitochondrial metabolism, and shows that mitochondrial oxidative capacity is essentially
preserved following trauma, with no evidence of any alterations that might reflect an ischémie event. Previous studies of cerebral mitochondrial function following CNS injury have concentrated on the effects of cerebral ischemia. Brain ischemia causes a reduction in RCR, indicative of uncoupling (Ozawa et al., 1967; Sims and Pulsinelli, 1987). The stimulatory effect of ADP addition on respiratory rate (state 3) is reduced (Fullered et al., 1984), particularly during and after an episode of incomplete ischemia (Rehncrona et al., 1979). This is unlike what we have described above for trauma, after which the RCR remains unchanged, and the oxygen consumption rate in state 3 does not decrease, but demonstrates a tendency towards increasing, albeit insignificantly, when compared to control animals. Our results are consistent with the observations of Yamakami and Mclntosh (1989), who have shown in this fluid-percussion model, at the level of injury used in the present experiments, that although significant decreases occur in regional blood flow at the injury site, these declines do not approach frank ischemia (i.e., 15-25 ml/100 g/min). The present studies have evaluated the mitochondrial respiratory activity at 4 h posttrauma. Previous studies have shown that this time is critical with respect to neurologic and biochemical recovery of animals following trauma (Vink et al., 1988b, c). For example, at 4 h posttrauma, the ratio of phosphocreatine to inorganic phosphate (Vink et al., 1988b) and the intracellular free magnesium concentration (Vink et al., 1988a) are linearly correlated to injury severity. Thus animals that demonstrate significant recovery posttrauma already have significantly improved bioenergetic status by 4 h posttrauma (Vink et al., 1988c). The present study demonstrates that the bioenergetic status at 4 h posttrauma is not related to mitochondrial 24
MITOCHONDRIAL METABOLISM AFTER BRAIN INJURY
respiratory function, which is essentially preserved at this time despite development of significant neurologic deficit following this level of injury (Mclntosh et al., 1989). Although no statistical differences are demonstrated for ADP/O ratio and state 3 respiration rate between control animals and injured animals, there were trends toward change that did not achieve statistical significance (p 0.08). With increased numbers of experimental animals, this p value would presumably achieve statistical significance. However, because the anesthesia alone tends to modify mitochondrial respiration, albeit insignificantly in itself (Ginsburg et al., 1977), the interpretation of the mitochondrial respiratory data necessitates comparisons with anesthetized control animals at similar times to the experimental group. In this respect, when compared to anesthesia controls, the ADP/O value did not demonstrate any trends toward change whereas the state 3 respiratory rate again showed trends toward an increase (p 0.08). Similar trends were also observed with state 4 respiration rates (Table 1). Respiration rate is a function of cytosolic phosphorylation potential (Ereckinska and Wilson, 1982), and decline in cytosolic phosphorylation potential (AGp) would tend to increase mitochondrial respiration rate. We have previously shown that traumatic brain injury in rats decreases AGp (Vink et al., 1988c). This decline is linearly related to the observed decline in intracellular free Mg2+ concentration (Vink et al., 1988c). It may be that the tendency toward increased respiration rate following trauma may be related to the profound fall in AGp. That these trends did not achieve statistical significance is puzzling in view of the magnitude of the decline in AGp observed. One can speculate that the effects of AGp on mitochondrial respiration are attenuated by a decline in total magnesium (Vink et al., 1988a) that would tend to decrease respiration rate (Ginsburg et al., 1977). Magnesium concentration is known to regulate respiratory transitions in mitochondria (Masini et al., 1983). Moreover, any alterations in free and total magnesium may also affect other mitochondrial functions, such as activity of the ADP/ATP translocase, ATP synthase, and mitochondrial creatine kinase, all of which are influenced by divalent cations, particularly magnesium (Lee et al., 1970; Saks et al., 1975; Klingenberg, 1977; Moreno-Sanchez et al., 1985; Fagian et al., 1986; Kramer et al., 1986). Determining which of these processes are being affected, resulting in decreased bioenergetic state, requires further investigation. =
=
ACKNOWLEDGMENTS This work was supported in part by Centers for Disease Control Grant 902269 to A.I.F., and a Veterans Administration Merit Review Grant to T.K.M. R. Vink is a recipient of a Queen Elizabeth II Fellowship and is supported by the Australian National Research Fellowships Advisory Committee.
REFERENCES ERECINSKA, M., and WILSON, D.F. (1982). Regulation of cellular energy metabolism. J. Membr. Biol. 70, 1-14. ESTABROOK, R.W. (1967). Mitochondrial respiratory control and the polarographic measurement of ADP:0 ratios. Methods Enzymol. 10,41-47. FADEN, A.I. (1986). Neuropeptides in central nervous system injury. Arch. Neurol. 43, 501-504. FADEN, A.I., DEMEDIUK, P., PANTER, S., and VINK, R. (1989). Excitatory amino acids, n-methyl-D-aspartate receptors and traumatic brain injury. Science 244, 798-800. FAGIAN, M.M., DA SILVA, L.P., and VERCESI, A.E. (1986). Inhibition of oxidative phosphorylation by Ca2+ or Sr2+: A competition with Mg2+ for the formation of adenine nucleotide complexes. Biochim. Biophys. Acta 852, 262-268.
GINSBURG, M.D., MELA, L., WROBEL-KUHL, K., and REIVICH, M. (1977). Mitochondrial metabolism following bilateral cerebral ischemia in the gerbil. Ann. Neurol. 1, 519-527. HILLERED, L., and ERNSTER, L. (1983). Respiratory activity of isolated brain mitochondria following in vitro exposure to oxygen radicals. J. Cereb. Blood Flow Metab. 3, 207-214. 25
VINK ET AL.
HILLERED, L., SIESJO, B .K., and ARFORS, K.E. (1984). Mitochondrial response to transient forebrain ischemia and recirculation in the rat. J. Cereb. Blood Flow Metab.
4, 438-446.
KLINGENBERG, M. (1977). Interaction of the ADP, ATP transport with the system of oxidative phosphorylation. In: Structure and Function of Energy-Transducing Membranes. K. Van Dam and F.F. Van Gelder (eds.). Elsevier: Amsterdam, pp. 275-282. KONTOS, H.A., and POVLISHOCK, J.T. (1986). Oxygen radicals in brain injury. CNS Trauma 3, 257-263. KRAMER, R., MAYR, U., HEBERGER, C, and TSOMPANIDOU, S. (1986). Activation of the ADP/ATP carrier from mitochondria by cationic effectors. Biochim. Biophys. Acta. 855, 201-210. LAI, J.C.K., and CLARK, J.B. (1979). Preparation of synaptic and nonsynaptic mitochondria from mammalian brain. Methods Enzymol. 55, 51-59. LEE, N.M., WIEDEMANN, I., JOHNSON, K.L., SKILLETER, D.N., and KUN, E. (1970). The dependence of oxidative phosphorylation and ATPase of liver mitochondria on bound Mg2+. Biochem. Biophys. Res. Commun. 40, 1058-1062.
LOWRY, O.H., ROSEBROUGH, N., FARR, A.L., and RANDALL, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MASINI, A., CECCARELLI-STANZANI, D., and MUSCATELLO, U. (1983). The role of Mg2+ in the regulation of the structural and functional steady states in rat liver mitochondria. J. Bioenerg. Biomembr. 15, 217-234. McINTOSH, T.K., VINK, R., NOBLE, L.J., YAMAKAMI, I., FERNYAK, S.E., and FADEN, A.I. (1989). Traumatic brain injury in the rat: Characterization of a lateral fluid percussion injury model. Neuroscience, 28, 233-244.
MORENO-SANCHEZ, R. (1985). Contribution of the translocator of adenine nucleotides and the ATP synthase to the control of oxidative phosphorylation and
arsenylation in liver mitochondria. J. Biol. Chem. 260,
12554-12560.
OZAWA, K., SETA, K., ARAKI, H., and HANDA, H. (1967). The effect of ischemia on mitochondrial metabolism. J. Biochem.
61, 512-514.
REHNCRONA, S., MELA, L., and SIESJO, B.K. (1979). Recovery of brain mitochondrial function in the rat after complete and incomplete cerebral ischemia. Stroke 10, 437-446. SAKS, V.A., CHERNOUSOVA, G.B., GUKOVSKY, D.E., SMIRNOV, V.N., and CHAZOV, E.I. (1975). Study of energy transport in heart cells. Mitochondrial isoenzyme of creatine phosphokinase: Kinetic properties and regulatory action of Mg2+ ions. Eur. J. Biochem.
57, 273-290.
SIESJO, B.K. (1981). Cell damage in the brain: A speculative synthesis. J. Cereb. Blood Flow Metab. 1, 155-181. SIESJO, B.K., and WEILOCH, T. (1985). Brain injury: Neurochemical aspects. In: Central Nervous System Trauma
Byrd Press/NINCDS: Bethesda. mitochondrial respiration in selectively vulnerable
Status Report 1985. D.P. Becker and J.T. Povlishock (eds.). William
SIMS, N.R., and PULSINELLI, W.A. (1987). Altered
subregions following transient forebrain ischemia in the rat. J. Neurochem. 49,
brain
1367-1374.
VINK, R., McINTOSH, T.K., DEMEDIUK, P., WEINER, M.W., and FADEN, A.I. (1988a). Decline in intracellular free Mg2+ is associated with irreversible tissue injury after brain trauma. J. Biol. Chem. 263, 757-761. VINK, R., McINTOSH, T.K., YAMAKAMI, I., and FADEN, A.I. (1988b). 31P NMR characterization of graded
Magnet Reson. Med. 6, 37-48. VINK, R., FADEN, A.I., and McINTOSH, T.K. (1988c). Changes in cellular bioenergetic state following graded traumatic brain injury in rats: determination by 31phosphorus MRS. J. Neurotrauma 5, 365-380. WILSON, J.E. (1987). Should nagarse be used during the isolation of brain mitochondria? Neurochem. Res. 9,831-834. YAMAKAMI, I., and McINTOSH, T.K. (1989). Effects of traumatic brain injury on regional cerebral blood flow in rats as measured with radiolabeled microspheres. J. Cereb. Blood Flow Metab. 9, 117-124. YANG, M.S., De WITT, D.S., BECKER, D.P., and HAYES, R.L. (1985). Regional brain metabolite levels following mild experimental head injury in the cat. J. Neurosurg. 63, 617-621. YOUNG, W. (1987). The post-injury responses in trauma and ischemia: Secondary injury or protective mechanisms. CNS Trauma 4, 27-51. traumatic brain injury in rats.
26
MITOCHONDRIAL METABOLISM AFTER BRAIN INJURY
YUAN, X.-Q., PROUGH, D.S., SMITH, T.L., and DeWITT, D.S. (1988). The effects of traumatic brain injury on
regional cerebral blood flow in rats. J. Neurotrauma 5, 289-301.
Address reprint requests to: Robert Vink, Ph.D. Department of Chemistry and Biochemistry James Cook University of North Queensland Townsville, Queensland 4811 Australia
27
Mitochondrial Metabolism Following Traumatic Brain Injury in Rats ROBERT VINK,1 VALERIE A. HEAD,2 PETER J. ROGERS,3 TRACY K. McINTOSH,4 and ALAN I. FADEN2
ABSTRACT
Although a number of studies of traumatic brain injury have implicated mitochondrial dysfunction as a cause of altered posttraumatic energy metabolism, no studies to date have isolated mitochondria and measured their respiratory capacity following trauma. The present study sought to determine whether mitochondrial capacity for oxidative phosphorylation is adversely affected by fluid-percussion-induced traumatic brain injury in rats. Prior to brain injury, the mitochondrial respiratory control ratio was 4.3 ± 0.2 and the ratio of nmoles of ADP phosphorylated per natom oxygen consumed (ADP/O ratio) was 2.66 ± 0.09. After injury (2.8 atm; t- 4 h), there were slight but not significant alterations in ADP/O ratio (2.41 ± 0.07) and state 3 respiratory rate (ADP stimulated); however, there were no changes in the respiratory control ratio. These data suggest that traumatic brain injury, unlike ischemia, does not cause uncoupling of ATP synthesis from respiration, and that brain mitochondria are quite resistant to trauma-induced injury.
INTRODUCTION
following neurotrauma involves a secondary injury of biochemical changes including release of endogenous response that is made up of a series autodestructive factors such as opioid peptides, excitotoxins, oxygen free radicals, and membrane breakdown products (Siesjo, 1981 ; Siesjo and Weiloch, 1985; Faden, 1986; Kontos and Povlishock, 1986; Faden et al., 1989). Alterations in ionic fluxes, especially those of calcium and magnesium, have also been observed (Young, 1987; Vink et al., 1988a). Mitochondrial dysfunction has also been implicated as a possible factor in injury (Yang et al., 1985; Vink et al., 1988b), causing a decreased bioenergetic capacity of tissue and thereby limiting certain protective mechanisms. Despite the implications, no studies have actually isolated mitochondria following neurotrauma and determined their respiratory capacity.
Development
of irreversible tissue damage
'Department of Chemistry and Biochemistry, James Cook University, Townsville, Australia, department of Neurology, University of California, San Francisco, and Center for Neural Injury,
Administration Medical Center, San Francisco, California. 3School of Science, Griffith University, Nathan, Brisbane, Australia. 4Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut.
21
Veterans
VINK ET AL. A number of studies have, however, examined mitochondrial function following cerebral ischemia (Ozawa etal., 1967;Rehncronaetal., 1979; Hilleredet al, 1984; Sims and Pulsinelli, 1987). They have shown that ischemia causes uncoupling of ATP synthesis from respiration, leading to a reduced capacity for oxidative phosphorylation. Furthermore, significant declines in state 3 respiration (ADP stimulated) have been noted (Ginsburg et al., 1977), with the degree of depression tending to be associated with eventual outcome. Focal reductions in blood flow are also a feature of fluid-percussion-induced traumatic brain injury (Yuan et al., 1986; Yamakami and Mclntosh, 1989). Together with the well-characterized decline in bioenergetic parameters of brain following traumatic injury (Yang et al., 1985; Vink et al., 1988b,c), it becomes important to establish whether alterations in mitochondrial metabolism as observed in ischemia, occur similarly following brain trauma. In the present study we demonstrate in isolated mitochondria that there is no mitochondrial uncoupling or depression of state 3 respiration following traumatic brain injury in rats.
MATERIALS AND METHODS Induction
of Traumatic Brain Injury
Male Sprague-Dawley rats (350-450 g) were prepared and injured by lateral fluid percussion as described in detail elsewhere (Vink et al., 1988b; Mclntosh et al., 1989). Animals (n 21) were anesthetized with 60 mg/kg sodium pentobarbital, cannulated for monitoring of arterial blood pressure and gases, and a craniectomy was performed over the left parietal cortex. After surgery and throughout the posttraumatic experimental procedures, the animals were maintained on a constant infusion of sodium pentobarbital (15 mg/kg/h). Fluid-percussion injury was induced at 2.8 atm (« = 5). This form of injury consists of rapidly injecting a saline pressure pulse into the cranial cavity to produce a transient deformation of the brain, resulting in irreversible injury and associated neurologic deficit. In addition to the 5 injured animals, 5 animals served as noninjured, anesthesia controls, while another 11 animals served as "normal" controls (anesthetized and immediately decapitated). =
Isolation
of Mitochondria
Mitochondria were isolated at 4 h posttrauma using a modified version of the method described by Lai and Clark (1979). After they were decapitated, the brain was rapidly removed (< 30 s), dissected, and the left hemisphere placed in ice-cold isolation medium (0.225 M mannitol, 0.075 M sucrose, 5 mM Tris, 120 mM KC1,1 mM EGTA, 0.1 % BSA, pH 7.4) where it was finely chopped. After being washed twice, the tissue was resuspended in isolation buffer containing 0.5% Nagarse and 0.5% BSA and homogenized using a handheld teflon/glass homogenizer. The homogenate was then centrifuged twice (2000 g x 10 min; 5°C) before the supernatant was recentrifuged at 12,500 g for 10 min. After the pellet was resuspended in 3% Ficoll in respiratory buffer (20 mM potassium phosphate, 160 mM KC1,0.1 mM EGTA, pH 7.1), the suspension was layered onto a 6% Ficoll respiratory solution and centrifuged for 30 min (11,000 g). The resultant pellet was washed once before being finally resuspended in 50 jxl of respiratory buffer.
Protein Determinations All mitochondrial preparations were subject to protein determinations using Lowry assays (Lowry et al., 1951). Previous studies have shown that the use of Nagarse in isolating brain mitochondria may degrade a significant amount of mitochondrial protein; however, it does not have a detrimental effect on respiratory parameters (Wilson, 1987).
Respiratory Assays Mitochondria were assayed for rate of oxygen consumption using a Clarke-type oxygen electrode (Yellow Springs Instrument Co.) as described by Estabrook (1967). Mitochondrial protein (0.5-1.0 mg) was added to 600 pi respiratory buffer in the assay chamber (25°C). Substrate (glutamate/malate) was added to 5 mM final 22
MITOCHONDRIAL METABOLISM AFTER BRAIN INJURY state 3 respiration was induced by the addition of 100 or 150 nmoles of ADP. Each mitochondrial suspension isolated from individual animals was assayed at least four times. Respiratory control ratio (RCR) was calculated by dividing rate of oxygen consumption in state 3 (ADP stimulated) by that in state 4 (ADP depleted). ADP/O ratio was determined by dividing the number of nmoles of ADP phosphorylated during state 3 respiration by the number of natoms of oxygen consumed.
concentration, and
Data
Analysis
Data were analyzed for statistical significance using analysis of variance (ANOVA) followed by individual Student Newman-Keuls tests. A value for p of < 0.05 was considered significant. All data are expressed as mean ± standard error.
RESULTS Total mitochondrial protein yield from the left hemisphere of normal uninjured controls was 2.5 ± 0.2 mg. Results reported by others for brain mitochondrial isolations (Rehnchrona et al., 1979; Fullered and Ernster, 1983) show mean rat whole brain yields of 7.6-7.9 mg with and without Nagarse treatment, respectively. If we consider that in the present studies the cerebellum and part of the brainstem were removed, and only the left hemisphere was used for mitochondrial isolation, the present yield is comparable to the protein yields reported previously. Traumatic brain injury did not cause any significant changes in total mitochondrial protein yield at 4 h posttrauma (2.2 ± 0.1 mg), thus indicating no selective loss of damaged mitochondria following trauma.
Figure 1 shows a typical oxygraph trace obtained from mitochondria isolated from normal controls. Prior to 0.5 mg mitochondrial
protein
Glutamate/malate (5mM) 100 nmoles ADP
150 nmoles ADP
20 natoms O min
FIG. 1. Typical oxygen electrode recording of respiring brain mitochondria isolated from normal controls. Concentration of additions is final concentration in the sample chamber. All experiments were performed at 25°C.
23
VINK ET AL. Table 1. Respiratory Control Ratios (RCR), ADP/O Ratios, and Respiratory Rates in Brain Mitochondria Isolated from Normal Control Animals, Anesthetized Control Animals, and Brain-Injured Animals
State 3 Normal controls (t = 0 b.) Anesthesia controls (t 4 h) Injured animals (t 4 h) =
=
RCR
A DPIO
4.3 ± 0.2 4.1+0.2 4.2 + 0.2
2.66 ± 0.09 2.53 + 0.14 2.41+0.07
State 4
(natoms OImini mg protein) 105 + 5 103 + 5 120 + 7
26 ± 3 26 + 4 29 + 3
the addition of ADP, oxygen consumption was 26 ± 3 natoms (O/min/mg mitochondrial protein). With the addition of ADP, oxygen consumption increased to 105 ± 5 natoms (O/min/mg mitochondrial protein) before declining to approximate pre-ADP levels as ADP became depleted. The calculated RCR from normal controls was 4.3 ± 0.2, while the ADP/O ratio was determined to be 2.66 ± 0.09. These values are in excellent agreement with previously published values for isolated brain mitochondria (Lai and Clark, 1979; Rehncrona
etal., 1979).
The effects of pentobarbital anesthesia on brain mitochondrial respiratory parameters are summarized in Table 1. There were no changes in RCR, ADP/O ratio, or state 3 respiration rate induced by 4 h of pentobarbital anesthesia. Animals subjected to fluid-percussion-induced brain injury demonstrated similar pre-ADP basal rates of oxygen consumption (29 ± 3 natoms/min/mg protein) as controls. However, with the initiation of state 3 respiration, oxygen consumption rate showed a trend towards an increase that did not reach significance (120 ± 7 natoms [O/min/mg protein]; p 0.08). In a similar fashion, while indicating a trend towards a decline, the calculated ADP/O ratio of 2.41 ±0.07 was not significantly lower than that observed for the normal controls (p 0.08). There was no change in the calculated RCR value posttrauma (4.2 ± 0.2) as compared to normal controls (Table 1). =
=
DISCUSSION
Despite the suggestion that traumatic injury to the CNS causes mitochondrial dysfunction (Yang et al.,
1985; Vink et al., 1988b), no studies to date have isolated mitochondria from traumatized tissue and assayed them directly for respiratory capacity. The present study has examined the effects of traumatic brain injury in rats on cerebral mitochondrial metabolism, and shows that mitochondrial oxidative capacity is essentially
preserved following trauma, with no evidence of any alterations that might reflect an ischémie event. Previous studies of cerebral mitochondrial function following CNS injury have concentrated on the effects of cerebral ischemia. Brain ischemia causes a reduction in RCR, indicative of uncoupling (Ozawa et al., 1967; Sims and Pulsinelli, 1987). The stimulatory effect of ADP addition on respiratory rate (state 3) is reduced (Fullered et al., 1984), particularly during and after an episode of incomplete ischemia (Rehncrona et al., 1979). This is unlike what we have described above for trauma, after which the RCR remains unchanged, and the oxygen consumption rate in state 3 does not decrease, but demonstrates a tendency towards increasing, albeit insignificantly, when compared to control animals. Our results are consistent with the observations of Yamakami and Mclntosh (1989), who have shown in this fluid-percussion model, at the level of injury used in the present experiments, that although significant decreases occur in regional blood flow at the injury site, these declines do not approach frank ischemia (i.e., 15-25 ml/100 g/min). The present studies have evaluated the mitochondrial respiratory activity at 4 h posttrauma. Previous studies have shown that this time is critical with respect to neurologic and biochemical recovery of animals following trauma (Vink et al., 1988b, c). For example, at 4 h posttrauma, the ratio of phosphocreatine to inorganic phosphate (Vink et al., 1988b) and the intracellular free magnesium concentration (Vink et al., 1988a) are linearly correlated to injury severity. Thus animals that demonstrate significant recovery posttrauma already have significantly improved bioenergetic status by 4 h posttrauma (Vink et al., 1988c). The present study demonstrates that the bioenergetic status at 4 h posttrauma is not related to mitochondrial 24
MITOCHONDRIAL METABOLISM AFTER BRAIN INJURY
respiratory function, which is essentially preserved at this time despite development of significant neurologic deficit following this level of injury (Mclntosh et al., 1989). Although no statistical differences are demonstrated for ADP/O ratio and state 3 respiration rate between control animals and injured animals, there were trends toward change that did not achieve statistical significance (p 0.08). With increased numbers of experimental animals, this p value would presumably achieve statistical significance. However, because the anesthesia alone tends to modify mitochondrial respiration, albeit insignificantly in itself (Ginsburg et al., 1977), the interpretation of the mitochondrial respiratory data necessitates comparisons with anesthetized control animals at similar times to the experimental group. In this respect, when compared to anesthesia controls, the ADP/O value did not demonstrate any trends toward change whereas the state 3 respiratory rate again showed trends toward an increase (p 0.08). Similar trends were also observed with state 4 respiration rates (Table 1). Respiration rate is a function of cytosolic phosphorylation potential (Ereckinska and Wilson, 1982), and decline in cytosolic phosphorylation potential (AGp) would tend to increase mitochondrial respiration rate. We have previously shown that traumatic brain injury in rats decreases AGp (Vink et al., 1988c). This decline is linearly related to the observed decline in intracellular free Mg2+ concentration (Vink et al., 1988c). It may be that the tendency toward increased respiration rate following trauma may be related to the profound fall in AGp. That these trends did not achieve statistical significance is puzzling in view of the magnitude of the decline in AGp observed. One can speculate that the effects of AGp on mitochondrial respiration are attenuated by a decline in total magnesium (Vink et al., 1988a) that would tend to decrease respiration rate (Ginsburg et al., 1977). Magnesium concentration is known to regulate respiratory transitions in mitochondria (Masini et al., 1983). Moreover, any alterations in free and total magnesium may also affect other mitochondrial functions, such as activity of the ADP/ATP translocase, ATP synthase, and mitochondrial creatine kinase, all of which are influenced by divalent cations, particularly magnesium (Lee et al., 1970; Saks et al., 1975; Klingenberg, 1977; Moreno-Sanchez et al., 1985; Fagian et al., 1986; Kramer et al., 1986). Determining which of these processes are being affected, resulting in decreased bioenergetic state, requires further investigation. =
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ACKNOWLEDGMENTS This work was supported in part by Centers for Disease Control Grant 902269 to A.I.F., and a Veterans Administration Merit Review Grant to T.K.M. R. Vink is a recipient of a Queen Elizabeth II Fellowship and is supported by the Australian National Research Fellowships Advisory Committee.
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Address reprint requests to: Robert Vink, Ph.D. Department of Chemistry and Biochemistry James Cook University of North Queensland Townsville, Queensland 4811 Australia
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