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Experimental Isobaric Subarachnoid Hemorrhage: Regional Mitochondrial Function During the Acute and Late Phase Fulvio Marzatico, Ph.D., Paolo Gaetani, M.D., Vittorio Silvani, M.D., Daniela Lombardi, M.D., Elena Sinforiani, M.D., and Riccardo Rodriguez y Baena, M.D. Institute of Pharmacology, Faculty of Science, Department of Surgery, Neurosurgical Section, I.R.C.C.S. Policlinico S. Matteo, Institute of Neurology, I.R.C.C.S. Fondazione C. Mondino, University of Pavia, Pavia, Italy
Marzatico F, Gaetani P, Silvani V, Lombardi D, Sinforiani E, Rodriguez y Baena R. Experimental isobaric subarachnoid hemorrhage: regional mitochondrial function during the acute and late phase. Surg Neurol 1990;34:294-300.
ratio in all tested brain areas was demonstrated. The increased mitochondrial vulnerability in the delayed phases could be one of the biochemical correlates of posthemorrhagic encephalopathy.
Patients treated for aneurysmal subarachnoid hemorrhage show, in the long-term follow up, an elevated rate of cognitive disturbances that are mainly related to the impact of the initial bleeding: the neurotoxic effects of blood deposition in subarachnoidal spaces may result in a diffuse encephalopathy, but the intrinsic mechanism and the biochemical correlates are not known. In the present study we have evaluated mitochondrial function after experimental induction of subarachnoid hemorrhage. Mitochondrial function was evaluated in four different rat brain areas (frontal cortex, occipital cortex, hippocampus, and brain stem) after experimental isobaric subarachnoid hemorrhage in rats. Subarachnoid hemorrhage was induced by injecting 0.07 mL of arterial autologous blood into the cisterna magna. Intracranial pressure did not significantly increase. The nonsynaptic mitochondrial fraction was isolated from different rat brain areas, and the maximal rate of enzymatic reactions of some key enzymatic activities related to the Krebs cycle [nicotinamide adenine dinucleotide (oxidized form) (NAD+)-isocitrate dehydrogenase, citrate synthase, and succinate dehydrogenase] and of the electron transfer chain (cytochrome oxidase) were evaluated. The nonsynaptic mitochondrial fraction was utilized also to check parameters related to the mitochondrial respiration: state 3, state 4, uncoupled state, respiratory control ratio, and adenosine 5 '-diphosphate/oxygen ratio. The biochemical parameters were measured at 1 and 72 hours after the subarachnoidal injection of blood. Subarachnoid hemorrhage did not affect the mitochondrial enzymatic activities both at 1 and 72 hours, while the mitochondrial respiration parameters were significantly affected: in particular, a significant decrease of respiratory control
KEY WORDS: Experimental subarachnoid hemorrhage; Mitochondria; Enzymatic activities; Vasospasm
Address reprint requests to: Paolo Gaetani, M.D., Department of Surgery, Neurosurgical Section, University of Pavia, Policlinico S. Matteo, I 27100 Pavia, Italy. Received January 8, 1990; accepted May 11, 1990.
© 1990 by Elsevier Science Publishing Co., Inc.
Subarachnoid hemorrhage (SAH) induces severe changes in cerebral circulation, but clinical manifestations range between mild functional and metabolic dysfunctions o f diencephalic structures and complete ischemic damage due to severe arterial spasm [10,21,44]. In the follow up o f patients treated for aneurysmal SAH, while the incidence o f neurological deficits due to vasospasm is lowering, there is an elevated rate of cognitive disturbances and psychological maladjustment [40,41]. In long-term evaluations after the operation, many patients show a significant impairment in verbal shortterm m e m o r y and in consolidation o f verbal memory, and deficits o f abstract abilities and concept-building processes [41]. These findings suggest an impaired function o f frontotemporal structures: no differences in cognitive disturbances have been found between patients who experienced aneurysmal SAH and SAH of unknown origin; moreover, disturbances o f cognitive functions do not have a close relationship with the timing of surgery [40] and are mainly related to the impact of the bleeding "per se." The neurotoxic effect of blood deposition may result in a diffuse encephalopathy, but its intrinsic pathogenesis and the biochemical correlates are not known. The majority o f experimental studies [20,36,45,46] have focused on cerebral blood flow (CBF) modifications following SAH. More recently, a simpler and inexpensive S A H model in rats was described, giving a reliable and reproducible model o f acute and delayed 0090-3019/90/$3.50
Energy Metabolism After Experimental SAH
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phases of cerebral hypoperfusion related to arterial spasm [7]. Solomon et al [39] and Delgado et al [7] measured the regional CBF and glucose metabolism using the double-isotope autoradiographic technique with 14C iodoantipyrine and 3H-deoxyglucose. Biochemical analysis of metabolic dysfunctions from 2 to 12 hours after the hemorrhage was performed by Fein [10], and we recently studied some enzymatic activities related to energy transduction and nonsynaptic mitochondrial respiratory parameters in different rat brain areas after hyperbaric SAH [27]. Previous experiences with a SAH r a t model were performed with injection of autologous arterial blood, which induces a significant increase of intracranial pressure (ICP) to 15-18 mmHg and influences brain energy metabolism [22]. In the present report we have studied mitochondrial function after experimental "isobaric" SAH in order to check the effects of subarachnoidal deposition of arterial blood by excluding the influence of ICP, and to clarify the biochemical correlate of neuronal impairment due to the hemorrhage.
rats were anesthetized as described before. Arterial blood samples were drawn every 10 minutes by a femoral cannula to measure pH, PCO2, and Po2 while monitoring the arterial blood pressure (Indirect Blood Pressure Sensor 446420, Bel-Art Products, Penquannock, N.J.) (Table 1). All the parameters were kept constant in the physiological range. Autologous arterial blood (0.2 mL) was collected from the femoral artery, and an aliquot of 0.07 mL was injected into the cisterna magna via the subcutaneous catheter in about 1 minute.
Experimental Groups Animals were divided into the following experimental groups: (a) control group (rats without any manipulation), (b) sham-operated group (rats submitted to surgical procedure and injected with 0.07 mL of saline solution at 37°C), and (c) SAH group (rats subjected to SAH procedure with injection of 0.07 mL of autologous arterial blood). Biochemical evaluations were performed at 1 hour and at 72 hours after experimental SAH.
Materials and Methods
Experimental Induction of S A H Male Sprague-Dawley rats (Charles River, Italy), weighing 350-375 g, were used. Anesthesia was induced with 3% halothane ( 7 0 % : 3 0 % N 2 0 : O 2 ) and maintained with 0.75% halothane (in a gas mixture). A scalp incision on the dorsal midline and a burr hole, by refrigerated twist drill, at the interparietal/occipital suture connection were performed. The bone was carefully cleaned and a small catheter (polyethylene PB-10, 0.62 mm outer diameter) was inserted into the cisterna magna. Suitable placement of the catheter was assessed by (a) testing cerebrospinal fluid (CSF) flowing throughout the catheter and (b) observing (with magnification) the lower distal part of the catheter through the atlooccipital membrane. Two days after catheter implantation,
Table 1. PhysiologicalParameters in Experimental Isobaric Subarachnoid Hemorrhage in Rats (Cisternal Injection of O.07 mL of Autologous Arterial Blood) MABP Time
(ram Hg)
Control
130 138 125 120 130 122
SAH, 15 rain
Control S A I l , 30 rain Contr o l SAH, 60 rain
-+ 4 + 5 -+ 4 + 6 -+ 6 -+ 8
pH 7.40 7.41 7.39 7.38 7.39 7.38
+-+ -+ -+ -+
0.02 0.01 0.02 0.02 0.01 0.02
Poz (mm Hg)
Pco2 (mm Hg)
114 110 120 108 113 110
36.6 37.7 36.4 37.7 37.6 36.8
"4- 7 +- 8 -+ 10 -+ 7 -+ 6 -+ 5
+ ++ -+ +-+
2 1 1 1 2 1
Abbreviations: MABP, mean arterial blood pressure; SAH, subarachnoid hemorrhage. Values are the mean -+ SEM.
Mitochondrial Isolation and Biochemical Evaluations At the set time, rats were killed by decapitation. Brain was quickly removed (about 15 seconds) from the skull and immediately immersed in cold isolation medium (IM) (sucrose 0.32 M plus ethylenediaminetetraacetic acid I mM at pH 7.4). Dissection of the brain areas [12] was performed in a precooled (-5°C) box in about 6 0 90 seconds. All manipulations were then carried out at 0°-4°C. Brain areas were homogenized in IM (10 wt/ wt) in a precooled Potter Braun S Homogeniser (six strokes up and down, 600 rpm). The homogenate was then submitted to subcellular fractionation to obtain nonsynaptic mitochondrial fraction utilizing a Ficoll density gradient [24]. The maximal rate of enzymatic reactions (Vm~,) of the following enzymatic activities were evaluated on nonsynaptic mitochondrial fraction: for the Krebs cycle: nicotinamide adenine dinucleotide (reduced form) (NADH+)-isocitrate dehydrogenase [23], citrate synthase [43], and succinate dehydrogenase [1]; for the electron transfer chain: cytochrome oxidase [37]. Protein content was evaluated in nonsynaptic mitochondrial fractions with bovine serum albumin as a standard [26]. For each enzymatic assay, 3 0 100/zg of protein were used. Mitochondrial respiratory parameters [state 3, state 4, uncoupled state, respiratory control ratio (RCR), adenosine 5'-diphosphate/oxygen (ADP/O) ratio] were evaluated in the presence of glutamate plus malate and succinate plus rotenone as oxidative substrates, in isotonic medium, and in the presence
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Table 2. EnzymaticActivities
Brain areas Frontal cortex
Occipital cortex
Hippocamp us
Brain stem
Experimental conditions
Control Sham-operated SAH, I hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours
N A D +lsocitrate dehydrogenase
11.1 10.5 11.1 13.1 9.6 12.7 12.5 14.9 13.8 15.3 13.6 16.6 12.4 12.3 15.0 16.1
+ ± ± ± ± ± ± ± ± ± ± ± -+ ± ± ±
1.2 1.6 1.8 4.1 1.0 1.7 1.4 3.7 2.0 3.5 1.9 4.8 2.0 1.2 1.1 3.7
Citrate synthase 1042.2 1500.0 1252.8 1129.4 957.9 1230.1 944.5 981.9 1018.6 998.0 893.7 1092.4 971.4 1121.1 1265.1 1117.2
± ± ± ± -+ ± + ± + ± -+ ± ± ± ± ±
120.3 170.7 154.6 165.3 76.0 102.9 130.9 143.3 149.2 135.7 172.9 183.4 87.7 115.9 284.5 209.0
Succinate dehydrogenase 92.0 84.6 86.6 88.9 89.9 90.2 72.7 81.0 107.7 89.5 102.7 108.2 81.2 78.5 72.9 116.9
± ± ± ± ± -+ ± ± ± ± ± _+ ± ± ± ±
8.9 5.8 15.2 10.2 10.0 9.1 13.9 7.4 9.7 7.9 10.4 16.4 10.4 6.1 10.5 14.7
Cytochrome oxidase 1454.0 988.1 1254.1 1128.1 1278.6 1021.8 1104.1 1142.8 948.7 874.9 930.1 776.4 1484.4 1175.9 1082.8 1287.4
-+ -+ ± ± ± ± ± ± ± ± + ± ± + ± ±
134.7 92.7 180.1 172.8 179.4 64.8 187.2 89.7 lll.5 82.8 217.9 104.7 216.5 101.9 173.6 141.7
The maximal rate of enzymatic reactions of nicotinamide adenine dinucleotide (oxidized form) (NAD+)-isocitrate dehydrogenase, citrate synthase, succinate dehydrogenase, and cytochrome oxidase evaluated on nonsynaptic mitochondrial fraction obtained from different brain areas of rats subjected to isobaric subarachnoid hemorrhage (SAH). The enzymatic activities were evaluated as nanomoles per minute per milligram protein. The values are the mean -+ SEM of groups of six animals. No significant difference was found between sham-operated rats versus other experimental conditions.
of 5 mM o f K + at 23°C [3]. It is well known that state 3 reflects the oxygen uptake capacity of cells and relates well with the functional state of the electron transfer chain, depending on oxidative substrates. State 4 reflects the dissipation rate of mitochondrial electrochemical gradient and indirectly gives information about selective properties of mitochondrial membrane.
Statistical Analysis Analytical runs were performed on lots of six animals for each group, which consisted of hemorrhagic and nonhemorrhagic (sham-operated) rats. Statistical analysis of the results was carried out using the analysis of variance (ANOVA) at one way, where the ANOVA test gave significant differences, Tukey's test for multiple comparisons was applied. Statistical significance was accepted for p < 0.05.
Results Subarachnoid hemorrhage did not significantly affect the Vm~, of enzymatic activities related to aerobic metabolism in all four brain areas (Table 2). It should be stressed that the evaluation of Vm~, in vitro with substrate excess and in conditions abolishing the metabolic autoregulative mechanisms is not a realistic "mirror" of the actual activity of enzymatic activity in situ. How-
ever, the Vm~, of the regulatory enzymes that catalyze irreversible reactions or reactions far from the equilibrium can depict the potential activity of an enzymatic or a metabolic pathway [2,4,5]. The decrease in the RCR with glutamate plus malate in the frontal cortex, 1 hour after SAH induction (Tables 3 and 4) may be related to a decrease in state 3 together with a little increase in state 4; 72 hours after SAH state 3 reached control values (sham-operated), while state 4 markedly increased (Table 5). In the occipital cortex, changes in the RCR found 1 hour and 72 hours after SAH induction (Tables 3 and 4) were primarily related to a marked increase in state 4 (Table 5). In the hippocampus, changes in state 3 and state 4 found in the first hour after SAH were not sufficient to determine a significant decrease in the RCR (Tables 3 and 4), while at 72 hours after SAH the state 4 (Table 5) markedly increased and the decrease in the RCR reached statistical significance (Tables 3 and 4). In the brain stem, at 1 and 72 hours after SAH induction, the significant decrease in the RCR depended on a progressive increase in state 4 (Table 5). There were no significant variations in the RCR (with succinate plus rotenone) after experimental SAH, while the hemorrhage significantly affected the ADP/O ratio (measured with NADH-producing substrates) in the occipital and frontal cortex and in the hippocampus at 1 hour after SAH induction (Tables 3 and 4).
Energy Metabolism After Experimental SAH
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Table 3. Mitochondrial Respiratory Activity [nicotinamide adenine dinucleotide (reducedform) (NADH)-Producing Substrates] Glutamate + malate Brain areas Frontal cortex
Occipital cortex
Hippocampus
Brain stem
Experimental groups Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours
RCR 4.92 4.33 3.27 3.19 3.70 3.71 2.85 2.69 3.78 3.25 2.50 2.20 3.69 3.42 2.55 2.03
± ± -+ ± ± -+ ± ± ± -+ ± ± ± -+ -+ ±
0.18 0.21 0.29' 0.53 a 0.22 0.15 0.19' 0.31 ~ 0.26 0.27 0.22 0.32 ~ 0.43 0.36 0.20 ~ 0.27 °
ADP/O 2.61 2.50 2.04 2.25 2.47 2.31 1.87 1.77 2.27 2.00 1.61 1.77 2.30 2.07 1.55 1.77
± ± ± +± ± + ± ± ± ± ± ± ± ± ±
0.40 0.12 0.14 a 0.16 0.13 0.11 0.14 ~ 0.11 a 0.13 0.13 0.16 ° 0.11 0.17 0.10 0.15 0.16
State 3U 85.64 91.22 66.77 82.30 68.09 81.42 81.76 63.78 63.10 81.98 64.07 66.88 73.33 82.96 87.23 84.10
± ± ± + ± ± ± ± + ± ± ± + ± ± -+
9.8 4.4 12.6 8.7 10.5 10.1 10.8 8.1 12.6 8.1 12.8 6.9 16.0 11.5 9.9 20.1
Respiratory control ratio (RCR), adenosine 5'-diphosphate/oxygen (ADP/O) ratio, and uncoupled stimulated rspiration (state 3U) in nonsynaptic mitochondria isolated from brain areas of rats subjected to isobaric subarachnoid hemorrhage (SAH). State 3U is expressed as nat O per minute per milligram protein. The values are the mean -+ SEM of groups of six animals. Statistical significance: sham-operated rats versus other experimental conditions (p < 0.01).
Discussion
Ca 2+, and some neurotransmitters has been suggested as related to this fact, but the intrinsic biochemical correlate has not yet been investigated. Several authors have correlated the encephalopathy of SAH to diffuse hypoperfusion and arterial vasospasm [ 13,18,33,47,48], but clinical investigation and experimental studies [15,28,31,34] showed that global CBF reduction poorly
The neurotoxic effects of blood deposition in the subarachnoidal space after SAH may result in a diffuse encephalopathy and one of the pathogenetic events leading to long-term cognitive disturbances and psychological maladjustment. The altered homeostasis of K +,
Table 4. MitochondrialRespiratory Activity [FADH2-ProducingSubstrates] Succinate + rotenone Brain areas Frontal cortex
Occipital cortex
Hippocampus
Brain stem
Experimental conditions Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours
RCR 3.03 2.80 2.96 2.99 2.90 2.75 2.64 2.67 2.67 2.56 2.60 2.48 2.58 2.54 2.43 2.43
± +± -+ ± ± ± +± ± -+ ± ± -+ ± ±
0.15 0.11 0.15 0.23 0.24 0.17 0.17 0.14 0.10 0.19 0.14 0.12 0.06 0.13 0.13 0.05
ADP/O 1.51 1.45 1.19 1.30 1.44 1.44 1.09 1.24 1.39 1.27 1.03 1.13 1.34 1.30 1.07 1.18
±-+ ~± + + ± ± -+ -+ + ± -+ ± ± +
0.07 0.07 0.07 0.08 0.08 0.05 0.08 0.08 0.10 0.08 0.06 0.07 0.07 0.07 0.09 0.07
State 3U 122.13 134.60 128.92 151.58 103.81 131.58 146.11 137.98 107.24 127.00 117.08 129.89 118.77 120.54 142.27 115.28
± ± ± -+ ± ± ± -+ ± ± ± ± ± +± +-
14.6 14.9 28.6 10.8 12.8 5.2 20.1 11.4 22.4 5.7 19.5 7.5 21.4 13.9 14.6 17.6
Respiratory control ratio (RCR), adenosine 5'-diphosphate/oxygen (ADP/O) ratio, and uncoupled stimulated rspiration (state 3U) in nonsynaptic mitochondria isolated from brain areas of rats subjected to isobaric subarachnoid hemorrhage (SAH). State 3U is expressed as nat O per minute per milligram protein. The values are the mean -+ SEM of groups of six animals. No significant difference was found between sham-operated rats versus other experimental conditions.
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Table 5. Mitochondrial Respiratory Activity Glutamate + malate Brain areas Frontal cortex
Occipital cortex
Hippocampus
Brain stem
Experimental groups Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours
State 3 92.4 90.6 76.8 87.1 74.1 80.1 85.8 73.6 75.2 82.0 75.9 76.6 84.4 81.9 83.1 89.9
± -+ ± -+ ± + -+ + ± -+ -+ + -+ -+ -+ -+
l l.7 12.3 13,7 11.1 9.8 7.1 11.1 13.1 13.8 6.9 10.7 I1.8 12.6 3.5 5.8 16.4
Succinate + rotenone
State 4 19.1 20.7 25.1 29.6 20.5 21.5 30.2 28.1 21.1 26.6 31.5 36.9 26.5 24.1 33.8 47.3
-+ 2.7 -+ 2.5 -+ 5.4 ± 3.2" -+ 3.1 -+ 2.5 -+ 3.4 + 5.2 ± 5.1 -+ 2.3 ± 4.7 ± 6.7" -+ 5.8 ± 2.9 ± 4.2 ~ ± 7.2 b
State 3 97.8 99.1 111.5 123.4 88.8 99.5 122.0 117.2 93.5 117.1 106.7 123.6 107.1 105.5 137.1 127.3
-+ ± ± ± ± -+ -+ -+ ± ± ± ± 4-+ 44-
11.9 10.4 11.4 10.2 10.5 10.2 9.8 13.1 16.4 15.7 13.9 9.3 17.1 10.3 13.4 14.2
State 4 32.8 35.2 37.7 44.6 32.7 36.3 47.3 43.8 36.2 45.3 40.8 50.8 41.9 41.2 54.5 52.5
+ ± + + + + --± ± ± --+ ± + ± ±
5.6 3.4 3.3 5.1 5.7 3.4 5.6 2.8 6.8 3.1 4.0 6.0 6.8 2.9 3.9 5.5
State 3 and state 4 in nonsynaptic mitochondria isolated from brain areas of rats subjected to isobaric subarachnoid hemorrhage (SAH). State 3 and state 4 are expressed as nat O per minute per milligram protein. The values are the mean -+ SEM of groups of six animals. Statistical significance: sham-operated rats versus other experimental conditions (op < 0.05, hp < 0.01).
correlates with angiographical vasospasm. The discrepancy between CBF and the degree of arterial narrowing would be related to the fact that global and regional CBF reduction would follow changes in cerebral metabolism [6,10]. Modifications of regional CBF and metabolism could be related to long-term cognitive disturbances in patients admitted with diagnosis of SAH and discharged without fixed neurological deficits [25]. In previously published experimental SAH models with rats, the amount of arterial blood injected into the cisterna magna ranged between 0.07 and 0.5 mL [6,7,38], with different variations of ICP. After blood injection, ICP increases, and the hemispheric blood flow should remain unchanged until the cerebral perfusion pressure remains above 35-40 mmHg [49]. Under these conditions the energy balance is well maintained [35], despite biochemical evidence of a shift to anaerobic metabolism during moderate intracranial hypertension [22]. Angiographic studies have shown that injection of 0.3 mL of arterial blood into the cisterna magna did not have significantly different effects during both the acute and the late phase of vasospasm, if compared with arterial narrowing caused by injection of 0.07 mL of blood [7]. Recently, some authors reporting experiences using the 3H-deoxyglucose method in order to evaluate glucose uptake and phosphorylation in vivo showed a different effect on glucose utilization with a 0.07-mL injection [6], when compared with a 0.37-mL injection [38]. In the first study Delgado et al [6] demonstrated a general reduction of CBF (20%) with a general increase of
glucose uptake (30%), while Solomon et al [38] found a global depression of cerebral metabolism after injection of 0.37 mL of blood. In a previous experience, we have found a massive impairment of mitochondrial function in different rat brain areas after injection of 0.35 mL of autologous arterial blood, causing a rise in ICP from 4 to 5 mmHg to 17-20 mmHg [27]. We injected 0.07 mL of blood, excluding a significant influence of ICP on pathophysiological changes after SAH. In this condition the aerobic enzymatic activities evaluated in the nonsynaptic mitochondria were unaffected, showing that the enzymatic activities are not the limiting factors in energy metabolism during isobaric SAH. On the other hand, we have found that experimental SAH induction with 0.07 mL of blood causes significant changes in nonsynaptic mitochondrial respiratory parameters. The nonsynaptic mitochondrial function was extensively studied to check the cell damage during anoxic-ischemic brain insult [ 11,16,30,32] in nonsynaptic mitochondria isolated after a complete or incomplete ischemia, a reduction in state 3 respiration (oxygen consumption in the presence of exogenous ADP) with a minor impairment of state 4 (oxygen consumption in the absence of exogenous ADP) is the typical pattern [16,32] Our results suggest that after experimental isobaric SAH, the nonsynaptic mitochondrial impairment is area specific: a reversible inhibition of the NADH-oxidative branch of the electron transfer chain is evident only in the frontal cortex in the acute phase of isobaric SAH,
Energy Metabolism After Experimental SAH
since the decrease in state 3 with glutamate plus malate as substrates appears evident, without variations in state 3 with sucCinate plus rotenone as substrates. The widespread increase in state 4 in all rat brain areas tested suggests a marked impairment of proton electrochemical gradient [14,42] and a dielectric breakdown of mitochondrial membrane [29]. This is evident only with NADH-generating substrates: the 02 consumption without exogenous ADP (state 4) is much lower than in the presence of FADH2-generating substrates. The impairment of mitochondrial membrane selectivity (i.e., H +) may reflect a change in 02 consumption, but it will be sufficient to determine a significant variation in state 4 with succinate plus rotenone. The mitochondrial membrane impairment could be the result of cascade reactions started by Ca 2+ influx after the hemorrhagic insult [17]. The different behavior of the hippocampus could be related to a different timedependent accumulation of Ca2+ in this area during experimental cerebral ischemia [9]. Deshpande et al [8] showed that the calcium content in the hippocampus, after cerebral ischemia, did not change until 24 hours, but after only 48 hours of the ischemic insult, the increase of Ca 2+ reached statistical significance. A further increase was shown at 72 hours. In experiment SAH, we have shown an impairment of hippocampal nonsynaptic mitochondria (significant decrease in the RCR) only at 72 hours: we can draw the hypothesis that this fact is related to calcium overload in the hippocampus. Recently, Voldby et al [47] have shown that after aneurysmal SAH, the cerebral metabolic rate for oxygen is reduced more then the CBF: this would indicate an uncoupled relationship between CBF and the oxidative cerebral metabolism. Moreover, Jakobsen et al [19] have found that the arterovenous difference for oxygen did not increase with the increasing degree of vasospasm, suggesting that vasospasm is not the cause of the decrease of the cerebral metabolic rate for oxygen. We can suggest that this phenomenon could be related to the significant impairment of mitochondrial function. In conclusion the results of the present study provide the experimental evidence that after SAH, the decrease in cerebral oxidative machinery could be related to a severe impairment of mitochondria and is site and time dependent, and that mitochondrial impairment is quite independent from the amount of blood injected and is not related to variations in ICP. The increased mitochondrial vulnerability in the delayed phases could be related to neuronal impairment following the initial bleeding and could be one of the possible biochemical correlates to post-SAH encephalopathy, which causes long-term cognitive disturbances.
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The findings in this paper were presented at the Annual Meeting of The American Association of Neurological Surgeons, Toronto, Canada, April 24-28, 1988. This research was supported by a grant from the Regione Lombardia, Milan, Italy, 1984.
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20. Kamiya K, Kuyama H, Symon L. An experimental study of the acute stage of subarachnoid hemorrhage. J Neurosurg 1983;59:917-24. 21. Kassell NF, Sasaki T, Colohan ART, et al. Cerebral vasospasm following subarachnoid hemorrhage. Stroke 1985;16:562-72. 22. Kjallqvist A, Siesjo BK, Zwemow N. Effects of increased intracranial pressure on cerebral blood flow and on cerebrospinal fluid HCO3, pH, lactate and piruvate in dogs. Acta Phys Scand 1969;75:345-52. 23. Lai JCK, Clark JB. Preparation and properties of mitochondria from synaptosomes. Biochem J 1976;154:423-32. 24. LaiJCK, WalshJM, Dennis SC, ClarkJB. Synaptic and nonsynaptic mitochondria from rat brain: isolation and characterization. J Neurochem 1977;28:625-31. 25. Ljunggren B, Sonesson B, Saveland H, et al. Cognitive impairment and adjustment in patients without neurological deficits after aneurysmai SAH and early operation. J Neurosurg 1985;62:673-9. 26. Lowry OH, Rosebrough BJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75. 27. Marzatico F, Gaetani P, Rodriguez y Baena R, et al. Bioenergetics of different rat brain areas after experimental subaracbnoid hemorrhage in rats. Stroke 1988;19:378-84. 28. Millikan CH. Cerebral vasospasm and ruptured intracranial aneurysms. Arch Neurol 1975;32:433-49. 29. NichoUsDG. The influence of respiration and ATP hydrolysis on the proton-electrochemical gradient across the inner membrane of rat liver mitochondria as determined by ion distribution. Eur J Biochem 1974;50:305-15. 30. Ozawa K, Seta K, Araki H, Handa H. The effect of ischemia on mitochondrial metabolism. J Biochem 1967;61:512-4. 3 I. Petruck KC, West GR, Marriott MR, Mclntyre JW, Overton TA, Weir BKA. Cerebral blood flow following induced subarachnoid hemorrhage in the monkey. J Neurosurg 1972;37:316-24. 32. Rehncrona S, Mela L, Siesjo BK. Recovery of brain mitochondrial function in the rat after complete and incomplete cerebral ischemia. Stroke 1979;10:437-46. 33. Ropper AH, Zervas NT. Outcome I year after subarachnoid hemorrhage from cerebral aneurysm. Management morbidity, mortality, and functional status in 112 consecutive good-risk patients. J Neurosurg 1984;60:909-15. 34. Sahlin C, Delgado T, Owman C, Svengaard NA. Changes in cerebral blood flow and metabolism following intraarterial or local administration of nimodipine, before and after experimental subarachnoid hemorrhage in baboons. Stroke 1986;17:220-4. 35. Siesjo BK, Zwemov N. Effects of increased cerebrospinal fluid
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pressure upon adenine nucleotides and upon lactate and piruvate in rat brain tissue. Acta Neurol Scand 1970;46:187. Simeone FA, Trepper PJ, Brown DJ. Cerebral blood flow evaluation of prolonged experimental vasospasm. J Neurosurg 1972;37:302-11. Smith L. Spectrophotometric assay of cytochrome c oxidase. In: Glick D, ed. Methods of biochemical analysis.New York: WileyInterscience, 1955:427-34. Solomon AR, McCormack BM, Lovitz RN, Swift DM, Hageman MT. Elevation of brain norepinephrine concentration after experimental subarachnoid hemorrhage. Neurosurgery 1986;19: 363-6. Solomon RA, Lobo Antunes J, Chen RYZ, Bland L, Chien S. Decrease in cerebral blood flow in rats after experimental subarachnoid hemorrhage: a new animal model. Stroke 1985;16:5864. Sonesson B, Ljunggren B, Saveland H, Brandt L. Cognition and adjustment after late and early operation for ruptured aneurysm. Neurosurgery 1987;21:279-86. Sonesson B, Saveland H, Ljunggren B, Brandt L. Cognitive functioning after subarachnoid hemorrhage of unknown origin. Acta Neurol Scand 1989;80:400-10. Stucki JW, Ineichen EA. Energy dissipation by calcium recycling and the efficiency of calcium transport in rat liver mitochondria. EurJ Biochem 1974;48:365-75. Sugden PA, Newsholme EA. Activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase and alanine arninotransferase in nervous tissue from vertebrates and invertebrates. Biochem J 1975;150:105-11. Symon L. Disordered cerebro-vascuiar physiology in aneurysmal subarachnoid hemorrhage. Acta Neurochir 1978;41:7-22. Umansky P, Kasgi T, Shalit MN. Regional cerebral blood flow in the acute stage of experimentally induced subarachnoid hemorrhage. J Neurosurg 1983;58:210-6. Varsos VG, LiszackTM, Han DH. Delayed cerebral vasospasmis not reversible by aminophylline, nifedipine or papaverine in a "two hemorrhage" canine model. J Neurosurg 1983;58:11- 7. Voldby B, Enevoldsen EM, Jensen FT. Regional CBF, intraventricular pressure, and cerebral metabolism in patients with ruptured intracranial aneurysms. J Neurosurg 1985;62:48-58.
48. Weir B, Aronyk K. Management, mortality and the timing of surgery for supratentorial aneurysms. J Neurosurg 1981;54:14650. 49. Zwemov N, Kjallqvist A, Siesjo BK. Cerebral blood flow during intracranial hypertension related to tissue hypoxia and to acidosis in cerebral extracellular fluids. Prog Brain Res 1968;30:87-92.
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Experimental Isobaric Subarachnoid Hemorrhage: Regional Mitochondrial Function During the Acute and Late Phase Fulvio Marzatico, Ph.D., Paolo Gaetani, M.D., Vittorio Silvani, M.D., Daniela Lombardi, M.D., Elena Sinforiani, M.D., and Riccardo Rodriguez y Baena, M.D. Institute of Pharmacology, Faculty of Science, Department of Surgery, Neurosurgical Section, I.R.C.C.S. Policlinico S. Matteo, Institute of Neurology, I.R.C.C.S. Fondazione C. Mondino, University of Pavia, Pavia, Italy
Marzatico F, Gaetani P, Silvani V, Lombardi D, Sinforiani E, Rodriguez y Baena R. Experimental isobaric subarachnoid hemorrhage: regional mitochondrial function during the acute and late phase. Surg Neurol 1990;34:294-300.
ratio in all tested brain areas was demonstrated. The increased mitochondrial vulnerability in the delayed phases could be one of the biochemical correlates of posthemorrhagic encephalopathy.
Patients treated for aneurysmal subarachnoid hemorrhage show, in the long-term follow up, an elevated rate of cognitive disturbances that are mainly related to the impact of the initial bleeding: the neurotoxic effects of blood deposition in subarachnoidal spaces may result in a diffuse encephalopathy, but the intrinsic mechanism and the biochemical correlates are not known. In the present study we have evaluated mitochondrial function after experimental induction of subarachnoid hemorrhage. Mitochondrial function was evaluated in four different rat brain areas (frontal cortex, occipital cortex, hippocampus, and brain stem) after experimental isobaric subarachnoid hemorrhage in rats. Subarachnoid hemorrhage was induced by injecting 0.07 mL of arterial autologous blood into the cisterna magna. Intracranial pressure did not significantly increase. The nonsynaptic mitochondrial fraction was isolated from different rat brain areas, and the maximal rate of enzymatic reactions of some key enzymatic activities related to the Krebs cycle [nicotinamide adenine dinucleotide (oxidized form) (NAD+)-isocitrate dehydrogenase, citrate synthase, and succinate dehydrogenase] and of the electron transfer chain (cytochrome oxidase) were evaluated. The nonsynaptic mitochondrial fraction was utilized also to check parameters related to the mitochondrial respiration: state 3, state 4, uncoupled state, respiratory control ratio, and adenosine 5 '-diphosphate/oxygen ratio. The biochemical parameters were measured at 1 and 72 hours after the subarachnoidal injection of blood. Subarachnoid hemorrhage did not affect the mitochondrial enzymatic activities both at 1 and 72 hours, while the mitochondrial respiration parameters were significantly affected: in particular, a significant decrease of respiratory control
KEY WORDS: Experimental subarachnoid hemorrhage; Mitochondria; Enzymatic activities; Vasospasm
Address reprint requests to: Paolo Gaetani, M.D., Department of Surgery, Neurosurgical Section, University of Pavia, Policlinico S. Matteo, I 27100 Pavia, Italy. Received January 8, 1990; accepted May 11, 1990.
© 1990 by Elsevier Science Publishing Co., Inc.
Subarachnoid hemorrhage (SAH) induces severe changes in cerebral circulation, but clinical manifestations range between mild functional and metabolic dysfunctions o f diencephalic structures and complete ischemic damage due to severe arterial spasm [10,21,44]. In the follow up o f patients treated for aneurysmal SAH, while the incidence o f neurological deficits due to vasospasm is lowering, there is an elevated rate of cognitive disturbances and psychological maladjustment [40,41]. In long-term evaluations after the operation, many patients show a significant impairment in verbal shortterm m e m o r y and in consolidation o f verbal memory, and deficits o f abstract abilities and concept-building processes [41]. These findings suggest an impaired function o f frontotemporal structures: no differences in cognitive disturbances have been found between patients who experienced aneurysmal SAH and SAH of unknown origin; moreover, disturbances o f cognitive functions do not have a close relationship with the timing of surgery [40] and are mainly related to the impact of the bleeding "per se." The neurotoxic effect of blood deposition may result in a diffuse encephalopathy, but its intrinsic pathogenesis and the biochemical correlates are not known. The majority o f experimental studies [20,36,45,46] have focused on cerebral blood flow (CBF) modifications following SAH. More recently, a simpler and inexpensive S A H model in rats was described, giving a reliable and reproducible model o f acute and delayed 0090-3019/90/$3.50
Energy Metabolism After Experimental SAH
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phases of cerebral hypoperfusion related to arterial spasm [7]. Solomon et al [39] and Delgado et al [7] measured the regional CBF and glucose metabolism using the double-isotope autoradiographic technique with 14C iodoantipyrine and 3H-deoxyglucose. Biochemical analysis of metabolic dysfunctions from 2 to 12 hours after the hemorrhage was performed by Fein [10], and we recently studied some enzymatic activities related to energy transduction and nonsynaptic mitochondrial respiratory parameters in different rat brain areas after hyperbaric SAH [27]. Previous experiences with a SAH r a t model were performed with injection of autologous arterial blood, which induces a significant increase of intracranial pressure (ICP) to 15-18 mmHg and influences brain energy metabolism [22]. In the present report we have studied mitochondrial function after experimental "isobaric" SAH in order to check the effects of subarachnoidal deposition of arterial blood by excluding the influence of ICP, and to clarify the biochemical correlate of neuronal impairment due to the hemorrhage.
rats were anesthetized as described before. Arterial blood samples were drawn every 10 minutes by a femoral cannula to measure pH, PCO2, and Po2 while monitoring the arterial blood pressure (Indirect Blood Pressure Sensor 446420, Bel-Art Products, Penquannock, N.J.) (Table 1). All the parameters were kept constant in the physiological range. Autologous arterial blood (0.2 mL) was collected from the femoral artery, and an aliquot of 0.07 mL was injected into the cisterna magna via the subcutaneous catheter in about 1 minute.
Experimental Groups Animals were divided into the following experimental groups: (a) control group (rats without any manipulation), (b) sham-operated group (rats submitted to surgical procedure and injected with 0.07 mL of saline solution at 37°C), and (c) SAH group (rats subjected to SAH procedure with injection of 0.07 mL of autologous arterial blood). Biochemical evaluations were performed at 1 hour and at 72 hours after experimental SAH.
Materials and Methods
Experimental Induction of S A H Male Sprague-Dawley rats (Charles River, Italy), weighing 350-375 g, were used. Anesthesia was induced with 3% halothane ( 7 0 % : 3 0 % N 2 0 : O 2 ) and maintained with 0.75% halothane (in a gas mixture). A scalp incision on the dorsal midline and a burr hole, by refrigerated twist drill, at the interparietal/occipital suture connection were performed. The bone was carefully cleaned and a small catheter (polyethylene PB-10, 0.62 mm outer diameter) was inserted into the cisterna magna. Suitable placement of the catheter was assessed by (a) testing cerebrospinal fluid (CSF) flowing throughout the catheter and (b) observing (with magnification) the lower distal part of the catheter through the atlooccipital membrane. Two days after catheter implantation,
Table 1. PhysiologicalParameters in Experimental Isobaric Subarachnoid Hemorrhage in Rats (Cisternal Injection of O.07 mL of Autologous Arterial Blood) MABP Time
(ram Hg)
Control
130 138 125 120 130 122
SAH, 15 rain
Control S A I l , 30 rain Contr o l SAH, 60 rain
-+ 4 + 5 -+ 4 + 6 -+ 6 -+ 8
pH 7.40 7.41 7.39 7.38 7.39 7.38
+-+ -+ -+ -+
0.02 0.01 0.02 0.02 0.01 0.02
Poz (mm Hg)
Pco2 (mm Hg)
114 110 120 108 113 110
36.6 37.7 36.4 37.7 37.6 36.8
"4- 7 +- 8 -+ 10 -+ 7 -+ 6 -+ 5
+ ++ -+ +-+
2 1 1 1 2 1
Abbreviations: MABP, mean arterial blood pressure; SAH, subarachnoid hemorrhage. Values are the mean -+ SEM.
Mitochondrial Isolation and Biochemical Evaluations At the set time, rats were killed by decapitation. Brain was quickly removed (about 15 seconds) from the skull and immediately immersed in cold isolation medium (IM) (sucrose 0.32 M plus ethylenediaminetetraacetic acid I mM at pH 7.4). Dissection of the brain areas [12] was performed in a precooled (-5°C) box in about 6 0 90 seconds. All manipulations were then carried out at 0°-4°C. Brain areas were homogenized in IM (10 wt/ wt) in a precooled Potter Braun S Homogeniser (six strokes up and down, 600 rpm). The homogenate was then submitted to subcellular fractionation to obtain nonsynaptic mitochondrial fraction utilizing a Ficoll density gradient [24]. The maximal rate of enzymatic reactions (Vm~,) of the following enzymatic activities were evaluated on nonsynaptic mitochondrial fraction: for the Krebs cycle: nicotinamide adenine dinucleotide (reduced form) (NADH+)-isocitrate dehydrogenase [23], citrate synthase [43], and succinate dehydrogenase [1]; for the electron transfer chain: cytochrome oxidase [37]. Protein content was evaluated in nonsynaptic mitochondrial fractions with bovine serum albumin as a standard [26]. For each enzymatic assay, 3 0 100/zg of protein were used. Mitochondrial respiratory parameters [state 3, state 4, uncoupled state, respiratory control ratio (RCR), adenosine 5'-diphosphate/oxygen (ADP/O) ratio] were evaluated in the presence of glutamate plus malate and succinate plus rotenone as oxidative substrates, in isotonic medium, and in the presence
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Table 2. EnzymaticActivities
Brain areas Frontal cortex
Occipital cortex
Hippocamp us
Brain stem
Experimental conditions
Control Sham-operated SAH, I hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours
N A D +lsocitrate dehydrogenase
11.1 10.5 11.1 13.1 9.6 12.7 12.5 14.9 13.8 15.3 13.6 16.6 12.4 12.3 15.0 16.1
+ ± ± ± ± ± ± ± ± ± ± ± -+ ± ± ±
1.2 1.6 1.8 4.1 1.0 1.7 1.4 3.7 2.0 3.5 1.9 4.8 2.0 1.2 1.1 3.7
Citrate synthase 1042.2 1500.0 1252.8 1129.4 957.9 1230.1 944.5 981.9 1018.6 998.0 893.7 1092.4 971.4 1121.1 1265.1 1117.2
± ± ± ± -+ ± + ± + ± -+ ± ± ± ± ±
120.3 170.7 154.6 165.3 76.0 102.9 130.9 143.3 149.2 135.7 172.9 183.4 87.7 115.9 284.5 209.0
Succinate dehydrogenase 92.0 84.6 86.6 88.9 89.9 90.2 72.7 81.0 107.7 89.5 102.7 108.2 81.2 78.5 72.9 116.9
± ± ± ± ± -+ ± ± ± ± ± _+ ± ± ± ±
8.9 5.8 15.2 10.2 10.0 9.1 13.9 7.4 9.7 7.9 10.4 16.4 10.4 6.1 10.5 14.7
Cytochrome oxidase 1454.0 988.1 1254.1 1128.1 1278.6 1021.8 1104.1 1142.8 948.7 874.9 930.1 776.4 1484.4 1175.9 1082.8 1287.4
-+ -+ ± ± ± ± ± ± ± ± + ± ± + ± ±
134.7 92.7 180.1 172.8 179.4 64.8 187.2 89.7 lll.5 82.8 217.9 104.7 216.5 101.9 173.6 141.7
The maximal rate of enzymatic reactions of nicotinamide adenine dinucleotide (oxidized form) (NAD+)-isocitrate dehydrogenase, citrate synthase, succinate dehydrogenase, and cytochrome oxidase evaluated on nonsynaptic mitochondrial fraction obtained from different brain areas of rats subjected to isobaric subarachnoid hemorrhage (SAH). The enzymatic activities were evaluated as nanomoles per minute per milligram protein. The values are the mean -+ SEM of groups of six animals. No significant difference was found between sham-operated rats versus other experimental conditions.
of 5 mM o f K + at 23°C [3]. It is well known that state 3 reflects the oxygen uptake capacity of cells and relates well with the functional state of the electron transfer chain, depending on oxidative substrates. State 4 reflects the dissipation rate of mitochondrial electrochemical gradient and indirectly gives information about selective properties of mitochondrial membrane.
Statistical Analysis Analytical runs were performed on lots of six animals for each group, which consisted of hemorrhagic and nonhemorrhagic (sham-operated) rats. Statistical analysis of the results was carried out using the analysis of variance (ANOVA) at one way, where the ANOVA test gave significant differences, Tukey's test for multiple comparisons was applied. Statistical significance was accepted for p < 0.05.
Results Subarachnoid hemorrhage did not significantly affect the Vm~, of enzymatic activities related to aerobic metabolism in all four brain areas (Table 2). It should be stressed that the evaluation of Vm~, in vitro with substrate excess and in conditions abolishing the metabolic autoregulative mechanisms is not a realistic "mirror" of the actual activity of enzymatic activity in situ. How-
ever, the Vm~, of the regulatory enzymes that catalyze irreversible reactions or reactions far from the equilibrium can depict the potential activity of an enzymatic or a metabolic pathway [2,4,5]. The decrease in the RCR with glutamate plus malate in the frontal cortex, 1 hour after SAH induction (Tables 3 and 4) may be related to a decrease in state 3 together with a little increase in state 4; 72 hours after SAH state 3 reached control values (sham-operated), while state 4 markedly increased (Table 5). In the occipital cortex, changes in the RCR found 1 hour and 72 hours after SAH induction (Tables 3 and 4) were primarily related to a marked increase in state 4 (Table 5). In the hippocampus, changes in state 3 and state 4 found in the first hour after SAH were not sufficient to determine a significant decrease in the RCR (Tables 3 and 4), while at 72 hours after SAH the state 4 (Table 5) markedly increased and the decrease in the RCR reached statistical significance (Tables 3 and 4). In the brain stem, at 1 and 72 hours after SAH induction, the significant decrease in the RCR depended on a progressive increase in state 4 (Table 5). There were no significant variations in the RCR (with succinate plus rotenone) after experimental SAH, while the hemorrhage significantly affected the ADP/O ratio (measured with NADH-producing substrates) in the occipital and frontal cortex and in the hippocampus at 1 hour after SAH induction (Tables 3 and 4).
Energy Metabolism After Experimental SAH
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Table 3. Mitochondrial Respiratory Activity [nicotinamide adenine dinucleotide (reducedform) (NADH)-Producing Substrates] Glutamate + malate Brain areas Frontal cortex
Occipital cortex
Hippocampus
Brain stem
Experimental groups Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours
RCR 4.92 4.33 3.27 3.19 3.70 3.71 2.85 2.69 3.78 3.25 2.50 2.20 3.69 3.42 2.55 2.03
± ± -+ ± ± -+ ± ± ± -+ ± ± ± -+ -+ ±
0.18 0.21 0.29' 0.53 a 0.22 0.15 0.19' 0.31 ~ 0.26 0.27 0.22 0.32 ~ 0.43 0.36 0.20 ~ 0.27 °
ADP/O 2.61 2.50 2.04 2.25 2.47 2.31 1.87 1.77 2.27 2.00 1.61 1.77 2.30 2.07 1.55 1.77
± ± ± +± ± + ± ± ± ± ± ± ± ± ±
0.40 0.12 0.14 a 0.16 0.13 0.11 0.14 ~ 0.11 a 0.13 0.13 0.16 ° 0.11 0.17 0.10 0.15 0.16
State 3U 85.64 91.22 66.77 82.30 68.09 81.42 81.76 63.78 63.10 81.98 64.07 66.88 73.33 82.96 87.23 84.10
± ± ± + ± ± ± ± + ± ± ± + ± ± -+
9.8 4.4 12.6 8.7 10.5 10.1 10.8 8.1 12.6 8.1 12.8 6.9 16.0 11.5 9.9 20.1
Respiratory control ratio (RCR), adenosine 5'-diphosphate/oxygen (ADP/O) ratio, and uncoupled stimulated rspiration (state 3U) in nonsynaptic mitochondria isolated from brain areas of rats subjected to isobaric subarachnoid hemorrhage (SAH). State 3U is expressed as nat O per minute per milligram protein. The values are the mean -+ SEM of groups of six animals. Statistical significance: sham-operated rats versus other experimental conditions (p < 0.01).
Discussion
Ca 2+, and some neurotransmitters has been suggested as related to this fact, but the intrinsic biochemical correlate has not yet been investigated. Several authors have correlated the encephalopathy of SAH to diffuse hypoperfusion and arterial vasospasm [ 13,18,33,47,48], but clinical investigation and experimental studies [15,28,31,34] showed that global CBF reduction poorly
The neurotoxic effects of blood deposition in the subarachnoidal space after SAH may result in a diffuse encephalopathy and one of the pathogenetic events leading to long-term cognitive disturbances and psychological maladjustment. The altered homeostasis of K +,
Table 4. MitochondrialRespiratory Activity [FADH2-ProducingSubstrates] Succinate + rotenone Brain areas Frontal cortex
Occipital cortex
Hippocampus
Brain stem
Experimental conditions Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours
RCR 3.03 2.80 2.96 2.99 2.90 2.75 2.64 2.67 2.67 2.56 2.60 2.48 2.58 2.54 2.43 2.43
± +± -+ ± ± ± +± ± -+ ± ± -+ ± ±
0.15 0.11 0.15 0.23 0.24 0.17 0.17 0.14 0.10 0.19 0.14 0.12 0.06 0.13 0.13 0.05
ADP/O 1.51 1.45 1.19 1.30 1.44 1.44 1.09 1.24 1.39 1.27 1.03 1.13 1.34 1.30 1.07 1.18
±-+ ~± + + ± ± -+ -+ + ± -+ ± ± +
0.07 0.07 0.07 0.08 0.08 0.05 0.08 0.08 0.10 0.08 0.06 0.07 0.07 0.07 0.09 0.07
State 3U 122.13 134.60 128.92 151.58 103.81 131.58 146.11 137.98 107.24 127.00 117.08 129.89 118.77 120.54 142.27 115.28
± ± ± -+ ± ± ± -+ ± ± ± ± ± +± +-
14.6 14.9 28.6 10.8 12.8 5.2 20.1 11.4 22.4 5.7 19.5 7.5 21.4 13.9 14.6 17.6
Respiratory control ratio (RCR), adenosine 5'-diphosphate/oxygen (ADP/O) ratio, and uncoupled stimulated rspiration (state 3U) in nonsynaptic mitochondria isolated from brain areas of rats subjected to isobaric subarachnoid hemorrhage (SAH). State 3U is expressed as nat O per minute per milligram protein. The values are the mean -+ SEM of groups of six animals. No significant difference was found between sham-operated rats versus other experimental conditions.
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Table 5. Mitochondrial Respiratory Activity Glutamate + malate Brain areas Frontal cortex
Occipital cortex
Hippocampus
Brain stem
Experimental groups Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours Control Sham-operated SAH, 1 hour SAH, 72 hours
State 3 92.4 90.6 76.8 87.1 74.1 80.1 85.8 73.6 75.2 82.0 75.9 76.6 84.4 81.9 83.1 89.9
± -+ ± -+ ± + -+ + ± -+ -+ + -+ -+ -+ -+
l l.7 12.3 13,7 11.1 9.8 7.1 11.1 13.1 13.8 6.9 10.7 I1.8 12.6 3.5 5.8 16.4
Succinate + rotenone
State 4 19.1 20.7 25.1 29.6 20.5 21.5 30.2 28.1 21.1 26.6 31.5 36.9 26.5 24.1 33.8 47.3
-+ 2.7 -+ 2.5 -+ 5.4 ± 3.2" -+ 3.1 -+ 2.5 -+ 3.4 + 5.2 ± 5.1 -+ 2.3 ± 4.7 ± 6.7" -+ 5.8 ± 2.9 ± 4.2 ~ ± 7.2 b
State 3 97.8 99.1 111.5 123.4 88.8 99.5 122.0 117.2 93.5 117.1 106.7 123.6 107.1 105.5 137.1 127.3
-+ ± ± ± ± -+ -+ -+ ± ± ± ± 4-+ 44-
11.9 10.4 11.4 10.2 10.5 10.2 9.8 13.1 16.4 15.7 13.9 9.3 17.1 10.3 13.4 14.2
State 4 32.8 35.2 37.7 44.6 32.7 36.3 47.3 43.8 36.2 45.3 40.8 50.8 41.9 41.2 54.5 52.5
+ ± + + + + --± ± ± --+ ± + ± ±
5.6 3.4 3.3 5.1 5.7 3.4 5.6 2.8 6.8 3.1 4.0 6.0 6.8 2.9 3.9 5.5
State 3 and state 4 in nonsynaptic mitochondria isolated from brain areas of rats subjected to isobaric subarachnoid hemorrhage (SAH). State 3 and state 4 are expressed as nat O per minute per milligram protein. The values are the mean -+ SEM of groups of six animals. Statistical significance: sham-operated rats versus other experimental conditions (op < 0.05, hp < 0.01).
correlates with angiographical vasospasm. The discrepancy between CBF and the degree of arterial narrowing would be related to the fact that global and regional CBF reduction would follow changes in cerebral metabolism [6,10]. Modifications of regional CBF and metabolism could be related to long-term cognitive disturbances in patients admitted with diagnosis of SAH and discharged without fixed neurological deficits [25]. In previously published experimental SAH models with rats, the amount of arterial blood injected into the cisterna magna ranged between 0.07 and 0.5 mL [6,7,38], with different variations of ICP. After blood injection, ICP increases, and the hemispheric blood flow should remain unchanged until the cerebral perfusion pressure remains above 35-40 mmHg [49]. Under these conditions the energy balance is well maintained [35], despite biochemical evidence of a shift to anaerobic metabolism during moderate intracranial hypertension [22]. Angiographic studies have shown that injection of 0.3 mL of arterial blood into the cisterna magna did not have significantly different effects during both the acute and the late phase of vasospasm, if compared with arterial narrowing caused by injection of 0.07 mL of blood [7]. Recently, some authors reporting experiences using the 3H-deoxyglucose method in order to evaluate glucose uptake and phosphorylation in vivo showed a different effect on glucose utilization with a 0.07-mL injection [6], when compared with a 0.37-mL injection [38]. In the first study Delgado et al [6] demonstrated a general reduction of CBF (20%) with a general increase of
glucose uptake (30%), while Solomon et al [38] found a global depression of cerebral metabolism after injection of 0.37 mL of blood. In a previous experience, we have found a massive impairment of mitochondrial function in different rat brain areas after injection of 0.35 mL of autologous arterial blood, causing a rise in ICP from 4 to 5 mmHg to 17-20 mmHg [27]. We injected 0.07 mL of blood, excluding a significant influence of ICP on pathophysiological changes after SAH. In this condition the aerobic enzymatic activities evaluated in the nonsynaptic mitochondria were unaffected, showing that the enzymatic activities are not the limiting factors in energy metabolism during isobaric SAH. On the other hand, we have found that experimental SAH induction with 0.07 mL of blood causes significant changes in nonsynaptic mitochondrial respiratory parameters. The nonsynaptic mitochondrial function was extensively studied to check the cell damage during anoxic-ischemic brain insult [ 11,16,30,32] in nonsynaptic mitochondria isolated after a complete or incomplete ischemia, a reduction in state 3 respiration (oxygen consumption in the presence of exogenous ADP) with a minor impairment of state 4 (oxygen consumption in the absence of exogenous ADP) is the typical pattern [16,32] Our results suggest that after experimental isobaric SAH, the nonsynaptic mitochondrial impairment is area specific: a reversible inhibition of the NADH-oxidative branch of the electron transfer chain is evident only in the frontal cortex in the acute phase of isobaric SAH,
Energy Metabolism After Experimental SAH
since the decrease in state 3 with glutamate plus malate as substrates appears evident, without variations in state 3 with sucCinate plus rotenone as substrates. The widespread increase in state 4 in all rat brain areas tested suggests a marked impairment of proton electrochemical gradient [14,42] and a dielectric breakdown of mitochondrial membrane [29]. This is evident only with NADH-generating substrates: the 02 consumption without exogenous ADP (state 4) is much lower than in the presence of FADH2-generating substrates. The impairment of mitochondrial membrane selectivity (i.e., H +) may reflect a change in 02 consumption, but it will be sufficient to determine a significant variation in state 4 with succinate plus rotenone. The mitochondrial membrane impairment could be the result of cascade reactions started by Ca 2+ influx after the hemorrhagic insult [17]. The different behavior of the hippocampus could be related to a different timedependent accumulation of Ca2+ in this area during experimental cerebral ischemia [9]. Deshpande et al [8] showed that the calcium content in the hippocampus, after cerebral ischemia, did not change until 24 hours, but after only 48 hours of the ischemic insult, the increase of Ca 2+ reached statistical significance. A further increase was shown at 72 hours. In experiment SAH, we have shown an impairment of hippocampal nonsynaptic mitochondria (significant decrease in the RCR) only at 72 hours: we can draw the hypothesis that this fact is related to calcium overload in the hippocampus. Recently, Voldby et al [47] have shown that after aneurysmal SAH, the cerebral metabolic rate for oxygen is reduced more then the CBF: this would indicate an uncoupled relationship between CBF and the oxidative cerebral metabolism. Moreover, Jakobsen et al [19] have found that the arterovenous difference for oxygen did not increase with the increasing degree of vasospasm, suggesting that vasospasm is not the cause of the decrease of the cerebral metabolic rate for oxygen. We can suggest that this phenomenon could be related to the significant impairment of mitochondrial function. In conclusion the results of the present study provide the experimental evidence that after SAH, the decrease in cerebral oxidative machinery could be related to a severe impairment of mitochondria and is site and time dependent, and that mitochondrial impairment is quite independent from the amount of blood injected and is not related to variations in ICP. The increased mitochondrial vulnerability in the delayed phases could be related to neuronal impairment following the initial bleeding and could be one of the possible biochemical correlates to post-SAH encephalopathy, which causes long-term cognitive disturbances.
Surg Neurol 299 1990;34:294-300
The findings in this paper were presented at the Annual Meeting of The American Association of Neurological Surgeons, Toronto, Canada, April 24-28, 1988. This research was supported by a grant from the Regione Lombardia, Milan, Italy, 1984.
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