High intracranial pressure (ICP) and interdependent low cerebral perfusion pressure (CPP) are serious neurologic complications that influence patient outcome (1). In recent years, a new concept has emerged, suggesting that secondary ischemic and hypoxic insults, such as reduced O2 delivery, reduced blood flow, or increased cerebral metabolism, account for a significant portion of brain damage (2). These secondary mechanisms can worsen brain swelling and ICP, further impairing CPP and brain oxygenation (3,4). The resulting decrease in oxygen supply may lead to drastic alterations in cerebral energy metabolism and structural tissue damage.
Lactate accumulates as the end product of glycolysis, if O2 imbalance arises between tissue O2 supply and demand, and oxidative phosphorylation as well as the tricarboxylic acid (TCA) cycle is reduced (5–7). Lactate also accumulates during intense cerebral stimulation under normoxic conditions and is thus an unreliable indicator of tissue hypoxia (8). For the detection of anaerobic metabolism, the simultaneous evaluation of pyruvate is necessary, because pyruvate is reduced to lactate by lactate-dehydrogenase under anaerobic conditions. The lactate/pyruvate-ratio (L/P-ratio) characterizes the relationship between aerobic and anaerobic metabolism, and reflects the cytosolic ratio of the reduced/oxidized forms of nicotinamide adenine dinucleotide. For this reason, the L/P-ratio is a reliable parameter for estimating the energy state of the cells (7). Brain function is also dependent on the sufficient delivery of glucose to brain cells by perfusion and diffusion processes. The interstitial glucose concentration reflects the balance between blood supply and utilization by cells (9). Besides energy-related metabolites, brain function can be reflected by changes in substances that indicate cell structure alteration. l-glutamic acid (glutamate) mainly mediates excitatory transmission in the brain, and under conditions of ischemia and hypoxia, an increased extracellular glutamate concentration is intimately involved in the development of neuronal damage (10,11). Phospholipids are the major constituents of brain tissue and provide the cellular membranes with suitable fluidity and permeability. The degradation of membrane phospholipids may lead to the impairment of vital membrane functions, a well-known phenomenon in acute brain disorders such as ischemia and hypoxia (12). Glycerol is an end product of this degradation and is a useful indicator of lipid hydrolysis after cerebral ischemia (13).
Intracerebral microdialysis is a highly sensitive technique for the early detection of cerebral hypoxia and allows the determination of regional metabolic tissue concentrations (14). The aim of this study was to explore the changes in energy-related metabolites harvested by microdialysis during a defined increase in ICP. To accomplish this goal, simultaneous recordings of biochemical markers were analyzed in relation to ICP and CPP.
This study involved 11 male domestic pigs (German landrace) with a body weight ranging between 32 and 40 kg. The animals were randomly assigned to either the experimental or control groups before the start of the study. All efforts were made to minimize animal suffering and to reduce the number of animals used. Experiments were conducted in accordance with the Law on Animal Protection and approved by the local Institutional Animal Ethics Committee.
Before surgery, animals were fasted overnight with free access to water. After premedication with 10 mg/kg ketamine IM (Ketamin, Cefa Sanre Animale, Germany), anesthesia was subsequently induced IV with 2–4 mg/kg propofol (Propofol, Parke-Davis, Germany) and maintained with a continuous infusion of 20–30 mg · kg−1 · h−1 propofol and 1–3 μg · kg−1 · h−1 fentanyl (Fentanyl-Janssen, Janssen-Cilag, Germany). The dosages of propofol and fentanyl were guided by clinical signs to ensure an appropriate depth of anesthesia. The pigs were tracheally intubated and mechanically ventilated, maintaining O2 saturation (SaO2) of 98%–100%. Arterial carbon dioxide partial pressures (Paco2) were adjusted between 35 and 37 mm Hg. Fluid was replaced during the experiment with a crystalloid solution at a rate of 2 mL · kg−1 · h−1. Arterial blood pressure was measured by an invasive method (A. femoralis, Leader Cath®, Vygon, France). A rectal thermoprobe was inserted to monitor body temperature, which was maintained as close to 37°C as possible by covering the animals during the experiment.
The scalp of the animal was incised in the sagittal direction to expose the surface of the skull. A burr hole (℘5 mm) was drilled in the skull 1 cm lateral to the sagittal suture and 1 cm rostral to the coronal suture. A BOLT-Catheter (Licox IM3, Integra Neurosciences, Plainsboro, USA) was fixed in the burr hole. Another burr hole (℘2 mm) was drilled 4 mm lateral to the sagittal suture and 10 mm rostral from the bregma. A catheter was inserted via this trepanation into the ventricular system of the brain, and connected with a pressure transducer (Smith Medical, Germany) to a registration unit (AS/3, Datex-Engstrom, Finland). The same channel was used to increase ICP levels by a bolus and subsequently continuous infusion of mock cerebrospinal fluid (sodium 147 mmol/L, calcium 2.25 mmol/L, potassium 4.0 mmol/L, chloride 155.5 mmol/L, glucose 100 mmol/L) into the ventricular system.
The technique of microdialysis is based on sampling fluid via a double-lumen probe with an integrated semipermeable membrane in which the equilibration of substances in the extracellular space and perfusion fluid takes place by diffusion according to the concentration gradient. A double-lumen microdialysis probe with a membrane length of 10 mm, an outer diameter of 0.6 mm and a cut-off at 20.000 Da (CMA 70, CMA-Microdialysis, Sweden) was used in our experiments. The microdialysis catheter was connected by low-volume fluorinated ethylene propylene-tubing (1.2μL/10 cm) to a precision infusion pump (CMA 102, CMA-Microdialysis, Sweden) and was continuously perfused at a flow rate of 2 μL/min with a solution containing 147 mmol/L NaCl, 2.7 mmol/L KCl, 1.2 mmol/L CaCl2, and 0.85 mmol/L MgCl2 (Perfusion fluid CNS, CMA-Microdialysis, Sweden).
The exchange of substances across the microdialysis membrane is limited by the total area of the membrane, the perfusion flow rate, the characteristics of the diffusing substance, and the diffusion constant in the tissue surrounding the probe (14,15). The recovery rate expresses the relation between the concentration of the substance in the microdialysis probe effluent and the concentration of the medium (16). Before and at the end of the experiment, the in vitro recovery rates for each probe were determined in a calibration solution equilibrated to 37°C by continuing the perfusion with the settings used during the experiment. In all experiments, we found no significant changes in the in vitro recovery rate. The measured experimental values were adjusted according to the relative recovery rate to estimate the in vivo extracellular concentration of the substances in the immediate vicinity of the probes. In vitro recovery rates were 53.9 ± 1.8% for lactate, 56.7 ± 2.1% for pyruvate, 56.6 ± 2.1% for glycerol, 46.3 ± 2.0% for glutamate, and 42.5 ± 1.9% for glucose (n = 11).
ICP in the experimental group was adjusted to 10 mm Hg, then increased stepwise by approximately 10 mm Hg until reaching a final pressure of 50 mm Hg (Fig. 1). Every ICP level was maintained for 1 h and was controlled intermittently. No changes in ICP were made in the control group. CPP was calculated as difference between arterial blood pressure and ICP. Transient increases in metabolites caused by probe insertion damage were avoided by a stabilization period of 60 min after insertion into the brain. In both groups, microdialysis samples were collected in 20 min fractions, whereas the transit time through the evacuation tube was considered. Dialysate samples were analyzed enzymatically with a CMA 600 Microdialysis Analyzer (CMA-Microdialysis, Sweden) for lactate, pyruvate, glucose, glycerol, and glutamate. The blood levels of O2, carbon dioxide, pH, lactate, and glucose were checked hourly by blood gas analysis (Rapidlab 865, Bayer Diagnostics). At the end of the experiment, killing was induced with an overdose of propofol, fentanyl, and potassium chloride.
The results of the experiments are expressed as the mean ± se of the mean (sem). Statistical analysis of the differences within and between groups was evaluated using a Welch corrected Student's t-test (InStat, GraphPad Software, San Diego, USA). Values of P < 0.05 were considered significant.
All 11 animals were included into the statistical analysis. There were no significant differences in blood glucose (102.46 ± 2.51 vs 101.57 ± 2.18 mg/dL, P = 0.79) or blood lactate levels (1.25 ± 0.15 vs 1.14 ± 0.05 mmol/L, P = 0.53) detectable between the groups. The pH was comparable between the groups, as were the Pao2, Paco2, and body temperature (Table 1).
Lactate and L/P Ratio
Basal cortical lactate levels were stable and showed no significant differences between groups (0.93 ± 0.12 vs 0.94 ± 0.25 mmol/L). After induction of cranial hypertension, cerebral lactate values were significantly increased above control at ICP levels ≥30 mm Hg and a concurrent reduction in CPP below 50 mm Hg (P < 0.05). The increase continued throughout higher cranial hypertension, reaching a maximum at the end of the last ICP step, coinciding with the lowest CPP (Fig. 2A). The increase in lactate was 514% of the basal level in the experimental group. Blood lactate concentrations in both groups remained constant throughout the experiment, suggesting that the increase in cortical dialysates was not caused by circulating lactate released from other organs. Changes in the L/P-ratio followed different time courses in the two groups. In the control group, no significant changes in the L/P-ratio were observed. The cranial hypertension group showed a significant increase in the L/P-ratio from 17.45 ± 2.37 during an ICP level of 10 mm Hg to 142.82 ± 48.12 at 50 mm Hg (P < 0.05). The significant difference compared with the control group was found at ICP levels ≥30 mm Hg and CPP levels <40 mm Hg (Fig. 2B).
Blood glucose levels remained stable during the course of the experiment in all animals. Basal cortical glucose concentrations were nearly equal in both groups and ranged around 1.30 mmol/L. The increase of ICP and concurrent reduction in CPP causes a strong decrease in the cortical glucose level. The decreased dialysate glucose concentration was statistically different from control values at ICP levels ≥40 mm Hg and a CPP below 40 mm Hg (0.89 ± 0.19 vs 0.29 ± 0.11 mmol/L, P < 0.05). During further ICP increase, dialysate glucose decreased to 0.20 ± 0.08 mmol/L at the end of the experiment (Fig. 3).
Glutamate and Glycerol
Basal glutamate levels were similar in both groups. A reduction in CPP to <40 mm Hg in conjunction with an increase in ICP ≥40 mm Hg was associated with significantly higher glutamate levels than those found in the control group, reaching maximum values at an ICP of 50 mm Hg (34.58 ± 11.60 vs 6.86 ± 2.90 μmol/L, P < 0.05). Peak glutamate levels were 322% of basal levels (Fig. 4A).
Basal glycerol values in the experimental and in the control group did not differ. During the increase of ICP, an immediate and continuous increase in interstitial glycerol occurred in the experimental group. At ICPs ≥30 mm Hg, a statistically significant difference was found between groups (P < 0.05). Maximum glycerol levels (223.19 ± 25.64 μmol/L) were found at an experimental ICP of 50 mm Hg and were 275% of basal values (Fig. 4B). In the control group, stable glycerol levels were observed throughout the experiment.
In the present study, we analyzed the changes in extracellular metabolites, namely glucose, lactate, pyruvate, glutamate, and glycerol, during a controlled increase of ICP and determined the values of ICP and CPP at which brain damage could be expected. Several studies have been published demonstrating that a reduction in CPP, cranial hypertension, and subsequent ischemia causes disturbances in the brain microenvironment and leads to irreversible brain damage (17–19). These studies measured only some metabolic variables, and there was no defined time course in the increase of ICP. Our main goal was the simultaneous measurement of these extracellular markers, a comparison with perfusion variables and a defined increase of ICP.
In the past few years, numerous studies have demonstrated that microdialysis is an excellent tool for exploring brain metabolism directly in the region of interest (2,20,21). This extracellular measurement of energy-related metabolites reflects metabolic events in the intracellular compartment. Cranial hypertension leads to a reduction in cerebral blood flow, followed by decreased O2 tension in the brain tissue and global ischemia (22). In severe ischemia, the high-energy metabolites adenosine triphosphate (ATP) and phosphocreatine, together with glucose, are consumed during the first few minutes, as reflected by an increase of lactate and a severe decrease of glucose in the tissue (23).
During the increase of ICP, there is an increase of interstitial lactate. Previously, this increased lactate was viewed as a waste metabolite from anaerobic metabolism accompanied by a decrease in extra- and intracellular pH. The precise role of lactate in brain tissue is more difficult to explain, because lactate concentrations within the brain also increase after physiologic stimulation (8). Other studies have shown that during O2 and glucose deprivation mild acidosis is neuroprotective (24). This could be an effect of lactate, which is then transported to neurons, and subsequently used as a substrate in the TCA cycle. If glucose were the only energy substrate available, diminished ATP levels would be further depleted before the TCA cycle could restore ATP levels, because glycolysis itself requires ATP. In contrast, the metabolism of lactate through the TCA cycle does not require ATP. Although lactate produces less total ATP than did glucose, the strain on depleted energy levels would be reduced, improving neuronal survival (25). It is obvious that lactate levels alone are not suitable as a damage marker. The extracellular L/P-ratio is a reliable marker of disturbed energy metabolism in ischemia, reflecting an impaired cytoplasmatic redox state (26). In addition, the L/P-ratio is probably not influenced by alterations in the in vivo recovery, because the recovery is nearly equal for both substances. In this study, an increase in the extracellular levels of lactate without an increase in the L/P-ratio was observed up to an ICP of 30 mm Hg and above a CPP of 40 mm Hg. This suggests that, under these conditions, increased glycolysis with preserved oxidative phosphorylation occurs, and lactate serves as an energy substrate. At ICP levels ≥40 mm Hg and a CPP below 40 mm Hg, O2 supply is drastically reduced, as reflected by an increase of the dialysate lactate concentrations and the L/P-ratio.
The brain depends on O2 and glucose supplies to maintain a sufficient production of ATP. The glucose concentration in the extracellular fluid reflects the balance between supply from the blood and utilization by the cells (9). At ICP levels ≥40 mm Hg and CPP below 40 mm Hg, a decrease in glucose levels is accompanied by an increased L/P-ratio, which indicates severe ischemia and a reduction in glucose supply due to a reduction in cerebral blood flow. Many studies on cell metabolism during severe ischemia/hypoxia have shown that the high energy metabolites ATP and phosphocreatine, along with glucose and glycogen stores, are consumed during the first few minutes (2,23,27). The mechanism by which extracellular glutamate is increased may be vesicular release, decreased glutamate reuptake, a reversal of the sodium glutamate transporter, or leakage from damaged cells (28). At ICP levels ≥40 mm Hg and CPP below 40 mm Hg, an increase in extracellular glutamate levels occurs. When energy stores are severely depressed, energy-dependent ionic pumps fail, resulting in the depolarization of neurons, the release of excitatory amino acids from presynaptic terminals, and the failure of transmitter reuptake systems (29). The correlation between glutamate levels and the L/P-ratio indicates that ischemic neurons release glutamate into the extracellular space.
Since the increase in extracellular glycerol started earlier than the increase in glutamate, it seems that this release was not caused by structural damage. In brain tissue, glycerol can be derived primarily from the glycolytic chain via glycerol-3-phosphate or from a phospholipase-activated degradation of the glycerophospholipids of cell membranes. Cerebral ischemia leads to the degeneration of membrane phospholipids, followed by cell membrane dysfunction and cell death (30,31). Glycerol is an end product of phospholipids breakdown in brain tissue and has been shown to be a promising marker of cell damage in traumatic brain injury where irreversible neuronal damage occurs (13). In our study, an increase of interstitial glycerol could be found at ICP levels ≥30 mm Hg and a CPP ≤50 mm Hg. It could be suggested that the first increase in interstitial glycerol is caused by release from the glycolytic chain, and then simultaneously, with glutamate increase at higher ICP levels, from degradation of membrane glycerophosholipids. Support for this hypothesis could be the correlation in the time courses of lactate and glycerol changes on the one hand and the L/P-ratio and glutamate on the other. In the context of neuromonitoring, interstitial glycerol could be a reliable marker of irreversible ischemia, complementary to the L/P-ratio and glutamate, which are faster to react, but more transient (30). At least, it seems to be obvious that the point of severe damage could be found where the L/P-ratio, glutamate, and glycerol are significantly increased.
Our investigation has some methodical limitations, which must be addressed. The comparison of a locally determined measure of microdialysate levels with a globally determined measure of CPP is an unavoidable obstacle (32). Therefore, local ICP and CPP in the vicinity of the microdialysis probe could be different from the pressure measured by the ventriculostomy tip. To limit this error, each ICP step was held for 1 h to ensure that the pressure would be consistent in all brain regions. Inherent changes in the extracellular space volume by increased water content due to hydrocephalic conditions may have had effects on microdialysate concentrations and probe efficiency. However, we suggest that the time course of changes in extracellular microdialysate levels is a more dynamic process than the slow development of an extracellular edema. In addition, we assume that the infusion of mock cerebrospinal fluid into the ventricular system on the opposite side of probe placement lead to much less extracellular edema in the measurement region. Additionally, the calibration of microdialysis probes can cause some problems. Compared with in vitro calibration, the in vivo recovery of substances strongly depends on the surrounding tissue properties, especially extracellular volume and geometry, as well as on various release, uptake, and clearance processes (33). Therefore, calculations of metabolite concentrations based on in vitro recovery can result in under-estimating of the actual interstitial concentrations (33–35). However, we believe that the changes in extracellular microdialysate levels reflect the time course of the dynamic process of an increase in ICP and a reduction in cerebral blood flow.
The present results show that an increase of ICP to ≥30 mm Hg and a concurrent decrease in CPP below 50 mm Hg causes a significant increase in lactate and glycerol levels. At higher ICP and lower CPP levels, an additional increase in extracellular glutamate concentrations and the L/P-ratio occurs, which reflects a reduction in O2 supply and the start of neuronal damage. Our results suggest that changes found below a CPP of 70 mm Hg are not immediately severe and irreversible. In our model of defined increased cranial hypertension and concurrent reduced CPP, borderline ICP and CPP values of 30 mm Hg and 40–50 mm Hg, respectively, can be determined.
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