A technique for continuous bedside monitoring of global cerebral energy state

Anesthesia, mechanical ventilation, and surgical preparation

The porcine model of hemorrhagic shock has been previously described [15]. The animals were fasted overnight with access to ad libitum water. Sedation was achieved with a standard mixture of medetomedin (0.05 mg/Kg), midazolam (0.25 mg/Kg), and atropine (0.25 mg/Kg). Anesthesia was induced with midazolam (0.625 mg/Kg) and ketamine (12.5 mg/Kg) and maintained with infusion of midazolam (5 mg/Kg/h) and fentanyl (50 μg/Kg/h). The animals were intubated and volume-controlled ventilated (Siemens 900 Ventilator; Siemens Elema, Stockholm, Sweden) with a tidal volume of 10 mL/kg and FiO₂ of 0.30. PaCO₂ was kept between 4 and 6 kPa and body temperature around normal 38.5 °C.

Multimodal monitoring

After establishing anesthesia, one sheath was inserted into the carotid artery for blood pressure monitoring and blood gas sampling, while the external jugular vein was cannulated for insertion of a pulmonary artery catheter (CCOmbo; Edwards Lifesciences, Irvine, CA, USA) to monitor cardiac output (CO), core temperature, and central venous pressure (CVP). Arterial blood gases (PaCO₂, PaO₂, pH), blood glucose electrolytes, and lactate levels were measured every 30 min (ABL800 FLEX, Radiometer Denmark).

One femoral artery was cannulated for withdrawing and re-infusing blood during the induced hemorrhagic shock. Another sheath was placed in the contralateral femoral artery, and a microdialysis catheter (CMA 70 Bolt, M Dialysis AB, Stockholm, Sweden) was inserted. A small craniotomy was placed in the frontal bone in the midline above the superior sagittal sinus. The sinus was cannulated by a standard 18G peripheral venous catheter, and one microdialysis catheter (CMA 70 Bolt, M Dialysis AB, Stockholm, Sweden) was introduced in the posterior direction and placed in the posterior part of the superior sagittal sinus. The superior sagittal sinus was chosen for analysis of cerebral venous blood due to the anatomic characteristics of the experimental animal. In the pig, most of cerebral blood is drained via paraspinal venous plexa and only a minor part passes into the internal jugular vein [16]. A third microdialysis catheter (CMA 70, M Dialysis AB, Stockholm, Sweden) was inserted 20 mm into the left parietal lobe and one probe for monitoring brain tissue oxygenation (PbtO2) (Licox CC1SB, Integra Neurosciences Ltd., NJ, USA) was introduced 15 mm into the contralateral parietal lobe. A transducer for monitoring intracranial pressure (ICP) (Camino, Integra Neurosciences Ltd., NJ, USA) was placed in the right hemisphere. A bladder catheter was placed for urine collection. All animals were given a baseline dose of 200 U/kg of heparin and supplemented hourly with 100 U/kg for anticoagulation during the hemorrhage period. At the end of experiment, the anesthetized animals were killed with an i.v. injection of sodium pentobarbital 200 mg/mL in concentrated ethanol.

Experimental protocol

Following a 120-min baseline period allowing animals to stabilize, the following surgery hemorrhagic shock was achieved by bleeding the animals to a pre-defined MAP of approximately 40 mmHg at a rate of 2.15 mL/kg/min over 7 min and then 1.15 mL/kg/min over the remaining period [15]. Animals were kept at a MAP of about 30–40 mmHg by withdrawing or infusing shed blood that was stored in a citrated glucose solution at 37 °C. Following 90 min of hemorrhagic shock, the animals were resuscitated by re-infusing the shed blood at a rate of 120 mL/min until all blood was returned. The pigs were observed for 3 h after hemorrhagic shock. Microdialysis probes were perfused with artificial CSF (M Dialysis AB, Stockholm, Sweden) at a rate of 0.3 μl/min (CMA 106 MD pump, M Dialysis AB, Stockholm, Sweden). The dialysates were collected in microvials and immediately analyzed for glucose, lactate, pyruvate, glutamate, and glycerol every 30 min using an ISCUS^Flex analyzer (M Dialysis AB, Stockholm, Sweden). After insertion, all probes were allowed a minimum of 2 h for stabilization.

PbtO₂ data were collected using the AC3.1 monitor (Integra Neurosciences Ltd.) and recorded every 20 s. All Licox probes were tested against atmospheric air and against each other before insertion and after removal. After insertion, appropriate function was confirmed by an oxygen challenge test.

ICP was monitored continuously and data were collected by a CAM01 monitor (Integra Neurosciences Ltd., NJ, USA) and ICP. Cerebral perfusion pressure (CPP) was calculated as MAP − ICP.

Statistics

Data are given as median (interquartile range) unless otherwise noted. To test our hypothesis, the time course of the LP ratio in the superior sagital sinus and femoral artery was modeled utilizing a mixed-effects model for repeated measurements with time and location of microdialysis probe as random effects and each animal as fixed effect. The null hypothesis was that no difference in the LP ratio was found between the superior sagital sinus and femoral artery. A p value below 0.05 was considered significant. Data analysis was performed in Stata 11.1 statistical software (StataCorp, College Station, TX, USA).

PbtO₂ and intracerebral glucose

PaO₂ and b-glucose levels were kept relatively constant during the experimental period (Table 1). In spite of this fact, PbtO₂ and intracerebral glucose decreased to very low levels during the induced hypotensive shock and remained very low after re-infusion of blood (Table 1, Fig. 1). This pattern is compatible with that observed during cerebral ischemia and corroborates the intracerebral microdialysis findings (Table 2). Accordingly, although MAP returned to close to the initial level after blood re-infusion (Table 1), cerebral perfusion was obviously not sufficient for restoring energy metabolism. The finding is probably explained by the fact that a progressive increase in ICP caused a decrease of CPP to a low level (40 mmHg; Table 1). The reason for the progressive increase in ICP is probably because of a global postischemic cytotoxic edema and leakage of the blood-brain barrier. Due to insufficient perfusion, arterial blood glucose was virtually completely extracted which resulted in a very low glucose level in the sagittal sinus (Table 2).

Glutamate and glycerol

During clinical intracerebral microdialysis use, an increase in glutamate concentration is generally interpreted as insufficient astrocytic uptake of released glutamate due to energy failure [21, 22]. In the present study, intracerebral glutamate increased markedly during the hypotensive shock period and did not return to normal level after blood re-infusion (Table 2). Thus, the observed changes in intracerebral glutamate are in accordance with the interpretation above: hypotensive shock caused cerebral ischemia and energy failure that did not recover after blood transfusion.

The normal blood-brain barrier is not permeable to glutamate [21]. Under normal conditions, interstitial cerebral concentration is approximately 2 μmol/L while blood concentration is 100–200 μmol/L. Accordingly, glutamate level obtained in cerebral venous blood does not reflect the intracerebral level. In the present study, the high concentration of glutamate obtained before the start of the experiment (100–200 μmol/L; Table 2) documents that the microdialysis catheter was actually positioned in the superior sagittal sinus in each experimental animals.

Intracerebral glycerol measured by microdialysis is conventionally used as a marker of degradation of cellular membranes into free fatty acids and glycerol [23, 24]. In the present experimental situation, intracerebral glycerol increased to a very high level during hypotensive shock and remained at this high level after transfusion (Table 2). The finding supports the interpretation that induced hemorrhagic shock to MAP 30 mmHg for 90 min caused cerebral energy failure and decomposition of cellular elements. However, increase in glycerol in cerebral venous blood (Table 2) does not necessarily result from degradation of cerebral cellular elements. The intact BBB has a very low permeability for glycerol [25]. In many extracerebral tissues, triglycerides are important cellular components. During stress and increased sympathetic tonus, triglycerides are degraded, which is reflected in fat tissue and in the blood as an increase of free fatty acids and glycerol [26]. Accordingly, an increase in glycerol concentration was in the present experimental situation also obtained in the femoral arterial blood (Table 2).

Clinical relevance of the experimental model

The study indicates that it is possible to evaluate global cerebral energy state by simultaneous monitoring of the redox state (LP ratio) in a cerebral vein. Under clinical conditions, this could be performed by placing the venous microdialysis catheter in the internal jugular vein close to the jugular bulb. In this way, it might be possible to continuously evaluate cerebral energy state bedside without inserting an intracerebral probe. This technique would be valuable in various serious conditions in need of critical care.

After cardiac standstill and resuscitation, the possibilities of evaluating cerebral damage and prognosis are still limited [27–30]. In these patients, a bedside continuous technique might also be used to monitor the effects of various therapies (e.g., hypothermia). For this purpose, intracerebral microdialysis has been used in a few selected cases [31] but it is unlikely that this invasive technique will be used in clinical routine. In patients subjected to open-heart surgery with or without cardiopulmonary bypass, minor cerebral complications appear to be frequent [32–36]. In these patients, analysis of lactate concentration from a microdialysis catheter positioned in a central vein has been proposed [37]. Although this technique was shown to give reliable information regarding global venous lactate level, it will not give specific information regarding cerebral energy metabolism. In patients with hepatic failure (HF) leading to cerebral symptoms and coma, intracerebral microdialysis has shown that an increase in tissue LP ratio is correlated to increases in tissue glutamine and hypoxanthine [38, 39] and energy failure appears to be an important pathogenetic component of both acute and chronic HF and a potential target for therapy [40].

The technique of evaluating global cerebral energy/redox state from the LP ratio obtained from a microdialysis catheter positioned in the internal jugular vein might give important information in a multitude of severe clinical conditions when direct measurements of tissue biochemistry is difficult or impossible. However, it should be recalled that evaluation of the LP ratio in the draining vein will not give quantitative, correct information regarding cerebral extracellular LP ratio (Table 2). This is of limited importance. Under clinical routine conditions, an upper normal level for the LP ratio is utilized (usually 30 or 40) and the exact level of the LP ratio is often of secondary importance [11, 41]. During intracerebral microdialysis, the LP ratio and the concentration of pyruvate have also been used to differentiate between ischemia and mitochondrial dysfunction [8, 10, 11]. This kind of detailed analysis and interpretation may not be possible when the LP ratio is monitored in cerebral venous blood.

During neurocritical care, the cerebral interstitial levels of glutamate and glycerol are used as indicators of insufficient energy production and cellular degradation. However, if the microdialysis catheter is positioned in cerebral venous blood, these interpretations are not valid for reasons given above.

Limitations

In experimental hemorrhagic shock, it has been described that, in contrast to the systemic macrocirculation, cerebral microcirculation may be remarkably well preserved [42]. Though the implications of this finding have been questioned, it is still an open question when and to what degree cerebral energy metabolism is compromised during hemorrhagic shock under clinical conditions [43]. In a recent experimental study utilizing multimodal monitoring with simultaneous imaging of cerebral hemodynamics and NADH signals, the authors demonstrated the temporal relationship between compromised microcirculation and compromised oxidative metabolism [44]. In this model of severe hemorrhagic shock, the oxidative metabolism was not restored after re-transfusion of the extracted blood volume. From that study and the present data, it appears that during severe hemorrhagic shock cerebral energy metabolism is severely compromised exhibiting a biochemical pattern typical of ischemia. Further, if hypotension is protracted and severe enough, cerebral energy metabolism may not be restored after transfusion. In the present experimental study, the biochemical pattern and the progressive increase of ICP indicated permanent cerebral lesions. The present experimental model was chosen because it creates reproducible severe global cerebral ischemia. However, the chosen hypotensive level of MAP around 35 mmHg is somewhat lower that the recommendations by the European Society for Intensive Care Medicine expert panel [45]. It is therefore important to stress that the present study cannot be used to determine the optimal level of MAP after hemorrhagic shock. The purpose of the present study was solely to establish a technique for “non-cranial” invasive monitoring of cerebral energy state. As shown in Table 2, there is a quantitative discrepancy in the LP ratio between the cerebral microdialysis probe and the one placed in the sinus. This suggests a “washout” effect. The degree of metabolic derangement in the present study was severe. In a clinical setting, e.g., after cardiac standstill, a less pronounced metabolic derangement will be expected. Accordingly, it might not be possible to detect minor metabolic derangements in the venous jugular bulb due to the washout effect. Future clinical studies are needed to determine this.