Microdialysis

Idil Cavus

Charles L. Wilson

Introduction

Microdialysis is a method that allows continuous in vivo sampling and neurochemical analysis of brain extracellular fluid (ECF) for extended periods of time. Although brain extracellular (EC) space comprises only 15% of brain volume, the neurochemical changes in this compartment are critical for the functioning of the various cellular receptors and transporters. Brain microdialysis was first introduced in animal research in the 1970s³²^,¹²⁵ and applied to human research in the 1990s. This method has been used in numerous animal models of disease, including epilepsy, to measure the EC levels of ions, drugs, neurotransmitters, and other small molecules and peptides under anesthetized as well as awake behaving conditions. In humans, brain microdialysis is used primarily to monitor or study the neurochemistry of brain injury related to trauma, stroke, or ischemia in neurointensive care and tumors in the neurosurgical setting, and it has also been used during intracranial electroencephalographic (EEG) monitoring of epilepsy patients (for reviews, see Benjamin et al.¹¹ and Hillered et al.⁵¹). Recently, significant advances in clinical microdialysis research have allowed the technique to be used not only for the study of disease neurochemistry, but also as a bedside monitoring and diagnostic tool in neurointensive care patients.¹⁰

Microdialysis Methodology

The microdialysis setup is relatively simple, involving a probe/catheter, a perfusion pump, and a collection system. The microdialysis probe (∼1 mm in diameter) consists of a concentric or side-by-side dual catheter with a semipermeable membrane attached to its tip and connected to inlet and outlet tubing. The probe membrane is usually 1 to 3 mm long in rodent studies and 5 to 30 mm in humans, with typically 10- to 20-kDa molecular weight cutoff. Microdialysis membranes with small pore sizes have the advantage of acting as an additional barrier to likely infectious agents, and may be preferable in human research. A recently introduced probe for human research with 110-kDa molecular cutoff,⁵⁴ however, allows for sampling of larger proteins and can be useful in studying the proteomics of the brain microdialysate.⁷⁰ A microdialysis probe with even larger membrane cutoff (3,000 kDa) has been used in tumor patients to sample neurotrophins and inflammatory factors.¹³⁶ Usually, the commercially available CMA 70 (molecular cutoff 20 kDa; CMA Microdialysis, North Chelmsford, MA) has been the probe of choice for human research in brain trauma, stroke, and tumor patients. In human epilepsy research, however, in which stereotaxic placement and simultaneous electrophysiologic monitoring are desired, probes with smaller diameters and longer shafts are preferred. The earlier homemade designs³⁵ have been replaced by custom-made probes. These probes can be inserted inside the depth electrode (Spencer probe) for simultaneous EEG³⁵ or single unit activity monitoring.⁴³ In human epilepsy research, patients with refractory epilepsy who are evaluated with intracranial electrodes for localization of their seizure onset site are implanted with microdialysis or Spencer probes after obtaining institutional review board approval and the necessary informed consents.

Spencer probes can be stereotaxically implanted into the brain area of interest and their position confirmed with magnetic resonance imaging (MRI) and computed tomography (CT). The probe is perfused with sterile artificial ECF through the inlet at relatively slow rates (usually 0.1–2.5 μL/min) allowing for diffusion of molecules across the membrane down their concentration gradient. The dialysate fluid is collected either manually or automatically (typical sampling rates 1–60 min) and can be analyzed on-line or off-line using high-performance liquid chromatography (HPLC), mass spectroscopy, or enzyme-based methodologies.⁷⁸ The more recently developed and highly sensitive capillary electrophoresis analysis allows for relatively high (5–60 seconds) temporal resolution,⁷¹^,⁸⁰ which is more useful in tracking fast neurochemical changes during seizures.

The amount of brain chemical recovered in the dialysate represents only a fraction of its actual interstitial concentration. This relative recovery depends on many factors, including the molecular weight, charge, uptake, and metabolism of the measured compound, diffusion, tortuosity factors in the interstitial microenvironment, length and composition of the dialysis membrane, flow rate, temperature, and composition of the perfusion fluid.¹²^,¹⁵ Usually, higher relative recovery is obtained at lower perfusion rates and with longer membranes.¹²³ The absolute EC concentration of the measured neurochemicals can be estimated using quantitative microdialysis methods such as no-net-flux⁶⁵ and extrapolation-to-zero-flow methods.⁵⁵ These methods have been rarely used in clinical research, for example, in epilepsy patients,¹^,²⁰ and in neurointensive care,⁵³^,⁶² because of their cumbersome nature.

The insertion of the microdialysis probe causes local tissue damage, transient disruption in the blood–brain barrier, and gliosis around the probe tract.¹³^,¹³⁴ Some of this damage is attributed to the relatively large, nonsterile probes inserted in small animals’ brains. Postmortem studies in sheep brain using sterile probes approved for human use reveal minimal disturbance to the cerebral parenchyma.¹³⁴ Similarly, implantation of small, flexible, sterile catheters in epileptic patients³⁵ has been reported to leave only a small track with minimal gliosis in the brain tissue. After the probe insertion, there is an initial period of disturbed metabolic function that can last up to 24 hours.¹²^,¹⁴ The disruption in the blood–brain barrier usually heals within 2 hours,¹⁵^,⁵² although a widespread leakage of albumin into the rat brains 24 hours after probe implantation has been also reported.¹³¹ In humans, several clinical studies cite stabilization of dialysate levels within 20 to 60 minutes of probe insertion.⁵² In contrast to microdialysis in rodents, in which dynamic exchanges across the dialysis membrane are lost relatively rapidly after probe implantation and in which experiments frequently must be conducted on the same day as the
probe implantation, human microdialysis parameters appear remarkably stable over many days. This stability is attributed to the minimal tissue damage and gliosis associated with the implantation procedure, which is most likely due to the use of sterile, relatively small, and flexible probes that are floating rather than rigidly secured to the skull, decreasing the trauma to the brain parenchyma.³⁵

In epilepsy patients, microdialysis can be performed intraoperatively or during the intracranial EEG monitoring phase, on an intermittent or continuous basis (usually up to 2 weeks). In our experience, continuous bedside microdialysis for several days at low flow rates can be used safely in epilepsy patients and in neurointensive care.¹⁰^,⁴⁹^,⁵⁰^,⁸¹ The microdialysis probe can be also used for drug delivery to the brain (reverse dialysis), and this technique is often used in animal experimentation. In humans, drug delivery through microdialysis has been used for local brain tumor therapy⁹⁶ and for research purposes in epilepsy³⁷^,³⁸ and stroke patients.⁵⁸ Microdialysis can be applied simultaneously with other methods, such as positron emission tomography (PET)⁴¹^,⁵² and Doppler flowmetry,⁵⁶ to study the relationship between the neurochemical changes in the EC milieu and changes in brain metabolism and blood flow.

The Cellular Origin of the Dialysate Neurotransmitters

Microdialysis typically samples neurochemical changes and volume transmission in the immediate vicinity of the probe, and it may provide information only indirectly on the changes within the synapse.¹²^,³⁴^,¹³³ The probe is much larger than the synaptic cleft, and the time resolution is orders of magnitude slower than that for synaptic events. The extrasynaptic concentration of a transmitter reflects a balance between its rate of neuronal and glial release and reuptake, together with its diffusion outside the synapse. In addition, these parameters are affected by the neuroanatomic location and density of terminals, receptors, and transporter molecules. As such, the measured ambient neurotransmitter levels may be most relevant for the function of the extrasynaptic receptors.

Although there is evidence that dialysate dopamine, norepinephrine, serotonin, and acetylcholine are primarily of neuronal, calcium-dependent origin, the cellular origin of glutamate and γ-aminobutyric acid (GABA) has been disputed.¹¹⁷^,¹³² In most cases, glutamate and GABA in dialysate are insensitive to tetrodotoxin and to calcium depletion, raising the possibility of glial metabolic release.¹¹⁷ Indeed, recent evidence indicates that glia are capable of releasing both glutamate and GABA through a variety of mechanisms, calcium dependent and independent, including tonic nonvesicular release and transporter reversal, via the cystine/glutamate antiporter and in response to neuronal stimulation.⁹^,¹⁹^,³¹^,⁸⁹ Thus, the basal microdialysate levels of glutamate and GABA may be most relevant to our understanding of volume transmission and the regulation of tonic excitation and inhibition through activation of the extrasynaptic receptors, which can in turn affect excitability.¹⁹^,³¹^,⁸⁸ EC glutamate and GABA may be also of neuronal origin, particularly in response to stimulation. Synaptically released glutamate and GABA can spill over into the EC space and become accessible to the microdialysis probe during intense physiologic and electrical stimulation, particularly when glial reuptake or coverage is reduced.³^,³⁴ Improvements in microdialysis method (smaller probes, increased temporal resolution, minimal tissue trauma) and in analytical procedures may enhance the detection of synaptically released neurotransmitters.³⁴^,⁸⁷

Role of Microdialysis Studies in Animal Models of Epilepsy

Although the primary utility of microdialysis in animal models has been to further our understanding of the basic mechanisms of epilepsy, these studies have also provided data that may have potential diagnostic value for patients.¹⁷ Measurement of brain anticonvulsant levels in animal models and their effect on electrographic, neurochemical, and behavioral expression of seizure activity only provides approximations of effective brain levels of antiepileptic drugs (AEDs) because of differences in AED absorption and breakdown between rodents and humans.¹³⁰ Although the effective levels may differ, the mechanisms of action can more easily be determined in animal models of epilepsy, which provide important information for identification of AED targets by measurement of neurotransmitters and transporters that are modulated by seizures. This provides a means for evaluating the potential for effective use of an AED in patient treatment²⁵^,²⁶ or identification of receptor agonists or antagonists with anticonvulsant potential by providing in vivo data complementary to in vitro electrophysiology.¹⁰⁹

Initial in vivo microdialysis studies of neurotransmitter modulation in epilepsy published in the 1980s and 1990s largely focused on the amino acids (AAs) glutamate, aspartate, taurine, and GABA in attempts to determine their role in seizure genesis as reflected by changes in AA release in rodent brain during seizures evoked by excitotoxins or stimulation (for review, see Chapman²¹). The use of microdialysis specifically in animal epilepsy research has increased from about 90 studies in the first 10 years of its use to >170 animal studies in an equivalent period since 1997, and these studies have focused on an ever-wider range of topics related to a variety of epilepsy-related neurotransmitters and neuromodulators, receptor agonists, and antagonists, as well as AEDs.

The animal models employed in these microdialysis studies are extremely varied but can be roughly divided into acute, chronic, and genetic. Acute models (such as systemic, intra-cerebral, or reverse dialysis administration of substances that induce depolarization, afterdischarge, and status epilepticus) are useful for evaluation of anticonvulsants, whereas chronic models (such as kindling models, which usually require several weeks of electrical or chemical stimulation; the status epilepticus model; and the genetic models) have the potential of identifying antiepileptic properties of drugs and perhaps more faithfully simulate the interictal and ictal clinical expression of epilepsy.

Microdialysis Research in Epilepsy in Animal Models

Amino Acid Studies

Investigation of the roles of excitatory and inhibitory amino acids in seizure generation has been a primary focus of microdialysis research. Naive rats generally show little change in AAs in response to a single acute electrical stimulation or chemical administration of picrotoxin (PTX), bicuculline (BMI), pentelynetetrazol (PTZ), or kainate (KA).⁷⁴^,¹⁰⁶ Systemic administration of 4-aminopyridine (4-AP)⁷² is effective in evoking a two- to sixfold increase in hippocampal glutamate or glutamine, and local reverse dialysis of pilocarpine produces significant release of glutamate as well as dopamine,¹⁰⁸ accompanied by seizures. Seizure-related increases in EC glutamate similar to the ones observed in humans³⁹^,¹³⁵ have been observed more reliably in chronic models of epilepsy, such as kindling with electrical stimulation¹²⁰ or intrahippocampal¹³⁵ or amygdalar KA injection.¹¹⁹ Paradoxically, in a PTX kindling model, ictal
discharges were associated with initial decrease in glutamate and aspartate,¹⁰⁴^,¹⁰⁵ which was followed by delayed, long-lasting increase in glutamate,¹⁰⁷ again indicating the need for rapid measurement to detect transient glutamate change and suggesting an important role for glia in slower glutamate modulation.

Repeated bursts of high-potassium stimuli in the rat hippocampus¹¹⁸ or electrical stimulation in kindled rats¹²¹ were associated with graded increases in glutamate release, suggesting that repeated short-term increases in EC glutamate levels results in enhancement of excitatory neuronal systems, causing propagation of seizure activity and culminating in secondary generalized seizures. In KA-kindled rats, the hippocampus had elevated interictal glutamate, which increased sharply in response to potassium-induced depolarization,¹¹⁹ findings that were again similar to reports in humans.²⁰^,³⁹ Ueda and colleagues¹¹⁹ identified a parallel downregulation in hippocampal glial glutamate transporters GLAST and GLT-1 as the basis of the increased interictal basal glutamate levels.

Not only are kindling models more responsive, but chronic rat models and genetic models also show AA increases that are absent in naive rats.⁶⁴^,⁸³^,⁹⁹^,¹¹³ Changes in metabotropic glutamate receptors have also been identified as important mediators of AA release.²²^,⁷⁶^,¹⁰⁹

Amino Acid Transporter Studies

Given the normally rapid reuptake of glutamate by its transporters and the resistance to glutamate elevation during transient evoked seizures in normal rat brain,⁶³^,⁷³ the observation of glutamate elevation with microdialysis during spontaneous seizures in the human hippocampus³⁷^,³⁹^,¹³⁵ has generated interest in the possible reduction or loss of glutamate transport in epilepsy. The glutamate transporters GLAST, GLT-1 (glial), and EAAC1 (neuronal) have received substantial study using the combination of microdialysis to detect changes in glutamate or GABA levels and quantitative immunoblotting or in situ hybridization to measure changes in transporters that might be associated with seizure models.⁷³ The genetically epilepsy-prone rat (GEPR) shows reduced GLAST, GLT-1, and EAAC-1 mRNA but not protein,⁵ whereas results of kindling in normal rats have been variable depending on the measure used,⁶^,⁴⁴^,⁷⁵^,¹²⁰ but usually showing less change in GLT-1. Rats receiving chronic administration of antisense oligonucleotide probes, which drastically reduce the expression of glial glutamate transporter GLT-1, respond with elevated EC glutamate levels and signs of excitotoxic neuronal degradation, whereas rats receiving antisense probes against the neuronal glutamate transporter EAAC-1 show no elevation of EC glutamate but do develop seizures.⁹⁷ Chronic KA¹¹⁹ or iron chloride¹⁰⁰ rat models are consistent in showing reduced GLT-1, and although most glutamate uptake is mediated by GLT-1 into glia, GLT-1 also transports glutamate into neuronal terminals,¹¹² making interpretation of transporter data more complex.

Antiepileptic Drug Pharmacokinetics and Effects on Neurotransmitter Release

Microdialysis has been used for pharmacokinetic analysis of AEDs,⁴⁶^,¹²⁹^,¹³⁰ and methodologic issues related to such studies have been discussed extensively by de Lange et al.²⁸^,²⁹^,³⁰ Studies using microdialysis in rat models of epilepsy to investigate the change in glutamate and GABA levels in response to AEDs usually report decreased glutamate levels and variable effects on GABA.³^,⁴^,⁵⁷^,⁶¹^,⁹⁸^,¹⁰⁷ Monoamine response to AEDs has also been investigated⁴^,²⁷^,⁷⁹ to determine whether increases in serotonin or dopamine may be associated with AED efficacy. Although changes in serotonin have been associated with action of these AEDs, dopamine’s role is less clear. Monoaminergic changes were preceded and are generally secondary to the role of GABA increase or Na⁺ channel blockade by AEDs.

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