Anker Jon Hansen

Determination of Brain Interstitial Concentrations by Microdialysis

Abstract: Microdialysis is an extensively used technique for the study of solutes in brain interstitial space. The method is based on collection of substances by diffusion across a dialysis membrane positioned in the brain. The outflow concentration reflects the interstitial concentration of the substance of interest, but the relationship between these two entities is at present unclear. So far, most evaluations have been based solely on calibrations in saline. This procedure is misleading, because the ease by which molecules in saline diffuse into the probe is different from that of tissue. We describe here a mathematical analysis of mass transport into the dialysis probe in tissue based on diffusion equations in complex media. The main finding is that diffusion characteristics of a given substance have to be included in the formula. These include the tortuosity factor (λ) and the extracellular volume fraction (α). We have substantiated this by studies in a welldefined complex medium (red blood cell suspensions) as well as in brain. We conclude that the traditional calculation procedure results in interstitial concentrations that are too low by a factor of λ2/α for a given compound.

Clinical restitution following cerebral ischemia in hypo-, normo- and hyperglycemic rats.

Rats with different levels of blood glucose concentration were exposed to 10 min of complete brain ischemia achieved by compression of neck vessels by a pneumatic cuff.

Beyond the role of glutamate as a neurotransmitter

Glutamate is the principal excitatory neurotransmitter of the central nervous system, but many studies have expanded its functional repertoire by showing that glutamate receptors are present in a variety of non-excitable cells. How does glutamate receptor activation modulate their activity? Do non-excitable cells release glutamate, and, if so, how? These questions remain enigmatic. Here, we review the current knowledge on glutamatergic signalling in non-neuronal cells, with a special emphasis on astrocytes.

Spreading depression is not associated with neuronal injury in the normal brain

This study was performed in order to evaluate whether waves of spreading depression (SD) induces irreversible neuronal injury. SD was elicited by topical application of 3 M KCl to the exposed cortex for 4-5 h and the resulting change of the cortical electrical potential showing the occurrence of SD, was recorded by glass microelectrodes. Histological examination of cerebral cortex revealed no signs of neuronal injury outside the area of KCl application as examine after 4 days recovery. The results indicate that recurrent waves of SD do not induce irreversible neuronal injury in the otherwise normal rat brain.

Cortical spreading depression causes and coincides with tissue hypoxia

Cortical spreading depression (CSD) is a self-propagating wave of cellular depolarization that has been implicated in migraine and in progressive neuronal injury after stroke and head trauma. Using two-photon microscopic NADH imaging and oxygen sensor microelectrodes in live mouse cortex, we find that CSD is linked to severe hypoxia and marked neuronal swelling that can last up to several minutes. Changes in dendritic structures and loss of spines during CSD are comparable to those during anoxic depolarization. Increasing O2 availability shortens the duration of CSD and improves local redox state. Our results indicate that tissue hypoxia associated with CSD is caused by a transient increase in O2 demand exceeding vascular O2 supply.

Characterization of Cortical Depolarizations Evoked in Focal Cerebral Ischemia

Cortical tissue surrounding acute ischemic infarcts undergoes repetitive spontaneous depolarizations. It is unknown whether these events are episodes of spreading depression (SD) elicited by the elevated interstitial K+ ([K+]e) in the ischemic core or whether they are evoked by transient decreases of the local blood flow. Electrophysiologically, depolarization caused by SD or by ischemia (ID) can be distinguished by their characteristic patterns of [K+]e rise: During SD, [K+]e rises abruptly, while in ID, this fast rate of increase is preceded by a slow rate lasting minutes. To characterize the depolarizations, we occluded the right middle cerebral artery (MCA) in rats and inserted two K+-sensitive microelectrodes into the cortex surrounding the evolving infarct. Repeated increases in [K+]e arose spontaneously following MCA occlusion. [K+]e increased during these transients from a resting level of 3–6 to 60 mM. One-third of these transient increases in [K+]e were biphasic, consisting of a slow initial increase to 10–12 mM, which lasted for minutes, followed by an abrupt increase, a pattern characteristic of ID. The remaining two-thirds exhibited a steep monotonic increase in [K+]e (>10 s), characteristic of SD. The duration of the transients was a function of the pattern of [K+]e increase: ID-like transients lasted an average 10.7 ±5.1 min, whereas the duration of SD-like transients was 5.7 ± 3.4 min. Both types of K+ transients occurred in an apparently random fashion in individual animals. A K+ transient was never observed solely at one electrode. In 40% of the cases, the K+ transients occurred simultaneously at the two electrode sites, and in the remaining a temporal separation of 20–420 s was observed. Our data suggest that the majority of the spontaneous depolarizations evoked by focal ischemia are SD-like phenomena probably evoked by the high values of [K+]e or glutamate in the ischemic focus, while the rest are elicited by independent foci of low blood flow within the ischemic border areas.

Regional changes in interstitial K+ and Ca2+ levels following cortical compression contusion trauma in rats

Brain trauma is associated with acute functional impairment and neuronal injury. At present, it is unclear to what extent disturbances in ion homeostasis are involved in these changes. We used ion-selective microelectrodes to register interstitial potassium ([K+]e) and calcium ([Ca2+]e) concentrations in the brain cortex following cerebral compression contusion in the rat. The trauma was produced by dropping a 21 g weight from a height of 35 cm onto a piston that compressed the cortex 1.5 mm. Ion measurements were made in two different locations of the contused region: in the perimeter, i.e., the shear stress zone (region A), and in the center (region B). The trauma resulted in an immediate increase in [K+]e from a control level of 3 mM to a level >60 mM in both regions, and a concomitant negative shift in DC potential. In both regions, there was a simultaneous, dramatic decrease in [Ca2+]e from a baseline of 1.1 mM to 0.3–0.1 mM. Interstitial [K+] and the DC potential normalized within 3 min after trauma. In region B, [Ca2+]e recovered to near control levels within 5 min after ictus. In region A, however, recovery of [Ca2+]e was significantly slower, with a return to near baseline values within 50 min after trauma. The prolonged lowering of [Ca2+]e in region A was associated with an inability to propagate cortical spreading depression, suggesting a profound functional disturbance. Histologic evaluation 72 h after trauma revealed that neuronal injury was confined exclusively to region A. The results indicate that compression contusion trauma produces a transient membrane depolarization associated with a pronounced cellular release of K+ and a massive Ca2+ entry into the intracellular compartment. We suggest that the acute functional impairment and the subsequent neuronal injury in region A is caused by the prolonged disturbance of cellular calcium homeostasis mediated by leaky membranes exposed to shear stress.

The effect of glutamate receptor blockade on anoxic depolarization and cortical spreading depression

We examined the effect of blockade of N-methyl-d-aspartate (NMDA) and non-NMDA subtype glutamate receptors on anoxic depolarization (AD) and cortical spreading depression (CSD). [K+]e and the direct current (DC) potential were measured with microelectrodes in the cerebral cortex of barbiturate-anesthetized rats. NMDA blockade was achieved by injection of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate [MK-801; 3 and 10 mg/kg] or amino-7-phosphonoheptanoate (APH; 4.5 and 10 mg/kg). Non-NMDA receptor blockade was achieved by injection of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX; 10 and 20 mg/kg). MK-801 and APH blocked CSD, while NBQX did not. In control rats, the latency from circulatory arrest to AD was 2.1 ± 0.1 min, while the amplitude of the DC shift was 21 ± 1 mV, and [K+]e increased to 50 ± 6 mM. All variables remained unchanged in animals treated with MK-801, APH, or NBQX. Finally, MK-801 (14 mg/kg) and NBQX (40 mg/kg) were given in combination to examine the effect of total glutamate receptor blockade on AD. This combination slightly accelerated the onset of AD, probably owing to circulatory failure. In conclusion, AD was unaffected by glutamate receptor blockade. In contrast, NMDA receptors play a crucial role for CSD.

Regulation of Sodium Channel Function by Bilayer Elasticity: The Importance of Hydrophobic Coupling. Effects of Micelle-forming Amphiphiles and Cholesterol

Membrane proteins are regulated by the lipid bilayer composition. Specific lipid–protein interactions rarely are involved, which suggests that the regulation is due to changes in some general bilayer property (or properties). The hydrophobic coupling between a membrane-spanning protein and the surrounding bilayer means that protein conformational changes may be associated with a reversible, local bilayer deformation. Lipid bilayers are elastic bodies, and the energetic cost of the bilayer deformation contributes to the total energetic cost of the protein conformational change. The energetics and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel–bilayer hydrophobic interactions link a “conformational” change (the monomer↔dimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (β-octyl-glucoside, Genapol X-100, Triton X-100, and reduced Triton X-100) that make lipid bilayers less “stiff”, as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change in gA channel lifetime. Cholesterol-depletion, which also reduces bilayer stiffness, causes a similar shift in sodium channel inactivation. These results provide strong support for the notion that bilayer–protein hydrophobic coupling allows the bilayer elastic properties to regulate membrane protein function.

Calcium accumulation by glutamate receptor activation is involved in hippocampal cell damage after ischemia

ABSTRACT‐ Rats exposed to 10 min of complete cerebral ischemia develop necrosis of the CA‐1 region of the hippocampus after 2–3 days. We studied the involvement of synaptic transmission for this process by ablation of the afferent input (which is mainly glutamatergic) to CA1 by bilateral destruction of CA‐3 neurons (Schafferotomi). The deafferentiation completely prevented the ischemic nerve cell destruction as revealed by histological studies after 6 days. The role of intracellular Ca++ overload was assessed by measurement of the interstitial Ca++ concentration. In control animals the interstitial Ca++ concentration decreases abruptly to 10% of the initial value 1.6 min after the onset of ischemia. The denervated hippocampi, however, showed no decrease during the 10 min of ischemia and hippocampi injected with 2‐amino‐5‐phosphovalerate (APV), a competitive antagonist of the glutamate N‐methyl‐D‐aspartate (NMDA) receptors, displayed a significantly reduced decrease (45% of the initial value) during ischemia. It is concluded that calcium influx via the glutamate‐operated channels during the ischemic period is an important link in the development of ischemic brain cell damage.