Cerebral oedema occurs as a consequence of perinatal HI brain injury, both in asphyxiated infants and in some perinatal animal preparations. In asphyxiated newborn infants, computerised tomography scanning and measurements of intracranial pressure suggest that brain swelling is maximal at 36-72 hours of age, and is associated with the development of cerebral necrosis which, in turn, implies a poor prognosis (Lupton BA et al 1988; Clancy R et al 1988). In the perinatal rat, cerebral oedema, determined by measuring brain water content at different time points following hypoxia-ischaemia, peaks 3 days post-ischaemia and is still present upto 6 days following the insult (Mujsce DJ et al 1990; Vannucci RC et al 1993).
Since 1956 cerebral impedance measurements have been used to measure the effect of hypoxia-ischaemia on the ion shifts between extracellular and intracellular compartments (de Boer Jet al 1989). Increases in brain volume, as a consequence of increased intracellular water content, are predominantly due to intracellular swelling in the glial cells of the grey matter (Williams CE 1991). Intracellular swelling of these cells, due to failure of ionic pumps, reduces the extracellular space and thus increases tissue impedance. Persistent increases in impedance following hypoxia-ischaemia indicate tissue damage both in vitro and in vivo (Williams CE et al 1991).
In fetal sheep, studies have clearly demonstrated that there is a biphasic increase in cortical impedance (Cl) following hypoxia-ischaemia (Williams CE et al 1991). A rapid early increase in Cl occurs during the insult, and a delayed phase commences several hours later. The early increase occurs as a consequence of inhibition of oxidative phosphorylation causing energy failure, depolarisation and a loss of ion homeostasis. Failure to recover extracellular space after the initial insult indicates severe tissue damage and is directly related to histological outcome assessed at 3 days post-ischaemia (Tan WK et al 1993).
The delayed increased in Cl is associated with a severe global ischaemic insult lasting for more than 20 minutes and is inevitably associated with the development of a significant degree of cerebral injury.
Histological evidence suggest that secondary intracellular swelling occurs when the injury is severe enough to cause tissue necrosis (Williams CE et al 1992). It indicates a delayed deterioration of membrane fimction suggestive of secondary tissue damage. Secondary cell swelling is coupled with cell death following transient ischaemia in vitro and is associated with loss of ionic homeostasis, oedema and neuronal death. Epileptiform activity is likely to contribute to the delayed increase in Cl in fetal sheep by increasing the metabolic demand in an already compromised brain. However, even in the presence of MK-801, which completely abolishes delayed cortical seizures, a secondary increase in Cl still occurs (Tan WK et al 1992). Continuous measurement of Cl can therefore demonstrate the time course of intracellular oedema and this can be related to derangements in ionic homeostasis that occur as a consequence of alterations in cellular energy metabolism.
2.3.1 Principles of the technique
The principle of techniques measuring changes in tissue impedance depend on changes in extracellular space (Williams CE 1991). Intracellular swelling reduces extracellular space and increases tissue impedance, and the changes in impedance can therefore be used to estimate changes in extracellular space (Heroux P and Bourdages M 1994). Following cerebral hypoxia-ischaemia, increases in Cl can be used to measure the time course of cytotoxic oedema that occurs as a consequence of derangements in energy metabolism and loss of ionic homeostasis across cellular membranes (Hossmann K A 1971).
Practically, tissue impedance can be measured by injecting a small electrical current through a pair of electrodes and measuring the potentials generated by a further pair of electrodes. The Maxwell relationship can then be utilised to determine the impedance or resistivity (R) of a tissue according to the electrode geometry (G), voltage signal (V) and the injected current (I) (Williams CE et al 1991). The relationship assumes tissue homogeneity:
Techniques for the experimental investigation ofperinatal brain injury
R - G x V / I
With a constant current source and fixed electrode geometry the relationship becomes:
R = Arx V
where A: is a constant. With these assumptions, changes in tissue impedance are proportional to changes in voltage amplitude.
Given the heterogeneity of the brain, the assumption of the modified Maxwell equation fails, and the extent to which it fails depends, in part, on the size of the electrode array with respect to the structure investigated. A structure will appear homogenous if it is much larger than the dimensions of the electrode array.
Given the large size of the cortex as a whole and the high impedance boundaries, the changes of impedance will reflect the changes of resistivity of the parasagittal grey matter. A small electrode array with a defined geometric factor (G) would need to be used to accurately measure the absolute resistivity of the parasagittal grey matter.
2.3.2 Technical approach for measuring Cl
A four electrode technique is used to measure changes in impedance that occur concomitantly with changes in extracellular space within the parasagittal cortex (Robillard P and Poussart D 1979; Williams CE et al 1991). The four electrode approach diminishes the artifacts arising fi'om electrical polarisation at the medium-electrode interface induced by electrode contact in a two electrode system.
An isolated current source is used to inject a sinusoidal current of ±0.2 pA at 150 Hz bilaterally via one pair of electrodes through the parasagittal cortex. The other pair of electrodes record the subsequent voltage signal. The voltage recording electrodes are
connected to a high impedance amplifier (10^^ ohm) to minimise currents at the electrode interface, that also generate polarisation artifacts. In addition, capacitive loading of the voltage electrodes is reduced by lowering the fi'equency of the current source. The subsequent impedance signal is superimposed on the unfiltered EEG signal, and an eighth order 30 Hz low pass filter is used to ensure that this does not contaminate the ECoG signal. The low amplitude impedance signal is then extracted with a powerful phase sensitive detection technique. This extracts the 'in phase' correlated impedance signal by averaging out the uncorrelated 'noise' (figure 2.5).
Spatial resolution is determined by the geometry of the electrodes, chosen to resolve impedance changes in the parasagittal cortex as a whole. The total measurement, as opposed to local measurement, is done to reduce the variability of the measurements as a consequence of the heterogeneity of damage within the parasagittal cortex. The high resistivity of white matter (700 ohm cm'^) compared to the grey matter (300 ohm cm'^) creates boundaries that further focal the impedance current through the parasagittal grey matter (Williams CE 1991).
Impedance techniques are limited in the estimation of absolute values of extracellular space. Fixed electrode geometry and an homogenous media are assumed and the changes in extracellular space are estimated from impedance measurements. Although the geometric factor for the electrodes is not known, it is taken to be constant for each study and similar between animals.
Techniques for the experimental investigation ofperinatal brain injury
Figure 2.5 Measurement of cortical impedance in the fetal brain
Isolated current source Synchronous detector
-ni
Impedance
output
Figure 5 shcfws the technique o f impedance measurements through the parasagittal cortex. A small 150Hz current (isolated) is injected through one pair o f electrodes, the potentials generated by the resitivity o f the tissue are measured by the second pair. To resolve the small signal obscured by electroencephalographic activity, a synchronous detector is utilised