4 CARACTERIZACIÓN DEL ÁREA DE INVESTIGACIÓN 4.1 AMBITO DE ESTUDIO
4.2. CARACTERISTICAS DEL AREA DE INVESTIGACION COMUNIDAD DE CHOCCO QUELICANI.
This chapter deals with the the measurement of cerebral oedema using the specific gravity technique. The results from the study were then correlated with a previous NMR/CBF study which was completed shortly before the work for this thesis was begun, but which had used the same period of ischaemia (30 minutes) and the same time points of reperfusion. The specific gravity (SG) work was done as a preliminary study before embarking on the use of NMR imaging to monitor water changes in the brain during and after ischaemia.
7.1 Introduction
The work presented in the previous chapters has used NMR spectroscopy in conjunction with the hydrogen clearance technique to measure CBF, in order to examine the relationship between cerebral energy metabolism and blood flow during ischaemia and hypoxia. The next chapter deals with the use o f NMR imaging to investigate changes in brain water during and after ischaemia. The work presented in this chapter forms a link between these two sections of the thesis, in that it deals with the study of cerebral oedema by the microgravimetric, or specific gravity technique. This is in order to gain a quantitative idea of changes in brain water during and after ischaemia, before embarking on the NMR imaging studies, where data would be more qualitative.
Cerebral oedema is a common and potentially fatal consequence of ischaemia, and can be caused by both lethal and relatively minor stimuli. The development and resolution of ischaemic brain oedema is a complex problem. Even following very brief periods o f ischaemia brain damage may occur in selectively vulnerable areas (see section 2.2.11, pg 40), with glial and vascular cells unaffected.
During ischaemia the failure of cellular energy metabolism leads to a failure o f ionic homeostasis and an increase in brain osmolality, and water tends to move into the
brain to balance this (Siesjo & Wieloch, 1985). To a large extent this cellular oedema results from translocation of water from the extracellular to the intracellular space, and a net increase in tissue water content may be absent or small. The amount of water accumulating during ischaemia depends upon the level of flow during ischaemia (Crockard et al, 1980), its duration (Ito et al, 1979), and whether the ischaemia is complete or incomplete (Schuier & Hossmann, 1980). In prolonged ischaemia, astrocytic swelling becomes pronounced and net oedema develops. Reestablishment of blood flow to an ischaemic region may lead to an increase in brain water, occurring either immediately (Iannotti & Hoff, 1983) or later, in association with breakdown of the blood-brain barrier (BBB) (Ito et al, 1979). This process worsens the brain damage incurred following permanent occlusion o f a cerebral artery, as in stroke. Another factor which may accentuate oedema formation on reperfusion is lactic acidosis (Siesjo & Wieloch, 1985).
In this chapter the effects of a moderate insult (30 minutes bilateral carotid occlusion) on brain water content in the gerbil were examined. Oedema was measured using the specific gravity technique. These results were then correlated with an earlier study which was completed before the work for this thesis was begun, where CBF, as measured by hydrogen clearance, and brain metabolism using NMR spectroscopy were measured (Allen et a l, 1988). The same period of ischaemia and time points of reperfusion were sampled in both studies.
7.2 Materials and methods 7.2.1 Animal preparation
Twenty eight adult male gerbils (60-70 g) were anaesthetized with halothane/oxygen and both common carotid arteries were occluded for 30 minutes using aneurysm clips. Body temperature was maintained at 36.5 - 37 °C using a heating lamp. Ten minutes before removal of the clips 0.1 ml 2% Evans’ Blue was injected intraperitoneally. Animals were killed at 0, 5, 30, 60, 120, and 180 minutes o f reperfusion (n=4 for each time point). The brains were removed and dissected under kerosene into frontal, parietal and occipital cortex, and thalamus using an operating microscope (Figure 7.1). Note was made of any Evans’ Blue staining. In all cases pieces of brain were
OLFACTORY TRACT
FRONTAL
CORTEX C O R T E XPARIETAL OCCIPITALC O RTEX
TH A LA M U S BRAINSTEM
Figure 7.1
Areas in the gerbil brain used for specific gravity measurements
A gerbil brain seen from above (top), and after being longitudinally cut in half to illustrate the areas sampled to measure brain water.
taken from the left and right sides. The brain was exposed to the air for as short a time as possible (approximately 30 seconds) to prevent dehydration, and each set of 6 pieces was transferred to a small glass pot containing kerosene.
7 .2 .2 Calibration of SG columns
SG was determined using a linear column of organic solvents, after the method of Marmarou et al (1978). Full details of how the columns and the calibration standards were made are given in Appendix B (pg 214). Before each experiment the columns were calibrated with standards of known specific gravity within the range expected for brain tissue. The standards were made of potassium sulphate in distilled water at the following specific gravities; 1.0530, 1.0509, 1.0489, 1.0468, 1.0447, 1.0406. Generally two columns were used per animal. The calibrated columns were kept in a waterbath at ambient temperature to avoid fluctuations in temperature and thus prevent convection currents within the column. The temperature of the waterbath and the room was recorded.
30 jri o f each standard, starting with the 1.0530 solution were taken up into a micropipette, and the droplet released just below the fluid level. The six standards were put in at 30 second intervals and the depth at which each settled was recorded three minutes after insertion, that is, thirty seconds after the last standard was dropped in (1.0406), it was time to record the level o f the first (1.0530). After calibration the six numbers were fed into a programmeable calculator to give a coefficient of determination (r2) for the column. There was a linear relationship between SG and graduate division. 95% of the columns had an r2 > 0.9990 (Figure 7.2).
7.2.3 Determination of SG
Each cube o f brain was placed in a tiny dental spoon and held just under the surface o f the column. It was then knocked off with a fine dental pick. The 6 pieces were inserted at 30 second intervals and the depth to which they fell recorded after 3 minutes (Figure 7.3). Care was taken to aim the pieces o f brain so that they did not stick on a calibration droplet. When an experiment was finished the droplets and brain
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191 -E
— 173 - cE
E 15 4 -o
o C 136 - CL 0> Q 99 - 8 0 H 1---1---'--- 1---'---1--- '--- 1--- '---1 1 .0 5 4 0 1 .0512 1 .0 4 84 1 .0 4 5 6 1 .0 4 2 8 1 .0 4 0 0Speci f i c gr a vi t y
Figure 7.2Linearity of specific gravity column
Graph demonstrating the linearity of the specific gravity columns, showing that there is a linear relationship between SG and graduate division. Each point on the graph represents a SG calibrating standard. In this column the coefficient o f determination (r2) = 0.9992.
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