Introduction Aims:

1. To characterise the ischaemic lesion produced by the model, in particular the key properties of lesion size, location and reproducibility.

2. To characterise any effects on brain areas not within the MCA territory (i.e. sub- cortical grey matter, contralateral hemisphere).

3. To demonstrate the flexibility of the model to produce a range of ischaemic periods (15-90 minutes) followed by reperfusion.

4. To demonstrate that ischaemia and reperfusion occur at the time of vascular occlusion and de-occlusion respectively, rather than after a variable delay due to vasoconstriction or unreliable mechanical operation, as is often a problem with other MCAO models (Yanamoto et al., 1998).

This characterisation serves a number of purposes. Firstly it is essential to establish a basic physiological response to the insult under the specific conditions created by the model. The interpretation of all subsequent results and particularly any assessments of the effect of disease-process modifying interventions must be carried out with reference to this characterisation. Secondly, it has not previously been possible to make multi-parametric MRI measurements during the immediate reperfusion period in existing external, distal- MCAO models due to the inherent surgical limitations. For example, where reperfusion cannot be induced by remote control, the animal is not inside the magnet at the time of reperfusion. The new model allows remote-controlled occlusion and de-occlusion of vasculature and therefore NMR measurement can be made at all time-points.

Two experimental protocols were studied; permanent occlusion and ischaemia- reperfusion. The permanent occlusion experiments facilitated a systematic analysis of lesion size and the inter-subject variation inherent in the model. We conducted a small

number of 3 hour occlusion experiments where the brain was removed, cut into 1mm thick slices and stained using 2,3,5-triphenyltetrazolium chloride (TTC) at the end of the occlusion (ischaemia) period. This colourless chemical is reduced to a red/pink colour by functioning, active mitochondrial enzymes. Where mitochondria are dysfunctional or have begun to degenerate, as is the case following a significant period of ischaemia (e.g. 30mins (Bimbaum et al., 1997)), the stain is not reduced and the lesion therefore appears white, while normal tissue develops the pink/red colouration (Figure 7-1).

TTC staining has been shown to provide an accurate and reliable assessment of lesion size and location in animal studies of cerebral ischaemia (Bednar et ah, 1994) provided that sufficient time is allowed for the cellular changes to evolve. Literature reports show that, in total, a minimum 3 hours of survival (either permanent ischaemia or ischaemia- reperfusion) are required for the TTC-derived lesion size to match lesion size as determined by traditional light-microscopy methods (Park et ah, 1988; Minematsu et ah,

1992). There are some reports of differences between TTC histology and more

conventional H&E histology, but these are generally small and/or not statistically significant (Park et ah, 1988).

Figure 7-1 TTC stained brain slice from rabbit brain (Bednar et ah, 1994).

Functional mitochondria in unaffected tissue reduce the stain to a pink/red form. The ischaemic lesion appears white due to the degeneration and inactivity of mitochondrial enzymes (blue arrows).

Given the general acceptance of TTC histology as a robust means of determining lesion size, and its comparatively low dependence on specialised histological expertise or equipment, we adopted the technique to investigate reproducibility in the new model. Notwithstanding ongoing arguments within the research community regarding small differences between this method and significantly more complex alternatives, the

technique is appropriate and suitable for investigating reproducibility in a standardised 3 hour experimental protocol. Measurements of the hemispheric lesion volume (HLV) were obtained by determining the hemispheric lesion area (HLA) (Equation 7-1) on successive contiguous axial brain slices.

The hemispheric lesion area was determined from diffusion-weighted images acquired in the central slice (centred 0.5mm caudal to bregma) following 3 hours of MCA and CCA occlusion. It is known that three hours of occlusion is sufficient to produce an ischaemic lesion of maximal size, and equivalent to permanent occlusion in rodent models of this type (Kaplan et al., 1991). Intra-ischaemic DWI scans acquired at this time point are known to provide a reliable marker of histological lesion size and location in other MCAO models (Moseley et al., 1990; Van Bruggen et al., 1992; Dardzinski et al., 1993; van

Gelderen et al., 1994; Hoehn-Berlage et al., 1995). It should be noted that the

hyperintense area on DWI is a function of a reduction in ADC and lengthening of T% relaxation time (due to cytotoxic oedema) which evolve in tandem during ischaemia

(Calamante et al., 1999). The HLA as determined from DWI was compared to

measurement obtained from the equivalent TTC histology slice in order to compare the histological and MR methods.

To investigate the underlying tissue response to ischaemia and reperfusion using the new model, three ischaemia times of 15 minutes, 45 minutes and 90 minutes were used,

followed by 2 hours of reperfusion. During this time the ADC and tissue water T2 were

serially measured to yield information about cellular energetics and oedema formation.

A major criticism of the intra-luminal suture MCA occlusion model, the most common remote-controlled technique to be implemented in NMR studies, is that the degree and time-points of vascular occlusion and de-occlusion are unreliable and subject to frequent ‘failed attempts’. These may give rise to pre-conditioning effects, in addition to making exact specification of the occlusion time impossible. For this reason we investigated ADC changes immediately following remote-controlled vascular occlusion, to confirm that the vessels had in fact been occluded and that occlusion has a reproducible effect on cellular energetics. Data from 18 permanent ischaemia experiments were pooled for this analysis.