Introduction
The development of therapeutic agents for the treatment of ischaemic stroke crucially depends on their efficacy in pre-clinical animal models. The neocortical clip model of focal cerebral ischaemia in the rat involves the occlusion of the middle cerebral artery (MCA) at a point distal to the lenticulostriate perforators which produces a lesion confined to the cortex only. This model provides a more defined and potentially reproducible ischaemic lesion and it has been successfully used in novel neuroprotection studies (1). In an attempt to further improve its translational qualities we have characterised the neocortical clip model of cerebral ischaemia using serial in vivo Magnetic Resonance Imaging (MRI), behavioural and neurological assessment, and immunohistochemistry in order to evaluate the structural and functional properties of this lesion.
Methods
The MCA was occluded in spontaneously hypertensive rats for 0 (n=10), 60 (n=8) and 120 (n=5) minutes. MRI was performed 2 days pre surgery and 1, 3 and 7 days post-surgery. Behavioural assessment was performed 2 days pre-surgery and 3 and 7 days post-surgery whilst neurological deficits were monitored daily. After scanning was completed the animals were perfused with 4% paraformaldehyde to allow immunohistochemistry for neuronal loss (NeuN), astrocytosis (GFAP) and macrophage (ED1) infiltration.
Results
MRI analyses showed that occlusion of the MCA for 0 minutes (sham-controls) did not produce a lesion whereas occlusion for 60 minutes produced a lesion that remained relatively stable over time (1 day 60 ± 67 mm3, 7 days 66 ± 59 mm3). 120 minute occlusion caused a severe cortical infarct 1 day post surgery (285±77 mm3) which decreased by 7 days (174 ± 65 mm3, p < 0.05). Lesion volume was significantly larger with 120 minute occlusion than that of 60 minutes at all time points (1 and 3 days p < 0.001, 7 days p < 0.05). Behavioural assessment and neurological deficit scoring correlated with each other (r = 0.626, p < 0.05) and with respect to lesion volume (r = 0.648, p < 0.01 and r = 0.781, p < 0.01) and demonstrated that functional capacity is reflected in the size of the lesion. The success rate of cerebral infarction was 65% which was possibly due to the subtle but observed inconsistency of the MCA tree within individual animals. Ex vivo histology of the MR defined lesion demonstrated a loss of NeuN positive cells and around the border an upregulation of GFAP positive astrocytes, both indicative of tissue damage. Histology also indicated a large inflammatory response with the presence of macrophages in the lesion territory.
Conclusion
The neocortical clip model has produced ischaemic lesions when present, that are reliably restricted to the cortical territories of the MCA. The duration of occlusion also appears to dictate lesion severity and evolution which may prove to be useful for probing therapeutic interventions at different stages of stroke progression.