Fig 1.
(A) Placement of laser-Doppler probe at Antero-Posterior (AP) ~-1.0 and Medio-Lateral (ML) ~3.0 from bregma, to measure blood flow in the territory supplied by the right MCA. (B) Three different silicone coated filaments, coated for different lengths (L) and with different diameters of coating (⌀), used throughout this study. For simplicity, we have titled these ‘Short’, ‘Thin’ and ‘Thick’. (C) Surgical site during intraluminal filament MCAO identifying the External Carotid Artery (ECA), Internal Carotid Artery (ICA), Central Carotid Artery (CCA). (D) Underside of mouse brain, identifying the Middle Cerebral Artery (MCA), Anterior Carotid Artery (ACA) and Posterior Cerebral Artery (PCA). (F) Koizumi’s method (left) and Longa’s method (right) of the intraluminal filament MCAO (not to scale). Anterior choroidal artery (AChA), HTA, hypothalamic artery (HTA), pterygopalatine artery (PPA), occipital artery (OA), superior thyroid artery (STA), ventral thalamic artery (VTA).
Fig 2.
Representative laser-Doppler flowmetry during Koizumi or Longa intraluminal filament MCAO
(A) Representative laser-Doppler flowmetry during Koizumi’s method of intraluminal filament middle cerebral artery occlusion (MCAO). (B) Representative laser-Doppler flowmetry during Longa’s method of intraluminal filament MCAO. Perfusion Units (PU) are arbitrary units of cerebral blood flow. a. Baseline. b. Immediately post CCAO. c. Immediately pre-MCAO. d. Immediately post-MCAO. e. Immediately pre-MCA reperfusion. f. 5 min post-MCA reperfusion. g. CCA reperfusion (Longa method only).
Fig 3.
Post-reperfusion time-course of ischemic lesion volume detected by TTC following 60 min intraluminal filament MCAO using the Koizumi method
(A) Representative TTC stained brain sections indicating areas of healthy tissue (red) and ischemic injury (white) for each group. (B) Total volume of ischemic lesion in the ipsilateral hemisphere, expressed as a percentage of the total contralateral hemisphere volume, 30 min, 4 h, 12 h and 24 h after reperfusion following sham surgeries or 60 min MCAOs, with thin or thick silicone coated filaments, via the Koizumi method. (C) No alteration in neurological severity scores between animals assessed at 4 h, 12 h and 24 h after reperfusion, following a 60 min occlusion. (D) Body weight loss post-MCAO significantly increased from 24 h as compared to 4 h and 12 h after reperfusion. Each value represents the mean ± the standard error of the mean (SEM). *p < 0.05. N = 3–4 for sham surgeries and n = 5–8 for animals undergoing occlusion.
Table 1.
Relative alterations in CBF following CCAO, MCAO, during occlusion and following reperfusion, for thin and thick filaments at 30 min, 4 h, 12 h, and 24 h after reperfusion.
Fig 4.
Ischemic lesion volume detected by TTC following 15 min, 30 min, 45 min or 60 min intraluminal filament MCAO using the Koizumi method
(A) Representative TTC stained brain sections indicating areas of healthy tissue (red) and ischemic injury (white) for each group. (B) Total volume of ischemic lesions in the ipsilateral hemisphere, expressed as a percentage of the total contralateral hemisphere volume, measured at 4 and 24 h after reperfusion, following 0 min, 15 min, 30 min, 45 min or 60 min of MCAO with a thick silicone coated filament, via the Koizumi method. (C) Significant increases in neurological severity scores following 45 or 60 min MCAO, compared to 15 min MCAO. (D) Body weight loss significantly increased from 4 h to 24 h after reperfusion in every group. Each value represents the mean ± the standard error of the mean (SEM). *p < 0.05. N = 3 for sham surgeries and n = 4–7 for animals undergoing occlusion.
Table 2.
Relative alterations in CBF following CCAO, MCAO, during occlusion and following reperfusion for 15 min, 30 min, 45 min and 60 min occlusions measured at 4 h or 24 h after reperfusion.
Fig 5.
Ischemic injury detected after 4 h reperfusion by TTC following 60 min intraluminal filament MCAO via the Koizumi or Longa method
(A) Total volume of ischemic lesion in the ipsilateral hemisphere, expressed as a percentage of the total contralateral hemisphere volume, measured at 4 h after reperfusion, following 60 min MCAO via the Koizumi or Longa method with a thick or short silicone coated filament. (B) Representative TTC stained brain sections indicating areas of healthy tissue (red) and ischemic injury (white) for each group. (C) Body weight loss following either Koizumi or Longa surgeries measured at 4 h after reperfusion. Each value represents the mean ± the standard error of the mean (SEM). N = 3 for sham surgeries and n = 5–6 for animals undergoing occlusion.
Table 3.
Relative alterations in CBF following CCAO, MCAO, during occlusion and following reperfusion for Koizumi vs. Longa at 4 h after reperfusion.
Table 4.
Correlations of relative alterations in CBF following CCAO, MCAO, reperfusion, neurological score, weight loss, age and starting weight to ischemic infarct volume.
Fig 6.
Representative laser-Doppler flowmetry traces of SAH and premature reperfusion
(A) A typical representative laser-Doppler flowmetry during Koizumi’s method of middle cerebral artery occlusion (MCAO), during a 60 min occlusion. Insert: representative resulting ischemic lesion analysed by TTC staining. (B) A typical representative laser-Doppler flowmetry during Longa’s method of MCAO, during a 60 min occlusion. (C) Representative laser-Doppler flowmetry of subarachnoid haemorrhage (SAH) during the Koizumi method of MCAO, with resulting ischemic injury. (D) Representative laser-Doppler flowmetry of false-positive SAH. Inserts: image of the same animal, with a brain bleed resulting from vessel perforation, yet leading to no discernible ischemic injury. (E) Representative laser-Doppler flowmetry showing arbitrary units of CBF falling gradually over the course of a 60 min MCAO occlusion time, conducted via the Koizumi method. (F) Representative laser-Doppler flowmetry indicating premature reperfusion, due filament movement out of place, followed by immediate recovery of MCAO by repositioning the filament. Perfusion Units (PU) are arbitrary units of cerebral blood flow.