TTC, fluoro-Jade B and NeuN staining confirm evolving phases of infarction induced by middle cerebral artery occlusion

Abstract

Considerable debate exists in the literature on how best to measure infarct damage and at what point after middle cerebral artery occlusion (MCAO) infarct is histologically complete. As many researchers are focusing on more chronic endpoints in neuroprotection studies it is important to evaluate histological damage at later time points to ensure that standard methods of tissue injury measurement are accurate. To compare tissue viability at both acute and sub-acute time points, we used 2,3,5-triphenyltetrazolium chloride (TTC), Fluoro-Jade B, and NeuN staining to examine the evolving phases of infarction induced by a 90-min MCAO in mice. Stroke outcomes were examined at 1.5h, 6h, 12h, 24h, 3d, and 7d after MCAO. There was a time-dependent increase in infarct volume from 1.5h to 24h in the cortex, followed by a plateau from 24h to 7d after stroke. Striatal infarcts were complete by 12h. Fluoro-Jade B staining peaked at 24h and was minimal by 7d. Our results indicated that histological damage as measured by TTC and Fluoro-Jade B reaches its peak by 24h after stroke in a reperfusion model of MCAO in mice. TTC staining can be accurately performed as late as 7d after stroke. Neurological deficits do not correlate with the structural lesion but rather transient impairment of function. As the infarct is complete by 24h and even earlier in the striatum, even the most efficacious neuroprotective therapies are unlikely to show any efficacy if given after this point.

Fig.1

TTC staining at different time points of reperfusion. White indicates infarction; red staining indicates normal tissue. Times post-reperfusion are indicated in the bottom right corner. The infarct area increases from 1.5h to 24h (A,B,C,D), and remains stable from 24h to 7d (D,E,F). The ipsilateral ventricle cannot be seen at 24h (D), but re-emerges by 7d (F).

Fig.2

Quantification of infarct volumes based on TTC staining. Evolution of infarct volume over time in cortex (A), striatum (B), and total hemisphere (C). Infarct reached its peak at 24h in cortex and total hemisphere, and at 12h in striatum. N=7 animals/time point. *P < 0.05.

Fig.3

Relationship between neurological deficits and evolvement of infarct. (A) Diagrammatic representation of mouse brain depicting the concept that neurological deficits mostly reflect impairment of function (penumbra, white circle) but not necessarily a structural lesion (core, black circle) early in the course of stroke. Over time the penumbra shrinks while the core grows. (B) Neurological scores decreased over time since reperfusion. Neurological deficits were significantly improved by 24h of stroke. (C) Correlation analysis of neurological scores at 6 time points and equivalent infarct volumes. A significant negative relationship was seen between infarct volume and behavioral score. N=6 animals/time point. *P < 0.05.

Fig.4

Changes in Fluoro-Jade B and NeuN staining reflect TTC time course. (A) Coronal section of mouse brain stained with TTC 24 hours following transient MCAO. Boxed areas illustrate cortical or striatal regions represented in the Fluoro-Jade B and NeuN images. (B) Representative Fluoro-Jade B staining of cortex and striatum following MCAO. Arrows (1.5 h) and insets denote early Fluoro-Jade B positive neurons. (C) Representative NeuN immunostaining of cortex and striatum following MCAO. Asterisks and insets denote examples of normal NeuN immunostaining (contralateral). (B,C) Times post-reperfusion are indicated in the bottom left corner. Dotted lines represent the pial surface. N=4 animals/time point. Scale bars = 100 µM except Fluoro-Jade striatum, scale bar = 50 µM.

Fig.5

Quantification of the Fluoro-Jade B positive cells in cortex (A), striatum (B) and the total hemisphere (C) at different time points after stroke. Positive staining increased during the early period of stroke and peaked at 24 hr of stroke. Some neurons in the cortex of contralateral hemisphere also had evidence of Fluoro-Jade B positivity at 72h of stroke. N=4 animals/time point. P* < 0.05.