TTC, Fluoro-Jade B and NeuN Staining Confirm Evolving Phases of Infarction Induced by Middle Cerebral Artery Occlusion

Overview

Journal J Neurosci Methods

Specialty Neurology

Date 2009 Jan 27

PMID 19167427

Citations 145

Authors

Fudong Liu

Dorothy P Schafer

Louise D McCullough

Affiliations

Soon will be listed here.

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.

Citing Articles

Inhibition of IL-6 trans-signaling promotes post-stroke functional recovery in a sex and dose-dependent manner.

Hall C, Nguyen D, Mendoza K, Tan C, Chauhan A J Neuroinflammation. 2025; 22(1):52.

PMID: 40011978 PMC: 11866694. DOI: 10.1186/s12974-025-03365-y.

and Extracts Mixture Target Pyroptosis in Ischemic Stroke via the NLRP3 Pathway.

Ko G, Kim J, Hong Y, Jeon Y, Baek H, Lee D Int J Mol Sci. 2025; 26(2).

PMID: 39859214 PMC: 11765050. DOI: 10.3390/ijms26020501.

Escape of Kdm6a from X Chromosome Is Detrimental to Ischemic Brains via IRF5 Signaling.

Ngwa C, Misrani A, Manyam K, Xu Y, Qi S, Sharmeen R Transl Stroke Res. 2025; .

PMID: 39752046 DOI: 10.1007/s12975-024-01321-1.

Escape of Kdm6a from X chromosome is detrimental to ischemic brains via IRF5 signaling.

Ngwa C, Misrani A, Manyam K, Xu Y, Qi S, Sharmeen R Res Sq. 2024; .

PMID: 39399684 PMC: 11469404. DOI: 10.21203/rs.3.rs-4986866/v1.

Transcranial direct current stimulation enhances the protective effect of isoflurane preconditioning on cerebral ischemia/reperfusion injury: A new mechanism associated with the nuclear protein Akirin2.

Kong X, Lyu W, Lin X, Feng H, Xu L, Li C CNS Neurosci Ther. 2024; 30(9):e70033.

PMID: 39267282 PMC: 11393012. DOI: 10.1111/cns.70033.

References

Butler T, Kassed C, Sanberg P, Willing A, Pennypacker K . Neurodegeneration in the rat hippocampus and striatum after middle cerebral artery occlusion. Brain Res. 2002; 929(2):252-60. DOI: 10.1016/s0006-8993(01)03371-6. View

Aspey B, Cohen S, Patel Y, Terruli M, Harrison M . Middle cerebral artery occlusion in the rat: consistent protocol for a model of stroke. Neuropathol Appl Neurobiol. 1999; 24(6):487-97. DOI: 10.1046/j.1365-2990.1998.00146.x. View

Adkins D, Voorhies A, Jones T . Behavioral and neuroplastic effects of focal endothelin-1 induced sensorimotor cortex lesions. Neuroscience. 2004; 128(3):473-86. DOI: 10.1016/j.neuroscience.2004.07.019. View

Wolf H, Buslei R, Schmidt-Kastner R, Pietsch T, Wiestler O, Blumcke I . NeuN: a useful neuronal marker for diagnostic histopathology. J Histochem Cytochem. 1996; 44(10):1167-71. DOI: 10.1177/44.10.8813082. View

Garcia J, Yoshida Y, Chen H, Li Y, Zhang Z, Lian J . Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat. Am J Pathol. 1993; 142(2):623-35. PMC: 1886726. View

Juttler E, Kohrmann M, Schellinger P . Therapy for early reperfusion after stroke. Nat Clin Pract Cardiovasc Med. 2006; 3(12):656-63. DOI: 10.1038/ncpcardio0721. View

Ajmo Jr C, Vernon D, Collier L, Pennypacker K, Cuevas J . Sigma receptor activation reduces infarct size at 24 hours after permanent middle cerebral artery occlusion in rats. Curr Neurovasc Res. 2006; 3(2):89-98. DOI: 10.2174/156720206776875849. View

Igarashi T, Huang T, Noble L . Regional vulnerability after traumatic brain injury: gender differences in mice that overexpress human copper, zinc superoxide dismutase. Exp Neurol. 2001; 172(2):332-41. DOI: 10.1006/exnr.2001.7820. View

Ginsberg M . Neuroprotection for ischemic stroke: past, present and future. Neuropharmacology. 2008; 55(3):363-89. PMC: 2631228. DOI: 10.1016/j.neuropharm.2007.12.007. View

10.

Hoehn M, Nicolay K, Franke C, van der Sanden B . Application of magnetic resonance to animal models of cerebral ischemia. J Magn Reson Imaging. 2001; 14(5):491-509. DOI: 10.1002/jmri.1213. View

11.

Lust W, Taylor C, Pundik S, Selman W, Ratcheson R . Ischemic cell death: dynamics of delayed secondary energy failure during reperfusion following focal ischemia. Metab Brain Dis. 2002; 17(2):113-21. DOI: 10.1023/a:1015420222334. View

12.

Grabowski M, Brundin P, Johansson B . Paw-reaching, sensorimotor, and rotational behavior after brain infarction in rats. Stroke. 1993; 24(6):889-95. DOI: 10.1161/01.str.24.6.889. View

13.

Dirnagl U, Iadecola C, Moskowitz M . Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 1999; 22(9):391-7. DOI: 10.1016/s0166-2236(99)01401-0. View

14.

Devries A, Nelson R, Traystman R, Hurn P . Cognitive and behavioral assessment in experimental stroke research: will it prove useful?. Neurosci Biobehav Rev. 2001; 25(4):325-42. DOI: 10.1016/s0149-7634(01)00017-3. View

15.

Furlan M, Marchal G, Viader F, Derlon J, Baron J . Spontaneous neurological recovery after stroke and the fate of the ischemic penumbra. Ann Neurol. 1996; 40(2):216-26. DOI: 10.1002/ana.410400213. View

16.

Baird A, Benfield A, Schlaug G, Siewert B, Lovblad K, Edelman R . Enlargement of human cerebral ischemic lesion volumes measured by diffusion-weighted magnetic resonance imaging. Ann Neurol. 1997; 41(5):581-9. DOI: 10.1002/ana.410410506. View

17.

Chopp M, Zhang R, Zhang Z, Jiang Q . The clot thickens--thrombolysis and combination therapies. Acta Neurochir Suppl. 1999; 73:67-71. DOI: 10.1007/978-3-7091-6391-7_11. View

18.

Benedek A, Moricz K, Juranyi Z, Gigler G, Levay G, Harsing Jr L . Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats. Brain Res. 2006; 1116(1):159-65. DOI: 10.1016/j.brainres.2006.07.123. View

19.

Wahl F, Allix M, Plotkine M, Boulu R . Neurological and behavioral outcomes of focal cerebral ischemia in rats. Stroke. 1992; 23(2):267-72. DOI: 10.1161/01.str.23.2.267. View

20.

Sugawara T, Lewen A, Noshita N, Gasche Y, Chan P . Effects of global ischemia duration on neuronal, astroglial, oligodendroglial, and microglial reactions in the vulnerable hippocampal CA1 subregion in rats. J Neurotrauma. 2002; 19(1):85-98. DOI: 10.1089/089771502753460268. View