1394
Evaluation of 2,3,5 -Triphenyltetrazolium
Chloride Staining To Delineate
Rat Brain Infarcts
Kazuo Isayama, MD; Lawrence H. Pitts, MD; and Merry C. Nishimura, BS
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Background and Purpose: Accurate and reproducible determination of the size and location of
cerebral infarcts is critical for the evaluation of experimental focal cerebral ischemia. The
purpose of this study was to compare intracardiac perfusion of 2,3,5-triphenyltetrazolium
chloride with immersion of brain tissue in 2,3,5-triphenyltetrazolium chloride to delineate
brain infarcts in rats.
Methods: After 6, 24, or 48 hours of ischemia induced by permanent middle cerebral artery
occlusion, some rats were perfused with 2,3,5-triphenyltetrazolium chloride; other rats were
given an overdose of barbiturates, after which brain sections were immersed in 2,3,5triphenyltetrazolium chloride. Coronal sections were taken 4, 6, and 8 mm from the frontal
pole, and infarct areas in perfused and immersed sections were compared; subsequently, the
same sections were stained with hematoxylin and eosin.
Results: In rats subjected to 24 or 48 hours of occlusion, areas of infarction were clearly
defined with both 2,3,5-triphenyltetrazolium chloride staining techniques, and the infarct sizes
correlated well with the results of hematoxylin and eosin staining (r=0.85-0.94).
Conclusions: These results demonstrate that intracardiac perfusion of 2,3,5-triphenyltetrazolium chloride is an accurate, inexpensive, and efficient staining method to detect infarcted
tissue 24 and 48 hours after the onset of ischemia in rats. (Stroke 1991;22:1394-1398)
T
he oxidation-reduction indicator 2,3,5-triphenyltetrazolium chloride (TTC) has been used
successfully for early histochemical diagnosis
of myocardial infarction.1 The staining action of
tetrazolium salts is based on the presence of a
dehydrogenase.2-6 Tissue with normal levels of the
enzyme is stained red, whereas ischemic and infarcted tissue remains unstained owing to loss of the
enzyme. Infarcted cerebral tissue in cats and rats can
be differentiated from normal tissue by immersion in
TTC solutions.7-12 Infarct size determined by this
method correlates well with that determined by staining with hematoxylin and eosin (H&E).7 Studies in
our laboratory have shown that infusion of TTC into
brain by aortic injection results in better staining and
clearer delineation of ischemic and nonischemic tissue than does the immersion technique. In rats
subjected to permanent middle cerebral artery
(MCA) occlusion, however, compromise of cerebral
From the Department of Neurological Surgery, School of Medicine, University of California, San Francisco, Calif.
Address for correspondence: Lawrence H. Pitts, MD, Department of Neurological Surgery, c/o The Editorial Office, 1360 Ninth
Avenue, Suite 210, San Francisco, CA 94122.
Received March 16, 1990; accepted June 6, 1991.
blood flow may prevent TTC solution from reaching
the infarcted area.
To determine whether the perfusion method overestimates the size of infarcted regions compared with
the immersion method or standard H&E staining, we
compared infarct sizes determined by TTC perfusion
or immersion with those determined by H&E staining of adjacent sections in rats subjected to permanent MCA occlusion.
Materials and Methods
Thirty-six male Sprague-Dawley rats weighing
350-400 g were divided into six groups for study. All
rats underwent permanent MCA occlusion.1314
Rats were anesthetized by intraperitoneal injection of 4% chloral hydrate solution (320 mg/kg). A
curved 2-cm incision was made beginning superior to
the left orbit and extending down to the left zygoma.
Under an operating microscope, the temporalis muscle was reflected to expose the left MCA, the posterior part of the zygoma was resected, and a 4x4-mm
craniectomy was performed just anterior to the foramen of the mandibular nerve. After the dura mater
had been incised carefully with a 27-gauge needle
and reflected, the MCA was occluded by microbipolar coagulation at a low power setting under contin-
Isayama et al
TTC Staining of Rat Brain Infarcts
1395
FIGURE 1. Panel A: Coronal brain section stained by 2,3,5-triphenyltetrazolium chloride perfusion 24 hours after middle cerebral
artery occlusion. Panel B: Hematoxylin and eosin-stained section from the surface of coronal slice shown in panel A.
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
uous saline irrigation. The MCA was cauterized
beginning 2-3 mm medial to the olfactory tract and
extending distally to the rhinal fissure. A small piece
of Gelfoam was placed over the exposed brain tissue,
and the temporalis muscle was laid over the craniectomy site. The skin was closed with a running 4-0 silk
suture. After 6, 24, or 48 hours, each rat was reanesthetized, and TTC staining was performed as described below.
The TTC solution (2% by weight) was prepared
with 37°C phosphate buffer (0.2 M Na2HPO4 and 0.2
M NaH2PO4, pH 7.4-7.6) immediately before use.
Because TTC is light sensitive, solutions must be
masked with aluminum foil.
For staining by the perfusion method, a blunted
18-gauge needle was passed through a small incision
in the left ventricle to the proximal ascending aorta
and clamped into position. The right atrium was
incised, and the TTC solution was delivered by
gravity flow through the needle. The rats were then
wrapped in a 37°C warming blanket for 30 minutes.
The brains were removed, placed in 10% buffered
formalin, covered with aluminum foil, and refrigerated. Sections were photographed within 3-7 days.
For staining by the immersion method, the rat was
given an overdose of barbiturate. The brain was
removed, and 2-mm-thick sections were cut 4, 6, and
8 mm from the frontal pole with a customized
slicer.15 Edematous or soft brains were placed in a
freezer at -20°C for up to 20 minutes to facilitate
sectioning. The sections were put in a glass Petri dish
containing a shallow layer of 2% TTC, and glass
coverslips wetted with the TTC solution were placed
on top of each slice. To ensure even staining, the top
and bottom surfaces of the section were in contact
with the glass. The dishes were covered with aluminum foil to prevent exposure to light and incubated
at 37°C for 30 minutes. The TTC solution was then
replaced with 10% buffered formalin. To prevent
distortion, brain slices were kept flat in the Petri dish
overnight. The stained sections were photographed
within 3-7 days.
After the TTC-stained sections had been photographed, they were stained with H&E and photographed. Tracings of coronal sections 4, 6, and 8 mm
from the frontal pole and of infarcted areas were
made from all three sets of photographs and quantified by an imaging analysis system (MacTablet,
Planimeter software program, Reed College, Portland, Ore.). Infarcted regions from the TTC and
H&E images were evaluated by persons unaware of
the study groups.
The statistical significance of differences in infarct
size, expressed as a percentage of hemispheric area,
FIGURE 2. Panel A: Coronal brain section stained by 2,3,5-triphenyltetrazolium chloride immersion 24 hours after middle
cerebral artery occlusion. Panel B: Hematoxylin and eosin-stained section from the surface of coronal slice shown in panel A.
1396
Stroke
Vol 22, No 11 November 1991
TABLE 1. Infarct Size Determined by 2,3,5-Triphenyltetrazolium Chloride Immersion and Perfusion and by
Hematoxylin and Eosin Staining
Staining method
TTC
Group
6-Hour ischemia
1 (« =5)
2 (n=5)
24-Hour ischemia
3 (n=8)
4 (»=8)
48-Hour ischemia
5 («=5)
6 («=5)
Cortex plus
Method basal ganglia
P
I
P
I
P
I
Cortex
H&E
Basal
ganglia
Basal ganglia
plus cortex
Cortex
Basal '
ganglia
0
18.6±2.6
16.7±2.1
13.1±2.6
11.3±1.7
9.2±1.9
5.4+0.7
19.4±1.2
15.6±1.8
10.9±0.8
10.1±0.9
29.5 ±1.6
27.8 ±1.8
19.9±1.2
18.3±1.8
9.7±0.6
9.4±0.7
16.3 ±2.4
14.3±2.0
10.1+0.9
8.9±0.8
25.1 ±2.3
25.4±3.8
15.6±1.9
16.8±2.8
9.5±0.7
8.6±1.0
23.6±1.2*t
2.0±0.9$
17.6±1.1*
2.0±0.9§
30.4± 1.5||
25.7+1.5
26.4±2.6
23.1 ±2.7
6.1±1.2*
Values are mean+SEM, expressed as percentage of hemispheric area. TTC, 2,3,5-triphenyltetrazolium chloride;
H&E, hematoxylin and eosin; P, perfusion; I, immersion.
*P=0.01-0.05 vs. H&E.
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
t p < 0.005 group 1 vs. group 2.
tp<0.005 vs. H&E.
§/j=0.005-0.01 vs. H&E.
\\p < 0.05 group 3 vs. group 4.
between groups was determined by paired t test and
linear regression analysis.
Results
The TTC stained normal tissue deep red; infarcted
or ischemic tissue remained unstained. In tissue
stained by intracardiac perfusion or immersion 24 or
48 hours after MCA occlusion, the boundary between
normal and infarcted tissue was well defined and
distinguishable macroscopically (Figures 1A and
2A). In rats killed at 6 hours, neither TTC method
reliably differentiated between normal and infarcted
tissue. The infarct areas in all groups are listed in
Table 1.
In rats that underwent TTC perfusion 6 hours after
MCA occlusion (group 1), ischemic or infarcted
tissue not stained by TTC was poorly demarcated
from the surrounding normal brain. The border zone
between infarcted and normal areas was pale red.
Areas of infarction determined by TTC perfusion
were larger than those determined by H&E staining
(23.6±1.2% versus 18.6±2.6%, p<0.05, paired t
test).
In rats that underwent TTC immersion 6 hours
after MCA occlusion (group 2), immersed sections
were evenly stained, making it difficult to differentiate between normal and infarcted tissue. The infarct
areas determined by TTC immersion did not correlate with those determined by H&E staining
(2.0±0.9% versus 16.7±2.1%,p<0.005). Subsequent
H&E staining showed no difference in infarct size
between perfused and immersed sections (Table 1,
group 1 versus group 2).
In rats that underwent TTC perfusion (group 3)
and immersion (group 4) 24 hours after MCA occlusion, both techniques clearly defined infarcted areas,
although infarct sizes were significantly larger in
perfused than in immersed sections (30.4 ±1.5% versus 25.7±1.5%, p<0.05). H&E staining, however,
showed no difference in infarct size in the two groups
(Table 1). Infarct sizes determined by TTC perfusion
correlated well with those determined by H&E
(r=0.91, slope=0.95, v intercept=0.8%) (Figure 3).
Compared with H&E staining, the perfusion method
slightly overestimated and the immersion method
slightly underestimated infarct size.
In rats that underwent TTC perfusion (group 5) and
immersion (group 6) 48 hours after MCA occlusion, the
results at 48 hours were similar to those at 24 hours.
There were no differences between TTC and H&E
staining, and linear regression analysis showed a signif-
24 hrs regression analysis
36
• TTC perfusion
34
O TTC immersion
32
u 30
t 28
I 26
to
26
£ 22
'8
20
22
21
26
28
30
32
34
36
38
% infarction (H&E)
FIGURE 3. Infarct size determined by 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin and eosin (H&E)
staining in each sample 24 hours after middle cerebral artery
occlusion (TTC perfusion: r=0.91, slope=0.95, y intercept=0.8%; TTC immersion: r=0.&5, slope=0.91, y intercept=4.5%). Dashed line, line of identity.
Isayama et al
48 hrs regression analysis
•TTC perfusion
OTTC Immersion
\<T
12.5
IS
17.5
20
22.5
25
27.5
30
32.S
35
37.5
% Infarction (H&E)
FIGURE 4. Infarct size determined by 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin and eosin (H&E)
staining in each sample 48 hours after middle cerebral artery
occlusion (TTC perfusion: r=0.92, slope=0.80, y intercept=4.1%; TTC immersion: r=0.94, slope=1.33, y intercept =5.3%). Dashed line, line of identity.
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
icant correlation between infarct sizes measured by
TTC and those measured by H&E staining (Figure 4).
Discussion
Different methods have been used to detect the
morphological features of cerebral tissue after ischemic
injury.7-9."-13.16-19 One of these methods is TTC staining, which has been used to demonstrate irreversibly
damaged ischemic cerebral tissue in rats7 and cats.8'9-1'
In normal tissue, dehydrogenase reduces TTC to formazan, which stains red. In ischemic or infarcted tissue,
however, dehydrogenase activity is reduced or eliminated,20-21 and those areas remain unstained.
Consistent and reproducible results with TTC
staining depend on such factors as concentration of
the dye, mode of staining, and time after the ischemic
insult.7-12 Although morphological and biochemical
changes in the mitochondria occur within 60 minutes
after the onset of cerebral or myocardial ischemia,1-4-5-16-19-22"24 the duration of ischemia is crucial
for TTC staining by perfusion or immersion. In
several studies, irreversibly damaged brain was detected as early as 2-3 hours after the ischemic
insult,10-11 and in others only after 12-24 hours. 7912
In this study, infarct sizes determined by TTC immersion, TTC perfusion, and H&E staining after 6
hours of ischemia were inconsistent. The difference
between perfusion and immersion may be due to
postmortem artifacts; the differences between TTC
perfusion and H&E staining may be caused by mild
cortical spongiosis and neuronal shrinkage in the
H&E-stained sections; the difference between immersion and H&E staining may be due to inadequate
or incomplete diffusion of TTC.
At 24 and 48 hours, TTC perfusion gave consistently larger and TTC immersion consistently smaller
infarct sizes than those determined by H&E staining.
The larger infarct sizes may have resulted from
compromised accessibility of dye to all regions of the
TTC Staining of Rat Brain Infarcts
1397
MCA after the occlusion. The smaller infarct size
may be attributed to macrophage and glial cells. Glial
cells surrounding damaged tissue and macrophages
with intact mitochondria that infiltrate the infarcted
region also contain enzymes that reduce TTC, which
causes the tissue to be stained red.
The TTC perfusion technique has advantages over
both TTC immersion and H&E staining. It is technically easier, less expensive, and more rapid than
H&E staining; it also delineates the infarct borders
more clearly than either of the other techniques.
With the TTC perfusion technique, the brain is fixed
after staining and is therefore easier to slice into
uniform sections. For these reasons, we prefer to use
the TTC perfusion technique in our laboratory.
References
1. Sandritter W, Jestadt R: Triphenyltetrazoliumchloride (TTC)
als reduktionindikator zur makroskopischen diagnose des
frischen herzinfarktes. Verh Dtsch Ges Pathol 1958;41:165-177
2. Honjo J, Ozawa K: Lysolecithin inhibition of mitochondrial
metabolism. Biochim Biophys Acta 1968;162:624-627
3. Lippold HJ: Quantitative succinic dehydrogenases histochemistry: A comparison of different tetrazolium salts. Histochemistry 1982;76:381-405
4. Orten JM, Neuhaus OW: Human Biochemistry, ed 9. St Louis,
Mo, CV Mosby Co, 1975, pp 173-252
5. Ozawa K, Seta K, Araki H, Handa H: The effect of ischemia
on mitochondrial metabolism. J Biochem (Tokyo) 1967;61:
512-514
6. Schutz H, Silverstein RP, Vapalahti M, Bruce D, Mela L,
Langfitt TW: Brain mitochondrial function after ischemia and
hypoxia. Arch Neurol 1973;29:408-416
7. Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis
RL, Bartkowski HM: Evaluation of 2,3,5-triphenyltetrazolium
chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 1986; 17:1304-1308
8. Bose B, Jones SC, Lorig R, Friel HT, Weinstein M, Little JR:
Evolving focal cerebral ischemia in cats: Spatial correlation of
nuclear magnetic resonance imaging, cerebral blood flow,
tetrazolium staining, and histopathology. Stroke 1988;19:28-37
9. Bose B, Osterholm JL, Berry R: A reproducible experimental
model of focal cerebral ischemia in the cat. Brain Res 1984;
311:385-391
10. Han DH, Zervas NT, Geyer RP, Adams JS, Ropper AH,
Kennedy JK, Heros RC, Varsos VG, Borges L, Hedley-Whyte
T: Can perfluorochemicals reduce cerebral ischemia? in Reivich M, Hurtig HR (eds): Cerebrovascular Disease. New York,
Raven Press, Publishers, 1983, pp 409-425
11. Liszczak TM, Hedley-Whyte ET, Adams JF, Han DH, Kolluri
VS, Vacanti FX, Heros RC, Zervas NT: Limitation of tetrazolium salts in delineating infarcted brain. Acta Neuropathol
(Berl) 1984;65:150-157
12. Lundy EF, Solik BS, Frank RS, Lacy PS, Combs DJ, Zelenock
GB, D'Alecy LG: Morphometric evaluation of brain infarcts in
rats and gerbils. J Pharmacol Methods 1986;16:201-214
13. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL,
Bartkowski H: Rat middle cerebral artery occlusion: Evaluation of the model and development of a neurologic examination. Stroke 1986;17:472-476
14 Tamura A, Graham DI, McCullouch J, Teasdale GM: Focal
cerebral ischaemia in the rat: 1. Description of technique and
early neuropathological consequences following middle cerebral artery occlusion. / Cereb Blood Flow Metab 1981;l:53-60
15. Brain microdissection techniques, in Cuello AC (ed): IBRO
Handbook Series: Methods in the Neurosciences. New York,
John Wiley & Sons, Inc, 1983, p 8
16. Arsenio-Nunes ML, Hossmann KA, Farkas-Bargeton E:
Ultrastructural and histochemical investigation of the cerebral
1398
17.
18.
19.
20.
Stroke
Vol 22, No 11 November 1991
cortex of cat during and after complete ischemia. Ada Neuropathol (Berl) 1973;26:329-344
Brown AW, Brierley JB: The nature, distribution and earliest
stages of anoxic-ischemic nerve cell damage in the rat brain as
defined by the optical microscope. Br J Exp Pathol 1967;2:
87-106
Coyle P: Spatial relations of dorsal anastomoses and lesion
border after middle cerebral artery occlusion. Stroke 1987;18:
1133-1140
Farber JL, Chien KR, Mittnacht S: The pathogenesis of
irreversible cell injury in ischemia. Am J Neuropathol 1981;102:
271-281
Rehncrona S, Mela L, Siesjo BK: Recovery of brain mitochondrial function in the rat after complete and incomplete cerebral ischemia. Stroke 1979;10:437-446
21. Siesjo BK: Cell damage in the brain: A speculative synthesis.
/ Cereb Blood Flow Metab 1981;1:155-185
22. Ginsberg MD, Mela L, Wrobel-Kuhl K, Reivich M: Mitochondrial metabolism following bilateral cerebral ischemia in the
gerbil. Ann Neurol 1977;l:519-527
23. Schaper J, Mulch J, Winkler B, Schaper W: Ultrastructural,
functional, and biochemical criteria for estimation of reversibility of ischemic injury: A study on the effects of global
ischemia on the isolated dog heart. / Mol Cell Cardiol 1979;
11:521-541
24. Seidler E: New nitro-monotetrazolium salts and their use in
histochemistry. Histochem J 1980;12:619-630
KEY WORDS • cerebral ischemia • histology • rats
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts.
K Isayama, L H Pitts and M C Nishimura
Stroke. 1991;22:1394-1398
doi: 10.1161/01.STR.22.11.1394
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1991 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://stroke.ahajournals.org/content/22/11/1394
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Stroke can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office.
Once the online version of the published article for which permission is being requested is located, click Request
Permissions in the middle column of the Web page under Services. Further information about this process is
available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Stroke is online at:
http://stroke.ahajournals.org//subscriptions/