(Stroke. 1995;26:2328-2332.)
© 1995 American Heart Association, Inc.
Articles |
Immunohistochemical Detection of Ki-67 in Replicative Smooth Muscle Cells of Rabbit Carotid Arteries After Balloon Denudation
From the Department of Neurosurgery (M.A., H.W., K.H.) and the Institute for Medical and Dental Engineering (H.A.), Tokyo Medical and Dental University, and the Department of Cell Biology, Tokyo Metropolitan Institute of Gerontology (M.A., M.Y., K.Y.) (Japan).
Correspondence to Masaru Aoyagi, MD, Department of Neurosurgery, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Background and Purpose Ki-67 immunohistochemistry has been shown to be useful for paraffin sections from human species after wet heating. We applied Ki-67 immunohistochemistry to rabbit arteries after balloon denudation and compared the results with proliferating cell nuclear antigen (PCNA) immunohistochemistry.
Methods Monoclonal antibodies MIB-1 for Ki-67 and PC-10 for PCNA were used to detect replicative smooth muscle cells in rabbit carotid arteries during a period of 4 weeks after balloon endothelial denudation.
Results We demonstrated clear immunoreactivities for Ki-67 in paraffin sections of rabbit arteries after hydrated autoclaving. Ki-67–positive smooth muscle cells appeared throughout the thickened intima 1 week after endothelial denudation. At 2 weeks, Ki-67–positive cells were confined to the surface layer of the intima. PCNA-postive cells appeared in almost the same location by 2 weeks after endothelial denudation but were significantly greater in number than Ki-67–positive cells. PCNA-positive cells remained in the surface layer of the intima 4 weeks after endothelial denudation, while Ki-67–positive cells had almost disappeared from the intima.
Conclusions Our results indicate that Ki-67 immunohistochemistry using MIB-1 monoclonal antibody provides a powerful tool, even in rabbit species, for the detection of replicative smooth muscle cells during the repair of arterial injury and that it detects replicative cells more accurately than PCNA immunohistochemistry.
Key Words: arterial wall • immunohistochemistry • muscle, smooth • rabbits
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The proliferation and migration of medial SMCs in the intima may play a key role in the development of intimal thickening after arterial injury.1 2 The identification of replicative SMCs is essential for understanding the mechanisms of development of intimal thickening after arterial injury or during atherosclerosis. The incorporation of tritiated thymidine or bromodeoxyuridine has been used to estimate cells in the S phase of the cell cycle after arterial injury in experimental animals.3 4 Because these methods require the incorporation of the labeling molecules into the DNA during the S phase, prior incubation of tissues or in vivo administration is necessary.
The recent development of mAbs to proliferation-associated antigens offers promise for the detection of proliferating cells in tissues from experimental animals or humans.3 The nuclear proteins PCNA5 6 and Ki-67 antigen7 8 may be such candidate antigens. The sensitivity and specificity of PCNA immunohistochemistry for the detection of proliferating cells in vascular tissues after injury have been investigated.9 Ki-67 immunohistochemistry has been shown to provide a reliablemethod of evaluating the growth fraction of neoplastic tissues.10 11 The MIB-1 antibody, a recently developed mAb against a recombinant fragment of the Ki-67 antigen, has been shown to work well even in paraffin sections after wet heating.12 13 Although the Ki-67 antibody has been used mostly in human tissues,7 8 recent studies indicate that the MIB-1 mAb may be successfully applied to tissues from nonhuman species.14 15
In the present study we applied Ki-67 immunohistochemistry to formalin-fixed/paraffin-embedded sections of rabbit CCAs after balloon endothelial denudation. We used MIB-1 mAb in paraffin sections after hydrated autoclaving treatment. We demonstrate clear immunoreactivities for Ki-67 in replicative SMCs of rabbit CCAs after balloon denudation and compare the results with PCNA immunohistochemistry.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Arterial Endothelial Denudation Model
Ten-week-old male Japanese White rabbits (weight, 1.8 to 2.2 kg) were anesthetized with 25 mg/kg IV of sodium pentobarbital (Nembutal, Abbott Laboratories). The animals were subjected to endothelial denudation of the left CCA with the use of an arterial embolectomy catheter (12-040-3F/400 mm/3F, American Edwards Laboratories), as previously described.16 17 The procedure used in this study was in accordance with the animal welfare regulations of the Tokyo Medical and Dental University and the guidelines of the American Physiological Society. The embolectomy catheter was inserted in a retrograde fashion through a small incision made near the carotid bifurcation. The luminal surface of the artery from the origin to the balloon entrance was gently rubbed back and forth with the balloon filled with 0.15 mL of air. The right CCA was exposed but not subjected to endothelial denudation and was used as a control. Both CCAs were removed 3 days, 1 week, 2 weeks, or 4 weeks after endothelial denudation. CCAs were also removed from normal animals as controls. Six or more arteries were used for each time point. The specimens were fixed in 10% neutralized formalin for 24 hours at room temperature and embedded in paraffin. Morphological sections were obtained from the middle of the CCAs.
Immunohistochemistry
To retrieve the Ki-67 antigens and PCNA in
formalin-fixed/paraffin-embedded sections, we performed
autoclave pretreatment of sections according to the method of Shin et
al with some modifications.18 19 After deparaffinization
in xylene and rehydration in graded ethanol, 5-µm sections immersed
in a vessel filled with 10 mmol/L citrate buffer, pH 6.0, were
autoclaved at 121°C for 15 minutes. The primary antibodies used were
MIB-1 mAb (x50, Immunotech SA) for Ki-67 and PC-10 (x400, Dako) for
PCNA. Immunohistochemical detection was performed with the use of a
labeled streptoavidin-biotin method with an LSAB kit (Dako).
Endogenous peroxidase activity was blocked with 3%
hydrogen peroxide in water, and then nonspecific staining was blocked
by incubation for 30 minutes with 10% goat serum. The sections were
incubated with the primary antibody for 60 minutes, then reacted with
biotinylated link antibody (goat antibody to mouse and rabbit
immunoglobulins), and finally with peroxidase-labeled
streptoavidin. The sections were developed with diaminobenzidine.
Nuclei labeled positively for Ki-67 or PCNA were defined by visual determination in three or more randomly selected fields per cross section at x1000 magnification. The results were expressed as the mean number of labeled nuclei per intimal surface area. The intimal surface area was measured with an image analyzer (SPICCA-II, Olympus). The quantification of the labeled nuclei was performed independently by two investigators in a blinded manner. The labeled nuclei per intimal surface area for Ki-67 and PCNA were compared with the unpaired t test. The differences were considered statistically significant at the P<.05 level.
Cell types were identified in deparaffinized sections without antigen
retrieval by immunostaining with the use of a mouse mAb
directed against SMCs (
-smooth muscle cell actin, Dako) and a
rabbit polyclonal antibody against factor VIII (Dako) by a labeled
streptoavidin-biotin method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Balloon endothelial denudation of the rabbit CCAs caused an increase in intimal thickening over a period of 4 weeks. After hydrated autoclaving treatment, positive immunostaining for Ki-67 was clearly identified with the use of MIB-1 mAb in paraffin sections of arteries after endothelial denudation (Fig 1
). The staining was diffuse and/or
granular in the nuclei. No positive immunostaining was
observed in sections without exposure to the primary antibody, without
autoclaving treatment, or from control arteries.
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On day 3, positive immunostaining for Ki-67 was
observed in the medial cells, mostly close to the internal elastic
lamina. There was occasional minimal intimal thickening composed of
Ki-67–positive cells, although most sections of the vessels failed to
develop neointima. These cells did not
immunostain for factor VIII and were identified as
proliferating SMCs. At 1 week, when intimal thickening had clearly
developed, Ki-67–positive cells appeared throughout the thickened
intima but were still seen in the media close to the internal elastic
lamina (Fig 1a
). At 2 weeks, the number of
Ki-67–positive cells decreased in the intima, and the cells were
primarily confined to the surface of the intima. There were few
Ki-67–positive cells at the base (abluminal side) of the intima. At 4
weeks, Ki-67–positive cells were seen only occasionally in the surface
layer of the intima. No Ki-67–positive cells were seen in the media at
4 weeks (Fig 1c).
Positive immunostaining for PCNA was clearly seen in
paraffin sections of arteries after hydrated autoclaving treatment (Fig 1). PCNA staining was observed even without antigen
retrieval, but the staining intensity was not adequate. Diffuse weakly
staining cells were often seen in addition to the strongly stained
cells in PCNA immunohistochemistry. We regarded only strongly staining
nuclei as PCNA-positive nuclei. PCNA-positive SMCs appeared in almost
the same location as Ki-67–positive SMCs but were significantly
greater in number (Fig 1b and Fig 2;
P<.0001 at 2 weeks, unpaired t test).
PCNA-positive SMCs remained clearly observable in the surface layer of
the intima even 4 weeks after endothelial denudation
when Ki-67–positive SMCs had almost disappeared from the intima (Fig 1d; P<.0001).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine mAb of Ki-67 reacts with a human nuclear antigen that is present in proliferating cells but absent from quiescent cells.8 However, this antibody can be applied only on fresh or frozen tissues.10 11 The MIB-1 mAb, a recently developed mAb raised against a recombinant fragment of the Ki-67 antigen, has been shown to have a higher affinity for both the Ki-67 antigen alone and the Ki-67 antigen/DNA complex than the earlier Ki-67 mAb and to react well even with formalin-fixed/paraffin-embedded histological materials after wet heating.12 20
In this study we have demonstrated clear immunoreactivities for Ki-67 in replicative rabbit SMCs during repair after balloon endothelial denudation using the MIB-1 mAb and hydrated autoclaving. The minimal intimal thickening composed of Ki-67–positive SMCs appeared on day 3, but only sporadically in the cross sections of the arteries. At 1 week, when intimal thickening had clearly developed, Ki-67–positive SMCs appeared throughout the thickened intima. At 2 weeks, proliferating SMCs were restricted to the surface layer of the intima. SMCs in the thickened intima were almost quiescent by 4 weeks after endothelial denudation. These findings are consistent with previous reports of studies using thymidine or bromodeoxyuridine incorporation.21 22 23
To our knowledge, there have been no reports demonstrating Ki-67 immunoreactivities in vascular tissues derived from nonhuman species. We used hydrated autoclaving pretreatment of sections in place of the microwave treatment generally used as a method of antigen retrieval.13 19 In comparative studies of these treatments by us (M.A. et al, unpublished data, 1994) and others,24 a significantly better antigen retrieval was obtained by hydrated autoclaving than by microwave treatment. It is likely that the higher affinity of the MIB-1 mAb for the Ki-67 antigen and the hydrated autoclaving treatment may have contributed to the successful immunodetection in sections from rabbits.
PCNA immunohistochemistry has been shown to work well in sections from various species of experimental animals.9 25 26 PCNA immunohistochemistry for the detection of proliferating cells in vascular tissues after injury to the rat aorta has been investigated and shown to be more sensitive for the detection of proliferation than thymidine or bromodeoxyuridine incorporation.9 In the present study we compared Ki-67 immunostaining using MIB-1 mAb with PCNA immunostaining in sections close to each other. Diffuse, weakly staining cells were often seen in addition to strongly staining cells in PCNA immunohistochemistry. This weak staining may indicate nucleoplasmic PCNA staining, which Bravo et al27 have shown to be restricted to non-S phase cells. We regarded only strongly staining nuclei as PCNA-positive nuclei, but it was sometimes difficult to distinguish between strongly and weakly staining cells. PCNA-positive SMCs appeared in almost the same locations as Ki-67–positive SMCs but were significantly greater in number. Notably, PCNA-positive SMCs remained clearly observable in the surface layer of the intima even 4 weeks after endothelial denudation when Ki-67–positive SMCs had almost disappeared from the intima. We compared the labeled nuclei per surface area of the intima. Although looking at area versus labeled cells may be influenced in the mature neointima by the increasing space between cells due to the deposition of extracellular matrix, the labeled nuclei for PCNA were significantly greater at 2 and 4 weeks than those for Ki-67. This difference between the results of PCNA and Ki-67 immunohistochemistries has also been confirmed in brain tumor tissues by us (M.A. et al, unpublished data, 1994) and others.28 29
The Ki-67 nuclear antigen has been shown to be expressed in G1, S, G2, and M phases but not in the G0 phase.8 Recent studies20 30 have shown that Ki-67 antigen starts to be expressed during the S phase, progressively increasing through the S and G2 phases and reaching a maximum in the M phase; the amount of Ki-67 antigen in cells entering the G1 phase begins a progressive decline during this phase and finally disappears in cells with a long G1 phase. Thus, the Ki-67 antibody may not recognize all proliferating cells, such as those with the long G1 phase, those at the end of the G1 phase, or those entering the S phase. PCNA staining is most noticeable during late G1 and early S phases, but PCNA labeling occurs also in the G1 and G2 phases of the cell cycle, and the half-life of PCNA was reported to be 20 hours.5 27 31 It is therefore conceivable that cells remain PCNA-positive even after leaving the S phase. The fraction of PCNA-positive cells without detectable nuclear Ki-67 staining may represent cells passing a long period in the G1 phase after mitosis, cells in the late G1 phase, or cells entering the S phase. PCNA-positive SMCs without Ki-67 staining in the surface layer of the intima 4 weeks after endothelial denudation may represent cells passing a long period in the G1 phase after mitosis rather than cells in the late G1 or early S phase. From the findings by Lopez et al30 using lymphocytes, the half-life of immunodetectable Ki-67 antigen is at least within 2 hours. This may indicate a shorter half-life for Ki-67 than PCNA, providing another explanation for the more accurate detection of positive immunostaining for Ki-67 than PCNA.
Finally, although the cross-reactivity of the MIB-1 mAb in species other than rabbits and previously reported animals14 15 needs to be clarified, the immunodetection of Ki-67 in paraffin sections with the use of MIB-1 mAb and hydrated autoclaving may provide another useful tool with which to elucidate the mechanism for the development of intimal thickening after arterial injury in experimental animals as well as of atherosclerosis in vivo.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture, Japan (No. 06671378).
Received July 3, 1995; revision received August 17, 1995; accepted August 17, 1995.
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