Ref - Furukawa S, Morita S, Wingenfeld L, Matsuda W, Nakagawa T, Sakaguchi I,
Takaya A, Nishi K. Immunohistochemical staining of the brain tissue obtained
from a man with multiple focal brain infarctions and different staging. Anil
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Title
Immunohistochemical staining of the brain tissue
obtained from a man with multiple focal brain infarctions
and different staging
Satoshi Furukawa, Satomu Morita, *Lisa Wingenfeld,
Wakoto Matsuda, Tokiko Nakagawa, Ikuo Sakaguchi,
Akari Takaya, Katsuji Nishi
Department of Legal Medicine, Shiga University of
Medical Science, Shiga Japan
*Institute of Legal Medicine, Munich University, Munich,
Germany
Satoshi Furukawa
Department of Legal Medicine, Shiga University of
Medical Science, Shiga Japan
Setatsukinowa, Otsu, Shiga 520-2192, Japan
31041220@belle.shiga-med.ac.jp
TEL/FAX; +81775482200
Abstract
We encountered an autopsy case of a 65-year old male
with multiple infarct legions in his brain. The stage of
infarctions were different, that is, infarction with
bleeding, necrotic and cavity, respectively. We examined
morphological and immunohistochemical study of each
legion using antibodies against CIRP, RBM3, HSP70,
HIF1, VEGF, e-NOs, AIF1, p53, c-Fos, CCC9 and SIRT1.
The results indicate that CIRP and RBM3 may play an
important role of prevention of hypoxic status.
The reactivity of RBM3 in the infarct legions was
detected in early stage, and increased with progress of
the stages of infarcts. And RBM3 may also play an
important role in the angiogenesis in the infarct area.
SIRT1 was expressed in the neurons or glial cells
surrounding of infract legions especially in late stage,
showing the expression of SIRT1 was delayed than those
of CIRP and RBM3.
KeyWords: Multiple brain infarctions, Hypoxia,
Immunohistochemical staining,
Introduction
There were many studies on histological characteristics
of brain infarctions (1-4). We also made a study
concerning the immunohistochemical staining figures on
the chronic brain infarction(5). Although chronic
infarctions
with tissue defect in the brain were often
detected in the forensic autopsy cases from elder victims,
detection of the focal infarction with bleeding was rare.
We experienced an autopsy case in which a 65-year-old
male had multiple focal infarctions with different
staging, and we made immnuhistochemical staining of
the brain tissues using antibodies to resolve the roles of
hypoxia, ischemic and stress related antigens and
inflammation materials on the progress of the brain
infarction. It is widely accepted that in hypoxia and
ischemic cases an increase in the transcription level of
specific genes, including those encoding for
hypoxia-inducing factor 1(HIF1), apoptotic inducing
factors (AIF-1), and stress related factors containing Cold
inducing RNA binding protein (CIRBP) and RNA binding
motif 3 (RBM3) which are recognized as a cold stress
inducing protein and were intensively expressed in the
postmortem human heart muscle obtained from
accidental hypothermic death cases (1-6). Neuroglobin
(Ngb) , Complement component C 9 , c-Fos , Protein 53
(p53) , Sirtuin 1 (SIRT1) and Heat shock protein 70
(HSP70) were also expect to express in the hypoxia or
ischemic brain area(7-13).
Materials and methods
Autopsy case
A 65-year-old male was a victim. Although he has been
thinking that he remains unmarried for his life after his
parent’s death, he had a formal meeting with a view to
marriage the last year. The next day of the meeting he
married with 45-year-old female who had one sun. After
one week of his marriage he was found death in his living
room when his wife and sun came back to home after
looking their jobs. The police suspected the criminal.
The police investigation revealed that there was one
visitor who was his relative during their absence and he
has been suffering from diabetes, liver, kidney and heart.
At autopsy, the chronic infarctions in the right-frontal
lobe and right-cerebellum were recognized and subacute
one was detected right-basal ganglion, and additionally
newly infarction with small amount of bleeding was seen
in left-cerebral cortex.
Histology
Each brain tissue cut at autopsy was fixed in formalin
and prepared paraffin blocks within three days after
autopsy. Stainability of Macrophage, lymphocytes,
neurons and astroglia involved in brain infarction were
studied by immunohistochemistry using ABC technique
(Nichirei, Japan), and antibodies against CIRP, RBM3,
HSP70, HIF1, VEGF, e-NOs, AIF1, cFos, Ngb, Wnt,
CCC9, carbindinand SIRT1.Immunohistochemical
staining with antibodies was performed, according to the
manufacture protocol. The characteristics of the
antibodies used were described in Table 1.
Results
1: Fresh infarction with abundant bleeding
The infarcted area has severe bleeding containing large
number of intact erythrocytes and small number of
macrophages. Anti CIRBP, RBM3 and HSP70 stained
macrophage, but not neutrophills, erythrocytes, and
endothelium of blood vessels. The reactivity with these
antibodies was detected in the neurons and glias around
the infarcted area. The staining intensity by anti RBM3
was intensive among these antibodies. Anti HIF1 showed
weak reactivity with the neurons and glias around the
bleeding area, anti VEGF and e-NOs showed the similar
reactivity with the cells around bleeding area, the
staining intensity among these antibodies was
remarkable by anti e-NOs, and these antibodies showed
no reactivity with vascular endothelial cells. Anti AIF1,
Ngb, Wnt, cFos, CCC9, p53 and SIRT1 showed no
reactivity in the bleeding area. Anti SIRT 1 showed
moderate reactivity with neurons and glias around
bleeding. Anti CCC9 showed weak reactivity with these
cells. The other antibodies showed a feeble or no
reactivity.
2: Semi-chronic infarction
Semi-chronic infarction might be subdivided into two
phases.
2-1: Phase of acute infarction
HE staining showed the presence of coagulative necrosis,
spheroids, mononuclear cells, macrophages and
gemistocytes, however remarkable neo-angiogenesis and
inflammatory cells were not detected in this patient. Anti
RBM3 showed clear reactivity with nucleus of vascular
endothelial cells and other type of cells, such as
macrophages and mononuclear cells. Anti CIRBP and
HSP70 showed the similar stainability but were
relatively weak(Figure.１). Anti eNOs showed similar
reactivity with those by anti RBM3, however anti HIF1
and VEGF showed feeble or very weak reactivity in the
lesion (Figure.2). Anti SIRT1 showed weak or a feeble
reactivity with nucleus in the cell group containing giant
granular corpuscle cells in the infarct area. Anti CCC9
showed intensive reactivity with some cells that were
pyramidal shape and not able to identified, and the
antibody also showed clear reactivity with the giant
granular corpuscle cells in the infarction area. The other
antibodies showed feeble or no reactivity in the lesion.
2-2: Phase of subacute infarction
HE staining revealed amorphous, granular, pink
material、fibrous cells and gemistocytes in which the
ghost outlines of some necrotic neurons could be detected
in semi-chronic infarction area. Although angiogenesis
and abundant of macrophages were observed,
perivascular inflammatory mononuclear cells were not
remarkable in this patient. Anti RBM3 clearly stained
nucleus in the many type of the cells inside the infraction
area, and the cells that were adhered to the angiogenesis
capillary walls (Figure.3). Anti CIRBP and HSP70
showed the similar stainability with relatively weak.
Although anti HIF1 and VEGF showed weak reactivity
with the several kinds of cells inside the infarction area,
anti e-NOs showed moderate reactivity with these cells
(Figure.4). Anti SIRT1 showed clear reactivity with the
nucleuses of the cell group containing giant granular
corpuscle cells in the infarct area, while the antibody
showed weak reactivity the cells outside infarction area.
Anti AIF1, cFos and Ngb showed weak reactivity with the
cells inside the infarction area, p53 and Wnt showed
feeble or no reactivity. Anti CCC9 showed no reactivity in
the infarct area and clear reactivity with the giant
granular corpuscle cells outside the infarction area
(Figure.5).
3: Chronic infarction in the cerebellum (resorption phase)
Since this patient had the cerebellum with a large
cavitation, the infarct area in the cerebellum was used to
investigate. HE staining revealed a cystic space from the
resolvedliquefactive necrosis, hemosiderin pigments,
compound granular corpuscle cells and macrophages in
chronic infarction area. Anti CIRBP, RBM3 and HSP70
showed feeble reactivity with the scattered cells in the
liquefactive necrotic area, and anti RBM3 and CIRBP
showed weak reactivity with the nucleus of cells in the
arterial walls. These antibodies reacted with giant
granular corpuscle cells around the necrotic area. Anti
HIF1, VEGF and e-NOs showed the cells around infarct
area and no in the cavitation. Anti c-Fos, p53, CCC9 and
SIRT1 showed a feeble or no reactivity in this area.
Discussion
The report concerning to immunohistochemical study on
multiple brain infarction with several stages are very
few. In this case, patient with multiple infarct lesions
with bleeding, numerous macrophages, necrosis,
neo-vascularization and cavitation in infarction lesions,
respectively, presumed to die due to cardiac fibrillation,
since the small thrombosis in blood vessel near by fresh
infarct with bleeding and in right coronary artery were
detected, and then multi-focal infarctions presumed to be
originated from his heart.
First, we were able to group each lesion into three
categories according to Mena et al (14). However in this
case, peri-vascular cuffing was not clearly detected,
although morphological changes could be agreed to the
described findings.
In this study, anti RBM3 showed clear and intensive
reactivity in the infarct area. RBM3 was also expressed
in the nucleuses on the macrophage, and neutrophils and
CIRBP and HSP 70 reactivity were also recognized in the
glial and/or neural cells detected around the infarct area.
These results indicate the expression of RBM3, CIRBP
and HSP70 may relate to hypoxia or ischemic situation,
although RBM3 and CIRBP were accepted as cold
inducible antigens and these were expressed in the
cardiac muscle obtained from individuals who died due to
accidental hypothermia (11).
It is well accepted that HIF1 which closely relate with
hypoxia controls or manages to expression of VEGF, and
the expression of VEGF is connected with the expression
of e-NOs, since Qing et al have proposed that the
response to hypoxia is primarily mediated by the
transcription factor hypoxia-inducible factor-1 (HIF-1)
which leads to the induction of a variety of adaptive gene
products including vascular endothelial growth factor
(VEGF) and endothelial nitric oxide synthase (eNOS)
(15).
In this study the expression of RBM3 was detected in the
endothelial cells of the blood vessels in the acute or
subacute infarct area, showing similar to those in VEGF
and e-NOs. These results indicate that RBM3 may also
play an important role in the angiogenesis in the infarct
brain area.
Several studies have implicated the protection effect of
SIRT1 against ischemic insult in the heart. We also
showed that SIRT1 expressed in the hearts obtained from
individuals who died due to hypothermic and carbon
monoxide poisoning. Although expression of SIRT1 in the
brain was not or weakly detected in our previous studies
which were performed using the brains obtained from
victims who died due to self-strangulation, hanging
and/or manual strangulation, the existence in the
nucleuses in the cells of around infract area was clearly
recognized in this case. The prolonging of hypoxic and/or
ischemic situation occurred in his brain may bring out
the increasing of transcription of SIRT1. SIRT1 may also
play an important in neuroprotection.
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