Bystin (BYSL) as a possible marker of severe hypoxic-ischemic changes in neuropathological examination of forensic cases
Bystin (BYSL) as a possible marker of severe hypoxic-ischemic changes in neuropathological examination of forensic cases
Mieszko Olczak 0 1 2
Dominik Chutorański 0 2
Magdalena Kwiatkowska 0 1 2
Dorota Samojłowicz 0 1 2
Sylwia Tarka 0 1 2
Teresa Wierzba-Bobrowicz 0 2
0 Departament of Neuropathology, Institute of Psychiatry and Neurology , 9 Sobieskiego St., 02-957 Warsaw , Poland
1 Oczki St. , 02-007 Warsaw , Poland
2 Department of Forensic Medicine, Medical University of Warsaw
3 Mieszko Olczak
Bystin (BYSL) is a 306-amino acid protein encoded in humans by the BYSL gene located on the 6p21.1 chromosome. It is conserved across a wide range of eukaryotes. BYSL was reported to be a sensitive marker for the reactive astrocytes induced by ischemia/reperfusion and chemical hypoxia in vitro and is considered to be one of the common characteristics of astrogliosis. In our study we examined whether BYSL could be used as a marker for hypoxic-ischemic changes in forensic cases. Groups suspected of acute hypoxic-ischemic changes presented strong BYSL expression in the cytoplasm of neocortical neurons especially in layers 3-5, that seemed to be short-lasting. In the hypoxic-ischemic-reperfusion group we did not find BYSL expression. BYSL expression in the cytoplasm of cortical neurons was minimal in the control group (cardiac arrest). BYSL seems to be a promising early marker of severe hypoxic-ischemic changes in neuropathological examination of forensic cases and certainly requires further studies.
Bystin; Brain ischemia; Hypoxia-ischemia; Marker
Acute hypoxic-ischemic brain injury results in several
biochemical changes, which occur shortly (60–180 s)
after its onset. These include ATP depletion, anoxic
dep o la r iz a ti on a n d m ul ti d ir ectional i mpairment of
neurolemmal function causing extracellular sodium,
chloride and calcium decrease and potassium leak into
the extracellular space [
]. Calcium influx into the
neuron results in the activation of calcium-dependent
pathways (eg. calpain system activation), leading to the
onset of apoptosis . Local release of lactic acid and
hydrogen ions turns extracellular space acidic within
20 s of the hypoxic/ischemic insult [
release results in excitotoxic enzyme activation, free
radical formation and nitric oxide toxicity leading to
neuronal breakdown [
Those biochemical changes are accompanied by
mitochondrial damage [
], abnormal Golgi complexes formation [
depressed protein synthesis [
] and the induction of heat
shock protein production [
]. Neuronal cell death is a result
of three coexisting mechanisms: necrotic cell death, apoptosis,
and free-radical induced damage with autophagocytosis [
]. Cell death does not occur if the reperfusion is established
within the “revival period” .
Modern and specific markers of acute hypoxia in human
include decreased calbindin-D28k expression in
Neurological and neurobehavioral consequences of
hypoxicischemic brain injury in survivors include: seizures, movement
disorders eg. post-hypoxic parkinsonism, delayed post-hypoxic
leukoencephalopathy, disturbances of sensorimotor function,
cognitive, emotional and behavioral impairments [
Bystin (BYSL) is a 306-amino acid protein encoded in
humans by the BYSL gene located on the 6p21.1 chromosome
also conserved across a wide range of eukaryotes, including
]. BYSL was originally identified as a
cytoplasmic protein forming a complex with thophinin and tastin in
human trophoblastic embryonal carcinoma HT-H cells [
]. These proteins are expressed at the uteroplacental interface
or at implantation sites at early stages of pregnancy and
disappear from the placenta after the 10th week of pregnancy .
Analysis of bystin in human cancer cells and mouse embryos
indicates that bystin functions in the biogenesis of the 40S
ribosome and in cell growth [
]. Animal studies reported
presence and up-regulation of BYSL expression levels in
astrocytes following brain injury what was promoted by
proinflammatory mediators (lipopolisaccharide, interleukin-1β) and
nerve growth factor . Compared with GFAP, BYSL was
reported to be more sensitive marker for the reactive astrocytes
induced by ischemia/reperfusion and chemical hypoxia in vitro
and was considered to be one of the common characteristics of
]. In our study we wanted to examine if BYSL
might be a useful marker for hypoxic-ischemic changes in
Materials and methods
The study was carried out using 9 cases suspected of acute and
fatal hypoxic-ischemic changes of the CNS (hanging, carbon
monoxide fatal poisoning (HbCO > 50%), fatal external/
internal bleeding) - HI group; 7 cases suspected of acute
hypoxic-ischemic changes followed by reperfusion and a few
(2–5, average of 3.43) days of hospitalization prior to death
(hanging, drowning) - HIR group; 8 cases of sudden death
cardiac arrest - sudden and instantaneous death with no
cardiopulmonary resuscitation, used as a control group - C group; one
case of brain ischemic stroke with proven duration of at least
48 h studied by forensic pathologists at the Department of
Forensic Medicine, Medical University of Warsaw.
Brain specimens (frontal lobes) were collected during
forensic autopsies and fixed in buffered formalin, then
embedded in paraffin. Sections were stained histologically
(hematoxylin-eosin) and immunohistochemically with: anti
bystin (BYSL) (Merc-Millipore, 1:200), anti-glial fibrillary
acidic protein (GFAP) (Serotec, 1:700) and anti-human
macrosialin (CD 68) (Dako, 1:75) according to the IHC-P
protocols supplied by the manufacturers.
Microphotographs of brain sections were taken with an
Olympus BX41 microscope and the Olympus DP25 digital
camera. Microphotographs were taken with the same light
level for all cases.
To assess the BYSL expression, sections showing BYSL
reaction in the frontal cortex were analyzed. Microphotographs
of two adjacent areas were taken with magnification of 200 (area
of 141,808.92 μm2) for each single case, then converted to a
16bit grayscale. Percent of positive antibody reaction was counted
as a stained area fraction in the analyzed region, with ImageJ
1.41o software and used for statistical analysis. Area fraction
was automatically counted with the “Threshold” function of
the ImageJ program, which marks all the pixels of a selected
grey value and counts all the groups of marked pixels (e.g.
positive BYSL reaction) within the selected area. Two adjacent
areas for each case were analyzed. Values were averaged for
each case and used for statistical analysis [
The STATISTICA software package for Windows
(StatSoft, Tulsa, OK, USA) was used to analyze all data.
Probability (P) levels of less than 0.05 were considered
H&E staining revealed some neuropathological findings in
each experimental (HI, HIR) and control (C) groups. Control
group presented features of slight to moderate brain edema.
The astroglia morphology in GFAP staining appeared normal.
BYSL expression in cytoplasm of neurons in cortex was
minimal (Fig. 1a–c).
Study groups HI and HIR displayed brain edema and
ischemic neurons with perineuronal satellitosis, which was found to
be severe in the HIR group in H&E staining (Fig. 1d, g). Both
experimental groups presented astroglia proliferation and
clasmatodendrosis. Both were slightly more pronounced in
HIR group (Fig. 1e, h).
The HI group presented strong BYSL expression in the
cytoplasm of neocortical neurons especially in layers 3–5
(Fig. 1f). BYSL expression seemed to be most prominent in
ischemic cases (e.g. stab wound with fatal bleeding) and
moderate in hypoxic ones (e.g. CO poisoning) (data not shown). In
the HIR group we did not find BYSL expression at all (Fig. 1i).
The brains of the control group, and experimental (HI and
HIR) groups were quantitatively assessed for BYSL
expression. Results presented in Table 1 show that acute
hypoxiaischemia caused statistically significant increase in BYSL
expression as compared to the control group and HIR group
where no BYSL expression was observed. Mann-Whitney
U-test revealed that the amount of BYSL was significantly
higher in the IH group than in the control group (p < 0.01)
and HIR group (p < 0.01). The latter (HIR) was also lower
than the control group (p < 0.01).
As we found no BYSL expression in the hypoxic-ischemic
changes followed by reperfusion (HIR) group, we performed
additional staining to confirm that finding. We examined a case
of brain ischemic stroke with proven duration of over 48 h. No
BYSL expression was found in this case, similar to the HIR
group. Sample microphotographs are shown on Fig. 2.
Hypoxic-ischemic injury includes number of
neuropathological changes seen in the light microscope examination. Within
1 h of the insult microvacuolization with normal or shrunken
Fig. 1 Frontal lobes, magnification × 200. H&E and anti-BYSL staining in
the 3rd neuronal layer of the neocortex. GFAP marks white matter. a –
control group (sudden death), slightly pronounced brain edema; b
control group, normal view of astroglia; c – control group (sudden death),
white matter, minimal BYSL expression in neuron cytoplasm; d – HI group
- brain edema, ischemic neurons and perineuronal satellitosis; e – HI group,
astroglia proliferation and clasmatodendrosis; f – HI group, white matter,
nucleus, cell shrinkage, normal or slightly basophilic
cytoplasm, and perineuronal spaces around some neurons may
be observed. These changes cannot be distinguished from
postmortem autolysis [
]. For this reason we proposed
BYSL as a new marker of early ongoing neuronal changes
after hypoxic-ischemic episodes.
Typical neuropathological hypoxic-ischemic changes
include: ischemic cell change - seen in the first 6 h of survival,
with further neuronal nucleus and cytoplasm shrinkage and
pink cytoplasm staining; homogenizing cell change typically
seen around 24 h after survival; and a final stage termed
“ghost cell” with reduced or absent cytoplasm around a dark
and shrunken nucleus and eventual cell loss [
]. In our HI
C - control group; HI - acute hypoxia-ischemia group; HIR - acute
hypoxia-ischemia with survival/reperfusion group; AV-AF - average
stained area fraction (in %) of positive BYSL reaction in the frontal
cortex; (sample microphotographs shown on Fig. 1, pictures C, F, I);
p/C - significance comparing to control group; p/HIR - significance
comparing to HIR group
statistically significant (comparing to control group) increased BYSL
expression in cytoplasm of ischemic neurons; g – HIR group with brain
edema, ischemic neurons and severe perineuronal satellitosis; h – HIR
group, slightly more pronounced astroglia proliferation and
clasmatodendrosis; i – HIR group with no BYSL expression. Inserts b, e,
h shows astrocytes in higher magnification
group ischemic neurons were present despite the fact that
death occurred immediately after the ischemic episode. This
further emphasizes a need for a validated early marker of
In our study we documented that acute and fatal
hypoxicischemic brain injury results in increased BYSL expression in
cortical neurons, especially in layers 3–5. Similar findings
were documented previously in vitro [
] and in animal
]. BYSL expression seems to be more prominent in
ischemic episodes (e.g. fatal bleeding) and moderate in
hypoxic ones (e.g. CO poisoning), but it is possible that the length
of the episode plays an important role here. Minimal BYSL
expression in control group shows, that ischemic brain injury
in cardiac arrest may also be distinguished and recognized
with this technique. Previous studies describe minimal
BYSL expression in astrocytes of cerebral cortex in a
physiologically “normal” CNS [
]. Similarly, previous studies
showed that BYSL expression is a more sensitive marker for
reactive astrocytes and can be considered one of the common
characteristics of astrogliosis [
]. Here we documented
BYSL as a potential marker of hypoxic-ischemic brain injury
with a short survival time after the episode, which could be
useful for future forensic examinations. In spite of GFAP
expression not revealing differences between hypoxic-ischemic
and hypoxic-ischemic-reperfusion groups, BYSL expression
was increased and detectable (within ≤48 h) after the
hypoxicischemic episode, which could be potentially useful for dating
Fig. 2 Case of brain ischemic
stroke with duration of over 48 h;
magnification ×200. H&E
macrophages infiltration; CD68
microglia infiltration (arrows) in
the vicinity of blood vessel;
GFAP - astrocytes (arrows) in the
area of brain ischemic stroke,
poor GFAP expression; BYSL
no BYSL expression in the area
of brain ischemic stroke
of hypoxic-ischemic episodes. Further studies including
animal ones are needed to establish the precise dynamics of
BYSL expression. Previous studies prompted a hypothesis
that cell death results in decrease of BYSL expression [
This hypothesis, however, requires further studies. In in vitro
models 0.5 and 3 h ischemia induced significant BYSL
astrocyte expression while no differences between the control
group and 24 h ischemia and 24 h ischemia/reperfusion were
observed. Similarly mild chemical hypoxia induced
significant BYSL expression in vitro. These increases were not
accompanied by increased GFAP expression [
experimental animal models of brain injury elevated BYSL level was
observed more than 5 weeks after the injury [
] and it was
suggested that BYSL may be involved in glia activation and
differentiation as its increased expression was observed in vitro
during astrocyte IL-β transformation [
]. Studies in vitro
showed increased BYSL expression was observed positively
correlated with IL-1β, IFN-γ, NGF action [
BYSL seems to have a great potential, as an early marker of
severe hypoxic-ischemic changes in neuropathological
examination of forensic cases and certainly requires further studies.
Hypoxia-ischemia leads to enhanced, short-lasting induction
of bystin expression in cytoplasm of neocortical neurons.
No bystin expression was observed in the
Cardiac arrest leads to a minimal bystin expression in
Bystin may serve as an early marker of severe
hypoxicischemic changes in neuropathological examination of
Compliance with ethical standards
Conflict of interest All authors declare that they have no conflict of
Ethical approval For this type of study formal consent is not required.
Financial disclosure statement None.
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