https://doi.org/10.52973/rcfcv-e34454
Received: 20/05/2024 Accepted: 09/07/2024 Published: 19/09/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34454
ABSTRACT
Listeria monocytogenes is an intracellular, food–borne bacterium.
Silage is an important source of this pathogen causing listeriosis.
Listeriosis is an important health problem for both animals
and humans in the world. The disease comprises three clinical
syndromes: meningoencephalitis, septicemia and metritis with
abortion. Encephalitis is frequently observed and the factors
that play a role in its pathogenesis are the subject of research. In
this study, the immunohistochemical expression of MMP–2 and
MMP–9 together with TUNEL staining was investigated in the
pathogenesis of meningoencephalitis in sheep naturally infected
with L. monocytogenes. The brains of 25 sheep with Listerial
meningoencephalitis were used in this study. Brain material from
10 sheep provided from the slaughterhouse was also used as a
control. Tissue sections were stained immunohistochemically with
L. monocytogenes, MMP–2 and MMP–9 antibodies. Additionally, TUNEL
staining was performed to determine apoptosis in the disease. As a
result of the study, it was observed that TUNEL staining in neurons
and glial cells, MMP–2 and MMP–9 expressions in vascular endothelial
cells, inammatory cells, microglia and especially neurons in the
infected brain tissue were significantly increased compared to
controls. These results suggested that MMP–2 and MMP–9 play an
active role in the neurodegeneration and cell death that occur in
Listerial encephalitis.
Key words: Immunohistochemistry; Listeria monocytogenes;
MMP–2, MMP–9; TUNEL
RESUMEN
Listeria monocytogenes es una bacteria intracelular transmitida
por los alimentos. El ensilaje es una fuente importante de este
patógeno causante de listeriosis. La listeriosis es un importante
problema de salud tanto para los animales como para los humanos
en el mundo. La enfermedad comprende tres síndromes clínicos:
meningoencefalitis, septicemia y metritis con aborto. La encefalitis
se observa con frecuencia y los factores que influyen en su
patogénesis son objeto de investigación. En este estudio, se investigó
la expresión inmunohistoquímica de MMP–2 y MMP–9 junto con la
tinción TUNEL en la patogénesis de la meningoencefalitis en ovejas
infectadas naturalmente con L. monocytogenes. En este estudio
se utilizaron los cerebros de 25 ovejas con meningoencefalitis por
listeria. También se utilizó como control material cerebral de 10
ovejas procedente del matadero. Las secciones de tejido se tiñeron
inmunohistoquímicamente con anticuerpos de L. monocytogenes,
MMP–2 y MMP–9. Además, se realizó tinción TUNEL para determinar
la apoptosis en la enfermedad. Como resultado del estudio, se
observó que la tinción de TUNEL en neuronas y células gliales, las
expresiones de MMP–2 y MMP–9 en células endoteliales vasculares,
células inamatorias, microglía y especialmente neuronas en el tejido
cerebral infectado aumentaron signicativamente en comparación
con los controles. . Estos resultados sugirieron que MMP–2 y MMP–9
desempeñan un papel activo en la neurodegeneración y muerte celular
que se producen en la encefalitis por Listerial.
Palabras clave: Inmunohistoquímica; Listeria monocytogenes;
MMP–2; MMP–9; TUNEL
Immunohistochemical expression of MMP–2 and MMP–9 in the brain tissue
of sheep naturally infected with Listeria monocytogenes and relationship
with cell death in the Listerial encephalitis
Expresión inmunohistoquímica de MMP–2 y MMP–9 en el tejido cerebral de ovejas infectadas
naturalmente con Listeria monocytogenes y relación con la muerte celular en la encefalitis por listeria
Mehmet Önder Karayigit
1
* , Mehmet Halıgür
1
, Mehmet Ekici
2
1
Cukurova University, Faculty of Ceyhan Veterinary Medicine, Pathology Department. Adana, Türkiye.
2
Cumhuriyet University, Faculty of Veterinary Medicine, Physiology Department. Sivas, Türkiye.
*Corresponding author: karayigit09@hotmail.com
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INTRODUCTION
Listeria monocytogenes is a gram–positive, facultative anaerobic,
rod–shaped intracellular and ubiquitous bacterium that causes
Listeriosis, affecting both animals and humans [1, 2]. Ruminants
such as cattle (Bos taurus), goats (Capra hircus) and sheep (Ovis aries)
play an important role in the maintenance and spread of this pathogen
in the farm environment [3]. The bacterium may cause septicemia
by invading the intestinal tissue of herbivorous animals, and also
causes neurological disorders [2, 4]. Typical histopathological
ndings of encephalitic Listeriosis are microabscesses consisting
of macrophages and microglial cells and neutrophil leukocytes [4, 5,
6, 7]. Matrix metalloproteinases (MMPs), especially gelatinous MMPs
(MMP–2 and MMP–9) play a critical role during inammation and healing
processes in mammals [8, 9, 10]. In a healthy central nervous system,
small amounts of gelatinous MMPs are known to be expressed under
normal physiological conditions. However, a remarkable increase has
been reported under various neuropathological conditions [11, 12].
Gelatinous MMPs disrupt the structure of the extracellular matrix
(ECM) and therefore play a signicant role in the pathogenesis of
diseases in the nervous system. In general, gelatinous MMPs lead
to some pathological changes after decreased cerebral blood ow
including loss of ionic homeostasis, energy deciency increased
oxidative stress, apoptosis, irreversible tissue/organ damage, and
neurological disorders [13, 14]. It is shaped by the disruption of the
complex interaction of the inammatory reaction and the ECM model
after the decrease of blood ow in the cerebral tissue [15]. It has been
reported that MMP–2 and MMP–9 are expressed in vascular endothelial
cells, meninges, inammatory cells, microglia and especially neurons
in neuroinammatory changes in the central nervous system [16, 17].
Apoptosis is involved in the physiological processes of many
cells in the body during and after the embryonal period. These
physiological processes include embryonic development, organ
metamorphosis, cell cycle, development and activation of cells,
and cell aging. Thus, it is important to maintain tissue homeostasis
under normal physiological conditions [18, 19]. However, abnormal
apoptosis (either too little or too much) is a critical factor in the
explanation of pathogenesis of some pathological conditions
including neurodegenerative diseases, autoimmune disorders and
many types of cancer. The mechanism of Listerial encephalitis is a
complex process accompanied by many cellular interactions and
expressions. Although some of the cellular effects that play a role
in this mechanism have been revealed, it appears that there are new
factors that contribute to the neurodegeneration and cell death
that occur in the disease over time. In this context, the relationship
between gelatinous MMPs and apoptosis has not been previously
studied in Listerial encephalitis. Therefore, in this study, it was
aimed to evaluate the roles of gelatinous MMPs and apoptosis in the
formation of lesions in the central nervous system of sheep naturally
infected with L. monocytogenes in terms of pathogenesis.
MATERIAL AND METHODS
Ethical statement
Because the experiment did not involve any invasive procedures
for animal experiment Ethics Committee permission is not required.
A decision was taken from the Cukurova University Faculty of Ceyhan
Veterinary Medicine Research Ethics Committee stating that ethics
committee approval was not required for the study (Document Date
and Number: 01/02/2024–31330).
Tissue samples and histopathology
Twenty–ve sheep brain tissues samples were obtained from the
archive of the Department of Pathology, Faculty of Ceyhan Veterinary
Medicine, University of Cukurova. The samples were from sheep of
different breeds in different farms at different times. The sheep
were 2−5 years old. The breeds of sheep included Akkaraman (fteen
cases), Kivircik (one cases), Merinos (four cases) and crossbreed (ve
cases). Although symptoms such as droopy ear, drooping eyelid,
fever, lack of coordination, salivation were reported in sheep, circling
and silage history were common anamnesis information. Hyperemia
and opacication of the meninges were common necropsy ndings.
Listeriosis was diagnosed based on immunohistochemical and
histopathological ndings in the tissues. The 10 healthy animals
comprising the control group had been slaughtered for human
consumption and the heads of these animals were purchased from
the Slaughterhouse. The infected and control brain tissues were
comprised by cerebral cortex, midbrain, cerebellum and brain stem. All
brain tissues were xed in 10% buffered formalin solution for 48 hours
(Sigma, Darmstadt, Germany), then washed thoroughly in tap water
overnight. After dehydration in graded alcohols, were cleared in xylene
and embedded in paran (Merck, Darmstadt, Germany). Paran
blocks of cerebral cortex, midbrain, cerebellum and brain stem
were cut at 5 μm (Leica, RM 2125) and stained with hematoxylin and
eosin [20] (HE) Immunohistochemistry (IHC) method was performed
according to the manufacturer’s protocol. Stained sections were
examined and photographed using a trinocular light microscope
(Olympus BX51) with a DP25 digital camera (Tokyo, Japan). The severity
of L. monocytogenes infection in each animal was classied based
on occurrence of the following neuropathological changes: gliosis;
neuronal necrosis; perivascular cell inltration; and bacterial antigen
immunostaining in the neurons, leuykocytes and glial cells.
Immunohistochemistry
Immunohistochemical staining was performed using the
routine streptavidinbiotin–peroxidase technique according to
the manufacturer’s recommendations [Anti rabbit streptoavidin/
biotin immunoperoxidase kit (Histostain–Plus Kits, California,
USA). The selected 5 μm paraffin tissue sections were stained
immunohistochemically in order to elucidate the expressions of
anti–L. monocytogenes polyclonal antibody [Novus NB100–65667,
(diluted 1/250)], anti–MMP–9 [orb13583,Biorbyt (diluted 1/250)] and
anti–MMP–2 [GeneTex, GTX104577, (diluted 1/500)] The red color
reaction was enhanced using 3–amino–9–ethylcarbazole (AEC)
(Zymed AEC RED substrat kit, ABD) as the chromogen. All sections
were counterstained with Gill hematoxylin (HX71788774,Meck, USA)
solution and then washed in water. Coverslips were applied with
water–based mounting medium (Shandon Immuno–mounting). In
additon, all infected and control tissues were stained by TUNEL
method to determine apoptotic cells that undergo extensive DNA
degradation during the late stages of apoptosis (In Situ Cell Death
Detection Kit, Roche, Basel, Switzerland). Routine IHC period was
applied to the sections until the antibody stage, and then the ready–
use TUNEL kits were gently mixed and dropped onto the tissues and
kept at room temperature for 1 hour. Then In Situ Cell Death Detection
Kit–POD was added and left for 30 min, it was washed and stained
with AEC and covered with a coverslip using water–based adhesive
(Shandon Immuno–mounting).
TABLE I
Neuronal immunohistochemical expression of MMP–9, MMP–2, and TUNEL staining
MMP–9 MMP–2 TUNEL staining
Groups n Mean (Median) SD n Mean (Median) SD n Mean (Median) SD
Healthy control lambs 10 0.50 (0.50) 0.53 10 0.60 (1.00) 0.52 10 0.70 (1.00) 0.48
Listeria monocytogenes infected lambs 25 2.60 (3.00)*** 0.58 25 2.20 (2.00)*** 0.65 25 2.00 (2.00)*** 0.50
***:
P<0.001 shows signicance when comparing healthy control lambs to those infected with L. monocytogenes according to Mann–Whitney Test
TABLE II
Glial immunohistochemical expression of MMP-9, MMP-2 and TUNEL staining
MMP–9 MMP–2 TUNEL staining
Groups n Mean (Median) SD n Mean (Median) SD n Mean (Median) SD
Healthy control lambs 10 0.60 (1.00) 0.52 10 0.40 (1.00) 0.52 10 0.50 (0.50) 0.53
Listeria monocytogenes infected lambs 25 2.52 (3.00)*** 0.51 25 2.12 (2.00)*** 0.60 25 2.16 (2.00)*** 0.62
***:
P<0.001 shows signicance when comparing healthy control lambs to those infected with L. monocytogenes according to Mann–Whitney Test
FIGURE 1. Immunohistochemical expression of MMP–9, MMP–2, and TUNEL
staining in neuronal cells was assessed in both healthy and Listeria monocytogenes
infected lambs
FIGURE 2. Immunohistochemical expression of MMP–9, MMP–2 and TUNEL
staining in glial cells was assessed in both healthy and Listeria monocytogenes
infected lambs
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Histomorphometric analysis
Positive labelled neuronal and glial cells was measured using
a computerized image system comprising a Leica CCD camera
DFC420 (Leica Microsystems Imaging Solutions, Ltd., Cambridge,
UK) connected to a Leica DM4000 B microscope (Leica Microsystems
Imaging Solutions, Ltd.). Five representative elds were selected and
consecutive images were captured by Leica QWin Plus v3 software using
a 20× objective lens (N Plan; Leica Microsystems Imaging Solutions).
Same setting used for all integrated optical density of MMP–2, MMP–9
and TUNEL staining was measured and the mean stained area/total
area was calculated using Leica QWin Plus v3. All images were collected
under the same lighting conditions. An investigator blinded to the
identity of the sections quantied all sections.
RESULTS AND DISCUSSIONS
Statistics Result
The obtained data were statistically analyzed using the Mann
Whitney U test in the SPSS version 26 package program. The results
were presented in the format of mean (median) ± standard deviation
(SD). P–value less than 0.05 was considered statistically signicant
(TABLE I, TABLE II, FIG. 1, FIG. 2)
FIGURE 3. Histopathological ndings of Listerial encephalitis a) Control. Medulla oblongata, HE, bar: 100 µm. b) Thickened meninges and severe lymphocyte
inltration (arrows). Medulla oblongata, HE, bar: 100µm. c) Perivascular inamatory cell inltratration (arrows). Medulla oblongata, HE, bar: 100µm. d) Severe
inammation, inammatory exudate, and neutrophils (arrows) and macrophage inltration (arrow heads). Medulla oblongata, HE, bar: 25µm
Pathogenesis of Listerial Encephalitis / Karayigit et al. ______________________________________________________________________________
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The reason for the differences in neuronal and glial cells undergoing
apoptosis may depend on the relationship between the physiological
activities of the cells and neuropathology. The difference between
MMP–2 and MMP–9 expression may depend on the diversity of signaling
molecules and the number of cells expressing these gelatinous cells
in the affected area.
Histopathology Results
At histopathological examination, edema was detected in the
Virchow–Robin spaces and hyperemia was seen in the vessels in
the central nervous system. Common findings associated with
L.monocytogenes infection were found in the cerebellum and
brain stem especially in the pons and medulla oblongata. It was
observed that the lesions associated with the disease varied from
mild to severe leptomeningitis. Neurodegenerative reaction and
liquefaction necrosis were detected in all cases especially in the
brain stem. In addition, meningitis and perivascular inammatory cell
inltration including lymphocytes, plasma cells and macrophages,
mostly neutrophil leukocytes were observed in all cases (FIG. 3).
This inammatory cell inltration was mostly localized in the pons,
medulla oblongata and cerebellum. Multiple microabscesses, which
are the typical histopathological nding of the disease, were seen in
the same regions (FIG. 3).
Immunohistochemistry results
Immunopositive reactions for MMP–2 were detected in the
cytoplasm of neurons and glial cells (FIG. 4). Additionally, a strong
cytoplasmic immunopositive reaction of MMP–2 was observed in
perivascularly inltrated leukocytes (FIG. 4). However, weak MMP–2
expressions were seen in control brains (P<0.001) (FIG. 4).
MMP–9 immunoreactivity was detected in the cytoplasm of glial
cells, neurons, perivascularly inltrated leukocytes and endothelial
cells (FIG. 5). It was seen that the immunoreaction was very
strong in the pons and medulla oblongata. On the other hand, a
weak immunoreactivity was observed in the control preparations
(P<0.001) (FIG. 5).
Listeriosis is a seasonal disease in ruminants. Farm management
practices, animal health and hygiene, feed quality and storage play
an important role in the emergence of the disease. One of the most
common pathological ndings of Listeriosis is encephalitis. It is
FIGURE 4. Immunohistochemical staining for MMP–2 antibody of brain with Listerial encephalitis. a) Weak MMP–2 expression in control tissue. Medulla
oblongata, IHC, bar: 25 µm. b) Strong expression of MMP–2 in neurons (arrows) and glial cells (arrow heads). Medulla oblongata, IHC, bar: 100 µm. c) Strong
expression of MMP–2 in glial cells (arrows). Medulla oblongata, IHC, bar: 25 µm. d) MMP–2 positive perivascular leukocytes (arrows) and neuron (arrow head).
Medulla oblongata, IHC, bar: 25 µm
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assumed that disease agents reach the brain via the bloodstream or
via the branches of the trigeminal nerve ending in the oral cavity, nasal
cavity or conjunctiva [21, 22, 23]. Therefore, pathological examination
and immunohistochemical analysis in the central nervous system will
contribute to the diagnosis and treatment of the disease. Best of our
knowledge no reports of immunohistochemical expression of MMP–2
and MMP–9 and associated neuronal apoptosis in brain tissues of
sheep with naturally occurring Listerial encephalitis. In this context,
this is the rst immunohistochemical study to report the expression
of MMP–2 and MMP–9 and associated neuronal apoptosis in brain
tissues of sheep with naturally occurring Listerial encephalitis.
Cortical neuron and astrocyte cultures show that in pathological
conditions, astrocytes and neurons may express more MMP–2 and
MMP–9. [24]. Zeng et al. (2018) reported that the release of MMP–2
and MMP–9 from the brain increased during ischemia–perfusion in
rats and at the same time, apoptosis was occurred in neurons [25].
There are also reports that MMP–2 and MMP–9 expressions play an
important role in the destruction of the blood brain barrier [26]. Studies
have emphasized that various neurotropic disease agents develop
strategies to cross the blood–brain barrier. These strategies include,
crossing the blood brain barrier directly, crossing the blood brain barrier
via paracellular, and crossing the blood brain barrier with infected
leukocytes [27, 28] (Trojan horse mechanism). In the study, the presence
of leukocytes immunohistochemically positively labeled with listeria in
the inammatory area may be related to these strategies. In addition,
it was determined that macrophages also play an important role in the
penetration of the agents through the blood–brain barrier. It is observed
that in Listeriosis infection perivascular cellular inltration in the
vessels may cause ischemia and therefore lead to neurodegeneration.
The most interesting nding in this study was the strong MMP–2 and
MMP–9 expressions in neurons, glial and endothelial cells in the brains
of sheep with Listeriosis. It has been suggested that increased MMP–2
and MMP–9 expressions may contribute to neurodegeneration and
neuropathology in Listeriosis through extracellular matrix degradation
and associated endothelial cell damage.
Immunopositivity reactions of L. monocytogenes antigens were
detected in the cytoplasm of microglia, neurons and perivascular
inmamatory cells (FIG. 6). It was determined that TUNEL positive
cells mainly consisted of neurons and microglia especially in the
caudex cerebri (FIG. 6).
TUNEL positive areas were seen especially in the regions of
microabscess, perivascular and meningeal inammatory reaction
areas in Listeriosis. Weak TUNEL reaction was detected in the control
brain tissues (P<0.001) (FIG. 6).
FIGURE 5. Immunohistochemical staining for MMP–9 antibody of brain with Listerial encephalitis a) MMP–9 immunostaining in healty control tissue. Medulla
oblongata, IHC, bar: 25µm. b) Strong MMP–9 expression in neurons (arrows). Medulla oblongata, IHC, bar: 100µm. c) Strong MMP–9 expression in neurons
(arrows). Medulla oblongata, IHC, bar: 25µm. d) MMP–9 immunolabelling in perivascular leukocytes (arrows) and endothelial cells (arrow heads), Medulla
oblongata, IHC, bar: 25µm. e) Strong MMP–9 expression in glial cells (arrows). Medulla oblongata, IHC, bar: 25µm. f) MMP–9 immunolabelling in leukocytes
(arrows) and endothelial cells (arrow heads) and glial cells (thick arrows). Medulla oblongata, IHC, bar: 25µm
Pathogenesis of Listerial Encephalitis / Karayigit et al. ______________________________________________________________________________
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Previous studies have reported that neurological symptoms occur
in neuronal and glial damage or central nervous system.infections
[29, 30]. It has also been stated that infections cause apoptosis of
neural stem and progenitor cells, thus impairing neurogenesis [31].
Additionally, bacteria cause apoptosis in neural and immune system
cells in the central nervous system [32]. MMP–9 contributes to caspase–
mediated cytotoxicity that promotes apoptosis, nally leading to
brain cell death in focal and global ischemia [33]. Apoptosis has a
complex pathogenesis that occurs as a result of the interaction of
many factors. Disruption of blood brain barrier function may reverse
many physiological conditions in the brain and lead to consequences
that directly affect the microenvironment. One of these is that cells
FIGURE 6. Listeria monocytogenes and TUNEL immunostaining of brain with Listerial encephalitis. a) Control,, Medulla oblongata, IHC, bar: 25 µm. b)
Immunostaining of L. monocytogenes antigens in perivascular inamatory cells (arrows) and glial cells (arrow head), Medulla oblongata, IHC, bar: 25 µm. c)
TUNEL positive neurons (arrows) and glial cells (arrow head) of healty control animals, Medulla oblongata, IHC, bar: 25 µm. d) Strong TUNEL positive neurons
(arrows) and glial cells (arrow head), Medulla oblongata, IHC. Scale bar 25 µm
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are exposed to oxidative stress. Increased gelatinous MMPs causes
ionic homeostasis and energy deciency after decreased cerebral
blood ow [13, 34]. As a result, increased oxidative stress, apoptosis,
irreversible tissue/organ damage, and neurological and behavioral
disorders appear to occur [14]. There are former reports that show
oxidative stress triggers apoptosis in the pathogenesis of Listeriosis [5,
7]. The data obtained from this study showed that increased gelatinous
MMPs expressions in Listeriosis may be one of the rst steps in a chain
of neuropathological mechanisms that trigger each other.
CONCLUSION
In the presented study, MMP 2 and MMP 9 expressions were found
to be high in brain damage caused by encephalitic Listeriosis. The
damage in the brain cells was demonstrated pathologically by TUNEL
staining. There were also differences in expression between animals
in general. However, almost all of them showed higher expression
than controls. This difference may depending on the individual
sensitivity of the animals and the severity of infection. However,
statistically signicant results were obtained when compared to
controls. As a result of all neuronal changes, it was thought that
MMP–2 and MMP–9 were especially effective in neurovascular cells
and would shed light on the pathogenesis of L. monocytogenes
infection. In addition, using this information, it was concluded that
the prevention of MMP–2 and MMP–9 activation, which play a role in the
pathophysiology of neurodegenerative diseases such as Listeriosis,
that is, MMP–2 and MMP–9 inhibitors may be useful in the treatment.
It has been suggested that apoptosis occurring in the cells of the
central nervous system in L. monocytogenes infection may help the
diagnosis in terms of determining the level in the pathogenesis and
severity of the disease.
ACKNOWLEDGEMENTS
We thank to Biostatistics laboratory of Cukurova University for
providing us with the opportunity for our laboratory analyses.
Conict of Interest
The authors declare no conict of interest.
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