ADAMTS-13 and HMGB1-induced oxidative stress in Taenia multiceps-infected animals

This study investigated the cytotoxic effects of oxidative stress (OS), high mobility group box 1 (HMGB1), ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs), and neuropathology associated with coenurus cerebralis (Taenia multiceps). ADAMTS-13, HMGB1, glutathione reductase (GR), copper/zinc superoxide dismutase (Cu/Zn SOD), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression levels were studied. The study found that ADAMTS-13 (P < 0.005), HMGB1 (P < 0.005), GR (P < 0.005), Cu/Zn SOD (P < 0.005), and 8-OHdG (P < 0.005) levels were significantly higher in T. multiceps (c. cerebralis)-infected animals compared to healthy control animals. This study's most important finding was that HMGB1 up-regulation in neurons, endothelial cells, and glial cells can directly cause brain parenchymal destruction and that HMGB1-mediated oxidative stress plays a crucial role in the neuropathogenesis of coenurosis. The results also showed that increased levels of ADAMTS-13 may play a pivotal role in regulating and protecting the blood–brain barrier integrity and neuroprotection. These findings also suggest that ADAMTS-13 and HMGB1 compete in the prevention or formation of microthrombi, which was regarded as a remarkable finding. ADAMTS-13 and HMGB1 are valuable biomarkers for disease risk assessment, estimating host neuropathy following T. multiceps (c. cerebralis) exposure, and providing a new therapeutic target. This is the first study to show that HMGB1 and ADAMTS-13 are expressed in reactive cells and are associated with neuroimmunopathology in coenurosis.


Clinicopathologic findings
It was found that cyst volumes and clinicopathological findings in T. multiceps (c.cerebralis)-infected lambs and sheep had a very close relationship.The clinicopathological results became more severe as cyst volumes and numbers increased.The most common neurological symptoms observed in T. multiceps (c.cerebralis)-infected animals were lateral deviation of the head and circling motion around its axis, paralysis, incoordination, and ataxia.Other findings included nystagmus, torticollis, a lack of direct light reflex in the pupil, seizures, and coma.

Gross findings
Macroscopically, white-colored protoscolex bundles ranging from 23 to 164 were found within the thinwalled, transparent, and clear fluid-filled cysts (Fig. 1B-D).The collected cysts were observed to be 2.4 × 3.3 cm-5.8 × 7.6 cm in diameter and varied in size from a ping-pong ball to a baseball (Fig. 1C,B).Sometimes, a sizeable unilateral cyst was found; in others, two to three small or large cysts were observed in different locations (Table 1).In the evaluation made according to the cyst locations in the brain, there was a more widespread distribution predominantly in the right or left cerebral hemispheres.Cysts were found in the cerebellum in only three cases (16.6%) and in the thalamus in one case (5.5%).When the cyst locations in the cerebral hemispheres were examined, it was seen that the cysts were in the right or left parietal, frontal, temporal and occipital lobes (Table 1).Because of the pressure atrophy caused by the cysts occupying the brain parenchyma (Fig. 1A-C), it was observed that the cyst area became thinner and even the skull's inner surface softened in all cases.

Histopathologic findings
Hematoxylin and eosin-stained brain sections from healthy control animals exhibited standard architecture.Significant histopathological lesions were observed in the brain tissues of all animals.Histopathological examination revealed many multinucleated giant cells surrounding the cyst walls and lymphocytic reactions surrounded by inflammatory cells (Fig. 2A-D).Severe hyperemia and edema were detected in the meninges of T. multiceps (c.cerebralis)-infected animals.In addition, demyelination, liquefaction necrosis around cerebral cysts, degeneration of neurons, focal pressure atrophy, perivascular cell infiltrates, thrombus and fibrinopurulent meningoencephalitis were noted as the most important histopathological findings (Fig. 2A-D).

ADAMTS-13 and HMGB1 expressions
Expressions of ADAMTS-13 and HMGB1 in all brain regions of all animals in both T. multiceps (c.cerebralis)infected and healthy control animals were carefully measured.It was noted that ADAMTS-13 and HMGB1 expressions were increased, and the difference was statistically significant and relatively high when this increase was compared with the healthy control animals and interpreted quantitatively (P < 0.005) (Fig. 3 and Table 2).We detected weak ADAMTS-13 expressions in healthy control animals, glial cells and neurons (Fig. 4A).Contrary to these findings, ADAMTS-13 expressions in brain tissues of T. multiceps (c.cerebralis)-infected animals were significantly increased, especially in neurons (Fig. 4B-F).In addition, partial ADAMTS-13 immunoreactivities were detected in glial cells (Fig. 4C).Weak or deficient HMGB1 expressions were observed in neurons, endothelial and glial cells in healthy control animals (Fig. 5A).However, it was determined that the intensity of HMGB1 expression in the brain tissues of T. multiceps (c.cerebralis)-infected animals were higher than in the healthy control animals (Table 2).Pathological levels of HMGB1 expressions were found in glial and endothelial cells, especially in necrotic and healthy neurons (Fig. 5B-D).The most striking finding of this study is that the expressions of ADAMTS-13 and HMGB1 were significantly increased in the lesioned areas and the areas close to the cyst.The most striking finding of this study is that the expressions of ADAMTS-13 and HMGB1 are significantly increased in the lesioned areas and the areas close to the cyst.T. multiceps (c.cerebralis)-infected animal brains www.nature.com/scientificreports/showed enhanced ADAMTS-13 and HMGB1 levels and prolonged OS, which may contribute to neurotoxicity and parenchymal degeneration.These conditions handle severe neuropathology, such as demyelination and thrombus and permeability of the BBB.

Glutathione reductase expressions
In healthy control animals, some glial cells and neurons exhibited relatively weak or no immunoreactivity for GR (Fig. 6A).GR expression increased significantly in the vascular endothelial, glial cells, and necrotic/ healthy neurons (Fig. 6B-D), which was also substantially higher than the levels in the healthy control animals (P < 0.005).We detected that an important anti-oxidant marker, GR, was up-regulated considerably in T. multiceps (c.cerebralis)-infected animals (Fig. 3 and Table 3).

Cu/Zn SOD expressions
Moderate Cu/Zn SOD expressions were observed in neuronal and glial cells in healthy control animals (Fig. 7A).
Another conspicuous finding of the present study was that Cu/Zn SOD expressions were markedly increased in the endothelia, glial cells and microglia/macrophages (Fig. 7B-D), which was also significantly higher than the levels in the healthy control animals (P < 0.005).We detected that a very important anti-oxidant marker, Cu/Zn SOD, was significantly up-regulated in T. multiceps (c.cerebralis)-infected animals (Fig. 3 and Table 3).

8-OHdG expressions
In healthy control animals, some glial/neuronal cells showed weak immunoreactivity to 8-OHdG (Fig. 8A).8-OHdG expression was observed in the nucleus and in the cytoplasm of neurons, endothelial cells, glial cells, and microglia/macrophages (Fig. 8B-D), being more intense in the cytoplasm.8-OHdG expressions measurements in T. multiceps (c.cerebralis)-infected animals were significantly higher than in healthy control animals (P < 0.005) (Fig. 3 and Table 3).The most conspicuous finding of the present study was that 8-OHdG expression markedly increased in cyst walls.

Discussion
Coenurosis is a disease caused by coenurus cerebralis, frequently seen in small ruminants, caused by the larval stage of Taenia multiceps.Its zoonotic character poses a severe public health problem that cannot be ignored 1,2 .There are minimal studies on the neuroimmunopathogenesis of coenurosis, a significant parasitic disease.For this reason, the findings obtained in this study are significant in elucidating the neuroimmunopathogenesis of the disease.The study's most striking first finding, a significant increase in HMGB1 expression and a HMGB1mediated OS, has already been discussed.The second significant finding is the presence of ADAMTS-13, which is highly expressed in neurons and glial cells.It has thus been clearly demonstrated that the BBB's permeability increases, and its integrity is disrupted, contributing to the development of neuropathological findings.The third finding is that HMGB1 and ADAMTS-13 compete to form and prevent microthrombi, one of the most essential histopathological findings (Fig. 9).There is a link between oxidative stress and HMGB1 expression 41 .OS has been shown to regulate HMGB1 translocation, release, and activity significantly 41,42 .It has been found that HMGB1 functions as a critical regulator in OS-related infection, sterile inflammation, necrosis, and apoptosis 42 .In modeling studies of neurodegeneration in which HMGB1 inhibition is performed, it has been revealed that OS and neuronal cell death are significantly reduced and that it prevents neuropathology that may occur against BBB damage 20,43,44 .These studies show that neuropathology occurs in neurodegenerative diseases because of HMGB1-mediated OS.As the most important finding of this study, we showed that HMGB1 expressed in both necrotic and healthy neurons and glial cells at pathological levels are associated with OS.It is thought that a serious increase in HMGB1 expressions is among the causes of severe mitochondrial DNA (mtDNA) damage because of OS.In short, it has been clearly demonstrated that the most crucial trigger of OS-mediated neuropathology in coenurosis is HMGB1.The significantly increased levels of HMGB1 and HMGB1-associated OS shown in this study are thought to underlie the neuropathology seen in coenurosis.The development of therapeutic agents specifically targeting HMGB1 or its receptors may help to reduce the inflammatory response and OS associated with coenurosis.Inhibition of    HMGB1 release or blockade of downstream signalling pathways may contribute significantly to the reduction of neuroinflammation and related pathological processes.The blood-brain barrier is a physical and metabolic barrier that regulates and protects the microenvironment in the central nervous system (CNS) and separates the CNS from the peripheral circulation 45 .Considering the histopathological findings, it is seen that the permeability of the BBB increases in coenurosis.For this reason, the brain parenchyma and cells become sensitive due to the increase in BBB permeability during infection and play a critical role in triggering and exacerbating inflammation.For this reason, the brain parenchyma and cells become sensitive due to the increase in BBB permeability during infection and play a critical role in triggering and exacerbating inflammation.This study emphasises two reasons for the increase in BBB permeability.1) The first one is BBB damage occurs due to HMGB1 expression at the pathological level; 2) HMGB1-mediated OS increases BBB permeability and damages BBB integrity.Because it has been shown that increased HMGB1 expressions cause damage to brain endothelium and pericytes, disrupting BBB integrity 19,46 .Similarly, it has been reported that OS increases BBB permeability and that BBB damage caused by OS causes neurotoxicity, exacerbating neuropathology 37,38 .Another striking finding of this study was that ADAMTS-13 expression was significantly increased, particularly in neurons.
Our previous studies demonstrated that ADAMTS-13 is expressed at severe levels in endothelial cells, glial cells and neurons in Pestivirus-related neuropathology 25 , listeria encephalitis 31 , diabetic encephalopathy 32 and toxoplasmic encephalitis models 24 .Thus, ADAMTS-13 indicates BBB damage severity and increased BBB permeability.Similarly, we found that the severity of the existing damage can be followed up with ADAMTS-13, and it gives essential ideas for interpreting the severity in neuropathology.In addition, we revealed that ADAMTS-13 has protective effects on the BBB.We demonstrated in this study that the significantly increased ADAMTS-13 expression in the brain tissues of sheep with coenurosis has the neuroprotective effects described above and previously revealed, protects the brain from BBB damage, and provides information about the severity of the disease.In light of the studies, the role of ADAMTS-13 in investigating Coenurus cerebralis-related neuroimmunopathogenesis is significant.This and our previous studies suggest that ADAMTS-13 may play a role in modulating vascular integrity, inflammation or coagulation processes that may affect BBB permeability.Furthermore, examination of the relationship between ADAMTS-13 and BBB damage may help to elucidate the interactions between the cells that make up the BBB and its associates.
Coenurus cerebralis has been shown histopathologically to cause demyelination in the brain [47][48][49] .In this study, we also found demyelination in 5 sheep.Although demyelination is a significant neuropathology, no studies have been conducted to investigate the pathogenesis of demyelination in this disease.We previously investigated the role of ADAMTS-13 in preventing hydranencephaly, cerebellar hypoplasia, and, in particular, hypomyelinogenesis in Pestivirus-infected sheep.Furthermore, we discussed in the same study that it protects against the formation of myelin damage.The increase in ADAMTS-13 expression observed in this study may also play a role in demyelination prevention.It is obvious that when ADAMTS-13 expression levels are high enough, myelin damage will stop.Although demyelination is seen, the fact that it was not found in all sheep with coenurosis brains supports our theory.
The roles of ADAMTS-13 in thrombotic activity are significant.It has been revealed to prevent further deterioration of disease prognosis by cleaving ultra-large von Willebrand factor multimers into multimers with lower activity to reduce potential thrombotic activity and prevent microvascular thrombosis 50,51 .Mice with subarachnoid hemorrhage have shown statistically significant reductions in microthrombus areas and attenuated brain tissue damage after treatment with recombinant ADAMTS-13 27 .In contrast to ADAMTS-13, HMGB1 has been shown in an animal model of deep vein thrombosis to induce pro-thrombotic neutrophil extracellular trap formation and in vivo/in vitro platelet aggregation [52][53][54] .These studies show that increased HMGB1 activations at pathological levels may expose patients to a potential stroke.Thrombosis in coenurosis has also been linked to severe neuropathology 55,56 .This indicates that disturbances in thrombotic activity in coenurosis play an essential role in neuropathogenesis.At this time, it is believed that ADAMTS-13 and HMGB1 have a competitive interaction.Demyelination would not be interpreted this way if it were considered a universal neurohistopathological finding in all animals.However, the absence of demyelination in animals with coenurosis leads us to believe that ADAMTS-13 and HMGB1 compete as winners and losers.With HMGB1 promoting thrombus formation and ADAMTS-13 counteracting it, it is quite clear that an imbalance in favour of HMGB1 could lead to increased microthrombus formation and potentially contribute to tissue damage in coenurosis-related neuropathology.This demonstrated important finding suggests that targeting HMGB1 to reduce its pro-inflammatory and prothrombotic effects or increasing ADAMTS-13 activity to reduce microthrombus formation could be considered as potential interventions to limit disease progression.
In conclusion, this study revealed previously unknown neuroimmunopathogenesis of neuropathology in sheep with coenurosis.Damage to the BBB and increased permeability are visible.It is seen that HMGB1 alone and HMGB1-mediated OS are responsible for the damage and increased permeability of this BBB.In addition, the presence of mtDNA damage caused by OS was demonstrated by 8-OHdG expressions.In addition, it is thought that demyelination, an important neuropathological finding, is prevented with ADAMTS-13 expression.In addition, it was seen as a remarkable finding that ADAMTS-13 and HMGB1 play a competitive role in the prevention or formation of microthrombi.
Understanding the mechanisms underlying neuronal injury and inflammation in coenurosis provides important information for the development of neuroprotective interventions.With the findings obtained in this study, strategies that reduce neuronal plasticity and oxidative stress or promote tissue repair mechanisms can be investigated as potential therapeutic avenues.For example, targeting these factors, such as specific parasite proteins or antigens, may be among the first targets to reduce disease severity and promote recovery.

Ethics statement
Aksaray and Atatürk University's Animal Experiments Ethics Committee did not require approval.Because this study did not include any invasive procedures for animal experiments.The study samples comprised 18 dead lambs and sheep, ranging in age from 8 to 36 months.These animals were brought to Aksaray and Atatürk University, Faculty of Veterinary Medicine, Department of Pathology for routine necropsy.No animals studied were sacrificed for this study, and all procedures were performed with the permission of the animal owners.

Figure 2 .
Figure 2. Histopathological findings of T. multiceps (c.cerebralis)-infected animals.(A) Non-purulent meningoencephalitis, areas of necrosis around the cyst membrane and numerous foreign body giant cells (arrow) against the cyst wall.(B) Foreign body giant cells (arrow) against the cyst wall and inflammatory zone area (mononuclear cell infiltration) (asterisk).(C) Numerous foreign body giant cells (arrow) and extensive areas of necrosis (arrowhead) (D) Extensive areas of necrosis (arrowhead), numerous foreign body giant cells (arrow) against the cyst wall and large inflammatory zone area (mononuclear cell infiltration) (asterisk).

Table 2 .
Immunoperoxidase test results and statistical data for healthy control and T. multiceps (c.cerebralis)infected animals.

Table 3 .
Immunoperoxidase test results and statistical data for healthy control and T. multiceps (c.cerebralis)infected animals.