Shiga toxin 2 from enterohemorrhagic Escherichia coli induces reactive glial cells and neurovascular disarrangements including edema and lipid peroxidation in the murine brain hippocampus
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Shiga toxin 2 from enterohemorrhagic Escherichia coli is the etiologic agent of bloody diarrhea, hemolytic uremic syndrome and derived encephalopathies that may result to death in patients. Being a Gram negative bacterium, lipopolysaccharide is also released. Particularly, the hippocampus has been found affected in patients intoxicated with Shiga toxin 2. In the current work, the deleterious effects of Shiga toxin 2 and lipopolysaccharide are investigated in detail in hippocampal cells for the first time in a translational murine model, providing conclusive evidences on how these toxins may damage in the observed clinic cases.
Male NIH mice (25 g) were injected intravenously with saline solution, lipopolysaccharide, Shiga toxin 2 or a combination of Shiga toxin 2 with lipopolysaccharide. Brain water content assay was made to determine brain edema. Another set of animals were intracardially perfused with a fixative solution and their brains were subjected to immunofluorescence with lectins to determine the microvasculature profile, and anti-GFAP, anti-NeuN, anti-MBP and anti-Iba1 to study reactive astrocytes, neuronal damage, myelin dysarrangements and microglial state respectively. Finally, the Thiobarbituric Acid Reactive Substances Assay was made to determine lipid peroxidation. In all assays, statistical significance was performed using the One-way analysis of variance followed by Bonferroni post hoc test.
Systemic sublethal administration of Shiga toxin 2 increased the expressions of astrocytic GFAP and microglial Iba1, and decreased the expressions of endothelial glycocalyx, NeuN neurons from CA1 pyramidal layer and oligodendrocytic MBP myelin sheath from the fimbria of the hippocampus. In addition, increased interstitial fluids and Thiobarbituric Acid Reactive Substances-derived lipid peroxidation were also found. The observed outcomes were enhanced when sublethal administration of Shiga toxin 2 was co-administered together with lipopolysaccharide.
Systemic sublethal administration of Shiga toxin 2 produced a deterioration of the cells that integrate the vascular unit displaying astrocytic and microglial reactive profiles, while edema and lipid peroxidation were also observed. The contribution of lipopolysaccharide to pathogenicity caused by Shiga toxin 2 resulted to enhance the observed hippocampal damage.
KeywordsEncephalopathy HUS Inflammation Lipid peroxidation Hippocampus
Central Nervous System
Enterohemorrhagic Escherichia coli
Glial Fibrillary Acidic Protein
Hemolytic Uremic Syndrome
Ionized Calcium-Binding Adapter Molecule 1
Integral Optical Density
Myelin Basic Protein
Neuronal Nuclear Antigen
Shiga toxin-producing Escherichia coli
Shiga toxin 2
Thiobarbituric Acid Reactive Substances
Hemolytic uremic syndrome (HUS) is an illness characterized by a triad of events that include nonimmune haemolytic anaemia, thrombocytopenia and acute renal failure caused by Shiga toxin 2 (Stx2) from enterohemorrhagic Escherichia coli (EHEC). Patients may initially develop bloody diarrhea when EHEC succeeds to colonize the gastrointestinal tract. Once Stx2 reaches the circulation it may target endothelial, kidney and/or brain cells through the Stx2 globotriaosylceramide receptor (Gb3) causing cytotoxicity . Neurological impairment frequently occurs and is associated with a worse prognostic . Besides Stx2 pathogenicity, lipopolysaccharide (LPS) is another virulence factor that is also released form EHEC , for being a Gram negative bacteria. The action of LPS has been described as an enhancer of the deleterious effects of Stx2 in different cells  and organs such as the brain [4, 5].
Particularly in Argentina, post diarrheal HUS is endemic and possesses the highest rate of pediatric cases in the planet. 400 cases are reported annually with an incidence of 10 to 17 cases per 100,000 children under 5 years old, and among them lethality was reported between 1 and 4% . In addition, a high percent of the pediatric patients with HUS develop central nervous system (CNS) dysfunctions . As the grade of severity in HUS cases is usually associated with neurological dysfunctions, the mortality rate rises significantly between 2 to 3-folds when the CNS is involved . Moreover, it has been reported that about 9 to 15% showed neurological symptoms before the onset of HUS .
Although reports on cognitive dysfunctions have been reported in HUS patients, research in brain cognitive areas such as the hippocampus has been scarcely described. As mentioned, a case of cognitive dysfunctions in HUS patients occurred during a large outbreak of diarrhea-associated HUS in Germany 2011 . The responsible was an unusual enteroaggregative Shiga toxin-producing Escherichia coli (STEC) O104:H4. 2987 adults were registered with gastrointestinal infections. 22% of them underwent HUS, while more than a half of them acquired neurologic alterations and about 58% of those with neurologic involvement suffered from cognitive dysfunction, like trouble finding words, severe alteration of consciousness or late memory decline . In addition, other reports of Stx2-derived encephalopathy observed alteration of memory and consciousness, seizures and coma . As known, the hippocampus is involved in cognitive functions including memory formation . Functionally, the hippocampus is one of the brain areas found vulnerable to the deleterious actions of Stx2 . Neurons from the hippocampal CA1 region are mainly involved in memory tasks. Among them long-term potentiation and spatial learning is essentially controlled by these neurons . As Stx2 caused memory alterations including orientation deficits in patients , and as we demonstrated the presence of the Stx2-Gb3 cell receptor in neurons from the CA1 layer , we prompted us to study particularly the cellular events that occurred in such and surroundings layers of the hippocampus. In the current work, the deleterious effects of Stx2 and LPS are investigated in detail in hippocampal cells for the first time, providing conclusive evidences on how these toxins may damage in the observed clinic cases.
NIH male Swiss mice of approximately 25 g (30 days, housed under 12 h-light/12 h-dark cycles) were divided into 4 different groups according to their intravenous (i.v.) treatment: control (saline solution), LPS (800 ng, E. coli 055:B5, Sigma, St. Louis, MO, USA), Stx2 (0.5 ng or 1 ng, Phoenix Laboratory, Tufts Medical Center, Boston, MA, USA) and Stx2 + LPS (1 ng and 800 ng ± respectively). The total amount of i.v. solution injected was 100 μl per mouse and the Stx2 dose was about 60% of the LD50 (1.6 ng per mice). Food and water were provided ad libitum and the experimental protocols and euthanasia procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the School of Medicine of Universidad de Buenos Aires, Argentina (Resolution N° 046/2017). All the procedures were performed in accordance with the EEC guidelines for care and use of experimental animals (EEC Council 86/609).
Brain water content assay
The dry/wet weight method was applied to the brain of mice divided in groups according to their i.v. treatments as described above. The mice (n = 10) were sacrificed at 6, 12, 24 or 48 h post-i.v. treatment. The entire brain was used for this assay and they were weighted on a precision balance of 10 μg accuracy (Acculab ALC-110.4, Brooklyn, NY, USA) to obtain their wet weight. Then, they were dried in an incubator at 56 °C for 6 days to obtain their dry weight. The water content was determined from the difference between the wet weight and dry weight, according to Testylier et al. (2007) .
Histo and immunofluorescence assay
Mice (n = 4 for each treatment) were anesthetized with pentobarbital (100 mg/kg) and intracardiacally perfused with paraformaldehyde 4% diluted in phosphate buffer saline (PBS) 0.1 M, pH 7.4; at 2, 4, 7 and 20 days. Day 0 was set as the day of the i.v. treatment. The brains were removed from the skulls and post-fixed overnight at 4 °C with the same described fixative solution, then daily cryopreserved with increasing concentrations of sucrose diluted in PBS (10, 20 and 30%). Brain sections of 20 μm were cut in a cryostat. The brains slices were storage at − 20 °C in a cryopreservant solution (50% PBS, 30% ethylene glycol and 20% glycerol) until the day of histo and/or immunofluorescence assay.
The histofluorescence assay to detect the glycocalyx microvasculature was made with 10 μg/ml of biotinylated lectin from Lycopersicon esculentum (Sigma, St. Louis, MO, USA). Brain slices were first washed several times with PBS 10 mM to be incubated overnight with lectin at 4 °C. The slices were then washed several times with PBS 10 mM and incubated overnight with streptavidine Alexa Fluor 488 (Invitrogen Molecular Probes, Carlsbad, California, USA) at 4 °C followed by several washed in PBS to be mounted on slides for epifluorescence (Olympus BX50, Miami, Fl, USA) and confocal microscope (Olympus FV1000, Miami, Fl, USA) studies.
For the immunofluorescence assay, after several rinses with PBS 10 mM, brain slices were incubated with 10% fetal goat serum blocking solution in PBS 10 mM (Sigma, St. Louis, MO, USA) and 1% Triton X-100 (Sigma, St. Louis, MO, USA) for 1 h. The sections were immediately incubated with the following primary antibody (with Triton X-100 at 0,3%): rabbit anti-GFAP (1:500 - Dako, 225 Glostrup, Denmark), mouse anti-NeuN (1:250 - Millipore, Temecula, CA, USA), rabbit anti-MBP (1:500 - Dako, Glostrup, Denmark), goat anti-Iba1 (1:250 - Millipore, Temecula, Ca, USA) and rat anti-Gb3 (CD77, 1:250 - Serotec, Kidington, UK) overnight at 4 °C to identify astrocytes, neurons, oligodendrocytes, microglial cells and the Stx2 Gb3 receptor respectively. Once the sections were rinsed several times with PBS 10 mM they were incubated with their respective secondary antibodies (with Triton X-100 at 0,3%): goat anti-rabbit Alexa Fluor 555 (1:500 - Invitrogen Molecular Probes, Carlsbad, California, USA), goat anti-mouse Alexa Fluor 555 (1:500 - Amersham, GE, Piscataway, NJ, USA), donkey anti-goat Alexa Fluor 555 (1:500 - Millipore, Temecula, Ca, USA) and goat anti-rat FITC (Jackson Immuno Research, West Grove, PA, USA) overnight at 4 °C. Finally, all hippocampal brain slices were incubated with Hoechst 33342 (1:500 - Sigma, St. Louis, MO, USA) for 15 min at room temperature to show the nuclei of brain cells. Negative controls were made by omitting the primary antibody. The hippocampal CA1 area was observed with an Olympus BX50 epifluorescence microscope provided with a Cool-Snap digital camera and Olympus FV1000 confocal. Micrographs were obtained from CA1 hippocampal field, (− 1.70 and − 1.82 mm from bregma). Analysis of lectin histofluorescence, GFAP and Iba1 immunofluorescences were made in hippocampal CA1 stratum radiatum (Rad), while immunofluorescence to neuronal NeuN was analyzed in the CA1 pyramidal layer (Py). Finally, immunofluorescence to MBP was observed in the hippocampal fimbria (fi). The obtained micrographs were analyzed with the Fiji ImageJ software (NIH, MD, USA). GFAP and Iba1 expression levels were measured as the integral optical density (IOD) per cell, while histofluorescence to lectin and MBP expression levels were measured as total IOD of each micrograph.
For this purpose, all images were opened on Fiji ImageJ, the color channels were splitted and the one with the specific color was selected. In order to select only the immunopositive cells, the threshold tool was employed and the IOD was measured with the Analyze>Measure tool, and the “mean” was selected. The criteria to determine endothelial damage on lectin images were the number of lectin positive particles and the area occupied by the microvessels. For this purpose, the images were calibrated with a pre-set scale bar, the straight tool was employed to set a line of the same size of the scale bar and using the set measurements the length of the line was calibrated. After that, the color channels were splitted, the green color was selected and the same steps to analyze the IOD were done, with the difference that in this case, the measurements of “area” and “count” were selected. Finally, the measurement in 3 different places of the Py was made to determine neuronal damage in the Py layer. For this purpose, the scale bar was set as it was described before, and the thickness of the pyramidal layer was measured using the straight tool.
Thiobarbituric acid reactive substances assay
The malondialdehyde (MDA) content in hippocampus (n = 4) was evaluated as thiobarbituric acid reactive substances (TBARS) by a fluorometric assay with modifications at 12 h and 24 h following toxin treatments. Butylhydroxytoluene (4% w/v in ethanol) was used to prevent non-physiological TBARS formation during the sample processing . Results are expressed as pmoles of MDA per mg of protein. The MDA standard was prepared from 1,1,3,3,-tetramethoxypropane.
The data are presented as mean ± SEM. In all assays, the statistical significance were performed using the One-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (GraphPad Prism 4, GraphPad Software Inc.) between the 4 i.v. treatments (control, LPS, Stx2 and Stx2 + LPS). The criterion for significance was p < 0.05 for all the experiments.
Water content of murine brains was increased following systemic administration of a sublethal dose of Stx2
Stx2 increased the expression levels of glial fibrilar acidic protein (GFAP) in the murine brain hippocampus in a dose dependent manner
GFAP is a cytoskeletal protein used as a specific marker to identify astrocytes in a reactive state because of a wide variety of brain injuries . Two different sublethal doses of Stx2 were intravenously (i.v.) administered to determine whether deleterious effects of the toxin in brain cells may occur in a dose dependent manner (0.5 and 1 ng) and the day 4 was chosen to observe these effects. In our murine model, sublethal administration of Stx2 maximally induced reactive astrocytes and other events in the motor cortex and striatum at day 4 [5, 17]. At this context, the day 4 was initially chosen in order to establish the amount of Stx2 to be used in this work, and latter, a time curve was designed in order to determine the time Stx2 maximally induce reactive astrocytes but particularly in the murine hippocampal area CA1.
Systemic administration of Stx2 produced reactive astrocytes and the combination of this toxin with LPS resulted to enhance this effect
Stx2 decreased the area occupied by lectin-bound to glycocalyx microvessels and the co-administration with LPS enhanced this effect
The expression of neuronal NeuN was decreased in hippocampal CA1 pyramidal layer (Py) following the administration of Stx2
The expression of oligodendrocytic MBP myelin protein was decreased following Stx2 administration
Stx2 produced microglial reactivity and co-administration of Stx2 with LPS increased this event
Lipid peroxidation was determined following Stx2 and Stx2 together with LPS administration
The present results show for the first time the deleterious effects of systemic sub-lethal Stx2 in the hippocampus of murine brains at the cell level. Increased water content in the brain was determined 12 h until 48 h following Stx2 treatment. One possibility that may explain the observed increased in water content is the reported decrease in the expression of aquaporins involved in fluid elimination from the brain and the increased permeability of the BBB leading to brain edema . Stx2 provoked astrocyte reactivity in a dose dependent manner peaking at 48 h. This might have aggravated the observed BBB disarrangement. According with this observation, Stx2 and co-treatment of Stx2 with LPS led to the alteration of the endothelium evidenced by discontinuity of the endothelial glycocalyx which compromised the BBB integrity. As intact glycocalyx is necessary for the maintenance of normal vascular function  its discontinuity was reflected as a decrease in the expression of the glycocalyx . This disorder (increased BBB permeability) induced by Stx2 was already demonstrated by our group in the murine brain and cerebellum by using the Evans Blue staining [5, 28]. BBB alteration seemed to impact on CA1 hippocampal neuronal status; as the expression of NeuN was reduced, the thickness of the pyramidal layer was decreased in comparison to controls. Two possibilities may explain the decreased of the thickness in this layer as a consequence of toxin treatments. One possibility is the loss of fluid content in neurons and consequently the reduction of neuronal somatic size, while this was totally reversed after 20 days following the toxins treatments. Neuronal loss of fluid content has been previously reported by us in the murine striatum following the same characterized murine model of HUS-encephalopathy . Alternatively, a temporal inhibition in the expressions of NeuN and MBP might have occurred, which recovered after 20 days . These events could occur in those STEC-HUS patients that resolved acute symptoms from neurological disorders . To confirm this, further experiments would be conducted that escapes the current aim of the present work. In accordance to this, the observed lipid peroxidation may contribute to the reduction in the expression of neuronal NeuN from pyramidal CA1 layer. The assessment of lipid peroxidation has been determined in a model of renal failure following intravenous administration of Stx2 . Then, it was not surprised to detect lipid peroxidation in the hippocampus which may lead to cell death.
Stx2 may target neurons through Gb3 receptor. The presence of this receptor was previously immunolocalized in neurons, but not in astrocytes [14, 32]. Then the possibility that Stx2 may exert a direct neurodegenerative action through Gb3 is highly probable. How Stx2 may target astrocytes then? It was found that Stx2 releases high amounts of glutamate from neurons . This event generates excitotoxicity promoving astrocyte reactivity, an event by which an inflammatory process occurs . Then it could be inferred from the previous description that Stx2 influences astrocytes indirectly. Alternatively, Stx2 may target directly astrocytes not through Gb3 but by the presence of the TLR4 receptor. It has been reported that Stx2 activates neutrophils and release cytokines when it binds the TLR4 receptor . Then, astrocytes may respond in a similar way as neutrophils respond to Stx2.
Microglial reactivity was observed 2 days after toxin treatments. These cells might have contributed to the observed deleterious effects in the CA1 layer. Microglial activation possesses phagocytic properties, but produces and releases detrimental pro-inflammatory cytokines . In addition, reduction of oligodendrocityc MBP may have obeyed as a consequence of the observed astrocytic and microglial activations, and/or neuronal disarrangements. A previous work showed that incubation of Stx2 in cultured oligodendrocytes had no harming effects ; however, oligodendrocytes were damaged in a brain context. Then, communication between neurons and oligodendrocytes appears to be essential for healthy myelin . It has been reported that when oligodendrocytes extend its processes they are able to myelinate axons, keep axonal integrity, support axonal metabolism and neuronal survival . On the other hand, microglial cells may damage oligodendrocytes and/or the myelin sheath as they express pro-inflammatory cytokines such as TNFα and/or IL-1β [38, 39]. In addition, they produce ROS and NO radicals, both important sources of oxidative damage observed during the pathogenesis of demyelinating diseases . Lipid peroxidation may occur in oligodendrocytes. In summary, Stx2 reaches the brain affecting the BBB. Edema and lipid oxidation were earlier events. Microglial and/or astrocyte cells could be potential local inducers of the observed lipid damage. According to this, these cells were maximally activated at 2 days following toxin administration which coincided with the observed neuronal alteration (as the thickness of the CA1 pyramidal hippocampal layer decreased). A late event was observed in oligodendrocytic myelin sheath reduction that occurred 4 days following toxins administrations. Further studies should be carried out in order to determine possible mechanisms of cell plasticity observed at 20 days.
Stx2 damaged the neurovascular unit of the hippocampal area CA1, producing interstitial edema and a decrease in the profile of endothelial-glycocalyx expression that correlated with the disruption of the BBB. Additionally, Stx2 produced astrocytic and microglial cell reactivity and lipid peroxidation. Neurons and oligodendrocytic myelin sheath were found maximally damaged at day 2 and day 4 respectively following Stx2 administration. Co-administration of LPS enhanced the deleterious effect of Stx2 in the hippocampal area CA1. Then, LPS should be taken in consideration in HUS-derived encephalopathy models. The detrimental effects of these toxins were reversed at day 20. The present work could significantly shed light in the understanding of cell mechanisms that lead to hippocampal damage following sub-lethal administration of Stx2 and LPS. This knowledge could impact in the search for alternative treatments in the hippocampus of patients intoxicated with STEC strains.
We wish to thank Dr. Victoria Rosato-Siri for her invaluable advice and support in the preparation of this work.
This work was supported by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (PICT-2016-1175) and Universidad de Buenos Aires (UBACyT) (20020160100135BA), Argentina.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Conceived of the experiment: JG, CB, AP. Conducted the experiment: CB, AP, VC, DA, AC, PG. Analyzed the data: CB, VC, AP, DA, AC, PG. Wrote the manuscript: AP, CB, PE, JG. All authors read and approved the final manuscript.
Experimental protocols and euthanasia procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the School of Medicine of Universidad de Buenos Aires, Argentina (Resolution N° 046/2017). All the procedures were performed in accordance with the EEC guidelines for care and use of experimental animals (EEC Council 86/609).
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