Skip to main content
PMC home page
PLOS ONE logoLink to PLOS ONE
. 2020 Dec 3;15(12):e0242989. doi: 10.1371/journal.pone.0242989

Neurotoxicity of different amyloid beta subspecies in mice and their interaction with isoflurane anaesthesia

Laura Borgstedt 1, Manfred Blobner 1,2, Maximilian Musiol 1, Sebastian Bratke 1, Finn Syryca 1, Gerhard Rammes 1, Bettina Jungwirth 2, Sebastian Schmid 1,2,*
Editor: Stephen D Ginsberg3
PMCID: PMC7714346  PMID: 33270674

Abstract

Background

The aim of this study was to assess different amyloid beta subspecies’ effects on behaviour and cognition in mice and their interaction with isoflurane anaesthesia.

Methods

After governmental approval, cannulas were implanted in the lateral cerebral ventricle. After 14 days the mice were randomly intracerebroventricularly injected with Aβ 1–40 (Aβ40), Aβ 1–42 (Aβ42), 3NTyr10-Aβ (Aβ nitro), AβpE3-42 (Aβ pyro), or phosphate buffered saline. Four days after the injection, 30 mice (6 animals per subgroup) underwent general anaesthesia with isoflurane. A “sham” anaesthetic procedure was performed in another 30 mice (6 animals per subgroup, 10 subgroups in total). During the next eight consecutive days a blinded assessor evaluated behavioural and cognitive performance using the modified hole-board test. Following the testing we investigated 2 brains per subgroup for insoluble amyloid deposits using methoxy staining. We used western blotting in 4 brains per subgroup for analysis of tumour-necrosis factor alpha, caspase 3, glutamate receptors NR2B, and mGlu5. Data were analysed using general linear modelling and analysis of variance.

Results

Aβ pyro improved overall cognitive performance (p = 0.038). This cognitive improvement was reversed by isoflurane anaesthesia (p = 0.007), presumably mediated by decreased exploratory behaviour (p = 0.022 and p = 0.037). Injection of Aβ42 was associated with increased anxiety (p = 0.079). Explorative analysis on a limited number of brains did not reveal insoluble amyloid deposits or differences in the expression of tumour-necrosis factor alpha, NR2B, mGlu5, or caspase 3.

Conclusions

Testing cognitive performance after intracerebroventricular injection of different amyloid beta subspecies revealed that Aβ pyro might be less harmful, which was reversed by isoflurane anaesthesia. There is minor evidence for Aβ42-mediated neurotoxicity. Preliminary molecular analysis of biomarkers did not clarify pathophysiological mechanisms.

1. Introduction

The accumulation of amyloid beta (Aβ) in the brain is one of the key factors in the pathophysiology of Alzheimer’s disease (AD) [1]. Aβ is generated via processing of amyloid precursor protein (APP) in the amyloidogenic pathway. APP can be cleaved in various positions and several post-translational modifications have been identified resulting in different subspecies [2]. In the brains of people living with AD Aβ 1–40 (Aβ40) is most prevalent. However, the ability to accumulate and form oligomers is elevated in Aβ 1–42 (Aβ42) resulting in increased neurotoxicity [3]. Therefore, these two subspecies of Aβ have been investigated thoroughly [4–10]. Other modifications of Aβ by nitration or pyroglutamylation have been described [11, 12]. Aβ in AD patients contains 10–15% of pyroglutamated amyloid beta 1–42 (AβpE3-42, Abeta pyro) and it represents a dominant fraction of Aβ peptides in senile plaques of AD brains [13]. Abeta nitro (3NTyr10-Aβ) is a nitrotyrosinated (or nitrated) form of amyloid beta 1–42 [12] and is found in the cores of amyloid plaques in AD brains [14]. Their impact on neurobehavioural outcome parameters needs further evaluation [13, 14]. As mice and humans show similarities concerning behaviour, memory and learning [15–18] we chose this species in order to ultimately find the best anaesthetic regimen for people living with AD.

An increasing number of aged patients requires surgery and anaesthesia [19–22]. Consequently, more people living with AD undergo general anaesthesia [23]. It is controversially discussed whether or not anaesthesia can trigger or worsen AD in aged patients [24]. However, an interaction between anaesthetics and Aβ has been shown in various studies [4, 25–30]. Some studies suggest a possible link between anesthesia and AD in humans [31, 32], while more recent ones do not [33, 34]. Also, as stated by Lee et al. it is nearly impossible to discriminate the influence of general anesthesia from the effect of surgery itself on the development of AD [35].

The aim of this investigation was to further elucidate the effects of different Aβ subspecies on cognition in mice and their interaction with anaesthetics. Since there is no mouse model displaying pathology derived from post-translationally modified Aβ proteins, we decided to use the method of intracerebroventricular injection (ICV). Previous experiments have demonstrated that intracerebroventricular injection of Aβ oligomers leads to cognitive deficits [36], although this animal model is restricted to amyloidopathy. We assessed cognitive and behavioural function after ICV injection using the modified hole-board test (mHBT).

To further investigate the interaction between anaesthetics and different Aβ subspecies, mice were exposed to isoflurane anaesthesia with a minimal alveolar concentration (MAC) of 1.0. Isoflurane is one of the most extensively studied anaesthetic agents in animal research. It has been shown to induce caspase activation and increase levels of beta-site APP-cleaving enzyme (BACE) in vivo in C57/BL6 mice [27]. Furthermore, isoflurane leads to increased oligomerization of amyloid beta in vitro and therefore might interact with the different Aβ subspecies.

Since accumulation of amyloid beta leads to neuroinflammation, apoptosis and disruption of the glutamatergic system, we analysed the brain tissue for TNF alpha, caspase 3, NR2B, and mGlu5 as a secondary objective [37]. We looked for potential molecular mechanisms mediating cognitive and behavioural impairment and the interaction between isoflurane and Aβ.

2. Methods

This study was carried out in strict accordance with the recommendations of the Federation of European Laboratory Animal Science Associations (FELASA). The following experimental procedures on animals were approved by the Governmental Animal Care Committee (Regierung von Oberbayern, Maximilianstr. 39, 80538 Munich, Germany, Chair: Dr. B. Wirrer, Registration number: 55.2-1-54-2532-111-12, November 27th, 2012). All surgical procedures were performed under isoflurane anaesthesia and all efforts were made to minimize suffering. Animal welfare was assessed daily.

2.1 Surgical procedure: Implantation of intracerebroventricular cannula

60 male 10-week-old C57BL/6N mice (median weight 26.7 g) obtained from Charles River Laboratories (Sulzfeld, Germany) were housed under standard laboratory conditions (specific pathogen free environment, 12 h light/12 h dark cycle, 22°C room temperature, 60% humidity and free access to water and standard mouse chow) 14 days prior to the experiments for acclimatisation.

For induction of general anaesthesia mice were placed in an acrylic glass chamber that had been pre-flushed with 4.0 Vol% isoflurane and 50% of oxygen. After loss of postural reflexes mice were placed on a warming pad (rectal temperature was measured and maintained at 37.5°C) and the stereotactic frame was mounted. General anaesthesia was maintained with 1.6 Vol% Isoflurane (MAC 1.0) and a fraction of inspired oxygen of 50% (FiO2 0.5) administered via a nose chamber. Mice breathed spontaneously during surgery. The skin was shaved, disinfected and after local anaesthesia with 0.5 ml xylocaine 2% a midline incision was performed to expose the bone. Using a computer controlled motorized stereotactic instrument (TSE Systems, Bad Homburg vor der Hoehe, Germany) the insertion point of the cannula (1 mm lateral and 0.3 mm caudal of Bregma) was determined and a small hole (0.8 mm) was drilled. The cannula was placed with a depth of 3 mm using the stereotactic instrument. For further stabilisation a small screw was placed in the scalp and the cannula was cemented to the scalp and the screw. Wound closure was achieved using single stitches and 0.05 mg/kg of buprenorphine were injected intraperitoneally for pain treatment. The mice were then placed in the acrylic glass chamber with 50% oxygen, now without isoflurane, and were monitored until full recovery from anaesthesia. Afterwards the mice were placed in single cages.

2.2 Randomization and blinding

After successful implantation of the intracerebroventricular cannula the mice were randomly assigned to one of ten groups (n = 6 mice per experimental group) regarding Aβ subspecies or PBS and isoflurane anaesthesia or sham procedure using a computer-generated randomization list. The experimental groups were designed as follows: Aβ40/sham (n = 6 mice), Aβ40/isoflurane (n = 6 mice), Aβ42/sham (n = 6 mice), Aβ42/isoflurane (n = 6 mice), Aβ nitro/sham (n = 6 mice), Aβ nitro/isoflurane (n = 6 mice), Aβ pyro/sham (n = 6 mice), Aβ pyro/isoflurane (n = 6 mice), PBS/sham (n = 6 mice), PBS/isoflurane (n = 6 mice). The outcome assessor conducting the mHBT and the personnel performing the analysis of biomarkers were blinded to the group assignment.

2.3 Injection of amyloid beta

On day 14 after implantation of the cannula the mice were injected with 5 μl of either Aβ42, Aβ40, 3NTyr10-Aβ (Aβ nitro), AβpE3-42 (Aβ pyro), or PBS through the cannula. For this procedure a Hamilton® syringe connected to a plastic tube and a smaller cannula that was inserted into the ICV cannula were used.

Aβ42 (American Peptide Sunnyvale, CA, USA) was suspended in 100% HFIP (Sigma Aldrich, St. Louis, Missouri, United States) to 1 mg/ml and shaken at 37°C for 1.5 h. This solution was aliquoted to 5–50 μg portions and then HFIP was removed by evaporation for 30 minutes using a vacuum concentrator (Thermo Scientific Savant SpeedVac, Thermo Fisher Scientific, Waltham, Massachusetts, United States of America). When completely dry, the peptide aliquots were stored at -20°C. Before injection aliquoted monomeric Aβ42 was warmed in a water bath at 37°C for 10 minutes, then sonicated for 30 s, dissolved in NaOH (20 mmol/l, pH 12.2) and diluted in PBS (1:100) to start the oligomerization process and sonicated for another 30 s, mixed for 30 s, sonicated for 30 s and mixed again for 30 s before being placed on ice. The Aβ solution was used between 15 and 45 minutes after its preparation. It was brought to room temperature before use by loading it into the cannula 10 minutes before administration. Aβ42 concentration of the injected solution (5.0 μl) was 700 nmol/l resulting in a concentration of 100 nmol/l in the cerebrospinal fluid of the mouse. Aβ40 (American Peptide Sunnyvale, CA, USA), Aβ nitro (provided by Clinical Neuroscience Unit, Department of Neurology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany), and Aβ pyro (Bachem AG Bubendorf, Switzerland) were dissolved in PBS to reach concentrations of 3200, 700, and 11900 nmol/l in the 5 μl boli that were used for injection, respectively. This resulted in concentrations of 450, 100, and 1700 nmol/l of the corresponding substance in the cerebrospinal fluid of the mouse. The concentrations were chosen in order to reach equipotential concentrations in the cerebrospinal fluid of these four Aβ substances according to their effect on long term potentiation, excitatory postsynaptic potential, and spine density in vitro derived from other experiments [38].

2.4 Isoflurane anaesthesia

On day 4 after injection of the different Aβ-substances a 2-hour isoflurane anaesthesia was performed in 30 mice. The other 30 mice underwent a sham procedure (total n = 60 mice). After induction as described in 2.1 the mouse was placed with its nose in a nose-chamber and breathed spontaneously with a PEEP of 5 mbar. Temperature was monitored using a rectal probe and maintained at 37.5°C using a heating mat. A subcutaneous electrocardiogram was placed. Heart rate and impedance respiratory rate were monitored. Isoflurane concentration was maintained at 1.6 Vol% corresponding to a MAC of 1.0. Every 15 minutes the depth of anaesthesia was verified with a tail clamp that was kept in place for 1 minute [39]. The “sham” anaesthetic procedure included handling of the animals and placement in the induction box for 10 minutes without exposure to isoflurane.

2.5 Cognitive and behavioural testing

Starting on day 5 after the injection mice were tested for cognitive function, behaviour, and social interaction using the mHBT. This test is a combination of a classical hole-board with an open field test, according to an established protocol [40–42]. With this test, a total of 16 different parameters can be observed simultaneously. For the mHBT the hole-board is placed in the middle of the test arena. Ten cylinders are staggered in two lines on the board. Each cylinder contains a small piece of almond fixed underneath a grid that cannot be removed by the animals (S1 Fig). In addition, each cylinder is flavoured with vanilla to attract the animals’ attention. Three of the 10 cylinders are baited with a second–approachable–piece of almond and marked with white tape. The sequence of marked holes is changed according to a protocol every day. We performed testing for 8 consecutive days from day 5 until day 12 after the intracerebroventricular injection. Each mouse underwent four trials per day (300 s/trial).

We evaluated two different parameters regarding cognitive performance: Firstly, if mice visited non-baited holes or did not visit baited holes it was summed up as wrong choice total. A higher total number of errors can be interpreted as an impaired reference memory. Secondly, the total time needed to finish the task (time trial) was recorded as a marker for the overall cognitive performance. An extended duration represents a pathological finding. If an animal did not finish the task, i.e. did not find all three pieces of almond within 300 s, the trial was abrupted. Two parameters focussed on anxiety: the latency with which the mice first visited the area of interest (grey board with cylinders, S1 Fig) and the time spent on this board. An increased latency and reduced time on board represent avoidance behaviour and, therefore a higher level of anxiety. Arousal was evaluated by the total time mice spent grooming during the trial. The number of line crossings in the test arena served as an indicator for locomotor activity. The number of visits to a baited hole was counted as correct hole visits, with higher values being associated with increased direct exploratory motivation.

2.6 Sampling of brain and blood

On day 13 after ICV injection mice were deeply anaesthetized and brains were harvested by decapitation. The samples were stored at -80°C. Each brain was separated into hemicortices. One hemicortex was sliced into sagittal slides of 50 μm. The other one was separated into prefrontal motor cortex, sensory cortex and hippocampus.

2.7 Amyloid deposits

To detect amyloid deposits, a total of 20 (2 brains of each subgroup) brains were investigated. 50 μm thick sagittal brain slices (n = 21 per brain) including sensory cortex and hippocampus were fixed on microscope slides in −20°C acetone for 20 min. The staining protocol has been described previously [43–45]. After drying at room temperature, each slice was washed twice with wash solution (PBS/Ethanol denaturised with MEK in 1:1 ratio) for 10 minutes. Then methoxy-X04 solution (10 mg methoxy-X04 powder (Tocris, Bioscience) diluted in 100 μl Dimethylsulfoxid, mixed with 450 μl of 1,2-Propandiol, 450 μl of PBS, and 50 μl 1 N NaOH; 800 μl of this stock was diluted with 200 ml of a 1:1-PBS/ethanol solution) was applied to the slices on a shaker in the dark for 30 minutes. To remove the unbonded methoxy-X04, brain slices were washed three times with wash solution and twice with distilled water for 10 minutes per step. In a final step, brain slices were preserved in fluorescence mounting medium (DAKO, Santa Clara, California, USA). Methoxy-X04 has a high binding affinity for amyloid deposits. The stained brain slices were imaged by magnification using fluorescence microscopy in tile scan mode (ZEISS Axio Imager, ApoTome.2 and Zen 2012 Blue Software, Oberkochen, Germany).

2.8 Analysis of tumor necrosis factor (TNF) alpha, caspase 3, N-methyl-D-aspartate-receptor subunit 2B (NR2B), and metabotropic glutamate receptor 5 (mGlu5)

Sensory cortex and hippocampus of four animals per group (total n = 40) were suspended separately in grinding tubes (Sample Grinding Kit, GE Healthcare, Munich, Germany) and extraction-solution was added (1ml: 970μl Ripa Buffer; 20μl 50xComplete; 10μl 100xPhenylmethylsulfonylfluorid; 1μl Pepstation). After centrifugation the supernatant was stored at -80°C. The protein-concentration (by Bradford Assay) was standardized with Laemmli buffer (1.4ml, 4x times: 1ml NuPage LDS Sample Buffer (Invitro-gen NP0007); 400μl NuPage Sample Reducing Agent (Invitrogen NP0009)). The samples were transferred onto the gel (TGX Stain-Free™ FastCast™ Acrylamide Kit 10%; Bio-Rad Laboratories GmbH, Munich, Germany) in equal amounts (20μl) and equal protein-concentrations (1μg/μl) per lane for separation by gel electrophoresis and blotted onto a membrane (Amersham Hybond Low Fluorescence 0.2 μm Polyvinylidenfluorid-Membrane; TH Geyer, GmbH, Munich, Germany). The membrane was blocked for one hour and incubated afterwards with the first antibody (“TNF-alpha” ProSci XP-5284 1:1000, “Caspase 3” Cell Signaling #9662 1:1000, “NR2B” Cell Signaling #4207 1:1000, or “mGlu5” Abcam ab53090 1:1000) overnight at 4°C. After washing it with TBS/T (1l: 1l dH2O; 3g Tris, 11.1g NaCl; 1ml Tween 20) the membrane was incubated with the second antibody (“Anti-rabbit IgG” Cell Signaling #7076 1:10 000) for one hour. Following another washing step, the membrane was incubated in 1ml Clarity™ Western ECL Substrate (Bio-Rad Laboratories GmbH, Munich, Germany) for 1 minute. The labelled proteins were detected with camera imaging (Bio-Rad Molecular Imager® ChemiDocTM XRS+; Bio-Rad Laboratories GmbH, Munich, Germany). For analysis and normalisation ImageLab® was used in addition to the Stain-Free® Technology to assess the total protein amount (Bio-Rad Laboratories GmbH, Munich, Germany). A standard lane was included in every blot.

2.9 Statistical analysis

Neurocognitive and behavioural parameters were analysed using general linear models (GLM) comparing each substance (Aβ40, Aβ42, Aβ nitro or Aβ pyro) to PBS: We analysed the between-group factors subspecies for injection, anaesthesia (isoflurane or sham) and the within-group factor time and their interaction terms. Effects of time were analysed in a linear fashion due to the strictly monotonic decreasing character of learning curves in these tests. For determination of the effect size we calculated mean differences with 95% confidence interval and partial eta-squared with 90% confidence interval.

Regarding sample-size calculations, variables of the mHBT are considered relevant if two groups differ two times the given standard deviation. Based on a type I error of 0.05, a type II error of 0.20 and two-sided t-tests at the final test level of the hierarchical model 4 animals per group would have been appropriate. Our internal standard, however, suggests a minimal group size of six, which we used in our experiment.

In addition, we performed explorative studies on a limited number of brains on amyloid deposits and different biomarkers in order to detect possible mechanisms of interaction. Since distribution of the protein-concentrations of TNF alpha, caspase 3, NR2B and mGlu5 in the western-blot analysis were positively skewed, the statistical analyses were performed following logarithmic transformation. Western-blot results were analysed using analysis of variance (ANOVA) comparing each substance to PBS with the additional factors anaesthesia and the interaction term. The significance level was set at p < 0.05. Calculations were done with SPSS Statistics® (Version 24.0; IBM; New York; United States).

3. Results

3.1 Cognitive and behavioural testing

In mice injected with Aβ pyro the time required to complete the test, i.e. time trial, was decreased (Aβ pyro compared to PBS: mean difference (95% confidence interval): -41 s (-80 to -2 s); partial eta-squared (90% confidence interval): 0.198 (0.006 to 0.413); p = 0.038; Fig 1A). General anaesthesia with isoflurane led to an increase in time trial in those mice compared to control (Aβ pyro isoflurane vs. Sham: 55 s (17 to 94 s); 0.307 (0.055 to 0.508) p = 0.007; Fig 1A). Reference memory function, represented by the total number of wrong choices, was comparable between the different Aβ substances and PBS (Fig 1B).

Fig 1. Neurocognitive function after anaesthesia in mice injected with different Aβ subspecies compared to control.

Fig 1

Open in a new tab

A: Time Trial (overall cognitive performance), B: Wrong choices total (declarative memory); mean of all tests on two days and standard error (whiskers).

The time on board the exposed part of the test arena increased over time in mice injected with Aβ40, Aβ nitro, and Aβ pyro (partial eta-squared (90% confidence-interval): 0.340 (0.063 to 0.540), p = 0.007 (Aβ40); 0.226 (0.013 to 0.483), p = 0.029 (Aβ nitro); 0.320 (0.057 to 0.521), p = 0.008 (pyro); Fig 2A), but not in mice injected with Aβ42 (0.180 (0.000 to 0.416), p = 0.079; Fig 2A). Regarding the latency the mice first entered the board, another parameter for anxiety, there was no difference between the different Aβ substances and PBS (Fig 2B). In animals injected with Aβ pyro an isoflurane anaesthesia decreased the time on board (mean difference (95% confidence interval): -12 s (-22 to -2 s); partial eta-squared (90% confidence interval): 0.245 (0.021 to 0.460), p = 0.022; Fig 2A) and increased the latency until the animals first visited the board (33 s (2 to 65 s); 0.200 (0.007 to 0.416), p = 0.037; Fig 2B).

Fig 2. Anxiety-related behavioural changes after anaesthesia in mice injected with different Aβ subspecies compared to control.

Fig 2

Open in a new tab

A: Time on Board, B: Latency First Board Entry (both anxiety); mean of all tests on two days and standard error (whiskers).

Locomotor activity (line crossings) and direct exploratory motivation (correct hole visits), representing further behavioural parameters, did not differ between groups (Fig 3A and 3B).

Fig 3. Behavioural parameters of mice injected with different Aβ subspecies after anaesthesia compared to control.

Fig 3

Open in a new tab

A: Correct Hole Visits (exploratory motivation), B: Line Crossings (locomotor activity); mean of all tests on two days and standard error (whiskers).

3.2 Amyloid deposits

There were no insoluble amyloid deposits present on day 13 after the intracerebroventricular injection of the different subspecies in both groups, with and without isoflurane exposure.

3.3 Analysis of TNF alpha, caspase 3, NR2B, and mGlu5

There was no difference in protein concentrations of TNF alpha, caspase 3, NR2B, and mGlu5 in sensory cortex or hippocampus between the different subspecies compared to PBS. Isoflurane anaesthesia did not have an effect on these protein concentrations (Fig 4A–4D).

Fig 4. Total protein amount in sensory cortex and hippocampus.

Fig 4

Open in a new tab

A: Tumor necrosis factor alpha (TNF alpha), B: Caspase 3, C: N-methyl-D-aspartate receptor subunit 2B (NR2B); D: Metabotropic glutamate receptor 5 (mGlu5); median (horizontal lines), interquartile range (box) and range (whiskers).

4. Discussion

Modified hole-board testing revealed that, shortly after ICV injection, Aβ pyro may be less harmful, as it is associated with an enhancement of overall cognitive performance. This improvement was reversed by isoflurane anaesthesia, as the interaction between isoflurane and Aβ pyro led to decreased exploratory behaviour. The mice spent less time on board and took longer before entering the board or finishing the trial. Aβ42 led to increased anxiety. This might be explained by an elevated toxicity compared to the other Aβ subspecies, since Aβ42 is thought to be more pathogenic as it forms toxic oligomers more readily than other Aβ subspecies [46, 47]. We were not able to detect insoluble amyloid deposits in a limited number of brains. The preliminary analysis of biomarkers for apoptosis, inflammation, and the glutamate receptor subunits NR2B and mGlu5 did not reveal correlations to the different Aβ subspecies or isoflurane anaesthesia.

To further investigate the pathophysiology of AD and potential therapeutic options, different mouse models have been developed in the last years. Several transgenic mice are available, showing cognitive and behavioural impairment comparable to the pathological changes in human AD patients [48]. Whereas first animal models focussed on amyloidopathy, more recent transgenic mouse models also address aspects of tau causality [49]. In contrast, the ICV injection as a well-accepted method to simulate AD-like pathology is also restricted to amyloidopathy. Our data show that after a short period of five days after the injection of different Aβ subspecies in the lateral ventricle of mice there are only minor changes to cognitive or behavioural parameters. Our model was not able to display more complex neurological changes of AD like memory loss or learning impairment.

The main objective of our study was to elucidate the neurotoxicity of different Aβ subspecies and their interaction with isoflurane in vivo. Presently there is no AD mouse model available which represents an Aβ derived pathology including post-translationally modified Aβ proteins. Therefore, we decided to use the intracerebroventricular injection model being aware of its focus on amyloidopathy. In previous experiments we were able to show a cognitive impairment when testing of the mice began on day 2, 4, or 8 after intracerebroventricular injection [36]. Therefore, we performed isoflurane anaesthesia or a sham procedure on day 4 after the intracerebroventricular injection and started testing the following day. We expected a maximum interaction between anaesthesia and Aβ during this timepoint of maximum cognitive impairment. However, modified hole-board testing only showed minor alterations in cognitive performance. Mice injected with Aβ pyro showed an improved overall cognitive performance probably mediated by a decrease in anxiety over time. This finding of a supposedly reduced harmful effect on cognitive performance of Aβ pyro contrasts with other studies on the one hand. For Aβ pyro an increased potential to aggregate has been shown and therefore, it is considered as an Aβ subspecies with high neurotoxicity [38, 50]. In 2012, Nussbaum et al. published a potential mechanism by which Aβ pyro could trigger AD pathogenesis [51]. Their study showed a higher toxicity of Aβ pyro in wildtype (WT) mice neurons in vitro by co-oligomerization with excess Aβ42 as well as neuron loss and gliosis in WT mice at the age of 3 months in a tau-dependent manner. We examined the effect of Aβ pyro in 10-week-old mice without excess Aβ42 and in a tau null background which might explain the improvement in overall cognitive performance. On the other hand, we might have observed a neuroprotective effect of Aβ pyro. Emerging evidence suggests that Aβ might work in a neuroprotective way as an antioxidant, metal chelator, or by increasing synaptic plasticity, preventing excitotoxicity and stimulating learning and memory [52].

Furthermore, in our study we investigated the effect on cognitive performance after a very short period after ICV injection. At this early timepoint the increased aggregation-potential might not have led to clinically relevant oligomers, which are responsible for neurotoxicity [53]. Even on day 13 after injection we were not able to detect insoluble amyloid deposits.

In the mice injected with Aβ pyro, an exposure to isoflurane results in increased behavioural signs of anxiety. This impairs the improved overall cognitive performance and reduces it to baseline. A possible explanation might be an enhanced oligomerisation given the fact that an isoflurane anaesthesia enhances oligomerization and cytotoxicity of Aβ in vitro and has a negative effect on cognitive performance and mortality in vivo [4, 54]. A potential mechanism of interaction might lie in the ability of aggregated Aβ pyro to form membrane pores and the fact that Aβ pyro and isoflurane are both hydrophobic agents [55–57]. Several other studies report both favourable and non-favourable interactions between isoflurane and especially Aβ40 and Aβ42 [58–60]. We were not able to confirm these findings in our experiments using a non-transgenic mouse model.

Besides Aβ pyro, Aβ42 is also considered as one of the most neurotoxic subspecies with a high potential for aggregation [61]. Negative effects on cognitive function and behavioural parameters have been shown in several investigations [36, 62] but also dose-dependent effects have been reported. In 2017, Lazarevic et al. showed that 200 pM of Aβ40 and Aβ42 had a stimulating effect on neuronal synapses whereas 1 μM of Aβ40 and Aβ42 had a decreasing effect on active synapses [63]. In our study, increased anxiety in mice injected with Aβ42 was the only measurable effect of this Aβ subspecies. We did not see any effects on cognitive function, which contrasts with our former results, where we saw negative effects even very shortly after ICV injection [36]. This might be explained by an interference of increased anxiety with the cognitive testing in the mHBT [40]. A limitation regarding these findings is the fact, that in contrast to the other Aβ subspecies Aβ42 was dissolved in NaOH. Although the concentration was very low, NaOH might have had an additional inhibiting effect on the parameters of the mHBT.

We also did explorative studies on a limited number of brains on amyloidopathy, in order to detect targets for future research concerning the pathomechanism of different Abeta subspecies and the interaction of isoflurane and amyloid beta. As we were not able to detect insoluble amyloid deposits, again, the short time period between injection and testing might not be sufficient for Aβ42 to oligomerize.

As inflammation and apoptosis are considered as main driving forces behind the pathology of AD we examined the brains for changes in TNF alpha and caspase 3 as representative biomarkers [37]. Besides, alterations in the glutamatergic system like deficiencies in synaptic NR2B subunit phosphorylation and an accumulation and over-stabilization of mGlu5 are also considered as factors promoting the development of AD and might have been changed in our animals [64–66]. Since the intracerebroventricular injection only had minor effects on cognitive and behavioural parameters, not surprisingly, we did not detect significant changes in these biomarkers. These data should be considered preliminary as the primary endpoint of this study was the cognitive and behavioural outcome.

It might be considered a limitation of our investigation that we analysed biomarkers and amyloid deposits at one time-point after and not consecutively during the modified hole-board testing. However, we did not want to take series of blood samples or perform other analysis procedures which could have influenced neurocognitive testing. We performed the injection in 10-week-old mice, which might be another restriction to our experiments. There might be different interactions between the brain and the Aβ subspecies in older animals, as older brains show a higher amount of free metals as well as a reduction in antioxidative defence [67]. We limited our investigation to the four mentioned subspecies, although several other posttranslational modifications of Aβ have been identified. In addition, the interaction of other anaesthetics like desflurane, which was associated with less impact on Aβ in human cerebrospinal fluid, could be investigated in future research [68].

5. Conclusions

In conclusion, the model of intracerebroventricular injection is not suitable to simulate the complex symptoms of AD. Analysis of different Aβ subspecies revealed that shortly after ICV injection Aβ pyro might be less harmful, which was reversed by an exposure to isoflurane. There is minor evidence for an increased toxicity of Aβ42. Analysis of biomarkers in a limited number of animals did not clarify pathophysiological mechanisms.

Supporting information

S1 Fig. Modified hole-board consisting of test arena and hole-board with cylinders.

(PDF)

Click here for additional data file. (1.3MB, pdf)

Acknowledgments

We are indebted to Andreas Blaschke for performing parts of the analysis procedure.

Data Availability

All files with the original date are available from the mediaTUM database: https://mediatum.ub.tum.de/1579195.

Funding Statement

The authors received no specific funding for this work.

References

  • 1.Querfurth HW, LaFerla FM. Alzheimer's disease. N Engl J Med. 2010;362(4):329–44. 10.1056/NEJMra0909142 [DOI] [PubMed] [Google Scholar]
  • 2.Kummer MP, Heneka MT. Truncated and modified amyloid-beta species. Alzheimer's research & therapy. 2014;6(3):28 10.1186/alzrt258 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Hamley IW. The amyloid beta peptide: a chemist's perspective. Role in Alzheimer's and fibrillization. Chemical reviews. 2012;112(10):5147–92. Epub 2012/07/21. 10.1021/cr3000994 [DOI] [PubMed] [Google Scholar]
  • 4.Eckenhoff RG, Johansson JS, Wei H, Carnini A, Kang B, Wei W, et al. Inhaled anesthetic enhancement of amyloid-beta oligomerization and cytotoxicity. Anesthesiology. 2004;101(3):703–9. Epub 2004/08/27. 10.1097/00000542-200409000-00019 [DOI] [PubMed] [Google Scholar]
  • 5.Kayed R, Jackson GR. Prefilament tau species as potential targets for immunotherapy for Alzheimer disease and related disorders. Current opinion in immunology. 2009;21(3):359–63. Epub 2009/06/02. 10.1016/j.coi.2009.05.001 [DOI] [PubMed] [Google Scholar]
  • 6.Kheterpal I, Chen M, Cook KD, Wetzel R. Structural differences in Abeta amyloid protofibrils and fibrils mapped by hydrogen exchange—mass spectrometry with on-line proteolytic fragmentation. Journal of molecular biology. 2006;361(4):785–95. Epub 2006/08/01. 10.1016/j.jmb.2006.06.066 [DOI] [PubMed] [Google Scholar]
  • 7.Barghorn S, Nimmrich V, Striebinger A, Krantz C, Keller P, Janson B, et al. Globular amyloid beta-peptide oligomer—a homogenous and stable neuropathological protein in Alzheimer's disease. Journal of neurochemistry. 2005;95(3):834–47. Epub 2005/09/02. 10.1111/j.1471-4159.2005.03407.x [DOI] [PubMed] [Google Scholar]
  • 8.Yu L, Edalji R, Harlan JE, Holzman TF, Lopez AP, Labkovsky B, et al. Structural characterization of a soluble amyloid beta-peptide oligomer. Biochemistry. 2009;48(9):1870–7. Epub 2009/02/17. 10.1021/bi802046n [DOI] [PubMed] [Google Scholar]
  • 9.Nimmrich V, Grimm C, Draguhn A, Barghorn S, Lehmann A, Schoemaker H, et al. Amyloid beta oligomers (A beta(1–42) globulomer) suppress spontaneous synaptic activity by inhibition of P/Q-type calcium currents. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2008;28(4):788–97. Epub 2008/01/25. 10.1523/JNEUROSCI.4771-07.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Bernstein SL, Dupuis NF, Lazo ND, Wyttenbach T, Condron MM, Bitan G, et al. Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease. Nature chemistry. 2009;1(4):326–31. Epub 2010/08/13. 10.1038/nchem.247 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Mori H, Takio K, Ogawara M, Selkoe DJ. Mass spectrometry of purified amyloid beta protein in Alzheimer's disease. The Journal of biological chemistry. 1992;267(24):17082–6. Epub 1992/08/25. [PubMed] [Google Scholar]
  • 12.Guix FX, Uribesalgo I, Coma M, Muñoz FJ. The physiology and pathophysiology of nitric oxide in the brain. Progress in neurobiology. 2005;76(2):126–52. Epub 2005/08/24. 10.1016/j.pneurobio.2005.06.001 [DOI] [PubMed] [Google Scholar]
  • 13.Saido TC, Iwatsubo T, Mann DM, Shimada H, Ihara Y, Kawashima S. Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3(pE), in senile plaques. Neuron. 1995;14(2):457–66. Epub 1995/02/01. 10.1016/0896-6273(95)90301-1 [DOI] [PubMed] [Google Scholar]
  • 14.Kummer MP, Hermes M, Delekarte A, Hammerschmidt T, Kumar S, Terwel D, et al. Nitration of tyrosine 10 critically enhances amyloid beta aggregation and plaque formation. Neuron. 2011;71(5):833–44. 10.1016/j.neuron.2011.07.001 [DOI] [PubMed] [Google Scholar]
  • 15.Rosenthal N, Brown S. The mouse ascending: perspectives for human-disease models. Nat Cell Biol. 2007;9(9):993–9. Epub 2007/09/01. 10.1038/ncb437 [DOI] [PubMed] [Google Scholar]
  • 16.Fonio E, Benjamini Y, Sakov A, Golani I. Wild mouse open field behavior is embedded within the multidimensional data space spanned by laboratory inbred strains. Genes, brain, and behavior. 2006;5(5):380–8. Epub 2006/08/02. 10.1111/j.1601-183X.2005.00170.x [DOI] [PubMed] [Google Scholar]
  • 17.Peters LL, Robledo RF, Bult CJ, Churchill GA, Paigen BJ, Svenson KL. The mouse as a model for human biology: a resource guide for complex trait analysis. Nature reviews Genetics. 2007;8(1):58–69. Epub 2006/12/19. 10.1038/nrg2025 [DOI] [PubMed] [Google Scholar]
  • 18.Shultz LD, Ishikawa F, Greiner DL. Humanized mice in translational biomedical research. Nature reviews Immunology. 2007;7(2):118–30. Epub 2007/01/30. 10.1038/nri2017 [DOI] [PubMed] [Google Scholar]
  • 19.Vaupel JW. Biodemography of human ageing. Nature. 2010;464(7288):536–42. Epub 2010/03/26. 10.1038/nature08984 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Liu SS, Della Valle AG, Besculides MC, Gaber LK, Memtsoudis SG. Trends in mortality, complications, and demographics for primary hip arthroplasty in the United States. International orthopaedics. 2009;33(3):643–51. Epub 2008/05/08. 10.1007/s00264-008-0549-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Wang MC, Kreuter W, Wolfla CE, Maiman DJ, Deyo RA. Trends and variations in cervical spine surgery in the United States: Medicare beneficiaries, 1992 to 2005. Spine. 2009;34(9):955–61; discussion 62–3. Epub 2009/04/09. 10.1097/BRS.0b013e31819e2fd5 [DOI] [PubMed] [Google Scholar]
  • 22.Anger JT, Weinberg AE, Albo ME, Smith AL, Kim JH, Rodríguez LV, et al. Trends in surgical management of stress urinary incontinence among female Medicare beneficiaries. Urology. 2009;74(2):283–7. Epub 2009/06/09. 10.1016/j.urology.2009.02.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Quiroga C, Chaparro RE, Karlnoski R, Erasso D, Gordon M, Morgan D, et al. Effects of repetitive exposure to anesthetics and analgesics in the Tg2576 mouse Alzheimer's model. Neurotoxicity research. 2014;26(4):414–21. Epub 2014/06/15. 10.1007/s12640-014-9478-8 [DOI] [PubMed] [Google Scholar]
  • 24.Hussain M, Berger M, Eckenhoff RG, Seitz DP. General anesthetic and the risk of dementia in elderly patients: current insights. Clinical interventions in aging. 2014;9:1619–28. 10.2147/CIA.S49680 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Jiang J, Jiang H. Effect of the inhaled anesthetics isoflurane, sevoflurane and desflurane on the neuropathogenesis of Alzheimer's disease (review). Mol Med Rep. 2015;12(1):3–12. Epub 2015/03/05. 10.3892/mmr.2015.3424 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Xie Z, Dong Y, Maeda U, Alfille P, Culley DJ, Crosby G, et al. The common inhalation anesthetic isoflurane induces apoptosis and increases amyloid beta protein levels. Anesthesiology. 2006;104(5):988–94. Epub 2006/04/29. 10.1097/00000542-200605000-00015 [DOI] [PubMed] [Google Scholar]
  • 27.Xie Z, Culley DJ, Dong Y, Zhang G, Zhang B, Moir RD, et al. The common inhalation anesthetic isoflurane induces caspase activation and increases amyloid beta-protein level in vivo. Annals of neurology. 2008;64(6):618–27. Epub 2008/11/14. 10.1002/ana.21548 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Xu Z, Dong Y, Wu X, Zhang J, McAuliffe S, Pan C, et al. The potential dual effects of anesthetic isoflurane on Aβ-induced apoptosis. Curr Alzheimer Res. 2011;8(7):741–52. Epub 2011/01/20. 10.2174/156720511797633223 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Dong Y, Zhang G, Zhang B, Moir RD, Xia W, Marcantonio ER, et al. The common inhalational anesthetic sevoflurane induces apoptosis and increases beta-amyloid protein levels. Archives of neurology. 2009;66(5):620–31. Epub 2009/05/13. 10.1001/archneurol.2009.48 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Tian Y, Chen KY, Liu LD, Dong YX, Zhao P, Guo SB. Sevoflurane Exacerbates Cognitive Impairment Induced by Aβ (1–40) in Rats through Initiating Neurotoxicity, Neuroinflammation, and Neuronal Apoptosis in Rat Hippocampus. Mediators of inflammation. 2018;2018:3802324 Epub 2018/11/08. 10.1155/2018/3802324 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kuehn BM. Anesthesia-Alzheimer disease link probed. Jama. 2007;297(16):1760 Epub 2007/04/26. 10.1001/jama.297.16.1760 [DOI] [PubMed] [Google Scholar]
  • 32.Culley DJ, Xie Z, Crosby G. General anesthetic-induced neurotoxicity: an emerging problem for the young and old? Current opinion in anaesthesiology. 2007;20(5):408–13. Epub 2007/09/18. 10.1097/ACO.0b013e3282efd18b [DOI] [PubMed] [Google Scholar]
  • 33.Jiang J, Dong Y, Huang W, Bao M. General anesthesia exposure and risk of dementia: a meta-analysis of epidemiological studies. Oncotarget. 2017;8(35):59628–37. Epub 2017/09/25. 10.18632/oncotarget.19524 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Sprung J, Warner DO, Knopman DS, Petersen RC, Mielke MM, Jack CR Jr., et al. Exposure to surgery with general anaesthesia during adult life is not associated with increased brain amyloid deposition in older adults. British journal of anaesthesia. 2020;124(5):594–602. Epub 2020/03/17. 10.1016/j.bja.2020.01.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Lee JJ, Choi GJ, Kang H, Baek CW, Jung YH, Shin HY, et al. Relationship between Surgery under General Anesthesia and the Development of Dementia: A Systematic Review and Meta-Analysis. BioMed research international. 2020;2020:3234013 Epub 2020/04/28. 10.1155/2020/3234013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Schmid S, Jungwirth B, Gehlert V, Blobner M, Schneider G, Kratzer S, et al. Intracerebroventricular injection of beta-amyloid in mice is associated with long-term cognitive impairment in the modified hole-board test. Behav Brain Res. 2017;324:15–20. Epub 2017/02/15. 10.1016/j.bbr.2017.02.007 [DOI] [PubMed] [Google Scholar]
  • 37.Fricker M, Tolkovsky AM, Borutaite V, Coleman M, Brown GC. Neuronal Cell Death. Physiol Rev. 2018;98(2):813–80. Epub 2018/03/01. 10.1152/physrev.00011.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Rammes G, Seeser F, Mattusch K, Zhu K, Haas L, Kummer M, et al. The NMDA receptor antagonist Radiprodil reverses the synaptotoxic effects of different amyloid-beta (Abeta) species on long-term potentiation (LTP). Neuropharmacology. 2018;140:184–92. Epub 2018/07/18. 10.1016/j.neuropharm.2018.07.021 [DOI] [PubMed] [Google Scholar]
  • 39.Deady JE, Koblin DD, Eger EI 2nd, Heavner JE, D'Aoust B. Anesthetic potencies and the unitary theory of narcosis. Anesth Analg. 1981;60(6):380–4. Epub 1981/06/01. [PubMed] [Google Scholar]
  • 40.Ohl F, Holsboer F, Landgraf R. The modified hole board as a differential screen for behavior in rodents. Behav Res Methods Instrum Comput. 2001;33(3):392–7. 10.3758/bf03195393 [DOI] [PubMed] [Google Scholar]
  • 41.Ohl F, Sillaber I, Binder E, Keck ME, Holsboer F. Differential analysis of behavior and diazepam-induced alterations in C57BL/6N and BALB/c mice using the modified hole board test. J Psychiatr Res. 2001;35(3):147–54. 10.1016/s0022-3956(01)00017-6 [DOI] [PubMed] [Google Scholar]
  • 42.Gordan ML, Jungwirth B, Ohl F, Kellermann K, Kochs EF, Blobner M. Evaluation of neurobehavioral deficits following different severities of cerebral ischemia in rats: a comparison between the modified hole board test and the Morris water maze test. Behav Brain Res. 2012;235(1):7–20. 10.1016/j.bbr.2012.07.027 [DOI] [PubMed] [Google Scholar]
  • 43.Burgold S, Bittner T, Dorostkar MM, Kieser D, Fuhrmann M, Mitteregger G, et al. In vivo multiphoton imaging reveals gradual growth of newborn amyloid plaques over weeks. Acta Neuropathol. 2011;121(3):327–35. Epub 2010/12/08. 10.1007/s00401-010-0787-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Klunk WE, Bacskai BJ, Mathis CA, Kajdasz ST, McLellan ME, Frosch MP, et al. Imaging Abeta plaques in living transgenic mice with multiphoton microscopy and methoxy-X04, a systemically administered Congo red derivative. J Neuropathol Exp Neurol. 2002;61(9):797–805. Epub 2002/09/17. 10.1093/jnen/61.9.797 [DOI] [PubMed] [Google Scholar]
  • 45.McCarter JF, Liebscher S, Bachhuber T, Abou-Ajram C, Hubener M, Hyman BT, et al. Clustering of plaques contributes to plaque growth in a mouse model of Alzheimer's disease. Acta Neuropathol. 2013;126(2):179–88. Epub 2013/06/19. 10.1007/s00401-013-1137-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.DeToma AS, Salamekh S, Ramamoorthy A, Lim MH. Misfolded proteins in Alzheimer's disease and type II diabetes. Chemical Society reviews. 2012;41(2):608–21. Epub 2011/08/06. 10.1039/c1cs15112f [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Savelieff MG, Lee S, Liu Y, Lim MH. Untangling amyloid-β, tau, and metals in Alzheimer's disease. ACS chemical biology. 2013;8(5):856–65. Epub 2013/03/20. 10.1021/cb400080f [DOI] [PubMed] [Google Scholar]
  • 48.Esquerda-Canals G, Montoliu-Gaya L, Guell-Bosch J, Villegas S. Mouse Models of Alzheimer's Disease. J Alzheimers Dis. 2017;57(4):1171–83. Epub 2017/03/18. 10.3233/JAD-170045 [DOI] [PubMed] [Google Scholar]
  • 49.Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, et al. Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron. 2003;39(3):409–21. Epub 2003/08/05. 10.1016/s0896-6273(03)00434-3 [DOI] [PubMed] [Google Scholar]
  • 50.Dammers C, Schwarten M, Buell AK, Willbold D. Pyroglutamate-modified Abeta(3–42) affects aggregation kinetics of Abeta(1–42) by accelerating primary and secondary pathways. Chem Sci. 2017;8(7):4996–5004. Epub 2017/10/04. 10.1039/c6sc04797a [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Nussbaum JM, Schilling S, Cynis H, Silva A, Swanson E, Wangsanut T, et al. Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-beta. Nature. 2012;485(7400):651–5. Epub 2012/06/05. 10.1038/nature11060 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Carrillo-Mora P, Luna R, Colín-Barenque L. Amyloid beta: multiple mechanisms of toxicity and only some protective effects? Oxidative medicine and cellular longevity. 2014;2014:795375 Epub 2014/04/01. 10.1155/2014/795375 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Bao F, Wicklund L, Lacor PN, Klein WL, Nordberg A, Marutle A. Different beta-amyloid oligomer assemblies in Alzheimer brains correlate with age of disease onset and impaired cholinergic activity. Neurobiol Aging. 2012;33(4):825 e1-13. Epub 2011/06/21. 10.1016/j.neurobiolaging.2011.05.003 [DOI] [PubMed] [Google Scholar]
  • 54.Perucho J, Rubio I, Casarejos MJ, Gomez A, Rodriguez-Navarro JA, Solano RM, et al. Anesthesia with isoflurane increases amyloid pathology in mice models of Alzheimer's disease. J Alzheimers Dis. 2010;19(4):1245–57. 10.3233/JAD-2010-1318 [DOI] [PubMed] [Google Scholar]
  • 55.Perez-Garmendia R, Gevorkian G. Pyroglutamate-Modified Amyloid Beta Peptides: Emerging Targets for Alzheimer´s Disease Immunotherapy. Current neuropharmacology. 2013;11(5):491–8. Epub 2014/01/10. 10.2174/1570159X11311050004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Pavel MA, Petersen EN, Wang H, Lerner RA, Hansen SB. Studies on the mechanism of general anesthesia. Proceedings of the National Academy of Sciences of the United States of America. 2020;117(24):13757–66. Epub 2020/05/30. 10.1073/pnas.2004259117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Piccini A, Russo C, Gliozzi A, Relini A, Vitali A, Borghi R, et al. beta-amyloid is different in normal aging and in Alzheimer disease. The Journal of biological chemistry. 2005;280(40):34186–92. Epub 2005/08/17. 10.1074/jbc.M501694200 [DOI] [PubMed] [Google Scholar]
  • 58.Zhou R, Bickler P. Interaction of Isoflurane, Tumor Necrosis Factor-alpha and beta-Amyloid on Long-term Potentiation in Rat Hippocampal Slices. Anesth Analg. 2017;124(2):582–7. Epub 2017/01/19. 10.1213/ANE.0000000000001698 [DOI] [PubMed] [Google Scholar]
  • 59.Carnini A, Lear JD, Eckenhoff RG. Inhaled anesthetic modulation of amyloid beta(1–40) assembly and growth. Curr Alzheimer Res. 2007;4(3):233–41. Epub 2007/07/14. 10.2174/156720507781077278 [DOI] [PubMed] [Google Scholar]
  • 60.Zhang S, Hu X, Guan W, Luan L, Li B, Tang Q, et al. Isoflurane anesthesia promotes cognitive impairment by inducing expression of beta-amyloid protein-related factors in the hippocampus of aged rats. PLoS One. 2017;12(4):e0175654 Epub 2017/04/14. 10.1371/journal.pone.0175654 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Mroczko B, Groblewska M, Litman-Zawadzka A, Kornhuber J, Lewczuk P. Amyloid beta oligomers (AbetaOs) in Alzheimer's disease. J Neural Transm (Vienna). 2018;125(2):177–91. Epub 2017/12/03. 10.1007/s00702-017-1820-x [DOI] [PubMed] [Google Scholar]
  • 62.Lai J, Hu M, Wang H, Hu M, Long Y, Miao MX, et al. Montelukast targeting the cysteinyl leukotriene receptor 1 ameliorates Abeta1-42-induced memory impairment and neuroinflammatory and apoptotic responses in mice. Neuropharmacology. 2014;79:707–14. Epub 2014/01/25. 10.1016/j.neuropharm.2014.01.011 [DOI] [PubMed] [Google Scholar]
  • 63.Lazarevic V, Fieńko S, Andres-Alonso M, Anni D, Ivanova D, Montenegro-Venegas C, et al. Physiological Concentrations of Amyloid Beta Regulate Recycling of Synaptic Vesicles via Alpha7 Acetylcholine Receptor and CDK5/Calcineurin Signaling. Frontiers in molecular neuroscience. 2017;10:221 Epub 2017/08/09. 10.3389/fnmol.2017.00221 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Evans CE, Miners JS, Piva G, Willis CL, Heard DM, Kidd EJ, et al. ACE2 activation protects against cognitive decline and reduces amyloid pathology in the Tg2576 mouse model of Alzheimer's disease. Acta Neuropathol. 2020;139(3):485–502. Epub 2020/01/27. 10.1007/s00401-019-02098-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Um JW, Kaufman AC, Kostylev M, Heiss JK, Stagi M, Takahashi H, et al. Metabotropic glutamate receptor 5 is a coreceptor for Alzheimer abeta oligomer bound to cellular prion protein. Neuron. 2013;79(5):887–902. Epub 2013/09/10. 10.1016/j.neuron.2013.06.036 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Bilkei-Gorzo A. Genetic mouse models of brain ageing and Alzheimer's disease. Pharmacology & therapeutics. 2014;142(2):244–57. Epub 2013/12/24. 10.1016/j.pharmthera.2013.12.009 [DOI] [PubMed] [Google Scholar]
  • 67.McKee AC, Kowall NW, Schumacher JS, Beal MF. The neurotoxicity of amyloid beta protein in aged primates. Amyloid: the international journal of experimental and clinical investigation: the official journal of the International Society of Amyloidosis. 1998;5(1):1–9. Epub 1998/04/18. 10.3109/13506129809007283 [DOI] [PubMed] [Google Scholar]
  • 68.Zhang B, Tian M, Zheng H, Zhen Y, Yue Y, Li T, et al. Effects of anesthetic isoflurane and desflurane on human cerebrospinal fluid Abeta and tau level. Anesthesiology. 2013;119(1):52–60. Epub 2013/02/27. 10.1097/ALN.0b013e31828ce55d [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Stephen D Ginsberg

11 Jun 2020

PONE-D-20-10009

Neurotoxicity of different amyloid beta subspecies in mice and their interaction with isoflurane anaesthesia

PLOS ONE

Dear Dr. Schmid,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration by 2 Reviewers and an Academic Editor, all of the critiques of both Reviewers must be addressed in detail in a revision to determine publication status. If you are prepared to undertake the work required, I would be pleased to reconsider my decision, but revision of the original submission without directly addressing the critiques of the two Reviewers does not guarantee acceptance for publication in PLOS ONE. If the authors do not feel that the queries can be addressed, please consider submitting to another publication medium. A revised submission will be sent out for re-review. The authors are urged to have the manuscript given a hard copyedit for syntax and grammar.

==============================

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. 

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously? 

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Rationale of the study

The rationale of the study is partly explained, but many aspects of this study are not put in context in the Introduction. The Introduction could be improved by addressing the comments below.

• What is the relevance of Abeta-pyro and Abeta-nitro to AD? Literature should be included in the introduction (and referenced) to place this in context. Also, what species are Abeta-pyro and Abeta-nitro Abeta1-40 or 1-42?

• “to ultimately find the best anaesthetic regimen for … AD”, this statement in the introduction should be clarified as only one anaesthetic agent is included in this study. Furthermore, an explanation of why isoflurane chosen rather than another aesthetic to study, would strengthen the introduction.

• What is the rationale for investigating the interaction between different Abeta species and anaesthetics? Please include relevant literature in the introduction to address this.

• The rationale for why TNFalpha, caspase 3, NR2B and mGlu5 were chosen as biomarkers is explained in the Discussion, but it would benefit the reader if this information was presented earlier in the manuscript.

• The anaesthetics/AD literature is currently not adequately referenced in the Introduction (e.g. Line 50, 54, 56, 57, 269), this should be rectified.

Methods

The major concerns I have with this work is the experimental design; the study is likely underpowered, lacks some controls and uses different concentrations of the Abeta species across the experimental groups.

• This study is underpowered, behavioural data has n=6 per experimental group, amyloid deposit analysis n=2 per experimental group and WB analysis n=4 per experimental group. An n=2 per experimental group is not sufficient for analysis. Power calculations should be performed to determine the change that can be detected with 90% power for each dataset in the manuscript and this information needs to be included in the manuscript. This will allow the authors to comment on whether the study was sufficiently powered to detect a change, or whether the results a likely to represent false negatives.

• The Abeta 1-42 solution injected includes hexafluoroisopropanol and NaOH, whereas all other Abeta species are diluted in PBS. An additional control group with the same concentration of hexafluoroisopropanol and NaOH in PBS should be included in the study.

• Different concentrations of each Abeta species are used, the explanation for this is that the different concentrations of Abeta species will have equivalent “neurotoxicity” and a paper examining LTP, EPSC and spine density data from brain slices is references. Please define “neurotoxicity” in this context. How do the authors know that this measure of “neurotoxicity” from brain slices will be the same in vivo?.

• Insufficient detail in the methods:

o Where were the Abeta species purchased from or how were they made?

o Concentration of hexafluoroisopropanol in the Abeta 1-42 solution should be provided.

o What was the oligomerization status of all 4 Abeta species at the time of injection? Was it comparable?

o Concentrations of antibodies used for WB should be provided.

o Why were the hippocampus and sensory cortex chosen for WB analysis?

o How many saggital slices were analysed per mouse? Which brain regions were analysed?

Results and Interpretation

• The results are currently unclear and could be improved:

o Are the significant differences in the time trial data for the Abeta-pyro condition only for the 7-8th day of testing? Is the statistically significant difference between Abeta-pyro vs PBS for the sham or isoflurane condition or both?

o Are the data described in line 232 and 233 (Fig 2A) of the manuscript for the sham condition, the anaesthetic condition or both? Is the difference only present for the 7-8th day of testing?

• Abeta-pyro is by far the most concentrated Abeta species when injected, could this explain why the other Abeta species had no/limited effects? And explain the interaction of only Abeta-pyro and isoflurane in the behavioural testing data? Opposite effects of picomolar and micromolar Abeta1-42 and Abeta1-40 have previously been reported (Lazarevic et al., 2017, doi: 10.3389/fnmol.2017.00221). There are also many published papers reporting protective effects of Abeta that should be included in the discussion related to the Abeta-pyro results (i.e. Carrillo-Mora et al., 2014, doi: 10.1155/2014/795375).

• Is there a potential mechanism for the interaction between Abeta-pyro and isoflurane to explain the results?

• Abeta1-42 is injected containing hexafluoroisopropanol and NaOH, there is no control group that accounts for the hexafluoroisopropanol and NaOH, thus, the difference in the time spent on the exposed part of the arena in the Abeta1-42 experimental group (Figure 2A) may be due to the impact of hexafluoroisopropanol, NaOH, Abeta1-42 or a combination of the three.

• The lack of difference in the amyloid deposits and TNFalpha, caspase 3, NR2B and mGlu5 may be due to the study being underpowered or a non-optimal concentration of Abeta species being used. This should be discussed.

• What were the differences between the previous study Schmid et al., 2017 (Reference 8) and the current study?

The anaesthetic used for canula insertion surgery is different, the concentration of Abeta 1-42 injected is much higher, the number of mice per experimental group is higher, this warrants discussion.

Other

• The manuscript should use inclusive language for people living with dementia (i.e. “patients suffering AD” is not appropriate, “people living with AD” is appropriate). This should be revised throughout.

• Figure 1 – reduce the range of the y axis so that data and error bars can be seen clearly.

• Figure 1, 2 – marking of significance on graphs is unclear, which time point is this for? Please rectify.

• Line 232 remove the word “as”.

• Line 268 “…an inhibiting effect on behaviour” this should be reworded to be more specific.

• Paragraphs 2 and 3 of the Discussion could be integrated for a better justification of the mouse model used.

• “in vitro” and “in vivo” should be italicised throughout.

Reviewer #2: The present study investigated the effect of different amyloid beta subspecies on behaviour and cognition in mice and their interaction with isoflurane anesthesia. The main findings of the study are that Aβ pyro improved overall cognitive performance which seemed to be contrary to the previous studies, and isoflurane could counteract this improvement. Inflammation and apoptosis biomarkers such as tumor -necrosis factor alpha, NR2B, mGlu5, or caspase 3 were not involved in this process. There are several points which the authors should consider.

1. The biochemical endpoints appear to be limited to a single evaluation This would be informative to have additional time points to better understand the effects on these markers.

2. Isoflurane is rarely used clinically, as it is known for its adverse effects on cognitive functions. It would be more pellucid if the authors could illuminate the reason why selected isoflurane in this study.

3. The size of the experimental groups of mice in the study is unclear. Is the number of animals included based on power analysis? Please state the number of animals in each experimental group in Material and Methods

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

==============================

Please submit your revised manuscript by December, 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Stephen D. Ginsberg, Ph.D.

Section Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

PLoS One. 2020 Dec 3;15(12):e0242989. doi: 10.1371/journal.pone.0242989.r002

Author response to Decision Letter 0

4 Nov 2020

Response to Reviewers

Reviewer #1: Rationale of the study

The rationale of the study is partly explained, but many aspects of this study are not put in context in the Introduction. The Introduction could be improved by addressing the comments below.

• What is the relevance of Abeta-pyro and Abeta-nitro to AD? Literature should be included in the introduction (and referenced) to place this in context. Also, what species are Abeta-pyro and Abeta-nitro Abeta1-40 or 1-42?

Thank you very much for this important comment. Abeta-pyro, also known as AβpE3-42, has first been described in 1992 by Mori et al. [1]. It is a modification of Abeta 1-42. Total Abeta contains 10-15% of pyroglutamated amyloid beta (i.e. Abeta pyro) and it represents a dominant fraction of Aβ peptides in senile plaques of AD brains [2]. Abeta nitro (3NTyr10-Aβ) is also a nitrotyrosinated (or nitrated) form of amyloid beta 1-42 [3] and is found in the cores of amyloid plaques in AD brains [4]. Pyroglutamylation as well as nitrotyrosination of amyloid beta leads to increased oligomer stability and thus neurotoxicity in vitro as well as in vivo shown by neurodegeneration, premature mortality of mice, and disruption of calcium dyshomeostasis [5, 6].

Abeta1-40, Abeta1-42, Abeta-pyro and Abeta-nitro are all post-translationally modified forms of the peptide amyloid-beta.

We described Abeta pyro and Abeta nitro in lines 54 – 58 (unmarked version of the revised manuscript)/57 – 61 (marked-up copy of the revised manuscript) more clearly and cited relevant literature.

• “to ultimately find the best anaesthetic regimen for … AD”, this statement in the introduction should be clarified as only one anaesthetic agent is included in this study. Furthermore, an explanation of why isoflurane chosen rather than another aesthetic to study, would strengthen the introduction.

We are grateful to Reviewer #1 for this extremely helpful remark. With this sentence we wanted to express our long-term goal of understanding the pathophysiology underlying Alzheimer’s disease and its interaction with different anaesthetics. Since the aim of our study was to investigate the effects of different intracerebroventricularly administered Abeta subspecies (Abeta1-40, Abeta1-42, Abeta-pyro and Abeta-nitro) in vivo and their interaction with general anaesthesia, we chose to concentrate on one anaesthetic agent, i. e. isoflurane. Isoflurane has been shown to induce caspase activation and increase levels of beta-site APP-cleaving enzyme (BACE) in vivo in C57/BL6 mice [7]. Sevoflurane seems to induce cellular and histological effects comparable to isoflurane [8], while desflurane was associated with a decrease in Abeta 1-42 levels [9].

As recommended, we included parts of the paragraph above in the introduction (lines 78–82 unmarked version/lines 92–96 marked-up version of the revised manuscript) and discussed desflurane as a possible anesthetic for future research (lines 404-406 unmarked version/lines 429-431 marked-up version of the revised manuscript).

• What is the rationale for investigating the interaction between different Abeta species and anaesthetics? Please include relevant literature in the introduction to address this.

Thank you for this important critique which helps us to improve our manuscript. Alzheimer’s disease (AD) is the most common form of dementia worldwide and affects as much as three percent of men and women aged between 65 and 74 years [10]. Due to an ongoing medical progress this population is also very likely to undergo surgery, often conducted under general anesthesia [10]. It is still unclear whether general anesthesia contributes to the development of AD. Some studies suggest a possible link between anesthesia and AD [11, 12], while more recent ones do not [13, 14]. Also, it is nearly impossible to discriminate the influence of general anesthesia from the effect of surgery itself on the development of AD, as Lee et al. stated earlier this year [15]. To further illuminate the pathophysiology behind AD and the possible association of anesthesia and AD we wanted to investigate the potency of inducing AD and their individual interaction with isoflurane of the most prominent amyloid beta subspecies (i. e. Abeta 1-40, Abeta 1-42, Abeta pyro and Abeta nitro) one by one.

We included relevant literature in the introduction in lines 62, 65, 66, 68 (unmarked copy of the revised manuscript)/67, 70 - 73 (marked-up version of the revised manuscript).

• The rationale for why TNFalpha, caspase 3, NR2B and mGlu5 were chosen as biomarkers is explained in the Discussion, but it would benefit the reader if this information was presented earlier in the manuscript.

We included our reasons for investigating TNFalpha, caspase 3, NR2B and mGlu5 in the introduction (lines 83 - 85 (unmarked version)/102 - 104 (marked-up version)).

• The anaesthetics/AD literature is currently not adequately referenced in the Introduction (e.g. Line 50, 54, 56, 57, 269), this should be rectified.

Thank you for raising this important point. We corrected for this in the unmarked version of the revised manuscript in lines 52, 53, 59, 61, 62, 64, 317/in the marked-up copy of the revised manuscript in lines 55, 56, 63, 66, 67, 70, 340. We also reworded lines 314 - 317 in the unmarked copy of the revised manuscript/338 - 340 in the marked-up version of the revised manuscript.

Methods

The major concerns I have with this work is the experimental design; the study is likely underpowered, lacks some controls and uses different concentrations of the Abeta species across the experimental groups.

• This study is underpowered, behavioural data has n=6 per experimental group, amyloid deposit analysis n=2 per experimental group and WB analysis n=4 per experimental group. An n=2 per experimental group is not sufficient for analysis. Power calculations should be performed to determine the change that can be detected with 90% power for each dataset in the manuscript and this information needs to be included in the manuscript. This will allow the authors to comment on whether the study was sufficiently powered to detect a change, or whether the results a likely to represent false negatives.

We apologize for not describing the statistical approach in sufficient detail, especially the sample size considerations. Accordingly, the reviewer must have come to the impression of a power problem. We included our sample size considerations in the methods section (lines 245 - 249 in the unmarked version of the revised manuscript/ lines 268 - 272 in the marked-up version of the revised manuscript):

The primary endpoint of the study was the cognitive and behavioural outcome. The variables of the hole-board test are considered relevant if two groups differ two times the given standard deviation. Based on a type I error of 0.05, a type II error of 0.20 and two-sided t-tests at the final test level of the hierarchical model 4 animals per group would have been appropriate. Our internal standard, however, suggests a minimal group size of six, which has been used accordingly.

In order to facilitate readability of our findings in the modified hole-board test, we calculated the effect size of our findings and included mean differences with 95% confidence interval and partial eta-squared in the manuscript (unmarked version of the revised manuscript: lines 242 – 244, 262, 263, 275, 281/ marked-up version of the revised manuscript: lines 266, 267, 285, 286, 298, 304).

We agree with the reviewer, that our study was not sufficiently powered for amyloid deposit and western blot analysis. We did explorative studies on a limited number of brains on amyloidopathy and other biomarkers in order to detect targets for future research concerning the pathomechanism of different Abeta subspecies and the interaction of isoflurane and amyloid-beta. Since we could not detect any amyloid deposits, we did not perform statistical analyses regarding amyloidopathy. Although we are aware of the shortcomings, we think that the results of the amyloid deposit and the western blot analysis are valid and should be presented in the manuscript. However, we stated the preliminary character of these analyses more clearly in the abstract and the discussion of the revised manuscript (abstract: lines 25, 26, 32, 38 of the unmarked version/lines 25 - 27, 34, 40 of the marked-up copy; discussion: lines 317, 318, 382, 394, 411 of the unmarked version/lines 273, 341, 406, 419, 436, 437 of the marked-up copy).

• The Abeta 1-42 solution injected includes hexafluoroisopropanol and NaOH, whereas all other Abeta species are diluted in PBS. An additional control group with the same concentration of hexafluoroisopropanol and NaOH in PBS should be included in the study.

We are deeply grateful to Reviewer #1 for raising this important point and apologize for providing insufficient detail in the original version of the manuscript.

After suspending Abeta 1-42 in hexafluoroisopropanol (HFIP), it was removed from the stock solution using a vacuum concentrator (Thermo Scientific Savant SpeedVac, Thermo Fisher Scientific, Waltham, Massachusetts, United States of America). HFIP has been shown to increase cell permeability and to decrease cell viability [16, 17], so a protocol to purge HFIP from the Abeta 1-42 stock solution was established in our lab [18]. We reworded the respective part in the Methods section in the revised manuscript (unmarked version: lines 133 - 138, marked-up version: lines 154 - 159).

After Abeta 1-42 was dissolved in NaOH it was diluted 1:100 with PBS. Therefore, the amount of NaOH injected can be considered as minimal. We added the dilution ratio in the manuscript (unmarked version: line 140, marked-up version: line 162). As we did not shield the solution containing NaOH from air, NaOH reacted with CO2 in the 15 to 45 minutes before injection and formed sodium carbonate and sodium bicarbonate, which further reduced the amount of NaOH. In conclusion we consider the effect of the NaOH used to solve Abeta 1-42 minimal. To perform our experiments in accordance with the principles of the 3Rs (Replacement, Reduction and Refinement) in animal research we decided against an additional control group. However, in order to account for a possible bias, we added the use of NaOH in only one group as a possible limitation in the discussion of the revised manuscript (unmarked version: lines 378 - 381, marked-up version: lines 402 - 405).

• Different concentrations of each Abeta species are used, the explanation for this is that the different concentrations of Abeta species will have equivalent “neurotoxicity” and a paper examining LTP, EPSC and spine density data from brain slices is references. Please define “neurotoxicity” in this context. How do the authors know that this measure of “neurotoxicity” from brain slices will be the same in vivo?

Thank you very much for drawing our attention to this important point. Our definition of “neurotoxicity” in this context was the ability of Abeta 1-40, Abeta 1-42, Abeta pyro and Abeta nitro to induce alterations in behaviour, cognition and fine motor skills in mice and the ability to induce amyloid deposits in the brains, respectively. With our study we wanted to investigate whether the findings of Rammes et al. (Rammes et al., “The NMDA receptor antagonist Radiprodil reverses the synaptotoxic effects of different amyloid-beta (Aβ) species on long-term potentiation (LTP)” Neuropharmacology. 2018 Sep 15;140:184-192. doi: 10.1016/j.neuropharm.2018.07.021. Epub 2018 Aug 11.) concerning the AD-inducing effects of different Abeta subspecies in vitro could be transferred into a mouse model. Therefore, we chose to use the same concentrations as Rammes et al. We did not know if the effects on brain slices were to be the same in vivo but we wanted to find out with this study. In order to avoid confusion for the reader we replaced the term “neurotoxicity” in line 152 - 153 (unmarked version of the revised manuscript)/line 175, 176 (marked-up copy of the revised manuscript) and replaced it with the actual in vitro findings of the cited reference.

• Insufficient detail in the methods:

o Where were the Abeta species purchased from or how were they made?

We apologize for not stating this beforehand. Aβ1-40 and Aβ1-42 were both purchased from American Peptide Sunnyvale, CA, USA. AβpE3-42 was purchased from Bachem AG Bubendorf, Switzerland.

3NTyr10Aβ was provided to us by Clinical Neuroscience Unit, Department of Neurology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany. 3NTyr10Aβ was made as described in [4].

We included this information in lines 133, 145 – 148 (unmarked copy)/lines 154, 168 – 170 (marked-up copy) of the revised manuscript.

o Concentration of hexafluoroisopropanol in the Abeta 1-42 solution should be provided.

For aliquotation Abeta 1-42 was dissolved in 100% hexafluoroisopropanol at a concentration of 1 mg/ml. As described above HFIP was completely removed after aliquotation and therefore no HFIP was present in the Abeta 1-42 solution that was injected. We once again apologize for the incorrect information about HFIP in the first version of the manuscript.

o What was the oligomerization status of all 4 Abeta species at the time of injection? Was it comparable?

All 4 Abeta species were injected 15 to 45 minutes after preparation. We assume that at this early timepoint after dissolving the substances the oligomerization process has just started and we injected predominantly Abeta monomers. Further oligomerization then should have taken place in the brains of the animals. A part of our study was to investigate whether this process leads to formation of Abeta plaques. As we were not able to detect Abeta plaques, retrospectively it would have been interesting to investigate for formation of Abeta oligomers e.g. using SDS-PAGE. We will consider this interesting approach when planning our next experiments.

o Concentrations of antibodies used for WB should be provided.

We apologize for not including this in the manuscript in the first place. The antibodies for WB were as follows:

Primary antibodies

Anti-Metabotropic Glutamate Receptor 5 Antibody ab53090 1:1000 Abcam (Cambridge, UK)

TNFα Antibody ProSci XP-5284 1:1000

ProSci (Poway, CA, USA)

NMDAR2B Rabbit Antibody #4207 1:1000 Cell Signaling Technology (Danvers, Massachusetts, USA)

Caspase-3 Rabbit Antibody #9662 1:1000 Cell Signaling Technology (Danvers, Massachusetts, USA)

Secondary antibodies

Anti-rabbit IgG, HRP-linked Antibody #7076 1:10 000 Cell Signaling Technology (Danvers, Massachusetts, USA)

We corrected for this in lines 227, 228, 230 (unmarked copy)/lines 249, 250, 252 (marked-up copy) of the revised manuscript.

o Why were the hippocampus and sensory cortex chosen for WB analysis?

We wanted to investigate cognitive and behavioural changes in the modified hole-board test in wild type mice after intracerebroventricular injection of different Abeta subspecies as the primary endpoint of our study. As the hippocampus is important for working and reference memory and the sensory cortex plays a pivotal role in spatial orientation and movement planning, we decided to analyze TNF alpha, caspase 3, NR2B and mGlu5 as secondary endpoints in those brain regions.

o How many saggital slices were analysed per mouse? Which brain regions were analysed?

We analysed a total of 21 sagittal slices per mouse. The frontal and temporal lobe including sensory cortex and hippocampus were analysed.

We included the number of brain slices and the location in line 198 (unmarked copy)/line 220 (marked-up copy) of the revised manuscript.

Results and Interpretation

• The results are currently unclear and could be improved:

o Are the significant differences in the time trial data for the Abeta-pyro condition only for the 7-8th day of testing? Is the statistically significant difference between Abeta-pyro vs PBS for the sham or isoflurane condition or both?

Thank you very much for these important remarks. We analysed the data derived from the modified hole-board test using general linear models for the factors subspecies for injection, anaesthesia (isoflurane or sham) and the within-group factor time and their interaction terms. The results show differences that are present over the whole test period of 8 days and not only on the 7-8th day of testing. We agree with the reviewer, that the symbols marking significance in figures 1 und 2 could have been confusing for the reader, changed the figures accordingly and added the description “Effect over all days of testing” in the legend. Statistical analysis revealed a significant difference for the factor “subspecies for injection” Therefore, the difference between A-beta pyro and PBS is present for all animals. To make this clearer for the reader, we added mean difference and partial eta-squared for assessment of the effect size in the manuscript (unmarked version of the revised manuscript: lines 262, 263, 265, 275 – 278, 281 - 283/ marked-up version of the revised manuscript: lines 285, 286, 288, 289, 298 – 300, 303 - 306).

o Are the data described in line 232 and 233 (Fig 2A) of the manuscript for the sham condition, the anaesthetic condition or both? Is the difference only present for the 7-8th day of testing?

Regarding this comment please be referred to our answer on your comment above. The difference is present over all days of testing. We corrected figure 2 accordingly.

• Abeta-pyro is by far the most concentrated Abeta species when injected, could this explain why the other Abeta species had no/limited effects? And explain the interaction of only Abeta-pyro and isoflurane in the behavioural testing data? Opposite effects of picomolar and micromolar Abeta1-42 and Abeta1-40 have previously been reported (Lazarevic et al., 2017, doi: 10.3389/fnmol.2017.00221). There are also many published papers reporting protective effects of Abeta that should be included in the discussion related to the Abeta-pyro results (i.e. Carrillo-Mora et al., 2014, doi: 10.1155/2014/795375).

Although we cannot rule out that the different concentrations of the Abeta species, with Abeta-pyro being the most concentrated could be the reason for our results, we think that the distinctive properties of Abeta pyro might explain our findings and the interaction of only Abeta-pyro and isoflurane. Please be also referred to our answer to the following critique as well as the third Reviewer comment in the Methods section.

In order to improve the discussion of the Abeta effects we included the referenced studies in lines 351 – 354 and 371 – 374 (unmarked version)/lines 374 - 377 and 395 – 398 (marked-up version) of the revised manuscript.

• Is there a potential mechanism for the interaction between Abeta-pyro and isoflurane to explain the results?

The fact that most of the statistically significant results in our study could be found regarding Abeta-pyro might be due to the rather short time between the intracerebroventricular injection and the behavioural testing. Abeta-pyro has been shown to accumulate in the brain at early stages of AD [2, 19], with the hippocampus being one of the predominant regions. 12-week-old C57BL/6 mice showed an impairment in spatial working memory and delayed learning in Y-maze and Morris water maze tests after intracerebroventricular injection of aggregated Abeta-pyro within two weeks after injection [20]. Abeta-pyro and isoflurane are both hydrophobic agents [21, 22]. As aggregated Abeta-pyro forms membrane pores and thus seems to alter membrane permeability [23], we also see a potential mechanism for the interaction there.

We included our thoughts on a potential mechanism of interaction in the discussion (lines 363 – 365 (unmarked version)/lines 387 – 389 (marked-up version) of the revised manuscript).

• Abeta1-42 is injected containing hexafluoroisopropanol and NaOH, there is no control group that accounts for the hexafluoroisopropanol and NaOH, thus, the difference in the time spent on the exposed part of the arena in the Abeta1-42 experimental group (Figure 2A) may be due to the impact of hexafluoroisopropanol, NaOH, Abeta1-42 or a combination of the three.

Once again, we are deeply sorry for not providing sufficient detail in the Methods section and would like to refer to our answer to the second reviewer comment on the methods. The Abeta 1-42 stock solution was purged from hexafluoroisopropanol (HFIP). NaOH in a concentration of 20 mmol/l was diluted 1:100 with PBS and reacted to sodium carbonate and sodium bicarbonate, so we think that the results are due to the impact of Abeta 1-42 rather than HFIP or NaOH.

• The lack of difference in the amyloid deposits and TNFalpha, caspase 3, NR2B and mGlu5 may be due to the study being underpowered or a non-optimal concentration of Abeta species being used. This should be discussed.

We fully agree with Reviewer #1 and would also like to refer to our answer to the first and third Reviewer comment in the Methods section. The primary endpoint of the study was the cognitive and behavioural outcome. The explorative studies we did on a limited number of brains on amyloidopathy, neuroinflammation and receptor expression should be considered preliminary. We stated this in lines 32, 250, 317, 382, 411, 412 (unmarked version)/lines 34, 273, 341, 406, 436, 437 (marked-up version) of the revised manuscript.

• What were the differences between the previous study Schmid et al., 2017 (Reference 8) and the current study?

The anaesthetic used for canula insertion surgery is different, the concentration of Abeta 1-42 injected is much higher, the number of mice per experimental group is higher, this warrants discussion.

In the previous study of Schmid et al., 2017 mice were intracerebroventricularly injected with Abeta 1-42 or PBS. Subsequently neurocognitive and behavioural parameters were evaluated using the modified hole-board test. Mice were anaesthetized with a combination of midazolam, medetomidine and fentanyl intraperitoneally. The main differences between the study under review and the previous one are the different subspecies of amyloid-beta and the type of anaesthesia: since we wanted to investigate the effects of different amyloid-beta subspecies on cognition and behaviour, mice were intracerebroventricularly injected with not only Abeta 1-42 or PBS but with Abeta 1-40, Abeta 1-42, Abeta nitro, Abeta pyro and PBS. Also, we aimed to investigate a possible interaction between isoflurane anaesthesia and the respective amyloid-beta subspecies. We decided to also use isoflurane anaesthesia for cannula implantation to avoid any interaction of other anaesthetics and opioids like midazolam, medetomidine and fentanyl with the Abeta subforms.

We agree that the concentration of Abeta 1-42 used in our 2017 study was higher. However, the final concentration of Abeta 1-42 in the brain is comparable as the amount of Abeta 1-42 is identical: In our 2017 study we injected 3.5 µl of a solution containing 1 µmol/l Abeta 1-42 resulting in a total amount of Abeta 1-42 of 3.5 pmol [24]. In the current study we injected 5.0 µl of a solution containing 700 nmol/l Abeta 1-42 resulting in the same total amount of Abeta 1-42 of 3.5 pmol. We agree that it was not clear for the reader at first sight that we injected 5.0 µl of Abeta and added the exact volume in the methods section (line 144 (unmarked version)/line 166 (marked-up copy) of the revised manuscript).

We apologize for the confusion caused regarding the experimental groups. In the study of Schmid et al, 2017, a total of 24 mice was divided in 4 groups. The first group was injected with Abeta 1-42 and started testing on day 2, the second group (also injected with Abeta 1-42) started on day 4, the third group (also injected with Abeta 1-42) started on day 8 after the intracerebroventricular injection. The fourth group (mice injected with PBS) started on day 4 after the injection. In the current study we used 60 mice divided in 10 groups. In each study we used 6 mice per group. To make this clearer for the reader we included the exact number of animals per group in the manuscript (lines 122 - 125 (unmarked copy)/lines 143 – 146 (marked-up copy)).

Other

• The manuscript should use inclusive language for people living with dementia (i.e. “patients suffering AD” is not appropriate, “people living with AD” is appropriate). This should be revised throughout.

Thank you for this important comment. We revised the wording in lines 50, 60, 62 (unmarked copy)/lines 53, 64, 67 (marked-up copy) of the manuscript.

• Figure 1 – reduce the range of the y axis so that data and error bars can be seen clearly.

We apologize and revised figure 1 accordingly.

• Figure 1, 2 – marking of significance on graphs is unclear, which time point is this for? Please rectify.

We revised figures 1 and 2. Please be referred to our answer on your first comment in results and interpretation.

• Line 232 remove the word “as”.

We removed the word “as” in line 274 (unmarked version)/line 297 (marked-up copy) of the revised manuscript.

• Line 268 “…an inhibiting effect on behaviour” this should be reworded to be more specific.

We apologize for not expressing this more precisely and reworded the sentence in lines 313, 314 (unmarked copy)/lines 336, 337 (marked-up copy) of the revised manuscript.

• Paragraphs 2 and 3 of the Discussion could be integrated for a better justification of the mouse model used.

We integrated paragraphs 2 and 3 of the Discussion in lines 69 – 75 (unmarked version)/lines 70 – 85 (marked-up version) of the revised manuscript.

• “in vitro” and “in vivo” should be italicised throughout.

We italicised “in vitro” as well as “in vivo” in lines 80, 81, 153, 332, 347, 362, 363 (unmarked version)/in lines 94, 95, 176, 355, 370, 386, 387 (marked-up version) of the revised manuscript.

Reviewer #2: The present study investigated the effect of different amyloid beta subspecies on behaviour and cognition in mice and their interaction with isoflurane anesthesia. The main findings of the study are that Aβ pyro improved overall cognitive performance which seemed to be contrary to the previous studies, and isoflurane could counteract this improvement. Inflammation and apoptosis biomarkers such as tumor -necrosis factor alpha, NR2B, mGlu5, or caspase 3 were not involved in this process. There are several points which the authors should consider.

1. The biochemical endpoints appear to be limited to a single evaluation This would be informative to have additional time points to better understand the effects on these markers.

We absolutely agree with Reviewer #2 that it would have been very interesting to perform biochemical analyses at several timepoints throughout our experiments. For example, given the fact that the caspase activation may not last very long following isoflurane anesthesia [7], it would have been informative to test for caspase 3 activity early after isoflurane anesthesia. However, the primary endpoint of our study was cognitive performance and behavioural alterations in the modified hole-board test. Like any other test in animals, the modified hole-board test is also very vulnerable regarding any interference. Exposing the animals to additional stressful situations, i.e. taking multiple blood samples, during the eight consecutive days of the modified hole-board testing would have altered the test performance of the mice. Therefore, we decided to evaluate the biochemical endpoints one time at the end of the mHBT being aware of this limitation to the study. To state this more clearly for the reader we reworded the corresponding paragraph in the discussion and emphasized the limitation of a missing consecutive analysis of biochemical parameters (lines 396 – 399 (unmarked version of the revised manuscript)/ lines 421 – 424 (marked-up copy of the revised manuscript)).

2. Isoflurane is rarely used clinically, as it is known for its adverse effects on cognitive functions. It would be more pellucid if the authors could illuminate the reason why selected isoflurane in this study.

Thank you for this important comment. We are sorry for not explaining this more clearly beforehand. As Reviewer #2 correctly stated, we wanted to investigate the effects of different amyloid beta subspecies on behaviour and cognition after intracerebroventricular injection in male C57BL/6N mice and their interaction with anaesthesia. We are aware, that isoflurane has adverse effects on cognitive function and is not regularly used in first world countries anymore. However, isoflurane is one of the most extensively studied anaesthetic agents in animal research. It has been shown to lead to increased oligomerization of amyloid beta in vitro [25, 26] but not in vivo [27]. Since the above cited studies used human cell lines transfected with APP or a transgenic mouse model of Alzheimer’s disease, we decided to investigate the direct interaction of isoflurane and “extrinsic” amyloid beta in a mouse model of intracerebroventricular injection. We included our considerations regarding the use of isoflurane in the introduction of the revised manuscript (lines 78 - 82 (unmarked version)/ lines 92 – 96 (marked-up version)).

We agree with the reviewer, that it would be very useful to examine different anaesthetics in future studies. Especially desflurane could be an interesting anaesthetic as it did not increase amyloid beta and tau levels in human cerebrospinal fluid and therefore could be regarded as less “neurotoxic” than isoflurane [9]. We included this consideration for future research in the manuscript (lines 404 – 406 (unmarked copy)/lines 429 – 431 (marked-up copy)).

3. The size of the experimental groups of mice in the study is unclear. Is the number of animals included based on power analysis? Please state the number of animals in each experimental group in Material and Methods

We are deeply sorry for not stating this more clearly. We included the number of animals in each experimental group (6 mice per group, resulting in a total of 60 mice) in Materials and Methods lines 122 – 125 (unmarked version)/lines 143 – 146 (marked-up version).

Concerning a power analysis, please also be referred to Reviewer#1’s first comment in the Methods section. We apologize for not describing these very important considerations in the first version of our manuscript. In short, this study was designed as an observational study, with cognitive and behavioural outcome as primary endpoints. Therefore, sample size calculations were performed based on the following considerations: The variables of the hole-board test are considered relevant if two groups differ two times the given standard deviation. Based on a type I error of 0.05, a type II error of 0.20 and two-sided t-tests at the final test level of the hierarchical model 4 animals per group would have been appropriate. Our internal standard, however, suggests a minimal group size of six, which has been used accordingly. We added this information in lines 245 – 249 (unmarked version) of the revised manuscript/lines 268 - 272 (marked-up version) of the revised manuscript.

References

1. Mori H, Takio K, Ogawara M, Selkoe DJ. Mass spectrometry of purified amyloid beta protein in Alzheimer's disease. The Journal of biological chemistry. 1992;267(24):17082-6. Epub 1992/08/25.

2. Saido TC, Iwatsubo T, Mann DM, Shimada H, Ihara Y, Kawashima S. Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3(pE), in senile plaques. Neuron. 1995;14(2):457-66. Epub 1995/02/01.

3. Guix FX, Uribesalgo I, Coma M, Muñoz FJ. The physiology and pathophysiology of nitric oxide in the brain. Progress in neurobiology. 2005;76(2):126-52. Epub 2005/08/24.

4. Kummer MP, Hermes M, Delekarte A, Hammerschmidt T, Kumar S, Terwel D, et al. Nitration of tyrosine 10 critically enhances amyloid β aggregation and plaque formation. Neuron. 2011;71(5):833-44. Epub 2011/09/10.

5. Wirths O, Breyhan H, Cynis H, Schilling S, Demuth HU, Bayer TA. Intraneuronal pyroglutamate-Abeta 3-42 triggers neurodegeneration and lethal neurological deficits in a transgenic mouse model. Acta Neuropathol. 2009;118(4):487-96. Epub 2009/06/24.

6. Guivernau B, Bonet J, Valls-Comamala V, Bosch-Morató M, Godoy JA, Inestrosa NC, et al. Amyloid-β Peptide Nitrotyrosination Stabilizes Oligomers and Enhances NMDAR-Mediated Toxicity. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2016;36(46):11693-703. Epub 2016/11/18.

7. Xie Z, Culley DJ, Dong Y, Zhang G, Zhang B, Moir RD, et al. The common inhalation anesthetic isoflurane induces caspase activation and increases Aβ level in vivo. Annals of neurology. 2008;64(6):618-27.

8. Jiang J, Jiang H. Effect of the inhaled anesthetics isoflurane, sevoflurane and desflurane on the neuropathogenesis of Alzheimer's disease (review). Mol Med Rep. 2015;12(1):3-12. Epub 2015/03/05.

9. Zhang B, Tian M, Zheng H, Zhen Y, Yue Y, Li T, et al. Effects of anesthetic isoflurane and desflurane on human cerebrospinal fluid Aβ and τ level. Anesthesiology. 2013;119(1):52-60. Epub 2013/02/27.

10. Quiroga C, Chaparro RE, Karlnoski R, Erasso D, Gordon M, Morgan D, et al. Effects of repetitive exposure to anesthetics and analgesics in the Tg2576 mouse Alzheimer's model. Neurotoxicity research. 2014;26(4):414-21. Epub 2014/06/15.

11. Kuehn BM. Anesthesia-Alzheimer disease link probed. Jama. 2007;297(16):1760. Epub 2007/04/26.

12. Culley DJ, Xie Z, Crosby G. General anesthetic-induced neurotoxicity: an emerging problem for the young and old? Current opinion in anaesthesiology. 2007;20(5):408-13. Epub 2007/09/18.

13. Jiang J, Dong Y, Huang W, Bao M. General anesthesia exposure and risk of dementia: a meta-analysis of epidemiological studies. Oncotarget. 2017;8(35):59628-37. Epub 2017/09/25.

14. Sprung J, Warner DO, Knopman DS, Petersen RC, Mielke MM, Jack CR, Jr., et al. Exposure to surgery with general anaesthesia during adult life is not associated with increased brain amyloid deposition in older adults. British journal of anaesthesia. 2020;124(5):594-602. Epub 2020/03/17.

15. Lee JJ, Choi GJ, Kang H, Baek CW, Jung YH, Shin HY, et al. Relationship between Surgery under General Anesthesia and the Development of Dementia: A Systematic Review and Meta-Analysis. BioMed research international. 2020;2020:3234013. Epub 2020/04/28.

16. Capone R, Quiroz FG, Prangkio P, Saluja I, Sauer AM, Bautista MR, et al. Amyloid-beta-induced ion flux in artificial lipid bilayers and neuronal cells: resolving a controversy. Neurotoxicity research. 2009;16(1):1-13. Epub 2009/06/16.

17. Ennaceur SM, Sanderson JM. Micellar aggregates formed following the addition of hexafluoroisopropanol to phospholipid membranes. Langmuir : the ACS journal of surfaces and colloids. 2005;21(2):552-61. Epub 2005/01/12.

18. Rammes G, Seeser F, Mattusch K, Zhu K, Haas L, Kummer M, et al. The NMDA receptor antagonist Radiprodil reverses the synaptotoxic effects of different amyloid-beta (Aβ) species on long-term potentiation (LTP). Neuropharmacology. 2018;140:184-92. Epub 2018/07/18.

19. Sergeant N, Bombois S, Ghestem A, Drobecq H, Kostanjevecki V, Missiaen C, et al. Truncated beta-amyloid peptide species in pre-clinical Alzheimer's disease as new targets for the vaccination approach. Journal of neurochemistry. 2003;85(6):1581-91. Epub 2003/06/06.

20. Youssef I, Florent-Béchard S, Malaplate-Armand C, Koziel V, Bihain B, Olivier JL, et al. N-truncated amyloid-beta oligomers induce learning impairment and neuronal apoptosis. Neurobiol Aging. 2008;29(9):1319-33. Epub 2007/04/27.

21. Perez-Garmendia R, Gevorkian G. Pyroglutamate-Modified Amyloid Beta Peptides: Emerging Targets for Alzheimer´s Disease Immunotherapy. Current neuropharmacology. 2013;11(5):491-8. Epub 2014/01/10.

22. Pavel MA, Petersen EN, Wang H, Lerner RA, Hansen SB. Studies on the mechanism of general anesthesia. Proceedings of the National Academy of Sciences of the United States of America. 2020;117(24):13757-66. Epub 2020/05/30.

23. Piccini A, Russo C, Gliozzi A, Relini A, Vitali A, Borghi R, et al. beta-amyloid is different in normal aging and in Alzheimer disease. The Journal of biological chemistry. 2005;280(40):34186-92. Epub 2005/08/17.

24. Schmid S, Jungwirth B, Gehlert V, Blobner M, Schneider G, Kratzer S, et al. Intracerebroventricular injection of beta-amyloid in mice is associated with long-term cognitive impairment in the modified hole-board test. Behav Brain Res. 2017;324:15-20. Epub 2017/02/15.

25. Xie Z, Dong Y, Maeda U, Alfille P, Culley DJ, Crosby G, et al. The common inhalation anesthetic isoflurane induces apoptosis and increases amyloid beta protein levels. Anesthesiology. 2006;104(5):988-94. Epub 2006/04/29.

26. Eckenhoff RG, Johansson JS, Wei H, Carnini A, Kang B, Wei W, et al. Inhaled anesthetic enhancement of amyloid-beta oligomerization and cytotoxicity. Anesthesiology. 2004;101(3):703-9. Epub 2004/08/27.

27. Bianchi SL, Tran T, Liu C, Lin S, Li Y, Keller JM, et al. Brain and behavior changes in 12-month-old Tg2576 and nontransgenic mice exposed to anesthetics. Neurobiology of aging. 2008;29(7):1002-10.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (90.6KB, docx)

Decision Letter 1

Stephen D Ginsberg

13 Nov 2020

Neurotoxicity of different amyloid beta subspecies in mice and their interaction with isoflurane anaesthesia

PONE-D-20-10009R1

Dear Dr. Schmid,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Stephen D. Ginsberg, Ph.D.

Section Editor

PLOS ONE

Acceptance letter

Stephen D Ginsberg

25 Nov 2020

PONE-D-20-10009R1

Neurotoxicity of different amyloid beta subspecies in mice and their interaction with isoflurane anaesthesia

Dear Dr. Schmid:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Stephen D. Ginsberg

Section Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Modified hole-board consisting of test arena and hole-board with cylinders.

    (PDF)

    Click here for additional data file. (1.3MB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (90.6KB, docx)

    Data Availability Statement

    All files with the original date are available from the mediaTUM database: https://mediatum.ub.tum.de/1579195.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES