Chronic stress exposure following photothrombotic stroke is associated with increased levels of Amyloid beta accumulation and altered oligomerisation at sites of thalamic secondary neurodegeneration in mice

Abstract

Exposure to severe stress following stroke is recognised to complicate the recovery process. We have identified that stress can exacerbate the severity of post-stroke secondary neurodegeneration in the thalamus. In this study, we investigated whether exposure to stress could influence the accumulation of the neurotoxic protein Amyloid-β. Using an experimental model of focal cortical ischemia in adult mice combined with exposure to chronic restraint stress, we examined changes within the contra- and ipsilateral thalamus at six weeks post-stroke using Western blotting and immunohistochemical approaches. Western blotting analysis indicated that stroke was associated with a significant enhancement of the 25 and 50 kDa oligomers within the ipsilateral hemisphere and the 20 kDa oligomer within the contralateral hemisphere. Stroked animals exposed to stress exhibited an additional increase in multiple forms of Amyloid-beta oligomers. Immunohistochemistry analysis confirmed that stroke was associated with a significant accumulation of Amyloid-beta within the thalami of both hemispheres, an effect that was exacerbated in stroke animals exposed to stress. Given that Amyloid-beta oligomers, most notably the 30–40 and 50 kDa oligomers, are recognised to correlate with accelerated cognitive decline, our results suggest that monitoring stress levels in patients recovering from stroke may merit consideration in the future.

Introduction

Secondary neurodegeneration (SND) refers to the progressive death of brain regions that were connected to, but not initially damaged by, the primary infarction process. Over the course of several decades SND has been consistently observed in humans following stroke using MRI,^1–4 CT^5–8 and PET^9–11 imaging. In experimental stroke models, SND occurs without exception.^12–17 The most dominant histological features of SND is neuronal loss concomitant with intense microglial activation.¹²

We recently identified, using a preclinical model, that stress could exacerbate the extent of neuronal loss occurring at sites of SND.¹² We were motivated to examine this issue, as it is known that patients recovering from stroke report experiencing high levels of stress.^18,19 Moreover, several pre-clinical studies have demonstrated that exposure to chronic stress post-stroke can increase the severity of both motor and cognitive deficits.^20,21

While the ability of chronic stress to disrupt normal repair and recovery processes has been noted, our understanding of the specific mechanisms involved remains under-developed. In the current study, we were interested in examining whether stress may alter the formation and/or aggregation of Amyloid-beta (Aβ) at sites of SND. Numerous studies have demonstrated significant accumulation of Aβ within the thalamus (a major site of SND) after focal cortical stroke.^17,22–24 Aβ is known to be neurotoxic, and within the context of Alzheimer’s-like pathology increased levels of soluble forms of Aβ, and in particular Aβ dimers, trimers and dodecamers (Aβ × 56), have been linked to cognitive decline.^25,26

The major question that we wanted to address was ‘do animals exposed to chronic stress and stroke respond differently to those exposed to stroke alone?’ We were not interested in the effects of chronic stress by itself, as this phenomena has been examined.^27–29 The addition of a chronic stress alone condition would have substantially and unnecessarily reduced the overall level of experimental power. We therefore have chosen to run a study with three experimental groups: shams, stroke alone and stroke with chronic stress.

To our knowledge, no previous studies have examined whether exposure to chronic stress during the recovery period may influence the levels of Aβ accumulation at sites of SND post-stroke. Accordingly, in the current study, we adopted two complementary approaches to investigate whether exposure to stress during recovery from stroke altered Aβ oligomerization and accumulation within the thalamus. Firstly, we used fresh tissue analysis, using SDS-PAGE in combination with Western blotting, to assess the nature of soluble Aβ oligomer formation within the contra- and ipsilateral hemispheres.³⁰ Secondly, we undertook a fixed tissue immunohistochemical assessment of Aβ accumulation within both the contra- and ipsilateral hemispheres to assess the regional deposition of Aβ. We also quantified infarct volume and examined changes in the numbers of mature neurons (NeuN) within the thalamus.

Materials and methods

Animals

All experiments were approved by the University of Newcastle Animal Care and Ethics Committee, and conducted in accordance with the New South Wales Animals Research Act and the Australian Code of Practice for the use of animals for scientific purposes. This study also complies with the ARRIVE guidelines. Total of 48 C57BL/6 adult male mice (eight weeks old), each randomly allocated to one of the following groups: sham, stroke and stroke + stress (n = 16 per group). Mice were obtained from the Animal Services Unit at the University of Newcastle. Mice were maintained in a temperature (21℃ ± 1) and humidity controlled environment with food and water available ad libitum. Lighting was on a 12:12 h reverse light–dark cycle (lights on 19:00 h) with all procedures conducted in the dark phase under low-level red lighting (40 Lux). Mice were allowed to acclimate for a minimum of seven days prior to the start of the experiment.

Photothrombotic occlusion

Photothrombotic occlusion was performed as described.^12,31 Briefly, mice were injected intraperitoneally with 0.2 mL of 10 mg/mL of rose bengal 8 min prior to 15 min of illumination using a cold light source with a fibre optic end of 4.5 mm diameter placed 2.2 mm lateral of bregma (0.0 mm) onto the exposed skull. Irradiation of the translucent skull using a cold light source following injection with rose Bengal results in coagulation and consequently thrombotic microvessel occlusion. For the sham group, similar procedure was applied except that Rose Bengal was replaced with 0.2 mL of 0.9% saline.

Stress protocol

Mice were exposed to chronic unpredictable restraint stress protocol three days following the photothrombotic occlusion as described.¹² Chronic unpredictable restraint stress is the most widely used and extensively characterised model of chronic stress in rodents.³² Briefly, mice were placed in 50 mL conical tubes with ventilation holes for 2 h, 3 h, 4 h, 5 h or 6 h per day for five days per week randomly, an average of 20 h per week for six weeks in their home cage. The variation in the duration of exposure and time of delivery ensures that the stress response is persistence. This restraint protocol is a well-validated model, which has been shown to effectively induce stress-related behaviours in mice.³² Non-stressed mice had no access to food and water for the same period of time as stressed mice, and were handled for 2 min twice daily throughout the duration of experiment.

Tissue processing

At day 42, post-stroke mice were euthanized. For Western blot analysis, mice (n = 8 per group) were deeply anesthetized via intraperitoneal injection of sodium pentobarbitol and transcardially perfused with ice cold 0.9% saline for 2 min. Brains were dissected and rapidly frozen in −80℃ isopentane. Sections were sliced in a cryostat (−20℃) at a thickness of 200 µm and both contra- and ipsilateral thalamus (bregma −1.0 to −2.2 mm) were punched using a 2 mm tissue punch (Figure 2(a)). Thalamus samples were kept frozen at all times until protein extraction. For immunohistochemistry analysis, mice (n = 8 per group) were deeply anesthetized via intraperitoneal injection of sodium pentobarbitol and transcardially perfused with ice cold 0.9% saline for 2 min followed by ice cold 4% paraformaldehyde (pH 7.4) for 13 min. Brains were removed and postfixed for 4 h in the same fixative then transferred to a 12.5% sucrose solution in 0.1 M PBS for storage and cyroprotection. Serial coronal sections were sliced on a freezing microtome (−25℃) at a thickness of 30 µm and stored in cytoprotection solution at 4℃ until analysis.

Figure 2.

Detection of Aβ oligomers in the contra- and ipsilateral thalamus of sham, stroke and stroke + stress. (a) Schematic illustration of the thalamus.³⁵ The protein homogenates for Western blot analysis were taken across the full extent of contralateral (blue shade, CL Th) and ipsilateral (red shade, IL Th) thalamus. The infract hemisphere is identified with asterisks (*). (b) D3D2N and (c) 6E10 anti-amyloid β antibodies were used to detect soluble Aβ oligomers, specifically decamers (50 kDa), intermediate size oligomers (30–40 kDa), pentamers (25 kDa) and tetramer (20 kDa). The 6E10 antibody picked up additional 75 and 56 kDa bands. The loading controls were performed by the analysis of β-actin. NB: monomers present ( < 10 kDa) but not shown due to the need to use longer exposure times for visualization. D3D2N antibody is used for subsequent Western blot analysis.

Protein extraction and Western blot

Protein extraction and Western blot were performed as previously described with minor modification.³³ Thalamus samples were sonicated in 300 µL of lysis buffer (50 mM Tris buffer pH 7.4, 1 mM EDTA, 1 mM DTT, 80 µM ammonium molybdate, 1 mM sodium pyrophosphate, 1 mM sodium vanadate, 5 mM b-glycerolphosphate, 1 protease inhibitor cocktail tablet, 1 phosphatase inhibitor cocktail tablet, final concentration) with a UP50H microsonicator (Hielscher Ultrasonics GmbH, Germany) for 3 × 30 s pulses at 4℃. Samples were centrifuged at 14,000 g for 20 min at 4℃. The clear supernatants were collected and protein concentrations were determined by Pierce BCA protein assay kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Samples were diluted with lysis buffer to equalize protein concentrations (1.5 mg/mL) and stored at −80℃ until further analysis. Samples were mixed with sample buffer (2% sodium dodecyl sulphate, 50 mM Tris, 10% glycerol, 1% DTT, 0.1% bromophenol blue, pH 6.8) and 15 µg of total tissue protein samples were electrophoresed to Biorad Criterion TGC Stain-Free 4–20% gels. Gels were transferred to PVDF membranes by in transfer buffer (25 mM Tris, 200 mM glycine, 20% methanol, pH 8.3). PVDF membranes were washed in Tris-buffered saline with tween (TBST) (150 mM NaCl, 10 mM Tris, 0.075% Tween-20, pH 7.5) and blocked in 5% skim milk powder (SMP) in TBST for 1 h at 25℃. Membranes were incubated with primary antibodies (anti-amyloid β (D3D2N) #15126, Cell signalling Technology, 1:1000, anti-amyloid β, (6E10) #503002, BioLegend, 1:1000 and anti-β-actin-peroxidase, AC-15 #A3854, Sigma-Aldrich, 1:50,000) and secondary antibodies (horseradish peroxidase conjugated goat anti-mouse, #170-6516, Biorad, 1:10,000). In between each incubation step, membranes were washed in TBST. Membranes were visualised on an Amersham Imager 600 (GE Healthcare Life Sciences) using Luminata Forte Western blotting detection reagents (Merck Millipore). The density of the bands was measured using Amersham Imager 600 Analysis Software.

Immunohistochemistry

For immunoperoxidase labelling, free-floating sections were immunostained as previously described with minor modification.³⁴ Brain sections were identified by reference to the Mouse Brain in Stereotaxic Coordinates.³⁵ All reactions were run at the same time, with the same reagents, at the same concentrations, by an experimenter blind to treatment. All brain sections were incubated with 1% hydrogen peroxidase for 30 min at 25℃ to quench endogenous peroxidase activity and were then incubated with 3% horse serum for 30 min at 25℃ to block non-specific binding. Brain sections were incubated with primary antibody (anti-β-Amyloid (D3D2N) #15126, Cell Signaling Technology, 1:1000 or anti-NeuN (clone A60) MAB377, Merck Millipore) for 48 h at 4℃ and followed by secondary antibody (goat-anti-mouse biotinylated, #115-065-003, Jackson ImmunoResearch) for 1 h at 25℃. Next, brain sections were incubated for 2 h at 25℃ with avidin–biotin-peroxidase complex and then developed using DAB peroxidase substrate. Brain sections were washed with PBS in between each incubation step. After processing was complete, sections were mounted onto chrome alum-coated slides, and cover slipped.

Image acquisition and analysis

Images were acquired at 20× using Aperio AT2 (Leica, Germany). Mosaic images were acquired of the whole brain and sections of interest were identified. The % infarct area was evaluated in brain sections at bregma level + 1.0, 0.0 and −1.5 mm (Figure 1(a)). Area of contra- and ipsilateral hemispheres were traced with ImageJ software 1.50 a, NIH. % infarct size was determined by the equation: [(area of contralateral hemisphere – area of ipsilateral hemisphere)/area of contralateral hemisphere] × 100%.

Figure 1.

Evaluation of infract size after photothrombotic stroke. (a) Representative immunohistochemical staining images of coronal sections in sham, stroke and stroke + stress. The infract area is identified with asterisks (*) and scale bar represents 0.1 cm. (b) Regional distribution of the percentage infarct areas in coronal sections aling the rostrocaudal axis as measured from bregma. The percentage infarcted areas are determined by the equation: [(area of contralateral hemisphere – area of ipsilateral hemisphere)/area of contralateral hemisphere] × 100%. The data are shown as the means ± SEM (n = 8 per group). *p < 0.05, ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons).

The threshold analysis was evaluated as previously described.^12,31,36 Briefly, Matlab software R2015a was used to crop both contra- and ipsilateral thalamus at bregma level −1.5 from the mosaics. Pixel intensity level considered to be optimal for detecting genuine differences in immunoreactive signal was determined using ImageJ software to visualise thresholding of cropped regions at individual pixel intensities. The term ‘% of thresholded material’ refers to the percentage of pixels that were captured at and below pixel intensity 85 to the total number of pixels in each image (Figure 5(a)). These data were used to investigate between-group differences.

Figure 5.

The threshold analysis of Aβ accumulations in contra- and ipsilateral thalamus at bregma level −1.5 mm. (a) Aβ staining (left panels) and the material thresholded at pixel intensity (PI) 85 (right panels) of the ipsilateral thalamus. (b) The number of pixels that were captured at and below pixel intensity 85 was then expressed as a percentage of the total number of pixels in each image and this data were used to investigate between-group differences. The data are shown as the means ± SEM (n = 8 per group). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons).

Manual and blinded cell counts were obtained for mature neurons using NeuN in both contra- and ipsilateral thalamus at bregma level −1.5.

Data analysis

All data for sham, stroke, stroke + stress groups were expressed as mean ± SEM and were analyzed using Prism 6 for Windows Version 6.01, GraphPad Software. One-Way ANOVA was used to determine whether there were any significant treatment effects across the groups. Additional Tukey multiple comparisons were used to analyse differences between the mean of each group with the mean of every other group. The significant differences shown on the graphs with asterisks (*) refer to the post hoc tests. All differences were considered to be significant at p < 0.05.

Results

Chronic stress increased infarct size post-stroke

Photothrombotic occlusion within the (somatosensory) cortex was performed and resulted in relatively consistent infarct sizes for animals in the same treatment group (Figure 1(a)). At bregma +1.0 and 0.0 mm, occluded animals exhibited a significantly bigger infarct area compared to sham animals (stroke, p < 0.001; stroke + stress, p < 0.001), but no significant difference was visible between the stress and non-stress group (Figure 1(b)). At bregma −1.5 mm, animals subjected to stress after stroke exhibited significantly bigger infarct areas compared to animals within the sham (p < 0.001) and stroke alone group (p < 0.05). There was no significant difference between sham and stroke treatment.

Western blot detection of Aβ oligomers in the thalamic tissue

The protein homogenates for Western blot analysis were taken across the full extent of contra- and ipsilateral thalamus and were used to assess precise differences in soluble Aβ oligomers between groups (Figure 2(a)). To ascertain whether these soluble Aβ oligomers exist in wild type C57BL/6 mice, we compared the banding patterns of two different antibodies (anti-amyloid β (D3D2N) and anti-amyloid β (6E10)) using high-sensitivity and high-specificity immunoblots.³⁰ We found that both D3D2N and 6E10 antibodies revealed similar banding patterns at 20, 25, 30–40 and 50 kDa, which most likely representing specific Aβ oligomers of varying sizes (Figure 2(b) and (c)). The 6E10 antibody also picked up additional 75 and 56 kDa bands. Our analysis focused on the soluble Aβ tetramer (20 kDa), pentamer (25 kDa), intermediate size oligomers (30–40 kDa) and decamer (50 kDa), whose effects may be responsible for the cellular pathology associated with Alzheimer’s.³⁷ Monomers were present with both antibodies. However, the band was lighter and required separate exposure conditions to visualize.

Chronic stress increased Aβ oligomers post-stroke

The contra- and ipsilateral thalamic tissues from all groups (sham, stroke, stroke + stress; n = 8 per groups) were analysed by Western blot using anti-amyloid β (D3D2N) for soluble Aβ oligomers. The results for Aβ decamer, intermediate size oligomers, pentamer, tetramer and monomer levels were normalized to β-actin as loading control (Figure 3). The data for all groups were expressed as a fold increase of the mean ± SEM for each group relative to the mean of the sham group. We found a significant increase in Aβ oligomerization in the contralateral thalamus of stroke animals (tetramer, p < 0.05) and stroke + stress animals (decamer, p < 0.001; intermediate size oligomers, p < 0.05; tetramer, p < 0.001) compared to sham animals. We also found a significant increase in Aβ oligomerization in the ipsilateral thalamus of stroke animals (decamer, p < 0.001; pentamer, p < 0.05) and stroked +stress animals (decamer, p < 0.001; intermediate size oligomers, p < 0.05; tetramer, p < 0.05; monomer, p < 0.01) compared to sham animals. Additionally, stroked animals with stress have significant increase in Aβ oligomerization in the contra- and ipsilateral thalamus compared to without stress (Figure 3).

Figure 3.

Chronic stress increased Aβ oligomers in the contra- and ipsilateral thalamus post-stroke. The levels of Aβ (a, b) decamer, (c, d) intermediate size oligomers, (e, f) pentamer, (g, h) tetramer and (i, j) monomer in the contra- and ipsilateral thalamus. The data for all groups were expressed as a fold increase of the mean ± SEM for each group relative to the mean of the sham group. (n = 8 per group). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons).

Qualitative and semi-quantitative immunohistochemistry of Aβ accumulation in the thalamic SND site

Immunohistochemistry staining of anti-amyloid β (D3D2N) antibody was used at a single level (bregma −1.5 mm) to validate Western blot results and to better understand the spatial distribution of Aβ accumulation following stroke (Figure 4). Qualitatively, Aβ staining appeared to be granulated (punctuated). Aβ accumulations were sparsely distributed within the ipsilateral thalamus of stroke animals but densely distributed within the ipsilateral thalamus of strokes animals subjected to stress treatment (Figure 4(b) and (c)).

Figure 4.

Aβ immunostaining in the contra- and ipsilateral thalamus of (a) sham, (b) stroke and (c) stroke + stress at bregma level −1.5 mm. Adjacent high magnifying images depicted Aβ accumulations are sparsely distributed in stroke ipsilateral thalamus and densely distributed in stroke + stress ipsilateral thalamus.

Optical density of Aβ staining within both the contra- and ipsilateral thalamus was then semi-quantitatively assessed using the threshold analysis. Using ImageJ software, we identified that pixel intensity 85 detected genuine Aβ immunoreactive material (Figure 5(a)). The number of pixels that were captured at and below pixel intensity 85 was then expressed as a percentage of the total number of pixels in each image and these data were used to investigate between-group differences. We found a significant increase in Aβ accumulation in the contralateral thalamus of stroke + stress animals (p < 0.001) compared to sham animals (Figure 5(b)). We also found a significant increase in Aβ accumulation in the ipsilateral thalamus of stroked animals (p < 0.01) and stroke + stress animals (p < 0.001) compared to sham animals (Figure 5(b)). Additionally, stroked animals with stress have significant increase in Aβ accumulation in the ipsilateral thalamus compared to without stress (p < 0.05).

Chronic stress exacerbated the loss of thalamic neurons post-stroke

Immunohistochemical staining of mature neurons using anti-NeuN antibody was used at bregma −1.5 mm to investigate the loss of neurons within both the contra- and ipsilateral thalamus (Figure 6 (a)). We found no significantly difference in NeuN positive cells between any of the groups in the contralateral thalamus. We also found significantly lower NeuN positive cells in the ipsilateral thalamus of stroke animals (p < 0.001) and stroke + stress animals (p < 0.001) (Figure 6(b)). Additionally, stroked animals with stress have significant loss of NeuN positive cells in the ipsilateral thalamus compared to without stress (p < 0.05).

Figure 6.

Evaluation of neuron loss in the thalamus at bregma level −1.5 mm after photothrombotic stroke. (a) NeuN immunostaining in the contra- and ipsilateral thalamus of sham, stroke and stroke + stress. (b) NeuN positive cell counts in the contra- and ipsilateral thalamus. The data are shown as the means ± SEM (n = 8 per group). *p < 0.05, ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons).

Discussion

Stroke often produces profound changes in the patient’s life circumstances, with many patients left with severe impairments of motor function, communication and cognition. The suddenness and severity of these deficits can mean that recovery from stroke can be very stressful.^18,19 While the evidence base confirming the presence of high levels of stress in patients recovering from stroke is established,³⁸ remarkably little has been undertaken to determine exactly how exposure to stress during the recovery period influences brain repair processes. In the current study, we were interested in determining how exposure to stress during the recovery period altered the deposition and composition of Aβ at sites of SND. We were motivated to examine Aβ as it is deposited at sites of SND,^17,22–24 is neurotoxic and at high levels can impair synaptic function. Our primary finding was that chronic stress exposure following stroke altered the composition of Aβ oligomers, most notably the increased presence of decamer (50 kDa) and intermediate size oligomers (30–40 kDa), which have previously been linked to serious cognitive decline in the context of Alzheimer’s.^25,39,40 Exposure to stress in the weeks following stroke also resulted in significantly higher levels of Aβ deposition in both the ipsilateral and contralateral hemispheres. We further observed that higher levels of Aβ oligomerization and Aβ accumulation were associated with significant levels of neuron loss in the ipsilateral thalamus.

We and others, have previously identified that photothrombotic occlusion of the somatosensory cortex induces significant levels of SND within the thalamus, inducing pronounced levels of microglial activity and neuronal loss.^12–17 Consistent with these findings, our two complementary approaches used to investigate Aβ, clearly confirmed that the thalamus possessed visibly greater levels of Aβ oligomerization and Aβ accumulation. We further identified that animals exposed to chronic stress after stroke, relative to stroke alone, exhibited significantly higher levels of Aβ oligomerization and Aβ accumulation in both the contra- and ipsilateral hemispheres. To our knowledge this is the first time that this phenomena has been identified.

Aβ is predominately a 40 - or 42-amino acid peptide (monomer, ∼5 kDa). After release, Aβ monomers appear to have a propensity to aggregate into amyloid fibrils that are found in the core of plaques in Alzheimer’s. However, evidence suggests that soluble Aβ oligomers, rather than insoluble fibrils, may be responsible for the cellular pathology associated with Alzheimer’s.³⁰ Particular attention has been focused on the dodecemers (Aβ × 56), decamers, intermediate and low molecular weight oligomers in transgenic mice and Alzheimer’s patients.^25,39,41 To investigate the pattern of oligomerisation post-stroke, we utilised SDS-PAGE in combination with Western blotting. The primary advantage of this approach over immunohistochemical analysis is that it provides direct information on the relative levels of soluble Aβ in various oligomerisation states.³⁰ Several studies have shown that different commercially available antibodies can recognise different Aβ sequences and/or conformations.^25,30,39,42 As such we were first motivated to compare the performance of two of the most commonly anti-amyloid β antibodies; D3D2N, reactive to residues at the amino terminus of Aβ and 6E10, reactive to amino acid residue 1–16 of Aβ. The D3D2N and 6E10 antibodies revealed a highly correlated banding, particularly at decamers, intermediate size oligomers, pentamers and tetramers. For consistency, we used the D3D2N antibody for both Western blot semi-quantitative analysis and immunohistochemistry threshold analysis.

We observed that stroke irrespective of stress status was associated with a significant alteration in oligomerisation status. Specifically, we identified that there was a significant increase in tetramers within the contralateral hemisphere and a significant increase in the decamers and pentamers in the ipsilateral hemisphere of the stroke alone group. In addition, animals exposed to stress exhibited even greater levels of oligomerisation, specifically we identified that there was a significant increase in decamers, intermediate size oligomers and tetramers within the contralateral hemisphere and a significant increase in the decamers, intermediate size oligomers, tetramers, and monomers in the ipsilateral hemisphere. On the basis of these findings, it appears reasonable to speculate that chronic stress exacerbates Aβ accumulation and is associated with formation of higher molecular weight soluble Aβ oligomers in both the contra- and ipsilateral hemisphere thalamic territories at six weeks post-stroke.

In terms of accounting for greater levels of Aβ oligomerisation and Aβ accumulation, there are at least two explanations. The first is that exposure to stress induced fundamental disturbances in the mechanisms involved in Aβ production or clearance. The second is that exposure to stress exacerbated the severity of the primary injury, and therefore triggered greater levels of SND and thus simply represents a natural amplification of the initial stroke response.³⁸ In favour of the latter explanation we identified that, while the tissue loss from the ipsilateral hemisphere at the site of the primary or core infarction was not different, the hemispheric size at the more caudal location assessed was considerably greater. This finding is consistent with data reported by Kirkland et al.,²⁰ in which they observed that post-stroke stress increased infarct size but in a non-uniform manner. Given these findings, we suggest that greater levels of Aβ oligomerization and Aβ accumulation seen at sites of SND are mediated via an increased level of tissue loss. Of course this does not preclude the possibility that stress exposure also alters Aβ production or clearance and further investigations should be undertaken to evaluate this issue.

We further observed that Aβ oligomerization and Aβ accumulation were associated with neuron loss in the ipsilateral thalamus. Aβ monomers have the propensity to aggregate and form oligomers which activate glial cells, as part of the uncontrolled feedforward neurodegeneration loop in Alzheimer’s. Aβ oligomers from injured or dead cells can trigger pathological activation of microglia and astrocytes through various pathways, which in turn induce the production of reactive oxygen species and the expression of proinflammatory signals. These neurotoxic and neuroinflammation signals then lead to neuronal death.⁴³ Consistently, we and others have identified that photothrombotic occlusion of the somatosensory cortex induces pronounced levels of glial activity and neuron loss within the thalamus.^12–17,31 In the context of SND following stroke, the mechanism of uncontrolled feedforward neurodegeneration loop warrants further investigation.

Given the findings that chronic stress increases AB oligomers and AB accumulation in the thalamus following stroke and exacerbates the loss of neurons, it would be of interest in the future to block the signals generated during the stress response, specifically corticosterone (cortisol in humans). There are two common approaches to pharmacologically restricting the actions of corticosterone, the first is restricting the synthesis of corticosterone within the adrenal gland using Metyrapone and the second is inhibiting the glucocorticoid receptor using an antagonist RU-486.

The results from the current study clearly demonstrate that exposure to chronic stress increases the formation of higher molecular weight Aβ oligomers and Aβ accumulation in the thalamus following stroke and exacerbates the loss of neurons. To our knowledge, this is the first time that Aβ oligomerisation levels have been investigated at a site of SND post-stroke and is also the first study to examine whether stress is capable of altering Aβ accumulation at a site of SND. As the association between Aβ and cognitive decline is now reasonably well established, the increased levels of Aβ seen in animals exposed to stress may have clinical relevance. Indeed, given that stress is known to be both high and persistent in individuals that have suffered a stroke, some consideration may be given, in the future, to undertaking clinical research in order to assess stress loads in recovering patients.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article. This study was part supported by the Hunter Medical Research Institute, Faculty of Health and Medicine Pilot Grant and The University of Newcastle, Australia. We would like to thank International Brain International Brain Research Organization for supporting LKO with Young Investigator Travel Program.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

LKO, ZZ and FRW designed the experiment. LKO and ZZ performed the majority of the experiments. LKO and MK provided optimization and quality control of the Western blot protocol. LKO and FRW analysed the data and interpreted the results. ZZ, MK, and MN helped revise the manuscript and LKO and FRW wrote the paper.