Optimising the Photothrombotic Model of Stroke in the C57BI/6 and FVB/N Strains of Mouse

doi:10.21203/rs.3.rs-1122345/v1

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Optimising the Photothrombotic Model of Stroke in the C57BI/6 and FVB/N Strains of Mouse

https://doi.org/10.21203/rs.3.rs-1122345/v1

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The photothrombotic stroke model relies on the interaction between photosensitive-dye and light for clot formation. Interestingly, the relationship between the length of light exposure and stroke-outcome has never been examined. This model has yet to be established in the FVB/N strain, even though stroke-outcomes are strain-specific. Therefore, this study aimed to examine the effect of different lengths of light exposure in two strains of mice on photothrombotic stroke. Male FVB/N and C57Bl/6 mice were subjected to stroke using 15, 18, or 20-mins light exposure. Mice underwent functional testing for up to 7 days. Infarct volume was assessed with thionin staining, and cellular responses to injury analysed via immunofluorescence. Blood brain barrier (BBB) breakdown was assessed using Evans blue dye. Increasing light exposure from 15 to 20-minutes increased infarct volume but not functional deficit. Interestingly, there were strain-specific differences in functional outcomes, with FVB/N mice having less deficit on the hanging wire test than C57BI/6 after 15-minutes of light exposure. The opposite was seen in the adhesive removal test. There was no difference in number of neurons, astrocytes, microglia, macrophages, and T cells between the strains, despite FVB/N mice demonstrating greater BBB breakdown and an enlarged spleen post-stroke. Increasing light exposure systematically increases infarct volume but does not worsen functional outcomes. FVB/N and C57Bl/6 mice exhibit subtle differences in functional outcomes post stroke, which highlights the need to choose tests which are appropriate for the mouse strain being used.

Cellular & Molecular Neuroscience

Neurology

Stroke

photothrombotic stroke

FVB/N

C57Bl/6

strain comparison

ischemia

Stroke is a leading cause of both disability and death world-wide [1]. Despite having such a large impact on patients and an economic burden, there are very limited treatment options for patients. Therefore, there is a large need for the development of new therapies to treat this debilitating condition. Animal stroke models have allowed research and development of new therapies, however, none of these models are a perfect representation of human stroke and there has been a failure to translate these animal studies clinically [2]. Given that stroke is such a heterogenous disease, using a wider range of animal models and species/strains of animals has been recommended to improve these translatability issues [3]. The filament model of middle cerebral artery occlusion (MCA) is the most commonly used stroke model and is advantageous in that it closely mimics the location of human stroke [4]. However, this stroke model is very severe and has large variation in infarct size. The photothrombotic (PT) model of stroke is a recently developed method of inducing focal cerebral ischemia. This method involves injecting animals with the photo-sensitive dye, rose bengal, and then illuminating the skull to cause free radical formation, resulting in damage to the endothelium, platelet aggregation and blood clot formation [5]. An advantage of this model is that it has higher reproducibility compared to the filament model of MCAO. The length of light exposure varies depending on the protocol being followed and typically ranges between 15 and 20 minutes [5–8]. Surprisingly, the relationship between the length of light exposure and stroke-induced damage has not yet been systematically examined.

Additionally, it has been shown in the filament model of MCAO there are differences in functional outcomes and the immune response between C57Bl/6 and FVB/N mice, with C57Bl/6 mice having a greater T helper(Th)-1 compared to Th-2 response, whereas the opposite is seen in FVB/N mice [9]. Furthermore, FVB/N mice have a greater macrophage and neutrophil response compared to C57Bl/6 mice. This translates into FVB/N mice having less functional impairment than C57Bl/6 mice following MCAO. However, there are no reports on the effects of photothrombosis in the FVB/N strain of mice, and therefore it remains to be established whether the strain differences identified in the filament MCAO model of stroke are also evident in this milder model of cerebral ischemia.

Patients experience a breakdown of the blood-brain barrier (BBB) following stroke [10]. Given that the BBB protects the brain against toxins and maintains brain hemodynamics, its disruption results in increased edema and the potential for hemorrhagic transformation [11]. However, this breakdown can also be beneficial given that it allows drugs access to the brain which are not normally permeable across the BBB. In rats following PT stroke, BBB breakdown has been shown to occur by day 1 post-stroke, and increases at 3-days post-stroke [12]. In mice, disruption of the BBB has also been shown to occur around the peri-infarct area as soon as 3 hours [13], and are still seen at 3 days post-stroke [14], BBB disruption was maintained up to 21 days post-stroke in the PT model [15]. Interestingly, there is very limited characterisation of BBB breakdown in the PT model of stroke, and potential strain differences in the BBB response to stroke have never been examined in any model of cerebral ischemia.

Therefore, the aims of the current study were to establish the relationship between the length of light exposure and damage in the PT model, and to characterise the differences in functional outcomes, infarct size, cellular response and BBB breakdown between C57Bl/6 and FVB/N mice in this model of stroke.

Animals

All experimental procedures for this study were approved by a Monash University Ethics Committee (MARP/2017/144) and (MARP/23298) and performed in accordance with the National Health and Medical Research Council of Australia and ARRIVE guidelines for the care and use of animals in research. A total of 65 male FVB/N and 47 male C57Bl/6 mice (8 to 12 weeks old) obtained from the Monash Animal Research Laboratory were included in this study (Monash University, Clayton campus). All mice were maintained on a 12-hour light/dark cycle with free access to water and food pellets and were housed in specific pathogen free cages.

Inclusion criteria and blinding:

To be included in the current study, mice needed to survive for the 7 days post stroke and to have a measurable infarct. In total, 26 mice were excluded as they did not meet this criteria or were euthanised at an earlier time point due to animal welfare.

Experimenters were blinded to all treatments.

Photothrombotic stroke surgery

The PT stroke surgery was performed as previously described by Labat-gest and Tomasi[16]. However, to obtain a concentration-response to light exposure, the light source was turned on for either 15, 18, or 20 minutes in both the C57Bl/6 and FVB/N strains of mice. Sham mice were subjected to the same procedure but did not receive the intraperitoneal (i.p) injection of rose Bengal. For a full description of the PT surgical procedure, refer to the Online Resource.

Functional tests:

The hanging wire and adhesive removal tests were performed as an indicator of functional outcome. These tests were performed the day prior to stroke induction, to establish baseline performance, and then repeated at 24 hours, 72 hours, & 7 days post-stroke.

Hanging wire test:

The hanging wire test was used to assess forelimb strength and grasping ability, providing an indication of motor function. The test was performed as described by Hattori, et al.[17], where mice were suspended by their forelimbs on a wire between two posts 45cm above a foam pillow. The time in seconds (s) for the mice to fall from the wire was recorded with a maximum test period of 300s. The test was repeated three times per mouse, with a 5-minute break period between each test. An average of the 3 individual tests was calculated and recorded as the latency to fall for that time point.

The adhesive removal test:

The adhesive removal test was used to measure sensory function and motor coordination [18]. Mice were placed in an empty cage and allowed to acclimate for 2 minutes. Following this time, an adhesive sticker (8 mm diameter) was placed on each forelimb paw. The mice were then placed back in the empty cage and the time for the mouse to remove both adhesives was recorded. The maximum test period was 300s. The test was repeated three times per mouse with a 5-minute rest period between tests. The average of the 3 individual test periods was calculated and recorded as the time to remove adhesive for that testing period.

Harvesting of tissues:

On day 7 post-stroke, mice were euthanised with inhaled anaesthetic isoflurane (in 2 - 4% oxygen; Issorane, Baxter, Baxter Healthcare Pty. Ltd). Brains were removed from the skull and snap frozen using liquid nitrogen. The spleen and thymus were then removed, weighed, and snap frozen using liquid nitrogen. Tissue samples were then stored at -80°C. Spleen and thymus weight ratio was calculated using the formula: organ weight(mg)/body weight(g)

Preparing tissue sections:

Brain sections were cut using a cryostat (Leica Biosystems) and were thaw-mounted onto Superfrost Plus glass slides (Thermo Scientific; 25 X 75 X 1 mm) and stored at -80°C. To assess infarct volume, 30µm thick coronal sections were collected every 240µm across the span of the infarct. For immunofluorescence, a series of twelve 10µm thick coronal sections were collected every 240µm within the infarct.

Infarct size analysis:

Thionin staining was used to measure infarct volume. Thionin stains Nissl substances in soma and dendrites, where neuronal bodies stain dark blue, neuropil stains purple, whilst the infarcted tissue does not retain stain and appears white [19]. Brain tissue sections (30µm thick) were stained with 0.1% thionin (Sigma, Australia, diluted in acetic acid, Ajax Chemicals, Australia), for 2 minutes, rinsed in dH₂O water for 5s, placed in 70% and 100% ethanol for 2 minutes each, and then coverslipped with xylene (Merck, Australia) and distyrene plasticiser (DPX; Merck, Australia).

The sections were then photographed using a charge coupled device (CCD) camera (Cohu Inc., San Diego, CA, USA) mounted above a lightbox (Biotec-Fischer Colour Control 5000, Reishkirchin, Germany) using IS capture imaging software. Infarct volume was analysed using image analysis software (Image J, NIH, Bethesda, MD, USA). The area of infarct for each section was measured and the sum of the total infarct area for all the stained sections was multiplied by the distance between the sections to give an approximation of the total infarct volume. Oedema was corrected for using the following formula: Corrected infarct area = [left hemisphere area – (right hemisphere area – right hemisphere infarct area) [20].

Immunofluorescence:

For immunohistochemical analysis, 10µm thick tissue sections were taken from mice subjected to either sham surgery or 18 minutes of photothrombosis. The 18-minute light exposure was selected as it produced a sufficient functional deficit in both strains, with the area of damage still being conserved to the cortex. Cellular architecture was assessed using a: mouse anti-NeuN antibody (MAB377, 1:500 dilution; Merck Millipore, USA) to detect the number of viable neurons; goat anti-GFAP antibody (SAB2500462, 1:500 dilution; Sigma) to detect astrocytes; rat anti-F4/80 antibody (MCA497G, 1:100 dilution, Bio-Rad) to detect macrophages; rabbit anti-CD3 antibody (Ab16669, 1:200 dilution, Abcam) to detect T cells; goat anti-Iba1 antibody (Ab5076, 1:400 dilution, Abcam) to detect microglia in the brain following stroke.

NeuN, GFAP, and Iba1 images were captured at 400X magnification using an Olympus fluorescent microscope and CellSens software (Version 1.15; Olympus, USA), 3 images were taken from the infarct region, peri-infarct region, and undamaged region of the stroked hemisphere, and also from the non-stroke hemisphere, in the region corresponding to the infarcted area. For mice subjected to sham-surgery, the region that would be subjected to infarct was duplicated as the peri-infarct region as they are in the same brain region. The number of NeuN positive (+) cells per region were counted using Image J and averaged per region of the brain for each animal. The percentage area of positive staining of GFAP or Iba1 was measured using Image J and averaged per region of the brain for each animal.

For CD3 staining, each hemisphere of the brain was imaged and the total number of CD3 positive cells was counted per hemisphere due to a low number of cells in the brain.

For F4/80, the stroked and non-stroked hemisphere was imaged at 40X magnification, and the percentage area of positive staining was measured with Image J and averaged per region for each animal.

For detailed immunofluorescence protocols and an image of the different brain regions analysed, refer to Supplementary Fig. S1 online. Representative images can be found in the online Supplementary material, Fig. S2 to S5.

Evans Blue:

To assess permeability of the BBB, mice were subjected to 18 mins of PT stroke or sham surgery and were then injected with 2% Evans Blue dye (Sigma) in saline (4mL/KG) at 4 hours post-stroke (i.v). The dye was allowed to circulate for 30 minutes prior to culling . The animals were then euthanised and transcardially perfused with heparinised PBS (10u/mL; Heparin sodium, Hospira, Australia). The brain was removed and imaged.

To quantify the amount of evans blue staining, the % area of blue dye in the hemisphere was measured by tracing the area around the ipsilateral hemisphere and the area of evans blue extravasation. The % area was calculated by: area evans blue extravasation/ area of ipsilateral hemisphere X 100.

Statistical analysis:

All data was expressed as mean ± standard error of the mean (SEM). All statistical analyses were conducted using GraphPad Prism (version 9, GraphPad Software Inc., USA). Functional tests and weight measurements were analysed using two-way repeated measures (RM) ANOVA, followed by Tukey’s and Sidak’s post hoc tests. To assess evans blue extravasation, a one-way ANOVA test was used with Tukey’s post hoc test. To assess infarct volume and immunofluorescence, a two-way ANOVA was used, with Tukey’s and Sidak’s post hoc tests. Probability values of P<0.05 were considered as statistically significant.

Increasing the length of light exposure increases infarct volume in both strains of mouse.

There was a relationship between the length of light exposure and infarct volume (Figure 1, P<0.001). Indeed, mice given 20-minutes of light exposure had a significantly larger infarct volume compared to those with a 15-minute light exposure (C57Bl/6 15-minutes: 9.003mm³ ± 0.79mm³ vs. 20-minutes: 14.02mm³ ± 1.31mm³; FVB/N 15-minutes: 8.19mm³ ± 0.74mm³ vs. 20-minutes: 12.97mm³ ± 1.11mm³; Figure 1b, P<0.001). Interestingly, there was no difference in the infarct volume between the FVB/N and C57BI/6 strains of mice.

C57Bl/6 mice have greater motor impairment and stroke-induced weight loss than FVB/N mice. In contrast, FVB/N mice are more sensitive to the adhesive removal test.

The hanging wire test is a measure of grip strength and motor function post-stroke, with mice expected to fall off the wire faster following a stroke. All C57Bl/6 mice subjected to stroke fall off the wire significantly faster than sham-operated mice at 1-day post-stroke, regardless of the length of light exposure (P<0.001, Figure 2b). In comparison, in the FVB/N strain, only mice subjected to 18 or 20 minutes of light exposure have a hanging wire deficit compared to sham at 1-day post-stroke (P<0.05, Figure 2a). Nevertheless, both strains of mouse appear to have a similar amount of motor impairment when the length of light exposure is increased to 18 or 20 minutes (1-day post-stroke: C57Bl/6: 18 mins: 97.5s ± 30.4s, 20 mins: 85.6s ± 24.7s; FVB/N: 18 mins: 85.8s ± 10.1s, 20 mins: 108.2s ± 29.5s).

The adhesive removal test is a measure of somatosensory function post-stroke, with the length of time taken to remove the adhesives expected to be longer for mice with more somatosensory impairment. In this test, C57Bl/6 mice subjected to stroke took longer to remove the adhesives compared to sham. Increasing the length of light exposure did not influence performance in this test, with all C57BI/6 mice taking a similar amount of time to remove adhesives.

In contrast, in the FVB/N strain, all mice took significantly longer than sham to remove adhesives at 1 day post-stroke, and maintained this deficit at 7-days post-stroke (Figure 2c). Overall, FVB/N mice appear to have a greater sensorimotor deficit in this test compared to C57Bl/6 mice, with FVB/N mice exposed to 20 minutes of light taking 33.3s ± 7.8s to remove the adhesives at 1-day post-stroke. This is longer than the performance seen in the corresponding C57BI/6 group at the same time (20 min: 16.7s ± 5.3s).

In both strains of mouse, regardless of the length of light exposure, mice lost a significant amount of weight across the 7-day testing period compared to sham, with C57Bl/6 mice losing more weight at 1-day post-stroke than FVB/N mice (1 day: C57Bl/6: 15 mins 8.97% ± 1.09%, 18 mins: 9.42% ± 0.42%, 20 mins: 9.10% ± 0.51%; FVB/N: 15 mins: 5.49% ± 0.659%, 18 mins: 7.06% ± 0.56%, 20 mins 6.56% ± 0.62%; Figures 2e and f. 15-and 18-mins: P<0.005, 20-mins: P<0.05). Interestingly, FVB/N mice subjected to sham surgery lost a significant amount of weight at 1- (3.04% ± 0.79%) and 3-days (4.196% ± 1.04%, P<0.05) post-surgery (Figure 2e, P<0.05), however, this surgery effect was not observed in the C57Bl/6 strain (1-day 1.89% ± 0.752%, 3-days: 0.794% ± 1.121%, Figure 2f).

FVB/N mice have heavier spleens than C57Bl/6 mice following stroke.

FVB/N mice subjected to stroke have heavier spleens than C57Bl/6 mice (C57Bl/6: 15 mins: 2.59 ± 0.07, 18 mins: 2.81 ± 0.14, 20 mins: 2.65 ± 0.16; FVB/N 15 mins: 3.37 ± 0.14, 18 mins: 3.77 ± 0.40, 20 mins: 3.56 ± 0.14; Supplementary Fig. S2). Additionally, FVB/N mice given 18 or 20 minutes of light exposure have a significantly larger spleen/body weight ratio compared to sham (FVB/N sham: 2.99 ± 0.1; Supplementary Fig. S2). However, there is no stroke-induced difference in thymus weight between the two strains (Supplementary Fig. S2).

Cellular architecture

In sham-operated mice, there is a consistent number of neurons across the different regions of the brain. However, in mice subjected to stroke, there is a loss of neurons within the brain region directly affected by stroke (infarct region) (Infarct: C57Bl/6: sham: 95 cells ± 5 cells, 18 mins: 10 cells ± 3 cells; FVB/N sham: 110 cells ± 4 cells, 18 mins: 3 cells ± 2 cells; P<0.001, Figure 3a), but not on the peri-infarct region.

PT stroke causes an increase in astrocyte (GFAP) expression within the peri-infarct region of the brain compared to sham (C57Bl/6: sham: 1.56% ± 0.87%, 18 mins: 15.47% ± 2.78%; FVB/N sham: 0.94% ± 0.46%, 18 mins: 17.896% ± 2.95%, P<0.001, Figure 3b). There was also an increase in GFAP staining, albeit to a lesser extent, in the undamaged region of the brain on the stroked hemisphere, (undamaged: C57Bl/6 sham: 2.21% ± 0.72%, 18 mins: 9.02% ± 2.14%; FVB/N sham: 3.74% ± 1.6%, 18 mins: 10.67% ± 3.74%; P<0.05, Figure 3b). Interestingly, there was a trend for an increase in the non-stroked hemisphere compared to sham, although this was not significant (Figure 3b).

Similarly, microglial (Iba1+) expression was also increased in the peri-infarct region of the brain, however unlike astrocytes, this staining was specific to this region, with all other regions exhibiting almost no microglia (Peri-infarct: C57Bl/6: sham: 0.68% ± 0.19%, 18 mins: 32.68% ± 2.94%; FVB/N sham: 0.85% ± 0.16%, 18 mins: 29.64% ± 4.74%). An identical pattern of staining was observed with macrophages (F4/80), with an increase in staining visible in the peri-infarct region, (Infarct hemisphere: C57Bl/6: sham: 0.97% ± 0.50%, 18 mins: 14.04% ± 0.99%; FVB/N: sham: 1.56% ± 1.05%, 18 mins: 9.41% ± 1.59%; P<0.001. Supplementary Fig. S6). Furthermore, initially there appeared to be an increase in macrophages (F4/80) staining in the C57Bl/6 strain compared to the FVB/N strain (Infarct hemisphere: C57Bl/6: 14.04% ± 0.99% vs FVB/N: 9.41% ± 1.59%; Supplementary Fig. S6), this difference was lost once the area of staining is normalised to the infarct volume (C57Bl/6: 1.14% ± 0.12% vs FVB/N: 0.94% ± 0.19%; Supplementary Fig. S6).

Overall, the expression of CD3+ T cells was low in both sham-operated and stroked mice. There was an increase in T cells in the stroke hemisphere compared to the non-stroked hemisphere, although this is only significant in the FVB/N strain (C57 18 mins: non-stroked hemisphere: 5 cells ± 2 cells, stroke hemisphere: 17 cells ± 5 cells; FVB/N 18 mins: non-stroke hemisphere: 7 cells ± 3 cells, stroke hemisphere: 21 cells ± 8 cells; Figure 4).

Overall, there was no difference in any of the cellular markers between the two strains of mice following stroke.

FVB/N mice have greater BBB breakdown compared to C57Bl/6 mice after 18 minutes of PT

Mice subjected to sham surgery show no evidence of evans blue extravasation into the brain, whereas in the mice subjected to stroke, there is a clear increase in evans blue staining specifically around the region of damage. FVB/N mice have a greater percentage area of evans blue dye in the stroked hemisphere compared to C57Bl/6 mice (C57Bl/6: 38.44% ± 3.34% vs FVB/N: 51.61% ± 2.57%; P<0.01, Figure 5). This is most likely due to the fact that in the C57Bl/6 mice, the evans blue dye leakage was confined to the peri-infarct region of the brain, whereas there was leakage throughout the infarct and peri-infarct regions in brain taken from the FVB/N mice (Figure 5), which is reflective of a greater breakdown in the BBB.

The current study demonstrates that increasing the length of light exposure progressively increases the extent of damage in the brain, with longer light exposures resulting in larger infarct volumes. However, this worsening of brain damage was not correlated with more severe behavioural deficits, such that there was a disparity between infarct volume and functional outcomes. Interestingly, while there was no difference in infarct volume between the two strains of mouse used, there was a strain-related difference in performance on function tests. FVB/N mice performed better on the hanging wire test but showed a larger stroke-induced deficit using the adhesive removal test compared to C57Bl/6 mice, indicating that the adhesive removal test may be a better indicator of stroke damage in the FVB/N strain of mouse. While the FVB/N mice strain exhibited a more pronounced BBB breakdown compared to C57Bl/6 mice, this did not translate into enhanced immune cell infiltration into the brain.

In different models of stroke, the severity of brain damage can be manipulated. For example, in the filament model of MCAO, increasing the length of time the filament is in situ causes a more severe stroke [21]. Similarly, in the endothelin-1 model of MCAO, it is possible to increase the volume of stroke damage by increasing the amount of endothelin-1 administered [21]. The current study has shown that stroke severity can be altered by changing the length of light exposure. The PT model is an embolic stroke model where the interaction between light and rose bengal causes clot formation in the microvasculature. Extending the length of light exposure increases the reaction time, and amplifies the clot formation within the microvasculature, which in turn increases the area with reduced cerebral blood flow, and the infarct volume. The ability to manipulate the volume of damage diversifies this model of stroke and presents the possibility that this method of stroke induction could be used to target different brain regions, or to represent a more severe stroke. However, whilst increasing light exposures resulted in greater infarct volumes, this did not translate to a worsening of the physical symptoms of stroke.

Increasing the length of light exposure did not increase functional deficit in C57Bl/6 mice, despite there being an increase in infarct volume. This disparity between infarct volume and behavioural performance has been previously reported in this model of stroke in mice, such that mice with a larger region of damaged tissue do not necessary have poorer outcomes physically [6]. The fact that increasing light exposure does not translate into a generalised worsening of the functional outcomes, suggests that even the shortest length of light exposure (15 mins) completely damages the primary motor cortex (M1) and somatosensory cortex (S1), which are the regions of the brain involved in determining performance on both tests used in the current study. The increase in infarct volume facilitated by lengthening the time of light exposure most likely causes damage in regions adjacent to M1 and S1 which are not involved in performance on either the hanging wire or adhesive removal test. It is possible that a wider range of tests are needed to correctly predict behaviour according to infarct volume or vice versa.

It is known that mice have strain specific responses to injury [22], which may influence stroke outcomes. It is also understood that different mouse strains have different responses to stroke [23]. Indeed, the current study clearly demonstrates that FVB/N mice have better functional outcomes in the mild PT model of stroke compared to C57Bl/6 mice. Specifically, FVB/N mice had preserved motor function according to the hanging wire test following a mild stroke (15-minute light exposure) compared to their C57Bl/6 counterparts who had a significant deficit. This strain specific performance on the hanging wire test has also been demonstrated in the filament MCAO model of stroke. Following a 30 min occlusion of the MCAO, FVB/N mice are reported to have less deficit than C57Bl/6 mice on the hanging wire test, despite both strains having the same infarct volume [9]. In contrast, the FVB/N had much more variable performance on the adhesive removal test and appeared to have an exaggerated stroke induced deficit on this test compared to the C57Bl/6 mice. This clearly highlights the need to choose tests which are appropriate for the strain of mouse being used, for example, the hanging wire test is more sensitive to the stroke effect in C57Bl/6 mice, if 15-minutes of light exposure is to be used, whereas the adhesive removal test is better suited to FVB/N mice.

As expected, C57Bl/6 mice lost more weight post stroke compared to FVB/N mice, which has been previously reported in the MCAO model of stroke [9]. Surprisingly, FVB/N mice experienced surgery-induced weight-loss, such that even the sham group in the FVB/N strain lost a significant amount of weight over the experimental period. This surgery related weight loss is unlikely to be related to anaesthesia, as it has been previously shown that there is no strain specific susceptibility to isoflurane [24]. It is possible that the surgery-induced weight loss is a result of increased anxiety levels impacting feeding behaviour post-surgery. Indeed, it has been shown that FVB/N mice experience more stress-induced weight loss than C57Bl/6 mice after 2 hours of daily restraint over a 10-day period [25].

Despite the PT stroke being a mild model of stroke, there was a robust loss of neurons, as evident by a reduction in NeuN+ staining, in the infarct region. As expected, this loss of neurons was specific to the infarct region only, with all other regions, and sham animals having high neuronal staining. Both strains of mice had similar numbers of surviving neurons, which is consistent with having similar infarct volumes. Similarly, there was also a decrease in astrocytes, macrophages, and microglia in the infarcted region, which combined with the lack of neurons, suggests that this area has undergone necrosis. Furthermore, consistent to what has previously been reported in this model of stroke, astrocytes, macrophages and microglia were all upregulated in the peri-infarct region [26], indicating that these cells are all involved in the endogenous response to stroke. There were also no differences in the amount of positive staining for reactive astrocytes, macrophages, or microglia between the two strains. Interestingly, there was an increase in positive staining for astrocytes across the whole brain, even in regions remote to the site of injury, suggesting that even in a mild, focal model of stroke, the response to injury is a global event involving the entire brain.

Overall, we observed a low expression of T cells after photothrombosis, with a small increase in the number of T cells in mice subjected to stroke within the affected hemisphere. Whether such low numbers of cells are likely to have a physiological impact is unclear. We did observe a slight increase in T cell levels in FVB/N mice compared to C57Bl/6 mice subjected to stroke, which is the opposite to what has been previously shown [9]. It is possible that strain specific differences in the cellular responses to stroke seen previously were not observed because the current stroke model is not severe enough to produce a robust immune response. For noting, the previous study examined immune cells at 24-hours post-stroke, whereas our study looked at the cellular responses at 7-days post-stroke.

There is a greater increase in spleen size in the FVB/N mice following stroke compared to C57Bl/6 mice, despite the fact that both strains have similar spleen weights in the sham groups. In addition, the FVB/N mouse had a greater degree of blood brain barrier (BBB) breakdown than C57Bl/6 mice. This is the first time a difference in the BBB breakdown between these strains has been observed. Surprisingly, this did not correlate with changes in infarct volume, functional outcomes, or direct measurement of immune cell infiltration. However, BBB breakdown was measured at 4.5-hours post-stroke, whereas the other endpoints were assessed at 7-days post-stroke. It is possible that by the 7-day time point, any differences between the two strains had resolved. Indeed, it has been shown that FVB/N mice have elevated levels of neutrophils in the filament model of stroke [9], which is consistent with the elevated breakdown of the BBB, however, neutrophil levels were measured at 24-hours post-stroke.

In conclusion, we have shown that increasing the length of light exposure during PT stroke in causes an increase in infarct volume, but this does not translate into a worsening of functional outcome post-stroke. There are strain specific differences in functional outcomes, with the FVB/N strain of mouse being more resilient against the motor symptoms of stroke, but more sensitive to stroke impact on somatosensory tasks. Similarly, there are strain specific differences in BBB breakdown and spleen size, however, this was not associated with a difference in cellular response to stroke. Now we have more extensively characterised the PT model of stroke, it opens the opportunity to vary stroke severity with this model using different lengths of light exposure. Given that there are limited stroke models and that there has been a failure to translate pre-clinical studies into humans, being able to use photothrombotic stroke to model different levels of stroke severity or location may help improve this issue with translation.

Acknowledgements:
None

Author contributions:

All authors contributed to the study design. Experiments and analysis were performed by AK. The first draft of the manuscript was written by AK and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability Statement:

All data generated or analysed during this study are included in this published article and its supplementary information files.

Funding:

None

Ethics declarations:

Competing interests:

The authors declare no competing interests.

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Optimising the Photothrombotic Model of Stroke in the C57BI/6 and FVB/N Strains of Mouse

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