Previously titled : Distal middle cerebral artery occlusion does not result in depression-like behaviours

Stroke is a devastating neurological injury, which can result Background: in significant cognitive and behavioural deficits. Modelling the disease processes associated with stroke in animals is key to the development of novel therapeutic approaches. However, some aspects of stroke pathophysiology, including neuropsychiatric symptoms, do not translate well from humans to animals. Here, we aimed to investigate the development of post-stroke depression in a rodent model of stroke. The distal middle cerebral artery (MCA) was permanently Methods: occluded by electrocoagulation in adult male C57/Bl6/J mice. Animals were allowed to survive for 6 hours, 24 hours, 2 days, 5 days or 7 days prior to behavioural testing. Brains were taken to confirm lesion volumes at the above times. Behavioural tests studied basic exploration and motivation (open field and marble burying) as well as depression-like behaviours (tail suspension and sucrose preference). Animals developed robust and reproducible lesions in the cortex Results: but whilst stroke reduced activity in the open field, animals showed no associated behavioural deficits in any of the tests used for depression-like behaviours. The distal middle cerebral artery occlusion (MCAO) model Conclusions: results in a small cortical lesion which produces no depression-like behaviours. These negative data are important for those wishing to investigate the more cognitive and behavioural aspects of stroke. 1,2 2,3 2


Introduction
Major depression is an important neuropsychiatric consequence of stroke, and develops in approximately one third of stroke patients, often independent of functional deficits 1,2 . Modelling affective deficits in rodents is key to the development of novel therapeutic strategies, but has proved challenging thus far. Rodent models of post-stroke depression often combine middle cerebral artery occlusion (MCAO) models with models of chronic stress 3 . Whilst this results in a semi-reproducible set of animals showing both lesions and depression-like behaviours, it is not necessarily representative of the human condition.
The majority of ischemic strokes occur in the territory of the middle cerebral artery 4 . As such this is most commonly modelled in rodents 5 . The introduction of the use of endovascular treatment for acute ischemic stroke means that the transient model of stroke has good face and construct validity 6 . However, model variability still blights pre-clinical stroke research and direct translation of useful therapies has been limited 7,8 . The permanent MCAO model results in a consistent cortical infarct which shows reproducible evolution. This model is used to mimic human stroke without reperfusion, which represents the majority of clinical strokes.
The evolution of the lesion results in both local central nervous system (CNS) inflammation and changes in systemic immune reactivity 9,10 . MCAO is known to induce activation of microglia outside the territory of the MCA, even into the contralateral hemisphere 11 , a finding which translates to human stroke where microglial activation has been found in regions distant from the core infarct 12,13 . Inflammation within the CNS, both from internal activation of glial cells and from infiltrating immune cells and inflammatory mediators, is a known causative factor in depressive-like behaviours, both in rodents and humans [14][15][16] . Indeed in a number of CNS diseases, including stroke, widespread inflammation and depression are now being causally linked [17][18][19] . As such, the inflammation associated with the distal MCAO model, which has also been shown to extend into the contralateral hemisphere 20 could potentially result in a depression-like phenotype.
Studying affect in rodents provides its own challenges, with the most common models of depression involving either transgenic animals or models of chronic stress 21 . Combining these approaches with models of stroke results in a rodent model which shows very little face validity, but rather a complex model of vascular disease and stress. In addition, the use of methodologies commonly used to study depression, such as the forced swim test, may be incompatible with animals with limb deficits 22 and, as such, hampers our capacity to investigate depression-like behaviours in rodents.
Therefore, the aim of this study was to investigate the effect of stroke on some very basic tests of exploration, motivation and escape behaviours. We used the distal permanent model of MCAO (pMCAO), which gives consistent cortical lesions with minimal surgical trauma, and aimed to study behaviour and lesion volume to determine whether this model induced a depression-like phenotype.

Methods
Animals: Adult (8-10 weeks), male C57BL/6/J mice (Taconic Ltd., Ry, Denmark) were housed in individually ventilated cages with a standard sawdust and nesting material mix, and kept under diurnal lightning conditions with ad libitum access to food and water. Animals were housed in groups of 5 and an n of 5 was used per group, a single animal was considered as one unit. Animals were pre-assigned to time and sham/stroke groups (see Animal Numbers, Dataset 1 23 ). Experiments were approved by The Danish Animal Inspectorate under the Ministry of Food and Agriculture (permit number: 2013-15-2934-00924) and all efforts were made to minimize suffering, distress and lasting harm.
Surgery: All surgeries were performed under isoflurane anaesthesia (2% in O 2 ). The distal part of the left middle cerebral artery (MCA) was permanently occluded as previously described 24,25 . Briefly, an incision was made between the lateral part of the orbit and the external auditory meatus. A burr hole was made in the skull above the distal part of the MCA. The artery was occluded using bipolar forceps coupled to an electrosurgical unit (ICC 50, Erbe) causing local vessel coagulation, and ensuring a restricted cortical infarct. Animals received a local infusion of lidocaine at the injury site as well as subcutaneous saline/Temgesic and were allowed to recover in a heated environment for 30 minutes prior to being returned to normal housing conditions. No adverse events occurred during surgery.
Behavioural tests: All behavioural tests were performed during the light phase following the NC3Rs guidelines and we have used the ARRIVE checklist when writing our report 26 . Behavioural analysis was videotaped and analysed blind to surgery.
Open field: This test was performed as previously described 27 in a 45cm 3 box with observations taking place over a 10 minute period. Movement was tracked using automated software (SMART v3.0, Panlab, Spain) connected to a camera positioned above the box. The area is divided into peripheral and central units, and locomotion and rearing can be recorded in these units. In the open field, low levels of engagement and interaction with the environment (staying close to the wall, moving around little) are traditionally thought to be traits describing emotionality 28 . Locomotion, rearing and time spent in certain predefined areas of the open field can be measured automatically, rearing was measured manually.
Marble burying: This test was performed as previously described 29 in an open Perspex mouse home-cage (44 × 28 × 12cm) with 5cm of sawdust bedding. Twenty marbles were placed at 2cm intervals in 5 lines of 4. Animals were allowed to explore the cage for 30 minutes and the latency to start digging behaviours and the total number of marbles buried to at least 2/3 of their surface was counted. Latency was analysed manually post-hoc from video footage.
Tail suspension: Animals were individually suspended for a period of 6 minutes. Movement was analysed manually post-hoc from video footage. Immobility was defined as a period of no less than 3 seconds where no active movement was observed, escape activity was measured using a stop-watch and subtracted from the total time measured (6 minutes). Latency to the first period of consistent immobility (≥3 seconds) was recorded on a second stopwatch.
Sucrose preference: This test was performed as previously described 30 . Animals were acclimatized to sucrose with a 1% solution for 24 hours 7 days prior to surgery. No food or water deprivation was implemented. Baseline testing occurred the day prior to surgery. Mice were given 12 hours of free choice between either 1% sucrose or normal drinking water during the dark (active) phase of their cycle. This time frame excluded the possibility of using this test at the 6 hour survival time. The position of the solutions in the cage was switched at 6 hours to prevent side-bias. Percentage preference for sucrose was calculated at the end of the test using the following formula: Sucrose Preference = Volume Sucrose solution /(Volume Sucrose solution + Volume Water ) × 100.
Statistical analysis: All data were analyzed using Graphpad Prism software (version 7; GraphPad Prism Inc., La Jolla, CA). Analysis of data was performed using two-way analysis of variance (ANOVA) or one-way ANOVA, as appropriate. Data were considered significant at p<0.05. Tukey's multiple comparisons post-hoc testing was applied as appropriate. All data sets are presented as mean ± standard error of the mean (SEM); n are included in figure legends.

Distal pMCAO results in lesions which are largest at 24 hours
In order to carry out basic characterization of the distal pMCAO model over a short time, we took brains at 6 hours, 24 hours, 2 days, 5 days and 7 days post-surgery. At no time point did sham animals show any significant CNS histopathology. Distal pMCAO animals showed a variation in infarct volumes over the time course, peaking at 24 hours ( Figure 1A) with the infarct being restricted to the cortex at all time points. However, overall the ANOVA showed no statistical significance. Similarly weight loss in pMCAO animals was higher overall than sham animals ( Figure 1B; two-way ANOVA; p<0.05) but no single time-point was significantly different from its sham counterpart.   The digging behaviour seen in the marble burying test has been shown to be a motivational need in laboratory mice, rather than a direct measure of anxiety 32,33 . Here, all animals showed similar levels of motivation to dig the substrate material when compared to naïve animals, as measured by the latency to start digging ( Figure 2E) and the total number of marbles covered in substrate ( Figure 2F). At no point during the test were sham and pMCAO animals significantly different from each other.

Distal pMCAO does not increase depressive-like behaviours
The tail suspension test (TST) is a commonly used test to explore motivational escape-like behaviours in the study of depression 34 . Similarly, a lack of sucrose preference has been shown to be a good indicator of anhedonia, a depression-like sign in rodents 21 . In both the TST and sucrose preference tests there was no overall difference between post-surgery animals and naïve animals in any of the metrics analysed (Figure 3). In the TST, both latency to the first episode of immobility, and total duration of immobility were not affected by pMCAO, when these animals were compared to shams. Sucrose preference also did not decrease after surgery at any time. In order to determine whether preference was simply a facet of hypodipsia, total volume of liquid drunk was also measured. Whilst this was slightly decreased at 24 hours in all animals when compared to naïve, this did not reach significance, and at no time was the total volume of water missing from the bottles different in pMCAO and sham animals.

Discussion
The emotional disturbances associated with post-stroke depression can severely hinder recovery and, as such, are a crucial area for intervention. However, current anti-depressant therapies, even in uncomplicated major depressive disorder, are ineffective in up to 60% of patients 35 . Investigating new avenues for therapy in rodent models is a key way of addressing this. However, the rodent models used must be representative of the human condition. There is a long-held belief within the pre-clinical stroke community that the distal pMCAO model does not show any behavioural deficits which could constitute a depression-like phenotype but, to date, there are no citeable articles. This is likely due to a general publication bias in The distal pMCAO model produces a robust cortical lesion which varies slightly over time. This data is in line with others showing a large lesion at 24 hours 22,25 . This reduction in the manifest infarct is due to lesion resolution and the destruction of the core of dead tissue, largely by microglia and infiltrating macrophages 36 . The location of the lesion potentially contributes to the development, or lack thereof, of post-stroke depression, although results from studies thus far remain controversial 37 . In humans, there is some evidence for hemispheric bias but these results seem to be hampered by poor patient recruitment, inconsistent methodology and poor controls. Left-hemisphere lesions have been shown to be more commonly correlated with depressive symptoms than right hemisphere lesions 38,39 . However, a systematic review has shown that right-sided lesions may contribute in the sub-acute phase 40 . Whilst some degree of lateralization exists in the rodent brain 41 the degree to which this contributes to emotionality, is still under scrutiny. Rather, the location of the lesion within the cortex may be the cause for the lack of an affective phenotype. Studies using the focal endothelin-1 (ET-1) model of stroke, where the ET-1 was introduced into the pre-frontal cortex, have shown depressivelike behaviours at the chronic time point (>1 week) 42 . The prefrontal cortex is known to play a major role in depression in humans 43,44 and whilst its existence in rodents is controversial 45 , the shift in location from the more functional areas of the cortexsuch as the motor and sensory areas, to the PFC -may be the reason for the depression-like behaviours in the ET-1 model used by Vahid-Ansari and colleagues.
Emotionality in rodents has been traditionally challenging to study. Indeed, in models of depression controlling for slight variables in testing regime can significantly affect outcome 21 . This model of stroke has been repeatedly shown to have minor functional deficits in forepaw function 20,22 . In models of stroke, there has to be some consideration given to a lower degree of locomotor capacity. For example, the tail suspension test remains more appropriate than the forced swim test because of potential motor deficits shown by the MCAO animals. In this study, our data show that there is no significant change in anhedonic behaviours, either tail suspension or sucrose preference, even at 7 days post-stroke. In a bilateral model of global ischemia in the mouse, depression-like behaviours -as measured by sucrose preference and tail suspension -were not present at 7 days, but did develop at one month poststroke 46 . Liu and colleagues suggest that neuronal loss within the hippocampus and hypothalamus may be responsible for this change in behaviour, and that these manifest at later time points in their model. It is possible that there could be some degree of persistent inflammation which continues after 7 days 20 , which may result in the development of depressivelike behaviours, but this was beyond the scope of this study.
In conclusion, this study demonstrates that whilst the distal permanent MCAO model produces a robust cortical lesion and is known to demonstrate markers of the ischemic cascade, relevant to interventional studies 47 , up to 7 days there are no significant affective components to this model and as such, those wishing to investigate depression-like behaviours in rodents post-stroke should consider using alternate models. Whilst this study may have been slightly underpowered in terms of animal numbers, actively pursuing significance with higher numbers would be ethically questionable, and against the principles of reduction and refinement. This data provides citeable evidence that there is no overt depression-like phenotype in the acute phase of the distal MCAO model. Overall the study has several flaws. First the cohort size (5/group) is insufficient and this needs more replication (n = 10-15/group is typical, but larger may be necessary due to stroke variability) to detect behavioral changes. Second, the lesion site is not sufficiently characterized. Third, control measures of fine locomoter activity are not done. Fourth, the tests used are not effective in the strain or not appropriate to assess anxiety/depression. Fifth, only one test of depression test is done; ideally, 2-3 tests for each phenotype need to be done.

Specific comments:
One confound is whether multiple tests were done in the same day; this needs to be clarified: how many animals were used, what tests were done on each group, how many tests/day on each mouse? Fig. 1A: It would be important to show the size and location of the stroke at 24 hr. These strokes are notoriously variable, hence mapping the stroke is important. More details and quantification of the location (Bregma) of the incision and stroke need to be provided. Since the stroke is so large and variable (esp. at 24 h), additional studies to assess fine motor impairments are critical (e.g., ladder test, cylinder test, etc. Motor impairments can affect results in locomotor, anxiety and depression tests. Fig. 2: At none of the time points was any of the outcomes significant, only an overall significance. More data points are needed to test when there is impairment or recovery. Rearing is not specific measure for anxiety: validated tests need to be done (e.g., elevated plus maze, centre time in open field, etc.). Fig. 2: Marble burying was also maximal 20 marbles for all groups, thus may be to insensitive to detect motor impairments. Fig 3. In the sucrose preference test as performed, almost 100% of the mice show 100% preference for sucrose. This raises concern about the sensitivity of the test to detect anhedonia in C57BL6 mice: this strain of mice is unresponsive in this assay even after 9 weeks of chronic stress (Pothion, 2004). Hence the data are inconclusive. 1. Fig. 3: The data for TS are not convincing; the error seems too high since N value is too low. The dashed line needs to show N, mean ± SE of naïve mice: why are sham having more immobility than naïve? 1.

9.
issues which negatively affect the impact of this work in its present form. Group sizes of 5 are exceedingly low, but the sentence 'Whilst this study may have been slightly underpowered in terms of animal numbers, actively pursuing significance with higher numbers would be ethically questionable, and against the principles of reduction and refinement.' is absurd and unscientific. In fact, underpowered studies are unethical, as pointed out by Cressey D . An informal, post hoc power calculation on their data set reveals that they may have achieved only 10-20 % power (instead of 80 or 95%). This is particularly worrisome, as they report a NULL result, so false negatives are a major concern.
The study is lacking a justification of the sample sizes, and Type II error considerations. What are the effect sizes the study could have possibly detected?
In general, the test statistics of this study don't make a lot of sense. Although they are formally correct, in the absence of the definition of one primary outcome, and the fact that they performed at least 12 ANOVAs (for which they did not correct), as well as given the exploratory character of the study, test statistics should be avoided, and the focus should be on effect sizes and variance. This brings me to another problem: The authors use SEMs (a measure of precision, not of the spread of the data) and they use bar graphs, instead of dot plots (individual data points) and true measures of variance to illustrate their data. The authors do not mention how many animals went into the study (ARRIVE guidelines), they only mention group sizes (in the figures). Only downloading the full data set reveals that there obviously was no attrition, although there are unexplained missing data points. The authors are strongly advised to consult a statistician. External validity I: Left sided occlusion only! Kronenberg et al , in a mouse model of transient proximal occlusion demonstrated that left, but not right, middle cerebral artery occlusion leads to chronic 'Depression-Like' behavior, which was reversed by serotonin reuptake inhibition . This article, as well as a number of other relevant publications pertinent to the subject of modeling of PSD in rodents, is not cited. Although Gammelstrup Andersen used left sided occlusion, it at least demonstrates the potential of lateralization of behavioural symptoms in rodents. External validity II: Only male mice are studied, and of an age range and without comorbidities. This does not reflect the cohort of patients which are at risk for stroke and PSD. The title of the study 'Distal middle cerebral artery occlusion does not result in depression-like behaviors' is imprecise and should contain at least the species, but ideally more information on the model. I suggest: An exploratory investigation of 'depression-like' behaviours in a model of left-sided distal middle cerebral artery occlusion in young, male C57B6 mice.
In summary, I strongly recommend to change the graphical presentation of the data, redo the statistics (descriptive instead of test), discuss the limitations (in particular the exceedingly low power, and issues of external validity), and include some of the missing literature.

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly No competing interests were disclosed. Competing Interests: I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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