Neuroinflammation as common denominator in heart failure associated mental dysfunction
Gouweleeuw, Leonie
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10.33612/diss.122192415
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Gouweleeuw, L. (2020). Neuroinflammation as common denominator in heart failure associated mental
dysfunction: Studies in animal models. University of Groningen. https://doi.org/10.33612/diss.122192415
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CHAPTER 4
Sex specific associations between
microglia activity and behaviour
after cardiac surgery in rats
Leonie Gouweleeuw1, Hui Liu1, Raffaele Altara2, Ulrich L.M. Eisel1, Regien G. Schoemaker1,3 1 Department of Molecular Neurobiology, Groningen Institute of Evolutionary Life Sciences, University of Groningen, Groningen, the Netherlands.
2 Department of Pharmacology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands
3 Department of Cardiology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ, Groningen, the Netherlands.
Abstract
Background: Neuroinflammation, including microglia activation, is thought to be a mediator
in behavioural and cognitive changes after myocardial infarction (MI) or cardiac surgery. Studies in rodent models of MI have found neuroinflammatory and behavioural changes including depressive like behaviour and anxiety. In patients, women are more prone then men to develop depression or anxiety after MI, possibly caused by underlying differences in inflammatory pathways between men and women. Aim of this study was to investigate the link between (neuro)inflammation and behavioural changes in male and female rats undergoing sham or MI surgery.
Methods: Material and data collected from a previous study were used to study plasma and
CSF cytokines and microglia activation in hippocampus, amygdala and paraventricular nucleus of the hypothalamus in male and female rats after sham or MI surgery. Pearson’s correlations were performed between behavioural parameters and microglial activity, separated by sex.
Results: Plasma levels of IL-2, IL-6, MIP-1β, TNF-α, and sTREM-1 were found to be upregulated in male MI rats vs male sham rats, whereas in female MI rats, no changes in plasma cytokines were found. In CSF, IL-1α and IL-13 were upregulated in male MI rats, and IFN-γ was upregulated in female MI rats. Microglial activity was associated with behaviour, especially in male rats.
Conclusion: The results in this study indicate that peripheral inflammation following MI is
different in male vs female rats. We showed that the association between microglia activity and behaviour differs between male and female rats. Results suggest that inflammatory pathways following MI may be sex specific.
Background
Myocardial infarction (MI) and cardiac surgery are well recognized for their risk on changes in mood and cognition, in patients as well as animal models [1-3]. Major depressive disorder is one of the most common (up to 40%) co-morbidities in MI patients and adversely affects prognosis [4, 5]. As MI, surgery and depression share increased levels of the same circulating cytokines [6-8], we hypothesize that the inflammatory response might be the mediating factor in the neuro-cardiac interaction. Acute MI or cardiac surgery evokes activation of the innate immune system necessary for wound healing. This response is not limited to the injured area, as circulating cytokine levels increase as well [6-8]. Afferent nerve stimulation [9], in addition to leakage of the blood brain barrier [10, 11] contributes to a reflection of this inflammatory response in the brain, presented as neuroinflammation, which could lead to changes in mood and cognition. It is well accepted that cytokine signalling modulates brain functions, such as synaptic plasticity, neuroendocrine function, neurotransmitter metabolism, and the neural circuitry of mood [12]. Our previous studies have shed light on several mediators of inflammation in these processes. Thorax surgery as well as experimental MI in rats led to increased cytokine levels [13]. Furthermore, increased plasma and brain levels of the inflammatory marker Neutrophil gelatinase-associated lipocalin (NGAL) are observed after MI, and associated with behavioural outcome in male, but not female rats [14]. Interestingly, gender differences have been revealed in both neuropsychological and inflammatory response to MI. Women display a higher risk of developing depression in the general population, as well as among cardiac patients [15, 16]. Moreover, Tumour Necrosis Factor alpha (TNF-α), a cytokine indicated to play a major role in neuroinflammation and depression after MI [17-19] shows sex-related differences in mice [17] and rats [19].
In a model of post-operative cognitive decline, we showed correlations between hippocampal NGAL and behavioural outcome, which were associated with neuroinflammation, measured by microglia activity, in the hippocampus [20]. Also myocardial infarction can lead to long-term local changes in microglia activity in the brain in animal models [21-23], while microglia activation has also been associated with depression [24]. Recently, we developed a new method for measuring microglia activity based on morphological changes; cell body size to total cell size ratio [25]. In the present study, we aimed to investigate these morphological changes in microglia in male and female rats with MI and the relation with behavioural changes, using brain tissue obtained in our previous study [14].
Methods
Rats
The study was performed on brain material obtained from our previous rat study on sex differences in response to MI [14]. For details about the experimental procedures for behavioural testing and tissue processing we kindly refer to this publication. Briefly, young adult male and female Wistar rats (10 weeks old) were obtained from Envigo (Venray, the Netherlands). Animals were housed in groups (2 to 3 animals per cage) under standard conditions at reversed 12h light/dark cycle at animal facilities of the University of Groningen. All animals were fed standard diet (standard rodent chow, Hope Farms, Woerden, The Netherlands) and received food and water ad libitum. All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Committee for the Animal Experiments of the University of Groningen.
Surgery
Rats were randomly subjected to either permanent coronary artery ligation or sham surgery, as described previously in detail [14, 26]. Under isoflurane anaesthesia and artificial respiration, MI was induced by ligating the left descending coronary artery. In sham operated rats, the same surgery was performed without the actual ligation. Analgesia was performed intraoperatively by an intercostal block with Marcaine, and postoperatively with Buprenorphine (0.003 µg/kg, sc).
Behavioural tests
Behavioural tests were executed as described in detail previously [14]. Briefly, all tests were performed in the dark phase, starting three weeks after surgery. Exploratory behaviour was tested by the open field test, motivation by the free exploration test, and anxious behaviour was examined in the elevated plus maze. The open field test was performed in a circular open field with a diameter of 140cm. The rat was allowed to explore the new environment for 5 minutes. Distance walked was tracked with Ethovision 3.0 software (Noldus Information Technology). Time spent on exploratory behaviour (walking, rearing, sniffing) was scored using an in house designed software program to track behaviour manually. In the free exploration test, the rat in its home cage with the lid open was placed in the open field, and allowed to choose to explore the environment by climbing out of the cage. Latency to exit the home cage and time spent exploring the inside of the cage vs the outside environment was obtained as measure for motivation [27]. The elevated plus maze consisted of a plus-shaped maze with two opened and two closed arms (10x50cm) located 50cm above the floor. Rats were placed in the centre of the maze, facing an open arm, and allowed to explore the maze
for 5 minutes. Number of entries was recorded and the relative time spent in the open arms was taken as reciprocal degree of anxiety.
Sacrifice and tissue processing
Rats were terminated as previously described [14]. Briefly, rats were anesthetized with pentobarbital, a blood sample was taken through cardiac puncture and rats were transcardially perfused with 0.9% NaCl. Cerebrospinal fluid (CSF) was obtained and immediately frozen in liquid nitrogen. The brain was excised and fixated in freshly prepared 5% paraformaldehyde in 0.01M PBS for 72 hours at 4°C. Brains were cryoprotected in a 30% sucrose solution for 16 hours and then frozen in liquid nitrogen and stored at -80°C. The heart was dissected and weighted. A mid-ventricular slice was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for at least 72h, dehydrated and embedded in paraffin for measurement of infarct size [14].
Measurement of infarct size
Deparaffinized 5µm sections were stained with Sirius Red and fast green to distinguish infarct from viable tissue. Methods as well as the measurements of infarct size were described in detail by van Kerckhoven et al [21]. Infarct size was expressed as percentage of left ventricular circumference.
Cytokine measurements
To identify the potential differences in the change of cytokine levels in male and female rats, screening tests of plasma and CSF were conducted using a proteome profiler array (Proteome Profiler Cytokine Array Panel A; R&D Systems, Minneapolis, MN, USA) as previously described [28]. Since only up to 250 µl CSF per rat can be collected and 1 ml was necessary for analyses, four rats from each group were randomly chosen and their CSF or plasma samples were pooled. The pooled samples were centrifuged at 10 000g for 5 min at 4°C. Then the supernatant (1000ul) was conjugated with 36 different antibodies that were spotted in duplicate on a nitrocellulose membrane. The membranes were incubated with IRDye® 800CW Streptavidin (LI-COR®) and were scanned using an Odyssey® imaging system (LI-COR®). The density of each spot on the membranes represented the level of the corresponding cytokine. Cytokines/spots that had very low densities (<0.3 for males and <0.2 for females after normalization for the negative control) were excluded. An MI/sham ratio of the density value above 2.0 (or below 0.5) was considered to represent a relevant change.
Microglia staining
Brains were cut on the cryostat in the coronal plane into 30 µm thick sections. Microglia staining and analysis was performed as described in detail previously [25]. Briefly, free floating sections were stained with an anti-Iba-1 antibody (Wako, Neuss, Germany) diluted in phosphate-buffered saline, 0.01 M (PBS) containing 2% Bovine Serum Albumin and 0.1% Triton X-100. Immunohistochemistry followed the peroxidase method with a biotinylated secondary antibody (rabbit anti-goat, Jackson Immuno Research, UK, 1:500 dilution), ABC Elite reagent (Brunsing Chemie, Amsterdam, the Netherlands) and diaminobenzidine (DAB, Sigma Chemical Co., St. Louis, US) as a chromogen. Pictures were taken at 200x magnification using a Leica microscope. Image analyses were performed using Image Pro Plus 6.0 (Media Cybernetics, Inc. Rockville, USA). Appropriate gain and black level settings were determined on control tissues (sham animals). The total Iba-1 positive area, the number of microglia profiles, and the area of the cell bodies were measured in the amygdala, hippocampus and paraventricular nucleus of the hypothalamus (PVN). Subsequently, the cell body to total cell size ratio was calculated as an index of microglia activation [25].
Data analysis
Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using IBM SPSS Statistics 22 (IBM, Armonk, New York, US). Two-way ANOVA was used to evaluate specific differences between the MI group and the sham group and females and males. For comparison of means, rats with < 20% MI size were excluded from the above analysis, because of hemodynamic compensation [29]. Correlations between microglia parameters and behavioural measures were analyzed using Pearson’s correlation (one-tailed); all rats with a quantifiable MI were included in these analyses. A p-value of less than 0.05 was considered statistically significant.
Results
Heart failure parameters and behavioural data
Coronary artery ligation led to mortality in 9 out of 27 male rats and 10 out of 35 female rats. Heart failure related parameters are shown in Table 1. MI rats showed signs of heart failure evident from increased ratio of lung weight to body weight. We also observed a trend towards higher heart weight to body weight ratio. Outcomes of the behavioural tests are shown in Table 2. Sex effect is seen in all behavioural tests, with females showing a higher degree and motivation for exploration and less anxious behaviour. However, there were no significant differences between sham and MI regarding exploratory or anxious behaviour.
Table 1. Heart failure parameters, as adapted from Gouweleeuw et al. 2016 [14] Sham M MI M Sham F MI F Sex
effect F value MI effect F value Interaction effect F value N 22 13 19 15 - - -Body weight 408±11 416±20 239±4 248±6 249.8** 0.57 0.00 Infarct size (% of LV) - 33±3 - 36±2 - - -HW/BW 3.2±0.1 4.0±0.2 3.7±0.1 3.8±0.4 0.33 3.90# 2.33 LW/BW 3.3±0.1 5.7±1.0 4.7±0.3 6.5±0.9 3.50 12.47** 0.39 F=female, HW/BW=heart weight per body weight, LV= left ventricle, LW/BW=lung weight per body weight, M= male, MI= Myocardial infarction, SpleenW/BW=spleen weight per body weight. Data are presented as mean ± SEM. *=p<0.05 vs sham
Table 2. Behavioural parameters, as adapted from Gouweleeuw et al. [14] Sham M MI M Sham F MI F Sex
effect F value MI effect F value Interaction effect F value N 22 13 19 15 - - -Behaviour Interest in environment Exploration (% of total time) 88±3 88±3 94±1 91±1 7.87** 0.01 0.00 Total distance (cm) 3325±207 3264±179 4094±198 4339±261 17.4** 0.17 0.48 Time in center (% of total time) 13±2 13±3 16±2 16±2 2.25 0.03 0.03 Motivation
Explore ratio in/out 0.53±0.05 0.73±0.10 0.55±0.06 0.65±0.12 0.18 3.56# 0.42
Latency (s) 58±9 55±13 34±7 19±3 11.65** 1.07 0.47
Anxiety
EP Time in open arms (% of total)
10±3 13±3 22±5 28±4 12.94** 1.36 0.14
EP= elevated plus maze, F=female, HW/BW=heart weight per body weight, LV=left ventricle, LW/BW=lung weight per body weight, M=male MI=myocardial infarction, SpleenW/BW=spleen weight per body weight. Data are presented as mean + SEM. #p<0.10, **p<0.01
Cytokines
Among the 36 tested cytokines, 11 cytokines were expressed in sufficient density. From these, the levels of circulating IL-2, IL-6, MIP-1β, TNF-α, and sTREM-1 were increased over 2-fold in male MI compared with male sham rats. No cytokines were found to be consistently up-regulated over 2-fold in plasma of female MI compared to female sham rats. In CSF, IL-1α and IL-13 were up-regulated over 2-fold between male MI and sham rats, while in female rats, IFN-γ was up-regulated. None of the cytokines was downregulated more than 2 times in MI versus sham in either sex (Figure 1).
Microglia analysis
Microglia were analyzed in the hippocampus, amygdala and PVN. We measured the total surface area of the microglia per field of focus, the number of microglia and the activity score. Activity score was defined as the ratio of cell body/total cell size. No MI or sex effect was seen for these measurements in any of the regions analyzed, nor did we find a significant interaction effect. A representation of the data can be seen in Figure 2.
Correlations microglia activity and behaviour
To investigate possible associations between behaviour and microglia activity, Pearson’s correlations were performed for male and female rats separately. Since we did not find significant differences between MI and sham rats in behaviour or microglia data, data for sham and MI animals were pooled. Results of correlations between microglia activity and behaviour are shown in Figure 3. In male rats a higher microglia activity in the amygdala showed significant association with a lower degree of exploration in the open field test and a higher latency to exit the home cage in the free exploration test, but no association was observed with time in open arms in the elevated plus maze. Hippocampal microglia activity was not associated with behavioural outcome, though a trend could be observed regarding the correlation with latency to exit the home cage in the free exploration test (p=0.070). PVN microglia activity was only significantly associated with latency in the free exploration test, but not with other behavioural parameters. In female rats, a negative correlation was found between microglia activity in the hippocampus and time spent in open arms in the elevated plus maze.
Figure 1: Cytokine array measurements. A) Nitrocellulose membrane of the proteome profiler for plasma samples;
TNF-α spots are indicated as example. B) Results from male plasma. C) Results from female plasma. D) Results from male CSF. E) Results from female CSF. …..line of equal expression between sham and MI rats; ---line of twice as
much expression in MI versus sham. When ratio MI/sham was higher then 2, this was considered a relevant increase.
Figure 2: Example pictures of the microglia staining in the hippocampus in male sham (A), male MI (B), female sham
(C) and female MI (D) rats. E) Results of the total surface area of the microglia in the different brain areas. F) Results of the hand counted number of microglia in the different brain areas. G) Results of the ratio cell body/total cell size (microglia activity score) in the different brain areas. Mean ± SEM.
Discussion
Neuroinflammation is considered an important mediator for behavioural changes due to peripheral inflammatory events. Microglia activation is generally accepted as indicative for neuroinflammation. We previously showed an association between microglia activity and exploratory behaviour in rats undergoing abdominal surgery [20]. Thoracic surgery and myocardial infarction are associated with neuroinflammation, as well [13]. In the present study, we aimed to explore whether microglia activity is also associated with behavioural parameters in rats undergoing either sham surgery or coronary artery ligation. In our previous study, we observed sex differences in inflammation and behaviour in MI rats [14]. In the present study, no sex or MI difference was found in the total microglia surface area, number of microglia or microglia activity score in the amygdala, hippocampus or PVN. Others have shown differences in microglia activity between sham and MI rats, at least in the PVN, starting 24 hours after surgery and lasting at least until 16 weeks after surgery [21, 22]. In a previous study, we did find significant microglia activation in the hippocampus of MI rats compared to non-surgical controls, but not compared to sham surgery, 2 weeks after surgery. Sham surgery by itself already induced microglia activation [13]. Accordingly, a difference in microglia morphology between non-surgical controls and MI rats was also found by Rinaldi
Figure 3: Pearson’s correlations between microglia activity and the different behavioural parameters exploration
et al [23]. Moreover, atdifferent ages microglia morphology already differs between males and females [30], suggesting different starting conditions at the induction of MI. Because we observed differences between male and female rats, but not between MI and sham rats, we analysed association between microglia parameters and behaviour in males and females separately, but pooled the data for MI and sham rats. Amygdala and PVN microglia activity in male rats were associated with parameters of exploration and motivation, whereas in female rats microglia activity could only be associated with anxiety. This indicates that microglia could indeed be a mediator in myocardial infarction or surgery related behavioural changes, including anxiety and depression and that the association between neuroinflammation and behaviour is differentially regulated between male and female rats. This difference may then be reflected in the different expression patterns of plasma and CSF cytokines we observed. Several of these cytokines have been described as overlapping in heart failure and depression in patients, without taking sex differences into account [8]. In the literature usually only male rats have been used. In accordance with our findings, plasma TNF-α in male MI rats is elevated at least up to 6 weeks after MI [31, 32] and reflected in increased TNF-α expression in the hypothalamus, both at mRNA and protein level. Although we are not aware of any study reporting Il-1α in CSF, as we observed in our male rats, recent studies have identified this cytokine as a key danger signal triggering the inflammatory response after myocardial infarction [33, 34]. IFN-γ as generally known to polarize macrophages to a proinflammatory phenotype. Hence, increased IFN-γ expression in female CSF may represent general inflammation. Not so much is known regarding sex-related differences, but for TNF-α in animals clear male to female differences have been observed. Female rats seem to be more sensitive to TNF receptor 2 mediated effects [18], and less sensitive to TNF receptor 1 mediated effects [19].
Conclusions
In conclusion, although neither behaviour nor microglia activity showed MI-related differences, correlation analysis suggests associations between behaviour and neuroinflammation, which differ between sexes. More research is required to further establish the nature of these differences.
Acknowledgements
Many thanks to Jan Keijser, Wanda Douwenga and Bert Venema for their technical assistance. We also acknowledge Leroy Schreuder, Walderik Zomerman and Nienke de Wit for help in carrying out the behavioural tests and tissue processing and staining.
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