Neuroinflammation as common denominator in heart failure associated mental dysfunction
Gouweleeuw, Leonie
DOI:
10.33612/diss.122192415
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2020
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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 5
Differences in the association
between behavior and neutrophil
gelatinase-associated lipocalin
in male and female rats after
coronary artery ligation
Leonie Gouweleeuw1, Iris B. Hovens1, Hui Liu1,2, Petrus J.W. Naudé1,3, Regien G. Schoemaker1,4
1Department of Molecular Neurobiology, GELIFES, University of Groningen, Groningen, The Netherlands.
2Department of Neurology, Zhongnan Hospital, Wuhan University, P.R. China
3Department of Neurology and Alzheimer Research Centre, University Medical Centre Groningen, Groningen, The Netherlands.
Abstract
Heart failure is associated with an increased risk of developing depression and cognitive dysfunction, which negatively affects prognosis. Plasma levels of neutrophil gelatinase associated lipocalin (NGAL) are increased in heart failure and depression. Moreover, NGAL levels are associated with depression in heart failure patients. Since women are at a higher risk of developing comorbid depression with heart failure, the aim of this study was to examine sex differences in the link between NGAL and behavior in a rat model of heart failure.
In young adult male and female Wistar rats, myocardial infarction (MI) was induced by means of coronary artery ligation, while control rats received sham surgery. We analyzed aspects of cognition and depression/anxiety using various behavioral tests starting three weeks after surgery. Hemodynamic measurements were performed and hearts and lungs were weighed. NGAL levels in plasma, cerebrospinal fluid (CSF) and brain tissue were analyzed.
MI induced impairment in cardiac contractility and relaxation, and an increase in lung weight. NGAL correlated with signs of heart failure in male, but not female rats. Male MI rats displayed cognitive problems, but not depressive-like or anxiety-like behavior. No behavioral effects of MI were observed in female rats. Plasma NGAL levels were higher in male than female rats with higher concentrations in MI compared to sham. CSF NGAL was higher in MI rats compared to sham and higher in males compared to females. The number of NGAL positive cells in the paraventricular nucleus of the hypothalamus (PVN) was only increased in male MI rats. In male, but not in female rats, NGAL levels correlated with depressive-like behavior and cognitive dysfunction.
Data indicate that while MI increased NGAL levels in plasma, CSF and PVN, correlations of NGAL with behavior are sex-specific, but independent of whether sham or MI surgery was performed. This suggests that inflammatory processes related to thorax surgery and their potential effects on depressive-like behavior and cognition may be sex -specific.
Introduction
Heart failure is the ultimate result of many cardiovascular diseases and has a very poor prognosis. It is associated with a wide range of comorbidities including renal dysfunction, skeletal myopathy, COPD, sleep apnea, liver dysfunction, and diabetes, with inflammation as suggested common denominator [1]. Changes in mood and cognition, though just as common and detrimental as the comorbidities mentioned above, are often overlooked. Studies indicate that up to 27% of heart failure patients ≥ 50 years of age meet the criteria for major depression and up to 74% of heart failure patients report depressive symptoms. Moreover, depression in heart failure patients ≥18 years of age is associated with an increased risk of hospitalizations and mortality. Accordingly, several studies in animal models have found evidence that there is an association between behavior and cardiovascular parameters [2, 3].
Women have been reported to be at higher risk for heart failure associated depression [4, 5]. Similarly, a high number of heart failure patients suffer from cognitive problems [6, 7], which also negatively affect prognosis [8]. Sex differences in cognitive impairment have been reported in heart failure patients, but are inconsistent. While Ghanbari et al. reported a higher prevalence of cognitive impairment in female patients [9], Pressler et al. reported a higher prevalence of cognitive impairment in male patients [10]. Others found that only age, but not sex, was associated with cognitive impairment [11].
Since optimal cardiovascular treatment does not reduce depressive symptoms and effective treatment of depressive symptoms does not improve cardiovascular prognosis [12], the relationship between mental dysfunction and heart failure may be attributed to common risk-factors, rather than represent a causal relationship. Inflammation may provide an interesting candidate for this interaction [1], since both cardiovascular disease and mental morbidities have been associated with overlapping pro-inflammatory processes [13]. Hence, we are searching for specific markers that could link heart failure to alterations in mood and cognition.
Neutrophil gelatinase associated lipocalin (NGAL) is classically known as a marker for renal damage [14, 15], but has recently been shown to reflect inflammatory damage in a variety of tissues, including the brain and heart [16, 17]. NGAL plays a role in the innate immune system
of NGAL have been reported [16, 19, 20], and we recently described a link between NGAL and depressive symptoms in heart failure patients [21, 22]. The role for NGAL as potential mediator of depression and cardiovascular disease was extensively reviewed by Gouweleeuw et al. [23]. Therefore, NGAL may provide an interesting candidate to link neurocognitive dysfunction and heart failure.
The aim of the present study is to investigate the association of NGAL with behavior in heart failure induced by chronic myocardial infarction (MI) in rats. In this model, cardiac dysfunction and neuroinflammation have been previously documented in young adult male rats [24-26]. In these rats we previously showed behavioral changes that could be associated with depression from 3 weeks after infarction on [26, 27]. Since prevalence of depression is sex-dependent, both male and female rats were included in the present study.
Materials and methods
Animals and surgery
Male Wistar rats of 290-320 grams and age-matched female Wistar rats (around 10 weeks old) underwent either sham surgery or coronary artery ligation to induce MI as described previously [26, 27]. MI surgery was induced in 27 male rats and 35 female rats and sham surgery in 22 male rats and 19 female rats. Briefly, rats were anesthetized with 2.5% isoflurane in 0.9 L oxygen/air (1:2)/min. After intubation, the rats were put on a mechanical ventilator (frequency 90/min), a left-side thoracotomy was performed, and the left coronary artery was ligated. In sham-operated rats, the same surgery was performed, without the ligation. Analgesia was performed intraoperatively by an intercostal block with Marcaine, and postoperatively with Buprenorphine (0.003 µg/kg, sc). Rats were socially housed in groups of 2 or 3 per cage in a controlled environment with ad libitum access to standard rodent chow and tap water. After the surgery, rats were placed on a reverse 12:12 light/dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee.
Behavioral tests
Rats were subjected to behavioral tests to evaluate behavioral changes starting three weeks after the MI or sham surgery, when irreversibly damaged myocardium is replaced by scar tissue. All tests were performed during the dark (active) phase of the animals. Depressive-like behavior was obtained from interest in environment as measured by exploratory behavior in the open field and motivation to do so in the forced and free exploration tests respectively [26]. Anxiety was tested in the open field and elevated plus maze, while cognitive
performance was evaluated in the novel object and novel location test. Test results were evaluated blinded.
Interest in environment
As reduced interest in environment is one of the main symptoms of depression in humans, this aspect was tested in behavior in a novel environment for the rat; the open field. The open field test was conducted on day 24 after surgery and previously described and validated as a test for depressive-like behavior [28-31]. Open field was performed in 22 male sham rats, 13 male MI rats, 19 female sham rats and 15 female MI rats. The rat was placed into the middle of a circular open field with a diameter of 140cm. The arena was subdivided in the center (diameter 70cm) and border zone. Activity of the animals was recorded for 5 minutes with Ethovision 3.0 software (Noldus Information Technology) and analyzed for distance walked and time spent in the center and border of the open field, while time spent on different behavioral elements was measured using E-line software. The percentage of time spent on the behavioral elements sniffing, walking, exploration of walls and rearing were used as measures for exploratory behavior. A lower distance moved, less time in the center area, and less time spent on exploration were regarded as signs of reduced interest in the environment. The test was performed twice and results were averaged.
Motivation
The free exploration test in the open field was previously described as a test for motivation and exploration in a stress-free situation [32]. For this test we used an adapted version of the protocol described by Knardahl and Sagvolden [33], described in detail by Schoemaker et al. [26], which was performed in 18 male sham, 9 male MI, 15 female sham and 11 female MI rats. This test was performed on day 25 after surgery in the same open field arena as the forced exploration test, with the difference that the rat was now placed in the arena in its home cage, with the lid open, allowing it to choose whether or not to explore the unknown/ new area by climbing out of its open cage. To create a less aversive situation, the rat was allowed a 5-min exploration prior to the test. The percentage of time spent on resting and grooming in the home cage, exploration behavior inside the home cage, exploration of the outside (edges and space above its home cage), and the time before the rat climbed out of its cage into the open field (latency), were measured during a 5 minute period. Relatively less time spent on exploration of the outside and a longer latency to leave the home cage are
Anxiety
The elevated plus maze consisted of a plus-shaped apparatus with two open and two enclosed arms (50*10 cm), elevated 50 cm from the floor and previously described and validated by Pellow et al. [34]. The test was executed on day 35 after surgery. Rats; 22 male sham, 13 male MI, 18 female sham and 14 female MI, were placed at the intersection of the four arms of the maze, facing an open arm. An entry was counted when all four paws of the rat were in one arm. The number of entries and time spent in each arm was recorded over a 5min period. Time spent in the closed-arm time as a percentage of total time is is regarded an index of anxiety-like behavior of the rats.
Recognition and spatial memory
For the novel object and location test the rats were placed in a square Plexiglas box of 50x50x40 cm. The test was performed as previously described [35], on day 38 after surgery. For this, 8 male sham rats, 7 male MI rats, 8 female sham rats and 9 female MI rats were given 3 minutes per day for 2 days to familiarize themselves with the new environment before the test was started. The test began after the second 3 minutes habituation, followed by three 3-minute tests separated by 45 second pauses.
Baseline test: During the baseline test, the rat was presented with two identical objects
placed in two corners of the test box. The objects to be discriminated were stacked lego cubes and transparent glass bottles.
Novel object test: For the novel object test, one old object and one novel object were placed
in the box.
Novel location test: In the novel location test, two familiar objects were placed in the test
area, one in a familiar corner and one object in an opposite corner.
During the 45 second pauses the objects were cleaned with 70% ethanol. To control for possible effects of test-order and object preference, we alternated which object was replaced and also alternated test order (novel object or novel location first). The tests were recorded with a video camera and time spent exploring the objects was analyzed using e-line software. Exploration of the objects was defined as touching the object with the nose or pointing the nose in the direction of the object at close distance. The discrimination index was calculated as time spent exploring the novel/relocated object divided by total time exploring objects. A lower discrimination index is regarded as impaired memory function.
Hemodynamic measurements
Hemodynamic measurements were performed in 14 male sham rats, 10 male MI rats, 12 female sham rats and 4 female MI rats. One week after the final behavioral test, rats were anesthetized using pentobarbital. A Microtip pressure transducer (Millar, Houston, USA) was inserted into the left ventricular cavity via the right carotid artery and measured the heart rate (HR), left ventricular systolic pressure (LVSP), and left ventricular end-diastolic pressure (LVEDP). As indices of contractility and relaxation, the maximal rates of increase and decrease in LVP (dP/dtmax and dP/dtmin) were determined. The catheter was retracted into the aortic arch and mean arterial pressure (MAP) was recorded.
Sacrifice
Rats were anaesthetized with pentobarbital and blood was collected through cardiac puncture. After this, rats were transcardially perfused with 0.9% phosphate buffered saline (PBS) (0.01 M, pH=7.2). Cerebrospinal fluid was collected and immediately frozen in liquid nitrogen. The hearts were excised for determination of infarct size and fixated in freshly prepared 4% paraformaldehyde in PBS (0.01 M, pH=7.2) for a minimum of 72 hours at 4°C. The brains were removed, cut in half, and from one half the hypothalamus was removed, frozen in liquid nitrogen and stored at -80°C until further processing. The other half of the brain was immediately immersed in 4% paraformaldehyde for a minimum of 72 hours at 4°C. The brains were then transferred into a 30% sucrose solution for cryoprotection and left approximately for 16 h at room temperature. After dehydration, brains were stored at -80°C until they were further processed. Whole blood was centrifuged at 2600g for 10 minutes at 4°C and plasma was collected and stored at -80°C until use.
Protein extraction from brain tissue
Hypothalamus and hippocampus tissue was weighed and transferred to an Eppendorf tube. Per mg tissue, 5 µL lysis buffer (50mM Tris-HCl, 150mM NaCl, 0.002% Tween-20 and 1 tablet protease inhibitor/10mL buffer) was added. Samples were homogenized using a pellet pestle for 30 seconds. Brain homogenates were sonicated on ice for 2x 5 seconds, with a 5 second break. Samples were centrifuged for 15 minutes at 12.000 RPM at 4°C, after which the supernatant was collected. Protein concentrations were determined using a Bradford analysis and all samples were diluted to a final concentration of 2.5mg protein/ml and stored at -80°C until use.
ELISA
TNF-α (LEGEND MAX TNF-α rat ELISA Kit, Biolegend, San Diego, USA) and Interleukin-6 (IL-6; Rat IL-6 ELISA kit (thermos scientific)) were measured in 9 male sham, 10 male MI, 8 female sham and 10 female MI rats, according to manufacturers’ instructions, in undiluted plasma. NGAL was measured in plasma (14 male sham, 12 male MI, 12 female sham and 14 female MI rats), CSF (7 male sham, 5 male MI, 6 female sham and 5 female MI rats), hippocampus (9 male sham, 10 male MI, 7 female sham and 10 female MI rats) and hypothalamus (8 male sham, 9 male MI, 8 female sham and 9 female MI rats) tissue using the Rat NGAL ELISA kit (Enzo Life Science BPD-kit-046) according to manufacturers’ instructions. Prior to NGAL ELISA, plasma samples were diluted 1:10000 and CSF samples were diluted 1:1000 in the provided dilution buffer. The hypothalamus and hippocampus tissue homogenates were diluted 1:10 in dilution buffer to a concentration of 0.25mg protein/mL. Intra-assay Coefficients of variance were 3.2% for NGAL, 7.7% for TNF-α and 4.8% for IL-6.
Determination of infarct size
After dissection of the heart, a mid-papillary slice (2 mm) was fixated in 4% PFA, paraffinized and cut in 5µm sections on a Leica microtome. Sections were deparaffinized by incubating 2x 10 minutes in fresh Xylene solution, followed by 1 minute incubations in 100% ethanol, 96% ethanol (2x), 70% ethanol and demineralized water. After deparaffinization, sections were stained for 30 minutes in 0.1% Sirius Red in saturated picric acid (pH 2), followed by 1 minute incubations in 0.01M HCL (2x), tap water (2x) and demineralized water (2x). Sections were subsequently stained for 30 minutes in 0.1% fast green solution, again followed by 1 minute incubations in 0.01M HCL (2x), tap water (2x) and demineralized water (2x). After staining, the sections were dehydrated with 1 minute incubations in 70% ethanol, 96% ethanol, 100% ethanol and 100% xylene. Sections were mounted with DPX (Klinipath). Infarct size was expressed as the percentage of scar length to total left ventricular circumference, as described previously [26, 27].
Immunohistochemical staining of brain sections for NGAL
Brains were cut in the coronal plane in 30 µm thick sections using a Leica cryostat and stored until use in 0.01M PBS containing 0.1% sodium-azide at 4°C. Sections containing the PVN region were 3’3 Diaminobenzidene (DAB) stained using the free floating method with a specific antibody against NGAL (R&D Systems, Minneapolis, MN, US, 1:200 dilution). Prior to staining sections were incubated for 30 minutes in 0.3% H2O2 in PBS. Antibodies were diluted in phosphate-buffered saline, 0.01 M (PBS) and section were incubated in the antibody solution 2 hours at 37°C followed by 96 hours at 4°C. A biotin conjugated
secondary antibody was used (rabbit-anti-goat, Jackson laboratories, 1:500 dilution). Staining was followed by incubation with an avidin-biotin complex (Vector laboratories) and DAB staining (Sigma-aldrich) according to manufacturers’ instructions. In between the different staining steps sections were washed with 0.01 M PBS. Pictures were taken on a Leica microscope at 400x magnification and the number of cells per field of focus was manually counted. Pictures were analyzed for 10 male sham rats, 9 male MI rats, 8 female sham rats and 8 female MI rats.
Statistical analysis
Statistical analysis was performed using IBM SPSS statistics version 22. Difference between male and female sham and MI rats were analyzed with 2-way ANOVA, with data of rats with infarct sizes < 20% excluded because of hemodynamic compensation [36]. If a significant interaction effect was present, simple main effects analysis was performed. Associations were analyzed using Pearson correlations in male and female rats separately. For correlations between cardiovascular parameters and NGAL only MI rats (with infarct size >15% of LV) were analyzed, whereas for correlations between behavior and NGAL sham and MI rats were taken together. A p-value <0.05 was considered statistically significant.
Results
Heart failure parameters
MI induced mortality of 33% (9 out of 27 rats) in males and 29% (10 out of 35 rats) in females, and occurred mainly in the first 24h after surgery. Parameters related to MI are presented in table 1. After exclusion of rats with small infarcts (20%; 4 male and 10 female), average infarct size was similar in male and female MI rats. MI-induced cardiac dysfunction is evidenced from reduced systolic pressure and cardiac contractility, and impaired relaxation, with no significant sex-differences. Lung weight corrected for body weight was significantly increased in MI rats, indicating congestive heart failure.
Behavioral test outcomes
Data from the behavioral tests is shown in Table 2. No significant effects of MI were observed on depressive-like behavior, anxiety-like behavior or motivation, though in the free exploration testthere was a trend (p=0.065) for a higher ratio of exploration inside vs outside the home cage in MI rats.
Table 1: Characterization of experimental groups 5 weeks after MI/sham(Mean ± SD) 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 409±50 416±70 239±17 248± 22 249.8** 0.570 0.00 Infarct size (% of LV) - 33±11 - 36±9 0.964 HW/BW (mg/g) 3.2±0.3 4.0±0.8 3.7±0.4 3.8±1.7 0.331 3.90 2.33 LW/BW (mg/g) 3.3±0.4 5.7±3.5 4.7±1.4 6.5±3.6 3.51 12.47** 0.35 SpleenW/BW (mg/g) 2.0±0.4 2.1±0.4 2.1±0.4 2.3±0.3 3.56 3.30 0.07 N 14 10 12 4 Infarct size (% of LV) - 33±12 - 33±5 0.009
Heart rate (B/min) 366±44 372±63 365±52 349±27 0.660 0.097 0.497 MAP(mmHg) 89±15 86±20 100±20 78±26 0.083 3.816 2.16 LVSP (mmHg) 114±16 110±18 122±19 93±25 0.497 6.082* 3.272 LVEDP(mmHg) 2.5±2.7 5.7±7.0 1.5±3.3 3.0±2.3 1.403 2.249 0.300 dP/dtmin (mmHg/s) -9307±2232 -7936±2933 -9813±2917 -4770±1379 2.088 12.15** 3.98 dP/dtmax (mmHg/s) 10091±2283 8970±2965 10220±2657 5751±1088 2.98 9.77** 3.5
B/min= beats per minute, dP/dtmax= peak difference in pressure over time , dP/dtmin= minimum difference in pressure over time, F=female, HW/BW=heart weight per body weight, LV= left ventricle, LVEDP=left ventricular end diastolic pressure, LVSP=left ventricular systolic pressure, LW/BW=lung weight per body weight, M= male, MAP= mean arterial pressure, MI= Myocardial infarction, SpleenW/BW=spleen weight per body weight. Sham M n=14, MI M n=10, sham F n=12, MI F n=4. *p<0.05, **p<0.01.
Compared to male rats, female rats showed overall more exploratory and anxiety related behavior (sex effect in exploration and distance moved in the forced OF, latency to exit cage in free OF and anxiety behavior in EPM). Regarding the cognitive tests, in the novel object test no differences between groups were observed, whereas in the novel location test male MI rats performed significantly worse than male sham rats. This difference was not seen in female rats.
plasma TNF-α and IL-6 levels
TNF-α levels were undetectable in 4 out of 38 plasma samples and had values below the first standard curve point (7.8 pg/ml) for the other 34 samples (data not shown). Due to these results, TNF-α was not further analyzed. IL-6 levels were below detection level (23.5 pg/ml) in 22 out of 38 plasma samples (Fig. S1). Statistical tests were not carried out because of small group sizes and large variation.
NGAL levels measured with ELISA
Plasma levels of NGAL were significantly higher in males than in females (Fig. 1A) and higher in MI vs sham rats. Similarly, in the CSF both a sex and MI effect were found (Fig. 1B).
There were no statistically significant differences between the groups in the expression of hypothalamic NGAL (Fig. 1C), though there were trends for a sex (F=3.93, p=0.057) and an interaction (F=4.14, p=0.051) effect. No differences were found between groups in hippocampus NGAL levels (Fig. 1D). In both male and female rats, CSF levels correlated with plasma levels of NGAL (Fig. 1E), with plasma levels being around 20 times higher than CSF levels. Hypothalamic levels of NGAL correlated with plasma, CSF and hippocampus NGAL levels (Fig. 1F-1H). Similarly, hippocampus NGAL levels were associated with plasma and CSF NGAL levels (Fig. 1I-1J).
Table 2: outcome behavioral tests (Mean ± SD)
Sham M MI M Sham F MI F Sex effect F value MI effect F value Interaction effect F value Interest in environment Exploration (% of total time) 88±10 88±10 94±4 94±5 7.87** 0.01 0.00 Total distance (cm) 3325±971 3264±647 4094±864 4339±1011 17.4** 0.17 0.48 Time in center (% of total time) 13±8 13±10 16±7 16±9 2.25 0.03 0.03 Motivation
Explore ratio in/out 0.53±0.22 0.73±0.29 0.55±0.22 0.65±0.41 0.18 3.56 0.42
Latency (s) 58±42 55±45 34±31 19±13 12.36** 0.01 1.90
Anxiety
EP Time in open arms (% of total)
10.2±12.4 13.2±12.0 22.4±19.8 28.3±15.2 12.94** 1.36 0.14
Cognition
Novel object (DI) (%) 61±20 62±19 65±10 62±10 0.24 0.03 0.19
Novel location (DI) (%) 79±13 54±12+ 60±21 66±18 0.37 2.40 6.64*
DI=discrimination index, F=female, M= male, MI= myocardial infarction. *p<0.05, **p<0.01. +p<0.05 post-hoc
analysis sham vs MI per sex
Figure 1: expression levels of NGAL in (A) Plasma, (B) CSF, (C) hypothalamus, and (D) hippocampus, mean + SEM.
Correlations of (E) plasma and CSF NGAL, (F) plasma and hypothalamus NGAL, (G) hypothalamus and CSF NGAL, (H) hippocampus and hypothalamus NGAL, (I) hippocampus and CSF NGAL and (J) plasma and hippocampus NGAL. Open circles: male sham, closed circles: male MI, open squares: female sham, closed squares: female MI. CSF= cere-brospinal fluid, MI= myocardial infarction, NGAL=Neutrophil gelatinase-associated Lipocalin.
NGAL staining
Staining of NGAL was performed on brain sections from the hippocampus and the PVN. In the PVN, but not in the hippocampus, clear NGAL positive cells could be observed, as presented in figure 2. An MI effect and a sex*MI interaction in the number of NGAL positive magnocellular neurons in the PVN were observed. Post-hoc analysis revealed that only for male rats there was a significant difference between sham and MI, with a higher number of NGAL positive cells in MI. For the parvocellular part of the PVN, no differences between the groups were found.
Association of NGAL with cardiovascular disease
We observed an association of NGAL with parameters of severity of cardiovascular disease (infarct size, heart weight/body weight and lung weight/body weight) in infarcted rats. As cardiac function measurements were performed in a small subgroup, these were not included in the correlations. Correlations are depicted in table 3, with correlation graphs in supplementary figure 2 (Fig. S2). In male rats, infarct size was positively correlated with plasma NGAL levels. Moreover, increased lung weight corrected for body weight, and cardiac hypertrophy, expressed as heart weight corrected for body weight, were positively correlated with NGAL in plasma. In contrast, in female rats there was no correlation between the parameters of cardiovascular disease and plasma NGAL levels, neither did we find correlations between cardiovascular parameters and NGAL in CSF, hippocampus, hypothalamus or PVN in male or female rats (data not shown).
Figure 2: (A) Illustrations of the NGAL staining in the PVN. The upper panel shows the structure of the PVN and
the lower panel shows a higher magnification of the magnocellular part. (B) NGAL expression in the magnocellular part of the PVN (mean + SEM) *p<0.05. (C) NGAL expression in the parvocellular part of the PVN (Mean + SEM). M= magnocellular region, P=parvocellular region, MI= myocardial infarction, NGAL=Neutrophil-gelatinase-associated
Association of NGAL with behavior
Since behavioral test-outcomes were not different for sham and MI rats, with the exception of novel location recognition in male rats, associations between NGAL and behavior were analyzed for sham and MI combined. Plasma NGAL was associated with depressive-like behavior (shorter distance in open field, and higher ratio of exploration inside versus outside the home cage in the free exploration test) in male rats. Furthermore, plasma NGAL correlated negatively with spatial memory determined by the novel location test in male rats (Table 4, and Fig. S3). In male rats, the number of NGAL positive cells in the magnocellular area of the PVN, as well as hippocampal NGAL was associated with distance moved in the open field, but not other behavioral parameters.
In female rats, neither plasma, nor PVN NGAL was associated with behavior, but hippocampal NGAL was associated with lower distance moved in the open field.
Table 3: correlation (r) of plasma NGAL with parameters of cardiovascular disease in MI animals.
Male Female
r p r p
Plasma NGAL
Heart weight/body weight 0.830 0.000* 0.045 0.892
Lung weight/body weight 0.606 0.028* -0.064 0.822
Infarct size 0.662 0.014* -0.039 0.890
MI=myocardial infarction, NGAL=Neutrophil-gelatinase-associated Lipocalin. *p<0.05
Table 4: Correlations (r) between NGAL and behavior in all rats
Male Female
r p r p
Plasma NGAL
Interest-total distance -0.417 0.031* -0.283 0.137
Motivation-ratio exploration in/out 0.597 0.007* 0.164 0.489 Cognition-Novel location ratio -0.575 0.032* -0.192 0.472
PVN M NGAL
Interest-total distance -0.464 0.039* 0.548 0.081
Motivation-ratio exploration in/out 0.279 0.314 -0.045 0.896 Cognition-Novel location ratio -0.430 0.248 -0.496 0.212
hippocampal NGAL
Interest-total distance -0.458 0.049* -0.558 0.020* Motivation-ratio exploration in/out 0.509 0.091 -0.090 0.770 Cognition-Novel location ratio -0.232 0.519 -0.035 0.928 NGAL=Neutrophil-gelatinase-associated Lipocalin, PVN M= magnocellular region of the paraventricular nucleus. *p<0.05
Discussion
As heart failure and depression are closely linked, but treating the one does not affect the other, we hypothesized a common underlying mechanism rather than a causal relationship. We investigated NGAL as potential inflammatory marker linking these morbidities in a rat model. Because of the sex difference in the prevalence of depression in heart failure [37], male as well as female rats were studied.
Heart failure
Myocardial infarction was induced by chronic coronary artery ligation, an established model for heart failure [26, 38]. Infarct size was on average more than 30% and similar in male and female rats. Hemodynamic measurements in a smaller subgroup of rats confirmed decreased left ventricular function in MI rats, evidenced by a decrease in LVSP and changes in dP/dtmin and dP/dtmax. No statistical differences were observed for LVEDP, indicating relatively mild heart failure. Still, congestion was indicated by significantly increased lung weight in MI vs sham rats.
Cognition
Male MI rats displayed a significant decrease in the ability to discriminate object location in the novel location task compared to male sham rats, whereas female MI rats did not show this effect. This result suggests that male rats suffer from spatial memory impairment after MI, which is not present in female rats. Others reported moderate impairment in spatial memory in both male and female MI mice [39-41]. Cognitive dysfunction in patients with heart failure has frequently been reported and was extensively reviewed [6, 7]. The spatial memory deficits found in our study and other studies in rodents may reflect cognitive impairment found in heart failure patients.
Depression/anxiety
In the present study, no depressive-like behavior or anxiety-like behavior was observed in MI rats compared to sham rats in either males or females. This is in contrast with our previous studies in male MI Wistar rats at the same age and time point after MI, in which we showed decreased interest in environment measured as exploration in the open field and decreased social interest, as well as increased anxiety [26, 27]. Depressive-like symptoms, measured by increased immobility in the forced swim test [25] 2 weeks after MI and anhedonia [25, 42]
the case in patients, this might explain the lack of statistical differences between sham and MI rats shown in this study. Another explanation might be the relatively mild heart failure induced in this study and the short interval between surgery and behavioral testing. However, neither in animals [41], nor in patients [44], symptoms of depression or anxiety are related to the severity of heart failure (ejection fraction, NYHA classification, MI size).
NGAL and behavior
We observed that NGAL in plasma and the PVN correlated with depressive-like behavior in male, but not female rats, independent of MI or sham. This was in contrast to what we expected, as in patients depressive mood has been correlated with plasma cytokine levels in women, but not in men [45]. Studies in animal models, however, show that male rats are more susceptible to the neurobiological effects of chronic or acute stress [46, 47]. This may indicate a reversed sex difference in rodents compared to humans, as in humans women are more susceptible to stress and depression. The protective effect on acute and chronic stress could be attributed to estrogens inhibiting cytokine secretion by microglia [48, 49], preventing neuroinflammation and associated behavior. Estrogens have been reported to be important for the protection of heart failure in female rats [50, 51]. Alternatively, sex differences in the immune response to cardiac ischemia have been reported, particularly differences in TNF-α signaling [52, 53]. This may be important for the lack of behavioral effects in our female rats, as we hypothesize inflammation to be key in the link between cardiovascular disease and behavioral outcome.
In contrast to other studies [54], we were not able to detect increased TNF-α in plasma of our MI rats. However, this absence of TNF-α upregulation was observed concomitantly with absence of behavioral effects following MI, which may rather support our hypothesis of inflammation linking heart failure and behavior.
Novel location discrimination index showed a negative correlation with plasma NGAL in male, but not female rats. The correlation between NGAL and spatial memory is interesting, as NGAL has previously been linked to dementia [55]. As mentioned above, inflammation could be regulated differently in female compared to male rats, explaining the lack of correlation in female rats. However, our results did not show a statistically significant difference in hippocampal NGAL expression between groups. Hippocampal NGAL levels were associated with more depressive-like behavior in both male and female rats, which could be an indication that NGAL expression in the hippocampus is associated with behavior. As the correlation was found in sham and MI rats combined, this may also reflect mechanisms, unrelated to the
surgery performed. The hippocampus has been known as an area involved in cognition and emotion [56, 57]. In addition, it is an area highly sensitive to inflammatory changes [58, 59]. It is hypothesized that the hippocampus is involved in the cognitive and emotional changes seen in heart failure patients. Volume reduction of the hippocampus has been observed in heart failure patients compared to healthy controls [60]. Increased apoptosis in the hippocampus has been shown in MI animals, which can be partially reversed by interference with TNF-α [61] or by treatment with an anti-depressant drug [62]. The hippocampus plays an important role in spatial mapping of the environment [63]. Regarding this role of the hippocampus, higher NGAL in the hippocampus could have contributed to lower distance moved in the open field.
NGAL and cardiovascular disease
We found plasma and CSF NGAL levels higher in rats with MI. A sex effect was also present, with higher levels of NGAL in males than in females. In clinical studies, heart failure is generally associated with increased levels of NGAL, dependent on the severity of heart failure [16, 64]. Studies also show that NGAL in heart failure patients is associated with worse prognosis [65, 66]. Indeed in our study plasma NGAL levels in male rats were positively correlated to infarct size and parameters of heart failure. One reason that we failed to find this effect in female rats is because there is evidence that estrogens can inhibit vascular expression of NGAL [67], which could be an explanation for the lack of NGAL upregulation in female rats.
In a recent study [68] we found that the procedure performed in sham surgery, similar to the methodology described in the present study, caused substantially elevated NGAL levels. It may therefore be that the NGAL levels measured in our sham rats do not accurately reflect unstimulated NGAL levels of healthy subjects.
The NGAL plasma levels seem reflected in CSF, hippocampal and hypothalamic NGAL levels. This finding suggests that increased levels in the CSF and brain could be attributed to increased systemic circulating levels. Since the circulating NGAL levels are correlated to infarct size in males, this suggests that the NGAL levels in the hypothalamus and hippocampus reflect the severity of peripheral cardiac damage. In female rats, such a strong connection could not be obtained from our data.
subdivided in two parts, a magnocellular part and a parvocellular part. The former is involved in the production of the hormones vasopressin and oxytocin, while the latter is involved in the regulation of the sympathetic nervous system, reviewed by Badoer [70]. In male, but not in female rats, MI led to increased numbers of NGAL positive cells in the magnocellular part of the PVN, while no changes in NGAL expression was found in the parvocellular area. This is interesting, as previous studies found changes in inflammation mostly in the parvocellular area, linked to changes in sympathetic activation [24, 69]. The absence of NGAL upregulation in the parvocellular area could be linked to the relatively mild heart failure induced in our study, but that would not explain the different findings in the magnocellular area. Alternatively, it is possible that NGAL expression is not linked to the previously found microglia activation in this area, as the latter seems dependent on the distance from de 3rd ventricle [69]. The presence of higher NGAL levels in the magnocellular area of the PVN could be linked to its role in the production of vasopressin and oxytocin. These neuropeptides have previously been linked to behavior [71, 72]. It is unclear whether the NGAL signal represents local production of NGAL or uptake of NGAL from the extracellular space. It is possible that plasma or CSF NGAL diffuses into the PVN, and is subsequently taken up by specific cells, or alternatively higher concentrations of NGAL stimulate cells in the PVN to produce NGAL. We know from literature that cells in the CNS can both produce and take up NGAL [17, 73], and also that there is expression of NGAL receptors on the blood brain barrier [74], supporting both these hypotheses.
Limitations of the study
Several factors in this study could be considered as limitations. The model used, coronary artery ligation in healthy young adult rats, is commonly used in experimental heart failure. Although the pathophysiology of this experimentally induced MI is incomparable to the human situation, the healing process and hemodynamic consequences of this MI show relevant similarities to the clinical condition of MI-induced heart failure. Moreover, mental problems such as anxiety/depression and cognition, already extremely complex in patients, provide a real challenge to study in animal models. Hence, translation of the obtained results to heart failure patients has to be very cautious as patients are usually elderly and have comorbidities.
Regarding the sex-related aspects, as discussed, in rats some of the male/female differences may be opposite to the human situation. Furthermore, the largest part of female heart failure patients is post-menopausal while young female rats still have an estrus cycle, possibly influencing important outcomes. However, in a pilot study in 18 months old female rats, that
did not present an estrus cycle anymore, also no behavioral differences between sham and MI rats could be observed (unpublished results).
Moreover, depressive-like behavior and anxiety were not observed in this study, though other studies have found changes in depression and anxiety in experimental heart failure [25-27, 41, 75]. Although the relatively mild severity of heart failure and the short interval between surgery and behavioral testing might provide an explanation, usually depressive symptoms are reported independent of parameters of cardiac dysfunction.
Final limitation in the study is the group size for the hemodynamic measurements. Since the study was focused on behavioral aspects, hemodynamics were only obtained in a subgroup of rats in order to confirm signs of heart failure. Hence, for this aspect the study appeared statistically underpowered.
Conclusions
Since NGAL has previously been associated with heart failure severity [16] and with depression [21] in heart failure patients, we investigated the role of NGAL as potential mediator in depression and heart failure in male and female rats. In the present study, no increased anxiety/depressive-like behavior was observed in MI rats, and cognitive impairment was only seen in male MI rats. Irrespective of sex, increased plasma and CSF levels of NGAL were observed in MI versus sham rats. However, in male, but not in female rats, circulating NGAL was found correlated to parameters of cardiac dysfunction as well as behavior. Moreover, male, but not female, MI rats displayed increased number of NGAL positive cells in the magnocellular area of the PVN. To conclude, this study indicates that NGAL can be regarded a (neuro)inflammatory element that is associated with cardiac dysfunction in male but not in female rats, suggesting sex differences in the inflammatory response post-MI. The correlations between NGAL and behavior are sex specific, but irrespective of type of surgery (sham or MI) and might thus reflect phenomena not related to the MI induced inflammation.
Supplementary figure S2: correlations between (A) plasma NGAL and heart weight/body weight in male rats, (B)
plasma NGAL and lung weight/body weight in male MI rats, (C) plasma NGAL and infarct size in male rats, (D) plasma NGAL and heart weight/body weight in female rats, (E) plasma NGAL and lung weight/body weight in female rats and (F) plasma NGAL and infarct size in female rats.
Supplementary figure S3: correlations between plasma NGAL, PVN NGAL and hippocampusNGAL and behavioral
outcomes in male (A-I) and female (J-R) rats. Open circles: male sham, closed circles: male MI, open squares: female sham, closed squares: female MI. FE: free exploration, NL: novel location, OF: open field.
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