Ovariectomy, a model of menopause in rodents, causes a premature aging of the nervous and immune systems

Journal of Neuroimmunology 219 (2010) 90–99

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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Ovariectomy, a model of menopause in rodents, causes a premature aging of the nervous and immune systems I. Baeza a, N.M. De Castro a, L. Giménez-Llort b, M. De la Fuente a,⁎ a b

Department of Physiology, Faculty of Biology, Complutense University of Madrid, Madrid, Spain Department of Psychiatry and Forensic Medicine, Institute of Neuroscience, Autonomous University of Barcelona, Bellaterra, Spain

a r t i c l e

i n f o

Article history: Received 18 September 2009 Received in revised form 16 December 2009 Accepted 18 December 2009 Keywords: Ovariectomy Aging Behavior Chemotaxis Lymphoproliferation Natural killer activity

a b s t r a c t Ovariectomy in rodents is a good model for mimicking human ovarian hormone loss. This work studies the consequences of ovariectomy on the nervous and immune systems in the context of biological aging. Ovariectomy accelerates the process of aging by impairing the sensorimotor abilities (with loss of muscular vigor and impaired equilibrium and traction capacities) and the exploratory capacities (with reduction of vertical exploratory activity). It also leads to a premature immunosenescence with regard to chemotaxis index, lymphoproliferative response and natural killer activity, parameters investigated in the spleen and axillary nodes. Therefore, ovariectomy deteriorates homeostasis and may be a model of premature aging. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Aging leads to a significant deterioration of the physiological systems, including the immune, nervous and endocrine systems, as well as of the immune-neuroendocrine network (De la Fuente, 2008). The age-related decline in immunity, known as immunosenescence, involves innate and adaptive immune responses, but concerns especially T cell functions (Gruver et al., 2007; Kumar and Burns, 2008; De la Fuente and Miquel, 2009). As regards the endocrine system, aging involves a progressive decrease in the secretion of several hormones such as estrogens (Arlt and Hewison, 2004). These hormones, due to the widespread distribution of their receptors throughout the brain (Aloysi et al., 2006; Morrison et al., 2006), also play a key role in the neurobiology of aging (Morrison et al., 2006) as they exert a great influence on a broad array of brain regions, mostly areas associated with cognition, memory and emotional processing (as mood and affect). All these areas constitute important sites of agerelated neurodegenerative changes, such as neuronal loss and compensatory gliosis (Heinikken et al., 2004; Ferrari and Magri, 2008). Other physiological age-related changes commonly described among the elderly include balance dysfunctions, reduced speed, shorter step length and muscular weakness due to skeletal mass reduction (El Haber et al., 2008). ⁎ Corresponding author. Department of Physiology, Faculty of Biology, Complutense University of Madrid (UCM), c/ Jose Antonio Novais no.2, 28040 Madrid, Spain. Tel.: +34 91 394 49 86; fax: +34 91 394 49 35. E-mail address: [email protected] (M. De la Fuente). 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.12.008

According to all the above, one essential concept that must be taken into consideration when studying the aging process is the concept of “biological age,” which arises as a consequence of the different rates of physiological changes in the members of a population of the same chronological age and suggests that chronological and biological age do not necessarily coincide (Borkan and Norris, 1999; De la Fuente and Miquel, 2009). Therefore, the assessment of biological age requires the use of biomarkers to determine the level of senescence and life expectancy. The immune system has been proposed as a good marker of health, biological age and predictor of life span, since a good maintenance of several immune functions is related to a longer life span (Wayne et al., 1990; Guayerbas et al., 2002a,c; Guayerbas and De la Fuente, 2003; De la Fuente and Miquel, 2009). Although during the last decades of the 20th century human life expectancy in developed countries has increased from approximately 75 to 83 years, the age at which women encounter their major agerelated hormonal change, that is, menopause, has remained essentially constant at around 50 years (Miquel et al., 2006). Therefore, many women will spend over one-third of their lives in the postmenopausal state. Since estrogens have a regulatory role on many organs, the rapid decline in their circulating levels associated to menopause triggers many physiopathological reactions, making women more prone to experience disease and disability (Amir et al., 2003). Thus, chronic deficiency of sex hormones has many implications in a wide variety of non-reproductive functions, and among the most studied symptoms we can cite hot flashes, skin aging and high risk of osteoporosis and cardiovascular disease (Miquel et al., 2006).

I. Baeza et al. / Journal of Neuroimmunology 219 (2010) 90–99

There have also been described different psycho-emotional symptoms that somehow overlap with depressive symptoms and include disturbed sleep, lack of concentration, anxiety, irritability, frustration, mood lability, depression and fatigue (Rasgon et al., 2005; Sarkaki et al., 2008). Moreover, estrogens have been shown to influence the development, regulation and normal functions of the immune system (Islander et al., 2003; Rehman and Masson, 2005). These hormones modulate lymphoid cell growth, differentiation and proliferation, antigen presentation, cytokine and antibody production, NK activity and cell survival (McMurray, 2001). Thus, the repercussions of menopause on female health have become a subject of increasing interest. To tackle this question, a great deal of research has been done during the last few years in murine experimental models as a first approach to clarify the repercussions of menopause. Since rodents become anovulatory at a mature age (10–12 months old) but maintain a basal gonadal steroid secretion, in contrast to what happens in women (Nelson, 2008), ovariectomy in those animals became the best tool to mimic human ovarian hormone loss. Not surprisingly, during the last years there has been a great increase in the number of published work focusing on the consequences of ovariectomy, mainly in rats, on different physiological functions or systems, such as the central nervous system, vascular function, hepatocytes, bone, skin (Castillo et al., 2005, 2006; Perez-Martin et al., 2005; Tresguerres et al., 2008) and immune function (De la Fuente et al., 2004; Baeza et al., 2007, 2009). Since the neuro-immunoendocrine network is essential to coordinate adaptive mechanisms and ensure homeostasis preservation (Besedovsky and Del Rey, 2007; O'Connor et al., 2008; De la Fuente, 2008; De la Fuente and Miquel, 2009), we have analyzed the functions of two of the systems involved in this network, namely, the nervous and the immune systems, as well as the repercussions of ovariectomy on the estrogens levels. Thus, we have studied a group of mature female mice of 14 months of age submitted to ovariectomy (or shamoperated) at 12 months of age. Furthermore, in order to find out whether ovariectomy is a good model of premature aging, a group of intact female adult animals (6 months old) and another of intact female old animals (26 months old) have been included. 2. Materials and methods 2.1. Animals A group of 38 female ICR-CD1 mice (Mus musculus) was obtained from Harlan Iberica (Barcelona, Spain) and housed in polyurethane boxes at a constant temperature (22 ± 2 °C) on an inverted 12-h light/ dark cycle starting at 20:00 pm. They were fed standard laboratory diet (A04 Panlab, Barcelona, Spain) and water ad libitum. The animals were treated according to the guidelines of the European Community Council Directives 1201/2005 EEC. 2.2. Experimental design In the present study, mice of three different ages have been used: one group of 10 adult mice (6 months old), one group of 18 mice that were submitted to ovariectomy (n = 10) or sham-operation (n = 8) at a mature age (12 months old) and were studied when they reached 14 months, and finally a group of 10 old mice (26 months old). 2.3. Surgical procedure: ovariectomy Bilateral ovariectomy was performed in a group of animals 12 months old as follows: mice were anesthetized by an intramuscular injection with a mixture of Ketamine and Xilacine (80 mg/ 12 mg/kg) (Merial Laboratories S.A. and Química Farmacéutica BAYER S.A., Barcelona, respectively). The dorsal part of the lumbar region was

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shaved and then cleaned with ethanol. One small incision (1 cm) was made through the skin and the muscle wall on each side of the backbone, in the dorsal aspect. Ovaries were then located and a braided silk sterile suture (Lorca Marín, Murcia, Spain) was performed around the area of the uterine horns, that were sectioned thereafter, and the ovaries were removed. The wound was closed in two layers, i.e.: muscle and skin using sterile sutures (Lorca Marín, Murcia, Spain). Sham animals were also anesthetized, the skin and muscle layers were opened and the uterus and ovaries were manipulated but not excised. After surgery, mice were housed individually for some hours to allow recovery and then re-grouped in their home cages. 2.4. Behavioral tests The experiments were performed from 09:00 to 14:00 h in accordance with the Spanish legislation on “Protection of Animals Used for Experimental and Other Scientific Purposes” and the European Communities Council Directives (1201/2005) on this subject. Behavioral testing took place during four consecutive days. On the first day, animals were subjected to the whole battery of sensorimotor tests. On the second day, corner test and open field were performed. On the third day, animals were subjected to the holeboard test. Finally, the T-maze test was performed on the last day. The sequence of testing was based on previous reports by different authors (Johansson et al., 2001; Giménez-Llort et al., 2002). Behavior was evaluated by three independent observers. Mice were weighted before performing the tests, in order to be sure that all of them were active in the same way. Olfactory trails were removed by cleaning the surface of the apparatuses after each test. 2.4.1. Sensorimotor abilities 2.4.1.1. Visual placing reflex. The visual placing reflex was tested in order to evaluate the function of the visual system. For the performance of this placing response, the mouse was suspended by the tail and lowered toward a solid black surface. Complete extension of the forelimbs was considered a positive response. The mean response was rated in three trials. 2.4.1.2. Hindlimb extensor reflex. This reflex was evaluated during the previous test as the ability to perform complete extension of the hindlimbs when the animal was suspended by the tail. Such response was considered positive. The mean response was rated in three trials. 2.4.1.3. Wood and wire rod tests. In order to assess equilibrium and motor coordination, animals were placed in the centre of two different elevated rods (rod dimensions: 22 cm height, 80 cm length, divided in segments of 10 cm) with increasing difficulty: a wood rod (square section of 2.9 cm) and a wire rod (diameter of 0.7 cm). Each animal was submitted to two trials of 20 s. Equilibrium was measured by means of the ability of not falling off the rod. Adult animals are usually more prone to spend time exploring the new environment, and consequently they may not complete the task within the time given (although this would not indicate lower motor coordination ability, but a higher exploratory capacity). Due to this fact, we established two different criteria to evaluate motor coordination: criteria 1, as “percentage of mice that cover at least 1 segment”, and criteria 2, as “percentage of mice that complete the test”. 2.4.1.4. Tightrope test. This method is used to evaluate the vitality loss in aging mice by testing their muscular vigor, motor coordination and traction in two training trials of 5 s and a test trial of 60 s (Miquel and Blasco, 1978; Guayerbas et al., 2002a). Mice were hanged by its forelimbs in the middle of an elevated horizontal tightrope (40 cm height, 60 cm length and divided in segments of 10 cm). Muscular vigor was assessed as the percentage of mice falling off the rope and

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the latency to fall (in seconds). Motor coordination included the percentage of mice that walk at least 1 segment (criteria 1) and the percentage of mice that complete the test (criteria 2). Traction was evaluated by analyzing the different parts of the body that mice used to remain hanged (forelimbs, hindlimbs and tail) and, subsequently, the percentages of mice displaying the maximum (forelimbs, hindlimbs and tail) and minimum (forelimbs only) traction capacities were assessed within each group. 2.4.2. Exploratory and anxiety-like behavioral tests This group includes different tests that study the exploratory capacity and the anxiety-like behaviors in the animals: the corner, the open field, the holeboard and the T-maze tests. 2.4.2.1. Corner test. This test is aimed to study how the animal behaves in a new environment (in order to assess neophobia and emotionality) when placing the mouse in a new cage (220 × 220 × 145 cm) with bedding. Both horizontal (assessed as the number of corners of the cage visited) and vertical exploratory activities (studied as the performance of “rearing”, that is, when the mouse stands up on his hindlimbs so that his body becomes more perpendicular to the ground) were evaluated. The trial was performed in 30 s. For this purpose, the number of visited corners and rearing behavior (% mice that perform rearing, total number of rearing and latency of first rearing) were recorded, respectively. 2.4.2.2. Open field test. Immediately after the corner test, animals were assessed in the open field test. The open field arena consisted in a squared surface (80 × 80 × 30 cm) divided into 16 equal sectors (10 × 10 cm) brightly illuminated (75 W). Mice were placed in the centre of the apparatus and the test was carried out for 5 min. Both horizontal and vertical exploratory behaviors were measured as external ambulation (number of crossings on the 12 peripheral squares), internal ambulation (number of crossings on the 4 internal squares), percentage of mice that perform rearing and total number of rearing. Latency to leave the starting central point and that to reach the periphery were measured, both parameters related to the anxiety status of the animal (as anxious animals exhibit what is called “freezing behavior”: when you first place them in the apparatus, as they are afraid to move around in an unknown environment, they remain still and without moving for some seconds; therefore, the latency to leave the starting central point and that to reach the periphery are much higher in anxious animals with respect to nonanxious ones). Self-grooming behavior was recorded by means of latency of first episode and total number of grooming. Aging involves a decrease in the defecatory behavior and an increase of urine incontinence. Therefore, the number of fecal boli and presence of urine were also considered in the different groups of age (in an attempt to study whether ovariectomy in mature animals caused these behaviors to be more similar to those observed in aged animals). 2.4.2.3. Holeboard test. The holeboard consisted in a box (60 × 60 × 45 cm) with matte-painted metallic walls, divided into 36 squares (10 × 10 cm), bearing four equally spaced holes (3.8 cm of diameter) and brightly illuminated (75 W). All but the 20 peripheral squares were considered central. Green plastic objects were placed in each hole to attain mice attraction and drive their ‘goal-directed behavior’. The test was performed during 5 min and the parameters recorded for ‘non-goal-directed behavior’ were deambulation (horizontal locomotor activity) and rearing (vertical locomotor activity) while the number of head-dippings was used as a measure of ‘goal-directed behavior’. Self-grooming behavior, the number of fecal boli and presence of urination were also recorded. 2.4.2.4. T-maze test. The apparatus was a wooden enclosed T-maze with walls (short arm: 27 × 10 cm; long arm: 64 × 10 cm; walls: 19 cm

high) and brightly illuminated (75 W) (De la Fuente et al., 1998; Guayerbas et al., 2002a,b,c; Guayerbas and De la Fuente, 2003). No reinforce was used. Mice were placed inside the “vertical” arm of the maze with its head facing the end wall. Horizontal exploratory behaviors recorded included: time elapsed until the animal crosses with both hindlimbs the intersection of the T-maze and the exploratory efficiency (time elapsed until mice complete the exploration of all three maze arms). Regarding vertical exploratory activity, % mice that perform rearing and total number of rearing were measured. Self-grooming behavior was evaluated by means of the total number of grooming. 2.5. Collection of tissue samples and obtention of lymphocyte suspensions At the diestrous phase of the cycle (determined by vaginal smears) animals were sacrificed by decapitation, according to the guidelines of the European Community Council Directives 1201/2005 EEC. Uteri were removed, freed of fat and connective tissue and weighed. Uterine weight (g) was studied in all groups of mice, as this is a key parameter to confirm that ovariectomy has been correctly performed. Spleens and axillary nodes were also removed aseptically, freed of fat, minced with scissors and gently pressed through a mesh screen (Sigma, St Louis, and USA). The cell suspensions were centrifuged in a gradient of Ficoll-Hypaque (Sigma) with a density of 1.070 g/ml. Cells from the interface were resuspended in RPMI 1640 medium enriched with L-glutamine (PAA, Pasching Austria) and supplemented with 10% heat-inactivated fetal calf serum (Gibco, Canada) and 1% gentamicin (10 mg/ml, Gibco), washed, and the number of leukocytes was determined and adjusted to 1 × 106 cells/ml. Cellular viability, routinely measured before and after each experiment by the trypan-blue exclusion test, was higher than 95% in all experiments. All incubations were performed at 37 °C in a humidified atmosphere of 5% CO2. 2.6. Immune function parameters 2.6.1. Chemotaxis assay The assay was carried out following a modification of the method of Boyden (1962) according to the method previously described by Guayerbas et al. (2002b). Chambers with two compartments separated by a filter (Millipore, Ireland) of 3 μm pore diameter were used. Aliquots of 300 μl of leukocyte suspensions were placed in the upper compartment, and aliquots of 400 μl of the chemoattractant fMet-Leu-Phe (Sigma) at a concentration of 10− 8 M were placed in the lower compartment. The chambers were incubated for 3 h, the filters were fixed and stained and the chemotaxis index (CI, the number of lymphocytes in the lower face of the filter) was calculated by counting in an optical microscope and the total number of leukocytes in one-third of the lower face of the filters. All CI were assayed in duplicate. 2.6.2. Lymphoproliferation assay The proliferation of lymphocytes in response to the mitogen lipopolysaccharide (LPS) was assayed following a method previously described by Del Río et al. (1994). Bacterial lipopolysaccharide (LPS) is a potent stimulant of B cells and macrophages, which induces B cell proliferation and differentiation into antibody secreting cells (Venkataraman et al., 1999). Aliquots of 200 µl of the spleen or axillary node leukocyte suspensions were seeded in ninety-six well flat-bottomed microtiter plates (Nunclon, Denmark) and incubated in the presence or absence (controls) of LPS (Sigma, 1 µg/ml) during 48 h. Each sample was assayed in triplicate. Then, 0.5 µCi 3Hthymidine (ICN, Costa Mesa, USA) was added to each well and after 8 h the cells were harvested and thymidine uptake was measured in a beta counter for 1 min. The results were expressed as percentage of proliferation in response to LPS, with 100% being the thymidine uptake cpm in control (without mitogen) wells (stimulation index).

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2.6.3. Natural killer (NK) Activity assay An enzymatic colorimetric assay was used for cytolysis measurements of target cells (Cytotox 96 TM Promega, Boerinher Ingelheim, Germany) based on the determination of the activity of the enzyme lactate dehydrogenase (LDH), using tetrazolium salts as previously described by Ferrández et al. (1999). Briefly, target cells (YAC-1 cells from a murine lymphoma) were seeded in 96-well U-bottom culture plates (Nunclon, Denmark) at a concentration of 104 cells/well in 1640 RPMI medium without phenol red. Effector cells (leukocytes from the axillary nodes and spleen) were added at a concentration of 105 cells/well, thus obtaining an effector/target rate of 10/1. Each sample was assayed in triplicate. The plates were centrifuged at 250 g for 4 min to facilitate cell to cell contacts and then they were incubated for 4 h. After incubation, plates were centrifuged again at 250 g for 4 min and LDH activity was measured in the supernatants by addition of the enzyme substrate at 490 nm. Results were expressed as percentage of lysis. 2.7. Estradiol levels Blood was collected immediately after the sacrifice of the animals in heparinized tubes, and plasma was obtained by centrifugation (1000 g, 20 min). Plasma estradiol levels were analyzed on all groups of mice (adults, mature sham, mature ovariectomized and old) with a commercially available E.L.I.S.A. kit (Cayman Chemical Company, Ann Harbor, MI. USA). Results were expressed in pg/ml and each sample was assayed in duplicate. 2.8. Statistical analysis The data were expressed as the mean ± SD of the values. The normality of the samples was tested by the Kolmogorov–Smirnov test. The data were statistically evaluated by the two-way analysis of variance (ANOVA) for comparisons of parametric samples and U test Mann–Whitney for non-parametric samples. For qualitative data, the Chi-square test was used. p b 0.05 was taken as the minimum significance level. 3. Results Weight differences were only found between adults (30.96 ± 3.59) and the other three experimental groups (mature sham: 45.69 ± 4.80, mature ovx: 46.03 ± 3.99; old: 44.88 ± 4.72, p b 0.001). 3.1. Behavioral tests Tables 1 and 2 summarize the results obtained in the behavioral assessment of mice. 3.1.1. Sensorimotor abilities The different sensorimotor tests (Table 1) assessed reflexes, equilibrium, motor coordination, muscular vigor and traction, being all of them functions that get clearly impaired as the animal ages. At this respect, the very first thing we observe is the detrimental effect of aging on reflexes. Thus, adult and mature sham animals exhibited normal reflexes in contrast to mature ovariectomized and old animals where half of the individuals exhibited incomplete hindlimb extension and 20 to 10% did also show an impaired visual reflex. As illustrated in Fig. 1A, a 2-fold linear progression of age-related deficits in equilibrium was observed indicating that ovariectomy exacerbates the impairment related to maturity. However, an increase of difficulty in the tasks (wire bar) evidences the better balance abilities of adulthood while the other experimental groups display a similar performance. Also, motor coordination is more affected by age than ovariectomy. The tightrope test clearly showed a reduction of the

93

Table 1 Behavioral tests (A. Sensorimotor abilities). Results are expressed as percentage (%) or mean ± SD in the adults (n = 10), mature sham (n = 8), mature ovx (n = 10) and old mice (n = 10). ***p b 0.001, **p b 0.01, *p b 0.05 with respect to adult animals. •p b 0.05 with respect to mature sham animals. #p b 0.05 with respect to old animals. Adults Mature Mature Old sham ovx Visual placing reflex % Mice showing this response Hindlimb extensor reflex % Mice showing this response Wood rod test Equilibrium (% of mice falling off) Motor coordination (criteria — 1 segment) Motor coordination (criteria — completed test) Wire rod test Equilibrium (% of mice falling off) Motor coordination (criteria — 1 segment) Motor coordination (criteria — completed test) Tightrope test (60 s trial) Muscular vigor (% of mice falling off) Muscular vigor (latency, sec) Motor coordination (criteria — 1 segment) Motor coordination (criteria — completed test) Traction

100

100

80

90

100

100#

50*•

50*•

See Fig. 1A 90 50 50 0*

40* 10

40* 10

10 60 20

70** 0** 0

100*** 0** 0

70*

90**

20* 0**

10* 0**

62.5*# 12.5 0

20 62.5 See Fig. 1B 70 0** 60 0** See Fig. 1C

muscular vigor (see Fig. 1B) and traction capacity (see Fig. 1C) with aging, which was worsen after ovariectomy. 3.1.2. Exploratory and anxiety-like behavioral tests The results corresponding to the tests of exploratory and anxietylike behavioral test are shown in Table 2. In the corner test, the animals that display a high number of corners visited (horizontal exploratory activity) as well as a high number of rearing (vertical exploratory activity) are denominated non-anxious. On the contrary, when they do not show either horizontal or vertical exploratory activity we say that they display an anxiety-like behavior. In the present work, all groups of animals showed similar horizontal activity (assessed as the number of corners visited) but a 30% reduction in the number of animals exhibiting vertical movements (known as “rearing”) was found after ovariectomy and in the aged (p b 0.01, see Fig. 2A) with respect to adult and mature sham animals. The behavior of adult mice in the open field test was characterized by an intense horizontal exploratory activity with a high number of total crossings, covered exactly with the same proportion in peripheral than internal areas. All the animals exhibited vertical exploratory behavior, a mean of 4 defecation boli and lacked urination. In contrast, the aged showed reduced horizontal and vertical activities. Thus, they only covered 12 peripheral rounds as compared to the 18 done by adults, and the number of animals showing rearing activity was reduced to 70% (see Fig. 2B). The mature sham animals behaved like the adults (18 peripheral rounds, 100% of animals doing rearing) but ovariectomy led to a shift to the behavioral pattern exhibited by the aged (12 peripheral rounds and only 60% of animals with rearing). Self-grooming behavior involves the actions of an animal of rearranging its fur by cleaning their body surface. This behavior, concerned with the care of the body surface, constitutes an attempt to remove anxiety. In the present work we have found no differences in this behavior (either studied as the latency of the first self-grooming or the total number of grooming) in the four groups studied. Taking into account that aging involves a decrease in the defecatory behavior and an increase of urine incontinence, we recorded these behaviors and compared them to the mean observed in the adult group. The aim of studying such parameters was finding out whether ovariectomized mice were more similar to the old ones regarding this behavior with respect to sham mice of the same age. Our results show that, with aging, there is a gradual increase in the number of animals with reduced defecatory behavior which reached a

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I. Baeza et al. / Journal of Neuroimmunology 219 (2010) 90–99

Table 2 Behavioral tests (B. Exploratory and anxiety-like behaviors). Results are expressed as percentage (%) or mean ± SD in the adults (n = 10), mature sham (n = 8), mature ovx (n = 10) and old mice (n = 10). ***p b 0.001, **p b 0.01, *p b 0.05 with respect to adult animals. #p b 0.05 with respect to old animals.

Corner test Number of corners visited %Mice that perform rearing Number of rearing Latency of first rearing (s) Open field test Horizontal activity Latency to leave the starting point (s) Latency to enter into the peripheria (s) Total deambulation Internal deambulation External deambulation Vertical activity %Mice that perform rearing Total number of rearing Self-grooming behavior Latency of self-grooming Total number of grooming Defecation and urination behaviors Number of fecal boli % animals with less than 4 defecation boli % animals exhibiting urination Holeboard test Non-goal-directed behavior Horizontal activity Latency to leave the starting point (s) Total deambulation Vertical activity %Mice that perform rearing Total number of rearing Goal-directed behavior %Mice that perform head-dips Total number of head-dips Self-grooming behavior Latency of self-grooming (s) Total number of grooming Defecation and urination behaviors Number of fecal boli % animals with less than 4 defecation boli % Urine presence T-maze test Horizontal activity Time for crossing the intersection of the maze Exploratory efficiency Vertical activity % Mice that perform rearing Total number of rearing Self-grooming behavior Total number of grooming

Adults

Mature sham

Mature ovx

Old

8.7 ± 2.16 See Fig. 2A 5.2 ± 2.35 7.1 ± 4.82

9.5 ± 5.58

8.1 ± 2.80

7.2 ± 2.80

4 ± 2.27 11.8 ± 4.60#

2.2 ± 1.99* 18 ± 9.46*

1.7 ± 1.42** 21.1 ± 7.69**

3.5 ± 2.37 8.4 ± 6.43 447 ± 144 220 ± 83 227 ± 83

7.88 ± 4.15* 12.13 ± 8.56 349 ± 149 135 ± 78 214±102

13.8 ± 27.93 20.5 ± 28.34* 253 ± 162* 104 ± 79* 149 ± 96

7.3 ± 3.13* 17.2 ± 12.22* 274 ± 137 124 ± 78 150 ± 68

See Fig. 2B 14.1 ± 8.9

13.38 ± 11.96

5.2 ± 8.27

8.5 ± 10.05

163 ± 57 1.2 ± 0.63

153 ± 69 2.38 ± 2.20

157 ± 69 2.4 ± 1.26*

157 ± 69 1.5 ± 1.18

4.3 ± 2.54 40 0

3 ± 1.31 62.5 25

2.4 ± 2.41 70 20

2.4 ± 1.95 80 20

2.3 ± 1.77 317 ± 96

2.88 ± 1.26 293 ± 107

12.4 ± 31.84 235 ± 111

3.7 ± 2.11 197 ± 87

See Fig. 2C 11 ± 7.5

12 ± 11.4

7.3 ± 7.3

6.8 ± 8.35

100 17 ± 5

100 11 ± 3

80 8 ± 6**

100 12 ± 8

286 ± 23 0.5 ± 0.85

205 ± 70** 1.75 ± 1.39

170 ± 125* 2.3 ± 2.11*

203 ± 100* 1 ± 0.94

3.2 ± 1.87 60 10

2 ± 2.20 75 12.5

2.4 ± 2.12 70 10

2.4 ± 1.17 90 40

4.7 ± 2 25.2 ± 12.12

4.63 ± 3.24 23.13 ± 4.88

8.6 ± 7.93 41.3 ± 36.86

8.8 ± 4.49 30.2 ± 11.25

See Fig. 2D 1.7 ± 2.057

1.63 ± 1.60

1.4 ± 1.51

2.2 ± 2.48

0

0

0.2 ± 0.42

0.2 ± 0.42

2-fold higher value in the old group (80%) as compared to adults (40%). 70% of the mature ovariectomized animals showed reduced defecation while only 62.5% of the mature sham animals did so. With respect to urination, there was a clear age-dependent effect with equal presence in mature and old groups and absence in the adults. The day after, the animals were confronted with the holeboard test. A gradual age-dependent effect on both horizontal (total deambulation) and vertical (rearing) non-goal-directed behaviors was observed (see Table 2 and Fig. 2C). Surprisingly, the number of animals exhibiting goal-directed behavior (head-dips) was reduced to 80% in ovariectomized mice as compared to all the other groups with 100% with this behavior. Holes attain most of the attention of the animals during this test, and this may be the reason why rearing behavior decreases in all groups, going from 90% in adults to 70% in old mice. Head-dipping percentages are only under 100% in the ovariectomized group, since two animals within such group showed a much higher physiological deterioration with respect to the others. Self-grooming behavior increases with aging and there is also an agerelated significant decrease in the latency of self-grooming. Finally,

there is a slight age-related increase in the number of animals showing reduced defecation, while urine incontinence only appears at an old age. In the last day of the experiments, mice were subjected to the T-maze test. This test is based on the spontaneous tendency of mice to explore places, and the results show that aging is directly related to a tendency to reduce the horizontal exploratory behavior, as shown by the increased time that aged and ovariectomized animals need for crossing the intersection of the maze and exploring the whole apparatus (with respect to the adult and mature sham groups). With respect to the adult group, aged mice showed a drastically reduced vertical activity (p b 0.01 in mature sham and ovariectomized groups, see Fig. 2D) and an increased self-grooming behavior. 3.2. Immune function parameters 3.2.1. Chemotaxis assay Migration of leukocytes from the spleen and axillary nodes is represented in Fig. 3A and B (respectively) as the chemotaxis index

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Fig. 1. Equilibrium as % mice falling off the wood rod (A), muscular vigor studied as latency in seconds of falling in the tightrope test (B) and traction capacities displayed in the tightrope test (C; evaluated as maximum and minimum traction capacities). ***p b 0.001, **p b 0.01, *p b 0.05 with respect to adult animals. #p b 0.05 with respect to old animals.

(CI). The first thing we observe in both organs studied is a gradual impairment of this parameter due to physiological aging. Thus, the CI found in old mice is significantly reduced with respect to the adult and mature sham groups (p b 0.001 and p b 0.01, respectively). One interesting finding is that axillary node leukocytes from mature sham animals preserve their chemotaxis capacity at the same level of the adult groups. In the spleen, even though the value of CI in the mature sham group is lower than in adults, no statistically significant differences have been found between these two groups, and thus, we could say that mature sham animals maintain their CI at levels much more similar to the adults than to the mature ovariectomized mice. Regarding the effect of ovariectomy, CI decreases strikingly in leukocytes from mature animals in which ovaries have been removed (p b 0.001 and p b 0.01 for spleen and axillary nodes, respectively) with respect to sham-operated animals of the same age.

3.2.2. Lymphoproliferation in response to LPS Lymphoproliferation values of leukocytes from the spleen and axillary nodes are shown in Fig. 4A and B. In this parameter, the detrimental effect of aging is only relevant in the case of the spleen, while no statistically significant differences were found in the axillary nodes (Fig. 4B). With respect to the spleen, a progressive decrease of the proliferative capacity has been observed from the adult age onwards. In this sense, mature sham and ovariectomized animals show an impairment of this function (p b 0.001) with respect to adults, but this age-related deterioration becomes much more relevant regarding the old group (p b 0.001 with respect to the adult and mature sham groups). Ovariectomy clearly accelerates this detrimental effect, as

Fig. 2. % Mice that perform rearing in the corner (A), open field (B), holeboard (C) and T-maze tests (D). **p b 0.01, *p b 0.05 with respect to the adult group. ••p b 0.01, •p b 0.05 with respect to the mature sham group.

ovariectomized animals show a significantly lower lymphoproliferative response than their sham-operated counterparts (p b 0.01). 3.2.3. Natural killer activity Natural killer (NK) activity also shows a gradual decrease along the process of aging both in leukocytes from the spleen and axillary nodes (Fig. 5A and B, respectively). Mature sham animals show a more significant impaired percentage of lysis than that found in adult mice (p b 0.001 in both organs), which this parameter decreasing even more in the old groups. Once more, ovariectomy performed on mature animals leads to an acceleration of the age-related impairment of the present function, with statistical significant differences found in the axillary nodes (p b 0.01) but not in the spleen.

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Fig. 3. Chemotaxis index (CI) of leukocytes from spleen (A) and axillary nodes (B) in adult (n = 9), mature sham (n = 8), mature ovariectomized (n = 8) and old animals (n = 6). Each column represents the mean ± SD. ***p b 0.001, **p b 0.01 with respect to the adult group. •••p b 0.001, ••p b 0.01 with respect to the mature sham group. ###p b 0.001, ##p b 0.01 with respect to the old group.

Fig. 5. % Lysis of leukocytes from spleen (A) and axillary nodes (B) in adult (n = 7), mature sham (n = 7), mature ovariectomized (n = 7) and old animals (n = 6). Each column represents the mean ± SD. ***p b 0.001 with respect to the adult group. ••p b 0.01 with respect to the mature sham group. #p b 0.05 with respect to the old group.

3.3. Uterine absolute weight Uterine absolute weight was expressed in grams (Table 3). The results show that this parameter tends to increase with advancing age, with the aged group exhibiting significantly higher values than the adult mice group (p b 0.05). Mature sham animals have slightly higher values of uterine weight than their adult counterparts, although no significant differences between them were found. However, ovariectomy induced a striking uterine atrophy with respect to sham animals of the same age (p b 0.01) and adult animals (p b 0.001). 3.4. Estradiol levels As we can observe in Table 3, there is a progressive and significant decrease of estradiol levels in the process of aging (p b 0.001). Ovariectomy precipitates this loss of estradiol, and we have found that mature ovariectomized animals show significantly lower Table 3 Uterine weight (g) and estradiol levels (pg/ml). Results are expressed as mean ± SD in the adult (n = 10), mature sham (n = 8), mature ovx (n = 10) and old group (n = 7). ***p b 0.001, *p b 0.05 with respect to the adult group. ••p b 0.01, •p b 0.05 with respect to the mature sham group. #p b 0.05 with respect to the old group. Fig. 4. % Lymphoproliferation of leukocytes from spleen (A) and axillary nodes (B) in adult (n = 7), mature sham (n = 7), mature ovariectomized (n = 7) and old animals (n = 6). Each column represents the mean ± SD. ***p b 0.001. **p b 0.01 with respect to the adult group. ••p b 0.01 with respect to the sham group. ###p b 0.001 with respect to the old group. Basal cpm in the spleen: 442 ± 81 (adults), 607 ± 200 (mature sham), 628 ± 247 (mature ovx), and 688 ± 331 (old); basal cpm in the axillary nodes: 330 ± 94 (adults), 384 ± 107 (mature sham), 450 ± 285 (mature ovx), and 520 ± 301 (old).

Adult

Mature sham

Mature ovx

Old

Uterine 0.134±0.053 0.191±0.060 0.060 ±0.011**••# 0.310 ± 0.010*•• weight (g) 160.77±37.26 91.96 ±22.59***# 58.89 ±15.77***• 54.72 ± 8.88*** Estradiol levels (pg/ml)

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estradiol levels (p b 0.05) with respect to the sham-operated animals of the same age.

4. Discussion In previous experiments performed in rats we found that the three immune functions studied in the present work, namely the chemotaxis, the lymphoproliferative response and the natural killer activity, were profoundly affected by aging, and that ovariectomy accelerated the age-related deterioration of these parameters (De la Fuente et al., 2004). Following that line of research, the aim of the present work was to study the repercussions of aging and ovariectomy (a model of menopause) in rodents, over two key systems that maintain body homeostasis: the nervous (by means of performing behavioral tests) and the immune (by analyzing key immune function parameters). This work has been performed on mice in the context of the biological aging process and, to our knowledge, this is the first time that ovariectomy has been studied as one possible cause of acceleration of senescence. According to our results, two main facts become clear: first of all, the aging process produces a progressive damaging effect on the neural and immune mouse capacities. Secondly, that ovariectomy performed in these animals (with the associated chronic deficiency of sex hormones) has many implications in a wide variety of non-reproductive functions, such as the nervous and the immune functions. Uterine weight and plasma estrogen levels confirmed that ovary removal had worked as a successful model of surgical menopause. Uterine weight constitutes a typical marker for estrogenic action, since estrogens play a predominant role in inducing uterine weight gain (Frasor et al., 2003). Therefore, ovariectomy is directly associated with a striking uterine atrophy in murine models, a finding described by different researchers (Li and McMurray, 2006; García-Pérez et al., 2006) and also corroborated in the present mouse strain (ICR-CD1). However, ovariectomy has no effects regarding the total mouse weight, a fact that has also been observed in other studies (Pillet et al., 2006). The behavioral capacities of mice along the process of aging and after ovariectomy were assessed through a wide battery of behavioral tests. As expected, age-related impairments of sensorimotor abilities were clearly found in old mice as compared to adults, in agreement with the known physical deterioration associated with aging (Thompson, 2008). With maturity most animals maintained the abilities of adulthood but, when ovariectomy is performed, a shift of abilities towards those seen at older ages was observed, mainly on reflexes, equilibrium, vigor and traction. These results were not due to a difference in weight, as the ovariectomized mice did not differ from the mature sham or old mice. Considering the results of the open field and holeboard tests, it seems that both horizontal and vertical activities are affected by the aging process, but the vertical exploratory activity (rearing activity) is the one which shows the clearest effects due to ovariectomy. In fact, reduction of rearing activity is observed in all tests. When analyzing deambulation, the vertical component (rearing) is considered to be the best parameter for measuring non-goal-directed exploratory activity, while head-dips in the holeboard constitute a specific measure of novelty-seeking or goal-directed behavior (Escorihuela et al., 1999). Although the reduced muscular vigor and the hindlimb extension reflex could account for some of these effects, we have also observed that ovariectomy leads to a reduction of novelty-seeking in the holeboard, which suggests that it may be a general effect of the lack of female sex hormones on the interest or the ability of the animal to interact with the environment. Despite this fact, there is no clear evidence of a higher anxiety state with increasing age or ovariectomy as the variables that are closely related to it (as latency to leave the centre, latency of self-grooming and peripheral versus internal deambulation) are very similar among groups.

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In addition, it has been demonstrated that both the aging process and the lack of sex steroids affect immune responses, although published information about their stimulatory/inhibitory role is still contradictory. The migration of leukocytes towards the inflammatory focus constitutes one of the first steps of the innate immune mechanisms. In the present work, and corroborating previous studies of our group in rats (De la Fuente et al., 2004), we have seen the deteriorating effect of aging over this function both in the spleen and axillary nodes. Moreover, it is clear that female sex hormones play a role in the correct development of such a function, as ovariectomy results in an impairment of leukocyte migration with respect to shamoperated animals of the same age. A work published by Mo et al. (2005) points out that female sex hormones such as estradiol are involved in the expression of chemokine receptors in T cells, with ovariectomy leading to a sudden decrease in such receptors in CD4 T cells. Nevertheless, other studies have observed that estradiol inhibits monocyte migration to the inflammatory focus as a way of inhibiting the development of atherosclerotic lesions (Pervin et al., 1998; Miller et al., 2004). Although both events could be compatible, further investigation is required to clarify the molecular mechanisms involved in the chemotactic capacity of female sex hormones. Lymphoproliferation is a crucial event of the acquired immune response. Several studies carried out in mice, rats and humans have described an age-associated decrease in this function (De la Fuente, 2002; Douziech et al., 2002; De la Fuente et al., 2004; De la Fuente et al., 2008; De la Fuente and Miquel, 2009). This finding has been corroborated by the results obtained in the spleen, although axillary nodes are not affected by age and ovariectomy in the same way. NK activity is considered to be the first line of immune defense against viral infections and neoplasia (Stopinska-Gluszak et al., 2006), and despite the sometimes controversial effects of age on this parameter, it is a function that in most studies shows an age-related decrease (Guayerbas et al., 2002c; De la Fuente et al., 2004; De la Fuente et al., 2008; De la Fuente and Miquel, 2009). Our findings fully agree with these studies, as both in the spleen and axillary nodes there is a gradual impairment of this function with advancing age. With respect to the effect of estrogens over these two parameters, experimental data are still controversial. Some researchers have described an increase of the proliferative response in immune cells after ovariectomy (García-Pérez et al., 2006; Pillet et al., 2006), while others (as it is also our case when studying proliferation of splenocytes) have found impairments (Bilbo and Nelson, 2001; De la Fuente et al., 2004) or even no effects (Li and McMurray, 2006). With regard to NK activity, some studies performed in animal models of ovariectomy and postmenopausal women have described an inhibitory role of estrogens on this parameter (Yovel et al., 2001; StopinskaGluszak et al., 2006). Nevertheless, other studies claim to have found just the opposite (Yang et al., 2000; De la Fuente et al., 2004; Baeza et al., 2007). In the present work, splenocyte proliferation and NK activity were impaired in cells from ovariectomized mice with respect to sham animals, thus accelerating in some way the age-related effect. During the last few years, different animal experimental models have been developed as novel approaches to assess premature aging. This is the case of a model of premature aging validated in our laboratory in the ICR-CD1 strain of mice that relies on the differences in performance among mice when subjected to a T-maze behavioral test (De la Fuente et al., 1998; Guayerbas et al., 2002a,b,c). Animals that fail the test are “biologically older,” i.e., suffer premature immunosenescence, show a neurochemistry similar to mice of an older chronological age and display higher levels of anxiety and emotionality with respect to animals of the same chronological age that performed the test correctly (Viveros et al., 2007). Moreover, these prematurely aging mice showed a shorter life span than their non-prematurely aging mice counterparts (Guayerbas et al., 2002a; Guayerbas and De la Fuente, 2003; Viveros et al., 2007; De la Fuente and Miquel, 2009).

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Even though this work constitutes a first approach in the study of the physiological consequences of ovariectomy and further research is needed, we suggest that ovariectomy could constitute another good model for assessing premature aging, since ovariectomized mice show an older biological age than the sham-operated animals. Recent work has shown that replacement therapies with phytoestrogens could constitute useful strategies for boosting the impaired immune functions that result after ovariectomy (Baeza et al., 2007). Acknowledgements This work was supported by grants from the Spanish MEC (SAF200613642), MICINN (no. BFU2008-04336), UCM Research Group (910379ENEROINN) and RETICEF (RD06/0013/0003) from ISCIII. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneuroim.2009.12.008. References Aloysi, A., Van Dyk, K., Sano, M., 2006. Women's cognitive and affective health and neuropsychiatry. Mt Sinai J. Med. 73, 967–975. Amir, S.H., Kuhle, C.L., Fitzpatrick, L.A., 2003. Comprehensive evaluation of the older woman. Mayo Clin. Proc. 78, 1157–1185. Arlt, W., Hewison, M., 2004. Hormones and immune function: implications of aging. Aging Cell. 3, 209–216. Baeza, I., De Castro, N.M., Alvarado, C., Alvarez, P., Arranz, L., Bayón, J., De la Fuente, M., 2007. Improvement of immune cell functions in aged mice treated for five weeks with soybean isoflavones. Ann. N.Y. Acad. Sci. 1100, 497–504. Baeza, I., Alvarado, C., Alvarez, P., Salazar, V., Castillo, C., Ariznavarreta, C., FdezTresguerres, J.A., De la Fuente, M., 2009. Improvement of leucocyte functions in ovariectomised aged rats after treatment with growth hormone, melatonin, oestrogens or phyto-oestrogens. J. Reprod. Immunol. 80, 70–79. Besedovsky, H.O., Del Rey, A., 2007. Physiology of psychoneuroimmunology: a personal view. Brain Behav. Immun. 21, 34–44. Bilbo, S.D., Nelson, R.J., 2001. Sex steroid hormones enhance immune function in male and female Siberian hamsters. Am. J. Regulatory Integrative Comp. Physiol. 280, R207–R213. Borkan, A., Norris, A.H., 1999. Assessment of biological age using a profile of physiological parameters. J. Gerontol. 35, 177–184. Boyden, S.V., 1962. The chemotaxis effect of mixtures of antibody and antigen on polymorphonuclear leukocytes. J. Exp. Med. 115, 453–466. Castillo, C., Cruzado, M., Ariznavarreta, C., Lahera, V., Cachofeiro, V., Gil-Loyzaga, P., Tresguerres, J.A.F., 2005. Effects of ovariectomy and GH administration on body composition and vascular structure and function in old female rats. Exp. Biogerontol. 6, 49–60. Castillo, C., Salazar, V., Ariznavarreta, C., Vara, E., Tresguerres, J.A.F., 2006. Effect of isoflavone administration on age-related hepatocyte changes in old ovariectomized female Wistar rats. Phytomedicine 13, 468–476. De la Fuente, M., 2002. Effects of antioxidants on immune system aging. Eur. J. Clin. Nutr. 56, S5–S8. De la Fuente, M., 2008. Role of neuroimmunomodulation in aging. Neuroimmunomodulation 15, 213–223. De la Fuente, M., Miquel, J., 2009. An update of the oxidation-inflammation theory of aging. The involvement of the immune system in oxi-inflamm-aging. Current Pharm Design 15, 3003–3026. De la Fuente, M., Miñano, M., Victor, V.M., Del Rio, M., Ferrandez, M.D., Diez, A., Miquel, J., 1998. Relation between exploratory activity and immune function in aged mice: a preliminary study. Mech. Aging Develop. 102, 263–277. De la Fuente, M., Baeza, I., Guayerbas, N., Puerto, M., Castillo, C., Salazar, V., Ariznavarreta, C., Fdez-Tresguerres, J.A., 2004. Changes with aging in several leukocyte functions of male and female rats. Biogerontol. 5, 389–400. De la Fuente, M., Hernanz, A., Guayerbas, N., Victor, V.M., Arnalich, F., 2008. Vitamin E ingestion improves several immune functions in elderly men and women. Free Radic. Res. 42 (3), 272–280. Del Río, M., Herranz, A., De la Fuente, M., 1994. Bombesin, gastrin-releasing peptide, and neuromedin C modulate murine lymphocyte proliferation through adherent accesory cells and activate protein kinase C. Peptides 15, 15–22. Douziech, N., Seres, I., Larbi, A., Szikszay, E., Roy, P.M., Arcand, M., Dupuis, G., Fulop Jr., T., 2002. Modulation of human lymphocyte proliferative response with aging. Exp. Gerontol. 37, 369–387. El Haber, N., Erbas, B., Hill, K., Wark, J.D., 2008. Relationship between age and measures of balance, strength and gait: linear and non-linear analysis. Clin. Sci. 114, 719–727. Escorihuela, R.M., Fernández-Teruel, A., Gil, L., Aguilar, R., Tobeña, A., Driscoll, P., 1999. Inbred Roman high- and low-avoidance rats: differences in anxiety, noveltyseeking, and shuttlebox behaviors. Physiol. Behav. 67, 19–26.

Ferrández, M.D., Correa, R., Del Río, M., 1999. Effect in vitro of several antioxidants on the natural killer function of aging mice. Exp. Gerontol. 34, 675–685. Ferrari, E., Magri, F., 2008. Role of neuroendocrine pathways in cognitive decline during aging. Aging Res. Rev. 7, 225–233. Frasor, J., Barnett, D.H., Danes, J.M., Hess, R., Parlow, A.F., Katznellenbogen, B.S., 2003. Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) alpha activity by ERbeta in the uterus. Endocrinology 144, 3159–3166. García-Pérez, M.A., Del Val, R., Noguera, I., Hermenegildo, C., Pineda, B., MartínezRomero, A., Cano, A., 2006. Estrogen receptor agonists and immune system in ovariectomized mice. Int. J. Immunopath. Pharmacol. 19, 807–819. Giménez-Llort, L., Fernández-Teruel, A., Escorihuela, R.M., Fredholm, B.B., Tobeña, A., Pekín, M., Johansson, B., 2002. Mice lacking the adenosine A1 receptor are anxious and aggressive, but are normal learners with reduced muscle strength and survival rate. Eur. J. NeuroSci. 16, 547–550. Gruver, A.L., Hudson, L.L., Sempowski, G.D., 2007. Immunosenescence of aging. J Pathol. 211, 144–156. Guayerbas, N., De la Fuente, M., 2003. An impairment of phagocytic function is linked to a shorter life span in two strains of prematurely aging mice. Dev. Comp. Immunol. 27, 339–350. Guayerbas, N., Catalán, M., Victor, V.M., Miquel, J., De la Fuente, M., 2002a. Relation of behavior and macrophage function to life span in a murine modelo of premature immunosenescence. Behav. Brain Res. 134, 41–48. Guayerbas, N., Puerto, M., Fernández, M.D., De la Fuente, M., 2002b. A diet supplemented with thiolic antioxidants improves leucocyte function in two strains of prematurely aging mice. Clin. Exp. Pharmacol. Physiol. 29, 1009–1014. Guayerbas, N., Puerto, M., Victor, V.M., Miquel, J., De la Fuente, M., 2002c. Leucocyte function and life span in a murine model of premature immunosenescence. Exp. Gerontol. 37, 249–256. Heinikken, T., Puoliväli, J., Tanila, H., 2004. Effects of long-term ovariectomy and estrogen treatment on maze learning in aged mice. Exp. Gerontol. 39, 1277–1283. Islander, U., Erlandsson, M.C., Hasseus, B., Johnsson, C.A., Ohlsson, C., Gustafsson, J.A., 2003. Influence of oestrogen receptor α and β on the immune system in aged female mice. Immunology 110, 149–157. Johansson, B., Halldner, L., Dunwiddie, T.V., Masino, S.A., Poelchen, W., Giménez-Llort, L., Escorihuela, R.M., Fernández-Teruel, A., Wiesenfeld-Hallin, Z., Xu, X.J., Hårdemark, A., Betsholtz, C., Herlenius, E., Fredholm, B.B., 2001. Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor. Proc. Natl. Acad. Sci. U. S. A. 98, 9407–9412. Kumar, R., Burns, E.A., 2008. Age-related decline in immunity: implications for vaccine responsiveness. Expert Rev. Vaccines 7, 467–479. Li, J., McMurray, R.W., 2006. Effects of estrogen receptor subtype-selective agonists on immune functions in ovariectomized mice. Int. Immunopharmacol. 6, 1413–1423. McMurray, R.W., 2001. Estrogen, prolactin and autoimmunity: actions and interactions. Int. Immunopharmacol. 1, 995–1008. Miller, A.P., Feng, W., Xing, D., Weathington, N., Blalock, E., Chen, Y., Oparil, S., 2004. Estrogen modulates inflammatory mediator expression and neutrophil chemotaxis in injuried arteries. Circulation 110, 1664–1669. Miquel, J., Blasco, M., 1978. A simple technique for evaluation of vitality loss in aging mice, by testing their muscular coordination and vigor. Exp. Gerontol. 13, 389–396. Miquel, J., Ramírez-Boscá, A., Ramírez-Boscá, J.V., Alperi, J.D., 2006. Menopause: a review on the role of oxygen stress and favorable effects of dietary antioxidants. Arch. Gerontol. Geriatr. 42, 289–306. Mo, R., Chen, J., Grolleau-Julius, A., Murphy, H.S., Richardson, B.C., Yung, R.L., 2005. Estrogen regulates CCR gene expression and function in T lymphocytes. J. Immunol. 174, 6023–6029. Morrison, J.H., Brinton, R.D., Schmidt, P.J., Gore, A.C., 2006. Estrogen, menopause, and the aging brain: how basic neuroscience can inform hormone therapy in women. J. Neurosci. 26, 10332–10348. Nelson, H.D., 2008. Menopause. Lancet 371, 760–770. O'Connor, J.C., McCusker, R.H., Strle, K., Johnson, R.W., Dantzer, R., Kelley, K.W., 2008. Regulation of IGF-I function by proinflammatory cytokines: at the interface of immunology and endocrinology. Cell. Immunol. 252, 91–110. Perez-Martin, M., Salazar, V., Castillo, C., Ariznavarreta, C., Azcoitia, I., Garcia-Segura, L.M., Tresguerres, J.A.F., 2005. Estradiol and soy extract increase the production of new cells in the dentate gyrus of old rats. Exp. Gerontol. 40, 450–453. Pervin, S., Singh, R., Rosenfeld, M.E., Navab, M., Chaudhuri, G., Nathan, L., 1998. Estradiol suppresses MCP-1 expression in vivo. Implications for atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 18, 1575–1582. Pillet, S., D'Elia, M., Bernier, J., Bouquegneau, J.M., Fournier, M., Cyr, D.G., 2006. Immunomodulatory effects of estradiol and cadmium in adult female rats. Toxicol. Sci. 92, 423–432. Rasgon, N., Shelton, S., Halbreich, U., 2005. Perimenopausal mental disorders: epidemiology and phenomenology. CNS Spectr. 10, 471–478. Rehman, H.U., Masson, E.A., 2005. Neuroendocrinology of female aging. Gend. Med. 2, 41–56. Sarkaki, A., Amani, R., Badavi, M., Safahani, M., Aligholi, H., 2008. Effect of ovariectomy on reference memory version of Morris water maze in young adult rats. Iranian Biomed. J. 12, 123–128. Stopinska-Gluszak, U., Waligora, J., Grzela, T., Gluszak, M., Jozwiak, J., Radomski, D., Roszkowski, P.I., Malejczyk, J., 2006. Effect of estrogen/progesterone hormone replacement therapy on natural killer cell cytotoxicity and immunoregulatory cytokine release by peripheral blood mononuclear cells of postmenopausal women. J. Reprod. Immunol. 69, 65–75. Thompson, L.V., 2008. Age-related muscle dysfunction. Exp. Gerontol. 44, 106–111.

I. Baeza et al. / Journal of Neuroimmunology 219 (2010) 90–99 Tresguerres, J.A.F., Kireev, R., Tresguerres, A.F., Borras, C., Vara, E., Ariznavarreta, C., 2008. Molecular mechanisms involved in the hormonal prevention of aging in the rat. J. Steroid. Biochem. Mol. Biol. 108, 318–326. Venkataraman, C., Shankar, G., Sen, G., Bondada, S., 1999. Bacterial lipopolysaccharide induced B cell activation is mediated via a phosphatidylinositol 3-kinase dependent signaling pathway. Immunol. Lett. 69, 233–238. Viveros, P., Arranz, L., Hernanz, A., Miquel, J., De la Fuente, M., 2007. A model of premature aging in mice based on altered stress-related behavioral response and immunosenescence. Neuroimmunomodulation 14, 157–162.

99

Wayne, S.J., Rhyne, R.L., Garry, P.J., Goodwin, J.S., 1990. Cell-mediated immunity as a predictor of morbidity and mortality in subjects over 60. J. Gerontol. 45, 45–48. Yang, J.H., Chen, C.D., Wu, M.Y., Chao, K.H., Yang, Y.S., Ho, H.N., 2000. Hormone replacement theraphy reverses the decrease in natural killer cytotoxicity but does not reverse the decreases in the T-cell subpopulation or interferon-gamma production in postmenopausal women. Fertil. Steril. 74, 261–267. Yovel, G., Shakhar, K., Ben-Eliyahu, S., 2001. The effects of sex, menstrual cycle and oral contraceptives on the number and activity of natural killer cells. Gynecol. Oncol. 81, 254–262.