Chronic Immobilization Stress

Introduction
The hippocampus is a vital region of the brain that regulates major aspects of learning, memory and emotions. The hippocampus has also been linked in the regulation and control of anxiety response and conditioned fear (Yee et al. 2007) and yet it can also retain a high degree of plasticity (McEwen, Gianaros 2010). Looking at the structure of the hippocampus, one would find a curved arrow on either hemisphere. The curved region is known as the CA1-CA3 subfields which contain the pyramidal layer while the arrow known as the dentate gyrus (DG) is made up of a granular cell layer. The neurons in CA3 (the lower part of the curve) connects directly to CA1 (the upper part of the curve), which would receive input from the DG.
Another component of the hippocampus is the brain-deprived neurotrophic factor (BDNF) and its specific receptor TrkB. It has been found that in previous research that there is a high level of BDNF expression in the central nervous system in which it plays an important role in the survival, protection, maintenance and differentiation from insults to neural cells (Barde, 1989). In the hippocampus however, BDNF helps with long-term potentiation (Figurov, 1996). The majority of the neural effects of BDNF are regulated by binding to the TrkB receptor (Barde, 1989). The BDNF and TrkB could also play a role in the stress response in hippampal neurons (Tapia-Arancibia et al. 2004, Pardon et al. 2005, Sirianni et al. 2010).

Previous research has observed how BDNF and TrkB in the hippocampus play a role in the regulation of the HPA axis. Its role has to do with the termination of hypothalamopituitary-adrenocortical (HPA) axis responses to stress (Pizarro et al. 2004, McCormick et al. 2010), essentially the regulating the stress circuit to help maintain homeostasis (Herman et al. 1995). Other studies have found that this structure regulates the HPA axis through inhibiting its activity of BDNF and TrkB (Herman et al. 1996) and that hippocampal neurons can be hypersensitive to stress (Rot et al. 2009). They are especially vulnerable to long-term damaging influence of the glucocorticoids and can decrease expressions of BDNF (McEwen and Magarinos 2001, Bartolomucci et al. 2002, Murakami et al. 2005). It has been found that the consequences of long-term exposure to stress in the hippocampus can remodel hippocampal cells, which then can result in a malfunction of the affected area (Diamond et al.1996, Bremner 1999). Different types of chronic or acute stress can modulate the reduction of BDNF and TrkB mRNA expression in the hypothalamus and in the pituitary gland of adult male rats (Rage 2002, Murakami 2005, Givalois 2001).
Stress can play a more damaging role in the hippocampal regions of young and aged rats as well. Hippocampal vulnerability and reduction in neurotrophic factors have also been found in stressed and aging rats (Smith 1996, Li Yi 2009), and as well as in age-dependent rats that were postnatally exposed to maternal deprivation, which changed the BDNF expression in selected rat brain regions (Roceri, 2004). All of which can possibly affect the structural synaptic plasticity which is has been found to be preserved in the dentate gyrus of aged rats (Geinisman, 1992) and has been linked to certain mood disorders (Duman, 2000).
Many previous studies on the role of BDNF and TrkB in the hippocampus under certain stress conditions relied mainly on the observations of modifications in the matter of these proteins in adult animals during immobilization stress (Givalois et al. 2001, Rage et al. 2002, Marmigere et al. 2003, Reagan 2007). All of which has found a difference in the expression of BDNF in certain regions of the HPA axis in adult male rats, which again can lead to the dysfunction of that affected region. Furthermore, to my knowledge no reports have been published about chronic immobilization stress-induced responsiveness of BDNF and TrkB and their role in juvenile and aged animals exposed to immobilization stress during two critical stages of brain morphologic and functional transformations. The hypothesis of the proposed study is that juvenile rats will have a reduction of the hippocampal region than those of aged rats when exposed to chronic immobilization stress.
Methods
Animal
Twenty male Sprague-Dawley rats from two age groups would be used for this study. The first group described as juvenile –JUV (postnatal 28 days) and the second group described as aged –AGE (postnatal 360 days). They would be housed in groups of five animals per plastic cage in a room maintained under standardized light (8am to 8pm-hour light-dark cycle) and temperature (22±3°C) conditions. They would be housed at least 1 week prior to the experiment. The animals would receive free access to food pellets and tap water. The care and treatment of the rats would be in accordance with the guidelines for laboratory animals established by the National Institute of Health as well as by the Local Ethical Committee of the University of Massachusetts Amherst.
Experimental Procedure
All tests were conducted once a day in 60-minute sessions for 20 consecutive days at the same time, between 10:00am and 3:00pm. JUV and ADT animals would be divided into experimental groups (n=5), exposed to chronic immobilization stress and control non-stressed groups (n=5), which would remain in their home cages until perfusion. The control animals would be handled for a few minutes daily by the same operator. The chronic stress stimulation would begin for JUV rats at postnatal seven days old.
Chronic Immobilization Stress
During each session, the animals would be immobilized in accordance with a well-established protocol (Badowska-Szalewska, 2010). Rats are fixed on a wooden board (18?25 cm) in a supine position by means of a leather belt, after which each of their legs was fixed at an angle of 45° to the body midline with adhesive tape (Badowska-Szalewska, 2010). Experimental and control animals were sacrificed on postnatal day 28 (JUV) and 360 (ADT), 90 min after the final session. All animals will be deeply anesthetized with a lethal dose of a drug (choice pending) and then perfuse with 0.9% saline solution with heparin, followed by 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.4) (Badowska-Szalewska, 2010). The brains will be removed and kept overnight and serial coronal sections of brain (40-?m-thick) will be cut.
Immunohistochemistry
Bordering sections would be processed for BDNF and TrkB with immunohistochemistry. The free-floating sections will be blocked in 10% Normal Goat Serum (NGS) for 2 hours and incubated at 4?C for 3 days with the primary polyclonal rabbit anti-BDNF antibody and primary polyclonal rabbit anti-TrkB antibody (Badowska-Szalewska, 2010). After multiple rinses in a buffered saline (TBS), the sections will be incubated (2-3 hours, room temperature) with a secondary antibody. The controls for the immunohistochemical procedures will be processed with the same procedure with the exception of the primary or secondary antibodies. Therefore, no staining will be observed in the control slides.
Qualitative Analysis
An image analysis system will be used to analyze the number of BDNF-ir and TrkB-ir positive cells in the hippocampus. The total number will be divided into three (counted separately) areas of the hippocampus: CA1 subfield, CA3 subfield and dentate gyrus (DG). The cells that will be counted are the BDNF-ir cells or TrkB-ir cells in the pyramidal layer of CA1 and CA3, and in the granular layer of DG. The hemisphere and sampling will be chosen at random. The data will be analyzed by a two-way analysis of variance (ANOVA) for the factor groups (intact vs. chronic immobilization stress) and age groups (juvenile-JUV and aged-AGE).
Expected Results
Under chronic immobilization stress exposure, juvenile rats should have a significant decrease in the density of BDNF immunoreactive (ir) neurons and TrkB-ir cells should be observed in CA1, CA3 and DG. After chronic immobilization stress exposure of aged rats, the density of BDNF-ir and TrkB-ir cells should not decline in any of the sub-regions of the hippocampus.
Discussion
The present study would investigate the age-related changes in the density of BDNF-ir and TrkB-ir neurons in the under the exposure of immobilized stress. Prolonged forced swim has been found to affect the amount of BDNF-ir and TrkB-ir cells in the hippocampus of juvenile, not aged rats (Badowska 2010). The number of cells that contains these proteins would be higher in DG than in CA1 or CA3, which is mostly to be related to the various intrinsic and extrinsic interactions of the denrate gyrus (Amaral and Witter 1989). If proven, then the high levels of BDNF and TrkB that are present in the hippocampus indicate that these molecules have important physiological functions in different stages of life (Tapia-Arancibia et al. 2008). It is extremely important as demonstrated during adolescence; BDNF influences almost all aspects of development, including stimulation of growth, differentiation of neuronal stem cells and many other various roles (Mattson et al. 2004, Tapia-Arancibia et al. 2004). The flip side of the coin is that during aging, BDNF may play a protective role by preventing neurodegeneration, and stimulating sprouting in the hippocampus (Smith 1996, Tapia-Arancibia et al. 2004), or increasing neuronal repair (Smith et al. 1995). As for TrkB, it exerts positive influence on dendritic branching and dendrtic integrity/plasicity in the hippocampus (Sato et al. 2001), therefore a reduction in the level of this protein may be underlying factor in the synaptic changes that occur with age in the hippocampus (Geinisman et al. 1992). It is also known that adolescent rats, versus adult subjects, tend to be more susceptible to the influence of aversive stimuli, which is created by HPA axis activation (Avital and Richter-Levin 2005, Lupien 2009), by the prolonged secretion of glucocorticoid (Romeo et al. 2004, Cruz et al. 2008, McCormick et al. 2010). Also the expression of BDNF and TrkB is possibly regulated in opposite direction, meaning that the growth of BDNF content occurs with the fall in the level of TrkB in the hippocampal cells of aged rats (Frank et al. 1997, Nibuya et al. 1999, Sommerfeld et al. 2000, Silhol M et al. 2007, Tapia-Arancibia et al. 2008).Therefore, the higher density of BDNF-ir and the lower density of TrkB-ir cells in AGE group of experimental rats may signify their protective effects against hippocampal damage of aging animals in stress conditions. This study will demonstrate that hippocampal subfields of juvenile and aged rats show different density of BDNF and TrkB immunostaining cells. Chronic immobilization stress would influence the density of BDNF-ir and TrkB-ir in juvenile animals and the aged rats would be the determining factor in the changes in the density of BDNF-ir and TrkB-ir in the hippocampal regions.
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