- Email: [email protected]
Hippocampal neurokinin-1 receptor and brain-derived neurotrophic factor gene expression is decreased in rat models of pain and stress
Neuroscience 133 (2005) 999 –1006
HIPPOCAMPAL NEUROKININ-1 RECEPTOR AND BRAIN-DERIVED NEUROTROPHIC FACTOR GENE EXPRESSION IS DECREASED IN RAT MODELS OF PAIN AND STRESS V. DURIC AND K. E. MCCARSON*
tent inflammatory stimuli increase both NK-1 receptor (McCarson and Krause, 1994, 1995, 1996) and BDNF mRNA levels (Zhou and Rush, 1996), while nociception- or SP-evoked dorsal horn cell excitation is blocked by specific NK-1 receptor antagonists (Radhakrishnan and Henry, 1991; McCarson and Krause, 1996). Similar regulation of NK-1 gene expression may also occur in other CNS sites; the observation that NK-1 receptor antagonists have antidepressant effects (Kramer et al., 1998) suggests that the effects of pain and stress may converge in higher brain centers sensitive to both types of stimuli, such as restraint or immobilization stress, as well as peripheral inflammatory nociception. However, the regulation of NK-1 receptor expression and function in mood/affect and emotion-processing systems (e.g. amygdala, hippocampus and hypothalamus) is poorly understood (DeFilipe et al., 1998; Maeno et al., 1993). The functional similarities between neurokinin receptors in affective and sensory systems are undefined, as is the role of hippocampal NK-1 receptors in the context of chronic pain. From previous studies, it is clear that stress can have profound effects on the hippocampus, a central component of the limbic system and one of key regulators of affect. Stress hormones target the hippocampus, making it a particularly plastic and vulnerable brain structure (Sapolsky, 2000). The hippocampus is sensitive to, and can be damaged by, endogenous and/or environmental factors such as chronic stress, aging, stroke or head trauma (Sapolsky, 1992; McEwen, 1999). Studies using stress as a model of depression have shown that long-term exposure to stress is accompanied by neuronal atrophy of the hippocampus (Watanabe et al., 1992) and a decrease in the expression of BDNF (Smith et al., 1995; Duman and Charney, 1999). Furthermore, clinical brain imaging studies have demonstrated that the volume of the hippocampus is reduced in patients with chronic depression (Sheline et al., 1996, 1999) or post-traumatic stress disorder (Bremner et al., 1995, 1997; Gurvits et al., 1996; Stein et al., 1997). Conversely, long-term antidepressant administration causes an increase in the expression of both BDNF and TrkB genes in hippocampus (Nibuya et al., 1995, 1996, 1999; Vaidya and Duman, 1996; Duman et al., 1997). Chronic pain also may affect the hippocampus and initiate cellular mechanisms that resemble stress. Therefore, we hypothesize that nociceptive stimuli regulate NK-1 receptor as well as a BDNF gene expression in the hippocampus. This hypothesis was tested by quantifying NK-1 and BDNF gene expression in rats; results indicate that either stress or pain down-regulates expression of
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Mail Stop 1018, Kansas City, KS 66160, USA
Abstract—Acute or chronic stress can alter hippocampal structure, cause neuronal damage, and decrease hippocampal levels of the neurotrophin brain-derived neurotrophic factor (BDNF). The tachykinin substance P and its neurokinin-1 (NK-1) receptor may play a critical role in neuronal systems that process nociceptive stimuli; their importance in stressactivated systems has recently been demonstrated by the antidepressant-like actions of NK-1 receptor antagonists. However, the functional similarities between neurokinin receptors in the hippocampus and those in sensory systems are poorly understood, as is the significance of hippocampal NK-1 receptor in the context of chronic pain. Therefore, we investigated the effects of immobilization stress or inflammatory stimuli on NK-1 receptor and BDNF gene expression in the rat hippocampus. Rats received an acute or chronic immobilization stress, or an acute (formalin) or chronic (complete Freund’s adjuvant) inflammatory stimulus to the right hind paw. Subsequently hippocampal volume and specific gravity were measured and NK-1 receptor and BDNF mRNA levels quantified using ribonuclease protection assays. Results showed that either stress or pain down-regulates expression of both NK-1 receptor and BDNF genes in the hippocampus. Hippocampal volume was increased by either pain or stress; this may be due to edema (decreased specific gravity). Thus, BDNF and NK-1 receptor gene plasticity may reflect sensory activation or responses to neuronal injury. These data may provide useful markers of hippocampal activation during chronic pain, and suggest similarities in the mechanisms underlying chronic pain and depression. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: substance P, immobilization stress, nociception, formalin, CFA, edema.
The tachykinin neuropeptide substance P (SP) and brainderived neurotrophic factor (BDNF), as well as their preferred receptors, neurokinin-1 (NK-1) receptor and tyrosine kinase B (TrkB) respectively, have been well characterized as mediators of nociceptive sensory information in somatosensory pathways in the nervous system (Vaught, 1988; Henry, 1993; Cho et al., 1997). In the spinal cord, persis*Corresponding author. Tel: ⫹1-913-588-7519; fax: ⫹1-913-588-7501. E-mail address: [email protected] (K. E. McCarson). Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; cAMP, cyclic AMP; CFA, complete Freund’s adjuvant; CREB, cyclic AMP response element binding protein; HPA, hypothalamo–pituitary–adrenal axis; NK-1, neurokinin-1; SP, substance P; TrkB, tyrosine kinase B. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.04.002
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both NK-1 and BDNF genes in the hippocampus. The results suggest that NK-1 and BDNF genes provide useful markers of the hippocampal activation that may be coregulated during chronic pain or stress.
EXPERIMENTAL PROCEDURES Animal housing and handling Seventy-four young male Sprague–Dawley rats (Harlan Farms, Indianapolis, IN, USA), used for all experiments, were age matched (7– 8 weeks old) at the beginning of the treatments. The maintenance of the rat colony and all the animal treatments were in accordance with NIH laboratory care standards and approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee. Efforts were made to minimize animal suffering and to reduce the number of animals used in this study. Animals were housed (12-h light/dark cycle) in groups of three per cage with ad libitum access to food and water; they were mixed together so there is one member of each treatment group in every cage. All rats, including the sham control group, were handled the same way to reduce the effects of stress associated with handling on the results.
Experimental design Rats (200 –300 g) were subjected to either immobilization stress or injected with an inflammatory stimulus (Fig. 1). Acute stress (n⫽12) rats were immobilized in restraint bags (Rodent Restraint Cones, Harvard Apparatus, Inc., Holliston, MA, USA) once for 45 min, while chronic stress (n⫽12) rats were immobilized for 45 min once daily for 10 consecutive days. Acute inflammatory pain rats received a s.c. injection of 100 L of 5% formalin (Fisher Scientific Co., St. Louis, MO, USA) into the plantar aspect of the right hind paw. Animals were decapitated either at 2 h and 45 min (n⫽12), 6 h (n⫽7), or 24 h (n⫽7) after the initiation of the final (or only) treatment. Chronic inflammatory pain (n⫽12) rats received a s.c. injection of 50 L of complete Freund’s adjuvant (CFA) (Sigma Chemical Co., St. Louis, MO, USA) into the plantar aspect of the right hind paw. Decapitation followed 10 days later. Sham (n⫽12)
animals received no hind paw injection, but were momentarily restrained and their right hind paw manipulated. Otherwise, they were handled identically to the treatment animals and were decapitated on the tenth day. The animal handling consisted of identical housing regimens, daily transport from the animal facilities to the laboratory, and interactions with the handler, including being picked up by the tail for taking weight measurements. One set of animals (n⫽8 or 7, from each treatment group) was used for the analyses of gene expression and edema assessment by solution hybridization–nuclease protection assays and specific gravity studies, respectively. The remaining rats (n⫽4, from each treatment group; 6 h and 24 h formalin groups excluded) were used for analysis of hippocampal volume.
Solution hybridization–nuclease protection assays Immediately after decapitation, rat brains were removed and dissected along the saggital midline. Left and right sides of the hippocampi were dissected as a whole (including dentate gyrus and CA 1– 4 sub-regions) and total cellular RNAs were extracted from the hippocampal tissue samples using a rapid guanidinium isothiocyanate–phenol/chloroform extraction method. The resultant total RNAs were analyzed using solution hybridization– nuclease protection assays (Fig. 1). Antisense 32P-labeled cRNA probes were synthesized using [␣-32P]UTP (3000 Ci/mmol) according to procedures suggested by Promega Biotec. Subsequently the DNA template was digested using RQ1 DNase (Promega). Total RNA samples were assayed for NK1, BDNF or -actin mRNAs using solution hybridization–nuclease protection assays. Briefly, 2⫻105 d.p.m. of each specific 32P-labeled antisense cRNA probe was co-precipitated with 2– 80 g of total RNA, 100 –1000 pg cRNA quantitation standards, or E. coli tRNA. The RNA-[32P]RNA co-precipitates were each re-suspended in 10 l of hybridization buffer (40 mM 1,4-piperazine diethane sulfonic acid (PIPES), pH 6.4, 400 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA); 80% (v/v) deionized formamide) and incubated for 16 –20 h at 45 °C. Digestion by nucleases A (4 g/mL) and T1 (0.2 g/mL) was performed for 20 min at 37 °C, followed by a 15 min digestion at 37 °C with proteinase K (100 g/sample). The digestion reaction products were precipitated, re-suspended,
Fig. 1. Experimental procedures used for quantifying NK-1 receptor and BDNF mRNA levels in hippocampal tissues of rats. A detailed description can be found in Experimental Procedures.
V. Duric and K. E. McCarson / Neuroscience 133 (2005) 999 –1006 and electrophoresed on 6% polyacrylamide gels containing 7 M urea. Gels were fixed, dried, and exposed to phosphor plates for 16 –24 h. Densitometric images were generated and analyzed using a Molecular Dynamics PhosphorImager SF (Sunnyvale, CA, USA). Specific mRNA amounts were determined by comparison to cRNA quantitation standards. Data values are reported as pg specific mRNA/ng -actin mRNA [mean⫾S.E.M.].
Hippocampal volume analysis Frozen (⫺70 °C) rat brains were cut transversely into 150 m thick sections using a cryostat (Leica Jung Frigocut 2800). The sections were mounted on gelatin-coated slides, stained with Cresyl Violet, dehydrated, and coverslipped. A computer-controlled digital camera (Coho, Japan) was used to collect images of each section. Individual measurements of left, right and total hippocampal areas on each section were measured using Scion Image (NIH Software). The area encompassed by hippocampus on each section was analyzed and the hippocampal volume calculated by summing the areas of all individual sections containing hippocampus. Volume data values are reported as mm3 [mean⫾S.E.M.].
Specific gravity analysis Specific gravity analysis for hippocampal edema was performed using a kerosene/bromobenzene density gradient column prepared in a 1 L graduated cylinder. The column was made by gradually mixing a heavy solution (1.0650 g/cm3; 192 mL bromobenzene and 308 mL kerosene) with a light solution (1.0250 g/ cm3; 162 mL bromobenzene and 338 mL kerosene). Sodium chloride solutions of known specific gravity were used as quantitation standards to determine the linearity of the density gradient column. Six standard solutions, ranging from 1.0385 g/cm3 to 1.0563 g/cm3, were prepared by dissolving a known amount of NaCl into 100 mL of distilled water. Two drops (volume⫽10 – 15 L) of each standard were placed into the column and their location in the column was measured after 2 min using a cathetometer. Hippocampal samples were dissected under and stored in cold (4 °C) kerosene to reduce tissue water loss. Small tissue samples (approx. 2–3 mm3) were placed into the density column and their position was determined after 2 min. The specific gravity of the samples was calculated by interpolation using the NaCl standards curve; data values are reported as g/cm3 [mean⫾ S.E.M.]. A decrease in specific gravity is correlated with an increase in tissue water content (edema). Specific gravity values were used to calculate percent tissue volume change (Nelson et al., 1971) from the following equation: % change in tissue volume as water ⫽[((sp.gr.⫺1)cont. ⁄ (sp.gr.⫺1)exp.)⫺1]⫻100 Note: sp.gr.⫽specific gravity; cont.⫽control; and exp.⫽experimental.
Statistical analysis Data from all the experiments were analyzed using analysis of variance (ANOVA) with Fisher’s PLSD tests used for post hoc comparisons. Significance was considered to be P⬍(or equal) 0.05; all comparisons were made to naïve controls.
RESULTS To determine the effects of stress on the regulation of NK-1 receptor and BDNF gene expression in hippocampus, rats were subjected to either acute stress (45 min) once or chronically (10 consecutive days) by immobilizing them in plastic restraining bags. Hippocampal NK-1 receptor and
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BDNF mRNA levels were assessed 2 h post-treatment using solution hybridization–nuclease protection assays (Fig. 2). Bilateral levels of NK-1 receptor mRNA were significantly down-regulated by either acute or chronic immobilization stress (Fig. 3a). A single 45 min immobilization of the acute stress paradigm provoked a more robust response than chronic stress treatment (51% vs. 32% reduction in NK-1 receptor mRNA levels, respectively). Stress treatments evoked similar responses in regulation of BDNF gene expression. Bilateral levels of hippocampal BDNF mRNA were significantly reduced by either acute or chronic immobilization stress (Fig. 3b) as previously reported by Nibuya et al. (1999). Acute stress produced a slightly more robust response. The influence of peripheral inflammatory pain on the expression of NK-1 receptor and BDNF mRNA in the rat hippocampus was also assessed using solution hybridization–nuclease protection assays. Initially all the assays were performed on ipsilateral vs. contralateral side of the hippocampus, since the inflammatory stimuli were s.c. injected into the rat right hind paw. However, formalin or CFA did not produce any significant sided differences in expression of either NK-1 receptor or BNDF genes, so the mRNA levels are shown as hippocampal bilateral average. Fig. 4a shows that injections of either 5% formalin or CFA both evoked significant decreases in hippocampal NK-1 receptor mRNA levels; the largest changes were observed at 6 h and 24 h post-formalin injections, which produced reduction in NK-1 receptor mRNA by 63% and 54%, respectively. Likewise, hippocampal BDNF mRNA levels were also significantly reduced after either formalin or CFA treatments (Fig. 4b). The most robust decrease occurred at the earliest time point, 2 h and 45 min post-formalin injection, which produced a 67% reduction in BDNF mRNA level. Hippocampal -actin mRNA levels (data not shown), unaffected by either pain or stress, were measured to serve as gel loading controls and to ensure that the observed changes in NK-1 receptor and BNDF gene expression are not due to a global down-regulation of genes in the hippocampus. Volumetric studies (Fig. 5) showed that both pain and stress can have a profound effect on the size of the hippocampus. Formalin and CFA nociception paradigms increased hippocampal volume by 18% and 12%, respectively. Acute stress, similar to formalin in the duration of the treatment, increased the overall volume of hippocampus by 15%. Chronic stress also produced a slight increase in volume, but the data were not statistically different from the sham controls. The purpose of the specific gravity experiment was to determine one of the possible mechanisms underlying the volume increase; measuring the specific gravity of the hippocampal tissue allowed us to assess how much of the change in volume is due to edema. In edematous brain tissue, the decrease in specific gravity of the tissue occurs from the influx of fluid (Emerson et al., 1999). Neither acute nor chronic pain had any effect on the specific gravity of hippocampal tissue (Table 1); however, both acute and chronic immobilization stress models provoked robust decreases in specific gravity resulting in increases
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Fig. 2. Densitometric images of the representative solution hybridization–nuclease protection assays used for quantitation of (A) NK-1 receptor and (B) BDNF mRNA species in total RNA samples isolated from hippocampal tissues of rats exposed to either immobilization stress, inflammatory pain or sham paradigms. (A) NK-1: Each sample contained 40 g of total RNA. The arrows show the sizes of the undigested NK-1 receptor antisense probe (696 bp) and the RNA species protected by the NK-1 receptor mRNA or 100 –200 pg of message-sense NK-1 receptor cRNA quantitation standard (588 bp). (B) BDNF: Each sample contained 80 g of total RNA. The arrows show the sizes of the undigested BDNF antisense probe (597 bp) and the RNA species protected by the BDNF mRNA or 100 –200 pg of message-sense BDNF cRNA quantitation standard (539 bp). Each lane in these assay images represents an RNA sample from one individual experimental subject. Thus, treatment effects may not be fully appreciable when viewing a single assay gel. (Note: acute stress⫽one 45 min bout of immobilization; chronic stress⫽45 min immobilization daily for 10 consecutive days; acute pain⫽2 h and 45 min after formalin injection; chronic pain⫽10 days after CFA injection.)
in tissue water content of 25% and 14%, respectively. Such an observation could provide one possible explanation for the initial increases in hippocampal volume seen after stress.
DISCUSSION Chronic pain is characterized by many complex alterations in nociceptive pathways and is often accompanied by clinical depression (Ruoff, 1996; Fishbain et al., 1997; Gallagher and Verma, 1999; Bair et al., 2003; Currie and Wang, 2004). To date, little emphasis has been on the mechanisms contributing to the “affective” component of pain or its impact on cognitive function (Woolf and Salter, 2000; Julius and Basbaum, 2001). Beyond the physiological aspects of nociception, a negative impact on affect is required for accurate characterization of a stimulus as “painful.” In this study we compared the effects of inflam-
matory pain and immobilization stress on the hippocampus, a limbic region involved in regulation of affect. NK-1 receptors and BDNF have previously been implicated in nociception-induced spinal central sensitization, however the effects of pain stimuli on the expression of these two genes in the brain regions involved in the regulation of emotion and affect have not yet been characterized. Persistent pain may have an effect on the hippocampus and initiate cellular transduction mechanisms similar to stress and depression. This study addresses the plasticity of the hippocampal NK-1 receptor and BDNF gene expression in rat models of pain and stress. Using solution hybridization–nuclease protection assays, the results clearly show that NK-1 receptor and BDNF mRNA levels were significantly attenuated by both acute (once for 45 min) and chronic (10 day) immobilization stress (Fig. 3). The observation that stress down-regulates the expression of hippocampal BDNF is consistent with results of other
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Fig. 3. NK-1 receptor and BDNF mRNA levels in the rat hippocampus following acute or chronic immobilization stress. (A) Two hours after either single (acute stress) or repeated (10-day chronic stress) 45 min immobilization in a restraint bag, NK-1 receptor mRNA levels in the rat hippocampus were decreased bilaterally by 51% and 32%, respectively, compared with control animals. (B) Acute or repeated stress significantly decreased bilateral BDNF hippocampal mRNA levels, by 61% and 50%, respectively, vs. the control animals. All values are given in pg mRNA/ng -actin mRNA (mean⫾S.E.M.; n⫽8). * P⬍0.05 compared with the control group (ANOVA and Fisher’s PLSD).
studies (Nibuya et al., 1999). However, the current findings provide additional evidence that the NK-1 receptor is involved in stress- and depression-evoked plastic changes in the CNS, particularly in the limbic system. Acute and persistent peripheral nociception evoked a similar down-regulation of hippocampal NK-1 receptor and BDNF gene expression, as was seen after stress. Both formalin and CFA significantly decreased NK-1 receptor mRNA levels in hippocampus (Fig. 4a), contrary to the prominent increases previously seen in the spinal cord (McCarson and Krause, 1994), thalamus and cortex (McCarson, 1997). The role of BDNF in nociceptive processing in the CNS is still poorly understood. Our studies show reduced BDNF mRNA levels in the hippocampus after either formalin or CFA injections (Fig. 4b), further suggesting that peripheral nociception also modulates regulation of BDNF gene expression in the higher brain centers. Both NK-1 receptor and BDNF appear to be homogenously expressed throughout the hippocampal formation,
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Fig. 4. NK-1 receptor and BDNF mRNA levels in the rat hippocampus 2 h and 45 min, 6 h or 24 h after a unilateral injection of 5% formalin (s.c.) or 10 days after a unilateral injection of CFA (s.c.) into the right hind paw. (A) Hippocampal NK-1 receptor mRNA levels were significantly decreased bilaterally as early as 2 h and 45 min after formalin treatment, or as long as 10 days after CFA injection. The most robust decreases in NK-1 receptor mRNA levels occurred 6 h after formalin injection (63% decrease compared with controls). (B) Inflammatory nociception diminished BDNF mRNA levels in the bilateral rat hippocampus. The largest decrease in BDNF mRNA (67% decrease vs. control animals) occurred at 2 h and 45 min post-formalin injection. Note that BDNF mRNA levels remained decreased for as long as 10 days in rats receiving CFA injections. All values are expressed in pg mRNA/ng -actin mRNA (mean⫾S.E.M.; n⫽7 or 8). * P⬍0.05 compared with the control group (ANOVA and Fisher’s PLSD).
including the granule layer and the hilus of the dentate gyrus, as well as pyramidal neurons in CA1 and CA3 sub-layers (Wetmore et al., 1990; Maeno et al., 1993; Schaaf et al., 1997; Yan et al., 1997). Thus, it may be contemplated that the changes in mRNA levels measured with the nuclease protection assays for the hippocampus as a whole are most likely evenly contributed by all the hippocampal sub-regions. Although the duration of the observed changes in gene expression is not known, decreased mRNA levels could partially be due to the stressand pain-evoked neurotoxicity and overall injury and atrophy of hippocampal neurons. However, some of these changes occur early (at 2 h and 45 min or 6 h posttreatment), which is likely not enough time for robust
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Fig. 5. Changes in bilateral volume of the rat hippocampus following acute and chronic immobilization stress or inflammatory pain. Rats subjected to either acute immobilization stress, unilateral injection of 5% formalin or unilateral injection of CFA all showed significantly increased hippocampal volumes. Values are given in cubic millimeters (mean S.E.M; n⫽4). * P⬍0.05 compared with control group (ANOVA and Fisher’s PLSD).
changes in gene expression to be manifested as major alterations in cellular morphology or proliferation. Stress and pain are known activators of the hypothalamo–pituitary–adrenal axis (HPA), and stimulation of this system may contribute to the hippocampal plasticity observed in this study. Increased glucocorticoid activity has been shown to have an effect on BDNF levels in the hippocampus (Schaaf et al., 1997), while SP and the NK-1 receptor have been reported to interfere with the regulation and physiological effects of the HPA axis (Nussdorfer and Malendowicz, 1998; Taylor et al., 1998; Jessop et al., 2000; Kandere-Grzybowska et al., 2003). However, clinical evidence suggests that HPA axis dysfunction, rather than hyperactivity, may be the main physiological factor underlying the co-morbidity of chronic pain and depression (BlackburnMunro and Blackburn-Munro, 2001). Decreases in hippocampal NK-1 receptor mRNA levels after either pain or stress are potentially counterintuitive, as NK-1 receptor antagonists have been shown to have antidepressant-like actions (Kramer et al., 1998). The antidepressant mechanism of action of NK-1 antagonist drugs may be via interference with serotonergic and noradrenergic systems in limbic regions rather than via a classical antagonist-receptor functional deactivation of the NK-1 receptor itself. McCarson et al. (1998) have previously shown that long-term administration of an NK-1 receptor antagonist caused significant increases in NK-1 mRNA in the striatum; NK-1 antagonist drugs could
have similar effects in the hippocampus. Furthermore, an increase in receptor mRNA levels is not necessarily indicative of increased functional activation of that receptor. Nociception-evoked down-regulation of NK-1 and BDNF genes in the hippocampus is distinctly different from a very robust increases previously described in the spinal cord (McCarson and Krause, 1994). Apparent difference in regulation within components of the CNS may reflect the role of these genes in affective modulation in the limbic system during pain, contrary to the previously characterized function of these neuromediators in the development of central sensitization in the sensory components of the CNS. The emerging pattern of hippocampal NK-1 receptor and BDNF gene regulation may suggest that similarities in the modulation of expression of these two genes are dependent on the cyclic AMP (cAMP)-phosphokinase A (PKA)– cyclic AMP response element binding protein (CREB) cascade. NK-1 and BDNF genes may be coregulated by pain or stress through mechanisms dependent on activation of CREB, since there are cyclic AMP response element (CRE) sites present in the promoters of both of these genes (Nibuya et al., 1996; Cho et al., 1997; Duman et al., 1997; Abrahams et al., 1999; Duman and Charney, 1999). However, additional studies measuring pCREB and cAMP levels in the hippocampus following inflammation will have to be conducted to provide direct evidence for a common molecular mechanism for nociceptive affect and stress in the hippocampus. An additional goal of this study was to determine whether chemogenic inflammatory nociception or immobilization stress produces similar alterations in hippocampal volume. Our results show that acute stress (one-time 45 min immobilization), acute pain (2 h and 45 min postformalin) or chronic pain (10 days post-CFA) all produced significant increases in hippocampal volume (Fig. 5). These results were initially surprising, particularly since previous clinical brain imaging studies have provided evidence that hippocampal volume is reduced in patients with chronic depression or post-traumatic stress disorder (Bremner et al., 1995, 1997; Sheline et al., 1996, 1999). However, it is important to point out that these observations were made in patients with long-term history of depression using magnetic resonance imaging (MRI). The degree of hippocampal atrophy and decreased volume was correlated to the length of depression, suggesting that the morphological changes are not likely due to the acute effects of stress and glucocorticoid release. The increase in hippocampal volume observed after either stress or pain may represent mild signs of excitotoxicity specific to hip-
Table 1. Effects of acute or chronic immobilization stress, or inflammatory pain, on specific gravity of rat hippocampus
Specific gravity (g/cm3) % Tissue volume change as water
Control
Acute stress
Chronic stress
Acute pain
Chronic pain
409⫾6 0
328⫾13 ⫹24.7*
360⫾13 ⫹13.6*
392⫾9 ⫹4.3
417⫾8 ⫺1.9
Rats immobilized acutely (once for 45 min) or chronically (45 min once daily for 10 consecutive days) both showed an increase in water content (edema) of hippocampal tissue. Edema is associated with a decrease in specific gravity. Note that acute stress resulted in more severe edema than chronic stress. Data are given in g/cm3 [(Mean specific gravity⫺1)⫾S.E.M.⫻104; n⫽8] * P⬍0.05 compared to control group (ANOVA and Fisher’s PLSD).
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pocampus, since the volume of the brain as a whole did not change (data not shown). Stress or pain may provoke increased stimulation of glutamatergic pathways in hippocampus, leading to excess intracellular Na⫹ and Ca2⫹ concentrations resulting in initial cytotoxic osmotic swelling of neurons and glia. If the stress input persists, a vasogenic edema can develop as a result of disruption of blood– brain barrier and protein movement (Emerson et al., 1999). In order to access how much of the volume change is due to edema, we measured tissue specific gravity (Table 1); presence of edema is inverse-proportional to the increase in specific gravity. Acute and chronic stress increased the tissue water content (edema) by 25% and 14%, respectively (Table 1), apparent from decreased specific gravity of the hippocampal tissue. However, acute and persistent pain models failed to produce significant changes in the hippocampal density. Edematous changes in the hippocampal tissue seen in our models of stress are considerable, and of similar magnitude as the measurements of hippocampal edema after cholinomimetic drug-induced seizures observed by Terry et al. (1990). The variance between the volumetric changes and presence of tissue edema in pain and stress models used in this study suggests that the hippocampal volume changes have multiple compartments. It seems that, while acute stress induces significant enhancement in tissue water content, no similar increase was observed in the pain models. Similarly, while chronic stress causes an increase in tissue water content, no correlating increase in hippocampal volume was observed. The presence of edema during stress but not during pain may indicate a difference in the response of the hippocampus to the negative affective component of pain as compared with stress. During stress, neuroprotective cellular mechanisms may be overridden by neurotoxic events, including excessive glutamate and glucocorticoid activation, which can result in tissue edema and degeneration. In our stress models neuronal atrophy may also be occurring, but the data suggest that at initial time points atrophy may be masked by the increased tissue edema. Edema was most robust in the acute model of stress and appears to decrease with time (Table 1). Overt atrophy may be more robust later on, and in concert with edemarecovery processes stimulated by unknown compensatory cellular mechanisms, which may be responsible for lesser changes in hippocampal volume after chronic stress.
CONCLUSIONS The regulation of NK-1 receptor and BDNF gene expression in the hippocampus by inflammatory stimuli provides useful markers of hippocampal activation by chronic pain. Pain or stress-induced increases in hippocampal tissue volume may have significant physiological importance, particularly as indicators of cell and tissue damage or toxicity. Further investigation is required to determine the exact causes and cellular events leading toward formation of stress-evoked edema, although it could be hypothesized that stress initially causes neuronal injury and that plastic-
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ity of NK-1 receptor and BDNF gene expression may reflect a response to hippocampal injury. However, the results of this study present new information about molecular markers of the “affective component” of chronic pain and its relationship to stress and depression, and ultimately, may contribute to the identification of novel therapeutic approaches for the control of chronic pain. Acknowledgments—We would like to thank Drs. Thomas L. Pazdernik and Mitchell R. Emerson for their input regarding the design of specific gravity experiments, as well as Michelle Winter for her expert technical assistance. This project was supported in part by DA12505 (K.E.M.) and an ASPET SURF grant to Janie Benoit, who assisted with the hippocampal volume measurements.
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(Accepted 4 April 2005)
HIPPOCAMPAL NEUROKININ-1 RECEPTOR AND BRAIN-DERIVED NEUROTROPHIC FACTOR GENE EXPRESSION IS DECREASED IN RAT MODELS OF PAIN AND STRESS V. DURIC AND K. E. MCCARSON*
tent inflammatory stimuli increase both NK-1 receptor (McCarson and Krause, 1994, 1995, 1996) and BDNF mRNA levels (Zhou and Rush, 1996), while nociception- or SP-evoked dorsal horn cell excitation is blocked by specific NK-1 receptor antagonists (Radhakrishnan and Henry, 1991; McCarson and Krause, 1996). Similar regulation of NK-1 gene expression may also occur in other CNS sites; the observation that NK-1 receptor antagonists have antidepressant effects (Kramer et al., 1998) suggests that the effects of pain and stress may converge in higher brain centers sensitive to both types of stimuli, such as restraint or immobilization stress, as well as peripheral inflammatory nociception. However, the regulation of NK-1 receptor expression and function in mood/affect and emotion-processing systems (e.g. amygdala, hippocampus and hypothalamus) is poorly understood (DeFilipe et al., 1998; Maeno et al., 1993). The functional similarities between neurokinin receptors in affective and sensory systems are undefined, as is the role of hippocampal NK-1 receptors in the context of chronic pain. From previous studies, it is clear that stress can have profound effects on the hippocampus, a central component of the limbic system and one of key regulators of affect. Stress hormones target the hippocampus, making it a particularly plastic and vulnerable brain structure (Sapolsky, 2000). The hippocampus is sensitive to, and can be damaged by, endogenous and/or environmental factors such as chronic stress, aging, stroke or head trauma (Sapolsky, 1992; McEwen, 1999). Studies using stress as a model of depression have shown that long-term exposure to stress is accompanied by neuronal atrophy of the hippocampus (Watanabe et al., 1992) and a decrease in the expression of BDNF (Smith et al., 1995; Duman and Charney, 1999). Furthermore, clinical brain imaging studies have demonstrated that the volume of the hippocampus is reduced in patients with chronic depression (Sheline et al., 1996, 1999) or post-traumatic stress disorder (Bremner et al., 1995, 1997; Gurvits et al., 1996; Stein et al., 1997). Conversely, long-term antidepressant administration causes an increase in the expression of both BDNF and TrkB genes in hippocampus (Nibuya et al., 1995, 1996, 1999; Vaidya and Duman, 1996; Duman et al., 1997). Chronic pain also may affect the hippocampus and initiate cellular mechanisms that resemble stress. Therefore, we hypothesize that nociceptive stimuli regulate NK-1 receptor as well as a BDNF gene expression in the hippocampus. This hypothesis was tested by quantifying NK-1 and BDNF gene expression in rats; results indicate that either stress or pain down-regulates expression of
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Mail Stop 1018, Kansas City, KS 66160, USA
Abstract—Acute or chronic stress can alter hippocampal structure, cause neuronal damage, and decrease hippocampal levels of the neurotrophin brain-derived neurotrophic factor (BDNF). The tachykinin substance P and its neurokinin-1 (NK-1) receptor may play a critical role in neuronal systems that process nociceptive stimuli; their importance in stressactivated systems has recently been demonstrated by the antidepressant-like actions of NK-1 receptor antagonists. However, the functional similarities between neurokinin receptors in the hippocampus and those in sensory systems are poorly understood, as is the significance of hippocampal NK-1 receptor in the context of chronic pain. Therefore, we investigated the effects of immobilization stress or inflammatory stimuli on NK-1 receptor and BDNF gene expression in the rat hippocampus. Rats received an acute or chronic immobilization stress, or an acute (formalin) or chronic (complete Freund’s adjuvant) inflammatory stimulus to the right hind paw. Subsequently hippocampal volume and specific gravity were measured and NK-1 receptor and BDNF mRNA levels quantified using ribonuclease protection assays. Results showed that either stress or pain down-regulates expression of both NK-1 receptor and BDNF genes in the hippocampus. Hippocampal volume was increased by either pain or stress; this may be due to edema (decreased specific gravity). Thus, BDNF and NK-1 receptor gene plasticity may reflect sensory activation or responses to neuronal injury. These data may provide useful markers of hippocampal activation during chronic pain, and suggest similarities in the mechanisms underlying chronic pain and depression. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: substance P, immobilization stress, nociception, formalin, CFA, edema.
The tachykinin neuropeptide substance P (SP) and brainderived neurotrophic factor (BDNF), as well as their preferred receptors, neurokinin-1 (NK-1) receptor and tyrosine kinase B (TrkB) respectively, have been well characterized as mediators of nociceptive sensory information in somatosensory pathways in the nervous system (Vaught, 1988; Henry, 1993; Cho et al., 1997). In the spinal cord, persis*Corresponding author. Tel: ⫹1-913-588-7519; fax: ⫹1-913-588-7501. E-mail address: [email protected] (K. E. McCarson). Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; cAMP, cyclic AMP; CFA, complete Freund’s adjuvant; CREB, cyclic AMP response element binding protein; HPA, hypothalamo–pituitary–adrenal axis; NK-1, neurokinin-1; SP, substance P; TrkB, tyrosine kinase B. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.04.002
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both NK-1 and BDNF genes in the hippocampus. The results suggest that NK-1 and BDNF genes provide useful markers of the hippocampal activation that may be coregulated during chronic pain or stress.
EXPERIMENTAL PROCEDURES Animal housing and handling Seventy-four young male Sprague–Dawley rats (Harlan Farms, Indianapolis, IN, USA), used for all experiments, were age matched (7– 8 weeks old) at the beginning of the treatments. The maintenance of the rat colony and all the animal treatments were in accordance with NIH laboratory care standards and approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee. Efforts were made to minimize animal suffering and to reduce the number of animals used in this study. Animals were housed (12-h light/dark cycle) in groups of three per cage with ad libitum access to food and water; they were mixed together so there is one member of each treatment group in every cage. All rats, including the sham control group, were handled the same way to reduce the effects of stress associated with handling on the results.
Experimental design Rats (200 –300 g) were subjected to either immobilization stress or injected with an inflammatory stimulus (Fig. 1). Acute stress (n⫽12) rats were immobilized in restraint bags (Rodent Restraint Cones, Harvard Apparatus, Inc., Holliston, MA, USA) once for 45 min, while chronic stress (n⫽12) rats were immobilized for 45 min once daily for 10 consecutive days. Acute inflammatory pain rats received a s.c. injection of 100 L of 5% formalin (Fisher Scientific Co., St. Louis, MO, USA) into the plantar aspect of the right hind paw. Animals were decapitated either at 2 h and 45 min (n⫽12), 6 h (n⫽7), or 24 h (n⫽7) after the initiation of the final (or only) treatment. Chronic inflammatory pain (n⫽12) rats received a s.c. injection of 50 L of complete Freund’s adjuvant (CFA) (Sigma Chemical Co., St. Louis, MO, USA) into the plantar aspect of the right hind paw. Decapitation followed 10 days later. Sham (n⫽12)
animals received no hind paw injection, but were momentarily restrained and their right hind paw manipulated. Otherwise, they were handled identically to the treatment animals and were decapitated on the tenth day. The animal handling consisted of identical housing regimens, daily transport from the animal facilities to the laboratory, and interactions with the handler, including being picked up by the tail for taking weight measurements. One set of animals (n⫽8 or 7, from each treatment group) was used for the analyses of gene expression and edema assessment by solution hybridization–nuclease protection assays and specific gravity studies, respectively. The remaining rats (n⫽4, from each treatment group; 6 h and 24 h formalin groups excluded) were used for analysis of hippocampal volume.
Solution hybridization–nuclease protection assays Immediately after decapitation, rat brains were removed and dissected along the saggital midline. Left and right sides of the hippocampi were dissected as a whole (including dentate gyrus and CA 1– 4 sub-regions) and total cellular RNAs were extracted from the hippocampal tissue samples using a rapid guanidinium isothiocyanate–phenol/chloroform extraction method. The resultant total RNAs were analyzed using solution hybridization– nuclease protection assays (Fig. 1). Antisense 32P-labeled cRNA probes were synthesized using [␣-32P]UTP (3000 Ci/mmol) according to procedures suggested by Promega Biotec. Subsequently the DNA template was digested using RQ1 DNase (Promega). Total RNA samples were assayed for NK1, BDNF or -actin mRNAs using solution hybridization–nuclease protection assays. Briefly, 2⫻105 d.p.m. of each specific 32P-labeled antisense cRNA probe was co-precipitated with 2– 80 g of total RNA, 100 –1000 pg cRNA quantitation standards, or E. coli tRNA. The RNA-[32P]RNA co-precipitates were each re-suspended in 10 l of hybridization buffer (40 mM 1,4-piperazine diethane sulfonic acid (PIPES), pH 6.4, 400 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA); 80% (v/v) deionized formamide) and incubated for 16 –20 h at 45 °C. Digestion by nucleases A (4 g/mL) and T1 (0.2 g/mL) was performed for 20 min at 37 °C, followed by a 15 min digestion at 37 °C with proteinase K (100 g/sample). The digestion reaction products were precipitated, re-suspended,
Fig. 1. Experimental procedures used for quantifying NK-1 receptor and BDNF mRNA levels in hippocampal tissues of rats. A detailed description can be found in Experimental Procedures.
V. Duric and K. E. McCarson / Neuroscience 133 (2005) 999 –1006 and electrophoresed on 6% polyacrylamide gels containing 7 M urea. Gels were fixed, dried, and exposed to phosphor plates for 16 –24 h. Densitometric images were generated and analyzed using a Molecular Dynamics PhosphorImager SF (Sunnyvale, CA, USA). Specific mRNA amounts were determined by comparison to cRNA quantitation standards. Data values are reported as pg specific mRNA/ng -actin mRNA [mean⫾S.E.M.].
Hippocampal volume analysis Frozen (⫺70 °C) rat brains were cut transversely into 150 m thick sections using a cryostat (Leica Jung Frigocut 2800). The sections were mounted on gelatin-coated slides, stained with Cresyl Violet, dehydrated, and coverslipped. A computer-controlled digital camera (Coho, Japan) was used to collect images of each section. Individual measurements of left, right and total hippocampal areas on each section were measured using Scion Image (NIH Software). The area encompassed by hippocampus on each section was analyzed and the hippocampal volume calculated by summing the areas of all individual sections containing hippocampus. Volume data values are reported as mm3 [mean⫾S.E.M.].
Specific gravity analysis Specific gravity analysis for hippocampal edema was performed using a kerosene/bromobenzene density gradient column prepared in a 1 L graduated cylinder. The column was made by gradually mixing a heavy solution (1.0650 g/cm3; 192 mL bromobenzene and 308 mL kerosene) with a light solution (1.0250 g/ cm3; 162 mL bromobenzene and 338 mL kerosene). Sodium chloride solutions of known specific gravity were used as quantitation standards to determine the linearity of the density gradient column. Six standard solutions, ranging from 1.0385 g/cm3 to 1.0563 g/cm3, were prepared by dissolving a known amount of NaCl into 100 mL of distilled water. Two drops (volume⫽10 – 15 L) of each standard were placed into the column and their location in the column was measured after 2 min using a cathetometer. Hippocampal samples were dissected under and stored in cold (4 °C) kerosene to reduce tissue water loss. Small tissue samples (approx. 2–3 mm3) were placed into the density column and their position was determined after 2 min. The specific gravity of the samples was calculated by interpolation using the NaCl standards curve; data values are reported as g/cm3 [mean⫾ S.E.M.]. A decrease in specific gravity is correlated with an increase in tissue water content (edema). Specific gravity values were used to calculate percent tissue volume change (Nelson et al., 1971) from the following equation: % change in tissue volume as water ⫽[((sp.gr.⫺1)cont. ⁄ (sp.gr.⫺1)exp.)⫺1]⫻100 Note: sp.gr.⫽specific gravity; cont.⫽control; and exp.⫽experimental.
Statistical analysis Data from all the experiments were analyzed using analysis of variance (ANOVA) with Fisher’s PLSD tests used for post hoc comparisons. Significance was considered to be P⬍(or equal) 0.05; all comparisons were made to naïve controls.
RESULTS To determine the effects of stress on the regulation of NK-1 receptor and BDNF gene expression in hippocampus, rats were subjected to either acute stress (45 min) once or chronically (10 consecutive days) by immobilizing them in plastic restraining bags. Hippocampal NK-1 receptor and
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BDNF mRNA levels were assessed 2 h post-treatment using solution hybridization–nuclease protection assays (Fig. 2). Bilateral levels of NK-1 receptor mRNA were significantly down-regulated by either acute or chronic immobilization stress (Fig. 3a). A single 45 min immobilization of the acute stress paradigm provoked a more robust response than chronic stress treatment (51% vs. 32% reduction in NK-1 receptor mRNA levels, respectively). Stress treatments evoked similar responses in regulation of BDNF gene expression. Bilateral levels of hippocampal BDNF mRNA were significantly reduced by either acute or chronic immobilization stress (Fig. 3b) as previously reported by Nibuya et al. (1999). Acute stress produced a slightly more robust response. The influence of peripheral inflammatory pain on the expression of NK-1 receptor and BDNF mRNA in the rat hippocampus was also assessed using solution hybridization–nuclease protection assays. Initially all the assays were performed on ipsilateral vs. contralateral side of the hippocampus, since the inflammatory stimuli were s.c. injected into the rat right hind paw. However, formalin or CFA did not produce any significant sided differences in expression of either NK-1 receptor or BNDF genes, so the mRNA levels are shown as hippocampal bilateral average. Fig. 4a shows that injections of either 5% formalin or CFA both evoked significant decreases in hippocampal NK-1 receptor mRNA levels; the largest changes were observed at 6 h and 24 h post-formalin injections, which produced reduction in NK-1 receptor mRNA by 63% and 54%, respectively. Likewise, hippocampal BDNF mRNA levels were also significantly reduced after either formalin or CFA treatments (Fig. 4b). The most robust decrease occurred at the earliest time point, 2 h and 45 min post-formalin injection, which produced a 67% reduction in BDNF mRNA level. Hippocampal -actin mRNA levels (data not shown), unaffected by either pain or stress, were measured to serve as gel loading controls and to ensure that the observed changes in NK-1 receptor and BNDF gene expression are not due to a global down-regulation of genes in the hippocampus. Volumetric studies (Fig. 5) showed that both pain and stress can have a profound effect on the size of the hippocampus. Formalin and CFA nociception paradigms increased hippocampal volume by 18% and 12%, respectively. Acute stress, similar to formalin in the duration of the treatment, increased the overall volume of hippocampus by 15%. Chronic stress also produced a slight increase in volume, but the data were not statistically different from the sham controls. The purpose of the specific gravity experiment was to determine one of the possible mechanisms underlying the volume increase; measuring the specific gravity of the hippocampal tissue allowed us to assess how much of the change in volume is due to edema. In edematous brain tissue, the decrease in specific gravity of the tissue occurs from the influx of fluid (Emerson et al., 1999). Neither acute nor chronic pain had any effect on the specific gravity of hippocampal tissue (Table 1); however, both acute and chronic immobilization stress models provoked robust decreases in specific gravity resulting in increases
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Fig. 2. Densitometric images of the representative solution hybridization–nuclease protection assays used for quantitation of (A) NK-1 receptor and (B) BDNF mRNA species in total RNA samples isolated from hippocampal tissues of rats exposed to either immobilization stress, inflammatory pain or sham paradigms. (A) NK-1: Each sample contained 40 g of total RNA. The arrows show the sizes of the undigested NK-1 receptor antisense probe (696 bp) and the RNA species protected by the NK-1 receptor mRNA or 100 –200 pg of message-sense NK-1 receptor cRNA quantitation standard (588 bp). (B) BDNF: Each sample contained 80 g of total RNA. The arrows show the sizes of the undigested BDNF antisense probe (597 bp) and the RNA species protected by the BDNF mRNA or 100 –200 pg of message-sense BDNF cRNA quantitation standard (539 bp). Each lane in these assay images represents an RNA sample from one individual experimental subject. Thus, treatment effects may not be fully appreciable when viewing a single assay gel. (Note: acute stress⫽one 45 min bout of immobilization; chronic stress⫽45 min immobilization daily for 10 consecutive days; acute pain⫽2 h and 45 min after formalin injection; chronic pain⫽10 days after CFA injection.)
in tissue water content of 25% and 14%, respectively. Such an observation could provide one possible explanation for the initial increases in hippocampal volume seen after stress.
DISCUSSION Chronic pain is characterized by many complex alterations in nociceptive pathways and is often accompanied by clinical depression (Ruoff, 1996; Fishbain et al., 1997; Gallagher and Verma, 1999; Bair et al., 2003; Currie and Wang, 2004). To date, little emphasis has been on the mechanisms contributing to the “affective” component of pain or its impact on cognitive function (Woolf and Salter, 2000; Julius and Basbaum, 2001). Beyond the physiological aspects of nociception, a negative impact on affect is required for accurate characterization of a stimulus as “painful.” In this study we compared the effects of inflam-
matory pain and immobilization stress on the hippocampus, a limbic region involved in regulation of affect. NK-1 receptors and BDNF have previously been implicated in nociception-induced spinal central sensitization, however the effects of pain stimuli on the expression of these two genes in the brain regions involved in the regulation of emotion and affect have not yet been characterized. Persistent pain may have an effect on the hippocampus and initiate cellular transduction mechanisms similar to stress and depression. This study addresses the plasticity of the hippocampal NK-1 receptor and BDNF gene expression in rat models of pain and stress. Using solution hybridization–nuclease protection assays, the results clearly show that NK-1 receptor and BDNF mRNA levels were significantly attenuated by both acute (once for 45 min) and chronic (10 day) immobilization stress (Fig. 3). The observation that stress down-regulates the expression of hippocampal BDNF is consistent with results of other
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Fig. 3. NK-1 receptor and BDNF mRNA levels in the rat hippocampus following acute or chronic immobilization stress. (A) Two hours after either single (acute stress) or repeated (10-day chronic stress) 45 min immobilization in a restraint bag, NK-1 receptor mRNA levels in the rat hippocampus were decreased bilaterally by 51% and 32%, respectively, compared with control animals. (B) Acute or repeated stress significantly decreased bilateral BDNF hippocampal mRNA levels, by 61% and 50%, respectively, vs. the control animals. All values are given in pg mRNA/ng -actin mRNA (mean⫾S.E.M.; n⫽8). * P⬍0.05 compared with the control group (ANOVA and Fisher’s PLSD).
studies (Nibuya et al., 1999). However, the current findings provide additional evidence that the NK-1 receptor is involved in stress- and depression-evoked plastic changes in the CNS, particularly in the limbic system. Acute and persistent peripheral nociception evoked a similar down-regulation of hippocampal NK-1 receptor and BDNF gene expression, as was seen after stress. Both formalin and CFA significantly decreased NK-1 receptor mRNA levels in hippocampus (Fig. 4a), contrary to the prominent increases previously seen in the spinal cord (McCarson and Krause, 1994), thalamus and cortex (McCarson, 1997). The role of BDNF in nociceptive processing in the CNS is still poorly understood. Our studies show reduced BDNF mRNA levels in the hippocampus after either formalin or CFA injections (Fig. 4b), further suggesting that peripheral nociception also modulates regulation of BDNF gene expression in the higher brain centers. Both NK-1 receptor and BDNF appear to be homogenously expressed throughout the hippocampal formation,
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Fig. 4. NK-1 receptor and BDNF mRNA levels in the rat hippocampus 2 h and 45 min, 6 h or 24 h after a unilateral injection of 5% formalin (s.c.) or 10 days after a unilateral injection of CFA (s.c.) into the right hind paw. (A) Hippocampal NK-1 receptor mRNA levels were significantly decreased bilaterally as early as 2 h and 45 min after formalin treatment, or as long as 10 days after CFA injection. The most robust decreases in NK-1 receptor mRNA levels occurred 6 h after formalin injection (63% decrease compared with controls). (B) Inflammatory nociception diminished BDNF mRNA levels in the bilateral rat hippocampus. The largest decrease in BDNF mRNA (67% decrease vs. control animals) occurred at 2 h and 45 min post-formalin injection. Note that BDNF mRNA levels remained decreased for as long as 10 days in rats receiving CFA injections. All values are expressed in pg mRNA/ng -actin mRNA (mean⫾S.E.M.; n⫽7 or 8). * P⬍0.05 compared with the control group (ANOVA and Fisher’s PLSD).
including the granule layer and the hilus of the dentate gyrus, as well as pyramidal neurons in CA1 and CA3 sub-layers (Wetmore et al., 1990; Maeno et al., 1993; Schaaf et al., 1997; Yan et al., 1997). Thus, it may be contemplated that the changes in mRNA levels measured with the nuclease protection assays for the hippocampus as a whole are most likely evenly contributed by all the hippocampal sub-regions. Although the duration of the observed changes in gene expression is not known, decreased mRNA levels could partially be due to the stressand pain-evoked neurotoxicity and overall injury and atrophy of hippocampal neurons. However, some of these changes occur early (at 2 h and 45 min or 6 h posttreatment), which is likely not enough time for robust
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Fig. 5. Changes in bilateral volume of the rat hippocampus following acute and chronic immobilization stress or inflammatory pain. Rats subjected to either acute immobilization stress, unilateral injection of 5% formalin or unilateral injection of CFA all showed significantly increased hippocampal volumes. Values are given in cubic millimeters (mean S.E.M; n⫽4). * P⬍0.05 compared with control group (ANOVA and Fisher’s PLSD).
changes in gene expression to be manifested as major alterations in cellular morphology or proliferation. Stress and pain are known activators of the hypothalamo–pituitary–adrenal axis (HPA), and stimulation of this system may contribute to the hippocampal plasticity observed in this study. Increased glucocorticoid activity has been shown to have an effect on BDNF levels in the hippocampus (Schaaf et al., 1997), while SP and the NK-1 receptor have been reported to interfere with the regulation and physiological effects of the HPA axis (Nussdorfer and Malendowicz, 1998; Taylor et al., 1998; Jessop et al., 2000; Kandere-Grzybowska et al., 2003). However, clinical evidence suggests that HPA axis dysfunction, rather than hyperactivity, may be the main physiological factor underlying the co-morbidity of chronic pain and depression (BlackburnMunro and Blackburn-Munro, 2001). Decreases in hippocampal NK-1 receptor mRNA levels after either pain or stress are potentially counterintuitive, as NK-1 receptor antagonists have been shown to have antidepressant-like actions (Kramer et al., 1998). The antidepressant mechanism of action of NK-1 antagonist drugs may be via interference with serotonergic and noradrenergic systems in limbic regions rather than via a classical antagonist-receptor functional deactivation of the NK-1 receptor itself. McCarson et al. (1998) have previously shown that long-term administration of an NK-1 receptor antagonist caused significant increases in NK-1 mRNA in the striatum; NK-1 antagonist drugs could
have similar effects in the hippocampus. Furthermore, an increase in receptor mRNA levels is not necessarily indicative of increased functional activation of that receptor. Nociception-evoked down-regulation of NK-1 and BDNF genes in the hippocampus is distinctly different from a very robust increases previously described in the spinal cord (McCarson and Krause, 1994). Apparent difference in regulation within components of the CNS may reflect the role of these genes in affective modulation in the limbic system during pain, contrary to the previously characterized function of these neuromediators in the development of central sensitization in the sensory components of the CNS. The emerging pattern of hippocampal NK-1 receptor and BDNF gene regulation may suggest that similarities in the modulation of expression of these two genes are dependent on the cyclic AMP (cAMP)-phosphokinase A (PKA)– cyclic AMP response element binding protein (CREB) cascade. NK-1 and BDNF genes may be coregulated by pain or stress through mechanisms dependent on activation of CREB, since there are cyclic AMP response element (CRE) sites present in the promoters of both of these genes (Nibuya et al., 1996; Cho et al., 1997; Duman et al., 1997; Abrahams et al., 1999; Duman and Charney, 1999). However, additional studies measuring pCREB and cAMP levels in the hippocampus following inflammation will have to be conducted to provide direct evidence for a common molecular mechanism for nociceptive affect and stress in the hippocampus. An additional goal of this study was to determine whether chemogenic inflammatory nociception or immobilization stress produces similar alterations in hippocampal volume. Our results show that acute stress (one-time 45 min immobilization), acute pain (2 h and 45 min postformalin) or chronic pain (10 days post-CFA) all produced significant increases in hippocampal volume (Fig. 5). These results were initially surprising, particularly since previous clinical brain imaging studies have provided evidence that hippocampal volume is reduced in patients with chronic depression or post-traumatic stress disorder (Bremner et al., 1995, 1997; Sheline et al., 1996, 1999). However, it is important to point out that these observations were made in patients with long-term history of depression using magnetic resonance imaging (MRI). The degree of hippocampal atrophy and decreased volume was correlated to the length of depression, suggesting that the morphological changes are not likely due to the acute effects of stress and glucocorticoid release. The increase in hippocampal volume observed after either stress or pain may represent mild signs of excitotoxicity specific to hip-
Table 1. Effects of acute or chronic immobilization stress, or inflammatory pain, on specific gravity of rat hippocampus
Specific gravity (g/cm3) % Tissue volume change as water
Control
Acute stress
Chronic stress
Acute pain
Chronic pain
409⫾6 0
328⫾13 ⫹24.7*
360⫾13 ⫹13.6*
392⫾9 ⫹4.3
417⫾8 ⫺1.9
Rats immobilized acutely (once for 45 min) or chronically (45 min once daily for 10 consecutive days) both showed an increase in water content (edema) of hippocampal tissue. Edema is associated with a decrease in specific gravity. Note that acute stress resulted in more severe edema than chronic stress. Data are given in g/cm3 [(Mean specific gravity⫺1)⫾S.E.M.⫻104; n⫽8] * P⬍0.05 compared to control group (ANOVA and Fisher’s PLSD).
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pocampus, since the volume of the brain as a whole did not change (data not shown). Stress or pain may provoke increased stimulation of glutamatergic pathways in hippocampus, leading to excess intracellular Na⫹ and Ca2⫹ concentrations resulting in initial cytotoxic osmotic swelling of neurons and glia. If the stress input persists, a vasogenic edema can develop as a result of disruption of blood– brain barrier and protein movement (Emerson et al., 1999). In order to access how much of the volume change is due to edema, we measured tissue specific gravity (Table 1); presence of edema is inverse-proportional to the increase in specific gravity. Acute and chronic stress increased the tissue water content (edema) by 25% and 14%, respectively (Table 1), apparent from decreased specific gravity of the hippocampal tissue. However, acute and persistent pain models failed to produce significant changes in the hippocampal density. Edematous changes in the hippocampal tissue seen in our models of stress are considerable, and of similar magnitude as the measurements of hippocampal edema after cholinomimetic drug-induced seizures observed by Terry et al. (1990). The variance between the volumetric changes and presence of tissue edema in pain and stress models used in this study suggests that the hippocampal volume changes have multiple compartments. It seems that, while acute stress induces significant enhancement in tissue water content, no similar increase was observed in the pain models. Similarly, while chronic stress causes an increase in tissue water content, no correlating increase in hippocampal volume was observed. The presence of edema during stress but not during pain may indicate a difference in the response of the hippocampus to the negative affective component of pain as compared with stress. During stress, neuroprotective cellular mechanisms may be overridden by neurotoxic events, including excessive glutamate and glucocorticoid activation, which can result in tissue edema and degeneration. In our stress models neuronal atrophy may also be occurring, but the data suggest that at initial time points atrophy may be masked by the increased tissue edema. Edema was most robust in the acute model of stress and appears to decrease with time (Table 1). Overt atrophy may be more robust later on, and in concert with edemarecovery processes stimulated by unknown compensatory cellular mechanisms, which may be responsible for lesser changes in hippocampal volume after chronic stress.
CONCLUSIONS The regulation of NK-1 receptor and BDNF gene expression in the hippocampus by inflammatory stimuli provides useful markers of hippocampal activation by chronic pain. Pain or stress-induced increases in hippocampal tissue volume may have significant physiological importance, particularly as indicators of cell and tissue damage or toxicity. Further investigation is required to determine the exact causes and cellular events leading toward formation of stress-evoked edema, although it could be hypothesized that stress initially causes neuronal injury and that plastic-
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ity of NK-1 receptor and BDNF gene expression may reflect a response to hippocampal injury. However, the results of this study present new information about molecular markers of the “affective component” of chronic pain and its relationship to stress and depression, and ultimately, may contribute to the identification of novel therapeutic approaches for the control of chronic pain. Acknowledgments—We would like to thank Drs. Thomas L. Pazdernik and Mitchell R. Emerson for their input regarding the design of specific gravity experiments, as well as Michelle Winter for her expert technical assistance. This project was supported in part by DA12505 (K.E.M.) and an ASPET SURF grant to Janie Benoit, who assisted with the hippocampal volume measurements.
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(Accepted 4 April 2005)