Department of Neuroscience, University of Cadiz, Spain,
1 Division of Medicine, University of Bristol, BRI, Marlborough Street, Bristol BS2 8HW, UK
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Abstract |
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Methods. Animals were separated on the basis of behaviour in the learned helplessness (LH) paradigm into groups of LH(+) (i.e. animals which did not escape footshock) and LH(-) animals. Adjuvant-induced arthritis (AA) was subsequently induced in the LH(+) and LH(-) animals.
Results. Plasma corticosterone was significantly increased in response to the LH test in the LH(-) compared with the LH(+) rats. We observed an earlier onset and increased inflammation in the LH(-) rats in spite of the greater corticosterone response to the acute stress. We noted lower levels of plasma testosterone in the LH(-) animals suggesting a possible influence for this protective factor in AA.
Conclusion. These data suggest that increased onset and severity of inflammation in AA is not a simple consequence of an attenuated HPA axis response to stress as proposed in the Lewis rat. Indeed we have observed the converse. Together these data suggest that the balance of pro- and anti-inflammatory factors released in response to stress may influence the progress of AA.
KEY WORDS: Learned helplessness, Adjuvant-induced arthritis, Inflammation, HPA axis, Corticosterone, Testosterone, Behaviour.
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Introduction |
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We have extended these observations of differential effects of stress within a single rat strain using the learned helplessness (LH) paradigm, as we have noted a relationship between emotivity and the susceptibility to become helpless [9]. The LH paradigm is a behavioural test based on the observation that exposure to uncontrollable stress produces performance deficits in subsequent learning tasks including the ability to escape from a subsequent controllable stress in a shuttle-box when provided with the opportunity to escape [10]. Animals unable to escape are considered to have learned helplessness, i.e. LH(+). LH(+) rats exhibit loss of appetite and weight, decreased locomotor activity and poor performance in motivated tasks [11]. The LH paradigm has been used as an animal model of depression [911]. Outbred Wistar rats exhibit considerable heterogeneity within a population when evaluated for the LH paradigm, with less than 50% of rats becoming LH(+) [9, 12, 13]. The remainder, when re-exposed to the footshock, will escape if the opportunity arises, i.e. they do not become helpless [LH(-)].
The aim of this study was to utilize the LH paradigm to determine whether the differential response to stress observed in the LH(-) and LH(+) groups following avoidable footshock has any influence on the inflammatory disease of AA. Adult rats of the Wistar strain were divided into LH(-) and LH(+) groups. Blood samples were taken prior to initial exposure to uncontrollable shock and 30 min following exposure to the LH stressor to determine if there was a differential corticosterone response associated with the behavioural changes. AA was induced 4 h following the LH test and the differences in behavioural response correlated with the onset and severity of inflammation. The animals were examined at regular intervals and paw volumes measured on days 0, 4, 7, 10 and 14 as an index of severity of inflammation. Trunk blood samples were collected at the end of the study for assay of corticosterone and testosterone and brains for determination of corticotrophin-releasing factor (CRF) mRNA in the hypothalamic paraventricular nucleus (PVN). Spleens and thymuses were collected for the determination of neuropeptide contents [CRF, arginine vasopressin (AVP), adrenocorticotrophic hormone (ACTH) and ß-endorphin]. If the hypothesis proposed is valid [2], a robust response to footshock would protect against the onset and severity of inflammation in AA. Conversely, an attenuated corticosterone response to stress might be predicted to be accompanied by increased severity of inflammation.
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Materials and methods |
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LH induction and blood sampling
The LH paradigm consisted of an exposure to a session of unavoidable footshock followed, after 48 h, by a performance task (conditioned avoidance training). Tail vein blood samples (0.40.5 ml), for subsequent corticosterone determination, were collected 24 h prior to the unavoidable footshock session (basal values), and 30 min after the LH test, to assess each animal's responsiveness to this stressor.
Induction of LH was carried out as described elsewhere [10]. Unavoidable footshock was delivered in a 20 x 12 x 10 cm chamber with Plexiglas walls and cover. The floor was made of a stainless-steel grid (1.5 cm mesh). Sixty scrambled, randomized and inescapable shocks (duration 15 s, intensity 0.8 mA, intershock interval 60 ± 20 s) were delivered to the grid floor by means of a constant current shocker.
In order to evaluate escape and avoidance performance (conditioned avoidance training), 48 h after the inescapable shock session the animals were placed in an automated two-way shuttle-box, divided into two equal sized chambers by a Plexiglas partition with a 7 x 7 cm hole that provided access to the adjacent compartment. The animals were singly placed in the shuttle-box and subjected to 30 avoidance trials. During the first 3 s of each trial a light signal was presented. The animals were allowed to avoid shock during this period. If an avoidance response did not occur a shock (0.8 mA; 3 s) was delivered. The response required of the rat during each trial was to pass through the gate into the other compartment of the shuttle-box. The intertrial interval was 30 s. The number of escape failures was recorded during shock delivery. Rats that recorded the highest escape failures (22 or more out of the 30 trials) were included in the LH(+) group (animals that became learned helpless). Rats that recorded the lowest escape failures (five or less out of 30 trials) were included in the LH(-) group (animals that did not become learned helpless).
Four hours following the LH test, groups of LH(+) and LH(-) rats were further divided into two groups and given either an intradermal injection of vehicle (paraffin oil) or ground, heat-killed Mycobacterium butyricum in paraffin oil for the induction of arthritis [14]. At this time point, 4 h after stress, hormonal responses to the stress would have returned to baseline. Paw volume was measured by plethysmometry (Ugo-Basile, Milan, Italy), as an index of the severity of inflammation and was measured immediately prior to adjuvant administration (basal) and 4, 7, 10, and 14 days after. Hind paws were submerged to the level of the lateral malleolus. Measurements were accurate to 0.01 ml (instrument error <1%). The experiment was terminated after 14 days when we have consistently observed significant hind-paw inflammation in this model [14].
Fourteen days after adjuvant injection, the rats were killed by decapitation and brains, thymuses and spleens were taken, frozen on dry ice and stored at 80°C. Trunk blood samples were collected in chilled, heparinized tubes and plasma was separated by centrifugation at 4°C (4000 r.p.m.) and stored at 20°C until assayed.
Plasma samples were assayed for total corticosterone and testosterone following termination of the experiment. Corticosterone was measured using an in-house radioimmunoassay (RIA) with antiserum kindly donated by Dr G. Makara (Institute of Experimental Medicine, Budapest, Hungary). The intra-assay variation was 9%. Plasma testosterone was measured by direct electrochemiluminesence immunoassay on a Boehringer Mannheim Elecsys 1010 immunoassay analyser (Boehringer Mannheim Diagnostics, Lewes, UK). The intra-assay variation was 7.4%.
In situ hybridization procedure (ISHH)
Brains were sliced (12 µm thickness) through the parvocellular part of the PVN of the hypothalamus (pPVN) on a cryostat and thaw-mounted on to gelatin-coated microscope slides. ISHH was performed as previously described [15, 16]. Briefly, a synthetic 48-mer oligonucleotide probe directed to the exonic mRNA sequence of CRF was used. The specificity of the probe has previously been described [16]. The specific activity of the 35S-dATP-labelled probe was 1.12 x 1019 d.p.m./mol. All sections were hybridized in the same incubation reaction and exposed together with brain paste standards for quantitation. Quantitation of the hybridized signal was determined using a computer-assisted analysis system (Image 1.22 developed by Wayne Rasband, NIH, Bethesda, MD, USA) and run on an Apple Mac IICi.
Spleens and thymuses were weighed and homogenized by hand in a glass Dounce in 5 ml of ice-cold acetic acid (0.1 mol/l) containing 2-mercaptoethanol (0.1%w/v). After centrifugation, supernatants were heated for 15 min at 90°C to prevent proteolytic interference during RIA incubation. The samples were desiccated and reconstituted in assay buffer and subjected to RIA for CRF, AVP, ß-endorphin and ACTH [17]. Antiserum for ß-endorphin was the kind gift of Dr S. Medbak, Department of Chemical Endocrinology, St. Bartholomew's Hospital, London, UK. This antiserum did not cross-react with either -MSH (melanocyte stimulating hormone) or ACTH. ACTH antiserum did not cross-react with ß-endorphin or
-MSH (either the acetylated or des-acetylated forms).
Statistical analysis
All results were analysed using either Student's t-test for paired or unpaired data or two-factors analysis of variance (ANOVA) as required. The factors of variance were LH(+) or LH(-) and AA or control (CT). Individual treatment effects (differences between groups) were analysed by means of the StudentNewmanKeuls test.
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Results |
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Paw volume
The AA LH(-) rats exhibited an earlier onset and had developed a significantly increased (P<0.05) hind-paw inflammation as early as day 10. At this time point inflammation was not evident in the AA LH(+) animals and paw volumes in this group were not significantly different to those in the control animals (Fig. 3). There was a significant (P < 0.05) increase in hind-paw inflammation in both groups of AA rats at day 14 compared with controls (Fig. 3
). Hind-paw volumes were significantly (P < 0.05) greater in the AA LH(-) rats at both day 10 and 14 compared with the AA LH(+) rats. These data confirm our earlier observations (data not shown) when paw volumes were only measured at day 14. Correlation coefficient calculations showed no significant correlation between plasma corticosterone at the 30 min time point and paw volume 10 and 14 days after injection in individual rats (data not shown).
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PVN CRF mRNA
CRF mRNA levels in the PVN were significantly (P<0.01) lower in AA rats compared with their respective non-AA controls, irrespective of whether the animals were LH(+) or LH(-). There was a 50% decrease in CRF mRNA levels in the AA LH(-) compared with the AA LH(+) rats, but this did not achieve statistical significance (Fig. 4). However, regression analysis of CRF mRNA and paw volume of the AA rats at day 14 revealed a significant (P<0.01) negative correlation.
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Plasma testosterone
Plasma testosterone concentrations in the LH(-) non-AA group (4.6 ± 1.1 nmol/l) were significantly (P < 0.05) less than in the LH(+) group (9.1 ± 1.3 nmol/l). Inflammation (AA) resulted in a significant (P < 0.05) decrease in plasma testosterone in both the LH(-) (0.8 ± 0.2 nmol/l) and LH(+) (1.1 ± 0.2 nmol/l) rats, but no significant differences in plasma testosterone concentrations were shown between these two groups (Fig. 5).
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Peptide contents in spleen and thymus
Splenic ß-endorphin contents were significantly increased in AA LH(-) rats compared with LH(+) rats [LH(+) = 8675 ± 879 pg/g tissue wet weight, n = 12; LH(- ) = 11 769 ± 1419 pg/g tissue wet weight, n = 9; P < 0.05; unpaired t-test]. No significant differences in splenic contents of CRF, ACTH or AVP were found comparing LH(+ ) and LH(-) animals (data not shown). No significant differences in neuropeptide contents were observed between AA LH(+) and AA LH(-) groups for any peptides measured in the thymus (data not shown).
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Discussion |
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The hypothesis that an attenuated neuroendocrine stress response increases susceptibility to inflammatory autoimmune disease has arisen mainly from observations relating the neuroendocrine response with susceptibility to inflammatory diseases in different inbred strains of animals, in particular the immature female Lewis vs Fischer strains [5, 18]. However, the relationship between the neuroendocrine response to stress and susceptibility to arthritis is less straightforward in adult rats. Both the type of stress and the strain of animal used appear to play a crucial role in susceptibility to disease. It has been reported that adult Lewis rats can perform poorly or similarly to the Fischer strain depending on the type of stressor [3, 1922]. We have shown that the CFY, Wistar and SpragueDawley strains can all mount robust neuroendocrine responses to either restraint or i.p. hypertonic saline administration, but the CFY strain is relatively resistant to AA whereas AA is readily induced in Wistar and SpragueDawley rats [23]. Furthermore, a very robust corticosterone response to stress does not protect the Piebald-Viral-Glaxo strain from AA [24], although it has been proposed that this robust response can protect this strain from EAE [25]. This discrepancy between the neuroendocrine response and susceptibility and/or severity has also been demonstrated in the Fischer rat. Both the conventional F344 and the germ-free (GF) F344 had equally robust corticosterone responses to interleukin-1 (IL-1
), yet the F344 rats were resistant to streptococcal cell wall-induced arthritis while the GF F344 rats were susceptible [26]. Within a single population of rats we have shown that despite a marked difference in corticosterone responsiveness to open field stress between the high and low emotive rats, there was no difference in the severity of inflammation when these animals went on to develop AA [8]. These data suggest that a robust HPA axis response to stress does not necessarily protect humans or rodents from the onset or severity of inflammatory disease.
The use of a single strain of rat separated into subpopulations on the basis of their behavioural response to the LH paradigm confers a number of advantages over previous studies using different strains of inbred animals. First, inbred strains of rat are defined by their genotypic uniformity. Comparison between strains is therefore hampered by the inherent difference between the strains. In contrast, the use of an outbred strain guarantees genotypic heterogeneity and is much closer to the human condition than comparison between inbred strains. Second, a large body of evidence has accumulated comparing structure and function in subpopulations of outbred strains such as the Wistar in response to stress, behavioural challenges, in response to immune challenges, etc. Comparable data are lacking for inbred strains. This evidence is likely to prove invaluable to an understanding of the mechanisms modulating individual differences in susceptibility to disease. Understanding these differences will lead ultimately to methods of prevention and/or control. Differences between subpopulations of an outbred (Wistar) strain of rat have been exploited to determine susceptibility to inflammatory and infectious disease [27, 28]. Study of the mechanisms underlying behavioural differences in individuals within subpopulations will help to elucidate the complex interactions linking behavioural and neuroendocrine responses to individual susceptibility to autoimmune disease.
It has been previously proposed that endogenous glucocorticoids might play an important role in the acquisition of LH behaviour. Adrenalectomy increased the percentage of rats becoming learned helpless, while corticosterone replacement reversed the behavioural deficits, suggesting that a dysregulation in the HPA axis might be involved in LH behaviour [13]. The present data support this view, as the lower corticosterone response to stress was associated with LH(+) behaviour. It is possible that the increased corticosterone secretion seen in the LH(-) animals in response to the LH test may have been driven by the trend towards increased CRF mRNA levels seen in the PVN in the non-AA rats. It is of interest to note that in view of the recognition that the LH model is considered to be an animal model of depression, that the reduced response to stress in terms of corticosterone secretion observed in the LH(+) rats is consistent with the attenuated ACTH and cortisol secretion seen in depressed patients in response to both stressors of the HPA axis and administration of CRF [29]. The latter phenomenon may be due to negative feedback by the chronically elevated blood levels of cortisol in human depression. Basal corticosterone levels were slightly higher in the LH(+) rats, although not significantly so, which might account for their poorer response to footshock stress compared with the LH(-) group. Thus, our findings support the view that the LH model shares neuroendocrinological similarities with human depression.
Glucocorticoids are able to alter the T-helper cell function from a Th1 to a Th2 profile. Elevated corticosterone would therefore act to suppress the Th1 response mediating AA. The timing of the LH stress in relation to the injection of the adjuvant may therefore be important. We chose the 4 h time point as we, and others, have consistently demonstrated plasma corticosterone concentrations returning to baseline 12 h after onset of acute stress. It should be noted that the intradermal injection of the adjuvant is inherently stressful but this increase in plasma corticosterone does not affect inflammation.
We have noted increased plasma corticosterone associated with increased hind-paw inflammation 14 days following induction of AA in both LH(+) and LH(-) Wistar rats, confirming the chronic activation of the HPA axis reported previously in other strains [14]. The Wistar rats with AA also demonstrated a paradoxical decrease in the expression of CRF mRNA in the PVN as reported previously [14]. There was a further 50% reduction in CRF mRNA in the LH(-) rats, associated with the increased inflammation, compared with the decreased levels seen in the LH(+) animals. While this decrease did not achieve statistical significance, regression analysis revealed a significant negative correlation between the decrease in CRF mRNA and the increase in paw volume, suggesting a close relationship between these parameters, as has previously been reported [30]. These data are further evidence for a close correlation between HPA axis activity and severity of disease during the course of AA.
In the present study we only determined HPA axis activity at day 14 of AA and it is possible that a more detailed investigation at other time points might reveal differences in activity between the two groups as noted previously [31]. It is also possible that the increased inflammation observed in the LH(-) group may be driven by a mechanism other than an alteration in HPA axis activation. The neuroendocrine stress response and its association with the inflammatory response is extremely complex. The stress response involves not only the stimulation of the HPA axis but also stimulates the sympathetic nervous system and the secretion of a variety of other compounds, many of which, such as cytokines, ß-endorphin, AVP and prolactin, are pro-inflammatory [32]. In addition to the increased corticosterone secretion in AA, increases in peripheral tissue levels of CRF, AVP and IL-1 have also been reported [17, 33]. Thus, it may be the net effects of pro- and anti-inflammatory factors released in response to stress which may determine the effects of stress on an inflammatory condition, as has been reported for RA [34, 35]. These pro- or anti-inflammatory effects may be determined by the type of stressor, the specific neurochemical pathway(s) activated, and the nature of the compounds secreted into the circulation.
It has been suggested that gonadal steroids play an important role in the development and expression of RA [36, 37]. The important sexual dimorphism in RA, with a clear female preponderance which decreases with increasing age, and the efficacy of hormone treatment support this view [3840]. It has previously been shown that testosterone may play a protective role in animal models of arthritis [41, 42]. Under the present experimental conditions, the non-AA LH(-) rats had significantly lower levels of plasma testosterone compared with the LH(+) rats, suggesting this may be a possible contributory factor for the earlier onset and increase in inflammation in these animals. Our data also confirm previous observations of a significant decrease in plasma testosterone concentrations in AA [42, 43].
Because many HPA axis neuropeptides involved in the stress response are also synthesized within immune tissues and can modulate immune functions [44], we measured spleen and thymic contents of CRF, ACTH, AVP and ß-endorphin. Elevated levels of total splenic ß-endorphin in the LH(-) group compared with the LH(+) group may be evidence that this opioid peptide is involved in the mechanisms which mediate inflammation in these animals with increased severity of inflammation. ß-Endorphin contents are elevated in the spleen in AA [17], and a mechanism has been proposed whereby lymphocyte-derived ß-endorphin can be targeted to specific sites of inflammation to attenuate the degree of inflammation [45]. Increased expression of ß-endorphin within splenic lymphocytes might be triggered as a protective mechanism in response to exacerbated inflammation in the LH(-) group. In addition to their role in mediating inflammation, endogenous opioids such as ß-endorphin have also been implicated in the induction of LH [46, 47]. Our data are further evidence that opioid pathways are important in the LH animal model of depression and also in the onset and severity of AA.
In conclusion, our data show that increased onset and severity of inflammation in AA is not a simple consequence of an attenuated response to stress. Indeed we have found the opposite to be the case. Thus, the hypothesis of a causal relationship between these factors does not appear to hold true in this model. Other factors such as sex hormones or neuropeptides produced by immune tissues as a consequence of stress may play an important role in determining the severity of AA. The power of our experimental design allows basal and stress-stimulated plasma corticosterone concentrations together with subsequent inflammation to be measured within individual animals, thus permitting a direct comparison to be drawn between any variation in the response to stress and the severity of AA in the individual rat. This experimental model should prove of considerable value in extending our observations on the relationship between the HPA axis response to stress and susceptibility to disease. We believe that this will provide important information about the involvement of the HPA axis in susceptibility to disease and infection.
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Acknowledgments |
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Notes |
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References |
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