In the present study, we were able to demonstrate a time dependent profile in the levels of circulating IL-1β and spinal IL-1β, IL-6 and TNF-α in stressed animals compared with non-stressed controls. In addition, we found temporal changes in the expression of the astrocytic marker GFAP in spinal and DRG samples. We also identified that WA stress is associated with transient increase in blood-spinal cord barrier permeability.
We report increased plasma concentration of IL-1β at 1 hour post 1 WA, 24 hours post 1 WA and 24 hours post 5 WA. We found no change in other circulating pro-inflammatory cytokines (IL-6 and TNF-α) in response to stress at any time points. These results point towards a rapid increase in IL-1β in the circulation in response to a single stress session (within 1-24 hours post 1 WA stress session), which seems to be maintained after 5 WA sessions. The changes in circulating IL-1β were significant (p
<.05) though it should be noted that the amplitude of the changes does not correspond to an “inflammatory profile” per se as can be seen in models of peripheral inflammation for example [27
]. These findings are consistent with other reports showing that, while the effect of stress on the immune system and the pattern of stress-induced cytokine increase in the circulation and in the CNS are depending on the nature of the stressor, IL-1β appears to be one key cytokine that is consistently increased after stress exposure in both blood and brain in several stress models [28
]. It is unlikely that circulating IL-1β originates from a peripheral site of inflammation or injury since we previously demonstrated that the model of chronic WA stress is not associated with significant gastrointestinal structural alterations or inflammation [30
]. However, it is possible that our results reflect a cell-mediated immune response to stress as described in other studies [31
]. The effect of stress on cell-mediated immune response may include trafficking or redistribution of peripheral lymphocytes between different immune compartments, including the spleen, in which changes in the expression of cell adhesion molecules (CAM) play a significant role [32
]. Although we did not measure circulating lymphocytes or cytokines from extracted circulating lymphocytes, it is possible that they contribute to the increased circulating IL-1β in our stress model [33
]. Cytokines in the blood may also originate from chromaffin cells of the adrenal medulla [34
], or may derive from high CNS concentrations [35
In the spinal cord, we observed increased concentration of IL-1β and IL-6 at 1 hours post 1 WA only, while TNF-α was increased 24 hours post 1 WA and 24 hours post 10 WA only. Our results point towards a rapid and transient increase in pro-inflammatory mediators in the spinal cord in response to a single stress session (within 1-24 hours post 1 WA stress session) while the only cytokine showing changes after 10 days of stress was TNF-α. This cytokine profile in the spinal cord does not reflect the changes of IL-1β observed in the serum from day 1 to 24 hours after day 5, suggesting the lack of direct peripheral origin of spinal cytokines. In the CNS, cytokines may be of neuronal or glial origins (including microglia and astrocytes) and play an important role in the sensitization of nociceptive signals [36
]. In the present study, our data show no change in the protein level of IBa1 at any time point (commonly used as one marker of microglia). However, we have previously demonstrated that chronic WA stress leads to p38 activation in spinal microglia 24 hours post 10 WA and that spinal microglia play a functional role in the expression of visceral hyperalgesia [18
]. In addition, previous studies have reported the potential of glia to release mediators without change in Iba1 phenotype [37
]. It is also known that stress alone can promote glia activation in the CNS, or prime glia to further stimulation [8
]. Our present results also show a biphasic change in GFAP expression with increased GFAP protein level 24 hours post 5 WA followed by a decreased level compared with controls 24 hours post 10 WA. These results are consistent with our previous findings showing decreased GFAP protein level and GFAP immunohistochemistry (IHC) intensity 24 hours after 10 WA [36
]. Together, these results suggest a possible implication of spinal glia activation and the release of glia mediators in the CNS immune response to chronic stress. We also found decreased spinal glutamate transporter expression after stress and supporting evidence for a role of the glutamatergic system in our model [18
IL-1β and TNF-α have been implicated in the pathophysiological changes occurring in various disease states including rheumatoid arthritis, neuropathic pain or inflammatory bowel disorders [35
]. In the spinal cord, the central role of IL-1β to the development of chronic pain is well established [40
]. Elevated spinal IL-1β was reported in both human [13
] and rat [43
] following spinal cord injury. It has been proposed that the pro-nociceptive effect of IL-1β is mediated through up-regulation of other pro-nociceptive mediators (NGF, CGRP) or the modulation of neuronal excitability by affecting receptors such as TRPV1, sodium channels, GABA or NMDA receptors. In a recent paper, spinal IL-1β released from astrocytes was found to enhance NR1 phosphorylation to facilitate inflammatory pain [44
]. Similarly, increasing evidence suggest a critical role of TNF-α in the pathogenesis of pain, including neuropathic pain and inflammatory pain and while the peripheral effect of TNF-α on nociceptors sensitization is well established [45
] the role of TNF-α in central sensitization is increasingly supported by a growing literature. TNF-α expression is induced in spinal cord glial cells in several models of chronic pain [46
] and intrathecal injection of TNF-α or a TNF-α inhibitor have been found to trigger and inhibit hyperalgesic response, respectively [12
]. Recent work identified the critical role of TNF-α in spinal synaptic activity and in the control of NMDAR activity. [12
In the chronic WA stress model, TNF-α is increased in the spinal cord 24 hours after 1 WA and 24 hours post 10 WA. Interestingly, these 2 time points correspond to the time points at which we previously reported visceral hyperalgesia [30
] suggesting a possible link between spinal TNF-α and stress-induced sensitization of visceral nociception. Although our study is limited by 1) the lack of statistical correlation between the level of TNF-α and visceral hyperalgesia (observations in different rat groups from different experiments precluding correlation analysis) and 2) the lack of pharmacological studies evaluating the role of cytokine inhibitors on peripheral and spinal markers and visceral sensitivity, one may speculate about the role of IL-1β and TNF-α in the modulation of the visceral nociceptive response. Further mechanistic studies using transgenic animals or testing the effects of cytokines receptor antagonists or blocking antibodies are warranted to confirm this hypothesis.
While glia is accepted as a potential source of cytokines in the CNS, it remains unclear what signals are causing glia activation and release of pro-inflammatory cytokines, in particular in models of stress-induced CNS glia activation. Evidence exists supporting the ability of peripheral cytokines to transduce their signals across the blood brain barrier (BBB) [48
] and thereby, triggering a series of signaling pathways in the CNS. This has been described for IL-1β which is thought to induce elevation of CNS IL-1β through several indirect mechanisms such as stimulation of vagal or other nerve afferents [49
], immune cell trafficking from blood to brain with subsequent secretion [51
], induction of mediators release from BBB cells [52
], interaction with circumventricular organs [53
] or directly crossing the BBB. In a recent paper [54
], it was demonstrated that regional neural activation defined a gateway for autoreactive T cells to cross the BBB via an effect of IL-6 in endothelial cells in the spinal cord supporting a role for neural mechanisms in neuroimmune activation in the CNS.
In our study, we found simultaneous increase in IL-1β in the circulation and in the spinal cord in the early stress exposure, with increased circulating IL-1β outlasting changes in spinal IL-1β. In view of these results, it is difficult to conclude whether peripheral IL-1β may be a triggering factor in the increase of spinal IL-1β. However, it is possible that increase in spinal TNF-α, (which occurs only at 24 hours after 1WA and 24 hours after 10 WA) may be consecutive to the transduction of peripheral IL-1β signals from the periphery to the CNS. This is supported by our observations that blood spinal cord barrier permeability is increased in our model of chronic WA stress at 24 hours post 1 WA. We showed increased permeability to small molecules (4KDa) 24 hour post 1 WA and 24 h post 5 WA while permeability to Evans Blue (which binds to albumin (68KDa) was significantly increased 24h post 5 WA (which may be connected to the prior passage of smaller molecules enabled 24 hours post 1 WA). Surprisingly, the significant increase of BSCB permeability to large molecules matched the increased expression of spinal GFAP at 24 hours post 5 WA. It is possible that changes in astrocytes phenotype, although associated with increased GFAP, may influence BSCB permeability at day 5. The role of connexin 43 (gap junction protein involved in astrocytes networking) in the control of BBB permeability has been previously discussed [55
]. However, while connexin 43 is predominantly associated with astrocytic gap junctions, connexin 43 in endothelial cells may have cell autonomous effects on their responses to permeability enhancing signals, independent of any effects on intercellular communication (co-localization with the tight junction molecules occludin, claudin, and ZO-1) [56
], which may explain the different time course observed for stress-induced changes in BSCB permeability and changes in expression of spinal connexin 43 and GFAP in our model after 10 WA.
Our findings showing a trend for sustained increased levels of connexin 43 in DRGs throughout the 10 WA period and significantly higher levels of GFAP at 24 hours post 10 WA in DRGs, suggest that satellite glia cells (SGCs, glial cells present in sensory neurons in DRGs) may respond to input from either spinal or peripheral origin. It has been previously observed that increased gap-junction mediated coupling between SGCs (as indicated by increased connexin 43 expression) contributes to increased sensory neurons coupling and augmented neuronal excitability. Interestingly, SGCs were also found capable of releasing cytokines such as IL-1β and TNF-α [57
]. Although sensitization of DRG neurons is well documented in rat models of visceral pain induced by inflammation [59
], the role of SGCs in visceral pain has received little attention. A recent study showed that GAP junction blockers injected directly into DRGs abolish colonic inflammation-induced changes in SGCs and sensory neurons and significantly reverse visceral pain behavior suggesting a role of SGCs in the modulation of visceral nociception after inflammation [60
]. Our data indicate SGCs activation and increased coupling, suggested by increased connexin 43 in response to chronic WA stress in DRGs. The role of these changes in spinal glia activation and increase in visceral nociception in the chronic WA stress model needs to be further investigated.
In conclusion, the present study establishes a profile of circulating and spinal cytokines during the course of 10 days WA stress associated with changes in spinal astrocytes, BSCB permeability, and DRG SGC activation. While the data provide a mostly descriptive analysis of those changes, it provides a necessary framework to further evaluate the functional role of peripheral and spinal cytokines in stress-induced spinal glia activation and the development of visceral hyperalgesia.