Discussion
Consistent with previous studies using this model [17] we found
persistent weight-bearing asymmetry and mechanical allodynia of the
ipsilateral paw in animals with MIA-induced knee OA. These observations
are similar to the clinical features seen in patients with knee OA, in
that they typically experience pain with loading the affected joint and
a subset of patients also develop hyperalgesia in tissue distant from
that joint (referred pain) [25]. Both characteristics represent the
hypersensitivity state, as the pain is caused by mild, normally
non-noxious stimuli. Vortioxetine dose-dependently suppressed both forms
of pain-related behaviour observed in MIA-treated animals, suggesting
that it may counteract sensitization of pain pathways and exert efficacy
against pain in OA. Previously, we noted the analgesic efficacy of
vortioxetine in various models of neuropathic and inflammatory pain that
share a common feature of sensitization of pain pathways [12, 14].
Duloxetine was also effective in both behavioural tests in MIA-injected
animals.
We observed a pronounced increase in Ngf mRNA expression in knees
injected with MIA compared with sham-injected counterparts. Increased
NGF levels have been previously documented in the knees of rats
receiving intra-articular MIA [26] and in synovial samples from
patients with knee OA [27]. There is ample evidence for the
particular importance of NGF in the pathogenesis of OA pain and
treatments that inhibit its action (anti-NGF antibodies or small
molecules) have been or are being developed as a new class of analgesics
for the treatment of pain in OA (reviewed by Schmelz et al. [4) and
Wise et al. [5]). NGF is released in the affected joint by
chondrocytes, synovial fibroblasts, and macrophages in response to
various stimuli (e.g. mechanical stress and pro-inflammatory mediators
including IL-1β) [27-29] and participates in the development of
peripheral and central sensitization, in multiple ways [4, 5].
Vortioxetine profoundly reduced Ngf mRNA expression in
MIA-injected knees. Previously, vortioxetine was shown to have the
ability to inhibit nuclear factor (NF)-κB in isolated human macrophages
exposed to pro-inflammatory stimuli [16]. NF-κB is a transcription
factor that plays a central role in the expression of a variety of genes
involved in inflammatory responses [30]. It has been demonstrated
(in cultured human corneal epithelial cells exposed to hyperosmolar
stress) that IL-1β upregulates NGF and that the IL-1β-induced increase
in NGF production is NF-κB-dependent [31]. The reduction inNgf mRNA levels induced by vortioxetine could therefore be
related to the suppression of NF-κB. This effect can be attributed to
the main mechanisms of action of vortioxetine: SERT inhibition,
5-HT1B receptor (partial) agonism and
5-HT3 receptor antagonism [10]. Inhibition of SERT
(by fluoxetine or sertraline) has been shown to suppress NF-κB
activation (in isolated brain tissue from mice with neuronal injury and
in inflammatory cytokine-stimulated cultured glial cells) [32, 33].
This effect of increased extracellular 5-HT could be mediated at least
in part by activation of 5-HT1B/1D receptors, as their
selective agonist (sumatriptan) decreased NF-kB levels (in tongue
samples from rats with oral mucositis) [34]. In agreement with this,
we have shown that 5-HT1B/1D receptors are involved in
the antinociceptive effect of vortioxetine in a trigeminal model of
inflammatory pain in mice [14]. Vortioxetine can itself activate
5-HT1B receptors, and this direct effect could also
contribute to lowering NF-kB and NGF expression levels. On the other
hand, an increase in extracellular 5-HT may counteract these beneficial
effects via 5-HT3 receptors, as activation of
5-HT3 receptors has been shown to upregulate the
production of IL-1β (in isolated human monocytes) [35], which could
subsequently increase NGF levels. By blocking 5-HT3receptors, vortioxetine could prevent a 5-HT-induced increase in IL-1β
expression and thus maintain/enhance the suppressive effects of locally
elevated 5-HT on NGF levels via 5-HT1B/1D receptors. The
lack of direct effects on 5-HT receptors may explain why duloxetine
(serotonin and norepinephrine reuptake inhibitor, SNRI) did not markedly
reduce Ngf mRNA expression.
It is important to note that vortioxetine-treated MIA-injected animals
had Ngf mRNA at the level measured in sham-injected, healthy
animals. The major concern with anti-NGF treatment of OA pain is the
risk of rapid joint destruction and osteonecrosis, which has been
observed in both animals and humans [4, 5, 36, 37]. The risk appears
to be dose-dependent and is thought to be due to the disruption of
physiological functions of NGF, which include peripheral sensory neurons
survival, cartilage repair, and load-induced bone formation [4, 5].
By maintaining basal NGF levels, vortioxetine treatment might be devoid
of joint/bone deterioration risk, while providing effective analgesia.
Consistent with previous findings in animal models of OA [6, 38] we
found increased markers of oxidative stress in MIA-injected joints.
Oxidative stress, as a result of an imbalance between the production of
reactive oxygen species and their detoxification by the antioxidant
defense system [39] is highly pronounced in OA cartilage and is an
important cause of chronic inflammation, and vice versa [40]. The
contribution of reactive oxidative species to pain and hyperalgesia in
OA thus appears to be indirect. However, an oxygen radical scavenger
(rebamipide) has shown an anti-hyperalgesic effect in rats with
MIA-injected knees [41]. In our study, vortioxetine reduced
parameters of oxidative stress in the knees of rats treated with MIA.
Accordingly, vortioxetine prevented an increase in superoxide anion
(O2•-) production in isolated human
monocytes and macrophages exposed to an oxidative burst-inducing
substance [16]. The antioxidant effect of vortioxetine may be also
related to 5-HT3 receptor antagonism, as this has been
shown to protect against oxidative stress (reviewed in Gupta et al.
[42]). 5-HT3 receptor antagonist (tropisetron)
reduced oxidative stress and inflammation in colonic tissue in a rat
model of colitis after rectal administration [43], suggesting that
5-HT3 receptor blockade at the site of inflammation may
be responsible for these effects. We found a greater increase in
oxidative stress markers in the MIA-injected knees of male rats than in
those of female rats. Similarly, a lower defense capacity against
oxidative stress was observed in synoviocytes isolated from men with
knee OA than from women [44]. This may be the reason why
vortioxetine was less effective in reducing oxidative stress parameters
in male rats than in female rats in the present study.
Significantly increased mRNA levels of Il-1β , Tnf-α ,Ngf , Bdnf and Tac 1 (encoding substance P) were
recorded in L3-L5 ipsilateral DRGs and spinal cords of the MIA-injected
rats (except Ngf in the spinal cord), compared with saline
controls. These mediators originate from neurons and/or non-neuronal
cells (e.g. glial and T cells) that communicate with each other, and are
upregulated under conditions of intense/sustained activation of primary
nociceptive neurons (as in peripheral inflammation or nerve injury) and
could contribute to the development of central sensitization (reviewed
by Sandkühler 2013; Grace et al., 2014; Miller et al., 2014; Schmelz et
al. 2019; Yang et al., 2022; Cheng et al., 2014) [4,7,8 45-47].
Vortioxetine significantly reduced the expression of all upregulated
mediators in male rats, whereas in female rats the effect was mostly
absent. The sex difference in this effect of vortioxetine can be at
least partially explained by the finding of Sorge et al. [48], who
reported that different cells are responsible for the development of
central sensitization in mice with peripheral inflammation: in males it
depends on glial cells, whereas in females it is independent of glia and
probably depends on T cells. In a chronic pain model caused by
peripheral nerve injury in male rats, SERT inhibitor (fluoxetine)
suppressed the activation of microglia in the DRG/spinal cord and
exerted an anti-allodynic effect [49]. In male rats with nerve
injury, spinal administration of a 5-HT3 receptor
agonist resulted in glial hyperactivity, neuronal hyperexcitability and
pain hypersensitivity, which was attenuated by a 5-HT3receptor antagonist [50]. Inhibition of SERT and blockade of
5-HT3 receptors could therefore lead to glial cell
suppression and be involved in the downregulation of pain-related
mediators that we observed in male rats after treatment with
vortioxetine. With peripheral tissue inflammation, T cells (mainly Th1)
migrate to the spinal cord and release mediators that induce chronic
pain [46]. It has been shown in human and murine T lymphocytes that
among the different 5-HT receptor subtypes expressed on their surface,
the 5-HT1B receptors play the most important role in
their activation [51]. Thus, by directly and indirectly activating
5-HT1B receptors, vortioxetine could contribute to the
activation of T cells, which could explain why it mostly lack effect on
the expression of pain-related mediators in female rats. Vortioxetine
was more effective in reducing oxidative stress in MIA-injected knees in
female rats and conversely, it was more effective in reducing the
expression of pain-related mediators in DRGs/spinal cord in male rats.
This may explain why the anti-allodynic effects of vortioxetine did not
differ significantly between the sexes.
In this study, it was not possible to compare the efficacy of
vortioxetine with that of duloxetine as only two doses of each drug were
tested. (An extension of this study with additional doses, i.e. animal
groups, would not be ethically justifiable.) However, higher doses of
both antidepressants did not reach the toxic dose range, as they did not
impair motor functions/cause sedation, as observed in the rotarod test.
Converted to human doses [21], the doses studied correlate to
therapeutic doses (vortioxetine 2 and 10 mg/kg/day is equivalent to 19.5
and 106 mg/day for humans, therapeutic dose range is 5-20 mg/day
[10]; duloxetine 15 and 25 mg/kg/day is equivalent to 146 and 243
mg/day for humans, therapeutic dose range is 30-120 mg/day [52].
Thus, our result suggests that vortioxetine may be as effective as
duloxetine for OA pain. It is even possible to expect vortioxetine to be
more effective, as it can prevent/reverse NGF upregulation in the
diseased joint, whereas duloxetine cannot. Another advantage of
vortioxetine over duloxetine could be its multimodal analgesic effect,
i.e. the involvement of multiple pain-modulating systems in this effect
(serotonergic, adrenergic, cholinergic, cannabinoid, adenosine and
opioidergic) [14, 53], the improved cognitive performance and the
lack of negative effects on cardiac redox status and general well-being
that we observed in a previous study using the same model [54].
In conclusion, vortioxetine may be effective against chronic pain in OA.
This effect appears to be mediated, at least in part, by the regulation
of NGF expression in the affected joint, i.e. by reducing its expression
to basal levels. It would be worthwhile to investigate the effect of
vortioxetine in patients with OA, considering the beneficial effects it
has shown in an animal model.