4 DISCUSSION
Hypoxic
ischemic brain injury after CA is the primary cause of death of CA
survivors [36], thus it is imperative to develop an effective
strategy to combat brain injury for CA victims with successful
resuscitation. In this study, we investigated the neuroprotective
effects of FFA and the underlying mechanisms after experimental CA/CPR
in mice. We presented that FFA treatment
remarkably
improved 7-day survival and neurologic outcome, lessened
neuropathological impairment, reduced brain edema as well as mitigated
BBB breakdown. Additionally, pro-inflammatory microglia/macrophages
polarization was suppressed while anti-inflammatory
microglia/macrophages polarization was promoted in CA/CPR mice
administered with
FFA.
Lastly, we demonstrated that Trpm4−/− mice
exhibited comparable effect to FFA and Trpm4−/−mice treated with FFA showed no
benefit
of superposition after CA/CPR. Taken together, our results supported the
neuroprotection of FFA against brain injury resulting from CA/CPR,
probably depended on inhibition of the overactivated TRPM4 channel in
the neurovascular unit. Our study highlights the significance of TRPM4
in the development of
post-CA
brain injury and comprehensively evaluates the functions of FFA in
post-CA brain injury.
Numerous studies have revealed that FFA can be an ion channel modulator
[22-23, 37-38]. Since then, interest in FFA’s “off targets” (ie,
beyond its well-known effect of COX inhibition) protection has been
rekindled and pleiotropic effects of FFA have been reevaluated. These
intriguing findings
gave
rise to several studies indicating that FFA afforded neuroprotection in
the
central
nervous system (CNS) diseases, in term of dampening inflammatory
response, facilitating angiogenesis, preserving myelin and motor
neurons, inhibiting glial activation and so on [24-26]. Here, we
stepped forward to elucidate the role of FFA in the model of CA/CPR. Our
exciting findings in this study were that FFA treatment showed benefits
in improving survival, neurological function and lessening
neuropathological injuries following CA/CPR, which further supported FFA
as a lifesaving neuroprotectant to address CA/CPR-induced brain injury.
BBB disintegration is a threatening event in hypoxic ischemic
encephalopathy after CA, causing fatal brain edema closely associated
with poor prognosis. Osmotherapy is the conventional treatment for
cerebral edema, by employing hypertonic saline or mannitol. However,
under BBB dysfunction conditions, dehydrates also accumulate within the
brain, therefore potentiating edema formation. Virtually, previous
studies have indicated that hypertonic treatment fails to improve the
outcome when administered after CA/CPR [39, 40]. Besides,
osmotherapy is unlikely to alleviate, but may aggravate, the
pro-inflammatory response of the extravasated toxic substances [41].
From a clinical point of view, osmotherapy after CA/CPR remains an
understudied topic due to these counterproductive effects. In this
respect, preventing edema by enhancing BBB integrity is preferable to
osmotherapy for treating the already swollen brain. Accumulating studies
have demonstrated that targeting TRPM4 could be a new perspective for
maintaining BBB integrity [14, 16, 42]. FFA was reported to
ameliorate capillary fragmentation and secondary hemorrhage following
spinal cord injury through blocking TRPM4 [43]. In the current
study, we further offer encouraging evidence that FFA ameliorates BBB
disruption and consequent edema formation following CA/CPR, supporting
FFA as a viable agent in the rescue of BBB breakdown, which has
important clinical implications given
considerable
incidence rate of cerebral edema in CA/CPR patients.
Neuroinflammation serves as a cardinal role in hypoxic ischemic
encephalopathy resulting from CA/CPR [34]. Microglia may get
activated for many weeks and develop macrophage-like capacity to
initiate inflammatory cascades of the CNS after CA/CPR. Classically
activated microglia/macrophages secrete
adverse
cytokines that worsen brain injury and impede damaged tissue remodeling,
whereas alternatively activated microglia/macrophages release
anti-inflammatory mediators that hasten brain repair and potentiate
phagocytosis of dying cells [44]. Endowing activated microglia with
a neuroprotective phenotype improved outcome in the model of CA/CPR
[45, 46]. Moreover, a previous study demonstrated that microglia
depletion by intrahippocampal injection of liposome-encapsulated
clodronate was not sufficient to salvage neuronal degeneration after
CA/CPR [47], which further supports that the balance between the
numbers of reparative versus deleterious microglia/macrophages
phenotypes rather than indiscriminate suppression of microglia
activation may be instrumental in optimal brain repair and neurologic
recovery. A recent study found that administration of FFA inhibited
microglia activation [24]. Herein, we add to the current knowledge
that FFA transforms microglia/macrophages from a pro-inflammatory
functional status into an anti-inflammatory one after CA/CPR.
Strikingly, CA/CPR not only triggered an elevation in the percentage of
pro-inflammatory microglia/macrophages, but also increased the
percentage of
anti-inflammatory
microglia/macrophages, suggesting self-protection of the brain in
response to CA/CPR. Previous studies also found that anti-inflammatory
cytokines, such as TGF-β and IL-10 increased in the brain following
CA/CPR [45, 48].
Sustained confrontation of dying cells is a major stimulus for harmful
inflammatory reaction [49]. After CA/CPR, the selectively vulnerable
regions with extensive cellular debris and cell corpses act as a
reservoir for various cytotoxicity and pro-inflammatory cytokines,
conducing to overwhelming neuroinflammation and enlargement of secondary
injury. In addition to triggering inflammation, the necrotic tissue
hampers neural
reorganization
and repair. Considering that interfering with apoptosis and necrosis is
still difficult to achieve in the clinical real-world setting, promotion
of reparative microglia/macrophages phenotype associated with augmented
clearance function toward damaged cells and cellular debris may be
indispensable for timely eliminating the source of inflammation and
ultimately enabling effective functional recovery. We illustrated that
efferocytosis of microglia/macrophages was strengthened after FFA
administration, which could be a reasonable explanation for less damaged
cells accumulated in the brain. Besides, the adhesion molecule ICAM-1 is
largely exposed due to glycocalyx degradation during CA/CPR, which is
critical for mediation of neuroinflammation [50]. We found that FFA
treatment lowered expression of ICAM-1, which contributed to arrest
amplification of neuroinflammation after CA/CPR. Altogether, our data
reveal that FFA treatment after CA/CPR significantly optimizes the
tissue-reparative function of microglia/macrophages
to
allow for better brain cleanup and stronger capacity of
neuroinflammation resolution, thereby averting further neuronal injury
and favoring neurologic recovery.
TRPM4 channel is a nonselective monovalent cation that upregulates in a
variety of CNS diseases [14, 17, 43] and leads to excessive influx
of extracellular sodium, causing oncotic cell death and BBB breakdown,
while gene deletion of this channel affords neuroprotection in several
models of neurological disease [13, 14]. Beyond its predominant
effect of attenuating edema and preserving BBB function,
increasing
attention has been attracted to study TRPM4 channel as a novel target
for abating neuroinflammatory burden in several CNS diseases [51,
52]. Kurland
et
al. [21] indicated that in microglia in vivo and in vitro, gene
silencing of Trpm4 decreased pro-inflammatory gene expression
following TLR4 activation in microglia, hinting a delicate connection
between TRPM4 and microglia polarization. In particular, activation of
TRPM4 requires calcium overload, which is virtually occurs under
conditions of CA/CPR [4]. In line with the previous findings in the
model of CA/CPR [19, 53], the expression of TRPM4 was unregulated
and co-localized with the neurovascular unit. In the present study, we
found more favorable neurological outcome, lighter histological injury,
less IgG leakage, and more anti-inflammatory microglia/macrophages
polarization in Trpm4−/− mice after CA/CRR,
which were comparable to the effects of FFA. Since FFA shows high
potency in blocking the TRPM4 channel [22, 23], these results
together further confirm the vital role of TRPM4 in the pathogenesis of
post-CA brain injury. TRPM4, which can lead to catastrophic BBB
disruption and persistent neuroinflammatory response, could represent a
promising and clinical relevant target for the future treatment of
post-CA induced brain injury. As a step forward, we observed that FFA
treatment significantly inhibited the upregulation of TRPM4, and FFA
treatment combined with Trpm4 deficiency showed no additive
protective effect, which indicated that suppression of TRPM4 in the
neurovascular unit by FFA may be the main reason for better outcome
following CA/CPR.
Although there is no doubt about the fundamental role of endothelium in
forming a barrier to restrict BBB permeability, the interaction of
endothelium with the components of neurovascular unit should not be
overlooked. In contrast to pericytes and astrocytes, the role of
microglia in regulation of BBB function is only beginning to be defined.
Following ischemia, microglia form perivascular clusters, which display
the intricate interplay between microglia and brain vessel. The
communication of aggregated microglia with
endothelium
exerts both beneficial and detrimental effects in BBB function depending
on the microenvironment [54]. After BBB disruption, peripheral
immune cells and toxic substances are recruited into the brain and
whereby amplify neuroinflammation,
including
classically activated microglia/macrophages polarization [55]. These
classically activated pro-inflammatory microglia may hinder BBB
remodeling and result in vascular leakage, while anti-inflammatory
microglia assist in the recovery of BBB damage [11]. Besides,
neuroinflammation accompanying CNS diseases has been shown to accelerate
BBB breakdown [56]. On this basis, the cross-talk between BBB
dysfunction and microglia/macrophages polarization plays a key role in
the vicious circle contributing to the pathogenesis of post-CA induced
brain jury, which necessitates a significant conceptual shift from the
widely recognized emphasis on protecting neurons to a novel treatment
paradigm of targeting the entire neurovascular unit, especially
microglia/macrophages. We propose that the effect of FFA on maintaining
BBB integrity may not only be through directly suppressing TRPM4 on
endothelium, but also be derived from regulating the functional status
of microglia/macrophages, namely that FFA treatment is an effective
strategy to break the vicious circle. Additionally, the pathophysiology
of post-CA induced brain injury encompasses a heterogeneous cascade.
Thus, using agents that affect multifaceted targets simultaneously may
be more efficacious than modulating a single point to alleviate secondary
brain injury after CA/CPR. We are convinced that FFA is promised to
stand out as an attractive candidate drug for a
polyvalent
approach to stabilize the BBB following CA/CPR.
Apart from blockage of the TRPM4 channel, FFA also affects the activity
of other nonselective cationic channels [38]. In fact, specific
pharmacological inhibition of TRPM4 is not practicable at present,
probably due to structural similarities of TRPM4 to other channels. So
far, pharmacological inhibition of TRPM4 in several models by FFA has
been pursued [22-25]. Besides, TRPM4 has relatively higher affinity
to FFA than other ion channels [38]. It is suggested that low
concentrations of FFA (~10 μM) may be suitable to
determine the physiological effect of TRPM4 in situ, because it has
little to no influence on other ion channels whose FFA sensitivity is
much lower [38]. Accordingly, TRPM4 modulation may commonly account
for the effects of FFA given its upregulation in CA/CPR model and high
sensitivity to FFA. Of note, plasma concentrations of 4–12 μM, measured
in conditions of FFA clinical use are sufficient to potently inhibit
TRPM4 [57], which further supports FFA to be a TRPM4 inhibitor in
clinical translation. To our knowledge, other channels potentially
modulated by FFA are not be expected to alleviate microvascular
dysfunction, since this protective effect is specific for the TRPM4
channel. More importantly, as gene deletion of Trpm4 mimicked the
effect of FFA and no additional benefit was found in FFA-treatedTrpm4−/− mice, the
participation
of other ion channels is unlikely. Moreover, despite its primary
characterization as COX inhibitors, FFA showed lower effectiveness than
other non-steroidal anti-inflammatory drugs and it is reported that FFA
provided neuroprotection through inhibiting excitotoxicity and limiting
neuroinflammation, which was independent of COX inhibition [26, 58].
Collectively, our findings corroborated that TRPM4 is the most likely
regulator of the neuroprotective impact of FFA on CA/CPR-induced brain
injury.