Introduction
Ischemic stroke is a leading cause of mortality and disability, and there is an urgent need for therapies that can limit ischemic brain injury beyond the acute phase following ischemic stroke [
1‐
4]. Neuroinflammation mediated by innate immune cells of the CNS (microglia and CNS-infiltrating macrophages collectively called CNS mononuclear phagocytes or CNS-MPs) comprises of complex pro-inflammatory responses that promote neuronal injury as well as anti-inflammatory/neuroprotective responses [
3,
5]. Potentially protective anti-inflammatory CNS-MP phenotypes predominate in the first 1–2 days following ischemic injury whereas detrimental pro-inflammatory phenotypes increase after 48 h [
6‐
8]. Identifying the key regulators of detrimental and protective CNS-MP responses can facilitate discovery of novel targets for ischemic stroke. The recently proven efficacy of endovascular thrombectomy for acute ischemic stroke has created exciting opportunities to translate neuro-immunomodulatory and neuroprotective strategies that target disease mechanisms beyond the acute phase [
9,
10].
The diverse transcriptional profiles adopted by CNS-MPs have been recently revealed by transcriptomic studies which found an emergence of unique disease-associated microglial (DAM) profiles and downregulation of homeostatic genes in mouse models of chronic neuroinflammation, aging, and neurodegeneration [
11‐
13]. More recently, we identified molecular heterogeneity within DAM which is comprised of independently regulated pro-inflammatory and anti-inflammatory DAM sub-profiles [
13]. The potassium channel Kv1.3 was identified as a marker and regulator of pro-inflammatory DAM [
13], and inhibition of Kv1.3 channels was found to be beneficial in Alzheimer’s disease models as well as in the transient middle cerebral artery occlusion (tMCAO) ischemic stroke model [
13‐
15]. Although increased expression of Kv1.3 channels have been confirmed in acutely isolated CNS-MPs by electrophysiology, it is unclear whether microglia and/or CNS-infiltrating macrophages are the primary cell type with increased Kv1.3 channel expression [
14].
We have applied a novel flow cytometric assay of functional cell-surface Kv1.3 channels coupled with parallel phagocytic profiling of acutely isolated CNS-MPs [
16] at early and delayed timepoints following tMCAO, to define the temporal profiles of Kv1.3 channel expression within CNS-MP subsets.
Materials and methods
Reagents
Fluorescein-conjugated ShK-F6CA was purchased from Peptides International (Louisville, KY) [
17,
18]. Fluorophore-conjugated antibodies for flow cytometry were obtained from BD Biosciences (CD11b-APC-Cy7, CD45-PE-Cy7, CD45-FITC, Ly6c-PE) [
13]. Polystyrene phycoerythrin-fluorescent 1-μm microspheres (Thermo Fisher #F13083) were used for phagocytosis assays [
16]. Percoll was purchased from Sigma-Aldrich (#P1644).
Animals
Male C57BL/6 J mice (JAX 000664) aged 8–12 weeks were housed in the Department of Animal Resources at Emory University under standard conditions. Institutional Animal Care and Use Committee approval was obtained, and all in vivo studies were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health, and in compliance with the ARRIVE guidelines.
Transient middle cerebral artery occlusion (tMCAO) model
After anesthesia (isoflurane) and exposure of the carotid artery, a 6-0 silk suture was advanced from the external carotid artery into the internal carotid artery until the origin of the middle cerebral artery on the ipsilateral side [
19,
20]. The suture was then withdrawn after 30 min of cerebral ischemia. Laser Doppler flow was used to monitor cerebral perfusion at baseline, at time of MCAO, immediately after recanalization, and 5 min post-recanalization. Animals with > 75% decreased middle cerebral blood flow and > 75% reperfusion were included. In total, 32 mice were included for flow cytometric analyses (30 min:
n = 8, 24 h:
n = 7, 48 h:
n = 8, 72 h:
n = 4, 7 days:
n = 5). Sham surgery controls were performed (
n = 3 per condition). Transcranial Doppler flow data demonstrating effective MCAO and complete reperfusion for mice included in the study are shown in Additional file
1: Figure S1.
CNS-MP isolation
After tMCAO, mice were euthanized at 30 min, 24 h, 48 h, 72 h, and 7 days followed by cardiac perfusion. CNS-MPs were acutely isolated from ipsilateral and contralateral hemispheres using Percoll density (70%/35%) centrifugation as previously described [
16,
18]. After myelin removal, mononuclear cells were re-suspended in saline or Trizol. From each hemisphere, half was used for RNA extraction and the other half was used for flow cytometry.
Flow cytometry
Functional cell-surface Kv1.3 channel expression in acutely isolated CNS-MPs was measured using a validated fluorescein-conjugated ShK-F6CA assay [
13,
18,
21]. Cells were incubated with 10 nM ShK-F6CA along with fluorophore-conjugated CD11b (APC-Cy7), CD45 (PE-Cy7), and Ly6c (PE) antibodies for 30 min and then washed twice with cold PBS to remove any unbound antibodies. Compensation controls were run along with appropriate negative/isotype controls [
18]. Live mononuclear cells were first gated based on forward and side scatter profiles, and then single cells were gated and then further gated for CD11b
+ CNS-MPs. CD11b
+ CNS-MPs were gated into CD45
lowLy6c
low, CD45
highLy6c
low, and CD45
highLy6c
high subpopulations. Each CNS-MP subpopulation [
22,
23] was evaluated for ShK-F6CA labeling.
Immunohistochemical studies
Following 30 min MCAO, mice were euthanized at 1 h, 24 h, and 48 h timepoints and brains were post-fixed in 4% PFA for 24 h, then transferred to 30% sucrose for another 24 h, and then sectioned (30 μm) and preserved in cryoprotectant solution. Free-floating sections were blocked with 10% horse serum for 1 h, then incubated with primary antibodies (anti-Kv1.3 mAb 1:100 UC Davis/NIH NeuroMab clone L23/27, anti-Tmem119 rabbit mAb 1:100 #ab209064, or anti- Iba1 rabbit mAb 1:300 #ab178846) at 4 °C overnight. Sections were then washed and incubated with fluorophore-conjugated secondary antibodies (1:500) for 30 min and then mounted on slides and dried. Hard-mounting medium (with DAPI-VectorLabs # H1500) was used to mount sections which were then imaged on an immunofluorescence microscope (Microscope: Olympus BX51 and camera: Olympus DP70) at 20× and 60× (oil immersion) magnifications. The ipsilateral (stroke) hemisphere was marked prior to sectioning. At least five fields in the peri-infarct region ipsilaterally and corresponding region on contralateral hemisphere were imaged per mouse (n = 2–3 mice per timepoint). All immunofluorescence images were processed using ImageJ software (version 1.52a).
Phagocytosis assay
Acutely isolated CNS-MPs were incubated with phycoerythrin (PE)-conjugated microspheres (1:100) at 37 °C for 30 min, washed, and labeled with fluorophore-conjugated anti-CD11b (APC-Cy7) and anti-CD45 (FITC) antibodies. Microsphere phagocytosis in CNS-MPs was assessed as the proportion of cells taking up > 1 microsphere as previously reported [
16].
Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)
CNS-MP RNA extracted in Trizol was purified for qRT-PCR [
18,
24]. RNA was reverse-transcribed to cDNA (Ambion), and qRT-PCR was performed (7500 Fast RT-PCR instrument, Applied Biosystems) using cDNA, TaqMan PCR Master Mix and gene-specific TaqMan probes (Applied Biosystems) against Kcna3 (Mm00434599_s1), Kcnj2 (m00434616_m1), Ptgs2 (Mm00478374_m1), and Hprt (Mm03024075_m1) in duplicate. Relative gene expression was normalized to Hprt and calculated using the 2ΔΔC
T method [
25].
Statistical considerations
GraphPad Prism version 7.0 and SPSS Version 24 were used for statistical analyses. Pairwise comparisons were performed using T-tests (independent sample, two-tailed, unequal variance). Non-parametric Mann-Whitney U test was used for pairwise comparisons of flow cytometric data. The statistical significance level was set at p < 0.05.
Discussion
Microglial activation and CNS infiltration by peripheral myeloid cells following ischemic stroke can impact infarct expansion, edema, and neuronal survival [
31]. Disease-modifying roles for CNS-MPs in ischemic stroke are suggested by microglia depletion studies resulting in exacerbation of neuroinflammation and brain injury in focal ischemic stroke models [
32]. In neurodegeneration, microglia progressively transition from homeostatic to DAM states [
12,
33,
34] and within DAM, we have identified distinct pro-inflammatory and anti-inflammatory DAM sub-profiles with potentially opposing functional roles [
9,
13]. Although the relevance of the homeostatic and DAM paradigm to ischemic stroke is unclear, anti-inflammatory CNS-MP phenotypes have been observed in the first 1–2 days after which pro-inflammatory phenotypes predominate in rodent MCAO models [
7]. Therefore, selective neuro-immunomodulatory therapies for stroke are needed that inhibit pro-inflammatory CNS-MP responses and shift CNS-MPs towards protective anti-inflammatory and homeostatic states rather than non-specific anti-microglial strategies [
6,
32,
35,
36].
The Kv1.3 potassium channel has been identified as a promising therapeutic target to inhibit pro-inflammatory CNS-MP responses in neurodegenerative disease [
15,
24,
28] as well as ischemic stroke models [
14], resulting in improved neuropathological outcomes. Kv1.3 is highly co-expressed with pro-inflammatory DAM genes (Il1b, Tlr2, Hif1a, Ptgs2) and is a key regulator of pro-inflammatory CNS-MP responses [
13,
28,
29,
37]. Kv1.3 channels regulate membrane potential, calcium flux, immune signaling, and effector functions of effector memory T cells, memory B cells, and subsets of activated microglia and macrophages [
38,
39]. Neuronal Kv1.3 expression is limited to olfactory and cortical neurons as hetero-tetramers with other Kv1-family channels while immune cells exclusively express the homo-tetrameric channel [
38,
39]. This pattern of expression allows selective blockade of immune Kv1.3 channels by highly selective small molecule blockers (Pap1) and sea anemone toxin-based peptides (ShK analogs) without undesired off-target effects [
40,
41]. An early phase study of Kv1.3 channel-blocking ShK analog (ShK-186/dalazatide) in humans has demonstrated safety, supported by multiple pre-clinical studies showing safety and efficacy of Kv1.3 blockers in models of systemic autoimmunity, obesity, CNS demyelinating disorders, and neurodegeneration [
15,
18,
28,
42‐
44], suggesting that rapid translation of Kv1.3 blockers to ischemic stroke is feasible [
45]. To further build on the pre-clinical rationale of Kv1.3 channel blockade as a therapeutic approach in ischemic stroke, the key CNS immune cells targeted by Kv1.3 blockers and the optimal therapeutic window for Kv1.3 blockers need to be determined.
We have utilized a rapid flow cytometric assay of functional cell-surface Kv1.3 channels [
13,
18,
21] to phenotype acutely isolated CNS-MPs following tMCAO, and found that functional Kv1.3 channel expression is increased specifically in CD11b
+CD45
lowLy6c
low resident microglia as well as in the subset of CD11b
+CD45
highLy6c
low CNS-MPs, but not in CD11b
+CD45
highLy6c
high inflammatory monocytes. Therefore, it is highly likely that microglia are the primary cell types targeted by Kv1.3 blockers and that the beneficial effects of Kv1.3 blockers in stroke models are unlikely to be mediated via modulation of peripheral immune responses. We also found increased Kv1.3 expression in the CD11b
+CD45
highLy6c
low population of CNS-MPs which may represent an activated population of microglia or non-inflammatory patrolling monocytes that are recruited to the brain [
26]. We also observed that not all microglia increase Kv1.3 expression, suggesting that additional immune sub-profiling of microglia may provide novel biological insights into the regulation and roles of Kv1.3 channel expression in microglial activation [
46]. Increased functional cell-surface Kv1.3 channel expression at 48 h post-tMCAO was not accounted for by mRNA-level changes in Kv1.3, implicating post-transcriptional and post-translational processes in channel regulation in microglia [
46]. However, this interpretation is limited by the large variance in our qRT-PCR data, insufficient mRNA yields from CNS-MP subsets and the use of all CNS-MPs (which contain 60% CD11b
+ CNS-MPs), rather than enriched CD11b
+ CNS-MPs within the scope of this study. Based on increased Kv1.3 channel expression between 24 and 72 h followed by a decrease by 7 days post-tMCAO, we also define the therapeutic window for Kv1.3 blockers as the subacute/delayed phase of ischemic stroke rather than hyper-acute/acute timeframes.
Our tMCAO studies were limited to 30 min of vessel occlusion followed by reperfusion. This model was specifically selected to minimize mortality observed with longer durations of occlusion especially beyond 48 h post-tMCAO. Although we did not characterize Kv1.3 channel expression by flow cytometry in longer MCAO models, other groups have shown in the 60-min MCAO model that Kv1.3 protein expression was indeed increased between day 2 and day 8 post-MCAO, suggesting that our findings regarding delayed Kv1.3 channel expression by microglia seems to be consistent regardless of the duration of vessel occlusion [
14]. As compared to traditional electrophysiological methods such as a whole-cell patch clamp which has very limited sampling capabilities (< 40–50 cells per mouse), our flow cytometric approach to characterize Kv1.3 channel expression provides a rapid and comprehensive strategy which also allows us to assess Kv1.3 expression within CNS-MP subsets. Another interesting finding that we report is the downregulation of Tmem119 protein in microglia at 48 h post-tMCAO, which suggests that microglia downregulate homeostatic genes/proteins and may potentially transition towards activated phenotypes following MCAO [
11‐
13,
28]. Whether these delayed activated phenotypes share any similarities with DAM phenotypes observed in neurodegeneration remains to be explored. We also did not note any relationship between phagocytic activity in CNS-MPs and Kv1.3 channel expression. The overall increase in phagocytic activity in both hemispheres at 48 h post-tMCAO may indicate a global response although the mechanisms or implications of this observation remain unexplored. Our results with phagocytic uptake of latex microspheres also do not necessarily reflect the ability of CNS-MPs to clear cellular debris in the ischemic core or to phagocytose healthy neuronal synapses as these processes involve specific receptors including complement receptors [
3,
47]. Ongoing studies investigating specific mechanisms of phagocytosis in post-stroke CNS-MPs, as well as transcriptomic and proteomic efforts to characterize CNS-MPs in stroke models, will provide additional clarification.
From a clinical perspective, our findings in the transient 30-min MCAO model are relevant to acute large-vessel occlusion (LVO) stroke, which represent 25–40% of acute ischemic stroke [
1,
48]. With the advent of mechanical thrombectomy as a highly effective reperfusion strategy, acute LVO stroke patients can be treated up to 24 h post-onset of stroke symptoms. However, nearly 50% of patients still suffer significant disability [
9] and very few effective therapies exist beyond the 24-h window [
10,
49]. Since reperfusion can be established in LVO patients, the stroke field is ideally poised to revisit neuroprotective and neuro-immunomodulatory strategies such as Kv1.3 blockade to target disease mechanisms most relevant in the post-acute phases of ischemic stroke.
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