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Sex Differences in Poststroke Inflammation: a Focus on Microglia Across the Lifespan

Originally publishedhttps://doi.org/10.1161/STROKEAHA.122.039138Stroke. 2022;53:1500–1509

Abstract

Stroke is one of the leading causes of death worldwide and currently only few therapeutic options are available. Stroke is a sexually dimorphic disease contributing to the difficulty in finding efficient treatments. Poststroke neuroinflammation is geared largely by brain microglia and infiltrating peripheral immune cells and largely contributes to sex differences in the outcome of stroke. Microglia, since very early in the development, are sexually divergent, imprinting specific sex-related features. The diversity in terms of microglial density, morphology, and transcriptomic and proteomic profiles between sexes remains in the adulthood and is likely to contribute to the observed sex-differences on the postischemic inflammation. The impact of sexual hormones is fundamental: changes in terms of risk and severity have been observed for females before and after menopause underlining the importance of altered circulating sexual hormones. Moreover, aging is a driving force for changes that interact with sex, shifting the inflammatory response in a sex-dependent manner. This review summarizes the present literature on sex differences in stroke-induced inflammatory responses, with the focus on different microglial responses along lifespan.

See related articles, p 1432, 1438, 1449, 1460, 1473, 1487

Stroke is the second cause of death worldwide and one of the leading causes of disability, although the incidence and mortality show large geographic variations, and depend on a variety of risk factors present in different populations. Sex differences have been described in terms of risk, symptoms, severity, and outcome.1–3 Despite women present a lower stroke incidence than men throughout most of their lifespan, the overall prevalence of stroke is higher in women because of their increased longevity.4 Moreover, the incidence of stroke increases in women after menopause, when the cytoprotective effect of estrogen disappears.5 At the time, also the severity of stroke increases to the level comparable to men at similar ages.6

An important contributor to stroke pathology is postischemic inflammation, which is largely geared by concerted function of brain microglia and peripheral immune cells.7 It is now clear that sex differences in the inflammatory response exist also in the pathogenesis of several other diseases.8–10 This review summarizes the sex differences in microglia and their role in the inflammatory response after stroke, taking into consideration the effect of circulating hormones and age.

Sex Differences in Microglia in Healthy Brain

Microglia exists in region, time, and context-dependent states in the healthy adult central nervous system, presenting differential phenotypic characteristics and specific transcriptomic profiles in different brain areas.11,12 However, knowledge on sex differences in microglia states in healthy brain is scarce.

Most of the information on sex differences in microglia are derived from studies performed in rodents. Sex-related differences in microglial density and morphology exist already in early the development and, although these features change during the lifespan, some of the differences remain in the adult brain. Only few studies have addressed sexual dimorphism in adult rodents in different brain regions. Some studies show that females have a higher density of microglia in specific hippocampal regions compared with males,13,14 whereas others show a higher density of microglia in the cortex, amygdala, and hippocampus of male rodents.15–17 Not only the density but also microglial morphology (eg, number of branches, processes per branch, and branch thickness) is region-dependent. For instance, microglia in the dorsal hippocampi have a higher total number of processes and more processes per branch compared with the prefrontal cortex in male rats. On the contrary, microglia are reported to have a higher total number of processes and more processes per branch in the prefrontal cortex compared with the dorsal hippocampi in female mice.18 Microglia are more ramified in the prefrontal cortex in female rats compared with males,19 while microglia in hippocampus are more ramified in males compared with females.20 The contradictory results may be partly due to differences in the analyzed brain region, age, and species, but also due to the microglia markers and techniques that were used.21 This fact limits the comparison between studies, urging the researchers to use harmonized techniques to explore the sex-differences in microglia (Table 1).

Table 1. Sex Differences in Microglial Density and Morphology in Adult Brain

Species: ageDifferencesReference.
Differences in cell density
C57BL/6 mice: 3–4, 13–14, and 20–24 moDG and CA1: F>M at all the ages13
C57BL/6 mice: 3–4 moCx, Hp, Amy: M>F17
Str, Cb: M=F
SD rats: 2–3 momPFC, prelimbic region: M=F19
SD rats: 2–3 momPFC: F>M15
SD rats: 2–3 moPaCx, CA1, CA3, DG, Amy: M>F14
Differences in microglia morphology (number and complexity of microglial branches)
C57BL/6 mice: 3 moCA3: M>F in process volume and area, number of branches and intersections.20
C57BL/6 mice: 3–4 moCx, Hp, Amy: M>F in the size of the cell soma17
C57BL/6 mice: 3 moM: more complexity in the dHIP than in the PFC18
F: more complexity in the PFC than in the dHIP
SD rats: 2–3 moPaCx, CA1, CA3, DG, Amy: M>F in the thickness and length of microglia processes14
SD rats: 2–3 moCx: F>M in primed/ramified microglia19

Amy indicates amygdala; CA1, cornus ammonis 1; CA3, cornus ammonis 3; Cb, cerebellum; Cx, cerebral cortex; DG, dentate gyrus; dHIP, dorsal hippocampus; F, female; Hp, hippocampus; M, male; mPFC, medial prefrontal cortex; PaCx, parietal cortex; PFC, prefontral cortex; SD, Sprague-Dawley; and Str, striatum.

Of note, analysis of the expression of molecular patterns or transcriptomic programs in rodents reveals sex differences that cannot be quantified by morphometric analysis. Single-cell transcriptomics emerges as a powerful tool to analyze changes in cellular populations and states, yet, studies evaluating sex-differences at the single cell level are largely missing. In the recent years, a growing number of studies have demonstrated differences in the bulk transcriptomic and proteomic profiles of microglia depending on the brain region12,22 or age.23 Focusing on sex differences, transcriptomic analysis revealed 1109 differentially expressed genes in the hippocampus, 55 in the cerebral cortex and 46 in both regions between male and female mice, demonstrating brain region to be an important factor in microglial sex-diversity.17 Whole-brain microglia showed increased expression of 204 genes related to inflammatory processes in male mice compared with females, including regulation of cell migration, chemotaxis, and cytokine production.24 In agreement with transcriptomic studies, NF-κB (nuclear factor kappa B) is transcriptionally more activated in male microglia compared with female microglia.25 This suggests that male microglia are inherently more prone for inflammatory activation in pathological conditions compared with female microglia, consistently with higher expression of interferon-stimulated genes in males.26 On the other hand, females show higher expression of genes associated with development or morphogenesis that are under the control of transcription factors linked to the inhibition of the inflammatory response.24 Female microglia also express more sensome-related genes,26 a cluster of transcripts that code for proteins involved in sensing of endogenous ligands and microbes.25 In addition, proteomic profiling of microglia has demonstrated 263 proteins with significantly higher expression in males and other 96 proteins, preferably expressed in females.17 All these studies provide evidence that female and male microglia express different protein and RNA patterns even in the healthy brain which may contribute to the sex-differential response of microglia to stroke in rodent models (Table 2).

Table 2. Sex Differences in Mouse Microglial Gene Expression at Different Ages

Species and ageRegionDifferencesReference
C57BL/6 mice: 3-to 4-mo oldCx, HpGO of genes overexpressed in M cortex: transcription factor activity, histone demethylation and deacetylation.
GO of genes overexpressed in M hippocampus: regulation of defense response to bacteria, insulin receptor pathway, glial cell differentiation, and ATP binding
GO of genes overexpressed in F dataset: GABA and glutamate receptor activity, ubiquitin protein activity, and magnesium ion transport
17
C57BL/6 mice: 3-mo oldWhole brainGO of genes overexpressed in M: inflammatory response, leucocyte migration, regulation of response to wounding, chemotaxis, regulation of cytokine production. Many genes regulated by NF-kB
GO of genes overexpressed in F: movement of cell, nervous system development, cell migration, vasculature development, anatomic structure formation, actin cytoskeleton organization. Many genes regulated by transcription factors, linked to the inhibition of the inflammatory response.
24
C57BL/6 mice: 3-, 12- and 24-mo oldHpActivation of inflammatory genes in both sexes, but more evident in old F: higher expression of genes for the complement system and the microglial sensome with the age.27
C57BL/6J mice: 2- to 3-mo oldN/IF: higher expression of interferon-stimulated genes (S100a8, S100a9, Ifit1, Ifit2, Cxcl10, Ccl2, Irf1, Ccnd3, and Gbp5).
Differential expression of sensome genes between F and M.
26
C57BL/6 background mice: 3-, 12-, and 24-mo oldForebrainThe main sex differences in gene expression are observed at 24-mo old. 37 transcripts differentially expressed between M and F at 24-mo.
F: higher levels of Spp1, Gpnmb, Lgals3, ApoE, Ccl3, Clec7a, and Ccl4.
28

ApoE: apolipoprotein E; Ccl2, C-C motif chemokine ligand 2; Ccl3, C-C motif chemokine ligand 3; Ccl4, C-C motif chemokine ligand 4; Ccnd3, cyclin D3; Clec7a, C-type lectin domain family 7 member A; Cx, cerebral cortex; Cxcl10, C-X-C motif chemokine ligand 10; GABA, gamma aminobutyric acid; GO, gene ontology; F, female; Gbp5, guanylate-binding protein 5; Gpnmb, glycoprotein Nmb; Hp, hippocampus; Ifit, interferon-induced proteins with tetratricopeptide repeats; Lgals3, lectin galactoside-binding soluble 3; M, male; N/I, not indicated; NF-kB, nuclear factor kappa B; S100a8, S100 calcium-binding protein A8; S100a9, S100 calcium-binding protein A9; and Spp1, sporulation-specific protein 1.

In addition, there are differences in microglia across species. Microglial morphology is relatively conserved between humans, macaque, and rodents, but human microglia are more heterogeneous and display an organization in different subtypes, as revealed by single-cell RNA-seq.12,29 These subtypes do not correlate with sex-specific features but add an additional layer of complexity to the analysis of sex differences.

Sex Differences in Microglia Response to Stroke

Microglia are essential in responding to brain insults and play a central role in initiating and gearing the neuroinflammatory response.7 In the presence of a stimulus, such as stroke, these cells can exhibit a wide spectrum of activation states.30 Traditionally, microglia were divided into M1 and M2 phenotypes, with M1 type cells secreting pro-inflammatory cytokines, proteins of the complement system, reactive oxygen species and reactive nitrogen species, and the highly phagocytic M2 type microglia releasing anti-inflammatory and immunosuppressing cytokines. While this classification has been used extensively in the literature, we omit from using this over-simplistic dichotomy, and rather refer to microglial states that are both stimulus and context dependent and form a continuity between different states.30,31

The abovementioned sex differences in the gene transcription contribute to their differential responses to ischemic stroke. Differential stroke outcomes (short- and long-term) between sexes, as well as sex-specific inflammatory response are reported, which are critical to understand the consequences of sex-differences in microglial response to stroke and in the related outcomes (Table 3).

Table 3. Sex Differences in Infarct Size and in the Inflammatory Response

Infarct volume
M>FNeonatal: 3 dpi,32 30 dpi,33 90 dpi34
Young/adult: 1 hpi,24 1 dpi,24 2 dpi,24,35 3 dpi,36–38 4 dpi42
M=FNeonatal: 1 dpi,32 3 dpi34,39
F>MAged: 3 dpi40
Microglial density after stroke
M>F41
M=F34,42,43
Microglial reactivity/activation after stroke
M>FRamification44 and morphology39
Leukocyte infiltration
F>M45
M>F33,35,39,46
Pro-inflammatory serum cytokines
M>FTNF-α,32,33 IL-1β33

Dpi indicates days postischemia; F, female; hpi, hours postischemia; IL-1β, interleukin 1 beta; and M, male; and TNF-α, tumor necrosis factor alpha.

Sex Differences in Infarct Volume and Microglial Activation After Stroke

According to clinical data, the age-adjusted incidence of stroke is higher in men compared women, although this has changed during the recent years.47 In line, adult female rodents display smaller infarcts compared with males24,35–38,42 (Table 3). The protection in females also exists in neonatal ischemia, but the studies are less consistent32,34,39 The protective effect observed in young/adult females in their reproductive period of life is reversed with aging,40,48 as discussed in Section Interations Between Age and Sex: the Relevance of the X Chromosome.

To correlate microglia activation to the severity of stroke, different morphological parameters have been utilized, such as number of microglia and morphological changes from ramified to amoeboid morphology. Despite the lack of sex differences in the number of microglia after stroke in most of the studies, males display a more ameboid microglial morphology compared with females that has been interpreted as an indication of more robust activation.24,44 These studies suggest that microglial activation in young females is less pronounced compared with males. However, besides the morphological features, microglia can respond in many other ways depending on the cytokine profile they secrete, and this has led to a myriad of phenotypes with different anti- and pro-inflammatory properties.31

Stroke Induces Divergent Microglial Phenotypes Between Male and Female

Several studies have had a focus on the characterization of the microglial phenotypic response after stroke. After ischemic insult, microglial inflammatory phenotype polarizes differently between sexes. The expression of markers of pro-inflammatory microglia is higher in males compared with females, where the anti-inflammatory phenotype is dominant,35,37 revealing a clear predisposition of male microglia to trigger inflammation. The studies gathered in Table 4 are evidence of distinctive molecular pathways of triggering inflammation between sexes and are in line with transcriptomics studies that showed differential expression of inflammation-related genes in males and females in healthy brain (Table 4).

Table 4. Sex Differences in Microglia Markers After Hypoxia/Ischemia and Stroke

MarkerTechniqueModelReference
Pro-inflammatory phenotypeCD16/32+M>FmRNANeonatal pMCAO39
M=FProteinCortical injury in adults41
TNF-αM>FmRNANeonatal pMCAO39
COX-2M>FmRNANeonatal pMCAO39
M=FproteinNeonatal pMCAO34
IL-1βM=FmRNANeonatal pMCAO39
NOS2M=FmRNANeonatal pMCAO39
MHCIIM>FProteinNeonatal hypoxia-ischemic32
M=FproteinNeonatal hypoxia-ischemic33
Anti-inflammatory phenotypeYM1F>MProteintMCAO in adults35
F>MProteinpMCAO in adults24
Arg1F>MProteintMCAO in adults35
F>MProteintMCAO in adults34
F=MmRNANeonatal pMCAO39
F=MproteinNeonatal pMCAO34
M>FproteinCortical injury in adults41
CD206F>MproteintMCAO in adults37
F>MproteinNeonatal hypoxia-ischemia33
F=MmRNANeonatal pMCAO39
M-trans phenotypeArg1+/COX2F>MProteinNeonatal pMCAO34
OthersCX3CR1M=FmRNANeonatal pMCAO39
CD68M=FProteinNeonatal hypoxia-ischemia43

The changes are divided depending on if they are measured based on protein levels (indicating changes in the expression per cell or changes in the number of positive cells for this protein) or mRNA levels (showing mRNA transcriptional changes in microglia). Arg1 indicates arginase 1; CD206, cluster of differentiation 206; CD68, cluster of differentiation 68; COX2, cyclooxigenase 2; CX3CR1, C-X3-C motif chemokine receptor 1; F, female; IL-1β, interleukin 1β; M, male; MHCII, major histocompatibility II; pMCAO, permanent middle cerebral artery occlusion; tMCAO, transient middle cerebral artery occlusion; and TNF-α, tumor necrosis factor alpha.

The evidence of divergent microglia responses to stroke between sexes has also been demonstrated in prenatal ischemia, when the sexual hormones remain at the same levels in both sexes.39 This suggests that part of differences between males and females arise from inherent developmental differences before the sexual maturation. In this regard, male and female microglia display differences in their gene expression during brain development,20,26 some of them are associated with the X and Y chromosomes. The number of differently expressed genes increases in adulthood.26 For instance, adult females show a higher expression of IL (interleukin)-4 receptors, a well-known inducer of the anti-inflammatory phenotype, as well as higher levels of Ifr3 (interferon regulator factor 3) compared with males, which acts as an anti-inflammatory regulator of microglia.17,46 Similarly, the higher responsiveness to immunologic stimuli observed in males may be also related to the higher levels of proteins of the TLR (toll-like receptor) pathway and to the presentation of MHC (major histocompatibility complex) I and II reported in males.17,49

These sex-related differences could arise from the specific programs activated to drive the differential sexual maturation of the brain.50 Early in the development, between post-natal day 1 and post-natal day 10, the expression of pro-inflammatory-related genes (specifically PGE2 [prostaglandin E2]) triggered by the presence of estradiol aromatized from testicular testosterone drives the brain masculinization.51 Microglia participate in this process by both producing and responding to PGE2,50 and this might lead to a priming effect persisting long in the adulthood,52 as well as to higher male vulnerability observed in early-life inflammation.

Despite the scarce number of studies that explore the sex-differential response to stroke in microglia, all the previous evidence suggest that microglia acquire a sex-linked transcriptomic signature triggered by X- and Y-associated gene expression and brain sexual differentiation. This may render microglia imprinted even after the sensitive period,24,26 leading to differential inflammatory responses between sexes reported in brain pathologies. The sex differences in microglial response are also observed in human postmortem tissues from different diseases, including stroke,53 Alzheimer disease,54 or glioma,55 evidencing that the differential sex response is a conserved feature between species that also occurs in the human brain.

Influence of Circulating Sex Hormones in Stroke Outcome

Sex hormones have a profound impact on the outcome of stroke.56 There is clinical evidence demonstrating a higher stroke incidence and severity in women after the menopause,5 and for this reason, female sex hormones, specifically estrogen, have been attributed a cytoprotective role in ischemic stroke.57,58 Several studies consistently demonstrate that estrogens drive the sex-differential protective effect in young females, which has been confirmed in ovariectomized female rodents subjected to stroke and the protection by estrogen therapies.57,59,60 While the effect of estrogens is consistent across studies, the role of testosterone remains controversial with evidence of both detrimental and beneficial effects.61,62 The fact that the beneficial effect of testosterone is dose-dependent62 suggests that it is the balance between the sex hormones that matters.63 Besides this controversy, it has been suggested that estrogens have an anti-inflammatory role after stroke in which microglia may be involved.64

Estrogens and Microglia After Stroke

Estrogens initiate most physiological and pharmacological actions by binding to the ERα and ERβ (estrogen receptors α and β), and to the recently identified novel estrogen receptor denominated GPR30. The predominant and biologically most active form of estrogen is the 17β-estradiol (E2).65 At present, little is known about ER receptors in microglia, and there is an ongoing debate whether ERα or ERβ mediate signaling in microglia.58 Whether ERβ but not ERα expressed in microglia is controversial.66,67 However, transcriptomic studies show that there are no sex differences in ER subtype expression in microglia,68 suggesting that the cytoprotective effect of estrogens may rely on the amount of hormone rather than on the differential expression of estrogen receptors between females and males. In addition, a study carried out by Elzer et al59 proposes that the estrogen-elicited cytoprotection relies on neuronal ERα expression, suggesting that the protective mechanism of estrogens is mediated by neurons rather than microglia. Immunosuppressive effect of E2 on microglia and astrocytes is apparent: it limits microglial-inducible nitric oxide synthase activity, downregulates apoptotic factors and secretion of pro-inflammatory cytokines, such as IL-1β (interleukin 1 beta), IL-6 (interleukin 6), and TNF-α (tumor necrosis factor alpha). In addition, E2 mediates upregulation of pro-survival and anti-inflammatory factors, including TREM2 (triggering receptors expressed on myeloid cells 2) and IL-10 (interleukin-10; Figure). Indeed, several publications report an inflammatory resolving effect of estrogen.57,69

Figure.

Figure. Factors that contribute to the sex differences in microglial response to stroke. Yellow refers to females and blue to males. Among the factors that contribute to sex differences in microglia response are the peak of hormones. In males, this drives the brain sexualization and imprints microglia toward a pro-inflammatory predisposition (blue line). The presence of estrogen in females during adulthood results in cytoprotection after experimental stroke (orange line). The age-dependent increase of X-chromosome escapes in females, giving an extra dosage of X-related genes (gray line). Infarct volumes after stroke are smaller in adult females than in males. Microglia also adopts more anti-inflammatory phenotype in adult females compared with males. Aged females, however, shows a switch in the response and the infarct volume increases compared with young females, and microglia displays a more pro-inflammatory phenotype. E2 indicates 17β-estradiol; IL-1β, interleukin 1β; IL-6, interleukin 6; IL-10, interleukin-10; MHCII, major histocompatibility II; TNF-α, tumor necrosis factor alpha; and TREM2, triggering receptor expressed on myeloid cells 2. Created with www.BioRender.com

It is worth to highlight that, as previously mentioned, microglia display a differential response between males and females after postnatal stroke in the absence of sex hormones.39 Moreover, microglia maintains sex-specific expression of a significant number of genes in culture, in ovariectomized female mice, or after transplantation to the opposite sex.24 All this evidence supports the hypothesis of the microglia imprinting in early development, but it does not contradict the fact that estrogens play an important role in the post-ischemic inflammatory response by microglia.

Interations Between Age and Sex: the Relevance of the X Chromosome

Age is the main contributor for the outcome of stroke and has a complex and interactive effect with sex. Aging by itself is associated to changes in the immune system, such as a rise in inflammatory markers and diminished response to cytokine stimulation, which can modulate the stroke progression and outcome.70 In experimental stroke, while young females show smaller infarct volume compared with males, the outcome is inverted in aged males.16,40,48 It is still under discussion whether the worsened outcome of aged females compared with males is caused by the lack or dysregulation of estrogens, or due to a sex chromosome–linked features. Some studies highlighting the relevance of the X chromosome in aging suggest that these differences could rely on X chromosome dosage. The additional X chromosome is silenced in young female animals,71 thus no differences in the infarct volumes are observed between females with 1 and 2 X chromosomes (XX and XO).72 However, aging affects the stability of the silencing, and thus the X chromosome may escape from the inactivation, giving an extra dosage of X-related genes.73 The Four Core Genotype mouse model is a useful tool to dissect the effect of X-dosage from sex hormones along lifespan. In this model, the testis-determining gene sry is deleted from the Y chromosome and inserted on an autosome, allowing the generation of XXM mice (a gonadal male with XX chromosomal complement) and XYF mice (a gonadal female with XY chromosome complement).74 Manwani et al60 utilized this model to demonstrate that gonadal hormones are responsible for the cytoprotection in young females compared with males. The authors show that both XXF and XYF (gonadal females) display reduced infarct volumes compared with XXM and XYM (gonadal males), which is not observed when comparing males with ovariectomized females. Moreover, estradiol treatment not only reverses this but also exerts protective effect in males suggesting that circulating estrogen is the main driver for the protection during reproductive period of life. However, age is a game-changer. McCullough et al40 used the Four Core Genotype model in aged mice to demonstrate that aging changes the relative contribution of XX/XY chromosomes to ischemic damage when compared with young mice. XXF and XXM aged mice have significantly larger infarct volumes. Moreover, these mice show a more robust stroke-induced microglial activation pattern compared with XY female and XY male mice, revealing that the chromosomal complement shapes the inflammatory response as well as the outcome of stroke in aged mice.40 The combination of these 2 studies provide a wide perspective of the relative contribution of XX/XY chromosomes along lifespan.

Focusing on microglia specific features in aged animals, sex differences are scarcely studied. Transcriptomic analysis through the lifespan show activation of inflammatory genes in both males and females, but these changes are more evident in females, resulting in a sharper sex divergence in the elderly.27,28 These data suggest that the predisposition of pro-inflammatory signaling observed in young males is compensated by age in females, possibly via X chromosome escape as described above.53 In fact, microglia from aged female animals express higher levels of pro-inflammatory markers (MHCII [major histocompatibility II]) compared with male microglia, together with an increase of pro-inflammatory cytokines in the serum (TNF-α and IL-1β).40 Another example of this sexual divergence along the age is the differential expression of complement proteins in microglia of old females and males. The complement system is induced upon ischemic stroke and modulates of the poststroke inflammatory response.75 Microglia are described to release several complement effectors after stroke, such as C1qa (complement 1qa)76 and complement 3 (C3).77 Aging induces the transcription of C1qa and complement 1qc (C1qc) genes and the translation of C1q protein at higher levels in females compared with males.27 Similar to the complement system, there is a higher upregulation of the sensome genes in female microglia compared with males.27 In addition, the importance of X chromosome escape has been highlighted by a recent study on 2 X escapee genes, the lysine demethylase 5C and 6a (kdm5c and kdm6a respectively), which code for histone demethylases.53 These genes regulate microglial production of cytokines and are expressed at higher levels in microglia from aged females subjected to middle cerebral artery occlusion compared with males as well as in human postmortem tissue from stroke patients, suggesting that epigenetic modifications may play a role in poststroke sex differences.53

All these studies highlight the fact that sex differences in the inflammatory response are not static along lifespan and raise the need of more in-deep studies of the sex-differential inflammatory response after stroke in aged animals to achieve better therapies.

Sex-Specific Features of Microglia As a Therapeutic Target in Stroke

Sex differences in treatment efficacy are common in stroke, yet, only a few studies have reported sex-differences on microglia-based therapies. Minocycline and other PARP (poly [ADP-ribose] polymerase) inhibitors78 have been reported to alleviate poststroke neuroinflammation and ischemic damage only in male rats.79,80 Interestingly, minocycline was ineffective in ovariectomized female with estrogen levels similar to males. Consistent with experimental studies, Amiri-Nikpour et al81 showed that oral administration of minocycline was effective in male patients with stroke but not in women. Meta-analysis of 7 randomized-controlled clinical trials showed that although minocycline seems to be a promising cytoprotective agent in patients with stroke, further studies are needed to evaluate the efficacy and safety.82 Our cautious conclusion is that minocycline may fail to effectively modulate highly dynamic, complex and sex-specific stroke-induced microglial responses.

Indeed, to discover more effective microglia-targeted therapies for stroke, we need to gain a deeper understanding of microglia biology. An elegant work by Villa et al demonstrated a significant difference in the transcriptome profile between adult male and female microglia independent of hormonal cues. Female phenotype is retained after transfer of microglia into male brain and more importantly, female microglia protected the male brain from ischemic stroke.24 The higher expression of genes involved in cell plasticity, control of inflammation and brain repair in female mice could contribute to protection. These genes may provide novel targets for sex-related approaches for effective stroke therapy.

Going beyond the acute phases of stroke is also important. Peri-infarct cortex is highly plastic undergoing reorganization through the reactivation of neurodevelopmental mechanisms to compensate lost neuronal functions.83 Increase in dendritic spine production together with axonal sprouting is an integral part of remodeling and brain repair process. Here, stroke-induced microglia have a major role in elimination of spines and shaping neural networks in functionally relevant manner. In depth understanding of the sex differences in microglia modulation of the peri-infarct cortex during stroke repair process, similar to neurodevelopment, may offer novel therapeutic targets for long-term functional brain recovery.

Different molecular mechanisms evolve after stroke in time-dependent manner. These are potential targets for microglia-based therapies to reduce degeneration and alleviate neuroinflammation or later to enhance brain repair, when properly timed. High number experimental drugs and interventions are effective in stroke models, yet, they have not translated into clinic success.84 It is expected that emerging understanding of sexually dimorphic brain and stroke pathology improves translation of basic science results to sex-specific therapies. For translational success, both sexes and aged animals should be considered and used whenever possible as suggested by STAIR committee,85 despite budgetary constraints and complex study design. Microglial chimeric mice enable comparison of sex differences in human context.86

Conclusions and Future Directions

Sex has been shown to contribute to the progression of poststroke inflammation and outcome. Specific molecular programs are launched to drive the process of brain differentiation between sexes already in very early stages of development and specifically in microglia. The characteristic differences acquired during development will influence the inflammatory response in adulthood and particularly in case of severe brain insults such as stroke. These inherent differences need to be investigated and understood to ensure that the novel therapeutic paradigms translate into new treatments.

Recent advances in molecular biology and single-cell analysis have provided considerable insights into dynamic microglial responses in stroke. In-depth understanding of sex-dependent microglial responses in aging animals and both acute and long-term consequences aid in driving and accelerating development of sex- and microglia-specific, personalized treatment strategies for acute protection and brain repair in stroke.

Article Information

Nonstandard Abbreviations and Acronyms

C1qa

complement 1qa

ERα

estrogen-receptor α

ERβ

estrogen-receptor β

Ifr3

interferon regulator factor 3

IL-1β

interleukin 1 beta

IL-6

interleukin 6

IL-10

interleukin-10

MHCII

major histocompatibility II

PARP

poly-adp ribose polymerase

PGE2

prostaglandin E2

TLR

toll-like receptor

TNF-α

tumor necrosis factor alpha

TREM2

triggering receptor expressed on myeloid cells 2

Disclosures None.

Footnotes

*I.F. Ugidos and C. Pistono contributed equally.

For Sources of Funding and Disclosures. see page 1507.

Correspondence to: Tarja Malm, PhD, A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, Neulaniementie 2, 70211 Kuopio, Finland. Email

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