X, but not Y, Chromosomal Complement Contributes to Stroke Sensitivity in Aged Animals

Post-menopausal women become vulnerable to stroke and have poorer outcomes and higher mortality than age-matched men, and previous studies suggested that sex chromosomes play a vital role in mediating stroke sensitivity in the aged. It is unknown if this is due to effects of the X or Y chromosome. The present study used the XY* mouse model (with four genotypes: XX and XO gonadal females and XY and XXY gonadal males) to compare the effect of the X vs. Y chromosome compliment in stroke. Aged (18–20 months) and gonadectomized young (8–12 weeks) mice were subjected to a 60-min middle cerebral artery occlusion. Infarct volume and behavioral deficits were quantified 3 days after stroke. Microglial activation and infiltration of peripheral leukocytes in the aged ischemic brain were assessed by flow cytometry. Plasma inflammatory cytokine levels by ELISA, and brain expression of two X chromosome–linked genes, KDM6A and KDM5C by immunochemistry, were also examined. Both aged and young XX and XXY mice had worse stroke outcomes compared to XO and XY mice, respectively; however, the difference between XX vs. XXY and XO vs. XY aged mice was minimal. Mice with two copies of the X chromosome showed more robust microglial activation, higher brain-infiltrating leukocytes, elevated plasma cytokine levels, and enhanced co-localization of KDM6A and KDM5C with Iba1+ cells after stroke than mice with one X chromosome. The number of X chromosomes mediates stroke sensitivity in aged mice, which might be processed through the X chromosome–linked genes and the inflammatory responses.


Introduction
Stroke is a sexually dimorphic disease [1,2]. Both clinical and experimental studies have shown that ischemic stroke sensitivity is mediated primarily by gonadal hormones in Shaohua Qi and Conelius Ngwa contributed equally to this work.

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young populations, regardless of the sex chromosome complement [3,4]. However, after menopause, the incidence of stroke in women increases with the loss of estrogen [5][6][7]. Our previous studies using four core genotype (FCG) mice have found sex chromosomal complement contributes to the stroke sensitivity in aged mice [8]. However, to date, it is not clear yet which sex chromosome (X vs. Y) plays a more important role in mediating the sex difference in stroke. Specifically, the genes involved in the regulation of stroke sensitivity during aging remain elusive.
Emerging data has suggested that the epigenetic modification of stroke-related genes (transcription factors or cell signaling regulators) can promote post-stroke recovery [9], whereas aberrant epigenetic modification is associated with a high risk of stroke [10][11][12]. Previous studies [13] showed that X chromosome-linked genes play important roles in the epigenetic modification of genes related to post-stroke inflammation. In the present study, we hypothesized that the second X chromosome increases stroke sensitivity in aged mice, presumably through immune responses mediated by X chromosome-linked genes. The XY* mouse model was used for the study, in which there are four genotypes of mice that have different combinations of X/Y chromosomes (XX or XO gonadal females and XY or XXY gonadal males) [14]. The model was generated by mating C57/BL6 WT female mice (XX) with XY* males in which the Y chromosome has an aberrant pseudoautosomal region (PAR), so that the offspring has either one or two X chromosomes, each in the absence or presence of a Y chromosome which causes testes to develop [14,15]. This model is useful in dissociating the effect of X chromosome number from the effect of that Y chromosome. Aged mice were used, as the vast majority of stroke patients are over 65 years of age. This also minimizes the activational effects of gonadal hormones.

Mice/Animals
XY* mice (XO and XX gonadal females and XY and XXY gonadal males) were produced by crossing XY* males (provided by Dr. Arthur Arnold) with C57BL/6 females (Jackson Laboratory) [16,17]. The mice were housed until they reached 18-20 months old, at which point females have become acyclic. Another cohort of mice was bilaterally gonadectomized under general anesthesia at 3 weeks of age and was used at 8-12 weeks old (GDX cohort). All mice were housed in an ambient temperature and humidity-controlled vivarium, with a 12-to 12-h day-night cycle and free access to food and water. Animal protocols were approved by the University's Institutional Animal Care and Use Committee. All experiments were performed in accordance with the National Institutes of Health and the University of Texas Health Science Center at Houston (UTHealth) animal guidelines.

Fluorescence In Situ Hybridization
To determine the genotype of mice by counting the number of X and Y chromosomes, interphase fluorescence in situ hybridization (FISH) was carried out with RUO-Rab9b (XqF1)/WC Y mouse probe (#KI-30505, Leica Biosystems) according to the vendor's instruction. Briefly, peripheral blood cells were collected from the mouse tail and mixed with 0.075 M EDTA solution on ice and five rounds of icecold 3:1 methanol/acetic acid. A droplet of cells suspended in methanol/acetic acid fixative was released onto a microscope slide from 10 to 12 in. height, air-dried, and stored in − 80 °C. Slides were treated with 2 × SSC and 0.5% Igepal pH 7.0 (Sigma-Aldrich), at 37 °C for 15 min, and dehydrated in 70%, 85%, and 100% ethanol consecutively for 1 min each. Cells were denatured in 70% formamide/2 × SSC pH 7.0 (Fisher Scientific) at 72 °C for 2 min, dehydrated, and air-dried. The probe mix RAB9B (XqF1)/WC Y was denatured at 90 °C for 10 min and applied to air-dried slides, and then the slides were placed in a humidified chamber at 37 °C overnight. The slides were washed in 2 × SSC/0.1% Igepal for 2 min at RT and then in 0.4 × SSC/0.3% Igepal for 2 min at 72 °C and in 2 × SSC/0.1% Igepal for 1 min at RT. Fifteen microliters of DAPI at 1:1000 dilutions and Vectashield (10 µL, Vector Labs) were applied, slides were cover-slipped, and images of X or Y chromosomes were captured with a fluorescence microscope (Leica Application Suite X 3.5.5.19976).

Ischemic Stroke Model
Cerebral ischemia was induced by 60-min reversible middle cerebral artery occlusion (MCAO) under isoflurane anesthesia as previously described [18]. Rectal temperatures were maintained at approximately 36.5 ± 0.5 °C during surgery with an automated temperature control feedback system. A midline ventral neck incision was made, and unilateral MCAO was performed by inserting a 6.0-mm monofilament (Doccol Corp., Redlands, CA, USA) into the right internal carotid artery 6 mm from the internal carotid/pterygopalatine artery bifurcation via an external carotid artery stump. Reperfusion was performed by withdrawing the suture 60 min after the occlusion. Regional cerebral blood flow was measured in all stroke animals using Laser Doppler flowmetry. Animals that showed a regional cerebral blood flow reduction by at least 85% from baseline levels during MCAO were included for further experimentation. After MCAO, the mice were administered daily injections of 0.9% sodium chloride, provided with wet mash, and body weight was recorded. Behavioral assessments were performed 72 h after MCAO immediately prior to sacrifice. Sham-operated animals underwent the same surgical procedure, but the suture was not advanced into the middle cerebral artery.

Cresyl Violet and Immunohistochemical Staining
At 72 h after MCAO, the animals were anesthetized with tribromoethanol (Avertin® intraperitoneal injection at a dose of 0.25 mg/g body weight). Animals were perfused transcardially with ice-cold 0.1 M sodium phosphate buffer (pH 7.4) followed by 4% paraformaldehyde (PFA). The brain was removed from the skull and post-fixed for 18 h in 4% PFA and subsequently placed in cryoprotectant solution (30% sucrose). The brain was cut into 30-µm free-floating coronal sections on a freezing microtome, and every eighth slice was stained by cresyl violet (CV) (Sigma, St. Louis, MO, USA) for evaluation of ischemic damage [19]. Immunohistochemical (IHC) staining of fixed-frozen sections was performed as described previously [20]. Briefly, the brain sections were blocked in 0.1 M PBS with 0.25% Triton X-100 (Sigma) and 10% donkey serum for 2 h and incubated overnight at 4 °C with the following primary antibodies: goat anti-Iba1 (1:300; Novus Biologicals), rabbit anti-KDM5C (1:300; Novus Biologicals), and rabbit anti-KDM6A (1:300; Cell Signaling). After washing in TBS + 0.05% Tween 20, the sections were incubated with the indicated secondary antibodies for 1 h. The following secondary antibodies were used: donkey anti-rabbit IgG Alexa Fluor 647 conjugate (1:500; Invitrogen) and donkey anti-goat IgG Alexa Fluor 594 conjugate (1:500; Invitrogen). The nuclei were stained with DAPI (Invitrogen). Ten 63 × fields from each animal were analyzed in the peri-infarct area at the inner boundary zone of the infarct. Double-positive cells were counted manually by an unbiased, blinded investigator using ImageJ software (NIH, version 1.52a), and the cell numbers were normalized to sham groups.

Behavioral Testing
All behavioral tests were performed by a blinded investigator.

Neurological Deficit Scores
Neurological deficit scores (NDSs) were recorded at day 3 after stroke. The scoring system used was as follows: 0, no deficit; 1, forelimb weakness and torso turning to the ipsilateral side when held by tail; 2, circling to affected side; 3, unable to bear weight on affected side; and 4, no spontaneous locomotor activity or barrel rolling as described previously [21,22].

Corner Test
Sensorimotor activity was measured by the corner test 3 days after stroke as described previously [19]. Briefly, the mouse was placed between two pieces of cardboard (size of each: 30 cm × 20 cm × 0.1 cm). The two boards were gradually moved to close the mouse from both sides to encourage the mouse to enter into a corner of 30° with a small opening along the joint between the two boards. When the mouse entered the deep part of the corner, both sides of the vibrissae were stimulated together by the two boards. Animals with cerebral ischemia neglect the damaged side, and rear to the intact side (the right side) when the cardboard stimulates the vibrissae. The number and direction of rears were recorded for 20 trials, and the percentage of right turns was calculated. Only turns involving full rearing along either board were recorded [21].

Y-Maze Test
Spontaneous alternation using a Y-maze is a test for habituation and spatial working memory [23,24]. The symmetrical Y-maze consists of three white opaque plastic arms at a 120° angle from each other. After placing the mouse in the center of the maze, the animal is allowed to freely explore the three arms. Over the course of multiple arm entries, the subject should show a tendency to enter a less recently visited arm. The test consists of a single 5-min trial; spontaneous alternation (%) is defined as consecutive entries in three different arms (arms A, B, and C), divided by the number of total alternations (total arm entries minus 2) [25]. Mice with less than 8 arm entries during the 5-min trial were excluded from the analysis. An entry occurs when all four limbs are within the arm.

Open Field Test
The traveling speed in an open field measures spontaneous locomotor activity in a novel environment [26]. In this task, the total ambulatory activity of the mouse was assessed. The mouse was placed in the open field chamber (15 in. × 15 in.). Traveling speed was quantified by a computer-operated PAS Open Field system (San Diego Instruments, San Diego, CA, USA). Each testing session was 20 min long, and the data was collected in 60-s intervals. All the mice were acclimatized to the dark testing room for 10 min before the beginning of the test, and the activity was recorded immediately after the mice were placed in the open field apparatus.

Statistical Analyses
Data from individual experiments are presented as mean ± SD and assessed by two-way ANOVA with Tukey's post hoc test for multiple comparisons (GraphPad Prism Software, Inc., San Diego, CA, USA) except the NDS, which was analyzed with the Mann-Whitney U test. For two-way ANOVA, significant difference analysis by paired comparisons was conducted with the Holm-Sidak test, whereas the group analysis was compared using Tukey's post hoc correction. Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

The Cerebral Vascular Architecture Was not Different Between Genotypes in Aged XY* Mice
The genotypes of XY* mice were identified by FISH as shown in Fig. 1A, with red dots indicating the X chromosome and green dots representing the Y chromosome. To examine whether all XY* mice have similar large vessel structure, we injected DiI to XY* mice to stain brain vessels [30] and found the circle of Willis is not different between the XY* mice of the four genotypes (Fig. 1B, upper panel). Since stroke outcomes can be affected by the number of anastomoses between vascular territories, we also examined the middle cerebral artery (MCA), anterior cerebral artery (ACA), and posterior cerebral artery (PCA) territories and measured the line of anastomoses [31] (indicated by the distance of the line to the midline of the brain; Fig. 1B, lower panel) between the three territories. No significant difference in the line of anastomoses was found in any of the five measured areas (Fig. 1B, C). We also measured plasma levels of testosterone and estradiol-17β in these aged mice, and there were no significant differences between any of the four genotypes (Fig. 1D, E). These data suggest that the cerebrovasculature in XY* mice is similar between genotypes and that stroke outcomes by the MCAO model in these mice should be comparable.

X Chromosomal Effect Contributes to Stroke Sensitivity
We have previously used the FCG mouse model and found that aged XX mice showed worse stroke outcomes than XY mice with the same type of gonad, either testes or ovaries [8]. In the present study, we sought to determine which sex chromosome contributes to sex differences in stroke outcomes. Aged XY* mice were subjected to a 60-min MCAO; 72 h later, infarct volumes and neurobehavioral deficits were quantified. Comparisons between XX vs. XO and XY vs. XXY were examined for an X chromosomal effect and those between XO vs. XY and XX vs. XXY for a Y chromosomal effect ( Fig. 2A). We found XX and XXY mice had larger infarcts than XO and XY mice, respectively, in the cortex, the striatum, and the entire ipsilateral hemisphere (Fig. 2B). Larger infarcts were also seen in XXY vs. XO mice in all areas, and in XX vs. XY in the striatum and in the ipsilateral hemisphere. Correspondingly, worse behavioral deficits were seen in XXY vs. XY mice in the NDS (Fig. 2C), in the corner test (Fig. 2D), and in the open field test (Fig. 2E). XX mice also had higher NDS than XO mice. No difference was seen between XXY vs. XX and XO vs. XY mice either in the infarct measurement or in any behavior test. To rule out the activational effects of gonadal hormones [32], a cohort of XY* mice was included in which gonadectomy (GDX) was conducted at weaning age (21 days). These mice were subjected to MCAO at 8-12 weeks of age. GDX XX mice showed larger infarct in the ipsilateral hemisphere than XO mice, and XXY mice had larger infarct than XY mice in the striatum (Fig. 2G, H), accompanied by worse neurological deficit scores in XX vs. XO mice (Fig. 2I). Larger infarcts were also seen in XXY vs. XO mice in the striatum and hemisphere (Fig. 2H), but no difference was seen between XXY vs. XX and XO vs. XY mice. Taken together, these data clearly point to an X chromosomal effect in stroke sensitivity.

Microglial Activation Was Seen in Aged Mice with Two Copies of the X Chromosome
Microglial activation plays a pivotal role in initiating and perpetuating inflammatory responses to ischemia [33,34]. Our previous study showed that microglia with different combinations of X and Y chromosome exhibited different activation states [8], suggesting that microglial activation may also be subjected to regulation by X or Y chromosome genes. To elucidate which sex chromosome contributes to the sex difference in microglial activation, we performed flow cytometry on brain samples from XY* mice to examine microglial activation status after stroke. Microglia were gated as CD45 int CD11b + (Sup Fig. 1A). Microglial cell numbers were not significantly different between any two genotypes of XY* mice either in stroke or in sham groups (Fig. 3A). Next, we examined microglial activation markers, pro-inflammatory (TNFα, IL-1β, CD68) and anti-inflammatory (IL-4, IL-10, CD206) cytokines. The gating strategies for these cytokines are shown in Sup Fig. 1B-G. Interestingly, the expression of intracellular cytokines (TNFα, IL-1β, and IL-10) exhibited striking sex differences after stroke except for IL-4 ( Fig. 3B-E). Stroke mice with two copies of X chromosome had significantly higher levels of both TNFα and IL-1β compared to mice with one X chromosome. IL-10 exhibited an opposite pattern, i.e., lower levels were seen in mice with two vs. one X chromosome (Fig. 3E). No sex differences were seen in the cell membrane markers CD68 and CD206 (Fig. 3F, G). The Y chromosome does not seem to play an important role in microglial activation, as no differences in cytokine expression were seen between XO and XY or between XX and XXY mice. No sex differences were seen in sham groups.

Infiltration of Peripheral Immune Cells into the Brain of Aged XY* Mice Differed After Stroke
As shown in Fig. 3, we found an X chromosomal effect on microglial activation. To further evaluate the chromosomal effects on the immune cell response to stroke, we analyzed the infiltration of peripheral leukocytes in the ischemic brains by flow cytometry 72 h after stroke. Total peripheral myeloid cells were gated as CD45 high CD11b + and lymphocytes as CD45 high CD11b − (Sup Fig. A). From the total peripheral myeloid cells, monocytes were further gated as CD45 high CD11b + Ly6C + Ly6G − and neutrophils as CD45 high CD11b + Ly6G + (Fig. 4C). The infiltration of these cells was quantified as the percentage over total leukocytes in the ischemic hemisphere. In general, there were significantly more peripheral myeloid and lymphocytic cells in the ischemic brains of XX vs. XO mice, and XXY mice brains also had more lymphocytes than XY or XO mice (Fig. 4A, B). Neutrophils were significantly elevated in XX vs. XO or in XX vs. XY mice, and more monocytes were seen in XXY vs. XY or in XX vs. XY mice (Fig. 4D, E). Sham groups did not exhibit any sex difference.

Plasma Cytokine Levels in Aged XY* Mice After Stroke
Apart from immune cell inflammatory profiles, we also measured circulating cytokine levels in the plasma of XY* mice after stroke, including pro-inflammatory cytokines (TNFα, IL-1β, and iNOS) and anti-inflammatory cytokines (IL-4, TGF-β1, and IL-10). Sex differences were seen in stroke groups but not in sham mice. XX mice had significantly higher levels of TNFα and iNOS compared to XO or XY mice after stroke, and the same pattern was seen in XXY vs. XY or XO mice (Fig. 5A, C). We did not detect any significant differences in TGF-β1 or IL-10 (Fig. 5E, F). However, the anti-inflammatory cytokine IL-4 level was significantly higher in XY vs. XXY or XX mice. Surprisingly, the pro-inflammatory cytokine IL-1β also showed a similar pattern as IL-4 (Fig. 5D); the reason is unknown. Taken together, data from Figs. 3, 4, and 5 suggested that mice with two copies of X chromosome have an exacerbated immune response to stroke compared to mice with one X chromosome.

Microglial Expression of KDM5C/KDM6A in Aged XY* Mice
Our previous study suggested that two X-chromosome genes (Kdm5c and Kdm6a) that escape from X chromosome inactivation (XCI) in microglia contribute to the sexually dimorphic inflammatory response to ischemia [13]. To better understand sex differences in X escapee genes in microglia after stroke, we evaluated the expression of Kdm5c and Kdm6a in microglia from XY* mice after ischemia. IHC was performed on brain slices from XY* mice, and Iba1 fluorescence signal was co-labeled with antibodies against KDM5C or KDM6A. The ratio of double-positive cells (KDM5C + Iba1 + or KDM6A + Iba1 + ) to total Iba1 + cells was averaged from 8 peri-infarct areas from each mouse (Fig. 6A), and the mean values of each strain were compared. The results showed that in sham groups, both KDM5C and KDM6A ratios were significantly higher in XX or XXY vs. XO mice. However, after stroke, the second X chromosomal effect was seen in XX or XXY vs. XY mice (Fig. 6B, C, D, E). These data suggested that ischemic insult can impact microglial expression of the two XCI escapee genes.

Discussion
The present study used the XY* mouse model to examine the effect of sex chromosomes on stroke sensitivity, and several important findings were revealed. Firstly, the second X chromosome (but not the Y chromosome) contributes to sex differences seen in stroke. This effect is independent of the acute activational effect of gonadal hormones as the same sex differences were seen in the absence of gonadal hormones in a cohort of surgically gonadectomized mice. Secondly, sex differences in the inflammatory response to stroke are also impacted by the chromosome compliment. Significantly higher post-stroke inflammation was seen in mice with two vs. one X chromosome. Similar patterns were seen in the activation of microglia, the infiltration of peripheral immune cells in the ischemic brains, and the level of circulating cytokines. Thirdly, two X chromosome genes named Kdm5c and Kdm6a are more highly expressed in microglia having two vs. one X chromosome both before and after stroke. Based on our knowledge, this is the first study that specifically investigated the contribution of the second X chromosome to stroke sensitivity and post-stroke inflammation. This has further extended our understanding of the biology and etiology of sex differences in stroke.
Our previous studies have recapitulated the "aged female sensitive" stroke phenotype seen in stroke patients. Aged female mice have larger infarcts and worse neurobehavioral deficits compared to age-matched males after MCAO [5]. The conclusion was further confirmed in another study using aged FCG mice that also showed one copy of X chromosome [8]. However, these prior studies only suggested that sex chromosome contributes to the stroke sensitivity, but could not differentiate the effects of the X chromosome from that of the Y chromosome. The present study replicates and extends our previous finding in FCG mice by using the XY* mouse model. This model is designed to specifically examine the effect of an X or Y chromosome on stroke sensitivity by comparing XX vs. XO and XY vs. XXY (for X effect) or XX vs. XXY and XO vs. XY (for Y effect). Our data (Fig. 2) have clearly shown the Y chromosome has no effect on the stroke sensitivity, but the X chromosome does. We primarily used aged XY* mice as stroke mainly affects the elderly, and this also allowed us to minimize the effect of circulating hormones, as gonadal hormone levels were equivalently low in aged XY* mice of all four genotypes. However, hormonal activational effects [32] at puberty may still impact stroke outcomes. Therefore, we included a cohort of mice that were gonadectomized at weaning age to eliminate the activational effects of gonadal hormones. Similar sex differences as those seen in aged mice remained in GDX mice, i.e. XX and XXY mice had worse outcomes compared to XO and XY, respectively (Fig. 2G-I), suggesting the detrimental X chromosomal effect on stroke outcomes is independent of the activational effects of gonadal hormones. We cannot exclude organizational effects of hormonal steroid that occur before or after birth and are permanent [35]. Nevertheless, the consistency of the data in both our aged and GDX young cohorts strongly indicates a deleterious effect of X chromosomal dosage to stroke sensitivity. Some reports suggest that the Y chromosome plays a pivotal role in various diseases [36][37][38][39][40]. However, the present study suggests that Y chromosome does not contribute to stroke sensitivity. Nevertheless, the contribution of Y chromosome to the stroke sensitivity is not conclusive and warrants further investigation.
Microglia are the brain resident immune cells and are one of the first immune cells activated after brain injury [41]. Microglial activation after stroke has been increasingly recognized as a key element in initiating and perpetuating post-stroke inflammation [42][43][44]. Our data suggested that both pro-inflammatory (TNFα, IL-1β) and anti-inflammatory (IL-10) activations in microglia are also regulated by the second X chromosome (Fig. 3), and this effect extends to effects on peripheral immune cell infiltration (Fig. 4) and circulating cytokine levels. The immune responses to stroke are both causative and resultant to the ischemic injury [45] and lead to secondary neuronal death after stroke [46]. Studies by our laboratory and that of other groups have consistently revealed sex differences in the immune responses to stroke [13,27,[47][48][49][50]. The present study has further determined that X chromosomal effects could be the driving force for sex differences in post-stroke inflammation. The immune system has long been known to develop in a sexually dimorphic manner that results in a sex bias in infectious diseases, autoimmune and inflammatory diseases, and cancer [51]. Our data suggest that there is a mechanistic link between X chromosomal genes and sex differences seen in the inflammatory response to stroke.
The XX vs. XY differences in post-stroke inflammation and outcomes in aged mice could be explained by inherent differences in XX and XY cells. Our interest focuses on a group of genes that escape the XCI. Normally, males and females differ in sex chromosome content (XY vs. XX) and X chromosome imbalance is tolerated because of dosage compensation by XCI. The process of XCI reduces the expression of genes on one of the two X chromosomes randomly in each XX cell, bringing the dose of expression in XX cells to within the range of XY cells [52,53]. However, some X genes escape inactivation and are expressed by both X chromosomes, so that expression is constitutively higher in XX than XY cells [54]. We have previously found two X escapee genes, Kdm5c and Kdm6a, have higher expression in aged female vs. male microglia. KDM5C and KDM6A are histone demethylases of H3K4me3 and H3K27me3, respectively [55,56]. Demethylation of H3K4me3 and H3K27me3 by KDM5C and KDM6A to their me1 forms confers a repressive and activational effect on gene expression, respectively [57,58]. Since Kdm5c and Kdm6a escape XCI, the demethylation levels of the two histones are different between males and females, which might lead to sex-specific, epigenetic modification of inflammatory genes. Our previous study suggested that two important interferon regulatory factors (IRFs), IRF4 and IRF5, are targets of these KDMs [13], and IRF4/IRF5 is the key determinant of microglial anti-and pro-inflammatory activation, respectively [21,22,59,60]. The present study confirmed higher expression of these two KDMs in microglia having two vs. one X chromosome (Fig. 6), although our primary focus was on the determination of X vs. Y chromosomal effects on stroke sensitivity and post-stroke inflammation. Ongoing studies in the lab are using a conditional knockout mouse model in which Kdm5c or Kdm6a is specifically deleted in microglia, to investigate the molecular mechanistic link between the KDMs and IRFs.
The present study has several limitations that should be kept in mind when interpreting the data. For neuroinflammation profiles, we measured cytokine levels after stroke only in the plasma but not in the brain tissue due to the limited numbers of aged mice, and plasma cytokine concentrations may not reflect inflammatory events in the brain. However, the plasma cytokine data showed the similar X chromosomal effect as in microglial activation and peripheral leukocyte infiltration in the ischemic brain (Figs. 3 and 4). All experiments were performed at an acute time point post stroke, i.e., at 3 days after MCAO. Experiments at chronic phases to examine the long-term stroke outcome and neuroinflammation are warranted.
In summary, the present study employed the XY* mouse model and investigated the role of sex chromosomes in ischemic stroke outcomes and inflammation. We revealed that the number of X chromosomes determines the stroke sensitivity in aged mice and these effects are not attributable to circulating hormone levels. Microglial activation and the inflammatory responses to stroke are also impacted by the second X chromosome, which might be a driving source for the sex differences seen in aged stroke. The Y chromosome seems to play a minimal role in mediating stroke sensitivity, and X escapee genes likely initiate a downstream mechanistic cascade that leads to the sex differences in stroke.