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. Author manuscript; available in PMC: 2019 Jul 1.
Published in final edited form as: Stroke. 2018 Jun 4;49(7):1701–1707. doi: 10.1161/STROKEAHA.118.020627

Inhibition of miR-141-3p ameliorates the negative effects of post-stroke social isolation in aged mice

Rajkumar Verma 1, Rodney M Ritzel 1, Nia M Harris 1, Juneyoung Lee 2, TaeHee Kim 3, Gopal Pandi 3, Raghu Vemuganti 3,4, Louise D McCullough 1,2,5
PMCID: PMC6019570  NIHMSID: NIHMS965520  PMID: 29866755

Abstract

Background and Purpose

Social isolation increases mortality and impairs recovery after stroke in clinical populations. These detrimental effects have been recapitulated in animal models, although the exact mechanism mediating these effects remains unclear. Dysregulation of microRNAs (miRNAs) occurs in both stroke as well as after social isolation, which trigger changes in many downstream genes. We hypothesized that miRNA regulation is involved in the detrimental effects of post-stroke social isolation in aged animals.

Methods

We pair-housed 18-months-old male C57BL/6 male mice for two weeks prior to a 60-minute right middle cerebral artery occlusion or sham surgery and then randomly assigned mice to isolation or continued pair-housing immediately after surgery. We sacrificed mice either at 3, 7, or 15 days after surgery and isolated the perilesional frontal cortex for whole microRNAome (miRNAome) analysis. In an additional cohort, we treated mice one day after stroke onset with an ‘in vivo-ready’ inhibitor of miR-141-3p (antagomiR-141-3p) for three days.

Results

Using whole miRNAome analysis of 752 miRNAs, we identified miR-141-3p as a unique miRNA that was significantly upregulated in isolated mice in a time-dependent manner up to two weeks after stroke. Post-treatment with an antagomiR-141-3p reduced the post-isolation-induced increase in miR-141-3p to levels almost equal to those of pair-housed stroke controls. This treatment significantly reduced mortality (by 21%) and normalized infarct volume and neurological scores in post-stroke isolated mice. Quantitative PCR analysis revealed a significant up regulation of neuroprotective genes including transforming growth factor beta receptor 1 (Tgfβr1, a direct target of miR-141-3p) and insulin-like growth factor 1 (Igf-1) after treatment with antagomiR. Treatment also increased the expression of other pleiotropic cytokines such as interleukin 6 (Il-6) and tumor necrosis factor alpha (Tnf-α, an indirect or secondary target) in brain tissue.

Conclusions

miR-141-3p is increased with post-stroke isolation. Inhibition of miR-141-3p improved mortality, neurological deficits, and decreased infarct volumes. Importantly, these therapeutic effects occurred in aged animals, the population most at risk for stroke and post-stroke isolation.

Keywords: microRNA, stroke, neuroprotection, social isolation, miR-141-3p

Introduction

Emerging evidence from experimental and clinical studies suggests that social isolation is not only a risk factor for a stroke, but also contributes to increased stroke severity and delayed functional recovery1–3. Exacerbation of inflammation and a reduction in pro-survival growth factors mediate the detrimental effects of post-stroke social isolation3–5. Social interaction can overcome these negative effects by promoting adaptive behaviors and favorable neuroendocrine responses to biological stressors6. Social isolation is particularly relevant to the elderly as this population has a high risk of both strokes and isolation3, 7. We have previously found that aged mice that were socially isolated after stroke do not recover completely and exhibit continued deficits in memory/motor function and elevation in inflammation even months after ischemic injury3. Given the complex pathophysiology and additional contribution of aging and social isolation, there is a critical need to concurrently target multiple effector pathways involved in stroke pathology.

miRNAs are a class of small endogenously expressed non-coding RNAs that regulate gene transcription and/or translation to orchestrate mRNA and protein expression8. The role of miRNAs in stroke has been a subject of increasing interest since the first miRNA expression profiling study in cerebral ischemia was performed in 20089. Post-stroke recovery affects several miRNAs including miR-129, miR-141, miR-181a-d, and miR-200c, and treatment with mimics or antagomiRs of these miRNAs reduce injury and improve chronic behavioral recovery in young mice10. Similar studies are lacking in aged animals. miRNAs also mediate many aspects of social interaction. Social environments can directly influence miRNA expression, which then triggers expression of a plethora of downstream genes. For example, miR-124-5p is involved in social and behavioral deficits in frontotemporal dementia11, miR-200c in major depressive disorder12, and miR-181c-5p in the social withdrawal associated with autism13.

Post-stroke inflammation plays a critical role in stroke injury and recovery, which is mainly initiated by rapid activation of microglia5, 14, 15. Advanced age and social isolation enhance microglia-mediated inflammation either by disturbing the homeostatic balance between “pro-inflammatory” and “anti-inflammatory/reparative” cytokine secretion and/or by reducing its scavenger functions (e.g., phagocytosis), contributing to post-stroke pathophysiology16. Interestingly, several miRNAs play critical regulatory roles in microglial activation and function17. This led us to hypothesize that targeted manipulation of miRNAs, which concurrently regulate multiple effector pathways, prevent the detrimental effect of post-stroke social isolation on stroke recovery by altering the microglial response.

Material and methods

Data supporting the findings of this study are available from the corresponding or first author (raverma@uchc.edu) of the manuscript on reasonable request.

Materials

We purchased miRNA primers, Invivofectamine 3.0, antagomiR-141-3p, mirVana™ miRNA Inhibitor negative Control (or antagomiR negative control) and other qPCR related supplies for in-house miRNA analysis from Ambion, Life Technologies, (Camarillo, CA). Other lab chemicals and reagents were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Experimental animals

The Institutional Animal Care and Use Committee at the University of Connecticut approved all animal protocols, which we performed in accordance with National Institutes of Health guidelines. Aged C57BL/6 male mice (18–20 months old; 40 ± 2 g; National Institute on Aging, Bethesda, MD) were acclimatized for at least two months in the animal care facility at ambient temperature and humidity with free access to food and water. Of those 144 mice that were pair-housed for three weeks (2 mice/cage) with a daily compatibility examination (e.g., weight gain and absence of fight wounds), 10 pairs of mice were excluded due to incompatibility. Thus, 124 mice were randomly assigned to stroke or sham surgery. Immediately after surgery, we randomly assigned mice to one of three groups, as detailed previously3: Stroke and sham pair-housed (ST-SH) consisting of one stroke (called as ST-PH) and one sham mouse (called as SH-PH), stroke isolated (ST-ISO), and sham isolated (SH-ISO). The assigned housing conditions were maintained until sacrifice. If a mouse died in the pair-housed group, we excluded the partner from the study. A total of 92 surviving mice were used for the final analysis.

We conducted experiments in four separate cohorts: (1) sub-acute survival group for initial miRNAome assay at day 15, (2) post-stroke three and seven-day survival groups for temporal expression profiling of the miRNA, (3) in vivo treatment group for efficacy and target validation and (4) in vivo treatment group for neuroprotection (infarct volume and Neurological deficit score) analyses. Supplementary table II summarizes the number of mice used in each group of the four experimental cohorts.

Middle cerebral artery occlusion

To induce focal transient cerebral ischemia, a midline ventral neck incision was made under isoflurane anesthesia, and a 60-minute unilateral right middle cerebral artery occlusion (MCAo) was performed by advancing a 6.0 silicone rubber-coated monofilament (Doccol Corporation, Sharon, MA) 10–11 mm from the internal carotid artery bifurcation via an external carotid artery stump5. We monitored rectal temperatures (Fine Science Tools, Foster City, CA) and maintained animals at ~37°C with an automatic heating system. We used laser Doppler flowmetry (DRT 4, Moor Instruments, Devon, UK) to measure cerebral blood flow to confirm occlusion (i.e., reduction to 15% of baseline cerebral blood flow) and reperfusion. All mice were fed wet mash for 1 week after surgery to ensure adequate nutrition for chronic endpoints. In sham mice, we performed an identical surgery but did not advance the suture into the internal carotid artery. The reperfusion period was 3, 7, or 15 days.

Neurological deficit score

The neurological deficit (ND) score is an assessment of neurological impairments during post-stroke recovery. At several time points after stroke, investigators blinded to housing conditions recorded ND scores as follows5: 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.

Sample preparation and RNA isolation

We extracted total RNA from the perilesional ipsilateral cortex (Supplementary figure I) of stroke mice using miRNeasy Mini kits (Qiagen, Germantown, MD) for miRNAome analysis or mirVana miRNA isolation kits (Thermo Fisher Scientific, Waltham, MA) for other analyses, according to the suppliers’ protocols. We stored RNA at −80°C.

Real-time PCR of microRNA

We reverse transcribed 50ng RNA in 50µl reactions using the miRCURY LNA universal real-time (RT) microRNA complementary DNA (cDNA) system (Exiqon, Woburn, MA). We diluted the resulting cDNA (1:100) and assayed it in the microRNA ready-to-use PCR mouse and rat panel I and II (Exiqon) with the ExiLENT SYBR green master mix (Exiqon), according to the manufacturer’s protocol. We performed the amplification in a LightCycler 480 PCR System (Roche, Basel, Switzerland), and analyzed the data using the quantification cycle (Cq) method (Light Cycler software, Roche). For in-house miRNA analysis, we used the TaqMan reverse transcription kit (Thermo Fisher Scientific) and the TaqMan universal PCR master mix (Thermo Fisher Scientific), according to the manufacturer’s protocols.

miRNAome data analysis

We calculated the amplification efficiency using algorithms similar to LinReg software with Cq as the second derivative. We detected an average of 421 miRNAs per sample. To be included in the analysis, the assays were required to be detected with 5 Cqs less than the negative control and with Cq<37; for the Cq value of the global mean for each of the samples, see Supplementary Figure II. We normalized all data to the average of assays detected in all samples, which NormFinder software found to be the best normalizer.

Cresyl violet staining for infarct volume and tissue atrophy analysis

We measured tissue infarct volume after stroke as described previously5. Briefly, mice were sacrificed seven days after stroke surgery with an overdose of avertin (250 mg/kg by intraperitoneal injection). After blood collection by cardiac puncture, mice underwent trans-cardiac perfusion using cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Brains were fixed overnight, placed in cryoprotectant (30% sucrose in PBS) for 72 hrs, and cut into 30µm free-floating sections using a freezing microtome. Every eighth slice was mounted, stained with cresyl violet, and used for infarct volume calculations as described previously3.

Statistical analysis

We present data from individual experiments as mean ± SD. We statistically evaluated the data by the Student’s t-test (for comparison between two experimental groups) or by one- or two-way (housing condition and surgery as variables) analysis of variance (ANOVA) with a Bonferroni post-hoc test to correct for multiple comparisons (GraphPad Prism Software Inc., San Diego, CA). As ND scores are ordinal in nature, we used the Mann-Whitney U test/Kruskal Wallis test for statistical analysis for these experiments. A probability value of p<0.05 was considered statistically significant. An investigator blinded to the experimental groups performed the data analyses.

Results

MiRNAome analysis identifies miRNA leads involved in post-stroke social isolation (SI)

To examine the contribution of miRNAs to the detrimental effects of social isolation after stroke, we performed whole miRNAome analysis in aged mice subjected to ischemic stroke. After 15 days of reperfusion, we isolated RNA from perilesional ipsilateral brain tissue (collected as a 2 mm coronal section, from bregma 0.00 to 2.00mm) for whole miRNAome analysis. See figure 1 for a schematic of our approach. Using sham pair-housed mice as controls, we found several differentially expressed miRNAs across conditions (>2 fold up or down regulation, p<0.05).. Comparative analysis for the effect of surgery (stroke or sham) and housing condition (pair-housed or isolated) by a two-way analysis determined the number of miRNAs affected by surgery, housing or both conditions (p<0.05). Further validation by RT-qPCR brought our list down to four lead miRNAs that were modulated by post-stroke SI. Direct comparison between ST-PH (as control group) and ST-ISO confirmed our miRNAome finding (Figure 2 A &B)

Figure 1.

Figure 1

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Schematic of research design for (A) miRNA identification and (B) miRNA validation.

Figure 2.

Figure 2

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Shows lead miRNAs after post-stroke social isolation (A) Venn diagram illustrating differentially regulated miRNAs in the brains of aged mice during the stroke, social isolation (housing condition) and their overlap. (B) List of differentially regulated brain miRNAs, their p values, and their fold changes in aged male mice subjected to post-stroke pair-housed mice (ST-PH) compared with post-stroke social isolation (ST-ISO).

MiR-141-3p expression progressively increases after stroke

Among the four selected miRNAs, we were interested in miRNAs that showed a persistent increase or decrease in expression at different time points of ischemic/reperfusion (I/R) injury. We performed a similar experiment as above, using cohorts that underwent either three or seven days of reperfusion prior to RNA isolation. Among the four leads, miR-141-3p showed a progressive increase in expression (Figure 3A).

Figure 3.

Figure 3

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A) Temporal profile of miR-141-3p expression in mice subjected to post-stroke social isolation, as compared with their respective pair-housed controls. Data are expressed as mean±SD. (n=4 per group/time point; *p<0.05; ST-PH vs. ST-ISO, one-way ANOVA). B) AntagomiR-141-3p reduces miR-141-3p expression levels in socially isolated stroke mice. MiR-141-3p levels were higher in the brain following MCAo; antagomiR-141-3p reduced miR-141-3p levels, measured at seven days after stroke. Data are expressed as mean±SD and values are presented as fold change in gene expression of post-stroke socially isolated [NC (antagomiR negative control) and antagomiR-141] mice at day 7 against pair-housed stroke mice (#p<0.05 ST-PH vs. ST-ISO NC; *p<0.05 ST-ISO NC vs. ST-ISO antagomiR; n=8, n=6, and n=7 for ST-PH/SH-PH, ST-ISO NC, and ST-ISO antagomiR-141, respectively).

MiR-141-3p targets pathways involving cytokines and MAPK pathway genes in socially isolated mice after stroke

To identify the pathways that our lead miRNAs target, we used DIANA-mirPath vs. software and the microT-CDS database18. This approach identifies mRNA targets of input miRNAs within a KEGG pathway. This analysis predicted a total of 22 pathways significantly affected by the combined effect of the at least three out four lead miRNAs (Supplementary Figure IIIA). Upon performing a similar analysis for miR-141-3p, we identified four major biological functional categories (Supplementary Figure III B). Among these four, two pathways viz. gap junction and axon guidance pathways involve several cytokines and MAPK signaling pathway genes, which play a role in microglia-mediated inflammation and its resolution. This finding suggested that miR-141-3p is a strong candidate as a regulator of several common genes seen with post-stroke social isolation.

Inhibiting miR 141-3p improves stroke outcomes in socially isolated mice

We next examined the effects of inhibiting miR-141-3p on stroke outcomes in socially isolated mice. We treated mice with an antagomiR-141-3p (7 mg/kg, intravenously, i.v.) for three days starting 24 hrs after MCAo. We first validated the successful delivery and efficacy of the treatment by measuring its target gene expression. Indeed, following the treatment protocol, miR-141-3p levels reduced to almost 50% of the control levels, nearly equivalent to the pair-housed surgery controls (Figure 3B). After validating the in vivo efficacy of the antagomiR-141-3p, we assessed its effects on infarct damage and ND score. At 3 days post-treatment with antagomiR, the social isolation-induced increase in infarct volume and ND score were abolished (Figure 4A–C). Furthermore, antagomiR-141-3p treatment reduced mortality by 20% in post-stroke isolated mice at 7 days after stroke (Figure 4D).

Figure 4.

Figure 4

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AntagomiR-141-3p improves stroke recovery in socially isolated mice. (A) Representative coronal section of ST-PH, ST-ISO NC, and ST-ISO 141 anta-treated brains stained with cresyl violet. (B) AntagomiR treatment reduced infarct volume in post-stroke socially isolated mice to levels comparable to pair-housed stroke controls (#p<0.05 ST-PH vs. ST-ISO NC; *p<0.05 ST-ISO NC vs. ST-ISO antagomiR-141 ;). (C) Similarly, antagomiR treatment reduced the neurological deficit (ND)score to levels comparable to pair-housed stroke controls at day 7 of stroke (#p<0.05 ST-PH vs. ST-ISO NC; *p<0.05 ST-ISO NC vs. anatgomiR-141; Kruskal-Wallis Test, n=10, n=6, and n=7 for ST-PH/SH-PH, ST-ISO NC, and ST-ISO antagomiR-141, respectively). (D) Kaplan Meier survival curve shows that antagomiR-141 treatment reduced post-stroke mortality by 21% compared with the negative control (ST-ISO NC; *p<0.05) in socially isolated mice at day 7 after stroke.

miR-141-3p inhibition increases the expression of neuromodulator cytokines

We previously found that post-stroke social isolation increases M1-type ‘pro-inflammatory’ gene expression and reduces M2-type ‘anti-inflammatory’ gene expression5. Therefore, we hypothesized that antagomiR-141-3p treatment would increase several neuroprotective and anti-inflammatory gene targets of miR-141-3p. Indeed, several neuroprotective and anti-inflammatory genes such as Tgfβr1, a receptor for neuroprotective cytokine Tgfβ1 and Igf-1 were significantly upregulated in brain tissue from socially isolated stroke mice after miR-141-3p treatment (Figure 5). Besides the above, mRNA levels of Il-6, the most implicated cytokine in post-stroke social isolation, and Tnf-α were also increased by antagomiR treatment. Together, these data indicate that neuroprotective effect of antagomiR-141-3p might be mediated by increased expression of several neuromodulatory genes, including both direct (Tgfβr1, supplementary figure IV) and indirect or secondary miRNA targets (Il-6, Tnf-α, and Igf-1) of miR-141-3p.

Figure 5.

Figure 5

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Effect of antagomiR-141-3p treatment on anti-inflammatory gene targets. Bar graphs show that post-stroke social isolation decreases the expression of genes like transforming growth factor beta receptor 1 (Tgfβr1), insulin-like growth factor 1 (Igf-1), interleukin 6 (Il-6) and tumor necrosis factor alpha (Tnf-α) as compared with ST-PH mice. AntagomiR treatment reinstated the expression of the anti-inflammatory gene in post-stroke isolated male mice at 7 days after stroke. Data are expressed as mean±SD and values on the Y-axis were presented as fold change in target gene expression of post-stroke socially isolated mice (ST-ISO NC and ST-ISO antagomiR-141) mice at day 7 mice against pair-housed control stroked mice (ST-PH). ST-PH mRNA values were kept constant at 1 in determining fold change in respective genes expression of other groups (*p<0.05 ST –ISO NC vs. ST-ISO antagomiR-141; n=8, n=6, and n=7 for ST-PH/SH-PH, ST-ISO NC, and ST-ISO antagomiR-141, respectively).

Discussion

Social isolation is associated with increased mortality and morbidity in patients with the established vascular disease, including stroke. Experimentally, post-stroke social isolation increases ischemic damage and infarct volume in both young and aged mice3, 5, 19. Given that the deleterious effects of social isolation are mediated by post-stroke inflammation and that miRNAs are upstream of select inflammatory genes, we sought to examine the contribution of miRNAs to post-stroke social isolation. We identified several miRNAs, including some previously linked to recovery after stroke or social interaction (i.e., miR-181c-5p, miR-200c-3p, miR-141-3p, and miR-124-5p), that were modulated in post-stroke, socially isolated, aged mice. Among these, miR-141-3p expression increased consistently after stroke over a period of two weeks. An in vivo-ready antagomiR-141-3p was able to ameliorate the detrimental effects of social isolation following a stroke. Moreover, our data suggest that the neuroprotective effects of antagomiR-141-3p might be mediated by several neuromodulatory cytokines and growth factors.

Several in vivo and in vitro studies have reported dysregulation in miRNA expression after stroke10, 20, 21. miRNAs can regulate target genes directly through interactions with both conserved and non-conserved target recognition elements, which can lead to both decrease and increase in transcript abundance22. Researches have shown that miRNAs can be induced by proinflammatory stimuli23 and can also induce pro-inflammatory responses24. The detrimental effects of post-stroke social isolation are driven by several factors, including an enhanced inflammatory response, reduced anti-inflammatory cytokines and growth factors, changes in various gene expression that control long-term potentiation and long-term depression, as well as M1/M2-type pro- and anti-inflammatory switching3, 5, 25. With age, inflammatory cascades initiate more swiftly and aggressively after stroke in isolated mice compared with pair- housed mice3.

Among the 752 miRNAs evaluated in our study, two members (miR-200c-3p and miR-141-3p) of the miR-200 family were highly upregulated in mice subjected to post-stroke social isolation. We focused our attention on miR-141-3p due to the persistent increase in its expression for two weeks after stroke in isolated mice and its known role in the regulation of pleiotropic cytokines including Il-6, and Tnf-α26. Exacerbation of microglia activation and subsequent neuroinflammation mediate the detrimental effects of both social isolation4, 5, 15 and aging14, 27, 28 in stroke. We found that antagomiR-141-3p treatment reversed the reduced mRNA expression of several cytokines and cytokine receptors, including, Igf-1, Il-6, Tnf-α, and Tgfβr1 that were seen in isolated animals. Among these four genes Tgfβr1 and Igf-1 are implicated in inhibition of inflammation and homeostasis29. Given that Tgfβr1 is a direct target of miR-141-3p and Tgfβ family genes modulates secretion and activity of many other cytokines including Il-6, Tnf-α, and various other interleukins30 the neuroprotective of miR-141-3p may be mediated by Tgfβ signaling. Besides the Tgfβ family, increased microglial Igf-1 expression also promotes recovery following an ischemic injury31 possibly by increasing neurogenesis32 which is consistent with observed neuroprotection seen in present work. Increases in both Igf-1 and Tgfβr1 from stroke patient-derived endothelial colony-forming cells promote proliferation and differentiation of these cells to form mature endothelial cells and lead to vascular repair33, and is consistent with a potential neuroprotective role after stroke. Il-6 and Tnf-α were also increased in brain tissue. Although, Il-6 and Tnf-α are generally considered pro-inflammatory cytokines and are markers of injury in plasma/peripheral tissues, their role in the brain is more complex and diverse, and is dependent on region and timing of expression30. For example, Il-6 has been widely implicated in post-stroke social isolation, and Il-6 levels drop in the brain and increase in the plasma after isolation. Importantly, blocking the isolation-induced loss of brain Il-6 leads to improved outcome after stroke, suggesting that Il-6 signaling differs in the brain vs. the peripheral tissues3, 25. Il-6 also influences the balance between M1 and M2 type phenotype of microglia and macrophages34 and is an important determinant of alternative activation35. miR-141-3p inhibition by its antagomiR restored the decreased expression of Il-6 in the brain tissue of socially isolated mice, suggesting a possible indirect interaction between miR-141-3p and Il-6 as seen by others26. This suggests that miR-141-3p might be an indirect regulator for Il-6 as there was no direct miR-141-3p binding site at the 3′-UTR of Il-6. Tnf-α has a more complicated role in inflammation, as it has both pro- and anti-inflammatory effects36, 37, 38. A positive correlation between increased Il-6 and Tnf-α and neuroprotection after 7 days of stroke in aged mice suggest that the response of these cytokines might differ with age, tissue type, and duration of injury. The majority of experimental studies which have correlated increase inflammation/injury with elevated Tnf-α and Il-6 were conducted at acute time points (<3 days) in serum/plasma sample of young mice after stroke18, 37, 39. However, our study used aged mice and examined cytokine profiles in brain tissue at 7 days after stroke, a time point where the peak inflammatory phase is resolving and the resolution of inflammation occurs37, 40. The temporal profile of any cytokine expression is likely a key factor in the response41. Moreover, it is also becoming increasingly clear that cytokine responses and their downstream effects are altered with aging42. The potential also exists that the neuroprotection is mediated in a cytokine-independent manner. In sum, our findings suggest the antagomiR-141-3p treatment ameliorates the detrimental effects of social isolation and that restoration of Tgfβr1 may contribute to this effect. Future studies will examine this pathway in aged female mice, as social isolation may differentially affect females19, but will require additional miRNAome analysis to identify targets.

In conclusion, using miRNAome analysis, we identified miR-141-3p as a unique miRNA that is significantly upregulated in a time-dependent manner up to two weeks after stroke in isolated mice. Systemic treatment with an ‘in vivo, ready’ antagomiR of miR-141-3p at a clinically relevant time point reduced the isolation-induced increase in miR-141-3p to levels almost equal to that of pair-housed stroke controls. Post-treatment with this antagomiR significantly reduced mortality and infarct size after stroke, and increased the expression of neuroprotective Tgfβr1, a direct target of miR-141-3p, and modulated the responses of other cytokines implicated in social isolation. Overall, our data suggest a potential role for miR-141-3p in post-stroke isolation and suggests that the time window for intervention is wide. Importantly, these therapeutic effects occurred in aged animals, the population most at risk for stroke and post-stroke isolation.

Supplementary Material

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Inhibition of miR-141-3p ameliorates the negative effects of post-stroke social isolation in aged mice
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Acknowledgments

Sources of Funding

This work was supported by an American Heart Association grant (14POST20380612 to R. Verma) and the NINDS (NSO55215 and 1R01NS096493-01A1 to L.D. McCullough and RO1 NS099531 and RO1 NS101960 to R. Vemuganti).

Footnotes

Conflict(s)-of-Interest/Disclosure(s): None declared

References

  • 1.Henderson KM, Clark CJ, Lewis TT, Aggarwal NT, Beck T, Guo H, et al. Psychosocial distress and stroke risk in older adults. Stroke. 2013;44:367–372. doi: 10.1161/STROKEAHA.112.679159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Perissinotto CM, Stijacic Cenzer I, Covinsky KE. Loneliness in older persons: A predictor of functional decline and death. Archives of internal medicine. 2012;172:1078–1083. doi: 10.1001/archinternmed.2012.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Verma R, Harris NM, Friedler BD, Crapser J, Patel AR, Venna V, et al. Reversal of the detrimental effects of post-stroke social isolation by pair-housing is mediated by activation of bdnf-mapk/erk in aged mice. Scientific reports. 2016;6:25176. doi: 10.1038/srep25176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Craft TK, Glasper ER, McCullough L, Zhang N, Sugo N, Otsuka T, et al. Social interaction improves experimental stroke outcome. Stroke. 2005;36:2006–2011. doi: 10.1161/01.STR.0000177538.17687.54. [DOI] [PubMed] [Google Scholar]
  • 5.Verma R, Friedler BD, Harris NM, McCullough LD. Pair housing reverses post-stroke depressive behavior in mice. Behavioural brain research. 2014;269:155–163. doi: 10.1016/j.bbr.2014.04.044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Beery AK, Kaufer D. Stress, social behavior, and resilience: Insights from rodents. Neurobiology of stress. 2015;1:116–127. doi: 10.1016/j.ynstr.2014.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Steptoe A, Shankar A, Demakakos P, Wardle J. Social isolation, loneliness, and all-cause mortality in older men and women. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:5797–5801. doi: 10.1073/pnas.1219686110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed rnas. Science. 2001;294:853–858. doi: 10.1126/science.1064921. [DOI] [PubMed] [Google Scholar]
  • 9.Jeyaseelan K, Lim KY, Armugam A. Microrna expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke. 2008;39:959–966. doi: 10.1161/STROKEAHA.107.500736. [DOI] [PubMed] [Google Scholar]
  • 10.Ouyang YB, Stary CM, Yang GY, Giffard R. Micrornas: Innovative targets for cerebral ischemia and stroke. Current drug targets. 2013;14:90–101. doi: 10.2174/138945013804806424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Gascon E, Lynch K, Ruan H, Almeida S, Verheyden JM, Seeley WW, et al. Alterations in microrna-124 and ampa receptors contribute to social behavioral deficits in frontotemporal dementia. Nature medicine. 2014;20:1444–1451. doi: 10.1038/nm.3717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Belzeaux R, Bergon A, Jeanjean V, Loriod B, Formisano-Treziny C, Verrier L, et al. Responder and nonresponder patients exhibit different peripheral transcriptional signatures during major depressive episode. Translational psychiatry. 2012;2:e185. doi: 10.1038/tp.2012.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Hicks SD, Middleton FA. A comparative review of microrna expression patterns in autism spectrum disorder. Frontiers in psychiatry. 2016;7:176. doi: 10.3389/fpsyt.2016.00176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ritzel RM, Patel AR, Grenier JM, Crapser J, Verma R, Jellison ER, et al. Functional differences between microglia and monocytes after ischemic stroke. Journal of neuroinflammation. 2015;12:106. doi: 10.1186/s12974-015-0329-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Venna VR, Xu Y, Doran SJ, Patrizz A, McCullough LD. Social interaction plays a critical role in neurogenesis and recovery after stroke. Translational psychiatry. 2014;4:e351. doi: 10.1038/tp.2013.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Koellhoffer EC, McCullough LD, Ritzel RM. Old maids: Aging and its impact on microglia function. International journal of molecular sciences. 2017;18 doi: 10.3390/ijms18040769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Brites D, Fernandes A. Neuroinflammation and depression: Microglia activation, extracellular microvesicles and microrna dysregulation. Frontiers in cellular neuroscience. 2015;9:476. doi: 10.3389/fncel.2015.00476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lambertsen KL, Biber K, Finsen B. Inflammatory cytokines in experimental and human stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2012;32:1677–1698. doi: 10.1038/jcbfm.2012.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Venna VR, Weston G, Benashski SE, Tarabishy S, Liu F, Li J, et al. Nf-kappab contributes to the detrimental effects of social isolation after experimental stroke. Acta neuropathologica. 2012;124:425–438. doi: 10.1007/s00401-012-0990-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Bhalala OG, Srikanth M, Kessler JA. The emerging roles of micrornas in cns injuries. Nature reviews. Neurology. 2013;9:328–339. doi: 10.1038/nrneurol.2013.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Li Y, Mao L, Gao Y, Baral S, Zhou Y, Hu B. Microrna-107 contributes to post-stroke angiogenesis by targeting dicer-1. Scientific reports. 2015;5:13316. doi: 10.1038/srep13316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.O'Carroll D, Schaefer A. General principals of mirna biogenesis and regulation in the brain. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2013;38:39–54. doi: 10.1038/npp.2012.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Sheedy FJ, O'Neill LA. Adding fuel to fire: Micrornas as a new class of mediators of inflammation. Annals of the rheumatic diseases. 2008;67(Suppl 3):iii50–55. doi: 10.1136/ard.2008.100289. [DOI] [PubMed] [Google Scholar]
  • 24.Bott A, Erdem N, Lerrer S, Hotz-Wagenblatt A, Breunig C, Abnaof K, et al. Mirna-1246 induces pro-inflammatory responses in mesenchymal stem/stromal cells by regulating pka and pp2a. Oncotarget. 2017;8:43897–43914. doi: 10.18632/oncotarget.14915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Karelina K, Norman GJ, Zhang N, Morris JS, Peng H, DeVries AC. Social isolation alters neuroinflammatory response to stroke. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:5895–5900. doi: 10.1073/pnas.0810737106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Shen WS, Xu XQ, Zhai NN, Zhou ZS, Shao J, Yu YH. Potential mechanisms of microrna-141-3p to alleviate chronic inflammatory pain by downregulation of downstream target gene hmgb1: In vitro and in vivo studies. Gene Ther. 2017;24:353–360. doi: 10.1038/gt.2017.28. [DOI] [PubMed] [Google Scholar]
  • 27.Baumjohann D, Kageyama R, Clingan JM, Morar MM, Patel S, de Kouchkovsky D, et al. The microrna cluster mir-17 approximately 92 promotes tfh cell differentiation and represses subset-inappropriate gene expression. Nature immunology. 2013;14:840–848. doi: 10.1038/ni.2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Manwani B, Liu F, Scranton V, Hammond MD, Sansing LH, McCullough LD. Differential effects of aging and sex on stroke induced inflammation across the lifespan. Experimental neurology. 2013;249:120–131. doi: 10.1016/j.expneurol.2013.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Varin A, Gordon S. Alternative activation of macrophages: Immune function and cellular biology. Immunobiology. 2009;214:630–641. doi: 10.1016/j.imbio.2008.11.009. [DOI] [PubMed] [Google Scholar]
  • 30.Cavaillon JM. Pro- versus anti-inflammatory cytokines: Myth or reality. Cellular and molecular biology. 2001;47:695–702. [PubMed] [Google Scholar]
  • 31.O'Donnell SL, Frederick TJ, Krady JK, Vannucci SJ, Wood TL. Igf-i and microglia/macrophage proliferation in the ischemic mouse brain. Glia. 2002;39:85–97. doi: 10.1002/glia.10081. [DOI] [PubMed] [Google Scholar]
  • 32.Thored P, Heldmann U, Gomes-Leal W, Gisler R, Darsalia V, Taneera J, et al. Long-term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke. Glia. 2009;57:835–849. doi: 10.1002/glia.20810. [DOI] [PubMed] [Google Scholar]
  • 33.Navarro-Sobrino M, Hernandez-Guillamon M, Fernandez-Cadenas I, Ribo M, Romero IA, Couraud PO, et al. The angiogenic gene profile of circulating endothelial progenitor cells from ischemic stroke patients. Vascular cell. 2013;5:3. doi: 10.1186/2045-824X-5-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Luckett-Chastain L, Calhoun K, Schartz T, Gallucci RM. Il-6 influences the balance between m1 and m2 macrophages in a mouse model of irritant contact dermatitis. The Journal of Immunology. 2016;196:196.117–196.117. [Google Scholar]
  • 35.Mauer J, Chaurasia B, Goldau J, Vogt MC, Ruud J, Nguyen KD, et al. Signaling by il-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin. Nature immunology. 2014;15:423–430. doi: 10.1038/ni.2865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Zakharova M, Ziegler HK. Paradoxical anti-inflammatory actions of tnf-alpha: Inhibition of il-12 and il-23 via tnf receptor 1 in macrophages and dendritic cells. Journal of immunology. 2005;175:5024–5033. doi: 10.4049/jimmunol.175.8.5024. [DOI] [PubMed] [Google Scholar]
  • 37.Suzuki S, Tanaka K, Suzuki N. Ambivalent aspects of interleukin-6 in cerebral ischemia: Inflammatory versus neurotrophic aspects. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2009;29:464–479. doi: 10.1038/jcbfm.2008.141. [DOI] [PubMed] [Google Scholar]
  • 38.Kalliolias GD, Ivashkiv LB. Tnf biology, pathogenic mechanisms and emerging therapeutic strategies. Nature reviews. Rheumatology. 2016;12:49–62. doi: 10.1038/nrrheum.2015.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Doll DN, Barr TL, Simpkins JW. Cytokines: Their role in stroke and potential use as biomarkers and therapeutic targets. Aging and disease. 2014;5:294–306. doi: 10.14336/AD.2014.0500294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Hallenbeck JM. The many faces of tumor necrosis factor in stroke. Nature medicine. 2002;8:1363–1368. doi: 10.1038/nm1202-1363. [DOI] [PubMed] [Google Scholar]
  • 41.Ceulemans AG, Zgavc T, Kooijman R, Hachimi-Idrissi S, Sarre S, Michotte Y. The dual role of the neuroinflammatory response after ischemic stroke: Modulatory effects of hypothermia. Journal of neuroinflammation. 2010;7:74. doi: 10.1186/1742-2094-7-74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Hefendehl JK, Neher JJ, Suhs RB, Kohsaka S, Skodras A, Jucker M. Homeostatic and injury-induced microglia behavior in the aging brain. Aging cell. 2014;13:60–69. doi: 10.1111/acel.12149. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

Change of Authorship form
NIHMS965520-supplement-Change_of_Authorship_form.pdf (184.2KB, pdf)
Inhibition of miR-141-3p ameliorates the negative effects of post-stroke social isolation in aged mice
NIHMS965520-supplement-Inhibition_of_miR-141-3p_ameliorates_the_negative_effects_of_post-stroke_social_isolation_in_aged_mice.pdf (817.4KB, pdf)
Legacy Supplemental File
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