Obesity-induced Ly6C High and Ly6C Low monocyte subset changes abolish post-ischemic limb conditioning benefits in stroke recovery

Abstract

Remote limb conditioning (RLC), performed by intermittent interruption of blood flow to a limb, triggers endogenous tolerance mechanisms and improves stroke outcomes. The underlying mechanism for the protective effect involves a shift of circulating monocytes to a Ly6C^High proinflammatory subset in normal metabolic conditions. The current study investigates the effect of RLC on stroke outcomes in subjects with obesity, a vascular comorbidity. Compared to lean mice, obese stroke mice displayed significantly higher circulating monocytes (monocytosis), increased CD45^High monocytes/macrophages infiltration to the injured brain, worse acute outcomes, and delayed recovery. Unlike lean mice, obese mice with RLC at 2 hours post-stroke failed to shift circulating monocytes to pro-inflammatory status and nullified RLC-induced functional benefit. The absence of the monocyte shift was also observed in splenocytes incubated with RLC serum from obese mice, while the shift was observed in the cultures with RLC serum from lean mice. These results showed that the alteration of monocytosis and subsets underlies negating RLC benefits in obese mice and suggest careful considerations of comorbidities at the time of RLC application for stroke therapy.

Introduction

Intensive preclinical research on stroke has identified many potential targets for reducing stroke-induced brain injury. However, these neuroprotection-based strategies have shown limited to no efficacy in numerous clinical trials,^1,2 highlighting the need for a paradigm shift in addressing translational gaps. ³ One key issue involves the routine use of animals with normal metabolic states in many preclinical studies. Vascular risk factors, such as dyslipidemia, hypertension, diabetes, and obesity, are highly prevalent in human stroke patients.^{4 –6} Despite including the risk factors in recent animal stroke models that facilitated our understanding of how the comorbidities affect stroke pathology,^{7 –9} stroke-induced alteration of peripheral immunity, especially in comorbid conditions, are poorly understood. Obesity is a prevailing risk factor for stroke and leads to metabolic disorders (e.g., insulin resistance, diabetes, dyslipidemia, and elevated blood pressure). Enhanced inflammation and vascular dysfunction in these metabolic deregulated conditions exacerbate stroke-induced brain injury and delay recovery.^{10 –13}

Stroke elicits an acute inflammatory response that results in a massive infiltration of peripheral immune cells into the ischemic area. The process alters the peripheral immune system, influencing the extent of neural inflammation and infarct development. Despite the detrimental role of infiltrated CCR2⁺ monocytes in neural inflammation and tissue destruction, blocking the recruitment of these monocytes hinders subsequent stroke recovery. ¹⁴ The importance of early recruitment of inflammatory monocytes is supported by phenotype changes of the entered cells in the injured tissue. For instance, infiltrated monocyte-derived macrophages change their phenotype to alternatively activated reparative macrophages in the injured brain.^15,16 The critical role of circulating monocytes in acute pathology and stroke recovery thus suggests a potential influence of altered peripheral immunity on CNS injury development and repair processes.

Remote Limb conditioning (RLC), performed by intermittent interruption of blood flow to a limb, has been reported to alleviate brain injury and improve behavioral recovery when treated after ischemic stroke.^{17 –20} The role and mechanism of RLC in ischemic stroke are multiple. The protection is closely linked to inflammation, as the absence of an inflammatory mediator nullifies the conditioning benefit.^{19,21 –24} In addition, the application of RLC shifted the monocyte toward proinflammatory status in the blood, and the shift was required for RLC-induced neuroprotection and functional recovery. ¹⁹

In the paucity of intervention in stroke with comorbidity, this study investigated whether RLC provides neuroprotection and recovery in subjects with vascular risk factors. To closely mimic human vascular comorbidity, we utilized a high-fat diet (HD) to generate a diet-induced obesity in mice. Here, we report that obesity alters the number and subset of circulating monocytes, worsening stroke outcomes and neural inflammation. The altered peripheral immunity in obese conditions abrogates the RLC-mediated monocyte shift to pro-inflammatory status and functional recovery. Negating the RLC effect in obese stroke mice suggests that vascular comorbidity and metabolic status should be considered at the time of RLC application for clinical benefits.

Methods

Animals

Procedures for the use of animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Weill Cornell Medical College and reported in accordance with guidelines from the National Institutes of Health and Animal Research Reporting of In Vivo Experiments (ARRIVE). C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME) and bred and housed at Burke Neurological Institute Animal Facility. The facility monitors and maintains the temperature, humidity, and 12-hour light/dark cycle. A maximum of five mice were housed in a single cage with an individual ventilating system and irradiated bedding (The Anderson, Maumee, OH). Sterilized food and water were freely accessible in each cage.

High-fat diet (HD)-induced obese mice

Eight-week-old C57BL/6 mice were fed an HD (60% fat (S-3282), Bioserv, Flemington, NJ) for either 8 (males) or 12 weeks (females). Since female mice were resistant to diet-induced obesity, HD intervention was extended for female mice to obtain a similar degree of obesity between the sexes. Body weights (BW) were measured weekly, and glucose tolerance tests (GTT) were performed in overnight fasted mice one week before the end of the diet intervention. Mice received an intraperitoneal injection of D-glucose (2 mg/g BW), and blood glucose levels were measured at 0 (baseline), 15, 30, 45, 120, and 180 min using a glucometer (Ascensia Contour; Bayer, Whippany, NJ).

Transient middle cerebral artery occlusion (MCAO)

Mice were subjected to transient MCAO as previously described. ²⁵ Briefly, mice were anesthetized with isoflurane and connected to a Laser-Doppler Flowmeter (Periflux System 5010; Perimed) after a fiber-optic probe was glued to the parietal bone (2 mm posterior and 5 mm lateral to the bregma) for continuous monitoring of cerebral blood flow in the ischemic territory. For MCAO, a 6–0 Teflon-coated black monofilament surgical suture (coating length, 5–6 mm; diameter, 0.25 mm; Doccol, Redland, CA) was inserted into the exposed external carotid artery, advanced into the internal carotid artery, and wedged into the Circle of Willis to obstruct the origin of the MCA. The filament was advanced until a sufficient cerebral blood flow (CBF) drop was observed. Reperfusion was confirmed at the time of filament withdrawal. All the studies in normal chow diet (ND) and HD-fed mice used 30 min transient MCAO. The CBF was measured before the stroke, during the occlusion period, and 10 min after reperfusion. Animals exhibiting greater than 80% reduction of pre-ischemic baseline CBF (20% of pre-stroke baseline) during MCAO and greater than 80% of pre-stroke baseline at 10 min of reperfusion were included in the study. Accordingly, 9.2% of animals were excluded from this study due to insufficient CBF reduction and/or reperfusion. Mice were randomized to a specific diet and surgery. Animals’ identity and treatment were blinded to those who assessed stroke outcomes.

Remote limb conditioning (RLC)

Mice were performed RLC as previously described. ¹⁹ Briefly, RLC was performed on the left hindlimb 2 hours after MCAO by applying five cycles of inflation and deflation (200 mmHg, 5 min × 5 min interval between cycles) using a small blood pressure cuff (Hokanson). The sham group exposed to the exact duration of isoflurane was a control.

Brain atrophy measurement

Two months after MCAO, brains were isolated, cryosectioned at 20 μm thickness, and collected serially at 600 μm intervals. Collected brain sections were examined with phase-contrast microscopy to visualize. The extent of atrophy in the hemisphere was determined by integrating the volume from 13 serial sections and expressing it as a ratio to the contralateral side using ImageJ software.

Behavior assessment

Motor/gait analysis was performed before stroke (behavior baseline), and post-stroke behavior was assessed for 2 months. Spatial memory was evaluated at 9 weeks after stroke. Procedures on these behavior tests were adopted from previous publications with minor modifications.^26,27

For the rotarod test, animals were placed on a rotating rod (indented rod, 3.5 cm diameter) that accelerates from 4 to 80 rpm/min for 5 min. The latency to fall was used to assess motor performance. Animals were trained on the rotarod for five days with five daily trials and compared for baseline differences. At each time point after stroke, animals were tested in five consecutive trials, and the average was used as the final value. Gait changes were assessed using a Catwalk XT gait analysis system (Noldus Information Technology). The Catwalk measures the kinematics of gait while the mouse walks from one end of a flat surface to another. Footprints were identified and digitally recorded when the light changed with paw placement on the glass-walking surface. Running speed was used to gauge overall gait function. For detecting asymmetry, swing speed and stride length of the affected side of the body (left side) were used. Mice were pretrained for 3 days to cross an illuminated glass walkway three consecutive times, which is a necessary step to obtain quantifiable digital signals. For the open field test, mice were placed into a 40 × 40 × 40 cm field and allowed to explore the setup freely over 10 min. Animals were tracked by analyzing live video by software (ANY-maze, Stoelting Co.). The total distance covered by the animal was used to evaluate gross motor function and activity, and the center distance used for anxiety-related behavior.

Animals were assessed in the Water maze or Barnes maze task to assess spatial learning performance. For the place learning task, animals were released into the circular pool for Water maze (140 cm diameter, 50 cm depth) or circular platform (122 cm diameter, 1 cm thickness) containing 12 equally spaced holes (5 cm diameter) for Barnes maze and allowed to explore for a maximum of 90 or 150 sec. The test consisted of three trials per day for seven or five consecutive days. The results of three daily trials were averaged to get the daily means for latency to reach the platform. For the visible platform task, a platform marked by a visual marker flag was placed in the opposite quadrant of the previous target quadrant. Animals were tested in four consecutive trials to score their latency to reach the visible platform. ANY-maze (Stoelting Co.) software was used to record and analyze the data.

mRNA measurement

Relative mRNA levels were quantified with real-time reverse transcription-polymerase chain reaction (qRT-PCR) using fluorescent TaqMan technology. To obtain tissue that contains the entire infarct territory, an unbiased stereological sampling strategy was used according to the method described in the previous study. ²⁸ Three days after MCAO, brains were excised, frozen, and cut ∼6 mm tissue block rostrocaudal (roughly +2.8 mm and extending to −3.8 mm from bregma). Tissues between the 600 μm intervals were sectioned, cut in half, and collected for each hemisphere. Total RNA samples from the contralateral and ipsilateral hemispheres, excluding the olfactory bulb and cerebellum, were extracted using Tri reagent (Miltenyi Biotec), and 500 ng of total RNA was reverse transcribed using M-MLV Reverse Transcriptase (Life Technologies, Foster City, CA), according to the manufacturer’s protocol. PCR primers and probes specific to the genes in this study were obtained as TaqMan pre-developed assay reagents for gene expression (Life Technologies, Foster City, CA). Primers were used as follows: CCR2 (Mm00438270_m1), MCP-1 (Mm00441242_m1), and β-actin (Mm00607939_s1). β-actin was used as an internal control for the normalization of samples. The PCR reaction was performed in 20 µl total volume using FastStart Universal Probe Master Mix (ROX; Roche, Indianapolis, IN) by incubating at 95 °C for 10 min followed by 40 cycles of 15 sec at 95 °C and 1 min at 60 °C. The results were analyzed using 7500 Fast Real-Time PCR System software (Life Technologies, Grand Island, NY). To analyze the mRNA level, a delta-delta Ct (cycle threshold) method based on untreated samples and housekeeping gene (β-actin) was used. The overall formula to calculate the relative fold gene expression level was 2^−ddCt (ddCt = dCt (treated sample) – dCt (untreated sample), and dCt (delta cycle threshold) = Ct (gene of interest) – Ct (β-actin)).

Tissue/cell preparation for flow analyses of monocytes

To analyze monocyte infiltration in brain tissues, single brain cells from contralateral and ipsilateral hemispheres were prepared using Neural Tissue Dissociation Kit (P) with Debris Removal Solution (Miltenyi Biotec). An in vitro study was performed as previously described. ¹⁹ Briefly, sera were collected from lean or obese mice 24 hours after application of sham or RLC and were stored at −80°C until use. Naive splenocytes from lean mice were collected and incubated with each serum for 4 hours at 37 °C with 5% CO₂. After 4 hours of incubation, cells were collected. Cells were incubated with antibodies (anti-mouse REA) against CD11b (PE-Vio770), CD45 (Vio-blue), Ly6C (APC), CD36 (FITC) and a mixture of phycoerythrin-conjugated antibodies (PE; Lin; a mixture of phycoerythrin-conjugated antibodies against T cells (CD90.2), B cells (CD45R/B220), natural killer cells (NK-1.1 and CD49b), and granulocytes (Ly6G)) in PBS for 30 min in the dark. After washing with PBS, cells were immediately analyzed with a MACS Quant VYB flow cytometer (Miltenyi Biotec). For circulating immune cell analysis, monocyte numbers and subsets were analyzed according to the published gating strategy. ²⁵ Total CD45⁺ cells from 100 µl of sera were determined by fluorescence triggering of CD45⁺ event read to reduce the background due to numerous CD45 negative cells in the blood. Ly6C subsets were determined in CD45⁺/CD11b⁺/Lin⁻ cells. All antibodies for flow cytometry were purchased from Miltenyi Biotec and used at a 1:50 ratio.

Statistics

All in vivo data are expressed as the mean ± 95% confidence interval (CI), whereas all in vitro data is expressed as the mean±SD. The sample size for in vivo studies was determined based on predicting detectable differences to reach the power of 0.80 at a significance level of <0.05, assuming a 33% difference in mean and a 25% standard deviation at the 95% confidence level. Student’s t-test was used to compare differences between the two groups (i.e., brain atrophy, flow cytometric analysis). A log-rank test was performed to determine the survival curve. Multi-group analyses were performed using Two-way ANOVA using Prism 9.0 (i.e., BW change, behavior test). Bonferroni post hoc comparisons of the ANOVA were used to compare either stroke or RLC effects. The normality of data was analyzed using the D’Agostino-Pearson normality test.

Results

Obesity increases the number of circulating monocytes

Vascular risk factors such as hyperlipidemia, obesity, and diabetes are known to alter the number and composition of circulating immune cells. ²⁹ Thus, before addressing the RLC effect in obese stroke, we first determined the composition of circulating monocytes in obese conditions. Circulating monocytes were identified by their CD45 and CD11b expression but devoid of lineage marker (Lin) for T, B, NK cells, and granulocytes. The [CD45⁺/CD11b⁺/Lin⁻] population was further analyzed by Ly6C expression to define anti-inflammatory (Ly6C^Low) and pro-inflammatory (Ly6C^High) monocytes (Figure 1(a)). In non-stroked mice, obese mice showed increased total number of circulating monocytes (monocytosis) compared to lean mice (Figure 1(b)). The increases were also found in both Ly6C^Low and Ly6C^High monocyte subsets (Figure 1(c) and (d)). In mice with stroke, the increase in the number of circulating monocytes in total and both subsets were similarly increased in obese conditions (Figure 1(e) to (g)). The obesity-induced monocytosis before and after stroke thus demonstrates the influence of vascular comorbidity on changes in peripheral immunity.

Figure 1.

Circulating monocytes in non-stroked or stroked obese mice. (a) Gating strategy for monocytes (CD45⁺/CD11b⁺/Lin⁻ cells). Monocyte subsets in the blood were further analyzed by Ly6C expression. (b-d), The number of circulating monocytes (b), monocyte Ly6C^Low (c), and Ly6C^High (d) subsets in the non-stroked mice. (e-g), Number of circulating monocytes (e) and monocytes subsets (f, g) in the lean and obese mice at 3 days post-MCAO. Data are mean ± 95% CIs; Student’s T-test: *,**,****p < 0.05, 0.01, 0.0001 vs lean.

Obesity increases inflammation and monocyte infiltration in the post-ischemic brain

To investigate whether obesity-induced monocytosis in the blood affects neural inflammation in the post-ischemic brain, we determined the expression of MCP-1, a chemokine released from damaged tissue, and its cognate receptor CCR2 3 days after stroke. In lean mice, MCP-1 and CCR2 mRNA levels were increased on the ipsilateral side, and the increases were significantly greater in obese mice (Figure 2(a) and (b)). Subsequently, we determined infiltrating monocytes identified by CD45^High/CD11b⁺ population (Figure 2(c)). The analyses demonstrated the absence of the population in the contralateral hemisphere. On the other hand, both lean and obese mice had an increased number of CD45^High monocytes/macrophages (MM_Ø) in the ipsilateral hemisphere, with greater infiltration of these cells in obese condition (Figure 2(d)). The results indicate heightened neural inflammation in obese stroke.

Figure 2.

Effect of obesity on stroke-induced monocyte trafficking. (a, b), Assessment of MCP-1 (a) and CCR2 (b) gene expression in the lean and obese mice at 3 days post-MCAO. (c) Flow cytometry gating strategy to identify infiltrated CD45^High monocytes in the ipsilateral hemisphere. (d) Quantification of CD45^High MM_Ø counts. ctrl, contralateral; ipsl, ipsilateral; Data are mean ± 95% CIs; Two-way ANOVA, *,**,****p < 0.05, 0.01, 0.0001 vs ctrl, ^#,^###p < 0.05, 0.001 vs lean. MM_Ø, monocyte-derived macrophages.

Obesity exacerbates acute stroke outcomes and delays stroke recovery

To investigate the effect of obesity on stroke outcome in the chronic phase, stroke-induced body weight changes and motor/gait functions were determined. The stroke-induced weight loss against pre-ischemic baseline showed a greater loss during the acute phase and slower body weight gain for 2 months after stroke in obese mice (Figure 3(b)). Obese stroke mice also displayed a higher mortality rate (lean vs. obese; 13.6% (3 died out of 22) vs. 46.9% (15 died out of 32), p < 0.05) (Figure 3(c)). In survived lean and obese mice, the extent of brain atrophy at 2 months, an indicator of stroke severity, was similar between the groups (Figure 3(d)). Due to the significant difference in mortality between groups, we reasoned that functional outcome assessment in only survived animals would introduce sampling bias and mask the true effects of obese stroke. Thus, deceased mice were given the worst score registered in the surviving animals for brain atrophy at 2 months and behaviors at each time the death occurred. As a result, obese stroke mice showed greater brain atrophy than lean mice (lean vs. obese; 28.9 ± 11.4% vs. 39.9 ± 11.0%, p < 0.001) (Figure 3(e)).

Figure 3.

Assessment of chronic stroke outcomes. (a) Experimental timeline. (b, c), body weight change (b) and mortality rate (c) for 2 months after stroke. (d, Representative images and percentage of hemispheric brain atrophy in post-stroke mice at 2 months in surviving animals. (e) Brain atrophy including survived and dead animals. (f) Latency to drop from the accelerating rotarod. (g) Catwalk gait analyses: running speed, gait parameters for left hindlimb swing speed and stride length. (h) Total distance traveled for motor activity and central distance traveled for anxiety test in the open field test. (i) Cognitive function: platform learning task over seven days, showing learning curves and visible platform test for relearning task. All behavior tests are presented as a percentage of the pre-ischemic baseline. n = 22 (lean) or 32 (obese), Data show the mean ±95% CIs, Log-rank test for survival rate, ^*p < 0.05 vs. lean; Two-way ANOVA for BW change, atrophy and behavior tests, *,**,****p < 0.05, 0.01, 0.0001 vs lean.

For behavior outcomes, pre-stroke baselines were established in both groups. Despite obese mice showing a lower performance in rotarod tests, motor learning rates over the next five days were similar between the groups (Supplementary Fig. 1(a) and (b)). There were no differences between the groups in total or central travel distance in the open field test (Supplementary Fig. 1(c)). Thus, stroke-induced behavior outcomes were reported against individual pre-ischemic baseline. Analyses of behavior with the inclusion of deceased mice (assigned lowest registered scores within the cohort) revealed a greater motor deficit in the rotarod test (Figure 3(f)), slower running speed as well as impaired swing speed and stride length of the affected (left) hindlimb (Figure 3(g)) in obese stroke mice. Obese mice also showed less motor activity, spent less time in the center (Figure 3(h)), and were impaired in learning and relearning tasks (Figure 3(i)), suggesting that obesity aggravates acute behavior deficits and delays motor and cognitive recovery. When the behavior analyses were performed on only survived animals, the difference between groups was either absent or smaller (Supplementary Fig. 2). The results indicate the importance of the inclusion of dead animals in the analyses of stroke outcomes to capture the true effect, especially when mortality rates between groups were significantly different.

Obesity abrogates RLC-induced circulating monocyte shift to a pro-inflammatory status

There is a paucity of stroke interventions tailored to subjects with vascular comorbidities. RLC has been shown to provide cross-organ protection, including brain and RLC-induced functional benefits involving inflammatory mediators. ³⁰ In supporting the view, we reported that the shift of circulating monocytes to the proinflammatory Ly6C^High/CCR2⁺ subset underlies RLC benefits in ischemic stroke. ¹⁹ We first determined whether RLC causes a similar shift in the circulating monocyte subset in non-stroke obese mice. The analyses showed that the total number of monocytes, Ly6C^Low and Ly6C^High subsets in obese mice subjected to RLC were similar to those with sham conditioning (Figure 4(a) to (c)), resulting in no shift of monocyte subset to the pro-inflammatory status (Figure 4(d)). RLC also did not affect monocyte number and subset distribution assessed 3 days after stroke (Figure 4(e) to (h)). These results demonstrate that obesity with or without stroke, abrogates RLC-induced circulating monocyte subset change and monocyte shift to the pro-inflammatory subset.

Figure 4.

Circulating monocyte subsets in obese mice with RLC. (a-d), The number of monocytes (a), Ly6C^Low (b) and Ly6C^High (c) monocyte subsets, and ratios of Ly6C^High/Ly6C^Low (d) in the non-stroked obese mice applied with sham or RLC. (e–h), Number of circulating monocytes (e), monocytes subsets (f, g), and ratios of Ly6C^High/Ly6C^Low (h) in the obese mice with sham or RLC animals at 3 days post-MCAO. Sham, sham conditioning; RLC, remote limb conditioning; ns, non-significant.

To further confirm the effect of metabolic status on RLC-induced monocyte subset distribution, splenocytes from normal lean mice were cultured and incubated with sham or RLC sera from lean and obese mice. Analyses showed a selective increase of Ly6C^High subsets in cultures incubated to lean-RLC sera compared to lean-sham sera (Figure 5(b)), resulting in a significant shift of monocyte to proinflammatory status (Figure 5(c)). The observed RLC-induced monocyte subset changes and shift to Ly6C^High subsets, however, was absent in splenocytes exposed to obese-RLC sera compared to obese-sham sera (Figure 5(d) and (e)), confirming the in vivo finding of nullifying RLC-induced monocyte shift in obese condition. These results indicate the importance of metabolic states for the occurrence of RLC-mediated monocyte shift.

Figure 5.

Monocyte subsets in the splenocyte. (a) Gating strategy to identify monocyte subsets in the splenocyte from lean mice. (b, c), Counts of Ly6C subsets (b) and the ratio of Ly6C^High/Ly6C^Low (c) in splenocyte incubated with sera from sham or RLC-treated lean mice. (d, e), Sera were collected from obese mice with sham or RLC and treated to the splenocytes from lean mice. Counts of Ly6C subsets (d) and the ratio of Ly6C^High/Ly6C^Low subsets (e). Sham, sham conditioning; RLC, remote limb conditioning; ns, non-significant. Data are mean ±SD; Student’s T-test: **p < 0.01 vs Sham.

RLC does not attenuate obesity-enhanced neural inflammation in the post-ischemic brain

RLC applied in lean mice significantly reduced stroke-induced MCP-1 and IL-1β gene expression. ¹⁹ We, therefore, investigate whether the absence of RLC-induced monocyte shift directly influences neural inflammation in the post-ischemic brain in obese mice. Stroke increased MCP-1 and CCR2 mRNA levels in the ipsilateral side of the brain, and the extent of the increase was similar between sham vs. RLC groups (Figure 6(a) and (b)). In addition, the number of monocytes entered into the ischemic brain (CD45^High monocytes defined in Figure 2(c)) were also similar between sham and RLC groups (Figure 6(c)). The results collectively indicate that RLC does not mitigate stroke-induced neural inflammation in obese conditions.

Figure 6.

Effect of RLC on monocyte trafficking in the postischemic brain in obese condition. (a, b), Assessment of MCP-1 and CCR2 gene expression in sham and RLC treated obese mice at 3d post-MCAO. (c) Flow cytometric analysis of infiltrated CD45^High MM_Ø in the post-ischemic brain. ctrl, contralateral; ipsl, ipsilateral; Data are mean ± 95% CIs; Two-way ANOVA, *p < 0.05 vs. ctrl. MM_Ø, monocyte-derived macrophages.

RLC in obese condition does not improve stroke recovery

Enhanced motor and gait recovery during a chronic phase of stroke via the monocyte subset shift is a prominent benefit of RLC. ¹⁹ We thus determined whether the absence of RLC-induced monocyte shift in obese conditions nullified RLC-induced stroke recovery (Figure 7(a)). Stroke-induced acute weight loss and resistance to weight gain during the recovery period in obese mice were similar in sham and RLC groups (Figure 7(b)). As expected, obese stroke mice have high mortalities without any benefit in reducing the mortality rate in mice with RLC (37.5%; 6 died out of 16 vs. 41.2%; 7 died out of 17, sham vs RLC, ns) (Figure 7(c)). The extent of brain atrophy at 2 months was similar between sham and RLC groups, regardless of analyses only in surviving animals (Figures 7(d)) or in the inclusion of dead animals assigned the worst score registered within the cohort (Figure 7(e)). When the behavior analyses were performed only for survived animals, we did not observe differences between groups (Supplementary Fig. 3). We then assessed longitudinal behavior in animals with the inclusion of deceased animals (assigned worst score registered within the cohort). The analyses showed that RLC did not improve motor recovery (Figure 7(f)) and gait deficits (slower running speed, swing speed, and stride length (Figure 7(g)). There was no difference in motor activities in the open field test (Figure 7(h)), a learning curve to find an escape platform or probing time for the hidden platform and a visible platform task as an indication of relearning in the Barnes-Maze test (Figure 7(i)), suggesting no impact of RLC in obese mice on improving motor, gait, and cognitive function in stroke recovery.

Figure 7.

Effect of RLC on long-term motor and cognitive functions in obese condition. (a) Experimental timeline. (b, c), Percent of body weight change (b) and survival rate (c) for 2 months after stroke. (d) Representative images of brain sections from post-stroke at 2 months and percentage of hemispheric brain atrophy among survived animals. (e) Brain atrophy in survived animals with decreased animals with worst scores. (f-i), Functional recovery test for motor deficits by rotarod (f), gait analyses (running speed, left hind limb swing speed and stride length) (g), motor activities by open field test (h), and cognitive functions by Barnes maze test (platform learning and relearning) (i). All behavior tests are presented as a percentage of the pre-ischemic baseline. n = 16–17/group, Data show the mean ±95% CIs, ns, non-significant.

Discussion

Vascular comorbidities such as hyperlipidemia, diabetes, and obesity are prevalent among stroke patients and negatively influence stroke outcomes ^8,9,28,31 In the lack of intervention strategies tailored against these vascular comorbid conditions, the reported protective effects of RLC applied after stroke,^19,32,33 led the current investigation to address whether the endogenous protective mechanism could be used for stroke therapy in the presence of comorbidities. By using diet-induced obesity in mice to closely mimic human obesity, we found that RLC-induced benefits on stroke outcome are entirely abolished in obese mice and that obese-induced changes in peripheral monocyte number (monocytosis) and subset before stroke account for the absence of RLC benefits in stroke recovery.

In addressing the effect of obesity on histological and behavioral outcomes in stroke, the study encountered a significantly higher mortality in obese mice than in lean mice. Stroke-induced brain edema was shown to be a fatal complication during the development of infarction. With disproportionally enlarged brain swelling in animals with vascular comorbidities, obese conditions increase post-stroke deaths in the acute phase.^8,34 The observation compounded the interpretation of outcome analyses. In this study, the analyses of brain atrophy only in survived mice between lean and obese groups showed a similar extent of brain atrophy (Figure 3(d)). On the other hand, when deceased mice were assigned the worst value registered within each cohort, the analyses resulted in a significantly increased atrophy in obese mice (Figure 3(e)). Similarly, applying the mortality by including deceased animals in behavior analyses also revealed significantly worse motor/cognitive behaviors in obese mice (Figure 3(f) to (i)). The differences were smaller or minimized when analyses were performed only in survived animals (Supplementary Fig. 2). We further analyzed data in the study to determine the effect of RLC in obese mice. The mortality rate of sham-conditioned obese mice was not different from that of obese mice with RLC (Figure 7(c)). With the similar mortality rates between the groups, analyses on brain atrophy and behaviors performed only in survived animals were not different from those performed with the inclusion of deceased animals (Figure 7(d) vs. (e)) and behaviors (Figure 7(h) to (i) vs. Supplementary Fig. 3). Thus, this study underscores the importance of factoring in mortality during the analyses of stroke outcome when there are group differences.

Although stroke-induced brain injury is mainly considered a CNS event, the peripheral immune system profoundly influences the extent of brain injury. ³⁵ The changes in peripheral immunity influence the progression of post-ischemic inflammation and repair process. Studies in multiple species have shown that ischemic conditioning confers resistance to subsequent insults, an effect that is neither organ- nor tissue-specific. RLC with a blood cuff provides a practical and non-invasive way to remotely induce cross-tolerance in multiple organs.^17,32,33 Among proposed mechanisms involved in RLC protection, neural and humoral factors,^{36 –38} and pro-inflammatory mediators have been shown to play a role in mediating conditioning-induced protection.^{39 –41} Supporting the view, monocyte subset shift to pro-inflammatory cells has been demonstrated as an important underlying mechanism for RLC-induced benefit, as the absence of the shift in CCR2-deficient mice nullifies RLC benefits in lean mice. ¹⁹ In the current study, the immune-mediated mechanism of RLC in obese mice was addressed. Due to obesity-induced alterations of circulating monocyte number and composition before stroke, post-stroke RLC application could not shift monocytes to pro-inflammatory subsets in obese conditions and subsequently abrogate functional benefits. The study thus further confirms that the immune-mediated mechanism via circulating monocyte underlies RLC-induced functional benefit.

Consistent with our findings in obese stroke, others have shown the absence of RLC-mediated protection in subjects with comorbidities, including hyperglycemia and aging.^{42 –44} The age-related changes in the peripheral immune response, independent of infarct size, were shown to exacerbate neuroinflammation and behavioral impairment following stroke. ⁴⁵ Since obesity-induced changes in monocyte number and subsets occurred before and sustained after stroke, predisposed peripheral immune alteration related to comorbid conditions likely counteract the RLC-induced monocyte shift, negating RLC-induced stroke recovery.

The unresolved question is that if the shift to pro-inflammatory status underlies RLC-benefits, ²⁰ one would expect that obesity-induced increase Ly6C^High monocytes, even in the absence of Ly6C^High/Low ratio shift (Figure 1), would provide RLC-benefit in obese mice. A potential explanation of this paradoxical event may come from the contribution of vascular dysfunction. Aside from comorbidity-induced changes in peripheral monocytes, the conditions such as diabetes and obesity cause vascular dysfunction via excessive activation of VEGF signaling, enhanced BBB permeability, and enlarged brain edema in stroke.^8,31 Notably, VEGF functions differently in normal metabolic conditions as an anti-permeability factor to repair blood–brain barrier and reduce cerebral edema after stroke. ⁴⁶ Therefore, obesity-induced monocytosis and increasing both subsets without the shift to pro-inflammatory subset status may not be sufficient to overcome the predominant pathology of vascular dysfunction/BBB impairment and neural inflammation in obese stroke. Thus, the inability to exert RLC benefits in obese stroke suggests higher thresholds for RLC-induced protection in comorbid conditions. Although the adoptive transfer of CCR2 deficient immune cells in lean mice provided the causality of RLC-induced monocyte shift in functional recovery, ¹⁹ the potential contribution of other immune cells that express CCR2 such as subsets of in T cells,^47,48 warrantee future studies.

In summary, RLC-induced monocyte shift is a critical immune mechanism for functional benefits. In obese conditions, the increased number of monocytes and altered monocyte subset failed to induce the monocyte subset shift to pro-inflammatory status following RLC, resulting in the abrogation of the protective effects. The study showed the importance of monocyte subset shift for RLC-mediated benefits in stroke and suggests careful consideration of vascular comorbidity and peripheral metabolic status in applying RLC for stroke therapy.

Supplemental Material

sj-pdf-1-jcb-10.1177_0271678X231215101 - Supplemental material for Obesity-induced Ly6C^High and Ly6C^Low monocyte subset changes abolish post-ischemic limb conditioning benefits in stroke recovery

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X231215101 for Obesity-induced Ly6C^High and Ly6C^Low monocyte subset changes abolish post-ischemic limb conditioning benefits in stroke recovery by Il-doo Kim, Hyunwoo Ju, Joseph Minkler, Ahmed Madkoor, Keun Woo Park and Sunghee Cho in Journal of Cerebral Blood Flow & Metabolism