Prevention of Metabolic Impairment by Dietary Nitrate in Overweight Male Mice Improves Stroke Outcome

1. Introduction

The worldwide epidemic of obesity has dramatically increased the incidence of metabolic syndrome, type 2 diabetes (T2D) and related cardiovascular complications [1,2,3,4]. One of the major debilitating consequences of this is stroke, which is a major cause of adult disability worldwide [5,6]. Therefore, there is a large global medical need to reduce obesity and T2D, to consequently also reduce stroke risk and poor stroke outcome.

Weight loss has proven effective in reducing the risk for cardiovascular disease in the obese population, primarily via its effective prevention of weight-related co-morbidities such as T2D [7,8,9,10]. In recent years, effective pharmacological strategies, such as high-dose glucose-like peptide (GLP) 1 receptor agonists and dual GLP1R/glucose-dependent insulinotropic polypeptide (GIP) agonists, have been approved for weight loss [11,12,13]. However, current clinical guidelines only recommend the use of these drugs in individuals with obesity or with overweight and a diagnosed co-morbidity. Since most people with overweight have not yet developed weight-related co-morbidities [14,15], this leaves a large population at increased risk for stroke and poor stroke outcome with very limited options for intervention. Indeed, the only available strategy for these people are lifestyle changes (i.e. diet and exercise) that often face major problems with adherence, since they require considerable changes in habits and consequently most people revert to their old, unhealthy lifestyle [16,17]. In summary, sustainable efficacious interventions specifically targeting the overweight population in the preventive perspective to halt obesity/T2D and reduce stroke risk and stroke-induced disability are lacking.

Dietary nitrate (NO₃^-) is a novel strategy that effectively slows weight gain in obesity and T2D [18,19,20,21], making it a promising, non-pharmacological approach to evade obesity and T2D in the overweight population, thereby potentially also reducing stroke risk and persistent post-stroke disability. Upon ingestion, NO₃^- is metabolized via the nitrate-nitrite-NO pathway, which elicits a range of beneficial effects, such as improved insulin sensitivity and endothelial function, enhanced mitochondrial efficiency, and reduced oxidative stress, underscoring its multifaceted role in metabolic health [22,23]. Moreover, NO₃^- also exerts direct beneficial effects on brain vascular health [24,25,26] and improves stroke outcome in metabolically healthy rodents [27,28,29]. However, whether halting obesity and metabolic impairments by dietary NO₃^- in overweight improves stroke outcome has not yet been investigated.

Therefore, the aim of this study was to determine whether prolonged NO₃^- supplementation prevents the progression of overweight into obesity and T2D and consequently improves stroke outcome in a clinically relevant murine model. Moreover, the effects of prolonged NO₃^- supplementation on post-stroke neuroinflammation, neurogenesis and neovascularization were assessed.

2. Materials and Methods

2.1. Animals

Twenty C57BL/6JRj mice (Janvier Labs, France) were used in this study. Mice were housed in environmentally controlled conditions (22 ± 0.5 °C, 12/12 h light/dark cycle with ad libitum access to food and water). The mice were kept under pathogen free conditions in type III size individually ventilated cages with wood chip bedding and nest material.

This study was conducted according to the guidelines of the declaration of Helsinki and has been approved by the regional ethics committee of Karolinska Institutet (approval ID: 19666-2022). The work follows the 2010/63/EU directive and is reported according to the ARRIVE guidelines.

2.2. Sample Size Calculation

Group sizes were determined based on ≈ 20% effect size between groups in functional recovery after stroke, with α = 0.05 and a statistical power of 90%. The standard deviation (SD) used in sample size calculation was obtained from pilot experiments. Based on this analysis a minimum final sample size of n = 5 was determined. Taking into consideration the success rate of stroke surgery, mortality rate and likelihood of statistical outliers, the final sample size per group was set at n =10.

2.3. Experimental Design

Starting at 4 weeks of age, mice were exposed to high-fat diet (HFD; 60% energy from fat). Animals developed overweight after 6 weeks of HFD (defined as 15-25% weight gain compared to lean, age-matched mice). Thereafter, they were randomized in two groups: HFD and 10 mM/day NaNO₃^- (NO₃^-, n = 10) in drinking water and HFD and regular water (HFD, n = 10). Body weight and fasting glucose were monitored every 4 weeks until the HFD group developed obesity (defined as >50% weight gain compared to lean, age-matched mice) and hyperglycemia (defined as fasting glucose > 7 mmol/L). Then, insulin resistance was assessed via an insulin and glucose tolerance test, confirming the development of obesity and diabetes in the HFD group after 24 weeks of HFD. Then, mice were subjected to transient middle cerebral artery occlusion (tMCAO) to induce stroke, and functional recovery was tracked using the grip strength test (see below). An overview of the experimental design can be found in Figure 1.

2.4. Metabolic Assessments

2.4.1. Fasting Glycemia

Fasting glycemia was measured every 4 weeks during NO₃^- -supplementation via a tail tip puncture and a glucometer after overnight (ON) fasting.

2.4.2. Insulin Tolerance Test (ITT)

ITT was performed after 24 weeks of NO₃^- -supplementation. Briefly, mice were fasted for 2h and baseline glycemia was measured via tail tip puncture and a glucometer. Then, animals were injected with 0.5U/kg human insulin intra-peritoneally (i.p.) and glycemia was checked at 15, 30, 45, 60, 75, and 90 min after injection. Percentage of baseline glycemia was calculated and area under the curve was used for statistical analysis.

2.4.3. Glucose Tolerance Test (GTT)

GTT was performed after 24 weeks of NO₃^- -supplementation. Briefly, mice were fasted ON. Then, baseline glycemia was measured and mice were injected i.p. with 1 g/kg glucose. Glycemia was monitored at 15, 30, 60, 90, and 120 min after injection. Percentage change from baseline glycemia was calculated and area under the curve was used for statistical analysis.

2.5. Transient Middle Cerebral Artery Occlusion

Stroke was induced by tMCAO using the intraluminal filament technique as described previously [30,31]. Briefly, mice were anesthetized by inhalation of 3% isoflurane and throughout surgery, anesthesia was maintained by 1.5% isoflurane. Using a heated pad with feedback from a thermometer, body temperature of animals was kept at 37–38 °C. Left external (ECA) and internal (ICA) carotid arteries were exposed and a 7–0 silicone-coated monofilament (total diameter 0.17–0.18 mm) was inserted into the ICA until the origin of the MCA was blocked. The occluding filament was removed after 35 min. Cerebral blood flow in the vicinity of MCA was monitored by Laser Doppler Blood Flow Monitor (Moor Instruments Ltd, UK), and no differences between the groups were observed (data not shown). Stroke induction was considered unsuccessful when the occluding filament could not be advanced within the internal carotid artery beyond 7–8 mm from the carotid bifurcation, or if mice lacked symptoms of neurological impairment based on the neurological severity score [32]. After surgery, all mice were given an analgesic (Carprofen, 5 mg/kg) and soft food. After tMCAO, all mice were switched to normal chow, to mimic the clinical setting of a post-stroke balanced diet. 2 mice in the HFD-group and 1 mouse in the NO₃^- group were euthanized shortly after surgery because the humane endpoint was reached (HFD n = 8, NO₃^- n = 9).

2.6. Behavioral Assessment

To assess functional recovery after stroke, forelimb grip strength was tested as previously described [31,33]. Briefly, mice were held firmly by the body and allowed to grasp the grid with the paretic (right) forepaw. Hereafter, they were dragged backwards until their grip was broken. Grip strength was measured using a grip strength meter (Harvard apparatus, MA, USA) at 3 days and 1-2 weeks after tMCAO. Ten trials were performed by an experimenter blinded to groups, and the average of the two highest values was used for statistical analysis.

2.7. Tissue Collection

After a 4h fast, animals were anesthetized using an overdose of sodium pentobarbital. Hereafter, cardiac puncture was performed to collect blood, and then mice were perfused transcardially using PBS followed by a 4% ice-cold paraformaldehyde (PFA) solution. Brains were harvested and stored ON in 4% PFA at 4°C. After 24h of PFA-fixation, brains were transferred to a solution of PBS and 25% sucrose and stored at 4°C until they sank. Then, 30-μm thick coronal sections were cut using a sliding microtome, and sections were stored at -20°C in anti-freeze solution until further analysis.

2.8. Immunohistochemistry (IHC)

To stain brain-sections, the free-floating staining method was used. Briefly, sections were washed in PBS to remove anti-freeze solution. Sections were then incubated in a solution of 3% H₂O₂, 10% MeOH and PBS, for visualization with 3′-3 diaminobenzidine (DAB). For immunofluorescent staining with Ki67 and double-cortin (DCX), sections were then incubated 15 minutes in citric acid (pH = 6.0) at 95°C for antigen retrieval. For immunofluorescent staining with aminopeptidase N (CD13), podocalyxin (PDXL), a blocking step was performed with a 1h incubation in PBS supplemented with 0.25% Triton-X-100 and 5% serum at room temperature (RT). Hereafter, sections were incubated ON at 4°C in a PBS solution supplemented with primary antibody, 3-5% serum and 0.25% Triton-X-100. For NeuN, sections were incubated in primary antibody solution for 48h. The following primary antibodies were used: NeuN (1:500; RRID:AB_2298772), Ki67 (1:300; RRID:AB_443209), DCX (1:200; RRID:AB_10610966), Iba1 (1:1000; RRID:AB_2220422), CD206 (1:200; RRID:AB_2063012), CD68 (1:2000, RRID:AB_10975465), PDXL (1:200; RRID:AB_354858), CD13 (1:200; RRID:AB_323691). After incubation with primary antibody, sections were washed and incubated with secondary antibody solution consisting of PBS supplemented with secondary antibody, 3-5% serum and 0.25% Triton-X-100 for 2h at RT. The following secondary antibodies were used: biotinylated horse anti-mouse (1:200; RRID:AB_2313581), biotinylated horse anti-goat (1:200; RRID:AB_2336123), Alexa-488 conjugated horse anti-rabbit (1:200; RRID:AB_2336403), Alexa-594 conjugated horse anti-mouse (1:200; RRID:AB_2336412), Alexa-594 conjugated donkey anti-goat (1:500; RRID:AB_2340432), Cy5 conjugated donkey anti-rat (1:500; RRID:AB_2340671). A 1h incubation with avidin-biotin complex was next performed for the stainings for DAB-visualization according to manufacturer’s instructions (Vectastain Elite ABC kit, Vector Laboratories), followed by DAB-visualization. All quantifications of IHC-stainings were performed by experimenters blinded for groups.

2.9. Analysis

2.9.1. Quantification of Infarct Size

NeuN-labelled sections were displayed on a computer monitor using a 1.25x lens. Infarct size was determined using all serial sections containing visual ischemic damage. The volume of the contralateral, non-injured hemisphere and of the intact portion of the ipsilateral, injured hemisphere was measured using the Cavalieri Estimator probe (StereoInvestigator, MBF Bioscience) [34]. Then, the infarct size was calculated by subtracting the ipsilateral volume from the contralateral volume, to adjust for stroke-induced tissue shrinkage. Animals with extensive cortical damage (3 animals in the NO₃^- group) were excluded for analysis.

2.9.2. Quantification of Stroke-Induced Neural Stem Cell Proliferation (Ki67) and Early Neurogenesis (DCX)

Animals with extensive cortical damage (3 animals in the NO₃^- group) were excluded from these analyses. Manual counting of Ki67 in the subventricular zone (SVZ) and of DCX in striatum was performed as described previously [35]. Briefly, three coronal sections were manually counted using the Olympus BX40 microscope. The first section was selected based on its anatomical location along the rostral-caudal axis (approximately 1 mm from the Bregma). The second and third sections were 300 and 600 μm caudal from the first section. The number of Ki67⁺ cells in the SVZ and of DCX⁺ cells in striatum was manually counted in all three sections using a dry 40x lens. All counts were performed by experimenters blinded to the treatment groups. The sum of the counts in all three sections was used for statistical analysis.

2.9.3. Quantification of Neuroinflammation

The Fiji open-source software [36] was used to evaluate Iba1 immunoreactivity as described previously [35]. Animals with extensive cortical damage (3 animals in the NO₃^- group) were excluded from these analyses. Briefly, three coronal sections were included in the analysis. The first section was selected based on its anatomical location along the rostral-caudal axis (approximately 1 mm from the Bregma). The second and third sections were 300 and 600 μm caudal from the first section. Images of Iba-1 staining in striatum were acquired at 20x using the Olympus BX40 microscope. For each contralateral, undamaged hemisphere, 1 representative image of each section was acquired. For the ipsilateral, injured hemisphere, 3 images per section spanning > 90% of the whole striatum were acquired, accounting for 9 images in total for each animal. Then, images were converted into grayscale (8-bit) mode and a threshold was determined based on the lowest Iba-1 immunoreactivity in the contralateral striatum of the HFD group. The Iba-1⁺ area was then measured and expressed as a percentage of the total area that was analyzed.

The number of CD68⁺ cells in ipsilateral striatum and CD206⁺ cells in the peri-infarct area was counted manually in three coronal sections. Animals with extensive cortical damage (3 animals in the NO₃^- group) were excluded from these analyses. The first section was selected based on its anatomical location along the rostral-caudal axis (approximately 1 mm from the Bregma). The second and third sections were 300 and 600 μm caudal from the first section. For CD68⁺ quantification, positive cells were counted manually in the ipsilateral striatum using a 60x dry lens. The sum of the counts in 3 sections was used for statistical analysis. For the quantification of CD206 in the peri-infarct area, first the area of interest was delineated at 4x magnification and subsequently, cells were manually quantified using a 60x dry lens in the delineated area. Then, the number of positive cells per area was calculated and used for statistical analysis.

2.9.4. Quantification of Neovascularization

Animals with extensive cortical damage (3 animals in the NO₃^- group) were excluded from these analyses. Neovascularization was quantified using the open-source software Fiji [36]. Confocal images were acquired with a Leica DMi8 confocal microscope. For each animal, one brain section was selected, and one to two images were captured per section at 20× magnification in the peri-infarct area. This region was identified by comparing the immunofluorescence signal to previously acquired reference images showing the ischemic core based on NeuN staining. Image dimensions were 775 μm × 775 μm, with a z-stack depth of 10 μm and a step size of 0.5 μm. The same acquisition settings were used for all images. Vascular parameters were quantified on maximum intensity projections of thresholded images using Fiji’s area fraction measurement tool. The area density was expressed as the percentage of PDXL and CD13 of the total image area. Pericyte coverage was calculated by measuring the area of colocalized CD13 and PDXL signals and normalizing it to the total PDXL-positive area within the same image.

For vessel length quantification, maximum projected images were thresholded and skeletonized. The resulting skeletons were analyzed using the AnalyzeSkeleton plugin [37], as previously described [38]. To measure vascular diameter, we adapted the Vessel Analysis plugin [39,40]. Briefly, following maximum projection and thresholding, a Euclidean distance map was generated to indicate the distance of each pixel to the nearest background pixel. Simultaneously, skeletonized versions of the images were created. Multiplication of the distance map with the skeletonized images produced skeletons containing vessel thickness information, from which the average vessel diameter was calculated. All image analyses were scripted and automated using the ImageJ Macro language to minimize human error and bias. Macro scripts are publicly available at: https://github.com/carbui/image-analysis/.

2.10. Statistical Analysis

GraphPad prism version 10 (10.2.3 (347)) was used for statistical analysis. Data was checked for statistical outliers using the ROUT method, and for normality using the Shapiro-Wilk test. For body weight, weight change, fasting glucose and grip over time, repeated measures two-way ANOVA with two-stage linear set-up procedure of Benjamini, Krieger and Yekutieli was used. For AUC of ITT, GTT, and grip, fasting glucose, stroke volume, and CD68 quantification, unpaired t-test was used. For Ki67, DCX, Iba1, CD206, PDXL, CD13, CD13/PDXL ratio, vessel length and vessel diameter ordinary two-way ANOVA, with uncorrected Fisher’s LSD was used. All data are expressed as mean ± SD and results were considered significant when p < 0.05.

3. Results

3.1. Sustained NO₃^- Supplementation in Overweight Mice Attenuates the Development of Obesity and Hyperglycemia and Improves Stroke Recovery

Six weeks of HFD-feeding induced overweight compared to age-matched controls fed a standard diet (Suppl. Figure 1). Although NO₃^- supplementation did not prevent the development of obesity (Figure 2A), a 15% attenuation in weight gain was observed in the NO₃^- group (Figure 2B). Importantly, HFD-mice developed hyperglycemia during the duration of the study, reaching fasting glycemia levels of 9 mmol/L after 24 weeks (Figure 2C). On the contrary, fasting glycemia remained significantly lower in NO₃^--treated mice and below the diabetic threshold of 7 mmol/L (Figure 2C). No differences between the groups were observed when performing insulin- (Figure 2D,E) and glucose tolerance tests (Figure 2F,G), indicating that long-term NO₃^- did not improve insulin sensitivity.

These results indicate that although long-term NO₃^- supplementation did not prevent the occurrence of insulin resistance and obesity in overweight mice, it prevented the occurrence of hyperglycemia and slightly reduced weight gain, even under continuous HFD-feeding.

After stroke, grip strength was significantly greater in the NO₃^- group compared to HFD mice (Figure 2H, I) despite no difference in stroke volume between groups (Figure 2J). In accordance with previous studies [31,35], all mice lost weight after tMCAO, with no differences in the degree of weight loss between groups (Figure 2K,L). This rapid post-stroke weight loss resulted in attenuated hyperglycemia in both groups at 2 weeks after stroke (Figure 2M).

Taken together, these data show that a long-term supplementation with NO₃^- significantly improves functional recovery after stroke without affecting stroke size. Moreover, our results indicate that this improved stroke recovery was associated with an attenuation of HFD-induced weight gain and with the prevention of hyperglycemia before stroke.

3.2. Improved Stroke Outcome in NO₃^- Mice Was Not Associated with Increased Stroke-Induced Early Neurogenesis

NO₃^- has been shown to increase adult neurogenesis [41]. Since stroke-induced neurogenesis has been associated with improved stroke recovery [42], we next assessed the effect of long-term NO₃^- supplementation on this cellular process. The number of Ki67⁺ cells in the SVZ was not different between groups, showing that stroke-induced neural stem cell proliferation was not altered by NO₃^- supplementation (Figure 3A). Moreover, the amount of DCX⁺ neuroblasts in the ipsilateral striatum was significantly higher compared to the contralateral striatum, in both groups (Figure 3B). However, the number of DCX⁺ cells was not different between groups (Figure 3B). These results show that improved recovery in the NO₃^- group was not associated with differences in stroke-induced early neurogenesis.

3.3. Improved Stroke Outcome in NO₃^- Mice Was Associated with Decreased Post-Stroke Inflammation

Neuroinflammation plays a critical role in stroke recovery but is dysregulated by T2D [43]. Therefore, we investigated whether NO₃^- impacted post-stroke neuroinflammation. We quantified Iba1 immunoreactivity in both the ipsi- and the contralateral hemisphere to assess microglial infiltration. In accordance with previous studies, we found significantly higher Iba1 expression in the ipsilateral compared to the contralateral hemisphere, although in the NO₃^- group this was only a trend (p = 0.08) (Figure 4A). Importantly, the amount of Iba1 in the ipsilateral hemisphere was significantly lower in the NO₃^- group compared to HFD (Figure 4A), indicating a dampened inflammatory response in NO₃^- treated mice.

Next, we characterized whether this post-stroke neuroinflammation resembled either a more proinflammatory M1-type, indicative of persistent inflammation, or a reparative M2-type response. To identify M2-type immune cells, we quantified the number of CD206⁺ cells, a widely recognized representative M2 microglial marker [44,45,46], in both the contralateral hemisphere and the peri-infarct area of the ipsilateral striatum. NO₃^- significantly increased the number of CD206⁺ cells in the peri-infarct area compared to contralateral striatum, whereas we found no difference between hemispheres in the HFD group (Figure 4B), indicating that in addition to a decreased infiltration of microglia, NO₃^- also induced an M2-type reparative immune response compared to HFD-controls, which has been shown beneficial for stroke recovery [47]. In contrast, there was no effect of NO₃^- on the number of CD68-expressing microglia in ipsilateral striatum, which are indicative of activated M1-type microglia (Figure 4C).

Taken together, these results indicate that sustained NO₃^- supplementation improves stroke outcome in association with reduced post-stroke neuroinflammation.

3.4. Improved Stroke Outcome in NO₃^- Mice Was Not Associated with Improved Post-Stroke Vascularization

The beneficial effect of NO₃^- on vascularization is well-known [48,49,50]. Since post-stroke neovascularization plays an important role in stroke recovery, and diabetes hampers functional angiogenesis [51,52], we next assessed the effect of NO₃^- supplementation on post-stroke vascularization in the peri-infarct area. We found no difference between groups in PDXL staining density, indicating that NO₃^- did not alter the overall vessel area (Figure 5A). Moreover, there was no difference in the CD13⁺ area between groups (Figure 5B), nor in the ratio between CD13⁺/PDXL⁺ (Figure 5C), suggesting that NO₃^- did not alter the number of pericytes, or pericyte coverage of vessels after stroke. In addition, neither the total vessel length (Figure 5D) nor the average vessel diameter (Figure 5E) in the peri-infarct area was different between groups.

Taken together, these data indicate that NO₃^- supplementation did not affect post-stroke neovascularization.

4. Discussion

In this study, we showed that sustained NO₃^- supplementation in overweight mice attenuates weight gain, and prevents the occurrence of hyperglycemia, even during ongoing HFD-feeding. Importantly, these metabolic effects may contribute to enhanced functional recovery after stroke. Moreover, we showed that improved stroke recovery by NO₃^- was associated with reduced neuroinflammation, but not with improved post-stroke neovascularization or increased neurogenesis.

Overweight significantly increases the risk of stroke and worsens stroke outcome, predominantly by increasing the likelihood of obesity and T2D, both of which are known to raise the risk of stroke and severe post-stroke disability [53,54,55,56]. However, since overweight itself is not classified as a disease, pharmacological interventions are hard to justify in this group and current recommendations to halt the progression from overweight to obesity and T2D predominantly consist of generic lifestyle changes that often lack long-term adherence.

Dietary NO₃^- has been proven an effective and feasible non-pharmacological strategy to prevent obesity and metabolic complications [22,57,58]. In the current study, under continuous HFD-feeding, dietary NO₃^- did not prevent the occurrence of obesity in overweight mice. However, it did slow weight gain by 15% and significantly improved functional recovery after stroke. Interestingly, a 5-10% body weight reduction has proven sufficient to improve metabolic health in obesity [9,59,60] and decrease the risk of cardiovascular complications [61]. This suggests that even a modest effect in reducing weight gain as shown in this study could be important to improve stroke recovery. Moreover, the composition of HFDs used in murine studies, typically is more extreme than dietary patterns observed in humans with obesity [62,63,64]. Considering that NO₃^- is a dietary supplement and not a pharmacologically active drug, it is notable that even under concomitant exposure to an aggressive HFD, weight gain was still attenuated.

In the present study, NO₃^- did not prevent insulin resistance. Since insulin sensitivity closely correlates with weight gain, and both groups developed obesity after 24 weeks of HFD, this could explain the lack of difference in insulin sensitivity [65]. However, even though mice were insulin resistant, NO₃^- did prevent hyperglycemia entirely. Indeed, several studies show insulin-independent mechanisms by which dietary nitrate lowers glycemia, for instance through the stimulation of AMPK signaling and via enhancing of GLUT4 expression [66,67,68], thereby effectively enhancing glucose uptake and lowering glycemia. Given the well-established link between hyperglycemia and poor stroke outcome [69,70,71], it is conceivable that the improved stroke recovery by NO₃^- in our study was mainly mediated by the prevention of hyperglycemia.

In contrast to previous reports [28,29,72], we did not observe any acute neuroprotective effects of NO₃^- supplementation, as infarct size was not different between groups. NO₃^- was given in the drinking water, which limited control over its intake relative to the time of stroke induction. Given the relatively short half-life of NO₃^-, especially in mice [73], it is likely that we did not observe any neuroprotective effects simply because NO₃^- concentrations were insufficient at the time of stroke to mediate neuroprotection. Consequently, this suggests that the metabolic effects of NO₃^- before stroke likely were key contributors to the improved stroke recovery observed. How improved stroke recovery can be achieved in absence of changes in stroke volume remains unclear but may involve enhanced neuroplasticity during the recovery phase [74,75].

In the present study, NO₃^- administration was initiated at the onset of overweight and continued through the post-stroke period. While this experimental design reflects a clinically relevant scenario, it limits the ability to discern whether the improved stroke recovery observed with NO₃^- was due to pre-stroke, post-stroke, or combined effects. However, given the absence of differences in infarct size and post-stroke metabolism between groups, it is tempting to speculate that the beneficial pre-stroke effects of NO₃^- could have played a causal role in the improved recovery seen in the NO₃^- group. A follow-up study, restricting NO₃^- administration to the pre-stroke time frame would help clarify the specific metabolic mechanisms underlying this effect.

Neuroinflammation plays a crucial role in stroke outcome [76], and diabetes has been shown to exacerbate post-stroke neuroinflammation, thereby worsening stroke outcome [43]. In this study, we demonstrated that NO₃^- reduced microglial infiltration and promoted a shift to a more reparative M2-type inflammatory response, which has been associated with improved post-stroke recovery [47,77]. Indeed, microglia are key modulators of neuroplasticity, both under homeostatic conditions and in the post-stroke brain, when effective neuroplasticity is essential for the formation of new neuronal connections [78,79,80]. Our data suggests that the improved stroke recovery observed with NO₃^- supplementation may be mediated by enhanced neuroplasticity secondary to reduced neuroinflammation.

Post-stroke neovascularization is impaired in the context of T2D and is associated with poorer stroke recovery [52]. Nitrates, commonly used as vasodilators in the treatment and prevention of angina pectoris, have demonstrated various vascular benefits across differences diseases [48,49]. However, their role in post-stroke cerebral vascularization remains poorly understood. In our study, NO₃^- supplementation did not alter vascular parameters following stroke. Nevertheless, potential effects on the cerebral vasculature cannot be entirely ruled out. Hypertension is a well-established factor that worsens stroke outcome [81], and NO₃^- can reduce hypertension in the brain after ischemic injury [25,29,82]. In the present study, blood pressure was not monitored, leaving potential associations of improved stroke recovery by NO₃^- with lowered blood pressure unexplored.

Stroke-induced neurogenesis has been associated with improved stroke recovery, and some studies indicate positive effects of NO₃^- on neurogenesis [41,42]. However, we observed no effects of NO₃^- on post-stroke neurogenesis, at least at the time points assessed.

In conclusion, we demonstrate for the first time the efficacy of a previously unexplored strategy to improve stroke outcome using NO₃^- supplementation in overweight mice, even during continuous HFD feeding. If clinically validated, this prophylactic approach could benefit a population of individuals who are overweight and thus at a higher risk for the development obesity, T2D and subsequently for stroke and severe post-stroke disability. By receiving NO₃^-, such individuals may experience improved metabolic function, even in the presence of ongoing unhealthy dietary habits. This metabolic improvement could prove sufficient to lower their risk for poor stroke prognosis.