Regulation of Glycemia in the Recovery Phase After Stroke Counteracts the Detrimental Effect of Obesity-Induced Type 2 Diabetes on Neurological Recovery

Type 2 diabetes (T2D) and obesity are strong risk factors for stroke (1). It is estimated that by 2030, the number of people with T2D or obesity will reach 439 million (2) and 1.2 billion (3), respectively. These people comprise an enormous candidate group for stroke treatment/care. Despite these dire predictions, the effects of T2D/obesity on poststroke recovery are unclear. In fact, T2D worsens stroke outcome and is a strong predictor of dependency for activities of daily living after stroke (4–7). On the other hand, 85% of people with T2D are obese/overweight, and epidemiological studies have described a phenomenon called the “obesity paradox,” indicating that these people have improved clinical outcome after stroke (8). The validity of the obesity paradox is under debate (8–10) and especially questionable in people with T2D in whom worsened stroke outcome has been demonstrated. Thus, more studies aimed at understanding the interplay between obesity and T2D on poststroke recovery are needed.

Experimental stroke studies have shown a detrimental role of obesity-induced T2D on stroke outcome. For example, in genetic models (11), T2D decreases poststroke recovery. A detrimental role of obesity-induced T2D on stroke recovery was also shown in clinically relevant models using obesogenic diets (12,13). However, the exposure to obesogenic diets was extended into the poststroke recovery phase, thus not reflecting the likely situation of patients with T2D/obesity and stroke after hospitalization. Furthermore, it is known that even a short high-fat diet (HFD) exposure impairs glucose homeostasis, worsening stroke outcome (14). Therefore, the interplay between obesity and T2D on poststroke recovery should be further studied experimentally.

Hyperglycemia during acute ischemic stroke is detrimental to people with and without T2D (15). However, the role of chronic hyperglycemia in the stroke recovery phase is unknown. Furthermore, the most efficacious strategies that lead to an optimal glucose control in patients with T2D and stroke (and that would also facilitate recovery) are unknown. In fact, some of the most commonly used T2D drugs, such as insulin and sulfonylurea, can cause hypoglycemia (16), thereby increasing cardiovascular (CV) risk (17), which is already elevated because of T2D. Thus, hypoglycemic episodes should be especially avoided after a stroke by selecting the drugs with the lowest hypoglycemic risks.

Dipeptidyl peptidase 4 inhibitors (DPP-4is) are T2D drugs that inhibit endogenous glucagon-like peptide 1 (GLP-1) degradation, resulting in the prolonged insulin secretion with very low hypoglycemia risk (18). Previous data have shown that DPP-4is can also reduce stroke-induced brain damage in both normal and T2D rodents (19–22). However, their potential efficacy to improve poststroke neurological recovery is unknown.

The first aim of the current study was to determine whether obesity-induced T2D (defined as fasting glucose >7 mmol/L and weight gain >15%) after sustained HFD feeding worsens poststroke neurological recovery in a clinically relevant preclinical setting in which HFD was replaced immediately after stroke, and throughout the whole recovery phase, by a nutritionally balanced diet. We also investigated whether obesity-induced T2D affects neuroinflammation and stroke-induced atrophy of parvalbumin-positive (PV⁺) interneurons. These interneurons are important for stroke recovery (23), and we recently showed that their cellular body is atrophied by stroke under T2D (13). Finally, we investigated whether sustained glycemia regulation after stroke counteracts the effect of obesity-induced T2D in the poststroke recovery phase. To do so, we performed a head-to-head comparison in both T2D/obese and normal mice by using the DPP-4i linagliptin (24) and the sulfonylurea glimepiride (25). Sulfonylurea induces direct and sustained insulin secretion, bypassing the GLP-1/glucose-dependent insulinotropic polypeptide system.

One hundred male C57BL/6J mice were used in two studies (Fig. 1).

Five-week-old mice were randomly assigned to standard diet (n = 18, referred as non-T2D) or HFD (n = 54, referred as T2D) groups and after 7 months of exposure, were subjected to stroke (non-T2D n = 13, T2D n = 39) or sham (non-T2D n = 5, T2D n = 15) surgery. After surgery, the HFD in the T2D group was replaced with a standard diet to reflect the clinical setting of a balanced poststroke diet. One non-T2D and two T2D mice were removed from the study because of unsuccessful stroke, and one non-T2D and five T2D mice died after stroke surgery.

Three days poststroke, the remaining T2D animals were randomly assigned to the following experimental groups: vehicle treated (T2D) (0.5% natrosol solution; n = 11), linagliptin treated (T2D-Li) (10 mg/kg body weight per oral daily; n = 11), and glimepiride treated (T2D-Gli) (2 mg/kg body weight per oral daily; n = 10). The doses of linagliptin and glimepiride were determined in a pilot experiment that showed glucose-lowering effects without inducing hypoglycemia (Supplementary Fig. 6). Sham-operated T2D animals were also divided in vehicle-treated (n = 5), linagliptin-treated (n = 5), and glimepiride-treated (n = 5) groups. Within the first 4 weeks following stroke, 17 T2D mice were removed from the study for reaching ethical breakpoint (rapid weight decrease of >25% of presurgery weight coupled with dehydration and severe impairment of movement and feeding) (T2D n = 6, T2D-Li n = 6, T2D-Gli n = 5). The remaining stroke-operated (non-T2D n = 11, T2D n = 5, T2D-Li n = 5, T2D-Gli n = 5) and sham-operated (non-T2D n = 5, T2D n = 5, T2D-Li n = 5, T2D-Gli n = 5) animals were sacrificed 8 weeks after the surgery.

Twenty-eight 7-month-old standard diet–fed mice were subjected to stroke. Five mice were removed from the study because of unsuccessful stroke, and three mice died following surgery. Three days after, the remaining animals were randomly assigned to the following experimental groups: vehicle treated (n = 7), linagliptin treated (10 mg/kg body weight per oral daily; n = 7), and glimepiride treated (2 mg/kg body weight per oral daily; n = 6). Animals were sacrificed 8 weeks after stroke. All applicable guidelines for the care and use of animals were followed under ethical approval ID1126 (Karolinska Institutet).

Transient middle cerebral artery (MCA) occlusion (tMCAO) was used to model stroke by the intraluminal filament technique (26). To induce striatal infarct mainly affecting basal ganglia and with minimal cortical/hippocampal damage, the MCA was occluded for 30 min (see Supplementary Material). Sham surgery was performed in the same manner as the MCAO surgery but excluded the occlusion of the MCA by the filament.

Forelimb sensorimotor function was measured using a grip strength meter (Harvard apparatus) before, at 3 days after, and at 1–8 weeks after tMCAO (see Supplementary Material).

The mice were anesthetized and transcardially perfused with 4% ice-cold paraformaldehyde. The brains were then removed and after overnight postfixation, submersed in PBS with 20% sucrose until they sank. The brains were cut into 30-μm-thick coronal sections using a sliding microtome (see Supplementary Material). The Olympus BX51 microscope coupled with computerized setup for stereology (Visiopharm, Hørsholm, Denmark) was used for cell counting, as explained in detail in the Supplementary Material. All procedures were performed by experimenters blinded to experimental groups.

Stroke volume was evaluated on the basis of NeuN staining (see Supplementary Material). NeuN staining is a consistent method for quantifying neural damage because it exclusively stains neurons. Therefore, it is reliable to evaluate neuronal loss even several weeks poststroke, unlike ubiquitous cell markers, such as 3,5-triphenyltetrazolium chloride, that are accurate only within a few days after the injury as a result of later inflammatory cell infiltration and glial scar formation.

Active GLP-1 (K150JWC-1; Mesoscale, Gaithersburg, MD) and plasma DPP-4 activity were quantified by enzyme immunoassay and ELISA, respectively, on the basis of the manufacturer’s instructions.

The data were checked for normality using the Shapiro-Wilk normality test.

For body weight, fasting glucose, and grip strength, two-way repeated-measures ANOVA was used followed by Sidak or Tukey test to compare grip strength between the groups at each time point. To analyze the number of PV⁺, doublecortin-positive (DCX⁺), and ionized calcium-binding adaptor protein-1–positive (Iba-1⁺) cells, two-way ANOVA followed by Tukey test was performed.

For the statistical analysis of stroke volume, PV soma size measurement, CD68⁺, and CD68⁺/Iba-1⁺ colabeled cells, Mann-Whitney and Kruskal-Wallis with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli tests were performed. The following comparisons were used: in the T2D study, non-T2D versus T2D and T2D versus T2D-Li versus T2D-Gli, and in the non-T2D study, vehicle versus linagliptin versus glimepiride.

Data are expressed as mean ± SD. P < 0.05 was considered statistically significant. All data were analyzed using GraphPad Prism 8 software.

The data sets and critical resources supporting the results generated/analyzed during the current study and reported in the article are available from the corresponding authors upon reasonable request.

Forepaw grip strength was similar in non-T2D and T2D mice before tMCAO (Fig. 2A). Stroke significantly decreased forepaw grip strength at day 3 post-tMCAO versus pre-tMCAO in both groups (P < 0.0001), and this decrease was greater in T2D versus non-T2D mice (P < 0.0001). Moreover, non-T2D mice gradually recovered forepaw function over the post-tMCAO period and by 8 weeks, reached the levels close to pre-tMCAO. Recovery of forepaw function was also observed in T2D mice but less than in non-T2D mice (P < 0.0001) and never attained the pre-tMCAO status. The poststroke treatment (from day 3 after stroke) of T2D mice with linagliptin or glimepiride significantly improved forepaw grip strength recovery versus T2D mice from week 4 (P = 0.0329) and 5 (P = 0.0111). After week 5, the improvement was stronger in the T2D-Li–treated group and was significantly greater than in the T2D-Gli group. We recorded no difference in grip strength in the sham groups (Fig. 2D).

After 7 months of HFD feeding, T2D mice had significantly higher (20%) body weight (P < 0.0001) than non-T2D mice (Fig. 2B and Supplementary Fig. 1). After tMCAO and the diet change from HFD to standard diet, body weight in T2D animals rapidly decreased and by 3 days post-tMCAO, T2D mice did not significantly differ from non-T2D mice (Fig. 2B). Contrary to tMCAO-subjected mice, the diet change in sham-operated T2D mice led to a gradual decrease of body weight (first 5 weeks after surgery), while in the sham-operated non-T2D mice, the weight remained stable during the whole study (Fig. 2E).

The poststroke treatment (from day 3 after stroke) with linagliptin or glimepiride did not affect body weight in the poststroke recovery phase compared with the untreated T2D group (Fig. 2B). Moreover, no difference among the three groups was recorded in sham-operated T2D mice (Fig. 2E).

These results show that rapid weight loss (already 3 days poststroke) in T2D mice versus non-T2D mice was a consequence of stroke rather than of the switch from HFD to standard diet and that both linagliptin and glimepiride did not influence this factor. These data also suggest that changes in weight do not correlate with changes in poststroke neurological recovery.

Seven months of HFD led to a significant increase of blood glucose (fasting state) in T2D versus non-T2D mice (P < 0.0001) (Fig. 2C). After tMCAO and the change from HFD to standard diet, blood glucose significantly decreased in the T2D group but remained significantly higher versus non-T2D mice at 4 weeks post-tMCAO (P < 0.0001) (Fig. 2C). However, at 8 weeks after tMCAO, the fasting blood glucose in T2D mice decreased further, reaching the same levels of non-T2D mice (P = 0.839) (Fig. 2C). This effect was accelerated by linagliptin and glimepiride where euglycemia (5–6 mmol/L) was reached already at 4 weeks after tMCAO (P = 0.0002 and P < 0.0001, respectively) (Fig. 2C). In the sham-operated T2D mice, hyperglycemia also gradually decreased as a result of diet change/weight loss but remained significantly higher than in sham-operated non-T2D mice at 4 weeks (P = 0.004) and 8 weeks (P = 0.0395) (Fig. 2F) without reaching normoglycemia. Linagliptin and glimepiride accelerated the lowering of blood glucose, with linagliptin exerting a more pronounced effect already at 4 weeks (P = 0.0053) (Fig. 2F). The stronger effect of both drugs in tMCAO-operated versus sham-operated animals to reduce glycemia (Fig. 2C vs. Fig. 2F) was likely due to a more pronounced reduction of hyperglycemia under stroke versus sham conditions. This was in turn probably caused by the more rapid weight loss induced by stroke compared with sham-operated T2D mice (Fig. 2B vs. Fig. 2E).

These results show that during a large part of the poststroke recovery phase (at least for 4 weeks) T2D mice remain hyperglycemic, and only 8 weeks after stroke they reach similar glycemic levels as non-T2D mice. This effect is significantly accelerated (already at 4 weeks poststroke) by both linagliptin and glimepiride. We conclude that impaired poststroke neurological recovery in T2D mice is not associated with body weight change but rather with the regulation of fasting glycemia.

The extent of ischemic brain damage (determined on the basis of NeuN staining in striatum) was also compared between the groups. However, no significant difference was recorded (Fig. 3A). We also did not record changes in white matter damage (Supplementary Fig. 3).

Iba-1⁺ microglia cells were quantified in the contralateral and ipsilateral striatum at 8 weeks post-tMCAO in T2D and non-T2D mice. The results show an increased number of Iba-1⁺ cells in ipsilateral versus contralateral striatum in both T2D mice (P < 0.0001) and non-T2D mice (P = 0.0006) (Fig. 4A) at 8 weeks after stroke. However, the number of Iba-1⁺ cells was significantly greater in T2D mice (P = 0.0132). Remarkably, both linagliptin and glimepiride abolished this T2D-induced effect (P = 0.0003 and P < 0.0001, respectively) (Fig. 4A).

Microglia/macrophage activation was then analyzed by quantification of CD68⁺ cells. T2D significantly increased the number of CD68⁺ cells in the ipsilateral striatum at 8 weeks post-tMCAO (P = 0.012) (Fig. 4B). Interestingly, this effect was entirely counteracted by linagliptin and glimepiride (P = 0.0097 and P = 0.0421, respectively). We also quantified Iba-1⁺/CD68⁺ double cells to specifically evaluate activated microglia cells in striatum. Here, too, T2D significantly increased the number of activated microglia (P = 0.012) (Fig. 4C), and this effect was reversed by both linagliptin and glimepiride (P = 0.0033 and P = 0.009, respectively). These results suggest that at least 8 weeks after stroke, T2D increases microglia infiltration and activation, and these effects are blocked by both linagliptin and glimepiride treatment.

To investigate the underlying mechanisms of neuronal plasticity after stroke, we took advantage of a recently identified effect by which T2D decreases the cellular soma volume of GABAergic inhibitory interneurons in the peri-infarct striatum after stroke (13). Therefore, the soma volume and number of PV⁺ interneurons in the peri-infarct striatum (Fig. 5C and D) were quantified at 8 weeks after tMCAO. The soma volume of PV⁺ interneurons was significantly reduced in T2D mice versus non-T2D mice (P = 0.0275) (Fig. 5A). Both linagliptin and glimepiride prevented this reduction (P = 0.0103 and P = 0.0103, respectively). The survival of PV⁺ interneurons was reduced ∼50% in each group without significant differences (Fig. 5B). These results suggest that T2D induces PV⁺ interneuron atrophy in the peri-infarct striatum after stroke, and this effect is counteracted by linagliptin and glimepiride.

We assessed the formation of neuroblasts by quantifying the number of DCX⁺ cells and of new mature neurons (neurogenesis) by quantifying NeuN⁺/BrdU⁺ cells in striatum. Eight weeks poststroke, the number of DCX⁺ cells increased in the ipsilateral versus contralateral hemisphere in all groups (Fig. 6A), without a significant difference between T2D and non-T2D mice. However, we observed a greater increase in the number of DCX⁺ cells in T2D-Li mice compared with T2D and T2D-Gli mice in the ipsilateral hemisphere (P = 0.0013 and P = 0.046, respectively) (Fig. 6A). We found no difference between the groups when it came to number of NeuN⁺/BrdU⁺ cells in the ipsilateral striatum (Fig. 6B).

These results show that 8 weeks after stroke, T2D does not affect stroke-induced neurogenesis. Linagliptin increases the early step of neurogenesis by increasing neuroblast formation. However, this effect is not reflected in the formation of mature neurons.

In the non-T2D study, all mice gradually recovered forepaw function after stroke and reached the levels close to prestroke by 8 weeks. However, the recovery was initially accelerated in non-T2D-Li mice from week 1 until week 4 compared with non-T2D-Gli or non-T2D mice (P = 0.0278, P = 0.0229, P = 0.0096, and P = 0.0177, respectively) (Fig. 7A). We recorded no significant differences in body weight and glycemia among the groups (Fig. 7B and C). Stroke brain damage assessed by NeuN staining was also unchanged among the groups (Fig. 7D) as was the neuroinflammation response (number of Iba-1⁺ and CD68⁺ cells) (Fig. 7E–G), early neurogenesis process (number of DCX⁺ neuroblasts) (Fig. 7J), and number and size of PV⁺ cells (Fig. 7H and I). In conclusion, the results of the non-T2D study indicate that linagliptin can accelerate only the early neurological recovery after stroke, and this effect cannot be associated with changes in glycemia, body weight, neuroinflammation, PV atrophy, or neurogenesis.

We show that obesity-induced T2D worsens neurological recovery after stroke in association with hyperglycemia during the stroke recovery phase and with increased stroke-induced neuroinflammation and atrophy of PV⁺ interneurons 8 weeks after stroke. These effects were counteracted by glycemic regulation with DPP-4i or sulfonylurea despite the treatment being started 3 days poststroke. DPP-4i also accelerated early neurological recovery in non-T2D mice without affecting glycemia, body weight, neuroinflammation, PV⁺ interneurons, or neurogenesis.

Clinical studies have shown the detrimental effect of T2D on long-term neurological recovery after stroke (see introduction). However, the majority of people with T2D are obese, and results from some clinical studies have suggested a paradoxical effect of obesity leading to improved prognosis after stroke (see introduction). Therefore, more research, especially in obese patients with T2D, is highly needed. Several preclinical studies have demonstrated the detrimental role of T2D induced by obesogenic diets after stroke (12). However, the majority have focused on the effects of obesogenic diets on acute stroke injury (from 1 day up to 10 days [27–33]). The few addressing long-term recovery (including a recent study from our group [13]) have maintained the obesogenic diet during the recovery phase (34,35). Exposure to an obesogenic diet after stroke does not represent the likely clinical setting where patients with stroke will be put on a balanced nutrition plan. Moreover, in the first days/weeks after hospitalization, patients with stroke often face eating problems because of, among other reasons, dysphagia and/or reduced food intake or appetite (36). Therefore, aiming to characterize the detrimental effect of obesity-induced T2D on stroke recovery, further exposure to an obesogenic diet during the recovery phase might result in misleading conclusions, where the effects of prestroke and poststroke HFD could not be differentiated from each other. Therefore, we designed a preclinical study in which immediately after stroke, we replaced the HFD by a nutritionally balanced standard diet feeding throughout the entire duration of the stroke recovery phase (8 weeks). Under these experimental conditions, we confirmed the detrimental effects of obesity-induced T2D on neurological recovery.

Acute hyperglycemia after stroke in people with and without T2D is common and associated with worse outcomes (37). Moreover, poor glycemic control in people with T2D is associated with an increased risk of stroke and death (38). However, the specific role of chronic hyperglycemia in the stroke recovery phase of patients with obesity/T2D, beyond the first couple of days after stroke, has not been previously investigated. Furthermore, there is a paucity of evidence concerning the effect of interventions extended beyond the acute phase of stroke on functional outcome, and this represents a gap in knowledge that needs to be filled. Our data showing an association of decreased neurological recovery with hyperglycemia during the recovery phase suggest a detrimental role played by hyperglycemia after stroke not only acutely but also chronically. Similar effects have also been shown in the poststroke recovery phase in a type 1 diabetic mouse model (39,40). The potential importance of hyperglycemia in decreased neurological recovery is strongly supported by the fact that poststroke normoglycemia attained by both linagliptin and glimepiride correlated with a significant improvement of neurological recovery. Whether this effect is directly mediated by insulin (neurotrophic/neuroprotective effects) or secondary to reduction of brain damage as a result of the achievement of normoglycemia remains to be investigated. In this context, it is noteworthy that in later phases of recovery, linagliptin showed significantly greater effects than glimepiride and that it was also partly efficacious in non-T2D mice. These additional effects could be attributed to glucose-independent effects perhaps mediated by GLP-1 or additional DPP-4 substrates (41). Indeed, GLP-1 levels and DPP-4 were, respectively, increased and decreased by linagliptin in both studies (Supplementary Figs. 4 and 5). However, the fact that glimepiride was ineffective at improving neurological recovery after stroke in non-T2D mice (and linagliptin was only partially efficacious) supports a central role of plasma glucose level per se and of its normalization in the recorded effects in T2D mice. On the other hand, the possibility that both linagliptin and glimepiride contribute to the improvement of neurological recovery through the normalization of brain insulin resistance/sensitivity cannot be ruled out since HFD induces brain insulin resistance (42) and studies have shown that DPP-4i improves insulin sensitivity also in the brain (43). Thus, studies that address this question are warranted.

Glycemia-independent effects mediated by DPP-4i to increase neurogenesis and neuroplasticity have been shown in the past (44) and previously reviewed (22). On the basis of this literature, we hypothesized that linagliptin (but not glimepiride) could also improve poststroke neurological recovery independently of glycemia regulation. Our findings could not prove this hypothesis but, nevertheless, are not less interesting since they suggest the importance of poststroke glycemia regulation not only acutely but also chronically. Our results suggest that such a therapeutic strategy that is based on the regulation of glycemia could start several days after stroke, and these data perhaps represent the most important finding of this study since they provide the opportunity to select the best suited, lowest risk antihyperglycemic therapy. For example, an important clinical issue to consider when regulating glycemia in patients with T2D is the possibility that such treatments induce hypoglycemic episodes. This must be especially avoided after stroke since hypoglycemia increases CV risks, including recurrent strokes (17). DPP-4is have a very low risk of hypoglycemia in comparison with sulfonylureas (45,46). This class of drugs, therefore, could represent a better option when aiming to efficiently regulate glycemia and enhance poststroke neurological recovery.

Recent large outcome studies using different DPP-4is (22,47) have shown neutral results to reduce CV risk in patients with T2D. The results of our study suggest that future clinical studies should instead investigate the potential of these drugs to improve stroke outcome and long-term recovery as previously suggested (48). Indeed, a large number of animal studies indicated that both DPP-4is and GLP-1 receptor agonists decrease stroke-induced brain damage and facilitate recovery if systemically present close to stroke onset (49). Specifically, our group showed that DPP-4i (but not sulfonylurea) increases the survival of neurons after stroke in a model of obesity-induced T2D (19,41,50). Those studies together with the results presented in the current work suggest that a chronic DPP-4i–mediated therapy both before and after stroke could decrease stroke-induced brain damage and facilitate recovery in support of potential clinical translation.

When investigating potential cellular mechanisms at the basis of the decreased neurological recovery in T2D mice, we identified increased neuroinflammation and cellular atrophy of PV⁺ interneurons. It is known that neuroinflammation plays an important role in stroke recovery (51). While we could not find studies in T2D/obese models where microglia was assessed so late after stroke as in our study (8 weeks), two studies in genetic models of T2D/obesity have shown an aggravated proinflammatory response 2 weeks after stroke that was associated with decreased neurological recovery (11,52). When it comes to GABAergic PV⁺ interneurons, these cells are important contributors to neuroplasticity after injury (23). They specifically provide strong inhibitory control of the pyramidal neuronal activity, and their vulnerability after stroke can deeply affect the excitation/inhibition balance in brain circuits (53). We recently showed that sustained HFD feeding during the stroke recovery phase induces the persistent atrophy of these cells in correlation with decreased neurological recovery (13). In the current study, we confirmed this effect even after the replacement of obesogenic diet with a standard diet for 8 weeks after stroke. Importantly, our results show that both linagliptin and glimepiride completely normalized neuroinflammation and restored the normal cellular volume of PV⁺ interneurons 8 weeks after stroke. Therefore, although correlative, our results suggest that chronic hyperglycemia in the stroke recovery phase may play a detrimental role in stroke pathophysiology by inducing neuroinflammation and impairing neuroplasticity through dysfunctional PV⁺ interneurons. Importantly, these effects may be pharmacologically reversible by achieving normoglycemia during the recovery phase.

There are limitations in this study. The main one is in part justified by our original hypothesis (and thus experimental design) focusing on the glycemia-independent effects mediated by DPP-4i on neurological recovery rather than on the glycemia-dependent effects. Therefore, we did not collect plasma (e.g., weekly) during the stroke recovery phase, which could have provided important information (e.g., insulin or free fatty acids, glycated hemoglobin) to understand the mechanisms behind neurological recovery after stroke. These assessments will be needed in future studies. Neurological recovery assessed solely by measuring the forelimb sensorimotor function also represents a weakness of the study, and the results will have to be confirmed by using additional tests.

In conclusion, there is a high need for clinical studies that specifically address neurological recovery after stroke in patients with obesity/T2D. Our study supports the detrimental role of obesity-induced T2D on long-term recovery through chronic hyperglycemia together with increased neuroinflammation and atrophy of PV⁺ interneurons. The impairment is reversible because it can be strongly counteracted by effective glycemic control obtained by both DPP-4i and sulfonylurea treatments started 3 days poststroke. From a clinical perspective, this finding is very interesting because it provides the opportunity for delayed treatment onset after a stroke event. Since DPP-4i showed accelerated recovery also in non-T2D mice, this class of drugs could improve stroke outcome in patients without T2D as well. Although speculative, our results do not support the obesity paradox in the presence of T2D, and additional studies in obese mice without T2D will need to be performed in the future.

Acknowledgments. The authors thank Dr. Fuad Bahram (Södersjukhuset) for technical assistance and Dr. Hans Pettersson (Karolinska Institutet) for advice on statistical analyses.

Funding. Financial support was provided by Vetenskapsrådet (grant 2018-02483), European Foundation for the Study of Diabetes/Sanofi European Diabetes Research Programme in Macrovascular Complications, Hjärt-Lungfonden (grant 20190298), Diabetesfonden (grant DIA-2019-412), Svensk Förening för Diabetologi, Karolinska Institutet (Foundation for Geriatric Diseases and Karolinska Institutet Stiftelser och Fonder), Stohnes Stiftelse, O.E. och Edla Johanssons Stiftelse, Magnus Bergvalls Stiftelse, STROKE Riksförbundet, and Gamla Tjänarinnor Stiftelse and by the regional agreement on medical training and clinical research (ALF) between the Stockholm County Council and the Karolinska Institutet.

Duality of Interest. This study received funding from Boehringer Ingelheim Pharma GmbH & Co. KG. T.N. has received unrestricted grants from AstraZeneca and consultancy fees from Boehringer Ingelheim, Eli Lilly, Novo Nordisk, Merck, and Sanofi. T.K. is employed at Boehringer Ingelheim Pharma GmbH & Co. KG. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. I.L.A. and H.P. performed behavioral analysis, immunohistochemistry studies, and stereology analysis; acquired and processed images and figures; contributed to the discussion; and wrote the manuscript. M.L. provided expertise, contributed to the discussion, and edited the manuscript. C.K. helped with microglia experiments and edited the manuscript. T.N. provided expertise and resources, contributed to the discussion, and edited the manuscript. T.K. provided resources for the study, coordinated GLP-1 quantification, contributed to the discussion, and edited the manuscript. V.D. conceived and designed the study, performed the stroke experiments and diabetes tests, contributed to the discussion, and wrote the manuscript. C.P. conceived, designed, and coordinated the research plan; contributed to the discussion; and wrote the manuscript. V.D. and C.P. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.