Endocrine regulator rFGF21 improves neurological outcomes following focal ischemic stroke of type 2 diabetes male mice

Abstract

Background and Purpose:

The complexity and heterogeneity of stroke as well as the associated co-morbidities may render neuroprotective drugs less efficacious in clinical practice. Therefore, development of targeted therapies to specific patient subsets has become a high priority in translational stroke research. Ischemic stroke with type 2 diabetes (T2D) have a nearly double mortality rate and worse neurological outcomes. In the present study we tested our hypothesis that recombinant human fibroblast growth factor 21 (rFGF21) administration is beneficial for improving neurological outcomes of ischemic stroke with T2D.

Methods:

Type 2 diabetes db/db and non-diabetic genetic control db/+ mice were subjected into permanent focal ischemia of distal middle cerebral artery occlusion (dMCAO), we examined the effects of post stroke administration with rFGF21 in systemic metabolic disorders, inflammatory gatekeeper PPARγ activity at 3 days, mRNA expression of inflammatory cytokines and microglia/macrophage activation at 7 days in the peri-lesion cortex, and lastly neurological function deficits, ischemic brain infarction and white matter integrity up to 14 days after stroke of db/db mice.

Results:

After permanent focal ischemia, diabetic db/db mice presented confounding pathological features including metabolic dysregulation, more severe brain damage and neurological impairment, especially aggravated pro-inflammatory response and white matter integrity loss. However, daily rFGF21 treatment initiated at 6 hours after stroke for 14 days significantly normalized systemic metabolic disorders, rescued PPARγ activity decline, inhibited pro-inflammatory cytokine mRNA expression and M1-like microglia/macrophage activation in the brain. Importantly rFGF21 also significantly reduced white matter integrity loss, ischemic brain infarction, and neurological function deficits up to 14 days after stroke. The potential mechanisms of rFGF21 may in part consist of potent systematic metabolic regulation and PPARγ-activation promotion-associated anti-proinflammatory roles in the brain.

Conclusions:

Taken together, these results suggest rFGF21 might be a novel and potent candidate of the disease-modifying strategy for treating ischemic stroke with T2D.

INTRODUCTION

The complexity and heterogeneity of stroke as well as the associated co-morbidities may render neuroprotective drugs less efficacious in clinical practice ¹. Therefore, development of targeted therapies to specific patient subsets has become a high priority in translational stroke research ². Diabetic patients are 2–6 times more susceptible to ischemic stroke; about 30% of stroke patients are diabetic and more than 90% of them comprise type 2 diabetes (T2D)³. T2D stroke patients have nearly double mortality rates, worse neurological outcomes, and respond less favorably to tPA therapy due to the higher risk of hemorrhagic transformation^{4, 5}.

All diabetic stroke patients present post-stroke hyperglycemia and other metabolic disorders ⁶. From a translational perspective, development of new compounds with multiple pharmacological functions, particularly targeting T2D stroke-related metabolic dysregulation, ischemic brain damage and detrimental pro-inflammation, called disease-modifying approaches, would be novel and more effective for treating ischemic stroke with T2D ⁷. Fibroblast growth factor 21 (FGF21) might be one of optimal therapeutic candidates.

Fibroblast growth factor 21 (FGF21) is an endocrine member of the FGF family. It has a potent and central role in glucose and lipid metabolism, as well as in energy balance ⁸. Human FGF21 (MW: 19.5 kD) is highly homologous to mouse FGF21 (~75% identity) ⁹. The very low heparin-binding affinity makes FGF21 capable of crossing the blood-brain barrier (BBB) by simple diffusion ¹⁰. FGF21 exerts potent and multi-pleiotropic metabolic actions ^{11, 12}, with benefits that have been translated from rodents to obese humans with type 2 diabetes without causing side effects such as mitogenicity and hypoglycemia ^{13, 14}.

Growing experimental findings have demonstrated FGF21 is also a mediator of adaptive responses to tissue injury and repair in various pathological conditions ^{12, 15, 16}. In the present study we propose that FGF21 is a novel therapeutic candidate with both adaptive and protective pharmacological actions that protect against both metabolic and stroke stresses in T2D, that result in improvement of neurological outcomes. Using type 2 diabetes animal model, db/db mice and non-diabetic genetic control db/+ mice, we tested our hypothesis that recombinant human FGF21 (rFGF21) administration is beneficial for improving neurological outcomes of T2D stroke mice. Our experimental results are highly supportive to the hypothesis, and demonstrate that rFGF21 might be developed as a novel and potent disease-modifying approach for treating ischemic stroke with T2D.

MATERIALS AND METHODS

Details of materials and experimental procedures are available in the online-only Data Supplement. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Focal ischemia stroke animal model and experimental groups

10 weeks old male BKS.Cg-Dock7m +/+ Lepr db/J homozygous (db/db) mice, weighing 40 to 50 g, and their age-matched non-diabetic counterparts BKS-Cg-Dock7m +/+ Lepr db/J heterozygous (db/+) mice, weighing 25 to 30g, were purchased from Jackson Laboratory. Focal ischemic stroke was achieved with coagulation of the distal middle cerebral artery (dMCA), followed by 90 min bilateral common carotid artery (CCA) ligation as previously described with slight modifications ¹⁷. Animals were subcutaneously administrated with either 1.5mg/kg recombinant FGF21 or saline initiated at 6 hours after stroke, followed by twice daily subcutaneous injections (1.5mg/kg per time) with an interval of 10–12 hours for up to 14 days. All experiments were performed following protocols approved by Massachusetts General Hospital Institutional Animal Care and Use Committee in compliance with the NIH Guide for the Care and Use of Laboratory Animals. All experimental assessments were performed by investigators who were blind to the experimental groups.

Measurements of body weight, blood glucose, Hb1c (glycated hemoglobin), insulin and adiponectin levels

Detials are provided in the online-only Data Supplement.

Neurobehavioral assessments

The following sensorimotor function deficits were assessed before, and post-stroke day 1, 3, 5, 7 and 14. (1) Adhesive removal test result was represented by the ‘time-to-contact’ and ‘time-to-remove’ of each forepaw that was recorded, respectively as previously described ¹⁸. (2) Grip strength test result was quantified as percentage of each individual pre-ischemic baseline as previously described ¹⁹. (3) Foot fault test result was quantified as percentage of foot faults referring to the total steps as previously described ²⁰. Additionally, cognitive function deficit was assessed at post stroke day 14 by Y-maze test, and quantified with the percentage of alternations as we previously described²¹.

Reverse transcription and real-time polymerase chain reaction assay

Peri-infarct cortical tissues or corresponding cortical tissues in sham operated group were collected at 7 days after stroke and stored at −80°C until processing. Total RNA was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen) and real-time polymerase chain reaction (PCR) was performed as we previously described. Further detials are provided in the online-only Data Supplement.

Measuremment of PPARγ activity with Electrophoretic mobility shift assay (EMSA)

We performed EMSA by following a standard protocol as we previously described ²². Further detials are provided in the online-only Data Supplement.

Measurements of brain infarction and axon myelination level

At 14 days after stroke, brain sections were stained with hematoxylin & eosin for quantifing ischemic brain infarction size as we previously described ²³. Axon myelination level was detected with the luxol fast blue staining and quantified as previously described with slight modification²⁴. Briefly, myelination levels of external capsule were evaluated using quantitative scoring method. It assigned 4 for mice with sham operation (having normal myelin levels in external capsule area), whereas a fully demyelinated external capsule area was given a score of 0. Sections were measured and scored by two independent individuals who were blinded to the experimental group assignment.

Immunohistochemistry analysis

Immunohistochemistry was conducted using the standard protocol as we described previously ²¹. Further detials are provided in the online-only Data Supplement.

Statistical analysis

All data are expressed as mean ± SEM. For parametric and continuous variable measurements, we use analysis of variance followed by Tukey-Kramer post hoc tests. For nonparametric ordinal data such as neurobehavioral tests, we use nonparametric Kruskal-Wallis followed by post hoc Mann-Whitney tests. Overall, P<0.05 was considered significant.

RESULTS

1. rFGF21 redresses hyperglycemia, insulin resistance and adiponectin decline in diabetic db/db mice after focal stroke

After stroke, db/db mice maintained stable hyperglycemia (but body weight was not altered), had an elevated blood HbA1c level, hyperinsulinemia, and lowered serum adiponectin level (Figure 1). However, rFGF21 treatment significantly reduced blood glucose level by about 63% reduction (Figure 1A), and lowered HbA1c level from 8.9% to 7.1% (Figure 1B). The post-stroke hyperinsulinemia was also significantly diminished by rFGF21 treatment (71% reduction) (Figure 1C). Moreover, the stroke-induced lower serum adiponectin level was significantly rescued by rFGF21 treatment (60% increase) (Figure 1D). These data suggest that rFGF21 has potent therapeutic effects for metabolic improvement in hyperglycemia, post-stroke hyperinsulinemia and hypoadiponectinemia of db/db stroke mice. In a separate experiment we also tested rFGF21 effects in non-diabetic db/+ mice. There was no significant difference in both blood glucose levels and body weight of non-diabetic db/+ mice after ischemic stroke between rFGF21 treated and non-treated group , and rFGF21 did not cause hypoglycemia, indicating a relative safe profile of rFGF21 administration after stroke (Supplemental Figure I).

Figure 1. rFGF21 redresses hyperglycemia, insulin resistance and adiponectin decline in diabetic db/db mice after focal stroke.

Mice were continuously monitored for 14 days after stroke. A. Blood glucose levels and body weight changes. B. Blood HbA1c levels. C. Serum insulin concentrations. D. Serum adiponectin concentrations. Data are expressed as mean ± SE, n=8 per group (serum insulin and adiponectin) or 12 per group (blood glucose, HbA1c and body weight). *P＜0.05 stroke group versus sham control group; #P＜0.05 db/db stroke group versus db/+ stroke group; &P＜0.05 db/db stroke + rFGF21 group versus db/db stroke group; $P＜0.05 db/+ sham group versus db/db sham group.

2. rFGF21 attenuates neurological function deficits and reduced ischemic brain lesion size in diabetic db/db mice after focal stroke.

As expected, both db/+ stroke mice and db/db stroke mice showed significant deficits up to 14 days after stroke. The sensorimotor function deficits in db/db stroke mice were significantly worse than the non-diabetic db/+ stroke mice. Most importantly, rFGF21 treatment dramatically attenuated deficits in all three sensorimotor function tests of db/db stroke mice (Figure 2A).

Figure 2. rFGF21 attenuates neurological function deficits and brain infarct size in diabetic db/db mice after focal stroke.

A. Sensorimotor functions were assessed at 0, 1, 3, 5, 7 and 14 days post-stroke. Adhesive Removal Test consists of time to contact the adhesive tape (seconds), and time to remove the adhesive tape (seconds). Other two tests were Grip Strength Test and Foot Fault Test. B. Y-maze test was performed at 14 days post-stroke, the results were recorded as total number of arm entries, and alternations. Data are expressed as mean ± SE, n=12 per group. C. At 14 days after stroke, representative H&E staining images of coronal sections from db/+ mice, db/db mice and db/db mice treated with rFGF21 that show infarct area delineated by black dashed lines, and the infarct volume was quantified. D. Infarct area in eight consecutive coronal sections with 1 mm interval. Data are expressed as mean ± SE, n=6 per group. *P＜0.05 stroke versus sham; #P＜0.05 db/db stroke versus db/+ stroke; &P＜0.05 db/db stroke + rFGF21 versus db/db stroke.

The db/db sham mice were prone to motionlessness or moved slowly in Y-maze test. At 14 days after stroke, the further reduced total number of arm entries and lowered alternation ratio in db/db mice were all significantly rescued by rFGF21 treatment (Figure 2B). These results suggest a potent therapeutic potential of rFGF21 treatment for improving neurological function after ischemic stroke in T2D.

Ischemic brain infarct size was measured at 14 days after stroke. The db/db stroke mice had a significantly larger infarct (6.6% of contralateral side) compared to the db/+ stroke mice (2.1% of contralateral side), very excitingly rFGF21 significantly reduced infarct size of db/db stroke mice (44.1% reduction) (Figure 2C). We also found the 3rd coronal section (+1.3mm from bregma) represented the location of maximal cortical change in infarction after rFGF21 treatments, thus the 3rd coronal section was selected for histological analysis in this study (Figure 2D). There was no mortality in all stroke groups.

We also tested the effects of rFGF21 in neurological outcomes and ischemic brain lesion size of non-diabetic db/+ mice. rFGF21 treatment did not alter neurological functions up to 14 days after stroke (Supplemental Figure IIA), while slightly reduced brain infarction size in non-diabetic db/+ mice (12.5% reduction), but it was not statistically significant (Supplemental Figure IIB, IIC).

3. rFGF21 enhances nuclear transcriptional factor PPAR-γ DNA binding activity in diabetic db/db mice after focal stroke

The PPAR-γ DNA binding activity was significantly decreased at 3 days after stroke in both db/+ stroke (36.6% reduction) and db/db stroke mice (41.1% reduction). Importantly, rFGF21 significantly promoted the PPAR-γ DNA binding activity of db/db mice (34% increase) (Figure 3). These results demonstrate the dampened PPAR-γ activity in db/db mouse brains after stroke can be significantly elevated by post-stroke administration of rFGF21.

Figure 3. rFGF21 enhances nuclear transcriptional factor PPAR-γ DNA binding activity in diabetic db/db mice after focal stroke.

At 3 days post-stroke, DNA binding activity of transcriptional factor PPAR-γ was measured by electrophoresis motility shift assay (EMSA) in nuclear fractions. A. Representative image of EMSA gel. Loaded samples were lane 1–3: db/+ sham, lane 4: db/+ stroke, lane 5: db/db sham, lane 6: db/db stroke, lane 7: db/db stroke plus rFGF21. B. Quantification by densitometry of specific PPAR-γ DNA binding bands. Data are expressed as mean ± SE, n=7 per group. *P＜0.05 stroke versus sham; &P＜0.05 db/db stroke + rFGF21 versus db/db stroke.

4. rFGF21 suppresses pro-inflammatory cytokine expression in peri-lesion cortex of diabetic db/db mice after focal stroke

At 7 days after stroke, mRNA expression of inflammatory genes in the cortical peri-lesion area were examined by RT-PCR. We did not detect any significant differences between db/+ sham and db/db sham mice (Figure 4). However in four selected pro-inflammatory genes, ischemic stroke only significantly increased TNF-α and CCL3 mRNA expression in db/+ mice. While in db/db stroke mice, all four pro-inflammatory genes TNF-α, IL-1β, TLR4 and CCL3 mRNA expressions exhibited significantly higher levels, suggesting more dominant pro-inflammatory response in the ischemic brain of db/db stroke mice. Importantly, rFGF21 treatment significantly reduced the stroke-elevated pro-inflammatory gene mRNA expressions, demonstrating a potent inhibiting effect for pro-inflammation in the ischemic brain of T2D db/db mice (Figure 4A). In contrast, ischemic stroke significantly increased four anti-inflammatory cytokines or trophic factors mRNA levels for IL-4, IL-10, IFG-1 and TGF-β in both db/+ and db/db mice. Interestingly, there was a significant lower mRNA expression of IL-4 and IL-10 in the db/db stroke mice compared to db/+ stroke mice. Importantly, rFGF21 treatment to the db/db stroke mice significantly upregulated all four selected mRNA expressions of IL-4, IL-10, IFG-1 and TGF-β (Figure 4B). These data suggested there might be an impairment of anti-inflammatory and neuro-repair responses in the db/db mouse brains after stroke, which may be significantly restored by rFGF21 treatment.

Figure 4. rFGF21 suppresses pro-inflammatory cytokine expression in peri-lesion cortex of diabetic db/db mice after focal stroke.

Peri-infarct tissues were collected at 7 days post-stroke for RT-PCR assay. A Relative fold changes of pro-inflammatory cytokines/chemokines mRNA expression including: TNF-α, IL-1β, TLR4 and CCL3. B. Relative fold change of anti-inflammatory cytokines/tropic factors mRNA expression including: IL-4, IL-10, IGF-1 and TGF-β. Data are expressed as mean ± SE, n=5 per group. *P＜0.05 stroke versus sham; #P＜0.05 db/db stroke versus db/+ stroke; &P＜0.05 db/db stroke + rFGF21 versus db/db stroke.

5. rFGF21 suppresses pro-inflammatory microglia/macrophage activation in peri-lesion area of diabetic db/db mice after focal stroke

At 7 days after stroke, immunohistochemistry was performed to determine microglia/macrophage activation by counting Iba-1 positive cells, and M1-like pro-inflammatory microglia/macrophage by counting Iba-1/CD16 double positive cells. In the peri-lesion cortex, ischemic stroke significantly increased the Iba-1 positive cells and Iba-1/CD16 double positive cells; this increase was significantly higher in db/db mice than db/+ mice. However, rFGF21 almost completely eliminated the increases (Figure 5A, 5B) In the subcortex striatum areas, Iba-1 positive cell number was significantly increased in both db/+ and db/db stroke mice, but significantly higher Iba-1/CD16 double positive cells were only detected in db/db stroke mice. The increase in Iba-1/CD16 double positive cells can be significantly reduced by rFGF21 treatment (Figure 5C). Next, we determined M2-like anti-inflammatory microglia/macrophage by counting Iba-1/CD206 double positive cells. There were very low levels of Iba-1/CD206 double positive cells and no significant differences could be detected between groups. However, rFGF21 treatment significantly increased the Iba-1/CD206 double positive cells in peri-lesion cortex of db/db stroke mice (Figure 5D). These results further demonstrated a distressed pro-inflammatory response in the brain of db/db stroke mice; suppressing M1-like microglia/macrophage activation might be one of the anti-detrimental neuroinflammation mechanisms of rFGF21.

Figure 5. rFGF21 suppresses pro-inflammatory microglia/macrophage activation in peri-lesion area of diabetic db/db mice after focal stroke.

Mice were sacrificed at 7 days post-stroke and brain sections were stained for Iba1 or Iba1/CD16 double, or Iba1/CD206 double positive cells. A. In cortical and striatal peri-infarct area, representative immunofluorescence images of Iba1 or/and CD16 double positive cells. B. Number of Iba1 positive (Iba1⁺) or Iba1 and CD16 double positive cells (Iba1⁺ CD16⁺) in cortical peri-infarct area were quantified as % of db/+ sham control mouse brains per square millimeter. C. Numbers of Iba1 positive (Iba1⁺) or Iba1 and CD16 double positive cells (Iba1⁺ CD16⁺) in striatal peri-infarct area were quantified as % of db/+ sham control mouse brains per square millimeter. D. In cortical peri-infarct area, representative immunofluorescence images of Iba1 or/and CD206 double positive cells. Numbers of Iba1 and CD206 double positive cells (Iba1⁺ CD206⁺) were quantified as % of db/+ sham control mouse brains per square millimeter. Merged images under high magnification represent co-localization (white arrow) of Iba1 and CD16/32 or CD206, respectively. Scale bar = 50μm. Data are expressed as mean ± SE, n=6 per group. *P＜0.05 stroke versus sham; #P＜0.05 db/db stroke versus db/+ stroke; &P＜0.05 db/db stroke + rFGF21 versus db/db stroke.

6. rFGF21 ameliorates white matter injury in diabetic db/db mice after focal stroke

White matter integrity after stroke was examined at 14 days after stroke. Axonal injury was examined by immunohistochemistry of myelin basic protein (MBP) expression, and myelination score was determined by luxol fast blue staining at peri-lesion external capsule. Ischemic stroke significantly decreased more MBP expression (MBP loss) in db/db mice than db/+ mice, however we only detected a significant decrease of myelination score in db/db stroke mice. Very importantly, rFGF21 significantly rescued MBP expression decrease (Figure 6A, 6C), and elevated myelination score in db/db stroke mice (Fiure 6B, 6D). These results suggest an aggravated white matter integrity loss in db/db stroke mice, where rFGF21 significantly promoted white matter integrity by attenuating the axonal injury while promoting myelination, which might be in part responsible to the improved neurological outcomes.

Figure 6. rFGF21 ameliorates white matter injury in diabetic db/db mice after focal stroke.

Mice were sacrificed at 14 days post-stroke and coronal brain sections were stained for myelin basic protein (MBP) expression by immunohistochemistry, or stained with Luxol fast blue dye. A. Representative MBP immunofluorescence images. Loss of MBP displayed as dark area within the external capsule area adjacent to infarct core. B. Representative images of Luxol fast blue stained brain sections. Black arrow indicates the myelination of external capsule after stroke in each group of mice. C. Quantification of MBP fluorescence intensity within the external capsule area adjacent to the infarct core. MBP levels were expressed as % of average fluorescence intensity of db/+ sham mice. Scale bar = 20μm. C: cortex, EC: external capsule (the area between two dashed white lines), S: striatum, and IA: infarct area. D. Myelination levels were semi-quantified as myelination score. Data are expressed as mean ± SE, n=6 per group. *P＜0.05 stroke versus sham; #P＜0.05 db/db stroke versus db/+ stroke; &P＜0.05 db/db stroke + rFGF21 versus db/db stroke.

DISCUSSION

The major findings of the present experiments can be summarized that (i) compared with non-diabetic db/+ mice, diabetic db/db mice presented some of confounding pathological features of ischemic stroke including metabolic dysregulation, more severe brain damage and neurological impairment, and in particular aggravated pro-inflammatory response and white matter integrity loss. (ii) The db/db mice that received rFGF21 at 6 hours after stroke for 14 days showed significantly normalized systemic metabolic disorders, rescued anti-inflammatory gatekeeper PPARγ activity, inhibited pro-inflammatory cytokines mRNA expression and microglia/macrophage activation after stroke in the peri-lesion cortex. (iii) rFGF21 administration also significantly reduced white matter integrity loss, ischemic brain infarction, and neurological function deficits up to 14 days after stroke. These results demonstrate that rFGF21 might be a novel and potent candidate of the disease-modifying approach for treating ischemic stroke with T2D.

The most unique biological feature of FGF21 is its ability to exert potent and multi-pleiotropic metabolic effects ^{11, 12}, such as improvements in hyperglycemia, insulin resistance, lipid profiles and adiponectin production in obese and diabetic animals ^25–27. We found db/db stroke mice presented hyperglycemia, elevated post-stroke hyperinsulinemia and hypoadiponectinemia, each of these metabolic disorders has been demonstrated independently to be associated with poor clinical outcome after stroke ²⁸. Importantly, rFGF21 treatment, even when initiated at 6 hours after stroke remarkably eliminated these metabolic dysregulations. These potent metabolic modulation effects of rFGF21 may be one of its beneficial mechanisms for improving neurological outcomes after stroke in the db/db mice.

It is very important to determine whether this endocrine regulator rFGF21 is beneficial, or might cause detrimental hypoglycemia to the ischemic stroke control of non-diabetes. Experimental results showed there were no hypoglycemia and body weight decline during the 14 days treatment period of rFGF21 after stroke. Furthermore, rFGF21 treatment did not significantly alter neurological functions, but slightly reduced brain infarct size. These results suggest that rFGF21 is relatively safe and dose not induce hypoglycemia. However, the neutral results of rFGF21 treatment in long term outcomes of non-diabetes stroke mice might be in part due to the relatively smaller infarction and mild neurological deficits of the distal middle cerebral artery occlusion model, and relatively smaller sample size applied in current study ²⁹. The precise roles and mechanisms of rFGF21 in ischemic stroke outcomes of non-diabetes need to be fully investigated in future experiments including other ischemic stroke animal models. Importantly, in contrast to the protective effects of rFGF21 in type 2 diabetic db/db stroke mice, these results obtained from non-diabetic mice are also supportive to our hypothesis that rFGF21 is a novel and specific disease-modifying approach for treating ischemic stroke with T2D.

We next explored effects and potential mechanisms of rFGF21 in the brain of diabetic stroke mice. It has been known that a very low heparin-binding affinity makes FGF21 capable of crossing the BBB by simple diffusion ¹⁰. Our previous study showed subcutaneous injection with rFGF21 significantly increased its concentration in CSF, and phospho-FGFR1 (p-FGFR1) levels in mouse brain tissues, demonstrating rFGF21 could cross BBB and functionally activate FGF21-FGFR1 signaling in the brain²¹. Very interesting, FGF21 has been reported as a key mediator involving in the physiological and pharmacological actions of PPARγ ³⁰. PPARγ is a nucleus receptor that transcriptionally regulate gene expressions, especially functions as a master gatekeeper of cytoprotective stress response in brain injury and repair after stroke mainly by suppressing pro-inflammatory and neurodegenerative responses³¹. Since ischemic stroke in diabetic animals may exacerbate or sustain the pro-inflammatory response ^{5, 32}, thus we measured early transcriptional regulator PPARγ activity at 3 days after stroke, yet examined its downstream signaling pathways microglia/macrophage activation, and expression of inflammatory factors in the brain at late 7 days after stroke in type 2 diabetes db/db mice. However, the precise temporal causal relationship between PPARγ activity and inflammatory factor expression remains to be carefully elucidated.

We found a significant decrease of PPARγ DNA-binding activity at 3 days after stroke in peri-lesion cortex of T2D mice, but it was significantly elevated by post stroke administration of rFGF21. Although as a transcriptional factor, how the rFGF21-activated PPARγ regulate its targeting signaling pathways remain to be elucidated; accumulating experimental evidences have supported the critical roles of PPARγ activity for improving the chances of cellular survival and recovery of homeostatic equilibrium after ischemic stroke³¹.

PPARγ functions as a major transcriptional factor that inhibits pro-inflammatory responses after ischemic stroke³¹. Experimental evidence has demonstrated that in acute and subacute phase after ischemic stroke, the higher pro-inflammatory or lower anti-inflammatory cytokine expression profiles represent a relatively more detrimental environment in the ischemic brain that might contribute to the secondary brain tissue damage and neurorepair impairment after stroke ³². Our experimental data showed highly elevated M1-like pro-inflammatory microglia/macrophage activation in the brain of T2D db/db mice after stroke, which is consistent with the increased mRNA expression of pro-inflammatory cytokines. Very importantly, the potent effects of rFGF21 on inhibition of pro-inflammatory cytokine mRNA expression and M1-like microglia/macrophage activation, and elevation of anti-inflammatory gene mRNA expression, facilitate a strong anti-neuroinflammation role, and may be one of key underlying mechanisms for improving neurological outcomes in T2D stroke. The anti-neuroinflammation action of rFGF21, might be in part due to PPARγ activation promotion.

However, we can not exclude the possibility that rFGF21 might be able to suppress systemic inflammation and brain infiltration of inflammatory cells in diabetic stroke mice, thereby contribute to the promotion of neurological function recovery. Since in our previously published study, we showed rFGF21administration significantly reduced mRNA levels of pro-inflammatory cytokines IL-1β, IL-6, and TNFα in white adipose tissue, and completely eliminated the elevated blood plasma cytokine levels of TNFα and IL-1β, demonstrating a potent anti-systemic inflammation effect of rFGF21 in the high-fat diet (HFD) consumption-induced obese mouse model ²¹. However this systemic anti-inflammatory effects and mechanisms of rFGF21 in focal ischemic stroke of type 2 diabetes warrant further investigation.

Interestingly, white matter injury induced by ischemic stroke inside and beyond stroke lesions is one of the clinically pathological features of T2D, which has been considered one of the key mechanisms for worse stroke damage and poorer recovery ³³. A previous experimental study showed PPARγ activation by rosiglitazone treatment that facilitated microglial polarization toward the beneficial M2 phenotype, was considered at least in part, a contributor to the improvement of white matter integrity after cerebral ischemia in mice ³⁴. Importantly, we found rFGF21 significantly promoted white matter integrity by attenuating the axonal injury and increase myelination levels, which might be partially contributed to rFGF21 treatment-mediated PPARγ activation, and one of important underlying mechanisms for the improved neurological outcomes.

Although our experimental findings are both fundamentally and translationally significant, several limitations still exist. First, in this study we used C57BLKS-Leprdb type 2 diabetes mice (db/db T2D mice, Jackson Lab). We are aware that although the leptin receptor mutation does not reflect disease etiology in humans, this model has already given us insight into glucose metabolism and identified novel pathways of its complications ³⁵. However, there are variable pathogenic mechanisms between different T2D animal models ³⁶; testing rFGF21 in other animal models of diabetes with ischemic stroke should be pursued in the future. Second, in the present study, we only tested rFGF21 effects in PPARγ activation, cytokine mRNA expression and microglia/macrophage activation after focal stroke of T2D mice. However, our results suggested that rFGF21-mediated PPARγ activation might in part contribute to the proinflammatory inhibition in the brain of T2D mice. The causal relation and significance of the rFGF21-PPARγ activation in modulating neuroinflammation of T2D stroke needs to be defined by pharmacological and genetic approaches in future investigation. Third, there are multiple pathological factors that dynamically and interactively participate in T2D stroke brain damage evolution and recovery processes ³². Thus it would be very important to understand how rFGF21 may pharmacologically modulate these individual pathological mechanisms of tissue injury and repair, which requires more investigations ³⁷. Moreover, we acknowledge that it is likely impossible to truly separate each mechanism or signaling pathway of rFGF21, however, the multi-targeted property of rFGF21 might be a translational strength ¹⁵. Fourth, due to lack of available working antibody for immunohistochemistry to detect the active form of FGF21 receptor FGFR1 (phosphorylation-FGFR1), we were unable to define brain regions and cellular types for FGF21-FGFR1 activation that taking place, however spatial and time profile of exogenous FGF21-mediated receptor activation and consequent biological signaling pathways in the context of neurovascular unit warrant further investigation ³⁸. Fifth, since stroke patients while in hospital are treated with glucose lowering medications, it would be important to have them as rFGF21 controls, which was missing in present study. New experiments including the glucose lowering medication controls would be very informative to gain a broader cross-comparison of clinically relevant anti-diabetic agent. Lastly, this study was proposed as a proof-of-concept investigation, all translational aspects, include utilizing FGF21 analog, testing and comparing both male and female animals, should be further tested in a well-controlled preclinical translational setting ^{14, 37}.

In conclusion, we demonstrate rFGF21 administration is beneficial for improving neurological outcomes in T2D stroke mice. The potential mechanisms are, at least in part, by systemic metabolic modulation, brain tissue PPARγ activation-mediated pro-inflammation inhibition and whiter matter integrity promotion. rFGF21 might be developed as a novel and potent disease-modifying approach for treating ischemic stroke with T2D.