Endocrine Regulator rFGF21 (Recombinant Human Fibroblast Growth Factor 21) Improves Neurological Outcomes Following Focal Ischemic Stroke of Type 2 Diabetes Mellitus Male Mice

Abstract

Background and Purpose- The complexity and heterogeneity of stroke, as well as the associated comorbidities, may render neuroprotective drugs less efficacious in clinical practice. Therefore, the development of targeted therapies to specific patient subsets has become a high priority in translational stroke research. Ischemic stroke with type 2 diabetes mellitus has a nearly double mortality rate and worse neurological outcomes. In the present study, we tested our hypothesis that rFGF21 (recombinant human fibroblast growth factor 21) administration is beneficial for improving neurological outcomes of ischemic stroke with type 2 diabetes mellitus. Methods- Type 2 diabetes mellitus db/db and nondiabetic genetic control db/+ mice were subjected into permanent focal ischemia of distal middle cerebral artery occlusion, we examined the effects of poststroke administration with rFGF21 in systemic metabolic disorders, inflammatory gatekeeper PPARγ (peroxisome proliferator-activated receptor γ) activity at 3 days, mRNA expression of inflammatory cytokines and microglia/macrophage activation at 7 days in the perilesion cortex, and last 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 proinflammatory 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 proinflammatory 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 antiproinflammatory 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 type 2 diabetes mellitus.

Keywords: diabetes mellitus; inflammation; mice; stroke; white matter.

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.

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.

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.

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.

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.

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.

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.

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.