Mesencephalic astrocyte-derived neurotrophic factor reduces ischemic brain injury and promotes behavioral recovery in rats

Abstract

Mesencephalic astrocyte-derived neurotrophic factor (MANF), also known as arginine-rich, mutated in early stage of tumors (ARMET), is a secreted protein that reduces endoplasmic reticulum (ER) stress. Previous studies have shown that MANF mRNA expression and protein levels are increased in the cerebral cortex after brain ischemia, a condition that induces ER stress. The function of MANF during brain ischemia is still not known. The purpose of this study was to examine the protective effect of MANF after ischemic brain injury. Recombinant human MANF was administrated locally to the cerebral cortex before a 60-min middle cerebral artery occlusion (MCAo) in adult rats. Triphenyltetrazolium chloride (TTC) staining indicated that pretreatment with MANF significantly reduced the volume of infarction at 2 days after MCAo. MANF also attenuated TUNEL labeling, a marker of cell necrosis/apoptosis, in the ischemic cortex. Animals receiving MANF pretreatment demonstrated a decrease in body asymmetry and neurological score as well as an increase in locomotor activity after MCAo. Taken together, these data suggest that MANF has neuroprotective effects against cerebral ischemia, possibly through the inhibition of cell necrosis/apoptosis in cerebral cortex.

Fig. 1

Local cortical MANF pretreatment reduces cortical infarction induced by middle cerebral arterial ligation. A: MANF pretreatment, at a dose of 6 μg, reduced cortical infarction area (p=0.029, one-way ANOVA, * indicates p<0.05 Dunnett’s multiple comparison post hoc test). B: Effects of PBS and MANF, 6 μg, on infarction area per slice. The infarction area was significantly reduced in rats treated with MANF 6 μg as compared with PBS treatment (p=0.0008, treatment effect, two-way ANOVA). Arrows represent the sites of injection. C: Local cortical MANF administration reduced the area of infarction in the largest infarction slice. Area of infarction was significantly reduced in MANF (6 ug) treated rats (p=0.0053 one-way ANOVA treatment effect, * indicates p<0.05, **p<0.01 Dunnett’s multiple comparison post hoc test). D: Photomicrograph of first six slices of brains treated either with PBS (left) or MANF 6 μg (right).

Fig. 2

Effect of local cortical MANF administration on cortical TUNEL activity. A: TUNEL pixel density was not altered at a site distal to the MANF injection B: TUNEL pixel density was significantly reduced at the site proximal to MANF injection. C: Photomicrograph from the proximal cortical area of a rat treated with PBS. D: Photomicrograph from the proximal cortical area of a rat treated with MANF. Six sections were counted from each rat.

Fig. 3

MANF, 6 μg, pretreatment promotes behavioral recovery after cortical infarction induced by middle cerebral arterial ligation. Days 2, 7 and 14 indicates the day of the experiment after the stroke surgery. A: Statistically significant difference in total distance traveled, *** p<0.001, Student-Newman-Keuls. B Distance traveled in every 10 min is significantly increased after MANF injection in stroke rats. (*p=0.011, 2-way ANOVA). C: Statistically significant difference in body swing, *** p<0.001, Student-Newman-Keuls. D: Statistically significant difference in Bederson’s score, * p<0.05, Student-Newman-Keuls.

Fig. 4

Effects of PBS and MANF on cerebral blood flow (CBF). A: MANF did not alter CBF before MCAo. Δ CBF = (CBF at post-injection − basal CBF) / basal CBF × 100%. No difference in Δ CBF was found between the animals treated with PBS (15.2±8.7 %, n=5) and MANF (13.8±14.1%, n=6). B: MANF did not alter CBF during MCAo. Δ CBF = (CBF during MCAo − CBF before MCAo) / CBF before MCAo × 100%. No difference in Δ CBF was found between the animals treated with PBS (81.2±4.6%, n=5) and MANF (80.7±3.5%, n=6). C: A real time tracing of cerebral blood flow during MCAo in a rat pretreated with PBS.