Neuroprotection by leptin in a rat model of permanent cerebral ischemia: effects on STAT3 phosphorylation in discrete cells of the brain

Abstract

In addition to its effects in the hypothalamus to control body weight, leptin is involved in the regulation of neuronal function, development and survival. Recent findings have highlighted the neuroprotective effects of leptin against ischemic brain injury; however, to date, little is known about the role performed by the signal transducer and activator of transcription (STAT)-3, a major mediator of leptin receptor transduction pathway in the brain, in the beneficial effects of the hormone. Our data demonstrate that systemic acute administration of leptin produces neuroprotection in rats subjected to permanent middle cerebral artery occlusion (MCAo), as revealed by a significant reduction of the brain infarct volume and neurological deficit up to 7 days after the induction of ischemia. By combining a subcellular fractionation approach with immunohistofluorescence, we observe that neuroprotection is associated with a cell type-specific modulation of STAT3 phosphorylation in the ischemic cortex. The early enhancement of nuclear phospho-STAT3 induced by leptin in the astrocytes of the ischemic penumbra may contribute to a beneficial effect of these cells on the evolution of tissue damage. In addition, the elevation of phospho-STAT3 induced by leptin in the neurons after 24 h MCAo is associated with an increased expression of tissue inhibitor of matrix metalloproteinases-1 in the cortex, suggesting its possible involvement to the neuroprotection produced by the adipokine.

Figure 1

Systemic (s.c.) administration of leptin at the dose of 1 mg/kg, 3 h before the induction of ischemia, significantly reduces the brain infarct volume produced by 24 h permanent MCAo. By contrast, the same dose of leptin, administered 6 h or immediately before MCAo produces values of infarct volume comparable to those observed in vehicle-treated animals (a). ^##P<0.01 versus vehicle (one-way ANOVA followed by Dunnett's post-hoc test; n=5–6 rats per experimental group). (b) Representative TTC-stained brain slices and (c) corresponding values of infarct areas from rats subjected to 24 h permanent MCAo and treated (s.c.) with vehicle (PBS, 1 ml/kg) or leptin (1 mg/kg) 3 h before the induction of ischemia. Ischemic brain damage (pale area) involves the brain regions supplied by the middle cerebral artery, including the striatum and the cortex. Administration of leptin reduces ischemic brain damage in perifocal regions such as the medial caudate–putamen and the motor cortex. ^*P<0.05 and ^**P<0.01 versus corresponding brain section of vehicle-treated animals (Student's t test, n=6 rats per experimental group)

Figure 2

(a) Brain infarct volume assessed 3 and 7 days after permanent MCAo in rats treated (s.c.) with vehicle (PBS, 1 ml/kg) or leptin (1 mg/kg) 3 h before the induction of ischemia. ^#P<0.05 and ^##P<0.01 versus corresponding brain section of vehicle-treated animals (Student's t test, n=4–6 rats per experimental group). (b) Neuroscore and (c) body weight measured for 7 consecutive days after permanent MCAo in rats treated (s.c.) with vehicle (PBS, 1 ml/kg) or leptin (1 mg/kg) 3 h before the induction of ischemia. ^*P<0.05 versus corresponding score in vehicle-injected animals (Student's t test, n=4 rats per experimental group)

Figure 3

Changes in STAT3 and Akt phosphorylation following focal cerebral ischemia as determined by western blot analysis of phospho-STAT3 (Tyr705; p-STAT3), total STAT3, p-Akt (Ser473; p-Akt) and total Akt performed on brain cortical homogenates from the ipsilateral (I) and contralateral (C) cortex of rats treated with vehicle (Veh) or leptin (Lept) and killed 3 or 24 h after MCAo. The results are representative of three independent experiments. Histograms show the results of the densitometric analysis of the autoradiographic bands; ^*P<0.05 and ^***P<0.001 versus contralateral (C) side. ^#P<0.05 versus Sham 3 h

Figure 4

Changes in STAT3 and Akt phosphorylation following focal cerebral ischemia as determined by western blot analysis of phospho-STAT3 (Tyr705; p-STAT3), total STAT3, p-Akt (Ser473; p-Akt) and total Akt performed on the cytosolic and nuclear fractions of brain cortical homogenates from the ipsilateral (I) and contralateral (C) cortex of rats treated with vehicle (Veh) or leptin (Lept) and killed 3 or 24 h after MCAo. The results are representative of three independent experiments. Histograms show the results of the densitometric analysis of the autoradiographic bands; ^*P<0.05, ^**P<0.01 and ^***P<0.001 versus contralateral (C) side. ^§P<0.05 and ^§§P<0.01 (Leptin versus Vehicle). °P<0.05, °°P<0.01 and °°°P<0.001 (3 h versus 24 h)

Figure 5

Double p-STAT3/NeuN (a–l) and p-STAT3/GFAP (m–t) immunoreactivity in the contralateral (C) and ipsilateral (I) motor (a–p) and frontal (q–t) cortices of rats subjected to 3 h permanent MCAo. Leptin (1 mg/kg, s.c.) or vehicle (PBS, 1 ml/kg) were administered 3 h before the induction of ischemia. Phospho-STAT3 expression in the contralateral hemisphere of leptin-treated rats was not significantly different from the contralateral hemisphere of vehicle-injected animals shown in panel (a). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars=200 μm (a–l) and 100 μm (m–t)

Figure 6

Double p-STAT3/NeuN (a–p) and p-STAT3/GFAP (q–t) immunoreactivity in the ipsilateral motor (a–h), parietal (i–p) and frontal (q–t) cortices of rats subjected to 24 h permanent MCAo. Leptin (L, 1 mg/kg, s.c.) or vehicle (V, PBS, 1 ml/kg) were administered 3 h before the induction of ischemia. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars=100 μm

Figure 7

Changes in TIMP-1 expression following focal brain ischemia as determined by western blot analysis performed on brain cortical homogenates from the ipsilateral (I) and contralateral (C) cortex of rats treated with vehicle (Veh) or leptin (Lept) and killed 24 h after MCAo. The results are representative of three independent experiments. Histograms show the results of the densitometric analysis of the autoradiographic bands; ^**P<0.01 and ^***P<0.001 versus contralateral (C) side. ^§§P<0.01 (Leptin versus Vehicle)