Efficacy of leukemia inhibitory factor as a therapeutic for permanent large vessel stroke differs among aged male and female rats

Abstract

Preclinical studies using rodent models of stroke have had difficulty in translating their results to human patients. One possible factor behind this inability is the lack of studies utilizing aged rodents of both sexes. Previously, this lab showed that leukemia inhibitory factor (LIF) promoted recovery after stroke through antioxidant enzyme upregulation. This study examined whether LIF promotes neuroprotection in aged rats of both sexes. LIF did not reduce tissue damage in aged animals, but LIF-treated female rats showed partial motor skill recovery. The LIF receptor (LIFR) showed membrane localization in young male and aged rats of both sexes after stroke. Although LIF increased neuronal LIFR expression in vitro, it did not increase LIFR in the aged brain. Levels of LIFR protein in brain tissue were significantly downregulated between young males and aged males/females at 72 h after stroke. These results demonstrated that low LIFR expression reduces the neuroprotective efficacy of LIF in aged rodents of both sexes. Furthermore, the ability of LIF to promote motor improvement is dependent upon sex in aged rodents.

Keywords: Aging; Cytokine; Ischemia; Neuroprotection; Stroke.

Figure 1.

Kaplan-Meier curves show survival among (A) aged male rats and (B) aged female rats (sham-operated, PBS-treated, and LIF treated up to 72 h after MCAO. There was no significant difference in survival between sexes or treatment groups. n=9–17 animals per treatment group.

Figure 2.

(A) MRI scans were used to assess edema (T2) and infarct volume (DTI) at 72 h after MCAO. Although there was not a significant overall difference in (B) edema or (C) infarct volume between PBS- or LIF-treated male and female rats, there was a trend towards decreased tissue damage after LIF treatment in aged female rats. n=9–17 animals per treatment group.

Figure 3.

Four tests were used to determine motor skill function at 72 h after MCAO. (A) For the Circling test, there was a trend towards improvement in aged female LIF-treated rats that approached significance compared to PBS-treated females, but this trend was not observed in aged males. (B) A significant two-way interaction was observed between sex and drug treatment in the EBST results; LIF-treated rats performed significantly better than PBS-treated females and LIF-treated males. (C) LIF-treated females showed significant improvement compared to PBS-treated females in the Paw Extension test, but male LIF-treated rats showed no improvement compared to PBS-treated males. (D) Neither males nor females showed improvement according to the Step Test results.

Figure 4.

SOD activity was measured in the ipsilateral brain tissue samples from aged male and female rats. Neither drug treatment (PBS or LIF) had any significant effect on SOD enzyme activity at 72 h after MCAO.

Figure 5.

(A) Representative ipsilateral brain tissue sections from PBS and LIF-treated young rats were stained with antibodies against LIFR (red) and MAP2 (green). DAPI (blue) was used to label cell nuclei. LIFR immunoreactivity was apparent in the cytosol and membrane regions of neurons at 72 h after MCAO. Arrows indicate representative cells. Scale bar = 20 µm. (B) After LIF treatment, membrane localization of LIFR was apparent in the ipsilateral cortex, but nuclear localization of LIFR was more prominent in the contralateral cortex. Arrows indicate representative cells. Scale bar = 50 μm

Figure 6.

(A) Representative ipsilateral brain tissue sections from PBS- and LIF-treated aged male and female rats were stained with antibodies against LIFR (red) and MAP2 (green) to visualize LIFR localization after MCAO. Nuclei were labeled with DAPI (blue). Stained contralateral tissue served as a negative control. LIFR localization in neuronal nuclei and cytosolic/membrane regions was found in cortical neurons from rats in all treatment groups. Arrows indicate representative cells. (B) DAB-stained tissue sections also showed membrane localization of LIFR in the ipsilateral tissue of all treatment groups. Arrows indicate representative cells. Scale bar = 50 μm.

Figure 7.

(A) Primary neurons were treated with PBS or LIF and subjected to 24 h OGD. LIFR mRNA was significantly elevated in neurons treated with LIF compared to those treated with PBS (*p<0.05). n= 3 wells per group. (B) However, LIFR expression in the ipsilateral tissue of aged rats of both sexes was not altered by LIF treatment at 72 h after MCAO.

Figure 8.

Brain tissue sections from young male and aged (male and female) LIF-treated rats were stained with antibodies against LIFR (red) and vWF (green) to visualize LIFR localization to cerebral endothelial cells. DAPI (blue) was used to label nuclei. LIFR- positive endothelial cells were visualized in after LIF-treated in the brains of young male rats. LIFR immunoreactivity was less prominent in the brains of LIF-treated aged male and female rats compared to young male rats. Scale bar = 50 μm.

Figure 9.

LIFR protein levels were measured in the ipsilateral brain tissue from the young male, aged male, and aged female rats after MCAO. LIFR levels were significantly decreased in aged male (*p<0.05) and aged female (*p<0.05) compared to young male rats. There was also a significant decrease in LIFR levels between aged male and aged female rats after MCAO. n = 4–6 animals per treatment group.