Reactive Gliosis Contributes to Nrf2-Dependent Neuroprotection by Pretreatment with Dimethyl Fumarate or Korean Red Ginseng Against Hypoxic-Ischemia: Focus on Hippocampal Injury

Abstract

Recently, dimethyl fumarate (DMF) and Korean red ginseng (ginseng), based on their purported antioxidative and anti-inflammatory properties, have exhibited protective potential in various neurological conditions. Their effects on cerebral ischemia and underlying mechanisms remain inconclusive; however, increasing evidence indicates the involvement of the transcriptional factor Nrf2. This study evaluated the preventive effects of DMF and ginseng on hippocampal neuronal damage following hypoxia-ischemia (HI) and assessed the contributions of reactive gliosis and the Nrf2 pathway. Adult wild type (WT) and Nrf2^-/- mice were pretreated with DMF or ginseng for 7 days prior to HI. At 24 h after HI, DMF or ginseng significantly reduced infarct volume (52.5 ± 12.3% and 47.8 ± 10.7%), brain edema (61.5 ± 17.4% and 39.3 ± 12.8%), and hippocampal CA1 neuronal degeneration, and induced expressions of Nrf2 target proteins in WT, but not Nrf2^-/-, mice. Such hippocampal neuroprotective benefits were also observed at 6 h and 7 days after HI. The dynamic attenuation of reactive gliosis in microglia and astrocytes correlated well with this sustained neuroprotection in an Nrf2-dependent manner. In both early and late stages of HI, astrocytic dysfunctions in extracellular glutamate clearance and water transport, as indicated by glutamine synthetase and aquaporin 4, were also attenuated after HI in WT, but not Nrf2^-/-, mice treated with DMF or ginseng. Together, DMF and ginseng confer robust and prolonged Nrf2-dependent neuroprotection against ischemic hippocampal damage. The salutary Nrf2-dependent attenuation of reactive gliosis may contribute to this neuroprotection, offering new insight into the cellular basis of an Nrf2-targeting strategy for stroke prevention or treatment.

Keywords: Astrocyte; Astrogliosis; Microglia; Oxidative stress; Stroke; Transcriptional factor.

Fig. 1. Pretreatment with DMF or Ginseng reduces ischemia-induced infarct volume, brain edema, and hippocampal CA1 neuron loss 24 h after HI in WT mice, but not in Nrf2^−/− mice.

(a) Representative images of TTC-stained brain sections (n=4–6 per group). (b and c) Quantitative analyses of infarct volume and brain edema. (d) Representative images of CV-stained hippocampus (n=3 per sham group and n=4–5 per ischemic group). (e) Quantitative analyses of hippocampal CA1 neurons indicated by CV staining. 24 h after HI, ischemia-induced infarct volume, brain edema, and hippocampal CA1 neuronal loss were significantly reduced in WT, but more importantly, not in Nrf2^−/− mice pretreated with DMF or Ginseng, whereas Nrf2 deficiency significantly exacerbated the deterioration process. *P<0.05, **P<0.01, ^#P<0.05. TTC: 2,3,5-triphenyltetrazolium chloride; CV: Cresyl violet. Scale bar, 2 mm (a), 500 μm (d, hippocampus) and 50 μm (d, CA1 sub-region).

Fig. 2. In WT but not Nrf2^−/− mice pretreated with DMF or Ginseng, Nrf2 target antioxidant protein are significantly increased 24 h after HI.

(a, b) At 24 h after HI, the tissue from the ipsilateral hippocampal CA1 region was used for analysis of Nrf2 target proteins levels. Pretreatment with DMF or Ginseng significantly increased the expression levels of Nrf2 target antioxidant proteins NQO1, HO1, GPx1, and SOD2. More importantly, such benefits were not detected under Nrf2 deficiency. n=3–4 per group. *P<0.05, ^#P<0.05.

Fig. 3. Pretreatment with DMF or Ginseng attenuates ischemia-induced reactive gliosis in microglia and astrocytes, including astrocytic dysfunction of glutamate metabolism and water homeostasis 24 h after HI in WT but not Nrf2^−/− mice.

(a) Representative images of Iba1, GFAP, GS, and AQP4 stained hippocampal CA1 regions (n=4–5 per group). (b–e) Quantitative analyses of (a). In normal conditions, microglia and astrocytes tiled the whole CA1 region in a regular distribution pattern in both WT and Nrf2^−/− mice, mostly exhibiting a nonreactive status with small soma and fine processes. Ischemia triggered apparent proliferation 24 h after HI as indicated by increased immunoreactivity of glial cells, with hypertrophic soma and highly stained processes. Pretreatment with DMF or Ginseng significantly attenuated such process in WT mice, but failed to do so in Nrf2^−/− mice. In addition, pretreatment with DMF or Ginseng significantly protected against the sharp increase of GS and overt decline of AQP4 expression levels in WT but not Nrf2^−/− mice. n=4–5 per group. *P<0.05, **P<0.01, ^#P<0.05, ^##P<0.01. Iba1, ionized calcium-binding adapter protein 1; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; AQP4, aquaporin 4. Scale bar, 50 μm.

Fig. 4. The early phase of reactive gliosis and ischemia-induced neuronal injury are profoundly mitigated by pretreatment with DMF or Ginseng, whereas Nrf2 deficiency reduces such effects.

(a) Representative images of CV-, Iba1, GFAP, GS, and AQP4 stained hippocampal CA1 regions at 6 h after HI (n=4–5 per group). (b–f) Quantitative analyses for (a). The early phase of ischemia-induced hippocampal CA1 neuronal injury was significantly attenuated in WT but not Nrf2^−/− mice pretreated with DMF or Ginseng, whereas Nrf2 deficiency exacerbated such an injury. Consistently, reactive gliosis in microglia and astrocytes including astrocytic dysfunction of glutamate metabolism and water homeostasis was significantly reduced by pretreatment with DMF or Ginseng, but not under Nrf2 deficiency, as revealed by the intensity of Iba1, GFAP, GS, and AQP4 staining. *P<0.05, ^#P<0.05. CV: Cresyl violet; Iba1, ionized calcium-binding adapter protein 1; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; AQP4, aquaporin 4. Scale bar, 500 μm (a, hippocampus) and 50 μm (a, CA1 sub-region).

Fig. 5. The attenuated reactive gliosis contributed to the late-stage of Nrf2-dependent neuroprotection by pretreatment with DMF or Ginseng, but not under Nrf2 deficiency.

(a) Representative images of CV-positive neurons in hippocampal CA1 neurons. (b–f) Quantitative analyses of (a). It appears that in the late phase of ischemia, hippocampal CA1 injury had a slight recovery 7 days after HI. After pretreatment with DMF or Ginseng, WT but not Nrf2^−/− mice display a significantly better recovery process, as revealed by the average density of hippocampal CA1 neurons. However, Nrf2 deficiency significantly worsened the injury compared to ischemic WT controls. n=5–7 per group. *P<0.05, ^#P<0.05. CV: Cresyl violet; Iba1, ionized calcium-binding adapter protein 1; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; AQP4, aquaporin 4. Scale bar, 500 μm (a, hippocampus) and 50 μm (a, CA1 sub-region).

Fig. 6. Schematic representation of dynamic reactive gliosis in response to hypoxia-ischemia insults.

Following hypoxia-ischemia, acute ischemic insults promptly trigger activation of microglia and astrocytes, characterized by hypertrophic soma with thickened and retracted processes. With the rapid ischemic injury progression, the activated glial cells proliferate especially at the lesion sites to limit the injury and promote the repair. Nrf2 deficiency markedly exacerbates the progression above, along with the more severe neuronal degeneration in ischemic hippocampal CA1 region. In contrast, Nrf2 activation by DMF or Ginseng dramatically attenuates reactive gliosis, correlated well with the neuronal protection.