The spleen contributes to stroke-induced neurodegeneration

Abstract

Stroke, a cerebrovascular injury, is the leading cause of disability and third leading cause of death in the world. Recent reports indicate that inhibiting the inflammatory response to stroke enhances neurosurvival and limits expansion of the infarction. The immune response that is initiated in the spleen has been linked to the systemic inflammatory response to stroke, contributing to neurodegeneration. Here we show that removal of the spleen significantly reduces neurodegeneration after ischemic insult. Rats splenectomized 2 weeks before permanent middle cerebral artery occlusion had a >80% decrease in infarction volume in the brain compared with those rats that were subjected to the stroke surgery alone. Splenectomy also resulted in decreased numbers of activated microglia, macrophages, and neutrophils present in the brain tissue. Our results demonstrate that the peripheral immune response as mediated by the spleen is a major contributor to the inflammation that enhances neurodegeneration after stroke.

Fig. 1. Histological pathology of infarct in brain tissue of splenectomized rats 96 hr after MCAO

a: Fluoro-Jade staining shows a large area of damage in the ipsilateral hemisphere in the MCAO only group (center), whereas splenectomy reduces Fluoro-Jade-labeled degenerating neurons (right) and is similar in staining to tissue from sham-operated animals (left). b: Thionin staining of Nissl in living neurons in sections from sham-operated animals (left), MCAO-only animals (center), and splenectomized rats that underwent MCAO (right). Although there was extensive damage in the MCAO-only group as determined by thionin staining (center), splenectomized rats (right) that were subjected to stroke show reduced loss of tissue (scale bar = 5 mm, a,b). When infarct size was measured, there was significantly less damage in the brain of splenectomized rats after MCAO than in the MCAO-only group as determined with both Fluoro-Jade (c; *P < 0.001) and thionin (d; *P < 0.001) histological procedures. Significance was determined by ANOVA followed by Bonferroni’s post hoc test.

Fig. 2. Immunohistochemical labeling of microglia and infiltrating macrophages at the level of infarction

a: There were few isolectin IB4–labeled microglia or macrophages in brain sections from sham-operated rats. Arrows point to intact fasciolae of the striatum. b: Box denotes area labeled with isolectin IB4 in which activated microglia and macrophages are found. The arrow points to endothelial cells of a blood vessel labeled by isolectin IB4. c: Isolectin IB4 labeling of brain sections from splenectomized shows reduced microglia and macrophage labeling localized to a smaller area, as seen inside the box. Scale bar = 400 µm.

Fig. 3. MPO staining of neutrophils in the infarct zone

a: Immunohistochemical labeling of brain sections for MPO shows no neutrophils in the striatum of the sham-treated rats. b: MPO staining in brain sections from rats treated with MCAO only show many neutrophils in the striatum and a disappearance of fasciolae. Arrows denote labeled neutrophils. c: Brain sections from rats treated with splenectomy before stroke also had intact fasciolae similar to the sham-treated animals, but unlike the MCAO-only animals, there were few neutrophils within the striatum. Scale bars = 200 µm. d: MPO-positive neutrophils expressed as a function of infarct volume show a tendency for there to be more neutrophils in the MCAO group compared with the splenectomy–MCAO group. ANOVA followed by Bonferroni’s post hoc test showed no significant difference between these groups (P = 0.234). e: The linear regression of the correlation of the percentage of infarct volume to number of MPO-positive cells shows that the number of neutrophils significantly increases as infarction volume increases (R² = 0.6038, *P < 0.001).

Fig. 4

White blood cell profiles during treatments. White blood cell profiles were determined at 0, 48, and 96 hr after stroke for rats that underwent (a) MCAO only; (b) splenectomy followed 2 weeks later by MCAO; (c) sham MCAO; or (d) splenectomy and sham MCAO. e: White blood cell profiles were also determined during the 2-week recovery period between splenectomy and stroke. ANOVA followed by the post hoc Bonferroni’s post hoc test showed no significant differences between the groups.