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. 2012 Mar 22;73(6):1100-7.
doi: 10.1016/j.neuron.2012.01.020. Epub 2012 Mar 21.

Neuroprotection from stroke in the absence of MHCI or PirB

Jaimie D Adelson  1 , George E Barreto, Lijun Xu, Taeho Kim, Barbara K Brott, Yi-Bing Ouyang, Thorsten Naserke, Maja Djurisic, Xiaoxing Xiong, Carla J Shatz, Rona G Giffard
Affiliations

Neuroprotection from stroke in the absence of MHCI or PirB

Jaimie D Adelson et al. Neuron. .

Abstract

Recovery from stroke engages mechanisms of neural plasticity. Here we examine a role for MHC class I (MHCI) H2-Kb and H2-Db, as well as PirB receptor. These molecules restrict synaptic plasticity and motor learning in the healthy brain. Stroke elevates neuronal expression not only of H2-Kb and H2-Db, but also of PirB and downstream signaling. KbDb knockout (KO) or PirB KO mice have smaller infarcts and enhanced motor recovery. KO hippocampal organotypic slices, which lack an intact peripheral immune response, have less cell death after in vitro ischemia. In PirB KO mice, corticospinal projections from the motor cortex are enhanced, and the reactive astrocytic response is dampened after MCAO. Thus, molecules that function in the immune system act not only to limit synaptic plasticity in healthy neurons, but also to exacerbate brain injury after ischemia. These results suggest therapies for stroke by targeting MHCI and PirB.

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Figures

Figure 1
Figure 1
KbDb KO mice recover better than WT cohorts following MCAO. (A) Top: Example of sections of Cresyl Violet stained brains 7d post MCAO. Bottom: KO animals have significantly smaller infarct areas at 7d (p=0.03), but not 24hr (p=0.45), post MCAO. (B, C) KO animals outperform WT cohorts on motor tasks post MCAO. Average Rotarod (B) and Foot Fault (C) performances were assessed on days 2 and 7 post MCAO in all animals, and cohorts of each genotype were kept through 1 month survival with additional testing on days 14, 21 and 28. P values for the comparisons between genotypes on Rotarod on days 21 and 28 were 0.056 and 0.057 respectively, reflecting the reduced numbers of surviving animals. Groups were 19–25 mice (days 2 and 7), and 7–10 mice (days 14–28). P<0.05 refers to A–C. Data are represented as mean +/− SEM. See also Figure S1.
Figure 2
Figure 2
Expression of Kb and Db increases in brain of WT mice following MCAO. (A) Kb and Db gene expression was measured using qPCR at 24hr (n=7 for each experimental condition) or 7d (n=7 MCAO; n=4–8 Sham) post MCAO (Ipsi=Damaged hemisphere, Contra=undamaged hemisphere). Negative control data from KbDb KO samples demonstrates primer specificity (Kb:GAPDH=0.06 control relative to sham; Db:GAPDH=0.01 control relative to sham, p<10−8 for both). *P≤0.05, **p≤0.01. (B) Western blots showing protein expression of Kb 7d post MCAO in synaptosome-enriched preparations and (C) in synaptoneurosomes C=contralateral to injury; I=ipsilateral to injury; M1-4=different MCAO animals; S1-4=shams; KO=KbDb KO. (D, E) Immunohistochemistry of L5 in cortical penumbra using OX18 antibody. D: Majority of MHCI immunostaining (OX18; red) colocalizes with neuronal marker NSE (green) 7d post MCAO. E: GFAP (green; astrocytes) or Iba1 (green; microglia) 7d post MCAO. Scale bars D, E, 50μm. qPCR data represented as mean +/− SEM. See also Figure S2.
Figure 3
Figure 3
PirB KO enhances recovery and dampens astrocytic response; MCAO increases PirB proximal signaling. (A) KO animals have smaller infarct areas 7d post MCAO (n=10 per genotype), but not 24hr (n=7 for WT, n=8 for KO). Infarct area decreased 51% in KO mice at 7d vs 1d post MCAO (p=0.003). (B, C) KO animals outperform WT cohorts on motor tasks after MCAO. Average Rotarod (B) and Foot Fault (C) performances at 2d and 7d post MCAO. N=10–20 animals per condition. Rotarod performance of PirB WT was not significantly different at 7d compared to 2d, p=0.61. Data represented as mean +/− SEM. (D– F) Astroglial activation is diminished in cortical penumbra 7d post MCAO in PirB KO mice. (D) Cresyl violet stained section; rectangle indicates cortical area used for cell counting. (E) Representative images of astrocytes immunostained for GFAP at a distance of 400μm from the ischemic core. (F) Quantitation of GFAP+ cells in WT vs KO. *P<0.05. N=5 animals per experimental condition. Scale bars, 50μm. (G– J) Enhanced PirB expression and PirB proximal signaling following MCAO. (G) Protein extracts from 7d post MCAO (M) or sham (S) brains were immunoprecipitated with anti-PirB antibody followed by Western blotting for PirB. In each experiment (n=2), 4 hemispheres from sham (S1–4; S5–8) or MCAO brains (M1–4; M5–8) were pooled for protein extraction. C=contralateral to injury; I=ipsilateral to injury. (H) Expression of Kb, β2m and β-tubulin in brain lysates from (G). (I, J) Aliquots of protein extracts from (G) were subjected to immunoprecipitation for phospho-tyrosine (I) or SHP-2 (J) to assess levels of tyrosine phosphorylation of PirB, and SHP-2 recruitment to PirB. See also Figures S1, S3.
Figure 4
Figure 4
Diminished neuronal death in CA1 region of hippocampal organotypic slice cultures from KO mice following in vitro ischemia. PI-immunofluorescence (bright white) in CA1 region (top) and cumulative histograms of neuronal damage as measured by fluorescence intensity at 24hr after OGD (bottom) show that deletion of Kb and Db (A), or PirB (B), reduces cell death compared to WT. For cumulative histograms, each point is one slice (KbDb KO: n=55 vs WT n=52 slices from 5 animals; PirB KO: n=27 vs WT n=30 slices from 4 animals). Arrows indicate measured CA1 area. Scale bars, 800μm.
Figure 5
Figure 5
Increased corticospinal tract fibers in PirB KO vs WT mice 28d post MCAO. (A) Representative images of BDA-labeled crossed CST fibers originating in undamaged motor cortex and terminating in the red nucleus on the damaged side. (Midline is to right of each image.) Note more extensive labeling in KO. (B) The uncrossed CST projection to the red nucleus terminating on the undamaged side. (Midline is to left of each image.) Note similarity of labeling in KO vs WT. (C–E) Quantification of crossed fibers from undamaged CST by (C) total fiber length (WT=6.5±6.0mm, KO=9.9±1.3mm; p=0.032), (D) fiber number (WT=81.9±9.7, KO=118.1±11.1; p=0.036), or (E) number of fibers crossing the midline (WT=7.9±1.0, KO=11.2±0.6; p=0.024). N=5 KO, n=6 WT, 2–3 sections per animal analyzed. Scale bars, 100μm. Data are represented as mean +/− SEM.
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