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Poststroke delivery of MANF promotes functional recovery in rats

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Science Advances  23 May 2018:
Vol. 4, no. 5, eaap8957
DOI: 10.1126/sciadv.aap8957
  • Fig. 1 AAV7-MANF injection after ischemia results in enhanced behavioral recovery.

    (A) The rats underwent dMCAo surgery, and on day 1, they were divided into two groups, balanced according to the severity of their neurological deficit assessed by the EBST and BNST. On day 2, the animals received intracerebral injections of either AAV7-MANF or AAV7-GFP, and their behavioral recovery was evaluated on days 7 and 14. (B) Expression patterns of hMANF 2 weeks after intracerebral AAV7-MANF injection. Coronal sections of the lesioned cerebral hemisphere were immunostained using anti-hMANF antibody. Sections from positions close to injection sites (at approximately A/P +1.6 and A/P −0.4) are presented. The core of the ischemic lesion is outlined. Black and gray arrows mark the dorsal and lateral peri-infarct cortex, respectively, where the deeper layers are AAV7-transduced. (C) Close-up of hMANF expression in the peri-infarct region encompassing cortex (ctx), striatum (str), and external capsule (ec). Scale bar, 100 μm. (D and E) The behavior of rats was assessed with BNST (D) and the EBST (E) on days 7 and 14 after the dMCAo surgery (n = 9 in both groups). *P ≤ 0.003 by Mann-Whitney U test. (F) Forepaw use bias of AAV7-GFP– and AAV7-hMANF–injected rats as determined using the cylinder test. According to a two-way analysis of variance (ANOVA) [F(1,32) = 2.2056, P = 0.1613], the differences in right forepaw use were not statistically significant. Average ± SEM is shown.

  • Fig. 2 Chronic delivery of rhMANF results in enhanced behavioral recovery.

    (A) The rats underwent dMCAo surgery, and on day 2, they were divided into two groups, balanced according to the severity of their neurological deficit assessed by BNST. Intracerebral infusion of PBS (n = 5) or rhMANF (n = 5) was started 1 day later, with the cannula implanted into the peri-infarct region. On days 7, 14, and 24 after the dMCAo surgery, the behavior of rats was assessed with the EBST (B) and BNST (D). #P = 0.0317 by Mann-Whitney U test. (C) Forepaw use bias of the rats was assessed in the cylinder test on days 2, 14, and 24 after dMCAo. *P = 0.0257 by Bonferroni’s multiple comparisons test, following two-way ANOVA [effect of treatment: F(1,24) = 8.103, P = 0.0089]. Average ± SEM is shown.

  • Fig. 3 AAV7-MANF injection after ischemia results in enhanced reversal of behavioral deficits but does not reduce lesion size.

    (A) The rats underwent dMCAo surgery, and on day 2, they were divided into three groups, balanced according to size of ischemic lesion, as determined from T2-weighted MRI images. On the same day, rats received intracerebral injections of PBS (n = 9), AAV7-GFP (n = 7), or AAV7-hMANF (n = 9). The severity of their neurological deficit was assessed on day 16 using BNST (B) and the EBST (C). *P ≤ 0.0043 in Dunn’s multiple comparisons post hoc test, following Kruskal-Wallis test (P ≤ 0.0002). (D) T2-weighted MRI images representative of the development of lesion size from days 2 to 16 after dMCAo surgery. (E to G) The volume of ischemic lesion was measured from T2-weighted MRI images taken on days 2, 9, and 16 after the dMCAo surgery. For days 9 (E) and 16 (G), the lesion volume is expressed in relation to the size of the lesion in the same animal on day 2. P ≥ 0.1079 according to one-way ANOVA. Average ± SEM is shown.

  • Fig. 4 AAV7-MANF treatment increases the number of phagocytic immune cells in the subcortical regions.

    (A) qPCR analysis of Emr1, C3, Cfp, and Cd8a mRNA levels in the peri-infarct cortex at day 4 after ischemic injury (n = 4 to 5). ***P ≤ 0.0004 by Tukey’s multiple comparisons post hoc test, following one-way ANOVA (P < 0.0001). (B and C) Density of CD68+ objects in the corpus callosum/external capsule underlying the infarct at day 4 after ischemic injury. ***P = 0.0001. (D) Density of CD68+ objects in the dorsal striatum. **P = 0.0099. (E and F) Density of CD68+ objects in the lateral peri-infarct cortex. P = 0.7504. (G and H) Density of Arg1+ objects in the corpus callosum/external capsule. **P = 0.0053. (I) Density of Arg1+ objects in the dorsal striatum. **P = 0.006. (C to I) The individual points on graphs present the average of two coronal sections, taken from positions A/P +0.1 and −0.7. AAV7-GFP (n = 6) or AAV7-hMANF (n = 7). Representative image is shown for each area. Scale bars, 100 μm. (J) qPCR analysis of Manf mRNA levels in the injured and contralateral (contral.) cortex of Manffl/fl and NestinCre/+::Manffl/fl mice at 2 days after focal ischemic injury (n = 4 to 6). **P < 0.002 by Tukey’s multiple comparisons post hoc test, following one-way ANOVA (P < 0.0001). (K) Infarct volumes (averaged with SEM) in Manffl/fl and NestinCre/+::Manffl/fl mice sacrificed 2 days after a permanent MCAo. Lesion volumes were quantified from triphenyl tetrazolium chloride–stained brain sections. Student’s t test was used for the analysis of statistical significance. Average ± SEM is shown.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaap8957/DC1

    Supplementary Materials and Methods

    fig. S1. The expression patterns of MANF and GFP 2 weeks after intracerebral AAV7 injection.

    fig. S2. Postischemic AAV7-MANF injection does not affect spontaneous motor activity.

    fig. S3. AAV7-MANF injection after ischemia does not reduce lesion size.

    fig. S4. Analysis of anti-laminin and anti-synaptophysin immunoreactivity in the peri-infarct area.

    fig. S5. Analysis of anti-MBP immunoreactivity in the peri-infarct area.

    fig. S6. Analysis of anti–glial fibrillary acidic protein immunoreactivity in the peri-infarct area.

    fig. S7. AAV7-MANF injection after ischemia results in rapid hMANF expression and enhancement of behavioral recovery.

    fig. S8. Analysis of MBP and CD68 colocalization in the external capsule.

    fig. S9. Analysis of immune cell number on day 14 after AAV7-MANF treatment.

    fig. S10. qPCR analysis of alternative activation marker TGFb1 and Mrc1 (CD206) mRNA levels in the peri-infarct cortex at day 4 after ischemic injury (n = 4 to 5).

    fig. S11. Endogenous MANF has no effect on C3, Emr1, Arg1, or Mrc1 expression in the cortex after cerebral ischemic injury.

    table S1. Top hits from RNA sequencing analysis performed to find genes expressed differentially in the peri-infarct region of AAV7-GFP–treated (n = 3) versus AAV7-hMANF–treated (n = 3) rats.

    References (2530)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. The expression patterns of MANF and GFP 2 weeks after intracerebral AAV7 injection.
    • fig. S2. Postischemic AAV7-MANF injection does not affect spontaneous motor activity.
    • fig. S3. AAV7-MANF injection after ischemia does not reduce lesion size.
    • fig. S4. Analysis of anti-laminin and anti-synaptophysin immunoreactivity in the peri-infarct area.
    • fig. S5. Analysis of anti-MBP immunoreactivity in the peri-infarct area.
    • fig. S6. Analysis of anti–glial fibrillary acidic protein immunoreactivity in the peri-infarct area.
    • fig. S7. AAV7-MANF injection after ischemia results in rapid hMANF expression and enhancement of behavioral recovery.
    • fig. S8. Analysis of MBP and CD68 colocalization in the external capsule.
    • fig. S9. Analysis of immune cell number on day 14 after AAV7-MANF treatment.
    • fig. S10. qPCR analysis of alternative activation marker TGFb1 and Mrc1 (CD206) mRNA levels in the peri-infarct cortex at day 4 after ischemic injury (n = 4 to 5).
    • fig. S11. Endogenous MANF has no effect on C3, Emr1, Arg1, or Mrc1 expression in the cortex after cerebral ischemic injury.
    • table S1. Top hits from RNA sequencing analysis performed to find genes expressed differentially in the peri-infarct region of AAV7-GFP–treated (n = 3) versus AAV7-hMANF–treated (n = 3) rats.
    • References (25–30)

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