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Monitoring the Protective Effects of Minocycline Treatment with Radiolabeled Annexin V in an Experimental Model of Focal Cerebral Ischemia

¹ Department of Neurology, University of California, San Francisco, and San Francisco Veterans Affairs Medical Center, San Francisco, California; ² Department of Anesthesia, Stanford University, Stanford, California; and ³ Department of Radiology, Stanford University, Stanford, California

View larger version (25K): [in this window] [in a new window]   FIGURE 1.  Minocycline treatment reduces infarct volume. (A) Representative cresyl violet–stained brain sections of mice subjected to ischemia show a smaller infarct in minocycline-treated mouse (b) compared with vehicle-treated mouse (a). (B) Representative mouse brain SPECT images at 1, 3, 7, and 30 d after stroke show less annexin V uptake (right side of image) after minocycline treatment compared with vehicle-treated controls. (C) Infarct sizes increased with a peak at 7 d (#P < 0.05 vs. other time points). Infarct volume was reduced by minocycline treatment (black bars) compared with vehicle-treated controls (white bars) on 1, 3, 7, and 30 d after stroke (**P < 0.01, n = 5/group). (D) Annexin V binding was increased after ischemia with peaks at 1 and 7 d. Minocycline treatment (black bars) reduced binding at all time points compared with vehicle-treated controls (white bars). Data for each hemisphere at each time point are graphed as average of maximal lesion uptake (ROI normalized to background).

View larger version (14K): [in this window] [in a new window]   FIGURE 2.  Minocycline treatment improves neurologic function. (A) Corner test: Each mouse was tested 20 times, and data were converted to the number of turns toward the ipsilateral ischemic hemisphere for 10 trials. Minocycline-treated animals (black bars) showed less turning to lesion side than vehicle- treated animals (white bars) (**P < 0.01). Compared with sham-operated animals (gray bar), minocycline-treated animals performed no differently on the corner test. (B) Ladder test: Each mouse was tested 3 times and counts were averaged. Minocycline-treated animals (black bars) showed fewer forelimb faults per 100 steps than vehicle-treated mice (white bars) (*P < 0.05).

View larger version (45K): [in this window] [in a new window]   FIGURE 3.  Annexin V binds to different cell types over time. Comparison of annexin V staining when substrate was applied directly to tissue sections (A) or injected into animals 1 h before sacrifice (B) (100 µg/kg protein per animal, intraperitoneally). The annexin V binding pattern (green) for both methods is similar, with some background staining and staining of some intracellular organelles seen in both. Clearly, the staining pattern in both methods is distinct from that seen after ischemia (C) (DAPI, blue). (C) Representative images of annexin V binding to different types of cells after ischemia. Brain sections were stained with Alexa Fluor 488–conjugated Neun, GFAP, or FITC-labeled tomato lectin (n = 5/group) and then colabeled with annexin V conjugated to Cy3.18. Three days after stroke, annexin V bound primarily neurons (Neun, arrowheads), whereas 30 d after stroke, annexin V bound microglia (Lectin, arrows). Rare astrocytes (GFAP) bound annexin V (representative image shown here 7 d after stroke). (D) Annexin V binds neurons acutely and microglia subacutely after experimental stroke. Double-labeled cells in 6 consecutive nonoverlapping x400 fields within periinfarct zone were counted and expressed as fraction of all cells labeled for the respective cell-type marker. One and 3 d after stroke, annexin V bound primarily neurons (solid line) but, at later time points (7 and 30 d), annexin V bound primarily microglia/monocytes (dashed line). There were few annexin V–positive astrocytes, and this pattern did not change over time (dashed–dotted line). *P < 0.05, **P < 0.01 vs. other cell types at a given time point. (A–C, scale bar = 75 µm).

View larger version (60K): [in this window] [in a new window]   FIGURE 4.  Minocycline reduces numbers of TUNEL-positive cells. (A) Representative TUNEL-stained images taken from periinfarct regions of ipsilateral ischemic cortex of minocycline-treated and vehicle-treated animals 3 d after stroke (inset shows representative cell with apoptotic nuclear morphology; x1,000 magnification), scale bar = 100 µm. (B) Minocycline treatment significantly reduces all TUNEL-positive cells per microscopic field (black bars = minocycline, light gray bars = vehicle; #P < 0.01) as well as numbers of TUNEL-positive cells displaying characteristic apoptotic nuclear morphology (dark gray bars = minocycline, white bars = vehicle, *P < 0.01) in ischemic hemisphere 1, 3, 7, and 30 d after stroke (n = 3 per group). HPF = high-power field.

View larger version (70K): [in this window] [in a new window]   FIGURE 5.  Minocycline treatment reduces monocyte/microglial activation and infiltration. Representative images 7 d after stroke show abundant activated microglia/monocytes within periinfarct area of vehicle-treated mouse (A) compared with few positive cells in minocycline-treated mouse (B) (arrows; IB4 stain). Scale bar = 215 µm. (C) Compared with vehicle (white bars), minocycline treatment (black bars) significantly reduced monocyte/microglial activation and infiltration into brain parenchyma around infarct area (*P < 0.001, n = 3/group). HPF = high-power field.