Platelets cause microvascular occlusion and delayed neurological deficits after subarachnoid hemorrhage in mice

Fig. 1. Schematics of the experimental timelines.

Fig. 2. Plasma PAF and TXB2 are elevated on days 1 and 5 post-SAH. a–d Levels of plasma PAF (a), thrombin (b), TXB2 (c), and PF4 (d). Female mice were used (n = 6 per group per time point). *P < 0.05 versus sham, #P < 0.05 versus SAH day 1, †P < 0.05 versus SAH day 3, and ‡P < 0.05 versus SAH day 5.

Fig. 3. Exogenous PAF worsens outcomes after SAH in mice. a–c PAF administered at 1-h post-SAH causes higher mortality (a Sham + PAF n = 7 per sex, SAH + saline n = 18 per sex, SAH + PAF n = 18 per sex) and more brain microthrombi (c n = 5 per sex per group) than saline-treated female and male mice. Neuroscore (b) was similar for SAH + saline (females: n = 18, males: n = 18) and SAH + PAF (females: n = 11, males: n = 9), and both SAH groups had neuroscores significantly lower than Sham+PAF (females, n = 7; males, n = 6). d–f In female mice, PAF administered 3 days after SAH causes worse neurological behavior (d mean with s.e.m., sham + PAF n = 9–10, SAH + saline n = 16–26, and SAH + PAF n = 13–26), a higher rate of DND (e sham + PAF n = 10 and SAH n = 26) and more brain microthrombi (f n = 6 per group). Representative images of MSB-stained brain slices (c and f scale bar=100 μm) showing microthrombi (selected microthrombi marked with yellow asterisks) are best observed when zoomed in. *P < 0.05 sham + PAF versus SAH + saline, #P < 0.05 sham + PAF versus SAH + PAF, and †P < 0.05 SAH + saline versus SAH + PAF.

Fig. 4. Delayed platelet activation causes DND after SAH. a, b LIGHT−/− mice, which develop platelet activation 3 days after injury, have a higher DND incidence than WT mice. For the neuroscore (a mean with s.e.m.), sham n = 7 per sex, female WT SAH n = 19–25, female LIGHT−/− SAH n = 14–30, male WT SAH n = 19–25, and male LIGHT−/− SAH n = 21–29. For the incidence of DND (b), WT SAH n = 25 per sex, female LIGHT−/− SAH n = 27, and male LIGHT−/− SAH n = 29. *P < 0.05 LIGHT−/− sham versus LIGHT−/− SAH, #P < 0.05 WT sham versus WT SAH, and †P < 0.05 LIGHT−/− SAH versus WT SAH.

Fig. 5. Platelet depletion reduces DND after SAH. a–c, After SAH, mice depleted of platelets (PF4-DTR + DT), compared with nondepleted (WT + DT) mice, had better neuroscore (a mean with s.e.m.), less DND (b), and fewer brain microthrombi (c) (scale bar = 100 μm, representative images of MSB-stained brain slices with yellow asterisks marking selected microthrombi (best observed zoomed in)). All mice received the entire DT regimen. For neuroscore (a), sham n = 8 per sex, female WT SAH n = 20–30, female (PF4-DTR) SAH n = 27–29, male WT SAH n = 25–30, and male (PF4-DTR) SAH n = 30–31. For DND (b), WT SAH n = 30 per sex, and (PF4-DTR) SAH n = 29 female and n = 31 male. *P < 0.05 WT sham versus WT SAH, #P < 0.05 (PF4-DTR) sham versus (PF4-DTR) SAH, and †P < 0.05 WT SAH versus (PF4-DTR) SAH.

Fig. 6. Platelet antagonists can improve outcomes after SAH. Mice were euthanized 2 days after SAH. a Day 1 neuroscore (n = 9–10 per sex per group). b Day 2 brain microthrombi count (n = 6 per sex per group) (scale bar = 100 μm, representative images of MSB-stained brain slices with yellow asterisks marking selected microthrombi are best observed zoomed in). *P < 0.05 versus sham and #P < 0.05 versus SAH + vehicle 1 or 2, as appropriate.

Fig. 7. Tirofiban reduces microthrombi and DND after SAH. Female mice were allowed to survive for up to 7 days post-SAH. a For neuroscore, sham n = 10, SAH + vehicle 1 n = 10–20, SAH + vehicle 2 n = 15–20, SAH + daltroban n = 15–20, SAH + WEB2086 n = 14–20, and SAH + tirofiban n = 18–20. *P < 0.05 sham versus SAH + vehicle (1 or 2 as appropriate), #P < 0.05 sham versus SAH + daltroban, †P < 0.05 sham versus WEB2086, ‡P < 0.05 SAH + vehicle 1 versus SAH + daltroban, §SAH + vehicle 1 versus SAH + WEB2086, £SAH + daltroban versus SAH + WEB2086, @P < 0.05 sham versus SAH + tirofiban, and &P < 0.05 SAH + vehicle 2 versus SAH + tirofiban. b For DND, SAH + vehicle n = 40, and n = 20 for each antiplatelet group. c Day 7 brain microthrombi count (n = 6 per group; scale bar=100 μm, representative images of MSB-stained brain slices with yellow asterisks marking selected microthrombi are best observed zoomed in).

Fig. 8. Tirofiban reduces platelet activation in human aneurysmal SAH samples. Representative images show the platelet morphology changes (activated platelets). Selected activated platelets are marked with a yellow arrow. *P < 0.05 versus SAH + saline and #P < 0.05 versus SAH + tirofiban.