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. 2011 Dec 16;3(1):16.
doi: 10.1186/2040-7378-3-16.

Sex-independent neuroprotection with minocycline after experimental thromboembolic stroke

Md Nasrul Hoda  1 , Weiguo Li, Ajmal Ahmad, Safia Ogbi, Marina A Zemskova, Maribeth H Johnson, Adviye Ergul, William D Hill, David C Hess, Irina Y Sazonova
Affiliations

Sex-independent neuroprotection with minocycline after experimental thromboembolic stroke

Md Nasrul Hoda et al. Exp Transl Stroke Med. .

Abstract

Background: Minocycline provides neurovascular protection reducing acute cerebral injury. However, it is unclear whether minocycline is effective in females. We tested minocycline in both sexes and aged animals using a novel embolic stroke model in mice that closely mimics acute thromboembolic stroke in humans.

Methods: Five groups of mice were subjected to thromboembolic stroke: adult males, aged males, adult females, aged females, and adult ovariectomized females. They were treated with phosphate saline (vehicle) or minocycline (6 mg/kg) immediately after stroke onset. Behavioral outcomes, infarct volumes and cerebral blood flow were assessed. The effect of minocycline on expression and activity of MMP-9 was analyzed.

Results: The model resulted in reproducible infarct in the experimental groups. As expected, adult females were significantly more resistant to cerebral ischemic injury than males. This advantage was abolished by aging and ovariectomy. Minocycline significantly reduced the infarct volume (P < 0.0001) and also improved neurologic score (P < 0.0001) in all groups. Moreover, minocycline treatment significantly reduced mortality at 24 hours post stroke (P = 0.037) for aged mice (25% versus 54%). Stroke up-regulated MMP-9 level in the brain, and acute minocycline treatment reduced its expression in both genders (P < 0.0001).

Conclusion: In a thromboembolic stroke model minocycline is neuroprotective irrespective of mouse sex and age.

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Figures

Figure 1
Figure 1
Thromboembolic stroke model in C57BL/6 adult male mice. A) The clot is inserted at the MCA origin. Clots were visualized with Evan's Blue dye (arrowhead). B) Regional cerebral blood flow (CBF) over time as measured with Laser Doppler flowmetry (means ± SD, n = 7). The CBF declines to 18 ± 4% of baseline following embolization, which was associated with a reproducible sized infarct of the MCA territory (115 ± 22 mm3) at 24 post stroke hours. C, D) Representative infarct area at 24 hours as determined by T2 diffusion-weighted MRI (C) and with 2,3,5-triphenyltetrazolium chloride staining of the coronal brain sections (D). Additional representative images of thromboembolic stroke model are shown in the Additional file 1 (Figure S1), Additional file 2 (Figure S2), Additional file 3 (Figure S3), Additional file 9 (Additional Figure Legends) and Additional file 10 (Additional Methods).
Figure 2
Figure 2
Representative effect of minocycline in adult (A) and aged (B) males. Coronal TTC sections, outlining the infarct area in representative subjects treated by the phosphate-saline buffer (vehicle) or minocycline at 24 hours, are showed in the left panels. The corresponding change of regional CBF during the first 6 hours after stroke is showed on the right panel. The value of CBF is presented as means ± SD.
Figure 3
Figure 3
Representative effect of minocycline in adult (A), retired (B), and OVX (C) females. Coronal TTC section, outlining the infarct area in a representative subjects treated by vehicle or minocycline at 24 hours, are showed on the left panel. The corresponding change of regional CBF during the first 6 hours after stroke is showed on right panel. The value of CBF is presented as means ± SD.
Figure 4
Figure 4
Minocycline reduces brain tissue injury in males (A) and females (B). The infarct size of ischemic MCA territory was estimated as the percent volume of the total ischemic hemisphere. Regardless of treatment adult females had significantly smaller infarct volumes (both P < 0.0001, ***) than aged and OVX females who were not different than each other (P = 0.77). All data expressed as means ± SD.
Figure 5
Figure 5
Minocycline improves neurologic scores in males (A) and females (A). Neurologic evaluation was performed for surviving animals at 24 hours. Regardless of treatment adult females had significantly improved neurologic scores (both P < 0.0001, ***) than aged and OVX females who were not different than each other (P = 0.42). All data expressed as means ± SD.
Figure 6
Figure 6
Minocycline reduces level of MMP-9 in both genders. A) Densitometric analysis of immunoreactive band intensities and representative Western Blot showing expression of 92 kDa-MMP-9 in ipsilateral hemispheres of adult male and OVX female mice (n = 6-7 animals per group) at 6 hours after thromboembolization. Values are expressed as relative intensity normalized to 42 kDa-β-actin intensity. The saline-treated mice had higher levels of MMP-9 than minocycline treated animals (P < 0.0001, ***). Minocycline and sham groups were not significantly different from each other (P = 0.77). B) Densitometric analysis of brain MMP-9 activity and representative zymography. The brain injury shows increased variability of MMP-9 activity relative to sham, but there are no sign differences between treatment or sex groups. All data expressed as means ± SD.
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