Increased Plasma Matrix Metalloproteinase-9 Levels Contribute to Intracerebral Hemorrhage during Thrombolysis after Concomitant Stroke and Influenza Infection

Cerebrovasc Dis Extra. 2016;6(2):50-9. doi: 10.1159/000447750. Epub 2016 Aug 25.


Background: Thrombolysis is the only approved therapy for acute stroke. However, life-threatening complications such as intracerebral hemorrhage (ICH) can develop after intravenous administration of tissue plasminogen activator (tPA). Both infection and thrombolysis during cerebral ischemia disrupt the blood-brain barrier (BBB). tPA can induce matrix metalloproteinase-9 (MMP-9), which is known to be involved in BBB disruption. However, it has still not been investigated whether preexisting influenza virus infection during thrombolysis after acute stroke affects systemic levels of MMP-9 and its inhibitor TIMP-1 and whether increased systemic MMP-9 levels affect ICH. This study aimed to investigate the influence of influenza virus infection on plasma levels of MMP-9 and TIMP-1 after thrombolysis in acute stroke, and to determine whether the infection correlates with intracerebral bleeding.

Methods: C57BL/6 mice were infected by administering 1 × 105 plaque-forming units of human influenza (H1N1) virus intranasally. After 3 days of infection the middle cerebral artery was occluded for 45 min and then reperfused. Intravenous tPA (10 mg/kg) treatment was started 10 min after stroke onset. Twenty-four hours after stroke onset, mice were deeply anesthetized with ketamine, venous blood was drawn from the caval vein and centrifuged at 2,000 rpm, and the supernatant was collected and frozen at -80°C. Plasma levels of MMP-9 and TIMP-1 were quantified by using ELISA.

Results: After stroke, plasma MMP-9 was significantly increased in mice with a concomitant influenza infection that were treated with tPA (9.99 ± 0.62 ng/ml, n = 7) as compared to noninfected control mice that were treated with tPA (4.74 ± 0.48 ng/ml, n = 8). Moreover, plasma levels of TIMP-1, an inhibitor of MMP-9, were also significantly increased in mice treated with tPA after concomitant infection and stroke (42.17 ± 7.02 ng/ml, n = 7) as compared to noninfected control mice that were treated with tPA after stroke (20.22 ± 2.12 ng/ml, n = 8). MMP-9 values significantly correlated with intracerebral hemoglobin levels in animals treated with tPA after stroke (p = 0.028, r = 0.76, n = 8) and after concomitant stroke and infection (p = 0.039, r = 0.78, n = 7).

Conclusion: Preexisting influenza A virus infection led to increased plasma MMP-9 and TIMP-1 levels in mice undergoing thrombolysis after induced stroke. MMP-9 levels closely correlated with intracerebral bleeding after thrombolysis during concomitant infection and stroke. Thus, our data indicate that thrombolysis may be dangerous during influenza infection. MMP-9 inhibitors might be considered to reduce the side effects of thrombolysis during concomitant infection and stroke.

MeSH terms

  • Animals
  • Blood-Brain Barrier / drug effects
  • Cerebral Hemorrhage / enzymology
  • Cerebral Hemorrhage / etiology*
  • Cerebral Hemorrhage / physiopathology
  • Humans
  • Influenza A Virus, H1N1 Subtype
  • Male
  • Matrix Metalloproteinase 9 / blood*
  • Mice, Inbred C57BL
  • Orthomyxoviridae Infections / complications*
  • Random Allocation
  • Stroke / complications
  • Stroke / drug therapy*
  • Stroke / pathology
  • Thrombolytic Therapy / adverse effects*
  • Tissue Inhibitor of Metalloproteinase-1 / blood
  • Tissue Plasminogen Activator / adverse effects
  • Tissue Plasminogen Activator / therapeutic use


  • Timp1 protein, mouse
  • Tissue Inhibitor of Metalloproteinase-1
  • Tissue Plasminogen Activator
  • Matrix Metalloproteinase 9
  • Mmp9 protein, mouse