Hyperglycemia promotes tissue plasminogen activator-induced hemorrhage by Increasing superoxide production

Abstract

Objective: Risk of intracerebral hemorrhage is the primary factor limiting use of tissue plasminogen activator (tPA) for stroke. Clinical studies have established an association between admission hyperglycemia and the risk of hemorrhage with tPA use, independent of prior diabetes. Here we used an animal model of tPA-induced reperfusion hemorrhage to determine if this clinical association reflects a true causal relationship.

Methods: Rats underwent 90 minutes of focal ischemia, and tPA infusion was begun 10 minutes prior to vessel reperfusion. Glucose was administered during ischemia to generate blood levels ranging from 5.9 ± 1.8mM (normoglycemia) to 21 ± 2.3mM. In some studies, apocynin was administered to block superoxide production by nicotinamide adenine dinucleotide phosphate (NADPH). Brains were harvested 1 hour or 3 days after reperfusion to evaluate the effects of hyperglycemia and apocynin on oxidative stress, blood-brain barrier breakdown, infarct volume, and hemorrhage volume.

Results: Rats that were hyperglycemic during tPA infusion had diffusely increased blood-brain barrier permeability in the postischemic territory, and a 3- to 5-fold increase in intracerebral hemorrhage volumes. The hyperglycemic rats also showed increased superoxide formation in the brain parenchyma and vasculature during reperfusion. The effects of hyperglycemia on superoxide production, blood-brain barrier disruption, infarct size, and hemorrhage were all attenuated by apocynin.

Interpretation: These findings demonstrate a causal relationship between hyperglycemia and hemorrhage in an animal model of tPA stroke treatment, and suggest that this effect of hyperglycemia is mediated through an increase in superoxide production by NADPH oxidase.

Figure 1. Experimental design

(A) Hyperglycemia was induced by glucose injections at the designated time points. tPA infusion was begun 10 minutes before end of MCA occlusion. Controls received tPA vehicle. (B) Arterial blood glucose concentrations before, during, and after MCA occlusion. Filled symbols, tPA treatment; open symbols, vehicle treatment.

Figure 2. Hyperglycemia increases tPA-induced brain hemorrhage

(A) Each column shows coronal sections of a representative brain from the designated treatment group. Arrows denote some of the hemorrhage areas. Scale bar = 1 cm. (B) Hemorrhage is quantified as the volume of blood per brain. Results from two separate experiments are combined. n = 6–7; P < 0.02 for effects of both glucose and tPA on hemorrhage volume and P < 0.01 for linear trend between glucose and hemorrhage volume in the tPA-treated groups.

Figure 3. Hyperglycemia-induced superoxide production in re-perfused cortex is blocked by apocynin

(A) Brains were harvested from normoglycemic (5 mM glucose) and hyperglycemic (15 mM glucose) rats 1 hour after reperfusion with tPA. Photomicrographs show superoxide production identified by ethidium fluorescence (red) in representative sections. Scale bar = 40 μm. (B) Histogram shows normalized ethidium fluorescence of cells in cortex of a representative brain from each treatment group, with arrows indicating the median values. Graph shows mean (± s.e.m.) of the median values from the 4 rats in each group; *P < 0.05. (C,D). In a separate experiment, hyperglycemic rats treated were treated with apocynin or vehicle during tPA reperfusion, and brain sections were evaluated as in A,B. n = 5–7; *P < 0.05.

Figure 4. Apocynin reduces hemorrhage in tPA-treated brains

Hyperglycemic rats were pre-treated with apocynin (or vehicle) prior to reperfusion with tPA, and brains were harvested 3 days later. (A) Each column of coronal sections shows a brain from the designated treatment group. Scale bar = 1 cm. (B) Hemorrhage is quantified as volume of blood per brain. n = 6; **P < 0.01.

Figure 5. Effects of hyperglycemia on infarct size and blood-brain barrier

Brains were harvested 3 days after ischemia-reperfusion. (A) Infarct size in tPA-treated rats was increased by hyperglycemia and reduced by apocynin. n = 6–7; * P < 0.05 vs. 5 mM, ^#P < 0.05 vs. 15 mM. (B) Blood-brain barrier disruption identified by immunostaining for IgG (dark brown) in representative brain sections. (C) Graph shows quantified IgG staining. P < 0.02 for an effect of glucose on IgG staining in both tPA and vehicle-treated brains, with P < 0.01 for linear trend between glucose and IgG staining. (D) Representative images from a separate experiment in which hyperglycemic rats were pre-treated with apocynin or vehicle prior to tPA reperfusion. (E) Graph shows quantified IgG staining. n = 6–7; *P < 0.05. Panels A and C show results combined from 2 separate experiments.