Isoflurane posttreatment reduces brain injury after an intracerebral hemorrhagic stroke in mice

Abstract

Background: Intracerebral hemorrhage (ICH) is a devastating stroke subtype affecting 120,000 Americans annually. Of those affected, 40%to 50% will die within the first 30 days, whereas the survivors are left with a lifetime of neurobehavioral disabilities. Recently, it has been shown that volatile anesthetics such as isoflurane can reduce brain injury after an ischemic stroke. As a result, in this study, we investigated the effects of isoflurane as a posttreatment therapeutic modality in ICH-injured mice. Specifically, we investigated whether isoflurane posttreatment can preserve the structural integrity of the brain by reducing apoptotic damage and, in turn, improve functional outcome by amelioration of brain edema and neurobehavioral deficits.

Methods: Male CD1 mice (n = 53) were divided into the following groups: sham (n = 14), ICH (n = 14), ICH treated with 1.5% isoflurane posttreatment for 1 hour (n = 15), and ICH treated with 1.5% isoflurane posttreatment for 2 hours (n = 10). The blood injection ICH model was adapted; this involved extracting autologous blood from the mouse tail and injecting it directly into the right basal ganglia. One hour after surgery, treated mice were placed in a glass chamber maintained at 37°C and infused with 1.5% isoflurane for 1 or 2 hours. At 24 hours postinjury, mice were assessed for neurobehavioral deficits using the Modified Garcia Score and then killed and assessed for brain water content. Double immunofluorescent staining was performed using neuronal marker MAP-2 and TUNEL under a fluorescent microscope to assess for apoptosis.

Results: Our results indicated that after 1-hour 1.5% isoflurane posttreatment, there was a significant reduction in brain edema, a decrease in apoptotic cell death, and a significant improvement in neurobehavioral deficits.

Conclusions: Our results suggest that isoflurane may be an effective posttreatment therapeutic option for ICH because of its ability to reduce structural damage and subsequently preserve functional integrity.

Figure 1. Isoflurane Posttreatment Reduces Brain Edema

(A) Brain edema 24hrs after operation in sham, vehicle, and isoflurane-treated mice (p-value vehicle vs. treatment group = 0.002). # Significant Difference vs. Sham (p<0.05); * Significant Difference vs. Vehicle (p<0.05); (B) Gross pictures of the mouse brain displaying the needle site with corresponding hematoma in the right basal ganglia

Figure 2. Isoflurane Posttreatment Reduces Neurobehavioral Deficits

(A) Modified Garcia test 24hrs after operation in sham, vehicle, and isoflurane-treated mice (p-value vehicle vs. 1hr treatment group =0.003; vehicle vs 2hr treatment group 0.17). # Significant Difference vs. Sham (p<0.05); * Significant Difference vs. Vehicle (p<0.05); (B) Neurological outcomes were assessed by a blinded observer at 24hrs post-intracerebral hemorrhage (ICH) using the Modified Garcia Score, a 21-point sensorimotor seven –test assessment system with scores of 0–3 for each test (max score = 21).

Figure 3. Isoflurane Posttreatment Reduces Apoptotic Cell Death

(A) Representative photographs of immunofluorescence staining using TUNEL and MAP-2 staining; (B) Corresponding cell death quantification (p-values for vehicle vs treatment group = 0.002 and 0.003); # Significant Difference vs. Sham (p<0.05); * Significant Difference vs. Vehicle (p<0.05);

Figure 3. Isoflurane Posttreatment Reduces Apoptotic Cell Death

(A) Representative photographs of immunofluorescence staining using TUNEL and MAP-2 staining; (B) Corresponding cell death quantification (p-values for vehicle vs treatment group = 0.002 and 0.003); # Significant Difference vs. Sham (p<0.05); * Significant Difference vs. Vehicle (p<0.05);

Figure 4. Reductions in Cell Death Correlate with Reduced Neurobehavioral Deficits

TUNEL+/MAP2+ cells were plotted against neurobehavioral scores. A Lowess regression line (tension parameter of 0.5) was added to assess the shape of the relationship.